Advances in Microbial

Control of Insect Pests

Advances in Microbial
Control of Insect Pests
Edited by

Rajeev K. Upadhyay
Directorate of Plant Protection, Quarantine and Storage
Faridabad, India

Springer Science+Business Media, LLC

ISBN 978-1-4419-3395-9 ISBN 978-1-4757-4437-8 (eBook)
DOI 10.1007/978-1-4757-4437-8

©2003 Springer Science+Business Media New York

Origina1ly published by Kluwer AcademiclPlenum Publishers, New York in 2003

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PREFACE

The insect!1 remain in symbiotic associations with a tremendous number of

microorganisms, and some of them could be classified as parasitic/pathogenic. Without
question, insect pathogens act as natural mortality agents and represent the third leg of
the triad of biological control which is an environmentally sound alternative to chemical
control. The virulence and pathogenicity of an insect parasite i.e. disease agent are
determined by the microbial genome as a result of the coordinated expression of a
concert of genes. These genes may be organized as cassettes and be associated with
transmissible DNA. The acquisition of these domains or pathogenicity islands, may be
sufficient to develop a transgenic virulent pathogen. The insect pathogens are very
specific and this property can be exploited in making insects sick. However, rarely have
field applications of highly virulent strains of viruses, fungi, bacteria, protozoa resulted in
massive insect population reductions or induced widespread, persistent epizootics as the
same is also governed by host susceptibility regulated by genetics, age, sex and
physiological state of the host. Insect pathogens causing acute or chronic diseases must
be able to persist in the environment, to multiply in the host, and to spread to other
susceptible hosts. In this book, I have attempted to bring together all recent studies
regarding both fundamental and more applied research aspects related to
entomopathogens, bacteria, viruses, fungi and nematodes in order to facilitate their
development and commercial exploitation. There are 16 chapters written by 37 leading
research scientists working in their respective fields from allover the world.

Chapter 1-4: Bt and Bsp

The discovery of a bacterium with specific insecticidal activity was made with
Bacillus thuringiensis (Bt) in 1991 and the first attempt to use BT in insect control was
reported as early as the 1930s. The insecticidal activity of Bt and Bacillus sphaericus
(Bsp) is due to the presence of parasporal protein inclusion bodies, also called crystals,
produced during sporulation. These inclusions are composed of one of several specific
crystal protoxins (Cry, Crt and Bin toxins). The protoxins upon ingestion by insect larvae
get solubilized in the insect midgut releasing active toxins, which interact with receptors
resulting larval death. In these chapters, structure and binding of Cry toxins, genomics of
Bt, and their application in developing transgenic crops have been included, as well as
the mechanism of action of toxins and implications for resistance development.

Chapter 5-7: Baculoviruses

These chapters elaborate the structure, life cycle, genetics, genomics, mechanism of
action including molecular aspects, enhancement of virulence, commercial production
and delivery system ofbaculoviruses. The review papers are from three different leading
laboratories in China, USA and New Zealand and give new insight into the use of
baculoviruses in biological control of insect pests.

Chapter 8-12: Entomopathogenic Fungi

Two chapters i.e. 11 and 12 give a comprehensive account of various research

activities taking place and finding thereof in PR China and Latin America on
entomopathogenic fungi and their exploitation in biological control programs. Tsetse
flies, thrips in horti and floriculture, and brassica root flies have been taken up as case
studies in three different chapters i.e. 8, 9, 10 along with their biological control using
entomopathogenic fungi. These chapters have many new facets of knowledge.

v
Chapter 13-15: Entomopathogenic Nematodes (EPNs)

EPNs are biologically fascinating and economically important biological control

organisms that are extremely amendable to genetic manipulation. The information on
improving entomopathogenic nematode beneficial traits or eliminating weakness by
means of genetic manipulation using genetic engineering besides selection, selective
breeding, hybridization, and mutagenesis has been included in these chapters. An
overview of use of EPNs in biological control along with their biology, taxonomy,
production, commercialization and application has been discussed.

Chapter 16: Models of Pest Control

This chapter deals with the use of mathematical models to predict the effects of
parasites (virus, bacteria and fungi) on the dynamics of their hosts. Different kinds of
mathematical models given in this chapter could be used as vital tool in biological pest
control program.
Much work has been done on entomopathogens in recent years; however, a lot of
work still has to be done to improve their successful use in insect control. Moreover,
biointensive IPM programs desperately require more use of suitable microbes including
genetically engineered ones as still microbes do not have a significant share in pesticide
trade. There is an urgent need to develop coordinated interaction program using expertise
of entomologists, microbiologists, biochemists, molecular biologists and geneticists to
make use of effective microbes in biological control programs.
This book has been designed to be a reference book for university teachers,
researchers and advanced students of entomology, microbiology, biochemistry, microbial
genetics, molecular biology, zoology, pest management and biotechnology.
During the summer of 2000, I spent a couple of weeks in P.R. China particularly in
Wuhan City of Hubei State. In fact, extreme enthusiasm and optimism towards use of
microbes as biopesticides especially in younger scientists in P.R. China is the main
driving force in bringing out this book and for which I owe to them.
I should like to take this opportunity to thank all authors, and hope that contributions
made by them will enhance the interest for the larger public in entomopathogens and also
to Messers Neelam Graphics, New Delhi for meticulously preparing the ready to print
material. I am thankful to Kluwer Academic/Plenum Publishers for gladly agreeing to
publish this work, and I gratefully acknowledge the help, and collaboration given by
Miss Joanna Lawrence as publishing editor.

Rajeev K. Upadhyay
Faridabad, India

vi
LIST OF CONTRIBUTORS

I. Ananda Kumar, P. The Royal Veterinary and Agricultural

Principal Scientist, NRC for
Plant Biotechnology
Indian Agarciultural Research Institute
New Delhi-II 00 12, India
Tel: 091-11-5824787x240
Fax: 091-11-5766420
Email: polumetla@hotmail.com
2. Alves, Sergio Batista
Departmento de Entomologia
Fitopatologia e Zoologia Agricola
ESALQIUPS, Caixa Postal 9, \3418-900
Piracicada, SP, Brazil
Email: sebalves@esalq.usp.br
3. Brey, Christopher W.
Department of Entomology
Rutgers University, New Brunswick
New Jersey 08901, USA
Tel: (732) 932-9590
Fax: (732) 932-7229
Email: brey@rcLrutgers.edu
4. Boughton, Anthony, J.
Departmento de Entomology
Iowa state university
407 A, Science-II, Ames
lowa-50011-3222, USA.
Tele: (515) 294-1989
Fax: (515)294-5957
University, Thorvaldsensvej 40
DK 1871,Frederiksberg
C. (Copenhagen) Denmark
Tel: +4535282692
Fax: +4535282670
Email: joergen.eilenberg@ecol.kvl.dkor
jei@kvl.dk
9. Fairbairn, Jonathan
Department of Biological Sciences
University of Stirling, Stirling, FK9-4LA
Scotland, UK
Tel: 44(0) 1786,467819
Email :j.p.fairbaim@stir.ac.uk
10. Feng, Ming Guang
Director, Research Institute of Microbiology
College of Life Sciences, Zhejiang University
Hangzhou 310029, PR China
Tel: 86-571-86971129
Fax: 86-571-86971129
Email : mgfeng@cls.~u.edu.cn or
mgfeng@zjuem.zju.edu.cn

5. Bonning, Bryon C. II. Fenton, Andrew

Associate Professor, Department of
Department of Biological Sciences
Entomology, Iowa State University
407 A Science-II, Ames University of Stirling, Stirling FK9-4 LA
lowa-50011-3222, USA Scotland, UK
Tel: (515)294-1989 Tel :44(0)1786467819
Fax: (515)294-5957
Email: bbonning@iastate.edu Email: acf1@stir.ac.uk

6. Chen, Xinwen 12. Herren, R. Hans

Professor, Wuhan Institute of Virology
Chinese Academy of Sciences
Wuhan 430071, P.R. China
Tel: 86-27-87641106
Fax: 86-27-8761072
E-mail: chenxw@pentium.whiov.ac.cn
7. Ekesi, Sunday
Insect Pathologist, International Centre
oflnsect Physiology and Ecology (lCIPE)
P.O. Box 30772, Nairobi, Keyna
Tel: +254-2-861680 Ext 3061
Cellular: +254-72-527532
Fax : 254-2-860 II 0/803360
E-mail: sekesi@icipe.orgorsekesi@usa.net
8. Eilenberg, Jorgen
Department of Ecology
Insect Pathologist, International Centre
oflnsect Physiology and Ecology (lCIPE)
P.O. Box 30772, Nairobi, Kenya
Tel: +254-2-861680-4 Ext. 3061
E-mail: sekesi@icipe.orgor
sekesi@usa.net
13. Harrison, Robert L
Departmento de Entomology
Iowa state university
407 A, Science-II, Ames
Iowa-50011-3222, USA.
Tele: (515)294-1989
Fax: (515)294-5957

VII
 14. Hashmi, Sarwar 21. Laveissiere, Claude
Laboratory of Parasitology Institut de Recherche pour Ie Development
Lindsley F. Kimble Research Institute OCEAC, B.P. 288, Yasunde, Cameroun
New York Blood Centre, New York, USA
Tel :212-570-3119 22. Lopes,R.B.
Fax: 212-570-3121 Departrnento de Entomologia,
Email: shashmi@nybc.org Fitopatologia e Zoologia Agricola,
ESALQIUSP
15. Hu, Zhihong Caixa Postal 9, 134 18-900
Director General Piracicaba, SP, Brazil
Wuhan Institute of Virology Email: rblopes@carpa.usp.br
Chinese Academy of Sciences
Wuhan430071, P.R. China 23. Maniania, Jean Nguya Kalemba
Tel: +86-27-87641229, +86-27-87641072 Insect Pathologist, International Centre
Email: huzh@pentium.whiov.ac.cn of Insect Physiology and Ecology (IC1PE)
P.O. Box. 30772, Nyayo Stadium, Nairobi, Kenya
16. Hudson, Peter Tel: 254-2-82501/3/9(0),254-2-724901 (R)
Department of Biological Sciences Fax :254-2-803360/860110
University of Stirling, Stirling Email: nmaniania@icipe.org
FK9 4LA, Scotland, UK
Tel: +44(0) 1786 467778 24. Mracek, Zdenek
Email: p.j.hudson@stir.ac.uk Laboratory of Insect Pathology
Institute of Entomology
17. Hussaini, S.S. Czech Academy of Sciences, Branisolveska 31
Principal Scientist 37005, Ceske Budejovice, The Czech Republic
Project Directorate of Biological Control Email: mracek@entu.cas.cz
P.B. No. 2491, H.A. Farm Post
Bellary Road, Bangalore-560024 25. Meadow, Richard
Kamatak, India Department of Entomology and Nematology
Tel: +91-80-3411982 The Norwegian Crop Research Institute
Fax: +91-80-3411961 Plant Protection Centre, N 1432 As, Norway
Email: pdblc@kar.nic.in Email: richard.meadow@planteforsk.no
or sshblr@yahoo.com
26. Norman, Rachel
18. Hyink, Otto Department of Computing Science and
Department of Microbiology Mathematics, University ofSteriling
School of Medical Sciences FK9 4LA, Scotland, UK
University of Otago, P.O. Box 56 Tel: +44(0) 1786-497466
Dunedin, New Zealand Fax: 44(0) 1786 6464551
Tel: +64(0)3479-7307 Email: r.a.norman@cs.stir.ac.uk
Fax: +64(0)34798540 or ran@maths.stir.ac.uk
Eamail : hyiot 575@student.otago.ac.nz
27. Odulaja, Adedapo
19. KaImakoff, James Insect Pathologist, International Centre of
Professor, Department of Microbiology Insect Physiology and Ecology (ICIPE)
School of Medicines, University Otago P.O. Box 30772
PO Box. 56, Dunedin, New Zealand Tel: 254-82501/3/9
Tel: +64(0)34797720 Fax: 254-2-803360/860 II 0
Fax: +64(0)347978540 Email: nrnaniania@icipe.org
Email: james.kalmakoff@stonebow.otage.ac.nz
28. Roberto, M.Pereira
20. Jin,Hailin Research Entomologist
Departmento de Entomology USDA-ARS, Centre for Medical
Iowa state university Agricultural and Veterinary Entomology
407A, Science-II, Ames 1600 SW 23rd Drive, Gainesville
lowa-50011-3222, USA. Florida-32604, USA
Tele: (515)294-1989 Tel: (352)374-5769
Fax: (515)294-5957 Fax :(352)374-5818
Email: rpereira@gainesville.usda.ufl.edu.

viii
29. Sato, Ryoichi 36. Zhirning, Yuan
Laboratory of Molecular Mechanism of Wuhan Institute ofYirology
Bio-Interaction, Graduate School of Wuhan 430071, Hubei, P.R. China
Bio-Applications & Systems Engineering Tel:+862787869120
Tokyo University of Agriculture & Technology Fax: +862787641072
Nakamachi, Koganei, Tokyo 184-8588, Japan Email: yzm@pentium.whiov.ac.cn
Email: ryoichi@cc.tuat.ac.jp or yuanzm@hotmail.com

30. Sneddon, Katherine M.B. 37. Ms Zhang, Lei

Department of Microbiology
School of Medical Sciences, University
of Otago, P.O. Box 56, Dunedin, New Zealand
Tel: +64(0)3479-7303
Fax: +64(0)3 479-8540
Email: kate.caradocdavies@stonebow.otago.ac.nz
College ofUfe Science and Tecnology
Huazhong Agricultural University
Wuhan, Hubei-430070, PR China
Tel: 86-27-87283455
Fax: 86-27-87280670
Email: zhanglei2612@263.net

31. Sun, Ming

College ofUfe Science and Technology
Lab of Bacillus Molecular Biology
Department of Microbiology, Hazuhong
Agricultural University, Wuhan
Hubei-430070, P.R. China
Tel: 86-27-87384609(R), 86-27-87283455(0)
Fax: 86-27-87280670
Email: m IOsun@public.wh.hb.cn or
m98sun@mail.hzau.edu.cn

32. Sun, Xiulian

Associate Professor
Wuhan Institute ofYirology
Chinese Academy of Sciences
Wuhan 430071, P.R. China
Tel: +86-27-87886239
Fax: +86-27-87641072
Email: sunxl@pentium.whiov.ac.cn

33. Tarnai, Marco Antonio

Departmento de Entomologia,
Fitopatologia e Zoologia Agricola,
ESALQIUSP, Caixa Postal 9,
13418-900, Piracicaba, SP, Brazil
Email: maatamai@carpa.ciagrLups.br

34. YU,Ziniu
College of Life Science and Technology
Huazhong Agricultural University,
Wuhan, Hubei 430070, PR China
Tel: 86-27-87280802
Fax: 86-27-87393882
Email: yz41@public.wh.hb.cn

35. Ward, Yermon K.

Department of Microbiology
School of Medical Sciences
University of Otago, P.O. Box 56
Dunedin, New Zealand
Tel: +64(0)3479-7303
Fax: +64(0)3479-8540
Email: vemon.ward@stonebow.otago.ac.nz

ix
CONTENTS

Aminopeptidase N as a Receptor for Bacillus thuringiensis Cry Toxins ........................................................... I

Ryoichi Sato

Molecular Biology of Bacillus thuringiensis ................................................................................................... 15

Ming Sun, Lei Zhang, and Yu Ziniu

Bacillus sphaericus: Mechanism and Application as a Mosquito-Larvicide .................................................... 41

Yuan Zhiming

Insect Pest Resistant Transgenic Crops ............................................................................................................ 71

P. Ananda Kumar

Molecular Biology of Insect Viruses ................................................................................................................ 83

Zhihong Hu, Xinwen Chen, and Xiulian Sun

Genetic Enhancement of Baculovirus Insecticides ........................................................................................ 109

Bryony C. Boning, Anthony J.Boughton, Hailing Jin, and Robert L. Harrison

Baculovirus Genomics: A Resource for Biocontrol.. ..................................................................................... 127

Vernon K.Ward, Katherine M.B. Sneddon, Otto Hyink, and James Kalmakoff

Entomopathogenic Fungi as Potential Biocontrol Agents for Tsetse Flies .................................................... 145
Nguya K. Maniania, Claude Laveissiere, Adedapo Odulaja, Sunday Ekesi, and Hans R. Herren

Metarhizium anisopliae: An Effective Biological Control Agent for the

Management of Thrips in Horti-and Floriculture in Africa ............................................................................ 165
Sunday Ekesi and Nguya K. Maniania

Fungi for Biological Control of Brassica Root Flies, Delia radicum. and Deliafloralis ............................... 181
J0rgen Eilenberg, and Richard Meadow

Use ofEntomopathogenic Fungi in Latin America ........................................................................................ 193

S.B. Alves, R.M. Pereira, R.B. Lopes, and M.A. Tamai

Microbial Control of Insect Pests with Entomopathogenic Fungi in China: A Decade's

Progress in Research and Utilization .............................................................................................................. 213
Ming-Guang Feng

Use of Entomopathogenic Nematode (EPANs) in Biological ControL ........................................................ 235

Zdenek Mracek

Entomopathogenic Nematodes for the Control of Crop Pests ........................................................................ 265

S.S. Hussaini

Genetic Improvement of Entomopathogenic Nematodes for Insect Biocontrol... .......................................... 297

Christopher W. Brey, and Sarwar Hashmi

Mathematical Models of Insect Pest Control ................................................................................................. 313

R.A. Norman, A.C. Fenton, J.P. Fairbairn, and P.J. Hudson

Index ............................................................................................................................................................... 323

xi
 AMINOPEPTIDASE N AS A RECEPTOR FOR BACILLUS THURINGIENSIS
CRY TOXINS

Ryoichi Sato,

Laboratory of Molecular Mechanism ofBio-Interaction, Graduate School ofBio-

Applications & Systems Engineering, Tokyo University ofAgriculture and Technology,
Nakamachi, Koganei, Tokyo 184-8588, Japan

1. INTRODUCTION

Bacillus thuringiensis, a Gram-positive bacterium, produces various insecticidal proteinaceous

crystal inclusions during sporulation (Hofte and Whiteley, 1989). These inclusions consist of one or
more protein protoxins that are grouped into 30 classifications (Cry 1-32 and Cyt1-2) according to
their amino acid sequences (Crickmore et al., 1998, 2001). When susceptible insects ingest this
bacterium, the crystal inclusions are solubilized in the alkaline environment ofthe insect midgut and
processed proteolytically to yield smaller active Cry toxins (Gill et al., 1992). The Cry toxins bind
specifically to receptor molecules in the midgut epithelial cells ofhost insects (Hofmann et al., 1988a;
Hofmann et al., 1988b; Van Rie et al., 1989, 1990), altering the ion permeability of the midgut cell
membranes (Harvey and Wolfersberger, 1979). A net influx of ions and an accompanying influx of
water cause the cells to swell and lyse (Luthy and Ebersold, 1981; Knowles and Ellar, 1987). The
formation ofeither cation-selective (Knowles et al., 1989; Lorence et al., 1995; Slarin et al., 1990)
or small nonspecific pores in the membrane has been proposed as a possible mechanism for the toxin
action (Carroll et al., 1993).
Various APN isoforms or APN-like proteins have been suggested as candidate receptors for
B. thuringiensis Cry toxins in several insect species (Knightetal., 1994; Valaitisetal., 1995; Gillet
al., 1995; Yaoi etal., 1997). For example, 3 such proteins have been reported in Bambyx mari
(Hua et aI., 1998a; Tsukamoto et al., 1998; Yaoi et al., 1999a), 3 in Manduca sexta (Knight et al.,
1995;Cahng et al., 1999),3 inPlutellaxylostella (Denolf et al., 1997; Denolf, 1997; Chang et al.,
1999),2 in Heliothis virescens (Gill et al., 1995; Oltean et al., 1999),2 in Lymantria dispar
(Garner et al., 1999), and 2 inHelicoverpapunctigera (Emmerling et al., 1999a,b,c). A phylogenetic
tree of APN isoforms based on sequence-similarity data shows that APN molecules may be divided
into at least 4 classes (Oltean et al., 1999). In addition, it has been reported that different classes of
Cry toxins bind to APN molecules from different classes. For example, inM sexta, Cry 1C binds to
a 106-kDa APNbut not to a 1I5-kDa APN, and CrylAc binds to a 1I5-kDa APN but not to a
106-kDaAPN (Luo etal., 1996). rnL. dispar, CrylAc binds to the 120-kDa APNI , but CrylAa
and Cry lAb bind to a different molecule (Valaitis et al., 1995; Lee et al., 1996). Since APN diversity
may affect the susceptibility of an insect to Cry toxins, we have been studying the diversity of APN
isoforms of the midgut of B. mori and P. xylostella.

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002
 Experiments using membranes reconstituted with APN suggest that APN promotes insertion of
Cry toxins into membranes and pore formation by these toxins (Sangadala et a!., 1994; Luo et a!.,
1997; Schwartz eta!., 1997; Cooperetal., 1998). APN has been shown to function as a Cry toxin
receptor in vitro, but it is unknown whether APN functions as a receptor on epithelial cell membranes
in the environment ofthe insect midgut. Furthermore, cadherin-like proteins (CLPs) have also been
proposed as candidate receptors for Cry lAa and Cry 1Ab toxins (Vadlamudi et a!., 1995; Nagamatsu
et a!., 1998a; Nagamatsu et a!., 1998b; Ihara et a!., 1998). Our observations have confirmed that
Cry 1Aa and Cry 1Ab bind to both APN and recombinant cadherin-like protein in B. mori (data not
shown). However, the multifunctional binding mechanism of these toxins is still unknown, and it is
uncertain which candidate receptor is important for B. mori susceptibility. To elucidate the mechanisms
by which Cry toxins show differential toxicities in different insects, we have examined these issues by
a variety of approaches.

2. CLONING AND CLASSIFICATION OF APN ISOFORMS FROM B. MORI

AND P. XYLOSTELLA

A phylogenetic tree for available APN isoforms was constructed with ClustalX (Thompsonet
a!., 1997) by the neighbor-joining method based on sequence similarity data. APN isoforms from
insects were shown to separate into at least 4 classes (Figure 1). The group including B. mori APNI
(110 kDa)(Yaoi et a!., 1999), M sexta APNI (120 kDa)(Knight et a!., 1995; Luo et a!., 1999),
and H. virescens APN (170 kDa) (Oltean et a!., 1999) was tentatively named class 1. The isoform
group including P. xylostella APNI and M sexta APN2 (Denolf et a!., 1997) was named class 2,
the group including H virescensBTBPl (Gill eta!., 1995)andL. dispar APNI (120kDa)(Garner
et aI., 1999) was named class 3, and the group including APN-like protein fromP. xylostella (GenBank
accession no. AJ222699) (Denolf, 1997) was named class 4. As shown in Figure 1, in all insect
species examined, 1, 2, or 3 classes of APN isoforms or APN-like proteins are present, and no
insect has been reported to have all 4 isoforms. Accordingly, we addressed the question of whether
every insect species has APN s from all 4 classes, and if not, how many APN isoforms an insect
species typically possesses.
When amino acid sequences of APNs from insects and mammals were compared, several
conserved regions were observed. Cloning of an APN isoform cDNA from the B. mori midgut was
conducted by PCR with degenerate primers based on the conserved amino acid sequences. The 5'
and 3' ends ofthe resulting cDNA fragment were amplified by RACE, resulting in acquisition of the
complete cDNA. Alignment of the deduced amino acid sequence with those of other APNs followed
by construction of a phylogenetic tree showed that the cloned cDNA encodes a novel class 3 APN
(BmAPN3) (Figure 1). Thus, a novel cDNA encoding a class 3 APN from B. mori was cloned,
adding to the complement of APN class 1,2, and 4 cDNA fragments already reported from B. mor
I (Nakanishi et a!., unpublished).
We next cloned a complete APN isoform cDNA from P. xylostella midgut in the same way.
Alignment of the deduced amino acid sequence with those of other APN s followed by construction
of a phylogenetic tree showed that the cDNA encodes a novel class 3 APN (PxAPN3) (Figure 1).
Thus, an APN class 3 cDNA fragment was cloned, adding to the class 1,2, and 4 APN cDNA
fragments already reported. These results show that B. mori and P. xyloste lla express in the midgut
all 4 APN classes, suggesting that closely-related insect species, and possibly even Manduca,
Lymantria, Heliothis, and Helicoverpa, might express all 4 classes as well.
Although BmAPN4, HpAPN2, and PxAJ222699 were all grouped into the same class, the
similarity between PxAJ222699 and the BmAPN4 and HpAPN2 subclass is not high (Figure 1). As
more APN s are discovered, a reclassification of some of these APN s may become necessary, and
class 3 may be split into 2 or more classes. In fact, we have cloned a partial cDNA fragment
encoding a new APN -like protein that appears to be a fifth APN isoform in P. xylostella. Further,

2
as described later in this report, we have found in B. mori a 120-kDa protein that binds to Cry 1Aa
toxin but does not react with any class-specific APN antisera. Therefore, B. mori and P. xylasteUa
may have 5 or more APN isoforms in their midgut epithelial cells.

Class 4

Figure l. Protein sequence similarity among insect and mammalian aminopeptidase N isoforms. Sequences were
aligned using ClustalX (Thompson et aI., 1997), and a phylogenetic tree was drawn using PHYLIP (Joe Felsenstein).
GenBank accession numbers for the amino acid sequences shown are: B. mori APNI (BmAPN1), AF084257 (Yaoi,
1999); B. mori APN2 (BmAPN2), AB011497 (Hua, 1998 CBPBB); B. mori APN3 (BmAPN3), AF352574; B. mori
APN ABO 13400 (BmAPN4), AB013400; M sexta APNla (MsAPNla), AF123313 (Lou, 1999); M sexta APN2
(MsAPN2), X97877 (Denolf etaI., 1997); H virescens 170-kDaAPN (Hv170kDaAPN), AFI73552 (Oltean, 1999); H
virescens BTBPI (HvBTBP 1), U35096 (Gill, 1995); He/icoverpa punctigera APN 1 (HpAPN 1), AF217248 (Emmerling
et aI., 1999a); H punctigera APN2 (HpAPN2), AF217249 (Emmerling etal., 1999b); H punctigeraAPN3 (HpAPN3),
AF2172450 (Emmerling et aI., 1999c); P. xylostella APN-A (PxAPN-A), AF020389 (Chang, 1999); P xylostella
APN I (PxAPN I), X97878 (Denolf et aI., 1997); P. xylostella APN (PxAJ222699), AJ222699 (Denolf, 1997); L. dispar
APN 1 (LdAPN I), AF 126442 (Gamer, 1999); L. dispar APN2 (LdAAPN2), AF 126443 (Gamer, 1999); P interpunctella
APN (PiAPN), AF034483 (Zhu, 2000); Epiphyas postvittana APN (EpAPN), AF276241 (Simpson, 2000); H
contortus APN (nematoda APN), X94187 (Smith, 1997); M musculus APN (mouse APN), BC005431; R. norvegicus
APN (rat APN), M267 10 (Malfroy, 1989); F. catus APN (cat APN), U58920 (Tresnan, 1996); S. scrofa APN (pig
APN), Z29522 (Delmas, 1994); H sapiens APN (human APN), X13276 (Olsen, 1988); R. norvegicus aminopeptidase
A (rat APA), AF 146044 (Lee, 2000); H sapiens aminopeptidase A (human APA), Ll4721 (Nanus, 1990).

3. STRUCTURE OF B. MORI APN ISO FORMS

The structural characteristics of 4 APN isoforms from B. mori are shown schematically in
Figure 2. In each of the mature peptides, Zn2+-binding motifs ofthe gluzincin metalloprotease type
are found at amino acid positions 310 - 320 (Hooper et ai., 1994). This motif consists ofHis-Glu-
X-X-His, followed 19 residues downstream by a Glu residue. A Gly-Ala-Met-Glu-Gln motif at
position -36 upstream of the His-Glu-X-X-His sequence distinguishes gluzincin aminopeptidases
from other gluzincins (Laustsen et aI., 1997). APN activities have been reported for BmAPNl,
BmAPN2, and BmAPN4 (Yaoi etal., 1997; Hua et aI., 1998a,b,c).
Glycosylphosphatidylinositol (GPI) anchor signal sequences (Englund et aI., 1993), consisting
on small amino acids and a stretch of 19 - 21 hydrophobic residues, are found at the C termini of all
4 B. mari APNs. Since B. mari BmAPNI can be cleaved from the midgut membrane by
phosphatidylinositol-specific phospholipase C (PI-PLC) (Yaoi et aI., 1997; Hua et aI., 1999b),
BmAPNI is probably linked to the membrane by a GPI anchor.
Potential n-glycosylation sites of differing types are present in all 4 APN isoforms of B. mari,

3
and NetOglyc 2.0 (Hansen, 1995), a program for o-glycosylation site prediction, predicts potential
o-glycosylation sites in all four isofonns. For the class I APN of H virescens, Olteanet al. (1999)
reported a discrepancy between the molecular mass estimated by SDS-P AGE (170,000) and that
deduced from the cDNA sequence (113 ,000). This discrepancy may be due to extensive 0-
glycosylation. For BmAPN3, II such sites are predicted. The molecular weight ofBmAPN3, as
deduced from the cDNA sequence, is 107,500. Its molecular weight has not yet been examined by
SDS-PAGE, but it is likely that it will appear much larger by this method due to glycosylation at its
many predicted sites.

o o 00
BmAPNl

BmAPN2

BmAPN3

BmAPN4

I Signal sequence

•
N
Zinc binding moli!
potential N·glycosylation site
o Mature peptide

o predicted O·glycosylation site

Figure 2. Structural comparison of 4 APN isoforms from Bambyx mario

4. BINDING OF CRYl TOXINS TO APN ISO FORMS OF B. MORI

To detennine whether each of the 4 APN isofonns of B. mori can act as a receptor for Cry I A ,
we studied the binding of each ofthese molecules to Cry I Aa, Cry 1Ab, and Cry 1Ac. Since the 4
APN isofonns are similar in molecular weight to each other and to other unidentified toxin-binding
proteins, it is difficult to identify each APN isofonn by SDS-PAGE. Hence, protein fragments
corresponding to non-conserved regions of the APN isofonns (amino acids 57 - 173 ofBmAPNI)
were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli cells. The
fusion proteins were purified and used to raise APN class-specific antisera in mice. Proteins from
midgut brush border membrane vesicles (BBMV s) of B. mori were separated by SDS-PAGE and
immunoblotted with the APN class-specific antisera to identify the APN isoforms. The anti-BmAPNl,
-BmAPN2, and -BmAPN4 antisera bound to 11 O-kDa, 90-kDa, and 1OO-kDa proteins, respectively
(Figure 3) (Nakanishi et aI., unpublished). These observations are consistent with previous reports
(Yaoi et aI., 1997; Huaet aI., 1999a,b,c).
We used ligand blotting to identify the proteins binding to Cry 1Aa, Cry I Ab, and Cry 1Ac.
Cry 1Aa bound mainly to 230-, 120-, and 11 O-kDa proteins at 10 nM, suggesting that Cry I Aa binds
to BmAPN 1 and other unidentified proteins (Figure 3). Cry 1Ab was not observed to bind to any
protein at 10 nM, but at 50 nM, it bound to 230- and II O-kDa proteins, suggesting that Cry 1Ab
binds to BmAPN 1 and one unidentified protein. Cry 1Ac was not observed to bind to any protein at
10 nM, but at 50 nM, it bound to a 230-kDa protein, suggesting that Cry 1Ac does not bind to any
of the identified APN isofonns at low concentration. These observations indicate that the 3 Cry 1A

4
toxins bind APN isofonns in different manners with different affinities. On the other hand, CLP,
which yields a band of approximately 180 kDa by SDS-PAGE (Nagamatsu et aI., 1998b; Ihara et
aI., 1998), did not give a signal when ligand blotted with Cry 1Aa, b, or c, although the band stained
in Western blots with anti-cadherin antiserum. The IC so values for Cry 1Aa and Cry 1Ac protoxins
against B. mori 3rd instar larvae were reported to be 1.9 and 174.5Ilg/g diet respectively (Shinkawa
et aI., 1999), and the ICso value for Cry lAb protoxin against B. mori 4th instar larvae was reported
to be 175.8 Ilg/g (lharaet aI., 1993). It seems likely that this variation in susceptibility is at least
partially due to differences in the Cry-binding properties of the various APN isoforms

I 2 3 4 5 6 7

- 116
- 97

kDa

Figure 3. Identitication of Cry IA toxin-binding proteins. BBMV proteins were separated on a 6 % polyacrylamide
SDS-PAGE gel and ligand or Western blotting was conducted. Lanes 1-3, ligand blotting with 50 nM Cry lAc, 50
nM Cryl Ab, and 10 nM Cry I Aa respectively; lane 4, Western blotting with anti-full-length BmAPN I antiserum
that cross-reacts with all APN isoforms; lanes 5-7, Western blotting with BmAPNl -specific antiserum, BmAPN4-
specific antiserum, and BmAPN2-specific antiserum respectively.

5. IDENTIFICAnON OF THE CRY TOXIN-BINDING DOMAIN OF B. MORI APN

ISOFORMS

Cry I Aa toxin can bind to full-length recombinant BmAPNI produced in E. coli (Yaoi et aI.,
1997), indicating that its native glycosylation state is not required for formation ofthe binding epitope.
Thus, the recombinant protein has been used as a model for studying the binding epitope. Protein
fragments from various parts of BmAPNI were produced as GST fusion proteins in E. coli, and
Cry 1Aa-binding ability was assessed by ligand blotting. As a result, the Cry I Aa binding region of
BmAPNI was narrowed to the region Ilel35 - Pro198 (Yaoi et aI., I 999b)(Figure 4). The same
region of the P. xylostella class 3 APN, PxAPN3, was also shown to bind to Cry I Aa (Nakanishi et
aI. , 1999) (Figure 4). Thus, the conserved residues in this region may be important for CrylAa
binding. A comparison of this region ofBmAPN I and PxAPN3 with sequences from other APNs
from several insects showed that this region includes both conserved and variable subdomains; about
81 % of the residues common to BmAPNI and PxAPN3 are highly conserved in the other insect
APNs (Nakanishi et aI., 1999). Since Cry 1Aa toxin does not bind to the 120-kDa APN, LdAPN1,
from L. dispar BBMV (Lee et al., 1996), small sequence differences in this region are likely to have
a crucial effect on Cry 1Aa-binding affinity and insect susceptibility.
In the present study, toxin-binding regions from each APN class of B. mori and P. xylostella
were produced as GST fusion proteins, and ligand blotting was conducted to compare their binding
affinities for CrylAa. At 10 nM, CrylAa bound to the regions derived from class 1,2, and 4
molecules of both B. mori (BmAPN1, BmAPN2, and BmAPN4, respectively) and P. xylostella
(PxAPN-A, PxAPN 1, and PxAJ222699, respectively). At a higher concentration (1 00 nM), Cry I Aa
also bound to the class 3-derived binding regions from both B. mori (BmAPN3) and P. xylostella
(PxAPN3) (Nakanishi et aI., unpublished). Therefore, the conserved region in each ofthe 8 APN
isoforms appears capable of recognizing Cry 1Aa. No significant differences in amino acid sequence
of the conserved subdomain are present in the toxin-binding regions of the eight isoforms, and

5
therefore the reason for the low affInity ofthe class 3 molecules (BmAPN3 and PxAPN3) for Cry lAa
is unclear (Figure 4). Most of the conserved amino acids within the B. mori andP' xylostella APNs
are also conserved in human APN, suggesting that Cry 1Aa may bind not only to APN from insects
other than Bombycidae and Plutellidae, but also to mammalian (human) APN, although this supposition
has not yet been tested.

BmAPNl
'" '"
BmAPN2
BmAPN3 ,.,
116 '0'
11.
BmAPN4
PxAPN-A
'"
,01
PxAPNl
PxAPNJ
",0
'"
PxAJ222699 III 19'
human APN ... VIlLICG 11'

Figure 4. Comparison of the amino acid sequences of the Cry 1Aa-binding regions of APN isoforms from B. mori,
P. xy/ostella, and H. sapiens. Residues conserved in more than half of the 9 APN isoforms are shaded.

Cry 1Aa bound with high affinity to the toxin-binding regions from BmAPN2 and BmAPN4
when they were produced as GST -fusion proteins, but it did not bind to full-length BmAPN2 and
BmAPN4 separated from BBMV by SDS-PAGE. Midgut BBMV may contain only a small amount
ofBmAPN2 and BmAPN4 , or some feature ofthe full-length APNs may disturb the binding of
Cry 1Aa to their toxin-binding regions. In any case, this result demonstrates that APN isoforms do
not always function as receptors even in the presence of the potential binding epitopes.
Molecules of the integrin family bind to specific amino acid motifs. For example, integrin a
V~3 binds to the Arg-Gly-Asp motif of many proteins (Cheresh et aI., 1989; Gehlsen et aI., 1988).
Similarly, Cry 1Aa and Cry 1Ab can bind not only to APN but also to CLP (Vadlamudi et aI. , 1995;
Nagamatsu et aI., 1998a, 1999; Ihara et aI., 1998), suggesting that CLP and APN might have
consensus motifs responsible for Cry lA binding. However, we did not [md any consensus motifs for
the 2 molecules. Hence, we conclude that CrylAa recognition does not require a specific sequence
motif, or, alternatively, that Cry 1Aa toxin has two distinct binding sites, one for APN and the other
forCLP .
Cry 1Aa, Cry 1Ac, and Cry8Ca can bind to at least 9 non-APN-like proteins, including bovine
pancreatic RNase A, bovine carbonic anhydrase, and E. coli B-galactosidase. Bovine carbonic
anhydrase inhibits binding of Cry lAa to BmAPNl, and phosphatidyl inositol-specific phospholipase
c digests ofBBMV, which should contain solubilized APN, inhibit the binding between Cry 1Aa and
bovine pancreatic RNase A or E. coli ~-galactosidase. Furthermore, carbonic anhydrase reduces
Cry 1Aa cytotoxic activity in vitro when preincubated with Cry 1Aa (Kadotani et aI., unpublished).
From these results, we conclude that all these proteins bind to the same or neighboring sites on
CrylAa.
In order to determine the CrylAa toxin-binding regions of bovine carbonic anhydrase and
bovine pancreatic RNase A, these proteins were hydrolyzed with acid or protease to yield
small fragments. Testing of these fragments showed that Leu 189 - Lys261 of bovine carbonic
anhydrase and Argl 0 - Thr70 of bovine pancreatic RNase A contain CrylAa-binding epitopes
(data not shown). No consensus sequence was found for these Cry 1Aa-binding epitopes. As
shown in Figure 5, Argl 0 - Thr70 of bovine pancreatic RNase A contains helices, ~-strands, a
random coil, and loops (Birdsall and Mcpherson, 1992). No contribution of carbohydrate to the
binding of Cry 1Aa has been reported (Masson et aI. , 1995; Luo et aI., 1997), and E. coli ~­
galactosidase does not contain mammalian-type glycosylation. These observations suggest that the
Cry\ A-binding epitope of bovine pancreatic RNase A must consist of a simple structure made of
non-glycosylated amino acids.

6
Figure 5. Molecular model of the Cry toxin-binding structure of bovine RNase A. The model was generated from
PDB file I RTB (Birdsall and McPherson, 1992) with RasMol v. 2.6 (Sayle and Milner-White, 1995). A, secondary
structure of the Cry 1Aa-binding fragment Argl 0 - Thr70. Band C, full-length RNase A molecule with the Cry 1Aa
binding surface highlighted.

Figure 6. Candidate epitope for binding of monoclonal antibodies 1B 10 and 2C2 to Cry 1Aa toxin. The putative
binding domain at lieS 14 - Asp61S is shown in gray in a space-filling representation. Other amino acids that may
contribute to formation of the epitope are shown in black or white.

Figure 7. Amino acid residues conserved for B. mori Cry 1Aa, Cry 1Ac, and Cry9Da toxins. The 3-dimensional
structure ofCrylAa is shown (PDB accession no. 1CBY) (Grochulski eta!., 1995), with the consensus amino acids
superimposed in black. Domain III is shown in gray.

6_ BINDING SITE FOR BMAPNI ON CRYlAA

BALB/c mouse was immunized with Cry 1Aa toxin and monoclonal antibodies were screened.
The binding of Cry 1Aa to BmAPN 1 is inhibited by 2 different monoclonal antibodies, 1B 10 and

7
2C2, suggesting that their binding sites and the BmAPNI binding site are located very close together
on Cry I Aa. To localize these epitopes on Cry IAa, Cry I Aa deletion mutant proteins were produced
in E. coli and used in Western blotting with IB 10 and 2C2. The 2C2 antibody bound to the fragment
Ile514 - Asp615 in domain III ofCry IAa and to fragment Metl - Val 5 12, but notto Metl - Gln506
(Atsumi et aI., unpublished). A reasonable interpretation of these results is that the 2C2 epitope is
composed of amino acids from both Ile514 - Asp615 and Ile507 -Va1512. In fact, the Ile507 -
Val512 segment is adjacent to Ile514 - Asp615 in the structure. A similar epitope is probable for
IBIO, since IBIO bound to Metl-Val512 and Ile514 -Asp615, but not to Metl-Gln506 or Metl-
Pro256.
To further map the IB 10 and 2C2 epitopes, a series of8-amino acid polypeptides (overlapping
by 6 residues) from domain III of Cry I Aa were synthesized using a MULTIPJNIM peptide synthesis
kit (CHIRON). The ability of these peptides to bind to IB 10 and 2C2 was assessed by an enzyme-
linked immunosorbent assay. Antibody IB 10 bound to the Val582-Val589 peptide (Atsumi et aI.,
unpublished). Since this peptide abuts a segment (Ile507 - Val512) of the candidate epitope, the
amino acids contained in these 2 strands may constitute the IB 10 epitope (Figure 6). On the other
hand, the 2C2 epitope is clearly different from that of I B 10, since 2C2 bound to synthesized peptides
Gln520-Arg527 and Phe570-Ser577 in addition to Val582-Val589. Thus, amino acids from both
Va1582-Val589 and Ile507-Val512 may form the 2C2 epitope, since these 2 segments are close
together. Since I B I 0 and 2C2 inhibit each other's binding to Cry IAa, and each antibody almost
completely inhibits binding of Cry IAa toxin to BmAPNI, these antibodies appear to bind to single
sites within close proximity on Cry I Aa. Hence, it is most likely that Val582-Val589 contains the
epitope-forming amino acids. On the other hand, G1n520-Arg527 and Phe570-Ser577 are contained
in Ile5 14-Asp61 5 but far from Ile507-Val512. However, even ifG1n520-Arg527 or PheS70-SerS77
is part of the 2C2 epitope, 2C2 might be able to inhibit the binding of both APN and I B I 0 to the
toxin. Consequently, we cannot rule out the involvement of amino acids from GlnS20-Arg527 or
PheS70-SerS77 in forming the 2C2 epitope.
These experiments suggest that the binding sites for IB I 0 and 2C2 are on domain III ofCry IAa
(Figure 6). On the other hand, several other monoclonal antibodies that bound to domain III but did
not inhibit the binding of the toxin to BmAPN 1 were found in the same experiment. Consequently,
our results with antibodies 1B I 0 and 2C2 suggest that the BmAPN I binding site is on the specific
segment ofdomain III, on the domain adjacent to domain III, or at the inter-domain region consisting
of domain III and another domain. Domain III is reported to be important for binding specificity or
host specificity (Lee et all., 1995; de Maagd et al., 1996a,b; 1999). Jenkins et al. (1999) reported
that mutagenesis ofGlnS09, ArgSII, or TyrS 13 of Cry I Ac toxin resulted in elimination ofbinding to
the M sexta 120-kDa APN. In addition, Burton et al.(1999) reported that mutagenesis of AsnS06,
GlnS09, or TyrSI3 of the toxin reduced binding to APN. These reports are not in conflict with our
data discussed above.
In our ligand blotting experiments, in which BBMV proteins were separated by SDS-PAGE,
Cry I Aa, Cry lAc, and Cry9Da bound to BmAPNI, and binding ofeach was inhibited reciprocally
by the other 2 toxins (Shinkawa et al., 1999). These results suggest that these distantly related Cry
toxins bind to the same site on BmAPNI. Ifthis is true, then the structures conserved in these three
toxins may be important for APN binding. The sequences of Cry I Aa, Cry lAc, and Cry9Da were
compared, and the consensus amino acid residues were plotted onto the 3-dimensional structure of
CrylAa (Figure 7). Five main islands of consensus were observed: (I) an interdomain region
(domains I, II, and III) consisting ofLeu236, Arg265, Glu266, Tyr268, Glu288, Ile29 1, Gly490 and
Pro491; (2) an adjacent interdomain region (domains II and III) consisting of Arg292 , Pr0294,
His295, Leu296, His433, Leu48 I , Gly484, Val487, and Leu48 I ; (3) an interdomain region on the
opposite face (domains I, II, and III) consisting ofGln262, Arg430, His4S6, Arg4S7, G1n472, and
Lys477; (4) a region in domain III containing Arg521, Tyr522, Arg523, Arg525, andPhe570; and
(5) a region in domain II containing Asp409, Leu411, Pro416, Arg424, and Thr269. Conserved
region 3, which lies between domains II and III, forms a concave site consisting of Arg292, Pro294,

8
His29S, Leu481, Gly484, and Val487 that is in close proximity to the segments comprising the
candidate monoclonal antibody epitope, GlnS06-ValS12 and Va1S82-ValS89 (Figures 6 and 7). In
addition, the conserved region in domain III contains 2 conserved amino acid residues, ArgS21 and
ArgS23 , which are contained in the other candidate epitope for antibody 2C2. Hence, these conserved
amino acid residues may form at least part of the APN recognition site on Cry I A.

7. DISTRIBUTION OF APN IN THE MIDGUT OF B. MORI

Functional receptors for the Cry toxin must exist on the surface of the brush border membrane
of midgut epithelial cells and in the epithelial cell region that undergoes disruption upon ingestion of
Cry toxin. These Cry toxin receptors must be capable ofassembling toxin molecules into a multimeric
ion channel. Hence, we examined the distribution of APN in the midgut of B. mari and the affmity of
this APN for Cry toxins.
Deparaffinated and hydrated midgut tissue sections from 2nd instar B. mari larvae were treated
with anti-BmAPNl antiserum and immunocytochemically stained using the chromogenic reagent
VECTOR R VIP Substrate (Vector Laboratories). Only the brush border of the midgut columnar
cells was stained (Hara et aI., unpublished). Furthermore, when an entire midgut tissue was excised,
fixed, and immunocytochemically stained as above, all areas of the inner surface ofthe midgut were
stained. Thus, APN seems to be distributed in the brush border of all areas of the midgut. When the
2nd instar larvae ingested Cry IAa, columnar cells from every part oftheir midguts were damaged.
The distribution of susceptible midgut cells appeared to correlate with the distribution of APN. Since
the antiserum against BmAPN I cross-reacts with at least 3 APN classes (classes I, 2, and 4) in
Western blots, the staining observed in the above experiments could be due the presence of APN
mo lecules of any of these classes or a combination thereof.
When an entire midgut was immunocytochemically stained using anti -CLP antiserum, all areas
of the inner surface of the midgut were stained. In addition, staining of a midgut section showed that
only the brush border from midgut columnar cells was stained (Hara et al., unpublished). Cadherins
are believed to mediate calcium-dependent cell-cell adhesion (Takeichi, 1991), but the CLP of B.
mari appears to be present only at the midgut brush border. Transfected Sf9 cells expressing CLP
are reported to acquire Cry lAa susceptibility (Nagamatsu et al., 1999). Hence, CLP may function
as a receptor for CrylAa toxin in the midgut of B. mario
Dissociation constants for Cry 1Aa binding to BmAPN 1 and CLP from B. mari were reported
to be approximately 7 (Yaoi et aI., 1997; Shinkawa et aI., 1999) and 2.6 nM (Ihara et aI., 1998),
respectively. Although these values cannot be directly compared, since the methods used in the 2
studies were different; they do demonstrate that the affinity of the receptors for the toxin may be high
enough to allow receptor-mediated assembly of toxin molecules into functional ion channels at the
cell surface.

8. CONCLUSIONS

Cry toxins can bind to many kinds of proteins, including APN, CLP, pancreatic RNase A,
carbonic anhydrase, and others. Indications are that the binding sites of APN and other proteins on
Cry toxins overlap or exist in close proximity. Localization of the binding sites on the toxin and
elucidation ofthe mechanism for binding ofmultiple proteins to the toxin are important in understanding
the origin ofspecificity of Cry toxin binding.
APN isoforms are abundant in the brush border membrane distributed throughout the insect
midgut, where they correlate with sites damaged by the toxin. Furthermore, APN isoforms can
promote membrane insertion or pore formation by Cry toxins. Consequently, it is possible that APN
isoforms function as Cry receptors in the midgut of some or all Cry-susceptible insects. At least 4

9
kinds of APN isoforms are known, and each isoform binds with different affinity to each toxin.
Further complicating matters, CLP may also contribute to insect Cry toxin susceptibility. This
complexity makes the determinants ofCry toxin susceptibility difficult to explicate. As further steps
in understanding Cry toxin specificity, the identities of the functional Cry toxin receptors in the midgut
of susceptible insects must be determined and their roles must be studied.

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delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid
bilayers, FEBS Lett. 244: 259-262.
Laustsen, P.G., Rasmussen, T.E., Petersen, K., Pedraza-Diaz, S., Moestrup, S.K., Gliemann, J., Sottrup-Jensen, L.,
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Lee, H., Tomioka, M., Takaki, Y., Masumoto, H., and Saido, T.e., 2000, Molecular cloning and expression of
aminopeptidase A isofonns from rathippocampus(1), Biochim. Biophys. Acta 1493: 273-278.
Lee, M.K., Young, B.A., and Dean, D.H., 1995, Domain III exchanges of Bacillus thuringiensis CryIA toxins affect
binding to different gypsy moth midgut receptors, Biochem. Biophys. Res. Commun. 216: 306-312.
Lee, M.K., You, T.H., Young, B.A., Cotrill, 1.A., Valaitis, A.P., and Dean, D.H., 1996, Aminopeptidase N purified
from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis Cry lAc
toxin, Appl. Environ. Microbiol. 62: 2845-2849.
Lorence, A., Darszon, A., Diaz, e., Lievano, A., Quintero, R., and Bravo, A.. 1995, Delta-endotoxins induce cation
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FEBS Lett. 360: 217-222.
Luo, K.E., Lu, Y.-1., and Adang, M.1., 1996, A 106 kDa form of aminopeptidase is a receptor for Bacillus thuringiensis
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791.
Luo, K., Sangadala, S., Masson, L., Mazza, A., Brousseau, R., and Adang, M.J., 1997, The Heliothis virescens 170
kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis CrylA delta-
endotoxin binding and pore fonnation, Insect Biochem Mol Bioi. 27: 735-743.

11
Luo, K., McLachlin, I.R., Brown, M.R., and Adang, MJ., 1999, Expression of a glycosylphosphatidylinositol-
linked Manduca sexta aminopeptidase N in insect cells, Protein. Expr. Purif. 17: 113-122.
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Nagamatsu, Y., Toda, S,. Koike, T., Miyoshi, Y., Shigematsu, S., and Kogure, M., I 998a, Cloning, sequencing, and
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FEBS Lett. 460: 385-390.
Nakanishi, K., Yaoi, K, Shimada, N., Kadotani, T., and Sato, R., 1999, Bacillus thuringiensis insecticidal CrylAa
toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary
success, Biochim. Biophys. Acta 1432: 57-63.
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human intestinal aminopeptidase N as deduced from cloned cDNA, FEBS Lett. 238: 307-314.
Oltean, 0.1., Pullikuth, A.K., Lee, H.K., and Gill, S.S., 1999, Partial purification and characterization of Bacillus
fhuringiensis CrylA toxin receptor A from Heliothis virescens and cloning of the corresponding cDNA,
Appl. Environ. Microbiol. 65: 4760-4766.
Sangadala, S., Walters, F.S., English, L.H., and Adang, MJ., 1994, A mixture of Manduca sexta aminopeptidase
and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+
efflux in vitro, J Bioi. Chem. 269: 10088-10092.
Sayle, R.A., and Milner-White, EJ., 1995, RASMOL: biomolecular graphics for all, Trends Biochem. Sci. 20: 374.
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receptors, FEBS Lett. 412: 270-276.
Shinkawa, A., Yaoi, K., Kadotani, T., Imamura, M., Koizumi, N., Iwahana, H., and Sato, R., 1999, Binding of
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importance of Cry toxin's conserved structure in receptor binding, Curro Microbiol. 39: 14-20.
Simpson, R.M., and Newcomb,R.D., 2000, Binding of Bacillus thuringiensis delta-endotoxins CrylAc and Cry I Ba
to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles
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12
 membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins,
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Bioi. 30: 215-224.

13
MOLECULAR BIOLOGY OF BACILLUS THURINGIENSIS

Ming Sun, Lei Zhang, and YuZiniu,

College ofLife Science and Technology, Huazhong Agricultural University, Wuhan,

Hubei 430070, P. R. China

1. INTRODUCTION

Bacillus thuringiensis (Bt) is a Gram-positive bacterium, belonging to Genus 2, Bacillus,

in Group 18, as classified in Bergey's Manual of Determinative Bacteriology (ninth Edition). One
of the characteristics of Bt is that proteinaceous crystals appear in the cells during sporulation.
The parasporal crystals have been extensively researched and wildly applied in the world because of
its specific insecticidal activities against many insects in the orders of Lepidoptera, Diptera, and
Coleoptera, as well as some pests like plant/animal parasitic nematodes, mites, protozoan and
fluke. According to the variation of flagellum antigen and other characteristics, 70 serotypes and
83 subspecies of B. thuringiensis were identified (Lacedet et ai., 1999; Li Rongsen,
unpublished). Different subspecies, even different bacterial strains, possess quite different kinds
and quantities of insecticidal crystal proteins, resulting exuberance in the diversity of the toxicity.
In recent ten years, some review articles elucidated the insecticidal crystal proteins and their
genes in many aspects (Feitelson et al., 1992; Gill et ai., 1992; Lereclus et ai., 1989b, 1993;
Aronson, 1993; Visser et ai.,1993 ; Yamamoto and Powell, 1993; Knowles, 1994; Schnepf
et aI., 1998).

2. LOCATION OF BACILLUS THURINGIENSIS INSECTICIDAL CRYSTAL

PROTEIN GENES

2.1 Recognition of Gene Location

The relationship between the insecticidal activity and the parasporal crystal wasn't
revealed until 1953 since Ishiwata had found Bacillus thuringiensis in 1901. Then it was
identified that theparasporal crystal was composed of proteins (Hannay and Fitz~James, 1955). This
kind of proteins is generally named d-endotoxin. In further research, it was found that Bt lost
its biosynthesis ability of the parasporal crystal easily, but could not regain it. This is similar to the
trait ofthe plasmid-coding mode. In tphe next few years, plasrnids were found to commonly exist in
B. thuringiensis and the composition of plasmids were polymorphous, with the size between
2-200kb and the numbers ranged from 1 to 12 (Gonzalez et aI., 1980, 1981, 1982; Gonzalez and
Carlton, 1980).

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 15
2.2 Confirmation that the Crystal Protein Genes are Located on Plasmids

Although the biosynthesis of parasporal crystals had been found to relate to the plasmids, and
the existence of plasmids was very common, it was still rather difficult to confirm that crystal protein
genes are located in the plasmids. However, it was later found that insecticidal crystal protein genes
are located on large plasmids. But it was very different to detect large molecular weight (> 30MD).
Gonzales et al. (1981) developed a strategy to detectthe plasmids rapidly, especially for the large
ones. It showed great progress on the relationship of crystal protein genes and plasmids.

2.2.1 Curing of Plasm ids. One of the methods to eliminate Bt plasmids is cultivating the bacteria
under high temperature of 42°C. The comparison of the parasporal crystal formation and the
appearance of plasm ids among the mutants demonstrated that the formation of the parasporal
crystal has straight connection with the plasmids of 105MD, 50MD and 130MD in some strains
(Gonzalez et aI., 1981;1982).

2.2.2 Trans-Conjugation. The plasmids get transferred between the bacteria by the process of
trans-conjugation when B. thuringiensis cells are mixed in the culture. In some bacteria, the frequency
of trans-conjugation reached 70-80%. Further trans-conjugation experiment shows that the plasmids
(50MD, 75MD and 44MD) have close relationship with the parasporal crystal biosynthesis (Gonzalez
et al.,1982; Gonzalez and Carlton, 1982).

2.2.3 Molecular Hybridization. Plasmid-curing and trans-conjugation experiments have strongly

supported the opinion that there are certain plasmids which are larger and trans-conjugative, and
these plasmids must be directly concerned in the crystal formation. But it doesn't exclude the possibility
that these plasmids just have regulator genes for the synthesis of the crystal proteins, and the structure
genes are located on the chromosomes instead. In 1983, Kronstad et al. used crylAal gene as
probe and gave more demonstration to the conclusion that most B. thuringiensis crystal protein
genes are located on plasmids. The result of molecular hybridization was largely identical with that of
the plasmid-curing and trans-conjugation. But some bacteria have more plasmids which carry toxin
genes. For examples, there are two plasmids with toxin genes in strain HD-l, while in B. thuringiensis
subsp. wuhanensis (H) the toxin genes are located in the chromosome.

2.3 New Recognition of Gene Location

By the above three strategies, it can be confirmed that most of the crystal protein genes are
located on the larger and conjugative plasmids. But recognition of the gene location was not complete
due to lack of enough crystal protein genes that appeared at that time. One strain has one, or two or
up to five crystal genes. These multiple genes are perhaps dispersed on one or in several different
plasmids. For example, strain HD-l possesses five crystal protein genes, in which cry lAa, crylAc,
cry2Aa and cry2Ab are on the llOMD plasmid, and crylAb is on the 44MD plasmid (Carlton and
Gonzalez, 1985). B. thuringiensis subsp. israelensis also has five crystal protein genes-cry4A,
cry4B, crylOA, cry11A and cytl A. These genes are all located on the plasmid of7 5MD (Gonzalez
and Carlton, 1984; Hofte and Whiteley, 1989)
Molecular hybridization shows that the non-flagellum B. thuringiensis subsp. wuhanensis has
its parasporal crystal protein genes on the chromosomes. This subspecies produces at least four
crystal proteins (140kDa, 133kDa, I 28kDa and 65kDa, unpublished). The probe used by Kronstad
et al. (1983) was taken from crylAa, so it didn't definitely hybridize all the four crystal protein genes
of B. thuringiensis subsp. wuhanensis. The conclusion ofKronstad' s proved that the crystal protein
genes which could hybridize the crylAa gene probe were on the chromosomes. In fact, B.
thuringiensis subsp. wuhanensis at least has the crystal protein genes cry lAb and cry1 D (Iizuka,
unpublished). Carlson and Kolsto (1994) constructed a chromosomal physical map ofB. thuringiensis

16
subsp. HD-2 based on NotI digestion and detennined the size of the chromosome (5425kb). Except
these, they found that cry] A -like genes only could be detetable from the chromosomes in some
strains, and some of genes had more than one copy.

3. MOLECULAR BIOLOGY OF BACILLUS THURINGIENSISPLASMIDS

3.1 Functions of B. thuringiensis Plasmids

In the big family of B. thuringiensis, the number and size of plasmids are various in different
subspecies or different strains. So the functions of the plasmids probably exhibit notable abundance
and diversity. In the cells of some strains, the plasmid DNA even contributes 10-20% to the cell total
DNA (Aronson, 1993; 1. Baum, personal communication). Since there are so many kinds and so
large an amount of plasmids in the cells that it has become difficult to know: How do they work on
the survival ofthe cells in their environment and the evolution? How can a cell accommodate such an
amount of plasmid DNA? The functions of the B. thuringiensis plasmids have not been adequately
reported up to date, only some of them are discovered for their functions.

3.1.1 Bearing Toxin Genes. Not only most of the crystal genes are carried on the plasmids, but
also the biosynthesis of thuringiensin (b-exotoxin) concerns the plasmids. The research work carried
on by Levinson et al. (1990) showed that the synthesis ofthuringiensin had close connection with the
plasmids; the productivity of thuringiensin can also be trans-conjugated into the strains without the
ability of producing this toxin. Besides, the biosynthesis genes of thuringiensin and the decision-
factors of crystal protein biosynthesis are located on the same plasmid.

3.1.2 Harboring Transposable Elements. There are many insertion sequences and transposable
factors in B. thuringiensis. Most of them are on the plasmids and are frequently contiguous to the
crystal protein genes (Mahillon et al., 1994; Baum, 1994). For examples, the 65kb plasmid of B.
thuringiensis subsp. thuringiensis strain berliner 1715 has insertion sequences IS23]A, IS23] B,
IS2 3] C and IS2 32A, but in B. thuringiensis subsp. israelensis, it is the 112kb (75MD) plasmid that
carries IS23IF, IS23] W, IS240A and IS240B. Transposon Tn4430 exists in B. thuringiensis subsp.
kurstaki and other subspecies.

3.1.3 Trans-Conjugation. Although many large plasmids inB. thuringiensis are trans-coqiugative,
the mechanism ofthis phenomenon is unclear. During the course of trans-conjugation, some smaller
ones are always induced to trans-conjugate simultaneously (Carlton and Gonzales, 1985).

3.1.4 Other Functions. Besides the above functions, B. thuringiensis plasmids can also control
the mode of cell division and affect the morphology ofthe colonies. This characteristic is connected
with the 43MD plasmid and was transferrable to other strains like B. thuringiensis subsp. thuringiensis
strain HD-2, B. thuringiensis subsp. israelensis strain HD-567 and B. cereus strain T. What's
more, linear plasmids, such as the -1 OMD plasmid in strain HD-2, can be found in some strains.
They are probably the genomes of bacterium-phages (Gonzales and Carlton, 1982).
Finally, some species carry the genes controlling self-auto-agglutination reaction (Jensenet al.,
1996).

3.2 Distribution ofthe Plasmids

The plasmids ofB. thuringiensis show great variation in number and size. How many kinds of
plasmids are included in B. thuringiensis and what is the classification standard? Should they be
divided according to their size or function? However, sizes are not stable as well as functions. The

17
factors that maintain the stability ofplasmids, such as the replication origins (or replicons) ofplasmids,
could be taken into consideration. The plasmid replication fragments from B. thuringiensis subsp.
kurstaki HD-73 and HD-263 have been cloned. Functional analysis shows that the necessary region
for replication is limited in the sizes of 1. 7~2.3kb. With these fragments, the constructed plasmids can
survive at least eighteen generations in B. thuringiensis under the condiftion of no antibiotic-selection
(Baum et ai., 1990; Baum and Gilbert, 1991; Gamel and Piot, 1992; Lereclus et ai., 1989a). So
plasmid replication origin is the factor determing the stability ofthe plasmids.

3.2.1 Isolation of the Plasmid Replication Origins

3.2.1.1 Cloning ofthe Plasmid Replication Origins ofB. thuringiensis subsp. kurstaki. Baum
et al. (1990) cloned seven plasmid replication origins from strain HD-263, three from large plasmids
(60MD, 44MD and 43MD) and four others from small ones (7.5MD, 5.4MD, 5.2MD and4.9MD).
The functional regions for plasmid replication in the three large plasmids were demonstrated to locate
in the fragments of2.3~2.8kb. Sequencing analysis showed that every one ofthese fragments contained
a protein open reading frame (ORF) and a ribosome binding site (RBS) (Baum and Gilbert, 1991).
No homology was found between any of the seven replication origins. A plasmid replication origin
was isolated from the 15kb plasmid pHTl 030 from strain HD-73. It resides in the 2872bp Ban
fragment. The existence of this fragment can fully guarantee the stability ofthe constructed plasmid
(such as pHT31 01) surviving in B. thuringiensis (Lereclus and Arantes, 1992). One replication
origin of the 75kb plasmid from strain HD-73 has been cloned. It has completely the same sequence
to that of the 44MD plasmid in strain HD-263 (Gamel and Piot, 1992). Two plasmid replication
origins were cloned from kurstaki strain YBT -1520. Plasmid pBMB2062 with the length of2062bp
and high copy number has an ability to code replication protein and replication initiation protein. Its
HincH fragment has the full function for stable replication (Sun et ai., 2000; GenBankJEMBL
AF050 161). A 6.6kb plasmid from strain YBT -1520 was cloned and its replication region was
located on a 2.3kb segment. The sequence analysis showed that this fragment had 6,578 base pairs
(GeuBankJEMBL AF201532) and coded a novel replication protein (Sun M, unpublished).

3.2.1.2 Cloning ofthe Plasmid Replication Origins from B. thuringiensis subsp. israelensis.
At least two plasmids (PTXI4-1 and pTXI4-3) from B. thuringiensis subsp. israelensis have been
cloned and sequenced up to date. The lengthofpTXl4-3 is 7649bp with the shortest fragment of
283 bp for the necessity of replication (Madsen et ai., 1993).

3.2.1.3 Cloning ofthe Plasmid Replication Origin in B. thuringiensis subsp. tenehrionis. A

10kb EcoRl DNA fragment containing the plasmid replication region was isolated from B.
thuringiensis subsp. tenebrionis strain 165, and its physical map was constructed (Ming Sun,
unpublished). The definite replication region is in determination. It was shown thatthe plasmid replication
origins from B. thuringiensis subsp. kurstaki have no homology with that from B. thuringiensis
subsp. israelensis and tenebrionis (Baum and Gonzales, 1992). Utilizing plasmid replication origins
from different subspecies could be beneficial to overcome the potential incompatibility when
constructing recombinant strains with two or more plasmid vectors.

3.2.2 Distribution ofthe Plasmid Replication Origins. Difference of plasmid replication can at
least reflect the diversity of the plasmid no matter how they represent the types ofthe plasmids. Baum
and Gonzalez (1992) detected the distribution of the replication origins of the three large and two
small plasmids in strain HD-263 among more than 400 plasmids in forty-four strains belonging to
twenty-seven subspecies. Results showed that these replication origins are not randomly distributed.
Plasmids of B. thuringiensis subsp. israelensis and B. thuringiensis subsp.tenebrionis were not
found to hybridize to any ofthe above replication origins. Additionally, the other six replication origins
had similarity in their distribution.

18
 The non-random distribution of the plasmid replication origins indicates that the genetic diversity
still exists among the subspecies in B. thuringiensis, and insect host of B. thuringiensis is related to
serotypes (or subspecies) to a certain extent.

4. INSECTICIDAL CRYSTAL PROTEIN GENES OF B. THURlNGIENSIS

4.1 Cloning ofthe Insecticidal Crystal Protein Genes

While Gonzalez et ai. (1981) were studying the relationship between the plasmids and crystal
protein genes by curing plasmid, Schnepfand Whiteley (1981) cloned the first toxic insecticidal gene
from strain HD-l and identified the location ofthe structure gene usingtransposon TnS. The nucleotide
sequence ofthis gene was published subsequently, with its ORF coding a protein of 133.2kDa
(Schnepfet al., 1985). It was named crylA(a) in 1989 (Hofte and Whiteley, 1989) and was designated
as cry lAal in the new nomenclature system (Crickmore et aI., 1998). Owing to appearence of the
first cloned gene, many crystal protein genes were isolated. The crystal protein genes specific to
dipteran larvae are mostly from subsp. israelensis. Gene cytlA was firstly cloned by Ward et aI.,
1984. In 1983, crystal protein gene active to coleopteran insects was isolated from B. thuringiensis
subsp. tenebrionis (H8ab), termed as gene cryIIIA (laternamed cry3Aa). Afterthose years, crystal
protein genes were isolated with novel toxicity to mites, nematodes, and protozoa (F eitelson et a!.,
1992).

4.2 Classification of the Insecticidal Crystal Protein Genes

According to the diversity of the amino-acid sequences and the toxicity, Hofte and Whiteley
(1989) divided the published forty-two crystal proteins into fourteen subgroups offive groups. The
27kDa cytolytic protein was designated Cyt, and the other thirteen subgroups only having insecticidal
activity were designated crystal protein. cry genes were divided into four groups: cryI, cryII, cryIII,
and cryIVwhich were active to lepidopteran, lepidopteran and dipteran, coleopteran, and dipteran
larvae, respectively. In addition, the activities of B. thuringiensis isolates against other insect orders
(Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and against nematodes, mites, and protozoa
were reported (Feitelson et aI., 1992; Feitelson, 1993). Owing to this enlarged host range, the word
"insecticidal" was replaced by "pesticidal". Each gene has its definite position in this classification
system at that time. Therefore the classification system proposed by Hofte and Whiteley (briefly
addressed as HW system in this article) was widely accepted and applied and further greatly improved
the research work on B. thuringiensis. Since then, new genes were discovered continuously. Some
of them were comparatively easier to get a position in.the system, while some other genes were not
because they don't possess the two prerequisites simultaneously. Thereafter, many researchers had
tried hard in the classification of the genes (Feitelson et a!., 1992; Yamamoto and Powell, 1993;
Knowles, 1994), but no improvement was made because their work still based on the old systems.
New nomenclature principles were proposed by the B. thuringiensis Pesticidal Crystal Protein
Nomenclature Committee represented by Dr. D. H. Dean in the Annual Meeting of Society for
Invertebrate Pathology in 1995. So that the classification of B. thuringiensis crystal genes tried to be
perfected. A list ofthe updated crystal protein genes is given in Table 1. Conditions of being accepted
by this classification system are described as : (i) the crystal protein genes in B. thuringiensis code
products that have insecticidal activity, such as most ofthe insecticidal genes now existing, (ii) the
crystal genes code toxic proteins against some targets, such as cry l5A, (iii) the insecticidal crystal
protein genes have similar sequences to the accepted crystal protein genes, such as cry l8Aai from
Bacillus popilliae and cryl7Aal from Clostridium species, (iv) the nucleotide sequences or the
protein sequences must have been registered in one of the main international data bank (such as
EMBL, GenBank or PIR).

19
 Table 1. Pesticidal crystal protein genes of Bacillus thuringiensis *
Name Name Name Name
cry/Aa/-/2 cry/Hal cry5Ab/ cryl7Aa/
cry/Ab/-/5 cry/Hb/ cry5Ac/ cry/8Aa/
cry / Ab-like /-3 cry/Hlike cry5Bal cryl8Bal
cry/Acl-13 crylIal-8 cry6Aal cryl8Ca/
cry/Adl-2 cryllb/ cry6Ba/ cry/9Aa/
cry/Ae/ crylIcl cry7Aa/ cryl9Bal
cry/All cry lId/ cry7Ab/-2 cry20Aa/
crylAg/ cryllel cry8Aal cry2lAal-2
cry/Ah/ cryll-like cry8Ba/ cry22Aa/
cry/A like I crylJal cry8Cai cry23Aai
cry/ Bal-3 crylJb/ cry9Aal-2 cry24Aai
cry/ Bbl crylJcJ cry9Bai cry25Aai
cryl Bel crylJdl cry9Cal cry26Aai
crylBdl CrylKal cry9Dal-2 cry27Aai
crylBel cryl-like cry9Eal-2 cry28Aal-2
crylBll cry2Aal-9 cry9Ebi cry29Aai
cry/Ca/-8 cry2Abl-4 cry9like Cry30Aai
crylCbl~2 cry2Acl-2 cry/OAal-2 Cry3lAai
cryl Dal-2 cry2Adi cryllAal-2 Cry32Aai
crylDbl-2 cry3Aal-7 cryllBal cytIAa/-4
cryl Eal-6 cry3Bal-2 cryllBbl cytlAbl
cryl Ebl cry3Bbl-3 cryl2Aal cytl Bal
cry I Fa/-2 cry3Cai cryl3Aai cyt2Aai
cry/ Fbl-5 cry4Aal-2 cryl4Aai cyt2Bal-8
crylGal-2 cry4Bal-4 cryl5Aai cyt2Bbi
crylGbl-1 cry5Aai cryl6Aai

* Modified from the literature of Crickmore et al. (1998), and updated on September 28, 2001, at http://
www.biols.susx.ac.uklHomelNeil_Crickmore/Bti. The sequences for toxins originally designated cry / Ca6
and crylCa7 were subsequently withdrawn by the database managers.

The classification principles were definite. The prevailing classification system differs from the
1985 system for its principles that this new system classify the genes only according to the homology
of the amino acid sequences of crystal protein. After comparing the homology of the amino acid
sequences of the crystal proteins and drawing dendrogram (Figure 1) in the computer, four division
were established by the similarity values of 45%,78% and 95%.
Two hundred and eighteen genes belonging to thirty-four classes have been listed by the time of
September 28 of2001. The cry genes take up thirty-two classes and the cyt genes take up two
classes (Table 1). The first class including 113 individuals is the biggest one. It was extended and
established upon the basis of cryI in HW system, and enlarged from cry I A to cry I K. Gene cryII in
HM system is now designated cry2A and has been divided into subgroups a, b and c. Gene cry3
corresponds to the original cryIIIwith cryIIIC (cry 7A being the new designation) excluded, including
four subgroups, three groups. Gene cry4A is in coincidence with the original cryIV, except that
cryIVC is newly designated crylOA. The original cryIVD was designated cryllA in the new
nomenclature system.
As to the original cyt genes, they were divided into two groups, cytJ and cyt2, owing to that the
corresponding original CytA and CytB only had the homology identity of39% (Koni and Ellar,
1993,1994).
The proteins in the parasporal crystal but without insecticidal activity are not included, such as
the 40kDa protein of B. thuringiensis subsp. thompsoni and the 20kDa help protein of B.
thuringiensis subsp. israelensis (Table 2). Relative articles about classfication have been published
in 1998, and new genes also can be acquired at the address maintained by Dr. N Crickmore, http:/
/www.biois.susx.ac.ukihome/Neil_Crickmore/Bti. Henceforth, the nomenclature of anew gene must
undergo the following process. Firstly, the DNA or protein sequence should be sent by E-mail or

20
computer soft disk to Dr. D. Zeigler, Bacillus Genetic Stock Center (BGSC), The Ohio State University,
USA. He will analyze the sequence there and make further decision.

l - CyrlAa
f - CyrlAg
CyrlAd
l - CyrlAb
-1 I - CyrlAe
CyrlA!
I !
CyrlAc
CyrlAh
CyrlO.

r---1.
CyrlOb

.-
I
- CyrlHa
CyrlHb
CyrlF•
CyrlFb
I~ CyrlGa
CyrlGb
CyrlC.
l{ CyrlCb

r
I.
--- CvrlE•
-
CyrlEb
CyrlJa
CyrlJb

-
CyrlJc
CyrlJd
,- - - r--- ~ CyrlBe
~
f - CyrlB!

rI 1-1
CyrlB.
CyrlBd
f - CyrlBc
CyrlBd
-- '---
CyrlK.
CyrlLb
I--- CyrlLa
-!~
"
I--- CyrlLc
I--- CyrlL.a
CyrlLd
I--- Cyr7Aa
-
r

l r-r
' l-
I--
-
Cyr7Ab
Cyr9Eb
l- I Cyr1lEa
Cyr90a
Cyr9Ca

- l-
Cyr9Ba
Cyr1lAa

. ~
f-
CyrBAa
Cyr8Ba
CyrBC •
Cyr'26Aa
Cyr28Aa
Cyr4Aa
Cyr4B.
Cyr32Aa
Cyr3Aa

,-: . Cyr3C •
I - - Cyr3B.
I - Cyr3Bb
CyrI9B.
CyrlQAa
c.
[-r CyrlOAa
Cyr30Aa
Cyr29Aa
Cyr24Aa
.- Cyr25Aa

=
'-1 Cyr.20Aa
Cyrl6Aa
,I CyrT/Aa

, -
Cyrl7Aa
- ' CyrSAa
Cyr5Ac
CyrSAb
Cyr5S.
I Cyrl2Aa
L-...t Cyr21Aa
Cyrl4Aa
Cyrl3Aa
r-
--_..-'
=.
Cyr2Ab
Cyr2Ad
Cyr2Aa
I
I
I I
- Cyrl8C.
Cyrl8Aa
Cy"IS.
CyrllBb
Cy"lAa
Cyr31Aa

Figure I. Main lineage of crystal protein gene sequences

21
 Table 2. Bacillus thuringiensis toxin genes or toxin-like genes unlisted in the classification
system.
Name Accession Year Reason
CrylAb-like AF327924 2001 Uncertain sequence data
Cry 1Ab-like AF327925 2001 Uncertain sequence data
Cry 1Ab-like AF327926 2001 Uncertain sequence data
CrytA-like AF327927 2001 Uncertain sequence data
CryIH-like AF182196 1999 Insufficient sequence data
Cryll-Iike 190732 1998 Insufficient sequence data
Cry1-like 190729 1998 Insufficient sequence data
Cry9-like AF093107 1998 Insufficient sequence data
40kDa M76442 1992 No reported toxicity
cryC35 X92691 1995 No reported toxicity
cryTDK D86346 1996 No reported toxicity
cryC53 X98616 1996 No reported toxicity
p21med X98794 1997 No reported toxicity
ET34 AF038049 1998 No reported toxicity
vip3A(a) lA8811 1996 Not a crystal protein
vip3A(b) lA8812 1996 Not a crystal protein

4.3 Distribution ofthe Crystal Protein Genes

There are many strategies to test the distribution of crystal protein genes among subspecies
and even strains.

4.3.1 DNA Probing. After cloning of the first insecticidal crystal protein gene, Kronstad et al.
(1983) detected the existence of the homologous DNA fragments in twenty-two B. thuringiensis
strains from fourteen subspecies, using the 726 bp EcoRI fragment from cry1Aal gene as probe. The
result of hybridization indicated that eighteen ofthe strains with toxicity to lepidopteran insects had
cry1A gene and the other four without toxicity had no signals.

Table 3. Distribution of insecticidal crystal protein genes in B. thuringiensis

Serotype Subspecies/Strains Toxin Genes *
I thuringiensis HD-2 crylAh?crylB
berliner crylAb
3a a/esti crylAh
3abc kurstaki HD- I ** crylAa?crylAb
3abc kurstaki HD-263 crylAh?crylAc
3abc kurstakiYBT-1520 crylAa?crylAh?crylAc
4304b sotto crylAa?crylAb
4304c kenyae crylAh?crylAc?crylE
6 entomocidus crylAb
7 aizwailPL cry 1Aa?cry lAb?cry lC?cry I D
7 juroi crylAa?crylAb?crylC?crylD
8308b morrisoni crylD
9 to/worthy crylAh?cryl E
10 darmstadiensis crylAh?crylD?crylE
ll30llb toumanoffi crylD
203020c pondicheriensis crylAb
23 japonensis crylAa?crylAh?crylD
24 neo/eonensis crylAa?crylAh?crylAc
25 coreanensis crylAa?crylAh?crylAc
0 wuhanensis crylAh?crylAc?cryJD
0 chinensis CT-43 crylB?crylAa. cry2A
• Detected by PCR assays, most of the data were provided by T. Iizuka. Some genes may not be included.
•• Errors might occur.

22
4.3.2 Monoclonal Antibody Detection. Monoclonal antibodies were used to detect the
distribution ofCry lA, Cry lB and Cry1C proteins in twenty-nine strains ofeleven subspecies, through
which they found out that the insecticidal crystal protein gene cry1A had a wide distribution but
cry1B and cry1C were not so common. As to the latter two genes, they mainly existed in B.
thuringiensis subsp. aizawai and subsp. entomocidus (Hofte et aI., 1988; Hofte and Whiteley,
1989).

4.3.3 PCR Detection. Two sets of primers, one for the detection of cry1A-cry1F and another
for the detection of cry2A and cry4, were designed to test the distribution of crystal protein genes
(Kalman et al., 1993; Asano et al., 1993; Iizuka, unpublished data). Thereafter, a series ofuniversal
or specific primers have been designed for the detection ofthe gene distribution (Carozzi et al., 1991;
Bourque et al., 1993; Ceron et al., 1994; Chak et al., 1994; Juarez-Perez et aI., 1997; Ben-Dov et
a!., 1997). Table 3 shows the distribution of genes cry1 and cry2A in part ofB. thuringiensis strains.

5. STRUCTURE OF THE INSECTICIDAL CRYSTAL PROTEIN GENES

5.1 Location ofthe Insecticidal Crystal Protein Genes

5.1.1 Relationship between the Crystal Proteins and the Plasmids. As was stated above,
the overwhelming majority ofthe crystal protein genes are located on large and conjugative plasmids
except for a few crystal protein genes that are on the chromosomes.

5.1.2 Relationship between the Crystal Protein Genes and the Transposable Elements.
A large amount of experiments have proved that the crystal protein genes are closely connected with
the transposable elements (Kronstad and Whiteley, 1984; Lereclus et a!., 1983; 1984; Mahillon et
a!., 1994). A total of seventeen insertion sequences, including two transposable elements as well as
IS231 group, IS232 group and IS240 group, have been isolated from B. thuringiensis. Only two
transposons were identified in B. thuringiensis. Tn4430, the first isolated transposon from B.
thuringiensis, probably concerns in the trans-conjugation of the plasmids (Lereclus et al., 1983,
1993).1S231 is the largest group of insertion sequence in B. thuringiensis. Among them, there are
at least four (1S231 A,IS231 B, IS231 C and IS231 W) in close relationship with the crystal protein
genes which exist widely in B. thuringiensis in various forms. It is regarded that this diversity is in
coincidence with the close relationship between the insecticidal crystal protein genes and the
transposable elements. A point ofdoubt is that why the overwhelming majority ofthe crystal protein
genes are located on the plasmids while only few ofthem are on the chromosome; even the crystal
protein genes are highly related to the transposable elements and have diversity in distribution. It must
be pointed out that a lot ofplasmids without crystal protein gene also carry Tn4430 or 1S231 (Lereclus
et al., 1988; Mahillon et al., 1988; Mahillon and Seurinck, 1988). Therefore, the transposable elements
of B. thuringiensis are extensively distributed in different replication units.

5.1.3 Restriction Fragments Harboring Crystal Protein Genes. Kronstad et al. (1983) and
Kronstad and Whiteley (1986) found that the crystal protein gene categories were in relationship
with the sizes of HindlII restriction fragment to some degree when they were working on the location
of the insecticidal protein genes. This phenomenon mainly occurred in strains belonging to serotype
H3ab and HI. With an exception in H3ab, the HindlII fragment of cry1Ac1 0 in strain YBT-1520 is
6.8kb, but not typically 6.6kb (Sun et a!., 1996).

5.2 Structural Features ofInsecticidal Crystal Protein Genes

5.2.1 Primary Structures. Crystal protein genes have obvious expression structures such as the
promoters, ribosome binding sites (RBSs) and terminators. But some genes like cry2Aa without

23
promoter exist as part of an operon, and some genes like cry2Ab have a stem-and-loops on the
upstream ofcoding sequences working as terminators (Widner and Whiteley, 1989; Hodgman et al.,
1993).

5.2.2 Repeated Sequences. Inside and outside the ORF of the crystal protein genes, there are
many direct repeats (DRs) or inverted repeats (IRs). Altogether, ten inverted repeats reside inside or
outside of the cry I Aa gene, with the ability to form stem-and-loop structure. One of them is situated
at the downstream of the translation stop codon, functioning as the transcription terminator (Schnepf
et aI., 1985). In the downstream sequence of crylAc there are two continuous inverted repeats and
a poly(T) structure contiguous to the latter inverted repeat. These structures are obviously the
transcription terminators (Adang et al., 1985). As to other genes, whether such structures are found
is still unclear. Gene cry I Ac has three direct repeats. One pair of20bp are on the outside ofthe ORF
(one from -72 to -53, and the other from 73 to 92 at the downstream of the stop codon). The second
pair of 12bp are inside of the ORF (one from + 63 to +74, and the other from +865 to +976). The
third pairof8bp are at the edge of the ORF (-3 to +5 and +3910 to+3917, respectively) (Adang et
aI., 1985). Gene cry lAa also has at least two pairs of similar direct repeats (Schnepf et aI., 1985).

5.2.3 Special Structure. For cryI Aa or cryI Ac, the UTRs (untranslated regions) at 3' -terminal
and 5' -terminal of the mRNA have some special structures functioning as the tools of enhancing the
expression of the genes (Schnepf et aI., 1985; Adang et aI., 1985).
(i) Compared with the mRNA of most ofthe other bacteria, the mRNA of cry I Aa or crylAc
has long UTRs, with 80-90bp atthe 5' -terminal and 110bp atthe 3' -terminal.
(ii) There is a potential ribosome binding site before the stop codon in mRNA of cry I Aa or
crylAc. Following the stop codon at 4 base pairs, there are a start codon, four sense codons and a
stop codon. Such structures probably can assist ribosomes to stall at the 3' -terminal ofthe mRNA.
(iii) A pair of direct repeats (8bp individually) reside near to the start codon (-3 - +5) and the
stop codon (12-5 upstream ofthe stop codon). This structure may perform the function ofterminating
transcription. Because at the late phase of the sporulation a shorter RNA could be isolated which
terminated at the downstream repeat (corresponding to 3910 bp of cry1Ac and 4043 bp of cry1Aa).
It is unknown whether this termination-enhancing function is caused by increased degradation from
the 3' to 5' terminal or is caused by the occurrence of the regulation structures for RNA processing
or transcriptional termination.
(iv) At the upstream ofthe start codon (corresponding to 318-331 bp of crylAc) and at the
position near to the stop codon (81- 88bp downstream, corresponding to 4000-4012bp of cry lAc)
are situated an 8bp direct repeat and a 10bp inverted repeat, respectively, which lead to the alignment
of the UTRs. However, in this kind of alignment, RNA binding proteins or other components are
needed for help because the alignment is not strong enough for the formation of a stable duplex. If the
existence of this alignment is definite (either inter-molecular or intra-molecular), such a structure
would provide a means of channeling ribosomes from the end of crystal protein mRNA to the
translational start site.

5.2.4 The Homology ofthe Crystal Protein Genes

5.2.4.1 Recombination ofthe Crystal Protein Genes. Besides the high similarity between the C-
terminal halves, Cry 1 and Cry4 also have several conserved blocks in the N-terminal halves (Hofte
and Whiteley, 1989). Detailed analysis for Cry 1 proteins showed that some ofthe genes are found
very likely to be recombined by two other genes. Taking Cry I Ab for example, it seems to have been
from the recombination of Cry 1Ac and Cry 1Aa at the site corresponding to the 360th amino acid,
with twenty-six amino acids deleted in the C-terminal half. Likewise, Cry I E appears to have been
recombined between Cry 1C and either ofCrylAb or Cry 1Ac, at the site corresponding to the 350th
amino acid. Cry 1D is likely to be the recombining product of Cry I A and Cry I C, at the site of the

24
257th amino acid with some other variety taking place between the 500th and 600th amino acids. The
most divergent Cry IB among Cry I shows prominent homology with any other Cry 1 protein (such as
Cry 1Ab or Cry 1Ac) in the C-terminus of the toxin-core-fragments corresponding to the 500th -600th
amino acid (Visser et al., 1993).
Since the crystal protein genes of B. thuringiensis are located on plasmids and closely related
with the transposable element, can the above recombination be regarded as gene recombination
occurring during the course of the natural evolution? Yet no direct proof has been achieved here to
this point. Ifthings really happened like this, however, how to explain the phenomenon that there is no
continuous recombined genes occurrence.

5.2.4.2 Deletion ofthe 78bp sequence. Compared with the C-terminal halves of the other crylA
genes, cry1Ab has a sequence of78bp deletion. This sequence codes twenty-six amino acids including
four cysteine amino acids (Cry lAb has thirteen cysteine amino acids in total), and is at the position
between two direct repeats (Geiser et al., 1986). The 78bp fragments can notably affect the formation,
dissolution and even the toxicity of the parasporal crystals (Aronson et aI., 1993). The optimum
temperature for the growth of B. thuringiensis is 30°C?but CrylAb can hardly be formed into
crystal under this temperature while it can under 25°C (Minnich and Aronson, 1984). B. thuringiensis
subsp. aizawai HD-133 contains genes crylC and crylD on the plasmid of over 100MD, and
cry I Ab on the 45MD plasmid. The parasporal crystal protein coded by crylAb is toxic to Plodia
interpunctella (the toxicity comes from Cry 1C or Cry lAb) (Van Rie et aI., 1990). But solubility of
the crystal decreased when cryI C lost, leading to decrease in toxicity. If the parasporal crystal was
dissolved in vitro, the toxicity becomes fairly high. And when the cry lAb gene is transformed back
into the host, the solubility and toxicity ofthe crystal were regained.

5.3 Structural Features ofthe Insecticidal Crystal Proteins

5.3.1 Structural Profiles. Being the most adequately studied crystal protein, Cry 1 proteins are
composed of! , 100-1 ,200 amino acids with the molecular weight of 130-140kDa. The full-length
crystal protein is unnecessary for the toxicity. When the 600th -1150th amino acid residues of the C-
terminal half and the 28-29 amino acid residues at the N-terminal are cleaved off with trypsin, the
toxin-core-fragment of about 60kDa is formed. This fragment is both necessary and adequate for the
toxicity (Schnepf and Whiteley, 1985; Nagamatsu et al., 1984; Whiteley and Schnepf et al., 1986).
The C-terminal halves ofCry 1 crystal proteins are highly conservative with the homology surpassing
90%. The N -terminal halves have comparatively high diversity with the homology ranging from 40010
to 90%. These two parts have obviously different structural and functional characteristics. The
hydrophilic C-termina1 are exhibited in b-sheet, mainly supporting the formation ofparasporal crystal.
The 1N-terminal halves are mostly hydrophobic, forming a-helix and contributing to toxicity (Choma
et aI., 1990; Convents et aI., 1990).
In Cry 1Ac, all the sixteen of cysteine amino acids are concentrated within C-terminal half or 28
to 29 amino acids at N-terminal end, and with none in the toxin core fragment. Such structure also
exists in the other Cry 1crystal proteins. Crystal protein dipolymer can be formed between the protein
molecules through disulfide-bonds (Huber et al., 1981; Bietlot et al., 1990; Du et al., 1994). This can
further prove that the C-terminal half plays an important role in preventing the crystal from being
dissolved (Luthy and Ebersold, 1981).
Few studies about the structural characteristics have been carried out on the other types of
crystal proteins. To acquire more data, see the review articles ofHofte and Whiteley, 1989; Lereclus
etaI., 1993; Yamamoto and Powell, 1993; Knowles, 1994; Schnepfetal., 1998.

5.3.2 Conserved Blocks. The N-terminal halves in Cryl, Cry3A and Cry4 proteins, corresponding
to the toxin-core-fragment, share five conserved blocks (Hofte and Whiteley, 1989). Other crystal
proteins also have various conserved regions of their own (F igure 2). Except for Cry6 and Cry 15,

25
the other thirteen Cry insecticidal crystal proteins all have the same block I and block 2. For those of
the molecular weight over 130kDa, three consensus conserved blocks are contained (block 6, 7,8).

Domain I Domain /I Domain 11/

• ~ I •

CryJA

Cry3A
\ '\
Cry4A

Cry7A

Cry8A • •••
Cry9A • • •
CryJOA

CryJ9A

Cry20A

CryJ 6A

CryJ 7A

Cry5Aa

CryJ2A

CryJ4A

Cry2JA

Cry13 A

IT
Cry2A

Cry18A

CryJ lA
-
100 amino aCids

Figure 2. Position of conserved blocks among crystal proteins (Schnepf et a\. , 1998).

5.3.3 Functional Domains. The three-dimensional structure ofCry3A crystal protein has been
determined through X-ray (Figure 3). The partially degraded Cry 3A crystal protein of 67kDa can be
divided into three domains: Domain I is built up of seven antiparallel hydrophobic a-helixes, in which
helix 5 is encircled by the remaining helixes. Domain II is built of three antiparallel b-sheets jointed in
a typical 'Greek key" topology, arranged in a so-called b-prism fold; Domain III is a structure of
tightly packed b-sandwich with a "jelly roll" topology, with the C-terminal end packed in. The first
and the second conserved blocks are mostly in Domain I. The majority part of block 3, together with
block 4 and 5, is in Domain III. Functions of the three Domains can be deduced from their structural
characteristics. Domain I is the place where the toxin stretches into the cell membrane and exerts its
toxicity. Domain II, possessing quite a few kinds ofloop structures, is most likely to be involved in the
binding oftoxin to receptor. Domain III shows the ability to protect the C-terminal ofthe activated
toxin from being further degraded in the midgut of insect (Li et al., 1991; Schnept et al., 1998).
The determination ofthe spatial structure ofCry IAa and Cyt2A has completed now (Figure 3).
Cry I Aa and Cry3A proteins shares 36% identity in amino acid sequence, meanwhile their spatial

26
structure is very alike (Crickmore et aI., 1998). Cyt2Aa and Cry3A have the homology identity of
only 20%, with the spatial structure completely different.

CrylAa Cry3A

Figure 3. Three-dimensional structures of Cry lA, Cry3A, and Cyt2A (Schnepfet a!., 1998)

5.3.4 Functional Regions for Host Specificity. Different crystal proteins show different activity
to insects, not only in the insecticidal spectnun, but also in the toxicity strength, so that they exhibit the
insecticidal specificity. For examples, the toxicity of Cry lAa to Bombyx mori is 400 times higher
than that of Cry I Ab to B. mori, while is only one-tenth as it to Trichoplusia ni, and the toxicity is
similar for both Cryl Aa and Cry lAb to Manduca sexta (Ge et aI., 1989; 1991). By constructing
hybrid genes, the region specific to B. mori is found to reside between the 332nd to the 450'h amino
acids. The region with specificity to Heliothis virenscens is longer, surpassing the whole length of
Domain II and Domain III, the 335'h-615'h amino acids (Ge et aI., 1989). This explains why Cry I A
crystal proteins have multiple binding sites in the receptors of midgut of H virescens larvae 01 an Rie
et aI. , 1990a).
The Cry 1Ab crystal protein (such as Cry lAb 7) in B. thuringiensis subsp. aizawai strain IC I
has another type of insecticidal specificity (Haider et aI. , 1986; Haider and Ellar, 1987, 1988).
Typical Cry I Ab (eg. Cry 1Ab5) is only toxic to lepidopteran larvae, but Cry 1Ab? has extra toxicity
to mosquito larvae. Indeed, Cry 1Ab 7 and Cry I Ab5 differ from each other only in three amino acids.
The spectrum of variety is.caused by the shift of the 558'h-595'h amino acids leading to different
sensitivity to the proteinase in insect midgut (Haider et aI., 1989; Haider and Ellar, 1989).

6. EXPRESSION AND REGULATION OF THE INSECTICIDAL CRYSTAL PROTEIN

GENES

The biosynthesis ofthe crystal proteins begins following the second stage of sporulation resulting
in the crystal taking up the proportion of25% in the dry weight of the cells (Ribier and Lecadet,
1973; Agaisse and Lereclus, 1995).

6.1 Regulation on Transcription Level

6.1.1 Regulation by Promoter. Biosynthesis of the crystal protein during sporulation is

directed by the promoter of crystal protein genes. In 1983, two transcription promoters specific
in sporulation were firstly identified from crylAal gene (Wong et aI., 1983). The distance from
them to the start codon was 80-90bp.,Bt I, the promoter at the near end, had activity at the early
phase (t2-t6), and Bt II, at the far end, exerted activity at the late phase (after t s)' Two sporulation cr
factors of RNA polymerase, cr3S and cr28 , were also isolated. These two cr-factors would only be

27
synthesized during sporulation, so the transcription of crystal protein genes initiated during
sporulation and reached its highest level at the early phase (Brown and Whiteley, 1988; Adams
et aI., 1991). Therefore, the expression of the crystal protein genes was caused by the continuous
activation from spore-specific s factors, taking time into consideration. Ifthe Spo- mutation took
place at to and the sporulation was blocked, such hosts could not produce crystal. However,
when blocking occurred after t 2, the formation of the crystal would not be affected (Ribier and
Lecadet, 1981).
Three types of promoters of crystal protein genes have been identified in B. thuringiensis
(de Souza et aI., 1993; Lereclus et aI., 1989b). The first type is the promoter recognized by a 35
at the early phase of sporulation, such as promoter Bt I (eg. crylA gene) and Pbi(eg. cytlA) as
well as crylB, cry/VA and cry34 (i.e. cry15A)(Wong et aI., 1983; Waalwijk et aI., 1985; Ward
and Ellar, 1986; Brizzard et aI., 1991; Yoshisue et aI., 1993a; 1993b; Brown and Whiteley,
1992; Brown, 1993). These are the key promoters of crystal protein genes in B. thuringiensis.
The second type of promoters is recognized by sigma a 28 , such as Bt II of cry1A gene. And the
third types are the promoters with the s factors unidentified, such as PBc of cry3A and PbiII of
cytlA.
Meanwhile, the two promoters of cytlA, PBi I and Pbi II, are identified to be similar to Btl
(Ward et aI., 1988) and PBc (Waalwijket aI., 1985; Sekar et aI., 1987), respectively.

6.1.2 Regulation of Upstream Sequences of Promoters

6.1.2.1 Colepteran-Specific cry3A Gene. In B. thuringiensis, the expression of the coleopteran-

specific cry3A gene differs greatly from that of the other genes. The expression of cry3A begins in
vegetative growth and is at an activated state at the beginning of the stationary phase (Sekar, 1988;
Sekar et aI, 1987; De-Souza et aI., 1993). So does the expression of cry/HB (Donovan et aI.,
1992). The researchers in Ecogen Company considered that cry3A promoter is activated by the
factor analogous with SA (Malver et aI., 1994). The complete expression of cry3A needs a 1kb
fragment 400bp from the start codon at the upstream. This fragment largely enhances the expression
of cry3A within six hours from the beginning ofsporulation. Sequence analysis showed that no potential
amino acid coding sequence exist in this region, and this 1kb fragment contained a 220bp of A+T
rich region (De-Souza et aI., 1993; Agaisse and Lereclus, 1996). The expression of cry3A is
independent of sporulation and can be enhanced by the gene mutation (such as spoOA or spoOF)
occurring at the early phase of sporulation (Agaisse and Lereclus, 1994a,b; 1996; Malvar et aI.,
1994; Malvar and Baum, 1994). The enhanced expression may be caused by two reasons. One is
that the mutation at the early phase of sporulation makes the stationary phase lag and more time are
left for transcription and spore formation. The other reason of possibility is that the amount ofthe
sporulation-specific proteinase decreases due to mutation, so that the stability ofCry3A crystal
protein is enhanced.

6.1.2.2 cry 1 Genes. The upstreams of promoters in cry 1 genes have influence on the production
of gene expression. In different cry1 genes, the upstreams have different effect on gene expression,
such as the 208bp or the 780bp upstream fragments of cry1Ab and cry1C (Cheng et aI., 1999).

6.2 Post-Transcription Control

As was mentioned previously, each one of the crystal protein genes has a stem-and-Ioop
structure just at the downstream of coding sequence acting as a terminator. As early as in 1972,
Pasteur Institute noted that the half-life of the mRNA of crystal protein genes was as long as
ten minutes (Glatron and Rapoport, 1972). Wong and Chang (1986) regarded this stem-and-
loop structure as a kind of positive regulation factor protecting the 3' -terminal from being degraded
by the nuclease.

28
6.3 Post-Translation
6.3.1 20kDa Help Protein. In B. thuringiensis subsp. israelensis, a gene coding a protein of
20kDais settled in the downstream of cryllA. Being in the co-transcription unit with cryllA, this
gene is initiated to express by the promoter of cryllA (Adamset al., 1989).

6.3.1.1 Effectiveness in E.coli. Distinct effect ofthis 20kDa protein is that it can promote the
biosynthesis of Cyt 1A in E. coli (Adam et aI., 1989; Douek et aI., 1992; Mclean and Whiteley,
1987; Visick and Whiteley, 1991). Actually, the expression of cytlA has lethal effect on the host cell
of E. coli under the condition oflacking the 20kDa protein (Douek et al., 1992).

6.3.1.2 Effectiveness in Bacillus. When help protein gene is linked to gene cyt1A or cry11A, the
expression ofCytl A or Cry 11 A protein is detectable. But the expression level is very low (especially
when it is compared with that in the wild-type strain ofB. thuringiensis subsp. israelensis) and no
crystal formation can be observed (Adams et aI., 1989). At the same time, the 20kDa protein has
enhancing effect in Bacillus. For example, no crystal can be observed when cyt1A expresses in B.
mageaterium ifthere is no 20kDa protein (Donovan et aI., 1988a), and only mini crystal can be
found in B. subtilis (Ward et al., 1986). As to this effect, the two laboratories ofthe Insect Department
in Riverside University in US hold different opinions. Chang et al. (1993) thought that the high level
expression of cryllA and cytlA genes didn't need the 20kDa protein, because whether with or
without it, either Cyt 1A or Cry 11 A could be produced and form crystals. They also considered that
the stability ofCyt I A protein could be enhanced by Cry 11 A or the 20kDa protein in crystal negative
mutant strain CryB, if Cry 11 A or the 20kDa protein really existed in the strain. The 20kDa protein
has the ability to protect the survival of the cells and start the formation ofCytlA crystal (Wu and
Federici, 1993). When the 20kDa protein is absent, Cyt synthesized in strain CryB would kill the
cells, or the CytlA crystal formed in strain 4Q7 is very small. But when the 20kDa protein is present,
the expression of cyt1A results in a rhomboidal crystal that is much larger than in the strain of wild
type. Particularly, the CytlAcrystal can protect the survival of the cell when it is in strain CryB.
Pang et ai. (1999) cloned a p21zb gene from strain Bt2B522. This gene had 97.8% identity
with the p20 gene and had 85.1 % identity with p21med gene in B. thuringiensis subsp. medellin
(Thiery et al., 1997). Subsequently, five p21 genes were isolated from five other subspecies (HI7,
H33, H36, H4ac and H9).

6.3.1.3 Role of20kD Protein. In the first place, the existence ofthe 20kDa protein gene doesn't
affect the quantity of the specific mRNA of Cyt 1A. The mRNA of the 20kDa protein occurs two
hours earlier than that of cytlA but synthesis of both the proteins starts approximately at t2 during
sporulation. So the 20kDa protein must fulfill its function in the post transcription period. Second, the
20kDa protein was found to have no influence on the initiation of the cytlA gene translation through
gene fusion. Besides these, in the dissolved crystal sample from B. thuringiensis subsp. isrealensis
there is a 20kDa protein. And with the interaction of the antibodies (anti-20kDa protein or anti-
CytlA), the 20kDa protein always co-precipitates with CytlA (Visick and Whiteley, 1991). From
this phenomenon, it was deduced that the 20kDa protein functioned as a special molecular chaperone
commonly termed help protein.
Generally, the protein can fold into correct conformation automatically after it is biosynthesized.
But in many situations, especially when it is in vivo, the assembling of the protein demands the help
of the molecular chaperon (Ellis and Vander Vise, 1991). The 20kDa protein can bind with Cyt lA
when it is synthesized, protecting CytlA from the degradation and initiating the formation of the
crystal simultaneously (Liu et al., 1999). Pang et al. (1999) discovered that p21 gene could also
promote the expression of the silent cry1 genes.
Because CytlA has cytotoxic activity in vitro (Thomas and Ellar, 1983a,b), it is not hard to
give an explanation to the lethal effect of Cyt 1A to strain CryB. But the Cry- strain 4Q7, derivative

29
of B. thuringiensis subsp. israelensis, is ne~ to normal. That indicates the possibility that another
set of protection or help mechanisms occurs.

6.3.2 Other Help Proteins. At least three crystal protein genes have been found to reside in
operons. Gene cry2Aa is located at the end of an operon composed of three ORFs (Widner and
Whiteley, 1989), meanwhile gene cry15A is in an operon together with a gene encoding a 40kDa
protein (Brown and Whiteley, 1992; Brown, 1993), and gene cry11A is in an operon containing the
20kDa protein gene (Adams et al., 1989).
In the cry2Aa operon, gene ort2 affects the formation ofCry2Aa crystal by its product (Crickmore
and Ellar, 1992). ORF2 is a protein of29kDa with special structure of eleven random repeats of
fifteen amino acids, running at the position corresponding to that of the 50kDa protein as tested by
SDS-PAGE (Widner and Whiteley, 1989). Cry2Ab doesn't express under normal condition (Widner
and Whiteley, 1990; Hodgman et al., 1993). It has no promoter structure at the upstream of the
translation start codon, but has a stem-and-loop structure acting as a terminator (Hodgman et al.,
1993). Through gene fusion, ORF2 can also help Cry2Ab proteins to form crystal (Crickmore et al.,
1994). Therefore, ORF2 is also a molecular chaperon like the 20kDa protein.
Brown (1993) demonstrated that the Cry40 protein of 40kDa in B. thuringiensis subsp.
thompsoni with no toxicity was probably a molecular chaperon as well. Cry 15Aa could be solely
expressed in B. subtilis but the expression level evidently dropped. So Cry40 probably help regulating
the stability of Cry 15A. And now Cry 15Aa and Cry40 are proved to be binary toxin (Rang et al.,
1999).
Dervyn et al. (1995) found thatthe operon ofthe 20kDa protein gene and cry11A gene had
three ORFs altogether, with the 20kDa protein gene at the far end and a 19kDa protein gene (p 19)
at the upstream. We also found that p 19 had a promotion effect on the expression ofthe crystal
protein genes (unpublished).
Agaisse and Lereclus (1995) held that the 20kDa protein and the 19kDa protein as well as
ORF 1 and ORF2 in the cry2Aa operon all played principal roles in the synthesis of crystal proteins
or in the formation of the crystals. But the name "molecular chaperon" was not suitable for the time
being. It was better to term them as Help proteins or more accurately crystallization proteins.

6.4 Copy Numbers ofthe Genes

6.4.1 Differential Expression. Most B. thuringiensis strains contain multiple crystal protein
genes that show a certain degree of diversity in the expression level. It is easier to get a good
understanding for those genes with different promoters. Gene cyt1A have two different types of
promoters. CytlA has the highest expression production among all of the crystal proteins in B.
thuringiensis subsp israelensis (WU and Federici, 1993). But for those similar genes holding identical
promoters, the differential expression also exists. For example, the expression of cry1Aa, cry1Aab
and cry1Ac is not on the same level in strain HD-l , and their production is unequal in the amount
(Yamamoto et al., 1988a; Masson et aI., 1990a,b; Pusztai-Carey et al., 1994)(Table 4). So there
must be an another set of regulation system.

6.4.2 Diversity of Copy Number of Crystal Protein Genes. The different location ofthe crystal
protein genes on plasmids or chromosome leads to the difference in the copy numbers of the genes.
B. thuringiensis subsp. thuringiensis strain 407 has two of the cry1A genes, while cry1Aa gene has
a comparatively lower expression level. But when it was cloned in a crystal negative mutant strain
(cry-) with a low copy plasmid vector pHT31 01, expression of high level was acquired (Lereclus
et aI., 1989a). The expression variation may be caused by the low copy number of cry1Aa that led
to decrease in the ability ofcompeting for sfactors. Similarly, when crylAc gene was transferred into
other B. thuringiensis strains containing crystal protein gene, the expression dropped to a level
obviously lower than when it was transferred into the crystal negative B. thuringiensis strain (Baum

30
et al., 1990). It suggested that the production oftoxins in B. thuringiensis was not strictly related to
the copy nwnber ofthe cry genes. The capacity ofB. thuringiensis strains to produce crystal proteins
is limited (although at a high level) and reaches a maximwn at a certain nwnber ofcrystal protein gene
copies in the cell, above which there is no further increase in synthesis (Agaisse and Lereclus, 1995).

Table 4. Differential expression ofthe insecticidal crystal protein genes in Bacillus thuringiensis
Subspecies! Stains Genes Protein proportion(%)
kurstaki HD-I cryJAa 28
crylAh 39
crylAc 33
kurstaki NRD-12 crylAa 41
crylAb 36
crylAc 23
kurstaki A-20 crylAa 17
crylAh 17
crylAc 66
entomocidus cryJAa 40
crylAh <5
crylAc <5
crylB <5
crylC 50
kurstaki HD-73 crylAc 100
tenehrionis cry3A >90
israelensis cytlA 30
(+cry4)
Modified from the data the patent of Pusztai-Carey et al.( 1994).

Gene cry3A was studied for the expression by using plasmid vectors with different copy nwnbers
(Arantes and Lereclus, 1991). Cry3A had a low level of expression when it was cloned in a low
copy plasmid, while it reached to a quite high level when using high copy ones (copy nwnber of 15).
Adams et al. (1994) studied the expression of cry3A at the state of different copy nwnbers. As a
result of y-ray mutagenesis, a mutant strain NB 176 (B. thuringiensis subsp tenebrionis) with high
production of Cry3A protein was acquired, which had a production as high as 3-4 times to that of
parent strain NB 125. Southern hybridization exhibited that the mutant strain NB 176 not only had a
3kb HindIII fragment carrying cry3A gene at the 90MD plasmid just as the parent strain, but also
had another 4kb HindIII fragment carrying cry3A gene at a smaller plasmid. Sequence analysis
showed that at the downstream of the 3' -terminal of cry3A gene in the 4kb HindlII fragment there
was a transcriptase sequence that was identical with the sequence of Tn5401 (Bawn, 1994). So the
appearance ofthe 4kb HindlII fragment carrying cry3A gene was caused by the transposable element.
For further illustration ofthe correlation between the copy nwnber and gene expression, cry3A
gene was integrated into the genome ofNB 125 by using pCP 115, an integrative plasmid. Mutants
with various sizes ofcrystals were acquired through continuous enlargement of resistance selection.
The result showed that the sizes of the crystals were in proportion to the copy nwnbers of cry3A, but
the toxicity did not increase with the size ofthe crystal because the strain producing the biggest crystal
only had the toxicity near to that of the parent strain. The reason for the low toxicity may be that the
spores were unable to form normally so that the cells could not lyse and release the crystals. Or it
may be caused by the inability of the crystals for degradation. Although gene cry3A has no spore-
specific promoter, the increase of the copy nwnber of it still has influence on sporulation.

6. 5 Heterologous Expression ofthe Genes

After crystal protein genes are cloned, it is necessary to examine the expression and insecticidal
activity in model strains. The commonly used model strains include E. coli and B. subtilis as well as

31
some other strains. For example, the firstly cloned gene was examined for its expression and the
insecticidal activity in E. coli (Schnepf and Whiteley, 1981). The inclusion body formed in E. coli
could reach 10% of the crystal proteins to the total proteins in the cells (Shivakumar et aI., 1986).
The crystal proteins that were expressed with vectorpKK223-3 could even take up 48% of the total
cellular proteins (Ge et al., 1990). The expression in other hosts will not be discussed here.

7. MECHANISM OF ACTION FOR CRYSTAL PROTEINS

After sensitive insects take in the crystals, they enter insect midgut, and release the toxin-core-
fragments after solubilization and trypsin activation. Then the toxin-core-fragments act on midgut
epithelial cells and cause the swelling and lysing ofthe cells. Details ofthe histological and biochemical
process of the insecticidal activity have been reported. As to why different strains have different
insecticidal specificity or why they have different insect spectra and toxicity, the reasons also have
been reported in detail. Among these reasons, the specific binding of the toxins and the receptors on
the surface of midgut epithelial cells is critical (Luthy and Ebersold, 1981; Knowles, 1994; Schnepf
et aI., 1998).

7. 1 Environment in the Gut ofthe Insects

The crystals are mainly toxic to lepidopteran larvae and also have the toxicity to dipteran,
coleopteran insects as well as some other organisms. Half the weight oflepidopteran larvae is the gut,
and the midgut takes up most part of it. The pH is fairly high in midgut. For some insects, the pH is
higher than 12. The alkaline environment ofthe midgut benefits the dissolution ofingested crystals.
And the types of the proteinases determine the activation of the toxicity.

7.2 Release of the Toxins

7.2.1 Solubilization of Crystals. The crystals exert their toxicity only after the crystal proteins are
released under the alkaline conditions. Most of the crystals are dissolved and release the crystal
proteins after having their disulfide bonds linking the crystal proteins broken under the condition of
high pH and reduction. Experiments in vitro showed that the proteins Cry 1, Cry3, Cry4A, Cry4B
and CytlA in crystals could be easily dissolved and released when the pH was 9.5 and 5% of2-
sulphedryl ethanol or 10mllL ofDTT was contained. But the crystal proteins Cry2A and Cry 11A
didn't dissolve unless the pH was 10.5 and no reducing agent was contained. The pH is a bit below
7 in the guts ofChrysomelid insects?the sensitive objects ofthe Cry3A crystal proteins, and therefore,
it is supposed that some other factors may be involved in the solubilization of protein Cry3A (Slaney
et al. 1992).
The ability for insects to dissolve a certain kind of crystals can be measured by comparing the
toxic differences between the crystals and the protoxins (Jacquet et al., 1987). And the interactions
of different types of crystal proteins can also influence the solubilization ofthe crystals (Aronson,
1993).

7.2.2 Activation of Protoxins. The crystal proteins released after crystal solubilization are a kind
of protoxins that can not exert the toxicity directly. The protoxins are cleaved into anti-proteinase
toxic core fragments by the trypsin-like proteinases in the midgut. Only such activated fragments can
recognize the specific receptors on the surface of midgut epithelial cells, and fulfill its function of
exerting toxicity (Hofte and Whiteley, 1989). The Cry 1 crystal proteins have their C-terminal halves
and a small part of their N-terminus (28 to 29 amino acids) cleaved off in the course of being
activated, and leave themselves an activated fragment of 55-70kDa. Protein Cry 1Ab crystal protein
has the toxic core fragment from the 29th to the 607th amino acid residues with the molecular weight

32
about 60kDa (Hofte et aI., 1986; Hofte and Whiteley, 1989; Choma et al., 1990). The activation
process of Cry 1Ac crystal protein proceeds from the C-terminal half to the N-terminal. Through
reaction of the proteinases, seven short peptides of8-1 OkDa are cut off in order sequentially till the
reaction ends at the 623rd amino acid residue. Simultaneously, twenty-eight amino acids are cleaved
at the N-terminus and the anti-proteinase toxic core fragment of60kDa comes into being (Choma et
al., 1990). The activation ofother Cry 1 crystal proteins is similar to that ofthe CrylA crystal proteins.

7.3 Binding ofthe Toxins to the Receptors on Midgut Epithelial Cells

After a protoxin is activated, it reaches midgut epithelial cells through peritrophic membrane
and binds to one of the specific receptors on the brush borders membrane with high affinity, then it
exerts the toxicity. The interactions and binding ofthe toxins to the receptors were researched through
the preparation of the brush border membrane vesicles (BBMVs) or phospholipid vesicles (Knowles,
1994). Many researches have demonstrated that the binding of the toxins to the receptors is the first
interaction between the toxins and the midgut epithelial cells (Knowles, 1994). Two types ofbinding
sites with different affinity to the crystal proteins were identified in BBMVs and their insecticidal
specificity was found to have connection with the affinity. Although no quantitative relationship was
found between the binding and the toxicity, the binding to the receptors is indispensable for the
exertion of the toxicity and even determines the insecticidal specificity (Wolfersberger, 1990; Ferre et
aI., 1991; Garczynski et aI., 1991; VanRie etaI., 1989, 1990).

7.3.1 Toxin Receptors of Cry Crystal Proteins. Many assays attested that the receptors of
crystal proteins were glycoproteins of 120-180kDa (Garczynski et al., 1991; Knowles et al., 1991;
Oddou et aI., 1991). It was reported that the receptor of Cry lAc crystal protein identified in the
BBMVs of Manduca sexta was aminopeptidase N (Knight et al., 1994). The psychological fimctions
of the glycoprotein receptors and the interactions between the receptors and the toxins had been
unknown until the functional receptor genes were cloned and identified. But Knowles and Dow
(1993) took the view that three possible fimctions might be fulfilled by the receptors in the formation
process ofthe membrane pores: (i) they themselves acted as the transmembrane channels for the
toxins, (ii) they formed the membrane pores together with the toxins, (iii) they functioned as the
transmission media for the toxins to act on the cell membranes.

7.3.2 Toxin Receptors ofthe Cyt Crystal Proteins. The Cyt crystal proteins have extensive
cytolytic activity in vitro (Thomas and Ellar, 1983a). As the Cyt crystal proteins could bind with
unsaturated phospholipids and insert into the cell membranes automatically, it was considered that
the Cyt crystal proteins didn't need protein receptors during the course (Thomas and Ellar, 1983 b;
Drobniewski and Ellar, 1989; Chilcott et al., 1990), but may have definite receptors in vivo (Knowles
et aI., 1990).

7.4 Formation ofthe Pores in the Cell Membranes

When the toxins bind to the specific receptors on the brush border membranes ofmidgut epithelial
cells, they can insert into the plasma membranes rapidly and irreversibly. According to the structure
of Cyt 1A crystal proteins (Ward et aI., 1988) and the protease hydrolytic features of the insertion
toxins, Chilcott et aI. (1990) proposed a model of how Cyt 1A crystal proteins insert in the plasma
membranes. At the beginning, Cyt 1A proteins formed into an oligo-polymer composed of sixteen
toxic monomers, then the pores or the lesion in the plasma membranes came into being. Although
Cry crystal proteins and Cyt crystal proteins lacked homology from each other, Cry crystal proteins
could also insert in the plasma membranes and cause the lesion similar to that those caused by Cyt
crystal proteins (Knowles et al., 1989, 1992). According to the special structure of Cry3A crystal
proteins, two hypotheses were proposed about how the pores were formed by the Cry crystal

33
proteins. In Penknife model, the Cry proteins could form into helical hairpins in pair acting on the
plasma membranes and such helical hairpin structures formed into hexamers that made pores of
O.6nm in diameter (Knowles and Ellar, 1987; Hodgman and Ellar, 1990). The Umbrella Model was
proposed by Li et al. (1991), when they were detennining the spatial structure oftheCry3A proteins.
The main idea of this model was that the membrane pores were formed by oligomerization or by
further insertion of other helices from Domain I.

7.5 Cell Lysis

When the toxins induce the formation of the pores in the membranes of the columnar cells, the
ion gradient, such as the K+ electrochemical gradient that drives nutrient absorption, maintaining the
functions of the cell membranes, is damaged. And then some inter-membrane or outer-membrane
molecules that couldn't permeate the cell membranes now can freely pass through the cell membranes.
The columnar cells swell and are lysed finally. Many results on the cell dissociation have been reported,
but Knowles (1994) deemed that there should be two hypotheses that could illustrate the mechanism
of the damage in the midgut epithelial cells.

7.5.1 Proton Peril Hypothesis. This hypothesis was proposed for lepidopteran larvae and the
insects having high pH in their midgut. In the midgut epithelial cell membranes there were K+ pumps
that were driven by protons. The K+ selective channels formed in the columnar cell apical membrane,
making the columnar cells leaky to K +but not affecting active K+ pumping in the goblet cell (Harvey
et ai., 1986; Crawford and Harvey, 1988). Such results led to the suggestion that formation of a
cation leak in the normally K+-impermeable columnar cell apical membrane would result in
depolarization ofthe cell membranes and a consequent efflux ofhydrogen ions down along the steep
pH gradient (Knowles, 1994). The hydrogen ion concentration inside the membrane was as 10,000
times as that outside the membrane. Thus the pH rose in the cell plasma ofcolumnar cells, and then
the cells were killed, and the gut brokedown. Finally the insect was led to death (Harvey et al., 1986;
Wolfersberger, 1992).

7.5.2 Osmotic Lysis Hypothesis. The cells were inclined to take in water continuously because
of the large negative charged intracellular macromolecules like proteins and nucleic acids in the cells.
Slight leakage in the plasma membrane could be repaired by the activity of the ion pumps. But the
repression oflarge fissures or transportation would unavoidably bring to osmotic lysis of the cells
(Knowles and Ellar, 1987).

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Sekar, V., Thompson, D.V., Maroney, M.J., Bookland, R.G., and Adang, M.J., 1987, Molecular cloning and
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Sohesser, J.H., and Bulla, L.A., 1978, Toxicity of Bacillus thuringiensis spores to the tobacco homworm, Manduca
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40
BACILLUS SPHAERICUS : MECHANISM AND APPLICATION AS A MOSQUITO
LARVICIDE

YuanZhiming

Wuhan Institute ofVirology, Chinese Academy of Sciences, Wuhan 430071, China

1. INTRODUCTION

Bacillus sphaericus is a ubiquitous, cosmopolitan, aerobic and sporeforming bacterium, with a

terminal swelled sporangium and spherical spore. All strains have been grouped into 49 serotypes on
the basis ofthe flagella agglutination. Most strains are non-entomopathogenic with only nine serotypes
(HI, H2, H3, H5, H6, H9, H25, H26 and H48) exhibiting toxicity against mosquito larvae. The toxic
strains produce two kinds of toxins. One is a binary toxin, synthesized and assembled as the crystal
forms during sporulation. This kind oftoxin is composed of 42 and 51 kDa active peptides, and both
fragments are required for toxicity. The other kinds of toxins are mosquitocidal toxins (Mtx), which
are produced during bacteria vegetative growth. It has been shown that the highly toxic strains are
very toxic against Culex spp. followed by Anopheles spp., but have no or low toxicity to Aedes spp.
Strains ofB. sphaericus offer distinct advantages compared to bacillus spp. Strains of B. sphaericus
offer distinct advantages compared to Bacillus thuringiensis var. israelensis, having an increased
duration oflarvicidal activity against certain environments. In the last decades, B. sphaericus have
been must successfully use as a larvicide in many parts ofthe World, and it is considered a promising
agent for mosquito control, especially for Culex spp. in the suburbs of cities or small towns. This
Chapter will focus on biological properties, host range and toxicity, mosquitocidal toxins and the
encoding genes, genetically modification, production and field application ofB. sphaericus, as well
as the resistance problem in insects arose by the widely application of this bacterium.

2. TROPICAL DISEASE VECTORS AND THEIR CONTROL

2.1 Mosquito-Born Diseases

Mosquitoes not only transmit some of the world's most serious diseases, such as malaria,
encephalitis, filariasis, yellow fever, dengue etc., but also cause much nuisance to human being by
biting in some temperate regions, varying in effect from slight annoyance to severe discomfort and
itching. These blood sucking insects have plagued man since prehistoric times and they are vectors of
a multitude of diseases of man and animals through transmission of pathogenic viruses, bacteria,

Advances in Microbial Control o/Insect Pests

Edited by Rajeev K. Upadhyay. Kluwer Academic / Plenum Publishers, New York, 2002 41
protozoa and nematodes. In Family Culicidae, there are three sub-families, Culicinae, Anophelinae
and Toxorhynchitinae. Because the female adults ofToxorhynchitinae are not haematophagous, they
have no medical importance. The sub-family Culicinae is composed of 40 genera, including 3400
species, and some ofthem are medically important species, like the mosquitoes in genus Aedes,
Culex, Mansonia and Armigeres. Of them, Culex quinquefasciatus is an important vector of
filariasis, and C triaeniorhynchus transmits Japanese encephalitis. Members of the genus Aedes
are vicious biters and transmit yellow fever and dengue virus as well as filariasis in several parts ofthe
world. Anophelelinae has three genera, including 500 species. The most important species in genus
Anopheles, such as An. sinensis, An. dirus, An. gambiae, An. anthropophagus, An. funestus,
An. arabiensis etc., transmit Plasmodium species, filarial parasite (Wucheria bancrofti and Brugia
malayi), and a few arboviruses (Figure 1). Family Simuliidae consists of 1200 species, but only a
few can transmit Onchocera volvulus, the nematode responsible for river blindness caused by
accumulation of dead nematodes in eyes in infected people (Table 1).
As estimated by WHO, currently there are about 90 million cases of human lymphatic filariasis
worldwide, mainly occurred in the urbane parts in some developing countries where the environmental
conditions favor vectors and pest insects responsible for the transmission. Wucheria bancrofti,
transmitted by Culex sp., is probably the most widespread of filarial worm parasites of humans,
There are 300 million individuals infected with this nematode, which in severe cases gives rise to
gross deformity as elephantiasis. Furthermore, approximately 400 million people live in highly malarious
areas, and malaria has an annual incidence of about 270 million cases and killed 2 million people
worldwide and over 1 million persons in Africa alone. Alarmingly, not only some serious tropical
diseases that had been successfully controlled for last decades resurged, but also some new vector-
born diseases have been emerging in some parts of the world (WHO, 1995; 1996; Blanco Castro et
a!., 2000). More recently, West Nile virus, which was first noticed in Africa and transmitted by
Culex spp. has emerged in temperate regions of Europe and North America. Attack against the
mosquito is recognized as an effective approach for eradication ofthreat of these tropical diseases
(Miller, 1992).

Culicidae
(3400 species in 34 genera)
I I I
Anopheliae Culicidae Toxorhynchitinae
I
Anopheles
I I
Toxorhynchis
Culicinae
I (Not a disease vector)
Anopheles Culex
(about 400 species are Aedes
disease vectors Mansonia
e. g. malaria, Haemagogus
filariasis Armigeras
(Disease vectors,
e. g. filariasis, yellow fever, dengue)

Figure 1. Classification of mosquitoes and their impacts as disease vectors.

Before the appearance of chemical pesticides, people used smoke and some natural repellents
to keep mosquitoes away from them, also treated mosquito breeding sites with mineral or vegetable
oil and other biological agents. Since the beginning of40s in last century, the use ofchemical pesticides,
such as dichlorodiphenyltrichloroethane (DDT), gamrnanane, malathion, and chlordane, has been

42
one ofthe major methods for mosquito control. The use of DDT for vector control in the Global
Program for Malaria Eradication launched in 1955 by WHO, combining the successful use ofthe
anti-malarial and other anti-disease drugs, resulted in a dramatic reduction ofthe morbidity and
mortality caused by tropical diseases. However, malaria and other mosquito-borne diseases are on
the rise again in many tropical areas, mainly due to resistance in mosquito populations as well as
DDT's lack of selectivity affecting non-target populations, including mosquito competitors, predators
and pathogens. The emergence of pesticide- and drug-resistant mosquitoes, as well as the risks of
the powerful chemicals to mankind and the environment, has highlighted the need of some other
environment friendly alternatives. Among them, the biological control of mosquitoes is regarded as
one ofthe promising alternatives.

Table 1. The main tropical diseases and their vectors

Diseases Distribution Pathogens Main vectors
Malaria Asia, Africa and Protozoa An. sinensis, An. minmusi,
Latin America An. ganbiae, An. dirus
Filarasis Asia, Africa and Nematode C. quinquefasciatus,
Latin America C. pipiens, An. sinensis,
Aedes species
Onchocercose Asia, Mediterranean Nematode Simulium
and Latin America
Dengue India, Southeast Asia, Virus Ae. aegypti, Ae. albopictus,
Caribbean, Hawaii C. quinquefasciatus

Yellow fever West Africa, Virus Ae. aegypti, Ae. albopictus,

South America Mansonia, C.
quinquefasciatus
Encephalitis Japan, Korean Virus A. albopictus, Aedes species,
Peninsula, China C. tritaeniorhynchus,
and India C. quinquefasciatus etc.
Other encephalitis Worldwide Virus Aedes, Culex, Psorophora

2.2 BIOLOGICAL CONTROL OF MOSQUITO

Biological control is defined as using living organism directly and indirectly to control harmful
organisms, including the disease-carrying vectors. There are many organisms that can be employed
for mosquito control, such as predators, parasites, pathogens (including viruses, rickettsiae, fungi and
bacteria), and their mode of infection, site of replication, mechanism of pathogenicity, and efficacy to
the targets vary greatly (Roberts et aI., 1983; Federici, 1995).
Some fishes, insects and other animals are the naturally occurring mosquito enemies. About
250 fish species prey on mosquito larvae, and the most widely used species is Gambusia ajjinis.
Owing to its vicious feeding behavior, this fish has been applied for mosquito control in more than 60
countries (Vyonne and Walton, 1999). However, this species preys on not only mosquito larvae, but
also some other living organisms in water environment, arising the consideration of its impact to the
natural ecosystem (Gratz et aI., 1996; Rupp, 1996). In China, Cyprinus carpeo, Ctenopharyngodon
idella and Tilapia nilotica have been successfully used for An. sinensis and C. tritaeniorhynchus
control in rice field. In addition, there are some other mosquito predators, including the species in
orders of Coleoptera, Hemiptera, Odonate and Diptera. Some species in genus Toxorhynchites
were applied early in 30s in last century, because the larvae of these species can swallow other
mosquito larvae in the environment and satisfactory results have been obtained in controlling Ae.
albopictus, a dengue virus vector, in Hawaii (Swezey, 1930), andAe. aegypti andAe. albopictus
in the United States for many years (Gerberg, 1985). Besides, Mesocyclops aspericornis is another

43
effective mosquito larvae predator. This species is very suitable for controlling mosquito in clear
water and its efficacy to different mosquitoes in many parts had been evaluated.
Some nematodes are the main mosquito parasites. They can infect both adult and larvae, then
multiply inside and cause the death of mosquitoes. Nine genera of nematodes can infect mosquito
larvae, such as Romanomermis, Diximerimis, Octomyomermis etc., and only a few species can
infect adult mosquitoes, like Agamomermis, Perutilimermis etc. Romanomermis culicivorax has
been intensively studied and widely used for mosquito control in Cuba, Equator, the United States
and China. In 1985, two commercial mosquitocidal nematode formulations (Skeeter Doon®, Q-
Licide®) had been approved for field application in the United States (Finney-Crawley, 1985). Some
protozoa have a different pathogenicity to mosquito, including some species in family ofMicrosporidae
(Amblyospora, Pleistophora, Nosema, Varaia, Edhazardia, and Eugregarinidies)(Becnel, 1996).
Due to their weak pathogenicity against mosquitoes and the difficulty of maintaining the activity in
stocking, there is no report concerning its efficacy in field to date.
Several fungi and mosquito viruses have been investigated for their pathogenicity to mosquitoes.
Because the fungi take a long time for infection and action, these organisms are considered to have
little practical value in field application. Anyway, three genera might be potential for mosquito control
in clear water, such as Lagenidium, Culicimomyces and Coelomomyces (Jaronsky and Axtell,
1982; Sweeney, 1985). Several mosquito viruses have been isolated and characterized, and it was
indicated that these viruses had high virulence to C. quinquefasciatus (Federici, 1985; Moser et aI.,
1998; Baquerizo et aI., 2000). In consideration oftheir narrow active spectrum, the difficulty and
high cost of production, the possibility ofdevelopment of viral mosquitocidal formulation is limited
(Becnel et a!., 1998).

3. MOSQUITO PATHOGENIC BACTERIA

Except the mosquito control agents mentioned above, mosquito pathogenic bacteria are the
most extensively studied and successfully used agents, such as mosquitocidal B. thuringiensis subsp.
israelensis, B. sphaericus and Chlostridium bifermentans var malysia and others.

3.1 B. thuringiensis subsp. israelensis

Bacillus thuringiensis (Bt) is an aerobic, gram-positive bacterium that forms parasporal crystals
during the stationary phase of its growth cycle (Figure 1A), and 70 serotypes of Bt have been
identified on the basis of the flagella agglutination (Lecadet, 1999). It has been found that the insecticidal
crystal proteins (ICPs) have different activity against some insects belonging to 9 orders, nematodes,
mites, protozoa etc. Among the Bt subspecies, some have the specific toxicity to mosquitoes and
blackflies (Delecluse et a!., 1996). For nearly 40 years, various Bt strains have been favourably used
as biopesticides, mostly against pests in agriculture, horticulture, silviculture, vegetable and so on,
and commercialized successfully for pest control allover the world. B. thuringiensis subsp. israelensis,
Bti (HI4), the first Bt strain that was found to have toxicity against mosquitoes and blackflies, was
isolated from dead mosquito larvae in Israel (Goldberg and Margalit, 1977). Its toxicity against
targets is attributed to the parasporal inclusions deposited alongside the spore during sporulation
(Charles and de Barjac, 1981; Ibarra and Federici, 1986). These crystals mainly consist of four
crystal proteins designated Cry4 A, Cry4B, Cry 11 A and Cyt 1Aa according to the new nomenclature,
with molecular weight of 134, 128,72 and 28 kDa, respectively (http://www.biols.susx.ac.ukJHome/
Neil_crickmore!Btlindexlhtml). All four proteins, after ingested by target insects due to the action of
midgut proteases under alkaline pH, are processed into smaller activated fragments ranging around
40-45kDa for 128-134 kDa proteins (Angsuthanasombat et aI., 1992), and 30-35 for the 68 kDa
(Dai and Gill, 1993), and 25 for the 28 kDa (Koni and Ellar, 1993). The genes encoding the four
crystal proteins are located on a large plasmid of72 Mda.

44
 A

Figure 2. Sporulation of Bacillus thuringiensis (A) and B, sphaericus (8) (Provided by Dr. J ,F, Charles) S: Spore;
C: Crystal

The cloning and expression ofthese genes individually or in combination in different hosts
and the synergism among these toxins have been studied (Delecluse et aI., 1996), indicating that
Cry4A, Cry4B, CryllA or CytlAa had different levels of activity to the targets, depending on
the proteins and mosquito species, and that no individual component was as active as the native
Bli crystal. The maximum toxicity was exhibited by only the four components acting synergistically.
Although the unspecific cytolysic and haemolytic protein, CytlAa, has only low toxicity to
mosquito larvae, it has a strong synergism effort with other mosquitocidal crystal toxins (Wu and
Chang, 1985; Poncet et aI., 1995; 1997; Delec1use et ai, 1993; Sun et aI., 2001). Recent results
revealed that this protein might play an important role in suppression ofthe resistance developed
in insects to other insecticidal proteins (Wirth et al" 1997; 2000a; Thiery et al" 1998; Li et al., 2000).
The B. sphaericus resistance in C quinquefascialus was suppressed from> 17,000 to 2-fold with
a 3: 1 mixture of B. sphaericus and cytl Ab (Wirth et al. 2001). Laboratory and field evaluation
proved that Bli had a relatively wider active spectrum, having higher toxicity again Culex spp.
and Aedes spp., and relatively lower toxicity to Anopheles spp. It is also active against Simuliidae,
Tipulidae and Chironomidae larvae. Whereas, it has a disadvantage, having short persistence in
larval habitats, especially in organically enriched water, and its activity can last only a few days
(Priest, 1992; Thiery, et aI., 1996).
The application of Bli on a large-scale as larvicidal agent was undertaken only a few years after
its discovery. Owing to its high toxicity against Culex and Aedes, this bacterium has been routinely
used for mosquito control in many parts of the world (Ali et al., 1989; Ali, 1993; Thiery et al., 1996;
Becker et aI., 1992). In the Onchocerciasis Control Programme (OCP) launched on 1,300,000 km2
in 11 West African countries by WHO/TDR in 1974, because of the appearance of resistance in
Simulium damnosum, the vector of Onchocerca volvulus, to temephos, Bli formulation (Teknar®)
was first used for controlling this insect in the areas most affected by resistance problem in 1981 and
then alternatively with other chemical pesticides in 1985. Thousands of kilometers of river in these
countries have been treated with several hundred tons of Bli liquid concentrates each year. This has
resulted in a rapid decrease in the prevalence of this infection among the people protected by the
OCP, and the deserted villages in some regions devoured by the epidemic onchocerciasis were
rehabitated again (Guillet et aI., 1990; Hougard et aI. , 1997). In Brazil, two large programs are
currently undergoing using Bli to control mosquitoes and blackflies, the Simuliidae Control Program
(SCP) implemented in Rio Grande do Sui, Sao Paulo and other areas for S. perlinax control since
1983, and the PEAa-Program for Eradication ofAedes aegypli launched 1997 for fighting dengue
fever transmission through controlling Aedes species by temephos and Bli (Thiery et aI., 1996; Regis
et aI., 1995,2000). In Germany, over 1000 km2 of mosquito breeding area along the stretch of
Upper Rhine River were treated with Eli formulations for A. vexans control over 10 years, resulting
yearly reduction of mosquito population by more than 90% (Becker and Ludwig, 1993; Becker et

45
aI., 1995). In China, a Bli liquid fonnu1ation based on a local isolate 187 has been developed and
applied for Culex quinqueJasciatus, Anopheles dirus, Anopheles sinensis, Aedes albopictus and
other mosquito control in Hubei, Shangdong, Guangdong, Hainan Province for 20 years, and in last
decade about 50 tons each year has been routinely used to treat different mosquito breeding sites.
The density of adu1ts and the incidence of malaria got reduced by 90% in treated areas (Yuan et al.,
1995; Xu et aI., 1989). More recently, a wettable powder fonnulation has been produced, with a
potency of 4000IU/mg, and its efficacy in field is under evaluation.
In the USA, Bti has become one of the leading mosquito larvicides, and in Europe, tens of
tons of different Bli fonnulation are applied in mosquito control programmes in France, Hungary,
Italy, Russia, Spain, Switzerland, Slovenia and Yugoslavia, without any harmful impact on the
environment. In Indonesia and Colombia, a new Bli tablet was used effectively for A. aegypti control
in container.

3.2 Other MosquitocidaJ B. thuringiensis

Since the discovery of Bli, many other mosquitocidal BI strains have been isolated allover the
World. Based on mosquitocidal activity, polypeptide composition and their homology to Bli toxin,
these strains can be classed into three group. (Delecluse et aI., 1995), (Table 2).
Group I includes 8 strains, such as Bt subsp. morrisoni PG 14 (H8a8b), Bt subsp.
canadensis IIS2-1 (H5a5c), Bt subsp. thompsoni B175 (HI2), BI subsp. malayiensis IMR81.
1 (H36), K6, and B51. These strains exhibit the larvicidal and haemolytic activities as well as
crystal ploypeptides similar to those of Bli. Toxins from these strains appear to be the same as those
found in Bli (Earp and Ellar, 1987; Padua and Federici, 1990). Group II includes two strains,
Bt subsp.jegathesan 367(H28a28c) and Bt subsp. medellin 163-131(H30). The two strains
are nearly as toxic as Bti but produce different crystal polypeptides. The crystals of Bt subsp.
jegathesan are composed of 80, 72, 70, 65, 37, 26 and 16 kDa (Delecluse et aI., 1996), and
the genes encoding 80 kDa (CryllB), 65 kDa (CryI9A) and 26 kDa (Cyt2Bba) have been
found located on a plasmid ofl 05-120 kb (Kawalek, et al., 1995; Delecluse et aI., 1995; Rosso
and Delecluse, 1997; Wirth et aI., 1998); Bt subsp. medel/in is a specific mosquitocidal bacterium
isolated from Colombia in 1992 (Orduz et al., 1992). The crystals consist of94, 68 and 30 kDa
proteins and each one has different activity to mosquito larvae, and highest activity was observed
when the three toxin act in synergism (Orduz et aI., 1995; 1996). It was also found that the
Cyt I Ab I produced by this strain has a very high similarity to Cytl Aa of Bti (Orduz et aI., 1996,
1998; Thiery et aI., 1997, 1999). Group III includes 9 strains, such as Bt subsp. darmstadiensis
73EI0-2(HlOalOb), Bt subsp. Jukuokensis (H3a3d3e), Bt subsp. kyushuensis 74 F6-18
(Hllallc) and Bt subsp higo (H44). These strains are only weak active to the tested mosquito
larvae and their crystal toxins are different from those of Bti (Kim et al., 1996; Koni and Ellar, 1993;
Hwang et al., 1996).

3.3 Clostridium bifermentans

Clostridium biformentans was firstly described in 1918, and naturally presents in soil, water
and other anaerobic matrix. C. bifermentans var. malaysia (Cbm) CH 18 is the first anaerobic
isolate toxic to mosquito larvae (de Barjac et al., 1990). More recently, another new mosquitocidal
C. bifermentanls var. paraiba (Cbp) was isolated from a secondary forest floor in 1997 (Seleena
et aI., 1997). The active spectrum of both strains is similar and their toxicities are comparable to Bti
strains, although the toxic factor( s) are not the same as in Bli. They have high activity to Anopheles
larvae and less activity to Culex and Aedes. Simulium spp. are also sensitive to Cbm whole cu1ture,
but in a much less extent than the other Culicidae (de Barjac, 1990). C. bifermenlans is a very
diverse group of bacteria that includes some human pathogenic species. For this reason, it is needed
to take into consideration the safety of Cbm strain as a potential bioinsecticide. All tests conducted

46
with non-target invertebrates and vertebrates confirmed the innocuity ofthis isolate. This isolate is
only specifically active to mosquitoes, without any impact to human being or any other organisms
(Thiery et ai., 1992a; Yiallouros et al., 1994).

Table 2. Strains ofBacillus thuringiensis active to Dipteran larvae: serotype, activity and composition
of crystals.
Group Strains Activity against Proteins presented in
Serotypes A.aegypti A.stephensi C.pipiens crystals (kDa)*
israelensis 1884 HI4 +++ +++ +++ 135,125,68,28
morrisoni PGI4 H8a,8b +++ +++ +++ 144,135,125,68.28
kenyae LBIT·52 H4a,4c +++ NO +++ 135,125,68,28
canadensis II S2-1 H5a, 5c +++ +++ +++ 135.125,68,28
entomocidus LBIT·58 H6 ++ NO ++ 135.125,68,28
thompsoni IBI75 H12 +++ +++ +++ 135,125,68,28
malaysiensis IMR81.1 H36 +++ +++ +++ 135,125.68,28
AAT028 K6 +++ +++ +++ 135,125,68,28
I AAT021 B51 +++ +++ +++ 135.125,68,28
2 jegathesan 367 H28a,28c ++ +++ ++ 80,70-72,65,37,26,16
2 medellin 163-131 H30 ++ +++ ++ 94,2l!-70,~-1Q
3 kurstaki HO-I H3a, 3b, 3c ± + UO 130-135,66
3 Fukuokaensis H3a, 3d, 3e ± NO ± 90,86,82,72,50,48,37,27
3 galleriae 916 H5a,5b ± ± UO 130-\35,61
3 aizawaiICI H7 ± UO UO 130-135
3 darmstadiensis HIOa, lOb ± + + 125,83,79,69,50,27
3 kyushunsis 74E6-18 HIla, lIe ± ± ± 140,8,80,70,66,50,27,15,14
3 shandongiensis 89-ST-1-25 H22 ± NO NO 150, 60-70,25
3 Higo H44 NO ± ± 98,91,78,61,50,44,36,27

A TT : autoagglutinated strains that cannot be serotyped by flagella agglutination; +++: LC50 values similar to
those found for Bli crystal; ++, + and ±: LC50 values higher than those found for Bli crystals with ratios comprised
between 2-10, 10-50, and 50-1,500 fold, respectively; - non-toxic crystal for the species tested; ND: not determined;
'Underlined numbers indicated that proteins have high homology to Bli crystal proteins.

Unlike Bti and B. sphaericus, producing crystal insecticidal proteins during sporulation, Cbm
does not produce parasporal bodies or any other morphological phenotype that could be associated
with toxicity. Amorphous structures are observed close to the spores and it was suggested that they
could be responsible for the toxicity (de Batjac et al., 1990). Four major proteins, including a doublet
of 66-68 kDa and two other small proteins of 18 and 16 kDa, are likely to be responsible to the
toxicity of the strains (Nicolas et ai. 1993; Barloy et aI., 1996). The genes encoding those four
proteins have already been cloned and sequenced, and surprisingly, the genes encoding 68 and 66
kDa proteins are homologous to the cry16A and cry17A, respectively (Barloy et aI., 1998). The
expression results of these toxin genes revealed that these toxins were very unstable under some
chemical and physical conditions, and thus only a weak activity can be assayed in the final whole
cultures of strains.

3.4 Other Mosquitocidal Bacillus

Several other mosquitocidal Bacillus spp. have been studied for their activity to mosquitoes,
such as Brevibacillus laterosphorus, former Bacillus laterosphorus. Two B. laterosphorus
strains recently have been reported to be high mosquitocidal, having more toxicity to Aedes aegypti
and Anopheles stephensi and less toxicity to C. pipiens (Orlova et aI., 1998). The crystalline
inclusions of various shapes and sizes are likely responsible for their specific activity. And
another important mosquitocidal bacterium is B. sphaericus, which will be focused in the following
part.

47
4. PROPERTY OF MOSQUITOCIDAL B.SPHAERICUS

Bacillus sphaericus is a ubiquitous, cosmopolitan, aerobic and spore-forming bacterium, with

a terminal swelled sporangium and spherical spore (Figures 2B, and 3). This species is found in genus
Bacillus, family Bacillaceae, division Firmicutes (bacteria with a thick peptidoglycan layer) within the
kingdom Procaryotes.

Figure 3. The vegetative cell (x 13,000) (A) and sporulated cell (x50,000) (B), indicating the spore and parasporal
body (Zhang et aI., 1987)

All B.sphaericus strains have been grouped into 49 serotypes on the basis of the flagella
agglutination. Most strains are non-entomopathogenic with only nine serotypes (HI, H2, H3, HS,
H6, H9, H25, H26 and H48) exhibiting toxicity against mosquito larvae (Lecadet, 1996; Thiery et
aI., I 992b ) (Table 3). Since Kellen and Meyers (1964) isolated the K strain pathogenic to mosquito
larvae, numerous other strains with far greater mosquito larvicidal activity have been discovered in
many countries. Most of the highly toxic strains belong to serotype HS, H25, and H48 serotypes,
such 2362, 1593, C3-41, 2297 and IAB872 (Lecadet, 1996; Zhang et al., 1987). The highly active
strains can produce parasporal bodies during sporulation and not the low toxic strains, like SSII-l
and Kellen Q etc. (Kellen et aI., 1965). Based on the percentage of homology, five groups were
identified and all toxic strains share high DNA homology (>79%), belonging to DNA homology
Type IIA (Krych et al., 1980). Some other methods were also used for classification ofthis bacterium,
such as bacteriophage typing (Yousten et al., 1980), numerical classification (Alexander and Priest,
1990), esterase typing (Liu et al., 1987), ribotyping (Aquino de Muro et al., 1992), cellular fatty acid
composition analysis (Frachon et al., 1991), and random amplified polymorphic DNA analysis
(Woodburn et aI., 1995). The toxic strains have common characters. They are strict aerobic gram-
positive bacteria, lack the key enzymes of several biochemical pathways and thus cannot use sugar
or some other carbohydrates, whereas can use some metabolites ofTCA (Russell et aI., 1989).
They have the ability to denitrify or reduce nitrate to nitrite, no ability to produce the extracellular
enzyme (like amylase, chitinase etc.). All strain can grow on 40C O and biotin and thiamine are needed
for sporulation (Priest, 1992; Charles et aI., 1996).

5. MOSQUITOCIDAL TOXINS

The toxicity of B. sphaericus against mosquito larvae is caused by the mosquitocidal toxins
produced during their growth. The toxic strains produce two kinds of toxins. One is a binary toxin,
synthesized and assembled as the crystal forms during sporulation in all high toxic strains. The other
kinds of toxins are mosquitocidal toxins (Mtx), which are produced during bacterial vegetative growth,
such as Mtx 1 (l00 kDa), Mtx2 (31.8 kDa) and Mtx3 (3 S. 8 kDa) detected in most low toxic strains
and some high toxic strains.

48
5.1 Crystal Toxins

All high toxic strains and some medium active strains (for example LPI-G) can form parasporal
bodies alongside spores within the exosporium during sporulation. The crystals are composed of
equal molecules of 41.9 and 51.4 kDa proteins (named BinA and BinB respectively)(Baumann et
ai., 1991). The BinA and BinB begin to be synthesized onset of sporulation and then assembled as
crystals within exosporium in stage III of sporulation (Charles et al. 1993), which is in contrast to Bti
crystal toxins that are independent ofthe spore following lysis of the sporangium. Presence of equal
amount ofBinA and BinB is necessary for crystal formation and single BinA or BinB can only form a
crystalline structure when they are expressed in other Bacillus hosts (Arapinis et al., 1988; Baumann
and Baumann, 1991). BinA has low homology to BinB, only 25% homology (90 amino acids
identical) is found between BinA and BinB from strain 2362. It was also demonstrated that 4
conservative regions in the two fragments were noticed and deletion of2 or 3 ofthese regions will cause
the loss of toxicity of crystal protein(BaumannandBaurnann,1991;Broadwell et al., 1990) (Figure 4).

Table 3. Characteristics of mosquito-larvicidal Bacillus sphaericus strains.

Strains Resources Serotypes Bacterio- DNA Toxicity** Crystal Mix I Mtx2 Mtx3
phage homology protein
t:iEes t:iEes
KellenK USA HI IIA L + + +
KellenQ USA HI I IlA L + + +
B.S-197 India HI NA NA M + NA NA NA
SSII-I India H, 2 IIA L + + +
IAB881 Ghana H, NA H + NA NA
LP1.{J Singapore H, 8 IIA H + + NA NA
2362 Niger H, 3 IIA H + + + +
2317-3 Thailand H, **. NA H + + + NA
C341 China H, NA NA H + NA NA NA
47-6b China If. NA NA M + NA NA NA
IAB59 Ghana H, 3 NA H + + + +
31 Turkey If' 8 NA L + + +
2297 Sir Lanka H25 4 IlA H + + + +
2173 Inida H16 IIA M NA
IAB872 Ghana H.. 3 NA H + NA NA NA
14577 USA H, NA NA NA

Cited from Priest (1992), Charles et at. (1996), Lecadet (1996), Yuan et at. (I 999a) and the unpublished data;
** Based on the lethal concentration 50% after 48h on 4th instar larvae of C. pipiens: L::: 10-' dilution, M""IO-4,
dilution, H ~ 10"" dilution, * * * Strains not responding to any of bacteriophage tested; + Presence,- absence, NA-

....
Not determined.

; ...
v
rbs rbs Ter PSI
I I P42
51kDa 42kDa I '7.
rbs Ter v
Mtxl
31 87.
rbs Ter Mtx2 _ _
L I 291
rbs Ter Mtx3 _ _
r:::;
Ikb 100a&
'2'
Figure 4. Mosquito-larvicidal toxins and the encoding genes from Bacillus sphaericus; a: Crystal toxin; b: Mtx
toxins; Arrow point to the tryptic cleavage sites; rbs: Potential ribosome binding sites, and Ter: terminator (Charles
etal.,19%).

49
 Subcloning experiments and the bioassay results of the transfonnants revealed that single BinA
had only weak activity to target larvae and single BinB had no toxicity at all. The equal amounts of
two components are required for the maximal toxicity, so this toxin is a binary toxin. (Binary toxin,
Bin) (Baumann and Baumann, 1991). On the contrary, although the purified BinA is toxic to cultured
mosquito cells, and the existence of equal amount ofBinB does not increase the activity of the BinA
to the cells. This phenomenon may not be a true reflection of all the events occurring at the epithelium
in larval midgut and could be explained by lack of peritrophic membrane barrier and high affinity
receptors in cultured cells (Baumann and Baumann, 1991).
Amino acid sequences of the binary toxins from 2362,1593,2317-3 and C3-41 are totally
identical and only 1-3 and 5-8 amino acid differences could be found among BinA and BinB,
respectively, from strain 2297, IAB59, LO 1-0 and 2362 (Berry et al., 1989; Priest et al., 1997;
Humphreys and Berry, 1998; Yuan et al., 200 1). The change of amino acid in BinA likely plays a key
role in toxicity, especially the changes at the position around site 100. For example, the change of
phenylalanine to valine at site 99 and serine to alaline at site 104 ofBinA from strain 2297 leads to
increase the activity to the targets ofthe mutated toxin, comparable to that of binary toxin from 2362
(Berry et al., 1993; Shanmugavelu et al., 1998). In another study, it was found that the purified native
crystal toxin from strain LP I-g was not toxic to mosquito larvae because of the naturally occurred
change at site 93 of BinA from leucine to serine. On the contrary, replacement of the amino acid at
this site from serine to leucine leads to the recovery oftoxicity ofthe mutated protein. It is suggested
that the amino acid 93 of the BinA polypeptide is likely a key element in the fonnation ofthe BinAI
BinB complex, responsible for the toxicity and stability of B. sphaericus Bin toxins (Yuan et al.,
200 I). Despite the high level of identity observed for binary toxin sequences, only few amino acid
changes at the specific sites can affect its their structural integrity, leading to a partial or complete loss
of functionality and specific activity. Based on amino acid sequence and the toxicity, the crystal
proteins from B. sphaericus can be classed into four types (Table 4).

Table 4. Nomenclature and comparison of Bin Amino acid sequences from various Bacillus
sphaericus strains.

Type I Type 2 Type 3 Type4

Poly- Amino !AB59', 1593',2362', 2297' 2297'
Peptide Acid IAB88 I' 2317-3',BSEI8',
Position 9002 b C3-41 d, !AB872'
BinI A BinlA Bin2A Bin3A Bin4A
(41.9 kDa) 93 Leu Leu Leu Ser
g:) Val Val Phe Val
104 Glu Ala Ser Ser
125 His His Asn Asn
135 Tyr Tyr Phe Phe
267 Arg Arg Lys Lys
BinB BinlB Bin2B Bin3B Bin4B
(51.4 kDa) (f) Ala Ser Ser Ser
70 Lys Asn Asn Asn
110 lie Thr Thr lie
314 His Leu Tyr His
317 Leu Phe Leu Leu
389 Leu Leu Met Met
'Berry et al., 1989; b Humphreys and Berry, 1998; Priest et al., 1997;' Berry, C., personal communication; J
Yuan et al., 1998b;' Yuan et al. 2001 ;!Shi et al., 2001.

5.2 Mosquitocidal Toxin (Mtx)

The Mtx toxins have been found among some low toxic strains like SSII-1, Kellen Q and 2173
and some other high toxic strains like 2362,1593 and IAB872. In contrary to crystal protein, the

50
Mtx protein are produced in bacteria during vegetative growth stage, not related to the formation of
spores (Liu etal., 1993; Priest et aI., 1997; Thanabalu et aI., 1991; Thanabalu and Porter, 1996).
Fusion ofthe promoter gene ofmtx with lacZ gene also confirmed their vegetative expression, because
~-galactosidase activity was restricted to the early exponential phase in B. sphaericus (Ahmed et al.,
1995). Unlike the binary toxin, these toxins are relatively unstable, whose activity is easily destroyed
by heat, refrigeration, freezing and thawing.
Mtxl is a soluble toxin with a molecular weight of I 00 kDa, composed of870 amino acids.
This protein possesses a short N-terminal leader sequence that is characteristic of gram-
positive bacterial signal peptides (Figure 4). Mtxl can be further processed by gut proteases
into two fragments of 27- and 70-kDa that correspond to the N- and C-terminal regions,
respectively. The 70-kDa fragment has three repeated regions of -90 amino acids each and the
function of which is still to be known; the 27 -kDa fragment contains a short region corresponding to
a transmembrane domain, sharing weak similarity with the catalytic domain of several ADP-
ribosylating toxins. (Thanabalu et aI., 1992b). Deletion analysis suggests that the 27 -kDa fragment
could ADP-ribosylate itself, whereas the 70-kDa fragment is responsible for toxicity to cultured
C quinquefasciatus cells. However, both fragments are necessary for toxicity to mosquito larvae
(Thanabalu et aI., 1992b; Thanabalu and Berry, 1993). Although the purified Mtxl exhibits high
activity to mosquito larvae, with a LC50 ofl5 ng/ml to Cquinquefasciatus, the Mtx-producing
strains have only low activity to targets because of the poorly expression of this protein during the
vegetative growth phase and of the degradation of this polypeptide by endogenous proteases in
culture (Thanabalu et aI., 1995).
Mtx2 and Mtx3 toxins, with molecular weight of31.8 and 35.8 kDa, are composed of292 and
326 amino acids, respectively, and the genes coding these toxins were first cloned from B. sphaericus
strain SSII-1. These two toxins do not display homology to any crystal toxin and have high homology
to both the 33-kDa E-toxin from Clostridium perfrigens and the cytolysic toxin of31.68 kDa from
Pseudomonas aeruginos (Liu et aI., 1993; Thanabalu and Porter, 1996). Mtx2 and Mtx3 share
38% homology, and both have a putative transmembrane region and single peptide sequence similar
to those in gram-positive bacteria. Sequence analysis and larvicidal activity evaluation among the
Mtx2 toxins from 6 strains demonstrated that the amino acid at position 224 was the determinant for
toxicity and active spectrum of this toxin (Chan et aI., 1996).

5.3 Mode of Action

Many results revealed that the mechanisms of action of binary toxin against susceptible targets
included the following steps, (i) ingestion ofspore/crystals complex by mosquito larvae, (ii) solubilization
of the crystals by alkaline condition in midgut oflarvae (Charles, 1987; Charles and de Barjac,
1981), (iii) proteolytic cleavage of the 51 and 42 kDa to the activated 43 kDa and 39 kDa respectively
(Broadwell and Baumann, 1987; Clark and Baumann, 1990), (iv) binding ofthe activated proteins to
a specific receptor in epithelium cells oflarval midgut (Nielsen-LeRoux and Charles, 1992; Charles
et aI., 1997), (v) internalization ofthe both activated toxins to exert the toxicity through an unknown
mechanism, and (vi) death of mosquito larvae.
It has been demonstrated that the crystals can be dissolved and activated in both susceptible
and non susceptible mosquito larvae (Nicolas et al., 1990). The midgut alterations start as early as 15
min after ingestion of the B. sphaericus spore-crystal complex. Large vacuoles appear in C pipiens
midgut cells, whereas large areas oflow electron density appeared in An. stephensi midgut cells. A
generally occurring symptom is mitochondrial swelling in C pipiens andA. stephensi, as well as for
A. aegypti when it is treated with a very high dose of spore/crystal complex (Charles, 1987). The
midgut cells, especially those of the posterior stomach and the gastric caecae, are the cells most
severely damaged by toxin, and the delayed damage in neural tissue and in skeletal muscles has been
also observed (Singh and Gill, 1988; Cokmus et al., 1997). No lysis of midgut cells was observed
(Xu et aI., 1993; Lakshmi-Narasu and Gopinathan, 1988; Singh and Gill, 1988; Charles, 1987).

51
Ultrastructural effects have been reported in cultured cells of C. quinquefasciatus within a few
minutes oftreatrnent with soluble and activated B. sphaericus toxin, like swelling of mitochondrial
cristae and endoplasmic reticula., expanding ofvacuoles and condensation ofthe mitochondrial matrix
(Davidson apd Titus; 1987). Physiologically, the oxygen uptake by mitochondrial and the choline
acetyl transferase might be inhibited in B. sphaericus- treated larvae.
The tluorescently labeled toxin can bind the gastric caecae and the posterior stomach in susceptible
Culex sp. BinB does not bind to the midgut of A. aegypti, whereas BinA binds weak and
nonspecifically in this species (Davidson, 1989; Baumann and Baumann, 1991). Further studies
confirmed that only BinB could bind specifically to the caecum and posterior stomach, whereas BinA
binds nonspecifically throughout the midgut in C. quinquefasciatus. BinA can bind to the specific
areas only in the presence ofBinB. Deletion analysis of theses toxins revealed that the N-terminal of
BinB and the C-terminal ofBinA play key roles for the binding of the binary toxin to the specific
regions in susceptible larvae (Davidson 1989; Oei et al., 1991; Elangovan et al., 2000). These results
support the hypotheses that the N-terminal region ofBinB involves in regional binding ofthis protein
in the larval midgut and the C-terminal region ofBinB and the N- terminal region ofBinA involves in
the interaction ofthe two components that causes BinA to bind in the same region as BinB. However,
the mechanism ofaction after the binding is still to be unraveled.
The in vitro binding assays using 125I-Iabeled activated crystal toxin and midgut brush-border
membrane fractions (BBMF) isolated from either susceptible and or non-susceptible larvae indicated
a specific toxin receptor involved in this process. Direct binding with the susceptible C.
quinquefasciatus and C. pipiens BBMFs confirmed that the toxin bound to a single class of specific
receptor (Nielsen-LeRoux et al., 1992, 1995, 1997; Silva-Filha, 1997). No specific binding occurred
in BBMFs fromA. aegypti, which was consistent with the lack of specific binding in tluorescence-
labeling studies. The two components of crystal toxin could bind on BBMFs, but the linearity of the
Scatchard representation clearly confirmed that only one of them was bound to a receptor and this
result can be explained by the hypothesis proposed above. Based on that the single BinA is active to
the target and cannot specifically bind to midgut regions, whereas BinB is non-active and can bind
specifically to the midgut regions. Some authors assumed that the BinA component is the toxic moiety
and BinB is the binding component, and the crystal toxin is more likely to be similar too an AlB toxin
that too a binary toxin (Charles and Nielsen - Le Roux, 2000).
The presence of the specific receptor in midgut is still to be confirmed. It has proved that a kind
of a-glucosase, with a molecular weight of 60 kDa, might be involved in the binding process of
crystal toxin to midgut (Nielsen-LeRoux and Charles, 1992; Silva-Filha et al., 1999). The cloning
and expression of this protein gene revealed that this protein was composed of 580 amino acids with
a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus.
The amino acid sequence of the protein exhibited 39-43% identities with insect maltases (alpha-
glucosidases and alpha-amylases). These results support the view that this protein (Cmp 1) is an
alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the binary toxin
(Darboux et aI., 2001)
The Mtx toxins have been less studied than the crystal toxins, and their modes ofaction probably
differ.

6. MOSQUITOCIDAL TOXIN GENES AND THEIR EXPRESSION

6.1 Binary Toxin Gene (bin)

The cloning and characterization of binary toxin genes have been done from many B. sphaericus
strains with differentleve1s of toxicity (porter, 1993; Shi et al., 2001; Yuan et al., 1999b, 2001). In all
tested strains, the binary toxin genes are located on a 3.5-4.8 kb HindIII DNA fragment. The
operon of binary toxin gene from 2362, 1593,2317-3 andC3-41 iscomposedof3749 nucleotides.

52
One structural gene (binA) encoding BinA, beginning at position 496 and ending at position of
1839, is composed of 1344 nucleotides and encodes a peptide of 448 amino acids with a
molecular weight of 51.3 74 kDa. Another structural gene (binB), beginning at position of20 14 and
ending at 3127, encodes a peptide on 70 amino acids with a molecular weight of 41.873kDa. There
is a spacer of 176 bases between the two structural genes. Every structural gene has its own S-D
(Shine-Malganno) sequence similar to that of 16S ribosome RNA. The region upstream of the
open reading frame (ORF) for BinB protein clearly contains a promoter, but does not contain
sequence motifs that may be readily identified as being similar to any of the B. sublitis sporulation
promoters. Only one terminator is found in the downstream of binA, and no terminator between
the two structural genes. It is clear that the two structural gene share the same promoter and
this operon is an independent transcription unit. There is no much variation in nucleotide
sequence among the bin genes. The nucleotide sequences in their coding regions from strain 2362,
1593,2317-3, IAB872 and C3-41 are identical, and the greatest variation occurs among
strains IAB59, 2297 and LPI-G, with difference of7, 25 and 19 nucleotides from 2362 (Berry
et aI., 1989; Priest et aI., 1997; Yuan et aI., 2001). It was noticed that there were difference of
only 19 nucleotides in coding region of bin toxin operon from LP I-G in comparison with that
of2362, and the coded binary toxin was totally non-active against mosquito larvae (Yuan et aI.,
2001).
It is now clear that the binary toxin genes of some strains are located on the bacterial chromosome,
such as strain 2362, 1593, C3-41, LPI-G and 1691 (Aquino de Muro et aI., 1992; Porter, 1993;
Liu et aI., 1999; Priest et aI., 1997). One report indicated that the binary toxin gene of strain BS-l 0
was located on a large plasmid, but the result is needed to be verified (Liu et aI., 1990).

6.2 Mosquitocidal Toxin Gene (mtx)

The mtx genes are also detected in some toxic B. sphaericus strains (Priest et aI., 1997;
Liu et aI., 1996). An mtxl operon has been cloned and characterized from strain SSII-l, the
coding region begins at position 1207 and ends at position 3817, coding for a protein of 100.6
kDa and 870 amino acids. This toxin has no significant homology with the binary toxin.
Immediately upstream of the ORF is a ribosome-binding site with a 1O-base homology to the 3'
terminus of B. subtilis 16S rRNA. The ribosome-binding site is preceded by a putative
promoter which shows good homology to the consensus sequence for the 0'55 vegetative
promoter of B.subtilis (-10TATAAA and -35TTGACA). This gene can transcript and then
translate during the vegetative growth. mtx2 and mtx3 genes were also cloned from strain
SSII-l (Liu et aI., 1993; Thanabalu and Porter, 1996). Like mtxl, they have a region with high
homology to the 3'terminus of B. subtilis 16S rRNA and a putative promoter preceded this
region. It has been proved that the transcription of the two genes had no connection with the
formation of spore.

6.3 Expression of Toxins in Different Hosts

6.3.1 Expression in E. coli and Bacillus species. With the aim of evaluating the activity of
single BinA and BinB against mosquito larvae and studying the structure-function relationship, the
binary toxin from different strains has been expressed in different recipient hosts like Escherichia
coli, B. subtilis, oon- or low toxic B. sphaericus and crystal-minus Bti. Binary toxin can be expressed
at a low level in E. coli under control of different promoters, and the E. coli recombinants have only
low toxicity to mosquito larvae. Recently, the binary toxin was expressed in a high level in E. coli with
a pQE expression system (Ahmad et aI., 1998). Under the control of the promoters of B. subtilis or
the constitutive promoter of binary toxin, the binary toxin can be expressed successful and assembled
as acrystal inclusion inB. subtilis, and the activity ofthe recombinants have the same level as the wild
type B. sphaericus (Broadwell et aI., 1990).

53
 The binary toxin gene of B. sphaericus has been expressed from its own promoter by
using a B. subtilis plasmid (pGSPl 0) or shuttle vector (PBU4) in both a wild-type B. thuringiensis
subsp. israelensis strain 4Q2-71 and a crystal-minus strain 4Q2-81 cured of all resident
plasmids. Crystals of binary toxin are deposited alongside the spores outside exosporium in the
recombinant strains. However, the crystal-minus Bti recombinants expressing the only binary
toxin have same activity to the targets as B. sphaericus, having high toxicity against susceptible
C. quinquefasciatus and A. stephensi and very low-level activity to A. aegypti and B. sphaericus-
resistance C. quinquefasciatus. In recombinant Bli 4Q2-72, the expression of the binary toxin
and formation of crystalline inclusions did not greatly affect the production or deposition of the Bli
crystal, and the recombinants can produce two different kinds of crystals, all depositing outside the
exosporium. SDS-PAGE and Western blot results showed that the recombinant can produce Cry4A,
Cry4B, Cry I lA, Cyt1Aa, BinAand BinB during the sporulation. Although the binary toxin and the
Bli crystal proteins can be expressed in high levels, the recombinants have an active spectrum just
like the recipient Bti, having high activity against susceptible and resistant C. qUinquefasciatus, A.
aegypti and An. stephensi. There was no detectable additive or synergistic effects of expressing
both classes oftoxins in one cell compared with wide-type Bti strain (Bourgouin et al., 1990; Yuan et
aI., 1999a, b).
The I OO-kDa toxin (Mtx I), which is expressed during the vegetative phage of growth, has
been cloned and expressed in E. coli under the control of its own constitutive promoter. The
recombinant E. coli is about 10 times less toxic to A. aegypti and C. quinquefasciatus than that of
B. sphaericus SSII-I. The lower protein expression, instability or misfolding of polypeptide in E. coli
likely resulted in this difference of toxicity (Thansbalu et aI., 1991). A truncated 100 kDa toxin
lacking the nonessential N-terrninal putative signal sequence was expressed inE. coli and the purified
toxin from the recombinant strain had very highly toxicity against C. quinquefasciatus, with a LC50
of 15nglml, which was comparable with the value obtained for the binary toxin from high active strain
2362 (Thanabaluet aI., 1991; 1995). Thus, the low toxicity ofSSII-1 is more likely due to the poor
expression or enzymatic degradation of the toxins. Recently, the Mtx I was expressed in protease-
deficient B. sphaericus by electroporation, and the toxicity of the recombinant was almost as same
as that of2362, because the expressed Mtx I could not be degraded by protease in the recombinant
(Thanabalu and Porter, 1995).

6.3.2 Expression in Other Host. Caulobacter naturally occurs in every aquatic habitat and
is found predominantly in regions at or close the water surface, where larvae of many mosquito
species feed. In the flagella swarmer stage, Caulobacter cells are motile, thus allowing their
distribution throughout the habitat, and in both the swarmer and stalked cell stages they are
capable of attachment to solid particles. Furthermore, Caulobacter species are able to persist
and grow in adverse environments low in nutrients. The bin gene from strain 2297 and mtx]
gene from SSIJ-l were separately linked with the broad-host-range plasmid pRK248 and
then electroporated in Caulobacter cresentus CB15. The resulting recombinants expressing
binary toxin were very active to C. quinquefasciatus, with LC50 value of2x 10 5 cells/ml, which
is similar to the toxicity of the natural strain B. sphaericus SSII-l. However, recombinant
expressing the Mtx toxin has only weak activity to C. quinquefasciatus, and the lower expression
of toxin may be the main reason. It is likely to increase the expression in this bacterium by the
use of stronger promoters and ribosome-binding sites (Thanabalu et aI., I 992a) (Table 5).
Like Caulobacter, various species of cynobacteria are widely found near the water surface,
both in freshwater or saltwater environments. They have a wide temperature tolerance and a
limited nutritional requirement. The binary toxin gene was first express in Anacystis nidulans R2
with a shuttle vector and the cell protein extracts from the resulting recombinant exhibited only low
mortality to C. pipiens (de Marsac et aI., 1987). The effective expression of binary toxin in a
single-cell cynobacteria Anabaena sp. 7120 was done in 1993 and the resulting recombinant
had very high toxicities to the targets. Its efficacy for mosquito control in field had been evaluated

54
and it is proved that the engineered cynobacteria might be a potential agent for mosquito control,
with the persistence of more than one month (Xu et al., 1993; 2000). Using the shuttle vector
suitable for E. coli-Synechococcus, the binary toxin wa~ effectively expressed in another
single-cell Synechococcus PCC6301 (Sangthongpitag et al., 1997). Additionally, the binary
toxin and Mtx toxin were expressed in Asticcacaulis excentricus and the gaseous Ancylobacter
aquaticus (LiuetaI., 1996; Khampang etal., 1999; Yap etal., 1994). Comparing with the Gram-
positive host strains, these Gram-negative have some distinct advantages, growing well in
low-cost medium, staying longer near the water surface because of their mobility and the gas-
producing ability, persisting and growing in poor nutrient, lacking the protease that can degrade
the expressed toxins and having a better anti-UV ability. In one study, a sequence of a stronger
Expression Control System (ECS) was inserted into the plasmid containing bin gene, the
resulting recombinant plasmids were transferred into A. aquaticus and A. excentricus. The
toxicity of the recombinant A. excentricus to mosquito larvae was comparable with those of
2297 and 2317-3, 150 and 1000 times higher than the toxicity of the recombinantA. aquaticus and
C. crescentus expressing binary toxins, respectively (Liu et al., 1996; Yap et aI., 1994; Thanabalu
et al., 1992a). Recently, a C. crescentrius C2 recombinant expressing the binary toxin exhibited
11 and 7 times less toxicity to C. quinquefasciatus and A. albimanus, respectively, than the
final whole culture of B. sphaericus 2362 (Romero et al, 2001). It is suggested that the expression
of binary toxin in A. aquaticus and A. excentricus can be increased by substitution of promoter
and ribosome binding site.

7. HOST RANGE AND TOXICITY

Unlike Bti, B. sphaericus has a relatively narrow active spectrum, having toxicity only
to mosquitoes, not to blackflies or any other insects. Its activities to about 40 mosquito species
belonging to 8 genera (Culex, Anopheles, Aedes, Mansonia, Psorophora, Toxorhychitis,
Armigeres and Uranotaenia) have been evaluated in laboratory and in field conditions. It was
indicated that the high toxic strains exhibit high activity to Culex sp., Psorophora sp., Mansonia sp.,
medium activity to Anopheles sp. and poorly or no activity to Aedes sp. (Table 5). It has been
proved that B. sphaericus strains had different toxicity to mosquitoes belonging to different genus,
and the activity vary among different serotype strains, even among the strains in same serotype or
the strains producing the same kind of crystal toxins (Berry et aI., 1993; Davidson, 1981; Thiery
et aI., 1996; Zhang et al., 1994'; Yuan et aI., 1998). Strain 2362,1593,2317.3, IAB872 and
C3 -41 produce identical binary toxin, the main acting factor for their mosquitocidal activity, but
their active spectrum and activity vary greatly (Charles et al., 1996; Delecluse et al., 1996; Thiery
et aI., 1989; Shi et aI., 2001; Yuan et aI., 1999b). In general, the high toxic strains belong to
serotype H5, H25, H48, such as 2362, 2297, IAB872 etc., and the strains in serotype HI, H2
have only low activity to the targets.

Table 5. Active spectrums of several B. sphaericus strains.

Families Species Toxicity ofstrains*
IS2312362lC3:41 ZHl. IABS2 1£loG
Culicinae Culex spp. +++ +++ ++++ ++
Psorophora spp. +++ +++ NO NO
Monsonia uniformis +++ NO NO NO
Ae. atropalpus +++ +++ ++ NO
Aedes aegypti + NO
Anophelinae Anopheles spp. +++ ++ ++ ++
Simuliidae Simulium spp. NO

* The LC50 of the fmal whole culture: +++, '" I x 10-6; ++, '" I x 10.5; +, '" I x 10-1; -, > I 0.2; ND- not determined.

55
8. PRODUCTION AND APPLICATION

8.1 Semi-Solid Fermentation

Semi-solid fennentation procedures have been used for many years for the commercial production
of several microbial products, including fungal amylase, bacterial proteases and Bt formulations.
Under the semi-solid procedure, a coarsely divided matrix is moistened with a nutrient medium that
has been inoculated with the microorganism to be grown. The main advantages of this technique are
its simplicity of operation and low-cost for production. In consideration of the needs of mosquito
control in countryside in China as well as other developing countries, a semi-solid fennentation of B.
sphaericus has been conducted on shallow trays using a medium composed of bran, rice hulls, and
soybean flour (Dai et al. 1989; 1994). The potency of the dried culture was as high as 60-120 ITU/
mg in comparison with the standard RB80. This kind of fonnulation has a high activity to a variety of
mosquitoes and could be used for controlling mosquito larvae in field. This effective local production
technique can provide cost-effective products for local mosquito control activities, especially where
poor economic conditions prevail.

8.2 Deep-TankFermentation

Deep-tank fermentation of B. sphaericus has been undertaken in many parts of the world,
using techniques well developed for the production of B. thuringiensis pesticide for many years. B.
sphaericus does not use glucose and other carbohydrates for growth and lacks many of the enzymes
of sugar metabolism (Russell et aI., 1989), instead, it grows and develops well with organic acids
such as acetate, succinate, arginine and glutamate as sources of carbon and energy although gluconate
and glycerol can be used as sole carbon source. This feature ofthe physiology restricts the use of
agricultural products in fermentation media to those rich in protein/amino acids and prevents the use
of surplus, agricultural starchy materials. Industrially, the media mainly composed ofproteinaceous
substances are used for fermentation of B. sphaericus, but the biotin and thiamine are absolutely
required for its development. Cations, such as Mn2+ and Ca2+, favor sporulation and the associated
toxin formation, and can be supplied from local water supplies and media ingredients or can be
added if sporulation seems poor. In some company, a relatively high-cost peptides were used as
carbon and energy resources for B. sphaericus fennentation. However, many other low-cost materials
can be used for the fermentation of this organism, like agriculture by-product, fishery waste,
monosodium glutamate waste, fennented cowpea, Proflo (cottonseed meal), dextrose, yeast extract,
dried cattle blood and so on.
B. sphaericus is an obligate aerobe and an adequate air supply is needed for growth, initiation
of sporulation and toxin synthesis. In generally, B. sphaericus caused the pH to rise from nearly
neutrality to as high as pH 9.0. In China, the first experimental liquid formulation of B. sphaericus
was initially produced by deep tank fermentation with local isolate BS-l 0 in Jiangsu Province. The
potency of the formulation was 120 ITU/mg, calculated with International Standard RB-80 (Dai,
Personal communication). Subsequently, another more active liquid fonnulation based on strain C3-
41 was also developed by the Wuhan Institute of Virology , Chinese Academy of Sciences in 1988
(Liu et al. 1989). An optimal low-cost medium, mainly composed of agriculture by-products, such as
cottonseed flour, soybean flour, fishmeal, and yeast extract, was used in a large-scale fennentation.
Under the fermentation condition of30 ± 2oC, agitation ofl80rpm, aeration ofl: 1:0.25 to 0.48 (V/
V1M) at the early growth stage and 1: 1: 1.0 to 1.2 (V N 1M) at late growth stage, the strain C3-41
grew and developed well. It was found that 90 to 95% of the bacteria sporulated and produced the
parasporal crystal during this process. The average period of fennentation was 28.9 hrs. The number
ofheat-resistance spores and the larvicidal activity ofthe fennentation mixture in a seven-ton fennentor
were determined to be 58 x 108 cfu/ml and 260 lTIJ/mg (calculated according to RB80), respectively
(Liu et al. 1989).

56
8.3 Formulation

Since 1980, several experimental and commercialized flowable fonnulations, wettable powders,
granules, and sustained-release fonnulations have been developed in USA, Thailand, Brazil, India,
Philippines and Russia, and their efficacy have been evaluated in laboratory and ill fie'td worldwide. In
China, the C3-41 Flowable Fonnulation with an average potency of200 ITV/mg (Based on RB80)
was developed in 1989 (Liu et aI., 1989), now commercially registered as Jianbao®, with a potency
of80 ITU/mg calculated base on SPH88. Meanwhile, a new C3-41 concentrate with a potency of
500 ITV/mg (Based on RB80) was also prepared by concentrating and emulsifying the fennentation
mixture. A wettable powder and a sustained release granule formulation, with the potencies of 500
ITV/mg and 100 ITV/mg (Based on SPH88) respectively, have also been prepared, and their efficacies
in a variety of mosquito habitats have been evaluated as reported by Chen et al. (1994) and Zhang et
a1. (1994). In the past 20 years, about 2,000 tons ofa B. sphaericus flowable formulation was
produced in Jiangsu, Hubei, and Shandong Provinces, China (Dai and Shang, personal
communication). Recently, nearly 150 tons of this formulation was used for mosquito control each
year. At present, there are only a few commercial products which had been approved for field
application worldwide, such as Vectolex®, Spherimos®, Sphericide®, Spherico® and Spicbiomossllll,
Jianbao® (Table 6).

Table 6. The main experimental and commercial fonnulations of B.sphaericus.

Names Strains Fonnulations Quality Producers
(IU/mg)
Jianbaoill C341 Liquid &l Wuhan Xintai Biotech Co.
VectolexGiIl 2362 Granules 200 [USA] Abbott Lab.
VectolexAS<I> 2362 Liquid 250 [USA] Abbott Lab
Vectolex WP<I> 2362 Powder 1000 [USA] Abbott Lab
Spherimos<l> 2362 Liquid 120 [USA] Abbott Lab
Sphericideill 2362 Powder 450 [India] Biotech International Ltd
Spherico" 2362 Liquid I [Brazil] Geratec
Spicbiomoss<l> 2362 Liquid I [India] TuticorinAlkali
Chemicals and Fertilisers Ltd

8.4 Mosquito Control in Field

8.4.1 Culex spp. Control. In early 80s, the toxicity of B. sphaericus in laboratory and in field
was extensively evaluated in many projects supported by WHOITDR and desirable efficacy of this
bacterium against a multitude of mosquitoes in different habitats has been observed. It is tested
that B. sphaericus has a high activity against Culex spp, followed by Anopheles spp. and low or
no toxicity against Aedes spp. It~as also other advantages, having lest harmful effects on other
living orgariism:; or the environment. Importantly, the spores ofthis bacterium can be recycled to fonn
toxin and spores ili dead mosquito larvae in certain environments (Priest, 1992), thus persists longer
in mosquito larval habitats, which means that a long-term control can be achieved and time-span
between re-treatment could be extended and personal costs reduced. So the formulation based
on B. sphaericus is considered as a promising agent for mosquito control, especially for Culex
control in urban region in developing countries (Thiery et al., 1996; Priest, 1992; Das and AmaIraj,
1997). Early in 1987, one commercial product Spherimos@ was applied for C. pipiens control
in Mediterranean region in France. According to the types of mosquito breeding sites, water
quantity, with applicationrates of3-1 OOLIha and application frequency of2-3 times per month, the
toxicity can last for several weeks and the adult density can be reduced to a low level (Thiery et aI.,
1996; Sinegre et aI., 1994). The B. sphaericus larvicidal formulation was successfully used for C.
quinqueJasciatus control in different breeding sites in the United States, Spain and Germany, and
good results has been obtained (Becker and Ludwig, 1993; Ali et aI., 1993;

57
Rodrigues et aI., 1998; Dennett and Meisch, 2000). In some other tropical countries, like
Cameroon, Brazil, Cote d'Ivoir, India, Sri Lanka, Thailand and Tanzania, B. sphaericus was used
for filariasis vector, C. quinquefasciatus control for many years, and the density of adult mosquito
and the incidence of filariasis has been greatly decreased (Regis, 1995,2000; Zeze et aI., 1998;
Kumar et aI., 1996; Yadav et aI., 1997; Barbazan et aI., 1997; Mulla et aI., 1997, 1999; WHO,
1993; Kar et aI., 1997).
In China, about 150 tons of this formulation has been used as larvicidal agent each year in last
two decades. There are nearly 70 cities and towns in China using B. sphaericus larvicide to treat
sewage ditches, ponds, puddles, pit latrines, marshes, small tanks and a large number of periodically
inundated habitats. The major targeted species are C. quinquefasciatus in the south and C. pipiens
in the north. Approximately 8,000 hectares of different mosquito producing habitats were treated
with B. sphaericus each year. In certain southern cities in China, such as Shenzhen, Fushan and
Dongguan, where both temperature and relative humidity are favorable for year around mosquito
breeding, the B. sphaericus C3-41 formulation (now commercialized as lianbao®) has been used for
more than nine years. The mosquito population has been held at tolerable levels through widespread
use of this larvicide. In field trials, at application rates of 10-30 Llhectare for Culex sp., over 95%
mosquito larvae were killed within 3 days post-treatment (Zhang et aI., 1989; Yuan et aI., 2000).
Larvicidal activities as measured by bioassay lasted from 2 to 3 weeks. Moreover, it was demonstrated
that as dosage is increased, better activity and duration oflarvicidal activity occurred (Zhang et aI.,
1989). In small polluted-habitats, the toxicity ofthe C3-41 formulation lasted as long as five months
with one application. The mosquito density in areas managed with C3-41 exhibited a measured
reduction of85% (Zhang, Unpublished data).

8.4.2 Other Mosquito Control. As we know, malaria still remains an important disease in
certain parts of China. Anopheles sinensis, An. dirus, An. anthropophagus and other species are
the main malaria vectors. To control these malaria vectors, the efficacy of the C3-41 formulation
was evaluated against the above species in both laboratory and field investigations (Zhang et aI.,
1994; Chen et aI., 1994; Wang et aI., 1989; Li, 1989). It has proved that the new concentrated
C3-41 formulation, with a potency of 500 ITU/mg, was very toxic to these anopheline species.
The LC50 values ofthis formulation against the three species ranged from 0.458 to 2.836 mglL. In
small-scale field trials, treatment with 400Llhectares ofC3-41 against An. sinensis and An. dirus,
reduced larval density by 100% within one week (Zhang et aI., 1994) and the activity lasted several
weeks when higher application rates were used. In a large-scale field application of C3-41 conducted
in Zaoyang, Hubei Province and Qiongzhong, Hainan Province, effective control of the main
malaria vectors An. sinensis andAn. dirus was obtained with this new formulation. This formulation
was so effective that it was now suggested to be an alternative to chemicals for controlling
anophelines species in managing and possibly eliminating malaria from some parts of China. In the
Republic of Guatemala, 20 tons of B. sphaericus liquid formulation was used for evaluation of its
efficacy against malaria vectors. At an application of 1Omllm2, a total larval reduction of94.57% of
A. albimanus was observed and the malaria prevalence in treated areas got reduced by 50% (Blanco
Castro et aI., 2000). In India (Mariappan et aI., 1998), the United States (Kumar et aI., 1994;
Dennett and Meisch, 2000), Brazil (Rodrigues et aI., 1998), Mexico (Arredondo-limnez et aI.,
1990), Cameroon (Barbazan et aI., 1998), and other West African countries (Skovmand and Sanogo,
1999), different commercial and experimental B. sphaericus formulations have been applied for
Anopheles species control in different habitats, and a statistically significant larval reduction was
notice within one week. It has shown that this bacterium has high activity against An. jluviatilis, A.
culicifacies. A. darlingi. A. braziliensis. A. gambiae, A. muneztovari. A. quandrimaculatus
etc., thus can be used for malaria vector control.
Aedes mosquito species are not very susceptible to B. sphaericus formulations. For controlling
Aedes sp., such asAe. albopictus,Ae. aegypti, the application dosage had to be increased substantially
(Zhang et aI., 1989).

58
9. RESISTANCE IN MOSQUITOES

For many years, it was considered that larvicides based on B. sphaericus would not lead to
resistance in mosquitoes and this was one of the main advantages of microbial insecticides over
synthetic chemical insecticides. Because of the interaction ofthe four different toxins with putative
different modes of actions, there is no record offield resistance to Bti after it has been used for a long
time in controlling mosquito in field (Becker and Ludwig, 1993). However, the binary toxin is an one
site-acting molecule, because of the single receptor interaction with BinB component, thus, it is not
surprising to detect the resistance in mosquito to B. sphaericus. Under long-continuous selection
pressure, mosquitoes could have the ability to acquire resistance to B. sphaericus binary toxin both
in laboratory and infield, the resistance level depending on selection pressure and the time ofcontinuous
selection. Under different selection pressure, C. quinquefasciatus could develop 35- to 150000-
and 10- to 1O,OOO-foid resistance to B. sphaericus in laboratory (Georghiou et aI., 1992; Nielsen-
LeRoux etal., 1997; Rodcharoenetal., 1994) and in field (Silva-Filhaet aI., 1995; Rao etal., 1995;
Adak et aI., 1995; Sinegre et aI., 1994; Yuan et aI., 2000) respectively (Table 7). It is sure that
mosquitoes give a different response to the selection ofB. sphaericus strains and they easily developed
high-level resistance to the binary toxin-producing bacteria. In one study, the two field-collected C.
quinquefasciatus colonies were subjected to a selection pressure with three preparations of
B.sphaericus C3-41, 2362 and IAB59 strains in laboratory respectively. After 13 and 18 generation's
exposure to high concentration ofC3-41 and IAB59, a field-collected low-level resistant colony
developed a more than 144,000-fold resistance to C3-41 and only 46.3-fold to IAB59. This result
indicates that some strains, which may produce other kinds of mosquitocidal acting factors, can delay
the appearance of resistance in targets.

Table 7. Resistance evolutions of mosquitoes to B. sphaericus in laboratory and in field.

Target strain Countries Sites Selective Duration Resistance References
species Lab Fie Eressure Level (LC50)
Cq 2362 USA + LC"... FI2 100,000 Georghiou et aI., 1992
Cq 2362 France + Increased LC values F8 10,000 Nielsen-Leroux et aI., 1997
Cq 2362 USA + LC".... FIOO 35 Rodcharoen, 1994
Cq C3-41 China + LC..." 14 100,000 Yuan et al.
Cq IAB59 + LC".." 18 46 (Unpublish data)
Cq 2362 Brazil + 37 treatments 26 months 10 Silva-Filha et aI., 1995
Cq 1593 India + 35 treatments 2 years 146 Rao et aI., 1995
Cq 2362 India + 20-25 treatments 1 year 150 Adak et aI., 1995
Cq C3-41 China + 280 treatments 8 years 24,600 Yuan et aI., 2000
Cp 2362 France + 18 treatments 7 years >20,000 Sinegre et aI., 1994

The resistance level is calculated as the ratio of the LC50 values of the resistant population to that of susceptible
(or parental) colonies; Cq: c.quinquefaaciatus; Cp: C. pipiens.

In vitro binding between the toxin and midgut BBMF from the susceptible and different resistant
Culex colonies selected in laboratory or in field proved that there are two mechanisms of resistance.
For the high-level resistant Culex, no binding was detected, meaning the receptor was not functional
(Nielsen-LeRoux et aI., 1995); for a high-level resistant population from France and a low-level
resistant population from Brazil, no changes were found in binding kinetics, meaning the evolution of
resistance in these mosquito populations is not related to the changes of receptor. The genetical basis
of resistance have been studied on three high-level resistant populations from France, USA and
China, by crossing homozygous resistant colonies with susceptible colonies and backcross between
the F I and resistant and susceptible colonies. The results indicated that resistance is determined by a
major recessive gene, sex linked for the colony from France (Nielsen-LeRoux et aI., 1995, 1997)
but autosomal for the colonies from USA (Rodcharoen and Mulla, 1994; Wirth et aI., 2000b) and

59
China (Yuan et al., unpublished data). Because the resistant colony has weak biological fitness and
the resistance is recessive (Rodcharoen & Mulla, 1997), it seems that mosquito does not rapidly
develop high-level resistance in field, even with intensive applications (e.g. in Cameroon, Tanzania,
Brazil, India and China) (Charles et ai., 1996; Adak et ai., 1995; Regis et ai., 2000; Yuan et aI.,
2000). In the treated areas, the migration of susceptible mosquito, availability of untreated refuges
and constant supply of new temporary breeding sites formed and not immediately treated in some
areas after rainfall may favor the cross between susceptible and resistant colonies to form heterozygous
colony, being susceptible to B. sphaericus.
Whether in laboratory or in filed, mosquito developed high level resistance to strain 2362,
1593 and C3-41, all belonging to serotype H5 and producing the same crystal toxin. Bioassay
results of the resistant colonies to other mosquitocidal bacteria showed that these resistant
colonies had a high-level cross-resistance to 2297, 2362, C3-41 and other binary toxin-producing
bacteria (Rodcharoen and Mulla, 1996), and exhibited no or low cross-resistance to strain
IAB59, IAB872, LPI-G and 47-6b (Yuanetal., unpublished data). In contrary, alow-leve1 resistant
colony selected with IAB59 has a very high-level resistance to strain 2362, C3-41, meaning that
there is likelihood of another factor acting in these strains and their modes of action might differ
from that of crystal toxin. No cross-resistance in resistant colonies to Bli was detected, and even
more the colonies increase their susceptibility to this agent, and this is in agreement with the
finding that crystal toxin from B.sphaericus and the crystal toxins from Bli do not compete for
the same binding sites.
The appearance ofhigh-leve1 of resistance in mosquito is the threat to the future application
of B. sphaericus as mosquito control agents. In order to avoid appearance of resistance, it is suggested
to use B. sphaericus and Bli alternatively for control ofC. quinquefascialus in suburb of cities or
towns. In case the appearance of resistance in field, it is better to use other effective microbial agents,
such as Bli, other B. sphaericus strains having toxicity to the resistant colonies, to treat the larval
habitats, having mosquito recovered from its susceptibility to B. sphaericus.

10. PERSPECTIVES

10.1 Isolation of High Toxic Strains

Until now, all B. sphaericus isolates have a similar active spectrum, with high toxicity to
Culex sp., medium to Anopheles sp, and low or no activity to Aedes spp. (Lecadet, 1996; Sun et
ai., 1996; Thiery et ai., 1992b; Thiery and de BeIjac, 1989; Yuan et al., 1997; Luo et al., 1994), high
toxic strains producing a stable toxin (Bin) in large amount and a mosquitocidal toxin (Mtx). No
other mosquitocidal toxin has been detected among the B. sphaericus strains, so it is worth
screening new strains with specific activity to targets. In reality, the preliminary results shown that
there might exist a new kind of toxin in strains LPI-G, 47-6B and lAB 59 (Yuan et ai., 2000). Except
B. sphaericus and Bli, other Bl strains have been demonstrated to have activity to mosquito larvae
and their active spectrum and the level of activity varied with the strains. It is likely to screen
more active Bl strains and these strains will be the candidates for development of new
mosquitocidal formulation.

10.2 Mode of Action and Mechanism of Resistance

Although the mode of action and the structure/function of binary toxin has been preliminarily
investigated, indicating the binding of binary toxin to a specific protein receptor at brush border
membrane in midgut, there are still much to be unwind concerning the molecular mechanism of the
toxin. The understanding of these will be of help to design or construct new engineered larvicidal
strains with high activity and wider active spectrum.

60
10.3 Improvement of Formulation

The efficacy of a larvicide in field mainly depends on the active factor in it, but also on the
chemical and physical properties of the formulation, as well as on the technique of its application.
Except only a few powder and granule formulations, the most widely used B. sphaericus formulation
is a flowable, it is needed to develop other formulations suitable for mosquito control in different
larval habitats, such as the ones covered by vegetation, salty water, flowing water etc. The native B.
sphaericus can express binary toxin in a high-level, having very high activity to Culex sp. (LC!IO to C.
quinquefasciatus is of about 1x 102 spore-crystal complex!ml, LD!IO is of80 spore-crystal complex!
larvae) (Yuan et aI., 1992). It does not leave much space for increasing the toxicity of strain by
conventional methods. The crystals deposited alongside of spore within exosporium get easily settle
down and leave the feeding zone of mosquito with the particles in formulation, therefore it has a
relatively shorter persistence in larval habitats. There are several choices to overcome this problem,
improving the property of the formulations (decreasing the size of medium particles), using the
asporogenous mutants to decrease the settlement rate (England et al., 1997; Liu et al., 1999; Idachaba
and Rogers, 2001 ), or importantly expressing the binary toxin and other mosquitocidal toxin in aquatic
gram-negative bacteria with effective expression system. Although the expression of binary toxin is
relatively low, the mosquitocidal engineered carbolerbacteria and cyanobacteria will be the best
alternatives for future mosquito control, especially in rice field or in other clear water (Xu et aI.,
2000).

10.4 Broadening the Active Spectrum

B. sphaericus has a relatively narrow active spectrum, having high toxicity to Culex sp. followed
by Anopheles and low or no toxicity to Aedes. Unlike the Bti, it has not a least activity to the filariasis
vector (Similium). In contrary, Bli has high toxicity to Aedes and Culex, medium toxicity to Anopheles,
and is very effective to Similium. Although the Bti recombinants expressing binary toxin has wider
active spectrum and higher larvicidal activity in comparison with B. sphaericus or Bti (Bourgouin et
aI., 1990; Yuan et aI., 1999a), the persistence of toxicity of these recombinants in water is not
improved, because the two kinds of parasporal bodies produced during sporulation are deposited
outside the exosporium and later released into the medium. To widen the active spectrum or prolong
the persistence, the appropriate strategy for mosquitocidal strain engineering is to transfer cry4A,
cry4B, cry11A or cyt1Aa in combination into B. sphaericus or other hosts (Poncet et al., 1993,1995;
Servant et aI., 1999). These resulting recombinants should be of wider active spectrum and might
less prone to the development of resistance in target populations owing to the multiplicity of toxins.
The techniques increasing the levels ofMtx 1 synthesis and increasing its stability by replacing its
constitutive promoters with other stronger promoters from other B. sphaericus genes, and integrating
the foreign larvicidal toxin genes in chromosome for stable expression will be the alternatives for
future modification of mosquitocidal bacteria (Bar et al., 1998).

11. REFERENCES

Adak, T., Mittal, P .K., and Raghavendra, K., 1995, Resistance to Bacillus sphaericus in Culex quinquefasciatus
Say 1823, Curr Science 69: 695-698.
Ahmad, S., Selvapandiyan, A., and Bhatnagar, R.K., 1998, Increased toxicity of modified mosquitocidal binary
toxins of Bacillus sphaericus expressed in Escherichia coli, Appl. Microbial. Biotechnol. 49: 164-167.
Ahmed, H.K., Mitchell, W.J., and Priest, F.G.,1995, Regulation of mosquitocidal toxin synthesis in Bacillus
sphaericus, Appl. Microbial. Biotechnol. 43: 310-314.
Alexander, B., and Priest, F .G., 1990, Numerical classification and identification of Bacillus sphaericus including
some strains pathogenic for mosquito larvae, J. Gen. Microbial. 136: 367-376
Ali, A., and Weaver, M.S., 1989, Cotsenmoyer E. Effectiveness of Bacillus thuringiensis serovar. israelensis
(Vectobac 12 AS) and Bacillus sphaericus 2362 (ABG-6232) against Culex spp. mosquitoes in a dairy

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INSECT PEST RESISTANT TRANSGENIC CROPS

P. AnandaKmnar

National Research Center for Plant Biotechnology, Indian Agricultural Research

Institute, New Delhi-110 012, India

1. INTRODUCTION

Insect pests are the major scourge of agriculture down the ages. It is estimated that 14%
of crop productivity is lost to insect pests on a global scale (Krattiger, 1997). Agronomically
important crops and their high-yielding genotypes are highly susceptible to insect pests. Introduction
of chemical pesticides has brought about a significant change in the pest management practices but,
unfortunately, resulted in adverse effects on human health, other biological organisms and
environment. Figure 1 depicts the amount of money spent annually on pesticides on the global scale.
Although complete elimination of pesticides is neither feasible nor advisable, it is imperative to
reduce drastically the consmnption of pesticides in agriculture and environment for practising safe
and sustainable farming. Effective alternatives are now available in the form of genetically
engineered crops resistant to insect pests that can be integrated in agricultural ecosystems (Schuler
et aI., 1998). Many insecticidal proteins are available in nature which are highly specific to
agronomically important insect pests but at the same time harmless to man, mammals and other
organisms including beneficial insects. These proteins can be expressed in plant systems in
sufficient quantities so as to confer insect resistance.

Table 1. Insecticidal proteins having potential application in insect pest management.

Insecticidal protein Source
l. Insecticidal crystal proteins Bacillus thuringiensis
2. Vegetative insecticidal proteins Bacillus thuringiensis
3. Protease inhibitors Plants and animals
4. a-amylase inhibitors Plants
5. Lectins Plants
6. Cholesterol oxidase Streptomyces sps.,
7. Chitinases Plants and insects
8. Tryptophan decarboxylase Plants
9. Isopentenyi transferase Agrobacterium
10. Insecticidal toxin complex Photorhabdus sps.,
II. Peroxidase and lipoxygenase Plants

Advances in Microbial Control of Insect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 71
 Insecticidal crystal proteins found in a soil bacterium, Bacillus thuringiensis (Bt) form an
important part ofthis arsenal (J(umar et aI., 1996). Currently Bt-transgenic crops (pest resistant)
such as cotton, com and potato are under cultivation in many countries. Transgenic plants expressing
various kinds of insecticidal proteins other than Bt 8-endotoxins have been reviewed (Schuler et al.,
1998; Jouanin et aI., 1998). The present review deals with the role ofBt-transgenic plants in insect
pest management in agriculture.

Maize
620 (8·"')

Total Insecticide Use is U $ 8, 110 million

Figure 1. Global insecticide use on major crops, 1994 (Adopted from Kratitiger, 1997)

2. TOXINS OF BACILLUS THURINGIENSIS

The bacterium B. thuringiensis (Bt) was first discovered in Japan in 1902 in a silkworm rearing
unit. In 1911, it was again isolated in a flour moth population and characterized by Berliner in Thueringen
(Germany). Bt is a Gram-positive bacterium that synthesizes insecticidal crystalline inclusions during
sporulation. The crystalline structure of the inclusions is made up of protox in subunits called 8-
endotoxins. Most Bt strains produce several crystalline proteins (Cry proteins), each of which shows
a rather narrow host range (Kumar et aI., 1996; de Maagd et aI., 2001). About 100 genes encoding
protoxins from a wide range ofBt isolates have been isolated and sequenced. The genes have been
classified into 28 categories (www.biols.susx.ac.uklHomelNeil_CrickmorelBt/). Upon consumption
by insect larvae, the crystals ofBt are solubilized in the highly alkaline midgut releasing the protoxins.
The protoxins are activated by insect proteases which cleave the protein into a smaller polypeptide,
the toxin. This toxin binds to the surface ofepithelial cells in the midgut, causing lesions that destroy
the cells and lead to the death of the insect. Human beings, other mammals and organisms including
beneficial insects do not have the appropriate receptors for Bt toxins, which is the reason for their
innocuous nature. Bt has been under extensive use as a biopesticide for the past 55 years in agriculture,
horticulture, forestry and mosquito control in many parts of the world. However, an elegant and most
effective delivery system for Bt toxins is the transgenic plant. The major benefits of this system are
economic, environmental, and qualitative. In addition to the reduced input costs to the farmer, the
transgenic plants provide season-long protection independent of weather conditions, effective control
ofburrowing insects difficult to reach with sprays, and control at all ofthe stages of insect development.
The important feature of such a system is that only insects eating the crop are exposed to the toxin.
Genetic transformation of almost all the major crop species is now feasible with the development of
an array oftechniques ranging from the Agrobacterium approach to electric discharge-mediated
particle acceleration procedure (Pattanayak et aI., 2000).

3. BT EXPRESSION IN HIGHER PLANTS

The first Bt-transgenic plants were made in 1987 (Barton et al., 1987; Fischhoff et aI., 1987;
Vaeck et aI., 1987). The plants expressed full-length or truncated Bt toxin genes (cry 1A) under the

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control of constitutive promoters. The expression of the toxin protein was very poor in the tobacco
plants and the mortality of M sexta larvae was only 20%. Truncated cry lA genes coding for the
toxic N-terminal fragment provided better protection to the tobacco and tomato plants. When
compared to the plants transformed with full-length genes, the plants expressing truncated genes
were more resistant to the larvae, and the highest reported level of toxin protein expression was
about 0.02% oftotalleaf solubleprotein. Despite these low levels ofexpression, many ofthe plants
were shown to be insecticidal to the larvae ofM sexta. However, many ofthe noctuid lepidopterans,
which constitute a very serious group of insect pests, need higher amounts ofBt toxins for effective
control. Gene truncation as well as the use ofdifferent promoters, enhancer sequences, and fusion
proteins resulted in only limited improvement in Bt gene expression (Barton et al., 1987; Carozzi. et
aI., 1992; Vaeck et aI., 1987).
The toxin genes ofBt are not properly expressed in higher plants because of various factors
such as prokaryotic codon usage, high AT content and polyadenylation signals within the coding
region. The nature of the coding sequences ofBt genes distinguishes them from plant genes. In
particular, Bt genes are very rich (about 62%) in adenine (A) and thymine (T) while plant genes and
most bacterial genes which have been expressed in plants are on the order of 45-55% A+T. While
within any organism genes have similar A+T content, the A+T content can vary from organism to
organism. Due to the degeneracy of the genetic code and the limited number ofcodon choices for
any amino acid, most of the "excess" A+T of the structural coding sequences of some Bacillus
species are found in the third position of the codons. That is, genes of some Bacillus species have A
or T as the third nucleotide in many codons. Thus, A+T content in part can determine codon usage
bias.
Codon usage in the native o-endotoxin genes is considerably different from that found in typical
plant genes. Native endotoxin genes tend to have a low G + C content of around 37%. Plant genes
in general have a higher G + C content, as exemplified by maize (Murrayet al., 1991). Truncated 0-
endotoxin gene transcripts in transgenic plants could result from a number ofevents relating to their
high A + T content. These include premature transcriptional termination or polyadenylation in regions
of high A + T content or inappropriate splicing or cleavage. Instability of the mRNA could be the
result ofendonucleolytic or exonucleolytic degradation at specific sequences that destabilize the
message during transcription or create pauses due to the formation ofsecondary structures. Instability
of mRNA could also be the result of inefficient translation due to poor codon usage.
To address the above problems and to achieve high expression ofBt transgenes in plants attempts
have been made to modify the nucleotide sequence of the native genes without altering the protein
product. In designing a synthetic gene for expression in plants the following factors are taken into
consideration.
(i) Sequences which interfere with the efficiency of gene expression should be eliminated.
Such sequences include plant polyadenylation signals [AATAAA, AATGAA, AATAIT,
GATAAAandAATAAG].
(ii) Polymerase II termination sequence CAN7-9AG1NNAA. This sequence was shown to
be next to the 3' end of the coding region of the U2 snRNA genes ofArabidopsis thaliana
and is believed to be important in transcription termination upon 3' end processing. It is
necessary to modify such sequences in Bt genes, if present.
(m) CUUCGG hairpins are responsible for extraordinarily stable RNA secondary structures
associated with various biochemical processes. The exceptional stability ofCUUCGG
hairpins suggests that they have an unusual structure and may function in organizing the
proper folding of complex RNA structures. These sequences which affect mRNA
processing in plant cells need to be modified.
(iv) Plant consensus splice sites 5'= AAG:GTAAGT and 3'= TTTT(Pu)TTT(Pu)T(Pu)
T(Pu)TGCAG:C(where Pu is purine) may be present in Bt genes, which should be
eliminated.
(v) Modifications in nucleotide sequence ofBt gene coding region are to be made to reduce

73
 A + TcontentinDNA-base composition. The synthetic Bt is designed to have anA + T
content ofaround 50% in keeping with values usually found in plants.
(vi) When synthesizing a gene for improved expression in a foreign host cell, it is desirable
to design the gene such that its frequency of codon usage approaches the frequency
of preferred codon usage of the host cell. Frequency of preferred codon usage refers
to the preference exhibited by the host in usage of a particular nucleotide codon over
other degenerate codons to specify a given amino acid. The frequency of usage of a
particular codon in a given gene is thus the ratio of the number of occurrences of the
codon to the total number of codons specifying the same amino acid. The percent
deviation of the frequency of usage of a single codon from that of the host cell followed
by obtaining the average deviation over all codons. In general terms, the overall
average deviation of the codon usage of a synthetic gene from that of a host cell is
calculated using the equation:

(xn-Yn)
X 100
Z x
A= L
N=1 Z

where xn = frequency of usage for codon n in the host cell; yn = frequency of usage for codon n
in the synthetic gene where n represents an individual codon that specifies an amino acid, the total
number of different codons is z. The overall deviation of the frequency of the codon usage for all
amino acids should preferably be less than about 25% and more desirably less than about 10%. With
the above factors in consideration, Perlak and others at Monsanto designed Bt genes and made a
pioneering study of the expression of partially modified and fully modified (synthetic) crylAb and
crylAc genes in cotton (Perlak et aI., 1990, 1991).
Perlak et al. (1991) followed two approaches to modify the crylAb and crylAc genes.
One approach included selective removal of DNA sequences predicted to inhibit efficient
expression ofBt gene expression at both translational and MRNA levels by site-directed mutagenesis.
These genes were termed partially modified (PM) genes. The other approach was to generate a
synthetic gene with a fully modified (FM) nucleotide sequence, taking into account factors such
as codon usage in higher plants, potential secondary structure of mRNA, and potential
regulatory sequences. The PM-crylAb gene is approximately 96% homologous to the native gene
with a GC content of 41 %, with the number of potential plant polyadenylation signal sequences
(PPSS) reduced from 18 to 7 and the number ofATTTA sequences reduced from 13 to 7. The FM-
cry lAb is approximately 79% homologous to the native gene, with a GC content of 49% and the
number ofPPSS reduced to 1 and all ATTTA sequences removed. The toxin protein levels in
transgenic tobacco and tomato harboring these modified genes increased up to 100-fold over levels
seen with the wild-type Bt gene in plants. Perlak et al. (1990) made a gene construct in which the
first 1359 nucleotides were derived from FM-crylAb gene and the remaining sequence from PM-
crylAc gene. The variant gene was placed under the control of CaMV 35S promoter containing
a duplicated enhancer region. Cotton variety Coker 312 was transformed and the transgenic plants
were shown to have total protection from Trichop/usia ni (Cabbage looper), S. exigua, and H zea
(cotton boll worm). The maximum level oftoxin protein was 0.1 % oftotal soluble protein. The
Monsanto group also placed the FM-crylAc gene under the control of Arabidopsis thaliana
Rubisco small subunit promoter with its associated chloroplast transit peptide sequence (Wong et al.,
1992). Transgenic tobacco plants expressing this gene provided a 10- to 20-fold increase in
crylAc mRNA and protein compared to gene constructs in which CaMV 35S promoter with
duplicated enhancer region was used to express the same gene. The toxin protein was localized in
the chloroplast and in the tobacco plants that produce the Bt protein nearly 1% of the total leaf

74
protein had the highest levels ofBt toxin proteins yet reported. The enhancement ofBt toxin protein
levels in tissues in which Rubisco expression is highest may lead to very effective control ofcertain
insect pests that feed on leaves and other green tissues.
In the past few years, more than 30 plant species have been transformed by using a range of
modified Bt genes (Schuler et al., 1998; de Maagd et al., 1999). Some of the modified genes were
designed to suit the codon usage ofa particular plant species (Koziel et al., 1993). A variety of plant
promoters have been used in combination with cry genes, such as CaMV 35 S promoter, wound-
inducible promoter, chemically inducible promoters and tissue-specific promoters (Kumar et al.,
1996). A list of some important crop species carrying modified o-endotoxin genes is given in Table
2. Currently, three Bt crops (cotton, com and potato) are under cultivation (Table 3) in countries
such as USA, China, South Africa, Australia, Argentina, France, Indonesia and Mexico covering an
area of8.3 million hectares and comprising about 26% oftotal area under transgenic crops in the
year 2000 (www.isaaa.org). Commercial cultivation of Bt-cotton has been recently approved in
India.
An important finding of the studies conducted in China on Bt cotton was that the smallest
farmers, those farming less than 1 hectare, gained more than twice as much income per unit ofland
($ 400 per hectare) from Bt cotton, as the larger farmers ($ 185 per hectare). This finding is important
from an equity/distribution viewpoint and is deserving offurther investigation for Bt cotton that offers
promise to small resource poor farmers. It also has important implications in relation to the claim
often made by critics oftransgenic crops that they are inappropriate for small farmers. Indeed, by far
the largest benefits reported to-date from the studies reviewed here have been for small farmers who
can least afford the loss in yield due to pests, and stand to gain the most from increases in income and
suffer less health hazards resulting from fewer applications ofconventional insecticide
Vander Salm et al. (1994) developed transgenic tobacco and tomato plants expressing two Bt
genes, cry I Ab and cry I C, specific towards lepidopteran insects. Both of the genes were partially
modified to remove sequence motifs that affect mRNA stability in plant cells. The expression of a
crylAb-cryI C fusion gene resulted in protection against S. exigua, ofexpressing translational fusions
not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ
different gene classes in resistance management strategies. The second generation Bt crops carrying
multiple Bt genes will probably enter the market in near future.

Table 2. Some important examples of crop/plant species transformed with Bt genes.

Plant species Geneffoxin Target pest (s) Reference
Cotton cryIAb, cryIAc Bollworms Perlaketal.,1991
Maize cryIAb European corn borer Kozieletal.,1993
Cry9C European corn borer Jansens et aI., 1997
14 kD and 44 kD toxins Root worm Moellenbeck et aI., 2001
Potato cry3Aa Colorado potato beetle Perlak et ai., 1993
cryIAb Tuber moth Chakrabarti et aI., 2000
Rice cryIAc Yellow stem borer Nayak et aI., 1997
cryIAb Eight lepidopterans Shu et aI., 2000
cry2Aa Yellow stem borer Maqbool et aI., 1998
Basmati rice cryIAc Yellow stem borer Raina and Khanna, 2002
Tomato cryIAc Fruit borer Mandaokar et aI., 2000
Brinjai cryIAb Fruit borer Kumar et aI., 1998
Cabbage crylAc Diamondback moth Metz et aI., 1995
Broccoli cryIC Diamondback moth Cao et aI., 1999
Cano1a cryIAc Diamondback moth Stewart et aI., 1996
Alfalfa cryIC Beet army worm Strizhov et aI., 1996
Coffee crylAc Leafminer Leroy et ai., 2000
Peanut cryIAc Com stalk borer Singsit et aI., 1997

75
 Researchers at Calgene, in collaboration with Maliga and group expressed cry1Ac gene in
tobacco chloroplasts using chloroplast transformation vectors and particle bombardment technique.
The transplastomic tobacco expressed the Bt toxin at very high levels and achieved complete control
oflepidopteran larvae (McBride et al., 1995). This level ofexpression (3-5%) oftotal soluble protein
even provided protection against relatively Cry lAc-resistant pests. Recently, expression ofcry2Aa2
operon in transplastomic tobacco resulted in the formation ofcrystals and the foreign protein constituting
45% of total soluble protein (De Cosaet al., 2001).
The advantages of chloroplast expression system are manyfold:
(i) The Bt genes do not need any modification because the chloroplast transcriptional and
translational apparatus are typically prokaryotic.
(ii) It is possible to have many copies of the Bt gene in each cell.
(fu) The expression ofthe gene will be high if driven by promoters such as rbcl.
(iv) Because chloroplasts are maternally inherited, there is no risk ofpollen transfer of the Bt
gene to related plant species or weeds.

Table 3. Commercialized Bt-transgenic crops.

Crop Trade name (E.g.,) Gene Major target pest
Cotton BollgardlIngard crylAb/crylAc Bollwonns
Potato NewLeaf cry3Aa Colorado potato beetle
Maize Yieldgard crylAb European com borer
Maize Starlink cry9C European com borer
Tomato crylAc Fruit borers
Maize crylAc European com borer
Maize crylF European com borer

4. MANAGEMENT OF RESISTANCE TO BT

Past experience has shown that insects have developed resistance to many organic insecticides
and it can be assumed that resistance to Bt in transgenic crops will also develop eventually. Numerous
Bt-resistant populations were developed in the labomtory (Tabashnik, 1998). However, only one
insect species (Diamondback moth; P. xylostella) developed resistance under natural conditions
following sprays ofBt products (Shelton et aI., 2000).

4.1 Mechanisms of Resistance Development

Many possible mechanisms can be envisaged to explain resistance development in insects

(Frutos et al., 1999). The first is the activation of the protoxins by gut proteinases. The proteolytic
processing of protoxins into toxins is necessary for the insecticidal activity ofBt formulations. It
is also required by some transgenic plants expressing combinations ofBt toxins as fusion proteins or
toxins extending beyond the protease activation site. This mechanism of resistance was first
described in the Indian meal moth (Oppert et al., 1994, 1997). The Bt resistant strains of Plodia
interpunctella displayed slower processing and activation of Cry 1 protoxins. The slower protoxin
activation of the resistant strain led to a reduced quantity of toxin resulting in a survival
advantage. Some of the resistant strains lacked a major trypsin-like gut enzyme. Absence of the
gut proteinase and resistance to the toxin were genetically linked (Oppert et al., 1997). Similarly
a Bt-resistant strain of H virescens showed a slower activation ofthe protoxin as well as a faster
rate of degradation of the toxin by midgut extracts (Forcada et al., 1996). The most frequently
observed mechanism of resistance among insect pests is the modification of the receptor site
(Tang et aI., 1996). This mechanism was studied extensively on a large range of insect species
and toxins, and most of these studies pointed out a change in the level ofaffinity of the receptor for

76
the toxin or to a decreased number of receptor sites (Frutos et aI., 1999).
The first study of a mechanism of resistance in a field-evolved Bt resistant strain was made by
Ferre etal. (1991) using a colony ofP. xylostella from the Philippines. In the resistant strain, a loss
of specific binding to Cry lAb (Ferre et aI., 1991; Bravo et aI., 1992) suggested that the resistance
was due to a change in the Cry 1Ab binding site. A field-collected strain of P. xylostella from Florida
selected forresistance to HD-I was highly resistantto CrylAa, CrylAb, and CrylAc but not to
Cry IB, Cry 1C, and Cry 1D (Tang et al., 1996). Analysis of the binding characteristics ofbiotinylated
toxins on brush border membrane vesicles showed a loss ofbinding to Cry 1Ab in the resistant strain,
whereas binding properties of Cry 18 and Cry 1C remained unchanged (Tang et al., 1996).
The third mechanism of resistance is not related to reduced binding or altered protein activation
and corresponds to a still unknown mechanism or perhaps to several unknown mechanisms, and was
reported for the first time inH virescens (Gould et aI., 1992). Although this H virescens population
was selected for resistance to CrylAc, it also showed resistance to Cry 1Ab and Cry2Aa. The resistant
strain grew also faster than the susceptible one in the presence ofCrylAa, CrylB, and CryIC. This is
a broad resistance mechanism for which an insect species selected for resistance to only one toxin,
Cry lAc, can become resistantto other toxins sharing few sequence homologies (e.g., Cry 1Band
Cry2Aa). The lack of knowledge on the mode of action after receptor binding makes understanding
ofthis mode of resistance difficult and the determination of the number of different mechanisms that
could occur at this post-binding level. One can only assume that at least part ofthese reported cases
of resistance corresponds to altered post-binding events when resistance cannot be related to a
reduced binding or to a lower level of toxin activation. Such a mechanism could explain broad-
spectrum resistance if several toxins share common post-binding steps in their mode of action. A
detailed account of stability and genetics of resistance was given by Frutos et al. (1999).

4.2 Management Strategies

Many strategies to manage resistance to Bt toxins were proposed and reviewed (Gould, 1998;
Frutos et al., 1999). Field dataand experimental evidence are now available to help designing resistance
management strategies (Roush, 1997; Shelton et al., 2000). These strategies fall into several categories:

4.2.1 Tissue- or Temporal Expression of Toxins. Tissue- and temporal expression (including
expression controlled by wound-inducible or chemical-inducible promoters) oftoxins in transgenic
plants were proposed to limit production oftoxin to the most economically sensitive or most vulnerable
parts of plant or to specific time (Roush, 1997). This strategy does not require external refugia
because the plant itselfacts as such. However, efficient tissue or time-specific promoters are not yet
available (Roush, 1997). This strategy is dependent on the feeding behavior ofthe pests and can be
influenced by the feeding deterrent effect ofBt transgenic plants (Gould, 1994). Also, an "in planta"
refugia may be relevant for protection against a given member of a group ofpests but fail to provide
protection against a secondary pest attacking the plant at the same time but feeding on a different
part.
Several potential economical and sociological problems are related to the use of chemical-
inducible promoters (Roush, 1997). Chemicals to be used for induction might be costly and negatively
perceived with respect to environmental protection because the issue will be sprayed with a potentially
polluting chemical.

4.2.2 Gene StackingIPyramiding. This strategy relies on the simultaneous delivery of toxins
recognizing different binding sites. It is based on the assumption that the frequency ofindividuals with
two independent and rare resistance alleles is lower than that of individuals with one rare resistance
allele (Gould, 1998). The gene pyramiding strategy works best if there is no cross-resistance and,
whenever possible, selection of proper Cry proteins is done to minimize this risk. Experimental
evaluation of the potential ofcombinations of Cry toxins for delaying resistance is limited (Gould,

77
1998). Five resistance management strategies involving the use of combinations ofBt Cry toxins in
the presence or absence of external refuge were simulated (Caprio, 1998). These strategies were
sequential introductions of two toxins, annual alternation oftwo toxins, a mosaic distribution of two
toxins, a combination of two toxins. Although the strategies simulated by Caprio (1998) were based
on sprays, the combination approach was similar to pyramiding genes in a transgenic plant under the
control of strong constitutive promoters. The presence of a refuge when using a combination oftoxins
is an absolute requirement (Caprio, 1998; Gould, 1998).

4.2.3 Mixtures, Rotation or Mosaics of Plants. The status of these strategies has evolved
depending on conditions and availability ofmaterial. Although equivalent in efficiency to combinations
of toxins based on sprays (Roush, 1989), they were shown to be weak in delaying resistance when
applied to plants (Caprio, 1998). Rotation is based on the assumption that the frequency of resistance
alleles will decrease when the selection pressure is removed (Tabashnik, 1994). However, many
reports showed that resistance could remain stable or decrease slowly after removal of selection
pressure (Liu et al., 1996), making the use ofrotation inefficient. Mosaics correspond to the simultaneous
deployment in separate fields of varieties of the same crop containing different single toxins. The
mosaic strategy was not considered appropriate to deploy two toxin genes (Roush, 1997).

4.2.4 Combination of Toxins with Different Modes of Action. Protease inhibitors were shown
to act synergistically with Bt toxins (MacIntosh et aI., 1990), thus prompting jJe recommendation of
such combinations for resistance management. However, no field studies of such crops were carried
out. It was observed that Cytl Aa has overcome a 5000-fold resistance to Cry3Aa already established
in cotton wood leaf beetle Chrysomela scripta (F ederici and Bauer, 1998). This same protein was
shown to be very efficient in preventing evolution of resistance in mosquito larvae exposed to Bt
israelensis (Wirth et aI., 1997).

4.2.5 Refuge Strategy. Refuges or refugia are areas of crops or host plants free ofBt toxins or
insecticide treatment that allow part ofthe pest population to survive and to act as a reservoir of wild-
type susceptible alleles. By maintaining a refuge area close to the transgenic field, surviving individuals
that have been exposed to Bt toxins will mate with unselected individuals coming from the refuge,
thus diluting resistance alleles and reducing the intensity of selection pressure. The refuge strategy
was proposed for delaying resistance Bt toxins (Gould, 1998). Experimental data confirmed the
effectiveness of the refuge strategy (Liu and Tabashnik, 1997). The refuge should be maintained free
of any treatment with pesticides to ensure the presence of a sufficient number of susceptible individuals
for subsequent mating with survivors (Gould, 1998; Roush, 1997). Many ofthese requirements are
species related and refuge implementation will be a case by case approach, depending on conditions.
Spatial organization of refuges has been considered in different ways and the most efficient
spatial organization for refuge seems to be the external refuge. However, the solution will most likely
be a case by case decision based on pest biology and mobility. The size of the refuge is also a matter
of debate. Larger the refuge greater the delay of resistance. There is as yet no clear evidence ofthe
minimal size ofthe refuge and estimates rang.:: from IOta 50%, depending on crop, pest, and simulation
program (Liu and Tabashnik, 1997). Currently, a 4% refuge is mandated for Bt-cotton by the EPA in
the U.S. to manage resistance in H virescens (Gould, 1998).

4.2.6 "Trap Plants" Strategy. An approach closely related to the refuge system is the "trap
plant" strategy proposed by Alstad and Andow (1995). In this approach, the transgenic crop is not
considered as the main source of protection but as a trap. The Bt-transgenic plants are planted earlier
than the non-transgenic plants and owing to their advance in maturation attract the pests that are
killed after feeding, leaving the non-transgenic varieties relatively unharmed (Alstad and Andow,
1995). There is, however, no evidence that the basic key assumptions on movement and absence of
ovipositing preference will be verified in the field for this species or for another one. A similar computer

78
model designed on slightly different assumptions about feeding preference and movements led to the
conclusion that this strategy was failure-prone (Ives, 1996). Indeed, avoidance of transgenic plants
and ovipositing preferences were demonstrated (Arpaia et al., 1998) and even if an insect species
may be suitable for the "trap plant" strategy owing to its feeding preferences, the presence ofBt
toxins may adversely alter its feeding behavior.

4.2.7 High-Dose/Refuge Strategy. This strategy is considered the most efficient and promising
way of managing resistance to Bt toxins. This approach is in theory (Gould, 1998) and in practice
(Liu and Tabashnik, 1997) is very effective provided that a high level of insecticidal protein is produced
throughout the life span ofthe plant and all over the field. This may not happen because the production
of toxin is expected to decrease over time, due to plant senescence (Onstad and Gould, 1998). If the
level ofproduction ofBt toxin decreases toward the reproductive phase, heterozygous individuals,
which may often be slightly more resistant than susceptible homozygotes, might be able to survive
and transmit resistance alleles to the offspring. Another situation leading to the delivery ofa moderate
dose is that by which a larva displaying a high frequency ofmovement may feed alternatively on toxic
and non-toxic plants thus diluting the dose of the toxin. The importance of this effect will depend on
the behavior and intensity ofmovement ofthe pests and the spatial organization ofthe refuge. Use of
the combinations oftoxins along with the high dose-refuge strategy will be more effective. The use of
the combinations oftoxins both delivered at high dose will allow through the "redundant killing" effect
to control more efficiently heterozygous insects that might display a sufficient resistance level to resist
a single toxin, but more rarely to two highly active insecticidal proteins. However, even in such a
situation a refuge is necessary for a combination of toxins to delay resistance (Roush, 1998; Caprio,
1998).
A major problem for the high-dose refuge strategy using combinations oftoxins will be cross-
resistance. The control ofheterozygotes is an important factor and theoretically the simultaneous use
ofa combination ofhigh-dose toxins and a refuge will be the most efficient answer. Computer modeling
indicates that in the absence ofcross-resistance, a combination of~gh-dose toxins with a refuge is
expected to last over 160 generations, whereas the sequential use of the same toxins will delay
resistance for only 12 generations, which means that there is a lO-fold advantage ofusing a combination
(Roush, 1998).
A difficult situation might be that of multiple crops and multiple pests prevalent in tropical and
sub-tropical countries like India. A toxin may be highly active against a given pest but be moderately
active against another pest also present on the same crop or in the same area, and for which the dose
delivered by the transgenic plant does not correspond to a high dose. In that case, the second pest
will be exposed to a moderate dose and may develop resistance. This leads to another key feature of
insect resistance management, the use ofBt plants as a component ofIPM approach. The use ofBt-
based formulations within an IPM program was shown to be effective for controlling pests (Meade
and Hare, 1995). As there is no single answer or strategy to delay resistance, only a logical combination
ofvarious means ofpest control will provide sustainability. The high-dose refuge strategy, and especially
the association ofthis approach with combinations oftoxins is advocated by scientists, seed companies,
and administrations as the best way of controlling evolution of resistance (Peferoen, 1997; Gould,
1998; Roush, 1998; Fox, 1998). This composite strategy allows flexibility in terms ofspatial organization
ofrefuge areas and choice oftoxins that will help adaptation and implementation in various agronomic
systems. This approach is now required by the EPA in the U.S. (Fox, 1998). Although currently
limited to the U.S., this legal requirement for active resistance management by the producers will
have to be followed by other countries as well.
Transgenic crops have the potential to trigger evolution of resistance and it is essential that the
end users, authorities, and industry will be important players in the successful implementation of
resistance management and therefore sustainability oftransgenic crops (Kennedy and Whalon, 1995).
There is also a need for training, information and extension workers to playa major role in the
successful implementation of resistance management.

79
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MOLECULAR BIOLOGY OF INSECT VIRUSES

Zhihong Hu, Xinwen Chen and Xiulian Sun

Wuhan Institute ofVirology, Chinese Academy of Sciences, Wuhan 430071, P.R.

China

1. INTRODUCTION

Taxonomically, insect viruses can be classified into 18 different virus families, including 6 families
of DNA viruses and 12 families/groups of RNA viruses (Hunter-Fujita et al., 1998; Regenmorte1 et
aI., 2000). The DNA viral families are Ascoviridae, Baculoviridae, Iridoviridae, Parvoviridae,
Polydnaviridae and Poxviridae. Some of these families only occur in arthropods, such as Ascoviridae,
Baculoviridae and Polydnaviridae, others occur in vertebrates andlor plants. The twelve RNA viral
families/groups are Bimaviridae, Bunyaviridae, Caliciviridae, Cricket paralysis-like viruses, Flaviridae,
Metaviridae, Nodaviridae, Piconaviridae, Reoviridae, Rhabdoviridae, Tetraviridae and Togaviridae.
There are several unclassified insect viruses, such as Oryctes virus, Hz-l virus and various bee
paralysis viruses.
Many of the insect viruses are occluded in part oftheir replication cycle; this is a unique feature
of insect viruses. As many host insects are only available during a limited period ofthe year, the
occlusion bodies protected the virions from the damage ofthe environmental agents such as heat and
decay. The other function of occlusion body is to deliver the virus to the alkaline midgut where virus
gets access to susceptible larval tissues. It is known that some occluded viruses have survived for
decades in the absence of host insects and still be infectious. There are three types of occluded insect
viruses: baculoviruses, cypovirus and entomopoxviruses. As the baculovirus is the most well studied
insect virus group and has been most widely used for insect pest control, it will be the main topic of
this chapter. Following is a briefintroduction of the other insect virus groups.

1.1 Ascoviruses (Family - Ascoviridae)

The Ascoviridae is a family oflarge double-stranded (ds) DNA insect viruses. So far identified
species include the ascoviruses from SpodopteraJrugiperda (SfAVI), Trichoplusia ni (TnAV2),
Helicoverpa virescens (HvAV3), Diadromus pulchellus (DpAV4) and S. exigua (SeAV-5a). These
viruses are unique among insect viruses because the transmission among their lepidopteran hosts is
generally by being vectored mechanically by hymenopteran parasitoids. The shape of ascovirus
virions is allantoid, reniform or bacilliform with the size ofabout 130 nm x 400 nm. The genomes of
ascoviruses are circular and partially superhelical with the sizes of 100-200 kb (Chen et aI., 1999,

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 83
2000; Bigot et al., 1997; Fedirici et al., 1990), and contain interspersed repeats ofl-3 kbp (Bigot et
aI., 2000). DpA V DNA contains six to eight copies of an imperfectly repeated sequence of
approximately 500 bp. These sequences are interspersed in the genome (Bigot et aI., 1997). The
presence of an imperfect palindrome sequence and their interspersion suggest a possible functional
similarity to the baculovirus homologous repeat sequence, which might fimction as transcription enhancer
and origin of DNA replication. The DNA polymerase gene ofascovirus has been sequenced (Pellock
et al., 1996) and used for phylogeny analysis of the ascoviruses (Stasiak et aI., 2000). At least 12
polypeptides, ranging from 10-200 kDa, were detected in virion (Federici et aI., 1990).

1.2 Entomobirnavirus (Family - Birnaviridae)

So far only a few viruses have been identified in the Bimaviridae. The only known insect bimavirus
is Drosophyla X virus. Virus-infected flies died in 5-15 days and were unusually sensitive to anoxia
for 3-4 days before death. Most of the genetic information of the Birnaviridae comes from
Aquabirnavirus andAvibirnavirus. Virions are about 60 nm in diameter, single shelled, non-enveloped
icosahedrons. Virions contain two segments of dsRN A. Segment A contains two 0 RF s, encoding a
17kDa and a polyprotein which contains the pre-VP2, NS (protease) and VP3 (internal capsid
protein). Genome segment B encodes VPl (94kDa) which is the RNA-dependent RNA polymerase
as well as the genome-linked protein. There are no poly(A) tracts at the 3' ends ofthe RNA segements.

1.3 Bunyaviridae

Four out of the five genera of Bunyaviridae, i.e. Bunyavirus, Nairovirus, Phlebovirus and
Tospovirus, are capable of alternately replicating in vertebrates and arthropods. Viruses are generally
cytolytic in their vertebrate hosts, but cause little or no cytopathogenicity in their invertebrate hosts.
Various viruses are transmitted by mosquitoes, ticks, phlebotomine flies, thrips and other arthropod
vectors. Virions generally are spherical or pleomorphic, 80-120 nm in diameter. Virions contain 3
molecules of negative or ambisense RNA. The genome sizes are 11-20 kb. Terminal nucleotides of
each viral RNA species are base-paired forming non-covalently closed, circular RNAs. Viral mRNAs
are not polyadenylated. The virus-complementary L mRNA encodes the viral transcriptase-replicase
(L protein); the M mRNA encodes the envelope glycoproteins (G 1 and G2) and the S mRNA the
nucleocapsid protein (N).

1.4 Caliciviruses (Family - Caliciviridae)

Calicivirus occurs predominately in vertebrates. So far only one calicvirus has been identified in
insect, amyelosis chronic stunt virus, isolated from the navel orangeworm, Amyelosis transitella
(Lepidoptera). The virions are 30-38 nm in diameter with 32 cup-shaped surface depression arranged
in T=3 icosahedral symmetry. Virion contains a positive ssRNA, 7.4-7.7 kb in size. The virus is
infectious per os and is pathogenic to early instar larvae. Later instar larvae are more likely to develop
a chronic infection, resulting in stunted adults with reduced fecundity.

1.5 Cricket Paralysis-like Viruses

Cricket paralysis-like viruses have been isolated from insects cell cultures including insects
from Orthoptera, Hymenoptera, Lepidoptera, Hemiptera, and Diptera. Cricket paralysis virus (CrPV)
from Teleogryllus oceanicus and Drosophila C virus (DCV) from D. melanogaster have been
studied in some detail (Christian and Scotti, 1998). The virion is isometric, un-enveloped virions of
around 30nm in diameter and contains single-stranded (positive-sense), linear RNA of9-1 0 kb in
size. The 3' end of viral RNA is polyadenylated and in most species this is a protein, Vpg, covalently
linked to the 5' end of the genome. The genome conations two ORFs with the 5' untranslated regions

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of500-800 bp. The 5' ORF encodes the nonstructural proteins with sequence motifs related to the
helicase, protease, and replicase domains of the other positive sense viruses such as picornaviruses,
comoviruses and sequiviruses. The structural proteins are encoded by the 3' ORF and the translation
product is processed into at least three mature capsid proteins (Johnson and Christian, 1998; Moon
et aI., 1998; Sasaki et aI., 1998).

1.6 Cypoviruses (CPVs, Family - Reoviridae)

Cypoviruses have only been isolated from arthropods. In total, more than 230 cypoviruses
have been described, mainly lepidopteran cypoviruses, but also dipteran and hymennopteran
cypoviruses. Viruses frequently have a broad host range, which may include several families of
Lepidoptera. The majority of cypovirus infections produce chronic diseases often without extensive
larval mortality, many individuals reach the adult stage even though heavily diseased. Infected larvae
stop feeding as early as two days post-infection. Larval body size and weight are oftenreduced and
diarrhea is common. The host larval stage can be significantly increased. Larvae can recover from
cypovirus infection, possible because the gut epithelium cell has considerable regenerative capacity
and because infected cells are shed at each larval moult. In some cases, the cypoviruses can be used
as successful pest control agents.
Virions have a single shelled capsid (55-69 nm in diameter) with icosahedral symmetry. Viral
particles are occluded by a crystalline matrix ofpolyhedrin protein forming a polyhedral inclusion
body. Cypoviruses contain 10-11 dsRNA genome segments. So far the genomes ofthree cypoviruses
have been entirely sequenced. These are Lymantria dispar cypovirus 1 (Rao et aI., 2001a), L.
dispar cypovirus 14 (Rao et aI., 2001 b) and Trichoplusia ni cytoplasmic polyhedrosis virus 15
(Rao et aI., 2000). For L. disparcypovirus 1, the segment 1 is4164 bp and encodes for the putative
major core protein. Segments 2 is 3853 bp and encodes for RNA dependent RNA polymerase.
Segment 10 is 944 bp and encode forpolyhedrin. Segments 3, 4,5,6,7,8,9, with size of3846,
3262,2851,1792,1501,1331,1187 bp respectively, encode for unknown proteins. For L. dispar
cypovirus 14, the segment 1 is 4329 bp and encodes RNA dependent RNA polymerase. The segment
2 is 4065 bp and encodes for the putative major core protein. The segment 10 is 956 bp and
encodes forpolyhedrin. Segment 3, 4,5,6,7,8,9, with size of3921, 3339, 3159,1783,1391,
1250, 1141 bp respectively, all encodes for unknown proteins. T ni cytoplasmic polyhedrosis virus
15 has 11 segments, the segment 11 is only 200 bp. Viral replication and assembly occur in the host
cell cytoplasm. Particles are occluded within polyhedra apparently at the periphery of the virogenic
stroma, from about 15 hr post infection.
Cypoviruses are normally transmitted by ingestion ofpolyhedra on contaminated food materials.
The polyhedra dissolve within the high pH environment of the insect gut releasing the viral particles,
which then infect the cells lining the gut wall. Virus infection is generally restricted in larvae to the
columnar epithelial cells of the midgut, although goblet cells may also become infected. Cypovirus
replication in the fat body has been reported. In larva, virus infection spreads throughout the midgut
region. The production of very large numbers of polyhedra gives the gut a characteristically creamy-
white appearance. In the infected cell the endoplasmic reticulum is progressively degraded,
mitochondria enlarge and cytoplasm becomes highly vacuolated.

1. 7 Entomopoxviruses (EPVs, Subfamily - Entomopoxvirinae)

The subfamily Entomopoxvirinae infects only insects. The viral morphology can be either
brick-shaped or ovoid with a size of70-250 nm x 350 nm. Virions of several morphological types
have globular surface units that give a mulberry-like appearance. Mature virions are usually
occluded in spheroids comprised of a major crystalline occlusion body. A second type of
paracrystalline proteinaceous body spindle occurs in some lepidopteran and colepteran EPV s. Based
on virion morphology, host range and the genome size the Entomopoxviruses are further grouped into

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three genus, that of Entomopoxvirus A, Entomopoxvirus Band Entomopoxvirus C.
Entomopoxviruses have been reported from about 60 insect species within Lepidoptera, Coleoptera,
Diptera, Orthoptera and Hymennoptera. The fat body is the main site of virus replication but
some infections are systemic.
So far genomes of two entomopoxviruses have been entirely sequenced, that of Amsacta
moorei (AmEPV, Bawden et al., 2000) and Melanoplus sanguinipes (MsEPV, Afonso et al., 1999).
The 236,120 bp MsEPV genome consists of a central coding region bounded by 7-kbp inverted
terminal repeats (ITRs) and contains 267 open reading frames (ORFs). Genes predicting interactions
with host cellular mechanisms include homologues ofthe inhibitor ofapoptosis protein, stress response
protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF -hand
protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in
prevention and repair of DNA damage include a complete base excision repair pathway (uracil
DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase),
a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse
transcriptase, and a mutT homologue. The genome of AmEPV contains 232,392 bases encoding for
292 ORFs, with 279 unique ORFs in central core viral chromosome and 13 ORFs in each ofthe
ITRs. Novel AmEPV genes include those encoding a putative ABC transporter, a Kunitz-motif
protease inhibitor and subunits of viral poly(A) polymerase. There is no significant gene order
conservation between AmEPV and the MsEPY.

1.8 Flavivirus (Family - Flaviridae)

The family Flaiviridae contains three genera, that of Flavivirus, Pestivirus and Hepatitis C
like-virus. Most flaviviruses are arboviruses and are maintained in nature by transmission from
Hematophagous arthropod vectors (either mosquitoes or ticks) to vertebrate hosts when the arthropod
takes a blood meal. For certain isolates no arthropod host has been identified. Viruses may also be
passed trans-ovarially and trans-stadially. Transplacental and horizontal transmission between
vertebrates has been demonstrated for some viruses. Viruses replicate in susceptible species of both
vertebrates and arthropods. Virions are 40-60 nm in diameter, spherical in shape and contain a lipid
envelope. Flavivirus contains a single molecule oflinearpositive sense ssRNA with a genome size of
10.7 kb. The 5' end structure of the viral RNA has not been characterized. Except for a few strains
of the tick-borne encephalitis complex offlaviviruses, the genome RNA does not contain a poly (A)
tract at the 3' -end. The genome is the only viral message RNA. A single long ORF codes for a
polyprotein that is proteolyticaliy cleaved into all the virus-encoded proteins. The structural proteins
are located at the 5' end, non-structural proteins including proteases, helicase and polymerases, are
encoded at the 3' end. Two types of virus particles can be distinguished: cell associated virus and
extracellular virus. Extracellular virus contains the two envelope proteins E and M and an interval,
RNA-associated protein, C. Instead of the M protein, cell-associated virus particles contain a larger
precursor protein, termed preM, which is cleaved during or shortly after release of virus from infected
cells. Virus attachment is mediated by the viral E protein. After endocytosis and uncoating the virus
RNA is translated, the products processed and RNA replication endues. The viruses multiply in the
cytoplasm of infected cells and are associated with proliferation of rough and smooth endoplasmic
reticulum. Nucleocapsid has not been visualized in cells. Virus particles accumulate within lamellae
and vesicles. RNA replication occurs in foci in the perinuclear region through a negative strand
intermediate.

1.9 Iridoviruses (Family - Iridovidae)

The Iridoviridae occurs in many insect families and also in other invertebrates. Iridoviruses have
only been isolated from poikilothermic animals that have an aquatic stage in their life cycle. Invertebrate
iridoviruses are divided into two genera, iridovirus and chloriridovirus, based on the size of the

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icosahedral virions. The iridovirus or small iridescent viruses are 125-140 nm across whilst the
chloriridovirus or large iridescent viruses are 180-200 nm across. The normal route of infection is
unknown since it is very difficultto infect larvae per os and it is not known to cause epizootics. Ifthe
larvae become infected during the early instars, the disease is fatal. During viral replication, the nucleus
remains intact, and virions form paracrystalline arrays in the cytoplasm giving rise to an iridescent hue
in the infected host. Infected larvae and concentrated purified virus often produce a blue to purple
iridescence for iridovirus and a yellow-green color for chloriridovirus.
lridoviruses have large (about 200-250 kb) liner dsDNA genomes. Mosquito iridescent
virus has been reported to have a genome of 440 kbp. The viruses contain circularly permuted and
terminally redundant DNA. Chilo iridescent virus (CIV), the type species of the genus Iridovirus,
has been entirely sequenced (Jakob et aI., 2001). The 212,482 bp genome contains 468 ORFs.
Only a few genes have been identified with seque,nce homology, such as the two major subunits
of the DNA-dependent RNA polymerase, DNA polymerase, protein kinase, thymidine and
thymidylate kinase, thymidylate synthase, ribonucleoside-diphosphate reductase, major capsid
protein, and others.

1.10 Metaviridae

Metaviridae contains two genera: Matevirus and Errantivirus. Mateviruses have been reported
from yeasts, plants and invertebrates as well, but the viruses of genus Errantivirus have only been
reported in invertebrates so far. The morphology of particles is relatively poorly characterized and
capsomeric symmetry is unknown. The genomes ofretrotransposons in this family are positive strand
RNAs, which is composed oflong terminal repeats (LTRs) flanking a central unique domain. The
length of elements ranges from 4 kb to more than 10 kb. The LTRs are from 77 nts in the case of
Bombyx mori mag virus (BmoMagV) to greater than 2 kb, in the case of D. virilis Ulysses virus
(DViULV). The internal domain contains one to three ORFs, gag-pol ORF, two ORFs (gap and
pol), or three ORFs (gap, pol and env). D. Melanogaster Gypsy virus (DemeGypV) contains three
ORFs, while the type virus Saccharomyces cerevisiae Ty3 virus (SceTy3V) contains two ORFs
gag and pol. However the order of domains encoded in the ORFs is inferred to be 5' CA-(NC
where present)-PR{protease)-RT(reverse transcriptase)-RH(RNAseH) - IN(lntegrase)-
ENV( envelope)-3'. Envelope proteins are encoded downstream of the second ORF by spliced
mRNAs (Boeke et al., 2000).

1.11 Nodaviruses (Family-Nodaviridae)

Nodavirus is the only genus of the family Nodaviridae. The family is named after Nodamura
virus, which was isolated from mosquitoes in a village {now a city, Nodashi) in Japan. Virions are
unenveloped, roughly spherical in shape, 30 nm in diameter and have icosahedral symmetry (T=3).
The genome consists of two molecules of ssRNA molecules, both molecules are apparently
encapsidated in the same particle. Both molecules are capped at their 5' -end and lack a poly (A) tail
at their 3 '--end. The capsid consists of 180 protein subunits (protomers). The virus replicates in the
cytoplasm. RNA synthesis is resistantto actinomycin D. Infected cells contain three ssRNAs: RNAl
(3.lkb); RNA2 (1.4 kb) and a subgenomic RNA 3 of 0.4 kb. RNAl codes for protein A (112
kDa), which is probably a component of the viral RNA polymerase. RNA2 codes for the coat
protein precursor, alpha (44 kDa). RNA3 codes for protein B (10 kDa) which may playa role in
synthesis of positive-strand RNA. All species (seven so far), except striped jack nervous necrosis
virus (fish virus), were isolated from insects. Viruses are able to infect a range ofspecies, particularly
within the Lepidoptera and Coleoptera. In the laboratory, most nodaviruses can be propagated in
larvae of the common wax moth, Galleria mellonella. Some species have been reported to be able
to grow in mice or plants. Nodamura virus is transmissible to suckling mice by mosquitoes. Nordavirus
causes paralysis and death when injected into suckling mice or wax moth larvae.

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1.12 Parvoviruses (Family - Parvoviridae)
Viruses assigned to the subfamilily Densovirinae infect arthropods. They have small non-
enveloped virions with size of 19-24 nm and icosahedral symmetry. The ssDNA genome of virions is
either positive or negative sense with a size of 4-6 kb. Upon extraction, the complementary DNA
strands usually form dsDNA. There are four structural proteins. Viruses multiply efficiently in most of
the tissues oflarvae, nymphs and adult host species. Cellular changes consist of hypertrophy ofthe
nucleus with accumulation of virions therein to form dense, voluminous intranuclear masses. The
known host range includes members of the Dictyoptera, Diptera, Lepidoptera, Odonata and
Orthoptera. There is considerable variation in the biological characteristics ofthese viruses. Some
have very limited host range while others are able to infect species across several families.

1.13 Piconaviruses (Family - Picornaviridae)

The Picornaviridae includes small, ssRNA viruses that are common cause ofdiseases in mammals.
Virions contain positive ssRNA with 7-8.5 kb in size. A poly (A) tract is located at the 3' -teminal
sequence. A small protein of24 kDa is linked covalently to the 5' terminus. Virions are icosahedral
with no envelope. At least 25 small RNA viruses are isolated from various insect species. They are
described in the literature as piconaviruses, or piconavirus-like viruses. These includes bee acute
paralysis virus, bee slow paralysis virus, bee virus X, Drosophila C, P and A virus, sacbrood virus,
Queensland fruitfly virus and aphid lethal paralysis virus. Gonometa virus, isolated in Uganda from
Gonometa podocarpi which is a pest of pine trees, cause high mortality to the host and was shown
to be an effective control agent.

1.14 Polydnaviruses (Family - Polydnaviridae)

The polydnaviruses are unusual and complex viruses with a genome consisting of multiple
supercoiled DNAs ofvariable size from 2 to more than 28 kb. Estimates of genome size and complexity
are complicated by the presence of related DNA sequences shared among two or more genome
segements. Polydnaviruses have been isolated only from endoparasitic hymennopteran insects belonging
to the families Ichneumonidae and Braconidae. The Polydnaviridae, therefore, contains two genera:
/chovirus and Bracovirus. In nature, polydnavirus genomes are apparently transmitted as proviruses.
PolydIiavirus particles are injected into animals during oviposition; virus-specific expression leads to
significant physiological changes suppressing the host's immune response. These viruses are assumed
to be essential to the successful parasitism.

1.15 Rhabdoviruses (Family - Rhabdoviridae)

Many rhabdoviruses occur in insects but nearly all are transmitted by arthropods. The virions
are 100-430 nm long and 45-100 nm in diameter with a bullet shape. The virions contain a negative
ssRNA with a size of 11-15 kb. Only one virus, the sigma virus of Drosophila, is restricted to
insects. The virus is transmitted only vertically in nature and occurs in about 10% of the natural
population of Drosophila. Under normal circumstances it is not pathogenic but it confers sensitivity
to COz and the infected adults suffer fatal paralysis when exposed to the gas.

1.16 Tetraviruses (Family- Tetraviridae)

The family Tetraviridae contains two genera, "Nudaurelia capensis /3-like virus" and
"Nudaurelia capensis co-like virus". The virions are unenveloped, roughly spherical, about 40 nm in
diameter and icosahedral symmetry. The genome consists of ssRNA. The Nudaurelia capensis /3-
like viruses have a monopartite genome with a size of 5 kb. The Nudaurelia capensis co-like viruses
have bipartite ssRNA genomes. The complete sequence of the H armigera stunt virus has been

88
detennined and consists of RNA 1 of 53 11 nucleotides and RNA2 of2478 nucleotides. The viruses
are stable at low pH. All species were isolated from Lepidoptera. Individual viruses exhibit a broad
range of infection and pathogenicity. Infection leads to rapid death or growth retardation oflarval
stage. H armigera stunt virus is transmitted orally. It can grow slowly and without cytopathic effect
in cultured Drosophila and Spodoptera cells.

1.17 Togaviridae

Togaviridae contains two genera, Alphavirus and Rubivirus. Alphaviruses are transmitted
between vertebrates by mosquitoes and certain other hematophagous arthropods, while humans are
the only host for rubella virus. The virions are 70 nm in diameter, spherical, with a lipid envelope. The
genome consists ofa linear, positive sense, ssRNA with a size ofll-12 kb. The genomic RNA
serves as the mRNA for the non-structural proteins of the virus. The nonstructural proteins are
required for RNA replication. The nsPl protein ofalphavirus is thought to be involved in capping of
viral RNAs and in initiation of negative-strand RNA synthesis. The order of the genes of the non-
structural proteins in the genomic RNA is nsPl, nsP2, nsP3 and nsP4. These are made as polyprotein
precursors and processed by the nsP2 protease. The gene order in the other part of the genome is C-
E3 -E2-6K-E 1. The nsP2 functions as a protease to process the non-structural proteins and is
believed to be a helicase required for RNA replication. Protein nsP3 is also required for RNA
replication. Protein nsP4 is viral RNA polymerase.
Alphaviruses possess the ability to replicate and pass horizontally in mosquitoes, or transovarially
in certain vectors. Each virus usually has a preferred mosquito vector, but as a group the virus uses a
wide range of mosquitoes. In infected vertebrate cells, the infection is cytolytic and involves the
shutdown ofhost-cell macromolecular synthesis. In mosquito cells, alphaviruses usually establish a non-
cytolytic infection in which the cells survive and become persistently infected. The assembly ofvirions
in mosquito cells appears to differ from that for vertebrate cells wherein most virus assembly occurs
in association with intracellular membranes rather than by budding through the plasma membrane.

2. CLASSIFICATION OF BACULOVIRUSES

The Baculoviridae, a diverse family of more than 600 viruses, contains two genera, the
Nuc/eopolyhedroviruses (NPV) and the Granuloviruses (GV) (van Regenmortel et al., 2000).
NPV contains many virions occluded within occlusion body called polyhedra, while, on the other
hand, GV contains only one or, rarely, two or more virions in occlusion body called granules.
Nucleocapsid envelopment ofoccluded virion occurs either within the nucleus (NPV) or in the nuclear-
cytoplasmic milieu after rupture ofthe nuclear membrane (GV). Among NPVs, the viruses are further
designated as SNPV or MNPV depending on the single (S) or multiple (M) packaging of the
nUcleocapsids in the virion. The aggregation of nucleocapsids within the envelopes does not appear
to be phylogenetically significant, but is only oftaxonomic importance. On the other hand, phylogenetic
analysis based on several viral genes indicated that the NPV can be divided into two groups: group
I and II (Zanotto et al., 1993; Bulach et al., 1999). Group I contains the relatively well characterized
baculoviruses, like Autographa californica (Ac) MNPV, Orgyia pseudotsugata (OpMNPV) and
B. mori (Bm) NPV, while L. dispar (Ld) MNPV, S. exigua (Se) MNPV, H zea (Hz) SNPV, and
H armigera SNPV (HaSNPV or HearNPV) are included into group II (Chen et al., 2001 b). By
using GeneParityPlot (Hu et al., 1998), it has been revealed that the group I NPV and group II share
common genome structure (Chen et al., 2001a).
The virions are rod-shaped and contain a circular, double stranded DNA genome of80 to 180
kb depending on the species. Baculoviruses are pathogens that often cause fatal diseases in insects,
mainly in members ofthe families Lepidoptera, Hymenoptera, Diptera, and Coleoptera, but also in
Neuroptera, Trichoptera, Thysanura, Siphonoptera as well as in crustaceans (Decapoda). So far,

89
nineteen relatively well characterized baculoviruses have received species status including AcMNPV,
SeMNPV, LdMNPV, HzSNPV and T ni GV. The host families from which NPV s have been isolated
are listed in Table 1.

Table 1. Nucleopolyhedroviruses isolated from insect species *

Order Number Family
Coleoptera 5 Cerambycidae 2
Curulionidae 1
Dermestidae 2
Diptera T1 Calliphoridea 1
Chironomidae 1
Culicidae 20
Sciaridae 3
Tachinidae 1
Tipulidae I
Hymenoptera 31 Argidae I
Diprionidae 19
Pamphylidae 3
Tenthridinidae 8
Lepidoptera 455 Anthelidae 2
Arctiidae 22
Argyresthiidae I
Bombyciidae 4
Brassolidae I
Carposinidae I
Coleophoridae I
Cossidae I
Cryptophasidae I
Dioptidae I
Gelechiidae 3
Geometridae 63
Hepialidae 3
Hesperidae 5
Lasiocampidae 34
Limacodidae 1\
Lymantriidae 49
Lyonetiidae 1
Noctuidae 107
Notodontidae 12
Nymphalidae 15
Papillionidae 6
Pieridae 9
Plutellidae I
Psychidae 5
Pyralidae 23
Saturniidae 22
Sphingidae 14
Thaumetopoeidae 3
Thyatiridae 1
Tineidae 2
Tortricidae 2f,
Yponomeutidae 4
Zygaenidae
Neuroptera 2 Chrysopidae
Hemerobiidae
Siphonoptera Pulicidae
Thysanura Phaemachilidae
Trichoptera Limnephilidae

"From Adams and McClintock, 1991, with permission of the CRC Press.

90
 The current interest in the molecular biology of these viruses is fostered by their potential as
alternatives to chemical insecticides in the control of agricultural and forest insect pests (Moscardi,
1999; Cunningham, 1998) and also by their successful use as vectors for the expression of foreign
proteins (Possee, 1997; Korst and Condreay, 1999). The technological advancement associated
with the generation of expression vectors has also been successfully used to develop enhancing
baculovirus pesticide agents (Bonning and Hammock, 1996). Recently, baculovirus-derived vectors
have emerged as a possible tool for gene transfer into mammalian cells and holds a promise in gene
therapy (Hofmann et ai., 1995; Boyce and Bucher, 1995; Condreayet ai., 1999; Sarkis et aI.,
2000). An in-depth analysis ofthe baculovirus genome organisation, replication and gene expression
strategy is a prerequisite for optimal exploitation of these economically important applications. The
genetic analysis also leads to a better understanding ofbaculovirus replication and pathology.

3. BACULOVIRUS STRUCTURE AND INFECTION CYCLE

The baculovirus phenotypes appear to have evolved to suit the unique features needed to cause
horizontal and vertical infections of larvae. The replicate cycle is biphasic where two progeny
phenotypes are produced, the occlusion-derived virus (ODV) and the budded virus (BV). ODV is
encapsulated in a protein matrix composed predominantly of a single protein called polyhedrin (or
granulin in GV). The budded form ofvirus (BV) is not occluded. ODV is responsible for the initiation
of an infection in the insect. Although their nUcleocapsids are similar in structure, ODVsand BVs are
structurally distinct and have specific polypeptides (Figure I; Funk et aI., 1997).

Budded Virus Occlusion Derived Virus

(BV) (ODV)

Common VIrion ODV specific

Components Components

GP64

V.rlon EnY.~

...... -....-1"1
<PC ..
SPH 1U
PC t l).7
Pi 1U
os ...,
PE 1A

Figure 1. Structural comparison of the two baculovirus phenotypes, the budded virion, BV, and the occlusion
derived virion (OOV) (from Funk et aI., 1997; with permission from Kluwer Academic/Plenum Publishers). The
OOV structure represents the MNPV subgroup. Proteins common to both virion types are indicated in the middle
of the figure. Proteins specific to either BV or OOV are indicated on the left and right, respectively. The polar nature
of the baculovirus capsid is indicated in the diagram with the claw-like structure at the bottom and the ring-like
nipple at the top of the capsid. References for structural proteins are p6.9 (Wilson et aI., 1987); vp39 (Blissard et aI.,
1989; Pearson et aI., 1988; Russell et aI., 1991; Thiem and Miller, 1989); p80 (Lu and Carstens, 1992); MUller et aI.,
1990); pp78/83 (Russell et aI., 1997; Vialard and Richardson, 1993); polyhedrin (Hooft van Iddekinge et aI., 1983);
PEP (PP34 )(Gombart et aI., 1989; Russell and Rohrmann, 1990); OOV-E25 (Russell and Rohrmann, 1993); OOVE66
(Hong et aI., 1994); OOVE56 (Braungel et aI., 1996a; Theilmann et aI., 1996); OOVEI8, E35, and EC27 (Braungel et
aI., 1996b); gp41 (Whitford and Faulkner, 1992a, b); p74 (Kuzio et aI., 1989) and gp64 (Blissard and Rohramann,
1989; Whitford et aI., 1989). Lipid compositions of the BV and OOV envelopes derived from AcMNPV infected Sfl)
cells (Braunagel and Summers, 1994) are indicated (LPC, Iysophosphatidylcholine; SPH, sphingomyelin; PC,
phosphatidylcholine; PI, phsophatidylinositol; PS, phosphatidylserine; PE, phosphatidylethonalamine).

91
 Midgut Epithelium

-I
Occlusion 800.\'

I J ODV
,Ik. lin, pH •

NI
~/
I
I
Other Tissues \
.. I

11111 /b ~ ~
"."\ ~
I r------------~

~.(
I-~PM
\~1~IJj
I
/
8V
\L/
+
JI
I --CP54
r#"8V

Figure 2. The baculovirus infection cycle (From Blissard, 1996, with premission ofthe author and Kluwer Academic
Publishers). On the left, the infection of midgut epithelial cells by the occlusion derived virus (ODY) is illustrated.
After fusion of the ODY at the plasma membrane, two possible routes of nucleocapsid migration are indicated (a
and b). In what is thought to be the typical route (a), nucleocapsids (NCs) are transported to the nucleus where
gene expression, DNA replication, and assembly of progeny NCs occur. Newly assembled NCs then migrate from
the nucleus towards the plasma membrane. A portion of incoming NCs may bypass the nucleus (b), and migrate
directly to the basolateral plasma membrane. NCs bud from the basal side of the epithelial cell at sites where the
major envelope fusion glycoprotein (EFP) GP64 of the BY phenotype (indicated by arrows), has accumulated. The
right side of the figure represents the infection of insect cells, other than midgut epithelial, by budded virus (BY)
phenotype. After BY binding and uptake into the endosome, the acidification of the endosome triggers fusion of
the viral and endosome membrane, after which the NCs are released into the cytoplasm. Although the modes of
entry of the ODY and BY are different and specific for each virus phenotype, once NCs are released into the
cytoplasm, progression of infection appears similar. Except for midgut epithelial cells at the very late stage of the
infection cycle nucleocapsids become embedded within proteinaceous occlusion bodies (OBs) in the nucleus.
Upon death and liquefaction of the host, OBs are released into environment to initiate a new round of infection.

Transmission ofa baculovirus in an insect population occurs either via ingestion offood, via soil
contaminated with occlusion bodies by insect larvae or via ovipostion of eggs contaminated with
occlusion bodies (Blissard et aI., 2000). Infection is initiated when alkali sensitive occlusion bodies
are ingested by a susceptible host and dissolved by juices in the insect midgut. The dissolution is
aided both by the high pH and the presence of alkaline proteinases in the midgut lumen of most
Lepidoptera. The virions released from ODV then pass through the peritrophic membrane and fuse
with the midgut epithelial cell plasma membrane. The nuc1eocapsids are then released into the cytoplasm
and migrate to the nucleus, where transcription of viral genes and replication of the viral genome
takes place. The nUcleocapsids synthesized in the nucleus pass through the nuclear membrane and
bud and acquire a new envelope from the plasmalemma to become BV. The BVs produced in
epithelial cells of the midgut spread via the hemolymph (Granados and Lawler, 1981) and/or the
tracheal system (Engelhard et al., 1994) into all tissues ofthe insect causing a secondary infection, but
the predominant target is the fat body cell. Occlusion of virions does not occur in midgut epithelial
cells as they are sloughed offin to the gut lumen and replaced by regenerated cells in most lepidopterous
insects, or, where it does occur, it occurs only rarely (Flipsen et aI., 1993; Flipsen et aI., 1995b). In
the early stage of secondary infection, infected cells produce BVs which efficiently spread the infection

92
from cell to cell within insect tissues. At later stage of secondary infection, virions are occluded into
occlusion bodies in the infected cells. At the end of the infection, the cells and tissues of the dead
insects are disintegrated and occlusion bodies are released into the environment. Virus encoded
proteins such as fibrillin, chitinase and cathepsin have been reported to be involved in this process
(van Oers and Vlak, 1997; Hawtin et al., 1995; Hawtin et al., 1997; Slack et al., 1995; Hill et al.,
1995). The next infection cycle starts again when the occlusion bodies are ingested by other susceptible
insects (Figure 2).

4. GENOME ORGANIZATION

Baculoviruses contain a circular, supercoiled double-stranded DNA genome with a size ranging
from 80-180 kb depending on the species. Recently, DNA sequence analysis has begun to reveal the
distinctive feature of the baculovirus genomes and the extent oftheir diversity. So far, 9 NPV and
three GV genome sequences have been determined. These are AcMNPV (Ayres et al., 1994),
OpMNPV (Ahrens et al., 1997), LdMNPV (Kuzio et al., 1999), BmNPV (Gomi et aI., 1999),
SeMNPV (Ukel et aI., 1999), HaSNPV (Chen et al., 2001a), HzSNPV (Chen et al., 2001b), S.
litura (SpIt) NPV (Pang et al., 2001), Culex nigripalpus (Cuni) NPV (Afonso et al., 2001)and
Xestia c-nigrum (Xc) GV (Hayakawa et al., 1999) ,Plutella xylostella (Px) GV (Hashimoto et al.,
2000) and Cydia pomonella (Cp) GV (Luque et al., 2001). The sizes oftheses genomes range from
101 to 179 kb with 109-181 potential open reading frames (ORFs) in the different genomes (Table
2). The ORFs, in general, are tightly packed with minimal intergenic regions and with their orientations
almost evenly distributed along the genome. Both strands of the genome are involved in coding
functions and the genes are generally unspliced. Gene classes (early, late and very late genes) are not
clustered in baculovirus genomes.

Table 2. Characteristics ofdifferent baculovirus genomes.

Characteristic AcM an OpM LdM SeM HaS HzS SpIt Cuni Cp IX Xc

NPV NPV NPV NPV NPV NPV NPV NPV NPV Gv GI GI
Size(kb) 133.9 128.4 132 161 135.6 131.4 130.7 139.3 108.3 123.5 101178.7
Coding(%) 90 90 89 87 90 87 87 NK NK NK 86 88
TotalORFs 154 136 152 163 139 135 139 141 109 143 120181
UniqueORFs 14 4 26 47 17 20(0)* 24(4)* ']9 72 26 16 52
Numberofhr 9 7 5 13 4 5 5 17 4 0 4 9
G+Ccontent 41 40 55 58 44 39 39 42.7 50.9 452 40 41
Classification NPV NPV NPV NPV NPV NPV NPV NPV NK GI GI GI
I II II II II II
AcMNPV, Ayres et aI., 1994; BmNPV,Gomietal., 1999; OpMNPV, Ahrens etal., 1997; LdMNPV, Kuzio et aI., 1999;
SeMNPV, IJkel etal., 1999; HaSNPV, Chen etal., 2001a, b; PxGV, Hashimoto etal., 2000; XcGV, Hayakawaetal., 1999;
HzSNPV, Chen, 2001; CpGV, Luque et aI., 200 I, SpltNPV, Pang et aI., 2001, CuniNPV, Afonso et aI., 200 J. NK: not-
known.
• 20 unique HaSNPV ORFs have homologs in the HzSNPV genome.

4.1 Homologous Regions

One unique featureis that baculovirus genomes contain several homologous regions withrepeated
sequences (hrs). Hrs occur at multiple locations along the genome, ranging from 4 in PxGV to 13 in
LdMNPV. It has been demonstrated that hrs serve as putative origins ofDNA replication in transient
replication assays (Kool et al., 1995) and as enhancers of RNA polymerase II-mediated transcription
(Guarino and Summers, 1986; Guarino et al., 1986). It has also been reported that the conserved

93
imperfect palindromes within the repeated sequences are critical for their functions (AcMNPV).
However. no long palindromic sequence was found in the HaSNPV/HzSNPV hrs (Chen et al.,
2001 a, Chen 2001). Instead, these hrs are characterized by two types of direct repeats, similar to
the XcGV hrs, which also contain only imperfect direct repeats and have no palindromic structure
(Hayakawa et aI., 1999). In contrast to most of the ORFs, hrs do not share significant sequence
identity among different baculoviruses. Hence, hrs diverge significantly among baculoviruses both in
sequence and in structure. Recently CpGV genome sequence indicates that there is not a typical hr
along the genome. However, it contains one major repeat rejoin and 13 copies of a single 73-77 bp
imperfect palindrome (Luque et al., 2001).
Hrs might be hot spots of recombination within or between baculovirus genomes. Among
different baculovirus genomes, the most distinct regions are flanked by or contain a disproportionate
number of hrs (Hayakawa et al., 2000). Between HaSNPV and HzSNPV, which are the different
variants of the same virus species, the major insertion and deletion as well as the lowest sequence
identity between these two genomes are found in or near the hr regions. Also, most of the unique
HzSNPV 0 RF s map in the flanking regions of hrs (Chen, 2001). This supports the view that in
different variants of the same viral species hrs are hypervariable (Munoz et al., 1998).

4.2 Conservation ofBaculovirus Gene Content

Based on eleven lepidopteron baculoviral genome sequences, 63 ORFs were found conserved
among all baculoviruses, suggesting that they are required for the basic baculovirus features (Table
3). So far 36 of these 63 core genes have been assigned putative functions, while the functions ofthe
remaining 27 ORFs are still unknown. Seventeen of the former 36 genes encode proteins for DNA
replication, regulation oftranscription and late gene expression, e.g. genes essential for DNA replication
such as DNA polymerase, helicase, ie-I, lef-l, lef-2 and lef-3 (Lu et aI., 1997). A second set of
genes (15 ofthe 63) encodes structural proteins, including components ofpolyhedra, capsid, tegument
and envelope. Four genes that appear to provide the virus with a selective advantage in nature
function at the cellular or organism levels. It is worth noting that the genes involved in DNA or
nucleotide metabolism, e.g. DNA ligase, ribonucleotide reductase (two subunits) and dUTPase, are
not conserved in baculoviruses.

Table 3. Function of the 63 conserved baculovirus genes.

Replication I expression Structure Auxiliary Unknown
39K, dbpi dnapol, ac23,fp25K, gp41, alk-exo, fgf, 38K, 38.7K, ae22, ae29, ac38,
helicase, lefl, lef2 , odv-ei8, odv-e25, sod, ubiquitin ae53, ae66, ac68, ae75, ac76,
tej3, lef4, lefS, tef6, odv-ec17, odv-e56, ae78, ac81, ae82, ae92, ae93,
lefl, left, lefI I, iei, odv-e66, p6.9, p74, ac96, ae106, ac109, ael1O,
me53, p47, vlfI p9S, pki, poth, vp39, acllS, acIl9, ac142, ac145,
vplOS4 acI46, pi2, p40, p45
Bold indicates the gene is also conserved in CuniNPV.

In CuniNPV genome, a Diptera baculovirus, however, only has 36 ORFs which found
homologues in other baculoviruses, but their orientations and orders exhibit little conservation relative
to the genomes oflepidopteran baculoviruses (Afonso et al., 2001). CuniNPV genes homologous to
those from other baculoviruses include genes involved in early and late gene expression (lef-4, lef-5,
lef-8, lef-9, vlf-I, andp47), DNA replication (lef-I, lef-2, helicase-I, and dna-pol), and structural
functions (vp39, vp9I, odv-ec27, odv-e56, p6.9, gp4I, p74, and vpI054). Only one conserved
auxiliary gene, alk-exo, has a homolog in CuniNPV. In contrast to these conserved genes, CuniNPV
lacks apparent homologues ofbaculovirus genes essential (ie-I and lef-3) or stimulatory (ie- 2, lef-7,

94
pe38) for DNA replication. Also, baculovirus genes essential or stimulatory for gene transcription
(ie-O, ie-1, ie-2,pe-38, lef6, lefll, and pp31) are not identifiable. In addition, CuniNPV lacks
homologues of structural genes such as or/1 629, p87, p24, odv-e25, odv-e66, odv-e 18, and the
genes involved in polyhedra (polyhedrinlgranulin, p 10, and pp3 4). The absence ofhomologues of
occlusion-derived virion (ODV) envelope proteins and occlusion body (OB) protein (polyhedrin)
suggests that both CuniNPV ODV and OB may be structurally and compositionally different from
those found in terrestrial lepidopteran hosts. The striking difference in genome organization, the low
level ofconservation ofhomologous genes, and the lack ofmany genes conserved in other baculoviruses
suggest a large evolutionary distance between CuniNPV and lepidopteran baculoviruses.

4.3 Baculovirus Genome Diversity

One ofthe major diversity ofbaculoviruses is the genome size, which may vary from 100 kb
(PxGV) to 180 kb (XcGV) (Table 2). Although processes, such as deletion and insertion, transposition
and horizontal transfer ofgenes, might be involved, the major contributor to the genome size difference
is gene duplication. The largest genome, that ofXcGV, contains 30 repeated genes accounting for
37.5 kb or 20% of the genome (Hayakawa et al., 1999). Similarly LdMNPV contains 32 repeated
genes that account for 27.5 kb or 17% of the genome (Gomi et al., 1999). HaSNPV, which has an
average genome size, contains only 5 gene repeats, two iap and 3 bro genes (Chen et a1., 2001 a,b).
The most highly repeated gene family, bra, is present from one (AcMNPV) to 17 copies (LdMNPV),
but is absent in SeMNPV and CpGV. Functional analysis indicated that different bro genes might
have different functions (Kang et a1., 1999; Zemskov et al., 2000). Phylogenetic analysis also showed
that bros may have been acquired by either duplication or from a different source, and have been
adapted to their respective functions during the viral evolution. It is notable that most baculoviruses
have more than one copy of iap, suggesting that these genes are functionally important. They might
have been picked up from different genetic sources and may be active in different apoptotic pathways
or in specific cell types, tissues, or species.
Genomic comparison by GeneParity Plot analysis indicated that genomes of the group I
baculoviruses such as AcMNPV, BmNPV and OpMNPV, even though there are some inversions
and insertions (deletions), basically have a similar gene content and arrangement, while SeMNPV
and LdMNPV, which belong to group II, might have a common ancestor and be more distantly
related to group I (Hu et aI., 1998; IJkel et a1., 1999). The GV group is quite separate from NPV
group. However the gene organization in the "central" part of the baculovirus genomes is highly
conserved and is a key feature in the alignment ofbaculovirus genomes (IJkel et al., 1999; Heldens
et at, 1998).

5. GENE EXPRESSION OF BACULOVIRUS

Baculovirus gene expression is organized in a sequential, cascade-like fashion in which each

successive phase is dependent on the previous one (BUssard and Rohrmann, 1990). Regulation of
the baculovirus gene expression, including the timing and level, occurs at the transcriptional level.
Three phases, early, late and very late, are distinguished during a baculovirus infection. The products
ofearly viral genes function to both accelemte replicative events and to prepare the host cell for virus
multiplication. Specific early genes are also essential for virus-mediated regulation ofthe host, including
the control oflarval molting and evasion ofhost antiviral responses such as apoptosis.

5.1 EarlyGene

By definition, a baculovirus early gene is transcribed priortothe initiation ofviral DNA replication,
which begins 6 to 9 hours after infection. So, early transcription is independent of DNA replication

95
and late gene expression. Transcription ofmany early genes initiates immediately after virus inoculation.
It is possible that transcription begins as soon as the viral DNA is uncoated in the nucleus. Although
many early genes are transcribed into a single RNA species, early transcription is often characterised
by the synthesis of common 3' terminus but different 5' ends (Friesen and Miller, 1985; Lubbert and
Doerfler, 1984). The functional significance ofthis pattern is still unknown. Genes expressed during
the early phase of the infection are transcripted by host RNA polymerase II and associated host
transcription factors. Naked viral RNA is infectious, indicating that the host transcription system is
sufficient to initiate productive infection. Also, early viral transcription is blocked by the RNA
polymerase II inhibitor a-amanitin (Huh and Weaver, 1990). And the early promoters have a typical
eukaryotic consensus transcription motif, which is similar to that of RNA polymerase II -responsive
genes but strikingly different from the baculovirus late genes (Friesen, 1997).
Current evidence suggests that the promoters for early baculovirus genes are organized to
expedite immediate and regulated levels of transcription by mechanisms to exploit host RNA
polymerase II and associated host factors. Early promoters contain core transcription elements such
as a T ATA box and a ATCA(G/T)T (CIT) motif thatcooperate with auxiliary cis-acting elements
located either nearby or within distal transcriptional enhancers which are recognized by DNA sequence-
specific transcription factors. Several distinct baculovirus distal upstream activating regions motifs,
CTG motif with consensus sequence A(A/T)CGT(G/T), have been identified. These factors likely
function to stimulate the rate oftranscriptional initiation by the RNA polymerase II-complex. In
addition to the cis-acting elements, the baculovirus genome contains multiple copies of homologous
repeat sequences that function as transcriptional enhancers (Lu and Carstens, 1993; Nissen and
Friesen, 1989). To date, four viral proteins have been identified as transregulators which have the
capacity to stimulate early viral gene expression: IEO, IEl, IE2 and PE38. IEI encoded by the
immediate early gene ie-l is conserved among the baculovirus, a feature consistent with its central
role in virus replication. It is now known that many early baculovirus promoters, as well as heterogonous
promoters, can be affected to some extent by IE 1. IE 1 potently stimulates transcription of promoters
for the 39K, p35, gp64, p 143, dnapol, pe38, lef-l, lef-2 and lef-3 genes, as well as the ie-l
promoter itself(Pullen and Friesen, 1995).
The products of early viral genes function to prepare the host cell for virus multiplication and to
accelerate replicative events (Friesen, 1997). One ofthe important early genes is the immediate-
early gene 1 (ie-I). Its product is involved in the transactivation of many genes that are expressed
during the early and late phases ofa baculovirus infection. It is also required for viral DNA replication
in vitro.

5.2 Late and Very Late Gene

The late stage ofbaculovirus infection is defined as those events that occur following the initiation
of viral DNA replication and is usually subdivided into a late and a very late phase. These phases
coincide with the production ofBV and ODV, respectively (Lu and Miller, 1997). During the late
phase, genes encoding structural proteins of the viral nucleocapsid, such as vp39 and p6. 9, the major
capsid protein and basic core protein, respectively, are abundantly transcribed. Late genes are
transcripted by an a-amanitin-resistant RNA polymerase encoded by the baculovirus genes and this
transcription starts at a consensus AlGTAAG motif. Genes encoding occlusion-related polypeptides,
such as polyhedrin or granulin, are transcribed primarily during the very late phase. Another abundantly
expressed very late gene is pI 0, which encodes a small polypeptide that affects nuclear disintegration
in the final phases of cell death (van Oers and Vlak, 1997). These two very late genes (ph andp 10)
are expressed from very strong viral promoters and these promoters are extensively utilized in
baculovirus insect expression systems (Smith et al., 1983; Martens et al., 1995).
In vitro transcription assay found outthat eighteen genes, ie-I, ie-2, lefol-1I, dnapol,p 143,
p4 7, p3 5 and 39K, are required for optimal transcription of expression from the late vp39 and p6. 9
promoters and the very late gene polh and p I 0 promoter (Todd et al. 1995, 1996; Lu and Miller,

96
1994, 1995; Morris et al., 1994). These genes are referred to as late gene expression factor (LEF)
genes. Only two of these genes, ie-l and ie-2 also transactivate transient early gene expression.
Transient expression assay appears reflect the dependence ofbaculovirus lat gene expression on
DNA replication, although DNA replication also affects template copy number. Even the role of
some ofthese replication associated left such as dnapol, lef-3 and p143 are obviously related to
viral DNA replication, some of the left are probably not directly involved in DNA replication, while
others may have roles in both DNA replication and gene expression. IE 1 and IE2 are known to
activate expression from early promoters and their primary role:; may be to transactivate other left
including the early replicative left. Lef-2 may also be involved in DNA replication, late gene expression,
and very late gene expression (Merrington et al., 1996).
At least three ofthe transcription-specific left, lef-6, lef-8 and lef-9, are likely to be components
ofthe virus-encoded RNA polymerase (LuandMiller, 1994; PassarellietaL, 1994). LEF-8 and
LEF-9 contain amino acid sequence motifs that are conserved in prokaryotic and eukaryotic RNA
polymerase (Lu and Miller, 1994; Passarelli etal., 1994), while LEF -6 shares some sequence similarity
to the 19 kDa subunit ofthe vaccinia virus DNA dependent RNA polymerase (Passarelli and Miller,
1994). The remaining left, p74, lef-4, lef-5, 39K lef-i 0 and lef-ii, appear to have no significant
homology to genes in GeneBank database. One gene, very late expression factor-l gene (vlf-l), has
been found to regulate very late gene transcription (McLachiin and Miller, 1994; Todd et al., 1996).
A dependence oflate transcription on DNA replication is observed in normal infections. Treatment
of infected cells with aphidicolin, an inhibitor of both cellular and viral DNA polymerase, completely
inhibits late gene expression and blocks the infection at the transition point between early and late
gene expression (Rice and Miller, 1986).

6. DNA REPLICATION OF BACULOVIRUS

Cis and trans -acting elements associated with DNA replication have been identified in the
genomes of several baculoviruses using different strategies; Homologous regions (hrs) and non-hrs
have been identified as putative replication origins by the characterization ofdefective viral genomes
generated by serial passage of the virus in tissue culture (Kool et al., 1991, 1993) and from transient
replication assays that rely on the ability of plasmids containing these elements to be amplified when
transfected into baculovirus-infected insect cells (Ahrens et al., 1995; Leisy et al., 1995; Pearson
and Rohrrnann, 1995; Xie et al., 1995).
Homologous regions have been found in all the baculoviruses in which genome sequences have
been determined, except CpGV wherein only one repeat sequence was detected. The core sequence
of hrs contains an imperfect palindrome flanked by direct repeats. In AcMNPV, hrs consist of one
to eight copies of a repeated sequence composed of 30 bp palindromes flanked by 20 bp direct
repeats and separated by about 80-120 bp of DNA (Ayres et ai., 1994). The hr sequences have
been shown to confer upon a plasmid the ability to replicate in insect cells in an infection-dependent
manner using aDpnI based assay system (Ahrens et al., 1995). These analyses have also demonstrated
that a single palindrome from an AcMNPV hr can support limited plasmid DNA replication (Leisy et
al., 1995; Pearson et al., 1992). Elements flanking hr sequences have been shown to be necessary
for optimal infection-dependent plasmid replication (Leisy et ai., 1995; Pearson and Rohrrnann,
1995; Xie et al., 1995). However, there is no direct evidence that hrs ftmction as origin of viral DNA
replication in vivo up to now. Non-hr origins have been reported in AcMNPV, OpMNPV and
SeMNPV (Broer et al., 1998; Kool et al., 1994; Pearson et al., 1993). These regions contain unique
palindromic and repeat sequences that are not found in hr sequences and are relatively complex in
organisation, often involving multiple domains that collectively are necessary for maximal replication
efficiency.
Transient DNA replication assays have identified that 5 genes (p143, ie-i, lef-i, lef-2 and
lef-3) are essential for DNA replication and five are stimulatory genes (dnapol, p35, ie-2, lef-7)

97
(Lu and Miller, 1995; Kool et al. , 1994).pI43 gene encodes a predicted protein of143 kDa that
contains anumber ofmotifs characteristic ofhelicase including NlP-binding and DNA-RNA unwinding
motifs. P 143 has been shown to possess a both double-stranded and single-stranded DNA binding
(Laufs et al., 1997; McDougal and Guarino, 2001). In vitro experiments also show P143 has the
ATPase activity and the ability to unwinding a 40-mer annealed to ssM13 DNA strongly supporting
P 143 is DNA helicase (McDougal and Guarino, 2000, 2001; Bideshi and Federici, 2000). The
requirement for ie-J in baculovirus DNA replication may result from its function in activating the
expression of early genes, some of which are required for viral DNA replication; however, a direct
role in origin, binding and initiation ofthe early steps leading to the assemble ofa replication complex
is also possible. Alignment ofLEF-1 sequences from baculovirus identified four conserved domains,
of which three are also conserved in the DNA primase of several organisms, suggesting that LEF-l
may be a baculovirus primase. Yeast two-hybrid and glutathione S transferase interaction assays
both indicate that LEF -1 interacts with LEF -2, suggesting that these proteins may form a hetero-
oligomeric complex involved in replication (Evans et aI., 1997). LEF-3 might function as a single-
stranded DNA binding protein (Hang et aI., 1995). The presence of conserved amino acid motifs
found in all the replicative proteins strongly suggests that DNAPOL, P 143 and LEF-1 function within
the replisome complex as a DNA polymase, a helicase, and a primase, respectively. Since LEF-3
cooperatively binds to single-stranded DNA, its role in DNA replication may be to bind to single-
stranded DNA formed at the replication fork by the unwinding of parental duplex DNA by P 143,
while LEF-2 might function as a primase-associated protein.
Analysis ofhr-containing replicated plasmid NDA by partial digestion with restriction enzyme
that cuts the input plasmid at a single site resulted in DNA fragments that were multiples ofunit length,
suggesting that DNA was present as a concatemer containing a number of copies of the plasmid
(Leisy and Rohrmann, 1993, Xie et aI., 1995). Such a structure could be indicative of rolling circle-
type replication. In addition, defective genomes also appeared to consist of concatemers of sequences
from the HindIlI K region (Lee and Krell, 1992, 1994). The identification of viral replicati0n
intermediates and the mechanism by which a concatemeric structure could be resolved into unit
length, circular genome segments has not been report.

7. GENETIC ENGINEERING OF BACULOVIRUSES FOR PEST CONTROL

Baculoviruses are host specific for insects and can cause epizootic in nature which appear to
playa role in controlling insect populations. These viruses are, therefore, attractive biological control
agents and offer an alternative to wide-spectrum chemical insecticides (F AOIWHO Report, 1973).
Baculoviruses have been successfully used in most continents in the control of a variety of pest
insects, including the cotton bollworm, velvet bean caterpillar, tea moth, codling moth, pine beauty
moth, Douglas fir tussock moth, beet armyworm and fall army worm. To date, a number of them
have been registered as biological control agents in various countries. A few have been commercialized,
such as the MNPV of the beet army worm S. exigua (SpodexR, BioSys, USA) on cotton in the
USA; on shallot, garden pea, grape and Chinese kale in Thailand, and on flowers and ornamentals in
the Netherlands (Smits and Vlak, 1994; Kolodny-Hirsh and Dimmock, 1996). Others are produced
through govemment sponsored agencies, such as GypcheckR against L. dispar in the USA. In Brazil,
1,000,000 hectares of soybean are treated annually against the velvetbean caterpillar, Anticarsia
gemmatalis (Moscardi and Sosa-Gomez, 1992). In China about 100,000 hectares of cotton and
hot pepper are treated annually with the SNPV from H armigera (Zhang, 1989; Zhang, 1994).
Baculoviruses are also well suited for use in Integrated Pest Management strategies because of their
compatibility with other control agents, chemical or biological. They have a proven safety track
record with minimal effects on non-target insects, such as honeybees. They do not appear to present
safety or health hazards to humans and other vertebrates as encountered with chemical insecticides
and, lastly, they do not invoke resistance in insects (Persley, 1996).

98
 A major limitation to wider use ofbaculoviruses in insect control is theirrelative slow speed of
action, particularly in crops with low damage thresholds such as flowers and fruits. Normally, it takes
a few days for the virus to induce feeding inhibition in larvae when a more immediate insecticidal
effect is often desired. An other drawback is their relatively low virulence for the older instars which
cause the majority of damage in crops. Finally, baculoviruses are often too specific to meet the
commercial demands for broad spectrum activity.
Through genetic engineering the improved insecticidal properties, such as increased speed of
action, enhanced virulence and extended host range, might be achieved. Different strategies have
been tried to improve the baculovirus properties, mainly focused on increasing the speed of action.
These approaches include expressing physiological effectors (hormones, enzymes or antisense)
interrupting the normal metabolism of the insect, expressing insect specific toxins such as neurotoxin
and Bt, or deleting baculoviral genes that may interact with the reaction of the virus to the insect.
However, over- expression of insect genes that normally regulate key aspects of insect physiology or
development, such as diuretic (Maeda, 1989), prothoracicotropic (O'Reilly et aI., 1995) and eclosion
hormone (Eldridge et aI., 1992) and juvenile hormone esterase (JHE) (Hammock et aI., 1990) did
not significantly increase the baculovirus speed of action nor reduce the food consumption of the
infected insects. Insertion ofthe insect-specific neurotoxins holds the greatest promise for delivering
a commercially viable baculovirus insecticide (0 'Reilly and Miller, 1991; Stewart et a1, 1991; Tomalski
and Miller, 1991; Prikhod'ko etal., 1996; Popham et aI., 1997; Gershburg et aI., 1998).

Table 4. Physiological effectors that have been used for improving baculoviral pesticidal properties.
Viruses Efficac~
Genes Origin wtire Reduction in Host Reference
ST,o tested
Diuretic Tobacco 2nd_5 th Maeda, 1989;
hormone homworm BMNPVI 20% B.mori Maedaetal,
(DH) Manduca BmDH5 (injection) 1991
sexta
Juvenile
hormone Heliothis AcMNPVI feeding and
esterase virescens AcRP23.JHE growth neonate Hammock et aI.,
(JHE) inhibition Tni 1999
AcMNPVI 2nd instar
JHE H AcUW2(B). slight longer H McCutchen
virescens JHE virescens et aI., 1991
AcMNPVI 24% for
JHEI H vEGTDEU vJHEEGTD; neonate Eldridge
EGT virescens vJHEEGTZ 20% for T ni etal.,1992
deletion vJHEEGTD vEGTDEL
AcMNPV
JHE-KK H pJO-JHE-KK slightly longer neonate Jarvis et aI., 1996
virescens ieJ-JHE-KK H. virescens
Anti sense H aberrant 5th instar H. Hajos et a!.,
JHE virescens AcMNPV morphogenesis virescens 1999
Prothoracci-
cotropic B. mori AcMNPVI Increase LC,. S. Jrugiperda O'Reilly et a!.,
hormone vEGTDEL 1995
(PITH)
Pheromone
biosynthesis AcMNPVI neonate and Maetal.,1998
activating H. zea AcBX-PBA 26% 3,d instar

neuropeptide N4 19% Tni

(PBAN)

99
 Baculovirus encodes an EOT that specifically inactivates ecdysteroid honnone by conjugating
with glucose or galactose. Previous results ofdifferent baculoviruses indicated that deleting egt from
the genomes could increase the viral killing speed up to 30010 and cause a significant reduction in the
amount offood consumed (O'Reilly and Miller, 1991; Siavicek et aI., 1999; Chen et aI., 2000).
Expression of insect-selective toxins in the baculovirus system has proved to be highly successful for
increasing virus efficacy in insect-pest control. The insect-selective toxin derived from the scorpion
Buthus eupeus (BelT) was first introduced into AcMNPV (Carbonell et aI., 1988). However, for
unknown reasons, this toxin did notincrease the efficacy ofthe recombinant virus. Also expression of
the toxins from Bacillus thuringiensis did not improve baculovirus pesticidal properties (Martens et
at, 1990; Merryweather et aI., 1990). Expression of the insect-specific toxin AalT from the North
Africa scorpion Androc/onus australis in baculovirus produced the most positive results (Stewart et
aI., 1991; McCutchenet aI., 1991; Chen et ai., 2001). This toxin acts on the neuronal sodium
channel, causing presynaptic excitatory effects. Larvae infected with these recombinant baculoviruses
exhibited dorsal arching and increased irritability and ceased feeding. Lethal times are reduced by
25-40% compared with those of the wild-type virus, and the feeding damage by larvae infected with
the recombinant virusreduced by 50% compared with damage caused by larvae infected with wild-
type viruses. The other toxins which also enhanced recombinant baculovirus insecticidal efficacy
include TxP 1 from the straw itch mite, Pyemotes tririei (Tomaski and Miller, 1991, 1992), LqhITl
and LqhIT2, scorpion depressant toxin from scorpion Leiurus qulnquestriatus hebreus (Oershburg
et al., 1998).
The effectiveness ofthe genetically modified baculoviruses has been tested in the field and has
shown their potential to enhance crop protection. In 1994, Cory and the co-workers reported the
first field trial of a genetically improved AcMNPV that expresses AaIT. When artificially infested
cabbage loopers, T ni, were treated with recombinant virus and the wild-type at different doses, at
11 days after treatment, peak mortality had occurred in the recombinant treatment, but the peak
mortality had not occurred in the wild-type treatment until 16 days after treatment. Insect infected
with recombinant virus consumed 23- 29% less than the equivalent wild-type treatment within 16
days depending on the doses that were provided (Cory et aI., 1994).
Greenhouse studies conducted against H virescens on cotton showed that hastened virulence
exhibited by an egt deletion variant of AcMPNV (vEOTDEL) led to improved plant protection
versus wild-type AcMPNV. For example, following 5 weekly sessions offoliar application and H
virescens artificial infestation, cotton treated with wettable powder ofvEOTDEL and the wild-type
at 2.5 x 10 12 PIBslha averaged 25.7 and 61.8% damaged flower buds, respectively. However, in a
cotton field situation the differences in efficacy between vEOTDEL and the wild-type virus against T
ni were marginal and not statistically significant (Treacy et aI., 1997). In contrast to egt deletion
variant ofAcMNPV, AaIT insertion recombinants have notably increased field efficacy and resulted
in considerably reduced crop damage (Black et aI., 1997). Thus the recombinant baculovirus holds
possible commercial potential.

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insects, Arch. Insect Biochem. Physiol. 22:303-313.
Treacy, M.F., All, J.N., and Ghidiu, G.M., 1997, Effect of ecdysteriod UDP-glucosyltransfease gene deletion on
efficacy ofa baculovirus against Heliothis virescens and Trichoplusia ni (Lepidoptera: Noctuidae), JEcon.
Entomot.90:1207-1214.
Van Meer, M.M.M., Bonning, B.C., Ward, V.K., Vlak, 1.M., and Hammock, B.D., 2000, Recombinant, catalytically
inactive juvenile hormone esterase enhances efficacy ofbaculovirus insecticides, BioI. Controtl9: 191-199.
Van Oers, M.M., and Vlak, J.M., 1997, The baculovirus lO-kDa protein,Jlnvertebr. Pathol. 70: 1-17.
Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, J.,
Mayo, M.A., McGeoch, OJ., Pringle, c.R., and Wickner, R.B., 2000, Virus Taxonomy, Academic Press, San
Diego, USA.
Vialard. J.E., and Richardson, C.D., 1993, The 1,629-nucleotide open reading frame located downstream of the
Autographa californica nuclear polyhedrosis virus polyhedrin gene encods a nucleocapsid-associated
phosphoprotein, Journal ofVirol. 67:5859-5866.
Watkins, Kiyatkin N., Hughes, D., Beadle, D., and King, L., 1997, Study on the biological properties ofa novel
recombinant baculovirus, in : Microbial Insecticides: Novelty or Necessity? BCPC Symposium Proceedings
68:279-284.
Whitford, M., Stewart, S., Kuzio, 1., and Faulkner, P., 1989, Identification and sequence analysis of a gene encoding
gp67 an abundant envelope glycoprotein of the baculovirus, Autographa californica nuclear polyhedrosis
virus,} Virol. 63:1393-1399.
Whitford, M., and Faulkner, P., I 992a, Nucleotide sequence and transcriptional analysis of a gene encoding gp41,
a structural glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus, J Virol.
66:4763-4768.
Whitford, M., and Faulkner, P., I 992b, Structural polypeptide of the baculovirus Autographa californica nuclear
polyhedrosis virus contains O-linkedN-acetylglucosamine,J Virol. 66:3324-3329.
Wilson, M.E., Mainprize,T.H., Friesen, P.O., and Miller, L.K., 1987, Location, transcription, and sequence ofa
baculovirus gene encoding a small arginine-rich polypeptide, J Virol. 61 :661-666.
Xie, W.O., Arif, B.M., Dobos, P., and Prell, P.J., 1995, Identification and analysis ofa putative origin of DNA
replication in the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus genome, Virology
209:409-419.
Zanotto, P.M., Kessing, B.D., and Maruniak, J.E., 1993, Phylogenetic interrelationship among baculoviruses:
Evolutionary rates and host associations, JInvertebr. Pathol. 62: 147-162.
Zemskov, E.A., Kang, W., and Maeda, S., 2000, Evidence for nuclear binding ability and nucleosome association
of Bombyx mori nucleopolyhedrovirus BRO proteins,} ViroI74:6784-6789.
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Environment in the Yangtze Val/ey3:1-6.

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 GENETIC ENHANCEMENT OF BACULOVIRUS INSECTICIDES

Bryony C. Bonning, Anthony J. Boughton, Hailing Jin, and Robert L. Harrison

Department of Entomology and Program in Genetics, Iowa State University, Ames, IA, USA

1. INTRODUCTION

Baculoviruses are arthropod-specific viruses that infect species mainly within the order
Lepidoptera (Adams and McClintock, 1991). The two genera nucleopolyhedrovirus (NPV) and
granulovirus (GV), within the family Baculoviridae, are identified by occlusion body morphology
with single (GV) and multiple (NPV) virions occluded in granules or polyhedra respectively.
Baculoviruses are regarded as safe and selective insecticides, but although they have been used
successfully for management ofa number ofagricultural and forestry pests (Federici, 1999; Moscardi,
1999), their use as microbial pesticides has not met their potential. Two of the main reasons for their
limited use are the slow speed of kill of the targeted pest relative to classical chemical insecticides,
and a narrow host range that may exclude many of the pest species found on a given crop. Genetic
engineering has been used successfully to improve the speed ofkill ofbaculovirus insecticides (Harrison
and Bonning, 2000a; van Beek and Hughes, 1998). The baculovirus Autographa californica
multicapsid nucleopolyhedrovirus (AcMNPV) optimized by genetic engineering for improved
insecticidal efficacy has shown performance under field conditions comparable to that of classical
chemical insecticides (Black et al., 1997; Treacy, 1997; Treacy et al., 2000; DuPont, 1996).

In this chapter, we outline genetic modifications made to baculoviruses to enhance insecticidal

efficacy, and review progress toward increasing host range and virulence. We also discuss the role
baculovirus genomics may play for further improvement ofbaculovirus insecticides, and review risk
assessment studies ofrecombinant baculovirus insecticides.

2. NUCLEOPOLYHEDROVIRUS LIFE CYCLE

Baculoviruses possess a double-stranded circular DNA genome which is contained within an

enveloped, rod-shaped virion. VIrions are enclosed within a protective crystalline matrix consisting of
the viral protein polyhedrin to form polyhedra. On ingestion of polyhedra by a host insect, the
polyhedrin matrix dissolves in the alkaline conditions ofthe insect midgut and the infectious virions are
released. These occlusion-derived viruses cross the peritrophic membrane lining the gut, and initiate
infection by binding and fusing with columnar cells of the host (Horton and Burand, 1993). DNA

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002 109
uncoating and virus replication takes place in the nucleus. The infection is disseminated throughout
the host insect by a second viral phenotype called budded, or non-occluded virus. Polyhedra are
produced late in infection and are released from the dead insect. A single cadava may contain 10 10
polyhedra (Entwistle and Evans, 1985).lnfected larvae exhibit negative geotropism before succumbing
to the virus infection, thereby facilitating widespread dissemination ofpolyhedra. Polyhedra can survive
outside the host in soil, in crevices of plants or other refugia for years (Jaques, 1975), and are
ubiquitous.

Figure J. Life cycle of the nucleopolyhedrovirus. Polyhedra ingested by a lepidopteran larva dissolve in the
alkaline environment of the midgut, releasing infectious virions. Virions infect midgut cells and virus replication
ensues in the nucleus. Initially, budded or non-occluded virus is produced, which disseminates the virus within
the insect host. Subsequently, pre-occluded virions become embedded in the polyhedrin matrix and polyhedra are
formed. Figure reproduced with permission from Biotechnology and Genetic Engineering Reviews, Volume 10,
1992 (Figure I, page 458) ISSN 0264-8725 © Intercept Limited, PO Box 716 Andover, Hampshire SPIO lYG, UK.

3. GENETIC MODIFICATION TO REDUCE THE SURVIVAL TIME OF

BACULOVIRUS-INFECTED LARVAE

Several baculoviruses in addition to AcMNPV have been engineered for improved insecticidal
performance to reduce the amount of time taken by the virus to kill the host insect (Table 1). These
include Rachiplusia nu (Anagrapha falcifera) MNPV (RoMNPV)(Harrison and Bonning, 2000b),
and Helicoverpa zea SNPV (HzSNPV) (Popham et ai., 1997; Treacy et ai., 2000), in addition to

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Bombyx mori NPV (BmNPV) that has been used as a model system (Maeda, 1989; Maeda et al.,
1991 ). Most of the strategies employed to increase the speed of action ofbaculovirus insecticides
have involved insertion ofa transgene into the baculovirus genome. On replication ofthe baculovirus
within the cells ofthe infected host, the product of the transgene, usually a toxin or physiological
effector, is expressed along with the baculovirus proteins. Expression ofthese agents results in paralysis,
or in disruption of homeostasis ofthe host insect. Hence the baculovirus serves as a delivery system
for the toxic agent, and death results from expression of the toxin rather than from the baculovirus
infection. The agents expressed by recombinant baculovirus insecticides fall into two main categories
(Table I): neurotoxins that target the insect nervous system, or physiological effectors that disrupt a
physiological process such as water balance, or hormonal regulation of development. Most of these
genetic alterations have been discussed elsewhere (Harrison and Bonning, 2000a; van Beek and
Hughes, 1998). Of the recombinant baculoviruses developed to date, the viruses expressing insect-
selective toxins (Elazar et al., 200 I; Zlotkin et al., 2000), and the protease cathepsin L (Harrison and
Bonning, 200 I), have the greatest potential for insect pest controI.lndeed, the insecticidal potential
ofHzSNPV and AcMNPV expressing the toxin LqhIT2 was assessed for commercial viability for
control of the cotton bollworm Helicoverpa zea Boddie by DuPont Agricultural Products (DuPont,
1996; Smith et al., 2000a), while AcMNPV expressing the toxin AalT was assessed by American
Cyanamid Company (Black et aI., 1997; Gard, 1997). These insect-selective toxins are derived
from the venom of the scorpions Leiurus quinquestriatus hebraeus Hemprich & Ehrenberg, and
Androctonus australis Hector, respectively. Both of these corporate programs that were established
to develop recombinant baculovirus insecticides for commercialization have been discontinued, because
of internal changing priorities and competing technologies.

3.1 The Basement Membrane as a Novel Target

Based on evidence that secondary, systemic infection oftissues by budded virus is restricted by
the basement membrane, baculovirus expression of enzymes that degrade specific components of
the basement membrane was recently tested as a novel strategy to facilitate the spread of virus
infection (Harrison and Bonning, 200 I). Basement membranes are extracellular sheets of proteins
that surround the tissues ofall animals, providing structural support, a filtration function, and a surface
for cell attachment, migration and differentiation. Engelhard et al. (1994) observed that viral infection
of host tissues distant from the midgut was mediated by viral replication and movement through the
epidermal cells ofthe tracheal system. The tracheoblasts that penetrate basement membrane provide
a means by which baculoviruses can bypass this barrier. Three proteases that degrade basement
membrane components were tested for their ability to improve the insecticidal performance of
AcMNPV against a host insect (Harrison and Bonning, 200 I). Expression of cathepsin L derived
from the flesh fly resulted in a significant decrease in survival time oflarvae infected with the recombinant
baculov.irus relative to larvae infected with the wild type virus (Harrison and Bonning, 2001). The
reduction in feeding damage caused by larvae infected with the cathepsin L -expressing baculovirus
was similar to that caused by larvae infected with a recombinant baculovirus expressing LqhIT2.

4. GENETIC MODIFICATION TO ENHANCE VIRULENCE AND HOST RANGE

The "virulence" of a microorganism is defined as the relative capacity of that microorganism to

overcome the body defenses of the host, or as the degree of pathogenicity within a group or species
(Poulin and Combes, 1999; Steinhaus and Martignoni, 1967). The time taken by a virus to kill the
host insect generally decreases with increasing quantity of virus delivered to the host (Entwistle and
Evans, 1985; van Beek et aI., 1988), or with increasing virulence. The host range of a virus is
determined by its ability to enter the cells of a host organism, replicate and produce new infectious
virus. Virulence and host range are intrinsically linked. Enhanced virulence i.e. reduced LD50' against
insects that are semi-permissive to baculovirus infection will effectively expand the host range ofthe
virus under field conditions.

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Table 1. Genetic modifications used for enhancement ofbaculovirus insecticides

TransgenelAlteration Parental virus Reference

Toxins
AaIT (scorpion) AcMNPV McCutchen etal., 1991; Stewart et aI., 1991; Jarvis et
aI., 1996; Kunimi et aI., 1996; Kunimi et aI., 1997;
Hernandez-Crespo et aI., 1999; Treacy et aI., 2000;
Harrison and Bonning, 2000b
BmNPV Maedaetal.,1991
RoMNPV Harrison and Bonning, 2000b
HzSNPV Treacy et aI., 2000
LqhlTl (scorpion) AcMNPV Gershburg et aI., 1998
LqhIT2 (scorpion) AcMNPV Gershburg et aI., 1998; Harrison and Bonning, 2000b

RoMNPV Harrison and Bonning, 2000b

tox34 (TxP-I; mite) AcMNPV Tomalski & Miller, 1991; Tomalski and Miller, 1992;
Lu etal., 1996; Burden etal., 2000
HzSNPV Popham et al., 1997
Dol m V (A and B forms; hornet) AcMNPV Tomalski et aI., 1993
mag4 (Il-Aga-1V; spider) AcMNPV Prikhod'ko et aI., 1996
sat2 (As II; sea anemone) AcMNPV Prikhod'ko et aI., 1996
ssh \ (Sh I; sea anemone) AcMNPV Prikhod'ko et aI., 1996
DTX9.2 (spider) AcMNPV Hughes et aI., 1997
TalTX-\ (spider) AcMNPV Hughes et aI., 1997
Bt subsp. kurstaki HD-73 0 endotoxin AcMNPV Merryweather et aI., 1990
BtCryIA(b) AcMNPV Martens et aI., 1995
T-urfl3 (URF\3; maize) AcMNPV Korth & Levings, 1993

Physiological effectors
Juvenile hormone esterase (wild-type) AcMNPV Hammock et aI., 1990, Bonning et aI., 1992; Eldridge et
al.,1992a
Juvenile hormone esterase (mutated) AcMNPV Bonning et aI., 1995; Kunimi et aI., 1996; Jarvis et aI.,
1996; Kunimi eta!., 1997; Bonning eta!., 1997; Bonning
etal.,1999
Deletion of viral ecdysteroid UDP AcMNPV O'Reilly & Miller, 1991; Eldridge et aI.,
glucosyltransferase 1992a;O'Reillyetal., 1995; Treacy et aI., 1997
Diuretic hormone BmNPV Maeda, 1989
Ec1osion hormone AcMNPV Eldridge et aI., 1992b
Prothoracicotropic hormone AcMNPV O'Reilly et aI., 1995
Pheromone biosynthesis activating AcMNPV Ma et a!., 1998
neuropeptide (PBAN)

Proteases
Cathepsin L (flesh fly) AcMNPV Harrison and Bonning, 200 I
"Activated" stromelysin-I (rat) AcMNPV Harrison and Bonning, 200 I
Gelatinase A (human) AcMNPV Harrison and Bonning, 200 I

Miscellaneous
Chitinase (tobacco homworm) AcMNPV Gopalakrishnan et aI., 1995
Trehalase (mealworm beetle) AcMNPV Sato et aI., 1997
Antisense fragment of c-myc gene (Human) AcMNPV Lee et aI., 1997

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 In general, nucleopolyhedroviruses infect only members of the genus, or in some cases the
family ofthe host insect species from which they were originally isolated. There are a few exceptions
to this rule however, including AcMNPV (payne, 1986), RoMNPV (AnagraphaJalciJera MNPV)
(Harrison and Bonning, 1999; Hostetter and Puttler, 1991), and Mamestra brassicae MNPV
(MbMNPV)(Doyle et aI., 1990). These viruses are known to infect 30 or more species of
Lepidoptera. A baculovirus with a broad host range is optimal for use in agricultural pest management
for suppression ofmultiple pests affecting a single cropping system. Forty-three species of Lepidoptera
within 11 families are reported to be susceptible to AcMNPV (payne, 1986). Although AcMNPV is
highly infectious for several major crop pests including the tobacco budworm Heliothis virescens
(Fabricius), the beet armyworm Spodoptera exigua (Hubner) and the cabbage looper Trichoplusia
ni (Hubner), the susceptibility of other major cotton or vegetable pests such as the Egyptian cotton
leafurorm Spodoptera littoralis (Boisduval), H zea, the pink bollworm Pectinophora gossypiella
(Saunders) and the black cutworm Agrotis ipsilon (Hufnagel), is so much lower that doses of virus
would have to be increased significantly to provide adequate control.
There are numerous examples of selection ofbaculoviruses for increased virulence against specific
pest species. For example, five passages of an HzSNPV isolate through the species from which it
was originally obtained resulted in an 80% increase in virulence, which was stable for a further 11
passages (Shapiro and Ignoffo, 1970). Seven passages of Orgyia pseudotsugata MNPV
(OpMNPV) in T ni resulted in adaptation of this virus for T ni (Martignoni and Iwai, 1986). The
first five passages resulted in localized infection and the seventh passage resulted in systemic infection
ofT ni larvae. Repeated passage ofChoristoneuraJumiJerana MNPV (CfMNPV) through T ni
and the greater wax moth Galleria mellonella (L. )(Stairs et aI., 1981), and OpMNPV, CfMNPV
and Lymantria dispar MNPV (LdMNPV) through the saltmarsh caterpillar Estigmene acrea (Drury)
increased the virulence of these viruses to these host insects (Shapiro et al., 1982). Twelve to IS-fold
increases in virulence were seen for Helicoverpa armigera MNPV (HaMNPV) passaged through
T ni andS. exigua (Tompkins et al., 1981, 1988) and a IS-fold increase was seen on passage of
AcMNPV through the diamond back moth Plutella xylostella (L.) (Kolodny-Hirsch and Beek,
1997).
Despite numerous examples of selection being used to enhance baculovirus virulence, in no
case were the physiological or genetic bases for enhanced virulence ofthe selected viruses determined.
Hence, information resulting from such studies that might have been useful for genetic enhancement of
baculovirus virulence is unavailable.
There is one example ofa genetically engineered baculovirus that was shown to have enhanced
virulence. Polyhedra are surrounded by an electron-dense layer called the polyhedral envelope. This
envelope prevents loss of virions from the polyhedrin matrix and prevents aggregation of polyhedra
(Gross, 1994; Oers and Vlak, 1997; Whitt and Manning, 1988). Disruption of the gene encoding the
polyhedral envelope protein (PEP) in AcMNPV resulted in increased sensitivity to weak alkali
compared to the wild type virus and virions were more readily released from polyhedra (Zuidema et
aI., 1989). A mutant virus produced by disruption of the pep gene (Zuidema et al., 1989) was six-
fold more virulent to first instar T ni (Ignoffo et al., 1995). However, disruption ofpep in Lymantria
dispar MNPV (LdMNPV) did not cause any significant difference in virulence against the gypsy
moth, L. dispar (Bischoff and Slavicek, 1999). Removal of the polyhedral envelope may facilitate
dissolution ofpolyhedra at sub-optimal midgut pH, thereby enhancing virulence against semi-permissive
insect species. However, potential loss ofvirions from the polyhedrin matrix may negate any advantage
that pep disruption may confer.

4.1 Barriers to Baculovirus Infection

There are several potential barriers to infection of an insect host by a baculovirus that may affect the
host range or virulence of a virus against a particular host insect species. These barriers include:
midgut pH that may be unsuitable for dissolution ofthe polyhedrin matrix; penetration ofthe peritrophic

113
membrane; the ability of host cells to support virus replication; recognition of the virus and inactivation
by the immune system of the host insect. As more is learned of these processes, manipulation ofthe
virus genome to optimize the host range under field conditions becomes an increasingly viable prospect.

4.1.1 The Midgut pH as a Barrier to Infection. The midgut pH is an important parameter in

determining virulence. The extreme alkalinity within the midgut lumen of some lepidopteran larvae is
thought to protect larvae from toxic tannins in the diet (Berenbaum, 1980), but also facilitates baculovirus
infection. Polyhedra require a highly alkaline environment of pH 9.5 to 11.5 for dissolution (Granados,
1980; Horton and Burand, 1993). Species with lower midgut pH are likely to be less susceptible to
baculovirus infection.

4.1.2. The Peritrophic Membrane. The peritrophic membrane is an extracellular fibrous matrix
consisting of chitin, glycosaminoglycans and protein, which surrounds food in the midgut and may
present a barrier to baculovirus infection of midgut cells (Brandt et aI., 1978; Richards and Richards,
1977; Tellam, 1996; Volkman, 1997). Application ofStreptomyces griseus chitinase to the peritrophic
membrane caused roughening of the surface, and production of numerous holes (Brandt et al., 1978).
Inclusion of an unspecified bacterial chitinase in a formulation ofLdMNPV resulted in a five-fold
decrease in the LC so for L. dispar (Shapiro et aI., 1987). A metalloprotease, called enhancin, that
has been isolated from the occlusion bodies of Trichoplusia ni granulovirus (TnGV) (Derksen and
Granados, 1988; Gijzen et aI., 1995) facilitates infection of AcMNPV in several noctuid species by
degrading mucin, which is a component ofthe peritrophic membrane (Gallo et aI., 1991; Lepore et
al.. 1996; Wang and Granados, 1997; 1998; Wang et aI., 1994). Several enhancin genes have
been cloned and sequenced, from TnGV(Hashimoto et aI., 1991), Helicoverpa armigera GV,
Pseudoletia unipuncta GV (Roelvink et aI., 1995) and from LdMNPV (Bischoff and Slavicek,
1997). Disruption of two enhancin genes in the LdMNPV genome resulted in a 12-fold loss of
virulence of this virus (Popham et aI., 2001). Expression and localization of chitinase or enhancin in
the nucleus ofan infected cell, and subsequent incorporation into the polyhedrin matrix ofa baculovirus
may facilitate penetration of the peritrophic membrane by virions. The enzymes released on virus
dissolution in the midgut, would degrade the peritrophic membrane, making midgut cells more accessible
to virions.

4.1.3 Ability of Host Cells to Support Baculovirus Replication. Lepidopteran cells vary widely
in their ability to support replicatio~ of a particular baculovirus and can therefore have a major impact
on host range. Increased understanding of factors involved with determination of host range will
enhance the likelihood ofdeliberate modification ofhost range by genetic engineering (Thiem, 1997).
There are a number of cases where insertion or alteration of a single gene affects the host range
of the virus. For example, AcMNPV does not normally infect cells or larvae ofthe silkworm Bombyx
mori (L.). Modification ofthe AcMNPV p143 gene, which encodes a DNA helicase homolog,
enabled AcMNPV to infect B. mori (Argaud et aI., 1998; Crozier et aI., 1994; Kamita and Maeda,
1997). TnGV and AcMNPV both replicate in T ni, but the DNA helicase of TnGV could not
substitute for that of AcMNPY. This negative result may be because of inadequate protein-protein or
protein-DNA interactions necessary for a functional replication complex (Bideshi and Federici, 2000).
AcMNPV does not normally infect L. dispar cell lines and larvae, but will when engineered with the
hrf 1 (host range factor 1) gene of LdMNPY. The engineered AcMNPV was less virulent than
LdMNPV against L. dispar however the presence of hrf 1 also resulted in a slight enhancement of
AcMNPV virulence against H zea (Chen et aI., 1998). Hrf-l overcomes an apparent inhibition of
tRNA synthesis in L. dispar cells infected with AcMNPV (Mazzacano et aI., 1999). Efficient
replication of AcMNPV in T ni cell lines is dependent in part on the presence of the gene hc.f-l (host
cell-specific factor 1). This gene is not needed for replication of AcMNPV in as. frugiperda cell line
however (Lu and Miller, 1996). Hcf-l was also necessary for infection of T ni larvae when virus
was injected into the hemocoel, and mutation of hc.f-l resulted in a 20-30% increase in time taken by

114
the virus to kill the host insect when infected orally. The positive effect of hcf-l on virulence of
AcMNPV in T ni larvae would confer a significant competitive advantage to this virus in nature.
Other examples of host range genes include the A~MNPV tef-7 (late expression factor 7), which is
required for efficient infection of as. Jrugiperda and a S. exigua cell line, but not a T ni cell line
(Chen and Thiem, 1997), the baculovirus anti-apoptotic gene p35 that allows AcMNPV to replicate
in a S. Jrugiperda cell line and in larvae, but is not required for replication in T ni cells and larvae
(Clem et al., 1991, 1994; Clem and Miller, 1993), and finally, AcMNPV ORF 603, which is important
for infection of S. Jrugiperda larvae but not for infection of T ni larvae (Gearing and Possee, 1990;
Popham et at., 1998). The fact that AcMNPV has at least one gene that specifically facilitates
replication in T ni larvae and at least one gene that specifically facilitates replication in S. Jrugiperda
larvae suggests that the ability to infect multiple species confers a selective advantage to the virus in
nature.
It is highly likely that multiple genes are involved in determining infectivity ofaparticular virus to
a specific host (Miller and Lu, 1997). None of the host range factors identified thus far have
demonstrated practical application for expansion of host range or enhanced efficacy against semi-
permissive hosts under field conditions. In addition to the importance of the presence or absence of
a particular gene with a demonstrated role in host range, adaptation of other gene sequences to
promote replication in a particular host may also be important.

4.1.4. Baculovirus Inactivation by the Immune System of the Host Insect. The immune
systems of some Lepidoptera are able to recognize and inactivate AcMNPV. Washbum et al. (1996)
reported that the physiological basis of resistance ofH. zea to AcMNPV infection is based on
encapsulation of virus-infected cells. In addition, the immune response of the tobacco homworm
Manduca sexta (L.) appears to contribute to the low susceptibility of this species to AcMNPV
(Washbum et al., 2000). A greater understanding ofbaculovirus-host interaction and determinants of
virus recognition in these cases may allow engineering ofthe virus to circumvent the immune response
of the host insect, thereby increasing the host range ofthe virus. The lack of susceptibility ofH. zea to
AcMNPV is problematic foruse ofthis virus for control ofmultiple heliothine pest species on a given
crop (Treacy et aI., 2000).

5. POTENTIAL ROLE OF VIRUS GENOMICS IN FURTHER ENHANCEMENT OF

BACULOVIRUS INSECTICIDES

Baculoviruses have circular covalently closed, double-stranded DNA genomes from 90 to 180
kb. The genomes of 12 baculoviruses have been sequenced (Table 2). The genomes vary as a result
of deletions, gene insertions, inversions and duplications. The mechanisms underlying these
rearrangements are unclear, although transposable elements appear to have played a role in some
cases (Friesen, 1993; Jehle, 1996; Jehle etal., 1997). The differences in size between the genomes
ofdifferent baculoviruses are mainly due to gene duplications includingthe "baculovirus repeat ORFs"
or bro genes (Gomi et al., 1999; Hayakawa et al., 1999). A number of unique genes also contribute
to variation in genome size, particularly for the granuloviruses. The basic organization of genes in
NPV genomes is remarkably conserved, with rearrangements of genomic segments and presence or
absence ofauxiliary genes contributing to their diversity.
The continued study ofbaculovirus genes and their functions, along with comparative genomics
has the potential to enhance baculovirus-based insecticides. This is particularly true of genes that
control baculovirus host range and virulence. Such genes might be manipulated to augment
characteristics ofbaculoviruses significant to their application as control agents.
A promising approach to identifying such genes was outlined by Dall et al. (2001). In an effort
to find genes that are specifically involved in the relationship between insect viruses and their hosts,
Dall et aI. (2001) compared the gene content ofrepresentatives ofthree families ofviruses containing

115
insect-infecting members (Poxviridae, Baculoviridae, and Iridoviridae) to find genes that (i) were
present in insect-infecting members of the family but not vertebrate-infecting members, and (ii) were
present in members of all three virus families. The authors found six groups of genes, four of which
occur in baculoviruses (Dall et al., 2001). One ofthese gene groups, the fusolinlgp37 group, encodes
proteins that facilitate infection (Hukuhara et al., 1999; Mitsuhashi et al., 1998; Xu and Hukuhara,
1992). Further analysis of the other three groups may reveal functions relating to host range, immune
response evasion, or primary and systemic infection that can be modified for improvement ofbaculovirus
insecticidal efficacy.

Table 2. Baculovirus genomes sequenced

Baculovirus Reference
Autographa cali/ornica MNPV Ayres et aI., 1994
Bombyx moriNPV Gomietal.,1999
Orygia pseudotsugata MNPV Ahrens et aI., 1997
Spodoptera litura MNPV Pang et aI., 2001
Spodoptera exigua MNPV Ijkel et aI., 1999
Lymantria dispar MNPV Kuzioeta!.,1999
Rachiplusia nu MNPV (= Anagrapha Harrison and Bonning, unpublished
falci/era MNPV)
Helicoverpa armigera SNPV Chen eta!., 2001
Culex nigripalpus NPV Afonso et aI., 200 I
Xestia c-nigrum GV Hayakawaeta!.,1999
Plutella xylostella GV Hashimoto et aI., 2000
Cydia pomonella GV Luque et aI., 200 I

6. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

REQUIREMENTS FOR REGISTRATION OF MICROBIAL PEST CONTROL
AGENTS

Owing to their relatively high specificity, the use ofwild type and recombinant baculoviruses will
result in fewer deleterious effects on nontarget organisms in natural systems than the use of broad-
spectrum chemical insecticides. However, to assess the potential risks associated with a new microbial
control agent such as a recombinant baculovirus insecticde, the United States Environmental Protection
Agency (EPA) has established guidelines for analysis of (i) residues from the pest control product, (ii)
mammalian toxicity and pathogenicity, (iii) nontarget organism toxicity and (iv)environmental expression
(United States Environmental Protection Agency, 1996). Protocols have been designed to take into
account that microbial control agents have the potential to replicate and increase in numbers once
introduced into the environment, and potentially to infect and cause disease in other living organisms.
The EPA determines whether additional data are required for registration of genetically modified
microorganisms on a case by case basis. The guidelines use a tier testing scheme that is designed to
ensure that the minimum data are generated for scientifically sound regulatory decisions. Tier I testing
consists of maximum dose single species hazard tests on nontarget organisms. If adverse effects are
detected in Tier I tests, ecological exposure tests are performed (Tier II testing) to quantifY the levels
of the control agent to which the susceptible nontarget organism may be exposed. If Tier II tests
indicate that there may be significant exposure to the control agent, Tier III studies are adopted to
determine a dose-response effect and whether other factors would decrease adverse effects in the
environment. Pathogenic effects occurring at Tier III or beyond would reduce the likelihood of
registration of the control agent. Tier IV tests are designed to address problems that are not resolved
by lower tier testing. Tier I toxicology and nontarget organism toxicity tests are conducted in preparation
for registration of a new microbial pest control product.

116
 The purpose of nontarget organism testing is to assess potential hazard of the control agent to
terrestrial wildlife, aquatic animals, plants, and beneficial insects. Nontarget organisms are exposed
to the control agent for a period ono days where possible, in Tier I tests, or until control mortality
rises above 20%. For testing of nontarget insects, Tier I testing is used to assess the toxicity and
pathogenicity ofthe agent to the honey bee and to three species of predaceous and parasitic insects.
Predaceous and parasitic insects are selected from at least two of the following groups: parasitic
Diptera, parasitic Hymenoptera, predaceous Hemiptera, Coleoptera, Neuroptera and predaceous
mites. The species tested should be of some importance in the ecosystem to be exposed to the
control agent. For virus control agents, species that are known to attack the targeted host, or share
the same ecological habitat should be selected. If data generated from Tier I testing indicate no
adverse effects, no further testing would normally be required.
Before granting permission for the small-scale field trials «1 0 acres) conducted by American
Cyanamid Company and DuPont Agricultural Products (Black et aI., 1997; DuPont, 1996; Gard,
1997), the EPA required data on the effects of the recombinant viruses on nontarget insects, and on
environmental fitness, soil persistence, and host range of the recombinant baculovirus. For large-
scale field trials (10 - 5000 acres), an experimental use permit would have been required, necessitating
more extensive toxicological and ecotoxicological studies. Extensive testing of wild type baculoviruses
has shown that they are not toxic, pathogenic or allergenic to mammals, birds, or fish (Groner, 1990).
Twenty-six different baculoviruses have been tested against 10 different mammalian species including
humans (Doller, 1985). An important considemtion is that large numbers ofbaculoviruses are already
present on vegetables in the human diet (Heimpel et aI., 1973), and this continuous exposure without
ill effect underscores their safety. The insertion ofa gene encoding an insect-selective toxin, hormone
or enzyme will not change the safety ofthe virus to nontarget vertebmtes. Toxicity tests ofa baculovirus
expressing AaIT have been conducted on rats and guinea pigs, with no adverse effects detected in
the test animals (Possee et aI., 1993).

7. RISK ASSESSMENT OF RECOMBINANT BACULOVIRUS INSECTICIDES

A theoretical framework has been proposed which can be used to assess the risks posed to
nontarget organisms in natural systems by the use ofbaculovirus insecticides (Richards et aI., 1998)
and requires considemtion oftwo separate issues: impact evaluation and exposure identification. For
impact evaluation the direct and indirect effects on organisms in the environment arising from contact
with baculoviruses are determined. For exposure identification the ways in which organisms in the
environment might come into contact with baculoviruses are assessed. This framework recognizes
that for there to be a risk posed to organisms in the environment from the use ofbaculoviruses, there
must.be both a negative impact on susceptible organisms and also some likelihood of exposure of
susceptible organisms to the baculovirus. For impact evaluation, the potential for detrimental effects
on nontarget organisms arising from the use ofbaculoviruses is assessed. The nontarget species that
have typically been examined play beneficial roles within agricultural systems, suchas insect predators,
parasitoids and pollinators. Pollinators, predators and parasitoids are not susceptible to infection by
baculoviruses. However the possibility exists that insect predators and parasitoids that come into
contact with lepidopteran larvae infected with recombinant baculoviruses, may suffer adverse effects
as a consequence of contact with the foreign protein expressed by the virus.
Although a study on the hemipteran predator Nabis roseipennis Reuter (Ruberson et aI., 1991)
documented adverse effects arising from consumption of prey infected with wild type baculoviruses
relative to consumption ofuninfected prey, the vast majority of studies on insect predators have failed
to document any adverse effects on predator life history traits arising from consumption of wild type
baculovirus-infected prey (Young and Hamm, I 985a, b; Vinson, 1990). No detrimental effects on
insect predators arising from consumption of prey infected with recombinant baculoviruses have
been detected relative to consumption ofprey infected with wild type baculoviruses. Consumption of

117
H virescens larvae infected with AcMNPV AaIT had no detrimental effects on adults ofthe insidious
flower bug, Orius insidiosus (Say), or larvae of the green lacewing, Chrysoperla carnea Stephens,
relative to consumption ofwild type virus-infected larvae (Heinz et al., 1995). In addition, injection of
the recombinant virus into honey bees, Apis mellifera L. failed to cause adverse effects (Heinz et al.,
1995). Studies with five recombinant AcMNPV and HzSNPV viruses expressing AalT or LqhlT2
failed to show any adverse effects on the adults of three generalist predators (Li et ai., 1999).
H virescens larvae infected with these recombinant baculoviruses, were fed to red imported fire
ants, Solenopsis invicta Buren, big-eyed bugs, Geocoris punctipes (Say), and convergent ladybird
beetles, Hippodamia convergens (Guerin-Meneville), but no impacts on survival, fecundity, travel
speed, or rate of food consumption were detected relative to predators fed H virescens infected
with wild type virus. Field trials in cotton, in which predator densities were investigated following
applications of AcMNPV AalT and HzSNPV AalT to control H virescens and H zea, failed to
detect any differences in predator densities or diversity between recombinant and wild type virus
treatments (Smith et ai., 2000a). Studies with two recombinant AcMNPV viruses, one expressing
AalT and the other expressing a mutated juvenile hormone esterase failed to show any adverse
effects on a generalist predator and two species of scavenging insects (Lee and Fuxa, 2000). T ni
larvae infected with these recombinant baculoviruses, were fed to the predatory spined soldier bug
Podisus maculiventris (Say), the flesh fly Sarcophaga bullata (Parker), and the house cricket
Acheta domesticus (L.), but no significant impacts on survival were detected relative to insects fed
T ni infected with wild type virus.
The social wasp, Polistes metricus Say, is a generalist predator in many ecosystems. When
P metricus adults were fed S. Jrugiperda larvae infected with recombinant baculoviruses expressing
the mite neurotoxin TxP-l or the scorpion toxin AalT, no detrimental effects on wasp life history
traits were seen (McNitt et al., 1995). When fed S.Jrugiperda larvae infected with a baculovirus
expressing a marker enzyme under control ofthe HSP70 promoter, the marker enzyme was detected
inP metricus. The HSP70 promoter is a constitutative promoter and is therefore active in cells that
are non-permissive to virus infection. The marker enzyme was not detected when expression was
driven by a baculovirus very late promoter. Seven recombinant baculoviruses expressing various
reporter genes under control of the baculovirus ETL early promoter or polyhedrin late promoter,
were used to examine baculovirus infection and replication in 23 insect species in 17 families from
eight orders (Huang et ai., 1997). Although reporter gene products from at least one of the viruses
were detected in each of the 10 lepidopteran species examined, no reporter gene products or other
evidence ofbaculovirus replication were detected in any of the 13 non-lepidopteran species examined.
In contrast to the situation with most predators, impact evaluation has shown adverse effects of
wild type baculoviruses on parasitoids parasitizing the same host. These deleterious effects are not
due to infection of parasitoids by baculoviruses, but arise because the parasitoid and virus compete
for host resources. In addition, if a host dies prematurely due to virus infection, so does the developing
parasitoid larva. Whether or not a developing parasitoid can survive baculovirus infection ofthe host
is typically dependent upon the time that elapses between parasitization and virus infection of the
host. Ifparasitization occurs two or three days before infection, the parasitoid larva will often survive.
However if the gap is less, parasitoid survival is low.
There are two reports on the effect of recombinant baculovirus infection of a host on parasitoid
survival. Although development time and adult size ofMicroplitis croceipes (Cresson) were reduced
in wasps parasitizing H virescens larvae infected with AcMNPV AalT or a recombinant virus
expressing modified juvenile hormone esterase, parasitoid survival was not significantly different to
that seen in hosts infected with wild type virus (McCutchen et al., 1996). Survival, adult size and sex
were recorded for M croceipes parasitizing H virescens larvae that were exposed at two or four
days post-parasitization to field application rates of AcMNPV LqhIT2 and HzSNPV LqhIT2 or
wild type virus controls (Smith et al., 2000b). No differences were seen in emergence and sex ratios
between paras ito ids among recombinant, wild type and control treatments. Significantly more
parasitoids emerged from hosts parasitized four days before virus infection, but these wasps were
significantly smaller than wasps from the two-day cohort, regardless of treatment.

lIS
 Exposure identification studies have been concerned primarily with aspects ofbaculovirus ecology,
namely replication, stability and dispersal which determine the likelihood of exposure of susceptible
organisms to baculoviruses. Baculoviruses can persist for many years in soil (Jaques, 1975), but they
are rapidly degraded upon exposure to ultraviolet radiation (Hunter-Fujita et a1., 1998). Baculoviruses
are dispersed primarily by abiotic factors such as rain (Entwistle, 1986), but may a1so be dispersed
by biotic factors such as birds and grazing mammals (Entwistle et a1., 1993; Fuxa, 1991), and insect
predators, parasitoids and scavengers (Lee and Fuxa, 2000; Li et aI., 1999; Vasconcelos et ai.,
1996; Vinson, 1990; Young and Yearian, 1989) . Genetic engineering may cause significant changes
in the biological characteristics of recombinant baculoviruses and such changes may influence the
exposure of susceptible organisms to the virus. For example, recombinant baculovirus infection results
in lower production of polyhedra relative to the wild type viruses, typically due to earlier death of the
host (Cory et ai., 1994; Kunimi et ai., 1996). Following infection with recombinant baculoviruses,
lysis of the insect cadavers is delayed relative to lysis after infection with wild type baculoviruses
(Fuxa ct ai., 1998). The lower production of polyhedra combined with delayed lysis of the cadaver,
reduces the spread of virus and likelihood of exposure of nontarget organisms to the recombinant
baculovirus.

8. CONCLUSIONS

Considerable advances have been made in optimizing baculovirus insecticides by genetic means,
particularly with respect to enhancing the speed with which the viruses act on their hosts. Continued
research on the biology of host-virus interaction, determination of the functions ofbaculovirus genes,
and comparison of insect virus genomes, will promote the optimization of virulence and host range.
Given the efficacy of the recombinant baculoviruses that have been developed, corporate programs
are expected to be profitable. Indeed, risk assessment studies with recombinant baculoviruses suggest
that they will provide an environmentally benign alternative to chemical insecticides for insect pest
management.

ACKNOWLEDGEMENTS

We thank Dr. Elliot Krafsur and Dr. John Obrycki for critical reading of this chapter. This
project was funded in part by grants from USDA (97-02936 and 2000-04035). Journal Paper No.
1. - 19672 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project
Nos. 3301 and 6535, and supported by Hatch Act and State oflowa funds.

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BACULOVIRUS GENOMICS: A RESOURCE FOR BIOCONTROL

Vernon K. Ward, Katherine M.B. Sneddon, Otto Hyink, and James Kalrnakoff

Department of Microbiology, School of Medical Sciences, University of Otago, P.O. Box 56,
Dunedin, New Zealand

1. INTRODUCTION

Baculoviruses are the most studied group of invertebrate viruses and are unique among the
invertebrate viruses in how important they have become to many other fields of research. This has
largely come about through their development as one of the premiere eukaryotic protein expression
systems available today (Kost and Condreay, 1999). Despite this wide use ofbaculoviruses, much
of their biology remains to be deciphered. By combining the knowledge being derived from baculovirus
genome analysis with studies into the biology of these viruses and their hosts, many advances are
being made in our understanding of how these viruses work. Baculovirus genome analysis is also
providing the basis for elucidating the roles ofmany baculovirus genes. This is giving new leads into
how baculoviruses can be better utilised or improved, and supplying genes that have the potential to
be used effectively in other pest control strategies.
Baculoviruses are characterised by circular double-stranded DNA genomes that range in
size from 80-180kbp (van Regenmortel et a!., 2000). This genome may carry up to 200 genes, the
majority of which have no proven function. The genome is packaged into rod-shaped nucleocapsids
that are enveloped. A defining criterion ofthe baculoviridae is their biphasic life cycle. Infection of an
insect commences with the ingestion ofpolyhedral inclusions bodies (PIBS) that dissolve in the alkaline
midgut ofa susceptible host. The released virions, occlusion derived virus (ODV), must penetrate the
peritrophic matrix lining the insect midgut, attach to midgut cells, and infect those cells. The virus must
then replicate in the nucleus ofthe infected cell, avoiding any host defences such as apoptosis and/or
sloughing. For most baculoviruses the infection then spreads to many tissues in the infected larva.
This dissemination phase requires the virus to bud from the cell rather than be occluded in polyhedra.
This alternate budded virus (BV) form of the virus has a modified host membrane envelope and
disseminates via the tracheal system to get beyond the gut barrier. The virus is then free to infect,
replicate and spread throughout much of the host insect. Late in the infection cycle in each cell, a
switch to very late gene expression leads to de novo envelope synthesis and occlusion of progeny
virions to generate more ODY. One of the final acts of the infectious process is the dissolution of the
infected larva by proteolytic and chitinolytic enzymes to release the PIBS into the environment where
they are free to re-infect another susceptible host.

Advances in Microbial Control of Insect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers. New York. 2002 127
 Baculoviruses are very well adapted to their niche with the biphasic life style allowing significant
viral amplification within a host and an array of viral responses to augment and influence the infection
process. The series of complex interactions that occur within the infected host are influenced by the
genetic complement ofbaculoviruses. Analysis of both nucleopolyhedrovirus (NPV) and granulovirus
(OV) genomes has represented a significant part of the study ofbaculoviruses in the last two decades.
Significant strides are being made into the function of individual viral genes (Miller, 1997). Many of
these studies have had far ranging implications, such as the apoptosis inhibitors originally identified by
Miller et al (Clem et aI., 1991). Other studies into gene function are providing new opportunities for
using baculovirus genes for pest control. This chapter looks at the current status ofbaculovirus
genome analysis and from that basis investigates four gene groups that are highly conserved in
baculoviruses and are important in different stages ofthe host-pathogen interaction. These genes are:
the enhancins, that are important in infection establishment; chitinases, that are important for host
dissolution and may playa role in infection establishment; ecdysteroid glucosyltransferases, that are
important in manipulating the biology of the host at an organismallevel; and other genes such as
apoptosis inhibitors, that are important in avoiding an intracellular host response.

2. BACULOVIRUS GENOMES

The sequencing ofthe genomes ofmany different species ofbaculoviruses has been undertaken,
with a total of 12 baculovirus genomes now fully sequenced (Table 1). This list of sequenced genomes
includes group I and group II NPV s, OVs and at least one non-lepidopteran NPV. The sequenced
dipteran NPV has a very different genomic composition to the lepidopteran NPV s. These sequencing
studies have allowed the identification of genes which are core to all baculoviruses and those which
characterise certain groups and species ofbaculoviruses.lt has also created a large resource of
information about the genetic make-up ofbaculoviruses that has been used to facilitate rapid mapping
and analysis ofless well studied baculovirus genomes (Hu et al., 1998; Hyink et al., 1998; Sadler et
al.,2000).

Table 1. Baculoviruses for which the complete genome has been sequenced

Baculovirus Acronym Genome Reference and GenBank

Size (bp) Accession Number
Autographa cali/ornica MNPV AcMNPV 133,894 (Ayres et aI., 1994): L22858
Bombyx mori MNPV BmMNPV 128,413 (Gomietal.,1999):L33180
Orgyia pseudotsugata MNPV OpMNPV 131,990 (Ahrens eta!., 1997): U75930
Epiphyas postvittana MNPV EppoMNPV 118,584 (Hyink et aI., 2002): AY043265
Lymantria dispar MNPV LdMNPV 161,046 (Kuzio etal., 1999): AF081810
Spodoptera exigua MNPV SeMNPV 135,611 (Ijkel etal., 1999): AF169823
Spodoptera litura MNPV SpltMNPV 139,342 (Pang et aI., 200 1): AF325155
Heliocoverpa armigera SNPV HaSNPV 131,403 (Chen etal., 2001): AF271059
Culex nigripalpus NPV CuniNPV 108,252 (Afonso et aI., 2001): AF403738
Cydia pomonella GV CpGV 123,500 (Luque et aI., 2001): 053466
Plutella xylostella GV OOV 100,999 (Hashimoto et aI., 2000): AF270937
Xestia c-nigrum GV XecnGV 178,733 (Hayakawaetal., 1999): AF162221

2.1 The Core Genes of Baculoviruses

The number ofcore genes, those with homologues in all baculoviruses, is decreasing as more
baculovirus genomes are sequenced. Hayakawa et al. (2000) identified 67 genes conserved between

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the seven lepidopteran baculovirus genomes sequenced at that point oftime. The addition of sequences
from four further lepidopteran baculoviruses, Heliocoverpa armigera NPV (HaSNPV), Cydia
pomonella GV (CpGV), Epiphyas postvittana NPV (EppoMNPV) and Spodoptera litura
(SpltMNPV), have reduced this number to 62. With the addition of the first sequence available from
the dipteran baculovirus Culex nigripalpus NPV (CuniNPV) this number reduced to 27 genes
(Afonso et aI., 200 1). This dipteran baculovirus clearly has a very different genome to those of
lepidopteran baculoviruses with only 36 of the 109 ORFs identified in CuniNPV having homologues
in other baculoviruses. However, with the elucidation of functions for unknown genes in CuniNPV it
may become apparent that the number of related genes is higher, for example, a homologue of the
major occlusion body protein has not been identified in CuniNPV, despite this virus being occluded.
Despite the large divergence in the genomes ofNPVs from insect orders other than the
Lepidcptera, the conserved genes provide insights into the unifYing genes of all baculoviruses (Table
2). These include genes associated with transcription, replication and structural proteins as well as
genes that manipulate the host such as the p3 5 apoptosis inhibitor and an alkaline exonuclease. There
are 7 genes conserved in all baculoviruses for which no known function exists, yet these must be of
central importance to baculoviruses. Elucidating the function ofthese unknown genes is likely to
provide interesting insights into baculovirus biology. A comparison of genes in different groups and
genera ofbaculoviruses is shown in Table 3.

Table 2. Genes present in all baculoviruses (adapted from Afonso et aI., 200 1).

Functional infonnation available p47, lel-8, lej9, vlf-I, lel-4, lel-5, lel-2, leI-I, dna-
pol. hel-I, vpI054, gp4I, vp39, vp9I, p6.9, p74,
odv-ec27, odv-e56, p35, alk-exo
No known function (AcMNPV orfnumbers) 23,68,81,96,98,109,115

2.1.1 Methods of Comparing Genomes. A number of methods have been employed to compare
the different baculoviruses. The earliest methods were based on the phenotypic appearance of the
virus particles. This grouped the baculoviruses into nucleopolyhedroviruses (NPV s) and granuloviruses
(GVs). A further phenotypic distinction in the NPV group was made on how they were embedded in
the occlusion bodies, as either single (SNPV) or multiple (MNPV) nucleocapsids. The large amount of
sequencing undertaken on baculoviruses has brought about the need for methods to compare viral
genomes. Phylogeny based approaches using single gene sequences have commonly used the polyhedrin
gene (polh) ofNPVs and the granulin gene (gm) ofGVs for phylogenetic analysis (Zanotto et aI.,
1993). As more baculovirus genomes are sequenced, it may be that the current grouping ofbaculoviruses
breaks down and a more continuous spread of baculovirus phylogeny emerges. Another commonly
used gene has been ecdysteroid glucosyltransferase (egt) (Caradoc-Davies et aI., 2001; Clarke et aI.,
1996). A phylogenetic tree ofbaculoviruses based upon the polh gene is shown in Figure 1.
These single gene phylogenies have been extremely useful in identifYing relationships between
baculoviruses but have the disadvantage of measuring gene phylogeny rather than whole organism
phylogeny. An additional problem with this approach becomes apparent with the sequence of the
first non-lepidopteran baculovirus. The NPV from the mosquito Culex nigripalpus (CuniNPV)
apparently lacks a homologue of the polyhedrinlgranulin gene.
A number of approaches have been used to compare whole genomes ofbaculoviruses. Hemiou
et al. (2001) used a number ofapproaches including: construction of a single sequence ofall conserved
proteins in nine different baculoviruses, breakpoint analysis identifYing discontinuities in genes order,
and gene content comparisons. These approaches provided similar results and are a good basis from
which to compare the genes present upon baculovirus genomes.
An alternative to the phylogeny inferences described above is graphical representation of
baculovirus gene relationships. Gene parity plots have become popular and offer a simple method of
comparingbaculovirusgenomes(Huetal., 1998). ThecomparisonsofgenecontentbetweenEppoMNPV,

129
Table 3. Core Genes oflepidopteran baculoviruses.

All' NPV' GroupP GV"

lef-2' Ac8l egt p24 p24
polh/grn Ac82/tlp20 lefiO Acl8 sod
pkl p95 gp37 Ac5l Acl50
38.7K vp39 Ac76 cg30 p l3/glycogenin
lef-l lef-4 Acl15 p26 helicase2
Ac22 Ac92 chitinase Ac74 dna-ligase
Ac23 Ac93 vcath Aclll Xecn2
dbpl p25/odv-e25 plO lef12 Xecn7
lef6 helicase ieD Ac43 XecnS
Ac29 Ac96 orfl629 Ac56 Xecnl7
fgf 38K Ac60 Acll7 XecnlS
vubi lef-5 Acl7 gpl6 Xecnl9
39K1pp3l p6.9 Acl9 Ac4 Xecn25
lefll p40 arifl Ac5 Xecn26
Ac38 pl2 pkip Acll Xecn29
p47 p45 Ac26 Ac44 Xecn34
odv-e66 AclO6 Ac34 Acl20 Xecn40
left! AclO9 Ac55 I1.3K Xecn47
Ac53 AcllO Ac57 Ac9l Xecn54
vplO54 Acll9 Ac59 pe38 XecnS5
tp/25K alk-exo iap2 ptpl XecnS6
lef9 p74 vp80/pS7 Ac2 Xecn90
dnapol meS3 AclOS odv-e26 Xecn1l3
Ac66 Acl42 pp341pep iapl Xecn1l6
lef-3 odv-el8 Ac30 Xecnl36
Ac68 odv-ec27 gta iap5
Ac75 Acl45 Ac72 Xecn142
vlfl Acl46 Ac73 Xecnl43
Ac78 iel Ac1l4 Xecnl65
gp4l odv-e56 GroupU' Acl24 Xecnl69
Ac52 len Xecnl72
Ld55 gp64/67 Xecn173
Ldl29 Acl32 Xecnl7S
Ldl411parg ie2

'Genes conserved in all lepidopteran baculoviruses, 'genes conserved in all lepidopteran NPVs with genes
only found in NPVs highlighted in bold, 3genes found in all group I NPVs with those genes unique to group J NPVs
in bold, 'genes found in all group II NPVs with those genes unique to group II NPVs in bold, 'genes found in all
GVs with those genes unique to the GVs in bold, 'genes are identified by common abbreviation(s) where known
or by orf number in the baculovirus where first identified; Ac, Autographa cali/arnica MNPV; Ld, Lymantria
dispar MNPV; Xecn, Xestia c-nigram GY.

a group I baculovirus, and selected group I and group II baculoviruses and a granulovirus, are shown
in Figure 2. To generate the graphs shown in Figure 2, the orfs of the selected viruses are sequentially
renumbered using the polh or grn genes as orf 1. The position of the homologous genes are then
plotted against each other with non-homologous genes plotted on the X or Y axis as appropriate.
The overall similarity between genomes can be identified rapidly as can gene orientation within each
genome. The group I NPV s are a tightly clustered group with a highly conserved genome content, as
shown by the EppoMNPV and OpMNPV gene parity plot. In comparison, the group II NPV s are
more diverse. The polyhedrin gene is also reversed in group II NPV s, leading to a reversal in the
relative gene order in these viruses. This is shown clearly in the gene parity plot analysis between
EppoMNPV and SeMNPV in Figure 2. The more distantly related granuloviruses show further
disparity by gene parity plot analysis with less homologous genes and more apparent disparity in gene
order. A cluster of conserved genes (Heldens et al" 1998) can also be observed in the middle of the
genomes as oriented from the polh gene.

130
 .----MbNPV
""'---LeseNPV
r~_~=====P~NPV
MaeoMNPV
,L--jliiiii=== TnSNPV

r : - - - - SeMNPV
Group II NPVs '""'---StMNPV
9 ' - - - - SpltMNPV

r---1ijiiL== HaSNPV
HzSNPV
3 .----MdMNPV
=---MnNPV
4

ChroNP,
PenuNP,
' - - - - OpMNPV
' - - - - ArceNPV
. - - - - EppoMNPV
....,..-- CfDEF
2
' - - - - ThorNPV
':-'--- AgMNPV
Group I NPVs L--l:9i:== AnpeNPV
;- ArNPV
, - - - - HycuNPV
.----1·100 llI!I._ _ ArnalMNPV

'--'--- AnfaMNPV
' - - - - LoMNPV
' - - - - BmMNPV

'----AsNPV
. - - - - CrleGV
GVs ........_ - CpGV
' - - - - HabrGV
' - - - - CfDY
'----AsGV

r -:-----fi:iiiL== TnGY
XeenGY

'-1-O--PibrGV

Figure I. Phylogenetic tree of baculoviruses based upon the polyhedrin and granulin amino acid sequences.
Numbers represent the number of times the tree branches at that point in 100 bootstrap replicates. Group I and
Group II NPVs and GVs are indicated. Species abbreviations are as described by van Regenmortel et at. (2000).

Of the many genes conserved between baculovirus genomes, some are known to be involved in
manipulation ofthe host insect and their conserved nature implies an important role in the pathogenicity
of these viruses. Several of these target genes would be expected to have significant potential in
manipulating insects for biocontrol purposes, even in the absence of other viral factors. Some of
these target genes and their potential for use in insect biocontrol are discussed below.

3. BACULOVIRUS ENHANCING PROTEINS

3.1 Enhancins from Granuloviruses

The phenomenon of synergism between baculoviruses has been known for many years. The
ability of a granulovirus and a nucleopolyhedrovirus to act synergistically was first reported in 1956

131
between two viruses infecting the annyworrn Pseudaletia unipuncta (Tanada, 1956). This relationship
was further refined to identify a factor in the capsule of the granulovirus (PsunGV) that was capable
of enhancing the NPV infection rate (Tanada and Hukuhara, 1971). An interesting observation was
that not all strains ofPsunGV showed this enhancement ofNPV infection (Tanada and Hukuhara,
1968) and this is likely to have contributed to the slow development ofthe application ofviral synergism
to biological control programmes.

EppoMNPV vs OpMNPY
16 0
14 0
.,,,,,,,,,,,1..'"
120
i;:10 ""","
Z ",' "
~ 80
o ",,'"
60 .'
.-
40 , , ,
,"
20
."",,,,,,,

o 20 40 60 80 100 120 140

EppoMNPY

EppoMNPY vs SeMNPY
16o,--,--,---,---,---,---,---,
1401___-+_-+_-+_-+_-+_--+--::,-,-,---1
,"
> 1201-,---t----:+---+--t--t----t----j
Q.. .....
~1001----+--+-.c:...,+--+--+--+----I
"
~ 8ffi--+--~-~~.~,~-+_-+_----I

6ili--+--~-~-~·~"~~+_-+_----I
4~~~-~-~-~-~~~-~

2~~~--~~~-+_--+_~+-~

o'~--+-~~-~--~--~--~~w
o 20 40 60 80 100 120 140
EppoMNPY

160,--.---.---E..:.;ppro-M-N-P..
V-vs-C-'p..
G-Y-,-----,

140 t--~t---+-,--t---t---t---r----j
1201---+-'--+-~+--+--+--+----1

1001--t---t---t--"'-"'''':+--+--+---1
> ......
~ 80 I--+-~+_-+_-+_'---+_-+_--j
u
60~-~-+_-+_-+_~+--+_--j
.'.
40r--t---r.--t---t---t---t-----1
201---~-+_-+_-+_-+_-+_---j

o ~--~--~--~~~~~~~~
o W ~ W W 100 IW I~
EppoMNPY

Figure 2. Gene parity plot comparison of the EppoMNPV genome with the genomes of a group I NPV (OpMNPV),
a group" NPV (SeMNPV) and a GV (CpGV)

Significant progress has been made in elucidating the mechanism of action of the synergistic
factor found in some granuloviruses over the last 15 years. Derksen and Granados (1988) showed
that the granulovirus of Trichoplusia ni (TnGV) contains factors that cause biochemical and structural
changes to the peritrophic matrix and this has been correlated with the presence of the synergistic

132
factor. In addition, a series of results were obtained confirming that these GV derived "enhancins"
were effective at enhancing the infectivity ofnucleopolyhedroviruses. These included increased mortality
and decreased survival times of insect larvae for the TnGV enhancing factor (Greenspan-Gallo et al.,
1991; Wangetal., 1994).
While there is an apparent linkage between peritrophic matrix breakdown and increased infection
rates in the presence of a GV derived enhancin, an ability to enhance fusion of budded NPV virions
to cells in culture has also been observed. Increased infection of SpodopteraJrugiperda cells (Sf)
by both budded and occlusion-derived virions ofTnMNPV has been shown in the presence of the
enhancing factor from PsunGV (Hukuhara and Zhu, 1989; Kozurna and Hukuhara, 1994), although
the exact role of fusion remains to be elucidated with Wang et al. (1997) finding no increase in cell
binding or fusion when testing TnGV enhancin.
One of the key steps in the development of enhancins has been the isolation and cloning of the
gene for the enhancin from TnGV and the subsequent proof that a homologous gene lies in other GVs
(Hashimoto et aI., 1991). Comparison to other enhancin sequences showed 98% and 80% identity
to PuGV and HaGV enhancins respectively (Roelvink et al., 1995). The high level of identity between
these sequences suggests that they are likely to have very similar modes of action. Similar genes have
also been identified in Lymantria dispar MNPV (LdMNPV) through sequence analysis (Bischoff
and Slavicek, 1997). These genes have been showed to be very important in the potency ofLdMNPV
(Popham et aI., 2001), however, most NPVs sequenced to date do not have homologues ofGV
enhancins.
GV enhancins are metalloproteases that specifically target an intestinal mucin associated with
the peritrophic matrix (Lepore et al., 1996; Wang and Granados, 1997) playing an important role in
limiting baculovirus infection (Wang and Granados, 1998). Combined, these observations indicate
that enhancins are proteases that degrade mucins in the peritrophic matrix, thus overcoming one of
the major barriers to infection by a baculovirus. The role of enhancins in increasing fusion to target
cells and thus facilitating the initiation of infection is less clear.
The future role ofGV enhancins in developing new biological control strategies remains to be
determined. There is a significant attraction to the idea of using enhancins as formulation additives but
sufficient amounts will need to be produced. The use of transgenic plants is also attractive but the
processed nature of the enhancin protein may pose some problems. This remains to be determined.
Enhancins have been shown to improve the efficacy ofBt toxins (Lepore et al., 1996) indicating that
these genes may have a significant role in biological control strategies other than baculoviruses.

3.2 Spindle Protein Homologues from Baculoviruses

The synergism originally identified forthe GVs has also been observed in other viral combinations.
In particular, entomopoxviruses have been shown to enhance baculovirus infections and this enhancin
activity has been localised to the entomopoxvirus spindle, which is composed ofthe fusolin protein
(Graves et al., 1998; Xu and Hukuhara, 1992; 1994). The spindle proteins ofsome entomopoxviruses
have been sequenced (Hayakawa et al., 1996) and successfully expre~sed in both plants and bacteria
(Hukuhara et aI., 1999; Hukuhara et aI., 2001). Insect feeding upon leaves of transgenic plants
expressing fusolin were more susceptible to NPV s than control larvae, indicating that these genes
may have an important role in future biological control programmes involving baculoviruses.
One of the results to come from the entompoxvirus spindle research is the presence ofa
homologue of the fusolin protein in NPV s. This protein is commonly called GP3 7. The protein is
expressed late in the infection cycle and is a glycosylated protein with a predicted chitin binding
domain (phanis et al., 1999). The role of this protein is yet to be determined, however, it is tempting
to speculate that it will have a similar role to the entomopox spindle. GP37 has been shown to
produces spindle shaped inclusions in the cytoplasm ofOpMNPV and AcMNPV infected cells
(Gross et al., 1993)and this protein has been discovered in PIBS (Vialard et al., 1990), suggesting a
role in the establishment of viral infection. This is consistent with a role as an enhancin. It has also

133
been observed that the interaction between Wiseana entomopoxvirus (WEPV) and AcMNPV was
syncrgistic with both WEPV and AcMNPV showing enhanced infection rates in the semipermissive
host Heliocoverpa armigera (Graves et al., 1998), suggesting that AcMNPV possesses an enhancin-
like factor. The factor( s) contributing to this association were not identified, but GP37 is one candidate.
GP37 is an ideal candidate gene for further analysis. Its close relationship with fusolin proteins,
which have been shown to be active enhancins, provide a strong lead into functionality. The protein is
significantly smaller than the GV enhancin molecules with much less processing involved. This small
size and minimal processing make it an ideal candidate for use in transgenic plants or other recombinant
organisms.

3.3 Chitinase

Chitin is composed of polymers ofN-acetylglucosamine and is the most abundant nitrogen-

bearing organic compound in nature (Muzzarelli, 1999). Chitin is found in insect exoskeletons,
peritrophic membranes and cocoons. It is also common in crustacean shells, cuttlefish, squid, and
molluscs and is particularly abundant in fungal cell walls. It has been estimated that 10 gigatons of
chitin is synthesised and degraded per year (Muzzarelli, 1999).
Because ofthe broad abundance of chitin, significant research effort has been devoted to analysing
chitinases. These enzymes have the potential for degrading chitin containing structures such as fungal
cell walls and peritrophic membranes, thus providing new approaches in the biocontrol offungi and
insects. Indeed, antagonism via chitinases is recognised as being important to biological disease
control.
A number of attempts to develop chitinase-based biocontrol agents have been made. The
chitinase of Manduca sexta has been used to enhance the susceptibility of A. cali/arnica larvae to
disease (Gopalakrishnan et aI., 1995). Chitinases have also been shown to enhance the activity ofBt
toxins and baculoviruses against a variety of insects (Morris, 1976; Shapiro et aI., 1987; Smimoff,
1974). For example, chitinase increases the infectivity of LdMNPV for gypsy moth larvae 5-fold
and this has been linked to disruption of the chitin component of the insect peritrophic membrane
(Brandt et aI., 1978).
One of the more interesting developments is the role of chitinases in plants, where they have
been directly linked to disease resistance (Chemin et aI., 1996; Davison, 1988). The introduction of
chitinases into the plant environment should aid in resistance to diseases and insects and a number of
approaches have been attempted. The S. marcesans chiA gene inserted into E. coli enhanced
Sclerotium rolfsii control (Oppenheim and Chet, 1992). This chiA gene has also been introduced
into symbiotic Rhizobium melliloti to provide fungal biocontrol on alfalfa roots (Sitrit et aI., 1993).
Another approach has been to utilise endophytes such as Pseudomonas jluorescens to deliver
chitinase to roots. This approach has been used successfully in the control of Rhizoctonia solani
(Downing and Thomson, 2000).
These observations indicate that there is a potential widespread application of chitinases in the
field of biological control and that new and varied forms of chitinase may have an important role. Of
particular interest are the chitinases ofbaculoviruses. Most baculoviruses sequenced to date contain
a chitinase, however, only the chitinase of AcMNPV has been studied in any detail (Hawtin et aI.,
1995, 1997). The AcMNPV chi gene has 60% identity to the S. marcesans chiA, however, it is
distinguished by possessing both endochitinase and exochitinase activity within the same enzyme.
Preliminary studies in our laboratory with the EppoMNPV chitinase gene indicate that it too has both
endo- and exochitinase activity, suggesting that this is likely to be a common feature ofbaculovirus
chitinase genes.
The AcMNPV chi gene is 1653bp in size, making it relatively simple to manipulate. There are
no known unusual post-translational modifications ofbaculovirus chitinases, hence their use in a
variety of systems should be feasible. The enzyme is broadly active from pH 4-10, with activity
retained to pH 12 (Hawtin et aI., 1997). The combination of high pH tolerance and both exo- and

134
endochitinase activity in the one enzyme make this a particularly appealing target for enhancement of
biological control agents. Modification ofspecific amino acids by site-directed mutagenesis has shown
that the relative preference of the enzyme to act as an endo- or exochitinase can be manipulated
(Thomas et al., 2000), offering potential of manipulating the enzyme for specific applications and
niches.
How important possessing both activities will be in the effectiveness ofbaculovirus chitinase
genes in a variety ofbiocontrol applications remains to be elucidated. Despite this uncertainty it
seems likely that this joint activity will provide an aggressive chitinase that will be well worth testing in
an array ofbiocontrol strategies against diseases and insects, as well as in combination with insecticidal
agents such as Bacillus thuringiensis insecticides (Wiwat et aI., 2000). The chitinase of M sexta
has been compared in insect feeding with the chitinases from Streptomyces and Hordem species
(Ding et al., 1998). The insect derived chitinase was shown to be more effective than non-insect
chitinases in other biocontrol strategies. Baculoviruses cause the rapid liquifaction oftheir hosts late in
virus infection. Chitinase is one ofthe key enzymes involved in this liquifaction process (Hawtin et al.,
1997), supporting the contention that baculovirus-derived chitinases are ideal candidates for
development in insect control strategies.

4. ECDYSTEROID UDP-GLUCOSYLTRANSFERASE

The baculovirus ecdysteroid UDP-glucosyltransferase (EGT) was identified through the analysis
of a non-essential region ofthe AcMNPV genome which was deleted during passage ofthe virus in
tissue culture (Kumar and Miller, 1987). Sequence analysis identified a gene was deleted that had
striking similarity to mammalian glucuronosyltransferases. Investigation ofEGT (O'Reilly and Miller,
1989) showed that the baculovirus EGT is secreted into the haemolymph of an infected insect,
compared to the mammalian UGTs which are membrane bound by a C-terminal anchor region. The
baculovirus EGT was shown to catalyse the conjugation of sugar groups from UDP-sugars to the
insect moulting hormones, the ecdysteroids (Figure 3) thus leading to their apparent inactivation.
From a biocontrol perspective, insects infected with an egt deletion mutant died earlier (O'Reilly
and Miller, 1991; Slavicek et aI., 1999). The deletion of this gene was as considered an acceptable
method of improving baculoviruses as biological pesticides, because not only did the deletion ofegt
lead to a virus with a quicker kill time, but it also provides a locus for the addition of genes to the viral
genome; e.g. insect specific scorpion toxins. As recombinant baculoviruses are developed, the
combination ofanumber ofchanges to the viral genome may lead to cumulative improvements. Using
the egt locus could have the added benefit of removing the egt gene as a component ofconstructing
a recombinant virus.

OH OH

~(jJ
: tHPH
OH
Ecdysone
22-0-[3-D-
HO" Glucopyranoside

Figure 3. Conjugation of glucose to ecdysone. EGT catalyses the transfer of a sugar group, such as glucose, from
a UDP-sugar to C22 of ecdysteroids such as ecdysone, thereby inactivating the hormone.

EGT is expressed early in the baculovirus infection cycle. The 60 kDa protein is secreted from
infected cells into the haemolymph where it circulates. EGT is glycosylated and the AcMNPV EGT

135
has been shown to form multimers ofbetween 3-5 subW1its (Evans and 0 'Reilly, 1999). A hydrophobic
leader sequence of 18-20 amino acids is cleaved from the active enzyme. EGT has been identified in
all baculoviruses fully sequenced except for the slow-killing XecnGV and CuniNPY. A signature
motif found in all glucosyltransferases, including those from mammals, plants and bacteria, has been
identified in a C-terminal region ofthe protein EGT can conjugate glucose and galactose to a range
of ecdysteroids providing there is a hydroxyl group at the carbon-22 position (O'Reilly et al., 1992).
An observed feature ofbaculovirus infection is that larvae infected in the final instar failed to
pupate and earlier instar larvae displayed abnormal development (Burand and Park, 1992). This can
be attributed to the presence of the viral EGT during the infection process of the larvae (O'Reilly and
Miller, 1991). To fully understand this gene and its influence on a baculovirus infection in vivo, the
effect of the hormone on the developing larva must be examined.
The role of ecdysteroids in lepidopteran larval development is to control the outcome of a
moult, whether a larval-larval or larval-pupal moult. The biosynthesis of ecdysteroids is a controlled
cascade of secretion and subsequent activation (Nijhout, 1994). The corpora allata produces the
prothoracicotropic hormone (PITH), which stimulates the prothoracic gland to produce the hormone
precursor, 3-dehydroecdysone(3DE), 3DE is converted to the prohormone ecdysone in a metabolite
cell where it becomes hydroxylated at carbon 20 to become the active hormone, 20-hydroxyecdysone
(20-HE). 20-HE is then transported to a target cell where it enters the cell and interacts with a
nuclear receptor assembly to initiate a cellular response. A variety of cell types are involved in this
biosynthesis pathway. Metabolite cells include those of the midgut, tracheal system and malpighian
tubules (Lafont and Connat, 1989). Target cells are any larval tissue, such as the epidermal cells,
neurones, midgut cells and many others. The responses of the target cells varies greatly from massive
cell proliferation, in preparation for a larval-larval moult, or massive cellular death, for a larval-pupal
moult. As levels of20-HE increase, a negative feedback loop starts to influence ecdysteroid production
by the prothoracic gland, causing levels of production to decrease and then stop (Sakurai and Williams,
1989).
By contrasting the route of a baculovirus infection with the ecdysteroid biosynthesis pathway
there are many tissues which are critical for an efficient baculovirus infection which may become
disrupted by ecdysteroid-induced events. Infection of a lepidopteran larva occurs through the midgut
epithelium, if there is amass cell death of this epithelial layer, then the infection would be abortive. By
producing an enzyme which inactivates the hormone, the baculovirus not only affects the host at the
cellular level, but it also displays a level of control at the whole animal level.
A mechanism for the inactivation of the ecdysteroids has recently been proposed where the
conjugation ofa sugar moiety to the ecdysteroid prevents it from entering a cell (Figures 4,5) (Caradoc-
Davies et al., 200 I). This mechanism is supported by the abnormally high levels of ecdysteroids that
have been reported in the haemolymph of infected Lymantria dyspar larvae (Park et aI., 1993,
1996), indicating that the negative feedback loop of ecdysteroid biosynthesis has been disrupted.
There have also been reports of ecdysteroid titres not rising above a basal level, this could indicate
that the biosynthesis of ecdysteroids is also disrupted during infection.
Kinetic analysis of purified EGT showed that AcMNPV EGT had a greater specificity for 3DE
and ecdysone than for the active 20-HE (Evans and O'Reilly, 1998). This could in part be due to
steric hindrance by the hydroxyl group at the C20 position, or it may be advantageous for an enzyme
to have a greater affinity for the inactive precursors ofthe hormone, rather than the active hormone,
which is only required at relatively low levels to have a detrimental effect on tissues supporting
baculovirus replication.
EGT is an example of a non-essential or auxiliary gene which increases virus PIB production
and its absence can sometimes increase the efficacy of a knockout virus as a biocontrol agent. The
functional analysis ofEGT has shown the direct application of functional genomics developing new
approaches in biocontrol. In addition, the egt gene is a spare locus for the addition of insect specific
toxins or other foreign genes. Genomic analysis of the position and context of egt within its locus is
also important to ensure that engineering of this site does not interfere with adjacent genes such as the
lef] gene.

136
 9
8
7
6
~5
!-
'0 4
~3
2
J
o
Cellular Cytoplasmic Nuclear

Figure 4. Effect of sugar conjugation upon ecdysteroid localisation. The uptake of ecdysone (hatched bars) or
conjugated ecdysone (black bars) into Sf21 cells. Conjugation prevents cellular uptake of ecdysone. Ecdysone
localises to the cytoplasm ofSf21 cells because they lack the receptor necessary for nuclear uptake (adapted from
Caradoc-Davies el al. ,200 1).

f:son~GT
~

Negative
Feedback

Midgut epithelia etc.

Figure 5. Proposed mode of action for EGT -based regulation of insect development. EGT dependent conjugation
of glucose or galactose prevents ecdysone from entering metabolite cells, thus preventing the production of 20-
OH ecdysone, which in turn does not provide negative feedback to the prothoracic gland. This provides a
possible mechanism for the increase in ecdysteroid titres that have been observed in late instar infected insects
(Parketal., 1993; Park et aI., 1996).

5. APOPTOSIS INHIBITORS

Apoptosis (programmed cell death) is a conserved cellular process that is required for nonnal
development of multicellular organisms (Vaux and Korsmeyer, 1999). Numerous stimuli and signalling
pathways converge to bring about the demise of the cell via activation of cysteine aspartic proteases
(caspases), the key effectors of cell death. The role of apoptosis in eukaryotes is extremely diverse,
however, one role is as a defence against viral infection. Viruses must avoid apoptosis ofthe host cell
if they are to complete their replication cycle successfully (O'Brien, 1998; Uren and Vaux, 1997).
Baculoviruses are no exception, they carry a range of genes designed to block apoptosis of host cells
in the insect.
There are two clear types of apoptosis inhibitor in baculoviruses, the p3 5/p49 genes and the
inhibitor of apoptosis genes (iaps). The p35 inhibitor was first identified in AcMNPV (Clem etaJ.,

137
1991) and is a stoichometric inhibitor of caspases (Bertin et ai., 1996; LaCount et ai., 2000). The
baculoviral P35 inhibitor is a general caspase inhibitor capable ofblocking apoptosis in a wide variety
of organisms including insects, nematodes and mammals (Beidler et aI., 1995; Bump et al., 1995;
Hay et ai., 1994; Sugimoto et al., 1994). Significant research into the structure and function ofP35
has also occurred (Eddins et ai., 2002; Zoog et ai., 1999). More recently a homologue ofthe p35
gene has been identified in Spodoptera littoralis MNPV (SpliMNPV) (Du et aI., 1999).
lAPs were first discovered in baculoviruses based on their ability to maintain host cell survival
during viral replication (Clem and Miller, 1994; Crook et aI., 1993). Subsequently, a number of
mammalian homologues have been identified and characterised (Verhagen et aI., 2001). All lAP
proteins contain at least one copy of a Baculoviral lAP Repeat (BIR) domain and a RING domain.
The BIR domains mediate association with other proteins and each BIR has a distinct interaction
profile (Holcik and Korneluk, 200 I). For instance some BIRs bind to and inhibit caspases directly,
while others interact with upstream regulatory molecules. The BIRs of the active lAP from OpMNPV
(OpIAP) interact with each other and with cellular apoptotic effectors (Hozak et ai., 2000). In
mammalian lAPs, RING domains have been implicated in protein ubiquitination and removal ofthe
RING domain enhances the anti-apoptotic activity of cellular lAPs (Yang et aI., 2000).
Baculoviral lAPs exhibit a number of different properties to their mammalian counterparts.
Mammalian lAPs interact with caspases directly to inhibit apoptosis (Roy et aI., 1997). Op-IAP
(Op-iap-3), the most well studied baculoviral lAP, has been shown to inhibit apoptosis in SF21 cells
by preventing the activation of pro-Sf-caspase-I (LaCount et aI., 2000; Seshagiri and Miller, 1997).
The requirement for RING domain is also intriguing. In baculoviral lAPs this domain is essential and
removal inactivates the lAP (Hozak et aI., 2000; Maguire et aI., 2000). Why the RIN G requirement
is different between baculoviral and cellular lAPs remains unknown.
There are 5 iap groups based upon sequence similarities. However, until recently only the iap-
3 class of inhibitor had been shown to possess any apoptotic activity. A study into EppoMNPV
showed that the IAP-2 inhibitor from this virus was capable of inhibiting apoptosis and that the iap-
1 gene encoded a protein that could delay but not prevent apoptosis (Maguire et aI., 2000). No
activity was observed for the IAP-3 and lAP-4 class inhibitors ofEppoMNPV. This study indicated
that the role of the various iap genes in baculoviruses is likely to be in apoptosis inhibition, except for
iap-4, for which no apoptosis function has been observed and it lacks a key BIR domain. Why
baculoviruses often carry so many inhibitors is unknown.
Apoptosis inhibitors are thought to play an important role in host range determination so mUltiple
inhibitors may function in alternate host and/or cell types. One mechanism that could explain this is the
dominant interference observed by BIR domains (Hozak et aI., 2000). Some viral lAPs may interact
negatively with cellular factors so that a range of lAPs with varying BIR sequences may improve the
likelihood of apoptosis prevention in a range of cells and hosts. Another possibility is that varying
expression of different lAPs may occur in different cells, for example, the expression ofIAP-2 from
EppoMNPV requires a second upstream gene, though it can function as an apoptosis inhibitor if
expressed from a constitutive promoter (Maguire et aI., 2000). Expression of supposedly inactive
iap genes may not be occurring, hence no inhibition of apoptosis is observed.
A substantial amount is known about the biochemistry of apoptosis inllibitors which can not be
covered in this manuscript. In contrast, the effect upon baculoviral biology is less well studied and
how apoptosis inhibiting genes could be exploited in other biocontrol systems is hard to determine.
That apoptosis has a role in host range determination is clear. AcMNPV grows extremely poorly in
S. litloralis cell lines (Chejanovsky and Gershberg, 1995) and its growth can be rescued by the P49
apoptosis inhibitor ofSpliMNPV (Du et aI., 1999). The lack of apoptosis inhibition does not block
replication completely but reduces the yield of virus substantially, correlating with a large decrease in
late and very late gene expression (Hershberger et aI., 1992). This observation is also noticed in
vivo (Clem and Miller, 1993). One of the findings related to this observation is the development of a
persistent AcMNPV infection through the deletion ofthe p35 gene (Lee et ai., 1998). This is one
mechanism by which persistent baculovirus infections may become established in the field.

138
 Whether apoptosis inhibition is a mechanism that can be used to augment biocontrol agents with
narrow host ranges remains to be determined. In contrast, a wide acting baculovirus such as AcMNPV
may be restricted in its host range by removal ofan apoptosis inhibitor gene such as p35. lbis may be
attractive where more specific biocontrol agents are desirable. Apoptosis inhibitors have also been
used to stably transform cell lines to make more robust cell culture systems for biotechnology
applications (Mastrangelo and Betenbaugh, 1998). The long term effectiveness of these applications
of apoptosis inhibitors remains to be determined, but they offer a number of interesting application
possibilities. The study of apoptosis owes a lot to the baculoviral apoptosis inhibitors and they are
sure to provide advances in the future.

6. SUMMARY

There are too many genes in baculoviruses to cover all of them in this chapter. Despite this
limitation, it is clear that baculovirus genornics has provided much ofour understanding ofbaculoviruses
and is providing important leads into the interactions of these viruses with their hosts. These studies
are in turn providing new avenues for augmenting and improving baculoviruses and other biocontrol
agents. Many of the genes discussed in this chapter will have applications beyond baculoviruses,
such as the potential of chitinases for fungal biocontrol on plants and enhancement ofother gut active
pest control agents. Any of the genes ofbaculoviruses that manipulate the host at the subcellular,
cellular or organismallevel have potential for development with host range factors, proteinases, cell
cycle regulators and growth factors all offering potential for application in other biocontrol programmes.
The functional analysis ofthese genes, and the array ofbaculovirus genes for which no function has
been determined, will undoubtedly provide new insights into many areas ofbiology and new approaches
to biocontrol.

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Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwell, J. D., 2000, Ubiquitin protein ligase activity ofiAPs
and their degradation in proteasomes in response to apoptotic stimuli, Science 288:874-877.
Zanotto, P. M., Kessing, B. D., and Maruniak, 1. E., 1993, Phylogenetic interrelationships among baculoviruses:
Evolutionary rates and host associations, J. Invertebr. Pathol. 62: 147-164.
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143
ENTOMOPATHOGENIC FUNGI AS POTENTIAL BIOCONTROL AGENTS FOR
TSETSE FLIES

Nguya K. Manianial , Claude Laveissiere2, Adedapo Odulaja l , Sunday Ekesi l And

Hans R. Herren l

IIntemational Centre ofInsect Physiology and Ecology (lCIPE), P.O. Box 30772
Nairobi, Kenya; 2Institut de Recherche pour Ie Developpement, OCEAC, B.P. 288,
Yaounde, Cameroun

1. INTRODUCTION

The tsetse fly (Glossina spp) has been labelled 'Africa's scourge or bane'. It is the one pest
largely preventing full utilisation ofthe best agricultural/grazing lands on the continent (Nash, 1969;
Offori, 1981; Rogers and Randolph, 1988; Cattand, 1995). About one-third ofthe continent, or
nearly 9 million km2is infested. The flies feed exclusively on vertebrate blood and are responsible for
the transmission of protozoan parasites of the genus Trypanosoma, which cause human and animal
trypanosomosis, otherwise referred to as sleeping sickness and nagana, respectively.
With regard to human trypanosomosis, an estimated 200 foci of infection occupied by nearly 60
million people in inter-tropical Africa have been reported, and each year more than 300,000 new
cases are reported (Cattand, 1994). A spectacular recrudescence of the disease has occurred since
1970. Epidemiological data indicate that the current foci of infection follow sites of the historical ones
(Gouteux et aI., 2000); the situation is comparable to that of 1925-1930 (OMS, 1996). The words
of Simon Gould ofMedecins sans Fronitieres (Doctors Without Borders) depicts the situation: " ...
the entire African villages have been depopulated. In the Sudanese village ofEzo, for example, the
incidence of sleeping sickness skyrocketed from less than 1% in 1989 to 43% in 1997. This is
extremely depressing, and is like setting back the clock 60 years". Animal trypanosomosis is also
often fatal, especially in cattle. Direct losses in animal production have been estimated at USD 800 to
1600 million armually (World Bank, 1999). Between 60 to 90 million cattles are at risk, as well as
tens of millions of goats, sheep, and camel. This undermines food security, as sick animals produce
less milk and meat, in addition to reproducing less. There is also a considerable reduction in the
economic self-reliance of affected states.
Although studies have shown the beneficial impact oftsetse control on sleeping sickness and
cattle mortality, control of these trypanosome vectors has failed to keep pace with other pest control
measures in agriculture and forestry. Some of the control measures used for tsetse control over the
years range from destruction ofvegetation and vertebrate hosts to application of different insecticides,
all with varying degrees of success. Three releases of the parasitoid Nesolynx spp. (formerly

Advances in Microbial Control of Insect Pests

Edited by Rajeev K Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002 145
Syntomosphyrum) were attempted over 70 years ago in Malawi, Nigeria and Tanzania but did not
achieve any substantive control of tsetse (Lamborn, 1925; Lloyd et aI., 1927; Nash, 1933).
The initial success of insecticide sprayings was so overwhelming that their side effects and
potential long-term consequences were not considered in advance. More recently, less environmentally
contaminating methods have been developed that involve the attraction of flies to traps or other
devices which have been treated with insecticides. The sterile insect technique (SIT) has been tested
on a large scale and the possibility of 'eradication' of an isolated tsetse population has been
demonstrated in (Politzar and Cuisance, 1982; Williamson et al., 1983; Oladunmade et aI., 1990;
IAEA, 1997), but only after having used other techniques to considerably lower the vector populations.
The use of the sterile male technique, however, requires colossal investments, and the cost oferadication
may override the long-term benefit.
Jordan and co-workers (1979), Hargrove and Langley (1990), and Langley and colleagues
(1999) proposed sterilisation of tsetse by insect growth regulators or juvenile hormone mimics as
alternati ves to chemical insecticides. These hormones are known to have unquestionable advantages
such as good persistence and the possibility of transmission ofthe product from male to female during
mating. Rather than killing the insects, they affect their capacity to reproduce. Consequently, in epidemic
foci, the disease cannot be stopped quickly, which goes against the principle of rapid anti-vectorial
control.
The question now is what remains in the arsenal of available control methods for 'Glossinologists' ,
considering that any control technique must be effective, fast, cheap and at the same time be easily
applicable in all circumstances? One approach could be to exploit insect pathogens that occur naturally
in insect populations. Among the various pathogens alternatives, viruses, bacteria and protozoa need
to be ingested ifthey are to infect the host. Entomopathogenic fungi, whose infection occurs through
the cuticle, offer a unique opportunity and this characteristic could be exploited. Such fungi are able
to kill the insect and can be transferred from one individual to another by simple contact. Used in
combination with the existing trapping technology, could entomopathogenic fungi be the up-and-
coming solution that may finally encourage decision-makers to apply this anti-vectorial control measure
on a scale large enough to have significant impact?
The present chapter reviews the progress made over the last few years on the development of
entomopathogenic fungi as biocontrol agent for tsetse. A previous review by Laird (1977) assesses
the future for biological methods in integrated control. Two bibliographies on pathogens of medically
important arthropods (Roberts and Strand, 1977; Roberts et aI., 1981) contains authored by Nolan
(1977) and Nolan and Daoust (1981), respectively, on pathogens of Glossina.

2. TSETSE PATHOGENS

Although insect pathogens (fungi, bacteria, protozoa, viruses and nematodes) can cause lethal
or debilitating diseases in a range of pests that attack crops and disease vectors, little information is
available concerning the entomopathogens associated with tsetse flies. The monitoring of the natural
environment that should have provided data on the host spectrum of entomopathogenic fungi, their
relative frequency and their role in regulating natural populations of Glossina spp. (Remaudiere et aI.,
1981) has been neglected. There are only three comprehensive ecological investigations that have
been reported: in Tanzania, Nigeria and the Central Africa Republic. Nash (1933) reported that a
Phycomycetes fungus caused up to 20% mortality in wild populations of G. morsitans, particularly
during periods of heavy rain and flood. Lester (1934) observed a 11-33% mortality in G. morsitans
caused by an unidentified fungus. Vey (1971) reported that 45-50% of pup aria ofG. congolensis

146
that failed to hatch were mycosed with Absidia repens and Penicillium lilacinum. Other than these
studies, conducted over 30 years ago, a few other initiatives by individuals have produced a series of
isolated contributions to the understanding of pathogen-tsetse relationships. Table I gives the list of
fungal pathogens reported from Glossina spp. With the exception of Beauveria bassiana, most of
the fungal species listed are facultative or opportunistic fungi.

Table 1. Fungal pathogens reported from Glossina spp.

Fungus species Glossina spp. Country References

Cicadomyces sp. G. tachinoides Congo Roubaud, 1919
Cicadomyces sp. G. pa/palis Congo Roubaud,1919
Cicadomyces sp. G. morsitans Germany (laboratory) Nogge, 1974
Cicadomyces sp. G. pa/palis Uganda Wallace, 1931
Candida sp. G. morsitans Portugal (laboratory) Oliveira and Nobre, 1970
Cryptococcus a. morsitans Portugal (laboratory) Oliveira and Nobre, 1970
TorulopSis sp. a. morsitans Portugal (laboratory) Oliveira and Nobre, 1970
Rhodotorula sp. G. morsitans Portugal (laboratory) Oliveira and Nobre, 1970
Beauveria bassiana G. pallidipes Kenya Maniania (unpub!.)
B. bassiana G.fuscipes Kenya Maniania (unpub!')
Absidia repens G. congolensis Central Africa Rep. Vey,1971
Penicillium lilacinum G. congolensis Central Africa Rep. Vey,1971
Penicillium sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Aspergillus niger G. pallidipes Kenya Kaaya and Okech, 1990b
A. Jlavus sp. a. pallidipes Kenya Kaaya and Okech, 1990b
A. ochraceus Glossina sp. Chad Vey, 1974 (cited by Nolan, 1977)
Aspergillus sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Fusarium sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Fusarium semi- Glossina sp. South Africa Doidge, 1950
tectum var. majus
Mucor sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Rhizopus sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Trichoderma sp. G. pallidipes Kenya Kaaya and Okech, 1990b
Phycomycetes G. palpalis Tanzania Swynnerton, 1936
Phycomycetes a. morsitans Tanzania Nash, 1933
Phycomycetes Glossina sp. Tanzania Nash, 1970
Phycomycetes a. brevipalpalis Somali Moggridge, 1936
Ascomycetes G.juscipes Uganda Carpenter, 1912
Fungi Imperfecti G.fuscipes Uganda Carpenter, 1912
Unidentified sp. a. palpalis Ghana Macfie, 1916
Unidentified sp. G. palpalis Nigeria Lester, 1934
Unidentified sp. G. palpalis DR Congo Van Hoof and Henrard, 1934

3. INSECT-FUNGUS RELATIONSHIPS

Roberts and Humber (1981) have defined different types of relationships between insects and
fungi:
Pathogens cause early death ofthe host by penetrating and proliferating inside the host, which is
killed by being deprived ofnutrients in its hemolymph, by the invasion or digestion of its tissues, and!
or by the release of toxins from the fungus. Examples ofentomopathogenic fungi include members of
the Beauveria and Metarhizium genera.
Ectoparasites are parasites that may impair host activities and/or cause severe debilitation, but
do not cause death. A fungal example is Laboulbeniales.
Facultative pathogens include weak pathogens that usually attack only old, weakened, diseased,

147
or wounded hosts. The fungus may be capable of penetrating the cuticle but does so only occasionally.
Examples are the Conidiobolus species.
Wound pathogens are incapable of penetrating the insect's cuticle but can invade the haemocoel
through the wound. They are lethal to the insect if entry is accomplished. Examples are Phythium,
Mucor and Trichothecium spp.
A broad variety of fungi are commensals and symbiotes. Trichomycetes, for example, are found
attached to the gut of many insects and other arthropods, but no evidence suggests any parasitic or
pathogenic role.
We consider here true fungal pathogens that cause early death of the host.

3.1 Fungal Infection and the Disease Development Process

Entomopathogenic fungi generally infect their host through the external cuticle. This mode of
infection is unique among all the other entomopathogenic microorganisms (bacteria, viruses and
protozoa) which penetrate the host through the mid-gut. Three phases are recognised in the
development of fungal infection process and disease development:

3.1.1 Adhesion and Germination. Contact between a fungal spore and its insect host is the prerequisite
for the establishment ofa mycosis. The epicuticle of the host integument is the site for the initial fungus-
host interaction. The role of specific molecular receptor interactions at the spore-cuticle interface has
been emphasised (Locke, 1884). In many cases, attachment of the spores to the insect results from an
apparently passive mechanism (Zacharuk, 1970; Michel, 1981). The degree of adhesion depends on the
fungus species. Most of the dry spores are passively attached to the host and may thus be readily
removed. Spore adhesion has also been frequently correlated with virulence or host specificity of a
fungal species. Fargues (1981) was able to correlate host specificity ofM. anisopliae with the ability of
spores to attach to the cuticle of scarabid larvae. The importance of spore adhesion was also revealed
by Al-Aidroos and Roberts (1978) using a mutant of M. anisopliae, which was hypovirulent when
compared to a virulent wild type because of inability of the conidia of the former to attach to mosquito
larvae.
Fungal adhesion was hypothesised by Fargues (1984) to involve electrostatic forces. At the initial
contact, adsorption would involve interaction of charged groups of both the spore and host surfaces
(Pendland and Boucias, 1984), implicating hydrogen bonds or van der Waals forces. This finding has
prompted the idea of formulating fungal conidia electrostatically (Underwood, et aI., 1999). Once the
spore has attached to the insect, it will germinate and produce a germ tube, which will then penetrate
the host cuticle. In addition to serving as the penetrant hypha, the germination structures also playa role
in strengthening the adhesion of the fungus to the insect cuticle. Germinating spores of several
entomopathogenic species produce an appressorial cell at the germ tube-epicutic1e interface (Zackaruk,
1970; Brobyn and Wilding, 1977; Trav1and, 1979; Michel, 1981).
Germination is strongly dependent on rnicroclimatic factors, especially temperature and humidity.
Within a species, some strains are able to germinate using only their own nutrients, whereas the
conidia of other strains need to draw nutrients from their environment.
It is at the level of the cuticle that host specificity may first be manifested. Brobyn and Wilding
(1977) reported that conidia of Neozygites fresenii germinated on susceptible and resistant aphid
hosts but infected only the former. Fargues (1981) also observed that conidia of M anisopliae
isolated from two related hosts germinated only on the cuticle of homologous hosts. Some cuticular
compounds, especially the C s' Cs' and C9 fatty acids, can have an inhibitory effect on germination
(Smith and Grula, 1982). On the other hand, the polar compounds soluble in methanol, containing
salts, amino acids, proteins and phenolic compounds, stimulate the germination of conidia of M
anisopliae in vitro.

3.1.2 Penetration ofthe Host Integument. The cuticle represents the main barrier to fungal infection.
The insect cuticle consists of proteins, chitin, lipids and phenolic complexes (Richards, 1978).
Entomopathogenic fungi are known to produce exocellular proteolytic, chitinolytic and lipolytic enzymes

148
in vitro, and several histological studies suggest that enzymatic activity occurs during penetration (Brobyn
and Wilding, 1977; Lambiase and Yendol, 1977; Grula et a!., 1978). In some instances, high levels of
lipase, protease and chitinase have been correlated with the aggressiveness of entomopathogenic fungi.
For example, chitinase-negative and lipase-negative strains of Beauveria brongniartii are not able to
infect Melolontha melolontha larvae (Paris and Ferron, 1979), and the virulence ofVerticillium lecanii
has been associated with high extracellular chitinase activity (Jackson et a!., 1985). St. Leger et a!.
(1986a-d) reported that all virulent strains of M. anisopliae produced high amounts of proteases. The
application of protease inhibitor to the cuticle surface caused a significant delay in mortality when
compared with the control, thus confirming the involvement of proteolytic enzymes in penetration of the
fungus to the cuticle (St. Leger et a!., 1986b). In some other cases, no correlation was found between
enzymatic activities and virulence.
Not all the conidia that have germinated will penetrate the host cuticle. Delmas (1988) reported
that an aggressive strain of P fumosoroseus germinated and penetrated the epicuticle, while a non-
aggressive strain grew extensively over the cuticle surface with no penetration.

3.1.3 Intra-Haemocoelian Development of the Fungus. Once the cuticular barrier has been
breached, penetrant hyphae bud off blastospores within the haemocoel. However, the ability of the
fungus to develop within the haemocoel depends on its capacity to overcome the immunodefensive
mechanism of the insect. The main immune reaction in weakly or nonpathogenic isolates is the cellular
encapsulation and phagocytosis of the fungal propagules, which are immediately melanised upon
penetration into the haemocoel (Vey and Gotz, 1986). In locusts, granulocytes adhere to entrapped
conidia followed by plasmatocytes; lysis takes place, and within 6-12 h, the entrapped conidial mass
becomes melanised (Miranpuri et a!., 1991). In highly pathogenic isolates, fungi overcome encapsulation
and free living blastospores can be observed in the hemolymph within 24-48 h. Nutrient-rich hemolymph
allows for production of secondary metabolites such as oxalic and citric acids, as well as toxins including
destruxins and beauveracin (Roberts et aI., 1992).
Tsetse, however, like other dipterans exhibit humoral encapsulation characterised by formation
of melanotic capsules around foreign objects (Kaaya et al., 1986). No cellular immune reactions
were observed in tissues infected with M anisopliae and B. bassiana (Kaaya et al., 1991).

4. FACTORS AFFECTING THE EFFICACY OF FUNGI AS BIOCONTROL AGENTS

The key factors that are involved in the cause, the initiation and the development ofdiseases in
insects are the pathogen popUlation, the susceptibility of the host population to the pathogen and the
environmental conditions (Benz, 1987; Tanada and Fuxa, 1987; Watanabe, 1987; Ferron et al.,
1991 ).

4.1 Pathogen Population

The ability to cause infection in a host varies with the different fungal species. Glossina spp. has
been reported to be susceptible to fungal infection (Table 2); however, a strong difference in virulence
has been found among fungal species and strains within species. Poinar et al. (1977) reported that
Hirsutella sp. and Nomuraea rileyi were not pathogenic to adult G. morsitans, and B. bassiana,
M anisopliae and Paecilomyces farinosus were only slightly pathogenic. On the other hand, Kaaya
(1989) found that B. bassiana and M anisopliae were more highly pathogenic to adult G. morsitans
than P fumosoroseus and P farinosus. Within-strain differences in virulence have been reported
with B. bassiana and M anisopliae against G. morsitans (Kaaya, 1989).

149
 Virulence of the pathogen is often measured by the response of the host to a known pathogen
inoculum. Generally, mortality is directly dependent on the inoculum dosage and is measured by
parameters such as LC so ' LD so ' and LTso values. Dosage and concentration values are difficult to
evaluate in dipterans compared to foliage feeding insects because oftheir behaviour. However, Kaaya
(1989) and Maniania (1994) have demonstrated dose-response relationship in Glossina spp. Time-
mortality relationships were also important with the fungal isolates tested (Kaaya, 1989; Maniania,
1994). Metarhizium anisopliae isolate ICIPE 30 was found to kill rapidly with a LTso value of5.0
days (Maniania, 1994).

Table 2. Fungal pathogens tested against Glossina spp. in the laboratory and field

Fungal species Glossina sp. Reference

Beauveria bassiana G. morsitans Poinar et aI., 1977; Kaaya, 1989; Kaaya and
Okech, 1990a,b; Kaaya et aI., 1991; Langley,
1995; Kaaya and Munyinyi, 1995
Metarhizium anisopliae G. morsitans Poinar et aI., 1977; Kaaya, 1989; Kaaya and
Okech, 1990 a, b; Kaaya et al. 1991; Kaaya
and Munyinyi, 1995; Maniania, 1994, 1998
G. centralis Maniania and Odulaja, 1998
G·fuscipes Maniania, 1998
G. pallidipes Maniania, 1998
Nomuraea rileyi G. morsitans Poinar et aI., 1977
Hirsutella sp. G. morsitans Poinar et aI., 1977
Paecilomyces fumosoroseus G. morsitans Kaaya, 1989
Paecilomyces farinosus G. morsitans Poin'lr et aI., 1977; Kaaya, 1989
Entomophthora muscae G. palpalis Vanderyst, 1923
Entomophthora sp. G. palpalis, G. Roubaud, 1911
thachinoides, G.
longipalpis

4.2 Host Population

The susceptibility ofthe host population to fungal infection varies according to host species and
has been demonstrated in many insect groups (Puttler et a!., 1976; Ignoffo, 1981; Boucias et a!.,
1982; Maniania and Fargues, 1984). In the case of Glossina, few studies have been conducted to
evaluate the variability in the susceptibility of these insects to entomopathogenic fungi. Maniania and
Odulaja (1998) found no difference in susceptibility between adult G. morsitans and G. centralis to
M anisopliae. Although not tested in the laboratory, field populations of G. juscipes, G. austeni,
G. pallidipes and G. longipennis were susceptible to the same fungal isolate (Maniania, unpub!.).
On the other hand, in another study, Maniania (unpub!.) observed that under the same experimental
conditions, G. brevilpalpis was less susceptible to fungal infection than G. austeni and G. pallidipes.
Roubaud (1911) reported that adult G. palpalis, G. tachinoides and G. longipalpis were not
susceptible to a strain of Entomo phthorales isolated from Stomoxys calcitrans (a closely related
species to Glossina), while Musca domestica succumbed to the infection.
All the stages of insect development are generally susceptible to fungal infection, and may vary
within one host species according to sex and age. Results with Glossina spp. showed that adult flies
are more susceptible to fungal infection than larvae and puparia, which are refractory (Poinar et a!.,
1977; Kaaya, 1989; Kaaya and Okech, 1990b). However, postponed mortality by mycosis of flies
emerging from treated puparia has been observed (Kaaya and Okech, 1990a,b; Kaaya and Munyinyi,
1995). This phenomenon could result from contamination of emerging flies through the puparium as

150
demonstrated with noctuid egg masses (Fargues and Rodriguez-Rueda, 1980).
Male G. morsitans adults have been reported to be more susceptible to fungal infection than
females (Kaaya, 1989; Kaaya and Okech, 1990a,b). However, Maniania and Odulctia (1998) found
that female adults of both G. morsitans and G. centralis were more susceptible than male flies
(Table 3). The higher susceptibility of male flies was attributed to their smaller sizes (Kaaya, 1989;
Kaaya and Okech, 1990a,b) or to their weaker immune response compared to female flies (Kaaya
and Darji, 1988). According to Maniania and Odulaja (1998), the difference between these two
results could also be explained by the mode of contamination of flies. Insects were dipped into
conidial aqueous suspensions (Kaaya, 1989; Kaaya and Okech, 1990 a,b), while the substrate was
treated in the other experiment (Maniania and Odulctia, 1998). The male genitalia are characterised
by the presence of the button-like hypopygium and sclerotized plates surrounding the anus while the
valve characterises the female genitalia. The tarsi and genitalia are generally in contact with any
substrate. The hypopygium of the male fly is likely to pick up many conidia from the substrate, but
may also loose conidia during friction with the substrate. On the other hand, female genitalia will pick
up few conidia, but the adherence will be high since conidia are lodged in the anus and valve.
The susceptibility of tsetse to fungal infection has also been found to vary with the host age.
Maniania and Odulaja (1998) reported that younger flies (less than 1 day old) of G. morsitans and
G. centralis were more resistant to infection by M anisopliae than older flies (20-and 40-day-old)
and accounted for the largest variability in mortality in the bioassays (Table 3).
Factors such as species, sex, and host age do not respond independently to fungal infection but
do interact, as demonstrated in G. centralis and G. morsitans (Maniania and Odulaja, 1998) (Table
3).

Table 3. ANOVA means squares for differential susceptibility between fly species, sex, and age to
the entomopathogenic fungus Metarhizium anisopliae (Maniania and Odulaja, 1998)

Days after treatment

Source dJ. T3 T4 Ts T6 T7 T8 T9 TIO

Species (P) 0.07* 0.12' 0.34' 0.09'" 0.00'" 0.02"' 0.0"' 0.0'"
SexeS) 0.14* 0.06'" 0.05'" 0041* 0.39'* 0.19' 0.26" 0.09'"
Age (A) 2 0.25" 0.36'* 0.82*** 1.29*'* 1.25"* 1.10'" 1.09*" 0.40***
P"S I 1.03**' 0.48" 0.11'" 0040" 0.15'" 0.17" 0.24'* 0.02'"
P*A 2 0.69'** 0.67*" 0.51** 0.77'*' 0.53*" 0.21 " 0.17" 0.06'"
S *A 2 0.56*** 0.37*' 0.13'" 0.09'" 0.10'" 0.07'" 0.08'" 0.03'"
P *S *A 2 0.18*'* 0.38** 0.43** 0.19'" 0.12'" 0.06'" 0.07'" 0.0'"
Error 48 0.03 0.06 0.06 0.07 0.05 0.03 0.03 0.03

"'Not significant; *Significant at P < 0.05; "Significant at P < 0.01; "-Significant at P < 0.001; dJ.: degrees of
freedom; T3: day 3 after infection: ....etc.

4.3 Environmental Factors

Entomopathogenic fungi are subject to a plethora of biotic and abiotic factors that influence
their survival and ability to cause diseases in their host (Benz, 1987). To better predict efficacy under
field conditions, the effect of environmental constraints must be determined in order to accurately
identify control windows. Ofthe various environmental parameters that affect insect fungal pathogens,
temperature, humidity and solar radiation are probably the most severe.

4.3.1 Temperature. Temperature is an abiotic limiting factor that affects rate of germination, growth,
sporulation and survival of entomopathogenic fungi. It also acts not only on the pathogen alone and

151
on the host, but also on the pathological interaction as a whole (Fargues et aI., 1992; Maniania and
Fargues, 1992). Numerous studies have established temperature optima for infection, growth and
sporulation at temperatures between 20-30° C (Walstad et aI., 1970; Roberts and Campbell, 1977;
Hall and Papierok, 1982). However, variation in temperature tolerance within strain also exists, and
generally, fungal strains originating from tropical regions are more thermo-tolerant than strains from
temperate climates (Fargues et aI., 1992; Ekesi et aI., 1999). Such variation permits selection of
isolates tolerant to the temperature range found within the ecosystem in which the pathogen is to be
used. Thus, in order to develop fungal pathogens for the control ofthe elm bark beetle, Scolytus
scolytus, and the black vine weevil, Otiorhynchus sulcatus, both of which inhabit temperate regions,
Dorbeski (1981) and Soares et al. (1983) selected strains with pathogenic activity below 15°C. On
the other hand, the LUBILOSA project selected strains with pathogenic activity above 30° C for the
control of the desert locust in West Africa (McClatchie et aI., 1994). The therrmal death point of
conidia was assessed as 50-70° C for several minutes (Robert and Campbell, 1977). Walstad et ai.
(1970) recorded 10 min at 49 and 50° C as the thermal death point for M anisopliae and B.
bassiana, respectively.

4.3.2 Moisture. Fungal germination and sporulation requires saturated or near saturated moisture or
free water, and many pest control failures have been attributed to dry weather conditions. However,
dry conditions have been shown to be less critical for infection than was previously thought (Ferron et
aI., 1991). Metarhizium anisopliae var. acridium (=M flavoviride) can infect the desert locust at
relative humidities as low as 13% and the fungus can even produce spores within cadavers under dry
weather conditions (Fargues et aI., 1997). The ability of fungi to infect under dry conditions has been
attributed to the persistence of moisture in the microhabitat, such as on abaxial leaf surfaces and in the
intersegmental membrane of the cuticle. Although it is now established that the moisture effect is less
detrimental than was thought before, it is still very important in horizontal transmission of many
Entomophthoralean species and for infection of blastos pores of V lecanii.

4.3.3 Solar radiation. The B component of ultraviolet radiation (UV-B; 280-320 nm) of the solar
spectrum is highly detrimental to entomopathogens (Tevini, 1993). Half-lives of less than 2 days have
been reported (Ferron, 1991). However, the differences in susceptibility to irradiation vary with fungal
species and strains within species (Fargues et aI., 1996). On soybean foliage, N. rileyi and B. bassiana
persisted for 2-5 days under field conditions and on cabbage and pigeon pea plants, the half-life of N.
rileyi decreased to 3.6 h (Ignoffo et aI., 1976; Gardner et aI., 1977). In protected locations within plant
canopies, irradiation is substantially reduced relative to exposed surfaces (Inglis et aI., 1993; Ekesi et
aI., 2000), however even under shade, propagules will eventually be killed (Smits et aI., 1996). Addition
ofUV-B protectants may help increase persistence (Moore et aI., 1993; Inglis et aI., 1995). Modifying
application practices as by spraying in the evenings to avoid midday sun can also lessen the adverse
effect ofuItraviolet radiation (Clarkson, 1992). In autoinoculator devices, propagules are even likely to
survive for a longer period since most devices provide adequate protection against direct sunlight.

5. APPLICATION STRATEGIES

5.1 Design of Laboratory Infectivity Bioassays

Experimental infections, induced under controlled conditions, allow testing of the pathogenic
potential ofdifferent fungal strains and the susceptibility of different target species (Hall and Papierok,
1982). Insects that are not susceptible to fungal infection in nature can be tested for their susceptibility
in the laboratory. Diverse modes of inoculation are used to perform bioassays: spraying conidia on
the host organisms; exposing insects to treated leaves; dipping insects into titrated conidial suspensions;
and treating the rearing substrate. The usual mode of inoculation of adult dipteran hosts with
hyphomycetes fungi has so far been either by dipping insects into titrated conidial suspensions (Kaaya,
1989) or by exposing them to dry conidia (Clark et aI., 1968; Poinar et aI., 1977; Rizzo, 1977). In

152
all cases, the flies were completely covered either by wet or dry conidia. Maniania (1994) discussed
the relevance of these two modes of contamination in the light ofthe behavior ofdipteran flies, which
stand on the tarsi. It is through tarsal contact that flies would pick up conidia from the substrate in
nature. A simple laboratory technique for infecting adult tsetse flies with fungal pathogens was
subsequently developed by the author. The technique uses a nitrocellulose filter membrane (Millipore)
as substrate for the conidia and which thereby allows accurate estimation of the inoculum on the
substrate. Flies are also allowed to walk freely on the contaminated substrate (Maniania, 1994).

5.2 Design of Field Delivery Systems

Inundative and augmentative releases are the main methods employed for introduction of
entomopathogens, including fungi, into the ecosystem (Lacey and Goettel, 1995). However, a new
strategy is currently being considered, whereby insect pathogens are disseminated among target pest
popUlations by using devices that attract insect pests into a focus of the pathogens (Vega et al.,
2000). Autoinoculating devices have been developed for the introduction ofB. bassiana against the
dusk beetle, Carpophilus lugubris (Vega et al., 1995) and the bark beetle, Ips btypographus
(Vaupel and Zimmermann, 1996), M anisopliae against the Japanese beetle, Popi/liajaponica
(Klein and Lacey, 1999), and Zoopththora radicans against the diamondback moth, Plutella
xylostella (Pell et al., 1993; Furlong et al., 1995).
Since males and females of many tsetse fly species respond strongly to visual and olfactory
cues, in addition to their low rates of reproduction and low population densities, several different
traps have been developed as a means of controlling them (Dransfield et al., 1990; Laveissiere et al.,
1990; Wall and Langley, 1991). Flies that are attracted to the trap are forced to enter a polyethylene
bag mounted on top of the trap, where they are killed due to suffocation or extreme temperatures.
Maniania (1998) provided the first attempt at developing autoinoculating devices containing fungal
spores that replace the polyethylene bag as a means ofinfecting male and female flies entering the
trap. The author tested several inoculating chambers of different sizes (Maniania, 1998). Each chamber
was made ofclear Plexiglas with the inner side covered with a metallic wire mesh lined with a nylon
mesh. With the exception of one c~ber, all the chambers had the top part open to allow the flies to
exit the chamber. The other inoculating device had the top part closed to prevent rain from entering
the chamber, but the four sides ofthe chamber were perforated with holes (7 mm diam.) that served
as exit holes for the flies (Maniania, 1998). A circular hole (3.0 cm diameter) made in the centre of
the bottom part of the chamber was fitted with a cone (4 x 2.5 cm), that served to connect the
chamber to a biconical trap ofChallier et al., 1977. The autodissemination devices were tested on
natural populations ofG. pal/idipes and G. longipennis at Nguruman in the Rift Valley, southwestern
Kenya, and G.fuscipes at ICIPE's Mbita Point Field Station on Ljike Victoria in western Kenya.
The experiments were intended to (i) assess the rate offungal infection in the flies crossing the chamber,
(ii) test whether trapped flies can exit the infection chamber, and (iii) select the best inoculating chamber,
in terms of inducing high fungal infection. Biconical traps bearing auto inoculator chambers were
deployed in the woodland areas in a linear fashion along the lakeshore. Between 1-1.5 g of dry
conidia ofM anisopliae were smeared on the nylon mesh lining the inner sides ofthe chambers. The
amount of conidia varied according to the size of the chamber. Each chamber was fitted with a
collector to capture flies exiting the system. Flies were transferred from the cage to a PVC tsetse-
rearing cage and brought to the laboratory where they were maintained at ambient conditions.
Results indicated that the three tsetse species (G. pallidipes, G. /ongipennis and G. fuscipes)

153
that were attracted to the traps successfully entered the autoinoculator device, and were contaminated
with Metarhizium conidia and became infected. However, the numbers of flies entering the
auto inoculator and the numbers of insects that died from fungal infection varied according to the type
of chamber. One auto inoculator outperformed the others and was selected for further studies after
minor modifications. The modifications included lining the inner sides of the chamber with a velvet
material to increase adherence ofconidia on the surface of the velvet, and fitting a small basket on the
upper side of the chamber to reduce the exit space for the flies while increasing their contact with the
conidia (Figure I).

Figure I. Infection chamber for tsetse (A, B). (B) improved infection chamber with a small basket for fungal
conidia at the top that acts as a barrier to reduce the exit space for flies and increases their contact with the conidia;
the top piece of the chamber shows the movement of flies. Flies that enter the trap are forced to enter the chamber,
which is mounted on the top of a biconical trap.

The auto inoculator was, however, found to be both complex and expensive to construct and,
therefore, impractical for use by peasants. A simpler autoinoculator has now been developed using a
clear plastic mineral water bottle (Figure 2) (Maniania, in press). The bottle is divided vertically into
two equal sections by nylon mosquito netting. The bottom of the lower section is lined with a raw
strip of sheep's wool, and a square hole (4 x 4 cm) that connects the unit to the apex of the trap
(Figure 2). A 2-cm diameter hole made below the mouth ofthe bottle serves as the fly exit. The
positioning of the exit hole on the underside prevents entry of rain. The lower surface of the mosquito
netting running through the length ofthe bottle is also woolen-lined to enhance contamination of the
ventral and dorsal surfaces of the escaping flies. This newly developed auto inoculator chamber was
named' Maniania's contamination device' (or Maniania's Cd). It has also been tested in the field on
Rusinga Island near Mbita Point Field Station, following the same protocol as described earlier.
Observations on flies that had passed through the autoinoculator at Rusinga Island revealed
100% mortality caused by M anisopliae. The time spent by flies in the auto inoculator device and the
number of conidia subsequently collected varied between 5-240 seconds; however, approximately
58% of the flies spent less than 60 seconds in the autoinoculator device. The mean number of conidia
picked up by flies varied between 1.6 x 105 and 40.5 x 105 conidia per fly. Since 100% mortality was
observed among flies that passed through the Cd, it could be assumed that the inoculum at the lowest
concentration, i.e. 1.6 x 105 conidia per fly, was lethal. This amount ofconidia collected by flies in the
autoinoculator is in the range ofthe inoculum required for fly-to-fly contamination in the laboratory
(Maniania, unpublished). This suggests that a fly passing through the auto inoculator will be able to
pick up a lethal dose of conidia and pass them to healthy flies during mating and/or casual contact.

154
 The viability of conidia in the auto inoculator was also investigated. After a 31-day exposure in
the field, germination of conidia dropped from 86% at day 0 to 62% at day 31. The lost of viability
did not, however, affect the infectivity of the fungus, which caused over 90% mortality (by growth of
the fungus on the surface of the cadavers) amongst G. jitscipes flies.

Figure 2. Inoculating device (Maniania's Cd) made from a clear plastic mineral water bottle, divided into two equal
sections longitudinally by nylon mosquito netting, and mounted on the top of a biconical trap of Challier et aI. ,
1977 (Maniania, in press)

6. LARGE-SCALE FIELD SUPPRESSION TRIALS AND PROSPECTS OF THE

INOCULATING DEVICE FOR TSETSE CONTROL

The prospects of using Maniania's Cd for population suppression oftsetse and autodissemination
offungal conidia were tested under large-scale field conditions on three islands (Mfangano, Nzenze
and Ngodhe) in Lake Victoria, Suba District, western Kenya, between 1999 and 2000. Annual
rainfall in this area ranged from SOO-1 000 mm and mean annual maximum temperature in the region
is within 2S-30°C. The three islands harbour G.fuscipes as the main species of tsetse fly.
Conidia of M anisopliae were produced following the technique described by Maniania (1998).
Dry conidia ofthe fungus (1.S-2.0 g/Cd) were used to smear the surfaces of the woolen strips.
Approximately 160 pyramidal traps (Gouteux and Lancien, 1986) mounted with autoinoculators
were deployed along the lakeshore and rivers on Mfangano Island at 200 to 300-m intervals. Three
pyramidal traps were deployed as the conventional 'trap and kill' population suppression method
(Gouteux et al., 1986; Dransfield et al., 1990) on Nzenze Island, at approximately the same trap
density as on Mfangano Island. The third island, Ngodhe, remained untreated. Traps were serviced
monthly: autoinoculator devices were emptied ofloose conidia and were recharged; killing bags
were also exchanged. Apparent changes in population size on the three islands were monitored
weekly using biconical traps (Challier et aI., 1977). Twelve biconical traps were set at Mfangano
Island, three on Ngodhe Island and two on Nzenze Island, the number of traps approximating the
relative sizes of the islands. Grease was applied to the poles supporting the traps to prevent ants from
damaging the catch. Prior to the setting up of the experiments, samples of flies from the islands were
tested for any existing fungal infection in the population, but none was found. '
Results revealed that population catches of both female and male G.fuscipes in the fungus-
treated and 'trap and kill' islands were relatively low compared to the untreated island (Figure 3). A
substantial reduction offlies was observed in 'trap and kill' treatment during the first S weeks following
the initiation ofthe trial. However, the fungus treatment generally maintained a more consistently low
population than the 'trap and kill' treatment.

155
 300

250

r::
&' 100

50 .

o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
Week

: - - Cd - - NZEN ~ CONT '

Figure 3. Catches of male and female G/ossinaJuscipes/trap/day on Mfangano (Cd), Nzenze ('trap and kill') and
Ngodhe (untreated control) islands. Data after 2,3 and 5 months following the termination of the treatments are
presented as week 51,52 and 53, respectively

The prevalence of infection caused by M anisopliae in tsetse flies on the fungus-treated island
was low during the first weeks but increased thereafter (Figure 4). Fly population catches in the 'trap
and kill' treatment built up faster than in the fungus treatment 3 months after removal ofthe treatments,
suggesting the presence and persistence ofthe fungus in the tsetse populations (Figure 3). The results
are indicative ofthe potential ofusing an autoinocuiating device as a control method for tsetse flies. A
more extensive testing, however, is required to truly quantify the advantage of the Cd.
100 , - - - - - - - - - - - - - - - - - - - -

90

~ 80
"B
~ 70
.::
~ 60
~
~ 50

40

6 8 10121416182022242628303234363840424446485052
Week
Figure 4. Fungal infection by Metarhizium anisop/iae in Glossinafoscipes population during the trial (1999-2000)
on Mfangano Island in Lake Victoria

7. EFFECTS OF FUNGAL PATHOGENS ON NONTARGET ORGANISMS

The safety of fungal pathogens to nontarget arthropods has recently been reviewed by Goettel
et aI. (1990), Vinson (1990), Roy and Pell (2000) and to vertebrates by Saik et aI. (1990) and Siegel
and Shadduck (1990).
Among insect pathogens, fungi have the widest spectra ofhost ranges. For instance, Metarhizium
spp. have been reported on more than 200 hosts (Veen, 1968). However, Goettel et al. (1990)
cautioned on any conclusions being drawn based on such host lists, since many records are based on
a single specimen with dubious identification of both host and pathogen, and the fact that host ranges
have rarely been verified experimentally. Furthermore, laboratory studies have shown that different

156
isolates ofthe same species have varying degrees of specificity (Fargues, 1976; Maniania and Fargues,
1984).
Extensive studies conducted on the safety of fungal pathogens to beneficial arthropods (Goettel
et al., 1990; Vinson, 1990; Roy and Pell, 2000) indicate the positive nature of the interactions between
arthropod natural enemies and entomopathogenic fungi.
Springate et al. (2000) studied the effects of application ofM anisopliae used to control tsetse
flies on the taxonomic and guild composition of the non-formicid hymenopteran communities at
Nguruman over a 1O-week period during and after the application of M anisopliae. Ten biconical
traps mounted with the 'Maniania Cd' were loaded with conidia, as described earlier, and deployed
at 1OO-m intervals along the riverbank. It was intended that the tsetse as well as the nontarget organisms
passing through the inoculator would be infected with conidia and would be dispersed into the
environment. Hymenopteran populations were monitored using Malaise traps. Familial diversity was
similar before and after fungal application. However, significant differences were found in species
richness before and after application ofM anisopliae, likely due to the increasing aridity during the
experimental period than to fungal application (Springate et a!., 2000). No significant differences
were noted between the richness of ectoparasitoids pre- and post-application.
Metarhizium spp. has never been reported to infect humans (Siegel and Shadduck, 1990).
Rats, mice and rabbits given this entomopathogen by inhalation, oral administration, s.c. injection, i.p.
injection, and topical administration had no signs of infection or illness.

8. CONCLUSIONS

With a growing world population and the resulting demand for more and better food, improved
health and the need for a more sustainable production/consumption pattern, there is a need to seek
new strategies for the control of many vector-borne diseases that affect both animals and humans.
The two kinds of tsetse-borne trypanosomoses- human sleeping sickness and the animal disease
nagana- are part of the problem of underdevelopment in Africa, and their control will bring about
the needed reliefto allow for major strides forward in tenus ofsustainable and equitable development.
While there are major undertakings underway in the control of malaria, sleeping sickness and
nagana have been neglected over the past 20 years. It is the latest outbreaks of the disease both in
humans and animals that have rekindled interest from donors and governments that are now leading
to new programmes. Other key factors in the resurgence of interest in controlling the diseases are
two recent successful projects: (i) the tsetse control campaign in Ethiopia, where the Government has
successfully controlled the fly over an area of approximately 40,000 km2 using a trapping method.
This has been achieved With the technical backstopping ofICIPE and funding from the European
Union, and (ii) the announcement by the International Atomic Energy Agency (IAEA) and FAO that
eradication of tsetse on the island of Zanzibar has been achieved using SIT (IAEA, 1997).
The above approaches ~e not new, and have been tried before with mixed results; neither
approach has brought about a lasting solution. The SIT approach is very expensive, the results less
than promising (except in the very special case of an island situation) and is 'top-down', with little if
any community participation. One ofthe premises for a successful SIT campaign is the long-term
commitment by the governmellt of the affected country, but this has never materialised in any project.
The environmental, social and economical aspects have not been considered in full, nor the biological
and ecological constraints posed by the tsetse fly which make it more difficult to control, than say, the
fruit fly or the screwworm, both of which have been successfully controlled by SIT.
It is therefore a major challenge to find an alternative strategy that will respect the basic principles
ofsustainable development: the approach being implemented must be environmentally safe, socially
and economically sound and affordable, and within the technical and financial reach of the communities
or farmers. Tsetse, like any other insect, will require methods that try to 'outsmart' it, not to beat it to
death with excessive force (e.g. SIT). A combination of approaches that will not lead to the

157
development of escape mechanisms nor have side effects that in the end are worse than the original
problem is what is needed. The use ofa combination of baited tsetse traps with an entomopathogenic
fungus may be a key component in a new sustainable tsetse control strategy under the many different
ecological and social conditions where both sleeping sickness and nagana do occur. The proposed
use of M anisopliae, given its potential as an environmentally friendly biopesticide, will go a long
way in expanding the tsetse control toolbox. The experimental data available to date need further
confmnation and more field research is needed on the deployment and integration of these new tools
with older ones. Areas that need such investigation include the effect of the Cd on pregnant and
teneral flies that are usually not readily susceptible to trapping.
Yet another issue to be resolved when using entomopathogens relates to policy and registration.
Entomopathogens are naturally occurring organisms that need to be treated in a different manner than
synthetic pesticides. The governments of African countries need to work closely with bodies such as
the OUAIIAPSC to standardise the regulations and speed up the registration procedure in order to
give a fair chance for these products to be produced and used on a large scale.
The use of entomopathogenic fungi in the control of tsetse has reached the point where more
research, building on the latest advances, will bring about a major move forward, in particular in the
formulation and application of the product. Given that only an integrated approach to vector
management will bring about the needed results (just as in the case ofIPM for crop pests), the time
is right to promote entomopathogens in the combat against the major disease vectors, including the
tsetse.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. D.J. Nadel for his contribution to the work, Dr. A.N. Mengech
(ICIPE Science Editor) for reviewing the draft of this chapter, and to J.O. Adino and J.O. Op~re for
technical assistance. This work received financial support from the European Union (EU) and Austrian
Development Co-operation (ADC).

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163
METARHIZIUM ANISOPLIAE: AN EFFECTIVE BIOLOGICAL CONTROL AGENT
FOR THE MANAGEMENT OF THRIPS IN HORTI- AND FLORICULTURE IN
AFRICA

Sunday Ekesi and Nguya K. Maniania

International Centre ofInsect Physiology and Ecology (ICIPE), PO Box 30772,

Nairobi, Kenya

1. INTRODUCTION

The legume flower thrips, Megalurothrips sjostedti (Trybom) [Thysanoptera: Thripidae], the
onion thrips, Thrips tabaci Lindeman [Thysanoptera: Thripidae] and the western flower thrips,
Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae] are major pests of vegetable and
ornamental crops in Africa. Megalurothrips sjostedti is considered as the major pest attacking the
reproductive structures of cowpea (Vigna unguiculata (L.) Walp) [Leguminosae], an important
pod and fodder crop that provides more than half the plant protein in human diets on the continent.
Damage by M sjostedti on cowpea is characterised by distortion, malformation and discolouration
ofleafbuds, flower buds and flowers, leading to necrosis and/or abscission (Ezueh, 1981). Yield
losses vary between 20 to 100% in different parts of Africa (Singh and Allen, 1980).
Onion thrips causes serious damage to cultivated crops, mainly on the Alliaceae plants and
Brassicas, which are important income-generating crops for multitudes of small-scale farmers. The
insect causes direct damage by destroying the epidermal cells, causing the leaves to completely
whiten (Ghabn, 1948), and indirect damage by transmission of viruses (Sakimura, 1963). Significant
yield losses (up to 50%) can result (Ampong-Nyarko and Sithanantham, unpubl.).
The westem flower thrips is a polyphagous and widely distributed pest attacking pepper, cucumber,
eggplant, onion, tomato, grape, strawberry, peach, nectarine, gloxinia, chrysanthemum and other
vegetable and flower crops. Feeding damage causes a flecking on growing leaf surfaces, flowers and
fruits. Feeding on growing leaves causes distortion and mottling of the foliage while fully expanded
leaves take on a characteristic silvery appearance as the dead cells are filled with air. In addition to
direct damage, western flower thrips also serves as the vector ofTornato Spotted Wilt Virus (TSWV)
and Impatiens Necrotic Spot Virus (INSV) which can cause destruction of the entire crop (Gill et al.,
1998).
Chemical insecticides are the major control measures used in practice for all three species of
thrips. In most cases, and particularly on western flower thrips, treatment applications are often
repeated, causing residue accumulation and resistance problems. The western flower thrips is believed
to have developed resistance to all the major classes of chemical insecticides (Jensen, 2000). The

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 165
world-wide tendency towards reducing chemical insecticide usage because ofthe associated problems
of environmental contamination, adverse effects on non-target organisms, and the demand for
pesticide-free foods, among others, has necessitated the search for other viable control strategies for
thrips management. Fungi have a role to playas microbial insecticides for thrips control because they
are known to be the pathogens most closely associated with thrips in nature (Butt and Brownbridge,
1997). Because of their sucking behaviour, insects such as thrips are prone to fungal attacks almost
exclusively, because only these pathogens possess the necessary enzyme to penetrate the insect
cuticle, and do not need to be ingested to infect their host.
Within the last decade, the development and use of fungi as microbial insecticides have increased
considerably with the discovery of new strains and genetic improvement of others (Lacey and Goettel,
1995). Stable products that are easily tank -mixed for spraying and that are cost -competitive with
chemical insecticides have been developed and are now available in different parts ofthe world,
including Africa (Shah and Goettel, 1999). This paper reviews work on the microbial control of
thrips undertaken at the International Centre of Insect Physiology and Ecology (lCIPE), particularly
with regard to the use of Metarhizium anisopliae (Metsch.) Sorok. as a mycoinsecticide.

2. LABORATORY EXPERIMENTS

2.1 Strain Selection

The successful development of entomopathogenic fungi as microbial insecticides requires careful

and appropriate selection of the most efficacious strains (Soper and Ward, 1981). Twenty-two
isolates of entomopathogenic fungi (obtained from the lCIPE Germplasm Centre) were bioassayed
against the adult stage of the target insect, M sjostedti. Using a standard concentration of I x 10 8
conidia ml- I , all the strains were found to be pathogenic to adult M sjostedti; however, considerable
variation was detected between both species and strains within species (Table 1). Two strains of
Beauveria bassiana (Bals.) Vuil. (ICIPE 53 and Technical powder - GHA) and four strains of M
anisopliae (ICIPE 30, 66, 69 and 74) were the most pathogenic, causing 100% mortality in 7 days
with LT jO ranging between 2-3 days (Table 1). The LC jO were within the range of 1.3-7.9 x 10 6
conidia ml- I ) (Ekesi et aI., 1998a). Strains of Verticillium lecanii (Zimm.) Viegas andPaecilomyces
fumosoroseus (Wize) Smith and Brown were generally less pathogenic.
It is a general consensus that strains of entomopathogenic fungi are most pathogenic to the
species of insect from which they are isolated. This was not found to be true for ICIPE 83, which was
isolated from M sjostedti. Apart from ICIPE 30 and 74, the most pathogenic strains were isolated
from a soil substrate using the 'Galleria bait method' (Zimmermann, 1986). This suggests that although
widely distributed, fungi such as B. bassiana and M anisopliae could be relatively host-specific as
pathotypes. Other environments such as the soil also offer the potential as sources for highly virulent
isolates and warrant further exploration.
Temperature is one of the environmental factors that influences fungal growth and disease
development in insects (Benz, 1987). Increased interest in the use of pathogens in pest management
programmes necessitates the selection of strains tolerant to the temperature range found in the
ecosystem which the target pest inhabits (Ferron et aI., 1991). Field studies have revealed that active
populations of the legume flower thrips can be found at temperatures as low as 15°C and as high as
30 DC (Alghali, 1991; Ekesi et aI., unpublished). Identifying a strain with a broad temperature range
was therefore found necessary for a rational approach to M sjostedti management. The germination,
growth and pathogenic activity of the six strains selected above were tested at 15,20,25,30 and
35°C. The six strains were found to grow and germinate at all temperatures tested, but germination
and growth were slower at 15 and 35°C, with the optimum occurring at 25 and 30°C. Strain ICIPE
69 showed pathogenic activity over a broad range of temperature compared with the other strains
(Ekesi et aI., 1999a) (Table 2), and was therefore selected for further studies. The optimum temperature
range between 25 to 30 DC for growth and pathogenicity to M sjostedti compared favourably with

166
the optimum range of27 to 2~C for high thrips numbers observed under field conditions (Alghali,
1991).

Table 1. Pathogenicity ofentomopathogenic fungi to adult Megalurothrips sjostedti: percentage

mortality rates and LT50 values of different strains at a concentration of 1 x 108 conidia ml- l at 7
days post-treatment
Fungal species Strain Percentage LT,o in days
mortality ± SE (95% Fiducial limit)
Beauveria bassiana ICIPE48 70.0±4.1c 5.5 (5.3-5.9)
ICIPE53 IOO.O±O.Oa 2.9 (2.8-3.0)
ICIPE59 28.8±6.3e 7.9(7.6-8.1)
ICIPE77 43.8±2.5d 8.1 (7.8-8.5)
ICIPE78 31.3±4.8e 8.4 (8.0-8.6)
ICIPE82 43.8±2.4d 7.5 (7.3-7.7)
ICIPE83 71.1 ±4.2c 5.4 (5.2-5.5)
TP-GHA 100.0±0.Oa 2.7 (2.5-3.0)
Metarhizium anisopliae ICIPEl8 62.5±2.9c 6.1 (5.8-6.2)
ICIPE20 53.8±2.5d 6.8(6.6-7.1)
ICIPE30 IOO.O±O.Oa 2.5 (2.4-2.6)
ICIPE60 62.5±2.9c 6.1 (5.9-6.2)
ICIPE62 93.8±9.5b 3.3(3.1-3.5)
ICIPE63 93.8±9.5b 3.4(3.2-3.6)
ICIPE66 IOO.O±O.Oa 2.7 (2.5-2.8)
ICIPE67 85.0±9.lb 4.2 (4.0-4.4)
ICIPE69 100.0±0.Oa 2.4 (2.2-2.6)
ICIPE74 100.0±0.Oa 2.5 (2.2-2.6)
ICIPE75 91.3±4.9b 3.3 (3.0-3.4)
Verticillium lecanii ICIPE85 67.5±5.0c 8.4 (8.1-8.7)
ICIPE86 28.8±4.8e 9.5 (9.3-9.8)
Paecilomyces fumosoroseus MY613 12.5±2.7f 8.7 (8.5-8.9)
Means (± SE) within a column followed by the same letter do not differ significantly by Student- Newman-
Keuls' test (P < 0.05).

Table 2. Effect of temperature on virulence of strains ofBeauveria bassiana and Metarhizium

anisopliae on adult Megalurothrips sjostedti

% mortality caused by fungus

Species Strain 15°C 200C 25°C 30°C

B. bassiana ICIPE53 27.4± I.3c 88.4± 1O.7c IOO.O±O.Oa 65.1 ± 11.7c

TP-GHA 33.9± 10.lc 85.2± 1.4c 96.2±4.7ab 100.0±0.Oa
M anisopliae ICIPE30 64.0± 1.6b 94.6± l.3c 96.2±4.lab 100.0 ± O.Oa
ICIPE66 88.9±5.9a 100.0±0.Oa 92.2± 1.6b 74.7± 1.9b
ICIPE69 72.1 ±9.9b 100.0 ± O.Oa 100.0±0.Oa 100.0±0.Oa
ICIPE74 34.5±2.5c 97.4±3.8b 100.0±0.Oa 100.0±0.Oa

Means (± SE) within a column followed by the same letter do not differ significantly by Student~Newman­
Keuls' test (P <0.05).

All insect developmental stages are generally susceptible to fungal infection (Ferron et al., 1991).
When M anisopliae isolate lCIPE 69 was evaluated against different developmental stages (Larva
II, pupa and adult) of thrips, all stages were found to be susceptible to infection, although the larval
and pupal stages were less susceptible to.infection than the adult stage (Table 3). The differential
susceptibility at various life stages can be attributed to interaction between the fungus and ecdysis of
larval and pupal stages. Ecdysis is known to be an important factor in insect resistance to fungal

167
infection, particularly when the time interval between successive ecdysis is short (Vey and Fargues,
1977). Although mortality at the larval stage was reduced, adult thrips surviving infection as larvae
produced fewer numbers of eggs and showed reduced egg hatchability and longevity than untreated
insects (Ekesi et aI., 2000a).

Table 3. Susceptibility of different developmental stages of Megalurothrips sjostedti to

Metarhizium anisopliae ICIPE 69: percentage mortality and LT so at 8 days post-treatment

Concentration (1 x 10' conidia mi")

Stage 106 10' 108 LT,o(days)'
Larva II IS.2 ± 1.6Bc 20.3±2.5cB 25.7± 1.8cA 9.5 ± 1.2a
Pupa 20.S ± 3.4bC 38.2± 1.9bC 45.6± 5.3bA 7.2± 1.3b
Adult 64.1 ±7.SaC 72.7±S.8aB 100aA 3.1 ±O.4c
Means (± SE) within-column bearing the same lower case letter and within-row bearing the same upper case
letter are not significantly different by Student-Newman-Keuls' test (P < 0.05) test. ' LTso values are based
on a cqncentration of 1 x 10' conidia mi".

2.2 Mass Production and Formulation

Mass production of conidia is a prerequisite to large-scale field application. This can be achieved
by liquid fermentation or submerged culture and/or solid substrate fermentation. Until recently,
submerged conidiation was unknown for Metarhizium spp., but has now been achieved with
Metarhizium anisopliae var. acridum (=Mflavoviride Gams & Rozyspal) obtained from a range
of orthopteran hosts (Jenkins and Prior, 1993). There are also preliminary indications that M
anisopliae ICIPE 69, isolated from the soil, can also be produced in submerged cultures (D. Stephan,
pers. comm.). In our studies, however, we adopted the solid substrate fermentation systems based
on methods developed in Brazil by Mendon9a (1992) for the production ofICIPE 69. This involves
production of blastospores in shake flasks by liquid fermentation followed by surface conidiation on
the solid substrate in plastic bowls. The solid substrate consisted of ground maize/vermiculite that had
been autoclaved for I hat 121°C. The substrate was then transferred to the bowls (33 x 25 x 13
em), inoculated with a 3-day-old cultUre of blastospores (50 ml), and covered with a sterile polythene
bag. Cultures were incubated for 21 days at ambient conditions. The polyethylene bag was then
removed, and the culture allowed to dry for 5 days at room temperature. Conidia were harvested by
sifting the substrate through a sieve (295 flm mesh size). The conidia were stored in a refrigerator (4-
6°C; 40-50% RH) before being used in the field.
No formulation was performed on harvested conidia until field applications. For spray application,
conidia were suspended in water containing 0.05% Silwet L-77® (organosilicone surfactant/emulsifier)
for high volume (HV) aqueous formulation. The ultra-low volume oil/aqueous formulation contained
50:50 com oil and water and 0.05% Silwet L-77. Nutrient agar (0.1 %), glycerine (0.1 %) and molasses
(0.5%) were added to the inoculum as protectant and bait to complete the formulation.

3. FIELD TRIALS

3.1 Efficacy of Metarhizium anisopliae against Megalurothrips sjostedti on Cowpea

Field trials were conducted at ICIPE's Mbita Point Field Station on the shores of Lake Victoria,
western Kenya. The first trial evaluated the efficacy of two formulations ofconidia: ULV oil/aqueous
formulation and HV aqueous formulation, each applied at two concentrations of 1 x 1011 and I x

168
10 13 conidia ml- ' during the September-December, 1996 and March-July 1997 rainy seasons_ The
treatments were laid out in a 6 x 6 quasi-complete Latin square design to ensure that uneven pest
invasion of the crop from one side did not bias the experiment, as each treatment appeared next to
every other treatment twice in both rows and columns (Bailey, 1984; Bailey et al., 1989). The fungal
treatments were compared to a synthetic insecticidal treatment using lambda-cyhalothrin (Karate®
17.5 EC) applied at the recommended rate of 1 litre ha- ' of the commercial formulation, containing
17.5 g a.i. ha- ' . In both seasons, three applications were performed. The ULV formulation was
administered with a Micron-Ulva spinning disk sprayer (Micron Sprayers Ltd) at an output of 1 litre
ha- ' and the HV formulation was applied with a CP 15 knapsack sprayer (Cooper Pegler) at an
outputof350 litre ha- ' .
Application of the fungus resulted in a significant reduction in thrips density with a concomitant
increase in grain yield (Table 4). A high proportion ofthe insects collected from the treated field died
from mycosis in both seasons (Ekesi et al., 1998b). The ecological niche associated with M sjostedti
within leafbuds and flowers ofcowpea evidently provided sufficient microclimate humidity for fungal
infection to take place (Benz, 1987), thus contributing to the high mortality. The HV formulation
generally performed better than the ULV formulation. It is probable that the spray droplets forced
under pressure from HV application into flowers (sites of high thrips numbers) enhance the direct
contact of the pathogen with the insect, hence the superior level of control (Brownbridge et aI.,
1996).
On-station trials using the same fungus against M sjostedti on cowpea have also been carried
out at the International Institute ofTropical Agriculture (IITA), Cotonou, using protocols supplied by
ICIPE. It is reported that the fungus was as effective as the standard chemical insecticide, and more
efficacious than papaya leafand neem extract at controlling thrips and protecting cowpea yield (UTA,
1999).

Table 4. Effect ofMetarhizium anisopliae application on mean (± SE) cowpea grain yield (kg
ha- ' )

Treatment First season Second season

Control (ULV) 572.6 ± 85.3c
Control (HV) 872.6 ± 52.4c 348.0±46.2b
Karate 17.5 ga.i. ha·' 1826.3±200.7a
ULV I x 10"conidiaha-' 1028.3 ± 170.7bc 1007.5 ±268.9ab
ULV I x 10"conidiaha-' 1021.1 ± 206.9bc 1013.4± 183.4ab
HV 1 x 10"conidiaha-' 1544.4 ± 198.8ab 1495.1 ±209.3a
HV 1 x 10"conidiaha-' 1897.2 ± 74.9a 1730.4±205.4a
Means within a column followed by the same letter are not significantly different by Student-Newman-
Keuls' test (P < 0.05).

Careful timing of application of chemical insecticides based on cowpea growth stages is an

important component of integrated pest management (IPM) and has been recommended for M
sjostedti control (Alghali, 1992, 1995). Normally two applications ofchemical insecticides (one at
the flower bud stage and the other at flowering) are required for the control of thrips (Afun et al.,
1991; Alghali, 1992). Compared to chemical insecticides, fungal pathogens are slower acting mortality
agents. Trials carried out to assess the timing of application of M anisopliae for thrips control on
cowpea showed that two applications of the fungus timed as above did not protect cowpea against

169
M sjostedti (Ekesi et a!., 2000b). Instead, one application of the fungus given at flower bud, and
two applications given at flowering were required to keep M sjostedti in check, as these stages are
most sensitive to thrips damage (Table 5).

Table 5. Effect of timing of application of Metarhizium anisopliae on cowpea grain yield (kg
ha- i ± SE)

Treatments * First season Second season

Control 338.1 ± 31.0e 453.3 ± 27.6e
37,44 387.5 ± 27.le 494.7± 31.3e
44,51 778.0 ± 37.5c 894.5 ± 48.3c
44,51' 1102.3 ± 98.7b 1231.6±85.8b
51,58 571.4±47.5d 635.1 ± 36.1d
37,44,51 806.3 ± 34.3c 902.8± 51.6c
44,51,58 1441.3±98.7a 1581.3± 103.2a

• Spray application at days after plant emergence: 37 (70-100% leafbud formation), 44 (40-60% flower bud
formation), 51 (30-50% flowering), 58 (70-90% flowering). Means within a column followed by the same letter
do not differ significantly by Student-Newman-Keul's test (P < 0.05). ' Karate treatment.

Most studies on the persistence of fungal pathogens have revealed that the conidia applied on
foliage have a short persistence and therefore a low infectivity (Gardner et al., 1977; Ignoffo et aI.,
1979; Smits et a!., 1996). On cowpea, Daoust and Pereira (1986) reported that conidia of M
anisopliae and B. bassiana persisted for 1-2 days. Persistence testing of M anisopliae ICIPE 69
was conducted by treating potted cowpea plants with a concentration of 1 x 107 conidia ml- i and
exposing them to maximum incident sunlight. Leafbuds and flowers were picked soon after the
fungal residue had dried (designated 'day 0' after treatment) and subsequently at 1,2,3,4,5,6, and
7 days after treatment. Thrips were exposed to the plant materials for 48 h and mortality was recorded
daily for 7 days after exposure to treated plant materials.

120 -+- Mellll'hizium anitiOplifle

- - Control
C 100
!. 80
c
C 60
;:
..
<II
u
<II
Q.,
40
20
0
0 2345678
Time (days)

Figure I. Persistence of Metarhizium anisopliae ICIPE 69 in terms of mortality on adult Megalurothrips sjostedti

The fungus remained active in the field for about 3-4 days (Figure 1). The position of conidial
deposition and plant architecture have been reported to influence the rate of inactivation of infective
propagules (Fargues et a!., 1988; Inglis et aI., 1993; James et aI., 1995). The rate of inactivation
observed in our study is slightly lower than that reported by other workers. Daoust and Pereira
(1986) used fully formed cowpea leaves that were attacked by cowpea leafbeetles, and the leaves
were directly exposed to sunlight. In this study, flowers and leafbuds, which constitute the habitation
sites of M sjostedti, were used. Although flowers of the variety of cowpea used in this study were

170
also exposed to direct sunlight, they open in the morning and evening and close in the daytime during
periods of high light intensities. Since the spray applications were administered in the evening, the
behaviour of the flowers ensures that the conidia, that are deposited inside the open flowers are
afforded some protection from direct sunlight. Cowpea leafbuds are also protected from high light
intensities by the large leaves ofthe upper canopy. Under these circumstances, the plant parts evidently
provide some protection to the conidia from inactivation by ultraviolet radiation, hence prolonging the
fungus persistence.

3.2 Efficacy of Metarhizium anisopliae Against Thrips tabaci on Onion

Gillespie (1986) reported that isolates of M anisopliae and B. bassiana were pathogenic to T
tabaci in the laboratory. The fungi were not tested in the field. The same author reported that V
lecanii provided good control of onion thrips on cucumber in the glasshouse. Although M anisopliae
ICIPE 69 was selected specifically for M sjostedti control, attempts were made during the long
rains of 1997 to evaluate the fungus for the control of onion thrips. The fungus was compared with a
recommended synthetic insecticide, dimethoate, in four treatment applications: (i) M anisopliae
applied weekly, (ii) M anisopliae applied biweekly, (iii) dimethoate applied biweekly, and (iv) control
(water spray). The fungus was applied at the rate of 1 x 1011 conidia ha· l and dimethoate was applied
at the rate of300 g.a.i. ha· l . Application ofthe fungus was observed to be as effective as dimethoate
in reducing thrips density (Figure 2) and increasing onion bulb yield (Table 6). Thrips population in
the weekly application of fungus declined steadily after the first spray but biweekly treatment of the
fungus and dimethoate increased at 2 weeks after treatment and thereafter the density declined. The
density in the control plots continued to increase over the observation time (Figure 2) .

...... ontro
:l -' - M. ani\'opliue weekly
i
C ............ M. tflli.,op/iue hi-weeki}'
2 - - Dimethoate
<>
i
'1:
-S
'E
g
~
2 3 4
Weeks after treatment

Figure 2. Effect of Metarhizium anisopliae application on the density (mean ± SE) of Thrips tabaci on onion

Onion thrips constitutes an exceptional opportunity for control with entomopathogenic fungi.
Their feeding damage is concentrated on the sheathing leafbase of the onion plant, where they
aggregate in large numbers. Spray applications of fungus directed at this region of the plant that is
protected from direct sunlight allows the host and pathogen to persist in some degree of balance. This
could favour the development of epizootics, because infected insects could serve as a source of
inoculum for infecting other thrips.

3.3 Efficacy of Metarhizium anisopliae Against Frankliniella occidentalis on Flowers,

French bean and Snowpea

The horticulture and floriculture industries in Africa are under increasing pressure to reduce
chemical pesticide residue levels in their crops. For example, the European Union, which is the main
importer of horticultural products from Kenya, recently enforced the minimum residue requirements

171
for vegetable imports from this region. Alanned by this reaction, flower and vegetable growers in
Kenya seeking alternatives to the use ofchemical insecticides for thrips control approached ICIPE in
1996 to identify other control methods that could eliminate or reduce pesticide application for western
flower thrips control in the vegetable and flower industries. A series oftrials were therefore conducted
on three different types of flowers (chrysanthemum cuttings, roses, and carnations), and on French
beans and snowpea to evaluate the efficacy of M anisopliae ICIPE 69 against F occidentalis.
Apart from the French beans trial that was carried out at the ICIPE Mbita Point Field Station, all
other trials were conducted in the greenhouses ofthe fanners selected by the Fresh Produce Exporters
Association of Kenya (FPEAK) and the Kenya Flower Council (KFC). Yoder Fanns (Embu) was
selected for trials on chrysanthemum cuttings, Finlay Flowers (Kericho) for trials on carnations,
Kijabe Flowers (Naivasha) for roses and VegPro Industry (Naivasha) for trials on snowpea. All
treatment applications were done by the fanns' technical staff with guidance from ICIPE.

Table 6. Onion bulb yield following treatment with Metarhizium anisopliae and dimethoate

Treatment Yield (kg ha·' ± SE)

Control (water spray) 14873.9± 1646.0b

M. anisopliae weekly 19758.1 ± 941.2a
M. anisopliae biweekly 16014.0± 1390.3b
Dimethoate biweekly 17826.1 ± 1222.8ab
Means within a column followed by the same letter do not differ significantly by Student-Newman Keul's
test (P < 0.05).

At Yoder Fann, two trials were conducted using chrysanthemum variety Bright Yellow
Mayshoesmith. The treatments consisted of (i) M anisopliae applied weekly (ii) M anisopliae
applied biweekly, (iii) Lannate (Methomyl90 WP) applied weekly, (iv) Lannate applied biweekly,
and (v) control (water spray). The fungus was applied at the rate ofl x 10 13 conidiaha- I and Lannate
was applied at the grower's recommended rate of 440 g.a.i. ha· 1 ofthe commercial formulation at an
output of 1000 I ha- I . Treatments were started when the plants were 3 week old and continued at
weekly or bi-weekly intervals. The fungus and the chemical insecticide were administered with a
motorised knapsack sprayer. On 7th day after treatment, results indicated a significant reduction in
thrips population density in both the fungal and Lannate treated plots, when compared with the
control. It was noted, however, that the fungus performed better on adult thrips than on the larval
stages, and that Lannate achieved better control on the larvae than on the adults.
An attempt was made in the second trial to combine both control agents at high and reduced
doses in order to obtain a better level of control of both developmental stages. Prior to this trial,
laboratory bioassays had shown that Lannate did not inhibit germination and growth ofM anisopliae.
Nine treatments were therefore applied in the field:
(i) M anisopliae applied at 1 x 1012 conidia ha- I ,
(ii) M anisopliae applied at 1 x 1013 conidia ha- I ,
(m) Lannate applied at 440 g a.i. ha- I ,
(iv) Lannate applied at 44 g a.i. ha- I ,
(v) M anisopliae applied at 1 x 10 12 conidia ha- I + Lannate at 440 g a.i. ha- I ,
(vi) M anisopliae applied at 1 x 10 12 conidia ha- I + Lannate at 44 g a.i. ha- I ,
(vii) M anisopliae applied at 1 x 10 13 conidia ha- I + Lannate at 440 g a.i. ha- I ,
(viii) M anisopliae applied at 1 x 10 13 conidia ha- I + Lannate at 44 g a.i. ha-I,
(ix) Control (water spray).
Spray equipment and applications were similar to the previous trial and three applications given

172
at 5-day intervals were made in the evenings between 1700 and 1830 hrs to lessen the adverse effect
ofultraviolet radiation.
At Finlay (carnations) and Kijabe Farms (roses), treatments were applied on commercial plots
with three treatments: (i) M anisopliae applied at 1 x 1013 conidia ha- I , (ii) Lannate at 440 g a.i.
ha- I , and (iii) control (water spray). Spray applications were made as in previous experiments.
On French bean, the fungus was compared with neem and Karate, and water as control. The
fungus was applied at rates similar to carnations and roses, neem was applied at 500 g a.i. ha-I , and
Karate was applied at the same rate as above at an output of3 50 I ha- I . On snowpea, M anisopliae
was compared with Lannate and with an untreated control. The fungus was applied at rates similar to
French bean and Lannate was applied at 440 g a.i. ha- I . Unlike in the other trials, where thrips
popUlations were allowed to establish on the plant (between 20-60 thrips/sampling size) before
administering the spray, on snowpea the treatment application started earlier at pest levels ofbetween
2 to 5 thripS/20 flowers. This was done to assess whether early (or prophylactic) treatments would
be more advantageous than curative applications for thrips management.
Results from Yoder Farm showed a reduction in larval (Table 7) and adult (Table 8) thrips
population densities receiving fungal, Lannate or a combination ofthe fungus and Lannate treatments
as from 7th day onward after treatment. Two treatment combinations (M anisopliae at 1 x 10 13
conidia ha· 1 + Lannate at 440g a.i. ha- I and M anisopliae at 1 x 10 13 conidia ha- I + Lannate at 440g
a.i. ha- I ) were, however, superior to all other treatments in reducing both developmental stages at 14
and 21 days after treatment (Tables 7 and 8). Many workers (Butt and Brownbridge, 1997 and
references therein) have reported positive interactions between fungal pathogens and chemical
insecticides. The combined use of chemical insecticides from different groups and with different
modes ofaction is one ofthe most effective strategies for managing insecticide resistance in arthropods
because it increases the number of independent targets that have to be overcome (Georghiou, 1994).
Anderson and Roberts (1983) suggested using a combination of B. bassiana and chemical ~ticide
for the management of resistant populations ofColorado potato beetle, so that individuals with genes
resistant to chemical insecticides could fall prey to the fungal pathogen. Resistant populations of
western flower thrips are now developing in Kenya (Brodsgaard, 1994), and resistance to Lannate
(the most commonly used insecticide against F occidentalis in Africa) has been reported in the US
(lmmaraju et al, 1992). The combined use of M anisopliae and Lannate reported here might help
reduce the selection pressure for resistance in African greenhouses, thereby delaying or preventing
the build-up of resistant populations.

Table 7. Frankliniella occidentalis larval population (mean ± SE)/20 cuttings following

treatment with Metarhizium anisopliae and Lannate on chrysanthemum

Days after treatment

Treatment 0 7 14 21

Fungus 10 12 conidialha 34.7±2.1a 21.7 ± 2.3b 13.8±2.5b 12.0± 1.6b

Fungus 10" conidialha 40.7± l.3a 29.3±3.5b 13.7±2.7b 12.7±2.1b
Lannate 440 g a.i./ha 43.7±9.8a 14.0± 1.8c 8.0±0.7c 8.1 ± l.lc
Lannate 44 g a. i./ha 40.0 ± 2.8a 20.0±2.1b 15.1 ±0.9b 13.1 ± l.5b
Fungus 10 12 + Lannate 440 g/ha 39.3± 8.7a 10.1 ±0.8c 5.7± l.lc 5.3±0.8c
Fungus 10 '2 + Lannate 44 g/ha 32.3 ±2.3a 10.1± l.lc 6.1 ±0.7c 6.3 ±O.4c
Fungus 10" + Lannate 440 g/ha 44.7±2.6a 5.3±0.7d 2.0±0.4d 1.0±0.2d
Fungus 10" + Lannate 44 g/ha 43.3 ± 8.7a 6.1 ± l.ld 2.7±0.2d l.3±O.ld
Control (water spray) 45.5± 1.8a 48.1 ±2.3a 37.1 ±4.5a 38.3±3.4a

Means within a column followed by the same letter do not differ significantly by Student-Newman-Keul's
test (P < 0.05). Day 0 denote pre-treatment samples.
All results from Finlay (carnations) and Kijabe Farms (roses) indicated that M anisopliae was
as effective as the standard chemical insecticide in reducing F occidentalis populations. Similarly,

173
spray applications on French bean showed that the fungus was as effective as neem and even more
effective than the chemical insecticide in increasing pod yield (Figure 3). The fungal application also
resulted in more exportable blemish-free pods due to western flower thrips damage at harvest than
either the insecticide or neem treatments.

Table 8. Frankliniella occidentalis adult population (mean ± SEY20 cuttings following treatment
with Metarhizium anisopliae and Lannate on chrysanthemum.

Days after treatment

Treatment 0 7 14 21

Fungus 10" conidialha 23.4±2.1a IOJ ± l.2b IS.2±2.1b 11.3 ± l.lb

Fungus 10 13 conidialha 27.7±3.8a 12.0 ± l.l b 10.8±2.1b 6.7 ± 0.7c
Lannate 440 g a.i.lha 28.7± 1.6a 10.0±0.8b 10J ± O.4b 6.8±0.2c
Lannate 44 g a.i.lha 33.3± 1.7a 13.7±0.Sb 12.3±0.7b 12.1 ± 0.8b
Fungus 10" + Lannate 440 g/ha 20.0±2.la 11.0± l.lb 10J ± 0.9b 6.0 ± 0.Sc
Fungus 10" + Lannate 44 glha 33J ± 1.9a 11.2± l.8b 13.7±0.2b 11.0 ± 0.lb
Fungus 10 " + Lannate 440 glha 28.7± O.5a S.7± OJb 6.0±0.lc 2.7± O.2d
Fungus 10" + Lannate 44 g/ha 34.3 ± 1.2a 7.3±0.2b 6.7 ± 0.4c 2.1 ± O.ld
Contro I (water spray) 34J ± l.Sa 22.1±1.1a 22.0±2.la lS.7 ± 3.1a

Means within a column followed by the same letter do not differ significantly by Student-Newman-Keul's
test (P < O.OS). Day 0 denote pre-treatment samples.

5000 r-
-
'~" 4000
3000 -- r--
."

~ 2000
~
1000

0 ContrOl Fungus Heern Lannate

Treatments

Figure 3. Pod yield (kg ha") of French beans following application of Metarhizium anisopliae, Neem and Lannate
against Frankliniella occidentalis

60r--------r-.
~ Control
~ 50 - ':- Lannate

c
~ 40
.....,..... M. olli.wpliae

= 30
~ 20
.......'1:
:.d
10

14 21
Days after treatment

Figure 4. Frankliniella occidentalis population (mean ± SE) following three applications of Metarhizium
anisopliae and Lannate on snowpea. Day 0 denote pre treatment samples

On snowpea, spray applications began when the thrips density was < 5 thrips!20 flowers. One
noticeable effect of this early treatment was that the thrips population remained at less than 3 thrips!
20 flowers in the fungus-treated plots, whereas in the control, up to 49 thripsl20 flowers were recorded
at 21 days after treatment (Figure 4). This was a clear indication that early or prophylactic treatment

174
with M anisopliae may be more beneficial than curative treatment for exporters of vegetable and
flowers, who in most cases do not tolerate damage of any kind.

4. FUNGAL INTERACTION WITH GENETIC HOST PLANT VARIATION FOR

THRIPS CONTROL
The use of resistant varieties is an important component of integrated pest management (rPM)
that is generally assumed to be compatible with other pest management measures (Duffey and Bloem,
1986). Little is known about the compatibility of host plant resistance with entomopathogens for
thrips management. Studies in other systems show a certain degree of compatibility between host
plant resistance and some biological control agents for the control of certain insect pests (Bong et al.,
1991 ; Meade and Hare, 1994), although exceptions to these observations are also common (Ramoska
and Todd, 1985; Felton et a!., 1987). The susceptibility of M sjostedti to M anisopliae rCIPE 69
when reared on three varieties of cowpea, ICV 2, rcv 7 and ICV 8, known to be susceptible,
tolerant and moderately resistant, respectively (Pathak and Olela, 1986), was examined at different
temperatures in the laboratory. The objective was to determine the compatibility of the fungus with
host plant resistance.

Table 9. Mortality (% ± SE) of adult Megalurothrips sjostedti exposed to direct sprays of

Metarhizium anisopliae at different temperatures on three cowpea varieties

Concentration Cowpea Temperature

(conidialml) variety I SoC 2O"C 2SoC 30"C

Control Mod. resist. 17.5 ± 4.la 20.4 ± 2.4a 20.6±3.3a 24.S±2.6a

Tolerant 1.2± O.Sb O.O±O.Ob 3.S±0.7b IS.0±2.3b
Susceptible 4.6±2.3b 3.7± l.3b S.6± I.2b l2.2±4.5b

I xlO' Mod. resist. 46.7±6.0a 4S.0±S.Sa 46.7±3.3a 60.1 ± S.Sa

Tolerant 6.1 ±2.9c 11.7± 7.4c 17.0±2.7c 13.1 ±4.2c
Susceptible IS.3± 1.7b 2S.7±7.4b 33.4±8.7b 3S.4±7.2b

I x 106 Mod. resist. S8.3 ± 1O.7a 66.7± 11.7a 70.2± IS.Sa 7S.3 ± 16.la
Tolerant 11.2±S.6c 10.3±3.3c 23.1 ±3.8c 27.3± S.4c
Susceptible 2S.3 ± 1.9b 3S.0±2.Sb 4S.3 ± 16.7b 63.3±9.6b

I x 10' Mod. resist. 6S.0±2.9a 75.0± 12.3a 95.4± 12.Sa lOOa

Tolerant IS.3±5.0c 13.3 ± 9.7c 20.5 ± 11.7c 15.7±4.4c
Susceptible 45.0±2.9b 50.1 ± l2.lb 63.3± 11.4b 7l.7±10.3b

Means within a column followed by the same letter do not differ significantly by Student-Newman-Keul's
test (P < 0.05).

Results showed that mortality of thrips was higher on the moderately resistant variety at the
different concentrations of inoculum and temperatures compared with the susceptible and tolerant
varieties (Table 9). Lethal time and lethal concentration values on the resistant varieties were shorter
and lower, respectively, compared to the susceptible and tolerant varieties. This shows that a faster
kill ofthrips could be achieved on the moderately resistant variety with low concentrations ofinoculum
compared to the other varieties. Thrips feeding on moderately resistant cowpea varieties have a
longer developmental time, lower body weight and lower reproductive potential (Ekesi et al., I 998c).
These effects increase the susceptibility ofthrips to the fungal pathogen due to the physiological and
metabolic stress imposed on the insect by feeding on a sub-optimal host (Butt and Brownbridge,
1997). This result therefore suggests that a combined use of moderately resistant varieties of cowpea
and M anisopliae may be a compatible rPM strategy for M sjostedti management.

175
 Contrary to the above results, thrips raised on the tolerant variety incurred an exceptionally low
level ofmortality. Germination of spores exposed to volatiles emanating from the tolerant variety was
highly reduced, indicating that the leaf surface chemistry of this variety affected the viability of the
fungus (Figure 5). A hexane crude extract of the same variety was found to be inhibitory to fungal
growth (Ekesi et aI., 2000c ).

120
.,"
.~
100
·s;;" 80
OJ] 60

.....
~ 40

=-" 20
Control Resistant Tolerant Susceptible

Treatment

Figure 5. Germination of Metarhizium anisopliae ICIPE 69 conidia on Saboraud dextrose agar plates after 24 h
following exposure to volatiles of three cowpea varieties

A vast majority oflegumes including cowpea produce anti-microbial phenolic substances generally
referred to as phyloalexins (Allen, 1983). These compounds are known to be involved in multi-
faceted disruption of fungal metabolisms, reSUlting in the suppression of growth. Pathak and Olela
(1986) noted that the tolerant variety used in this study is also tolerant to most plant diseases. It is
therefore probable that anti-microbial factors affording protection to this cowpea variety against
plant pathogens can also directly or indirectly affect spores ofentomopathogenic fungi on plant surfaces.
These findings indicate that while M anisopliae ICIPE 69 has displayed successful control ofthrips
in some varieties of cowpea, flowers crops, French beans and snowpea, the fungus may not be
compatible with all varieties of the host plants that the three thrips species under investigation attack.

5 COMPATIBILITY OF METARHIZIUM ANISOPLIAE WITH NON-TARGET

ORGANISMS AND AGROCHEMICALS

In addition to entomopathogens, biological control agents such as predators and parasitoids

can playa significant roles in the reduction of insect pests through natural occurrence or by augmentative
or classical introductions. Selection of biological control agents should therefore depend not only on
their individual efficacies, but also on the potential for complementary action against the target insect
(Lacey et aI., 1997). In the trials in which we assessed the efficacy of M anisopliae within cowpea
intercrop and within the onion agroecosystem, impact assessment of the fungal application on
biodiversity within these cropping systems was also undertaken. Predator populations encountered
in the cowpea trial included coccinellid beetles (Scymnus spp. and Cheilomenes spp.), ants (Dorylus
spp. and Camponotus spp.), predatory bugs (Orius spp.), spiders (Araneidae, Theridiidae and
Thomisidae), staphylinid beetles (Paederus spp.) and earwigs. Unlike chemical insecticide treatment,
no significant adverse effect of the fungus on predator populations was observed when compared
with the controls in the cowpea intercropping trails (Ekesi et aI., 1999b). On onion, apart from
spiders, the results were consistent with the above (Maniania et aI., 2001). Rath and co-workers
(1995) observed that application of M anisopliae for the control of the scarab Adoryphorus couloni
(Burmeister) in pasture had no adverse effect on non-target invertebrates. Stolz (1996) treated cassava
plants bearing mealy bug with M anisopliae var. acridum and introduced the parasitoidApoanagvrus
lopezi de Santis into the plant. No significant effect on mortality and reproduction was observed.
Maniania and Knapp (unpubI.) did not observe any effect when M anisopliae was sprayed on
predatory mites. Entomopathogenic fungi are known to be frequently more specific to target organisms

176
under field conditions and especially under epizootics (Goettel and Johnson, 1992), and this specificity
reduces the level of hazard to non-target organisms. Some ofthe taxa of predators and parasitoids
recorded in our studies are known to readily accept thrips as prey (Lewis, 1973; Matteson, 1982;
Kyamanywa et aI., 1993; Tamo et al., 1997). These organisms, together with the fungus, may
complement each other in the overall management ofthrips.
Because thrips are not the only pest problem on vegetable and flower production, different
chemical pesticides may be needed to suppress pests not targeted by M anisopliae, or control
certain plant diseases or weeds. Knowledge of the compatibility of the selected fungus with different
agrochemicals becomes important in order to allow farmers to select the appropriate chemical and to
determine the time of application of the compound to reduce harmful effects on the fungus. In
consultation with various vegetable and flower growers under FPEAK and KFC, 19 fungicides, four
insecticides and two acaricides which are commonly used by them were supplied and evaluated in
vitro in the laboratory for their effects on M anisopliae ICIPE 69. The fungicides included Acrobat
[0.2% Dimethomorph + Mancozeb], Alliette [0.3% Fosetyl Aluminium], Bavistin [0.1 % Carbendazim],
Baycor [0.2% Bitertanol], Benlate [0.1 % Benomyl], Bravocarb [0.1 % Chlorothalonil +Carbendazim] ,
Daconil [0.13% Chlorothalonil], Delan [0.2% Dithioanon], Dithane M45 [0.25% Mancozeb], Kocide
[0.2% Metallic copper], Metaltox [0.25% Dodemorph], Nimrod [0.2% Bupirimate], Polar [0.05%
Polyoxin], Previcur N [0.1 % Propamocarb hydrochloride], Ridornil Mz [0.25% Mancozeb/Metalaxyl]
Rovral WP [0.15% Iprodione], Rubigan (0.035% Fenerimol], Saprol [0.15% Triforine], Stroby
[0.05% Kresoxinmethyl] and Thiovit [0.3% Sulphur]. The insecticides tested were Fastac [10%
Cypermethrin], Karate [1.75% lambdacyhalothrin], and Talstar [10% Bifenthrin]. The acaricides
were Peropal [1.5% Azocyclotin] and Orthene [1.0% Acephate]. The fungus was exposed to different
rates ofapplication of the insecticides (recommended rate, Y2 the recommended rate and 1110 ofthe
recommended rate over different periods of time (less than 2 h, 24 h and 48 h). The result showed
that Acrobat Daconil, Delan, Dithane M45 and Ridomil inhibited growth at all rates out of the 19
fungicides tested. All other fungicides and the insecticides and acaricides had no deleterious effect on
fungal germination and growth. Although pesticides that are inhibitory in the laboratory do not always
exhibit the same action in the field (Easwaramoorthy et al., 1978; Jaros-Su et al., 1999), precautionary
measures are often important to avoid control failures. Based on the results reported here, it should
therefore be possible to schedule the application of the pesticides observed to be harmful to M
anisopliae in order to minimise their adverse effect on the fungus. For example, Benomyl and V
lecanii have been successfully scheduled for disease and aphid control on chrysanthemum in
greenhouses (Gardner et aI., 1984).

6. CONCLUSIONS

Butt and Brownbridge (1997) have stressed the need to exploit entomopathogenic fungi as
important biological control agents in the strategic approach to the management ofthrips. The range
of data presented here show that control of M sjostedti, T tabaci and F occidentalis with M
anisopliae holds considerable promise. The fungus is amenable to large-scale mass production on
rice and maize/vermiculite. Production is not a high-input technology, and is thus feasible under low-
input agriculture as commonly practised in Africa. Efficacy data must, however, still be generated in
large field plots in addition to mammalian safety testing. Evidence indicates that the fungus is compatible
with non-target organisms within target ecosystems and some agrochernicals, but the performance of
the fungus on different host plant varieties warrants further investigation. The adoption ofthis biological
control agent is likely to reduce chemical insecticide usage, lower production costs and help growers
in Africa meet the increasing environmental demands of European countries importing vegetables and
flowers from Africa. However, acceptability of any new technology is crucial to the development of
that technology; training of national research organisations, plant protection and extension officers,
NGOs and farmers will therefore be necessary in the adoption process ofmycoinsecticide for thrips
management

177
ACKNOWLEDGEMENTS

We acknowledge with thanks the encouragement of Dr. K. Ampong-Nyarko (Alberta

Agriculture, Edmonton, Alberta, Canada), B. Lohr (lCIPE) and I. Onu (Ahmadu Bello University,
Zaria-Nigeria) to carry out this work. We thank E. Wesonga, D. Nyagol and F. Gitonga for their
contribution to this research. We are also grateful to Drs. A Ng'eny-Mengech and AJ. Ngi-Song
for their critical review of the manuscripts. This research was supported by grants from the German
DAAD, Fresh Produce Exporters Association of Kenya (FPEAK), the Kenya Flower Council (KFC)
and lCIPE's core.

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180
 FUNGI FOR BIOLOGICAL CONTROL OF BRASSICA ROOT FUES DELIA
RADICUM and DELIA FLORALIS

J0rgen Eilenberg i and Richard Meadow 2

iDepartment of Ecology, The Royal Veterinary and Agricultural University,

Thorvaldsensvej 40, DK 1871 Frb.C. ,Denmark; 2 Department ofEntomology and
Nematology, The Norwegian Crop Research Institute, Plant Protection Centre, N
1432 As, Norway.

1. INTRODUCTION

Root flies that attack Brassicae, Delia spp. (Diptera, Anthomyiidae) cause considerable economic
damage on outdoor vegetables in the temperate regions ofthe world (Europe, North America). They
damage white cabbage, cauliflower, broccoli, radish, Chinese cabbage, Brussel sprouts, rutabaga,
canola and other cruciferous vegetables due to the larval feeding in the roots (Hill, 1987; Finch,
1993; Broatch and Vernon, 1997). Feeding and damage sometimes extend to the aboveground
parts of the plant. The two species, the cabbage root fly (Delia radicum) and the turnip fly (Delia
jloralis) have much biology in common and control is directed against both species at the same time.
D. radicum has the widest distribution and was introduced to North America (where it is known as
the cabbage maggot) from northwest Europe (Biron et al., 2000).
Traditionally, control has been established by using synthetic insecticides, while a range of
alternative methods are also in use or under development: plant resistance (Dosdall et al., 2000),
tillage (Dosdall et al., 1998), repellents (den Ouden et al., 1996), fiber barriers (Hoffmann et ai.,
2001), mulching (Hellquist, 1996). Despite some effect of such protection methods, the larvae still
cause serious damage and these species are regarded as one of the most important pest problems in
organic vegetable production (Langer, 1995). Naturally occurring antagonists of the two root fly
species include insect predators and parasitoids from the Coleopteran families Carabidae (several
species) and Staphylinidae (Aleochara spp.) and a Hymenoptera (Tryb/iographa rapae) (Jonasson
et al., 1995; Fournet et al., 2000; Neveu et ai., 2000); steinernematid nematodes (Nielsen, 2000);
fungi (Eilenberg, 2000; Eilenberg and Michelsen, 1999; Klingen et ai., 2000); bacteria and
microsporidia (Larsson et al., 1995; Eilenberg et al., 2000). Investigations on biological control have
so far included studies on the use of the coleopteran predators and parasitoids (Finch, 1993; Fournet
et ai., 2000; Neveu et ai., 2000), nematodes (Schroeder et al., 1996; Nielsen, 2000), Bacillus
thuringiensis (Havukkala, 1988; Vlinninen et al., 1999b), and hyphomycetous fungi (Vanninen et
ai., 1999a, Meadowet al., 2000; Klingen et al., 2002b).
The aim ofthis chapter is to provide a status offour fungi naturally occurring on these two Delia
spp. with potential for their biological control with emphasis on the most promising biological control

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 181
strategy for each fungal species. The four strategies for biological control are (Eilenberg et al., 200 1):
classical biological control, inundation biological control, inoculation biological control and conservation
biological control. Classical biological control will not be considered since D. radicum and D.floralis
are both native to Europe, which is where they are most important as pests.

2. METARHIZIUM ANISOPLIAE

M anisopliae (Deuteromycota) has a world wide distribution and is commonly found in soil in
temperate regions (Steenberg, 1995; Vanninen, 1996; Roddam and Rath, 1997; Bidochka et al.,
1998; Klingen et al., 2002a). It is known as an insect pathogen naturally occurring on many insect
species from different orders (Zimmermann, 1993). In temperate regions it is mainly a pathogen of
insects in or on soil (Glare, 1992; Steenberg et al., 1995; Eilenberg et al., unpubl.). Recent phylogenetic
analyses based on DNA suggest that M anisopliae can be divided into ten genetically distinct clades
(Driver et al., 2000). This may reflect isolate differences in geographical distribution, habitat
association, life cycle, and natural insect hosts, and M anisopliae may show to consist of a number
of cryptic species (Bidochka et aI., 2001). M anisopliae has long been studied with respect to the
use in biocontrol of a number of insect pests and extensive reviews have recently appeared, for
example Butt et al. (2001). Below we give solely attention to aspects of particular reference to
brassica root fly control.

Table 1. Natural occurrence of Metarhizium anisopliae, Beauveria bassiana, Entomophthora

muscae and Strongwellsea castrans in Delia radicum and D. floralis and their predators*.

Insect M anisopliae B. bassiana E. muscae Strongwellsea spp.

Delia radicum, adults
Delia floralis, adults
Coenosia tigrina, adults
Carabidae, adults
Carabidae, larvae
Staphylinidae, adults
+ ++ E.muscae +++ S. castrans +++
E. muscae +++ S. castrans +++
+ E. muscae 1 + + Strongwellsea sp. 2 +++
++
+ +
+ +++

+ In few specimens, ++ Regularly, +++ Common, natural epizootics recorded.

'E. muscae from C. ligrina differs from E. muscae from Delia spp. based on peR
profiles, 2Strongwellsea sp. from C. tigrina is an undescribed species from the genus
"Data from Eilenberg, 1999; Eilenberg and Michelsen, 1999; Jensen and Eilenberg, 2001; Jensen et al.
(accepted), Klingen et aI., 2000; Steenberg et aI., 1995.

M anisopliae has rarely been found as a natural pathogen in Diptera (Zimmermann, 1993). In
an extensive survey on adult flies associated with cattle (Steenberg et al., 2001), no natural infections
with M anisopliae were found among 4079 sampled and incubated individuals. It was only found in
one instance as a natural pathogen on one adult D. radicum during extensive surveys with more than
4000 sampled and incubated individuals (Table 1) (Eilenberg et al., 1994; Eilenberg and Michelsen,
1999). M anisopliae should therefore be regarded as a fungus that very rarely naturally occurs on
adult Delia spp. No records of natural occurrence on larvae of brassica root flies exist, despite the
fact that these larvae must naturally be exposed to this fungus while feeding around the roots. The
interactions between M anisopliae and D. radicum and D. jloralis. are shown in Figure 1.
In vitro isolates of M anisopliae can be obtained with simple method from insects (Goettel
and Inglis, 1997) and soil (Vanninen, 1996). A high number of isolates exist in strain collections, but
none from brassica root flies (Humber 1992; Eilenberg et al., unpubl.). Studies on biocontrol have

182
therefore been performed with isolates from other sources, especially isolates from soil (V1inninen et
aI., 1999a,b). In both laboratory studies, M anisopliae isolates were able to cause increased larval
mortality of D. radicum and D. floralis (V1inninen et al., 1999a; Chandler and Davidson, 2001). In
the field, isolates of Manisopliae gave a lowernumber of pupae of the two Delia spp. and higher
yields of cauliflowers than untreated plots, but chemical control gave significantly better control
(Viinninen et aI., 1999b). D. radicum adults were susceptible to M anisopliae in laboratory tests
(Meadow et aI., 2000) and also other adult dipterans have proven susceptible (Poprawski et aI.,
1985; Castillo et aI., 2000).

Adult insects (Diptera) present

above the soil get infected 1)
during emergence from pupae by
conidia in the soil or 2) via adult
fly - adult fly transmission

Soil surface

Insects in or on soil get The released conidia

infected by conidia. contribute to the
Conidia are produced population of conidia
by cadavers in or on the soil

The conidia in the soil

survive over long
periods and are
infective to several
insect orders

Figure I. Life cycle of Metarhizium anisopliae and Beauveria bassiana in temperate agro-ecosystems with
particular reference to interactions with root flies (The fungi are mainly present in the upper soil layers. The
number of genotypes present in each field is limited. Upon emergence from pupae in the soil, adult Diptera can get
infected, rarely with M anisopliae, less rarely with B. bassiana. When the infected flies die, they drop to the soil
surface, and conidia from these insects also contribute to the amount of conidia on or in the soil. The natural
exchange of inoculum between different insect host species is unknown).

In soil, it is possible to quantifY the number of conidia over time, showing that the fungus persists
over prolonged periods (more than three years) after application (V1inninen et aI., 2000), even more
than seven years (Rath et aI., 1997). Recent studies in rape (Inyang et aI., 2000) demonstrated that
oil formulation increased the persistence of M anisopliae after application in this cruciferous crop.
Concerning non-target effects in vegetable fields, M anisopliae occurs naturally but not
commonly on beetles from Staphylinidae and Carabidae (Table 1), but in experimental work in the
field, only very low levels of infection among these non-targets occurred (Vestergaard and Eilenberg,
2000). Hokkanen and Zeng (200 I) challenged large carabid predators from the genus Carabus to
M anisopliae, but the beetles did not receive infection. After application to control pests in brassica
crops, only few non target effects on the natural enemies of the insect pests are therefore to be
expected (Lynch et ai., 2001; Zeng et aI., 2001).

183
 A summary of the biological and ecological properties of M anisopliae relevant for brassica
root fly control is shown in Table 2. Due to its lack of significant natural interaction with these Delia
spp., neither inoculation nor conservation would be recommended strategies. On the other hand, a
highly virulent isolate of M anisopliae (still to be found) will have potential for inundation biological
control with few expected non-target effects.

3. BEAUVERIA BASSIANA

Much of the biology of B. bassiana is similar to M anisopliae. B. bassiana is also commonly

found in soil, and has been isolated from soil in various cropping systems in the temperate regions
(Steenberg, 1995; Vfuminen, 1996; Klingen et aI., 2002a). It seems to be relatively more common
than M anisopliae further north (Vanninen, 1996; Bidochka et aI., 1998). The fungus occurs
commonly in insects from several order. In the temperate parts of the world it is often found in insects
in or on soil (Steenberg et aI., 1995; Eilenberg, unpubl.). The fungus can be readily isolated in vitro
from infected insects (Goettel and Inglis, 1997). B. bassiana occurs regularly in adult dipterans
(Steenberg et aI., 2001), which is also the case of D. radicum, Table 1 (Eilenberg et aI., 1994;
Eilenberg and Michelsen, 1999.), but the fungus has never shown ability to establish natural epizootics
in adult brassica root flies. It has not been reported naturally occurring on larvae of these flies, but an
isolate in vitro from a pupa of D. radicum (Humber, 1992) indicates that natural infection ofjuvenile
stages can occur. As it appears, B. bassiana is not so bound to the soil environment as M anisopliae.
D. radicum and D. floralis larvae were subjected to B. bassiana by Viinninen et al. (1999a,b).
They found the B. bassiana isolates tested gave an effect on the larval population, while chemcial
control gave significantly better results. Adults of D. radicum were susceptible to B. bassiana in
laboratory tests performed by Meadow et al. (2000). They proved further that exchange of inoculum
of the fungus among adult flies ofD. radicum was possible. Autodissemination in the field between
adult flies either in natural situations or after application is therefore a possibility, greatly widening the
potential of this fungus for biological control. It is an open question whether exchange of inoculum
between adults and larvae of bras sica root flies will take place in the field. A study on a similar fungus
indicates a strong host instar - pathogen relationship in the field. Studies on the identity of Beauveria
brongniartii in adults and larvae of the Coleoptera Melolontha melolontha (Col.) showed that the
genotypes of B. brongniartii on adults were different than the genotypes found on larvae (Enkerli et
aI., 2001). This may reflect that the natural exchange of inoculum between adults and larvae is very
low or even zero.
B. bassiana did not persist as long as M anisopliae in the study by Vanninen et al. (2000),
since most fungal propagules disappeared after one winter. The fungus occurs regularly in soil dwelling
beetles (Steenberg et al., 1995) and some non-target effects upon direct exposure can be expected.
B. bassiana certainly has prospects as a biological control agent of these Delia spp. (Table 2).
Against larvae the strategy should be inundation with no expectation of a secondary spread of the
inoculum, as for M anisopliae. Concerning adults, B. bassiana occurs naturally and was proven
successful in the laboratory with respect to autodissemination Using a trap to attract adults, this
method could potentially be applied in the field (Meadow et al., 2000). Therefore inoculative release
is a good possibility.

4. ENTOMOPHTHORA MUSCAE

E. muscae belongs to the Entomophthorales (Zygomycota). It is known as a naturally occurring

pathogen from many fly species from many families thoughout the world (MacLeod et aI., 1976;
Balazy, 1993). E. muscae is now regarded as a complex of species, which are under redescription
or new formal description. The original host, house fly Musca domestica, is now recognised as the

184
host forthe "true" E. muscae (Keller et aI., 1999). The fungus found on D. radicum and D.floralis
should so far still be tenned E. muscae (Keller, subm.), while molecular data suggest that a revision
ofthe species complex will result in the description of anew species from Delia (Jensen and Eilenberg,
2001 ; Jensen et aI. , accept.).

Table 2. Summary of biological properties ofthe fungi Metarhizium anisopliae, Beauveria

bassina, Entomophthora muscae and Strongwellsea castrans with particular focus on their
potential use in brassica root fly control

Metarhizium anisopliaei Entomophthora muscae/

Presence in insects in
temperate regions
Epidemics in brassica root
flies
Disease transmission
Presence in soil, including
winter survival stages
Methods for isolation of
spores from soil
Method for isolating in
vitro from insects
Spore production in vitro
Target instar
Ecological host range
Physiological host range
Expected non-target effects
Potential strategies for
biocontrol of root flies
Beauveria bassiana
In soil dwelling insects ITom many
orders and occasionally in adult flies
Not observed in the field
Insect to insect or ITom conidia
in the soil
Conidia, measurable quantities
Via insect bait or directly ITom soil
to selective growth medium
Solid medium,
simple method
Large scale conidia production
possible with simple media and
methods
Larvae (Ma and Bb), adults (Bb)
Broad, if non-targets are directly
affected, otherwise narrow
A single isolate can infect several
insect orders
Few
Inundation (Ma and Bb) and
inoculation (Bb)
Strongwellsea castrans
In adult higher flies
Observed yearly
Insect to insect
Resting spores, not measurable
quantities
No method available
Liquid medium, complicated
method
Small scale conidia production
possible (Em), not documented (Sc)
Adults
Each epidemic is restric-ted to one
host species
A single isolate can infect several
families (Em) or one genus (Sc)
Very few or none
Inoculation (Em) and conservation
(Em and Sc)

E. muscae has a life cycle with strong and indispensable interactions with the host population
(Figure 1). Adult flies are infected and after the fungus-induced death, conidia are discharged to
infect other adult individuals. Towards the end of the season, resting spores are fonned (Thomsen
and Eilenberg, 2000), and flies dying with these resting spores drop to the soil surface, where the
spores survive the winter and are assumed to genninate with infective conidia the following spring.
No method to quantifY the number of resting spores in the soil has been published. The fungus can
establish rapidly developing epizootics in brassica root flies with prevalences well above 50 percent
(Eilenberg, 2000; Klingen et aI., 2000). Isolation in vitro in simple, liquid media is possible, and a
number of in vitro isolates from D. radicum exist in the collection at The Royal Veterinary and
Agricultural University in Copenhagen and at the USDA in Ithaca, NY, USA (Humber, 1992; Eilenberg,
unpubl.). No conidia from E. muscae isolates from D. radicum or D. floralis have yet been produced
in vitro, while a limited conidia production in vitro was obtained with an isolate of E. muscae from
the host Musca domestica (Eilenberg et a!., 1990). No experiment to control brassica root flies
using E. muscae have been reported. Experiments using E. muscae and the similar species E.
schizohoporae to control M domestica indoor have shown that it is actually possible to induce

185
epizootics using these fungi (Geden et aI., 1993; Steinkraus et aI., 1993; Six and Mullens, 1996;
Kuramoto and Shimazu, 1997). Obviously, outdoor conditions provide us with divergent temperature,
sub-habitats, vegetation, and moisture, thus making an inoculation more unpredictable compared
with indoor releases.
While prevalence studies and molecular data suggest a strong interaction between one genotype
and the host species, laboratory studies show that members of the E.muscae-complex can be
transmitted from one host species to another, also across dipteran families (Steinkraus and Kramer,
1987; Steenberg et aI., 2001). It is unclear, if such transmission to other host species really takes
place in the field (Figure 2) It seems anyway fair to conclude that no or very few, insignificant non-
target effects will occur in the field after release of E. muscae.
The strong ability to create natural epizootics and the strong population interaction with the host
points towards exploitation in inoculation biological control. A release of fungal material early in the
season could result in a more rapid development of epizootics. Further, enhancement of natural
epizootics as conservation biological control could be done by leaving field boundaries undisturbed,
allowing more resting spores to survive the winter. Table 2 summarises the present status of E.muscae
for root fly control.

Figure 2. Life cycle of Entomophthora muscae in the host, cabbage root fly, Delia radicum
I. Pupae of D. radicum overwinter in the soil.
2. During spring, it is assumed that infective conidia of E. muscae are produced and discharged from resting
spores in the soil.
3. Adult D. radicum emerge from pupae during spring and become infected by conidia of E. muscae.
4. After the incubation period, E. muscae kills D. radicum. The dead fly is attached to the vegetation by rhizoids
and/or legs. Conidiophores emerge from the abdomen.
5. Conidia are discharged from the cadaver and infect other adult D. radicum. Several successive infection
cycles can take place during one season in the host population.
6. After midsummer, some E. muscae-infected D. radicum develop resting spores instead of conidia. After the
incubation period, flies die with resting spores and drop to the soil surface (S). The abdomen is filled with
resting spores (assumed to be azygos pores ).
7. Thick walled resting spores survive in the soil layers during winter. The following year they genninate and
discharge primary conidia and the cycle is completed.
8. Hypothetically, an alternative, closely related host species from Diptera (for example other Delia spp.) may
receive infection from D. radicum
9. Conidia are discharged from the dead alternative host and infect D. radicum.

186
5. STRONGWELLSEA CASTRANS

The fungus S. cas trans also belongs to the Entomophthorales. It is peculiar in the sense that
during the infection, one or two abdominal holes develop, from which conidia are discharged from
active adult fly (Figure 3). Therefore the lethal time is longer than the incubation period and the fimgus
is able to disperse by means of the flying hosts. Towards the end of the season thick walled resting
spores are formed (Eilenberg and Michelsen, 1999) and infected flies drop to the soil surface, where
the resting spores are deposited. It is assumed that they germinate to form infective conidia the
following spring (Figure 3). S. castrans occurs naturally in brassica root flies, both in northern Europe
and North America (Strong et aI., 1960; Eilenberg and Michelsen, 1999; Klingen et aI., 2000). It is
able to establish epizootics in D. radicum and D. floralis with occasionally high prevalences (Lamb
and Foster, 1986; Eilenberg, 2000; Eilenberg and Michelsen, 1999; Klingen et aI., 2000), but the
epizootics seem to develop less rapidly than epizootics of E. muscae. Attempts to isolate S. castrans
in vitro have been successful, but only in liquid media, and the biomass produced was very limited.
No conidia were ever obtained in vitro (Eilenberg et aI., 1992). It has so far not been possible to
obtain isolates in vitro from the soil, or to quantify the number of spores in the soil.

Figure 3. Life cycle of Strongwellsea castrans in the host, cabbage root fly Delia radicum
I. Pupae of D. radicum overwinter in the soil.
2. During spring, it is assumed that infective conidia of E. muscae are produced and discharged from resting
spores in the soil.
3. Adult D. radicum emerge from pupae during spring and become infected by conidia of E. muscae.
4. Flies infected with fungi from Strongwellsea discharge conidia from abdominal holes while still alive.
5. Conidia are discharged from the cadaver and infect other adult D. radicum. Several successive infection
cycles can take place during one season in the host population.
6. After midsummer, some E.muscae-infected D. radicum develop resting spores instead of conidia. After the
incubation period, flies die with resting spores and drop to the soil surface (S). The abdomen is filled with
resting spores (assumed to be azygospores).
7. Thick walled resting spores survive in the soil layers during winter. The following year they germinate and
discharge primary conidia and the cycle is completed.

Strongwellsea spp. have only been found on various adult flies from Cyclorrhapha (Eilenberg
and Michelsen, 1999). Morphologically, it seems that the fungus from the genus Strongwellsea

187
commonly occurring on the predator of adult flies, Coenosia tigrina, is a different species than S.
castrans (Table 1). Furthennore, no transmission experiments from one fly family to another have
been successful with Strongwellsea spp. (Eilenberg, unpub\.). Therefore, no non-target effects can
be expected from S. cas trans. Despite the very attractive biological features of S. cas trans: high
host specificity, natural occurrence in the host, epizootiological properties, no non-target effect, the
fungus is far from exploitation. Based on the present, limited infonnation about S. castrans, this
fungus has only potential in conservation biological control (table 2). For example, undisturbed field
boundaries can support the overwintering survival ofresting spores and thus the initiation ofepizootics
the following year.

6. CONCLUSION

The four naturally occurring fungi have potential as biological control agents against brassica
root flies. Each should be given further attention but in relation to different strategies: inundation (M
anisopliae and B. bassiana), inoculation (B. bassiana and E. rnuscae) and conservation (E. rnuscae
and S. castrans). The knowledge on the development ofepizootics in the field, and practical possibilities
to enhance these, is still very limited. Ecological studies using PCR may yield significant new knowledge
allowing a much better understanding. The lack ofeffective in vitro growth methods severely hampers
the production of E. rnuscae and S. castrans.
Further development offungi for biological control ofroot flies should also be seen in light of the
development of other non-chemical control methods and compatibility with these methods.

ACKNOWLEDGEMENTS

Charlotte Nielsen, Tove Steenberg and Susanne Vestergaard gave valuable inputs to the
manuscript. Parts of the studies were supported by The Ministry of Agriculture, Fisheries and Food,
Copenhagen, Denmark.

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Kuramoto, H., and Shimazu, M., 1997, Control of house fly populations by Entomophthora muscae (Zygomycotina:
Entomophthorales) in a poultry house, Appl. Entomol.Zool. 32: 325-331.
Lamb, D.J., and Foster, G. N., 1986, Some observations on Strongwellsea castrans (Zygomycetes:
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of control methods, Biol.Agric. Hart. 12: 151-171.
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(Linnaeus, 1758)(Diptera, Anthomyiidae), EuropJ Protistol. 31: 275-285.
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Hokkanen, I., Thomas, M.B., Tommasini, G., Waage, J.K., Lenteren, J.C. van, and Zeng, Q.-Q., 2001, Insect
biological control and non-target effects: A European perspective, in: Evaluating Indirect Effects ofBiological
Control, E., Wajnberg, J.K. Scott and PC. Quimby, eds., CAB International, pp. 99-125.
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Meadow, R., Vandenberg, J.D., and Shelton, A.M., 2000, Exchange of inoculum of Beauveria bassiana (Bals.)
Vuill. (Hyphomycetes) between adult of flies of the cabbage maggot Delia radicum L. (Diptera: Anthomyiidae),
Biocont. Sci. Techn. 10: 479-485.
Neveu, N., Krespi, N., and Nenon, J.P., 2000, Host-stage selection of Trybliographa rapae, a parasitoid of the
cabbage fly Delia radicum, Entomol. Exp.Appl. 96: 231-237.
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Anthomyiidae),J.AppI.Entomol. 120: 427-432.
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Workshop on Soil Invertebrates, DJ. Rogers and L.N. Robertson, eds., pp. 78-80.
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bassiana from subantarctic Macquarie Island, Jlnvertebr.Pathol. 69: 285-288.
Schroeder, P.C., Ferguson, C.S., Shelton, A.M., Wilsey, w.T., Hoffmann, M.P., and Petzholz, C., 1996, Greenhouse
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USE OF ENTOMOPATHOGENIC FUNGI1N LATIN AMERICA

S.B. Alves!, R.M. Pereira2, R.B. Lopes! and M.A. Tamai!

!
Departamento de Entomologia, Fitopatologia e Zoologia Agricola, ESALQIUSP,
Caixa Postal 9, 13418-900, Piracicaba, SP, Brazil; 2USDA-ARS, Center for
Medical, Agricultural and Veterinary Entomology, 1600 SW 23rd Drive, Gainesville,
FL 32604, USA

1. INTRODUCTION

Microbial control of insects, and the use of entomopathogenic fungi for this purpose, has been
researched and used in small scale in Latin American countries since the early 1900's. However, the
large scale commercial production of taese organisms is a recent phenomenon, the use of
entomopathogens in pest control is still in its infancy in Latin America, just as in world. Although,
commercial products can be found in most, if not all, countries in the region, the market share for
these products is still minimum, with some notable exceptions for certain crops in some
countries.Despite its small share in the pesticide market, microbial control in general and the use of
entomopathogenic fungi has received much attention from some research institutions throughout Latin
America. The factors that have stimulated the development ofeFltomopathogenic fungi as pesticides
in Latin America are as variable as the economies in the region, .their crops and pest problems, and
technological expertise. However, some common trends can be identified as described in this,chapter.
Because many local small producers exist in different countries, it would be impossible to gather
information on all products and uses of entomopathogenic fungi in Latin America Wehave gathered
here information on some of the most important projects, including information on some commercial
products. Many of these projects are collaborative efforts among researchers or companies in
different countries. Products are exported and imported among Latin American countries, in an
exchange that is constantly increasing the knowledge basis on the use of fungi for control of insects.
As can be seen in some examples presented in this chapter, the use of Metarhizium anisopliae and
Beauveria bassiana is well represented in Latin American countries. Some ofthe know-how developed
in the region is now being exported and used in many other regions of the world. Latin American
countries are among the leaders in the development ofbiopesticidal use of entomopathogenic fungi.
World researchers and entrepreneurs Jook at these countries as guiding examples for the development
of new products and uses for entomopathogenic fungi.

Advances in Microbial Control o/Insect Pests

Edited by Rajeev K Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 193
2. FACTORS AFFECTING ENTOMOPATHOGENIC FUNGUS USE

Several factors have influenced the use ofentomopathogenic fungi in Latin America, where the
existence of many commercial products is a proof of the importance of microbial control in insect
pest management. Among these factors are the favourable climate for fungal infection and the need
for locally produced pesticides. These factors have a very favourable influence on the development
of entomopathogenic fungi and other pathogens for the control ofpests. This effort is supported by a
relaxed regulatory system, but deterred by the low level of initial capital investment and research on
product development.

2.1 Climate

Humid tropical climate prevalent over large portion of Latin America is conducive to fungal
infections of insects. The common observation offungal epizootics in insect populations, and the easy
identification of the causal agent in these epizootics are probable causes for an emphasis on fungal
entomopathogens as microbial control agents in Latin American agriculture. In areas of tropical
climate, natural epizootics of the fungi Aschersonia spp., Verticillium iecanii, Hirsutella spp.,
Paecilomyces spp., Nomuraea rileyi, M anisopliae, B. bassiana, Cordyceps sp., and those in the
order Entomophthorales are relatively ofcommon occurrences in Latin American countries. Relative
humidities between 40 and 90% and temperatures between 19 and 30°C, which are common
throughout South America, favour these epizootics. Observations ofthese natural epizootics serve
as incentives for the development of control programs using fungi as biological control agents.

2.2 Relaxed Regulatory System

One important obstacle to the development of entomopathogens as commercial products

throughout the world has been the high cost of registration and necessary testing. These high costs,
especially in North American and European countries, have prevented the development of a cottage
industry that produces microbial control agents. However, many Latin American countries have a
less rigid regulatory system that allows the development and survival of small commercial producers
of microbial products which reach the market in shorter time, with lower capital investment, and the
companies are able to generate cash flow that can be reinvested in product development and
improvements in the production system. The regulatory system in many North American and European
countries require heavy investment in product development, testing, and marketing before any revenue
can be generated from sales. Although this system prevents the production and use of ineffective
products, it also prevents the development of industry as businesses develop in many other areas of
the economy. A relaxed regulatory system eliminates the need for large investments in the microbial
biopesticide industry, reduces the need of outside investors, and maintains control of decisions in the
hands of those developing the products. These are usually the researchers or other personnel involved
directly in the control of pests or production of fungal entomopathogens.
An unfortunate consequence of a relaxed regulatory system is the possibility of products with
very low quality (low efficacy and high contamination risks) reaching the market. Users of these
products may be discouraged by the lack of efficacy against the pest populations, and avoid future
use of any biological pesticide. To avoid the development of a bad reputation for all biopesticides,
the regulatory agencies must find a balanced approach to biopesticide regulations that do not prevent
the development of products and companies, but prevents the commercialisation of these low quality
products. This can be accomplished by a system that combines a low cost of product development
and registration, with strict attention to quality of the products. Low cost of development and
registration can be accomplished by a greater flexibility in the governmental regulations on registration
ofbiopesticides as is done in Brazil, where some tests required in the registration process can be
satisfied by presentation of information available in the scientific literature. This approach takes

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advantage of the accumulated experience with use of entomopathogenic fungi as biopesticides in the
Latin American countries, and their safety records, in the regulation of new products.
In Brazil, after comprehensive study of the biopestidide regulations from USA, Canada and
European countries, the new regulations for biopesticide registration were approved in 1997. The
regulations apply to products fonnulated with naturally occurring fungi, bacteria, viruses, etc, for
control of insect and plant diseases. The products containing organisms derived from recombinant
DNA procedures are analysed by the Biosafety Technical Committee before registration can be
granted. Brazil, Argentina, Paraguay, and Uruguay, through the Phytoprotection Committee
(COSAVE), are looking into ways to standardise the biopesticide regulations in these countries. A
standing committee on Biological control (GTP-CB), has been created and has issued documents
with nonns and regulations for biopesticide registration, which in the future can be sold in these 4
countries (De Nardo et aI., 1998).

2.3 Low Level of Private Investment

Although the low level of initial capital investment favours the development ofthe biopesticide
industJy in Latin American countries, it also has a detrimental effect on the quality of products and
product marketing. Product research and development is limited, at least initially, and the full potential
of some fungal entomopathogens may not be properly realised. Fonnulation development is probably
the area most strongly affected by the low capital investment. Primitive fonnulations, without the
necessary characteristics to allow long survival in storage and after field application, have been used
frequently, with a consequent low product efficacy. Also, production systems have been limited, both
in installed capacity and technological advancements, that would lower production cost and allow
capture oflarger market shares by biopesticidal products.
Many biopesticidal products tend to remain regional in market penetration, due to improper
marketing and the lack of sufficient production capacity to supply national or international markets.
Although, many products survive in these limited markets for extended period oftime, further product
and market development restricts the benefits that can be derived from large-scale use ofbiopesticides.

2.4 Need for Locally Produced Pesticides

Most emerging economies in Latin America have suffered at one time or another the need to
substitute imported products by locally produced ones that would not drain the hard currency reserves.
Because of political or economic conditions, several Latin American countries decided against the
importation of either products or technology that would increase their dependency on foreign capital
and know-how. Also, because of increasing environmental concerns in developed and developing
countries, environmental authorities in several countries search for ways to avoid the pollution problems
that were first felt by European and North American countries. These conditions have created the
need for pesticides that can be produced locally in the countries in Latin America. Several researchers
and entrepreneurs considered the entomopathogenic fungi as ideal candidates as substitutes for several
pesticides, and great incentive was created for the studies in this area.
Besides economic and environmental reasons, populations in Latin American countries also see
the locally produced pesticides as an instrument to exercise national independence. The feeling that
countries from the northern hemisphere have missed the opportunity to develop strong programs in
microbial control of insects has served as the backdrop for much work with entomopathogenic fungi
in Latin America. Nationalistic feelings combined with economic, political, and environmental forces
have had a very positive effect on the development ofentomopathogenic fungi as pest control agents
in Latin America. In the special case of Cuba, the economic embargo imposed by the USA has
forced the development of important programs in biological control. Consequently, Cuba has become
more developed in some areas of biological control, especially the use of entomopathogenic fungi,
than the USA.

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3. TYPES OF COMMERCIAL PRODUCTION

Initial development of the fungal bioinsecticide industry in Latin America was dominated by
govemmental agencies followed by cottage industries. High-technology operations have only recently
entered the market of entomopathogenic fungi for insect control. Some characteristics ofthese industry
segments are as follows:

3.1 Governmental Agencies

Govemmental and other official agencies have been the initial points for most of the large-scale
entomopathogenic fungus production in Latin America. The need to produce large quantities offungi
to conduct large field research, and to meet the demand of the interested public for a solution to very
immediate pest problems have been the forces behind the quasi-commercial production of
entomopathogenic fungi by these agencies. In some cases in which the sale ofbioinsecticidal products
by these govemmental agencies may be prohibited, the free distribution ofentomopathogens to control
specific pests represented a form of subsidy to the agricultural sector. Examples of official agencies
involved in the large-scale production ofentomopathogenic fungi include: CODECAP, IPA, Instituto
Biol6gico (Brazil); CIAT, (Colombia); International Potato Institute - IPC, SENASA (Peru);
CONICIT (Venezuela);ANAPO (Bolivia); CREE (Cuba), INIA(Chile), among others. The farmer's
cooperatives and other private entities also can be included in this class ofestablishments for production
ofentomopathogenic fungi. The involvement of many ofthese entities in the production ofbiopesticides
evolved from the close relationship between these operations and the research agencies. Like the
governmental agencies, focus of these producers was in the delivery of a service to the clientele,
sometimes with disregard to production costs and other forces that are considered necessary in
commercial production units. Examples ofthese entities include: ASPLANA, COPERSUCAR (Brazil),
CENICAFE (Colombia) and others.

3.2 Cottage Industry

Entrepreneurs aware of potential commercial value ofentomopathogenic fungi as insect control

agents have started low-input industries for production of these microorganisms. Many of these
businesses are operated by personnel related to the research agencies involved in the study and
development of fungi as bioinsecticides. The cottage industry model prospers under a system oflow
regulatory pressure where standards for production facilities and products are flexible. The cottage
industry represents an important step in the development of the biopesticide industry, by providing
opportunities for testing of non-traditional production systems and products. The variable range of
products and services that can be provided by cottage producers ofentomopathogens offer alternatives
for agricultural systems that require low-cost or non-chemical pest control methods. Production
systems, formulations and marketing strategies tend to be less technologically advanced, with high
labour and low capital input. Examples ofthis level of commercial production of entomopathogenic
fungi are found throughout the Latin American countries as well as in many other regions of the globe.

3.3 High Technology

Technologically advanced systems for production of entomopathogenic fungi may result from
the evolution ofthe cottage industries or from new investments in the sector. Entomopathogenic fungi
with high potential as commercial products attract the attention of capital investors and/or other
producers of agricultural products. This new investment usually results in the production of
entomopathogenic fungi under the highest levels of quality control and the formulation/marketing of
final products employing the models used for chemical pesticides. Although this approach is certainly
desirable, some ventures fail due a strong reliance on marketing techniques that work adequately

196
with chemical products but are not adaptable to biopesticides and the special consumer market for
these products. Examples of this class of businesses include Itaforte Bioprodutos that maintains an
excellent quality control program in the production of products containing fungal isolates selected by
the Insect Pathology and Microbial Control Laboratory at ESALQlUniversity of Sao Paulo. Also
included here are those products imported from other countries including Europe and the United
States.

4. USE OF ENTOMOPATHOGENIC FUNGI BY INDIVIDUAL COUNTRIES

4.1 Brazil

The entomopathogenic fungi have been studied in Brazil for more than 70 years. However, it
was only after 1964, after an epizootic occurrence of M anisopliae on sugarcane spittlebugs, that
these pathogens received more attention from the researchers. In 1974, the first university course on
insect pathology was offered as a part of the curriculum ofthe Graduate Studies on entomology at the
ESALQlUniversity of Sao Paulo, in Piracicaba, Sao Paulo. The Insect Pathology and Microbial
Control Laboratory at ESALQlUniversity of Sao Paulo is responsible for the training ofthe leaders
in research and application of microbial control of insects in Brazil, Argentina, Costa Rica, Bolivia
and other American countries. Since then, several other institutions have been active in both basic
and applied studies in insect pathology and microbial control of insects. More than 50% of the
published studies on insect pathology in Brazil deal with the use of fungi. Approximately 90% of
these studies have been developed within the last 20 years.
Entomopathogenic fungi are used in Brazil for control of insects in several important crops. The
following examples are the principal projects in this country.

4.1.1 Sugarcane (Saccharum spp.). Sugarcane is grown in approximately 5.4 million ha in Brazil.
This crop generates large amount of business related to the export of sugar and the national program
for production of ethanol as automobile fuel. The spittlebugs Mahanarva posticata and Mahanarva
jimbriolata (Hemiptera: Cercopidae) are among the most important pests of the sugarcane in Brazil.
Mahanarva posticata is the principal species in the northeastern region and M jimbriolata is
important in the southeastern region of Brazil. In northeastern Brazil, M posticata occurs in
approximately 800,000 ha ofsugar cane, causing 11 % ofloss in agricultural yields (sugarcane weight),
and 15% ofloss in industrial yields (sugar weight).
The initial successes on the control ofM posticata with the fungus M anisopliae were conducted
in 1969, and in 1975, regional laboratories were constructed to produce the fungus in northeastern
Brazil (Marques et al., 1981). Initially, small quantities were produced for use in inoculative releases
in areas from where the fungus could disseminate. Between 1970 and 1972, 118 such areas were
established (±400 ha total area) by spray treatment with ground equipment and 9 areas (±112 ha
total area) by treatment with airplane-mounted sprayers. Fifteen to 20 days after initial application of
the fungus, greater than 80% mortality of nymphs and adults was observed in aerially treated areas.
Areas treated by ground application had only 30-40% nymph and 20-30% adult mortalities (Guagliurni,
1971; Guagliumi et al., 1974). Between 1970 and 1998, agencies involved with the production ofM
anisopliae (IAAJPlanalsucar, IPA, and others) in the state of Pernambuco produced 40.000 kg of
conidia. This material was applied to an area of 500,000 ha of sugarcane.
In the state of Alagoas, also in the northeastern region of the country, approximately 670,000
ha infested with the spittlebugs were sprayed with M anisopliae between 1977 and 1991. Besides
a reduction of72% in the infestation rates of the pests, a secondary benefit from this fungal application
was the reduction on the use ofchemical pesticides in the crop from an initial area of 150,000 ha/year
to 30,000 ha/year. In recent years the average area treated with chemical pesticides has been reduced
to 12,000 ha, or less than 10% of area initially treated with chemical pesticides (Marques, 1992).

197
 The strain of fungus used in the region was selected through bioassays, starting from a large
number of isolates collected in the region and characterised by biological and biochemical methods.
The optimum timing for the fungal application is the beginning of the raining season when young
spittlebugs get established in the sugarcane fields. A minimum population of the pest (5 nymphs per
plant, not counting the ones on the whorl) must be present at the time of application. Applications are
made at 30-day intelVals, ideally during the time when most nymphal movement occurs.
The initial application of the fungus and other conidial sources present in the field selVe as the
inoculum for contamination ofthe initial wave ofnymphs and adults. The population of infected adults
moves to new plants and die in the whorl of the sugarcane plant or under the leaf. These protected
spots selVe as a moist environment for sporulation of the fungus on the cadavers. The conidia are
carried by rain, dew, wind and other means to other parts of the plant. Nymphs that ec10se from eggs
laid on old leaves on the soil surface or the lower part of the plants climb the sugarcane stalk and
encounter a large fungal inoculum. The spittle or foam produced by the nymphs selVes as an ideal
environment for the development of the fungus, and many nymphs succumb to the fungal infection.
The nymphal stage is most susceptible to the fungus, especially due to the different opportunities for
contamination with the fungus. These cadavers represent the primary foci for future dissemination of
the disease in the sugarcane field. SUlViving nymphs are contaminated as they develop into adults
that will also disseminate the fungus to other areas.
Symptoms of M anisopliae infection in spittlebugs vary. Nymphs have slower movements and
may abandon their protective foam. The adults are also slow, stop feeding, reduce their flight distances,
and females will not oviposit. However, adults may die at considerable distances from the point
where they contacted the fungal inoculum.
Presently, the augmentation strategy is used in the application of M anisopliae, because the
fungus is already present in the sugarcane fields. Doses between 2 and 5 x 10 12 , that is, approximately
200-500 g of conidiaiha, can be used. The cost for the products is approximately US$ 22 for a dose
of3.5 x 1012 conidia. The applications are done with either ground equipment or with aerial application.
Typically the product is applied in 50-200 litre of water per ha for ground applications and 20-30
litrelha for aerial applications, with the aeroplane 2-5 meters above the sugarcane tops.
The use of M anisopliae in sugarcane crops has been very advantageous in the northeastern
region of Brazil. The sugarcane crop is semiperenial and occupies large areas where the continuous
application of chemical pesticides, a common practice in previous years, can cause irreparable
ecological damage. The use of microbial control prevents the environmental degradation and the
elimination of several other biological agents that help control pests of sugarcane. The results from the
application of M anisopliae depend on the location ofthe treated fields and the occurrence of rain.
Table 1 shows examples of the spittlebug nymph and adult mortality and fungal prevalence levels
after application of M anisopliae in sugarcane crops near several sugarcane mills. The area treated
with M anisopliae has decreased in recent years due to the removal of a federal subsidy for the use
ofthe fungus for control ofthe spittlebugs. The subsidy was eliminated after the extinction ofPlanaisucar,
the agency responsible for the centralisation of sugarcane research in Brazil. Presently, 10 laboratories
produce M anisopliae in northeastern Brazil.

Table 1. Percent nymph and adult mortality for Mahanarva posticata and prevalence of
Metarhizium anisopliae in some sugarcane mill areas in 1980.

Sugarcane Mills Mortality Percent

Nymphs Adults M. anisopliae*
Sao Jose, PE 7.5 20.6 15.4
Pedrosa, PE 9.7 32.1 25.8
Salgado, PE 26.5 31.3 30.6
Trapiche, PE 21.3 40.4 67.8
Santa Maria, PB 23.1 58.4 50.7
Media 17.6 36.6 38.0
'Percent sugarcane stalks with M anisopliae-killed spittlebugs.

198
 The root spittlebug M fimbriolata has gained importance recently as a sugarcane pest in
southeastern Brazil. The reasons for the increase in attack by this pest are not yet known, but it has
coincided with modifications in the cultural practices, especially those related to the harvest. In recent
years, machine harvest has eliminated the need for previous burriing of the sugarcane foliage. The
increase in spittlebug importance has cause the growers to use an increasing amount to chemical
pesticides, a practice that may interfere with the biological control ofthe sugarcane borer, Diatraea
saccharalis (Lepidoptera: Crambidae), by parasitoids. According to data from industries involved
in the production of M anisopliae, and the sugarcane grower cooperative (Copersucar),
approximately 5,000 ha of sugarcane are treated each year in southeastern Brazil with M anisopliae.
The application of 1.8x 1011 conidia/ha has produced excellent results, with up to 80% control. With
increase in the incidence of this pest and a growing demand for organic sugar, the use ofM anisopliae
will probably increase in the coming years. The consequent decrease in the use ofchemical pesticides
is an essential element in the integrated pest management program in sugarcane crops.
The sugarcane borer is also susceptible to M anisopliae and B. bassiana. M anisopliae kills
naturally approximately 10% ofthe borer in northeastern Brazil, and the fungus is effective against all
life stages of the pest, especially 1-2 day old eggs and newly-eclosed larvae. In field experiments
using 10 13 conidialha, M anisopliae killed 58% of the borer larvae, whereas B. bassiana killed
44%. In the state ofMaranhilo, in northern Brazil, 780 ha of sugarcane have been treated with M
anisopliae against sugarcane borer eggs and newly-eclosed larvae (Mendonya et al.1996). Another
important pest of sugarcane in Brazil, the giant borer Castnia ficus (Lepidoptera: Castniidae), is also
susceptible to M anisopliae, B. bassiana and Beauveria brongniartii. Studies by Villas Boas and
Alves (1988) demonstrated the possibility of controlling up to 40% of the larvae of this insect with
Beauveria spp., in association or not with chemical insecticides.
The subterranean termites that attack sugarcane cause severe yield losses. Among the genera
that are considered sugarcane pests, Heterotermes (Isoptera: Rhinotermitidae) is the most common
and important. This insect damages the roots, the planted stalks and the base of the plants, opening
galleries in the plant and causing the plant death. The damage occurs in the first or in any subsequent
harvesting cycle (usually 2-4 cycles before the field is replanted), causing loss of stand and lower
yield.
The Insect Pathology and Microbial Control Laboratory at the ESALQlUniversity ofSllo Paulo
has developed attractive bait/traps (Termitrap®) treated with isolates of entomopathogenic fungi
selected for each of the target termite pests. The fungus can also be associated with a compatible and
non-chlorinated chemical pesticide (e.g., imidacloprid, fipronil, and insect growth regulators) for
control oftermites (Almeida et aI., 1997; 1998). Another efficient control method is the use of
attractive bait/traps to lure large number of termites that can then be inoculated with selected fungal
isolates. The use of attractive baits is interesting because it has low cost in comparison to the broadcast
application of insecticides. Besides the sugarcane crop, these baits can be used in several other crops
including forestry areas planted with Eucalyptus spp. A minimum amount of the pesticides (biological
or chemical) is used to eliminate a large number of insects that are attracted to the baits. In field
experiments, it has been demonstrated that the termites are either killed or stressed by the chemical
pesticides. Stressed insects are more susceptible to the fungus in the bait, and the fungus that grows
on cadavers killed by either the chemical or the biological agent represents an inoculum source for
infection of other termites in the area. The hygienic behaviour of some termite species and the
exchange of material by trophalaxis can favourthe dissemination offungal conidia among the termite
population.

4.1.2 Pastures (Gramineae). The spittlebugs in the generaMahanarva, Deois and Zulia (Hemiptera:
Cercopidae) are the most important pests in Brazilian pasture, attacking approximately 10 million ha
annually. These insects are responsible for up to 60% reduction in the grazing capacity of pasture,
with an average reduction of 15% ofthe sugarcane biomass. These pests, as is the case of sugarcane
spittlebugs, are susceptible to infection by M anisopliae. The fungus is applied using wettable powder

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fonnulations with a minimum dose of5 x 10 12 conidialha, or approximately 500 g of pure conidia.
Ground application equipment is typically used with 200-300 litres of spore suspension per ha. The
applications are timed to coincide with the eclosion of the second or third generation of nymphs,
since the immature fonns are the most susceptible to the fungus. The fungus is most efficient when
applied to grass that is 25-40 cm high, and provides protection to the fungus against the detrimental
ultraviolet radiation. A high relative humidity and temperatures around 25-28°C are the necessary
conditions for success of M anisopliae application for pasture spittlebug control. In cases where
there is a large population of adults, this should be knocked down with the use ofselective chemical
insecticides that are not harmful to the fungus and other natural enemies of the spittlebugs. Nonnally,
the level of control obtained varies between 10 and 100%, depending on the application and the
environmental conditions. These levels ofcontrol are obtained with a single application of the fungus
with relatively low doses (100-500 g ofconidialha). Some examples of control levels obtained with
application of M anisopliae against several important spittlebug pests in Brazil are presented in
Table 2.
Several companies produce M anisopliae for control ofpasture spittlebugs in Brazil. Among
these, ltaforte Bioproducts has an excellent quality control program and its product (Metarril PM) is
used in approximately 50,000 ha at a cost ofUS$ 8/ha. Some fungi in the order Enthomophthorales,
such Furia sp. and Batkoa sp., cause natural epizootics in pasture ecosystems, especially on the
spittlebugs Deois schach and M jimbriolata. Conservation methods such as the use of selective
chemical pesticides, pasture management, and others are important in the occurrence ofthese epizootics.
Also, the dissemination ofpest cadavers infected with this fungus can be used to increase the incidence
of the pathogen in spittlebug populations (Alves, 1998; Leite et aI., 2000ab).

Table 2. Field results for applications of Metarhizium anisopliae against the principal species
ofpasture spittlebugs in Brazil.

Species Location If Efficacy Reference

(%)
Aeneolamia selecta Carpina, PE 65-96 Veiga (unpubl. data)
Zulia entreriana Janauba,MG 5-35 Teixeira Alves (unpubl. data)
Deois jlavopicta Brasilia,DF ffi Teixeira Alves (unpubl. data)
Z. entreriana Janauba, MG 17 Reis et al. (1983)
D. jlavopicta Rondon6polis, 44-66 Alves (1998)
Z. entreriana MT
Deois incompleta Moreno,PE 68 Ramos (1986)
A. selecta
D. jlavopicta Several Ranches 60-100 itaforte BioProdutos
Z. entreriana

Tennites in the genus Cornitermes (Isoptera: Terrnitidae) are also important pests in Brazilian
pastures. These insects feed on the plants and build large nests that reduce the availability of grazing
to the cattle and make cultural practices very difficult. From 1990 to 1996, several studies demonstrated
the efficacy of M anisopliae (for summer applications) and B. bassiana (for winter applications) in
controlling these tennites. The fungal application is done in an inundative approach, with application
oflarge doses of the fungi into the nests to prevent the counter action of any physical, chemical, or
behavioural defence by the tennites against the pathogen. A dust applicator nonnally used for
application offonnicides has been adapted with a mixing chamber in the discharge hose that improves
the distribution of the fungus inside the tennite nests. Depending on the size of the nest, 3-6 g ofpure
conidia (6-12 g of fonnulated material) are used. A hole is made on top of the nest mound, usually
using a tractor-mounted drill, and the fonnulation is blown into the nest with a power duster. An
unifonn distribution ofthe product is an essential element in obtaining complete control. All the galleries
and chambers in the nest must be covered with the fungal fonnulation so the insects contact the
pathogen that kills most of the colony.

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 The conidia germinate on the insect cuticle and, 2 days after the application, 20-30% mortality
can be observed. Many cadavers are packed by the termites in nest galleries, in an attempt to limit
the disease spread. After 6-30 days, the mortality can reach 100% depending on the size of the
colony. Small nests are killed easily and in short time (1-2 months), but large nests require very good
distribution ofthe fungal formulation and may take up to 3 months to be eliminated. At this time, many
cadavers are observed on the bottom part of the nest where a dark paste with foul odour is the result
of the invasion ofthe nest by bacteria and other microorganisms (Fernandes and Alves, 1991; Alves
et al., 1995). Other invaders such as flies, ants and mites may attack sick termites and contribute to
the destruction of the nest. Three months after application of the fungus, the nest mounds can be
destroyed with a tractor since these abandoned mounds are less compact than living nests. Also,
after 2-3 years, the application should be repeated targeting nests that were not observed during the
first fungal application. The efficacy of this strategy for termite control depends on the conditions in
the termite nests. Thousand of individuals are concentrated inside the nest, protected by compacted
soil walls, and subjected to a controlled temperature kept at approximately 20°C by a sophisticated
venting system. The humidity is maintained at almost 100% and these conditions favour the growth of
the entomopathogenic fungi, especially because these are protected from the ultraviolet radiation.

4.1.3 Citrus (Citrus spp.). The chemical pesticides, which predominate in the Brazilian citrus industry,
can have detrimental effects on entomopathogenic fungi, depending on the strain and the species, the
dose, and the chemical nature ofthe active and other formulation ingredients. Inhibition of vegetative
growth and sporulation, and the genetic mutation of the fungi are common effects ofthese chemical
pesticides that cause a decrease in virulence against certain pests (Alves et ai., 1993). The chemical
pesticides commonly used in Brazil have been classified based on their effect on entomopathogenic
fungi (Alves et al., 1998a; 2000). The classification system uses the effect of the products on the
vegetative growth and sporulation of fungi in vitro.
To protect the entomopathogenic fungi present in citrus crops, growers must consider a
conservation strategy by using chemical pesticides rationally, rotating products and applying pesticides
only after pest population monitoring. When chemical and microbial pesticides are used in association,
information on the toxicity can help determine application intervals and possibility of tank mixtures.
The careful selection of chemical pesticides allows the preservation of beneficial fungi like
Colletotrichum gloeosporiodes, Myriangium, Myiophagus, Nectria spp., Fusarium spp.,
Verticillium spp., and Aschersonia spp. that occur naturally in populations of scales, whiteflies and
mites in citrus.

4.1.4 Rubber Tree (Hevea brasiliensis). Hevea brasiliensis, from which the raw material for the
production of natural rubber is obtained, is originally from South America. In Brazil, more than
200,000 ha are planted with this crop, distributed in several regions of the country, but especially in
the central-western and the southern regions (Ortolani, 1986; Abreu, 1996). The key pest in this
crop is a stinkbug Leptopharsa heveae (Hemiptera: Tingidae) that causes the destruction ofleaf
tissue as it feeds on the plant, reducing plant growth (height and diameter) (Carrera, 1973; Moreira,
1985). In the 1980's, natural epizootics of the fungus Sporothrix insectorum were observed inL.
heveae populations, and the fungus was cultured and mass production on a large scale was initiated
for application in commercial rubber tree plantations. Presently, this fungus is applied to 35,000 ha
annually. Production ofthis fungus is usually on solid media (rice or wheat bran), and the conidia are
separated from the medium by successive washing procedures. A suspension of 10' conidiaJml is
applied using atomisers (300-400 l/ha) to target the underside of the leaves where the insects are
found. Using these parameters, the applications cause 70-100% control of nymphs and adults in 2
months (Celestino Filho and Magalhaes, 1986). The success of the applications is based on inducing
early epizootics before the insect has a chance to develop into large populations.
More recently, selected strains of B. bassiana have been recommended for areas where the
mite Calacarus hevea (Acari: Eriophyidae) occurs. This fungus can infect both the lacebugL. heveae
and the mite, which is not infected by S. insectorum (M. Tanzini, pers. comm.).

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4.1.5 Banana (Musa spp.). Bananas (Musa spp.) are grown in approximately 481,000 ha in Brazil,
the second largcst producer of bananas in the world. The main pest in this crop is the banana weevil,
Cosmopolites sordidus (Colcoptcra: Curculionidac), which is found in all the banana-producing
regions. Both adults and larvae burrow into galleries in the rhizome and the pseudo stem and cause
size rcduction of banana bunches, breakage of plants, and favour the attack of fungal pathogen that
causes Panama disease (Fusarium wilt). Populations of C. sordidus are controlled by use of baits
prepared by slicing the pseudo stem ("cheese" bait), or cutting 50-cm sections of pseudostem and
splitting these sections in half ("tile" bait). Baits are trcated with B. bassiana conidia or a paste of
conidia, mycelium and rice medium left in the field. As the adults enter the baits, they contact a large
inoculum and arc colonised by the fungus.
The Pernambuco Institute for Agricultural Research and the Sao Paulo Biological Institute
recommend 50-100 baitslha. The baits may be treated by submersion in suspension prepared with 1
kg offungus (mycelium + conidia + rice medium) in 10 litres of water. However, the preferred
treatmcnt is by spreading 50 ml of a paste prepared by blending the same amount of fungal material
with just 1 litre of water (approximately 5 x 109 conidia/bait). The treated surface ofthe bait is placed
against the soil surface in the banana field, and is replaced at every 15th day. Treatment must continuc
until the number of insects observed in each bait averages to less than 5. Under laboratory condition,
85-90% of adults exposed to these baits are killed by the fungus. In field experiments, a 61 % decrease
in the weevil population has been observed by Biological Institute personnel. In natural infestation,
Beauveria amorpha is frequently found infecting the banana weevil, causing 18% of adult mortality
(Batista Filho et a!., 1995). M anisopliae is also pathogenic to C. sordidus but the use ofthis fungus
has not resulted in control as efficient that obtained with B. bassiana.

4.1.6 Coffee (CojJea arabica). Coffee production (in 2.2 million ha) and processing reprcsents
approximately 5% of Brazilian exports in the last 10 years, with US$ 2-3 billion/year. The coffee
berry borcr, Hypothenemus hampei (Coleoptera: Scolytidae), is the most important pest in the
crop, attacking fruits at all stages of maturation. Until 1995, control of this pest was by chemical
pesticides only, but since then the use of B. bassiana has grown in importance. The fungus is used
during the "transit" period, when the insects move from the previous season's fruits to the current
scason's new fruits. Approximately 2-5% of the fruits is attacked at this time. After application of the
fungus, the female borer contacts the pathogen as it crawls on branches and foliage or when attcmpts
to burrow into the fruit. Often, the death of the insect occurs before the coffee borer penetrates the
fruit. B. bassiana occurs enzootically in several regions in Brazil, causing up to 20% of mortality of
adults. The fungus is used commercially in approximately 1,000 halyear, generally in organic plantations,
with applications of200-300 liters of spore suspensioniha. Several strategies for the large-scale use
of this fungus have been proposed, including a conservation approach with selected fungicides with
little harmful effect on B. bassiana used whenever plant diseases need to be controlled in the crop.

4.1. 7 Other Projects Under Development

4.1.7.1 Greenhouse. The most important pests in greenhouses are thrips, whiteflies, aphids,
caterpillars and mites, all of which are controlled by chemical pesticides (Oliveira, 1995). These
products do not always reduce the pest populations to economic levels and can cause other problems
such as pesticide resistance, elimination of natural enemies, pesticide poisoning, toxic residues in
produce, and others. Studies are under way on the use of pathogens in the suppression of insect and
mite populations in protected crops. Great potential has been demonstrated for the fungi B. bassiana
and M anisopliae for control of Tetranychus urticae (Acari: Tetranychidae) (Table 3), Frankliniella
occidentalis (Thysanoptera: Thripidae), and Bemisia tabaci (Hemiptera: Aleyrodidae) (Alves et
aI., 1998b; Tamai et a!., 1999; Lopes et a!., 2000; Ramos et a!., 2000). The research program
involves the selection of isolates of different fungal species for high viru1ence and conidial production,
compatibility with chemical pesticides, survival under storage conditions and development cycles for

202
the fungi on the pests. The use ofinundative releases and entomopathogen conservation and protection
are recommended for greenhouse environments. Favourable conditions related to temperatures
(between 20 and 30°C), high relative humidity, and low exposure to ultraviolet radiation, enhance the
survival ofentomopathogens in greenhouses.

Table 3. Mean population of Tetranychus urticae on chrysanthemum leaves sprayed with

standard chemical pesticides or with Beauveria bassiana (isolate PL63) sprays during the
initial 18 days (Alves et aI., 1998b).

Days Number of mites/leaf

Control Chemical Pesticides B. bassiana
0 2.3 3.4 1.8
7 2.2 1.5 0.6
14 2.9 0.8 0.1
21 5.2 2.4 0.1
28 6.0 1.7 0.1

At present, there are commercial products available in the Brazilian market containing B. bassiana
and M. anisopJiae for use against mites and insects in greenhouses. Between 2,500 and 12,000 kg of
these products are sold annually. The efficient use of these products in greenhouses is only possible
with the implementation of an integrated pest management system. No single program can be used
with the different crops, cropping systems, location, technical expertise of the growers, and diverse
pest problems of diffferent plants.

4.1.7.2 Papaya (Carica papaya). This crop occupies approximately 40,000 ha in Brazil,
concentrated in the states ofBahia and Espirito Santo. These regions have climate that is very favourable
to the entomopathogenic fungi. For example, the area near Linhares, state ofEspirito Santo, where
papaya is grown for the export market, the minimal monthly temperature is 20.5 °C and the maximum
is 26 °C. The mean relative humidity is always above 80%. The mite T. urticae is the most important
pest in the production region, being responsible for a large portion of the production costs. The use
ofchemical pesticides in this crop makes export ofthe fruit difficult due to the presence of residues on
the fruits. Growers have been interested in the use of B. bassiana to control this pest to avoid
problems with chemical residues. This fungus can control the pest efficiently, and substitute the chemical
pesticides. The formulation Boveril PM, produced by ltaforte Bioproducts under strong quality control
and containing a selected isolate is applied in more than 1,000 ha of commercial papaya production.

4.1. 7.3 Soybean (Glycine max). The soybean caterpillar, Anticarsia gemmatalis (Lepidoptera:
Noctuidae), a key pest in Brazil, is susceptible to several entomopathogens. The most important
pathogen used against this pest is the nuclear polyhedrosis virus (NPV), which is used in more than 1
million ha in southeastern Brazil, and regions ofParaguay, Uruguay, and Argentina. Due to this large-
scale program, other natural enemies ofthe soybean caterpillar are preserved. One ofthese enemies,
the fungus Nomuraea rileyi, causes natural epizootics that complement the use of the virus. Under
favourable conditions (temperatures between 26 and 27°C, and relative humidity above 60%), early
occurrence ofthis fungus can disrupt the growth oftheA. gemmatalis populations, making the use of
the viral product and other pesticides unnecessary.

4.1. 7.4 Fire Ant. The fire ants Soienopsis spp. (Hymenoptera: Formicidae) is an important urban
pest. Researchers from ESALQIUSP and the University of Florida have studied the control ofthese
ants with the entomopathogenic fungus B. bassiana (isolate 447). Encouraging results have been
obtained under controlled conditions. Field experiments with the species S. saevissima produced
better results than with the species S. invicta, a species that has become an important pest in North

203
America and other regions of the world. New studies conducted to develop new application
methodologies, powder and bait formulations (Stimac et al., 1989; Stimac and Alves, 1994).

4.1.7.5 Grasshopper. The grasshopper Rhammatocerus schistocercoides (Orthoptera: Acrididae)

causes serious problems in the state ofMato Grosso, in the cerrado region just south of the Amazonic
forest. In 1993, the agricultural research service of the Brazilian Ministry ofAgriculture, with support
from FAa, initiated a biological control program for grasshoppers using a Brazilian isolate of
Metarhizium anisopliae var. acridum (=Metarhiziumjlavoviridae). Under field conditions, this
pathogen, formulated in vegetable oil, is applied at a dose of 1x 1013 conidia/ha. Control of more than
85% of the insect nymphs 14 days after application has been obtained with this dose (Magalhlies et
al.,2000).

4.2 Peru

Biological control was first used in this country in 1904, but since 1960 it has gained importance
with the implementation ofthe National Program on Biological Control by the National Agricultural
Protection Service (Servi90 Nacional de Sanidade Agraria - SENASA). Peru has a long tradition in
biological control, especially in the use of parasitoids and predators. Recently, a large laboratory
specialised on insect pathology and microbial control was built in Lima, and the federal govemment
maintains an agreement with 19 private companies that produce entomopathogens for commercial
use in the country. The largest project on the use of entomopathogenic fungi in this country is for
control of the Andean potato weevil, Premnotrypes spp. (Coleoptera: Curculionidae), with the
fungus B. brongniartii in the potato crop (Ortiz et aI., 1996; Torres et al., 1993). A product known
as "White fungus" is produced on autoclaved cereal grains in plastic bags or trays. The fungus is
produced in 12 centres managed by SENASA, universities and experimental stations, with an average
of 14,000 kg/year for the period between 1996-1999 (A Vera, pers. comm.). The product is applied
to moist soil at a rate of2 kg/m2 at locations used for potato storage and is mixed into the soil to a
depth of 10 cm, ensuring contamination oflarvae entering the soil. The tubers being stored are then
placed directly on the treated soil surface. The pathogen infects the larvae that leave the potatoes to
pupate in the soil. In storage facilities, B. brongniartii has resulted in 78 to 92% weevil control
(A. Vera, pers. comm.). Studies on Andes weevil control are developed by the International Potato
Centre (!PC) and SENASA The direct application ofthe fungus to concentrations of hosts in protected
areas, the low cost of the product, and good acceptability by the farming community are key factors
for program's success (Jackson et aI., 2000). Other programs are listed on Table 4.

Table 4. Use of entomopathogenic fungi in Peru (mod. from Ramirez, 1998).

Fungus Crop Target Pest

B. bassiana Coffee H. hampei
Banana C. sordidus
Rice Spodoptera frugiperda
(Lep.: Noctuidae)
Cotton and sweet B. tabaci
potato
B. brongniarlii Potato Premnotrypes spp.
Adioristus sp. (Col.: Curculionidae)
Epilrix sp. (Col.: Chrysomelidae)
M. anisopliae Vegetables PluleUa xylosteUa (Lep.: Plutellidae)
Several crops Migratory grasshoper
Stenoma sp. (Lep.: Stenomidae)
Leptopharsa gibbicarina
(Hem.: Tingidae)
V. lecanii Vegetables, sweet B. tabaci
potato, cotton
Several crops Aphids and whiteflies

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4.3 Colombia

Colombia also has a long tradition in the use of biological control, especially using parasitoids
and predators. There are more than 45 biological pesticides available in the market including pathogens
such as Bacillus thuringiensis, B. bassiana, M anisopliae, plant extracts, etc. The production of
these biological pesticides involves varying degrees oftechnological development, from simple cottage
industry to modem industrial operations.
A product called "Micobiol Completo", found in the Colombian market, is a mixture of the
fungi B. bassiana, M anisopliae, Nomuraea rileyi, Paecilomycesfumosoroseus and the bacterium
B. thuringiensis. It is a wettable powder formulation containing Ixl0 9 viable spores of each
entomopathogen per gram of product. Use of this product is recommended for control of immature
and adult Lepidoptera, Coleoptera, Hemiptera, Diptera, and mites at a rate of I kg/ha in 200 liters of
water. Another product, "Micobiol H.E.", is a mixture of the entomopathogenic fungi B. bassiana,
M anisopliae, M jlavoviridae, N. rileyi, P fumosoroseus, Paecilomyces lilac in us, V lecanii
and Hirsutella thompsonii. The wettable powder formulation also contains lx10 9 viable spores of
each fungus per gram of product and is used to control ofthe same pests targetted by "Micobiol
Completo" plus nematodes. The recommended dose varies from 1 to 2 kg/ha applied in 200 litres of
water (Velaquez, 1998).
The fungus B. bassiana is produced by the CENICAFE for control of H. hampei.
Approximately 200 tons ofthis product are prepared per year and up to 75% control ofthe coffee
berry borer is achieved by the application ofthis fungus. The agrochemical company Agrevo developed
a product called Conidia WG, formulated as a dispersible granule containing 2.5 x 10 10 conidia/gram
of product, and recommended for control of coffee berry borer. More than 50,000 ha of coffee crop
are treated with this product per year (Ferrer, 1998; Sosa-Gomez, 1999). The company Laverlam
sells several products containing entomopathogenic fungi. These products include 2 with B. bassiana
(Bauveril, recommended for control of Coleoptera, and Brocaril, for use against coffee berry borer),
I with M anisopliae (Destruxin for control of several spittlebugs and other pests), 1 with V lecanii
(Vertisol, for control of whiteflies). This company also advertises a product formulated with
Entomophthora virulenla, which is also recommended for use against whiteflies. Table 5 lists the
most important uses of entomopathogenic fungi in Columbia.

Table 5. Use of entomopathogenic fungi in Colombia.

Crop Fungus Target Pest

Coffee B. bassiana H. hampei
M. anisop/iae H. hampei
Rice and Sorghum N. ri/eyi S. Jrugiperda
Tomato B. bassiana Neo/eucinodes e/eganta/is
(Lep.: Pyralidae)
M. anisopliae N. e/eganta/is
V lecanii Heliothis spp. (Lep.: Noctuidae)
Pastures B. bassiana Col/aria sp. (Hem.: Miridae)
M anisopliae Zu/ia spp.
Oil Palm B. bassiana Stenoma sp.
M anisopliae L. gibbicarina
Ornamentals B. bassiana Phyl/ophaga sp. (Col.: Scarabaeidae)
M anisopliae Ancognata sp. (Col.: Scarabaeidae)
Cacao B. bassiana Mona/onion dissimu/atum
(Hem.: Miridae)
Vegetables and others V lecanii B. tabaci

4.4 Cuba

The use of microbial control, or biological control in general, in the Caribbean region has great
potential due to the climatic conditions, the characteristics of crops and small farm sizes typical of this
region. In Cuba, microbial control has developed mainly because ofthe embargo of chemical pesticides

205
imposed by the USA. Historically, biological control in this country was initiated in 1930 when
parasitoids were introduced for control of sugarcane and citrus pests. Since 1960, when this country
imported commercial formulations ofBt, new methodologies for production of bacteria and fungi
have been developed. In 1988, a national program on biological control was initiated with the creation
of220 laboratories called CREE (Centros de Reproduyao de Entomofagos e Entomopatogenos).
These laboratories were responsible for mass production, marketing and distribution ofnatural enemies,
which are applied to approximately 800,000 ha. The principal products found in Cuba and their
recommended use are the following: Basisavl Bb (B. bassiana, 1 kg/ha), used in sweet potato,
banana, citrus, sugarcane and rice for control of beetles; Vertisav 57 (V lecanii, 1 kg/ha), used in
pastures, vegetable, fruit and ornamental crops, for control of whiteflies, aphids and mites; Metasav-
11 (M anisopliae), used in rice, banana and pastures for control of aquatic Coleoptera, C sordidus,
caterpillars and spittlebugs.
Integrated pest management program, that include the entomopathogenic fungi in tomato and
curcubit crops, in which V lecanii is used to obtain 70-85% control of B. tabaci, Brevicoryne
brassicae and Lipaphis erysimi (Hemiptera: Aphididae) (Vazquez, pers. comm.). Presently, Cuban
researchers are working on the development of new formulations, quality control and lowering
production cost for the entomopathogenic fungi (Vega, 1998).

4.5 Mexico

The use of biological control on Mexico initiated around 1870-1900 with the identification of
entomopathogens for control of grasshoppers, as well as parasitoids and predators for control of
other pests. In 1991, a national standard centre for biological control (Centro Nacional de Referencia
de Controle Biologico - CNRCB) was created to standardise the biological control activities in the
country and facilitate technology transfer in this sector. Mexico has 45 centres for production of
beneficial organisms including 10 producing B. bassiana, 8 producing M anisopliae, 5 producing P
fumosoroseus and 2 producingA. aleyrodis. Together, these centers produced approximately 23,000
litres of entomopathogenic fungi in 1996, sufficient to treat about 20,000 ha and benefit 8,295 farmers
(Bernal, 1998). One of the programs of microbial control is the effort to manage the spittlebugs
Prosapia spp. and Aeneolamia spp. (Hemiptera: Cercopidae) in pastures and sugarcane with
application of M anisopliae to approximately 50,000 ha.
The company Agrobiologicos del Noroeste produces several commercial products including
powder and liquid formulations of products containing P fumosoroseus (for control of whiteflies),
M anisopliae (for control of weevils and soil pests), and B. bassiana (for control of boll weevil,
whiteflies, coffee berry borer, and others). The fungus V lecanii is formulated in a liquid product
used for control of aphids, whiteflies and thrips.

4.6 Argentina

Microbial control has only recently received attention in Argentinean research centres such as
IMYZA-CICA-INTA. Twelve species of entomopathogenic fungi have been reported as having
potential for control of insects and mites in Argentina. Epizootiological studies with N rileyi have
been conducted with the objective of causing early natural epizootics ofthis fungus inA. gemmatalis
populations in soybean crops. Studies are also conducted on the control of Ciclocephala signaticol/is
(Coleoptera: Scarabeidae) with encouraging results (Alves and Lecuona, 1996). Also, irrigation is
studies as a environmental manipulation practice in sugarcane crops to favour the development of the
fungus Cordyceps sobolifera which controls Proarna bergi (Hemiptera: Cicadidae) in the northern
region of Argentina (Sosa-Gomez and Moscardi, 1991).
At the research centre INTA - Sao Pedro, experiments have been conducted on the possibility
of controlling Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), Myzus persicae and Aphis
gossypii (Hemiptera: Aphididae) in tomato and green pepper crops. The B. bassiana formulation

206
Naturalis-L (2.3 x 10 7 conidia/ml) is used for these pests. This fungus is also used for control of
Triatoma infestans (Hemiptera: Reduviidae), with oil or water-based formulations and a dose of 5
x 10 12 conidialha. Argentina maintains collaborative projects with the Cenargen unit of the Brazilian
agricultural research agency (EMBRAPA) on the control of Musca domestica (Diptera: Muscidae)
and Anthonomus grandis (Coleoptera: Curculionidae) usingB. bassiana CR. Lecuona, pers. comm.).

4.7 Other Latin American Countries

4.7.1 Ecuador. The few microbial control laboratories are usually associated with sugarcane mills,
the National Institute for Sugar Research, or some universities. Few studies on microbial control are
reported from this country, but the fungi P lilacinus, applied against Faustin us apicalis (Coleoptera:
Curculionidae) in citrus crops, and N rileyi, applied against Sibine fosca (Lepidoptera: Limacodidae)
in oil palm, are used in small scale.

4.7.2 Venezuela. The company Probioagro has had tremendous success with the production of
entomopathogens. Since 1986, this company, although still in a cottage industry level, has produced
M anisopliae that is formulated into a product called Cobican-l. This product is used with good
results to control Aeneolamia varia (Hemiptera: Cercopidae) in approximately 50,000 ha of
sugarcane. The same product is also used against Sipha jlava (Hemiptera: Aphididae) and Podischnus
agenor (Coleoptera: Scarabaeidae). Probioagro also commercialises products with V lecanii, N
rileyi and B. bassiana which are used for control of B. tabaci, Spodoptera frugiperda (Lepidoptera:
Noctuidae) and many other pests. Most fungi are produced in a biphasic process using rice as the
sporulation medium. The products are wettable powders with approximately 1010 conidia/g.

4.7.3 Uruguay. The institutions involved in microbial control studies in Uruguay are the Ministry of
Livestock, Agriculture and Fishery (MGAP), the National Institute of Agricultural Investigations
(INIA) and the Agricultural College at the Universidad de la Republica. During the 1980's, field
technicians that work in soybean crops were trained to monitor conditions in the fields and implement
measures that favour the fungus N rileyi. This fungus controls natural populations ofA. gemmatalis
and Rachiplusia nu (Lepidoptera: Noctuidae), if environmental conditions are favourable to its
development. In leguminous plants used as cattle forage, the fungus Zoophtora radicans is being
studied as a control measure against Epinotia aporema (Lepidoptera: Olethreutidae) (Stewart, pers.
comm.). In greenhouse crops, studies of entomopathogenic fungi used against T vaporariorum are
conducted by graduate students. Also, several pathogens are being isolated and identified from beetles
in the families Scarabaeidae and Curculionidae. Most isolations are represented by the fungi Cordyceps
sp. and M anisopliae, as well as nematodes, which are evaluated as potential agents for use in
crops. For instance, the fungus M anisopliae is being evaluated for its effect on the consumption of
wheat seedlings by Diloboderus abderus (Coleoptera: Scarabeidae) (Castiglioni, pers. comm.).

4.7.4 Panama. The fungus M anisopliae has been used in the central sugarcane producing region
(Engenho Santa Rosa - Municipio de Aguadulce) to control the root spittlebug Aeneolamia lepidior
(Hemiptera: Cercopidae). The product Biotech, imported from Brazil, is applied at a rate of 5 x 10 12
conidialha. Besides the fungus, yellow traps treated with Stykem Special (40 traps/ha) are used to
collect adults. These traps collect millions of adults per year, causing a reduction of hundreds of
thousands of eggs in the sugarcane crop.

4.7.5 Paraguay. The School of Agricultural Sciences ofthe Universidade Nacional de Assunc,;ao is
involved in research on the use of B. bassiana and M anisopliae for control of workers, gardeners
and soldiers of the leaf-cutting antAtta spp. (Hymenoptera: Formicidae).

4.7.6 Dominican Republic. The fungus V lecanii, imported from Colombia, is used for the control
of B. tabaci in several crops in this country. Also, two other programs involve the use of the

207
entomopathogenic fungus B. bassiana in applications to 8,000 to 2,000 ha of citrus and coffee
crops. In citrus, products manufactured by a citrus-grower cooperative are used for control of
Diaprepes spp. (Coleoptera: Curculionidae). Coffee-berry-borer control is done with the formulations
Brocaril and Bauverial produced by Laverlan in Colombia. The Instituto Superior de Agricultura is
also involved in this research project (Gomez, pers. comm.).

4.7.7 Guatemala and Honduras. These countries use the fungus M anisopliae for control of
Phyllophaga sp. and Aeneolamia sp. in sugarcane. In Guatemala, the company Produtos Ecologicos
has 2 commercial products: Salivase for control of spittlebugs, and Bichoxe for control of whiteflies.

4.7.8 Nicaragua. The fungus B. bassiana is used to control H hampei in coffee crops whereas the
fungus M anisopliae, imported from Colombia, is used for control of spittlebugs in sugarcane.

4.7.9 Costa Rica. M anisopliae is used for control of Aeneolamia postica, Zulia vilios and
Prosapia simulans (Hemiptera: Cercopidae). Use of this fungus produced by the Extension service
responsible for the sugarcane crop has increased from approximately 350 ha treated with 115 kg of
pure fungus in 1989 to almost 6,000 ha treated with 1650 kg of fungus in 1999.

4.7.10 Trinidad and Tobago. M anisopliae has been used since 1910 for control of the spittlebugs
Aeneolamia spp.in approximately 5,000 ha of sugarcane. An oil formulation containing this fungus
was developed for this purpose by the National Institute of Higher Education, Research and
Technology.

5 MICROBIAL PESTICIDES FORMULATIONS

Approximately 36 commercial formulations ofentomopathogenic fungi are used in Latin America,

including formulations of M anisopliae, Beauveria spp., V lecanii, Pfumosoroseus, Sporothrix
insectorum, N rileyi, and E. virulenta (Figure 1). Fifty percent ofthese formulations are produced
by small to medium producers. In general, these products are simple wettable powder formulations
prepared with the ground growth substrate (rice, wheat, etc), a clay carrier, and, in some rare cases,
a wetting agent. Other products ar~ prepared at research centres and universities, and contain the
liquid culture medium or solid substrate plus the fungus. Both products from research centres and
those from industrial facilities need improvements in the formulations to make them more effective.
However, few researchers are dedicated to the formulation aspect, but the investments by the industrial
producers are very limited in this area. This situation prevents technological developments in
formulations, and the final products remain a weak link in the chain from research to application of
entomopathogenic fungi. Large multinational companies have shown little interest in the development
ofentomopathogenic formulations. However, with and increasing public interest in agricultural products
with minimal chemical residues, organic agriculture, and a more sustainable agriculture, the traditional
pesticide companies may be forced to change their position in relation to the use ofentomopathogens
in biopesticidal products. When this change occurs, the number of products containing
entomopathogenic fungi is certain to increase.

6 PERSPECTIVES FOR ENTOMOPATOGENIC FUNGUS USE IN LATIN

AMERICA

The entomopathogenic fungi are, in general, wide spectrum pathogens, capable ofcausing diseases
in several insect and mite species, initiating natural epizootics. These pathogens are versatile and can
infect different stages of the hosts. Penetration of the host can be through different body parts or

208
tissues, but mainly by cuticular penetration. The typically high conidial production of these pathogens
can be very important in the determination of a high horizontal transmission rate. Although,
entomopathogenic fungi were the first organisms studied and used as biocontrols for insect pests, this
group of insect pathogens is perhaps the most complex in terms of development of commercial
products for use as microbial pesticides. Much research still is needed to elucidate the etiology and
epizootiology of the most important groups of entomopathogenic fungi. Also, research is necessary
for the development of effective and stable formulations that can be used as a part of integrated
management of individual pests.
In Latin America, microbial control is more advanced in Brazil, Colombia, Cuba and Peru.
Other countries need further training oftechnical personnel specialised in this area. In general, the
limitations on the development of microbial control in Latin America are the following: (a) low level of
capital investment in this sector; (b) few institutions involved in research; (c) lack of qualified technical
personnel; (d) lack of continuity in research and implementation projects; (e) deficiencies on the
transfer of microbial control technology to the end user. On the other hand, the examples reported in
this chapter, demonstrate the great potential for the use ofthe entomopathogenic fungi in Latin America.
Favourable climatic conditions, including temperature and relative humidity, are common throughout
most countries in Latin America and contribute for the success offungi as insect control agents in the
region. Also, the great genetic variability found in the entomopathogenic fungi allows the selection of
isolates that can be most efficient against individual pests.

Figure 1. Commercial Latin American bioinsecticides containing entomopathogenic fungi, according to the active
ingredient (fungal species or species group) in the formulation.

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Leite, L.G., Alves, S.B., Takada, H.M., Batista Filho, A., and Roberts, D.W., 2000a, Occurence of Batkoa sp. and
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Lopes, R.B., Alves, S.B., and Tarnai, M.A., 2000, Fungo Metarhizium anisopliae e 0 controle de Frankliniella
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Magaihaes, B.P., Lecoq, M., Faria, M.R., Schmidt, F.G.v., and Guerra, W.D., 2000, Field trial with entomopathogenic
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Oliveira, M.R.V., 1995, Controle Biol6gico em casas de Vegetarlio com Especial Referencia a Trialeurodes
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Ramos, I.M., 1986. Atua9ao do fungo Metarhizium anisopliae sobre as cigarrinhas Deois incompleta (Wlk.) e
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 MICROBIAL CONTROL OF INSECT PESTS WITH ENTOMOPATHOGENIC FUNGI
IN CHINA: A DECADE'S PROGRESS IN RESEARCH AND UTILIZATION

Ming-Guang Feng

Research Institute of Microbiology, College of Life Science, Zhejiang University,

Hangzhou 310029, P. R. China

1. INTRODUCTION

More than 700 species of fungi are associated with insect diseases, and thus called
entomopathogenic fungi (Pu and Li, 1996). Those with potential to be utilized for control of insect
pests usually fall within two group: Hyphomycetes in Deuteromycotina and Entomophthorales in
Zygomycotina (F eng, 1998a). Of those, however, only a very few number of species have been
studied intensively for microbial control of various insect pests. Over the past decade a great progress
has been made worldwide in the development ofentomopathogenic hyphomycetes into commercially
registered mycoinsecticides. Recently registered products include various formulations of Beauveria
bassiana, B. brongniartii, Metarhizium anisopliae, Nomuraea rileyi, Paecilomyces
fumosoroseus, and Verticillium lecanii, which are used to control a wide range of insect pests such
as homopterans, coleopterans, lepidopterans, orthopterans, and dipterans (Shah and Goettel, 1999).
Techniques involved in mass production, formulation, and application offungal agents have greatly
advanced worldwide and reviewed recently by numerous authors (Burges, 1998; Caudwell and
Gatehouse, 1996; Cliquet and Jackson, 1997; Hedgecock et aI., 1995; Ibrahim et aI., 1999; Jackson
et aI., 1997; Kleespies and Zimmermann, 1998; Lacey and Goettel, 1995; Lacey and Kaya, 2000;
Milner, 1997; Wraightand Carruthers, 1999).
Back to 1980s, Chinese scientists contributed a great deal to the development of fungal
formulations against insect pests, particularly the large-scale use of Beauveria bassiana for control
of pine caterpillars. Non-registered B. bassiana products for agaisnt forest pests were produced in
more than 10 state-owned workshops supported by the Ministry ofForestry. The earlier information
on B. bassiana has been reviewed by Feng et al. (1994). Over the past decade, dramatic changes
have occurred in China. Accompanied with the transformation of economic system from state plan-
directed system to market-driving one, B. bassiana products are considered to be economically
marginal and become less attractive to investors. Governmental and enterprise input into research
and development for fungal formulations is lacking. Due to misuse or overuse of chemical pesticides
everywhere, production of agricultural products with pesticide residues at tolerable levels has become
a great challenge to the whole country. The fact that farmers do not consume vegetables they produce
for sale has frequently become a focus in news media and perplexes city consumers. Despite the

Advances in Microbial Control of Insect Pests

Edited by Rajeev K. Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002 213
austere situation, there are groups of Chinese scientists who have been devoting themselves to basic
and applied studies on entomopathogenic fungi and making their contributions to better utilization of
these fungi for microbial control of insect pests. However, most oftheir contributions are published in
Chinese language journals, which usually are difficult or inaccessible to non-Chinese language readers.
This review is a retrospect to the recent 1O-year progresses made in China. It is aimed to bridge the
language gap between Chinese and western scientists and facilitate their communication and
cooperation in microbial control of insect pests with entomopathogenic fungi.

2. HYPHOMYCETES

2.1 Selection of Candidate Agents

Entomopathogenic hyphomycetes have a wide range of host insects that adapt to a diversity of
environments and thus differ greatly, at species or strain level, in host specificity, virulence, and potential
for utilization for a specific purpose (Feng et al., 1994). For microbial control oftarget insects concerned,
selecting 'best' or 'most virulent' species or isolates is always a routine, but laborious and time
consuming, procedure that involves in rearing insects under controlled conditions (Lacey and Kaya,
2000). Theoretically, extracellular enzymes such as proteases and chitinases that may involve in
penetration of host cuticle in fungal infection are potentially useful for selection of candidate isolates.
However, results from the correlation of virulence of different B. bassina isolates to their enzyme
activities are often conflicting (Fan andHu, 1996; Hu and Fan, 1996; TangandLi, 1996). Quantifying
the relationship between the virulence index, LT50 , and the extracellular protease and lipase activities
of 17 B. bassiana isolates from different hosts and locations, Feng (1998b) found that only a small
part of variation in LT50 was attributed to the activities of protease (38%) and lipase (15%). As
known as so far, there remains no better approach to selecting candidate isolates for microbial control
than the conventional bioassay method, which allows the most promising isolates to be recognized or
selected largely based upon their virulence indices such as LD 50 and LT 50'
Data from bioassays of microbial agents including hyphomycetes are commonly in the time-
dose-mortality form. Virulence indices are usually estimated by processing bioassay data. Finney's
(1971)' Probit Analysis' is used as the' know how' for analysis of bioassay data. In the past few
years, a novel time-dose-mortality model has been applied to evaluating the responses of microbial
agents to target insects (Feng et aI., 1996, 1998; Nowierski et aI., 1996) and proved greatly
advantageous over the classic probit analysis (Feng and Poprawski, 1999). The time-dose-mortality
modeling technique includes the use of two independent variables, i.e., dose and time, in the model
and enables the generation of parameters that describes not only the separate effects of dose and
time on the tested agent-insect relationship, but also the reasonable interaction between the two
variables. In contrast, the probit analysis is based on the linear regression of a single independent
variable (logarithm-transformed dose for LD50 and time for LT50) against probit-transformed mortality,
and unable to measure the interaction between dose and time, which apparently affect the mortality
of target insects tested. Due to biological and mathematical robustness (Feng and Poprawski, 1999),
the modeling method has become a very useful tool for replacement ofthe probit analysis for evaluating
the potential of microbial agents in insect control. The whole modeling process including estimation of
lethal dosages varying with time and lethal times varying with dosages is performed using DPS software
(Tang and Feng, 2002).

2.2 Mass Production ofInocula

Technology for mass production of entomopathogenic hyphomycetes has been developed in

China as well as elsewhere in the world. For hyphomycetes infecting foliar insects, aerial conidia are
inoculated close to those causing natural infection in the field, and are superior to mycelia, blastospores,

214
and submerged conidia or a mixture of them produced in liquid culture because oftheir greater
tolerance to processing and environment (Feng et aI., 1994). Thus, a diphasic technology involving
the use ofliquid-phase fermentation for mycelial production and solid-phase one for conidiationhas
been widely applied to conidial production of B. bassiana and other fungal agents using substrates
(such as wheat bran and rice chaff) inexpensive and available everywhere (Feng et aI., 1994). Separation
of conidia from solid substrates is achieved by a specifically designed harvest machine (Chen et aI.,
1990), from which high-quality conidial powder flows out. During the first few years of 1990s, much
effort was made toward standardization of mass production of B. bassiana conidia in order to
guarantee quality, viability, and shelflife of conidial powder (Yin et aI., 1996a,b). The standardized
diphasic technology includes the use of a liquid medium (w/v: 7% rice flour, 2% peanut dregs, 2%
what bran, 0.5% peptone, and 2% sucrose) and solid substrates, i.e., rice chaff and wheat bran
mixtured at the ratio of 6:4. A production cycle takes about 10 days, including 2-day submerged
fermentation in tank and 7- or 8-day conidiation on solid substrates. With the improved technology,
the products of B. bassiana conidia can be produced as 'crude powder' containing 2.50xl0 1o
conidia/g and 'pure powder' containing 1.2xlOli conidia/g (Ym et aI., 1996a). The cost for production
of the 'pure powder' is less than RMB ¥50,000/ton (""US$6,025/ton) and presently is sold at a
price ofRMB ¥1 OO,OOO/ton (H.T. Ma, pers. comm.).
The practicability of using rice as a solid substrate for producing aerial conidia of B. bassiana
and Paecilomyces fumosoroseus was recently reevaluated in our laboratory. In state-owned grain
supply centers distributed in all provinces of China, a huge volume of rice is of poor quality due to
over three-year storage for' strategic reserve' or contamination with microbes in storage and has to
be displaced yearly with newly harvested grains at a great cost. It is a challenge to utilize the poor-
quality rice, which usually is sold at a price as low as or even lower than wheat bran for components
of animal feed. With cost and availability in consideration, the poor-quality rice could be utilized to
produce conidial powder offungal agents. Following a method described by Alves and Perceira
(1989) in preliminary experiments, we obtained a yield of ca. 30 g high-quality conidial powder
(> 10 II conidia/g) per kilogram of rice using B. bassiana and P fumosoroseus isolates selected for
control of homo pteran pests (unpublished data). With our experiences, the yield of conidia could be
further enhanced by optimizing rice firmness (water content and cooking time), thickness in pan, and
environmental conditions (temperature, relative humidity and illumination) during fungal growth and
conidiation. Utilizing the poor-quality rice for mass production of conidial powder may become
economically acceptable only if technology involved is consistently reliable enough to gain a yield of
30-50 g/kg. The use of rice as a solid substrate for conidiation is conspicuously advantageous over
the use of other solid substrates such as wheat bran and rice chaff. First, harvest of conidia is easier
with a vibrating sieve apparatus, which needs much less investment than a harvest machine for separation
of conidia from dust-rich substrates. Second, conidial powder harvested from rice is virtually pure,
not containing any non-fungal dust. Finally, the used rice that remains rich in nutrients and fungal
conidia and mycelia can be re-utilized, at least partially, in the following cycle of conidial production
or used as materials for control of underground insect pests after milling and formulization. This
warrants further study.

2.3 Fonnulation

Selection of a formulation for hyphomycetes-based mycoinsecticides to large degree depends

on the ease of field application to target insects within their habitats and the enhancement of shelf-life
and environmental persistence after application (Feng et aI., 1994). Since spraying is the most effective,
conventional approach for applying fungal agents to foliar insects, formulating conidial powder into
emulsifiable suspension and wettable powder is usually the first choice for control of sucking pests
such as aphids, whitefies, planthoppers, leafhoppers, thrips, and mites (Shah and Goettel, 1999).
This is also a direction toward which we are developing formulations of B. bassiana and P
fumosorseus isolates against aphids on vegetables and leafhoppers on tea. An oil formulation, namely

215
'951', developed in 1990s (Li et a!., 1996a) is applied to spray B. bassiana conidia onto pine
caterpillars in forest canopy with an ultra-Iow-volmne technique. The oil formulation is obtained by
adding 50 g of conidial powder to 45 rnl ofan undisclosed solvent oil, then supplemented in total (wi
v) with 5% antioxidant, 0.025% activating agent, 0.01 % synergist, and 0.01 % UV absorbent. Major
quality indices for the oil formulation include a content of:2:5x I 010 conidia/ml, a conidial viability of
:2:90%, a water and volatile content of :::;4%, a conidial suspensibility of:2:80% in a 20-fold dilution of
diesel oil at 30°C, a pH range of 6.2-7.0, and a specific gravity of 1.025-1.035 (Li et a!., 1996b).
The Asian com borer, Ostrinia nubilalis, is a devastating pest threatening to the crop throughout
the China, particularly in northern provinces. Early in 1990s, three experimental formulations of B.
bassiana conidia were developed against 0 nubilalis, including a wettable powder for liquid spray,
a flowable powder for dusting, and granules for hand application to plants at whorl stage (Zhang et
a!., 1990). The three formulations had little difference in field efficacy but all effectively controlled the
borer populations by 79-86% in an area of800 ha (Zhang et a!., 1990). Thus, the wettable powder
fonnulation suitable either for spraying or for dusting was further improved by optimizing its components
(data undisclosed), and turned out a product containing 5x 10 10 conidia/g with both viability and
suspensibility of>85% (Zhang et aI., 1992).
Effort has also been directed toward the formulization of B. brongniartii against the grubs,
Holotrichia spp., that make serious damage to peanuts underground in growing season. A conidia-
based, powdery formulation contained 4x 10 9 conidia/g (Deng et a!., 1995) while mycelia-based
ones are in the form ofalginate pellets or simply dessicant mycelia mixed with wheat bran and attapulgite
(Nong et a!., 1998). These formulations are designed for use in soil at the time of seeding, weeding,
or fertilizing.
In our laboratory, much effort is also being made to develop an emulsifiable formulation of B.
bassiana conidia for control of sucking insects such as aphids and leafhoppers. A mineral oil was
selected as inert carrier, sucrose esters of fatty acids as an emulsifier, sodimn carboxymethyl cellulose
as a stabilizer, and vitamin C as a UV protectant based on wettability, suspensibility, stability, and,
most importantly, biological compatibility with B. bassiana conidia (Ying and Feng, 2001). An
emulsifiable formulation that has been developed for pilot field experiments consists of 10% conidial
powder (> 1011 conidia/g), 89.4% mineral oil, and 0.6% in total of the other additives (Feng et a!.,
unpublished data).

2.4 Shelf-life

Commercial registration of a fungal formulation cannot be permitted unless it has a market-

acceptable shelf-life, which may differ from country to country. Since active components of a real
mycoinsecticide are always viable inocula (conidia, blastospores or mycelia) which are sensitive to
processing procedures in drying and formulation and environmental factors (Chen and Feng, 2002),
shelf-life is a limiting factor for its registration. For this reason, no hyphomycetes-based formulations
to date have been officially registered in China though much efforts have been made for this purpose
and non-registered products of B. bassiana have been widely applied (Feng et a!., 1994). With the
oil formulation '951' for control of pine caterpillars, for example, conidial viability decreased from
>90% at start to 80% after five-month storage at ambient temperature and then fell down sharply to
only 40% by the end of another three-month storage (Li et a!., I 996a,b ). The relatively short shelf-
life remains an obstacle to registration of the oil formulation. The wettable powder of B. bassiana
conidia developed by Zhang et al. (1992) has a longer shelf-life than the oil formulation. Stored at
ambient temperature (I 0-20°C), it kept a conidial viability of about 85% just as started after eight
months, and had only a 10% decrease in viability after 10 months (Zhang et aI., 1992). Neither
alginate pellets nor powdery formulation ofB. brongniartii mycelia can be stored at room temperature
for longer than six months (Nong et al., 1998).
Factors that influence the shelf-life ofa fungal formulation are complex. Generally, water content
in formulation, biological compatibility of additive components with inocula formulated, and

216
environmental conditions for storage are often speculated to be closely associated with shelf-life. A
fonnulation with longer shelf-life at ambient temperature is always pursued. However, it is difficult to
make as dry fonnulations as possible at a low cost with no loss of viable inocula, and to find detailed
infonnation on the correlation offungal shelf-life to potentially influential factors. In a recent experiment,
vacuurn-freeze-dried conidial powder of B. bassiana with 4.0% water content were half-monthly
examined for viability during one-year storage at 4 and 20°C, respectively (Feng and Ying, 2002;
Ying and Feng, 2002). The viability of considia stored at 4°C decreased slightly from 99.0% to
90.2% at the end of one year, however, at 20°C, the viability declined slowly during the first 5.5
months but thereafter decreased dramatically, becoming nearly undetectable at the end ofeight months
(Figure 1). A modeling analysis showed that the time for the conidial powder to lose 50% viability
was 1006 days at 4°C and 197 days at 20°C, respectively. Clearly, even with water content below
5%, the conidial powder of B. bassiana can be safely stored for one year or longer only at low
temperature but no longer than 6 months at ambient temperature.

100

80
'"""'

I
~
t<I
:a
·S 60
0
u
...... ~".
0

g 40 l
:.c.~ \

°
>
20

,
o ~~~~~~~-r~~~~-r~~~'~~~~~~~
o 60 120 180 240 300 360
Number of Days in Storage

Figure 1 Variation in the viability (II) of vacuum-freeze dried B. bassiana conidia over time (d) during one-year
storage at 4 and 20°C

Further examination ofB. bassiana conidial viability and internally reserved nutrients including
saccharides and proteins have been inspiring while fresh powder with water content of 58. 9% and
vacuurn-freeze-dried powder with water content of7.4% were stored at 4°C and 20°C (Feng and
Ying, 2002). During one-month storage, water content and temperature were found interactively
affecting the levels of conidial viability and the contents of reserved saccharides and proteins, which
in turn correlated considerably well to the viability. Stored at 4°C and 20°C, the vacuum-freeze-
dried conidia lost their saccharides by 13.4 and l4.l %, proteins by 39.2 and 38.2%, and viability by
32.0 and 55.8% when germinating in water only, and 6.7% and 10.4% when germinating in 2%
glucose solution plus 0.5% peptone, respectively. In contrast, the four pairs of estimates for the fresh
conidia stored at 4°C and 20°C decreased by 42.4 and 43 .2%, 66.3 and 65.4%, 96.4.8 and 99.4%,
and 9.9 and 98.4%, respectively. Apparently, water content in B. bassiana conidia affected the
range of variation in their viability and content of internally reserved nutrients while temperature for
storage influenced the rates ofvariation for both. However, depletion ofthe internally reserved nutrients
did not necessarily inactivate the conidia. Instead, such conidia may germinate at relatively high level
as long as supplied with sufficient nutrients.

217
2.5 Application

Following success in 1980s (Feng et aI., 1994), non-registered B. bassiana fonnulations have
been used for control of pine caterpillars in southern provinces of China despite limited governmental
input and fast-growing labor cost that have influenced the scale ofthat practice. Forest protection
largely gets benefit from the use of B. bassiana products. In forest areas where B. bassiana conidia
are sprayed with more or less supplement of chemical insecticides, pine caterpillars can be consistently
suppressed to a very low level, accompanied by more stable and diversified cornmunity of arthropods
and insect fungi (Li et aI., 1998). In 2001, more than 70 tons of 'crude powder' of B. bassiana
(2.5x 1010 conidia/g) were produced and sprayed against pine caterpillars in forest areas ofGuangdong
Province alone (H.T. Ma, pers. cornm.)
Spraying conidial suspensions of B. bassiana on piles of com stalks after harvest was an effective
approach to reduce the populations of overwintering larvae and pupae of the com borer, Ostrinia
nublalis over ten years ago (Feng et aI., 1994). Nowadays, this practice is still effective in some
areas but not in so large scale as before. It is effective only when all farmers simultaneously treat their
com stalk piles after harvest. However, not all ofthem are willing to take an action for control ofcom
borers that may not necessarily make damage to their own crop in the following year. To stimulate
farmers' enthusiasm toward the use of B. bassiana products against 0. nublatis, pilot field experiments
with three B. bassiana fonnulations were conducted in Changtu County, Liaoning Province during
the com-growing season (Zhang et aI., 1990). In an 800-ha area, a field efficacy of reducing 0.
nublalis larvae by 79-87% was achieved by spraying, dusting, or applying granules of B. bassiana
to plants at whorl stage. With efficacy and efficiency in consideration, a wettable powder of B.
bassiana conidia suitable for spraying was then recommended for field application against com
borers (Zhang et aI., 1992).
A preparation of B. brongniartii conidia was tested for efficacy for protection of peanuts from
damage by white grubs during 1992-1994 (Deng et al., 1995). The resulting field efficacy depended
on the time and dosage of application. During the growing season, the preparation mixed with soil
was applied by hand at the rate of7 j-15.0x 1013 conidia/ha, followed immediately by hoeing. The
damage of white grubs to peanuts was reduced by 70-80% in a 300-ha area in Shandong and
Jiangsu provinces. This efficacy was close or similar to that given by application of 40% isofenphos-
methyl at the rate of 1.2 kglha. When applied at the time of seeding, however, a larger dosage of B.
brongniartii conidia was needed to gain the same level of control.
Fan and Li (1996) investigated the potential of Metarhizium anisopliae preparations for control
of the peach fruit borer, Carposina nipponensis, in apple orchards. Based on counts of colony-
fonning units (CFU) in sterile and non-sterile soil samples, the half-life of initial inocula after soil
inoculation was measured as 26.9 and 23.8 days for conidial preparation (2.0x 109 conidia/g), and
26.9 and 19.6 days for mycelial preparation (l.Ox 109 CFU/g), respectively. Introduction of
overwintering C. nipponensis larvae, with their cocoons, into soil inoculated 100 days before may
greatly enhance the level ofinocula in the soil with the recovery CFU counts being 1000 times greater
on day 278 after inoculation than those in the non-larval soil on day 271 after inoculation. The
mortality of C. nipponensis larvae attributed to fungal infection was beyond 90% by day 131 after
introduction and declined to 9.3% by day 237 but zero in uninoculated soil. In plot trials, 97-100%
of C. nipponensis larvae were killed by application of M anisopliae preparation in soil at the
dosages of 15-37.5 kg/ha with the percentage of bored fruits being reduced to 2.7-5.0%. In pilot
experiments in a 670-ha area in north ofShaanxi Province, the percentage of bored fruits was reduced
from 30% in untreated orchards to 2.4% in orchards treated with the M anisopliae preparation at
the rate of22.5 kg/ha. This work is a good example for the use of M anisopliae preparation for
control of fruit borers that stay in soil for a stage to complete their life cycle.
Numerous species of aphids are highly susceptible to B. bassiana in laboratory bioassays
(Feng and Johnson, 1990; Feng et aI., 1990a,b, 1996; Liu et aI., 1999a,b; Xu et aI., 2002) though
the fungus rarely causes epizootic of aphids in nature (Feng et aI., 1991). We recently tested field

218
efficacy for two emulsifiable suspensions of B. bassiana conidia with one containing'" 1010 conidia/ml
only (ES-I) and another made by adding 1% (w/v) of 10% imidacloprid WP to ES-I (ES-II). The
chemical insecticide added in the ES-II was biologically compatible to B. bassiana very well (Xu et
aI., 2002). Three dilutions of both (1 07 , 106 , and 105 conidia/ml) were sprayed onto cabbage plants
for population control of the green peach aphid, Myzus persicae, in randomized block plots of a field
located in Kunming, Yunnan Province from late June through late July 200 1. During a 28-day period
of sampling at intervals of3 or 4 days, the ES-II sprayed at the concentration of I 07 conidia/ml
effectively controlled aphid population with an efficacy being consistently above 90% from day 7
after spray (Ying et aI., 2002). At the same concentration, the ES-I also yielded a good control of the
pest with an efficacy on day 7 after spray reaching 85%, remaining >70% in the following two
weeks, but declining to 64.4% on day 24 and 52.6% on day 28, respectively. At the lower
concentrations sprayed, both of the formulations resulted in a significant control of the aphid pest but
the ES-II consistently had a better control than the ES-I (Figure 2). The summer weather ofKunming
characterized with mild temperature and frequent slight-rainy days was highly favorable to the use of
B. bassiana for aphid control.

300 300
--<>- CK - .. - 10' (A) (B)
'-'9-0" 10' -'0--- 10' C
!! 240
...
~, ao ES- II
"C .;;
:g, 120
-<
{
-<
120
~..}.:j
C
Z 60
C
Z 60 -<f,··t
. !. .. .:-.;.~:::::s:::::.~~:.~.=:~._ ..f _. -f
o 12 16 20 24 28 12 16 20 24 28

Number of Days ane r pra y Number of Day .ner pray

30 (D) ___ max

G
.=...
~2 5

'; 20
~
E
~ 15

12 16 20 2. 28

Num ber Of D ay' an .. Spray

Figure 2. The efficacy of two emulsifiable suspensions of B. bassiana conidia for control of M persicae populations
on cabbages in a field-plot experiment in Kunming, Yunnan during July 2001 (Ying ef al., 2002). (A) and (8)
Fluctuation of aphid densities after spray of ES-J and ES-II at varying dosages. (C) Relative efficacy for aphid
control at different dosages of each suspension. (D) Local records for temperature and precipitation during the
experiment.

The predominant insect pests infesting tea leaves and shoots in southern China are small green
leafhoppers, including mainly the false-eye leafhopper Empoasca vitis, the lesser green leafhopper
E. flavescens, and other less abundant Empoasca species (Zhang et a!., 1997; Zhao et a!., 2000).
Dwelling on new shoots of tea plants, the leafhoppers make serious damage to tender leaves by
sucking, causing a loss of> 25% in tea yield and dramatic decrease in tea quality due to curling and
parching of the leaves damaged (Zhu et a!., 1993). To control the tea pests, chemical insecticides

219
including fenvalerate, cyfluthin, and cypermethrin (Zhuang, 2000) are frequently sprayed leading to
high-level residues of pesticides in tea. In June 1999, the European Union issued new standards for
tea safety control, i.e., maximum residue level (MRL) for pesticides for imported tea products (Zhuang,
2000). The new MRL standards that have been effective since July 2000 strictly limit the export of
Chinese tea products to Europe. For instance, the new MRLs for fenvalerate and dicofol are no
more than 0.1 mg/kg, greatly lower than the previous 10 and 20 mg/kg, respectively. To meet the
new standards for international tea trading, tea pest control that has relied primarily upon chemical
pesticides for long must turn to a new approach that may minimize the level of pesticide residue in tea
products. Under this situation, the two emulsifiable suspensions of B. bassiana conidia were
preliminarily tested for their potential for control oftea leafhoppers (unpublished data). Spraying of
500-, 1000-, and 1500-fold dilutions of both ES-I and ES-II in a tea garden in Menghai County,
Yunnan Province significantly reduced the leafhopper populations compared to that in plots of water
spray for control (Figure 3A and B). During a 28-day period of sampling after spray, the estimates of
field efficacy were 50-83% and 33-49% for 500-fold dilutions ofthe ES-II and ES-I, 30-69% and
37-48% for 1OOO-foid dilutions, and 26-56% and 25-60% for 1500-fold dilutions, respectively
(Figure 3C). Apparently, adding a very small volume of chemical insecticide (much far below its
sublethal dose) into the emulsifiable suspension of B. bassiana could enhance the infection level of
the tea leafhoppers under field conditions. Noticeably, it was not rainy during the first five days after
spray though relative humidity from 2:00 a.m. to 9:00 a.m. and temperature seemed to be suitable for
B. bassiana infection during the experiment (Figure 3D and E). At the same dosage sprayed, both of
the emulsifiable suspensions controlled M persicae on cabbages (Figure 2) more effectively than the
leafhoppers on tea.

3. ENTOMOPHTHORALES

Entomophthoralean fungi are usually characteristic with active discharge of primary conidia
from insect cadavers, which is of great significance in their epizootiology. In nature entomophthoralean
fungi frequently cause epizootics that take important part in natural control of pest insect populations,
e.g., cereal aphids under irrigated conditions (Feng et aI., 1991, 1992). The large potential for
Entomophthorales-caused epizootics to collapse insect populations during a limited period of time
makes it imaginable to consider them as important agents for microbial control of insect pests.
Nevertheless, formulating entomophthoralean fungi into commercial products is technically far more
difficult than formulating hyphomycetes and remains a dream at present though mycelial production
of some species in Pandora and Zoophthora can be quite easy (Liu and Feng, 2001). Contributions
of Chinese colleagues to entomophthoralean study in the past decade are summarized as follows.

3.1 Entomophthoralean Resources in China

With long-term effortofLi 's group in Anhui Agricultural University, Hefei, an important monograph
'Flora Fungorum Sinicorum, Vol. 13, Entomophthorales' has recently been published by Science
Press in China (Li et aI., 2000). Following Humber's system for entomophthoralean classification
(Humber, 1989), the monograph demonstrates that about 340 species described in the world can be
correctly assigned to the order Entomophthorales though there are numerous species that more than
240 synonyms are documented in literature. Ofthose, 59 species that since mid-1980s have been
recorded or described by Li and his colleagues in China are classified into 12 genera of 4 families,
including (i) Ancylistaceae: Conidiobolus Brefeld; (ii) Basidiobolaceae: Basidiobolus Eidam; (iii)
Entomophthoraceae: Batkoa Humber, Entomophaga Batko, Entomophthora Fresenius, Erynia
(Nowakowski ex Batko) Remaudiere & Hennebert, Furia (Batko) Humber, Pandora Humber,
Strongwellsea Batko & Weiser, Tarichium Cohn, and Zoophthora Batko; and (iv) Neozygitaceae:
Neozygites Witlaczil. Entomophthoraceous species originally described or revised by Li's group on

220
the basis of Chinese specimens including Erynia chironomis (Fan & Li) Fan & Li, Er. gigantea Li,
Chen & Xu, Furia Jujiana Huang & Li, F gloiospora (Vuilllemin) Li, Huang & Fan, F
shandongensis, F triangularis (Villacarlos & Wilding) Li, fan & Huang, Pandora athaliae (Li &
Fan) Li, Fan & Huang, P bibionis Li , Huang & Fan, P borea (Fan & Li) Li, Huang & Fan, P
cicadellis (Li & Fan) Li, Fan & Huang, P shaanxiensis Fan & Li, Tarichium syrphis Li, Huang &
Fan, Zoophthora anhuiensis (Li) Humber, and Z. pentatomis (Li, Chen & Xu) Li, Fan & Huang.
This milestone work provides a major avenue into resources of enomophthoralean fimgi in China as
well as in the world.

~Coolrol - <0 - 1500X 6

- v _ IOOOX .-0... 500X
(A)

ES- I

o 8 12 16 20 24 28 o 12 16 20 24 28
Number orOays aner Spray Number of [}ays afrer Sl1ray

_ JS ·I, " '

c:==l i~ -#I"' \ c:::::lI IJI. . u a.lln\: ~ I)o_n~\: (C)

10~--.---.--,---.---.--,---.
o 12 16 20 24 28
S:IImpling Oal~ anti'" Spny Number of Days after Spray

::
.__ ._. 2;00 - 9:00 .•- .•.. 1":00 ·--- --· 20;00 -l\II!AIII

---. ' .. /' ....-..-_.. _....._... ~ : ;.~..- ..

100
~~ ~-.
~ 90
~ /. /. 'L.: \ .~ :', ~ ;' ,r. ":I
'0
'e 80
"t" ,
/,
I'
i, '
\ / '-.,.,. 1 ,~;,!~ i.
" I \
.I~ . " I ~
•
12
-
_.
~
"
:<: \.... .! i \ i ~ .r~\ I \ ~ . i t g'
-'. ,.'.)1 .,< 1~ 1""'/'; ~, ~:'" ii
"
.~
70 ·
;; !
60
~ ,; ; : :,': (E)' ;'; '"
50 l !lq!!~ , I +-L , ~!!j 'f i
o 4 8 12 16 20 24 28
Number or Days aner pray

Figure 3. Control of tea leafhoppers by spraying two emulsifiable suspensions of B. bassiana conidia in Menghai,
Yunnan in autumn 2001 . (A) and (8) Fluctuation ofleafhopper densities after spray ofES-! and ES-II, respectively.
(C) Relative efficacy at varying dosages ofES-1and ES-II. (D) and (E) Local records for temperature and moisture
during the experiment. (Feng et aI., unpublished data)

3.2 Routine Maintenance and Preservation of Cultures

While Li's group devoted themselves to searching for entomophthoralean resources, our
laboratory focuses more upon understanding biological aspects of selected Pandora and Zoophthora

221
species and their interactions with aphids and environmental factors. To perform basic and applied
research on these fungal pathogens, safe culture maintenance and preservation tUlder routine laboratory
conditions are a prerequisite. Only professional laboratories such as Plant, Soil and Nutrition Laboratory
in Ithaca, New York, USA presently are capable of maintaining these cultures in liquid nitrogen (-
180°C) for long-term preservation. Researchers involved in entomophthoralean study often encotUlter
problems with variations or loss of their cultures. In an attempt to resolve such problems, we have
developed a simple, inexpensive method that can be easily used for routine maintenance and
preservation of Pandora and Zoophthora cultures with conventional equipments (Feng and Xu,
2001; Xu and Feng, 2001a). The method involves the use ofOS-SDAY, a modified SDAY (l %
peptone, 4% glucose, and 1.5% agar plus 1% yeast extract) medium containing 0.5% sesame oil and
0.1 % sucrose fatty acid esters, a foodstuff emulsifier. Slant cultures ofnumerous isolates ofP delphaics,
P neoaphidis, Z. anhuiensis, and Z. radicans can be safely preserved at 3°C on OS-SDAY for at
least six months. Some P neoaphidis isolates are still recoverable from the slant cultures preserved
for 14 months with no visible change in biology and virulence. Currently, more than 100 Pandora
and Zoophthora isolates are preserved in our laboratory with the above method, which guarantee
our study on all aspects of the fungal species.

3.3 Biological and Epizootiological Aspects

The genera Pandora and Zoophthora in the family Entomophthoraceae include species that
are of great potential for utilization in integrated pest management, e.g., the aphid-specific pathogen
P neoaphidis and the much less host-specific pathogen Z. radicans, both of which are well known
and globally distributed (Feng, 1997; Feng and Poprawski, 1998; Feng et aI., 1999; Glare and
Milner, 1991). Recent effort in our laboratory is emphasized on understanding biological and
epizootiological aspects of P de/phacis and Z. anhuiensis, the two species that are poorly known in
biological details elsewhere in the world. An approach to mass production and formulation oftheir
mycelia is also explored.

3.3.1 Pandora delpbacis Morphologically, P. delphacis infecting rice planthoppers and leafhoppers
is very difficult to be distinguished from P. neoaphidis on aphids though P. neoaphidis may infect
cereal aphids such as Sitobion avenae on late-season rice plants in paddy fields where P. delphacis
simultaneously infects planthoppers and leafhoppers (tUlpublished data). Both fungal species have
similar shape and size of primary conidia but more or less differ in culture features. Isolates ofP.
delphacis derived from rice planthoppers or leafhoppers in Hangzhou, China are characteristic with
much faster and easier growth on the plates ofSEMA (Sabouraud egg yolk-milk agar) and OS-
SDAY or in liquid culture (Feng and Xu, 2001; Xu and Feng, 2001 b). Entire darkness and longer
photophase are respectively favorable to its liquid and plate cultures (Xu and Feng, 1998). With my
own experience with Entomophthorales, P. delphacis is more competitive for microbial control of
homopteran insects than P. neoaphidis with the following reasons: (i) easiness of handling and mass
production; (ii) a wider host range; (iii) ecologically adapting to a wider range of temperature (l0-
30°C); and (iv) high potential for aphid control.
Two P delphacis isolates, F95127 and F95129, derived from mycosis-killed planthoppers
collected in Hangzhou, Zhej iang Province were bioassayed for their virulence to the brown rice
planthopper, Nilaparvata lugens (Xu et aI., 1999). Batches of 4- or 5-instar nymphs were exposed
to time-varying (dosage-different) showers of primary conidia discharged from sporulating plates of
mycelia from liquid culture and then were maintained on rice plants at 25°C tUlder a photophase of
12L: 12D for daily examination of mortality. Based on analysis of the resulting time-dose-mortality
data with a modeling technique (Feng et aI., 1996, 1998; Feng and Poprawski, 1999), the estimates
ofLDso for days 6-8 after exposure were 327, 122 and 46 conidialmm2 for F95129 and 409, 133
and 74 conidialmm 2 for F95127, respectively. The LTSG estimated for F95127 was 7.6 d at 95
conidialmm 2 whereas that for F95129 decreased from 6.6 d at 172 conidialmm 2 to 5.6 d at 525

222
conidialmm2• The virulence indices indicate that the two isolates ofP. delphacis had moderate virulence
to N lugens with no significant difference between them. .
On the other hand, P. delphacis F95129 and P. neoaphidis F97006, an aphid-derived isolate,
were tested for comparison oftheir virulence to M persicae, and repeated after 14-month preservation
on OS-SDAY slants as above (Xu and Feng, 2000). Batches of :S;3-day-old nymphs on detached
cabbage leaves were exposed to conidia showers of varying time lengths for inoculation with 88-345
nymphs per shower (dosage). The nymphs after exposure were maintained at the regime of 18-20°C
and L12:D 12 with high humidity and were examined daily for mortality. Subsequent time-dose-
mortality modeling resulted in similar slopes for dose effects of both species but faster time effect of
P. delphacis than P. neoaphidis. In other words, P. delphacis killed M persicae more rapidly than
did P. neoaphidis. The estimates ofLD 50 on days 3-7 after exposure decreased from 18.1 to 0.9
conidialmm2 for P. delphacis and from 17.0 to 0.04 conidialmm2 for P. neoaphidis, respectively.
The estimates ofLTso for the two pathogens were similar (about 3.5 days) at the dosages of>5
conidialmm2•
Successful infection ofP. delphasis to host insects depends on the germination ofconidia attached
to host integument, where biochemical substances relating to cuticular composition and host secretion
or excretion may have an effect upon the process of germination. In tests including amino acids, fatty
acids, carbohydrates, vitamins, minerals, and extracts of aphids or planthoppers (Xu et at., 2001),
the formation of infective germ tubes from primary conidia was significantly stimulated by cysteine,
asparagine, behenic acid (C22:0), trehalose, frU(~tose, glycerol, maltose, ascorbic acid, thiamine (VB I),
folic acid, ZnSO4 ' FeSO4' and extracts of aphids and planthoppers. Of these many were inhibitory
to the formation of secondary conidia from primary conidia, which in turn was stimulated by nutrients
not conducive to the germ tubes, including tyrosine, alanine, glucose, galactose, sucrose, glycogen,
inositol, pyridoxal (VB6), NaCI, and KCt.
Based on electron microscopic examination of insects infected, P. delphacis infected both N
lugens and M persicae very rapidly after exposure to conidia shower (Feng and Xu, 2002a; Xu,
1999). About 30-40% of the conidia attached to M persicae cuticle germinated within 4 hours in
three modes, i.e., one germ tube with an appressorium, two or more germ tubes with appressoria,
and branched germ tube only. In contrast, less than 10% of the conidia attached to N lugens cuticle
germinated during the same period oftime after exposure. Few secondary conidia were observed
forming on the cuticles ofboth insect species. In the following 6-1 0 hours, the fimgus began penetrating
aphid cuticle either by germ tubes or by appressoria, and planthopper cuticle by appressoria only.
Upon entry into host haemocoel, the fimgus proliferated rapidly in the form of protoplasts and, at
25°C, took 3 and 6 days to kill M persicae and N lugens, respectively. The dying host was filled
with fimgal protoplasts, which became hyphal bodies shortly before death apparently due to exhaustion
ofhaemocoel nutrition by the fimgus.
The survival of P. delphacis conidia was largely affected at different regimes of temperature
and relative humidity. As shown with fluorescence microscopy (Xu and Feng, 200 1c), primary conidia
on glass slides at 10-30°C survived the first hour by 44-97% at 51 % RH, 94-100% at 74% RH, 95~
100% at 85% RH, 93-100% at 90% RH, 96-100% at 95% RH, and 100% at 100% RH, respectively.
Maintained at 10-30°C for over 4 months, the conidial survival remained 55-74% at 51 % RH, 52-
87% at 74% RH, 38-73% at 85%RH, 1-65%at90%RH, 7-21% at 95% RH,and 1-5% at 100%
RH, respectively. Combinations of high humidity (~90% RH) with high (~25°C) or low temperature
(10°C) proved detrimental to P. delphacis conidia.
The infection of P. delphacis was confirmed to influence aphid survival rate, reproductive rate
and innate capacity for increase (Xu and Feng, 2002). In the experiment, fifth-instar nymphs ofM.
persicae were inoculated with a dosage of conidia higher than LD 50 and then maintained at different
regimes oftemperature (10-30°C) and relative humidity (74-100% RH) in a modified device (Feng
et al., 1999). Every three fifth-instar nymphs so inoculated were included. The results indicated that
infected aphids produced significantly fewer nymphs than those uninoculated in controls. The innate
capacity for increase differed greatly among the temperature regimes and was ofparabolic type with

223
a peak at 25 0e. No significant effect was detected on the innate capacity for increase among the RH
treatments. Inoculated aphids at 15-25°C had an innate capacity for increase significantly smaller
than uninoculated ones. During the 30-day development of mycosis in aphid colonies after inoculation,
the mortality of aphids (adults and progenies) due to infection was significantly higher at regimes of
25-30°C and 95-1 00% RH than at other regimes, suppressing the increase of aphid population by
>80%.
Epizootiological potential of P. delphacis in M. persicae colonies was evaluated recently by
Feng and Xu (2002b). One fresh cadaver was introduced to each colony including 10 second-instar
nymphs on a detached cabbage leaf and every eight colonies (replicates) so prepared were maintained
at each of25 regimes of temperature (I 0-30°C) and humidity (74-100% RH). Daily records of
survivals and cadavers were made for 30 days after introduction. The resulting counts for living and
disease-killed aphids were significant different among the 25 regimes. An epizootic attributed to P
delphacis established at all regimes but developed much more rapidly in colonies at the regimes of
higher temperature (20-30°C) and humidity (~95% RH), at which progenies were effectively infected
by contacting the conidia discharged from apterous cadavers. Compared to the increase ofM persicae
colony not contaminated with the fungal agent, the efficacy of control at all humidity regimes of30°C
was best: >60% on day 4 and 100% on day 16. Second to the best, the increase of colony size at 20
and 25°C was controlled by >30% on day 8 and >80% on day 20 at all humidity regimes with
occasional exceptions. The efficacy of control at 10 and 15°C was usually inferior to those at higher
temperatures but to less degree associated with relative humidity. Variation of daily mortality was
depicted quite well by temperature, relative humidity and the number of days after introduction (r2=0.85,
P<O.O 1) based on polynomial regression.

3.2.2 Zoophthora anhuiensis

The fungal species was first described as Erynia anhuiensis Li (Li, 1986) and revised to the
current name by Humber (I 989). So far recorded only in China, Z anhuiensis frequently causes an
epizootic in aphid populations, particularly on cruciferous vegetables in southern China from mid-
autumn through early winter (Feng et al., 1995). Based upon its biological and epizootiological features
learned in the past few years, Z anhuiensis is distinguished from Z radicans with two major
differences. Firstly, Z anhuiensis is an aphid-specific pathogen with no other insect hosts in records
whereas Z radicans is capable of infecting a wide range of insect hosts including Homoptera,
Lepidoptera, Coleoptera, Diptera, and Hymenoptera (Feng and Li, 1995; Humber and Hansen,
2001). Secondly, Z anhuiensis ecologically adapts to temperatures from 5 to 25°C, with a preference
to 15°C, whereas Z radicans usually adapts to temperatures from 15 to 30°C (Glare and Milner,
1991). The preference of Z anhuiensis to a lower range oftemperature phenologically coincides
well with its occurrence in aphid populations from October through December. Aphid cadavers
collected from the field in December are commonly filled with resting spores, which may interrupt
natural infection until the following autumn (uppublished data).
This fungal pathogen can be isolated from aphid cadavers and cultured on agar plates or in
liquid medium as easily as Z randicans. In a study on Z anhuiensis sporulation (Li and Feng,
2000), mycelia produced in liquid culture sporulated very well on water agar plates at 10-20°e.
Sporulation at 15°C was most rapid and abundant and was little affected by photophase. At 5 and
30°C, however, few conidia were produced from the mycelia on water agar plates. At 10,20, and
25°C, sporulation was significantly higher in light than in dark. A humidity required for sporulation
from M persicae cadavers was ~98% RH. The duration of exposing the cadavers to high humidity
was a key factor influencing sporulation. Exposed to 100% RH for> 12 h and then moved to a lower
humidity (85-90% RH), the cadavers remained capable of discharging conidia at the lower humidity.
The germination pattern of Z anhuiensis primary conidia discharged from sporulating cultures
was shown in relation to temperature and relative humidity (Li, 2000). The germination occurred at
5-25°C with the optimal temperature falling within 10-20°e. A relative humidity of about 100%,

224
even with free water, was necessary for the germination. Also, the interaction of temperature and
humidity comprehensively influenced the germinating behavior. eapilliconidia formed from the
germination of primary conidia were abundant on water agar plates at 10-20 o e whereas vegetative
germination mostly occurred at the regime of 100% RH and 25°C. Humidity seemingly was more
influential on the germination mode of the conidia than temperature. Germination of the primary
conidia on water agar plates usually led to capilliconidiation at 5-25°C. Both capilliconidiation and
vegetative germination occurred at regimes of 10-25°e and 100% RH with the proportions being
affected by temperature. The former predominated at lower temperatures (1 0-1 5°C) while the latter
predominated at higher temperatures (20-25°C). Vegetative germination occurred only at 98% RH.
On the cuticle of M persicae, the primary conidia of Z anhuiensis were observed germinating
within 4 h after attachment and started penetrating into the cuticle when germ tubes formed, based on
scanning electron microscopy CLi, 2000). During the following 2-h peiod, most of the germinating
conidia formed an initial structure of capilliconidiophore, followed by numerous caplliconidia on the
cuticle. Some of the capilliconidia produced infective germ tubes for penetration. This indicates that
capilliconidia may play an important role in the aphid-infecting process of Z anhuiensis as well as
primary conidia.
To quantitatively assess the infection rate of Z anhuiensis againstM persicae, a two-step
method was developed with isolate F97028 (Xu and Feng, 2000). The first step was to establish a
standard time-dose-mortality relationship with data from bioassay 1 at varying dosages of conidia
(0.4-10.4 conidialmm2). This relationship can be used to yield an estimate of expected mortality
probability at a specific time under a given dosage. The second step was to perform bioassay 2 by
simultaneously exposing six :5A-day-old nymph colonies to a shower of Z anhuiensis conidia at
each offour dosages (resulting from exposures of0.3-8.0 min). Subsequently, the colonies on detached
cabbage leaves were separately immersed ina 0.1 % chlorothalonil solution for 0.5 min to disinfect all
surviving conidia on the host integument at intervals of 1-12 h after exposure. The infection rate
during a specific period from the end of the exposure to the immersion was then estimated as the ratio
of the observed mortality over the expected mortality probability at a particular dosage. With this
method, it was found that the infection of Z anhuiensis to M persicae was highly rapid and had little
difference between the aphid colonies maintained at 15 and 20 0 e before being immersed in the
fungicidal so lution after exposure. The first 6-h period after exposure was most crucial to successful
infection of the fungus with the infection rate greatly depending on the dosages received. It took::;1 h
to infect >50% ofthe aphids at a dosage of> 1.5 conidalmm2 and >90% at>50 conidialmm2 .
There exists a difference in virulence among Z anhuiensis isolates tested. Based on time-dose-
mortality modeling analysis of bioassay data for isolate F95136 on M persicae (Feng etal., 1998),
the LD 50 was estimated as 87,44, and 34 conidialmm2 on days 5, 6, and 7 after inoculation, respectively.
The LTso was dependent upon the dosages of conidia received, decreasing from 6.7 days at 37
conidialmm2 to 4.5 days at 198 conidialmm2• A much higher virulence was seen in bioassays with
isolate F97028 to the same aphid species (Xu et ai., 2000). The LDso estimated ranged from 0.4
conidialmm2 on day 7 to 34.8 conidialmm2 on day 3 with an LTso decreasing from 2.9 days at 10.4
conidialmm2 to 6.0 days at 0.42 conidialmm2 . A
The impact of temperature and relative humidity on the infection ofZ anhuiensis to M persicae
was examined at regimes of varying temperature constant (10, 15,20,25, and 30°C) or fluctuating
(1.5-16.6°e and 8.5-20.2°C) and relative humidity (50, 65, 80, 90, 95, and 100% RH). After
exposure to conidia shower ofF95136 (79-90 conidialmm2) for inoculation, 42 batches of aphids
with each including 7-day-old 30-60 individuals pre-reared at 20°C were first maintained at 20°C
with nearly saturated humidity overnight and then separately transferred to each of the above regimes
(Liu and Feng, 2000). During a 19-day period of observation, death of the aphids due to Z anhuiensis
infection occurred at all the regimes with the resulting cumulative mortality differing greatly among the
regimes of temperature (F=7.46, P <0.01) or relative humidity (F=12.54,P <0.01) (Figure 2). The
optimal temperature for the development of Z. anhuiensis-caused infection was 20°C constant or
8.5-20.2°e fluctuating daily (12.4°e on average), at which the mortality tended to increase with

225
relative humidity. Among the regimes of 10-25°e with 100% RH, increase in temperature had little
effect on the cumulative mortality but greatly affected the developmental rate of mycosis with the
esitmates ofLT50s being 8.4, 7.1, 4.0, and 23.4 days at 10, 15,20, and 25°C, respectively. Linear
regression of lILTso against temperature showed a threshold temperature of 1.65°e for the
development of Z anhuiensis infection. Production of conidia from aphid cadavers required ~80%
RH at I 0-15°e and the two fluctuating temperatures, and ~90% RH at ~20°C. Resting spores of
the fungus were not found in aphid cadavers from any of the 42 regimes concerned.
The latent period of another Z anhuiensis isolate, F97029, in M persicae apterae inoculated
at the dose of about 60 conidiaJmm 2 was on an average 7.2, 5.3, 4.9, and 3.9 days at 10,15,20,
and 25°C, respectively (Li and Feng, 2001 a). The above latent periods were significantly correlated
to the temperatures (r2=0.94). During the latent periods, infected apterae remained capable of
producing nymphs but their fecundity was significantly under the influence oftemperature. The fecundity
estimated was 7.97 nymphs per aptera at 10°C, 11.20 at 15°C, 11.86 at 20°C, and 11.20 at 25°C.
In contrast to those uninoculated, the fecundity decreased by 56.45%?41.58%?39.98%?and 49.02%
at the four temperatures, respectively. Based on the life-fecundity table established with daily
observations during the latent periods, the net reproductive rate (Ro) of the infected apterae dropped
by 58.32%?45.54%, 43.11 %?and 50.84% at the temperatures considered. These contributed to
decreases oftheir intrinsic increase rate (r m) by 24.28%,16.98%, 14.12%, and 20.13%.
To explore the impact of initial inocula and host density on the development of Z anhuiensis-
caused epizootic in M persicae population, colonies, each inluding six apterae, were initiated with
inoculatedapterae and uninoculatedones at ratios of 0:6, 1:5, 2:4,3:3,4:2,5:1, and 6:0 (Fengand
Li, 2001). They were then allowed freely for reproduction, fungal infection and mycosis dissemination
on cabbage leaves at the optimal regime of 15°C and 100% RH. As a result, high-level epizootic
occurred in the colonies initiated with ~50% apterae inoculation. As shown in Figure 2, the density of
living aphids (apterae and their progenies) in the colonies never exceeded 50 aphids per 90 cm 2
during a 22-day period of observation whereas the density in the control (0:6) reached 656 aphids
per 90 cm 2 at the end of the period. In the colonies initiated with fewer inoculated apterae (1:5 and
2:4), aphid density decreased by 46-68% (356 and 207 aphids per 90 cm2) compared to that in the
control though the epizootic was insufficient to suppress the increase ofthe colonies. The development
offungal epizootic in each of the colonies was well described using a modified Gompertz model
(r~0.97) in the form
M,=Kexp[-B·exp(-Rt)]
where M, represents the variable for cumulative mortality, t the time (number of days) for
mycosis development, and K, B, and R are parameters to be estimated in modeling. This model is
usually used for modeling plant epidemic. Biologically, the parameter K is depicted as capacity of
epizootic in a specific pathogen-host-environment system and R the apparent infection rate that is a
ratio ofnewly increased infection over the previous infection at a given time interval. The initial proportion
of apterae inoculated in the colonies was highly correlated to R (r=0.89) and K (r=0.90), indicating
that initial inocula and host density are highly important for the development of M persicae epizootic
induced by Z anhuiensis (Figure 3)
With the above method, the influence oftemperature and relative humidity on the development
of Z anhuiensis-caused epizootic was examined (Li and Feng, 2001 b). At the same ratio of three
pathogen-free apterae over three ones inoculated with conidia of Z anhuiensis F97029, M percisae
colonies initiated were maintained on detached cabbage leaves for free reproduction and fungal
infection at the regimes of 10-25°e and 90-100% RH. It was shown that the development of Z
anhuiensis caused mycosis in the colonies was rapid at 15 or 10°C regardless of the humidity range
concerned. The cumulative mortality attributed to high-level epizootic was 72.9-98.2% at 10°C on
day 26, and 78.7-94.4% at 15°C on day 24, as shown in Figure 4. At 20°C, a high-level epizootic
occurred only at 100% RH with a cumulative mortality of71.2% on day 24, contrasting to a much
lower mortality (5.1-12.8%) at the other three humidity regimes. However, the maximal mortality at
25°C on day 20 was only 27.2% even at 100% RH. At the lower temperatures, obviously, Z

226
anhuiensis caused aphid epizootic more easily and to less degree was affected by humidity. The
disease prevalence (represented by the cumulative mortality) was correlated quite well to temperature,
relative humidity, the number of days from initiation, and interaction among the variables (r2=0.82,
P<O.OI).

MS 600 ---+- 0:6

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4 8 12 16 20

Number of Days after Initiation

Figure 4. The development of six-aptera colonies of M. persicae on detached cabbage leaves initiated with
different ratios of pathogen-free and Z. anhuiensis-inoculated apterae.

•
?-.-:.
~ 100 1:5 (A) 0 0 ~1.0
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Number of Days after Initiation Arcsin (Ratio)

Figure 5. The cumulative mortality (A) in M. persicae colonies, initiated with 6 apterae including different ratios
of Z. anhuiensis-inoculated individuals over pathogen-free ones (symbol: observed values; lines: fitted values
from Compertz model), and the regression of parameters K and R from fitting of Gompertz model against the
arcsine-transformed ratio of Z. anhuiensis-inoculated apterae over uninoculated ones (B).

3.4 Mass Production and Formulation

Mycelial production of Pandora and Zoophthora species in submerged culture is not difficult
at present and can be improved by optimizing composition ofliquid medium and conditions for
fermentation (Xu and Feng, 2000; Liu and Feng, 1999). Among Pandora and Zoophthora species,
P delphacis seems to be a very easy species to deal with. It can be grown well at 15-30 o e but
poorly at 35°e in Sabouraud dextrose broth (Xu, 1999). Maximal yields of dry mycelia are 23.0±4.0
mg/ml for 96-h culture at 15°e, 40.3±4.4 mg/ml for 96-h culture at 20 oe, 31.1±4.5 for 72-h culture
at 25°e, and 15.2±3.6 mg/ml for 36-h culture at 30 o e, respectively. The estimates of mycelial
biomass well fitto a logistic growth model (r2>0.97, P<O.Ol) at a given temperature. However, the
best time for harvesting vigorous mycelia from the liquid culture is around 60-72 h after initiation at

227
 420 420
IO"C+90% RH ) 10"C+95O/. RH
350 350
- - 8.PF-aptera colonly
280 280
210 -- 3+3 colony
210
140 140
70
a ~'1"f""""""". 70 --f-··· .. ···-f····!·__ ..:····f.___ •

o 4 8 12 16 20 24 8 12 16 20 24

350 300
10"C+91% RH 1 O"C+ 1 00% RH
280 250
200
210
150
140
100
70 50
O+-:-,~~~~~·--;·f,--···-:i_····c;:f·::::"'~'.~"_
4 12 16 20 24 o 12 16 20 24
1200
1000
800
600
400
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1000 900

~'=L
750
600
450
300
150

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1000
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1200 1200
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1000 1000
800 800
600 600
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1200 1200
25~C+98% RH
1000 1000
800 600
600 800
400
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j. . . [ 400
200
O~~~~~~------~
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Number of Days after Initiation of Colony

Figure 6. Dynamics of living aphids in M persicae colonies with each starting from 3 apterae inoculated with Z.
anhuiensis and 3 pathogen-free apterae (3+3 colony) in comparison to that of control (6-PF"aptera colony) at
varying regimes of temperature and relative humidity.

228
15°C, 48-60 h at 20°C, and 24-36 h at 25-30°C, respectively. This schedule guarantees a reasonable
balance between mycelial biomass and viability crucial to production and discharge of conidia. To
produce in large quantity the mycelia of P. delphacis, an inexpensive medium including (w/v) 1%
yeast extract for industrial use, 1% fish meal, 1% wheat bran, 1.5% com meal, 0.1 % ~PO4' 0.3%
(NH4)N03, and 0.025% MgS04·7H20 has been screened out (Liu and Feng, 2001a). Optimized
conditions for submerged culture are a combination of25°C, initial pH 6.5, and 10010 ofinitial inoculum
(liquid culture containing mycelia). With this medium and the conditions considered, a 48-h culture
may produce a considerably high yield of dry mycelia (>25 mg/rnl) with desirable capacity of spore
production.
A polyacrylamide-starch gel powder has been tested for gelatinization ofP. delphacis mycelia
produced in liquid culture and proved to be biologically compatible with the fungus (Liu et al., 2001;
Liu and Feng, 2001 b). The gelatinized mycelia are in the form of small beads or film, containing
nutrients or not. Sporulation capacity was compared between the gelatinized mycelia and those
naked on water agar plate during a 9-day period of observation. The sporulation capacity ofthe
mycelia gelatinized with nutrients (1910.9 conidialmm2) was 3.14 and 4.69 times greater than those
ofthe mycelia gelatinized without nutrients (608.5 conidialmm2) and the nake4 mycelia (387.6 conidial
mm2). Colonies of M persicae on detached cabbage leaves were exposed to a shower of the
conidia discharged from the mycelia-gelatinized film for inoculation. A dosage of2.23 conidialmm2
from I-min exposure caused a cumulative mortality of82.3% in the colonies within 7 days after
exposure. After being slowly dried, and stored, in silica chamber at 4, 14, and 24°C for 35 days, the
sporulation capacity decreased by 22.7-92.8% in the film-like gels, which then were different in
water content. The nutrient-supplemented, gelatinized mycelia stored at 14°C had a water content of
6.1-7.4% at the end of time and much less loss of sporulation capacity (with a remaining capacity of
845 conidialmm2) than those gelatinized without nutrients or stored at other temperatures. Based on
the numbers ofdischarged conidia before and after storage, the above results for P. delphacis mycelia
gelatinized with polyacrylamide-starch gel seem to be more encouraging than those obtained by
entrapping P. neoaphidis mycelia in alginate polysaccharide polymers (Shah et al., 1999).
Formulation of entomophthoralean fungi for practical use in insect control remains far away
from success and a great challenge in the future study despite considerable ease ofmycelial production.
With our experience, further effort should be made toward searching for such materials that are
biologically compatible with fungal species concerned, technically favorable to entrapping of fungal
mycelia, and most importantly, capable of trapping or keeping moisture. Since entomophthoralean
conidia are too fragile and life-short to be formulated, an ideal formulation for those fungi perhaps is
to gelatinize liquid-cultured mycelia into granules with materials like polyacrylamide-starch gel. So
formulated granules are able to not only discharge conidia when moistened, functioning as aphid
cadavers in the field, but also trap moisture when relative humidity in air is low (Feng, 1998). An even
greater challenge lies in exploring a way to properly desiccate the granules for storage and field
application.

4. SUMMARY

Historically, research and utilization of entomopathogenic fungi for insect control in China is
directed much more toward protection offorest than crops. No other microbial agents but fungi can
be considered against sucking insects such as aphids, leafhoppers, planthoppers, whiteflies and mites.
They are major pests on a wide range of crops in China. Out-of-control misuse of pesticides for
control of resistance-increasing sucking insects on vegetables, teas, and fruits leads to high-level
residues ofpesticides in agricultural products, which frequently cause accidents in Chinese societies.
Not until recently, public consciousness of food safety calls an urgent need for the development of
mycoinsecticides against sucking insects and will facilitate the progress in formulation technology in
the coming future.

229
 Registration of fungal formulations, based on viable inocula instead of toxins, for control of
insect pests on crops needs new regulations that reasonably judges, not only tactically but also
strategically, advantages and disadvantages of their release and relax some standards or limits made
for registration of chemical insecticides. It is unfair to compare fungal formulations with chemical
insecticides only on a basis oftheir efficacy and cost (Laceyet al., 2001). Advantages of using fungal
formulations are enormous when environmental benefits including safety for humans and other non-
target organisms, reduction of pesticide residues in food, increased activity of most other natural
enemies, and increased biodiversity in managed ecosystems are taken into consideration (Han and
Li, 1996a, b; Lacey et ai., 2001). Disadvantages such as slower kill speed and greater reliance on
environmental humidity for action can be overcome or minimized by incorporation oflow- or ultralow-
dose chemical insecticides as synergic agents into the formulations, use of biologically compatible
carriers and additives in formulation, selection ofcrop ecosystems and seasons favomble to action of
fimgal agents, and regulation through routine management measures (Feng, 1998; Quintela and McCoy,
1997). As an era of reliance on the use of chemical insecticides against insect pests passes away,
sustainable agriculture pursues control ofinsect pests at so tolerable levels that the residues ofchemical
insecticides must be greatly reduced and the safety of agricultural products must be secure. In the
following decades, development of mycoinsecticides with the advantages to meet the needs not only
for pest control but also for increasing safety in agricultural products will have a great chance in China
and elsewhere in the world. It deserves continuous inputs to improve techniques involved in mass
production, formulation, and application for minimization oftheir disadvantages.

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USE OF ENTOMOPARASITIC NEMATODES (EPANS) IN BIOLOGICAL CONTROL

Zdenek Mracek

Laboratory of Insect Pathology, Institute of Entomology, Czech Academy of Sciences,

370 OS, Eeske Budijovice, The Czech Republic

1. INTRODUCTION

Nematodes and insects can be considered as the most successful groups of invertebrate organisms
in the nature. Nematodes have colonized all type of ecosystems (excluding the air ecosytem where
they may occur only as phoretic organisms) and a wide range of different habitats. In contrast to
insects they inhabite even salt sea water. Nematodes are categorized as being free-living in marine,
freshwater, and soil ecosystems and as parasites of plants and animals. A large group of nematodes
is specialized for parasitism of insects. Relationships between nematodes and insects vary from
simple phoresis, symbiosis, and commensalism to facultative and obligate parasitism. Nematode
parasites either kill or seriously damage, e.g. sterilizing their insect hosts. Target pest resurgence and
secondary pest outbreaks which result from the disruptive effects of chemical pesticides on natural
enemies have caused increased interest in microbial control measures in pest management ecosystems.
For such control, entomoparasitic nematodes (EPAN s) offer promise as easily manipulated mortality
factors against insect pests. Of these, the most effective are entomopathogenic nematodes (EPNs)
belonging to the families Heterorhabditidae and Steinemematidae. They were fIrst used to decrease
an outbreak of the Japanese beetle in the end of the 1930's whenSteinernema glaseriwas introduced
and colonized in New Jersey (Glaser and Farrell, 1935). However, they have become commercially
available since 1970's when the rearing artifIcial medium was successfully established. Of the
nematodes associated with insects those belonging to the orders Mermithida, Aphelenchida, Tylenchida
and Rhabditida have been most intensively studied. However, at present only the rhabditid genera
Heterorhabditis and Steinernema are widely used for insect control due to their high and rapid
infectivity and pathogenicity and easy manipulation. Others, out ofthose mentioned above, are difficult
to culture on artifIcial media and their field introduction brings technical obstacles. Rhabditids are
amenable to mass-rearing techniques, and in a high percentage of reported field experiments their
utilization has resulted in increased parasitization levels, significant reduction in pest-population
densities, and adequate plant protection.
These nematodes have a ubiquitous world-wide distribution. Indigenous nematode species
significantly reduce natural populations of insects and together with other parasites and pathogens
they help to hold down pest outbreaks. The main groups ofEPANs are mentioned in the general
subchapters. They also comprise groups used for insect control in the past or with a good potential
for the future use. However, in the survey concerning field applications, those rhabditids (with several
exceptions) reported recently and those in present use are mentioned.

Advances in Microbial Control ofInsect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Publishers, New York, 2002 235
2. POSITION OF EPANS IN THE PHYLUM NEMATODA

Phylum Nematoda: Chitwood 1950 Order: Aphelenchida Siddiqi, 1980

Class: Adenophorea von Linstow 1905 Family: AphelenchoididaeParamonov, 1953
Order Mennithida Genus: Parasitaphelenchus Fuchs, 1929
Family: Mermithidae Braun 1883 Bursaphelenchus Fuchs, 1937
Family: Tetradonematidae Cobb, 1919 Ektaphelenchus Fuchs, 1937
Class: Secementea von Linstow 1905 Order: Tylenchida Thome, 1949
Order: Rhabditida Oeriy, 1880 Family: Neotylenchidae Thome, 1949
Family: Steinemematidae OlltooodaniChitwood, 1937 Genus: Deladenus Thome, 1941
Genus: Steinernema Travassos, 1927 Family: Sphaerulariidae Lubbock, 1861
Neosteinernema Nguyen and Smart, 1994 Genus: Sphaerularia Dufour, 1837
Family: Heterorhabditidae Poinar, 1976 Tripius Chitwood, 1935
Genus: Heterorhabditis Poinar, 1976 Family: AJlantonematidae Chitwood and
Chitwood, 1937
Genus: Allantonema Leuckart, 1884
Heterotylenchus Bovien, 1937
Howardula Cobb, 1921

3. OBLIGATE PARASITES AMONG EPANS AND THEIR LIFE CYCLES

3.1 Merrnithidae

3.1.1 Species Found in Aquatic Habitats (in mosquitoes, black flies, chironomids) - Preparasitic,
free-living, juvenile mennithid stages enter insect larvae by cuticular penetration, rarely via the intestine.
The parasitic larvae feed and develop in the host body. This stage lasts usually days and larval
mermithids escape the host before it pupates. Postparasitic larvae molt to adults, mate and females
deposit eggs which hatch in one week to preparasitic larvae. The entire life-cycle takes about one
month, depending on temperature.

3.1.2 Terrestrial Species (parasit;.cin a wide range ofinsects, e.g. grasshoppers, ants, moths).
The preparasitic, infective stage, is ajuvenile which hatches from the egg. Preparasitic larvae search
out hosts in the soil and enter into their bodies through the cuticle using stylet. Parasitic development
lasts for weeks. Hosts die shortly after the mermithid juveniles exit and the juvenile then molts to an
adult in the soil. The life-cycles take months. An exception is Mermis nigrescens which has an egg-
infective stage. Females of this species lay eggs on vegetation and these are ingested by insects.

3.2 Steinernematidae

Steinemernatids are soil-inhabiting, and have free-living, parasitic, and saprophytic stages. The
infective third stage juveniles (Dauer stage) survive outside the insect, and search for hosts (cruisers)
or wait for hosts to pass by (ambushers). They enter the insect host through any opening (mouth,
anus, spiracles), rarely penetrating the host's thin intersegmental membranes and grow into parasitic
stage. Death ofthe insect due to nematode parasitism is caused by bacterial symbiotes ofthe nematode.
Nematodes of the genus Steinernema exist in a symbiotic relationship with a brown-yellow-colored
Gram-negative bacterium which is released when infective juveniles enterthe hemacoel. The bacteria,
which are a food source for the nematodes, produce septicaemia and kill the host within one to
several days. The infective juveniles serve as a disease vector which introduces the bacteria into the
host's body cavity. The level of septicaemia rises rapidly and kills the host usually within 3 days.
Normally two nematode generations are completed in the host. Adult nematodes, larvae Ll ,2, 3-

236
(when the nematode is cycling within the cadaver), and LA are saprophytic and live in the dead insect
host. Infective juveniles (L3) are the free-living, soil inhabiting stage which rnigrateto the environment
when host nutrients are depleted.

3.3 Heterorhabditidae

These nematodes have a similar life cycle to that ofthe Steinernematidae. The only exception is
a hermaphroditic first generation occuring in the host. The second generation is bisexual. The symbiotic
bacteria form rod-colored colonies. Infective juveniles possess a tooth-like cuticular projection on
the head that enable penetration through the cuticle of hosts.

3.4 Aphelenchoididae

This family contains many ofthe phoretic species, but some species of Parasitaphelenchus,
Bursaphelenchus, and Ektaphelenchus are parasites of insects. The infective stage is the third stage
larva which moults and stays in the body cavity of the host larvae, pupae, and adults. Fourth stage
larvae leave the host. The free-living males, females and, 1st and 2nd stage larvae are mostly found
in the frass in bark and wood galleries or the soil where they feed on fungal hyphae. There are few
exceptions to this life-cycle.

3.5 Neotylenchidae

Species of this family are free-living or plant or anima1 parasites. The genus Deladenus attacks
woodwasps and its life-cycle is characterized by two separate self-perpetuating mycetophagous and
parasitic cycles. In the former, all developmental stages feed together on, with the siricids symbiotically
associated, fungus Amylostereum aerolatum growing in the wood galleries bored by the siricid
larvae. This cycle can last for months and then some ofthe fungal-feedingjuveniles develop into
infective 4th stage female larvae which findasuitableinsect. These4thstage infective female enter the host
body, using a stylet, for puncturing the cuticle. Female gonads mature during host pupation. Nematode
eggs hatch inside the female parent, migrate out and enter host's body where they accumulate in the
reproductive organs and penetrate into developing eggs. Eggs are sterilized within the host ovaries.
Egg-laying siricid females disseminate nematode larvae which then begin to feed on the fungus.

3.6 Sphaerulariidae

The most commonly known member ofthis family is Tripius sciarae, parasitizing in fungus
gnats. The infective stage is the fertilized female which retains its last larval cuticle and thus is able to
attach to the host cuticle and penetrate into the host body cavity. Eggs are laid in the haemocoel and
the nematode larvae develop into the 4th stage by the time they exit the dying host. Nematode larvae
mature in the soil, adults mate and infective females search for new hosts. Life-cycle lasts for 2-3
weeks. Larval hosts usually die and parasitized adults are sterilized.

3.7 Allantonematidae

Species of the two genera, Heterotylenchus and Howardula have been the most intensively
studied. H autumnalis parasitizes the face fly, Musca autumnalis. Fertilized, infective females live
in the dung and manure where they attack developing fly larvae. Females penetrate the host cuticle
and deposit eggs in the haemocoel. Eggs hatch and nematode larvae develop into the parthenogenetic
generation. Parthenogenetic females deposit thousands ofeggs, and young developing larvae migrate
to the adult fly oviducts and are passed from the host during oviposition. The life-cycle is completed
externally in the face fly habitat. The host may die, if there is a mass invasion by nematodes or

237
sterilized due to damage to the reproductive organs.
Howardula spp. also cause sterility of their insect hosts which usually include the orders Diptera,
Coleoptera, and Thysanoptera. H husseyi parasitizes the mushroom phorid, Megaselia halterata.
After mating in the soil the fertilized infective-4th stage larval females penetrate the cuticle of phorid
larvae and develop into the parasitic adult stage. During the hosts' pupation the nematode females
begin to deposit eggs. Hatched larvae molt to the second stage, penetrate the hosts' ovaries and
migrate to the oviducts from which they are liberated when fly eggs are layed. In soil the nematode
larvae molt twice to become the infective female larvae or three times to become adult males. The
entire life-cycle lasts days and is well adapted to the fly development.

4. DISTRIBUTION IN THE FIELD AND HABITAT PREFERENCE

EPANs occur worldwide. However, different species/genera/families of these parasites occur

in different habitats/ecosystems depending also on their insect hosts. Water mermithids are widely
spread in the wetlands of boreal ecosystems such as tundra, in streams and lakes of the temperate
zones, and swamps and rivers of tropical regions. Rate of parasitism varies considerably. Bailey and
Gordon (1975) recorded 4-5% of Newfoundland populations of black flies parasitized by
Neomesomermis jlumenalis. The pH of the habitat may significantly influence mermithid occurrence.
Paine and Mullens (1994) found a mermithid, Heleidomermis magnapapula, to be widespread in
manure-polluted habitats of California, but absent in two saline and alkaline habitats. Parasitism of
biting midge larvae ranged from 0 to 69%.
Tylenchid and aphelenchid nematodes are often specialized to a small host range or even specific
for one host. Their occurrence is strictly limited to the living sites of their hosts and the wood insect-
larval galleries where the nematode food, various fungal species grow; e.g. Bursaphelenchus
xylophilus was found in seven species of beetles (cerambycids, curculionids, and buprestids) in
Missouri (Linit et a!., 1983). Howardula husseyi seems to be specific for the mushroom phorid, M
halterata. Nematode developmental stages occur in the host or in the compost, its breeding site
(Richardson et a!., 1977). Bedding and Akhurst (1978) studied the geographical distribution and
host preference of Deladenus species which are important parasites ofsiricid woodwasps. D. wilsoni
was remarkable for its wide occurrence in all parts of the holoarctic while of the other six species,
three were confined to the nearctic region and rest to the palearctic. The authors assumed that insect
host specificity, climatic factors, or even different host trees may well play an important role in these
geographicallirnitations.
Heterorhabditids and steinernematids were found in many areas of all continents, excluding
Antarctica. Some research suggests that the steinernematids are more common in cooler, temperate
regions while heterorhabditids are in warmer, tropical conditions (Hominick et aI., 1996). These
families inhabit most terrestrial habitats, but their occurrence has been evaluated mainly in relation to
soil type and habitat (Hominick and Briscoe, 1990). In Germany (Sturhan, 1999), the rate ofprevalence
of steinernematids was highest in woodland (50.3%) where S. ajfinis, Sfeltiae, S. intermedium,
and Steinernema sp. B were the predominant species. Similarly, heterorhabditids were equally
abundant in turf and weedy habitats, but never found in closed-canopy forest (Stuart and Gaugler,
1994). Moreover, Hominick et a!. (1995) demonstrated that at least some steinernematids show a
distinct habitat preference that may reflect the distribution of suitable hosts which are adapted for the
habitat.
Even though, these nematodes are ubiquitous, their recovery from the field is influenced by a
number of biotic factors, including host range that is dependent on the suitability for penetration of
different insect hosts by nematodes, possibility offinding a suitable host in the habitats (e.g. leaf-
feeding insects cannot be readily attacked in the natural habitat), and by the natural population
density of the nematodes creating epizootics in outbreak sites (Peters, 1996). MrM:ek and Beevc10
(2000) emphasized an essential impact of host aggregations on the incidence ofEPNs. In their

238
experiments, the high percentage, about 70%, ofsampling sites with insect aggregations were nematode
positive.

5. COLLECTION AND LABORATORY REARING

5.1 Parasitized Insects

The field recovery of insects invaded by rhabditid nematodes is rare. Cadavers are hidden in
the soil and decompose rapidly. Host aggregations and insect outbreaks enhance the likelihood for
obtaining parasitized specimens. In sawflies, bibionids, and sugarcane scarabaeid aggregations the
nematodes may increase the occurrence of epizootics (e.g. Akhurst et al. 1992). Collection in such
localities often provides new isolates of wild EPNs (Mraeek and Sturhan, 2000). Water-living stages
of insects such as mayflies, mosquitoes, blackflies, chironomids etc. are attacked by mermithids.
Invaded specimens possess white spirally wound larval parasites in the thoracic and abdominal parts
of the body. These specimens can be collected on the under side of stones, submerged vegetation,
and by water straining. Adult mermithids, gathere on the bottom of pools and streams, and form
characteristic coils. Terrestrial, larval mermithids are found incidentally, mostly in the period before
the last molt, in pupating, and in adult insects. Mature merrnithids live in the soil and can be extracted
mostly by sieving of soil samples from the localities of aggregation of the most suitable hosts, such as
grasshoppers, grubs, Colorado potato beetles, various moth larvae etc. Tylenchid and aphelenchid
nematodes parasitize insects mostly in the larval stage. They occur frequently in bark beetles, siricid
wasps, house flies, chrysomelids, etc. They can be obtained by dissection of parasitized insects.
Adult nematodes can be collected from the frass of galleries eaten out by insects (e.g. bark and
wood boring insects) and from an insect breeding substrate (e.g. manure, compost, organic soil).

5.2 Galleria Baiting

The most frequently used technique for the detection of rhabditid EPNs is Galleria baiting.
Bedding and Akhurst (1975) buried Galleria larvae in soil in the field. These "in situ" tests provided
several entomopathogenic rhabditid isolates. However, there are some obvious disadvantages, such
as the necessity to return to the sampling site, vulnerability of Galleria larvae to predation and the
unsuitability ofthe method at low soil temperatures unless soil samples are assayed in the laboratory.
A simple modification was recommended by Mr<i(:ek (1980). Five soil samples are taken (each 1
dm3 in volume) from each locality, (minimun size of sampling site is 100 m2), mixed, transported to
the laboratory in plastic bags and then placed into Petri dishes (25 cm diameter) together 10 last
instar of Galleria larvae serving as a bait for the nematodes. To prevent Galleria escaping from the
Petri dish, it is placed in a steel mesh pouch. Parasitized insect larvae are characteristically brown-
yellow (with Steinernema) or red (with Heterorhabditis) colored. Checking for host mortality is
done within 7 days at laboratory temperature.

5.3 Direct Extraction

A sieving-decanting method can be employed, with [mal isolation of the rhabditid EPNs from
the sieving debris using a Baermarm funnel with cotton filter. For this method, which is commonly
applied for the extraction of plant-parasitic and soil nematodes (Southey 1986),250 ml soil samples
were used. The nematode suspensions obtained are fixed, checked for the presence ofEPN s using
an inverted light microscope, and the number of Heterorhabditis and Steinernema specimens
determined. The Galleria baiting technique is generally less effective and mixtures of species can be
frequently undetected. The direct extraction method provides quantitative estimates of infective-
stage juvenile density but no information on their infectivity or on morphological characters ofadults,
and nematode cultures cannot necessarily be established (Sturhan and Mra€:ek, 2000).

239
5.4 Laboratory Culturing

The commonly used method of rhabditid EPN propagation is based on Dutky et aI. (1964).
Several hundred IJs in insect physiological solution are applied to filter paper in the Petri dish. Usually,
five to ten last instar of the Greater wax moth, Galleria mellonella larvae are placed in the dish and
kept for two to three days. Dead insect larvae are replaced on the water trap when a new generation
ofI1s leave the cadaver and migrate into the surrouding water. Some improvements in production,
rapid harvest, and storage for S. carpocapsae were described by Lindegren et aI. (1993). "In vivo"
production can be affected by IJ inoculum rate and host crowding (Flanders et al., 1996). Commonly,
the laboratory temperature of20 to 22 EC is an optimum for the successful IJ invasion and nematode
development. But some warm active or cold active nematode species/strains need higher/lower
temperature ranging over 25EC/ 10 to 15EC, respectively. As alternative hosts some moth or
mealworm larvae can be applied to nematodes.
Tylenchid and aphe1enchid nematodes which have their life-cycle connected with feeding on
symbiotic fungi are multiplied on agar plates cultured fungus. For example, Hunt and Hague (1976)
cut elytra of Scolytus scolytus containing attached immature females of Cryptaphelenchoides scolyti,
removed any dauerlarvae present and placed them on the fungus culture of Ceratocystis ulmi.
Mostly females became active and began feeding on the fungus. A new nematode generation appeared
within 5 weeks.
Laboratory "in vitro" methods for rearing of water mermithids are based on field-collected
larvae of black flies, moquitoes, midges, etc. and mostly were developed by Petersen and Willis
(1972) using Romanomermis culicivorax in an aquarium separated into two parts by a screen
which kept mosquito larvae above while postparasitic mermithids leaving hosts droped. Postparasites
escape into the sand and molt within 2 to 3 weeks to adults. After mating the females lay eggs which
are stored viable in moist sand until needed. Recently, Mullens and Velten (1994) cultured
Heleidomermis magnapapula in larvae ofthe midge, Culicoides variipennis, in enamel pans
containing nutrient-rich water and polyester pads as a substrate. Fertilized female mermithids were
added to the substrate when the host larvae were in late second/early third stage. A mass-rearing of
terrestrial mermithid, Filipjevimermis leipsandra, a parasite of chrysomelid beetles was described
by Creighton and Fassuliotis (1982). They produced about 5 million eggs of the mermithid weekly
over a 6-months period. More detailed field and laboratory techniques for manipulation with EPN s
are described in Kaya and Stock (1997).

6. FIELD BEHAVIOUR AND INTERACTION WITH FIELD BIOTA

6.1 Safety and Impact for Non-Target Animals

EPN s including their bacterial symbionts have been proved safe for warm-blooded vertebrate
animals including humans (Boemare et aI., 1996). Recently, Bathon (1996) mentioned the infection
of2-day-old tadpoles of Rana esculenta by H bacteriophora, but only under artificial laboratory
conditions in Petri dishes. A lot ofstudies tested sensitivity of invertebrate animals to EPNs. Earthworms
(Capinera et aI., 1982) and slugs were reported as resistent. The only exceptions were observed by
1aworska (1993), highly sussceptible slugs (Deroceras spp.) to steinemematids an heterorhabditids
in the laboratory. However, the field parasitism was not checked, but may positively control these
serious plant pests when they occur.
Beneficial effect, 75-100% parasitism of S. carpocapsae on a non-target animal, the female
tick Boophilus annulatus was reported by Glazer and Samish (1993). Steinernematids and
heterorhabditids were pathogenic to engorged female ticks, but not to unfed or engorged larvae,
nymphs, males and unfed females. Parasitized female ticks died within 7.5days but no IJs development
occurred inside the ticks cadavers (Hill, 1998).

240
 In the laboratory and in field tests non-target arthropod predators were less succeptible to
EPNs and they did not adversely affect the numbers ofsoil arthropods (Georgis et al., 1991). Poinar
(1989) compiled results concerning the effects ofEPNs on specimens ofthe invertebrate Arachnida,
Crustacea, Diplopoda, Gastropoda, Symphyla, and the vertebrate Amphibia at extremely high
dosages. Generally, if the nematodes kill such hosts they are rarely infective, mostly inhibited by host
defense reactions, and unable to reproduce. Mortality of non-target animals can occur in the field,
but will be only temporal, spatially restricted, affecting a part of population, and its impact can be
considered negligible (Bathon, 1996).
However, some parasitoids may be affected by EPNs. Battisti (1994) recorded 66.6%
emergence reduction of ichneumonidXenochesisjUlvipes after field application of nematode against
web-spinning sawfly. Another ichneumonid, Ctenopelma lucifer seemed to be more resisitent to
nematode invasion. Two species ofichneumonid parasitoids of the codling moth larvae were able to
discriminate between untreated and EPNs treated moth larvae that were parasitized at considerably
lower rates (Unruh and Lacey, 2001).

6.2 Susceptibility ofinsect Hosts and Infection Process

Susceptibility of various pests to different EPNs varies significantly and is influenced first by
biotic factors, such as nematode species/strains, their origin, infective juvenile size, host suitability,
specificity to target host, host size, and second by abiotic factors such as, tolerance to temperature,
humidity, and soil texture. A knowledge of the behaviour of EPNs enables biological control
manipulation to be more successful. Gaugler et al. (1997a) categorized the infection process for
EPNs by using four sequential steps of host selection. Host-habitat finding ensures coincidence of
insects and nematodes in time and space so that they meet. Host finding covers a foraging ambush
and a cruise strategy. Host acceptance represents a nematode's assessment ofthe likely suitability of
a potential host before committing irreversibly to infection. Finally, host suitability is expressed by the
nematode's ability to overcoming the immune system ofits host. Understanding of the biology of
Steinernema and Heterorhabditis in the soil is very incomplete. The attraction ofsteinemematids to
their target hosts is complex, and secretions of plants and the microflora, in particular, appear to
change the nematode's behavour (Webster, 1998). For example, the altered responses ofIJs to
target cues following surface sterilization suggest that cues from the larval cuticle and seedlings root
may significantly influence their host-finding ability (Hui and Webster, 2000).
Abiotic factors influence greatly the infectivity and survival of infective juveniles of different
species in their host-fmding step. At 35EC S. riobrave was more than twice as infective to codling
moth larvae than S. carpocapsae and H bacteriophora. However, S. carpocapsae was the most
effective at temperatures ranging from 15 to 30EC that favour this nematode for control of this pest
under a variety of environmental conditions (Lacey and Unruh, 1998). Similarly, in a field treatment
against black vine weevil larvae S. carpocapsae caused 49.5 % and H megidis only 26% reduction
ofearly instar larvae (Kakouli-Duarte et al., 1997). In a temperate zone, the temperature may influence
results. Higher late spring (mid-May) temperatures may enhance the mortality of black vine weevil
larvae when compared with early spring (begining April), nematode application (Shanks and Agudelo-
Silva, 1990). The high soil temperature in the subtropical region of Texas was probably a reason for
the ineffectiveness of S. carpocapsae while indigenous S. riobrave provided parasitism of about
95% of the com earworm (Cabanillas and Raulston, 1996b).
High humidity is not so important in soil habitats where nematodes may survive in cryptobiosis.
However, it is an essential factor for a foliar nematode application. Different strains ofone nematode
species may differ significantly in their tolerance to desiccation. Glazer and Navon (1990) found that
S. feltiae All strain reduced to 20% survival at 50-70% RH, 25EC within 4 h and to 0% after 8 h
exposure on bean leaves. However, S. feltiae Mexican and Pye strains had a higher capability to
withstand the experimental conditions. Foliage application of the All strain resulted in only 40%
Heliothis armigera control whereas S.feltiae Mexican and Pye strains provided 75 and 95%

241
control, respectively.
Field efficacy is usually influenced simultaneously by both, biotic and abiotic factors. For
example, Legaspi et al. (2000) evaluated S. rio brave against Mexican rice borer, Eoreuma loflini.
High laboratory mortality of the host proved its suceptibility to parasitism, but the field results showed
that EPNs were ineffective in reducing borer incidence or sugarcane damage. The failure of the
nematode application may be due to desiccation caused by exposure to sunlight or to inadequate
humidity as well as to the difficulty of the nematode coming in contact with the borer larvae hidden in
stalk internodes.

6.3 Synergism with Chemical Pesticides

Using chemicals and EPNs together must recognize that it is essential to have high toxicity for
insects and non-toxicity for nematodes. Moreover, among nematode species/strains exist great
differences in efficacy against insect hosts. There was no advantage in combining two EPN s species
against western spotted cucumber beetle larvae (Choo et aI., 1996). It is more effective to combine
the EPNs with chemical insecticides. Suppresion of western com rootworms by the EPNs and
Terbufos ih separate experiments (Jackson, 1996) was equivalent in the case of the chemical and H
bacteriophora, but not so when S. carpocapsae was used.
Some experiments combined sub-lethal doses of chemicals and EPANs for pest control.
KoppenhOfer and Kaya (1998) found a strong synergistic effect of imidacloprid and H
bacteriophora. They gave significantly higher control of white grubs in turfgrass when applied
simultaneously. The degree of interaction, however, varied with nematode species, and it was synergistic
for S. glaseri and H bacteriophora, but oly additive for S. kushidai (KoppenMfer et aI., 2000a,b).
Riceland agrichemicals did not seriously inhibit parasitism of Culex quinquefasciatus by R.
culicivorax, when previously treated with postparasites and adults (Walker and Meek, 1987). Zhang
et al. (1994) tested the toxic effect of 14 organophosphates, 7 carbamates, 4 synthetic pyrethroids,
cartap, and imidacloprid on the S. carpocapsae. Only 3 insecticides provided high mortality ofIJs
(48-83%). Other insecticides caused low or no Us mortality but some of them apparently inhibited
infectivity ofIJs to Spodoptera litura larvae.
Three fertilizers, fresh cow manure, composted manure, and urea were tested for their effect on
the virulence of S. carpocapsae against G. mellonella. In field experimets, only fresh manure reduced
the high nematode virulence which was higher in a soil with a rich level of organic matter than in the
soil with reduced organic matter (Shapiro et aI., 1996). The herbicide, glyphosate, significantly reduced
infectivity of S. carpocapsae to Galleria larvae on days 3 and 8 after nematode application, but no
differences were detected by day 15 after treatment (Gibb and Buhler, 1998).

6.4 Applications with Other Microorganisms and Nematodes

A combination of S. carpocapsae and Spodoptera exigua multinucleocapsid nuclear

polyhedrosis virus produced significantly higher larval mortality of S. exigua (61.7%) compared with
either S. carpocapsae (24.8-35.1 %) or polyhedrosos virus (41.5-49.0%) alone (Agra Gothama et
aI., 1995). Larvae of scarabaeid, Cyclocephala hirta, infected by Bacillus popi/iae were significantly
more susceptible to EPN infection than non-infected larvae (Thurston et aI., 1993). Diamondback
moth, Plutella xylostella, was suppressed by both Bacillus thuringiensis ssp. aizawai (44%) and
S. carpocapsae (41 %). Combined treatment of both agents at half the rate resulted in an increased
control to 58% (Baur et aI., 1998). However, another field treatment that combined S. feltiae and B.
thuringiensis ssp. kurstaki against artichoke plume moth, Platyptilia carduidactyla, did not result
in significantly greater control than that achieved by the nematode used alone (Bari and Kaya, 1984).
Contrary, Koppenhoffer et aI. (2000b) found as the most prornissing combination of B. thuringiensis
ssp.japonensis with EPNs against white grub larvae. Laboratory tests of H bacteriophora against
the soilborne, S. exigua, previously exposed to Beauveria bassiana, resulted in the significatly

242
higher total mortality than treatments with either nematode alone or fungus alone. The inundative
release ofEPN s where B. bassiana occurs may result in greater control of insect pest than application
of nematodes alone (Barbercheck and Kaya, 1991).
Number of galls caused by Meloidogyne incognita on cucumber seedlings were reduced to
50% when Aphelenchus avenae or S. carpocapsae were applied to the cucumber roots. However,
A. avenae suppress infection (Ishibashi and Choi, 1991) of the turnip moth, Agrotis segetum, but
the common cutworm, Spodoptera litura, was not suppressed by S. carpocapse.

6.5 Role of Antagonists

Contrary to reports oflow EPN effects on non-target arthropods there are situations when
these animals feed on nematodes and even an abundance of such arthropods may increase after
application of nematodes (Epsky et al. 1988). A similar negative role on EPN population has been
reported for nematode pathogenic fungi (Timper and Kaya, 1989). Beneficial antagonistic effects
were observed by Bird and Bird (1986) when replicative application ofEPNs reduced the ability of
the plant parasitic root knot nematode, Meloidogyne javanica. to attack plant roots. Ishibashi and
Kondo (1987) found that application of S. carpocapsae and S. glaseri decreased the number of
plant parasitic and increased free-living rhabditid nematodes in the soil. Kaya and KoppenhOfer
(1996) identified antagonistic factors for EPNs as antibiosis, competition, and natural enemies.
Antibiosis can rise due to release of plant chemicals produced by roots in soil which may adversaly
affect nematode host-finding ability. Similar chemicals produced by the host may reduce the
reproductive potential of nematodes. Intra-specific and inter-specific competition reduce the nematode
fitness leading to local extinction. Natural enemies comprise predators such as some collembolans,
mites and some species of nematophagous fungi.

7. PRODUCTION, COMMERCIALIZATION AND APPLICATION

7.1 Mass Production

Mass production is based on large-scale monoxenical production in solid cultures and by liquid-
fermentation method. Formerly developed by Bedding (1981, 1984) who used a chicken offal or
another protein-rich medium soaked in an inert carrier (sponge, polyether polyuretane). Such a rearing
substrate was autoclaved in a glass flask, plastic containers etc. and inoculated with the specific,
symbiotic bacterium. One day later surface-sterilized nematode IJs were added. Harvesting ofthe
new IJ generation was done in 2-4 weeks. This method is applicable for both, steinernematids and
heterorhabditids, but scaling up the production capacity brings increased labour costs (Ehlers, 1996).
Method for liquid fermentation was developed by Pace et al. (1986), but for commercial
exploitation it was improved by Biosys, Palo Alto, California. S. carpocapsae IJ yields in bioreactors
reached lx10 5/ml (Georgis, 1992). Currently, several species/strains of steinernematids and
heterorhabditids are produced commercially in the USA, Australia, some countries of western Europe
such as Germany, Belgium, Norway, Switzerland and also in Poland. In Asia the production was
established in China and the small "in vivo" rearing exists in Thailand.

7.2 Formulation and Storage

After IJs are harvested from the laboratory culture or fermentors the nematodes often need to
be stored for several weeks in a clear or inert space. Maximum survival and maximum stability of
their infectivity is a goal for the long-term storage. For laboratory experiments, the storage on wet
sponge or polyether polyurethane in small glass tubes at a temperature range of 4-8 DC provides a
satisfactory micro-envonmental condition. IJs may survive for months, but the plug must be perforated

243
for aeration. Large-scale produced nematodes can be stored in suspension for several days/weeks in
a refrigerated bubbled tank, though with a risk of contamination and high oxygen demand. More
convenient storage is carried out using either inert carriers (polyether polyuretane sponge, vemiculite)
or active materials such as alginate and flowable gels, water dispersible granules, and wettable powders.
Liquid concentrate enables transport of nematodes at ambient temperatures when proprietary
metabolic inhibitor and antimicrobial agents are added (Grewal, 1998). Storage stability from three
to six moths in the water-dispersal granules can be enhanced through the induction of partial
anhydrobiosis which reduces oxygen and lipid reserve utilization (Grewal, 2000). Shapiro and McCoy
(2000a) compared effect of culture methods (commercial liquid fermentation and in vivo rearing),
formulation liquids and water-dispcrsible granules on thc virulence of S riobrave to the diaprepes
root weevil, Diaprepes abbreviatus. There was no any significant effect of culture method or
formulation.
Heterorhabditids were found less adapted for the long-term storage than steinemematids.
However, Strauch et al. (2000) reported maximum suvival ofH indica at 15 0 C and H bacteriophora
at 7.5 0 C. Positive effects on the heterorhabditids survival was achieved at increase of the salt
concentration in aquous suspensions and low pH between 6 and 4. Enhancement of H indica survival
was recorded for ascorbic acid, cinnamon, and cloves. Survival and infectivity of these heterorhabditids
was not ncgatively affected over several weeks storing in attapulgite and bentonite clay and sponge.
The authors stated, that storage ofheterorhabditids at controlled conditions (temperature, pH and
osmolarity), in aerated watcr is superior to all other methods tested.

7.3 Regulations

The EPN-bacterium association has been exempted from registration process in many countries,
e.g. in USA by the Environmental Protection Agency (EPA), but some have restrictions e.g. to
introduce non-indigenous species/strains or require registration. In Great Britain the indigenous and
genetically unmodified EPNs and their bacterial symbionts do not have to be registered. But currently,
introduction of non-indigenous and modified nematodes are strictly controlled. The European
Commission is harmonizing procedures for the authorization of plant protection products, including
EPNs, under the provision of Council Directive 911414IEEC (Richardson, 1996). Other European
countries either have the mandatory regulation based on Council Directive or no registration is required.

7.4 Application Technologies

Nematodes must be applied under appropriate moisture and temperature conditions and in the
environment where they are able to meet their insects and against the most vulnerable host stage. For
each environmental situation in which they are used, special application approaches may be required
(Bedding et al., 1993). High soil moisture and slow desiccation in foliar habitats are esentially required
for U nematode survival, migration and persistence. Inundative application by sprinkling, spraying,
and injecting of an aquous suspension ofIJs are the widely used methods. Application in the rainy
season, in cloudy weather, and during the night and by burying nematode-infected insect cadavers is
more effective for prolonged nematode survival.
Hayes et al. (1999) applied S carpocapsae against black vine weevil larvae through a solid set
sprinkler or boom sprayer. More effective delivery ofIJs to the sample points was realized by the
boom sprayer. Hand applied S carpocapsae preparation resulted in a significantly lower root injury
by the com rootworm larvae, Diabrotica spp., than did a lateral-move irrigation system. However,
data indicate that application through the irrigation system was feasible for com rootworm larval
suppression (Ellsbury et aI., 1996). A practical treatment method for the control of Hylobius congener
was used by Eidt et al. (1995) when a suspension ofIJs in water was applied to seedlings in multipots
with a watering can. A very interesting mode of dispersal ofS glaseri for control of Japanese beetle
was suggested by Lacey et a1. (1995). The laboratory invaded adult beetles were introduced into

244
field experimental plots where S glaseri was later fmUld. Some beetles were marked, released, and
recaptured in lure-baited traps. Even though a low number of these beetles was caught, 33% of these
were infected by nematodes. Dispersal ofEPNs by flying beetles may open new areas for nematode
control feasibility.

7.5 Application Improvements

Improved efficacy can be achieved, in part, by using the most effective "wild type" nematode
against a particulat pest, selecting for desiderable traits, producing and releasing only physiologically
fit nematodes for pest suppresion, and selecting or producing the biotic and abiotic environment that
favors the survival of nematodes (Bedding et al. 1993). In some examples the use of exotic EPNs
species may provide bctter control than endemic ones (Berry et aI., 1997), but application of non-
indigenous nematodes is restricted in many countries (Richardson, 1996).
Effective concentration oflls can vary depending on nematode species/strain/isolate, controlled
insect and environmental conditions. Mostly, previous laboratory tests of efficacy at several nematode
concentrations have been done. In large-scale experiments Jx I 06/m2Jx 109/haor 2-5x 104/plant is
considered to be satisfactory. For example efficacy of S glaseri against grubs of Oriental beetle was
0/21/54171 % at rates 1.24/2.47/4. 91l2.4x 109fha, respectively (Yeh and AIm, 1995). However,
increased doses oflls in experiments have not lead to higher host mortality. The field study on the
Colorado potato beetle by Wright et al. (1987) noted that an increase in Ils of Sfeltiae from 93 to
I 55/cm2 application caused even lower host mortality (78.7%/64.8%, respectively). Similarly, a
dose of 6x lOs/plant did not decrease the corn root damage by Diabrotica virgifera or adult
emergence as compared with that of a single application at 2x 105 nematodes (Jackson, 1996).
Water retention agents arc used as adjuvants when EPNs are applied on plant foliage, trunks, and
branches or to thc soil with low contents of water. Schroeder and Sieburth (1997) used several
surfactants to increase survival of S riobrave in potted sour orange seedlings for control of Diaprepes
abbreviatus. Mortality of pests was significantly higher in treatments with the surfactants compared
with those using only nematodes. Another approach is immobilization ofIls in hydrophilic colloids,
such as calcium alginate gels (Kaya and Nelson, 1985) that encapsulate and thus protect nematodes
from dessication.
Application technology on foliage has an impact on target pest mortality. Lello et al. (1996)
compared full cone hydraulic nozzles, that disseminated a great number ofIjs and giving up to 98%
pest mortality, and a spinning disc that saved more than 90% ofIl s and the mortality reached 50%.
Low-volume spinning discs seem to be justified and may lead to more cost-effective applications of
nematodes. Increased spray pressure with S carpocapsae reduced the strawberry root weevil larval
population from 26.7 to 0.6 live larvae per sample (Simser and Roberts, 1994).
Effects of irrigation is considered to be substantial for successful application ofEPNs and has
been proven by many experiments. Downing (1994) achieved an excellent control when plots were
irrigated before and 24 h after nematode application, while irrigation only before treatment gave
inconsistent results with 0-92% control. The spray volumes had no significant effect on nematode
efficacy. Mortality of pink bollworm, Pectinophora gossypiella, increased after each irrigation in
soil samples from field plots treated by S riobrave but did not increase after exposure to soil samples
from plots treated with S carpocapsae (Henneberry et aI., 1996). Shetlar et al. (1988) consider
that a ca. 0.64 cm irrigation is needed to move nematodes to the grub active sites 2.5 and 5 cm in the
soil. Non-irrigated release of S glaseri/S feltiae against Ligyrus subtropicus grubs failed to reduce
numbers (16.6/2.7 %, respectively) of this pest in a sugarcane field. On the contrary, peat mulches
did not increase the percentage of maggots infected with S feltiae and S carpocapsae when applied
on the experimental surface (Sweeney et aI., 1998). An increased infectivity to the corn earworm
larvae (78%) was achieved when S riobrave was applied via-in furrow irrigation when compared
with spray application on the soil surface before (46%) or after (64%) irrigation (Cabanillas and
Raulston, 1996a).

245
 Sprinkling ofEPN suspension on above ground parts of plants as stems or leaves causes rapid
drying ofl1. Different improvents were tested to extend persistence ofIJs. Baur et al. (1997) found
some adjuvants that provided the best antidesiccant actvity were non-toxic to EPN s, and increased
nematode persistence on watercress leaves and efficacy against larval Plutella xylostella. Mac Yean
et al. (1982) used aqueous formulations containing inert thickeners and surfactants which increased
retention of nematodes on potato leaves. The nematode efficacy to kill the Colorado potato beetle
was raised from 10 (water suspension only) up to 60% by the use of antidesiccants. Also, evening
nematode applications (preventing 11 s exposure to direct sunshine) produced higher pest control
than those made during morning.
Timing of field experiments may enhance final efficacy. For example, the European com borer,
Ostrinia nubililas, was controlled under greenhouse and field condition (Ben-Yakir et aI., 1998).
Larvae of this pest bore of com plants and after hatching feed in the leafaxil where they stay for only
a few days. This short period is considered as the accurate timing of the application for effective
control.
Genetic selection has also been used to improve efficacy of EPNs. Tomalak (1994) selected a
strain of S. feltiae to enhance efficacy of nematode against the mushroom sci arid, L. solani, by 34
repeated passages through fly larvae in compost. The nematode infectivity was improved 4-times in
the laboratory tests. H bacteriophora was genetically enhanced for thermotolerance by introduction
of a heat -shock protein gene. However, there was no difference between transgenic and wild type
strains in their ability in the field persistence (Gaugler et al., 1997b).
Predictability of optimal field trial conditions, biotic and environmental manipulation may
significantly improve efficacy. GeOIgis and Gaugler (1991 ) examinated two EPNs in inundative releases
on turfgrass against Japanese beetle larvae. Failed tests were explained by the use of unsuitable
nematode species/strain or environmental conditions. S. carpocapsae appeared to be ill-adapted,
but HP88 strain of H bacteriophora provided control of the pest comparable with that by chemical
pesticides, but the appropriate season (fall), soil temperature (> 20DC), soil type (silty clay), irrigation
frequency (1-4-d intervals), and thatch depth« 10 mm) had to be ensured.

7.6 Behaviour after Application

After application into the soil environment the nematodes are spread and migrate downwards
searching for a host. Duncan and McCoy (1996) recovered one hour after application 55% of
infective juveniles in depth 0-1 cm and 33% in 3-15 cm. In following 7 d the number of nematodes
almost disappeared in depth 0-1 cm but remained constant at a deeper level. Nine weeks after, the
number of insect pests, D. abbreviatus at soil depth 0-45 cm was reduced by 77-90%. Introduced
nematode species persisted in the treated area for a long period. Parkman et al. (1994) released S.
scapterisci on golf courses in Florida against mole crickets. Even two years after releasing the
nematodes there were a reduced number of these pests. However, the extended persistence of
EPNs leading to colonization seems to be more complicated relationship. Gaugler et al. (1992)
assessed colonization ofS. glaseri introduced to New Jersey in 30 and 40 Years. They rarely found
S. glaseri, and consider that this colonization was unsuccessful probably due to intolerance ofthis
subtropical species to a temperate climate.
Smits (1996) summarized the knowledge on post-application persitence ofEPN s. The most
critical periods for survival are the first minutes and hours immediately after application when Us
losses of 40-50% may occur. The remaining nematodes accomodate in soil and their numbers gradually
decrease at levels of 5-1 0% per day. Parasites and predators of nematodes, desiccation, depletion
of energy bring the main mortality reasons in this period. Usually, after 2-6 weeks about 1% of
original nematode inoculum is still alive. If the re-cycling in insects exists, the nematodes may persist
for years at such low level. The level of persistence varies depending on the soil abundance of
suitable hosts.

246
8. FIELD CONTROL OF TARGET PESTS

Control efficacy with EPANs is influenced by nematode species, strain, production and storage
conditions, persistence in the introduced habitat, and susceptibility of target insects. Some examples
of biological control using EPN s on small and large scale to control insects belonging to Coleoptera,
Diptera, Lepidoptera and other groups have been given in Tables 1-4. However, some recent attempts
to control agricultural and forest pests by using steinernematids and heterorhabditids have proved to
be unsatisfactory in decreasing host numbers and in preventing crop depredation.

8.1 Control in Soil Ecosystems

8.1.1 Target Fly Hosts. Flies are serious pests in mushroom farms, ornamental and vegetable
gardens and glasshouses. Current controls are based on the use of chemical insecticides that result in
widespread fly insecticide resistence.
The mushroom, Agaricus bisporus, is one of the most valuable hoticultural crops grown. Fly
larvae of species in the families, Cecidomyiidae, Phoridae, and Sciaridae (e.g. Lycoriella auripila,
L. mali, L.solani, Megaselia halterata) feed intensively on this commercially produced mushroom.
S feltiae was found effective in the control of fly larvae, and at the application rate of3x 106 IJ s/tray
significantly increased yields. However, it is likely that the physical presence ofa higher concentration
of nematodes and its bacterial symbiont adversaly affect mushroom yields (Grewal et aI., 1993;
Grewal and Richardson, 1993). Also Rinker et aI. (1995) confirmed the negative effect ofH heliothidis
and S foltiae on a mycelial growth which may confound the benefit of fly control. However, nematodes
can substitute and provide the same or even better fly control than do some chemical pesticides, e.g.
diflubenzuron (Sheepmaker et aI., 1997). A genetically selected strain of S ftltiae was slightly more
effective to fly larvae and with longer persistence in casing material than unmodified strains, when
applied to high population of the pests (Grewal et a!., 1993; Tomalak, 1994).
Fungus gnats, Bradysia spp. commonly infest glasshouse ornamentals consuming roots of cuttings
and small plants, tunelling their stems, and damaging tissues. Moreover, they are a vector of plant
bacterial and fungal diseases. S ftltiae seems to be the most effective of the EPNs tested, providing
significant reduction of fly emergence (Gouge and Hague, 1995) which suggests that it might be an
economical alternative to conventional chemical control (Harris et aI., 1995). Nematode, Tetradonema
plicans, is a specific parasite for the root gnats. Natural populations ofthese pest larvae and adults
can be reduced up to 9% and experiments in I-liter containers provided control of sciarid populations
by 74 to 80% for 4 months (Peloquin and Platzer, 1993).
Cabbage maggots, Delia radicum and D. floralis (Anthomyiidae) are serious pests of cole
crops in North America and Europe. The overwintering populations damage seedlings of crucifers
such as cabbage and cauliflower. The first generation is the most serious pest on rutabaga, and
directly damages the roots and reduces crops. EPN s were tested in several field studies. However,
their potential for economical control on rutabaga was evaluated as poor due to short period exposition
of 1S'-instar larvae to H bacteriophora and S feltiae and the failure of nematodes to invade inside
root tissues (Bracken, 1990). However, in recent studies, Sfeltiae @2.5-5 billionlha was effective
in reducing D. radicum larvae on cabbage (Schroeder et aI., 1996) and increasing the yield of
cauliflower 2.3-fold (but only 19% ofthat ofdiazinon) (Vtinninen et aI., 1999).
Cone maggots, Strobilomyia spp., infest cones of spruce trees and their larvae can destroy
about 60% of seeds in infested cones. Females lay eggs between the cone scales during early stages
of cone development in spring. Larvae tunnel around the cone axis, feeding on developing seeds. In
mid-summer, fly larvae drop to the soil, form puparia and overwinter. Adults emerge next spring or
remain in diapause for an additional year. In the short time, before formation of puparia, they can be
attacked by EPNs (Sweeney and Gesner, 1995).lnfection level immediately after nematode application
reached maximally 95% (mean 53.5%) (Sweeney et aI., 1998).

247
Table 1. Some recent examples of biological control by EPNs on small and large scale - Lepidoptera

P lantltree/ substrate Pest Nematode Dose (Ijs) Efficacy Literature

apple Synanthedon Ssp 3-4xlO'/ 32-76% Kahounova and
myopaeformis callus mortality Mracek(1991)
Choristonerura Sc 2xl09lha 13-37% control Belair et al. (I 999)
rosaceana
Cydia Sc 50/cm' 83% reduced Lacey and Unruh
pomonella Sr 31 % emergence (1998)
Hb 43%
avocado Boarmia Sc 250/m' 98±2% control Glazer and
selenaria Wysoki (I990)
bentgrass Agrotis spp Sc 2.5x1O'lha significant Buhler and
Sg mortality Gibb(1994)
b lack currant Incurvaria Sc 58-86% larval Samersov
capitella mortality etal. (1998)
cherry (peach) Euzophera Hb 3.9x IOs/liter insignificant Kain and Agnello
semifuneralis Sf 6.5xlO'/liter control (1999)
citrus Phyllocnistis Sc 5-30xlO'/ 69% mines Beattie et al.
citrella liter reduction (1995)
clover Wiseana Hb 4xlO'/m' 25% Wright and
cervinata Sf 60% reduction Jackson (1992)
corn Ostrinia Sc 5x 104/plant 5-20% reduced Ben-Yakir et al.
nubilalis plant damage (1998)
Helicoverpa Sr 2xlO'iha 22-100% Cabanillas and
zea parasitism Raulston (1995)
Sc 2xlOs/m' ineffective Cabanillas and
Sr 82-97% pre- and Raulston (l996b)
pupal parasitism
Sr 3.7xlO'- 79-93% Feaster and
1.2xl07 /m2 mortality Steinkraus (1996)
cotton Spodop/era Sc 250/ml 61% Glazeretal.
lil/oratis (1992)
Earias insulana I25/ml 76% reduction
grass Popillia Hb 1.25- >80% control Downing (1994)
japonica 5xlO'iha
Cyclocephala
borealis
litchi Comoritis Hb,Sc 14-92% larval Xu JieLian et al.
albicapilla Sg,Sf mortality (2000)
spruce Zeiraphera Sc 4x I 06-7/liter 82% reduction Eidt and Dunphy
canadensis (1991)
Ac/ebia fenniea Se,Sf I x lOS/plant 30-80% pest West andVrain
survivals (1997)
squash Diaphania Se 7.4xlO'lha 85-97% larvae Shannag et al.
nitidatis infected (1994)
sugarcane Eoreuma loflini Sr 1.24xlO' ineffective Legaspi et al.
2.47xlO' ineffective (2000)
vegetable Plu/ella Hb,Se 33.9% mortality Yang Ping et al.
xylosstella (1999)
watercress Plutella Sc 41% control Baur (I 998)
xylostella

248
Table 2. Some recent examples of biological control by EPN s on small and large scale - Coleoptera

plant/tree/substrate Pest Nematode Dose Efficacy Literature

alfalfa Otiorhynchus Hb 2.5- significant Shields et a\.
ligustici 15x109lha reduction (1999)
apple Conotrachelus Sc 2xl09lha 5-85% damage Belair et a\.
nenuphar reduced (1998)
carrOl Listronotus Sc 4.4xlO'lha 59% reduced Belair and
oregonensis damage Boivin (1995)
citrus P achneus litus Sr 2xlO'/tree 64-89% Bullock eta\.
Diaprepes Sc 7-53% (1999)
abbreviatus
Diaprepes Hb 2xlO'ibeiow ineffective Duncan et al.
abbreviatus Sr tree canopy 77 -90% number (1996)
reduction
Hb 5xlO'/tree 58% reduction Schroeder (1992)
Sc
Asynonychus Sc 50-500/cm' 38-82% Morse and
godmani reduction Lindegren( 1996)
cranberry Otiorhynchus Hb 2xlO'I18m2 68.8% Simser and
ovatus Sc 74% mortality Roberts (1994)
date palm Matamasius Sc 8xlO'/palm 51 % mortality Giblin-Davis et al.
hemipterus (1996)
golf course Phyloperta Hb 0.5-1.5xI0' 40-83% grub Sulistyanto and
horticola, Hm /m' control Ehlers (1996)
Aphodius
contaminatus
mulberry Abirus fortunei Sf 45-75/cm 88-96% larval Wei furrows Hong
furrows mortality Yi et al. (1999)
pasture Popilia Hb 5x10'/m' 44-91% Simoesetal.
japonica Sc reduction (1993)
Sg
peanut Maladera Hb 0.25- 50-90% Glazer and
matrida Sc IxlO'/m' 40% Gol'Berg (1993)
Sg 50% reduction
rhododendron Otiorhynchus Sf 3x10'/plant 72-88% Mnkeketal.
sulcatus protection (1993)
spruce Hylobius Sc 2-4xI O'/plant significant Eidt et al.
congener control (1995)
strawberry Otiorhynchus Hm 1.2- 26% Kakouli-Duarte
sulcatus Sc 7.6x1091ha 49.5-65% et a1.1997)
Hma 2.5x1091ha 42-{57% Wilson plants
1.5xlO'iha 9% infested etal. (1999)
plants
sugar beet Temnorhinus Hb 7.5- suficient control Boselli et al.
mendicus Sc 25x10'/m' (1977)
sweet potato Cylas Hb 1-3.1xlO'Jha significant Jansson et al.
formicarius Sc 1.1- beetle reduction (1990)
4.9xIO'lha
Cylas Hb,Hsp 1.25- increased Mannion and
formicarius Sc, Sf Sg 3.75x1O'lha mortality Jansson (1993)
turffield Pop ilia Sg 5xlO'iha 44-{)6% Selvan et al.
japonica reduction (1994)
Pop ilia Sg 19. 7x \09fha significant YehandAlm
japonica reduction (1995)

249
Table 3. Some recent examples of biological control by EPNs on small and large scale - Diptera

Plan tltreel substrate Pest Nematode Dose Efficacy Literature

bean Liriomyza Sc 9xlO'/ha > 65% mortality Haraetal.
trifolii (1993)
cabbage Delia Hb.Sf IxW'-2x 10'1 reduced fly pupae Schroeder et al.
radicum Sc. Sr plant and root damage (1996)
cauliflower Delia Sf 3.5x10'1 2.3-fold increased Vanninen et al.
radicum plant yield (1999)
D·floralis
lettuce Liriomyza Sf 2xlO iO 82±5% mortality Williams and
hui dobrens is -lxIO"/ha Walters (2000)
fuchsias Bradysia Sf 7.8x105/m' 92% adult number Gouge and
paupera decreasing Hague (1995)
grass Tipula Sf IxHflm' 53% reduction Ehlers and
paludosa Gerwien( I 993)
manure Musca Hb 5xlO'/m' significant Belton et al.
domestica decrease (1987)
Fannia spp. Hb 8-9x10'/m' ineffective Mullens et al.
Muscina Sf (1987)
stabulans
mushroom Lycoriella Sf 3xl0'/tray 8 and 11% Grewal and
auripila increased weight Richardson
and number (1993)
L. so/ani Sf 1-3x10'/m' 91.I -92.7% fly Tomalak (1994)
emergence
reduction
L. mali Sf IxlO(i/m 2 82.8% Grewaletal.
Sf 72.8% reduction (1993)
Hh 28-1 I20/cm' 52- I 00% mortality Rinker et al.
Sf I I-I I20/cm' 38- I 00% morality. (1995)
L. auripila, Sf IxlO'/m' 95-97% mortality Scheepmaker et al.
Megaselia (1997)
halterata
poinsettias Bradysia Sf 2-4xl 0'/1 5- 75% larval, 30% Harris et al.
coprophila cmpot pupal mortality (1995)
rutabaga D.radicum Sf 5x I 05/plant 5.5-fold puparium Bracken (1990)
reduction
soil Ceratitis Sf l50-5000/cm' 76.5-95.8%
capitata mortality Lindegren et al.
(1990)

Manure breeding flies, Fannia spp., Musca domestica (Muscidae) develop in the fecal
droppings and associated detritus that accumulates as manure in caged layer poultry bams and cow
houses. Large populations of flies are a nuisance to workers and neighbours and are pests to the
chickens and live stocks as vector of fowl mites and bacteria. As an alternative to commonly used
chemicals several papers concerning their control with EPN s have been published. Belton et aI.
(1987) reported significantly 3 to 7-fold lower adult fly emergence after application of H
bacteriophora to plastic backs filled by chicken manure. However, Mullens et al. (1987) using
extremely high application rates of 8-9 million Us 1m2 of H bacteriophora and S. feltiae to wet
manure failed to reduce fly larvae numbers. This was attributed to a probable toxicity of manure
substrate that inactivated nematodes rather quickly under the very wet field conditions.

250
Table 4. Some recent examples of biological control by EPNs on small and large scale - other insects

Plant/tree/substrate Pest Nematode Dose Efficacy Literature

apple Hoplocampa Sc IxJO'/50- 0-100% damage Belairetal.
testudinea Oem-long reduced (1998)
branch
golf courses Seapteriseus Sse 2x105/m2 0-100% nypmhal, Parkman et al.
spp. adult infection (1994)
grass (locust) Anoplophora Sc,Sf > 90% parasitism Liu QinLang et al.
chinensis (1999)
house Blatella Sc 2xlO6lbait significant Appel et al.
germaniea reduction (1993)
larch Cephalcia Sf 5-2Ox1 O'/cm 3.4-29.4% Georgis and
lariciphila 200/cm' 17.3-61% Hague (1988).
reduction
mound (ant) Solenopsis Sf 2-6x J06/plot 7.9-27.3% Jouvenaz et al.
invicta reduction (1990)
soil Reticulitermes Sf Ix1O'/m2 significant Epsky and
tibialis reduction Capinera (1988)
spruce Cephalcia Sf IxlO2/cm2 56(32.3)% Battisti (1994)
arvensis Sk 36.4% reduction
squash Anasa tristis Sc 2.4xlO' 24.1-70.8% Huei-Jung Wu
/plant infected (1988)

Abbreviations used in Table 1-4.

Hb - H. bacteriophora Sk - S kraussei
Hm -H. megidis Sr - S riobravis
Hma - H. marelatus Sc - S carpocapsae
Sr - S riobravis Sf - Sfeltiae
Sc.. - S carpocapsae Sg - S glaseri
Sf - Sfeltiae Sk - S kraussei
Sg - S glaseri Ssp - Steinernema sp.
Sse - S scapterisci g- genetically selected

8.1.2 Target Beetle Hosts. Root weevils are worldwide in occurrence and seriously damage leaves
and root systems of many plants. Most of them are polyphagous and their density decreases after
effective nematode application in the controlled area. However, it can be enhanced again due to
weevil migration from neighbourhood habitats where they feed on different non-economically important
plants. Weevil larvae live in soil and feed on roots, while adults feed on leaves where the effective
application and persistence ofEPNs is limited.
Mraeek et al. (1993) performed preventative treatment to control of black vine weevil larvae in
three successive releases in two years. S. jeltiae was in a nursery of ornamentals. Highly effective
control was recorded in three experimental beds and even adjacent rhododendron plots were protected
by migrating nematodes up to 52-77%. Effective control in strawberry farms was achieved by Wilson
et al. (1999). Citrus damaging root weevils, Pachneus litus and Diaprepes abbreviatus, were
effectively controlled by EPNs (Bullock et aI., 1999). S. riobravis and two formulations of H.
bacteriophora reduced recovery of D. abbreviatus by more than 90% and 50%, respectively.
However, within 6 days the population densities of all nematodes declined to 5% on the day of the
inoculation (Duncan et a1.l996). Schroeder (1994) suggested S. rio brave as a better control agent
of the citrus root weevil in potted plants than S. carpocapsae. Shapiro and McCoy (2000b) indicated
control potential of D. abbreviatus higher than 90% under greenhouse condition and 50 to 75%
mortality in field caged D. abbreviatus larvae beneath mature citrus trees (Shapiro and McCoy
2000a).
Sweet potato weevil, Cylas jormicarius, develops within vines and roots of plants and such a
cryptic nature decreases the effectivness of conventional pesticides. Nematode infective juveniles are

251
able to migrate inside by the pest bored tunnels and invade the hosts. Jansson et al. (1990) found H
bacteriophora more effective than chemical insecticides and in their experiments nematodes reduced
weevil damage to fleshy roots. Mannion and Jansson (1993) found Heterorhabditis sp. FL212 the
most pathogenic strain to weevil larvae among 6 nematode species tested. In general, heterorhabditids
caused higher levels ofmortality within the roots than did steinemematids. The carrot weevil, Listronotus
oregonensis, is a pest of carrots in north-eastern North America. Larvae feed on the top roots and
can damage as much as 40% ofthe carrot crop. S. carpocapsae reduced ovipostion and survival of
this pest, but at RH lower than 80% the nematode application was ineffcetive (Belair and Boivin,
1995). Of other weevil species tested the successful control was reported for alfalfa snout beetle
larvae (Shields et al., 1999), sugar-beet weevil larvae (Boselli et aI., 1977), but only low efficacy was
achieved against pecan weevil larvae (Smith et al., 1993).
Seedling debarking weevils are pests of newly planted conifer seedlings in clearcut forest areas.
In N. America Hylobius congener, and in Europe H abietis, the adult beetles damage seedlings by
gnawing the bark of the lower stem, and larvae feed on roots of fresh stumps. Control of H congener
with S. carpocapase reached tolerable levels when the roots were treated before planting by a
suspensioin of nematodes (Eidt et aI., 1995). The effect ofS. carpocapsae upon the larval population
of H abietis on pine stumps was tested by Burman and Pye (1979). The results varied from zero to
66% pest reduction. In general, larvae of weevils seem to be more sensitive to parasitism than the
adults with EPNs due to their soft cuticle and soil-inhabiting life cycle.
Scarabaeid beetles are the most widespread and serious pests ofturfgrass. In USA the Japanese
beetle larvae, Popillia japonica, feed on roots of grass and is a great nuisance problem in pastures
and especially in green golf courses where a large areas are affected. EPNs differ in their efficacy
against Japanese beetle larvae, and in general, the cruiser species with very long Us as a host-finding
strategy, are successful control agents. S. glaseri is considered as the most effective to control P
japonica grubs. However, there are differences in pathogenicity even among isolates of S glaseri.
Selvan et al. (1994) found NJ -43 and SI-12 (genetically selected) strains significantly more virulent
to grubs than the NC strain. Shetlar et al. (1988) reached 53% and 73% grub control when H
bacteriophora and S carpocapsae respectively were applied, but water irrigation before and after
treatment was necessary. Even daily irrigation with 3.2 mm of water for 14 d resulted in grub reduction
below a threshold of8/0.09 m2 (Yeh and Aim, 1995). A different level ofEPN efficacy against white
grubs was achieved in experiments by KoppenhOffer et al. (2000a).1t was not clearly proved which
nematode among three nematode species tested was superior. S. kushidai performed somewhat
better than did S. glaseri and H bacteriophora.
A golf course turf in Germany was inhabited by a dense population of Phyllopertha horticola
and Aphodius contaminatus grubs (> 50 specimens/m2 ). Grass was damaged by grubs' feeding
and secondly by crows preying upon the grubs. H bacteriophora and H megidis significantly
reduced the grub popUlation, and indirectly decreased the turf damage by digging crows (Sulistyanto
and Ehlers, 1996). Cupreous chafer grubs, Anomala cuprea, injured roots of many ornamental
bushes and trees, peanuts, sweet potatoes, lawns etc. S. kushidai showed a significant lethal effect
for the cupreous chafer grubs and was able to persist on the experimental plot for two additional
years.
The Colorado potato beetle, Leptinotarsa decemlineata, is the most destructive insect pests
of potatoes worlwide. Larvae and beetles can cause up to 80-100% crop loss. Moreover, this pest
has developed resistance to many insecticides. Transgenic potatoes expressing Bacillus thuringiensis
endotoxin are promissing for this pest control. However, the potential of the build up of resistance to
transgenic potato varieties gives rise to the potential to use EPNs against soil hibernating adults. For
effective control, nematode application before larval burial is recommended (Wright et aI., 1987).
Inconsistant control or maximally 31 % Colorado potato beetle reduction was reported by Stewart et
al. (1998).
The northern com rootworm, Diabrotica barberi, is an important pest in com fields in North
America. Larvae feed on roots and weaken the root system, resulting yield reduction. EPNs were

252
incapable of entering the roots and invade the feeding larvae. Therefore, Thurston and Yule
(1990) directed S. jeltiae application against first-instar larvae before they entered the roots.
Nematodes significantly reduced the pest larvae, but the insecticide fonofos gave a higher level of
control.
Besides the manure rearing flies, there is another insect, lesser meal worm Alphitobius
diaperinus, living in the litter of commercial poultry houses. This pest represents a public
nuisance problem. It is known to harbor numerous pathogens of avian disease and able to tunnel
building insulation material. Geden et al. (1987) observed a modest but significant control when
S. jeltiae strain All was applied. Mortality of pest decreased from 86.6% in week 1 to 73.5% in
week 7 post-treatment. These results were encouraging but in the following year experiments on
the long-term control of this pest failed, probably due to very high overall mean temperature of
26.4°C that rapidly declined a nematode survival (Geden and Axtell 1988). Termites cause
serious public nuisance, and EPNs were found able to kill termite workers. However, very high
Us doses of S. jeltiae were needed for the effective control in feeding site traps and so applications
on the entire termite colony were suggested as a more effective method (Epsky and Capinera,
1988).

8.1.3 Target Butterfly Hosts. The black cutworm, Agrotis ipsilon, is a polyphagous insect that
often damages turfgrass in recreationally managed areas in the midwestern USA where the use of
chemical pesticides is under the public pressure. Cutworm larvae live in soil and are susceptible
to number of EPNs. S. carpocapsae and S. glaseri controlled caterpillars significantly, but
nematode persistence was lost 8 d after application (Buhler and Gibb, 1994). The corn earworm,
Helicoverpa zea, is a major pest of corn field and other cultivated and wild host plants. The
indigenous nematode, S. riobrave, was highly effective to control prepupae and pupae of this pest
in Texas (Cabanillas and Raulston, 1996b). Similar effectivity was achieved by Feaster and
Steinkraus (1996) when S. riobrave was evaluated for inundative control of this pest in Arkansas
corn fields. Gelechid pink bollworm, Pectinophora gossypiella, completes five generations during
the Arizona cotton growing season. Larvae enter the soil to pupate and EPNs may serve as
biological control agents. In field treatments performed by Henneberry et at. (1996) S. riobrave
provided better pest larval control than did S. carpocapsae. Low potential of control was
observed for adults of the pink bollworm which is often considered to be an unsuitable
developmental stage to control.
Most of the economically important sesiid species feed in the larval stage on above ground
growing parts of plants. The exception is the grape root borer, Vitacea polistijormis, which
damages grape roots and causes a gradiated reduction in grape vigor and substantial yield
reduction. Saunders and All (1985) used S. carpocapsae against first-instar borer larvae beforel
they reach and establish in root feeding sites.
Three EPNs were tested against codling moth, Cydia pomonella, cocooned larvae in leaflitter.
The most efficient, S. carpocapsae, provided 99 % larval mortality whereas the mortality by S.
riobrave was 80% and by H. bactgeriophora 83 %, respectively. Significantly lower effectivity
was observed with nematode applications on tree logs - 83, 31, and 43% respectively (Lacey and
Unruh, 1998).

8.1.4 Other Target Hosts. Sawflies from the families Pamphilae and Tenthredinidae belong to
serious forest pests and seem to be optimal hosts for nematodes because after the larvae fall from
the trees, they pupate, overwinter, and some species have a several years of diapuse in the soil.
During this relatively a long period they are invaded by steinernematids. Mraeek and David
(1986) used S. kraussei against the spruse web-spinning sawfly, Cephalicia abietis a dangerous
pest of spruce monocultures in Czechoslovakia. The nematode was applied in the autumn against
diapausing sawfly larvae. In spring the mortality in experimental plots reached 81-97 %.
Effectiveness of S. jeltiae against web-spinning larch sawfly was tested in Great Britain. Foliar
application against feeding stages of sawfly larvae resulted in only 29.4 % infection, while more
suitable soil application against prepupae achieved 61 %, and even 1 year later ranged from 4.8 to
14.7% (Georgis and Hague, 1988). For another spruce sawfly, C. arvensis, emergence was up to
56 % reduced when S. jetiae was applied to soil before the mature sawfly larvae dropped and
entered the soil in Italy (Batistti, 1994).

253
 Mole crickets, Scapteriscus spp., native to South America, are the most damaging insect
pests of golf courses turf in the southern part of the USA. The nematode S. scapterisci, described
from the mole cricket in Uruguay, was introduced to Florida where it got successfully established and
persisted for years in the mole cricket populations (Parkman et aI., 1993). The level of infection of
adults (25%) was significantly greater than that for nymphs (1.2%), and mean trap catch of mole
crickets was reduced by up to 68% the 2nd yr after treatment (Parkman et aI., 1994).

8.2. Control in Cryptic Habitats

Cryptic habitats are considered as most favourable, enhancing the infectivity, survival, and
persistence ofEPANs, because the cryptic environment minimizes nematode death from ultraviolet
radiation and desiccation. TIll present, field experiments in these habitats have provided mostly consistent
and efficacious results. One ofthe most excellent examples was performed in Tasmania where thousands
hectares of the pine tree, Pinus radiata, were infested by the woodwasp, Sirex noctilio, which
implants to the trees fungus, Amylostereum aerolatum. This fungus killed many susceptible pine
trees. A specific tylenchid nematode, Deladenus siridicicola, was mass-reared and introduced into
the trunks with siricid larvae. Results brought the surprisingly high effectivity. Usually, over 70% of the
emerging woodwasps was parasitized by nematodes which were established in most of the treated
areas, and got spread up to 19 km in a single year and reduced damage of the pine forests significantly
in the following years (Bedding and Akhurst, 1974).
Currant borer moth, Synanthedon tipuliformis, larvae feed extensively on pith within stem of
the host plants. Miller and Bedding (1982) reported up to 90% mortality of the larvae. Higher level
of parasitization by S. feltiae (= N bibionis) was achieved in old wood, probably due to rougher
bark and bigger entrance holes to tunnels, than in new wood. However, some new generation IJs
migrated from parasitized larvae to infect larvae in other tunnels. Apple clearing borer moth,
Synanthedon myopaeformis, larvae were suitable for the control by Steinernema sp. Nematodes
were sprayed onto infested calluses. Mortality ofthe pest increased within 4 week period from 32 to
76% even though the weather conditions were unfavourable during the experiment (Kahounova and
Mraeek, 1991).
Carpenterworms damage a lot of ornamental and fruit trees in USA and Canada. Lindegren et
al. (1981) injected S. carpocapsae into the gallery openings of fig trees in California. Within 2-6 days
there was a decreased feeding activity of the carpenterworrn larvae, and the final pest mortality
reached 44-92%. Suppression up to 100% of the carpenterworrn was reported by Forschler and
Nordin (1988) when EPNs were injected into galleries, but bark surface spraying provided variable
results.
The American plum borer, Euzophera semifuneralis, is one of the most important fruit tree-
boring pests of tart cherries and causes a significant decline in the life span of cherry orchards. It is
associated with bark wounds that provide an entry into the cambium layer where the larvae feed.
Laboratory bioassays proved high sensitivity ofEPNs to pest larvae. However, failure of S. feltiae
and H. bacteriophora to control the plum borer larvae may have resulted from inadequate larval
contact with the nematode spray (Kain and Agnello, 1999). Bark folds create a cryptic microhabitat
in which the codling moth, Cydia pomonella, larvae pupate, but EPNs can find the space protecting
them against desiccation. July applications resulted in a total mortality of32% whereas in October
and February application against overwintering population settled in corrugated paper bands around
tree trunks brought the increased mortality of80 and 95%, respectively. Kaya et ai. (1984) attributed
this high efficacy to rainfal during the autumn and winter period and the water retention properties of
the paper bands.
Effective application in the cryptic environment was recorded by Shannag et ai. (1994). S.
carpocapsae was able to enter blossom of squash plants, where they persisted for days and infected
pickleworrn, Diaphania nitidalis, feeding on buds, blossoms, vines, and fruits, here. Apple sawfly,
Hoplocampa testudinea, an introduced apple tree pest to Eastern North America females lay their

254
eggs into blossoms and larvae feed on developing fruits by burrowing the tunnels. Damaged fruits fall
on the ground where sawfly larvae enter the soil, pupate and overwinter. Foliar and soil application
ofEPNs is feasible. Vincent and Belair(l992) used the spray with S carpocapsae which was effective
in reducing secodary fruit tunnel-damage.
Finney and Walker (1979) did not find an effective control ofelm bark beetle, Scolytus scolytus,
during the overwintering period. EPNs enter the small hole galleries made by feeding ofthe bark
beetles. However, effective control of hundreds of galleries in every infested tree is not feasible due to
the high labor cost and difficulties of introducing nematodes into bark opening. West Indian sugar
cane weevil, Metamasius hemipterus, larvae are borers that seriously damage date and banana
palms. Larval tunelling wounds petioles, crown, stems and later extends into healthy leaf or stem
tissue. A crown drench of S carpocapsae resulted in 51 % larval mortality which was comparable
with chemical insecticides (Giblin-Davis et aI., 1996).
As cryptic habitats can be considered moisture-retaining stations that often contain food
attractants. lbis type of habitat can be developed and by inoculating with EPNs becomes a nematode-
impregnated pad. Under such conditions S carpocapsae survived for weeks, and significantly reduced
the numbers ofthe German cockroach, Blatella germanica in infested apartments (Appel et al.,
1993).

8.3. Control in Foliar Habitats

Field applications ofEPNs against foliage-feeding insect pests have resulted in varying efficacy
due to many problems arising from using these nematodes in an unfavourable environment. Because
nematodes frequently enter the hosts on the foliage by ingestion, they must remain viable as long as
possible. Nematodes require high relative humidity, water drops on leaves, and avoidance of direct
sunshine otherwise they desiccate rapidly. Tropical conditions and rainy periods enhance nematode
survival on the foliage, but in temperate and dry areas nematode application has limited success.
When weather conditions are virtually optimal the foliar spray can be effective. Eidt and Dunphy
(1991) reduced up to 82% the spruce budmoth, Zeiraphera canadensis, emergence with water
spray containing IJ s of S carpocapsae. In experiments of Belair et al. (1998) the results in 1992 and
1993 indicated highS carpocapsae spray effectivity, resulting in reduced primary apple fruit damage
of98 and 100%, respectively, but none of the treatments was effective in 1994. Simultaneously, the
plum curculio, Conotrachelus nenuphar, the pest causing up to 85% damage of fruits, showed very
inconsistent control results from EPN treatment ranging from 5% fruit damage to no significantcontrol
effect. The relative high application costs preclude EPNs use as a sole control tactic against these
pests.
Butterfly citrus leafminer, Phyllocnistis citrella, larvae mine immature foliage and destroy
epidermal cells. This pest is widely spread in sub- and tropical Asia and Australasia. Beattie et al.
(1995) showed that the leafrniner is controlled with S carpocapsae satisfactorily, but its use is not
commercially acceptable at the concentration tested. The oblique banded leafroller, Choristoneura
rosaceana, damages apple tree foliage in eastern North America. Relatively low control (13-37%)
with S carpocapsae against leafroller larvae is attributed to low survival rate ofIJs, the difficulties
of nematode access to larvae because the suspension could not penetrate the tightly woven larval silk
(Belair et al., 1999).
Fly leafrniners are highly polyphagous and damage a wide range ofvegetables and ornamentals.
Foliar application ofS ftltiae under glasshouse conditions when humidity was > 90% RH, suppressed
the outbreak of Liriomyza huidobrensis (Williams and Walters, 2000), but nematodes had to be
applied against the second/early third larval stage ofleafrniner. Similar mortality level of75% for L.
trifolii was carried out with Sfeltiae on chrysanthemum reported Broadbent and Olthof (1995).
Control of the same pest performed under greenhouse and foghouse tests provided similar levels of
control with up to 69% mean mortality by S feltiae and> 65% by S carpocapsae All strain, with
a humidity of92% RH (Hara et al. 1993). The control ofleafminers can be successful if the EPNs

255
enter leafinines before desiccation. Very few cone maggot larvae were infected when nematodes
were sprayed or injected into spruce cones infested by Strobilomyia appalachensis (Sweeney and
Gesner, 1995).
The squa,sh bug, Anasa tristis, seriously damages cucurbit vegetables by tissue sucking and
is one ofthe most difficult insects to control with insecticides. As a biocontrol measure S. carpocapase
was used to reduce the nUmber of this pest on squash plants. Inconsistent results were attributed to
the variable weather conditions that probably limited survival ofIJs in water droplets on the leaves
(Huei-Jung Wu, 1988).

8.4. Control in Aquatic Habitats

Mermithid nematodes are regarded as possible eventual candidates for control of water inhabiting
stages of medically important insect vectors such as mosquitoes, blackflies, and midges. Several
experiments also utilized steinemematid and heterorhabditid nematodes against the aquatic larvae of
these pests. However, these nematodes are not adapted to swim and their host-finding ability in
water is limited. Under simulated stream conditions the mortality oflate instar black fly larvae, caused
by S. carpocapsae, was up to 50%. The intensity of parasitism decreased within next fews days and
almost disappeared by day 6 after application. No nematode establishment or re-cycling was observed
(Gaugler and MoUoy, 1981). A range of mosquitoes has been found to be susceptible to invading by
steinemematids. However, attempts for effective field control have not brought satisfactory results,
e.g. Finney and Hardig (1981). There are other factors limiting efficacy of steinemematids or
heterorhabditids such as settlement ofIJs on the bottom, insufficient aeration, injury ofIJs in larval
mouthparts, and strong immune response ofthe host.
Mermithid nematodes appear to be frequent in water habitats and in the past served as important
mosquito reducing agents in small-scale field experiments. There were several pioneer field releases
of Romanomermis culicivorax by Petersen's group e.g. Petersen et al. (1972) and Petersen and
Willis (1974) who successfully reduced mosquitoe populations in rice fields in California and in small
ponds in Lousiana, respectively. Nickle (1979) released the mermithid nematode, R. culicivorax,
into mosquito-breeding ponds in Maryland. Mermithids, originating from the warmer areas ofLouisiana,
probably established and overwintered in the region where the temperatures go down to _190 C in
January. It was able to self-reproduce and gave the mosquitoes mortality to a level of 50-1 00%.
Molloy and Jarnnback (1977) released newly hatched preparasitic larvae of Neomesomermis
jlumenalis in small stream with a high density of black fly larvae, Simulium spp. Parasitism rate of
71.4% immediately below the treatment point decreased markedly downstream indicating poor
preparasite dispersion. Two biolaboratories produced R. culicivorax commercially for a few years.
However, the limited number ofpreparasites from "in vivo" mass rearing, poor methods of"in vitro"
culturing and problems with storage and shipment ofeggs stopped the long-term commercial use of
these mermithids.

9. CONCLUSION

Interest in the use of entomoparasitic nematodes (namely those belonging among

entomopathogenic, Heterorhabditidae and Steinemematidae) as a biological control agents of insect
pests has grown rapidly in recent years. Field evidence suggests that these nematodes effectively kill
and usually replicate in suitable hosts, may produce epizootics, and thus slowly substitute the use of
chemical pesticides. Simultaneously conducted research has shown a low impact on non-target
organisms. The successful establishment ofmass cultivation techniques and formulations for infective
juveniles of entomopathogenic rhabditids is a requisite for their application into the field against a
wide spectrum of soil inhabiting insect stages. Some insects living in cryptic habitats can also be
effectively controlled by these IJs. Insect species mining, for IJs migration, too narrow tunnels,

256
feeding on upper ground plant tissues, or living in aquatic habitats have a limited control ability.
However, the selection ofthe optimal nematode strain/species, better formulations, and an improvement
ofapplication technologies will enhance their efficacy.
Many recent field releases of steinemematids and heterorhabditids have given different levels
of mortality of target insect pests. However, in those cases exhibiting low decreases of insect pests,
the cause of failure cannot be attributed only to nematodes. A future goal should be to incorporate
EPNs more closely into pest management systems because they are compatible with generally used
chemical pesticides and can be combined with lower doses of the chemicals to enhance target pest
mortality.
For future prospects the use ofEPNs in soil and cryptic habitats provide the most favourite wet
space where Us may survive, persist, establish, re-cycle, and develop a long-term regulation of
insect populations. Occasionally, foliage-feeders of which many species are highly suceptible to EPNs,
can be controlled in the short-term. However, for such trials a high relative humidity is an essential
factor during the host-finding period, otherwise nematodes quickly desiccate. Control of insect pests
in aquatic habitats has shown relatively high target susceptibility. However, the use of sub-specific
mermithids has been postponed due to rearing complications, and rhabditids are poorly adapted for
the movement and invasion ofsuch hosts as mosquitoes or black flies larvae in the aquatic environment.
Regardless ofsome negative aspects field results using EPNs represent a significant biologicalaltemative
to conventional chemical pesticides.

ACKNOWLEDGEMENTS

The author wish to thank Czech Ministry of Education for partial support provided and Prof.
J .M. Webster and Dr. L.A. Lacey for their kind reading and comments to the manuscript.

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ENTOMOPATHOGENIC NEMATODES FOR THE CONTROL OF CROP PESTS

S. S. Hussaini

Project Directorate of Biological Control,PB No. 2491, H.A.Fann Post,

Bellary Road, Bangalore 560 024 , Karnataka, India

1. INTRODUCTION

Growing public concern over ill effects of chemical pesticides, particularly ground water including
food chain contamination, resistance development in target organism and threat to Human beings and
wild life has fuelled an intense search for safer alternatives of pest management. The use of natural
enemies, particularly parasitoids and insect pathogens, as biological insecticides has been considered
by many to be the most viable pest management alternative, being environmentally safe, and as an
essential component of sustainable agriculture. The annual growth rate in chemical insecticides
production and use is 1-2% whereas that of microbial insecticides 10-25% (Ahmed and Leather,
1994). Nematodes associated with insects, referred to as entomophilic, entomogenous or 'entomo-
pathogenic' are known to parasitize, cause disease and kill the insects. The entomopathogenic
nematodes (EPN s) are potential biocontrol agents, besides serving as vectors of bacteria. There are
presently 60 laboratories in 38 countries working on EPNs and the number is growing every day.
The explosion of interest since mid -1980's is an impressive attributes to this technology. EPNs are
ubiquitously distributed and comprise two families: Steinemematidae and Heterorhabditidae. The
families are not closely related phylogenetically but share similarities due to convergent evolution.
Despite their lethality to insects, lack of pathogenicity to mammals led US EPA to exempt all strains
and species belonging to Steinernema and Heterorhabditis and their associated bacteria from
registration requirements under the Federal Insecticide Fungicide Rodenticide Act (FIFRA). Although
many EPNs are recorded as naturally occurring on insects, detailed studies on bionomics and mass
multiplication of these nematodes have not been attempted (David et al., 1994) . Reports on most
entomophilic nematodes in India stand up to generic level only. Attempts have been made to study
the biology of the following entomogenous nematodes: Panagrolaimus migophilus and Pelodera
sp. (Geetha Bai and Sankaran, 1985); Parasitylenchus coccinella (Reddy and Rao, 1987);
Hcrassirostris (Yatham and Rao, 1981); Protrellus chauhani (Rao, 1980). A brief introduction
about important families ofEPNs is as under:

Allantonematidae. Members of this family are obligate parasites of insects but are ineffective
as natural control agents. Many ofthem cause little damage to the insect hosts; those causing extensive
damage are difficult to maintain in the laboratory. Life cycle and population dynamics of

Advances in ivlicrobial Control of Insect Pests

Edited by Ra;eev K Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002 265
Heterotylenchus hydrabadensis were studied (Reddy and Rao, 1980). Laboratory study on the
host parasitic relationship in tenns of biochemical and histochemical changes in the host body showed
that infection of Longitarsus belgaumensis by the nematode, Howardula sp. resulted in depletion
of carbohydrate and protein levels in the host body (Raj and Reddy, 1990); the levels offree aminoacids
in the haemolymph of females of L. belgaumensis were altered or decreased due to infection by H
belgaumensis (Daniel and Reddy, 1990). About 47.5% depletion in the protein content of fourth
instar larvae of Spodoptera, Howardula sp. (Devi and Reddy, 1990; Devi et a!., 1991; Raj and
Reddy, 1990; Reddy and Rao, 1981) has been recorded.

Neotylenchidae. Species of neotylenchids that parasitize insects possess two possible life cycles-
one free living feeding on fungi, and one parasitic on insects. Several Deladenus spp. are effective
insect parasites.

Sphaerularidae. Members are obligate parasites restricted to a single family ofhosts,Sciaridae.

They are capable of killing or sterilizing the host. However, only one species Tripeus sciariae has
been experimentally released.

Tetradonematidae. This is a family of five species of obligate parasites, which generally kill their
hosts. Although members of this group have promise as biocontrol agents, their host specificity seems
to be restricted to insects oflittle or no economic importance thus greatly limiting their usefulness.

Mermithidae. Menni thids are large ,filifonn and important group of nematodes. They are obligate
parasites of arthropods,principally insects, but have also been recorded from spiders,
crustaceans, leeches, and mollusks. They are usually specific to single species or to one or two families
of insects and are almost always to their hosts. Earliest reference to mennithids is found in the writings
of Aldrovandi in 1623 . Mennithids have been reported from arthropods in a variety ofenvironments,
and often infecting large percentages of host populations. The study of these nematodes has however
been slow to develop because most mennithid observations were made by Entomologists with little
training in Nematology. Thus much of the earlier work was limited to host-parasite relationship or on
incidence of parasitism. Mennithids are effective as they offer no environmental hazard, offer no
threat from competitive displacement ofother desirable organisms because of their life cycle and the
potential exists for inundative release to give high initial host reduction, or inoculative releases to
establish the nematode and give partial control for an indefinite period. Mermithid induced intersexes
have been noticed in chironomid hosts and often consist ofa midge with genital appendages, but with
front legs, wings, and antennae similar to females. Other subtle morphological changes also occur.
Larvue of Hexamermis arvalis are wrinkled and bulky in appearance and lighter in colour compared
to healthy larvae. Paily (1990) succeeded in improving mass culturing of Romanomermis iyengari,
a mennithid nematode parasitising mosquito larvae, using moist sand bed.

Aphelenchidae. Praecocilenchus Jerruginophorus, a member of this family, was described from

the haemocoel of adult RhynchophorusJerrugineus from Kerala ( Rao and Reddy, 1980). Three
distinct adult fonns were found, vennifonn males and females and swollen viviparous females. It is
distinguished from the only species of the genus, P raphidophorus, by the absence of needle-like
crystals in older wonns and by the arrangement of the oesophagal glands. This is the first record of
Praecocilenchus in India. Bedford (1974) in a survey of adults of R. bilineatus (Montr.) infesting
coconut palms in New Britain in 1969-70, found about 15% of914 weevils parasitised by the
nematode P rhaphidophorus. Parasitised females were capable of oogenesis and the fonnation of
mature eggs, but these were less than in unparasitised weevils.

Steinernematidae and Heterorhabditidae. The pathogenic potential of nematodes for insect pest
suppression has been recognized for more than 60 years since the discovery of infection of Japanese

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beetle with Steinernema glaseri (Glaser, 1932). Although 40 nematode families have been found
associated with insects, only two families, Heterorhabditidae and Steinernematidae have widely been
used as biological agents (Gaugler and Kaya, 1990). Nematodes in the generaSteinernema and
Heterorhabditis are found to be potential agents for control of insect pests (Poinar, 1979;Gaugler,
1981; Kaya, 1985; Klein, 1990). The free living, non feeding infective juveniles possess
chemoreceptors, are motile, highly virulent, have high reproductive potential, and have the ability to
seek out their hosts. They have the potential for long term establishment in soil through recycling on
infected insects (Gaugler, 1981 )and are compatible with other control strategies, pesticides, ease of
production and ease of application (Gaugler and Kaya, 1990; Georgis and Hom, 1992). Bedding
(1996) observed that the EPN s based pesticides account for most ofthe biopesticides marketed in
the West than all other organisms, apart from Bacillus thuringiensis. They are on the commercial
range.
Attempts have been made in this paper to review the status ofentomopathogenic nematodes as
biocontrol agents, their commercial production and future directions for enhanced use as bioinsecticides.
Another steinernematid, S. carpocapsae (DD-136) was extensively tested against the codling moth
and a number of insect pests in lab and field trials with encouraging results.

2. DISTRIBUTION

Soil surveys were conducted in temperate, sub-tropical and tropical countries after Bedding
and Akhurst developed the baiting technique in 1975. Australia, Britain, Canada, Czechoslovakia,
Egypt, Florida, Finland, Germany, Hawaii, Israel, Italy, Japan, North Carolina, Northern Ireland,
Norway, Oman, Peurto Rico, Republic oflreland, Spain, Sweden, Srilanka were found to have
these nematodes. In India Poinar et a!. (1992) detected Heterorhabditis indica in sugarcane fields
at Coimbatore. Extensive surveys by PDBC since 1996 detected Steinernema carpocapsae from
Bangalore, Madurai, Rajahmundry; S. bicornutum from Delhi; Heterorhabditis indica from
Bangalore,Coimbatore, Chidambaram, Kanyakumari, Aligarh. Detailed RFLP analysis showed the
presence of S. tami from Jorhat, S. abbasi from Delhi and unidentified Steinernema spp.SSL2
(PDBCEN 13.21 ) from Coimbatore and PDBCEN 14.1 from Aligarh (Hussaini et a!., 2001a).
Besides, S. thermophilum was described from Delhi (Ganguly and Singh, 2000). There are presently
30 species of Steinernema and 9 of Heterorhabditis on record in the World.
The baiting technique is now widely recommended for collection of indigenous nematodes.
These nematodes have been isolated from all the inhabited continents (Kaya, 1990). This widespread
distribution might have been achieved either by co-evolution or by spread through soil dumped from
ships or through potted plants or by human being (Poinar, 1990). Though distribution is primarily
limited by the availability of suitable host, some sort of geographical pattern is seen. S. carpocapsae
is most widely distributed species which has been isolated from Europe, Australia, New Zealand
,India and America followed by S. feltiae from Europe, Australia and New Zealand. Some of the
most narrowly distributed species are S. anomali, only from Russia, S. rara from Brazil, S. kushidai
from Japan, and S. scapterisci from Uruguay. S. feltiae is the most prevalent species in Britain and.
S. affinis has been found in Northern Europe (Poinar ,1990) and in Great Britain. Some evidence of
seasonality in occurrence has been found in different surveys. Habitat type had little effect on their
presence in Tasmania, Britain, Ireland, Korea, Portugal, Argentina and Spain .They were isolated
from coastal areas (O-m elevation) to high altitude areas (2000 m). Annual median temperature and
rainfall had little association with the presence of nematodes during our surveys. In Scotland, Boag et
al. (1992) found S. foltiae in permanent pastures than in arable farmland, coniferous forest or deciduous
woodland, whilst none was recovered from scrub or heathland.

3. DISPERSAL

Active dispersal by IJ s is minimal. Some ofthe passive dispersal may be by water, wind and
human activity, although dispersal by these agents especially wind is not adequately documented

267
(Kaya, 1990). Timper et al. (1988) speculated that infected adults ofthe beet armyworm, S. exigua
could provide an important dispersal mechanism. Dispersal through phoretic relationships with
meso fauna of the soil was found by Epsky et al. (1988) and Shapiro et al. (1993). Cabanillas and
Raulston (1994) found the movement ofS. riobrave in the same maize field to be on an average of
4 cm/day . Boffand Smits (2001) reported that Ijs dispersed faster and further at high densities than
at low densities .Dispersal was also influenced by the age ofIjs .Individuals stored for 1.5 and 4.5
weeks were shown to be more active than those stored for 2.5 and 3.5 weeks .The presence of a
host insect enhanced the dispersal.

4. HOST DEFENSE MECHANISM

Portals ofentry for infective juveniles include natural body openings-spiracles, mouth and anus
and through thin cuticle. Symbiotic bacterial cells found in the gut of infective juveniles are released
through anus as soon as juveniles are established in the hemocoel (poinar, 1966). Entrance ofjuveniles
does not ensure infection as the defense mechanism of host insect comes into operation. Nematodes
entering through oral and anal routes may be defecated. Those reaching the hemocoel are attacked
and melanized at site of penetration. Encapsulation ofjuveniles does not always provide safety to
insect host as the adhering bacteria may establish and kill the insect .A host insect fed on its natural
diet is more resistant to nematode attack (Cui et aI., 1993; Peters and Ehlers, 1994; Epsky and
Capinera, 1994). Thus the process of infection is the result of interaction between host, vector and
the bacterium.

5. DEVELOPMENT, REPRODUCTION AND VARIATION

Entomopathogenic nematode families Steinemematidae and Heterorhabditidae, though not closely

related phylogenitically but share similar life histories through convergent evolution (Poinar, 1993).
The only free living stage is a non-feeding, developmentally arrested infective juvenile (a dauer larva)
whose sole function is to seek a new host and initiate infection. This life stage is of interest for
inundative biological control considerations. Infective juveniles of Steinernema develop into
amphimictic females and males and never into hermaphrodites. The Heterorhabditis spp. always
develops into hermaphrodites in the first generation. Subsequent generations produce amphimictic
males and females in both genera. First generation adults are termed as giant adults due to their size
(Poinar, 1990). Lewis and Gaugler (1994) hypothesized that protandry would be a reproductive
strategy for EPNs consistent with the cruise foraging strategy, but not with ambushing. Some of the
eggs are laid through vulva and later the females become ovoviviparous (poinar, 1990). They complete
2-3 generations in the insect that depends upon food availability. With the depletion of food in insect
cadaver the juveniles develop into the dauer and move out in search of new host.
Different species and strains of pathogens and predators differ in their effectiveness as biological
control agents of insects. These differences have been well documented for predatory insects,
parasitoids and a number of entomopathogens (Messenger et aI., 1976). The importance of such
interspecific and intraspecific variations has been recognized and demonstrated for insect parasitic
nematodes (Bedding et aI., 1983) as well. Genotypic characterization of isolates will allow the
assessment of genetic diversity within the species and populations and form a basis for the collection,
maintenance, and selection of insect parasitic nematodes (Curran and Webster, 1989). Diversity
within has been analyzed morphologically and with isozyme analysis (Poinar, 1986; Akhurst, 1987).

6. BIOEFFICICACY

Among the factors limiting the efficacy of nematodes is the need for timing of application to
coincide with the phenology of susceptible stages (Jackson and Brooks, 1995). In India the work on

268
steinernematids started in the sixties .In the seventies, Singh and Bardhan (1974) and Singh (1977)
worked on mortality in laboratory and field trials, life cycle and compatibility ofDD 136 with insecticides
and fertilizers. Work on heterorhabditids is of recent origin.
In Rice, high mortality of cutworm, Pseudaletia separata and leaf folder, Cirphis compta was
obtained with DD 136 strain, S carpocapsae in lab, green house and field experiments (Israel et aI.,
1969). Ragi pink borer, Sesamia inferens, rice leaffolder, Cnaphalocrosis medinalis, rice borer,
Chilo suppressalis, stem borer, Scirpophaga (= Tryporyza) incertulas ans paddy gall midge,
Orseolia oryzae were found highly susceptible. At temperature above 30 C nematodes were
ineffective against S incertulas (Rao et aI., 1971). Srinivas and Prasad (1991) reported 98% mortality
of the rice leaf folder, C. medinalis with S carpocapsae. Enhancement in efficacy of spray was
achieved with the addition of2 % glycerin (Yadav and Rao, 1970). They were found compatible with
fertilizers and insecticides and tolerant to osmotic stress (Rao et aI., 1975; Das, 1977). Maize stem
borer, C. partellus was parasitised by Neoaplectana sp. (Mathur et aI., 1966).
In Tobacco, DD-136 strain caused 66% mortality of cutworm, Slitura (Gupta et aI., 1987).
Narayanan and Gopalakrishnan (1987) found the effect of S feltiae to the pre-pupa, pupa, and
adult of S litura. Complete mortality of S. litura pre-pupae was recorded at 10,000 and 1000 IJ.
The pupae were less susceptible to nematode infection than pre-pupae and adults. However, 20-
60% mortality was observed in the case of 7- 8 day old pupa. Infective-stage juveniles of S
carpocapsae entered Liriomyza trifolii through the oviposition puncture made by the female during
egg laying, or though an unnatural tear in the mine surface. Nematodes were unable to enter mines by
penetrating the intact leaf cuticle (LeBeck et aI. 1993).
In Groundnut, DD-13 6, Burliar, Melur and Cherikunnu strains of H bacteriophora were tested
by Bhaskaran et ai. (1994) on red hairy caterpillar, Amsacta albistriga in field. DD 136 was most
effective followed by cherikunnu and burliar strains. The Melur strain was ineffective. In Potato, the
cutworms, Agrotis ipsilon andA. segetum were found parasitized (Singh 1977, 1993). The DD-
136 strain caused 100% mortality of chafer grubs, Anomala sp. (Rajeswari Sundarababu et aI.,
1984). Brinjal fruit borer, Leucinodes orbonalis, mustard sawfly, Athalia proxima (Singh and
Bardhan, 1974), castor semi looper, Paralellia algira (Gupta et a!., 1987). Sivakumar et a!. (1989)
found adult grasshoppers (Orthacris simulans), larvae of beetle, Draterius sp., citrus butterfly,
Papilio aristolochiae and Ergolis merione were susceptible to H bacteriophora.
At PDBC, populations of S glaseri, Scarpocapsae PDBC EN 6.11, PDBC EN 1.3, Sabbasi
PDBC EN 3.1 and H indica PDBC EN 13.3 were tested against S litura, H armigera, 0.
arenosella, P xylostella and P operculella for evaluating bio-efficacy by soil column assay. Among
the isolates tested S abbasi PDBC EN3.1 and H indica PDBC EN 13.3 consistently recorded
highest mortality (80-100 %) against all insects tested. S glaseri, S carpocapsae PDBC EN 6.11,
S abbasi PDBC EN 3.1 and H indica 13.3 proved efficient for S litura with 80 % mortality after
96 h of inoculation. S abbasi PDBC EN 3.1 and H indica 13.3 caused 100% mortality of H
armigera larvae after 96 h of exposure. Against 0. arenosella, S glaseri and. S carpocapsae
PDBC EN 6.11 were found very effective compared to other isolates with maximum (100%) mortality.
Bio-efficacy of all the isolates tested against P operculella and P xylostella was found to be on par
with 80-100% mortality (Table 1 ). S carpocapsae PDBC EN 7.2, PDBC EN 6.11, PDBC EN
1.3, S tami PDBC EN 2.1, Sabbasi PDBC EN 3.1, S glaseri and H indica PDBC EN 13.3
were tested against Holotrichia sp. grubs in lab by soil column assay. At a dosage rate of750 IJ /
insect larva of Steinernema spp., S. glaseri infected successfully and multiplied in Holotrichia sp.
Virulence of H indica PDBC EN 13.3 and PDBC EN 6.71 was on par by 77-88 % mortality of
grubs 120 h after inoculation (Table 2).
Bioefficiency of indigenous isolates ofEPN s were tested against L. orbonalis in lab and field.
In vitro studies indicated that the isolates, S abbasi PDBC EN 3.1, H indica PDBC EN 13.3 and
S carpocapsae PDBC EN 6.11 recorded maximum mortality of L. orbonalis at 50 IJIlarvae in 48
to 72 h of exposure. Other isolates exhibited lowered mortality either at higher concentration or at
longer duration of exposure. Preliminary field trial with the isolates, S carpocapsae PDBC EN 6.11
and H indica PDBC EN 6.71 on brinjal indicated that higher the concentration of IJ per dose,

269
higher the reduction in borer holes on brinjal fruits and the results were comparable with sprays of
neem seed kernel extract. Between the two species evaluated, S carpocapsae PDBC EN 6.11 was
found to be more effective in reducing the fruit damage in terms of number of fruits with borer holes
and increase in yields (Hussaini et ai., 2000a).

Table 1 . Bioefficacy ofEPN isolates against different insect pests-Percent mortality oflast instar
larvae.

% Mortality of larvae hrs. after inoculation

Nematode S. /itura H armigera 0. arenasella P apercu/ella P xy/astella
Isolates 24 48 72 96 24 48 72 96 24 48 72 96 24 487296 24 48 72 96
S.glaseri 0 20 60 &l 0 40 60 6020 20 100 100 20 40 &l&l 20 &l 100 100
S carpocapsae 0 20 60 &l 0 40 &l &l 0 20 60 100 0 20 6O&l 20 60 100 100
POBCEN6.11
S carpocapsae 0 0.0 40 60 0 20 60 &l 0 20 60 &l 0 o 40 60 0 40 60 ro
POBCENIJ
Sabbasi POBCEN3.1 0 40 60 &l 0 40 &l 100 20 40 &l &l 20 60 &l100 60 100 100 100
Hindica POBCEN 13.3 0 60 ro &l20 &l100 100 20 60 &l &l 20 &l &l 0 &l 100 100 100

Table 2. Virulence of entomopathogenic nematodes againstA. ipsilon and A.segetum larvae

and pupae

Nematode isolate Percentage mortality 72 hrs. after inoculation

Agrotis ipsilon Agrotis segetum
Larva Pupa Larva Pupa
S abbasi POBCEN3.1 93.33 44.44 33.23 0.00
(75.08)' (41.80)' (35.24)d (O.OO)d
S bicornutum POBC EN 3.2 100.00 100.00 80.00 0.00
(90.00)' (90.00)' (63.34)' (O.OO)d
S tami POBC EN 2.1 6.67 66.67 13.33 88.89
(14.95)' (60.00)" (21.39)' (78.20)'
S carpocapsae POBC EN 6.61 53.34 100.00 86.67 100.00
(4690)b (90.00)' (68.58)' (90.00)'
Scarpocapsae POBCEN 13.1 40.00 33.33 33.33 0.00
(39.23)" (30.00)' (35.24)b (O.OO)d
Scarpacapsae POBC EN 6.11 46.67 55.56 26.67 0.00
(43.IO)b (48.20)'" (31.10)b (O.OO)d
H indica POBC EN 6.71 66.67 55.56 100.00 55.56
(54.72)" (48.20)'" (90.00)' (41.80)'"
H indica POBC EN 13.3 86.67 100.00 93.33 44.44
(68.58)' (21.39)' (75.08)' (36.50)'
Control 13.33 0.00 6.67 0.00
(21.39)' (O.OO)d (14.95)' (O.OO)d

• Means followed by the same alphabet do not differ significantly (P=0.05)

Figures in parentheses are arc sin tranformed values.

To evolve a virulent strain ofEPN and to compare its virulence against different stages of
Agrotis spp., six Steinernema spp. (S tami PDBC EN 2.1, Sabbasi PDBC EN 3.1, Sbicornutum
PDBC EN 3.2, S carpocapsae PDBC EN 6.11, PDBC EN 6.61 andSteinernema sp. PDBC EN
13.1) and two H. indica strains (PDBC EN 6.71, PDBC EN 13.3) were tested againstA. ipsilon
and A.segetum larvae and pupae separately. Sand column assays were performed at 200 IJs/stage.
Mortality ofthe larvae and pupae was determined 72 h after exposure. Virulence was measured in
terms of mortality of Agrotis spp. Sabbasi PDBC EN 3.1, Sbicornutum 3.2 performed better

270
with 91-100% mortality than H indica strains for A. ipsi/on larvae and vice-versa for A. segetum.
Among the steinernematids, Scarpocapsae PDBC EN 6.61 and S bicornutum 3.2 were most
effective in soil with 80-90% mortality for A. segetum. The effect of S carpocapsae PDBC EN
13.1 and PDBCEN 6.11 was on par for both Agrotis spp. larvae. S tami PDBC EN 2.1 that
performed least with the larvae was effective with 60-80% mortality for pupae of both the species.
For A. ipsilon pupae, Sbicornutum PDBC EN 3.2 and Scarpocapsae PDBC EN 6.61 gave
100% mortality. Sabbasi PDBC EN 3.1 & S. bicornutum 3.2 were not effective againstA. segetum
pupae(Hussaini, 2000 e ).
Nematode isolates differed in their rank for virulence between the stages of the insect and also
among the species of Agrotis tested ( Glazer and Navon, 1990). Overall, higher virulence of
heterorhabditids and a few steinernematids can be attributed to the cruiser foraging strategy. Lower
mortality of pupae may be due to their sedentary nature, and an increase in exposure time may
increase the mortality percentage. Moreover, pupae with thick chitinous covering may impair the
infectivity of the EPN to certain extent. As the nematode isolates differ in their virulence, combination
of the two with different foraging strategies will result in additive effect (Choo et al., 1996) as it takes
care of the active larval and sedentary pupal stages ofAgrotis spp. Moreover the pathogenicity of
the EPN to cutworm pupae has not been documented previously.
Virulence of nematodes is often related to plant variety or plant chemistry of the host fed upon.
Nematode progeny production was highest from rootworms (Diabrotica undecimpunctata) that
had fed on corn, lower for peanut, and lowest for squash rootworms fed on bitter squash was lower
than from non bitter squash (Barbercheck et al., 1995). Hussaini et al. ( 2000 f) found A. ipsilon
reared on chickpea leaves was more susceptible to S. abbasi PDBC EN 3.1 and H indica PDBC
EN 13.3 with 100% mortality 48h post exposure and those reared on tomato, castor and artificial
diet were susceptible to all isolates with 50-100% mortality at 72h. Agrotis larvae reared on
pumpkin were found least susceptible. The penetration rate was highest in artificial diet reared larvae
with 36% for S. bicornutum and 35% for H indica. Penetration rate in larvae reared on chickpea
and tomato was on par and it was drastically reduced when reared on pumpkin leaves. Progeny
production of S abbasi PDBC EN 3.1, S. carpocapsae PDBC EN 6.11 and H indica PDBC EN
13.3 from natural host plant reared larvae was on par ranging from 0.44-0.825 lakh. The yield of H
indica from pumpkin grown larvae was found to be 0.33 lakh whereas no yield was recorded for
Steinernema spp. Progeny production of Steinernema spp. and H indica isolates from Agrotis
larvae reared on artificial diet ranged from 2.4-3 .8lakhs that was much higher compared to those
grown on natural host plants. Susceptibility ofSitona lineatus to infection by the EPN, S. carpocapsae
was documented by Jaworska and Ropek (1994 ).Mortality of S lineatus was significantly greater
for larvae originally from peas than for those collected from faba beans. However, larvae of S
lineatus from beans appeared more favourable hosts for nematode multiplication than larvae from
peas because greater numbers ofjuveniles of S carpocapsae emerged from bean-fed S lineatus.
EPNs have been found to be compatible with commonly used pesticides. At PDBC the
effect of five commonly used pesticides including one botanical pesticide on the biological traits viz.,
activity, penetration rate, infectivity and progeny production of two S bicornutum and two H indica
isolates was assessed by using G.mellonella larvae (Table 3). Ijs of both the genera tolerated most
of the chemicals tested but the response to different pesticides was variable. The inactivity per cent
increased with increased time and concentration of pesticide and it was less than 40% and 35% for
heterorhabditids and steinemematids respectively at field recommended dosages. In general, infectivity
of pesticide exposed infective juveniles was not adversely affected (Figure 1). Exposure for 72 h to
pesticides impaired the penetration rate of S bicornutum and S tami isolates (30-40%) while
additive response was observed in H indica isolates. Overall, no additive or synergistic response
was observed in progeny production of pesticide exposed infective juveniles. Among the pesticides,
mancozeb and neem were safe to all the nematode populations while the latter was deleterious to H
indica PD BC 13.3. Fifteen out of twenty combinations tested were compatible and may be included
in any IPM schedule (Hussaini et al., 2000 c) (Table 4,5).

271
7. HOST PREFERENCE AND INTERSPECIFIC COMPETITION

The fitness of a parasite can be adversely affected by increasing population density within the
host. Density dependent effects include mortality, reduction in adult size, increased generation time,
reduced fecundity, failure to establish, delayed development, reduced oviposition and change in sex
ratio . Effects of high density appear to result from competition for limited nutrients within the host
(Burlingamme and Chandler, 1941 ; Roberts, 1961; Boray, 1969; Moss 1971 ; Benson, 1973; Rotary
and Gerling, 1973; Williams, 1973; Wylie, I973; Hasselberg and Andreasen, 1975; Chapel and Pike,
1976; Anderson and Michel, 1977; Rotary and Sandlan 1979; Uznanski and Nickol, 1982; Hominick
and Tingley, 1984; Taylor, 1988; Selvan and Muthukrishnan, 1988; Selvan et aI., 1993 ).

Table 3. Progeny production of pesticides exposed Steinemema spp. and H. indica isolates

Nematode % difference in progeny production over control of pesticide exposed nematodes

Endosulfan Malathion Carbofuran Mancozeb Nee
S. bicornulum .fJ6.75 -38.46 -5.38 -16.92 -14.33
PDBC3.2
S. lami -35.59 -21.66 -19.01 -1.56 -54.64
PDBC2.1
H. indica -35.30 .fJ6.67 -20.59 -27.61 -15.25
PDBC6.71
H. indica 46.74 -31.14 -25.17 -12.72 -15.94
PDBC 13.3
The means with the same letter do not differ significantly by DMRT (P=0.05)

e tlalathion
.,
c: ..,
"''' 80
(G 8ldosu~ an

-.,
.-
.., 0 60 ~ tlancozeb
'"
.=> '"E
40 mcarbofuran
20
• z
~

0
. Neem
In Control
S.bicornu/um S./ami POSC 2. 1 H. indica POSC H. indica POSC
POBC3.2 13.3 6. 71
Nematode Isolates

Figure 1. Penetraton rate of pesticide exposed infective juveniles in Galleria mellonella

Leplinolarsa decemlineata is less sU'lceptible to EPN than many other insect species. The
roles of host-finding ability by the nematode S. carpocapsae and host non-selfresponse (immunity)
toward the nematodes were determined to partially explain this low susceptibility. An agar-based
assay was used to assess the chemotactic responses ofS. carpocapsae IJ ~o host-derived cues. The
nematodes were attracted to CO 2 and faeces of G.mellonella and Tenebrio molilor but were
repelled by L. decemlineata faeces. Examination ofL. decemlineata larvae that survived exposure
to S. carpocapsae revealed that the nematodes were often enclosed in haemocytic capsules in the
haemocoel. When nematodes were injected directly into the haemocoel, an individual L. decemlineata
larva could encapsulate up to 21 nematodes, but at loads above 9 nematodes per larva at least one
always escaped encapsulation resulting in insect death. Thus, the low susceptibility ofL. decemlineata
to S. carpocapsae is attributed, in part, to repellence ofthe host faeces and to haemocytic encapsulation
of penetrating nematodes (Thurston et aI., 1994).

272
 The host finding ability is an integrated combination ofbehaviolU'S consisting ofactive dispersal
(Choo et al., 1989), nictation (Ishibashi and Kondo, 1990) and chemotaxis (Schmidt and All, 1978).
Substances such as organic solvent wash offofthe surface of G. mellonella larvae, nitrogen compounds
from faecal pellets, plant root metabolites, bacteria and carbon dioxide trigger the chemotaxic stimuli
(Schmidt and All, 1979; Bird and Bird, 1986; Pye and Burman, 1978; Gaugler et al., 1980),
heterorhabditids have a better host finding ability than the steinernematids (Choo et al., 1989). However
the host finding ability depends on motility and attraction, as G. mellonella and Apis mellifera
produced carbon dioxide at the same rate, the host finding ability of nematodes was significantly
different. Insects are one ofthe many sources ofcarbon dioxide in the environment and hence nematodes
musthave other mechanisms ofdistinguishing host from non-host. Although extremes ofsoil environment
can adversely affect survival, behaviour and efficacy of nematodes, biotic factors also are capable of
negatively affecting nematode populations (Epsky et al., 1988). Bird and Bird (1986) demonstrated
that S. glaseri, a close relative of S. foltiae was attracted to the meristematic region of plant roots.
Many root-feeding insects are primary targets for these nematodes. Soil temperatures influence the
host finding ability. Low soil temperature impairs nematode activity. At 22°C steinemematids and
heterorhabditids killed their target insects within one week than at 16°C.
S. glaseri cruises through the soil matrix in search of sedentary hosts, while S. carpocapsae
tends to ambush mobile hosts (Georgis and Gaugler, 1991; Lewis et aI., 1992, 1993). In most
steinemematid species, male nematodes find and penetrate the host first and are then followed by
females. Males are more sensitive to volatile host cues primarily CO 2 than females (Grewal et al.,
1993).
Steinemematids have the ability to follow and infect cabbage maggot larvae in roots. This
behaviour is stimulated by infochemicals from the radish roots, from the insect cuticle as well as from
excretory products of cabbage maggot (Scmiege, 1963; Schmidt and All, 1978, 1979; Pye and
Burman, 1981; Lei et al. 1992).
Histological and scanning electron microscopic observations were made to reveal the infection
of S. foltiae [N carpocapsae] in S. litura via routes other than the alimentary canal. The infective
juveniles (13s) invaded the larval, pupal and adult spiracles of G. mellonella, while they did not
invade the spiracles of the last-instar larva or 3-day-old pupa ofS. litura. Many 13s congregated in
the vesicle under the oesophagus of the S. litura larva and some 13s penetrated the thin cuticular
membrane of the vesicle. The nematode~ also penetrated wounded tissues on the insect integument.
A few 13s were assumed to infect the larva through its thin intersegmental membrane, but no direct
evidence was obtained. The firm framework of the 13s head may be responsible for the mechanical
invasion through the cuticular membrane (Kondo and Ishibashi, 1989).
Comparison of infectivity of indigenous isolates ofEPN in sand and soil column showed H
indica out competed Steinernema spp. irrespective of soil depth, types and time for mortality ofA.
ipsi/on. Combined populations had an additive effect over single population (Hussaini et al., 2000
d). Presence ofS. glaseri enhanced the performance ofS. carpocapsae when applied in combination.
Combined populations ofS. carpocapsae and S. glaseri in soil column againstA. ipsi/on had additive
effect over single population . There are certain distinct characteristics of Steinernema and
Heterorhabditis, which areprimarilyirnportantinproperutilization ofthese for insect control (Table 6).

8. ECOLOGY

The nitrogenous compounds produced during the decomposition of all organisms can affect
nematode ecology. Various nitrogenous compounds, depending upon the nematode species, chemical
concentration, and duration ofexposure may affect virulence, infectivity, and survival. Soil characteristics
as texture and moisture, nematode-searching strategy as ambush versus cruiser affects nematode
host finding and infection behaviours. Most research on host finding and infectivity has focussed on
nematode response to non-infected host, and relatively little attention has been given to what happens

273
after infection is initiated. Although it appears that most IJ s are capable of infecting hosts the actual
number invading depends upon the nematode/host ratio. Compounds emitted from infected hosts
may deter secondary invasion of the cadaver. Nitrogen compounds are known to be inhibitory or
detrimental to EPNs. Nitrogen released by an infected host may be one of the factors affecting
secondary invasion. Amount of ammonia contained in German cockroach, Blatta germanica feces
were found repulsive to H. bacteriophora (Grewal et al., 1993). Small amounts of ammonia, less
than 10 ~ were attractive to S. carpocapsae and greater amounts were not tested. Further various
nitrogenous fertilizers can reduce survival and virulence ofEPNs (Shapiro et al., 1996; Mullens et al.,
1997). Thus attraction or repulsion ofH. bacteriophora to an infected host may depend at least in
part on the dynamics ofof nitrogen concentration in the soil surrounding the host cadaver.

Table 4. Percentage inactivity ofSteinernema spp. isolates upon exposure to selected pesticides in
aqueous solutions

Pesticide Exposure Time (h)* Pesticidesconcentration(l-'g/l-'l)**

s. tami PDBC 2.1 S. bicornulUm PDBC 3.2 S. lami PDBC 2.1 S. bicornutum PDBC 3.2

24 72 120 24 72 120 1/2N N 2N 1I2N N 2N

Endosulfan 4.4 7.2 12.S 11.2 34.5 44.7 4.2 7.4 12.8 17.4 26.9 46.0
(12.8); (16.1)' (21.4)' (19.9)' (36.2)' (42.2)' (12.6); (16.2)' (21.4)' (25.0)' (31.5)" (43.0)'

Malathion 7.4 10.8 17.3 10.0 33.0 41.4 6.3 10.81 18.37 14.81 30.8 38.89
(16.3)' (19.6)& (24.9)' (18.9)' (35.3)' (40.3)' (15.1)' (19.5)' (25.7)' ~23.0)'(34.0)' (38.S)'

Mancozeb 3.2 6.5 11.8 3.7 9.6 16.0 4.4 7.1 10.0 4.7 8.8 15.7
(l1.°Y (15.3); (20.5)' (11.7Y (18.5)' (23.9)' (12.8); (15.9)' (18.9)' (13.2); (17.8)' (23.6),

Carbofuran 7.5 14.3 21.4 15.4 39.9 51.8 8.5 13.9 20.S 25.0 34.S 49.3
(16.4)' (22.6)' (27.9)' (23.5)' (39.4)' (46.3)< (17.5)' (22.3)' (27.4)' (30.3)' (36.3)' (44.9)

Neem 0.0 0.3 6.8 0.0 0.8 4.3 1.5 2.6 2.9 1.1 1.1 1.2
(4.1)' (4.S)' (15.5)'; (4.1)1 (6.5)' (12.6)ij (17.Sy (9.7Y (l0.6);j (7.0)k (S.3); (9.IY

Control I.3 3.1 4.0 0.0 3.5 5.3 2.9 2.8

(7.3)' (9.SY (10.8y (4.1)1 (11.4Y (13.71); (IO.SY (10.2Y

* CD (p=O.05) Pesticide x Nematode x Time =2.89.

** CD (P=O.05) Pesticide x Nematode x Concentration =2.47. N - Nonna! field recommended dosage.
Figures in parentheses are arc sin transformed values. Means followed by the. same letter do not differ
significantly.

9. SURVIVAL

Nematode dispersal and infectivity is affected by soil texture (Georgis and Poinar, 1983).
Dense roots adversely affect nematode movement and infectivity in the soil (Choo et al., 1989).
Choo and Kaya (1991) tested the host finding ability ofH. bacteriophora in humus, clay, loam and
sand with and without roots. The per cent infection in G. mellonella larvae was invariably more in
soils with roots and maximum in humus followed by sand loam and clay. The infection is influenced by
pore size, and presence ofroots affects the dispersal by altering soil structure, water or microtlora. In
addition root metabolites serve as cues for nematode attraction.
Storage capacity of different EPN isolates and species was evaluated at PDBC . S. biconutum
and S. carpocapsae survived better than H. indica isolates and recorded 90-98 % survival at room
temperature for a period of 8 weeks in distilled water. H. indica PDBC EN 6.71 and PDBC EN
13.3 recorded 78-82 % survival. At 15 oC, distilled water was better than glycerin and liquid paraffin

274
for all isolates. In general, steinemematids survived better than heterorhabditids. Liquid paraffin was
found most suitable for both at 8 and 24°C (Hussaini et aI., 2000 b ).

Table 5. Percentage inactivity of indigenous H indica isolates upon exposure to pesticides in aqueous
solutions

Exposure Time (h)' Peshe ideseon eentrati on(llg/lll)"

Pesticide H Indica POBe 6.7 H. indica POBe 13.3 H indica PO Be 6.71 H. indica POBe 13.3
24 72 120 24 72 120 112N N 2N 1/2N N 2N

Endosulfan 21.6 36.2 52.6 10.5 18.1 26.3 24.6 37.1 48.1 7.5 17.7 29.4
(28.0)"' (37.3)' (46.8)" (193)' (25.4)' (31.0)" (30.0)' (378)" (44.2)" (16.4)'"(252)" (33.1)"

Malathion 25.1 43.1 51.1 6.3 16.5 23.7 32.9 40.2 46.2 7.4 12.3 26.9
(30.3 )" (41.2)" (45.9)" ( 15.1)" (24.3)' (29.4)" (35.3)' (39.6)" (431)" (16.2)'"(20.7)' (31.5)"

Mancozcb 0.5 5.0 14.5 4.5 11.0 15.8 4.1 6.2 9.7 5.1 10.8 15.2
(5.6)' ( 13.5)' (22.7)" (13.0)' (19.7)' (23.8)' (12.2)" (14.9)" (18.6)' (13.7)h (19.6)' (23.3)'

Carbofuran LI 2.9 10.6 10.1 24.8 31.2 2.3 3.5 8.3 12.9 21.6 34.8
(6.6)' (102)" (19.4)' (21.6)' (30.2)" (34.2)' (9.3)' (11.4)' (17.1)' (21.4)' (28.1)' (36.4)'

Neem 00 0.3 1.4 1.4 33.1 40.8 0.5 0.2 0.8 6.8 26.5 41.4
(4.1)' (4.8)' (7.5)" (7.4)"' (35.4)' ( 40.0)" (5.6)' (4.8)' (6.1)' (l5.6)h (31.3)" ( 40.3)"

Control 00 0.9 1.33 0.00 2.66 6.22 0.74 2.96

(4.0)' (6.2)' (6.9)' (4.0)i (97)h (14.9)' (6.1) (10.6)'

• CD (P=0.05) Pesticide x Nematode x Time = 3.01

•• CD (P~0.05) Pesticide x Nematode x Concentration = 2.89 N - Normal field recommended dosage
Figures in parentheses are arc sin transformed values Means followed by the same letter do not differ
significantly.

Table 6. Characteristic features of entomopathogenic nematodes

Character Steinernema Heterorhabiditis
Host searching ability Poor Good
Proliferation rate in host Fast Slow
Yield Less More
Mass production medium Non-specific Specific
Shelf-life in storage Good Poor
Tolerant to desiccation Poor Good
Transportation Easy Difficult

Thermal adaptation ofEPN- niche breadth for infection, establishment, and reproduction has
been worked out for 12 species and strains ofEPN collected from diverse climatic regions. S
riobravis infected G. mellonella larvae at the widest temperature range (10-39°C), whereas S
feltiae at the narrowest (8-30°C). Thermal niche breadth for establishment within hosts was the
widest for S glaseri (1 0-37°C) and the narrowest for Sfeltiae (8-30°C). Thermal niche breadth
for reproduction was widest for S glaseri (12-32°C) and the narrowest for S carpocapsae (20-
30"C). S scapterisci (20-32°C), S riobravis (20- 35°C), and Steinernema sp. (20-32°C) were
more adapted to warm temperature reproduction, and Sfeltiae to cooler temperatures (10-25°C).
Although heterorhabditids are endemic to warmer climates, the upper thermal limits and temperature
optima for reproduction of H bacteriophora and H megedis were cooler than that of some of the
steinemematids from South America and the Caribbean. Thermal niche breadths did not differ between
conspecific populations isolated from different localities, but were different for different species isolated
from the same locality. It was concluded that EPN species have well-defined thermal niches which

275
may be unaffected by their locality (Grewal et al., 1994). Absolute mortality ofG.mellonella and
A. ipsilon was obtained at 320C with S carpocapsae PDBCEN6.11, Sabbasi PDBCEN3 .1 , S tami
PDBCEN 2.1 and HindicaPDBCEN 6.71 and 13.3 (Hussaini et al., 2001c). Ganguly and Singh
(2001) reported 35°C to be optimum temperature for S thermophilum .

10. BACTERIAL ASSOCIATION, PHASE VARIATION AND ROLE OF BACTERIA

Penetrating nematodes inoculate associated bacteriumXenorhabdus or Photorhabdus spp.

for steinemematids and heterorhabditids, respectively into the host. The nematode may appear to be
little more than a biological syringe for its associated bacterium. Although parasitic EPNs have not
severed their nutritional relationship with bacteria, growth and reproduction are dependent upon
conditions established in the host cadaver by the bacterium. Symbiotic bacteria are of fundamental
importance. Bacteria cannot survive in water and soil nor can reach the insect hemocoel on their
own. The nematode must carry the bacteria into the hemocoel of an insect host where they multiply
rapidly, kill the host by septicemia and serve as an essential food source for the nematode. The
bacteria cannot penetrate into the hemocoel of a host, and the nematodes do not grow and reproduce
in the absence of the bacteria (Poinar and Thomas, 1966). Phase variation occurs as primary (phase
I) and secondary (phase II). Pathogenic differences are not found between the two (Akhurst,
1980). Primary form is found superior in terms of nutrition as it enhances maturity, reproduction, and
production of antibiotics (Akhurst, 1980, 1982). Phase variation is suggested for survival of bacteria
as phase I is more susceptible to phage virus (Akhrust and Boemare, 1990). Nematodes do not
reproduce in axenic cultures. Specificity of bacteria-nematode association has been known.
Steinernema species reproduce best in the monoxenic culture of their natural symbiont though they
can utilize other bacterial species (Akhurst, 1983). Controlled mass culture of bacteria released
metabolites that are anti-fungal and anti-bacterial. Indole derivatives, stilbene derivative xenorhabdins,
xenocoumacins, xeroxodes, and nematophins are bioactive. They inhibit Bacillus subtilis,
Staphylococcus epidermidis, S aureus, Aspergillus fumigatus and A. flavus (Webster et aI.,
1996). Xenorhabdus nematophilus was found to inhibit Botrytis cinerea, Cerastomella ulmi, C.
dryocoetidis, Mucor piriformiS, Pythium colaratum, P ultimum, Penicillium notatum,
Rhizoctonia solani, Trichoderma pseudokoningii, and Verticillium dahliae and Photorhabdus
inhibited Botrytis cinerea, Cerastomella ulmi, C. dryocoetidis, M piriformis, P coloratum, P
ultimum, T pseudokoningii, V. dahliae (Chen et aI., 1994). The identity of Xenorhabdus and
Photorhabdus from Indian isolates of Scarpocapsae and H indica respectively was established
by morphometrics, cultural characteristics and electron microscopy (Nagesh et al., 2001).
Fisher et al. (1999) examined the taxonomic position of Photorhabdus strains through the
results of DNA relatedness (S 1 nuclease method) studies associated with the determination of
DELTATm' 16S rRNA phylogenetic inferences and phenotypic characterization, including
morphological, auxanographic, biochemical and physiological properties. Three genomic species
were delineated on a consensus assessment. One of these species corresponded to P luminescens,
since strains were at least 50% related to the type strain of this species with DELTATm <7°C. The
two other species were novel genomic species II and III, which were <40% related to each other
with DELTATm higher than 9°C. A comparison of the complete 16S rDNA sequences of several
representatives of genomic species II and genomic species III revealed that each of them formed a
stable lineage independent of the cluster generated by P luminescens strains. The genomic species
differed in their maximum temperatures for growth. A correlation with the ecological origin of the
bacterial samples was noticed. The heat-tolerant group I (maximum growth temperature 35-39°C)
corresponded to the symbionts of H bacteriophora groups Brecon and HP88 and H indica,
nematodes living in warm and tropical countries, respectively. Group IT (maximum growth temperature
33-35°C) encompassed symbionts from H megidis, H zealandica and group NCl of H
bacteriophora, nematodes isolated in temperate climates. Group III were bacteria isolated from
human specimens. Two new species, P temperata sp. nov. (type strain CIP 105563T) and P

276
asymbiotica sp. nov. (type strain ATCC 43950T), are proposed for genomic species II and III,
respectively. Species I and II can be separated into sub-groups on the basis of high DNA-DNA
relatedness (>80% DNA binding with DELTATm <1. 5°C), 16S rDNA branching and phenotypic
characters. Therefore, it is proposed thatthe two species P luminescens and P temperata should
be subdivided into subspecies as follows: P luminescens subsp. luminescens subsp. nov. (type
strain ATCC 29999T), P luminescens subsp. akhurstii subsp. nov. (type strain CIP 105564T), P
luminescens subsp. laumondii subsp. nov. (type strain CIP 105565T) and P temperata subsp.
temperata subsp. nov.

11. EFFECT ON NON TARGET ORGANISMS

Akhurst (1990) reviewed the available data on the effect of these nematodes on non-targets,
beneficial organisms and reported no negative impact. Tests conducted on rats, chicks, rabbits and
pigs showed no symptoms or mortality by nematodes and their associated bacteria by either oral,
intradermal, subcutaneous, or intraperitoneal inoculation (Poinar, 1989). In mammals and birds, a
body temperature of37 °c prevents nematode or bacteria from reproducing. Hence they have been
found safe against several non-target organisms despite their wide host range. Nematode biopesticides
are exempted from registration in USA. Earthworms were reported safe to nematode application,
however injured earthworms are attacked and nematodes reproduced (Capinera et al., 1982). Several
molluscs, arthropods, diplopods are infected and killed. Fishes, birds, reptiles and mammals are not
attacked. Frog tadpoles are susceptible .At high dosages, garden millipedes are affected (Poinar and
Thomas, 1985; Poinar, 1989).

12. GENETIC MANIPULATION

EPN s are extremely promising biological control agents but limitations like susceptibility to
environment extremes, and diverse host-finding behaviour hamper their complete utilization. One
solution to this problem is to develop genetically improved strains ofEPN with enhanced traits for
biological control. Strategies for genetic manipulation include artificial selection, hybridization and
genetic transformation. Selection and hybridization have been employed in developing genetically
improved strains. These approaches may fail ifthe available nematode population do not possess
sufficient genetic diversity for the beneficial traits (Glazer et al., 1991). A lack of genetic diversity may
be overcome by surveying natural population for the desired traits (Gaugler, 1987). Selection is
employed to find genetically superior nematode species. A steinernematid species with increased
host finding ability has been successfully selected. A heat tolerant strain ofH bacteriophora designated
IS 5 was discovered in Negev desert, Israel in 1996 and was found superior to HP 88, a commercially
available strain ofH. bacteriophora. Stability ofthe heat tolerance trait and fitness ofH. bacteriophora
IS 5 was tested in the lab after multiple passages through last instar G. mellonella larvae.
Hybridization is an alternative approach because of its relative simplicity and its speed in
transforming beneficial genes. Hybridization is used for genetic improvement of heat tolerance in H.
bacteriophora (Shapiro et aI., 1997). H. bacteriophora IS 5 hybridized readily with commercial,
H. bacteriophora HP 88.
To design EPN with specific traits requires the ability to introduce cloned genes of known
functions. Genetic transformation methods are required to produce transgenic nematodes carrying
the DNA of interest. The nematode Coenorhabditis elegans is a powerful genetic system to study
a large spectrum of biological questions (Riddle et a!., 1997). The generation of transgenic animals is
a key technique to identifY genes and to analyze their functions. At present transgenic nematodes are
produced by microinjecting individual worms which leads to the propogation of free concatemeric
arrays of DNA (Mello et aI., 1991). Hashmi et al. (1995) reported the first successful genetic

277
transformatiS)fi of an EPN by microinjection technique. Foreign genes were introduced into H.
bacteriophora HP 88 using vector carrying the C elegans genes. C elegans genomic DNA
containing 16 KDa heat shock promoter, rol-6 collagen gene (coding for roller phenotype) was used
for injection and they have succeeded in developing transgenic H. bacteriophora HP 88. This study
proved that a construct carrying a promoter from C elegans can be used for transformation ofH.
bacteriophora and genetic transformation by microinjection technique has practical application in
developing genetically altered strains of nematodes. Wilm et al. ( 1999) described a novel method to
transform the nematode Ce/egans , in which DNA coprecipated with gold particles is shot at worms
by means of a belium beam. Transformed worms were either identified by a dominant visible marker
or selected by a conditional lethal system.
Genetic selection can also be used to enhance the resistance ofEPN to nematicides. Glazer et
al. (1997) reported that genetic selection ofH. bacteriophora strain HP 88 to nematicides resulted
in an 8-9 fold increase in resistance to phenarniphos and avermictin and 70 fold increase in resistance
to oxamyi. The enhanced resistance was stable and continued even after the selection was relaxed.
Selection for heat tolerance proved to be effective, but was associated in deterioration of
reproductive potential. The ability to withstand desiccation by entering anhydrobiosis is important for
the survival of many nematode species. We are interested in the metabolic changes that occur during
dehydration in the semi-arid strain IS-6 of the insect parasitic nematode S.feltiae. These changes
may enable IS-6 to be more tolerant to desiccation than temperate strains. Genes ofIS-6 that exhibit
changes in transcript level during desiccation have been identified. These include glycogen synthase
which is the rate limiting enzyme in the synthesis ofglycogen, which is likely to playa role in desiccation
survival. A shift from glycogen to trehalose synthesis is observed during dehydration ( Gal et aI.,
2001). On the other hand selection for resistance to nematicides was very effective, and lasted when
the selection pressure was removed, and did not compromise with other parameters ofbiocontrol
efficacy. Screening of natural isolates resulted in the identification ofa heat tolerant strain IS-5. Using
genetic markers and cross hybridization it is demonstrated that the trait was dominant and transferable
to the commercial strain HP88 without concomitant reduction in biocontrol efficacy. Mutagenesis is
useful for generating both mutants displaying desired beneficial traits and marker mutations. The
utility ofthe latter is demonstrated. Finally, genetic engineering is a most promising tool for enhancing
beneficial traits in EPN. The success in genetic transformation ofEPN opens the way for generating
transgenic nematodes carrying genes conferring resistance to various environmental extremes, most
notably heat shock genes (Segal et al., 1999).

12.1 Mutations

The methodologies of classical genetics and genetic engineering can be used for the genetic
improvement ofEPNS and their symbiont bacteria. Mutagenesis has the effect of inactivating genes,
so it is not a very useful approach for introducing new genetic material into experimental organisms.
Mutagenesis is useful, however, for inactivating negative regulators of gene expression and can thus
be a means of obtaining the constitutive expression of repressed genes. Screening several thousand
infective juveniles (IJs) for a desirable phenotype can be very time consuming unless a suitable means
of mass screening is devised. Lethal screens and behavioural screens provide an easy method of
mutant selection. It is demonstrated that in situations where a suitable and non-labour intensive screening
method can be devised, chemical mutagenesis can provide an accessible, robust and inexpensive
means ofEPN strain improvement (Burnell et al., 2000).Mutagenesis aimed at induction ofbeneficial
mutations is also a viable approach for genetic improvement as a desiccation tolerant mutant of
H.megidis has been evolved(O'Leary and Burnell, 1997).

13. MASS PRODUCTION -ISSUES INVOLVED

Both steinernematids and heterorhabditids though acknowledged as potential bioagents for

insects, several issues contribute to their commercialization. Mass production and subsequent
formulation and use are important issues. Some critical factors for successful mass production and
utilization are detailed below.

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13.1 Identification of Isolates of Nematodes

Accurate, fast and easy methods of identification of entomopathogenic nematodes are required.
Identification ofthese nematodes to species level has always been difficult and conservative due to
their feeding habits and life cycle.

13.1.1 Molecular Techniques. The major focus of molecular methods in nematode taxonomy has
been to develop novel diagnostic characters for species and intrasubspecific groupings in economically
important nematode genera where morphology lacked sufficient discriminatory power. Any difference
in protein or isozyme phenotype, affinity for antibody, DNA restriction fragment length polymorphism,
DNA probe or sequence data for any gene or region ofthe genome are potential diagnostic characters.
Little use has recently been made ofprotein or immunological techniques and DNA analysis received
greater attention in nematode taxonomy. In the field ofentomopathogenic nematology, we are plagued
with animals that to the trained eye, look very similar. Many ofthe morphological characters are not
always reliable because, the wide range ofvariations when dealing with very small sample size, make
them virtually useless. On the other hand, DNA - based molecular techniques, such as restriction
fragment length polymorphism analysis, allow the examination of any stage of the life cycle, as the
genomic DNA ofan organism remains constant throughout its life. With the advent ofpCR technology,
it has become possible to screen many isolates in a single day with a high degree of accuracy, using as
little DNA as that extracted from a single infective stage or adult (Reid, 1994).

13.1.2 Cross Breeding Techniques. The most fundamental test, whether two populations can
crossbreed to produce fertile offspring, is particularly important both as means ofidentifying species
and as an indicator of those characters that show great divergence between species. Poinar (1967)
used hanging drop of insect blood for cross-breeding test, but technique most commonly used is the
one given by Akhurst and Bedding (1978) based on injecting individual G. mellonella larvae with
one IJ from a stock culture and another from the population being tested. Therefore with variable
morphology and overlapping ofcharacters within the genus Steinernema, crossbreeding experiments
appear to be most reliable method for species identification.

13.1.3 PCR (POlymerase Chain Reaction) Method. PCR techniques have been found having
wide spread application in molecular taxonomy. This technology has effectively replaced the analysis
of extracted and purified DNA of entomopathogenic nematodes by agarose gel electrophoresis and
Southern technique (Curran and Webster, 1989). The major advantage ofPCR is its ability to amplifY
DNA from small quantities of initial samples and the specific amplification of regions ofthe genome
that are taxonomically usefuL Two DNA primers (which hybridize to opposite strands of the DNA)
are required. These flank the DNA sequence to be amplified and successive rounds ofdenaturation
of DNA by heating, then cooling and annealing of primers and sequence extension by Taq DNA
polymerase in the presence of dNTPs, effectively doubles the amount of DNA present after each
cycle. After approximately 30 rounds of amplification, sufficient DNA is produced for subsequent
RFLP, dot-blot or sequence analysis. The DNA from the species to be identified and other
morphologically similar steinernematidl hererorhabditid species are amplified by PCR using primer
specific for the ITS (internal transcribed spacer) region using Reid and Hominick (1992) method.
The PCR products for each nematode are then digested with a range of restriction enzymes and the
fragments separated by agarose gel electrophoresis. The resulting combination ofRFLPs produced
by restriction enzymes is then compared from species to species for similarity.
An in-depth knowledge of inter-and intraspecific variations in DNA sequence with Steinernema
and Heterorhabiditis to determine the taxonomic level at which different degrees of sequence
divergence operate is required. The research group at CABI-IP, UK is making a major contribution
in this area ofresearch. Recently, they have been able to construct the phylogeny ofentomopathogenic
nematode species by RFLP analysis of the ITS region of the ribosomal DNA repeat unit (Reid et al.,

279
1997). Most ofthe species descriptions are now appended with DNA data alongwith morphometric
and crossbreeding studies (Cabanillas et aI., 1994; Tallosi et aI., 1995; Elawad et aI, 1997; Waturu
et aI., 1997). It is now possible to identify large number of isolates to species level and below in as
little as a single day using no more than the DNA contained in a single infective stage with the advances
made in the field of molecular biology in the past ten years. The possibility for use ofPCR technology
in the field of population biology, taxonomy and for phylogenetic purposes is almost limitless (Reid,
1994). Despite the rapid development of molecular approaches, morphology has been and will
remain an essential component ofany taxonomic study of nematodes (Curran, 1992). Also, molecular
techniques such as those described and reliably distinguishing species of the generaSteinernema
and Heterorhabditis by Joyce et al. (1994) and Reid (1994) will be used more frequently in taxonomic
investigations.

13.2 Selection of Suitable Strain

Selection of an appropriate strain ofEPN with potential traits for biocontrol efficacy is of prime
importance. The importance of interspecific and intraspecific variations has been recognized and
demonstrated for insect parasitic nematodes (Bedding et aI, 1983). Genotypic characterization of
isolates will allow the assessment of genetic diversity within the species and populations and form a
basis for the co llection, maintenance and selection of insect parasitic nematodes (Curran and Webster,
1989). Diversity within has been analyzed morphologically (poinar, 1986) and with isozyme analysis
(Akhurst, 1987). Genetic manipulation has been used for producing nematodes with desired traits.

13.3 Characterization of Symbiotic Bacteria

An appropriate characterization of symbiotic bacteria is essential.

13.4 Issues Involved in Mass Production

Steinemematid and heterorhabditid EPNs products are available world-wide for control of
insect pests largely due to the recent progress made in mass production, formulations and field efficacy.
The exemption of the nematodes from registration and ability to infect and reproduce on a broad
spectrum of insects, and safety to non-target. They may be reared in vivo in the lab. G. mellonella
is the most used host all over the world as it can be easily reared, very susceptible and an excellent
host for nematode reproduction.

13.4.1 In vivo Culturing. Axenic culture process for S. glaseri and S. carpocapsae was developed
by Glaser (1940). Subsequently beneficial effect of hemoglobin and cholesterol in a yeast and peptone-
based medium, which can be autoclaved was reported (Rothstein, 1974). Vanfleteren (1976)
developed an inexpensive medium for C. elegans and for the first time reported results of high yield
of nematodes in 10 liters fermentor. Aeration was performed by bubbling and slow stirring assisted
suspension. The symbiotic bacterium grows on a number of proteins (animal and plant) and the
nematode is fed on the bacterium along with other nutrients in the medi~. Production on a commercial
scale had been accomplished by using inexpensive protein and sterol sources. An inexpensive
monoxenic, 3 dimensional culture matrix using animal byproducts was developed (Bedding, 1984).
This method of nematode production is applicable for cottage industries or for scale up by biotechnology
companies. Steinemematids and heterorhabditids also had been produced monoxenically in large
liquid fermentors using a combination ofplant and animal proteins (Friedman, 1990).
The morphology, virulence, bacteria carrying and host searching ability were compared in S.
carpocapsae and S. anomali cultured in chicken broth and larvae of G. mellonella. Reproduction
of S. anomali was 3-5 times greater and its virulence was 3 times greater when cultured in larvae of
the pyralid compared with those cultured in chicken broth ( Xu and Xie, 1992).

280
 In the past, steinernematids and heterorhabditis have been cultured on a variety of substances;
Potato mash (MacCoy and Glaser, 1936), ground veal pulp (MacCoy and Girth, 1938), peptone-
glucose agar and pork kidney (Dutky et ai., 1964), homogenized animal tissue (Bedding, 1976), dog
food (House et ai., 1965; Haraet ai., 1981), chicken offal homogenate (Bedding, 1984)(Table 7).
In solid culture ofEPN, inoculum size is important for optimizing the final yield ofIjs. Heterorhabditids
demand 1O-fold increase in inoculum size when compared to steinemematids to get higher yield. The
optimum inoculum size is 107IJ slflask and 10 6 IJ slflask for heterorhabditids and steinernematids
respectively. Reproduction and population development depends on inoculum size. Investigation on
this relationship between inoculum size, population development and final Us populations should
improve the efficiency of commercial nematode production.
Monitoring of quality prior to production (diet and inoculum quality), during production and of
the final product by bioassay technique are important steps to be followed during mass production of
entomopathogenic nematodes. Another constraint in solid culture is establishment of monoxenic in
vivo cultures ofEPNs. It can be achieved by scaling up by surface sterilization of nematode egg or
by alkaline lysis of gravid females, which is a laborious process, to obtain bacteria-free stage nematode
Us.
Growth temperatures also influence the infectivity of Us produced by mass production.
Temperature of25-30 °C is recommended for Steinernema culture as more lipids are accumulated
at this range than lower temperature which improve the physiological quality of Us .The physiological
quality of in vitro products may be adversely affected by culture conditions that bear little relationship
to the natural environment (Womersley, 1993). Sub-optimal medium composition produce inferior
bacteria resulting in poor quality nematodes. Media supplemented with extractable insect lipids produce
yields 1.9 X higher than did beef fat and lard supplemented media. Moreover the developmental rate
was 1.7X faster. Hence addition of fatty acid mixtures that resemble natural host lipids wrere
recommended ( Abu Hatab and Gaugler, 2001); this could provide nematode quality similar to in
vivo produced nematodes.

13.4.2 Liquid Culture. Thus, mass production of EPNs has been evolved from the first large scale
in vitro solid media production by Glaser et at (1940), to the in vitro production by Dutky et at
(1964), to 3D solid media in vitro process (Bedding, 1981, 1984) and to the in vitro liquid
fermentation production method (Friedman, 1990). Currently, nematodes are produced monoxenically
using the solid media process developed by Bedding (1981, 1984) or the liquid-fermentation method.
The solid media process has successfully produced pathogenic steinemematids and heterorhabditids,
but the high labour costs limit economies of scale. This technology is most adaptable for countries
where labour costs limit economies of scale. This technology is most adaptable for countries where
labour costs are minimal (Bedding, 1990). The liquid fermentation process, highly efficient for several
steinernematid species but not for heterorhabditids (Gaugler and Georgis, 1991) is economical for
industrialized countries. The latter process is highly efficient for several steinernematid species and
although heterorhabditids can also be produced in liquid fermentors, their quality, thus far, is poor in
comparison to steinernematids.
The cost of mass production using these methods is a major constraint for nematode
commercialization. Adverse effect of increased medium depth and poorly mixed media indicated
that oxygen transfer was a potential limiting factor in large volume cultures. Of particular concern
was the possibility that the agitation resulting from adequate aeration oflarge volume cultures would
damage the nematodes or prevent them from mating. Nematode reproduction occurred in both
rotary shaker and vigorously air sponged systems and the nematodes appeared normal (Beucher and
Popiel, 1989). Major problems related to EPN liquid culture mass production remain unsolved
(Ehlers, 2001). Physiological parameters that cause one dauer juvenile (OJ) to respond to the bacterial
food signal and another to remain in the DJ state remain unknown. One other source for process
instability results from the phase transition ofthe bacteria. Both fields deserve further investigation in
order to enhance process stability and yields.

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 Table 7. Synthetic media for mass rearing ofEntomopathogenic nematodes.

Synthetic medium Nematode species Incubation Temp.at Nematodes Reference

period harvest
Wheat bran S fe/tiae 3 weeks 25°C IQ4 Abe (1987)
Wheat bran + Salad oil S fe/tiae 3 weeks 25°C 10' Abe (1987)
Dog food agar medium S fe/tiae 10'/g Hara et al.
of medium (1981)
Beef extract, peptone, S fe/tiae Li(1984)
corn meal, water on sponge
Nutrient broth, yeast H. heliothidis 4 weeks 25°C IOxlO'1250 Wouts
extract, veg oil, flour coated ml flask (1981)
on spone
Soyapeptone 3%+ Yeast S g/aseri 1O,0511week Tarakanov
extract 3%+Chick embryo for 93 days (1980)
extract 10% medium
Kidney/fat homogenate S fe/tiae 2-3 weeks 25°C 38x106/30 flask
(1981) S bibionis 29x 106n3 flask
Sg/aseri 8x106/11 flask
H. bacteriophora 36x 106/10 flask
H. he/iothidis (NZ) 32xl0 61l5 flask
Modified dogfood medium S carpocapsae 6.6 J 64.66 Hussaini
Stami 20.45 et a/2000 f
S carpocapsae J3. J 32.69
S carpocapsae6. JJ 5.69
Modified egg yolk S carpocapsae 6.6 J 42.63
S tami 69.70
S carpocapsae J3. J 27.72
Wouts medium S carpocapsae 6.6 J 32.82
Stami 38.76
S carpocapsae J3. J 31.49
S carpocapsae 6. II 26.98
Modified wheat flour S carpocapsae 6.6 J 15.46
Stami 11.39
S carpocapsae J3. J 13.56
Egg yolk I S carpocapsae 6.6 J 22.41
Stami 58.29
Scarpocapsae J3. J 20.67
Eggyolk2 S carpocapsae 6.6 J 17.98
Stami 29.87
S carpocapsae 6. II 7.88
Dog biscuit+bacteria Steinernema sp. 30 days 24"C 1O.14x 10' Hussaini
et a/., 2000 g
Dog biscuit + peptone (SSL2) PDBC EN 12.2x 10'
13.21
Dog biscuit + beef extract 18.4x 10'
Dog biscuit+peptone+ beef 24.5 x 10'
extract
Wouts'medium 30.6x 10'

There are 20 nematode producing companies worldwide, half are in Europe and the US., and
the majority of the others in Asia. In the US, nematode production is carried out primarily at the
cottage industry level (less than 5 employee) and continues to be based on in vivo culture methods
(Gaugler et ai., 2000). Most of these companies base their production on G. mellonella, which is
available commercially and is available in large quantities. Most of the smaller companies do not
have expertise and capital to invest in liquid fermentation. Currently there are only two large companies
(Thermotrilogy, Ecogen) in US who are using large and expensive industrial liquid fermentation

282
technology. Insecticidal nematodes have been commercial products for twenty years, however the
competition with chemical pesticides remains fierce. Two reasons are: Non - competitive costs
compared with chemical pesticides and concerns of inconsistent nematode quality. End users will
not adopt biological control agents that do not provide efficacy. Advances in nematode application
timing and delivery, and particularly in selecting optimal target habitats and pests, have narrowed the
efficacy gap between chemical and nematode agents. Proposed changes in nematode production
and marketing strategies will allow nematodes to compete with chemicals on a more even footing.
Biologicals like nematodes are living organisms requiring special needs and possess limited shelf
life. Whereas chemicals are cheap to produce, stable i.e., have a long shelflife and are easy to use
and scale up. Small companies producing nematodes in customized batches with quick tum over for
a localized clientele can overcome the problems of biologicals. Companies such as these will be able
to deliver a fresh product that uses local nematodes to control local pests.
Gaugler et al. (2000) conducted a detailed analysis of Quality Assessment of Commercially
produced EPN s in USA. S. carpocapsae and H bacteriophora remain the most widely available
and used species. These two nematode species were selected for the evaluation. EPNs were ordered
from Thermo Trilogy, Integrated BioControl Systems (IBCS), Biologic, Green Spot, Hydro-gardens,
and M&R Durango. Packaging method and condition, arrival time, formulation, labels, and instructions
for use, and handling, coolant temperature, and cost were recorded. Scores were composite
judgements reflecting multiple, evenly weighted criteria. Service ratings were made separately based
on accessibility, knowledgeable staff, Order accuracy, and timeliness of delivery. Packaging was
assessed based on sturdiness, insulation capability. Taxonomic identification based on standard
descriptions, viability of infective juveniles, and nematode pathogenicity was assessed in lab. Results
showed that ease of accessibility was excellent, order accuracy was an issue with some, delivery
timeliness varied, most recommendations provided by the companies were highly accurate. Application
rate recommendations were occasionally dubious ;great disparity in cost was noticed. Product purity
was an issue with two firms. It is believed that 9 out of the 10 products evaluated were manufactured
using in vivo technology. The most discouraging aspect was the prevalence oflower than expected
nematode numbers in many shipments. The current paradigm for commercializing biopesticides is
based on an inflexible and unimaginative chemical model as it emphasizes major crops and is based
on cheap, stable products that are easy to scale up and use. As biocontrol agents are none of the
above, and therefore fit the model poorly. Reliable shelflife for a few weeks falls well short of the
conventional chemical standard of two years, necessitating special handling and distribution, and
contributing to inconsistent performance. Strains efficacious when freshly produced and applied by
researchers frequently perform poorly when tested on an industrial scale. Most troubling, commercial
nematode products too frequently contain mixed species, modest pathogenicity, inaccurate
numbers, or are unavailable.

14. PRODUCT DEVELOPMENT AND ECONOMICS

Biological pesticide must be able to compete in relative terms with chemical pesticide in field
efficacy, cost and application methods (Georgis, 1990b). The development of cost effective mass
production technology led to the availability of nematode products comparable with standard
insecticides in market. Quality control strategy has to ensure preservation of high nematode viability
and virulence during large-scale production and formulation. Factors affecting quality included provision
of essential nutrients and appropriate bacteria, delivery of oxygen, production and storage temperature
and contamination control (Grewal, 1996).

15. FORMULATION AND STORAGE

Formulation of nematode into a stable product has played a significant role in commercialization.
Active nematodes are immobilized to prevent depletion of their lipid and glycogen reserves.

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Immobilization has been accomplished by maintenance in aqueous suspension at low temperature
(5- I 5 DC). Steinemematids can be stored at 4-10 DC for 6-12 months without much loss of activity
whereas heterorhabditids do not store well and 2-4 months of storage at 4-10 DC is considered
good. A major constraint of commercialization of these nematodes is their poor storage capacity.
They can be stored under refrigeration in oxygenated water for up to 5 years (Friedman, 1990). But
this methodology is not commercially feasible. Nematode metabolism is temperature driven and
warm temperatures (20-30 DC) increase metabolic activities, reducing nematode viability (Georgis,
1990a). Subsequently, nematodes formulated in various carriers such as alginate, clay, vermiculite
and gel forming polyacrylamides can be stored for atleast 6 months under refrigeration and atleast 3
months at room temperature. Further advancement has been made with the development ofa flowable
concentrate. This technological advancement, if commercially successful, will revolutionize nematode
applications and eliminate many of the constraints associated with current formulation, namely the
dissolution of the carrier or separation of the nematodes from the carriers.
Formulation of nematodes is important and can affect handling, application, persistence and
stability in storage. Most common formulation is made in alginate, polyacrylamide gels, clay,
vermiculite, activated charcoal (Georgis, 1990b). Formulation stability is also achieved by partially
desiccating and immobilizing Ijs in specific carriers.
Compared to many biocontrol agents, nematodes as biopesticides have a good shelflife. Most
can be stored under refrigeration in aqueous suspension under adequate supply ofoxygen. Temperature
requirement depends on species. General range for steinernematids is 5-1 ODC and I 0-15 DC for
heterorhabditids (Georgis, 1990b). The introduction of commercial nematode formulations has
accelerated in recent years, but new formulation technology that produces stable, effective and
consistent products would expand markets even further (Georgis 1990a; Georgis and Hague, 1991).
Spray application using a nematode suspension in water is straightforward, inexpensive and often
effective. Novel alginate capsules containing nematodes (Kaya and Nelson, 1985; Kaya et aI.,
1987; Poinar et aI., 1985), wheat bran bait pellets (Capinera et aI., 1988) and alfalfa-wheat bait
pellets (Capinera and Hibberd, 1987) have been developed. Kaya and Nelson (1985) suggested
that encapsulation ofEPN with seeds has potential to protect roots from insect attack. Nematodes
escaped from the capsule within 7 days, as the seed germinated, it has an opening for the nematodes
to escape. Pesta, granular product with entrapped nematodes based on cohesive dough made
wheat flour, fillers and additives was developed by Connick et al. (1993).

'16. APPLICATION TECHNOLOGY

Many factors affect the ability to place quantities of nematodes on or in close proximity to the
target host to produce optimal results at the lowest possible cost. To overcome the impact of abiotic
and biotic factors on nematode efficacy and persistence, the inundative application ofhigh concentration
of a specific nematode species has been used as a primary control strategy. Application of nematodes
is mostly targeted to the soil and against cryptic habitats. Inundative and inoculative approaches have
been tried (parkman and Frank, 1992). In the field biotic factors like host susceptibility, pest population
levels, host behaviour, different nematode species and host cues affect the nematode behaviour.
Other than this susceptibility to environmental extremes reduces their field efficacy (Kaya and Gaugler,
1993). In soil application one or two pre-and post application irrigation have been tried to provide
enough moisture (Shetlar et al., 1988). As with chemical insecticides, spraying nematodes directly
onto the soil surface is the most commonly used application method. This broadcast method provides
good coverage and is simple and quick.
Timing soil applications ofnematodes with the life cycle oftarget insect is a key factor. Cabanillas
and Raulston (1996) found that the efficacy ofS. riobrave was higher on Helicoverpa zea in corn
when applied at 200,000 11fme through furrow irrigation or post irrigation than when nematodes
were sprayed on to the soil before irrigation. Thus S. rio brave was effective in suppressing corn

284
earwonn under field conditions of high temperature with irrigation. Presently they are being applied
through conventional equipment such as high volume sprayers, drip irrigation, food baits or through
sound trap (Georgis et a!., 1989; Parkman and Frank, 1992). Antidesiccants have been used
successfully to retard evaporation of the nematode suspension on foliage in foliar application of
EPNs, and thus reduce desiccation of nematodes (Welch and Briand, 1961; Webster and Bronskill,
1968; Shapiro et a!., 1985; Glazer and Navon, 1990). Glycerin 10% has been the most effective
adjuvant for increasing survival and activity of nematodes on foliage (Nash and Fox, 1969). However
with the high cost of glycerin and the risk of phytotoxicity at high temperatures, search for more
suitable adjuvant continued. Excellent control of Japanese beetle, Pjaponica and the northern
masked chaffer, Cyclocephala borealis was obtained with 1.25-5.0 billion Ijs/ha when plots were
irrigated before application and again within 24 h after treatment (Downing, 1994). Bauer et aI.,
(1997) studied the effect of a number of adjuvants on entomopathogenic nematodes persistence and
efficacy against Plutella xylostella. In laboratory, TX 7719, Rodspray oil, and Nufilm P provided
the best anti desiccant activity and in greenhouse, TX 7719 + Blankophor BBH increased nematode
persistence on waterstress leaves and efficacy against P xylostella significantly. Addition of
phagostimulants enhanced the effectiveness of S. carpocapsae against the 4th instar larvae of S.
litura feeding on sunflower head in lab and field tests (Sezhian et a!., 1996). EPNs are usually
applied to foliage using standard application mechanics and this leads to a very inefficient loading of
spray droplets and thus small numbers of nematodes reaching the target area. Considerable
improvements have been achieved through optimization of spray conditions using standard hydraulic
equipment and spinning disc (SD) sprayers, yet further modifications are required before nematode
application gives effective and consistent control oftarget insects. The need to reduce the quantity of
nematodes and carrier liquid is paramount ifEPNs are to compete directly with chemical control
methods. This can only be achieved ifthe quality of the spray produced is at an optimum for nematode
carriage. Here, an assessment is given for the droplets suitable for the application ofEPNs (carriage
droplets) and modifications to a SD apparatus that have led to an improvement in the spray quality of
nematodes. Not only has the loading of droplets been improved but there has also been an improvement
in the operation of the disc by the discovery and amelioration of a problem unique to this fonn of
apparatus and control agent, namely the clumping of the nematodes during emission (Piggott et aI.,
2000). Optical brighteners Ran HI,Ran 2B ,Ran Bvn, Ran MM, Ran S and PABA were found to be
good UV protectants for Steinernema (SSL2) PDBCEN 14.1,13.21 and H indica PEBCEN 14.3
and 13.22 (Hussaini et aI., 2001 d).

17. TRANSPORT

Steinemematid infectives are stored for extended periods (5 years) under cool, moist conditions
(Bedding, 1981). For industrial storage, the Ijs are placed onto clean, crumbed polyether-polyurethane
sponge @ 250-500 million per 1OOg of dry sponge and maintained at I-2°C in aerated polyurethane
tubes. During transportation, for trips less than 12 hrs, oxygen is added to the tubes before transport.
For longer transport the tubes are aerated with a battery operated air pump. These techniques result
in less than 10% mortality ofIjs in storage or transport. The heterorhabditid Us are best stored in
culture flask above 12°C. Transport is difficult and expensive due to additional weight ofthe culture
flask. During shipment, appropriate bacteria, delivery ofoxygen, production and storage temperature,
and contamination control are required. The quality of nematodes that survive the rigors of the
manufacturing process is analyzed by determining their shelflife and virulence potential. Nematode
shelflife is predicted from storage energy reserves (eg., dry weight, or total lipid content) ofIjs,
whereas virulence potential is measured using 1: 1 bioassay against wax moth.

18. INTEGRATION WITH OTHER ORGANISMS

In an IPM schedule integration ofEPN s has been reported. S. carpocapsae and NPV (SeM
NPV ) in combination produced additive effect against S. exigua in soybean than either of the

285
organisms alone ( Gothama et aI., 1995).Progeny ofSjeltiae contained virus particles in the lumen.
Kaya and Burlando (1989) found successful development and reproduction in 68% of moribund
larvae of beet armyworm. Competitive interaction between nematode and NPV in a single host may
not occur. S carpocapsae may infect SeNPV infected larva ,which is then unsuitable for the virus
development since the nematode development kill the larva in 1-2 days.
The reproductive success of S carpocapsae or H bacteriophora in soil borne larvae of S
exigua previously exposed to B. bassiana was assessed. Treatments with H bacteriophora and B.
bassiana resulted in higher total mortality of S exigua in soil than with either nematode or fungus
alone. For S carpocapsae plus B. bassiana, insect mortality was not significantly different compared
with S carpocapsae alone. In treatments with nematode and fungus, per cent mortality of S exigua
from S carpocapsae and H bacteriophora was reduced relative to treatment with nematodes alone.
Inundative release of EPN s where B. bassiana occurs, may result in greater control of soil borne
insect pests than application of nematodes alone( Barbercheck and Kaya 1991). Gill and Raupp
(1994) reported that bagworm, Thyridopteryx ephemeraeformis on Thuja occidentalis was
controlled using 2 formulations of neem, carbaryl, acephate, cyfluthrin, formulations ofS carpocapsae
and S. jeitiae, and Bacillus thuringiensis var. kurstaki. Neem gave the least control (36-56%
reduction). The nematodes, either alone or with oil or antidessicant, gave 91-100% control.The
synthetic pyrethroid cyfluthrin gave 100% control; carbaryl and acephate also gave acceptable control
(83 and 86%, respectively). The biological control agents, Bacillus and the nematodes, provided
intermediate levels of control. Both species of Steinernema were effective.
S feltiae and H heliothidis and the fungus B.bassiana possess broad and overlapping host
ranges. The impact of B. bassiana and nematodes on host period oflethal infection (PLI) and on
nematode and fungus progeny production in a single host was investigated. In general, the PLI for
larvae exposed to nematodes and B. bassiana simultaneously was shorter than that for larvae exposed
to either pathogen alone, or exposed to both sequentially. Even though infection with both pathogens
decreased host PLI, B. bassiana and nematodes rarely co-produced progeny in duly infected hosts.
In treatments with nematodes alone, or B. bassiana and nematodes applied at the same time or 1
day later, Xenorhabdus bacterium was observed in host haemolymph 12 h after the application of
the nematodes. Xenorhabdus or nematodes prevented or inhibited the growth of B. bassiana in the
insect if nematodes were applied within 24 h after application of B. bassiana. B. bassiana was
detrimental to the development ofS feltiae and H heliothidis when applied to the insect more than
48 h before nematodes. Temperature influenced the outcome of development in duly infected larvae.
In early sequential treatments B. bassiana was more likely to develop to the exclusion of nematodes
at 15°C, while nematodes were more likely to develop in these treatments at 22 and 30°C. It is
concluded that, even though dual infections can result in a decreased PLI compared to singly infected
hosts, antagonistic interactions between B. bassiana and EPN in duly infected hosts can adversely
affect pathogen development and progeny production (Barbercheck and Kaya, 1990).
Cypermethrin, B. thuringiensis subsp. kurstaki, S feltiae, NPV, and B. bassiana were evaluated
against H zea in the laboratory and greenhouse. In the lab, cypermethrin and B. thuringiensis were
significantly different from control initially despite concentrations. Sfeltiae showed a significant higher
mortality rate than the control later. The virus was highly effective between 2 and 7 days a:fiertreatment,
killing all larvae. In the greenhouse, cypermethrin was significantly more effective than no treatment
(Carrano-Moreira and All, 1995). Pasteuria penetrans, a bacterium parasitic on juvenile and
females of plant parasitic nematodes particularly Meloidogyne spp. is often used a a biocontrol agent
by soil application. The infectivity of an Indian isolate of Ppenetrans (PMI-I) to isolates of the
EPN, Steinernema sp. and Heterorhabditis sp. was tested in aqueous suspensions (Somasekhar
and Mehta, 2000). Spores of P penetrans did not attach to any of the EPNs even after 72 hours of
exposure at 25°C, while in the case of Me 10 idogyne incognita, from which the bacterium was
originally isolated, spore attachment was observed after 24 hours of exposure. These results indicate
that P pene/rans, a parasite of phytonematodes, does not adversely affect entomopathogenic
nematodes.

286
19. A PARADIGM FOR BIOLOGICALS

A paradigm shift is needed for the next century, one that is based on the realities of biological
systems(Gaugler 1997). Facets like industry, growers/extension, and researchers should be
considered. A new paradigm for the biopesticide industry might emulate business sectors that have
successfully overcome biological limitations; for example, the dairy industry has demonstrated that
low stability need not be a fatal flaw. Similarly, the micro brewery industry has demonstrated that
there is a strong market for fresh "product" sold without preservatives. The key has been local, small
- batch, custom production coupled with high quality and quick tum over. This concept can be
adapted to biological pesticides. It is desirable to develop local production technology for insecticidal
nematodes as more than 70% of the expense concerns formulation, storage, transport, waste disposal,
middlemen, and capital. Local production using in vivo methods eliminates these steps, providing
improved costs. Local production would imply that only "fresh" biologicals would be applied, providing
improved performance; and the primary outlet could be the existing cottage industry.
Conservation of natural populations in agro-ecosystems or the fate ofapplications has received
almost no attention. The few attempts at inoculative releases have met with some success but this
strategy may be inappropriate for some situations. Determination of whether to use inundative or
inoculative release strategies need to be made on case to case basis but pests ofhigh value commodities
with low economic thresholds will usually be relegated to inundative efforts. Biological methods of
conservation are designed to protect and maintain natural or introduced enemies of pests. With
EPNs, these can include application practices that will favour survival and lor establishment of
introduced individuals. It may also involve crop management practices that have a positive effect on
existing natural populations .The use ofEPNs has overwhelmingly involved inundative releases yet
the origins and future ofthe use ofEPNs are in inoculative and conservation biological control.

20. DISCUSSION

The future ofEPNs in this country is bright. Most of the pesticides have proved a liability in one
form or other. A number ofEPNs species are now known to occur naturally particularly species like
Scarpocapsae that is more versatile and most widely used worldwide is present in the country.
Besides India being a megadi versity country many more species could be detected through intense
efforts.
The quality of commercial nematode products is critical ifEPNs are to realize their full potential
as biological insecticides. The quality of nematodes produced -in vivo, in vitro, solid or liquid diets-
varies. Hence a suitable mass production method should be selected depending on the species. A
shift in paradigm is required that suits our need. Not mere numbers but quality of nematodes produced
with suitable fitness attributes is of prime importance. The concept 'one size fits all' is outdated and is
not applicable to EPN s. It is also worth considering whether anything less than perfect produce is
permissible. This transition requires growers having better education about biological control
technologies. The current paradigm for commercializing biopesticides is based on an inflexible and
unimaginati ve chemical model. The paradigm emphasizes major crops and is based on cheap, stable
products that are easy to scale up and use. Biocontrol agents are none of the above, and therefore fit
the model poorly.
A paradigm shift is needed for the next century, one that is based on the realities of biological
systems. Considering facets like Industry, growers/extension and researchers.
Most nematode cultures have focused on growth parameters such as temperature, and inoculum
size. In vitro medium has emphasized on yield and medium cost with little emphasis on nematode
physiological and biochemical quality. In vitro cultured nematodes could be adversely affected by
culture conditions that have little relationship to natural environment. Little consideration has been
given to this problem even though it may explain quality differences between in vitro and in vivo

287
products. Inappropriate medium composition would produce inferior quality nematodes with low
pathogenicity, or low storage ability. Aseptic conditions during the production cycle on artificial
media is very important for getting predicted yields.
Expanded use ofEPNs in biological control cannot be expected unless their field efficacy is
increased. Matching nematode species might bridge the efficacy gap between the nematodes and
chemicals and strains against those insects they are best adapted. Attention should be given to protecting
the genetic variability of new isolates, preventing the loss of alleles through laboratory adaptation.
Prediction models may have to be developed so that nematodes could be used as and where they are
likely to be effective. Distinction to be made between 'lab adapted' and 'field adapted' populations.
Simulation models can improve insight in the ecology ofEPNs and their potential as biocontrol
agents of soil pests. Models that include environmental and behavioural factors that are critical for the
efficacy of an inundative release, may be used for guiding application strategies, EPNs biocontrol-
product formulation and genetic engineering ofEPNs. Multi-generation population dynamical models
provide a tool for estimating persistence, thus allowing assessment of risks associated with releasing
genetically modified EPNs into the environment. Constructing a simulation model for one or more
species ofEPNs requires the retrieval and integration ofexisting knowledge ofthe quantitative ecology
and behaviour. Additional process-oriented research must be carried out to fill gaps in the available
knowledge. The validation ofsimulation models will involve experiments under field conditions (Werf
et ai., 2000).

21. CONCLUSION

It may be inferred from the above discussion that EPNs are potential biocontrol agents amenable
for mass production, handling and application on a large scale in the field for control of insect pests.
Indigenous isolates identified in the country could be utilized in different ecosystems. For expanded
use, there is a need to exploit the potential biodiversity in the country by continued surveys. Mixing
of nematodes collected from diverse habitats in the lab to produce a 'supernematode' may have to
be attempted. Conservation ofthe original true population with deterioration of any genetic trait is
important.
Biological control is essentially an ecological understanding closely associated with the theory,
experimentation and modeling ofvarious aspects ofecology especially foraging ecology and population
biology. Understanding EPNs' soil ecology is fimdamental to their effective use as biocontrol agents At
this moment, EPNs are stepping out of laboratory; the potential markets will only demand nematode
products when these are available at lower price. Although the price has been cut by halffollowing
the introduction ofliquid culture technology, it is still considerably too high to permit any application
on low-value crops. The continous scale-up of bioreactor volumes will bring further reduction in the
production cost. However, this development must be accompanied by furhter progress in improving
process stability and downstream processing in measures to extend EPN shelflife and in improving
transport 10gistics.Ifthis can be achieved, EPNs will further substitute insecticides (Ehlers, 200 I).

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GENETIC IMPROVEMENT OF ENTOMOPATHOGENIC NEMATODES FOR INSECT
BIOCONTROL

Christopher W. Breyl, and Sarwar Hashmi 2

I Department of Entomology, Rutgers University, New Brunswick, New Jersey, USA;

lLindsley F. Kimble Research Institute, New York Blood Center, New York, USA.

1. INTRODUCTION

Entomopathogenic nematodes (families Heterorhabditidae and Steinemematidae) are the most

important biological-control agents for soil-dwelling insect pests (Gaugler and Kaya, 1990). Several
species ofHeterorhabditids and Steinemematids are produced commercially and used as biotic
insecticides. Their unique association with symbiotic bacteria (Xenorhabdus for Steinemematidae
and Photorhabdus for Heterorhabditidae) enables them to rival the efficacy ofchemical insecticides.
Other key attributes include mass production, broad host range, and capacity to kill a host within 1 to
4 days. Furthermore, entomopathogenic nematodes are not pathogenic to plants or mammals, and
have therefore been exempt from government regulation and registration requirements (Kaya and
Gaugler, 1993). Nevertheless, there are ongoing efforts to improve entomopathogenic nematode
beneficial traits or eliminate weaknesses by means of genetic manipulation in the areas of increased
environmental tolerance, target specificity, enhanced host finding, mass production (Bedding, 1984),
and increased storage-life (Burnell and Dowds, 1996). Strain improvement using selection (screening
a natural population), selective breeding, hybridization, and mutagenesis has been successful in many
entomopathogenic nematode laboratories worldwide. Also, genetic engineering has shown some
success, but it has not been widely utilized in strain improvement programs.
Entomopathogenic nematodes belong to the same order Rhabditida as the free-living species
Caenorhabditis elegans. Caenorhabditis elegans is the best understood metazoan organism known
for studying molecular and classical genetics of higher eukaryotic animals (Brenner, 1974; Wood,
1988). The genetic and physical maps of its six chromosomes have been constructed and the entire
genome sequence containing 19,000 predicted genes has been determined (The C. elegans Sequencing
Consortium, 1998). Moreover, C. elegans researchers have developed molecular tools that have
advanced our understanding of how genetic information specifies the development, anatomy, and
behavior of this simple animal (Riddle et aI., 1997). In theory, molecular techniques and sequence
information developed by the C. elegans group could be with minimal effort transferred to the
entomopathogenic nematode research community. However, few researchers have utilized this rich
genetic resource. In this chapter we review the current state ofentomopathogenic nematode classical
and molecular genetics. In addition, we discuss potential C. e/egans research methodologies that
may be applied to further advance entomopathogenic nematode genetic strain improvement.

Advances in Microbial Control of Insect Pests

Edited by Rajeev K Upadhyay. Kluwer Academic / Plenum Publishers. New York. 2002 297
2. BIOLOGY AND LIFE-CYCLE

Entomopathogenic nematodes are obligate soil-dwelling rhabditids capable of infecting a broad

range of insect pests. The only stage that can survive outside of a host is the non-feeding third stage
infective juvenile or dauer juvenile. The infective juvenile has adapted to survive in harsh soil
environments, locate and infect a suitable host through natural openings (mouth, spiracles, anus)
before developing into adult male, female or hermaphrodite (Figure 1). The infective juveniles are
closely associated with their symbiotic bacterium which when released kills their host within 1-4 days
after infection (Thomas and Poinar, 1979). Inside a host, a Heterorhabditis infective juvenile feeds
on Photorhabdus bacteria and host tissues prior to maturing into a self-fertilizing hermaphroditic
female, which latter give rise to one or more generations of amphimictic males and females and
infective juveniles (Strauch et aI., 1994). At the end of second and third generation or as nutritive
conditions are depleted the infective juveniles emerge from the host's cadaver (Johnigk and Ehlers,
1999). In contrast, Steinernema juveniles feed onXenorhabdus bacteria and host tissues before
maturing into amphimictic females and males. After several life-cycles, a new generation of infective
juveniles emerge and begin to search for a new host.
Similar to its insect-parasitic counterpart Heterorhabditis, C. elegans, a free-living, bacteriovore
nematode is found in soils and undergoes hermaphroditic reproduction. In the laboratory it has a
rapid developmental growth potential and can develop from egg to adult within 36 h at 25°C. This
rapid reproductive strategy leads to overcrowding and habitat depletion. Under these conditions the
non-developing dauer stage, similar to the third-stage infective juvenile ofentomopathogenic nematode
is formed (Riddle et aI., 1997). Dauer larvae can survive for many months without food, and when
they encounter food they resume deVelopment to become self-fertile female hermaphrodites. The
evolutionary success of this stage in both free-living and insect-parasitic species is for dispersal to
more abundant nutrient rich habitats since a single hermaphrodite can establish a new popUlation.

3. GENETIC IMPROVEMENT

Strategies of genetic improvement include (i) screening for desired traits (natural population),
(ii) hybridization, (iii) selective breeding, (iv) mutagenesis, and (v) molecular genetics: recombinant
DNA technology (Gaugler, 1987). Screening natural entomopathogenic nematode populations for
desirable biological traits, while very laborious, is feasible and has shown some success in isolating
heat and cold tolerant strains (Glazer et a!., 1996; Mracek et al., 1998). As demonstrated for heat
tolerance, once a desired trait has been isolated cross hybridization with a commercial strain may be
performed (Shapiro et al., 1997). Because desired traits are likely to result from the interaction of
many genes, an effective method for genetically improving such traits is by selective breeding. Examples
of successful selective breeding include host finding (Gaugler et aI., 1989; Gaugler and Campbell,
1991), host pathogenicity (Peters et aI., 1998), efficacy (Tomalak, 1994a; Grewal et aI., 1993), cold
and heat tolerance (Grewal et aI., 1996a; Shapiro et al., 1997; Mracek et aI., 1998), and nematicide
resistance (Glazer et al., 1997). In situations where a few regulatory genes may control a desired
trait, mutagenesis maybe appropiate. Mutagenesis has been successful in the isolation of strains with
increased desiccation tolerance (O'Leary and Burnell, 1997). Recently, with the explosion of
knowledge and technology in molecular biology generated by studies conducted on C. elegans. the
improvement ofentomopathogenic nematodes by genetic engineering may have the greatest promise.

3.1 Natural and Artificial Selection

Most beneficial traits are polygenetically inherited, being controlled by many segregating loci
each with only a small additive effect on the phenotype. For these traits natural and selective breeding
are two processes most conducive to genetic improvement. For examples ofnatural selection, several

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entomopathogenic nematode strains endemic to cold and hot climates have been isolated and targeted
in genetic improvement programs (Glazer et aI., 1996; Mracek et aI., 1998).

Us penetie into host 3'1

Y!trI-~

IJ host finding:
ambush,
intermediate, Steinernema Heterorhabditis
or cruise (amphimictic) (hermaphrodite)
foraging

Reproduction and development

(0-2 additional amphimictic generations)

IJ production and emergence

Figure l. Life-cycle of entomopathogenic nematodes (source: Koppenhtifer and Kaya, 2001). Ambushers are sit-
and-wait strategists e.g., Steinernema, and cruisers are highly active species e.g., Heterorhabditis that seek out
a suitable host.

A successful selective breeding program must have for a desired trait either a moderately high
heritability value (f12 > OJ) or a very large genetically diverse base population from which to select

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(Glazer et aI., 1997). A phenotypic trait with a low heritability value has only a slight phenotypic
difference within an ecologically diverse (presume, genetically diverse) population and thus would be
impractical for selective breeding. A phenotype with a high heritability value has a wide variation
between population isolates and thus would be more responsive to selective breeding (Gaugler et al.,
1989). Furthermore, if there are substantial differences between phenotypes, the desired trait may
already be in one of the population isolates and thus inbreeding would be a method for stabilizing the
phenotype (Gaugler et aI., 1989). Selective breeding for strain enhancement of Steinernema spp.
are routinely performed by the hanging drop method (Poinar, 1967) or by injection of infective
juveniles into a living Galleria mellonella host (Akhurst and Bedding, 1978). For Heterorhabditis
spp., selective breeding is conducted by mating second generation amphimictic females and males on
an agar plate or in Photorhabdus treated G. mellonella cadavers (Dix et aI., 1992).
Artificial selection for enhancement of entomopathogenic nematode host-finding and infectivity
has been demonstrated in several selection programs. Despite an increase in the desired trait when
selected under laboratory conditions, these improvements did not always translate to increased field
efficacy. For example, Gaugler et al. (1991) achieved a 72-fold increase in host-finding ability of a
strain of S. carpocapsae (previously selected against G. mellonella) against the Japanese beetle
Popillia japonica, but when assayed in the field and laboratory no difference in infectivity was
detected when compared to the wild-type strain (Gaugler et aI., 1994). Gaugler et al. (1991) attributed
improved host-finding to the increased sensitivity of infective juveniles to CO2 expelled by the target
insect. In the case of Japanese beetle larvae, the nematode orientates towards the host spiracles, but
the spiracles are covered with sieve plates and thus prohibit nematode entry (Gaugler et aI., 1994).
As Gaugler et al. (1994) points out when selecting an entomopathogenic nematode for a specific
desired trait begin first with a species suited for the target host. Tomalak (1994a) used artificial
selection under natural conditions to enhance the infectivity of Steinernemafoltiae against the sciarid
fly Lycoriella mali, in mushrooms. Artificial selection has also been successful in selecting
entomopathogenic nematode for increased infectivity at low temperatures (Griffm and Downes, 1994;
Grewal et aI., 1996a), enhanced heat tolerance (Grewal et aI., 1996b), and increased nematicide
resistance (Glazer et aI., 1997).
Challenges to selective breeding are (i) the selected strain may become laboratory adapted, (ii)
relaxed selection may lead to loss of a field adapted trait, and (iii) an overall fitness cost to the animal.
Several ways to protect a selected strain are to cryopreserve a subsample of the isolate in liquid N2
(Nugent and Burnell, 1994; Popiel and Vasquez, 1991) and reapply selection pressure at regular
intervals.

3.2 Hybridization

The transfer of beneficial genes by hybridization can provide a powerful approach for genetic
improvement ofentomopathogenic nematodes. Once beneficial traits are found in a natural population,
and shown to be heritable, those traits can be combined through hybridization to produce superior
strains (Shapiro et aI., 1997). A trait for heat tolerance was transferred from H. bacteriophora
strain designated ISS discovered in the Negev Desert in Israel (Glazer et al., 1996) to the wild-type
H. bacteriophora HP88 strain. The hybrid progeny were confirmed by use of a marker mutant
isolate (Hp-dpy-2) and by backcrossing. More importantly there was no loss of fitness in the hybrid
progeny relative to the parental strain (Shapiro et aI., 1997).

3.3 Ethyl Methansulfonate Mutagenesis (EMS)

The isolation ofmutant nematodes with distinct morphological characteristics can serve as genetic
markers for the analysis of mutants displaying desired traits or for the mapping of beneficial genes
(Koltai et aI., 1994). Heterorhabditis bacteriophora is especially suited for mutagenesis because it
has a short generation time and is a self-reproducing hermaphrodite (Glazer et aI., 1991; Zioni et

300
aI., 1991). Homozygous development from a single parent allows pure lines to be obtained and
maintained easily, and the appreciable ma1e population of subsequent generations provides the vehicle
for the exchange of genetic material between individuals (Zioni et aI., 1991). The C. elegans
mutagenesis protocol (Brenner, 1974) using ethyl methanesulfonate (EMS) has been routinely used
in the development of entomopathogenic mutants (Zoni et a1., 1992; Rahimi et a1., 1993; Tomalak,
1994b; Koltai et aI., 1994).
Mutagenesis induced by exposing young H. bacteriophora hermaphrodites to EMS has been
documented (Koltai et aI., 1994). Three dumpy mutant strains were isolated from a mutagenized
population and shown to be recessive when back crossed to wild-type males. Complementation
tests between the three mutants gave F1 offspring of both wild-type and dumpy phenotypes indicating
that these recessive mutations affect different genes. The ratio of dumpy to wild-type phenotypes in
the F2 generation was approximately 1.0 suggesting that the three genes were also linked (Koltai et
aI., 1994). A similar mutagenesis study was conducted on S.feltiae by Tomalak (1999), who
described 19 different mutations representing nine independent genes. Although, none ofthe isolated
mutants were any more effective as a biologica1 control agent than the wild-type strain. Nonetheless,
the broad range of mutant phenotypes provided excellent comparative materia1 for the study of
morophological and behavioral characters associated with entomopathogenic nematode activity.
Desiccation tolerant mutants ofHeterorhabditis megidis have a1so been isolated by EMS mutagenesis
(O'Leary and Burnell, 1997). Overall the use of mutagenesis has been underexploited in EPNs
entomopathogenic nematode (Burnell et aI., 1998). However, the enormous amount of information
available on mutations and genes in C. elegans should be invaluable for studying corresponding
mutations and genes in entomopathogenic nematodes (Glazer et a1., 1994).

3.4 Genetic Transformation

Genetic engineering ofentomopathogenic nematodes offers a significant advantage over selective

breeding and mutagenesis, making it possible to produce small defined beneficial changes in the
genotype (Gaugler and Hashmi, 1996). Microinjection of foreign DNA into the gonad of a young
adult female or a hermaphrodite is a popular method of gene transfer for both C. elegans and
entomopathogenic nematodes (Hashmi et a!., 1999; Vellai et aI., 1999; Stinchomb et a!., 1985). A
second less common helminth transformation procedure is via ba1listic DNA bombardment (Davis et
aI., 1999). This technique was originally for plant cell transformation. In the more common
rnicroinjection technique, the injected DNA may be maintained by either integrating into the genome
or as an extrachromosomal array or it may be transiently expressed for just one or two generations.
Recombinant DNA is rarely integrated into the genome, but rather is maintained as a concatamer of
the exogenous sequence. Stinchcomb et al. (1985) were the first to describe the successful
transformation ofC. elegans, and Hashrni et a1. (1995) were frrst to report the successful transformation
of H. bacteriophora. Vellai et al. (1999) have recently deVeloped a genetic transformation system
for S.feltiae.
Hashmi et a!. (1998) transferred a heat-inducible C. elegans heat-shock gene, hsp 70 A, into
H. bacteriophora HP88. The 70-kDa heat shock protein encoded by the gene enables cells to
eliminate or renature proteins damaged by high temperatures. Overexpression of the gene results in
an enhanced thermotolerance in transgenic nematodes. In addition, no developmental or growth
differences were observed between transgenic and wild-type nematodes (Hashmi et a!., 1998).
Vellai et al. (1999) transformed S.feltiae with the yeast trehalose-6-phosphate synthase gene
(tps-I). Treha1ose-6-phosphate is a key enzyme involved in the biosynthesis oftreha1ose, a soluble,
non-reducing disaccharide of glucose that accumulates to high levels in organisms that natura1ly survive
dehydration. This common osmoprotectant molecule has specific properties that protect membranes
and proteins when in response to desiccation and other stresses (Crowe and Crowe, 1992).
Desiccation and osmotic tolerance was enhanced in adult S. feltiae nematodes transformed using a
heat-inducible promoter derived from C. elegans and the tsp-l gene (Vellai et aI., 1999).

301
 The short-term goal of genetic transformation of entomopathogenic nematodes is to develop
strains with enhanced traits for biological control. An lli1derstanding ofthe molecular determinants of
stress resistance should accelerate the evaluation and the transformation of other beneficial genes.
Moreover, in the future, genetic transformation will be useful to investigate expression and regulation
of other advantageous genes in entomopathogenic nematodes at the molecular level.

3.4.1 Selectable Markers. The two initial transformations of entomopathogenic nematodes utilized
enzymatic selection to differentiate transformed and nontransformed nematodes. The Escherichia
coli Lac Z encoded enzyme 13-galactosidase (13-gal) was used as the reporter molecule to transfom
nematodes (Vellai et aI., 1999; Hashmi et aI., 1995). However, 13-gal requires a lengthy staining procedure,
which kills the organism (Sihavy and Beckwith, 1985). Thus, more efficient transformation systems
with better selectable markers are being investigated. A potential selectable marker that has been used
extensively in C. elegans is the green fluorescent protein (GFP) of the jellyfish Aequora victoria.
The green fluorescent protein does not require other proteins or cofactors for expression, so
genes can be expressed and monitored in virtually any living cell as a fluorescent product (Chalfie et
aI., 1994). Under the control of a mec-4 promoter Hashmi et al. (1997) observed a readily detected
green fluorescence in the touch receptor neurons in the tail region of yOlli1g adult H bacteriophora.
However, the fluorescence was not observed in second or third-stage juveniles. Hashmi et al. (1997)
suggested that the lack of fluorescence was probably because the cells were not fully developed or
the cells were difficult to detect at this early stage. Although successful introduction ofgfp expression
in entomopathogenic nematodes has been attained, additional gfp constructs with promoters that
express in different cells and throughout the life-cycle of the nematode are needed.
Behavioral markers are routinely used to identifY C. elegans transformants. The Twitcher marker
produces an antisense sequence complimentary to a portion of the C. elegans unc-22 gene, causing
the nematodes to twitch (Moerman et aI., 1988). Roller, rol-6 (sui 006), a dominant allele, causes
transformed C. elegans to roll along its longitudinal axis, resulting in a circular motion (Kramer et al.,
1990). Attempts at transforming H bacteriophora and S. feltiae with the roller marker have so far
been unsucccssful (Hashmi et aI., 1995;Vellai et aI., 1999). Hashmi et al. (1995) did observe the
presence of the rol-6 gene in the progeny by Southern analysis, but they did not observe the roller
behavior. They suggest that the rol-6 gene may not interact with the basement membrane of H
bacteriophora in the same manner as it does in C. elegans.

3.4.2 Potential Genes. Identification of genes that encode useful traits (improve biological control
potential) for genetic engineering of entomopathogenic nematodes is a major area of research. The
limitation ofall entomopathogenic nematode species is sensitivity to desiccation (Kaya and Gaugler,
1993). Yet, nematodes of some species are capable of anhydrobiosis and can survive in a dormant
state for many years (Cooper and Van Glli1dy, 1971; Barrett, 1991). The general termanhydrobiosis
refers to those organisms that are able to undergo a reversible, physiologically arrested state of
dormancy after a significant water loss (95-98% of their body water). Steinernematids and
Heterorhabditids have been classified as slow dehydration strategists that are capable of only partial
dormancy or quiescent anhydrobiosis (Womersley, 1990). The biochemical and physiological changes
associated with the induction of this state are not fully lli1derstood. The most frequently reported
biochemical change in anhydrobiotic organisms, including nematodes, during the desiccation process
is the accumulation of trehalose (Womersley, 1981, 1987; Behm, 1997). An important property of
trehalose is that it protects biolological membranes by hydrogen bonding between the hydroxyl groups
of the sugar and the phosphate head groups of the phosopholipid membrane. By this association
trehalose protects the biological membrane by replacing water molecules that normally associate
with the phospholipid membrane (Crowe et. aI., 1992; Behem 1997).
Recently, considerable effort has been directed toward the identification of trehalose related
genes that enhance entomopathogenic nematode desiccation tolerance. Zitmann-Gal et al. (2001)
isolated the glycogen synthase (sf-gsy-l) gene, a rate limiting enzyme in the synthesis of glycogen,
from S. feltiae IS-6. They discovered changes in the steady-state level of sf-gsy-l transcripts upon

302
desiccation. They suggest a shift from glycogen to trehalose synthesis upon dehydration, which may
be in part regulated by glycogen synthase transcription. Solomon et al. (2000) identified a unique
heat stable, water-stress-related protein designated DESC47 encoded by the desc-47 gene in
dehydrated S foltiae IS-6. The 10-fold appearance ofDESC47 protein in infective juveniles exposed
to 97% RH for 72 h was accompanied by elevated levels of trehalose. Furthermore, DESC47
protein exhibits a biochemical property similar to water-stress-related LEA proteins in plants. LEA
proteins are thought to prevent cellular damage during desiccation in maturing seeds and in water
stressed higher plants by protecting membranes and protein structures or by renaturing unfolded
proteins (Dure, 1993; Close, 1996
Another source for potential genes that encode beneficial traits for genetic engineering of
entomopathogenic nematodes are nematode produced proteins associated with the disruption of the
insect cellular-immune system. Entomopathogenic nematodes can stimulate in the host rapid cellular
immune responses that result in encapsulation, melanization and nematode death (Wang et aI., 1994,
1995). For example in P japonica, Steinernema glaseri escapes cellular encapsulation and
melanization, but in Acheta domesticus only 10% evade immune encapsulation. Thus this ability to
escape is thought to be host dependent (Wang et aI., 1994). In part this capacity is also believed to
be conveyed by the release of anti-immune surface coat proteins from the nematodes (Wang and
Gaugler, 1999). Wang and Gaugler (1999) extracted nematode surface coat proteins from S.
glaseri and found that at least one protein (SCP3a) when injected into an insect host protects unrelated
nematode species from encapsulation and latex beads from phagocytosis. Therefore, potential
disruption of the insect cellular-immune response by genes that encode for anti-immune proteins
could be used to genetically engineer greater resistance to host immune systems in entomopathogenic
nematodes and thereby improve their host killing capacity.

4. CURRENT AND POTENTIAL MOLECULAR TOOLS ADOPTED FROM C.

ELEGANS

Over the past 25 years, the C. elegans research community has developed elaborate
methodologies such as transposon mutagenesis, expressed sequence tags (ESTs), creating and rescuing
mutant phenotypes, and RNA interference (RNAi) (Wicks et aI., 2000; Fire et aI., 1998; Epstein
and Shakes, 1995). Additionally, an extensive collection of mutants with diverse phenotypes, and a
variety of chromosome maps of mutant genes and molecular markers are available publicly (http://
elegans.swmed.edu). Since Heterorhabditis and Steinernema belong to the same superfamily
Rhabditioidea as C. elegans, the species within these families are phylogenetically closely related
(Blaxter et aI., 1998; Fodor et aI., 1990). Therefore, C. elegans not only provides a conceptual and
practical framework for parallel studies in parasitic helminths, but due to the vast knowledge available
on the biology and genetics of C. elegans, this model can be exploited particularly to insect-parasitic
nematodes.

4.1 Transposon Mutagenesis

Most, if not all, organisms contain discrete DNA sequences that are capable of integration and
excision into many different sites within their chromosome's coding region (Hsieh and Fire, 2000).
These DNA sequences usually range from several hundred to several thousand base pairs in size and
are collectively referred to as transposable elements or transposons (i.e., parasitic or selfish DNA).
Insertion of a transposon into a gene can disrupt that gene and lead to a mutant phenotype. The
majority of genes can serve as targets for transposons, and many insertion positions within genes are
candidates for potential target sequences. Caenorhabditis elegans contains six known families of
transposable elements (Moerman and Waterston, 1989). Among these, Tc 1 causes the majority of
gene inactivation in C. elegan high copy mutator strains (~ 300 per haploid genome) and is the best-

303
characterized and is used most often to map, identify and isolate genes (Moennan and Waterston,
1989; Plasterk and van Luenen, 1997). Tc 1 belongs to the Tel/ mariner superfamily of transposons
found in organisms ranging from fungi to chordates (Henikoff, 1992; Robertson, 1993; Radice et aI.,
1994; Avancini et al., 1996). The elements of the Tcl family are close to 1.6 kb in length, have short
54 base pair inverted repeats flanking a transposase gene that end with conserved sequence CAGT
and are flanked by TA target site, which is duplicated upon integration (Vos and Plasterk, 1994).
Transposons can be used to detennine the function of a particular gene (reverse genetics) and!
or identify a mutant phenotype (forward genetics)(Plasterk, 1992, 1994). Briefly, Tel mutagenesis
using reverse genetics is done by polymerase chain reaction (PCR) employing one primer that is
specific for the transposon, and one primer specific for the gene of interest. A PCR product is
obtained only when the Tc 1 primer is nottoo distant from the primer of the gene of interest. To detect
the PCR product one can combine this technique with freeze and recovery of C. elegans. That is
thousands of Tc 1 mutagenized nematode cultures are frozen in triplicate and screened by PCR to
detect the mutant gene of interest. Once the address of the nematode mutant has been identified, the
corresponding nematode culture is thawed and those surviving are analyzed by PCR to confirm the
mutant strain (Plasterk, 1994). A second approach (forward genetics) entails developing a mutant
phenotype by transposon insertion, followed by outcrossing, isolation, and characterization of the
gene of interest (Plasterk, 1994).
A few copies of a transposable element (faintly similar to MLEs described in C. elegans) have
been sequenced and cloned from H. bacteriophora (Greiner et aI., 1999). However, each copy
showed a series of mutations within the catalytic domain of the transposase indicating a non-active
element. This was further evident by the absence of transcripts in Northern blots of total RNA. No
transposable elements were found in the closely related genus Steinernema (Greiner et al., 1999).
The inability thus far to detect active MLEs in entomopathogenic nematodes should not be
taken as evidence that entomopathogenic nematodes do not have active MLEs in their genomes, but
that the sequences could be unlike the ones found in C. elegans. Therefore, the isolation ofan active
MLE from entomopathogenic nematodes is arguably within reach and would facilitate
entomopathogenic nematode molecular genetics tremendously.

4.2. Expressed Sequence Tags (ESTs)

Expressed sequence tagging is a quick and relatively inexpensive method for discovering new
genes for a particular organism when genomic sequencing is not practical or feasible. The generation
of nematode ESTs consists of (i) isolation of total or differentially expressed mRNA from specific
tissues or developmental stage, (ii) reverse transcription of mRNA to resultant cDNA using the
enzyme RNA reverse transcriptase, (iii) cloning of cDNA into plasmids to create a cDNA library,
followed by (iv) random selection of clones (250-500 bp long sequences which are taken at random
fonn the ends of cDNA clones) for sequencing and homology testing against other gene databases
(Waterson et. aI., 1997).
The genomic sequencing center parasitic nematode EST project (Washington University School
of Medicine, St. Louis, MO) has begun generating ESTs from 14 different parasitic and free-living
nematode species and expects to have >225,0005' ESTs by 2003. Additionally, the Sanger Center
and Epinburgh University will generate 80,000 ESTs from seven different nematode species within
the nllxt s~veral years. Through these combined efforts both institutions foresee the identification of
>80,000 new nematode genes. Sequences and project details are available at www.ncbi.nlm.nih.gov/
dbEST and www.NEMATODE.NET (Table 1).
Expressed sequence tagging is a simple strategy that could be adopted by entomopathogenic
nematode researchers. The discovery of new genes, as well as the genetic manipulation of these
novel genes could aid entomopathogenic nematode strain improvement.

304
 Table 1. Current animal parasitic EST survey projects.
Species Reference
Brugia malayi The Filarial genome project (1999)
Onchocerca volvulus Lizotte-Waneiwski et a!. (2000)
Schistosoma mansoni Williams et al. (1999)
Haemonchus contortus Hoekstra et al. (2000)
Toxocara canis Maizels et al. (2000)
Litomosoides sigmodontis Allen et al. (2000)
Necator americanus Daub et al. (2000)
Teladorsagia circumcincta A
Trichuris muris A
Ascaris suum A
A ~ hhtt:llnema.cap.ed.ac.uklnematodeESTs/small~enomes/overview.htm!.

4.3 Creating and Rescuing Mutant Phenotypes

Since the entire Caenorhabitis elegans genome has been annotated, C. elegans is particularly
suitable for transgenic rescue analysis, which provides a basic method ofcorrelating a specific mutant
phenotype with its physical gene map position (Janke et al., 1997). By vector transformation rescue
(Fire, 1986), the phenotype caused by a genetic mutation may be complemented or rescued (revert
to wild-type) by the addition of an extrachromosomal copy of the wild-type gene which may either
be done by direct injection into hermaphrodites heterozygous for the gene to be tested (Clarke and
Baillie, 1992) or by crossing a genetically marked strain into a mutant strain (McDowall and Rose,
1997). This allows researchers to extrapolate directly from a gene of interest identified on a genomic
map to mutations in that gene. Mutant rescue phenotype analysis has become a powerful tool for
investigating the physical and biochemical workings of C. elegan genes that do not show homology
to sequences in other genome databases. As new parasitic nematode genes are discovered, mutant
rescue phenotype analysis could be one technique used for gene characterization.

4.4 Gene Silencing: RNA Interference

The use of double stranded RNA (dsRNA) is a powerful molecular tool for interfering with
gene expression in a number of organisms (Bastin et aI., 2001; Sharp, 2000, 2001; Bosher and
Labouesse, 2000). Introduction of exogenous dsRNA may lead to specific degradation of
corresponding RNA transcripts that are homologous to a specific gene sequence through a phenomena
referred to as quelling in fungi (Cogoni and Macino, 1997; Cogoni and Macino, 1999),
posttranscriptional gene silencing in plants (Elmayan et aI., 1998; Furner et aI., 1998) and RNA
interference (RNAi) in animals and in some plants (Table 2).

Table 2. RNAi phenomenon in different species.

Organisms References
Plants Arabidopsis thaliana Chuang and Meyerowitz, (2000)
Invertebrates Hydra Lohmann et al. (1999)
T brucei Ngo et al. (1998)
Planaria Sanchez Alvarado and Newmark (1999)
Drosophila Kennerdell and Carthew (1998)
Misquitta and Paterson (1999)
C. elegans Tabara et al. (1998)
Ketting et al. (1999)
Cupiennius salei Schoppmeier and Damen (200 I)
Vertebrates Zebrafish Wargelius et a!. (1999)
Mouse Wianny and Zemicka-Goetz (2000)

305
 Fire et al. (1998) demonstrated that when dsRNA was injected into C. elegans the corresponding
gene products disappeared from the F, progeny and the behavior of the offspring mimicked the
known null-mutant phenotype with the effect usually lost by the F2 generation. Surprisingly, gene
interference is not observed when single-stranded RNA (ssRNA) is injected into the worm. In
addition, double and single-stranded DNA molecules are completely ineffective (Tabara et al., 1998).
Microinjection of dsRNA into the germ line or intestine is the most potent non-inheritable RNAi
strategy whereas both soaking (Tabara et aI., 1998) and feeding (Timmons and Fire, 1998) appear
to work less efficiently. A report by Tavernarkis et aL (2000) has shown that genetic interference is
heritable by in vivo synthesis of hairpin dsRNA in transgenic C. elegans. An interesting characteristic
ofRNAi is that two molecules per cell are estimated to effectively silence gene expression (Fire et
aL,1998).
The mechanism responsible for RNAi is not fully understood. One potential model for RNAi
proposes that dsRNA is processed to 21-23 nucleotides (referred to as short interfering RNAs) that
base pair with complementary mRNA followed by an endonuclease that cleaves the invading mRNA
thus achieving mRNA degradation (Bass, 2000). In the few years since the discovery ofRNAi, it is
now broadly viewed that the process is an ancient genome defense response to parasitic DNA (i.e.,
transposons) and foreign RNA sequences (i.e., viruses) (Cogoni and Macino, 2000). There is also
increasing evidence that RNAi may be involved in gene regulation (Sharp, 2001).
The discovery ofRNAi is one of the most exciting advances in molecular genetics within the last
ten years. RNAi is now the first method of choice for disrupting the function of genes in C. elegans,
the first multicelluar organisms to have its genome sequenced (Kuwabara and Coulson, 2000). Even
more astounding, RNAi can be applied as a tool for gene silencing across plant and animal kingdoms
(Mathilde et al., 2000; Cogoni and Macino, 2000). Given these results RNAi could easily be adapted
to study the function of genes involved in such pathways as infectivity, host finding, host immunity,
desiccation tolerance, and reproduction in entomopathogenic nematodes. For example, the function
of a gene that exhibits a distinct phenotype could be determined by injecting a portion of its
corresponding dsRNA, whereas before a lengthy mutagenesis protocol was needed to produce an
analogous mutant. As Kuwabara and Coulson (2000) point out it is imperative to begin with a gene
that is likely to exhibit a readily visible knockout phenotype. Also, the dsRNA should be 0.6 to 2.0
Kb in size and exon rich. RNAi has quickly become an indispensable tool for understanding gene
fimction ina wide variety ofeukaryotic organisms and could, in conjuction with EST parasitic nematode
genome projects, be adapted for furthering entomopathogenic nematode molecular research.

5. CONCLUSIONS AND FUTURE PROSPECTS

Genetic improvement of entomopathogenic nematodes has been an area of intense study for
several decades. Many species have been classically selected for (desirable traits), but so far none
have been transformed genetically for commercialization. The exponential growth in the molecular
genetics and the technological advances emerging from the C. elegans research community along
with data bases that are freely accessible to other researcher groups (e.g., entomopathogenic nematode)
provides an unprecedented reservoir of information. No other biological control agent boasts such a
comprehensive genetic model system. However, despite the abundance of C. elegans genomic
information, the entomopathogenic nematode research community will require parallel increases in
monetary and human resources.
What genetic information and molecular techniques can the entomopathogenic nematode research
community adopt from the C. elegans group to further advance its goal of producing genetically
enhanced insect parasitic nematodes? Entomopathogenic nematode genetic improvement has already
been advanced by databases such as WorrnBase (http://e1egans.swmed.edul), and by the numerous
parasitic genome EST databases. Even more important than the genomic information supplied by
these databases are the molecular techniques, such as transposon mutagensis, ESTs, creating and

306
rescuing mutant phenotypes, and RNAi that have been perfected in the C. elegans community. The
adoption of these molecular tools will be most useful for advancing entomopathogenic nematode
strain improvement.
In summary, entomopathogenic nematodes are biologically fascinating and economically important
biological control organisms that are extremely amendable to genetic manipulation. Entomopathogenic
nematodes, especially Heterorhabditis, share a life-cycle and developmental biology similar to C.
elegans ,thus suggesting that many of their genes and biochemical pathways are conserved.
Furthermore, understanding the genetic differences between C. elegans and entomopathogenic
nematodes should facilitate the genetic improvement of even more efficacious entomopathogenic
nematode biopesticide strains.

ACKNOWLEDGEMENTS
We are grateful to N. Cohen and A. KoppenhOfer, and R. Gaugler for critically reading the
manuscript.

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MATHEMATICAL MODELS OF INSECT PEST CONTROL

R.A. Nonnan l , A.C. Fenton2, J.P. Fairbairn2, and P.1 Hudson2

I Department of Computing Science and Mathematics, University of Stirling, Stirling

FK9 4LA, UK; 2 Department of Biological Sciences, University of Stirling, Stirling

FK9 4LA, UK

1. INTRODUCTION

1.1 Background

Over the past 25 years a vast literature has built up describing the use of mathematical models
to predict the effects of parasites (broadly defined here to include viruses, bacteria and parasitoids)
on the dynamics oftheir host(s ).The results of these models can be interpreted in two ways. In many
cases we are interested in controlling the disease for public health or economic reasons and there has
been extensive research in this area, particularly with reference to diseases such as measles. Models
of this type have even been used to determine UK government policy for di~eases such as BSE
(Bovine Spongifonn Encephalopathy) and Foot and Mouth disease.
An alternative interpretation of the results of these models is in tenns of the use of parasites to
control pest populations. In particular how we could use them as biologic;i!l control agents. The
majority of the literature on the models of host-pathogen interactions is deriv~d from the work of
Anderson and May which started in the late 1970s. Although models ofinfectiolls diseases had been
described before then they had assumed that the host population was constant, whereas Anderson
and May allowed the host population density to be a dynamical variable, chapg!p.g as a result of the
impact of the pathogen. Their models are simple sets of coupled non-linear diffen~ntial equations that
categorise members of the host population according to their infection status (su~c;eptible and infected
in the simplest case) and describe changes in abundance of the hosts in these categories over time.
Analysis of these models is usually limited to evaluating the long-tenn stability ofthe host-pathogen
interaction (Anderson and May 1978,1981; May and Anderson 1978). Many workers have expanded
the suite of models presented by Anderson and May (1981) in order to increase the level of biological
realism in the models and to determine the mechanisms which lead to stability ofthese systems.
Although there has been extensi ve work on the use of parasitoids as biological control agents
we will concentrate here on models of microparasites (viruses, bacteria, fungi and entomopathogenic
nematodes). The aim of this chapter is firstly to describe a simple, baseline model, which could be
effectively used to describe a micro parasitic-insect pest system, and, secondly to describe some of
the expansions of that model that have been studied to include more biological realism. We will then
discuss the benefits and limitations of using these models to detennine insect pest control strategies.

Advances in Microbial Control of Insect Pests

Edited by Rajeev K. Upadhyay, Kluwer Academic / Plenum Pub/ishers, New York, 2002 313
2. THE MODELS

2.1 A Simple Model

The following model is very similar to model G from Anderson and May, 1981 and includes a
density dependent birth rate and free living infective stages. Host individuals can either be susceptible
(X) or infectious (Y) and the free living infective stages ofthe parasite are given by W. The equations are
as follows:

dX
dt = (a-s(X + Y))(X + Y)-bX -vWX + yY,
(J)
dY
-=vWX-(a+h+y)Y (2)
dt

dW
- = A,Y -(,u+v(X +Y))W (3)
dt

Where a is the maximum per capita birth rate; s is a measure ofthe density dependent constraints
acting on the host population; b is the per capita natural death rate of the host; n is the transmission
rate of the disease; g is the rate at which infectious individuals become susceptible again; a is the per
capita death rate due to the disease; I is the rate at which free living particles are produced by
infectious individuals and m is the natural death rate ofinfectious particles.
The long-term dynamics of this model is analysed in a standard way (Anderson and May 1981).
Firstly we calculate the biologically relevant equilibria, i.e. the possible long-term population densities,
by setting each of the derivatives equal to zero. The stability ofeach of the equilibria is then tested by
calculating the eigenvalues of the Jacobian of the system at each equilibrium. If the eigenvalues all
have negative real parts then the system is said to be locally stable i.e. we would expect the population
to settle at these densities. The outcomes of these analyses are algebraic inequalities, in terms of the
parameter values, which determine when the different types oflong term behaviour occur.
In the model shown, the possible equilibria, written in the form (X,Y,W), are (0,0,0) i.e. nothing
present, (K,O,O) i.e. the host at it's carrying capacity, K, and no disease present (where K=(a-b)/s)
and (X', Y' ,Z') where the host and the microparasite are both present. (0,0,0) is never stable as long
as the host's birth rate is higher than the death rate (a>b) in other words, as long as the host population
can grow, it could invade a region in which there were no hosts. (K,O,O) is only stable when Hr>K
where Hr is the threshold host density for pathogen persistence given, in this case by

H, = (a+b+Y)JL (4)
v(A-a-b-y)

This inequality can also be written as

(5)

Where Ro is the basic reproductive rate of the disease defined as the number of secondary
infections achieved when a single infectious particle enters a totally susceptible population.
When K>HT i.e. Ro> 1 then the equilibrium with the host and pathogen persisting together is
either stable or we have stable limit cycles.

314
 12000 1
10000
stable limit cycles
8000
C\I
"t:l
.c 6000
E
..!l!
4000 stable coexistence
2000 ~ I ~K,O,O)
oI
0 5 10 15
alpha
Figure 1. Graph to show the regions in a-A parameter space for which different types of dynamical behaviour
occur. In this case parameters are: F2; b=l; 5=0.001; y=O.OOOI; y=0. I and M=3.

Clearly, although this model is relatively difficult to analyse mathematically, mainly due to the
inclusion of density dependence, we do have a good understanding of how these equations behave in
the long term. However, this is still a very simple system biologically. In the following sections we will
discuss how this baseline model has been expanded in several different ways to allow greater levels
of realism.

2.2 Two Hosts

When we consider the control of pests we are always concerned about the effects of our
control agent on other species both from a biodiversity point of view and in terms of the loss of
beneficial insects. Therc has therefore been an interest in looking at the effect ofpathogens on alternative
hosts, leading to the development of several host-host pathogen models. The first of these was by
Holt and Pickering in 1985 who formulated a model without density dependence or free living infective
stages but with two pathogen hosts who only interact via the directly transmitted pathogen. This
model was then modified to include density dependence (Begon et aI., 1992), free living infective
stages (Bowers and Begon, 1991) and competition between species directly as well as via the pathogen
(Bowers and Turner, 1997). We will ~oncentrate on the direct expansion of the model given in
equations 1-3, which is presented in Begon and Bowers, 1994.
When modelling the free living infective stages we could assume that we have a single population
offree living infective stages, so that any given infective particle is equally likely to be taken up by
either host species (Bowers and Begon, 1991). Alternatively, and more realistically, we can have a
population of free living infective stages which is divided according to whether the pathogen particles
were generated by one host or the other. This is the case which was presented in Begon and Bowers,
1994 and which we will discuss here. The equations for this system are as under:

dX]
- = ( a ] -s](x] +Y]»(x] +Y1)-bIX 1
dt
-V11X1W1 -v12X1W2 +YIYI (6)

(7)

315
 dX 2
- - = (a2 -S2(X Z +YZ ))(X 2 +Y2 )-b 2X 2
dt
-v 22 X 2 W Z -V2I X 2 W I +Y2 Y2 (8)

(9)

(10)

dW z
----;;t=AZYZ -(f-/z +V ZZ (X 2 +Y2 )+v 12 (X I +YI »)W2
(II)

Where the parameters are all as for equations 1-3, with the subscripts determining whether they
are for species 1 or 2. The transmission terms v II represents transmission from the pool of infectious
stages produced by species 1 to species I; v 12 represents transmission from the pool of infectious
particles produced by species 2 to species 1 with equivalent definitions forv z2 and V 21 • This model is
analysed in the same way as the single host-pathogen model. In this case the possible equilibria are (i)
neither species present; (ii) Species 1 alone at its carrying capacity; (iii) Species 2 alone at its
carrying capacity; (iv) Species 1 and 2 both present at their carrying capacities; (v) Species 1 and the
pathogen present without species 2; (vi) Species 2 present with the pathogen but without species 1
and (vii) both species present with the pathogen. The first three equilibria are never stable as long as
the species growth rates are both positive. When we consider the last three equilibria they can be
stable or replaced by stable limit cycles.
One interesting result of these two host models is that it is possible for one species to cause the
other to be excluded. This is a phenomenon known as apparent competition. In other words we do
not have direct competition between the two species but mediated via the pathogen. It is therefore
possible that the alternative host would either enhance or interfere with the effect of the pathogen on
the pest.
Following analysis of this model Begon and Bowers look at the application of the results to biological
pest control specifically in terms of "classical" biological control, where the pathogen is introduced once
(or at least rarely) and exerts long-term regulatory control on the pest's dynamics. By considering a range
of parameters which could appropriately describe a pest and an alternative host they found that the results
from this model suggest little chance of the non-target host undermining pest control, relatively little
chance of the non-target host enhancing pest control and a small but non-negligible threat to the non-target
host when parameter values are appropriate.

2.3 Models of Intermediate Complexity

In general there are two types of modelling approach. Firstly, the 'simple' models described in
the previous sections that do not have high levels of biological detail but which allow algebraic analysis
and can be applied to a wide range of systems. The alternative approach is to build a much more
biologically realistic model which is applicable to a specific system and has to be simulated. However,
in the I990s models of intermediate complexity were developed. These models include more biological
realism but still allow some algebraic results to be derived (Briggs and Godfray 1995a,b; Moerbeek
and Van den Bosch, 1997). Most often these models are stage structured (using delay differential
equations) and allow us to describe the fact that not all stages of the insect host are susceptible to the
pathogen.

316
 One result ofthese models is that the introduction of explicit time delays has little effect on the
model equilibria but strongly influence dynamics (Briggs and Godfray 1995b). It was found that
cycles with a period of one host generation could be observed, this is a type of population dynamic
not previously seen in insect-pathogen models (Briggs and Godfray, I 995b). It was also found that
time delays generally make systems more likely to cycle.
Other results from models of this type will be discussed below.

2.4 Transmission Terms

One of the implicit assumptions that the models discussed so far make, is that transmission is
proportional to host and virus density. This is represented by the vwx term in the equations and is
referred to as mass action or density dependent transmission. It assumes that host and pathogen are
randomly mixing and that the pathogen is distributed homogeneously across the landscape. This term
is used firstly for mathematical tractability and secondly because there has been limited evidence for
other relationships. However, in recent years some experiments have been carried out to test the
relationships between transmission and both host and parasite densities (Knell et aI, 1996, 1998;
Dwyer et al., 1997). In some systems, in which transmission works through a non-linear mechanism
such as cannibalism, the mass action term does not adequately describe transmission (Knell et al.,
1996, 1998). However, it has been shown in other systems that the mass action term is reasonable
(Fairbairn et al., 2002).
There have also been theoretical studies into the effect ofadding a transmission term other than
mass action into the models. Hochberg (1991) formulated a model which v is replaced by the general
formula v' X p W q where p is the 'susceptible response' and q is the 'infected response'. It was found
that the parasite is most likely to regulate its host if transmission efficiency increases with susceptible
host density (i.e. p>O) and has a small negative relationship to the density of infected hosts (i.e. -
I <q<0). Fenton et al. (2002b) reviewed the literature and estimated p and q for those insect-pathogen
systems for which suitable data was available. They found that in all cases -1 <q<0 and p>-0.5. This
means that if we expanded Our models to include more realistic transmission terms, of the form
described above, we would generally expect the resulting dynamics to be more stable than with the
simple mass action term.
In the stage structured models described in section 2.3 (Briggs and Godfray, 1995b) the models
are formulated with transmission either as a mass action term or as a non-linear function with vW
replaced by
kln[l+vW I k]
Using this non-linear function, the transmission rate increases with pathogen density but does so
at a decelerating rate. This introduces density dependence into the per capita efficiency ofthe pathogen
population. In general this function has a stabilising effect whichis related to the strength ofthe density
dependence. As discussed above when the Briggs and Godfray model has transmission by mass
action then cycles that are of one host generation length are seen. If the non-linear transmission rate
is used in the same model the region, in parameter space, for which the host and pathogen coexist in
a stable way is increased and generation cycles can disappear if the density dependence is strong
enough.
From these papers then it seems that we might expect biologically realistic forms of non-linear
transmission rates to have a stabilising effect on the predicted dynamics of a system. However, this is
not always the case, for example for entomopathogenic nematodes experimental data has been used
to estimate values of v', p and q for the transmission term from Hochberg, 1991. It is found that
using the predicted values makes an originally unstable system even more unstable (Fenton et al.,
2002b).In other words for a system in which we originally predicted the host population would cycle
we now predict less frequent cycles.

317
3. SHORT TERM DYNAMICS

In all of the cases described above the models are only analysed in terms oftheir long-term
dynamics and this may be of interest if we wish to consider 'classical' biological control in which the
pathogen persists and maintains the pest at lower levels. However, this analysis does not tell us
anything about how quickly this equilibrium is achieved or whether there are cycles or oscillations in
the transient dynamics. This is generally a problem if we are thinking about practical insect pest
control when it is more likely that short -term dynamics will be of interest. There are at least three
types of control that wc might be interested in, firstly indundative control in which we wish to apply
the control agent in large numbcrs in response to a perceived pest attack in the hope that we can
bring about a rapid decline in pest numbers. We would then expect to re-apply the agent in the face
of another attack. Secondly there is pre-emptive control in which we wish to apply the control agent
strategically in order to prevent invasion of an expected pest attack and finally there is long term
control where we apply the control agent continuously in order to maintain suppression of a pest
population. In each case, although the underlying model is the same, the methods of analysis are
different (Fenton et a!., 2001). Anderson and May (1981) considered this last type of control. They
modified their model G (free living infective stages and no density dependence) to include the
introduction of infecti ve stages into the environment at a constant rate. In order to eliminate the
pathogen with a relatively low rate of pathogen introduction they found that we must have a parasite
which is highly pathogcnic, long lived and has high transmission efficiency.
In a paper which looks at the use of entomopathogenic nematodes as pest control agents
(Fenton et aI., 200 I) studied all three of these types of control. A stage structured model was used
similar to that described in Briggs and Godfray (I 995b ). In this case the pest population was split into
juvenile and adult classes with only the juveniles susceptible to the disease. It was found that inundative
control would work best in a systcm in which the growth rate of the population is either very low or
very high, the death rate ofthe nematodes in the soil is low, the transmission rate is very high and the
crop has a high sustainable injury level. A good example ofthis would be the sciarid fly/mushroom
system. For pre-emptive control to be most successful we want a system in which the growth rate of
the pest is low, the death rate of the nematode in the soil is very low, the transmission rate is high and
the crop has a low sustainable injury level. An example of a system for which pre-emptive control
might be successful is a black vine weevil! ornamentals system. Finally for repeated application to be
preferable an ideal system would have a pest with a low reproductive rate, the nematode having a
low death rate in the soil, the transmission rate being mediurn/high and the crop having a high sustainable
injury level, for example Pjaponica/turfgrass.
Whilst these results were determined for the nematode systems the methods of analysis and
cven the results are applicable to any microparasitic pest control system.

4. SPATIAL MODELS

In all of the models we have discussed, interactions between pest and host do not take their
spatial relationship into account. There are several different ways to include this, for example a discrete
model for host-pathogen interactions can be developed and is used to represent the dynamics in
each patch within a landscape of n x n patches. Individuals then interact with their nearest neighbours
(Boots and Sasaki, 2000; Sato et aI., 1994; Durrett and Levin, 1993). These models are known as
cellular automata. However, they cannot be readily analysed algebraically and largely rely on simulation.
In addition the application of these models result to the practical aspects of insect pest control.
However, there has recently been some progress in the algebraic analysis ofthese types of models
using pair approximations (Boots and Sasaki, 1999,2000).
One result of these models which is of direct relevance to pest control is that if a pathogen has
a sublethal effect which results in large reduction in host fecundity then a new type of dynamical
behaviour is possible. A sublethal infection is one in which, if an individual is infected, they do not

318
necessarily become infectious. However, this resistance to the infection has some detrimental effect
on the host such as a reduction in fecundity. Generally sublethal effects are found to be destabilising
(Boots and Norman, 2000; Briggs and Godfray 1995c). In the spatial model, sublethal infection
which causes a large reduction in fecundity leads to a type of dynamical behaviour not normally seen
in a model with a mass action transmission term, this is deterministic extinction of the host (Boots and
Sasaki, 2002). This implies that we would expect a pathogen which castrates the host to be a highly
effective control agent. Although this is an intuitively obvious result, it is not one that would have
emerged directly from a model which does not include spatial factors.
Other ways of including spatial factors include coupled map lattice models, reaction diffusion
equations and metapopulation models. Coupled map lattice models are also based on a grid pattern,
but unlike cellular automata they allow more than one individual per cell. In coupled map lattice
models the usual temporal (differential equation) model is run in each cell with movement between
cells occurring at a set intervals (Comins et al., 1992; White et aI., 1996). Reaction diffusion equations
involve adding a diffusion term onto the end of the usual differential equations and do not rely on a
system of cells. However, they are less flexible than grid based models for allowing heterogeneities.
They often results in waves of infection with the interesting dynamics occurring behind the wave front
(Dwyer, 1992). Finally, metapopulation models assume that hosts live in patches with some migration
between them (Lynch and Bowers, 1998) and are possibly ofless relevance to pest control.

5. SEASONALITY

In many systems interactions between host and pathogen do not occur continuously in time as
most simple models assume. In fact they vary either due to season or to crop availability. One simple
way of adding seasonality has been used by several authors. The model is developed with two
separate components; (a) within season component which is the usual set of equations and (b) between
season component which maps the numbers of hosts and pathogens at the end of the season onto the
numbers present at the beginning of the following season. This method was used by Briggs and
Godfray (1995c) where their stage-structured model was within season component. This model was
analysed with both a mass action term for transmission and the non-linear term discussed in section
2.4 as well as including several other extensions such as release of pathogen particles prior to death
and a sublethal infection. It was found that the seasonal model, compared to the Anderson and May
(1981) model, is considerably less stable. However, that could also be due to the time delays in the
model.
Seasonal effects have also been taken into account in a spatial model. In White et aI., 1999 a
standard Anderson and May type model is run during the summer but during winter a discrete mapping
occurs as described above. It was predicted that a travelling wave of infection would sweep across
the landscape away from the point of pathogen introduction. However, seasonality could reduce the
range of disease spread compared to equivalent non-seasonal systems.

6. APPLICATION TO CONTROL

Despite the fact that models of host-pathogen interactions have been studied for the past 25
years the results of the models are often not interpreted directly in terms of optimal control strategies
or with reference to pest management techniques. There are some exceptions to this:
For example, another study of entomopathogenic nematodes has parameterised the model for
the sciarid fly-mushroom glasshouse system, and using available data on control experiments has
determined optimal strategies for control (Fenton et aI., 2002a). Since the mushroom crops have a
short lifespan (about 8 weeks), it is important to develop a model that can accurately take into
account the short term transient dynamics of the system. The model consists of a series of delay
differential equations describing a stage structured population, where again, only the larval stages are
susceptible to the nematodes. The full model is a modified version of that of Briggs and Godfray
(1995b). The model was fitted to data from one field trial and validated against data from a second

319
field trial before different control strategies were simulated. It was concluded that for a single dose it
is possible to achieve higher levels of control with only 2million nematodeslm2 than are observed with
the currently recommended dose of 3 million nematodes/m2 ifthe timing of the treatment is rigltt.
However, with a correctly timed split dose even greater control is achievable.

7. DISCUSSION

As we have seen, mathematical models have been applied to many aspects ofthe use of microbial
pest control and this chapter has only scratched the surface ofthe literature which is out there,
including several reviews (e.g Briggs et al., 1995).
We have not discussed many of the complex models that require simulation and have instead
concentrated on the models which are relatively simple biologically but which are algebraically
tractable. These are mainly based on the work of Anderson and May and the middle ground of
models of intermediate complexity. There are many other questions of biological interest which have
been studied but which are not described in detail here, these include studying the effect of resistance
to pathogens (Boots and Bowers, 1999) and the related trade offs in other life history traits (Bowers
et aI., 1994). The effects of host refuges (Hawkins et aI., 1993), pathogen persistence in the
environment (Thomas et aI., 1997) and secondary cycling ofthe pathogen (Thomas et aI., 1995)
Often the models described here have been discussed in terms of their theoretical interest
rather than in terms of their direct application to insect pest control or parameterisation for specific
pathogen-pest systems. However, as we have seen, if we know enough about a system, it is possible
to use models to describe a specific system, compare several control strategies and determine the
optimal one (Fenton et aI., 2002a). It is then possible to take the results ofthe comparisons and use
them to recommend control strategies in the field.
It seems clear that mathematical models can be enormously useful in helping us understand
many different aspects of how pathogens and insect pests interact and that there is huge scope for the
application of these models to answer specific pest control problems. The Fenton (2002a) paper
used simulation and comparison of particular pathogen application methods. A more rigorous
mathematical approach would be to develop a system of models that describe a range of different
control agents, pests and crops and to use techniques from control theory to analyse them. This
would allow us to determine the best strategies in terms of maintenance of biodiversity and pest
suppression for each ofthe different control objectives described in section 3. Techniques in control
theory are available to handle both single and multiple objective situations. It should therefore be
possible to find optimal control strategies for different generalised systems. We could, for example
categorise crops according to their growth period and the period between crops, we could define
pests according to which life stages are susceptible and how they move spatially. We could then look
at the effects of different control agents (or combinations of agents) with different application
techniques, and determine the conditions under which it would be best to apply a single dose or
several split doses and what the timing between those doses should be. These sorts of results could
then be applied to specific systems in order to inform integrated pest management approaches.
Biological pest control is an area in which it is hugely beneficial for mathematical modellers and
empirical biologists to work together closely. There is a two-way interaction in which laboratory
experiments can be designed to produce data that will inform the models and models are built and
analysed which will shed light on the biology of asystem.ln addition ifthe right sort of information can
be measured during field trials then it can be used to parameterise or validate the models.
In a time when the demand for organic or pesticide free produce is ever increasing, it is clear
that effective biological control methods must be found. We hope we have demonstrated that
mathematical models are a vital tool in this search.

320
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322
SUBJECT INDEX

A Anopheles spp., 42, 45, 55

Anopheles stephensi, 47,54
A. segetum, 243 Anopheles, 42,55,57,58,61
Abirus jortunei, 249 Anophelinae, 42
Acheta domesticus, 118, 303 Anoplophora chinensis, 251
Actebia jennica, 248 Anthonomus grandis, 207
Adioristus sp., 204 Anticarsia gemmatalis, 98, 203, 206, 207
Adoryphorus couloni, 176 Aphelenchus avenae, 243
Ae. aegypti, 42, 45, 46, 52, 54, 58 Aphis gossypii, 206
Ae. albimanus, 55, 58 Aphodius contaminatus, 249, 252
Ae. albopictus, 43, 46, 58 Apis mellifera L., 118
Ae. atropalpus, 55 APN isofonns, 1
Aedes species, 42, 43, 45, 52, 54, 55, 57, 58 Apoanagyrus lopezi, 176
Aedes, 43 Aquabirnavirus, 84
Aeneolamia lepidior, 207 Arabidopsis thaliana, 74, 305
Aeneolamia postica, 208 Arboviruses,42
Aeneolamia selecta, 200 Armigeres, 42, 55
Aeneolamia sp., 208 Ascaris suum, 305
Aeneolamia spp., 206 Aschersonia spp., 194,201
Aeneolamia varia, 207,302 Ascoviridae, 83
Agamomermis, 44 Asticcacaulis excentricus, 55
Agaricus bisporus, 247 Asynonychus godmani, 249
Agrobacterium, 71, 72 Atta spp., 207
Agrotis ipsilon, 253, 113 Autographa californica MNPV, 89, 109,
Agrotis spp., 248 128,130
Aleochara spp., 181 Autographa californica, 134
Allantonema, 236 A vibimavirus, 84
Alphitobius diaperinus, 253
Amblyospora, 44 B
Amsacta moorei, 86
Amyelosis transitella, 84 Bacillus, 49, 73
Amylostereum aerolatum, 237,254 Bacillus cereus strain T, 17
An. anthropophagus, 42, 58 Bacillus laterosphorus, 47
An. arabiensis, 42 Bacillus popiliae, 19, 242
An. dirus, 42, 43,46, 58 Bacillus sphaericus, 41, 44, 45, 47, 48, 49,
An. fluviatilis, 58 50,52,53,54,55,57,58,59,60,61
An. jimestus, 42 Bacillus subtilis, 53, 54
An. gambiae, 42, 43 Bacillus thuringiensis subsp. aizawai, 23
An. minmusi, 43 Bacillus thuringiensis subsp. alesti, 22
An. sinensis, 42, 43, 46, 58 Bacillus thuringiensis subsp. berliner, 22
Anacystis nidulans, 54 Bacillus thuringiensis subsp. chinensis CT.,
Anasa tristis, 251, 256 22,43
Ancognata sp., 205 Bacillus thuringiensis subsp. coreanensis,
Ancyclobacter aquaticus, 55 22
Androctonus australis, III Bacillus thuringiensis subsp.
Anomala cuprea, 252 dormstadiensis,22
Anopheles sp, 60

323
Bacillus thuringiensis subsp. entomocidius, Batkoa, 220
22 Beauveria amorpha, 202
Bacillus thuringiensis sUbsp.fokuokensis, Beauveria bassiana, 166, 167, 170, 171,
46 182,184,185, 188, 193, 194, 199,201,
Bacillus thuringiensis subsp. israelensis 202,203,204,205,206,207,213,215,
strain HD 567, 17 216,217,218,219,220,242,243
Bacillus thuringiensis subsp. israelensis, 16, Beauveria brongniartii, 184, 199,204,213,
17,18,20,30,41,44,45,47,59,60 216,218
Bacillus thuringiensis subsp.japonensis, 22 Beauveria spp., 208
Bacillus thuringiensis subsp.jegathesan, Bemisia tabaci, 202, 204, 205, 206, 207
46,367 Blatella germanica, 251, 255
Bacillus thuringiensis subsp.juroi, 22 Boarmia selenaria, 248
Bacillus thuringiensis subsp. kenyae, 18 Bombyx mori APN ABO 13400, 3
Bacillus thuringiensis subsp. kurstaki HD 1, Bombyx mori APN 1, 2, 3
22 Bombyx mori APN 2, 3
Bacillus thuringiensis subsp. kurstaki HD Bombyx mori APN 3, 3
263, 18,22 Bombyx mori NPV, 89, 116, 128
Bacillus thuringiensis subsp. kurstaki HD Bombyx mori, 1,2,3,4,5,6,7,9,87,99,
73,18 111, 114
Bacillus thuringiensis subsp. kurstaki HD Boophilus annulatus, 240
YBT, 15,20,22 Bracovirus, 88
Bacillus thuringiensis subsp. kurstaki, Bradysia coprophila, 250
17,18,22,242 Bradysia paupera, 250
Bacillus thuringiensis subsp. kyushuensis, Bradysia spp., 247
46 Brevibacillus laterosphorus, 47
Bacillus thuringiensis subsp. morrisoni, 22 Brevicoryne brassicae, 206
Bacillus thuringiensis subsp. neoleonensis, Brugia malayi, 42, 305
22 Budded virus (BV), 91
Bacillus thuringiensis subsp. sotto, 22 Bunyavirus, 84
Bacillus thuringiensis subsp. tenebrionis Bursaphelenchus xylophilus, 238
(H8ab),19 Bursaphelenchus, 236, 237
Bacillus thuringiensis subsp. tenebrionis, 18
Bacillus thuringiensis subsp. thompsoni, 20 c
Bacillus thuringiensis subsp. thuringiensis
strain 407,30 Caenorhabditis elegans, 297,303
Bacillus thuringiensis subsp. thuringiensis Calacarus hevea, 201
strain berliner, 15, 17 Camponotus spp., 06
Bacillus thuringiensis subsp. thuringiensis Carabus, 183
strain HD 2, 22 Carposina nipponensis, 218
Bacillus thuringiensis subsp. tolworthy, 22 Castnia licus, 199
Bacillus thuringiensis subsp. toumanofJi, 22 Caulobacter cresentus CB 15, 54
Bacillus thuringiensis subsp. wuhanensis, Caulobacter cresentus, 55
16 Cephalcia abietis, 253
Bacillus thuringiensis, 1,7,15,16, 18, 19, Cephalcia, 251
22,28,30,44,45,47,54, 71, 72, 181, Ceratitis arvensis, 25
135,252 Ceratitis capitata, 250
Bacillus, 49, 73 Ceratitis lariciphila, 251
Baculovividae, 83 Cheilomenes spp., 176
Basidiobolus, 220 Chloriridovirus, 86, 87
Batkoa sp., 200 Choristoneurafomiferana MNPV, 113

324
Choristoneura rosaceana, 248, 255 Culicimomyces, 44
Chrysomela scripta, 78 Cu1icinae,42
Chrysoperla carnea, 118 Cupiennius salei, 305
Ciclocephala signaticollis, 206 Cyclocephala borealis, 248
Clostridium bifermentans var. malysia Cyclocephala hirta, 242
(Cbm) CH 18, 46 Cydiapomonella GV, 93,116,128,129
Clostridium bifermentans var. malysia, 44 Cydia pomonella, 24, 248, 253
Clostridium bifermentans var. paraiba, 46 Cylasformicarius, 249, 251
Clostridium, 19 Cyprinus carpeo, 43
Coelomomyces, 44 cytlA, 19
Coenosia tigrina, 182, 188
Collaria sp., 205 D
Colletotrichum gloeosporiodes, 201
Comoritis albicapilla, 248 Deladenus siridicicola, 254
Conidiobolus, 220 Deladenus species, 181,238
Conotrachelus nenuphar, 249 Deladenus, 236
Conotrachelus nenuphar, 255 Deliajloralis, 181, 182, 183, 184,185,247,
Cordyceps sobolifera, 204, 206 250
Cordyceps sp., 194 Delia radicum, 181, 182, 183, 184, 185,
Cosmopolites sordidus, 202 186,187,247,250
crylAa?crylAb,22 Delia, 250
crylAa?crylAb?crylAc, 22 Densovirinae, 88
crylAa?crylAb?crylC?crylD,22 Deois jlavopicta, 200
crylAa?crylAb?crylD,22 Deois incompleta, 200
crylAal, 19,22 Deois schach, 200
crylAb and crylC, 75 Deroceras spp., 240
crylAb, 22, 76 Diabrotica barberi, 252
cry lAc, 76 Diabrotica spp., 244
crylB,22 Diabrotica virgifera, 245
crylD,22 Diadromus pulchellus (Dp AV 4), 83
crylE,22 Diaphania nitidalis, 248, 254
crylF,76 Diaphania, 248
cry2A,22 Diaprepes abbreviatus, 244, 245, 246, 249,
cry3A,28 251
cry3Aa, 19,76 Diaprepes spp., 208
cry9C, 76 Diaprepes, 249
cryI, cryIJ, cryIII, 19 Diatraea saccharalis, 199
cryIIIB,28 Diloboderus abderus, 207
cryIV, 19 Diximerimis, 44
Ctenopelma lucifer, 241 DNA ligase, 94
Ctenopharyngodon idella, 43 DNA polymerase, 94
Culex nigripalpus (Cuni) NPV, 42, 43, 93 Dorylus spp., 176
Culex nigripalpus NPV, 116, 128, 129 Drosophila melanogaster, 84, 87
Culex pipiens, 43, 57 Drosophila virlis, 87
Culex quinquefasciatus, 42, 43, 44, 45, 46, Drosophila, 84, 88, 89, 305
52,54,55,57,58,59,60,61,242 dUTPase,94
Culex sp., 52, 58, 60, 61
Culex spp., 41, 42,55,57 E
Culex triaeniorhynchus, 42, 43
Culex, 42 Earias insulana, 248

325
Edhazardia, 44 Galleria Inellonella 87
Ektaphelenchus, 236,237 Galleria Inellonella, 300
Elnpoascaj1avescens, 219 Galleria, 239, 242
Elnpoasca species, 219 Galnbusia afjinis, 43
Elnpoasca vitis, 219 Gonolneta podocarpi, 88
Entolnophaga, 220 Gonolneta virus, 88
Entolnophthora Inuscae, 182, 184, 185, 186, Granuloviruses (GV) 89, 109
188
Entolnophthora virulenta, 205, 208 H
Entolnophthora, 220
Entomopoxvirinae, 85 Haelnonchus contortus, 305
Entolnopoxvirus A, 86 Heleidolnerlnis Inagnapapula, 238, 240
Entolnopoxvirus B, 86 Helicoverpa arlnigera GV, 114
Entolnopoxvirus C, 86 Helicoverpa arlnigera MNPV, 113
Eoreulna loftini, 242, 248 Helicoverpa arlnigera SNPV, 89, 116, 128
Epinotia aporelna, 207 Helicoverpa arlnigera, 88, 98, 129, 134,
Epiphyas postvittana APN, 3 241
Epiphyas postvittana MNPV, 128, 129 Helicoverpa punctigera APN 1,3
Epitrix spo, 204 Helicoverpa punctigera APN 2, 3
Errantivirus, 87 Helicoverpa punctigera APN 3, 3
Erynia anhuiensis, 224 Helicoverpa punctigera, 1,3
Erynia chironolnis, 221 Helicoverpa virescens (HV A V3), 83
Erynia gigantea, 221 Helicoverpa zea SNPV, 110
Erynia, 220 Helicoverpa zea, 99, 113, 115, 118,248,
Escherichia coli, 4, 5, 6, 53, 54, 134, 302 253
Eucalyptus sppo, 199 Helicoverpa, 2, 248
Eugregarinidies, 44 Heliothis sppo, 205
Euzophera selnifuneralis, 248, 254 Heliothis virescens APN 2, 99
Heliothis virescens, 1, 3, 76, 77, 78, 99,
F 113, 118, 119
Heliothis virescens BT BPI, 2
Fannia sppo, 250 Hepatitis C like-virus, 86
Faustinus apicalis, 207 Heterorhabditis bacteriophora HP, 300,
Filipjevilnerlnis leipsandra, 240 301
Flaviridae, 86 Heterorhabditis bacteriophora IS 5, 300
Flavivirus,86 Heterorhabditis bacteriophora, 241, 242,
Frankliniella occidentalis, 165, 171, 172, 244,246,247,250,251,252,253,254,
173,202 298,300,301,302,304,307
Furia fojiana, 221 Heterorhabditis indica, 244
Furia gloiospora, 221 Heterorhabditis Inarelatus, 251
Furia shandongensis, 221 Heterorhabditis Inegidis, 241, 251, 252, 301
Furia spo, 200, 221 Heterorhabditis spo, 252
Furia triangularis, 221 Heterorhabditis sppo, 300
Fusariuln sppo, 201 Heterorhabditis, 235, 236, 239, 298,
Fusariuln wilt, 202 299, 303
Heteroterlnes, 199
G Heterotylenchus autulnnalis, 237
Heterotylenchus, 236, 237
Galleria, 242 Hevea brasiliensis, 201
Galleria Inellonella, 30, 87, 240, 242

326
Hippodamia convergens (Guerin-
Meneville), 118
Hirsutella spp., 194
Hirsutella thompsonii, 205
Hoplocampa testudinea, 254
Hoplocampa, 251
Howardula husseyi, 238
Howardula spp. 238
Howardula, 236, 237
Hydra, 305
Hylobius abietis, 252
Hylobius congener, 244, 249, 252
Hypothenemus hampei, 202, 204, 205, 208
I Melanoplus sanguinipes, 86
Ichovirus, 88
lncurvaria capitella, 248
lridoviridae, 83
lridovirus, 86, 87
L Metarhizium anisopliae lCIPE, 69, 168,
Lagenidium, 44
Leiurus quinquestriatus hebraeus, III
Leptinotarsa decemlineata, 252
Leptopharsa gibbicarina, 204
Leptopharsa heveae, 201
Ligyrus subtropicus, 245
Lipaphis erysimi, 206
Liriomyza huidobrensis, 250, 255
Liriomyza trifolia, 250, 255
Liriomyza, 250
Listronotus oregonensis, 249, 252
Litomosoides sigmodontis, 305
Lycoriella auripila, 250
Lycoriella mali, 250, 300
Lycoriella solani, 246, 250
Lymantria dispar APN 1, 2
Lymantria dispar APN 2, 3
Lymantria dispar cypovirus 1,85
Lymantria dispar MNPV, 89, 113, 114,
116, 128, 139
Lymantria dispar, 1,5,85,98, 114, 115,
133
M Nabis roseipennis, 118
Mahanarvafimbriolata, 197, 199,200
Mahanarva posticata, 197
Mahanarva, 199
Maladera matrida, 249
Mamestra brassicae MNPV, 113
Manduca sexta APN 1, 2
Manduca sexta APN 1a, 3
Manduca sexta APN 2, 3
Manduca sexta, 1,8,99, 134
Mansonia, 42
Mansonia, 43, 55
Matamasius hemipterus, 249
Matevirus, 87
Megalurothrips sjostedti, 165, 166, 167,
168,169,170,171,175,
Megaselia halterata, 238, 247, 250
Meloidogyne incognita, 243
Meloidogynejavanica, 243
Melolontha melolontha, 184
Mermis nigrescens, 236
Mesocyclops aspericornis, 44
Metamasius hemipterus, 255
170,176
Metarhizium anisopliae var. acridum, 168,
204
Metarhizium anisopliae, 166,167,168,169,
170,171,172,173, 175,176,182,
183,185,188,193,194,197,198,199,
200,202,205,206,207,208
Metarhiziumjlavoviridae, 204, 205
Metarhizium spp., 168
Microplitis croceipes (Cresson), 119
Monalonion dissimulatum, 205
Monsonia uniformis, 55
Musa spp. 202
Musca autumnalis, 237
Musca domestica, 184, 185, 207, 250
Musca, 250
Muscina stabulans, 250
Myiophagus,201
Myriangium, 201
Myzus persicae, 206, 219, 220, 223, 224,
225,227,229
N
Nairovirus, 84
Necator americanus, 305
Nectria spp., 201
327
Neoleucinodes elegantalis, 205
Neomesomermis jlumenalis, 238, 256
Neosteinernema, 236
Neozygites, 220
Nilaparvata lugens, 222, 223
Nodavirus, 87
Nomuraea rileyi 194, 203, 204, 205, 206,
207,208
Nucleopolyhedroviruses (NPV), 89, 100
Nudauretia capensis b-like viruses, 88
Nudaurelia capensis w-like viruses, 88
o 204,242,246,248
Occlusion-derived virus (ODV), 91
Octomyomermis,44
Onchocera volvulus, 42, 305
Orius insidiosus (Say), 118
Orius spp., 176
Orygia pseudotsugata MNPV, 113, 116,
128
Ostrinia nubilalis, 216 218, 246, 248
Otiorhynchus ligustici, 249
Otiorhynchus ovatus, 249
Otiorhynchus sulcatus, 249
p Pseudomonas jluorescens, 134
Pachneus titus, 249, 251
Paecilomyces fomosoroseus, 166, 167, 205,
206,208
Paecilomyces Ii/acinus, 205, 207
Paecilomyces spp., 194
Paederus spp., 176
Pandora athaliae, 221
Pandora bibionis, 221
Pandora borea, 221
Pandora cicadellis, 221
Pandora delphacis, 222, 223, 224, 227, 229
Pandora neoaphidis, 222, 223, 229
Pandora shaanxiensis, 221
Pandora, 220, 222,227
Parasitaphelenchus, 236, 237
Parvoviridae, 83
Pectinophora gossypiella (Saunders), 113,
245,253
Phlebovirus,84
Photorhabdus species, 71
Photorhabdus, 297, 298, 300
Phyllocnistis citrella, 248, 255
328
Phyllopertha horticola, 249, 252
Phyllophaga sp. 205, 208
Pinus radiata, 254
Planaria, 305
Plasmodium species, 42
Platyptilia carduidactyla, 242
Pleistophora, 44
Plodia interpunctella, 76
Plutella xylostella (Px) GV, 93, 116, 128
Plutella xylostella APN 1,2,3
Plutella xylostella APN 1, 3
Plutella xylostella, 1,2,3,5,6, 77, 113,
Plutella, 248
Podischnus agenor, 207
Podisus maculiventris, 118
Polistes metricus Say, 118
Polydnaviridae, 83
Polyhedrin or granulin, 91, 95
Popilliajaponica, 248, 249, 252, 300,303
Poxviridae, 83
Premnotrypes spp., 204
Proarna bergi, 206
Prosapia simulans, 208
Prosapia spp., 206
Pseudaletia unipuncta, 132
Psorophora spp., 55, 43
R
Rachiplusia nu MNPV, 116
Rachiplusia nu, 207, 110
Rana escu/enta, 240
Reticulitermes tibialis, 251
Rhammatocerus schistocercoides, 204
Rhizobium melli/oti, 134
Rhizoctonia solani, 134
Ribonucleotide reductase, 94
Romanomermis culicivorax, 240, 242, 256
Romanomermis,44
s
Saccharomyces cerevisiae Ty3 virus, 87
Sarcophaga bullata (Parker), 118
Scapteriscus spp., 254
Scapteriscus, 251, 254
Schistosoma mansoni, 305
Scolytus scolytus, 240, 255
Scymnus spp., 176 T
Sibine fusca, 207
Simulium spp., 43, 46, 55, 256 Tarichium, 220
Sipha flava, 207 Teladorsagia circumcincta, 305
Soienopsis invicta, 118,203,251 Teleogryllus oceanicus, 84
Soienopsis saevissima, 203 Temnorhinus men dicus, 249
Solenopsis spp., 203 Tetranychus urticae, 202, 203
Soienopsis, 251 Thrips tabaci, 165, 171
Sphaerularia, 236 Tilapia nilotica, 43
Spodoptera exigua MNPV, 116, 128 Tipula paludosa, 250
Spodoptera exigua, 113,242 Togaviridae,83
Spodopterafru,giperda (SfAVl), 83 Tospovirus, 84
Spodopterafru,giperda, 83, 99, 115, 118, Toxocara canis, 305
133,205,207 Toxorhychitis, 55
Spodoptera littoralis MNPV, 138 Toxorhynchitinae, 42
Spodoptera littoralis, 113, 138, 248 Trialeurodes vaporariorum, 206, 207
Spodoptera litura 242, 243 Triatoma infestans, 207
Spodoptera litura MNPV, 93, 116, 128, 129 Trichoplusia ni (Tn A V2), 83
Spodoptera, 89 Trichoplusia ni (TnGV), 90, 132
Sporothrix insectorum, 201, 208 Trichoplusia ni cytoplasmic polyhedrosis
Steinernema ajfinis, 238 virus, 15,85
Steinernema carpocapsae, 240, 241, Trichopiusia ni, 74, 85, 99, 113, 114
242,243,244,245,246,251, Trichuris muris, 305
252, 253, 254, 255, 256, 300 Tripius sciarae, 237
Steinernema feitiae IS 6, 302, 303 Trybliographa rapae, 181
Steinernemafeltiae, 238, 241, 242, 245,
246,247,251,253,254,255,300,301, u
302
Steinernema glaseri, 235, 242, 243, Uranotaenia, 55
244,245,246,251,252,253,303
Steinernema kushidai, 242, 252 v
Steinernema rio brave, 241, 242, 244,
245, 253 Varaia, 171
Steinernema scapterisci, 251, 254 Verticillium lecanii, 166, 167, 171, 194,
Steinernema sp., 238, 251, 254 204,205,206,207,213
Steinernema, 236, 239, 241, 298, 299, 303, Verticillium spp., 201
304 Vigna unguiculata, 165
Stenoma sp., 204, 205
Streptomyces griseus, 114 w
Streptomyces spp., 71
Streptomyces, 135 Wiseana cervinata, 248
Strobilomyia appaiachensis, 256 Wucheria bancrofti, 42
Strobilomyia spp., 247
Strongwellsea castrans, 182, 185, 187, 188 x
Strongwellsea spp., 182, 188
Strongwellsea, 187,220 Xenochesis juivipes, 241
Synanthedon myopaeformis, 248, 254 Xenorhabdus 297, 298
Synanthedon tipuiijormis, 254 Xestia c-nigram GV, 93, 116,128, 130
Synechococcus, 55

329
z
Zeiraphera canadensis, 248, 255 Zoophthora, 220, 222, 227
Zoophthora anhuiensis, 221, 222, 224, 225, Zulia entreriana, 200
226,227,248 Zulia spp., 205
Zoophthora pentatomis, 221 Zulia vilis, 208, 224
Zoophthora radicans, 207, 222 Zulia, 199

330