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Edited b Volurn+ {1}, April 2009

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Short views on
Insect Molecular Biology
Edited by
Raman Chandrasekar
UNIVERSITY OF KENTUCKY www.uky.edu
College of Agriculture Entomology Email: rpalli@uky.edu
S-225 Agricultural Science Centre
Building-North Tel. 859-257-7450
Lexington, KY 40546-0091 Fax: 859-323-1120
USA.
Foreword Message
Entomology as a science of interdepended branches like molecular entomology, insect

biotechnology; has made rapid progress in its attributes in the light of modern
discoveries.
This also implies that there is an urgent need to manage the available resources
scientifically for the good of man. In the past five decades, entomology in the country
has taken giant steps ahead. Continued research has evolved better pest management
through molecular approaches.
The editor of the this book Dr. Raman Chandrasekar have vast experience in research.
He is exposure to research in “Molecular Entomology” has added to his expertise for
mature thinking to conceive the editorship of present venture “Short Views on Insect
Molecular Biology”. The comprehensive review articles by the entomologist from around
the world in various research Organizations/ Universities enhanced the book’s reach
and appeal. I am quite confident that, this book will serve not only the needs of students
of entomology but also useful as reference book to the researcher across the globe.
I heartily congratulate the editor for the high standard and competent manner in which
he has achieved his goal.
Preface
The aim of this book “Short Views on Insect Molecular Biology” is to integrate
knowledge about molecular biology, biochemistry, physiology, genetics, developmental
biology and reproductive biology of insects.
While earlier studies on insect endocrinology have focused on juvenile hormone and
ecdysterioids, the focus during the last two decades shifted to physiological vital proteins,
neuropeptides, pheromone-binding proteins, and G-protein coupled receptors.
Genetically-engineered insects and microbial toxins are promising subjects for insect pest
management. Increased use of beneficial insects also forms an important part of this
biotechnological approach.
This book provides recent research from scientists around the world. The contributing
authors are recognized experts in their field of molecular entomology.
I would like to express my heartfelt gratitude to my former teacher Prof. M. Krishnan and
Prof. M. Kobayashi who supported me at the commencement of this “International Book
Mission Program”. Others who encouraged me during the research are Prof. Immo A.
Hansen, Prof. Seo Sook Jae, Dr. Emiel Janssen, Prof. M. Takeda, Prof. L.I. Gilbert, Prof.
Sumio Tojo, Prof. Keun Woo Lee, Prof. Y.E. Park, Prof. Young-Chang Kim. I have no
words to express my feelings for all those who provided valuable contributions and made
the completion of this book possible. I thank the Geyongsang National University
(GSNU), APSERI-08 Nagoya University, Japan, APMC9-South Korea and Department
of Science and Technology (DST), New Delhi, for financial assistance in terms of travel
grants and research fellowships. Further, I wish to recognize the emotional and moral
support extended by my father, mother, sister and brothers and for providing a peaceful
environment to pursue this work to the best of my abilities. My sister always told me what
Thomas Edison had quoted “Genius is 99 percent perspiration and one percent
inspiration”.
Last, but not least, I would like to thank all authors and the external reviewers for their
contributions. They have devoted their time and careful efforts in submitting reports and
reviews without any financial compensation. Their valuable evaluations have greatly
contributed to the quality of this book. I hope that this volume will inspire interest on the
diverse aspects of insect physiology and molecular biology in aspiring and established
scientists.
Raman Chandrasekar, Ph.D.,

Editor
Dedicated
to
Prof. M. Krishnan
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Short Views on Insect Molecular Biology, Vol. (1), 2009
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CONTENT Page No.
1. Perspective of Molecular Approaches to Entomology 1

Raman Chandrsekar
2. Molecular structure of insect clocks 21

Le Thi Dieu Trang, Qi-Miao Shao and Makio Takeda
3. Insect Hexamerin Storage Proteins: Biosynthesis, Utlization

and Evolution 49
Raman Chandrasekar, Muthu Meenakshi and Luiz Paulo MA.
4. Structural characteristics/ biosynthesis mechanisms of insect

Vitellogenin 69
Tufail Muhammad and Makio Takeda
5. Molecular mechanisms of insect Vitellogenin /lipophorin receptor 95

Tufail Muhammad and Makio Takeda
6. Nutritional signaling in adult mosquitoes 119

Immo A. Hansen and Geoffrey M. Attardo
7. Pheromone Biosynthesis Activating Neuropetide (PBAN) and

its G-protein coupled receptor 131
Rachel Bober and Ada Rafaeli
8. Molecular mechanisms of sex pheromone reception in Lepidoptera 147

Emmanuelle Jacquin-Joly and Martine Maïbèche-Coisné
9. Autophagic programmed cell death in the peripheral fat body tissues 159
of the silkworm, Bombyx mori L.
Sumithra P., Raman Chandrasekar and Krishnan M.
10. Apoptosis Regulation in Mosquito and its Importance to 175

Malaria Infection
Sylvester SL. Lyantagaye
11. Molecular mechanisms of cold hardiness in the European corn borer 191
(Ostrinia nubilalis, Hubn.)
Duško P. Blagojević, Mihajlo B. Spasić and Gordana N. Grubor – Lajšić
12. Molecular characteristics of the W chromosome, Z chromosome and

Retro transposable elements of the silkworm, Bombyx mori 203
Hiroaki Abe and Tsuguru Fujii
Appendix - A (Glossary and Terms) 229

Appendix - B (Author Index) 238
Contributing Authors
Prof. Makio TAKEDA Prof. Ada Rafaeli

Kobe University Department of Food Science &
Graduate School of Science & Technology Stored Products Unit
Japan. Agricultural Research Organization
The volcani Center
Prof. Krishnan, M. Israel.
Department of Environmental Biotechnology
School of Environmental Sciences Dr. Immo A. Hansen
Bharathidasan University Department of Biology &
India. Institute of Applied Biosciences
New Mexico State University
USA.
Dr. Tufail Muhammad
Kobe University, Dr. Hiroaki ABE
Graduate School of Science & Technology Department of Biological Production
Japan. Faculty of Agriculture
Tokyo University of Agriculture & Technology
Prof. Dusko Blagojevic Tokyo, Japan.
Department of Physiology
Institute for Biological Research Dr. Raman Chandrasekar
Serbia, YU College of Agriculture Entomology
2-255 Agricultural Science Centre Building – North,
University of Kentucky, Lexington, KY
Prof. Luiz Paulo Moura Andrioli USA.
Instituto de Biociencias
Universidade de São Paulo.
Cidade Universitária 05508-900 Dr. Sylvester Lyantagaye
Sao Paulo, SP, Brazil. Department of Molecular Biology and Biotechnology,
University of Dar es Salaam
Prof. Emmanuelle Jacquin-Joly P.O. Box 35179 - Dar es Salaam
UMR PISC INRA-UPMC-AgroParisTech Tanzania.
INRA centre de Versailles
Route de Saint-Cyr Dr. Geoffrey M. Attardo
F-78000 Versailles Yale School of Public Health and Epidemiology
France. 60 College Street, New Haven
USA.
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Short Views on Insect Molecular Biology, Vol.(1), 2009

External Supportive Members
Prof. Marc J. Klowden Prof. Randolf Menzel

Division of Entomology Freie Universität Berlin
University of Idaho Institut für Biologie - Neurobiologie
Moscow, ID 83844-2339 Königin-Luise-Str. 28/30
USA D - 14195 Berlin
Germany.
Prof. Enoch Y. Park
Innovative Joint Research Center, Prof. Frantisek Sehnal
Laboratory of Biotechnology Entomological Institute
Integrated Bioscience Section, Acad. Sci., Ceske Budejovice
Graduate School of Science and Tech. Czech Republic.
Shizuoka University,
Shizuoka, Japan. Prof. Reinhard F. Stocker
Institute of Zoology,
Prof. Lawrence I. Gilbert University of Fribourg
Department of Biology Pérolles, 1700 Fribourg
The University of North Carolina Switzerland.
USA.
Prof. Shalom W. Applebaum
Prof. Michihiro KOBAYASHI Mauerberger Professor Emeritus of
Division of Biodynamic Entomology
Graduate School of Biological Sciences Department of Entomology
Nagoya University Faculty of Agricultural, Food &
Japan. Environmental Quality Sciences
The Hebrew University – Rehovot Campus
Prof. Seo Sook Jae Isral.
Division of Applied Life Science
Gyeongsang National University Dr. Immo A. Hansen
South Korea. Department of Biology &
Institute of Applied Biosciences
Prof. Luiz Paulo Moura Andrioli New Mexico State University
Instituto de Biociencias USA.
Universidade de São Paulo.
Cidade Universitária 05508-900
Sao Paulo, SP, Brazil.
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Short Views on Insect Molecular Biology Lead Article
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(1), 1 – 20, 2009
Chapter – 1
Perspective of Molecular Approaches to Entomology
Raman Chandrasekar*
Department of Environmental Biotechnology, Bharathidasan University, Tamilnadu, India.
Abstract
Entomology today is a super science, having deviated considerably from the morphological-taxonomical approaches of
the first half of the century, embracing an interdisciplinary approach involving such aspects as biological diversity,
chemical ecology, circadian rhythms, neuroendocrinology, medical-entomology, molecular biology and biotechnology.
Molecular biology, integrated with genetics and biochemistry, has provided the necessary tools for transferring and
evaluating genetic characteristics not only for a host of insects, but also for related host plants. The molecular approaches
have enabled the study of physiological vital proteins and sensillar-neural complexes that are involved in pheromonal
studies. Such knowledge is vital to devise safe and specific agents for disrupting insect life cycles, thus increasing the
efficiency of efforts to manage agricultural pests and disease vectors.
Keywords: molecular biology, sensillomic, baculoviruses, display techniques, pheromone
*For Correspondence (email: koreanchandru@yahoo.com)
1. Introduction
Overview
Insects are the most successful species on
1. Introduction the planet. They have withstood the natural
2. Molecular and Biotechnology approaches calamities because of their extraordinary
3. Physiological vital Proteins diversity and adaptability. This vouches for the
(3a). Hexamerin storage protein obstacles that are encountered in the
(3b). Vitellogenin development of a foolproof strategy for pest
4. Trangenesis control. Apart from this, there are numerous
5. Sensillomics other factors that determine the effectiveness of a
6. Hormonomimetic Compounds pest control strategy. Most important of all is, to
7. Baculoviruses as Expression Vectors have an understanding of the significant
8. Seribiotechnology ecological roles played by “pest” species in both
9. Medical Entomology unmanaged and agricultural environments and a
10. Integrated pest management thorough knowledge of the physiological,
11. Concluding remarks morphological and biochemical aspects of the
12. Reference insect, despite this being an arduous task.
*Present Address: College of Agriculture Entomology,

S-225 Agricultural Science Center Building – North,
University of Kentucky, Lexington, KY, USA.
1
The biological knowledge will certainly provide the cue for developing a unique technology based
on the exceptional characteristic of the pest species. In short, if either biological knowledge or
technology is deficient, the control program will not be successfully accomplished (Pedigo, 1996).
Agricultural pest problems and action to alleviate them are nearly as old as the beginning of crop
cultivation. The earliest record of pest technology seems to be the use of sulfur and thereafter, the
advances made were tremendous. The, then popular insecticidal approach relied more on the ability to
kill the pests than plant protection and environmental protection. The conventional synthetic organic
pesticides are handicapped in the environmental context, by their long-term persistence, high toxicity
and propensity for bioaccumulation. Further, as a result of development of pest resistances to organic
pesticides, the concept of biological control increased. But, as natural enemies were non-dependable for
satisfactory containment, the concept of integrated control was developed. Methods of natural chemical
control to disrupt the pest's life cycle using semiochemicals or infochemicals (Dicke and Sabelis, 1988),
which refers to chemical signals that mediate interspecific and intraspecific interactions between
organisms, started gaining ground. This category includes pheromones and allelochemicals, which are
used to attract insects into traps, or confuse them or block some of their essential functions. Pheromones
mediate an interaction between organisms of the same species whereas allelochemicals mediate an
interaction between individuals that belong to different species.
The identification of neuropeptides such as allatotropins and allatostatins that regulate juvenile
hormone synthesis by corpus allatum has progressed considerably and promises new avenues of
neuroendocrine manipulation for insect control, in particular their interaction with the receptors of target
cells enabling hormonal imbalance in insects (Ananthakrishnan, 2001). Other insect hormones like
juvenile hormone analogs and ecdysones have also been exploited extensively. Considerable research in
the discovery and use of chemical inhibitors of juvenile hormone synthesis to cause chemical
allatotectomy, juvenoids and juvenile hormone antagonists to trigger developmental disorders and
ecdysone agonist/ antagonist to mediate insecticidal action, have been successful, but most of the time
they also have an adverse effect on beneficial insects. In recent years, increasing research has focused on
plant–derived insect antifeedants, which are non-pollutant, generally less toxic and easily biodegradable
especially if used as total or enriched extractives (Babu et al, 1996). The Sterile Insect Technique (SIT)
which involves the sterilization of the target pest by irradiation and sustained release of the large
numbers of sterilized insects to reduce the native population through infertile mating has been further
refined by recent progress in transformation systems that allow genetic engineering of diverse insects
(Wimmer, 2005). The new trend involves the use of fluorescent transformation marker for identifying
released insects, sex-separation methods based on female-specific expression of a conditional dominant
lethal gene and a transgenetic system that causes embryo-specific lethality after transmission to the
progeny, which could replace irradiation technique and produce more competitive sterile insects. There
is also a demand for increasing use of baculovirus and NPV in pest management for some of the
important agricultural pests.
In the past, singular strategies were used for the control of a pest species based on diverse aspects
of either biological, physical, chemical. Maybe that was the reason behind the failure of these strategies!
But now the trend is to integrate all individual techniques and tactics for insect pest management and use
them in a system that combines the most appropriate characters of each to produce an ecologically sound
and economically viable package called integrated pest management (IPM) (Ananthakrishnan, 2001).
This strategy is based on a clear understanding of ecological functioning of the agroecosystems. An
integration of pest monitoring and accurate timing of pesticide treatment, use of selective insecticides as
well as combined use of semiochemicals, host plant resistance, trap crops, oviposition deterrents,
antifeedents and of recent, biomimetics are important to manipulate the pest behaviour, effectively and
efficiently. This lead to the metamorphosis of the term: stimulo-deterrent diversionary strategy (SDDS)
(Ananthakrishnan, 2003).
2
2. Molecular and Biotechnology approaches
Biochemistry, genetics and

molecular biology have in recent Application of “OMICS” technology to
years become essential ingredients in
our understanding of entomological
Biotechnology
problems. With the molecular
biology approach, a different
emphasis has resulted from obtaining s ys
and handling DNA which has te m
sb
provided the necessary tools for io lo
transferring and evaluating genetic gy
o r k s
mo
characters from a host of insects. tw
e ne
le c
Genomics is a broadly used term gen metabolomics
u la
encompassing numerous scientific
rs
disciplines and technologies (Fig.1).
tr u
These disciplines include: genome
transcriptomics proteomics
c tu
sequencing; assigning function to
res
identified genes; determining genomics
genome architecture; studying gene
expression at the transcription level
(transcriptomics); studying protein Fig.1 Schematic diagram showing the branches of molecular
expression at the proteome level biology.
(proteomics); and investigating
metabolite flux (metabolomics)
(Fig.2). The polymerase chain reaching (PCR) enables repeated duplication of a trace amount of DNA
resulting in sufficient amount for detailed DNA analysis.
Fig.2 The Molecular basis of life.
The detection of specific fragments of DNA is made possible through the restriction fragment
length polymorphism (RFLP) technique. For identifying the degree of genetic variability of samples, the
random amplified polymorphic (RAPD) technique is used, in particular to characterize genera, species
and races, as in the studies of biotypes of mustard aphids and whiteflies from different regions.
Reproducibility of RAPD-PCR results, when verified, showed that RAPD patterns were consistent for a
given genotype (Dilawari and Gupta, 2004). Beside the routine DNA sequences, the use of microsatellite
3
loci (MSL) as genetic markers has become an established technique. With the molecular biology
approach, potentially resistant genes can be removed and transferred by a vector to a crop plant. A
common example is the incorporation of the Bacillus thuringiness insecticidal crystal protein gene into
host plants to have the toxic gene expressed in the host plant to give protection against several species of
caterpillars. Informational molecules such as proteins and nucleic acids are now believed to have high
taxonomic information, so that molecular approaches to systematics have become increasingly familiar
(Fig.3).
Nucleous
DNA (structural genomics)
Pre-mRNA
cytoplasam
mRNA
mRNA (Transcriptiomics)
Protein
Protein (Proteomics)
(Proteomics)
Functional
Genomics
Metabolites (Metabolomics)
Fig.3 Moving from targeted investigations of few genes to the investigation of many (all)
genes in untargeted experiments that use highly parallel approaches in order to
identify unknown genes.
3. Physiological vital Proteins

(3a) Hexamerin storage protein
In holometabolous insects, the larval period is an essential stage required for the accumulation of
nutritional reserves for the non-feeding pupal stage. Essential nutrients obtained during the larval stage
but needed by the non- feeding pupal stage must be sequestered and carried across stages until they are
mobilized. Storage proteins (SP) appear to be a special adaptation in insect molting, metamorphosis, and
cyclic reproduction, and have no known analogue in the vertebrates. The transformation from larval
caterpillar to non-feeding pupal and adult moth involves a complete remodeling and restructuring of the
insect, and its organs. The insect fat body is recognized as a complex multifunctional tissue: which
participates in multiple biochemical and physiological functions, and it is a major site for the synthesis
and storage of carbohydrates, lipids, proteins and nitrogenous components (Keeley, 1985; Haunerland et
al.,1990). Due to the existence of fat body heterogeneity and by correlation of their structure and
functions in Hyalophora cecropia (Tojo et al.,1978), G. mellonella (Bean and Silhacek, 1989), Heliothis
zea (Wang and Haunerland, 1992) and B. mori (Vanishree et al., 2005), Amsacta albistriga
(Chandrasekar et al., 2008a,b) these storage proteins are synthesized abundantly during the active
feeding larval period in the peripheral fat body (PF) tissues and subsequently the proteins are released
into the haemolymph. In a previous paper on the red hairy caterpillar, Amsacta albistriga we reported
on regionally differentiated fat bodies (peripheral and perivisceral) associated with the segregation of
synthetic and storage activities (Chandrasekar, et al.,2007; Chandrasekar, et al.,2008a). Additional
4
evidence of ultra thin sections and immunogold labeling studies during the spinning stage provided
direct evidence for the sequestering ability and huge accumulation of SP hexameric crystalline granules
which were observed in perivisceral fat body tissue (PVF) of A. albistriga (Chandrasekar et al.,2008b)
(Fig.4).
The appearance of storage

proteins in the haemolymph and
fat body of one day old sixth
instar larvae was in concert
with the findings of Yoshiga et
al. (1997). The storage proteins
showed fluctuation patterns
similar to that reported by many
researchers (Tojo et al., 1981;
Ryan et al., 1985; Nagata and
Kobayashi, 1990; Yoshiga et
al., 1997). Insects prepare for
the synthetic demands of
molting, metamorphosis and
reproduction by accumulating
storage proteins in their
haemolymph and fat body (Pan
and Telfer, 2001). The storage
proteins are basic proteins, that
are hexameric and have been
Fig.4 Electron micrograph of perivisceral fat body (PVF) tissues
best characterized with respect from day 2 pupa of Amsacta albistriga showing abundance of protein
to their role in metamorphosis granules. Scale bar - 3µm. PG – Crystalline protein granules;
(Telfer and Kunkel, 1991; Pan L – lipid droplet (From: Raman Chandrasekar, 2006).
and Telfer, 2001). Most proteins
destined for eggs must be
obtained during the larval stage and stored until synthesis of yolk proteins begins. The storage proteins
are obviously the storage reserve of amino acids (Wheeler et al., 2000, Chandrasekar, 2008b).
Physiological links between the amino acids stored in storage proteins and those amino acids used in
vitellogenesis may be common in insects. The uptake of hexamerins is an important process that allows
holometabolous insects to survive during the non-feeding pupal period (Fig.5). In addition, the dynamics
between sequestration and release of amino acids from storage proteins in the pupa/adult stage may be an
important feature enabling many of their diverse strategies of reproduction.
Peripheral fat body tissues

Perivisceral fat body tissues
Fig.5 Schematic diagram showing the synthesis

and uptake mechanism of hexamerin storage
protein from PF to PVF tissue during larval-
pupal transformation (From: Burmester and
Scheller, 1999).
5
(3b) Vitellogenin (Vg)

The developing embryo of oviparous animals and insects draws practically all of its requisite
nutrients from a cache of proteins, lipids, and carbohydrates stored within the egg as yolk (Sappington et
al., 1995, 1996). Yolk is the major internal food supply on which most embryos rely. Strict regulation of
its utilization is essential to provide nutrients at the right time to the developing tissues, and to ensure
survival of the embryo until it becomes a free-living organism, able to feed (Fagotto, 1995; Salerno et
al., 2002).
Vitellogenin (Vg) is a multifaceted protein and so,

analysis of this protein would serve diverse purpose. Many
behavioral, ecological and physiological factors are known
to induce a phenomenon called oosorption in various
insects. When oosorption is induced, constituents that had
accumulated in the oocytes are transported outside the
oocytes, by a mechanism that has not been fully elucidated.
A
Starvation induced oosorption in the females of Plautia
crossota stali, elucidated that the Vg already accumulated in
developing oocytes was released into the haemolymph
(Kotaki, 2003). But the mechanism needs to be studied.
Recently, fish Vg received attention as a biomarker for
estrogenic compound in aquatic areas. Normally, Vg is
observed in vitellogenic female serum however, male and
juvenile fish can also synthesize Vg when exposed to
B
exogenous estrogen (Specker and Sullivan, 1994) or to
substance that mimic estrogens. Therefore, Vg is used as a
biomarker for monitoring mimic estrogens in aquatic areas
(Fakuda et al., 2003). Another alternative use of Vg had
been reported by Amdam et al. (2003). Vg was found to be a
source for the formation of the proteinaceous royal jelly that
is produced by the hive bees which is used to feed larvae,
queen, workers, and drones (Koeppe et al.,1985). This
C
finding suggested that the evolution of a brood-rearing
worker class and a specialized forager class in an advanced
eusocial insect society has been directed by an alternative
utilization of Vg (Amdam et al., 2003).
The Vgs of most insects are members of a conserved

family of proteins that are present in organisms as diverse as
nematodes and vertebrates (Sappington et al., 2002). The Vg Mature
D
Yolk body
primary product has been thoroughly characterized from 14
insect species, six of them belonging to hemimetabola and
six belonging to holometabola (Sappington et al., 2002). Fig. 6. Schematic diagram showing
The comparison of the protein primary structures of Vgs the vitellogenin (Vg) uptake by the
from different insect species has shown that they are highly ovary in insect.
conserved and form a gene superfamily (Chen et al., 1997;
A. Receptor-mediated endocytosis,
Lee et al., 2000; Sappington et al., 2002). The insect Vgs B. Accumulation of Vg
have been shown to be homologous with the Vgs of other C. Formation of Transitional Yolk
invertebrates such as nematodes and those of oviparous Body, D. Mature Yolk Body
vertebrates such as amphibians and birds (Chen et al., (Courtesy: Prof. Keeley, L. L. , Texas
1994). Similarly, insect Vg share homology with Vgs of other A&M University)
arthropods, such as millipedes (Prasath and Subramoniam, 1991),
ticks (Chinzei et al., 1983; Rosell and Coons, 1991) and
Crustaceans (Chen and Chen, 1993).
6
The process of Vg uptake in the insect species present morphological peculiarities that are related
to their life strategies. Specific binding of Vg to oocyte membrane preparations and solubilized
membrane proteins has been demonstrated for insects, amphibians, fishes and birds (Opresko and Wiley,
1987; Stifani et al., 1990; Hiramatsu et al., 2002; Patino and Sullivan, 2002; Li et al., 2003). The initial
steps in the yolk uptake pathway are similar to those described for general receptor-mediated endocytosis
but in contrast to the degradation of the internalized ligands in lysosomes, the yolk proteins are stored as
yolk granules for later use during embryogenesis (Fig.6). The yolk granules appear to be modified
lysosomes with relatively high pH; during embryogenesis, the pH of the yolk granule drops to levels
more typical of lysosomes (Fagotto, 1995). The single layer of follicle cells that surrounds oocytes
constitutes an additional barrier to yolk precursors entering the oocyte. For invertebrates, the yolk protein
receptor is described for the fruit fly, D. melanogaster (Schonbaum et al., 1995), and Vg receptors
(VgRs) are described for the mosquito, A. aegypti (Sappington et al., 1996); the mosquito, Anopheles
gambiae ; the American cockroach, P. americana; the silkworm B. mori (NCBI) and the nematode,
Caenorhabditis elegans (Grant and Hirsh, 1999). In both insect and vertebrate VgRs, binding of Vg was
saturable, ovary specific, showed high Vg affinity, was inhibited by suramin and was sensitive to
changes in pH and Ca2+ concentration (Konig and Lanzrein, 1985; Osir and Law, 1986; Konig et al.,
1988; Dhadialla and Raikhel, 1991; Wang and Davey, 1992; Bujo et al., 1994). An integrative study on
the hormonal influence on the process of receptor-mediated endocytosis is required to clearly portray the
mechanism of regulation of this process so that it would provide a cue for the designing of a gene
delivery system.
In the perpetual arms race against the insect pests of agricultural importance, it is imperative to
develop novel strategies for pest control. But for this, it is indispensable to be equipped with a thorough
understanding of the biological system of the insect pest at the cellular and molecular levels, which apart
from opening new avenues in the development of novel strategies after molecular modeling of the SP-
SPR, Vg-VgR, interactions and Vg/SP analog-designing, might certainly contribute to the success of it
by overcoming possible loopholes. Nonetheless, such a basic yet in depth study will also find an
application in enhancing the favorable characteristics of a beneficial insect. So, in this context the recent
trend involves the identification and characterization of physiological essential proteins like apoLp-III,
ferritin, storage protein, vitellogenin, lipophorin and their receptors, which can be subjected to various
refinements and manipulations for their exploitation in combating the resistant pest species, in a target
specific and eco-friendly manner.
4. Transgenesis
The first Bacillus thuringiness (Bt) toxin gene from Bacillus thuringiensis Berliner was cloned in
1981 and the first transgenic plants were produced by mid-1980s. Since then, several crop species have
been genetically engineered to produce Bt toxins to control the target insect pests. Genes conferring
resistance to insects have been inserted into crop plants such as maize, cotton, potato, tobacco, rice,
broccoli, lettuce, walnuts, apples, alfalfa and soybean (Bennett, 1994; Federici, 1998; Griffiths, 1998).
The first transgenic crop was grown in 1994 and large-scale cultivation was taken up in 1996 in USA
(McLaren, 1998). Since then, there has been a rapid growth in the area under transgenic crops in USA,
Australia and China. Transgenic plants, with insecticidal genes, are set to feature prominently in pest
management in both developed and the developing world in the future. Among the developing countries;
China, India, Argentina, Mexico, Brazil, Pakistan and South Africa are pursuing the research on
transgenic crops vigorously. Entomologists, breeders and the molecular biologists need to determine how
to deploy this technology for pest management, and at the same time avoid or reduce possible
environmental risks. To achieve these objectives, it is necessary to have an appropriate understanding of
the insect biology, behaviour, its response to the insecticidal proteins, temporal and spatial expression of
insecticidal proteins in the plants, strategy for resistance management, impact of insecticidal proteins on
natural enemies and non-target organisms. Equally important are the issues concerning the transfer of
technology to the resource poor farmers. Development and deployment of transgenic plants with
7
insecticidal genes for pest control will lead to: (a) reduction in insecticide sprays, (b) increased activity
of natural enemies, and (c) IPM of secondary pests.
In recent years we have been witnessing a major revolution in entomological science, more
especially with increased understanding of biological systems at the cellular and molecular levels. I am
referring to the area of biotechnology relating to the fields of rDNA technology and genetic engineering
and transgenics-areas which have great potential application in agriculture. Much of the current interest
is centered on creation of pest resistant, herbicide resistant and disease resistant transgenic plants. The
discovery that the aerobic, Gram-positive spore forming bacterium, Bacillus thuringiensis Berliner (Bt),
which produces a toxic protein during sporulation, has introduced a new era of genetically engineered
biocontrol agents.
The successful adoption of Bt crops by farmers has led some to muse on the possibility that the
widespread use of Bt proteins in crops will lead to the development of insect populations that are
resistant to these proteins, thus rendering Bt crops and Bt sprays less effective in controlling the
destructive insects. The U.S. National Academy of Sciences recommended the development of insect
resistance management (IRM) plans for both Bt crops and sprayable Bt pesticides. The goal of these
plans is to ensure continued effectiveness of both the plant expressed and the sprayable formulations of
this family of pesticidal proteins. A coordinated scientific approach is used to establish management
practices that will minimize the risk of resistance and sustain the performance of Bt pesticidal proteins.
Recently developed IRM plans for Bt crops couple Bt plants with a structured "refuge" of non-Bt plants.
Refuge refers to a portion of the crop plants in or near a field that does not contain the Bt protein. A
typical IRM plan in corn requires that at least 20% of the grower's corn acreage be planted to a non-Bt
corn refuge within 1/2 mile of the Bt corn fields. The purpose of the refuge is to maintain a population of
target insect pests that are susceptible to the Bt protein. Those susceptible insects can mate with rare
resistant insects that may emerge from the Bt crop so that the resulting offspring will be susceptible to
the Bt protein.
Moving Bt insecticide genes from the original bacterium to plants through the mediation of a
vector, Psedomonus flourescens, accords crop protection from insect attack. By far the greatest research
effort in developing pest resistant transgenic crops has gone into the expression of Bt toxins or delta
endotoxins or crystalline proteins in different crop plants and tobacco and tomato provided the first
examples of genetically engineered insect resistance. Being related proteins, several strains of this
bacterium are known with distinct host ranges. At least ten genes encoding different Bt toxins such as
cry1Aa, cry1Ab, cry1Ac etc., have been engineered into plants and the different toxins have different
specifications for different orders of insects, notably Lepidopotera (Cry I), Coleoptera (Cry III) and
Diptera (Cry II). The heterogeneity in toxin production is responsible for some of the diversity in the
activity spectrum among strains (McGughey and Whalon, 1992). Currently there are other major groups
of plant derived genes used to confer insect resistance in crops and which are inhibitors of digestive
enzymes. These are the proteinase. Deciphering the chemical nature and information content of the
molecules that transmit information between insects, plants and natural enemies. Volatile
phytochemicals tend to promote or deter interactions between plants and phytophagous insects. Basic
volatiles related from leaf surfaces form constitutive chemical defense which often include
monoterpense, sequiterpenes and aromatics which accumulate considerably in plant parts. Chemical
signals and signaling systems in insects hold great promise in the utilization of semiochemicals in insect
control.
Recent research on induced defenses aims at generating intra- and inter-plant signals in plants due
to insect damage. An induced defense is ‘a special mosaic of defensive chemistry’. Some of the best
examples relate to the release ethylene, jasmonic acid and methyl jasmonate, salicylic acid and abscissic
acid in insect damaged plants, notably jasmonic acid which can induce the expression of proteinase
inhibitor gene even in nearby plants. Today we know that plants offer natural enemies with chemical
information about the presence and densities of insect hosts. To cite an example, terpenes such as beta-
ocimene, beta-farnescene, linanool and compounds like hexenyl acetate are released on damage and
8
several parasitoids and predators are known to exploit insect induced signals to detect their hosts. The
ability of host seeking insects to recognize and respond to such chemical cues is an essential aspect in
titrophic interactions. Such is the dependence of parasitoids on plants signals that even a slight genetic
change in plants may result in a decline of parasitism.
5. Sensillomics
The integration of the sensillar and neural complexes besides the biochemistry of chemoreception
involving molecular components naturally calls for a new approach designated as “Sensillomics”. In
order to fully integrate the sensillar and neural systems in chemoreception, a new approach designated as
“sensillomics” is required in addition to a biochemical one. While the morphometric approach involving
the ultrastructure of insect sensilla has been utilised to some extent, its functional counterpart, the
electrophysiological recording has not received the attention it deserves. The behavioural significance of
diverse chemical species that phytophagous insects encounter lies in the fact that neurons transmit
information between each other and throughout the neuronal network, via signals called nerve impulses.
These consist of spikes and action potentials. The ability to recognize specific molecules is inherent to
the receptor sites, such as those of a sexual pheromone selectively binding proteins in the olfactory
sensilla. Once the signal is detected it needs to be amplified in order to obtain a relevant response. The
axon from the olfactory sense cells end in an area of the brain called the glomeruli, considered to be
neuropile structures where integration of sensory inputs take place, thus enabling signal amplification.
The insect antenna (Fig.7) serves as a molecular ‘sieve’. Only those molecules absorbed in the sensory
hairs have a chance to cause a response in the dendrites of the receptor cells. Molecular absorption
increases the efficiency of such molecular trap. As the primary sites where an extensive integration of
olfactory inputs occurs, the glomeruli serve as centers in which inputs from the sensory cells with similar
response characteristics merge. The adaptive nature of a specific olfactory sensitivity has enabled a
better understanding of the response to chemical species. As a result, signals and signaling have become
the essential components in behavioural studies. The direct pathways for transmitting information are
located in the antennal lobe of the brain, while the general binding proteins occur in both sexes,
glomeruli are species-specific in terms of number, size and arrangement. Glomeruli have been mapped in
a number of insect species (Ryan, 1990). It is therefore in the understanding of the significance of the
sensillar-neural complexes, involved in the recognition of molecules such as pheromone binding proteins
that the composite field of sensillomics is concerned with. The cross-disciplinary field of sensillomics
thus puts an emphasis on the sensillar-neural complexes that handle signals arising from the interaction
between pheromones and specific binding proteins (Ananthakrishnan, 2003).
Fig.7 A pair of pectinated antennae

on the head of a male silkmoth. The
antennal branches are carrying
numerous cuticular expansions, the
sensilla.
Further, our understanding of pheromone production has evolved from identifying biochemical
pathways towards unraveling the molecular biology of key regulatory enzymes initiating a genomics
approach. Today we know of the pheromone binding protein (PBPs) and pheromone degrading enzymes
(PDEs) which inactivate pheromone signals within the sensilla. Biochemical transduction pathways are
now identified including all important olfactory receptor proteins. A marriage of molecular genetics and
genomics behaviour has resulted in a new understanding of how pheromones are detected. It is today
well known that olfaction and taste work via sensory neurons and that odour and taste molecules
stimulate these neurons by binding to receptor proteins.
9
6. Hormonomimetic Compounds
It is interesting that plants are also known to synthesize compounds that distrupt normal
development of the insects which feed on them. Examples of such compounds are phytoecdysone,
juvabione, anti-juvanile hormones or precocenes, and juvocimenes that mimic the functions of insect
hormones and are hence designated as hormonomimetic compounds. With advances in genetic
engineering techniques, it has been possible through structural elucidation of polypeptides to not only
identify the genes aiding in their synthesis, but also develop inhibitors of enzymes mobilizing such
synthesis. Incorporation of such genetic factors into host plants, employing baculoviruses as carriers,
may modulate production of hormones which can disrupt the physiology of the insect invader.
Genetically engineered transgenic plants with the ability to synthesize defensive proteins such as
phytoagglutinins, lectins and proteinase inhibitors are considered as alternate means to avoid excessive
use of biocides that also tend to disrupt the natural enemy complexes. Evidence also point to insects’
ability to overcome resistance based on single gene expressions (Van Emden, 1991), and therefore
emphasis is currently placed on the need to evolve multigene resistance mechanisms.
7. Baculoviruses as Expression Vectors
Baculoviruses are unique in that they form useful expression vectors. It has been suggested that by
introducing appropriate foreign genes into the baculovirus genomes, pathogenicity and insecticidal
effectiveness can be increased. In the formation of baculovirus insecticides, a recombinant baculovirus
of increased toxicity is made by introducing appropriate foreign genes into highly expressed polyhedron
gene sites. Infection with such recombinant viruses may cause direct toxicity, late behaviour or arrested
development of insects. Recent advances in recombinant DNA technology and the genome engineering
of insect baculoviruses have made possible the use of insect baculoviruses as expression vectors for
neurohormone genes. Neurohormones, by virtue of their peptidic nature are amenable to applications
using recombinant DNA and genetic engineering technology.
8. Seribiotechnology
Research on the silkworm Bombyx mori

has a long history, but Seri-biotechnology is an
emerging field of “Insect Biotechnology”. The
potential of silkworms to produce high levels
of exogenous proteins has attracted the
attention of academic and industrial
researchers. The silkworm is a useful model
for genetic, biochemical and physiological
studies, as an insect model system it occupies a
position next to the fruit fly, Drosophila
melanogaster, but is the system of choice
among the Lepidoptera. Hence, recently we
demonstrated that P-soyatose (hydrolyzed soy
bean protein) supplementation is of importance
in regulating the fibroin gene expression at
transcriptional level (Chandrasekar et al.,
2007b). This would be a practical contribution
to sericulture for maximizing economic traits
of economically important insect B. mori.
Insect offer many advantages for gene

regulation studies compared to vertebrates
or vertebrate cell culture. There is also a great Fig.8. Life cycle of NPV baculovirus in nature.
10
variety of applied problems with insect pests that need to be addressed. Differential gene expression
underlies a range of biological processes, including development, reproduction, and behavior (Harshman
and James, 1998). The silkworm has many advantages over other model organisms. However, in contrast
to D. melanogaster, B. mori lacks an efficient germline transformation system. Plasmids and viruses are
the contemporary vehicles for gene therapy and genetic vaccination and extremely promising results
have been reported from in-vitro, in-vivo and clinical studies. At present many recombinant proteins are
manufactured with a technology which has been directly transferred from laboratory to pilot scale
without further engineering (Fig.8). This approach has been more widely adopted in Asian countries,
including China, Japan and India, where silkworms are abundantly available and more laboratories have
experience in growing and maintaining larvae. The silkworm, B. mori, is one of the most attractive hosts
for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer
to mammalian cells. Baculovirus expression systems (BESs) are widely used to express heterologous
genes in cultured insect cells and insect larval hosts; gene expression is driven by the strong polyhedron
promoter (Kato et al.,2005; Gubitosi-Klug et al.,2005; Smith et al.,2007). Conventional preparation of
recombinant baculovirus requires at least 40 days. Now, it has been established that BmNPV bacmid
system provides the rapid protein purification in silkworm (as long as 10 days), is free from hazard, and
will be a powerful tool for the future production factory of recombinant eukaryotic proteins and
baculoviruses (Motohashi et al.,2005). Results have shown that the earliest pupal stages are the most
suitable for the production of viral proteins and probably for the expression of foreign genes under the
control of late viral promoters (Mikhailov et al., 1992; Park et al.,2008). In addition to recombinant
protein production, the surface display technology of recombinant protein on the surface of baculovirus
has been developed (Grabherr et al.,2001; Oker-Blom et al.,2003) and baculovirus displaying foreign
protein is used for monoclonal antibody production (Saitoh et al.,2007), subunit vaccines (Peralta et
al.,2007) and construction and screening of eukaryotic epitope library.
The display of proteins on the surface of

bacteria requires a mechanism by which a
desired, recombinant polypeptide: (a) is
exported from the cytoplasm, a process
typically involving the participation of the
protein secretory apparatus of the cell, (b) is
targeted to the outer membrane, and (c) can
transverse the outer membrane so that it
anchors to the external surface. Unfortunately,
the mechanisms that dictate targeting and
insertion of proteins within the outer membrane
are not well understood. Moreover, the
incorporation of aberrant proteins within the
outer membrane can be toxic to the cell. Poor
exposure of surface anchored heterologous
polypeptide sequences, typically resulting from
steric effects caused by the lipopolysaccharide
layer on the outer membrane, can prevent the Fig.9. Schematic diagram for the preparation of
interaction of surface-displayed heterologous functional bacterial magnetic particles (BacMP) using
proteins with antibodies and small molecule the protein display technique. To express a functional
protein onto BacMPs, the functional protein gene was
ligands. fused to an anchor protein gene. A plasmid containing
the fusion gene was transformed into M. magneticum
Baculovirus display strategies have AMB-1. Expression efficiency is strongly dependent on
also been used for modification of the viral the expression level of the anchor protein. The
surface to influence baculovirus-mediated functional BacMPs produced were extracted by
transduction of mammalian cells (Kost et disrupting cells. (From: Matsunaga et al. (2007) in
al.,2005). It has also been shown that TRENDS in Biotech..).
membrane proteins produced in infected
11
insect cells can be incorporated into baculovirus particles in a functional form. This was first observed
for the β-2-adrenergic receptor, which was recover in a functional form complexed with heterotrimeric
G-proteins (Loisel et al.,1997) and more recently for the human leukotriene B4 receptor (Masuda et
al.,2003). This approach has been used successfully to produce a functional γ-secretase complex on the
surface of baculovirus particles (Hayashi et al.,2004). Co-infection of Sf9 cells with viruses produced
virus particles with γ-secretase activity that was concentrated ~2.5-fold higher in the budded virus
particles as compared to Sf9 cell membranes. These studies show that baculovirus particles can provide
a unique scaffold for the assembly and enrichment of functional membrane bound protein complexes.
Current trends in the development of nanoparticles such as quantum dots, gold particles, and magnetic
particles recently have attracted research interest because of the uniqueness of their physical properties,
and they have been used a immobilization support (Phadtare et al.,2004; Yang et al.,2004), probes
(Akerman et al.,2002; Gao et al.,2004) or catalysts (Samuelson et al.,2002). These techniques have
been achieved using anchor molecules to display foreign proteins on the surface of microorganisms.
Foreign proteins also have been displayed on the surface of magnetic nanoparticles (Fig.9). Protein
display on bacterial magnetic particles was realized by the development of a fusion technique involving
anchor protein isolated from magnetic bacteria (Okamura et al., 2000). Recently, novel proteins tightly
bound to Bacterial Magnetic Particles (BMPs) were discovered in T. Matsunaga’s laboratory (Yoshino
and Matsunaga, 2006). These proteins were highly expressed in the lipid bilayer membrane covering
the BMPs.
In addition very high levels of expression of luciferase through recombinant BmNPV in the larval
caterpillar (10 mg recombinant protein per larva) were achieved, which resulted in the generation of
‘Glowing silkworms’ emanating significant luminescence on administration of the substrate luciferin
(Motohashi et al.,2005). A number of studies have documented enhanced protein production following
cotranfection with baculoviruses expressing chaperone proteins, which are know to aid in the folding
and modification of newly synthesized proteins. The expression of correctly assembled shaker
potassium channels in Sf9 cells was enhanced by coexpression of the calcium-binding, lectin-chaperone
calnexin together with substitution of the polyhedron promoter with the weaker basic protein promoter
to derive expression of the ion channel. Recent studies showed that co-transfected 3GnT2 of human
origin fused with GFP (green fluorescent protein) marker protein as a model protein and hCRT (human
calreticulin) as a model chaperone to assist in folding in endoplasmic reticulum (Park et al., 2008).
These studies demonstrate potential value of co-expressing chaperones to enhance functional protein
production.
Furthermore, the identification of new higher value functional nanoparticles displayed antibody
using BmNPV bacmid bioproducts is a chance for short term successes in industrial biotechnology.
Enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering
can contribute to develop new metabolic pathways, may be even for unnatural compounds. However,
the efficiency and stability of proteins displayed on functional proteins have been limited. Hence the
overall study required for development of strategies for displaying foreign peptides and proteins on
virus particles and the insertion of mammalian cell active expression cassettes in baculoviruses to
express genes efficiently into many different mammalian cell types (Sf-9 and Tn-5B1-4 cells). BmNPV
engineered to display foreign peptides and functional proteins on the viral surface have been proven
particularly useful as immunogens and surface nanoparticles display of fusion protein. The ability to
observe and count the antigen-bound gold nanoparticles is a novel method and suggests that antibody-
antigen binding was successfully observed at the molecular level, leading to high sensitivity. Protein-
based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as
selective drug delivery systems for specific cell types.
9. Medical Entomology
Applied entomology encompasses a broad range of research areas (agricultural, forestry, domestic
and medicolegal, etc.). Medical and veterinary entomology (MVE) is a key field that involves: (a)
12
communicable diseases, including parasitic (malaria, trypanosomosis, leishmaniosis, filariosis, etc.),

bacterial (plague, typhus, Lyme disease, etc.) and viral (yellow fever, dengue, hemorrhagic fever, West
Nile, etc.) forms; (b) allergic phenomena; (c) pests; (d) envenomation protection. Entomology also deals
with some domestic damage caused by insect pests (Dominique and Jean-Antoine 2004).
Arthropods—which are responsible for transmitting a broad range of diseases—have infinitely

varied behavioural patterns: blood feeding (by vectors and pests), inoculation of venomous toxins
(envenomation by stinging by black flies, hymenopterans, ticks, scorpions or spiders), and passive
transport of pathogens (anthrax, trachoma, salmonellosis). Moreover, epidemiological systems
(metazoonosis) and intravectorial cycles (anterograde or posterograde digestive infestations in vectors of
trypanosomosis, transovarian transmission of viruses and rickettsia) are highly complex.
In this context, MVE specialists require ‘broad-ranging scientific knowledge and extensive field
experience.’ Besides basic entomological training, they must have a full overall understanding of agents
that are transmitted (parasites, bacteria, viruses), epidemiological cycles (pathogen systems) and
concerned environments, in both natural ecosystems and ones changed by man (e.g. agrosystems).
Medical and veterinary entomologists are specialised to deal with this extraordinary pathogenic and eco-
epidemiological complexity (Geong, 2001; Mouchet and Bellec, 1990).
Chikungunya virus is geographically distributed in

Africa (Jawetz and Adelberg’s, 2004; Power et al., 2007),
Southeast Asia (Lam et al., 2001; Laras et al., 2005; Parola,
et al., 2006) and India (Chandrakant and Pradhan, 2006;
WHO Report, 2006). Sporadic cases are regularly reported
from different countries in the affected regions (Power et al.,
2007; Laras et al., 2005; Chandrakant and Pradhan, 2006;
Halstead et al., 1969; Warner et al., 2006). Aedes albopictus
(Fig.10) was implicated as a vector of the chikungunya virus
(CHIK) disease of humans, the hallmark feature of CHIKV
disease is a debilitating and prolonged arthralgic syndrome
that primarily affects the peripheral small joints. The pain Fig.10 Aedes albopictus
associated with CHIKV infection of the joints typically
persists for weeks or months causing serious economic and social impact on both the individual and the
affected communities. The most recent epidemic re-emergences were documented by International
Society for Infectious Disease’s Program for Monitoring Emerging Diseases and WHO in Kinshasa
(1999–2000), Indonesia (2001–2003), the Indian Ocean islands of Mayotte, Mauritius, Re´union, and the
(2005–2006), Singapore (2006), Hong Kong (2006), India (almost 1.4 to 6.5 million estimated cases in
2006–2007), Japan (2007) and Europe Spain, Gabon, Italy (Ravi, 2006; WHO Wkly Epidem Rec 2000;
Shailendra, 2007; Queyriaux, et al., 2008). Globalisation, and the increased travel and trade that it
brings, is also quickening the pace at which diseases can spread to new areas. In addition to the influence
of increasing temperatures (Global warming) and rainfall through warming of the oceans, and alteration
of the natural cycles that stabilise climate, one is inevitably drawn to the conclusion that Chikungunya
virus will continue to emerge in new regions (Power et al., 2007; Shailendra, 2007; Tyagi, 2007).
Scientifically, the large scope of the outbreaks has provided opportunities to accurately document
transmission and epidemiological patterns associated with movement of the virus an interest in
expanding basic and applied scientific knowledge. But, there is a critical lack of knowledge on the
biology of CHIKV, contrasting with related model alphaviruses like Sindbis virus (SINV), Semliki
Forest virus (SFV), and Ross River virus (RRV); this probably reflects the fact that CHIKV has mostly
afflicted persons in developing countries (Marion Sourisseau et al., 2007). Currently, no effective
therapeutic drugs or licensed vaccine is available for prevention against this pathogenic virus. Therefore,
there is an immediate need for the research on CHIKV cirus, for an effective vaccine besides
strengthening the existing diagnostic laboratory facilities.
13
The genome of CHIKV consists of a linear, positive-sense, single-stranded RNA of approximately

11.8 kb, and contains structural genes that encode three structural proteins; E1 and E2 of envelope, and
nucleocapsid protein (Samuell and Diamond, 2006). Viruses have developed amazing strategies to
exploit cellular machineries like vesicular and protein trafficking, biosynthesis and sorting machineries.
Alphaviruses attach to poorly characterized receptors on many different cell types in various species.
Individual viruses have different, but wide tropism, accounting somewhat for different disease patterns.
Recently, Cheu et al. (2006) has also dealt extensively with molecular characterization and analysis of
the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus West Nile Virus
(WNV) in A. albopictus. Very interestingly, they have identified αVβ3 integrin served as the functional
receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E)
was postulated to mediate virus binding to the cellular receptor (Lee et al., 2006). Further, the results
indicate that the WNV enters into the mammalian and mosquito cells C6/36 through clathrin-dependent
endocytosis and viral particles colocalize with both early and late endosomes (Chu and Nag, 2004a;
Chu and Nag, 2004b; Chu et al., 2006a,b).
This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the
fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive
the fusion reaction (reviewed more extensively in Margaret Kielian, 2006). Interestingly CHIKV virus,
E1 envelope protein is a class II fusion protein that mediates low pH-triggered membrane fusion during
virus infection. The E2 envelope protein is a type I transmembrane glycoprotein and has been known to
be responsible for receptor biding during the course of alphavirus cycle (Byungki Cho et al., 2008;
Seema and Jain, 2005). So far, alphaviruses have been mostly studied in murine and other animal cells.
In particular, the interaction of CHIKV with mammalian and mosquito cells C6/36 cells has not been
extensively characterized. Further molecular characterization of receptor and ligand-receptor binding
affinity by using modern-day techniques. The modulation of receptor gene(s) and/or protein(s) can be
used as a method for interfering with virus entry and can thus become a new method for disease
prevention. The importance of understanding the details of CHIKV virus entry-pathway by host cells
lies in its potential for exploitation in novel vector control strategies, and the molecular characterization
of (specific domain) the proteins involved has made the development of target oriented specific drug/
peptide for anti-vrial strategies a realistic possibility.
10. Integrated pest management

We are now at a stage where all
individual techniques and tactics for
insect pest management can be
Integrated Pest Management
integrated and used in a system that Indian Council of Agricl. Research

combines the most appropriate Pheromone
Technology
characters of each to produce an Indian Institute of Agril. Science
ecologically sound and economically

Intl. Center for Semi-Tropical Research
viable package, IPM. While several Sterile Technology
definitions exist for IPM, to succeed it DST, New Delhi
is critical to have detailed information Biological method
on the biology of the pest and how it CSIR, New Delhi
interacts with the environment, natural DBT, New Delhi

enemies and the crop itself. IPM has Molecular Target Orientated
Pesticidal Method
been misconstrued for reducing
pesticide inputs, but should be based on Insect Physiology
a clear understanding of ecological Insect Molecular Biology
functioning of the agro-ecosystems Developmental Biology
(Fig.9). What is important is pest
monitoring and accurate timing of
pesticide treatment, use of selective Fig.11. Schematic diagram showing the current scenario of
Integrated Pest Management approaches.
insecticides as well as combined use of
14
semiochemicals, host plant resistance, trap crops, oviposition deterrents and antifeedents to manipulate
pest behaviour. Today the term stimulodeterrent diversionary strategy is used.
11. Concluding remarks
The ideal transgenic technology should be commercially feasible, environmentally benign

(biodegradable), and easy to use in diverse agroecosystems as well as show a wide-spectrum of activity
against the crop pests. It should also be harmless to the natural enemies, target the sites in insects that
have developed resistance to the conventional pesticides, flexible enough to allow ready deployment of
alternatives (if and when the resistance is developed by the pest), and preferably produce acute rather
than chronic effects on the target insects (Sharma et al., 2000).
Transgenic crops have many of these requirements. Some of the criteria can be achieved by
exploiting genes that are based on antibody technology (Hilder and Boulter, 1999). Single chain
antibodies (ScFvs) can be used to block the function of essential pest proteins. The potential of plant
expressed antibodies or antibody fragments to serve as insect control agents against nematodes,
pathogens and viruses has also been described (Atkinson, 1993; van Engelen et al., 1994; Rosso et al.,
1996). This approach of controlling insects would offer the advantage of allowing some degree of
selection for specificity effects, so that pests, but not the beneficial organisms, are targeted. The
development of a delivery system from transgenic plants to the insect haemolymph will remove a key
constraint in the transgenic approach to crop protection. Incorporation of Bt genes will have a
tremendous effect on pest management. We need to pursue the management strategy that reflects the pest
biology, insect plant interactions and their influence on the natural enemies to prolong the life span of the
transgenics. Refugia can play an important role in resistance management and should take into account
the pest complex, the insect hosts and the environment. Expression of more than one gene (gene
pyramiding) and single chain antibody genes, which would be compatible with the likely trends in
pesticide discovery using biology derived target based methods. Emphasis should be placed on
combining exotic genes with conventional host plant resistance, and also with traits conferring resistance
to other insect pests and diseases of importance in the target region. Several genes conferring resistance
to insects can also be deployed as multilines or synthetics.
We have only touched upon the fringes of various aspects of research and it is needless to
emphasize that phenomenal progress has been made in all aspects of Entomology and with every passing
day new techniques in molecular biology are emerging to improve crop resistance to pests. What holds
greater promise for the millennium is the possibility of modifying pathways leading to semiochemical
production, enhancing the possibility of biotechnological production of man made “smart molecules”
preventing them from reaching their respective target tissues. This novel strategy can be applied to arrest
the insect pest’s physiology and development, thereby controlling the pest population in a natural
economical and eco-friendly way without using toxic pesticides. I may herein mention that one of the
most controversial and interesting topics of today pertains to insects and climate change, with the
assumption that global warming is centered on the increase of concentration of CO2 which influences
phytophagous pests. Rising levels of carbon dioxide have a potential to alter this state of terrestrial
communities, changing the relative abundance of species and in the long run, the species complexes of
local communities.
12. Acknowledgement
I would like to express my sincere thanks to Prof. Ada Rafaeli, Institute for Technology & Storage
of Agricultural Products, ARO, Israel, for a critical reading of this article. The author also thanks to
Prof. Justin Chu, NUS, Singapore and Prof. Y.E. Park, Shizouka University, Japan for their
encouragement and updating my knowledge in the field of medical and applied entomology.
15
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Short
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Short Views on Insect Molecular Biology Invited Review
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 Vol. (1), 00–Vol. 00, 2009
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(1), 21 – 48, 2009
IN
Chapter – 2
Molecular Structure of Insect Circadian Clocks

Le Thi Dieu Trang, Qi-Miao Shao, and Makio Takeda*
Graduate School of Agricultural Science, Kobe University,
1-1 Rokkodai, Kobe 657-8501, Japan.
Abstract
Circadian rhythms are observed in a wide range of organisms from cyanobacteria to insects as daily cycles of behavioral,
developmental, physiological, or biochemical events that are controlled by endogenous oscillators. Drosophila melanogaster
has contributed to molecular elucidation of circadian mechanism because of an ease for forward genetics approaches.
Many clock genes have been identified and cloned from D. melanogaster. Basically, the circadian oscillation is regulated by
interlocked feedback loops. Studies in other insects have shown that genes involved in circadian oscillation are conserved
but they are not always shared the same structures or functions. Interestingly, some components are more closely related
to those of mammals. In this review, we describe molecular structures of Drosophila circadian clock system in comparison
to that of other insect species in detail. The description is not only on genetic aspects but also in sub-cellular localization.
Similarities and discrepancies between Drosophila and other insects cast a new light for a diversity in the mode of the
circadian oscillation among organisms. A diversity was found also among closely related species in neurological structure.
Key words: circadian clock; Drosophila; insects; oscillator system; input pathway; output pathway
*For Correspondence (email: mtakeda@kobe-u.ac.jp)
Overview 1. Introduction
1. Introduction
Organisms living on this planet must cope
2. Molecular structure of Drosophila circadian clock with daily and seasonal changes in environment
such as UV light, high or low temperature, high
(2a) Molecular components of circadian pacemaker
or low humidity and abundance or scarcity of
(2b) Molecular circuitry of circadian oscillator and
roles of post-translational mediators food and natural enemies. To respond to these
(2b.1) Entrainment of two interlocked feedback loops in changes, a wide range of organisms employ
Drosophila endogenous clocks for temporal synchronization
(2b.2) Post-transcriptional regulation in the core
mechanism of circadian oscillation
of life processes to environmental changes. The
(2c). Circadian clock input pathway rhythms driven by these clocks free-run in
(2d). Circadian clock output pathway constant environmental conditions with a period
about 24 hours (Aschoff, 1960; DeCoursey,
3. Non-drosophilid insects and divergent models of circadian 1983; Pittendrigh, 1981; Saunders, 2002). The
system
(3a). Core oscillator system
endogenous oscillation underlying these rhythms
(3b). Input pathway
is thus called circadian oscillation; circa = about
(3c). Output pathway
and dian = a day. The circadian oscillation has
three fundamental features: (1) it free-runs in
4. Localization of insect circadian clock components: all in
their head? constant temperature, light or dark with a
5. Acknowledgements circadian period (τ); (2) it has a temperature
6. References compensation in τ at a wide range of
temperatures; and (3) it can be entrained by
Zeitgebers (Johnson et al., 2004). Functionally,
Ths article has been scientifically edited by Chandrasekar R.
21
Short Views on Insect Molecular Biology, Vol.(1), 2009 Invited Review
the circadian systems consist of a central circadian pacemaker (CPM), an input and output pathways
(Fig. 1). The input pathway transmits environmental signals to the CPM. Driven by the CPM, the output
pathway affects developmental, physiological or behavioral events of the organisms. Several model
systems have contributed to understanding of molecular structure of circadian oscillation such as the
blue green alga Synechococcus, the bread mold Neurospora crassa, the higher plant Arabidopsis
thaliana, the fruit fly Drosophila melanogaster and the mouse Mus musculus (Dunlap, 1999; Edery,
2000; Young and Kay, 2001). Although there are substantial differences among organisms, the central
mechanism of circadian system is supported in most cases by a negative auto-regulatory feedback loop.
Such a loop is highly conserved between mice and fruit fly (Allada et al., 2001; Stanewsky, 2002, 2003).
However, recent studies on cyanobacteria demonstrated that phosphorylation-dephosphorylation of clock
protein without transcription and translation processes could provide an alternative mechanism
generating circadian rhythms (Tomita et al., 2005; Nakajima et al., 2005).
Fig.1 Three functional subunits of the circadian clock system.
In the negative feedback loop, the transcription of clock genes is inhibited by their protein products
interfering the transcription regulators that bind a transcription enhancer element. In N. crassa, the first
cloned and best understood circadian clock gene is frequency (frq) (Feldman and Hoyle, 1973). Frq is
the central component in circadian clock of N. crassa (Aronson et al., 1994a, 1994b), encoding two
forms of FRQ proteins. white collar-1 (wc-1) and 2 (wc-2) encode the proteins that form a mono- or
heterodimer to activate transcription of frq (Crosthwaite et al., 1997).
Clock components of insects may be conserved but the mechanisms of circadian oscillation may
differ in each insect species. The PER/TIM complex translocates into the nucleus from the cytoplasm to
function as transcriptional mediators during night in D. melanogaster (Saez and Young, 1996).
Conversely, Antheraea pernyi PER-like signal was not strong in the nucleus of pacemaker cells
throughout the night due to masking of PER nucleic degradation and /or nuclear export and/or
cytoplasmic sequestration processes (Sauman and Reppert, 1996; Chang et al., 2003). Bombyx mori
shows a clear circadian rhythm in hatching (Tanaka, 1966; Niino and Yoshitake, 1982), eclosion
behavior (Shimizu, 1989) and photoperiodic response for diapause during egg and early larval stages
22
(Shimizu, 1982; Sakamoto and Shimizu, 1994). Several clock components such as Bmper, Bmtim,
Bmcyc, Bmdbt, BmCKs, Bmlark, and Bmcry were cloned and analyzed their expression (Markova et al.,
2003; Takeda et al., 2004; Iwai et al., 2006; 2007, 2008; Trang et al., 2006).
2. Molecular structure of Drosophila circadian clock
(2a). Molecular components of circadian pacemaker
D. melanogaster has been a leading edge for a forward genetic approach to molecular mechanism of
circadian oscillation. Many clock genes have been identified and cloned in this system and genetically
analyzed for their functions (a review by Hall, 2003) (Table 1). period (per) is the first clock gene
identified in D. melanogaster by Konopka and Benzer (1971) and the first cloned clock gene a decade
later of all the organisms. Subsequently, other genes were discovered and they were grouped according
to the molecular nature of their products as transcriptional activators, transcriptional repressors, protein
stabilizers or subcellular localizers, or protein degradators (Hardin, 2005).
Circadian oscillation is a product of two interlocked feedback loops. CLOCK (CLK) and CYCLE
(CYC) are the transcriptional activators of the first loop that contain basic-helix-loop-helix/ 2 PAS
domains. They are critical for circadian rhythmicity and transcription of per and timeless (tim) (Rutila
et al., 1998; Allada et al., 1998; Bae et al., 1998; Darlington et al., 1998). In both clk and cyc mutant
flies, per transcription is severely reduced. CLK and CYC form a heterodimer and bind to a
cis-regulatory sequence termed E-box to activate per and tim transcription. clk mRNA level cycles (peak
at ZT23-4) in anti-phase to those of per and tim (ZT13-16) while cyc mRNA level is constantly
expressed (Bae et al., 1998). PER/TIM represses their own transcription by interfering CLK/CYC.
Furthermore, a basic-leucine zipper transcription factor, Par domain protein 1ε (Pdp1ε) that activates clk
ε
transcription by binding V/P box of Pdp1 gene regulatory region (Cyran et al., 2003). CLK/CYC
activates Pdp1 ε transcription.
VRILLE (VRI) is a basic-leucine zipper transcriptional repressor (Cyran et al., 2003; Blau and
Young, 1999; Glossop et al., 2003). vri has an E-box in its regulatory region and is transcriptionally
activated by CLK/CYC. VRI represses clk transcription by binding V/P box of clk regulatory region.
This is the second loop. PER contains PAS domains that mediates homodimerization (Huang et al.,
1993) and heterodimerization with TIM (Gekakis et al., 1995). per mRNA is rhythmically expressed
with an early peak at ZT13-16), while its product PER displays an peak lagging behind the mRNA cycle
by 6-8h (ZT19-21) (Hardin et al., 1990; Zeer et al., 1990).
The second clock gene identified in D. melanogaster is called timeless. A null mutation of tim
resulted in arrhythmic behavior as with pero (Sehgal et al., 1994). TIM is rhythmically expressed with a
peak around ZT17-ZT19. TIM physically binds to PER forming a heterodimer to inhibit CLK-CYC
function. Both PER and TIM contain a nuclear localization signal (NLS) required for nuclear entry and
cytoplasmic localization domain (CLD) that promotes cytoplasmic retention. Remarkably, TIM also
binds to the CLD region of PER (Saez and Young, 1996). A new clock component, clockwork orange
(cwo) encodes a transcription factor synergizing with PER and inhibiting CLK-mediated transcription
(Kadener et al., 2007; Lim et al., 2007).
DOUBLETIME (DBT) is a protein stabilizer or subcellular localizer that destabilizes PER. This is
a Drosophila homolog to a mammalian casein kinase Iε (CKIε), destabilizing PER and affecting its
subcellular localization (Price et al., 1998; Kloss et al., 1998; Cyran et al., 2005). CASEIN KINASE II
(CKII) also destabilizes PER and makes a nuclear localization of PER (Lin et al., 2002; Akten et al.,
2003). Another kinase is the glucose synthase kinase 3 (GSK3) homolog SHAGGY (SGG) that
phosphorylates TIM to promote its degradation and the nuclear entry of PER/TIM (Martinek et al.,
2001). In contrast to the destabilization effects of protein kinases, protein phosphatase 2a (PP2a)
stabilizes PER via dephosphorylation (Sathyanarayanan et al., 2004), and PP1 stabilizes TIM from
proteasome-mediated degradation (Fang et al., 2007). The protein degrader group includes the
23
F-box/WD40 protein SLIMB (SLMB) which targets phosphorylated PER for degradation in the
proteasome (Ko et al., 2002; Grima et al., 2002). A further study showed that Cryptochrome (CRY) not
only functions as a photoreceptor for TIM degradation but also a transcription repressor for the
oscillation of peripheral circadian clocks in Drosophila (Collins et al., 2006). Recently, the
phosphorylation condition and stability of CLK were demonstrated to be controlled by DBT and protein
phosphatase (Kim and Edery, 2007).
(2b). Molecular circuitry of circadian oscillator and roles of post-translational mediators
Circadian oscillator consists of mutual coupled autoregulatory loops by which rhythmic

transcription of particular clock genes is controlled via feed backs from their own protein products. To
synchronize transcriptional feedback, the levels and subcellular localization of clock proteins are
regulated by post-transcriptional mechanisms.
(2b.1). Entrainment of two interlocked feedback loops in Drosophila
The molecular nature of the Drosophila clock was illustrated in Fig. 2. In the late morning, transcription
of per and tim genes is activated by the CLK/CYC proteins heterodimer which binds to an E-box of per and
tim genes to activate their transcription. Transcriptional activation results in increased per and tim mRNA with
their peaks at the beginning of the night (Allada et al., 1998; Rutila et al., 1998; So and Rosbash, 1997).
However, TIM reaches a maximum level at around midnight since it undergoes photodegradation by
CRY-mediated ubiquination and proteasome while accumulation of PER in cytoplasm proceeds more slowly
with the peak in the second half of the night (Marrus et al., 1996). This delay in PER accumulation is due to
destabilization by DBT and CKII activities which phosphorylate PER. Subsequently, TIM stabilizes
phosphorylated PER by forming PER/TIM heterodimer (Kloss et al., 1998; Price et al., 1998; Akten et al.,
2003; Nawathean and Rosbash, 2004). PER is also stabilized by PP2a, as suggested to remove the phosphates
added by DBT and CKII (Sathyanarayanan et al., 2004). The DBT/PER/TIM complex enters the nucleus upon
SGG-dependent phosphorylation and DBT/CKII-dependent PER phosphorylation (Lin et al., 2002; Akten
et al., 2003; Martinek et al., 2001; Kloss et al., 2001; Ashmore et al., 2003; Shafer et al., 2002; Cyran et al.,
2005). Once in the nucleus, the complex binds to a CLK/CYC dimer that removes CLK/CYC from the E-box
and inhibits per and tim transcriptions (Lee et al., 1998, 1999; Nawathean and Rosbash, 2004). PER appears to
be a more effective inhibitor of CLK/CYC dependent transcription than PER/TIM dimmer (Ashmore et al.,
2003; Shafer et al., 2002) consistent with the fact that TIM is degraded shortly after nuclear entry of the
DBT/PER/TIM complex probably through the action of SGG and possibly by other tyrosine kinases (Zheng
et al., 1996, Naidoo et al., 1999; Martinek et al., 2001; Hardin, 2005). The association between
DBT/PER/TIM and CLK/CYC complexes probably directs and facilitates phosphorylation of CLK by DBT.
PER and CLK are then destabilized via continuous DBT phosphorylation and degraded that forces the cycle to
start all over again (Kloss et al., 2001; Ashmore et al., 2003; Shafer et al., 2002; Cyran et al., 2005; Kim et al.,
2007; Nawathean et al., 2007).
This negative feedback loop is interlocked by another loop, in which the VRI regulates rhythmically the
transcription of clk gene. In this loop, the CLK/CYC heterodimer binds to an E-box to activate transcription
of vri and pdp1ε during the late day and early night (Cyran et al., 2003; Blau and Young, 1999; Glossop et al.,
2003). VRI accumulates and binds to VRI/PDP1ε box (V/P box) to inhibit transcription of clk gene.
Consequently, PDP1ε accumulates to high levels during the mid-to-late evening and activates clk transcription.
Thus, clk mRNA cycles in anti-phase to per and tim transcrips. In contrast to the rhythmically regulated clk
gene, cyc gene is constitutively expressed and CYC is abundant throughout the day. Therefore, CLK is the
limiting factor for CLK/CYC heterodimer and dictates the numbers of dimers available for activating per and
tim transcriptions. A similar feature of the per/tim and clk feedback loops is required for the CLK/CYC
activation. Since rhythmic transcription of CLK/CYC activated genes requires a feedback by PER/TIM, the
per/tim feedback loop is also needed for clk loop (Hardin, 2005). The clk loop also controls rhythmic
transcription of cry, which encodes a circadian photoreceptor that functions as a clock component in some
tissues (Stanewsky et al., 1998; Emery et al., 1998; Krishnan et al., 2001).
24
Table 1. Comparison of Drosophila and mammalian core clock components

Drosophila Mammals
Gene name Protein type Function Gene name Protein type Function
period (per) Circadian, PAS protein. Binds to TIM and inhibits CLK/CYC period 1 (per1) Circadian protein. PAS Physical associates with CRY
Transcriptional repressor. transcriptional activation. period 2 (per2) domain and inhibits CLK/BMAL1
–dependent transcription
activation.
period 3 (per3) Cyclic protein. PAS Not binding to DNA, may be an
domain indirect inhibitor. Possible clock
output pathway
timeless (tim) Circadian protein. Transcriptional Binds to PER to stabilize PER and timeless (tim) Constitutively expressed. Not clear in clock function
repressor. inhibits CLK/CYC transcriptional
activation.
clock (clk) Circadian, bHLH-PAS protein. Binds to CYC to activate other clock clock (clk) Constitutively expressed. Heterodimerizes with BMAL1 to
Transcriptional activator. Q-rich gene transcription. Transcription activator. activate other clock gene
expression.
cycle (cyc) Non-cyclic, bHLH-PAS protein. Binds to CLK to activate other clock bmal1 Cyclic RNA. bHLH-PAS Heterodimerizes with CLK to
Transcriptional activator. gene transcription. protein. Transcriptional activate other clock gene
activator. expression.
crypochrome (cry) Cyclic protein. Circadian Promote light-dependent degradation of cryptochrome 1 Cyclic RNA. Flavoprotein Associate with PER to stabilize
photoreceptor. TIM. (cry1) PER and inhibit CLK/BMAL1
Flavin binding domain cryptochrome 2 dependent transcription
(cry2) activation.
vrille (vri) Cyclic protein. Represses clk transcription. E4bp4 Cyclic RNA. bZIP Represses per1 transcriptional via
bZIP transcriptional repressor. transcriptional repressor. DBP-reponse element.
PAR domain protein Cyclic protein. bZIP-PAR Activates clk transcription. d-element binding Cyclic protein. bZIP-PAR Activates per1 transcription via
1ε (pdp lε) transcriptional activator. protein (dbp) transcriptional activator. DBP-response element.
Clockwork orange Cyclic protein. Synergizes PER to inhibit CLK/CYC
(cwo) Transcriptional repressor. transcriptional activation.
doubletime (dbt) Non-Cyclic protein. Ser/Thr Promotes degradation of PER and CLK Casein kinase 1ε Non-Cyclic protein. Affects PER stability via
kinase. via phosphorylation. Affects subcellular Ser/Thr kinase phosphorylation.
localization of PER and CLK. (CKIε)
25
Short Views on Insect Molecular Biology, Vol. (1), 2009 Invited Review
Casein kinase II α Non-Cyclic protein. Ser/Thr Promotes degradation and affects

(CKIIα) kinase. subcellular localization of PER.
Casein kinase Iiβ
(CKIIβ)
Shaggy (sgg) Non-Cyclic protein. Ser/Thr Phosphorylates TIM and enhances Glycogen Drosophila sgg homolog Not clear in clock function
kinase PER/TIM subcellular localization. synthase kinase
3β(SGK3β)
REV-ERBα Nuclear receptor Inhibitor of BMAL1 in nucleus.
Links feedback loop.
Protein Protein phosphatase Stabilizes TIM via dephosphorylation
phosphatase 1
(PP1)
Protein Protein phosphatase. Stabilize PER via dephosphorylation
phosphatase 2A Cyclic RNA in twins (tws) and
(PP2A) widerborst (wdb) regulatory
subunits.
26
Fig.2 A schematic illustration of interlocked feedback loops in Drosophila. CLK/CYC heterodimer binds to
E-box and activates transcription of per, and tim. PER is phosphorylated by DBT and CK2, leading to
its degradation. TIM binds to, and stabilizes phosphorylated PER. PER is also stabilized by PP2A.
TIM is phosphorylated by SGG and the TIM/PER/DBT complex enters the nucleus where the complex
binds to CLK/CYC and removes them from E-box. It releases the transcription inhibition of clock
genes. PER and CLK are then destabilized by DBT-mediated phosphorylation. Unphosphorylated
CLK forms heterodimer with CYC and another cycle of per and tim transcription begins. Similarly,
CLK/CYC binds to E-box of vri and Pdp1εto activate their transcription. Accumulated VRI binds to
V/P box on the promoter region of clk gene to inhibit clk transcription. PDP1εis accumulated later that
replaces VRI from V/P box and activates clk transcription. Solid lines with arrow: sequential steps in
the feedback loops; solid lines with bold bar: inhibition steps in the feedback loops; wave line: per, tim,
vri, Pdp1, or clk mRNA; dash lines: nuclear membrane.
27
(2b.2). Post-transcriptional regulation in the core mechanism of circadian oscillation
In addition to transcription regulation, the post-transcriptional modifications contribute to

stabilization of the circadian system (Curtin et al., 1995; Dembinska et al., 1997; Rosbash, 1997, Kim
et al., 2002; and Shafer et al., 2002). The best-studied case is the temporally gated phosphorylation of
PER and TIM (Kloss et al., 1998, 2001; Price et al., 1998; Scully and Kay, 2000; Martinek et al., 2001;
Lin et al., 2002; Nawathean and Rosbash, 2004) and a light-dependent clearance of TIM by the CRY
that synchronizes the clock with LD cycle (Stanewsky et al., 1998). SGG appears to phosphorylate TIM
and accelerates the nuclear accumulation of PER (Martinek et al., 2001; Cyran et al., 2005) while two
casein kinases, DBT and CKII, modify PER (Lin et al., 2002; Akten et al., 2003; Nawathean and
Rosbash, 2004). Although data indicates that DBT as well as CKII directly phosphorylates PER (Kloss
et al., 1998; Price et al., 1998; Lin et al., 2002), the possibility that one kinase regulates the activity of
the other cannot be ruled out. At least in vitro experiments show that period-altering dbt mutations
decrease the kinase activity of CKIs (Suri et al., 2000; Preuss et al., 2004). Phosphorylation targets
cytoplasmic as well as the nuclear monomeric PER in the absence of TIM and influences its
accumulation and degradation (Kloss et al., 1998; Price et al., 1998). Degradation proceeds through the
ubiquitin-proteasome pathway and is stimulated by Slimb that interacts preferentially with
phosphorylated PER (Grima et al., 2002; Ko et al., 2002). The PP2A keeps PER from DBT-mediated
degradation by dephosphorylating PER (Sathyanarayanan et al., 2004). It is considered that the kinase
activity delays the accumulation of cytoplasmic PER until lights turn off. Increased TIM levels results in
stable DBT-resistant PER/TIM/DBT complex, which is translocated to the nucleus, where continued
association of PER and TIM extends the cycle by interacting with CLK/CYC and repressing it for
transcriptional activation. Following light-dependent degradation of TIM, the nuclear level of PER falls
during the day that is most likely in response to phosphorylation-mediated degradation. Therefore,
combined activity of protein kinases delays the nuclear accumulation of PER and TIM until their
mRNAs reached the maximum levels. Moreover, the degradation of nuclear PER allows a resumption of
per and tim transcription (Price et al., 1998; Rothenfluh et al., 2000; Suri et al., 2000; Kloss et al., 2001).
However, this model is revised by recent studies. PER was detected in the nucleus of lateral
neurons at time when TIM was only cytoplasmatic (Shafer et al., 2002; 2004). Experiments using the
D. melanogaster S2 cells have indicated that PER can translocate to the nucleus without TIM and repress
transcription (Chang and Reppert, 2003; Weber and Kay, 2003; Nawathean and Rosbash, 2004; Cyran
et al., 2005). In S2 cells, DBT together with CKII phosphorylate PER and therefore directly increase
transcriptional repression activity of PER. The PER nuclear localization was suggested as an indirect
effect by the association of phosphorylated PER with DNA or chromatin (Nawathean and Rosbash,
2004). The latest in vivo study in D. melanogaster confirmed that PER was translocated to the nucleus
and repressed transcription in tim01 null mutants only if DBT kinase activity was inhibited. Thus, DBT
does not seem essential for transcriptional repression by PER in vivo. DBT not only stabilizes PER but
also regulates subcellular localization of PER. Phosphorylated PER was retained in the cytoplasm,
whereas unphosphorylated PER was accumulated in the nucleus (Cyran et al., 2005). Nuclear entry of
monomeric PER was prevented by DBT-mediated phosphorylation. TIM tended to stay in the cytoplasm
but SGG made TIM enter the nucleus and/or remain there. Although unphosphorylated PER does not
required TIM for the nuclear entry, TIM inhibits DBT-dependent phosphorylation of PER (Kloss et al.,
2001; Ko et al., 2002) and co-production of PER and TIM promotes nuclear accumulation of both
proteins, that is supported by SGG-dependent phosphorylation of TIM (Cyran et al., 2005).
Not only PER, CLK was shown existing as differentially phosphorylated proteins (Bae et al.,
2000; Kim et al., 2002; Lee et al., 1998). Recent studies suggested that PER brought DBT to the
CLK/CYC complex, which led to CLK phosphorylation and suppression of transcriptional activation
potential (Kim and Edery, 2007; Yu et al., 2006). The latest studies identified a conserved domain in
PER that is important for in vivo interaction with DBT. This domain is absolutely required for PER
hyperphosphorylation, nuclear localization and transcriptional repression (Kim et al., 2007; Nawathean
et al., 2007). After nuclear entry of phosphorylated PER, CLK phosphorylation was accelerated by
28
interactions between PER/DBT/CKII and CLK/CYC complexes (Nawathean et al., 2007). However, this
domain could also act as a docking site for other factors beside DBT for inhibiting CLK/CYC dependent
transactivation (Kim et al., 2007).
(2c). Circadian clock input pathway
As Zeitgebers, light intensity, temperature, food abundance, and social interactions could give a
temporal cue to a circadian system. Light must be perceived by the photoreceptor system, which then
alters the level of a cog of clock mechanism prone to photodegradation. This causes a phase-shift. If an
animal is transferred from an LD cycle to constant darkness (DD), light pulses given during the
subjective day cause little or no effect on phase, but those falling at early subjective night cause
phase-delays and those falling at late subjective night cause phase advance (Yang et al., 1998; Suri et al.,
1998; Konopka et al., 1989). The plot of a series of phase-shifts is called a phase response curve (PRC).
PRC shape is conserved in almost all organisms either diurnal, nocturnal or crepuscular (Johnson et al.,
2004).
In Drosophila, several photoreceptor molecules are known, one of which could trigger the
ubiquitin-proteasome dependent degradation of TIM via tyrosine phosphorylation, i.e., Crytochrome
(CRY) (Naidoo et al., 1999). Depending on tim mRNA levels, photo-degradation restores different
levels of TIM that affects PER level (Hunter-Ensor et al., 1996; Myers et al., 1996; Zheng et al., 1996;
Lee et al., 1996). Altered PER/TIM levels may release the suppression of transcription activators,
CLK/CYC. Late at night, light accelerates degradation of nuclear TIM, resulting in the release of
CLK/CYC repression by the PER/TIM dimmer, so that the per and tim can be transcribed again. Then,
translation follows, i.e., phase advance. In early evening, light reduces TIM accumulation, but tim
mRNA levels are high so that TIM protein level is rapidly restored by translation, resulting in phase
delays. During the subjective day, no phase shifting occurs. In Drosophila, at least three photoreceptor
organs are known to affect the light dependent phase shift in behavioral system. They are (1) external
photoreceptors in the compound eyes, (2) those of the ocelli, and (3) internal photoreceptors in the
Hofbauer-Buchner eyelet. The blue light photoreceptors, CRYs are responsible photopigment proteins
(Stanewsky et al., 1998; Emery et al., 1998). Unlike mammals, Drosophila has only one CRY species,
CRYb that is not involved in rhythm generation per se, at least in the pacemaker neurons. CRYb is
expressed in oscillator cells throughout the head (Klarsfeld et al., 2004) and plays an important role in
TIM degradation in response to light. CRYb possesses a highly conserved photolyase domain and a
unique carboxy-terminal domain (Cashmore et al., 1999). When activated by light, this C-terminal
domain is suggested to alter position or release an inhibitor for exposing a TIM binding site (Rosato
et al., 2001). It then binds TIM, and triggers its tyrosine phosphorylation and degradation by the
proteasome (Naidoo et al., 1999). CRY is also degraded by light, albeit more slowly. However, the
light-dependent TIM degradation mechanism of external and internal photoreceptors in neurons that
control behavioral rhythms has not been elucidated yet.
A recent study suggests that internal photoreceptors which express Rhodopsin Rh6 (extraretinal
H-B cluster) and the ventral lateral neuron photoreceptor (circadian pacemaker) are not required for
Drosophila larval response to light but have an exclusive role as a photoreceptor for circadian input
pathway (Hassan et al., 2005).
(2d). Circadian clock output pathway
In addition to the central mechanism based on transcriptional feedback loops, output pathway is
likely be regulated by clock dependent transcription of genes.
In Drosophila, pupal eclosion and adult locomotor activity depend on the lateral neurons (LNs).
The neuropeptide, pigment-dispersing factor (PDF) expressed within LNs of larvae and the small ventral
lateral neurons (sLNvs) regulate the rhythmicity of both events (review by Stanewsky, 2002).
29
Furthermore, a loss of function mutation in pdf gene results in short-period locomotor activity rhythms
that gradually become arrhythmic in Drosophila adult (Renn et al., 1999). In the larval LNs and adult
sLNv, cyc01 and clkJrk mutation affected pdf mRNA levels, suggesting a transcriptional regulation of the
gene by CLK and CYC (Park et al., 2000a, Blau and Young, 1999). However, this regulation is probably
indirect, since neither pdf mRNA levels are daily oscillating (Park and Hall, 1998) nor the E-box for
expression is required in promoter region of pdf mRNA (Park et al., 2000a). In addition, PDF expression
is post-transcriptionally regulated by PER, TIM and VRI. Thus, expression of pdf is both
transcriptionally and post-transcriptionally regulated by clock genes (reviewed by Stanewsky, 2002).
PDF expressed in LNs must regulate the eclosion or/and locomotor centers, though so far, the
mechanism remains to be understood. A potential downstream pathway, dependent on pdf and clock
gene function is the Ras/MAP-kinase pathway via Neurofibromatosis-1 (Nf1) that encodes a
Ras-GTPase activating protein. Mutation of Nf1 results in arrhythmic locomotor activity and an
overactivation of MAP-kinase signaling (William et al., 2001). In pdf01 mutations, levels of activated
MAP-kinase are downregulated, suggesting a role of this signaling pathway in the output of circadian
clock.
Another regulator of behavior rhythms is cAMP-dependent protein kinase A (PKA). Several

studies show the arrhythmic behavior in PKA mutated flies in catalytic and regulatory subunits (Park
et al., 2000b), and mutations in dunce, a gene encoding a cAMP specific phosphodiesterase, affect the
cAMP signaling in the input and output pathway of neuronal pacemaker in various organisms, including
mammals, probably due to an altered cAMP metabolism (Levine et al., 1995; Reppert and Weaver,
2002). The function of PKA in the output pathway seems specific for locomotor activity rhythms
(Majercak et al., 1997).
takeout (to) provides a link between feeding behavior and the circadian clock. It is a member of a
lipid-binding protein family including one that binds juvenile hormone (So et al., 2000; Claridge-Chang
et al., 2001; McDonald and Robash, 2001). Like pdf, to is indirectly regulated by CLK and CYC. to
requires no E-box for rhythmic expression, suggesting that other transcription factors, regulated by CLK
and CYC, mediate its transcriptional regulation (So et al., 2000). A 3’-UTR deleted mutation of to
probably produces an unstable transcript that leads to low TO protein levels (Sarov-Blat et al., 2000).
These mutations show abnormal locomotor activity rhythms and die earlier than wild type flies under
starvation.
Another clock output factor identified in D. melanogaster is lark. The null alleles of the lark gene
had dominant effects on pupal eclosion rhythm but did not affect circadian period or the clock regulation
of a distinct circadian rhythm in adult activity. However, increased lark gene dosage led to a late
eclosion phenotype (Newby and Jackson, 1993; 1996). Molecular analysis of LARK indicates that it is
an RNA-binding protein, which possesses two RNA recognition motif (RRM) along with a
retroviral-type zinc finger (RTZF) (Newby and Jackson, 1996). This protein did oscillate in abundance at
late pupal stage, even though its mRNA level did not (McNeil et al., 1998). Expression of LARK
oscillated in crustacean cardioactive peptide (CCAP)-containing neurons within the subesophageal
ganglion and the ventral nerve cord (VNC) of the third instar larvae and pharate adults (McNeil et al.,
1998; Zhang et al., 2000). LARK/CCAP brain neurons were shown to project from larval and pharate
adult brains to VNC; other neuritis terminated on esophageal muscles and in the prothoracic gland of late
pupae; VNC neurons contain both LARK and CCAP that send projections posteriorly to gut muscles
(Zhang et al., 2000).
In vertebrates, melatonin is widely accepted as a time-keeping hormone and an endogenous

mediator of photoperiodic information (Reiter, 1993). Melatonin level of Drosophila exhibited a
rhythmicity with a peak during day time (Hintermanm et al., 1996).
30
3. Non-drosophilid insects and divergent models of circadian system
(3a) Core oscillator system
A vast and still growing knowledge on Drosophila genetics, anatomy, physiology and behavior
has been great assets for biological clock research in other species.
In the Chinese oak silkmoth, Antheraea pernyi, per, tim, clk and cyc homologs have been cloned.
The per rescued an egg hatching rhythm in the per0 mutant of Drosophila. Based on S2 cell culture
assays, a circadian feedback loop model was supported in which per transcript translocates to cytoplasm
where it is translated into ApPER. ApPER enters the nucleus and inhibits ApCLK/CYC, thereby shutting
off its own transcription (Reppert et al., 1994, Levine et al., 1005; Chang et al., 2003). However, ApPER
and ApTIM were immunohistochemically detected primarily in the cytoplasm at all times of the day. It
may be due to a high rate degradation in the nucleus and/or nuclear export and/or cytoplasmic
sequestration of these proteins. In addition, antisense per RNA and perW, a homolog of about a half size
of Apper were expressed in Antheraea female (Sauman and Reppert, 1996; Gotter et al., 1999; Chang et
al., 2003).
Several homologues to Drosophila clock genes were identified in the commercial silkmoth
Bombyx mori such as Bmcyc, Bmper, Bmtim, Bmcry, Bmdbt, BmCKIα, BmCKIIα, BmCKII , and β
Bmlark (Iwai et al., 2006; 2007, 2008; Markova et al., 2003, Takeda et al., 2004; Trang et al., 2006). At
least three Bmper isoforms were isolated in B. mori genome with deletion/insertion sequence transcribed
from one locus has been reported in PER of Apis cerana also (Shimizu et al., 2001). Bmper mRNA and
BmPER showed a rhythmic expression in the brain (Takeda et al., 2004; Sehadova et al., 2004). Two
casein kinase I, (BmCKIα and Bmdbt) were cloned from B. mori brain and analysed. Their putative
ε
proteins showed highly homologous to insect and vertebrate homologs of CKI especially at ATP
binding, catalytic domain and nuclear localization signal (NLS) (Trang et al., 2006). In situ hybridization
revealed an identical localization of Bmdbt, bmCKIα, Bmper transcripts in the putative clock neurons
identified by antibodies against CRY, PER, CKIα, CYC and arylalkylamine N-acetyltransferase
(aaNAT) (Sehadova et al., 2004; Trang et al., 2006). Interestingly, the same cell that expressed high
level of dbt, CKIα and per mRNAs also expressed high level of dbt, CKIα, and per antisense transcripts
(Fig.3) (Trang et al., 2006). Following this study, two casein kinases II, CKIIα and CKII β, were cloned
and analyzed for their expression (Iwai et al., 2008). In situ hybridization analyses indicated that the
subunits were expressed in brain neurons expressing PER-like protein and surprisingly, antisense RNAs
of CKIIα and CKIIβ were also detected in the putative clock neurons (Fig.3). The lateral brain neurons in
A. pernyi female also co-expressed per sense and antisense transcripts which are derived from a per
locus on the chromosome W and therefore presumably attributed to the output mechanisms controlling
female-specific behaviors (Sauman and Reppert, 1996; Gotter et al., 1999). Antisense transcripts of
unknown function have been described for the Neurospora clock gene, frq (Dunlap et al., 1995). A
unique feature in B. mori is that the antisense signal was detected in both sexes and in the retina cells as
well. The functional meaning of antisense signals is important for understanding circadian system but
currently it is difficult even to speculate.
Levels of dbt mRNAs in the brain of B. mori showed no daily oscillation while CKIα mRNAs
significantly decreased at ZT12 in both transcripts. The constant accumulation of CKI-like antigen in the
cytoplasm of the clock neurons (Sehadova et al., 2004) may be caused by constitutive expression of
DBT. Steady levels of dbt mRNA and DBT protein were observed also in D. melanogaster, but
subcellular localization of Drosophila DBT oscillated (Kloss et al., 1998; 2001).
The honey bee, Apis mellifera is an interested organism for molecular analysis of circadian
rhythms because its clock system is implicated in a set of complex behaviors included time memory, sun
compass navigation, and dance language communication (von Frisch 1967; Moore-Ede et al., 1982). In
A. mellifera, putative homologs of per, tim, cry, cyc, clk, vri, and Pdp1 were cloned, and characterized
(Rubin et al., 2006). The structure of AmCYC and AmCLK is different from Drosophila but similar to
31
the mouse and A. pernyi in which the main transactivation domain of the CLK/CYC complex is in the C
terminal end of CYC. Amper and Amcry mRNA levels oscillate strongly with a similar phase, whereas in
Drosophila they are almost in anti-phase (Rubin et al., 2006; Emery et al., 1998; Glossop et al., 2003).
AmCRY and AmTIM lack domains and motifs thought to be essential for TIM and CRY function in the
Drosophila clock. Furthermore, AmCRY is structurally similar to mammalian CRY and includes
domains and motifs implicated in the transcription repressive function of these proteins (Rubin et al.,
2006).
Fig.3. An illustration for expression of clock genes and their proteins in the brain of B. mori (holometabola).
(A). Co-localization of B. mori clock proteins. (B). Co-localization of B. mori clock genes mRNA and
anti-sense signals.
32
In the migratory monarch butterflies Danaus plexippus, transcription of the per gene is under
circadian control (Froy et al., 2003), probably via the activity of CLK/CYC heterodimer (Zhu et al.,
2005). PER-ir exhibited a robust 24 hr rhythm that is under circadian control in the putative clock
neurons and that is abolished by constant light (Sauman et al., 2005). Two CRYs were discovered in the
monarch butterfly, one is Drosophila-type CRY and the other is mammalian-type CRY (Zhu et al.,
2005). However, PER is not detected in the nucleus of clock cells in the monarch brain. It is therefore
possible that the second CRY is an important transcriptional repressor for the central clockwork of
monarch butterflies, as well as in other nondrosophilid insects (Reppert, 2006). The second CRY
discovered recently is light insensitive and shares a common ancestor with the two mammalian CRYs
that have potent repressive activity on CLK/CYC-mediated transcription (Zhu et al., 2005). The
discovery of two distinct cry genes in the butterfly led to the recognition of the existence of two cry
genes in several non-drosophilid insects. A Drosophila-type CRY was co-localized with PER and TIM
in the dorsal lateral region and large neurosecretory cells in the pars intercerebralis (PI) of the monarch
butterfly brain, suggesting this CRY acts as a blue-light photoreceptor for the circadian clock of monarch
butterflies, as CRY does in Drosophila (Sauman et al., 2005; Stanewsky, 2003). However, the CRY-ir
cells in the lobula region of the optic lobe do not contain detectable PER.
Molecular analyses of structures of circadian clock have been accumulated gradually in various
insect species. per, tim, cyc, and cry have been cloned from the flesh fly, Scrcophaga crassipalpis (Goto
and Denlinger, 2002); tim from the mosquito Aedes aegypti (Gentile et al., 2006); cyc from the saw fly
Athalia rosae (Bembenek et al., 2007) and from the cricket Dianemobius nigrofasciatus (Shao et al.,
2008). per from P. americana, and so on. Several other studies have demonstrated expression of clock
proteins in brain of numerous insects included Periplaneta americana, M. sexta, D. nigrofasciatus,
Allonemobius allardi which are described below.
(3b). Input pathway
Light-dependent input systems to circadian oscillator in most animals are distinct from the visual
system and thus referred to as extraocular photoreceptors. In insects other than D. melanogaster,
extraocular photoreceptors have been postulated for the regulation of the circadian rhythms as well as for
photoperiodism (Truman 1976). The previous studies in A. pernyi (Williams et al., 1964, 1965; Williams
1969), the aphid, Megoura viciae (Lees, 1964; Steel and Lees 1977) and in the blowfly, Calliphora
vicina (Cymborowski and Korf 1995) indicated dorsal protocerebrum as a region essential for
photoperiodic reception. The most of CRY-positive cells in the cephalic ganglia of B. mori shows
extensive overlap with the staining for several components of the core oscillator, suggesting for
interactions of CRY with other clock proteins (Sehadova et al., 2004) as in D. melanogaster. Recently
the cerebral opsin-photoreceptor has been cloned in B. mori and designated as a Boceropsin.
Immunohistochemical localization of Boceropsin revealed several restricted clusters of cells in the
central brain of Bombyx larvae (Shimizu et al., 2001). Besides this extraocular opsin, two additional
opsins were isolated from the Bombyx compound eyes (Shimizu et al., 1998).
In Danaus butterflies, a Drosophila-type CRY was co-localized with PER and TIM in the
dorso-lateral region and large neurosecretory cells in the pars intercerebralis (PI), suggesting this CRY
acts as a blue-light photoreceptor for the circadian clock of monarch butterflies, as CRY does in
Drosophila (Sauman et al., 2005; Stanewsky, 2003). However, the CRY-ir cells in the lobula region of
the optic lobe do not contain detectable PER (Sauman et al., 2005). Ultraviolet (UV)-, blue- and
extraretinal long-wave-length (LW)-sensitive opsins were also immunohistochemically examined in
hawkmoths Manduca sexta, Acherotina atropos, Agrius convolvuli, and Hyppotion celerio. UV and blue
opsins were expressed in the adult retina and stemata while LW opsin was distinctly expressed in dorsal
and ventral neurons of the optic lobes. Together with adult stemata, the lamina, medulla, lobula and
lobula plate, accessory medulla and adjacent neurons that exhibited strong LW opsin immunoreactivity
were considered as extraretinal photoreceptors for detection of changes in ambient light (Lampel et al.,
2005).
33
(3c). Output pathway
A homolog of Drosophila output factor, lark was identified in the head and other peripheral tissues
of B. mori. Bmlark had two types of transcripts. However, Bmlark mRNA failed to show its rhythmic
expression both in circadian timing and developmental stages (Iwai et al., 2007).
As in vertebrates, melatonin and aaNAT activity were detected in numerous insects and
crustaceans. Melatonin level of the damselfly, Enallagma civile (Tilden et al., 1994) lacks rhythmicity,
while that of prawn, Macrobrachium rosenbergii (Withyachumnarnkul et al., 1992) exhibits a peak
during day. The compound eyes and brain of the locus Locusta migratoria (Viven-Roels et al., 1984),
the dragon fly, Ischnura verticalis (Tilden et al., 1994), moths, Trichoplucia ni (Linn et al., 1995), and
B. mori (Itoh et al., 1995) showed a peak of melatonin in the dark phase. P. americana also exhibits
melatonin-like labeling in several brain regions (Takeda et al., 1988) and NAT-like immunoreactivity
were detected in some neuronsecretory neurons in A. pernyi (Takeda et al., 1999). L’Helias group (1995)
has shown a circadian rhythm of NAT in the brain and stemata of of Pieris brassicae. Melatonin content
and aaNAT activity showed day/night fluctuation in P. americana in the brain, retina, hemolymph and
peripheral tissues (Bembenek et al., 2005), though melatonin affects several physiological and
behavioral responses in insects. Melatonin administered in drinking water affected the locomotor activity
of Acheta domesticus (Yamano et al., 2001). Both BmNAT and melatonin receptor signals were detected
in the clock neurons including the bilaterally coupling system of optic lobe CPM neurons in
P. americana (Hiragaki et al.; Bembenek et al., in prep.)
4. Localization of insect circadian clock components: all in their head?
In Drosophila, many organs express circadian clock proteins to regulate specific physiological
processes. Brain clocks are particularly important because they determine when animals rest or become
active. PER-expressing neurons in Drosophila brain can be roughly classified into 3 groups, that is,
dorsal neurons (DNs) located in the dorsal part of the protocerebrum, lateral neuron (LNs) located near
the optic lobe, and a group of neurons located in the lateral posterior protocerebrum (LPNs) (Kaneko and
Hall, 2000; Yoshii et al., 2005). The LNs are further classified into 4 categories according to their
locations and molecular and cellular characteristics, that is 4 PDF-positive s-LNvs and a single
PDF-negative s-LNvs (5th s-LNv) with small cells located ventrally, about 5 l-LNvs with larger
PDF-positive cell bodies located ventrally, and about 6 PDF-negative LNds located rather dorsally. The
DNs are classified into 3 categories according to their location, that is, most dorsally located DN1s, 2
DN2s and dorsolaterally located DN3s. Molecular and cellular study studies revealed that the ventrally
located LNs are responsible for driving the morning and a part of the evening peak while LNs plus the
PDF-negative 5th s-LNv are responsible for driving the rest of the evening peak (Rieger et al., 2006).
In D. melanogaster PER-, TIM-, CLK- and CYC-positive neurons are located in the LN as well as
in the DN (Helfrich-Forster, 2004). Two ventral groups of the LNs co-express PDF (Helfrich-Forster,
1995). The PDF-neurons show a wide fiber network on the surface of the optic lobe, connect both optic
lobes and run into the dorsal protocerebrum. This arborization pattern makes them suited to transfer
rhythmic signals to other brain areas, as well as to synchronize the cycling of all clock neurons.
The existence of a dorsal brain clock controlling rhythmic eclosion and flight activity was shown
for the silk moths, Antherea pernyi and Hyalophora cecropia (Truman, 1972). Sauman and Reppert
(1996) verified that PER-ir and per mRNA were both expressed in the cytoplasm of 8 neurons in the
dorsolateral region of the protocerebrum. Both PER and per mRNA exhibits prominent circadian
rhythms. PER protein peaked between ZT16 to ZT22 and disappeared between ZT 4 to ZT8. per mRNA
level peaked between ZT14 to ZT22 and disappeared between ZT4 and ZT10. Sehadova and coworkers
(2004) also showed that PER-ir, CYC-ir and DBT-ir were co-expressed in the 8 big neurons and some
small neurons of the dorsolateral region of the protocerebrum of B. mori (Fig. 3). PER protein showed a
robust oscillation with a peak level between ZT 8 to ZT16 and a low level between ZT 20 to ZT4.
34
Fig.4 Distribution of clock proteins in the brain of two related crickets (hemimetabola).
(A). D. nigrofasciatus. (B). A. allardi.
In crickets, the positions of the master clock are more divergent. Teleogryllus commodus has the
CPM in the proximal optic lobe, while Gryllus bimaculatus the master clock may reside in the distal
optic lobe (reviewed by Tomioka and Abdelsalam, 2004). On the other hand in A. domesticus, the pars
intercerebralis was reported to be the locus of the circadian clock. The neurosecretory cells in this area
35
show circadian rhythm in RNA and protein synthesis (Cymborowski and Dutkowski, 1968, 1969).
Moreover, careful examination of the role of PDF neurons in G. bimaculatus in combination of partial
removal of optic lobe with immunohistochemistry using anti-PDF antibody revealed that the PDFMe
neurons located near the accessory medulla are not the clock neurons (Okamoto et al., 2001): The
destruction of the distal area of the optic lobe without affecting PDFMe neurons eliminated the circadian
locomotor rhythm. We have reported that the immunoreactivities to three clock proteins, Cryptochrome
(CRY), PER, and Doubletime (DBT) differed substantially between two closely related cricket species,
D. nigrofasciatus, and A. allardi. In D. nigrofasciatus, the CPM is located in the aMe, whereas in
A. allardi the central brain and the suboesophageal ganglion (SOG) are the most likely loci of the CPM
(Shao et al., 2006) (Fig. 4). While immunohistochemical results of two transcriptional factors CYC and
CLK showed that SOG may play an important role in both the cricket species since they were
colocalized in the SOG (Shao et al., 2008).
In Leucophaea maderae, transplantation of the optic lobe to an animal that had been arrhythmic by
bilateral optic lobe removal restored circadian rhythm with a period close to that of the donor (Page,
1982). Stengl and her coworkers suggested that the accessory medulla area containing PDF
-immunoreactive neurons lying at the ventromedial edge is the most likely locus of the cockroach
circadian clock. The most convincing evidence for this notion is that the transplantation of the tissue into
optic lobeless cockroach restored the locomotor rhythms in L. maderae (Reischig and Stengl, 2003).
There are three clusters of PDF-immunoreactive neurons in the optic lobe of crickets and cockroaches,
i.e., (1) near proximal medulla with innervation of accessory medulla (PDFMe), (2) ventrally and (3)
dorsally near the outer chiasma (PDFLa). Stengl and coworkers stress that the PDFMe is the likely locus
of the circadian clock (Stengl and Homberg, 1994; Reichig and Stengl, 2003). Rather indirect evidence
includes that the rhythm reappeared in the optic stalk severed cockroaches in correlation with the
regeneration of PDF-immunoreactive fibers from the accessory medulla to median protocerebrum
(Stengl and Homberg, 1994). A similar correlation between the rhythm restoration and the PDF
innervations was found when an AMe from a rhythmic donor was transplanted to an arrhythmic host
cockroach (Reischig and Stengl, 2003). However, immunostainings with anti-Periplaneta PER antibody
in P. americana were not perfectly consistent with this pattern: PER-immunoreactivity was observed in
two clusters of small sized cells located dorsal and ventral to the outer optic chiasma in addition to the
neurons in the pars intercerebralis and pars lateralis in the central brain (Takeda et al., 2000).
The locomotor activity rhythm of L. maderae is controlled by bilaterally symmetric circadian

packmakers in the optic lobes (Nishiitsutsuji-Uwo and Pittendrigh, 1968a; Roberts, 1974; Sokolove,
1975; Page, 1982; reviews by Page, 1984; Chiba and Tomioka, 1987). In contrast to crickets, the
cockroach pacemakers are more strongly coupled (Page et al., 1977; Wiedenmann, 1984; Ushirogawa
et al., 1997). There is strong evidence that the accessory medulla (AMe), a small neuropil at the
ventromedial edge of the medulla is the circadian pacemaker (Stengl and Homberg, 1994; Reischig and
Stengl, 1996; Petri and Stengl, 1997). The AMe is densely innervated by neurites from PDH-ir neurons,
which are circadian pacemaker candidates in insects (Homberg et al., 1991; Helfrich-Förster, 1995;
reviewed by Helfrich-Forster et al., 1998). PDH-ir neurons contain circadian clock proteins, such as PER
and TIM in D. melanogaster (Helfrich-Förster, 1995; Helfrich-Förster et al., 1998). A comparison of the
morphology of PDH-ir neurons in crickets and cockroaches revealed that the numbers of PDH-ir
commissures were correlated with the length of the period, and the strength of bilateral coupling via
posterior optic commisure (Homberg et al., 1991; Stengl and Homberg, 1994). PDH-ir neurons
mediate a coupling pathway between bilateral pacemakers in the cockroach. Recently, a backfill
experiment by injecting HRP and dextran into one AMe, seven systems of commissures connecting both
optic lobes were found; three connected both AMe and two matched the arborization pattern of PDH-ir
neurons.
Various other insects including Archeognatha (bristletails), Zygentoma (firebrats), Ephemeroptera

(mayflies), Odonata (damselflies), Plecoptera (stoneflies), Caelifera (locusts), Blattaria (cockroaches),
Heteroptera (bugs), Coleoptera (beetles), Hymenoptera (bees), Lepidoptera (butterflies), Trichoptera
(caddisflies) and other Diptera (flies) have been analyzed for localization of their clock neurons with
antibodies against PER and PDF (Frich et al. 1996; Zavodska et al., 2003b; Bloch et al., 2003). Other
36
insects were examined exclusively for PER-positive neurons (Wise et al., 2002; Zavodska et al., 2003a)
or PDF-positive neurons (Homberg et al., 1991; Nassel et al., 1991; Sehadova et al., 2003). Similarly to
Drosophila, (a) PER- and PDF-positive neurons in the lateral protocerebrum, where the PDF-positive
neurons showed wide-field arborizations into the central brain as well as into the optic lobe and (b)
PER-positive neurons were found in the dorsal protocerebrum in most species. However, the
PDF-positive neurons in the optic lobe were not identical with the PER-positive optic lobe neurons in a
great majority of insects (Zavodska et al., 2003b). Co-localization of PDF and PER was only found in D.
melanogaster. Another important difference between D. melanogaster and most other insects was found
in the subcellular localization of PER: as predicted from the negative feedback loop described above,
Drosophila PER was entirely nuclear at certain times and cytoplasmic at other times. Contrarily PER ,
was detectable only in the cytoplasm in a great majority of insects. Exceptions were a few small neurons
in the dorsal protocerebrum of M. sexta (Wise et al., 2002), in the optic lobes of Pachymorpha
sexguttata (Frish et al., 1996) and in the optic lobes of Leucophaea maderae (Schneider and Stengl,
2005) that expressed PER in the nucleus. These differences indicate that the functional mechanisms
differ among insects in spite of the high structural conservation in clock proteins. The distribution of the
clock neurons so far described suggests that insects may possess two anatomically connected clock
centers: (i) an optic lobe clock and (ii) a central brain clock, while only one of these clocks appears to
be present in some species. The sphinx moth, M. sexta has PER-positive neurons only in dorsolateral
protocerebrum but PDF-ir is restricted in the PI (Wise et al., 2002; Homberg et al., 1991), and the
goldsmith beetle Pachnoda marginata had both types of neurons only in the optic lobe (Zavodska et al.,
2003b). Interestingly, this difference was observed between closely related species. The blow fly
Phormia regina had PER-positive cells only in the dorsal protocerebrum, but PDF-positive neurons in
the optic lobe and the dorsal protocerebrum (Zavodska et al., 2003a,b). Additional PER-positive neurons
were found in the SOG and PI in a few insects (Zavodska et al., 2003a,b; Shao et al., 2006). Additional
PDF-positive neurons have been localized in the distal optic lobe of some apterygote and exopterygote
insects (Siphlonurus armatus, Locusta migratoria, P. americana).
Neurons are not the only cells of the Drosophila brain showing circadian rhythms. Almost two
decades ago, it was shown that glia cells also rhythmically expressed PER (Siwicki et al., 1988). Even
more intriguing, Ewer and colleagues obtained evidence that weak rhythmic behavior can be observed
when PER expression is restricted to brain glia (Ewer et al., 1992). Recently, Suh and Jackson (2007)
explore the role of ebony (N-β-alanyl-biogenic amine synthase) gene in driving the normal cycle of
circadian locomotor behavior. Interestingly, Ebony protein appears to be exclusively localized to glia in
the adult brain (Richardt et al., 2002). Glia expression of Ebony undergoes dramatic changes over the
course of the day and Ebony levels cycle in harmony with locomotor activity: Ebony levels were the
highest during the day and lowest during the night. Expression of an ebony cDNA specifically in glia
was found to be sufficient to rescue ebony mutant defects in locomotor activity rhythms. The
neuropeptide PDF does not appear to be required to synchronize glial cell rhythms, suggesting that glia
rhythms might be cell autonomous. Self-sustained behavioral rhythms in activity can be observed even
when glias are the only brain cells with circadian function (Ewer et al., 1992).
In flies, a photopigment for circadian entrainment is CRY. It is expressed in at least a subset of the
clock neurons in the fly brain (Stanewsky et al., 1998; Emery et al., 1998, 2000; Klarsfeld et al., 2004).
CRY is expressed in most larval and adult neuronal groups that express PER protein, with the notable
exception of larval dorsal neurons (DN2s) in which PER cycles in antiphase to all other known cells
(Klarsfeld et al., 2004). Under LD cycles, CRY level remains very low in all brain neurons, consistent
with light-dependent CRY degradation (Emery et al., 1998, Lin et al., 2001). The accumulation of CRY
varied between neuronal groups; strong in four s-LNvs and two of DN1s. This does not generally
correlate with the strength of cry gene expression as inferred from cry-gal4 (Emery et al., 2000; Zhao
et al., 2003) and in situ hybridization data (Zhao et al., 2003). Intracellularly, CRY clearly accumulates
in both the nucleus and cytoplasm, including some neuritic projections at least in group. s-LNvs cells had
axonal and dendritic projections (Klarsfeld et al., 2004). Anatomical studies suggested that the
Hofbauer-Buchner (H-B) eyelet could be responsible for the synchronization of PER and TIM in the
s-LNv cells (Hofbauer and Buchner, 1989; Yasuyama and Meinertzhagen, 1999). The H-B eyelet
37
consists of 4 cells with photoreceptor properties, which are located between the retina and the lamina of
the fly eye. They send their projections into the accessory medulla, where their termini overlap with
those of the LNvs (Helfrich-forster et al., 2002; Malpel et al., 2002), suggesting of a functional
relationship between these cell groups.
The compound eyes are the only photoreceptor for the photic entrainment in cockroaches and most
crickets (Nishitsutsuji-Uwo and Pittendrigh, 1968b; Loher, 1972; Page, 1978; Tomioka and Chiba,
1984): removal of the compound eyes or sectioning of the optic nerves resulted in a loss of entrainment
even in the light- dark cycles. In G. bimaculatus, all parts of the compound eye contribute for the photic
entrainment because the partial reduction of the compound eye led to weaker entrainment (Tomioka
et al., 1990). The compound eye is the circadian photoreceptor also in a hemipteran insect, Graphosoma
lineatum (Nakamura and Hodkova, 1998). However, there are some reports suggesting for other
receptors. In species of Eghippiger, photic entrainment still occurs after the destruction of the compound
eyes and ocelli by electrocoagulation (Dumortier, 1972). Similar weak photic entrainment was observed
after the removal or occlusion of the compound eyes in the New Zealand weta Hemideina thoracica
(Wadder et al., 1990) and D. nigrofasciatus (Shiga et al., 1999). In these insects, extraocular
photoreception might be important in the photic entrainment. Recent studies demonstrated that GABA
and Mas-allatotropin are the neurotransmitters involved in the photic entrainment pathway in the
cockroach Leucophaea maderae (Petri et al., 2002). GABA-immunoreactive neurons innervate the optic
lobe including the medulla and accessory medulla, while Mas-allatotropin-immunoreactive neurons are
intrinsic and innervate the accessory medulla. Injection of these substances into the optic lobe induces
phase dependent phase-shifts and yielded phase response curves that are quite similar to that yielded by
light. Although PDF and serotonin also elicit phase shifts of the locomotor thythm in phase-dependent
manner, they mimic the action of light only a part of the circadian day (Petri and Stengl, 1997). These
findings suggest that the circadian clock receives photic information through different channels.
Interestingly, the sensitivity of the compound eye of crictkets and roaches, a principle circadian
photoreceptor, is controlled by the circadian clock (Tomioka and Chiba, 1982a; Wills et al., 1985). This
oscillatory input might contribute to stabilize the movement of the circadian clock.
5. Acknolwedgements
The work was supported by a JSPS Postdoctoral Fellowship for Foreign Researchers (ID No.
P 07159, and ID No. P07428), and JSPS Grant-in-aid (18380043).
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48
NAL B OO
Short
IO
Views on Insect Molecular Biology, Vol. (1), 2009 Review Article
Short Views on Insect Molecular Biology Review Article
T
TERNA
MIS
SIONË
 Vol. (1)., 00– Vol.
00, 2009
ËIN
(1), 49 – 68, 2009
Chapter – 3
Hexamerin Storage Proteins: Biosynthesis, Utilization and Evolution
Chandrasekar R1*., Muthu Meenaskhi P2., and Luiz Paulo AM3

1
Division of Applied Life Science, Geyongsang National University, Jinju, South Korea.
2
Department of Eco-Biotechnology, Bharathidasan University, India.
3
Departamento de Genética e Biologia Evolutiva, Instituto de Biociencias,
Universidade de São Paulo Cidade Universitária 05508-900, Sao Paulo, Brazil,
Abstract
The transformation from a lepidopteran caterpillar to a non-feeding pupa and adult moth involves
complete remodeling and restructuring of the insect and its organs. The synthesis of hexameric storage
proteins by the fat body, secretion into the larval hemolymph and reuptake by the fat body shortly before
pupation seemes to be a common process in the life cycle of all holometabolous insects. During
pupation, proteins are initially stored in protein granules and later proteolytically broken down to supply
amino acid resources necessary for the completion of adult development and also for reproduction.
Hexamerins are evolutionary related with arthropod hemocyanins and prophenoloxisases.
Key words: Hexameric storage protein; hemocyanin; peripheral fat body; perivisceral fat body;
endocytosis; storage protein receptor; metamorphosis
*For Correspondence (email: koreanchandru@yahoo.com )
Overview
1. Introduction a) Nutrient storage

2. Organization of the fat body b) Binding of hormones and other small
3. Regional and functional differentiation of organic compounds
fat body tissue c) Involvement of hexamerins in cuticle
4. Developmental differences formation
5. Types of Storage Proteins (SP) d) Possible role of hexamerins in humoral
a) Arylphorin immune defense
b) Methionine-rich storage proteins e) Other possible role of storage proteins
c) Juvenile hormone-suppressible proteins 8. Hexamerins uptake and Receptors
d) Tyrosine-rich proteins 9. Gene regulation
e) Riboflavin-binding proteins 10. Evolution
6. Biosynthesis, secretion and storage of 11. Conclusion
storage proteins (SP) 12. Acknowledgements
7. Role of storage proteins 13. References
______________________________________
*Present Address: College of Agriculture Entomology,
University of Kentucky, S-225 Agricultural Science Center
Building – North, Lexington, KY, USA.
49
Short Views on Insect Molecular Biology, Vol. (1), 2009 Review Article
1. Introduction
A common means of these reservoir amino acids in insects is ‘Storage Proteins’ (Wheeler et al.,
2000). These larval storage proteins have a molecular masses of about 500 kDa and are hexamers
composed of six subunits of approximately 72 ~ 80 kDa which may or may not be identical. They are
called hexamerins. Hexameric storage proteins appear to undergo special adaptation to insect molting,
metamorphosis, and cyclic reproduction and have no known analogue in vertebrates. The transformation
from larva to non-feeding pupa and adult involves a complete remodeling and restructuring of the insect.
The insect fat body is a complex multifunctional tissue which participates in multiple biochemical and
physiological functions and is a major site for synthesis and storage of carbohydrates, lipids, proteins and
nitrogenous components (Keeley, 1985; Haunerland et al., 1990). In most cases, the descriptions of
regional and temporal variations in the fat body have not been clearly illustrated expect for some
dipterans and lepidopterans. Fat body heterogeneity in structure and function has been shown in
Hyalophora cecropia (Tojo et al., 1978), G. mellonella (Bean and Silhacek, 1989), H. zea (Wang and
Haunerland, 1992), C. vicina (Hansen et al., 2002), B. mori (Vanishree et al., 2005), and Amsacta
albistriga (Chandrasekar et al., 2008). In lepidopterans hexamerins are synthesized abundantly during
the larval active feeding period in the peripheral fat body (PF) and subsequently released into the
haemolymph. During the larval-pupal transformation, these proteins are sequestered by the perivisceral
fat body (PVF) and stored in crystalline form until they are utilized during pupal and adult development
(Haunerland and Shirk, 1995; Chandrasekar et al., 2008). Hexamerins are important for insect
development and, therefore, numerous studies concerning their structure, biosynthesis, regulation and
evolution have been conducted during recent year (Haunerland, 1996; Burmester 1999, 2001;
Chandrasekar et al., 2008).
Although the synthetic, secretary and storage ability of fat body tissues insects are well
documented, the concept of heterogeneity in lepidopterans remains ambiguous as structural changes
takes place rapidly during development stages (Haunerland et al., 1990; Wang and Haunerland, 1992).
Storage proteins are a powerful model system to study genes that are proximally controlled by hormones
and distally regulated by photoperiod, gender, and nutrition (Telang et al., 2002). Because of their
physiological significance and patterns of developmental control, insect hexamerins have become
attractive for the study of gene regulation. With regard to their function, development, and evolution,
storage proteins are a complex family whose analysis promises to be very highly useful for the study of
many basic problems in insect biology.
2. Organization of the fat body
Generally, fat body tissue is organized in thick lobes of

highly tracheated tissue that is suspended in the haemolymph.
Cu PF
The tissue was described as leaf like bladder (Hydropsyche
sp., Buys, 1924), lace-like (Rhodnius prolixus, Wigglesworth,
1967), flat sheets or folded lobes (Calliphora erythrocephala, PVF
Thomsen and Thomsen, 1974) and ribbon like lobes (G. Gut
mellonella, Miller and Silhacek, 1982). In this form, the fat
body has ready to access nutrients, proteins, and hormones that PVF
are transported the haemolymph. The fat tissue close to the
epidermis is called the sub-cuticular or peripheral fat body
(PF), while the central region that surrounds the gut is known
as the perivisceral (Fig. 1) or visceral fat body (PVF). Fat
body is normally, composed mainly of adipocytes or
trophocytes (Keeley, 1985). Fat body color varies different Fig.1. Cross sections of paraffin-
insect species; in most cases, the fat body is a white tissue, but embedded A. albistriga stained with
it be yellow, tan brown, green, or blue. Color differences HE/mercuric bromophenal blue, as
within the individual insects have been reported in P. showing the location of the PF and
interpunctella (Shirk and Malone, 1989) and Helicoverpa zea PVF tissues. Cu- cuticle; G- gut.
(Haunerland et al., 1990).
50
3. Regional and functional differentiation of fat body tissue
Structural and functional differences between regions of the fat body have been described in
Diptera (Rizki, 1978, Hansen, 2002). The regionally differentiated fat body tissues were consequently
shown to be distinct in their ultra structure, biochemistry and gene expression pattern. In most cases,
however, protein expression has only been studied in the entire fat body, but not in the separate areas of
the tissue. Although some proteins are undoubtedly expressed in all regions, many genes may be
expressed exclusively or in specific parts of the fat body. Lauverijat (1977) observed that the cells of
peripheral and perivisceral regions of Locusta migratoria, show different responses to ovariectomy and
allatoectomy. Schin et al. (1977) observed that the sub-epidermal fat body is green while perivisceral fat
body is creamy white in Chironomus thummi. Regional differences in the color of the fat body were
noticed in H. zea. In this species, while most of the abdominal fat body become blue during pre-pupal
stage, (Haunerland and Bowers, 1986) during larval and pupal life it is creamy white in the peripherial
region. In this case, difference in color was shown to differential uptake of a protein that contained a blue
chromophore (Haunerland et al., 1990). In the study, Haunerland and colleagues observed that
perivisceral tissue become visible as it expan rapidly, turning brightly blue different from the peripheral
fat body that remain colorless and attached to the integument. Similar results were reported by Tojo and
Yoshiga (1994); Yoshiga et al.(1998) and Meenakshi, (2005) in Spodoptera litura, where the color of the
periviceral body is blue due to the biliverdin in the hemolymph. PVF tissue is principally involved in
pinocytosis of haemolymph proteins, including injected ferritin. The Indian meal moth, Poldia
interpunctella, has functionally and morphologically differentiated tan and white fat bodies were
observed (Shirk and Malone, 1989). De Loof and Lagasse (1970) reported that PF in Leptinotarsa
decemlineata, contains more glycogen than the PVF tissue, and therefore, may serve as a storage tissue.
Since the storage protein arylphorin is stored only in the perivisceral fat body, the author speculated that
a specialized perivisceral storage organ also occurred in other insect species that lack a colored storage
protein marker like the H. zea chromoprotein. During development the structure of the fat body changes
drastically, although just few studies investigate how differences during development changes fat body
functions.
4. Developmental differences
In many insects that undergo complete metamorphosis, the fat body dissociates into single cells
before it reaggregates into the adult form or the tissue is completely histolyzed and the new fat body
differentiates from stem cells. As the main biosynthetic and storage tissue of insects, the fat body is
crucial in all stages of insect’s life. During metamorphosis of holometabolous insects, virtually all organs
and tissues change and adult-specific proteins are expressed, while production of larval and pupal-
specific proteins is terminated. These changes in the fat body are usually immediately visible upon
observation of the gross anatomy: larval tissue is frequently arranged in sheets, whereas the adult fat
body has nodular clusters. The ultra structural data support that the biosynthetic function of PF in the
larvae: mitochondria, rough endoplasmic reticulum (RER) and plasma membrane reticular systems
(PMRS) are abundant in PF of actively feeding larva (day1 –5) of last instar. After SP synthesis has
ceased and the proteins have been released into the haemolymph, PF gradually disappears. Autophagic
vacuoles dominate its structure at the end of the last larval instar, the cell membrane and other (RER,
mitochondria) organelles are completely hydrolyzed during spinning stage (Wang and Haunerland, 1992;
Muller et al., 2004). At this time, peripheral fat body tissue consists in fragments of cell remainders.
Thus, PF fat body therefore seems to be a larval specific tissue, which is not needed in the adult stage.
Fat body remodeling has been extensively examined in C. ethlius (Dean et al., 1985) and it has been
confirmed in the other species of Lepidoptera such as Phylosamia Cynthia ricini (Walker, 1966); A.
kuhniella (Colln, 1973); G. mellonella (Dutkowski, 1974); H. cecropia (Tojo et al., 1978); B. mori (Tojo
et al., 1980); P. rapae (Kim et al., 1989b) and H. zea (Wang and Haunerland, 1992). Differences
between the PF and PVF are also obvious in the cellular ultra structure (Dean et al., 1985), particularly
in Lepidoptera and Diptera species. The appearance of electron-dense structures are noted only in the
PFV during the pre-pupal period. On the other hand, in day2 pre-pupal stage, the perivisceral fat body
has cytoplasm filled with numerous protein granules, many of which were crystalline or partly
51
crystalline, and mitochondrial and RER content increases during the larval-pupal transformation as well.
In the mid pupal and late pupal stage (day 6-8) crystalline protein granules a hydrolyzed and the amino
acid pool is utilized for adult structure formation and reproduction. Two fundamentally different
mechanisms have been suggested to explain these changes: (a) the complete destruction of larval fat
body and simultaneous synthesis of adult fat body from undifferentiated stem cells, and, (b) a process
called cells remodeling in which larval fat body tissue is dissociated to isolate cell that re-associate to
form the adult fat body (Larsen, 1976; Haunerland and Shirk, 1995; Lorenz and Anand, 2004).
5. Types of storage Proteins (SP)
The life cycle of insects is divided into distinct stages that are variously specialized for feeding,
dispersal and reproduction. Lepidoptera is a good example of that. Essential nutrients obtained in one
stage but needed in another must be sequestered and carried across stages until they are mobilized.
Similarly, if one specialized stage does not feed or restricts its diet; its activities must be supported by
nutrient intake during the stage that does feed. A common means of reserving amino acids in insects is
storage proteins (Wheeler et al., 2000).
Pupal Adult cuticle

Silk gland
SP2
SP2
2 Adult fat body

SP
SP1 & SP2 SP1 & SP2
SP Pervisceral SP1 SP1
Peripheral Haemolymph Haemolymph
fat body
fat body
tissues
Biosynthesis Accumulation Release Ovary Eggs

SP Storage
2 Reproductive
gland
SP1
SP1 Reproductive
Larval cuticule
accessory gland
Fig. 2. Schematic diagram showing the hexameric storage protein (SP) bio-synthesis, and its distribution in
Amsacta albistriga.
The term “storage proteins” implies uptake of SP from the haemolymph and their storage in fat
body tissue, which serves to storage the pool of amino acids resources for metamorphosis (Fig.2). In
holometabolous insects, storage proteins represent a major protein component of the larval haemolymph
(Levenbook, 1985; Roberts, 1987; Pan and Telfer, 2001; Hahn and Wheeler, 2003; Chandrasekar et al.,
2008). These proteins have been identified in a wide range of insects and consist of high molecular
weight hexamers composed of homologous or heterologous subunits with an average molecular weight
of 80kDa (Tysell and Butterworth, 1978; Kanost et al., 1990; Telfer and Kunkel, 1991; Burmester et al.,
52
1998; Burmester, 1999). SP are synthesized in large quantities by the larval fat body tissues during the
active feeding period and released into the haemolymph. Nevertheless, high hemolymph concentration is
found only during the last larval instar (Chrysanthis et al., 1981) and storage protein uptake occurs only
during a brief time period shortly before and after pupation, and this uptake is an endocytic process
(Ueno and Natori, 1987; Pan and Telfer, 1992; Haunerland et al., 1996; Burmester and Scheller, 1997a).
According to their biochemical properties, hexamerin subunits can be categorized in one of the
three major classes: 1) the arylphorins (74 and 76kDa) or proteins rich in aromatic amino acids (Fujii,
1989; Willott et al., 1989); 2) the female-specific methionine-rich proteins (SP 1, 82kDa) which contain
more than 4 % methionine (Ryan et al., 1985; Sakurai et al., 1988; Seo et al., 1998) typical in certain
Lepidoptera; and 3) specific hexamerins binding small organic compounds, such as riboflavin or juvenile
hormone (Telfer and Massy, 1987; Jones et al.,1990; Haunerland, 1996).
a) Arylphorin
In general, the arylphorin contains 1to 3% methionine and 16 -21% aromatic amino acids and
utilized for insect metamorphosis. Arylphorin has been positively identified in crystalline protein
granules in the fat body and it appears that these granules are gradually, but not completely, broken
down during the pupal stage (Levenbook and Bauer, 1985); in fact, many protein granules have been
detected in adult fat body and it has been suggested that hexamerin mainly serves as an amino acid
source for yolk protein production (Wang and Haunerland 1991).
b) Methionine-rich storage proteins

Methionine-rich proteins contain more than 4% methionine. In contrast to arylphorin, SP 1
proteins are not glycosylated and found in several, but not all, lepidopteran species investigated. In some
species, such as M. sexta and H. cecropia, two or more isoforms of methionine-rich proteins have been
found (Tojo et al., 1978; Wang and Hanuerland, 1992). Storage protein synthesis was reported in early
larval instar in M. Sexta, B. mori and G. mellonella (Riddiford and Hice, 1985; Ray et al., 1987; Fujii et
al., 1989). It is interesting to note, however, that in contrast to all other classes of storage proteins and
hexamerins, methoinine-rich proteins (82kDa) at least in M. sexta, are much more abundant in female
than in male larvae (Ryan et al., 1985). Subburathinam and Krishnan (1998); Chandrasekar et al. (2007)
reported that supplementation of hydrolyzed soy protein (p-soyatose) increased the accumulation of
storage protein and yolk proteins in the haemolymph of Bombyx mori. Recently we observed that SP1 of
A. albistriga is actively taken up as protein granules by the developing oocytes and serves as a yolk
protein during egg formation. It has long been suggested that the female-specific SP1 of B. mori supply
amino acids for the formation of the egg yolk protein precursor vitellogenin. (Engelmann, 2000; Ogawa
and Tojo, 1981). The SP1 of H. cunea has a high methionine content (6.0%) and low aromatic amino
acid content (8.5%) (Cheon et al., 1998). The relatively high methionine content of the B. mori SP1 may
be metabolized to cystine for chorion formation (Inokuchi, 1972; Zhu et al., 1986; Cheon et al., 2001
Chandrasekar, 2006). Cystathionine and lanthionine, which are metabolic intermediates from methionine
to cystine, remain low in males but increase prominently in silkworm females (Sumioka and Yoshitake,
1974). Both metabolic intermediates are finally incorporated into the egg shell protein (Inokuchi, 1972;
Shinbo, 1978), the major sulfur amino acid of which is cysteine (Kawasaki et al., 1971; Inokuchi, 1972;
Sumioka and Yoshitake, 1974).
c) Juvenile hormone-suppressible proteins
Expression of storage proteins is mostely confined to the last instar, a period where juvenile
hormone titers are low, and its has long been suspected proteins. Indeed, JH has been shown to suppress
the expression of some, but not all storage proteins. In addition to the above mentioned basic juvenile
hormone suppressible proteins, an acidic hexamerin has been characterized in T. ni (Jones et al., 1990)
53
and G. mellonella (LSP-82, Memmel et al., 1994). Further studies needed for fat and exact function of
these storage proteins.
d) Tyrosine-rich proteins
Tyrosaturins and related tyrosine-rich storage proteins are proteins with up to 27% tyrosine found
in coleopteran fat body (Delobel et al., 1992). Their synthesis and accumulation in fat body occurs
shortly before pupation, and these proteins are deposited in protein granules of pupal fat body. Later in
development these granules are partially broken down, suggesting that the tyrosine residues of
tyrostaurins are used for the biosynthesis of cuticular structures of the pharate adult. These poorly
soluble proteins appear to be deposited into protein granules immediately after being synthesized. So, far
no structural or sequence data are available for these interesting proteins.
e) Riboflavin-binding proteins
This proteins are glycosylated

and appear to contain a high
concentration of histidine and arginine.
These proteins are expressed strongly
only in the last larval stage, they are
not actively sequestered by fat body
before pupation and do not accumulate
in pupal fat body. Their hemolymph
concentration diminishes rapidly
during the pupal-adult eclosin, but it is
not known where these proteins are
simply hydrolyzed or taken up by fat
body, ovaries or any other tissue. To
date, no sequence information is
available for these proteins.
6. Biosynthesis, secretion and storage

of storage proteins (SP)
In many insect species, fat body

was shown to be the principle site for
biosynthesis of proteins during larval
stage. In the active feeding period, the
weight of the fat body tissues is
directly proportional to weight of the
larva. The synthetic activity of the fat
body reflects the cyclical nature of Fig. 3. Electron micrograph of shows a few hours before
ecdysis to the pupa of Amsacta albistriga, the protein has
growth and development. A few hours
begun to crystallize (0.5 µm - 2µm). (A) MVB leads to
after ecdysis the cells are small, with formation of storage protein crystalline (4µm), (B), (C) & (D)
little cytoplasm, in which the – Storage protein crystalline (smaller & larger), (E) Ellipsoid
mitochondrias are conspicuous, while shape crystalline protein granule. (From: Raman
little RER and GCs are small with only Chandrasekar, 2006).
a few transition vesicles and an
incomplete stack of saccules lacking
secretary vesicles (SV). During the first 2 days after ecdysis the cells become basophilic with many free
ribosome, an increasing amount of RER and larger GCs with saccules. Also lipid droplets arises and
glycogen appear. It was further shown that this preparatory phase was characterized by RNA synthesis.
Tysell and Butterworth (1978) showed in the larvae of D. melanogaster that RNA synthesis, RER
54
proliferation and mitochondrial division increased prior to heightened storage protein synthesis and
secretion. Time of peak synthesis of protein in the fat body coincided with steeply rising heamolymph
protein concentrations (Hahn and Wheeler, 2003). Furthermore, it was demonstrated that larval fat body
mRNA directed the synthesis of storage proteins in Lucilia sercata (Thomson et al., 1976); Calliphorin
sp. (Sekeris and Scheller 1977); D. melanogaster (Roberts et al., 1977); H. cecropia (Tojo et al., 1978);
G. mellonella (Ray, 1987) and C. vicina (Pau et al., 1979). It has been suggested that the subepidermal
(peripheral) part of the fat body is the site of the most active protein synthesis and there is no part in
sequestration of protein granules as they are programmed to undergo cell death during larval-pupal
transformation (Keeley, 1985; Haunderland et al., 1990; Wang and Haunerland, 1992; Muller et al.,
2004), while the main function of PVF in larvae and pre-pupae is storage of nutrients needed for later
development. To evaluate this proposal, localization and fate of the endocytosed storage protein and
storage protein granules in A. albistriga were examined by electron microscope, which indicated that the
storage proteins were crystallized in different forms as protein granules in PVF (Fig. 3). The reliability
of the identification of the storage protein granules was confirmed by immunogold labeling
(Chandrasekar et al., 2008a,b).
In addition to storage proteins, B. mori and M. sexta have a group of structurally related proteins
with a molecular weight of 30 kDa, known as micro vitellogenin, which accounts for 30% of total egg
protein, and are not stage or sex specific (Chen and Yamashita, 1990). The fat body of both sexes
initiates the synthesis of these proteins on day 2 of the 5th instar larvae. Like vitellogenin, micro vitellin
is also synthesized by the fat body, secreted into the haemolymph, and then taken up by the maturing
eggs (Kawooya et al., 1986; Sato and Yamashita, 1991). Micro vitellin is a single polypeptide, which
contains no lipids, carbohydrates or phosphate and plays a role in embryogenesis (Kawooya et al., 1986).
It was presumed that 30 kDa proteins contributed to storage function of haemolymph (Izumi et al., 1980,
1981; Tojo et al., 1981, Zhong 1999). Yolk proteins are degraded and used as raw materials for new
organ formation and as a source of energy during embryo development. Yolk proteins are synthesized by
fat body and released into hemolymph and sequestered by the growing ovary where Vg is transformed
into Vtn (Indrasith et al., 1987; Sappington and Raikhel, 1995; Chen et al., 2004).
7. Role of storage proteins
Storage proteins are the source of amino acids and energy for protein synthesis during
metamorphosis and reproduction (Ogawa and Tojo, 1981; Levenbook and Bauer, 1984; Roberts, 1987;
Chandrasekar et al., 2008a,b). They contribute to an array of functions for sustaining the life of insects
(Fig.4).
a) Nutrient storage
In insects, storage hexamers are secreted into larval hemolymph, where they can attain
extraordinary concentrations. The correlation between the appearance of fat body granules and the
disappearance of storage proteins from the hemolymph has been demonstrated repeatedly (Dean et al.,
1985; Burmester and Scheller, 1995; Wheeler et al., 2000; Chandrasekar et al., 2007, 2008a,b). In the
mid-pupal and late pupal stages (days 6–8), crystalline protein granules were hydrolysed and utilized as
an amino acid pool for adult structure formation (Kinnear and Thomson, 1975) and reproduction (Telfer
and Pan, 2003). In an earlier study over 99% of injected M-MtH was cleared from the hemolymph of
pharate pupae, compared with only 35% of ArH (Pan and Telfer, 1992). A consequence of this in both
Luna and Cecropia pupae is that the MtH’s are the most prominent proteins of fat body extracts while
ArH is the most prominent protein of hemolymph (Pan and Telfer, 1996, and Tojo et al., 1978). There
can also be differences in the disposition of proteins within the fat body, for in Cecropia the hexamerin
storage granules contain protein crystals embedded in an amorphous material (Tojo et al., 1978).
Differences in accessibility provide a plausible speculation on how V- and M- MtH, but not ArH, might
survive metamorphosis in sufficient amounts to support post-eclosion egg formation. A specific storage
function of hexamerins is also the most likely explanation for caste-specific accumulation in colony
founding and adult development of the ants (Martinez and Wheeler, 1994).
55
b) Binding of hormones and Other small

organic compounds
The expression of storage proteins is

mostly confined to the last larval instar, a
period where JH titers are low. In fact JH
has been shown to suppress the expression
of some, but not all, SPs. In addition to the
above-mentioned basic JH suppressible
proteins, an acidic hexamerin has been
characterized in Trichoplusia ni (Jones et
al., 1990).
Some hexamerins bind to small

metabolites like biliverdin (Miller and
Silhacek, 1992) or riboflavin (Magee et al.,
1994) with high affinity. It has been
suggested that these hexamerins serve as
specific transporters for these molecules, but
the exact physiological role of these
interactions is uncertain. Biliverdin was also
found associated with Vg, but its function
remains unclear (Maruta et al., 2002). For
the cockroach, L. maderae and P.
Americana, two major JH-binding proteins
have been identified: lipophorin (Lp) and
vitellogenin (Vg) (Englemann and Mala,
2000). There is a general non-specific Fig.4 Evolution of hexamerin function. Known functions
affinity of this protein to small organic of the hexamerins and hemocyanins are compiled and
compounds, or some role of hexamerins in arranged according to the phylogeny of these proteins
the detoxification of xenobiotics. (From Burmester, 1999).
c) Involvement of hexamerins in cuticle formation
Tyrostaurins and related tyrosine-rich storage proteins. The tanning of the insect cuticle involves
the incorporation of proteins into a complicated matrix and the subsequent cross-linking of these
proteins by various diphenols (Anderson, 1979). In the Diptera, the epidermis does not synthesize
hexamerins; therefore, their presence can be attributed to specific incorporation (Schenkel and
Scheller, 1986). Arylphorins play a role in the sclerotizing system during cuticle formation (Scheller et
al., 1980).
d) Possible role of hexamerins in humoral immune defense
The protection of the insect from infections and parasites is essential for the survival of the
animal. In recent years, several lines of evidence show that the arylphorins of the lepidoptera may be
specifically involved in immune protection and may act as cytotoxic effectors, which are specifically
induced by bacterial infections (Beresford et al., 1997).
e) Other possible role of storage proteins
• The methionine-rich storage proteins provide amino acids for the developing adults
especially for chorion proteins and vitellogenin (Ogawa and Tojo, 1981; Chandrasekar
et al., 2008).
56
• The arlyphorins bind and serve as carrier proteins for ecdysteroids (Reum et al., 1982).
The presence of lipids and aromatic amino acids led to the conception that hydrophobic
pockets may function in the binding and transport of ligands.
8. Hexamerins uptake and Receptors
The passage of macromolecules through biological membranes is an essential process for all
multicellular organisms. Insects have developed a mechanism different from that known for all other
eukaryotes investigated so far. Insect pupae do not feed during metamorphosis. Therefore, they depend
on materials that have been accumulated during the larval life. At the end of larval period, shortly
before pupariation, a rise in the ecysteriod hormones induces the incorporation of large fraction of a
storage protein (hexamerins) from the body fluid into the fat body cells. Hexamerin uptake has been
shown to be receptor-mediated endocytosis (Fig. 5).
Peripheral fat body Perivisceral fat body
Fig.5. Schematic diagram shows the receptor-mediated endocytotic uptake of storage proteins and
their utilization. (From: Haunerland, 1996)
A- protein granules; VHDL- very heavy density lipophroin; R – receptor; PG – Protein granules,
MVB- multivesicular bodies, BM- basal membrane
The presence of storage protein granules in larval tissue has been already observed by Bishop
(1922) in Apis mellifera, and later in D. melanogaster (Butterworth, 1965). Specific receptors were
postulated to explain the stage-specific uptake of hemolymph proteins by the insect larval perivisceral
fat body. It was also speculated that 20-hydroxyecdysone could stimulate the binding of storage
proteins to their receptors. It was subsequently demonstrated (Ueno et al., 1983) that the storage
protein receptor (SPR) is a protein of molecular range 125kDa, which is converted to active molecules
of 120kDa under the influence of 20HE (Burmester and Scheller, 1995, 1997b, 1999). The specificity
57
and efficiency of very high-density lipoprotein (VHDL; one of the storage proteins) uptake by
perivisceral fat body (PVF) of H. zea suggested a distinct endocytotic pathway involving a VHDL
receptor (Wang and Haunerland 1993; Burmester and Scheller 1999; Persaud and Haunerland 2004).
Such pathways have been demonstrated so far in the Lepidoptera (Wang and Haunerland, 1994a;
Kirankumar et al., 1997) and Diptera (Ueno et al., 1983; Burmester and Scheller 1995; Hansen et al.,
2003; Chandrasekar et al., 2008b) (Fig.6). Surprisingly, storage protein receptor also belongs to the
members of the hexamerin superfamily (Burmester and Scheller, 1999; 2001).
Fig. 6. Electron Micrograph of formation of coated pits in PVF, which shows the sequestration of storage
proteins via the receptor-mediate endocytotic pathway (From: Chandrasekar et al., 2008a).
9. Gene regulation
Several authors well documented the haemolymph proteins and their involvement in insect
development and reproduction. Storage proteins allow the study of genes that are proximally controlled
by hormones and distally regulated by photoperiod, gender, and nutrition. The classes of hexameric
storage proteins include JH suppressible, methionine rich, and arylphorin. The mechanisms that control
expression of developmentally regulated genes in holometabolous insects have been the subject of
research for many years, mostly because in these systems metamorphosis clearly separates the sets of
larval and adult specifically-expressed genes. The exact molecular mechanism behind the stage specific
expression of storage proteins in insects attracted a great deal of attention and the first investigation in
this direction was made by Kim et al. (1989b) in S. peregrina.
In B. mori and M. sexta, the upstream region of a sex –specific storage protein (SP 1) gene has
regions of sequence similar to an EcRE, and the first intron has sequence similar to a viral (SV40)
enhancer (Sakurai et al., 1988). The upstream region of an arylphorin storage protein (SP 2) gene also
has a sequence similar to the SV40 enhancer and another sequence associated with fat body-specific
expression in D. melanogaster (Fujii et al., 1989). A larva-specific B.mori storage protein (BmLSP)
gene has a TGATAAA heptamer (Fujiwara and Yamashita, 1992) that is typically found in the upstream
regions of the storage protein genes (Willott et al., 1989). The late larval mRNA developmental profiles
from a female-specific storage protein gene and a gender-undifferentiated storage protein gene were
determined in M. sexta (Riddiford and Hice, 1985). Allatectomy, used in conjunction with
administration of JH analog, demonstrated that female-specific storage protein (SP 1) gene expression
was specifically suppressed by JH (Webb and Riddiford, 1988). The D. melanogaster and L. migratoria
yolk protein genes are expressed in the fat body and ovarian follicle cells and are under control of
regulatory sequence responsive to tissue-specific factors, 20HE, and nutritional-mediated signals
(Bownes, 1994). Therefore, different sensitivity of storage protein genes to JH during insect
development, suggested the stage and tissue-specific and hormonal regulation on the expression of
storage protein in insects.
58
The nucleotide sequences at the 5’-end of the translation initiation site of TATA contain typical
promoter elements and putative regulatory sequences (Cherbas and Cherbas 1993; Kutach and
Kadonaga, 2000). Promoters of several genes expressed specifically in the fat bodies of insects,
including those encoding the D. melanoagastor and Ades atropalpus hexamerins, contain binding sites
for the GATA factors (Abel et al., 1993; Benes et al., 1996; Zakharkin et al., 2001; Attardo et al., 2003).
The exon/intron composition of SP 2 gene is remarkably similar to that of SP 1 gene. They observed
that the deuced primary structures of SP 1 and SP 2 exhibit nearly 30% homology, implying that two SP
genes of B. mori silkworm might have evolved from a common gene (Fig.7).
10. Evolution
The origin and the evolutionary

Spodoptera litura
relationships of this taxon is one of the (0.16220)
most debated issues in evolutionary H.cercopia
biology (Fortey and Thomas, 1997; (0.33333)
G.mellonella
Corpuz et al., 1991; Jones et al., 1993). (0.36310)
Hyphantria cunea
Recently, in Musca domestica,the (0.15923)
predicated secondary structure of the Manduca sexta
(0.24219)
polypeptides encoded by SP 1 (MdHex
Panulirus interruptus Hc
L1) and SP 2 (MdHe F2) revealed that
(2.2439)
the positions of several-helixes and Amsacta albistriga
(0.00)
sheets. Also, three domains orginally Partial sequences
described for proteins belonging to
Bombyx mori
hemocyanin superfamily are highly (0.11240)
conserved in the house fly hexamerins
(Moreira et al., 2004). Hemocyanins
and hexamerins are the members of the
D.Melanosgaster
same superfamily and it has been Aedes agypti (0.45536)
(0.41235)
estimated that hemocyanins from
primitive crustaceans diverged more
than 360 million years ago giving rise
to the insect hexamerins (Naumann and Fig..7 Radial tree of phylogenic analysis from Bombyx mori
Scheller, 1991; Beintema et al., 1994; (SP) and other species. Lengths of branches along the axis are
Burmester and Scheller, 1996; proportional to evolutionary distances calculated from the
pair-wise amino acid identity matrix.
Burmester, 1999, 2001). The entire
hemocyanin gene family-hemocyanin,
cryptocyanin, proph-enoloxidase and hexamerins-may participate to varying degrees in these two vital
functions of molting animals (oxygen binding and molting). Structural similarities between hemocyanins
and hexamerins also were observed for C. vicina LSP1 (Markl et al., 1992), D. melanogastor LSP1 and
LSP2 (Massey et al., 1997; Mousseron-Grall et al., 1997) and Aedes aegypti and Anopheles gambiae
LSP1 and LSP2 (Gordadze et al., 1999). Although hexamerins and hemocyanins are structurally similar,
the organization of the genes that encode these proteins are different (Markl et al., 1992). For example,
genes that encode arachnidan hemocyanins have eight introns that correspond to 96% of the total gene
nucleotides, while insect hexamerins have fewer and smaller introns. The genes that encode hexamerins
of the Lepdioptera B. mori and M. sexta contain four introns each and these correspond to 60% of the
total number of base pairs (Burmester and Scheller, 1996; Burmester, 2001, 2002). This hypothesis is
supported by the fact that the position of aromatic amino acid residues are not more conserved in
arylphorins than in the order hexamerins; in fact, when all insect hexamerins are aligned with the
Clustral W multiple aligment program, phenylalanine and tyrosine residues are common in many
positions in all hexamerins (Table 1). Much of this sequence conservation is located in regions that
have been shown in hemocyanin to form contacts between subunits and to lie near the beginnings
and ends of alpha-helical segments. In all three of the sequenced storage hexamers, only one of
the six copper- binding histidines is conserved. The divergence of the hemocyanin from
59
nonrespiratory serum proteins that have Vertabrates
very similar size and composition may

have occurred as early as 600 my BP, a
Craniata
currently agreed upon minimal age of the 75,000 species
arthropods. Nonrespiratory serum proteins Scorpion, mites, spider
(Arachnids) Amphioxus
are known from several crustaceans and Chelicerata
arachnids, and the insect hexamerins may Tunicata
be related more directly to these than to Myriapoda

13,000 species
the hemocyanins. Centipedes, millipeds
ta
6,000 species
Hexapoda
da
Echinoderms
950,000 species
or
A leading phylogenetics (Fig. 8)
Ch
Insects
Crustacea
hypothesis indicates a close relationship 40,000 species Cray fish,
between insects and crustaceans (Hwang et losters, crabs, shrimp
Ar
Ec
t hr
al., 2001; Giribet et al., 2001) suggesting 70,000 species
dy
Mollusks
soz
op
that fundamental changes in the mechanisms
oa
od
n
Nematodes
s
Annelids
s
of respiration must have accompanied the Ascaris, hookworms 12,000 species
Earthworm, leeches
diversification of insects from crustaceans Platyhelminthes
and their invasion of terrestrial and aerial 20,000 species

Liver fluks, tapeworm
environments. During evolution, hexamerins
Coelenterata
diversified according to divergence of the 10,000 species
Hydrozoa, Scyphozoa and Antozoa
insect orders, within the orders, there is a
Sponge
notable structural diversification of these 10,000 species
Diploblastic
proteins, which probably reflects specific
functions. Fig.8 Schematic diagram shows the evolutionary tree of
animal kingdom.
11. Conclusion
In the recent years, studies have focused on the biochemistry and physiology of lepidopteran
storage proteins, including the most intriguing problem of why the fat body first secretes storage proteins
into the hemolymph before sequestering them, apparently unchanged. It has been proposed that a
functional shift of the fat body takes place at the end of the last larval stage, from biosynthetic organ to
storage organ. The uptake of hexamerins is an important process that allows holometabolous insects to
survive during the non-feeding pupal period. In addition, the dynamics between sequestration and release
of amino acids from storage proteins in the pupa/adult stage may be important feature enabling many of
their diverse strategies of reproduction. This preliminary approach plays a pivotal role on insect
development and egg production and offers a promising tool to curtail the synthesis of storage protein or
preventing them from to reach the respective tissues. This novel strategy can apply to arrest the insect
pest’s physiology and development, there by controlling the pest population in a natural way with out
using toxic pesticides, which could be economical and eco-friendly.
60
Table. 1 Sequence information of insect hexamerin storage proteins.
Accession
Species Protein Name Reference
Number
Aedes aegypti (Yellowfever Hexamerin 1 gamma P90663 Caccone et al., 1998

mosquito) (Fragment)
Anopheles gambiae (African Hexamerin Precursor Q17020 Zakharkin, 1997
malaria mosquito)
Aedes aegypti Hexamerin 2 α P90664 Gordadze et al., 1999
Aedes aegypti Hexamerin 1γ U86079 Gordazde et al., 1999
Anopheles arabiensis Hexamerin A (Fragment) O76210 Caccone et al., 1998
Aedes aegypti Arylphorin-like hexamerin-1 Q5D0X2 Scharf M.E., 2005
(Yellow fever mosquito)
Anopheles arabiensis Hexamerin A AF020873 Caccone et al., 1998
Anopheles merus Hexamerin A AF020875 Caccone et al., 1998
Apis mellifera Riboflavin binding Burmester, 1998
(Honey bee) hexamerin
Apis mellifera Hexamerin 2 Q86G31 Schaefer, 2001
Aprona germari Hexamerin SP AY29387 Kim et al., 2004
Amsacta albistriga (groundnut Storage Protein-1 N-terminal amino Chandrasekar et al., 2008
pest) acid sequence
SwissProt #
P84632
Bracon hebetor Hexamerin 125974 Quistad & Leisy, 1996
Bombxy mori Sex-specific SP1 P09179 Sakurai et al., 1988
Bombyx mori Sex-specific SP2 P20613 Fuji et al., 1989
Blaberus discoidalis (Tropical Hexamerin U31328 Jamroz et al., 1996
cockroach)
Camponotus fetinatus Hexaermin 2 - Martinez & Wheeler .1994
Calliphora vicina LSP-1/Arylphorin P28513 Naumann & Scheller, 1991
(Blue blowfly)
Calliphora vicina LSP-1/Arylphorin (partial) X63340-X63344 Fischer & Scheller, 1991
Corcyra cephalonica Juvenile hormone binding Q9GQ56 Braun and Wyatt, 1996
(Rice moth) protein
Corcyra cephalonica Allergen Cr-PI Q9U5Y8 Kort and Koopmanschap 1995
Camponotus festinatus Cyanoprotein α protein Q94607 Miura, 1998
Choristoneura fumiferana SP-1 AAC35428 Palli et al., 1998
Choristoneura fumiferana SP-2 AAC35429 Palli et al., 1998
Drosophila melanogaster (fruit LSP-1 α (partial) X03872 Delaney et al., 1986
fly)
Drosophila melanogaster LSP-1 β U63556 Massey et al., 1997
Drosophila melanogaster LSP-1 γ(partial) AF016033 Bauer & Aquadro, 1997
Drosophila melanogaster LSP- 2 X97770 Mousseron-Grall et al. 1997
Drosophila melanogaster Fat body protein 1 Q04691 Maschat et al., 1990
Eurypelma californicum Hemocyanin a P02242 Voit and Feldmater-Fuchs
1990
Eurypelma californicum Hemocyanin d P02241 Sehartau et al., 1983
Eurypelma californicum Hemocyanin c S06701 Voit and Feldmater-Fuchs
1990
Galleria mellonella Larval Hemolymph Protein L21997 Memmel et al., 1994
(Wax moth) 82
Galleria mellonella Arylphorin M73793 Memmel et al., 1992
Hyalophora cercopia Riboflavin-binding AF032397 Massey, 1995
hexamerin
61
Hyalophora cercopia Moderately Met-rcih AF032398 Massey, 1995

hexamerin
Hyalophora cercopia Very Met-rcih hexamerin AF032399 Massey, 1995
Hyalophora cercopia Arylphorin AAB86647 Burmester et al., 1998
Hyphanteria cunea SP-1 U60988 Mi et al., 1998
Hyphanteria cunea SP-2 U60988 Mi et al., 1998
Helicoverpa armigera (cotton Arylphorin-like hexamerin Q86G31 Kim, 2003
bollworm)
Locusta migratoria Cyanoprotein beta subunit Q25461 Miura, 1998
Leptinotarsa decemelineata LSP-1γ Q24995 Koopmanschap et al., 1995
Manduca sexta Arylphorin α P14296 Willot et al., 1989
Manduca sexta Arylphorin β P14297 Willot et al., 1989
Manduca sexta Methionine-rich storage L07609 Wang et al., 1993
protein
Manduca sexta Prophenoloxidase L42556 Hall et al., 1995
Musca domestrica LSP-1 U72651 de Capurro et al., 1997
Periplaneta americana Hexamerin AAB09629 Wu et al., 1996
Perla marginata (Stone fly) Hexamerin 4 A4Q991 Hagner-Holler 1007
Perla marginata Hexamerin 70c A4Q004 Martin 2007
Plutella xylostella (Diamon black Hexamerin 2 Q56DL4 Ashfaq et al. , 2005
moth)
Panulirus interruptus Hemocyanin a Po4254 Bak and Benitema 1987
Panulirus interruptus Hemocyanin b P10787 Jekel et al., 1988
Plodia interpunctella (Indianmeal LSP-1 AF356842 Zhu et al., 2002
moth)
Plodia interpunctella (Indianmeal LSP-2 AF356843 Zhu et al., 2002
moth)
Ochlerotatus atropalpus (rock- Hexamerin AF430247 Jinwal et al., 2006
pool mosquito)
Reticulitermes flavipes (Eastern Hexamerin SP-1 EG325041 Zhou et al., 2006
subterranean termite)
Reticulitermes flavipes Hexamerin SP-2 EG327184 Zhou et al., 2006
Riptortus clavatus (Bean bug) Cyanoprotein α D87272 Miura et al., 1996
Riptortus clavatus Cyanoprotein β D87273 Miura et al., 1996
Scarcophaga peregrina LSP-1/Arylphorin A24941 Matsumoto et al., 1986
Sesamia nonagrioides Methionine-rich storage DQ147770 Spyliotopoulos et al., 2008
protein (SP2)
Spodoptera litura SP-1 CAB55603 Zheng et al., 2000
Spodoptera litura SP-2 CAB55604 Zheng et al., 2000
Trichoplusia ni Acidic JH-suppressible P22327 Jones et al., 1990
(cabbage looper) protein
Trichoplusia ni Basic JH-suppressible Q06342 Jones et al., 1993
protein α
Trichoplusia ni Basic JH-suppressible Q06343 Jones et al., 1993
protein β
Tenebrio molitr Hexamerin storage protein -- Cho et al., 1999
(yellow meal worm)
SP1- storage protein 1; SP2- storage protein 2; LSP 1 – Larval Serum Protein 1;
LSP 2- Larval Serum Protein 2
62
12. Acknowledgements
The authors are grateful to Dr. Immo Hansen (Biology Dept., New Mexico State University) for
critical reading of this article. The author thanks the Department of Science and Technology, New Delhi
(MK. No. Lr. No. SP/SO/C24/99 DE 08/01/2002) for partial financial support and APMC9-South Korea,
APSERI-08 Nagoya University, Japan for providing travel grant for fruitful discussion with
Prof. Seo Seook Jae, Prof. M. Kobayashi and Prof. S. Tojo.
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 Vol. (1), 69 – 94, 2009
Chapter - 4
Insect vitellogenins: Structural features, biosynthesis, and

uptake mechanisms
Muhammad Tufail* and Makio Takeda
Graduate School of Science and Technology,
Kobe University, Nada, Kobe 657-8501, Japan.
Abstract
This chapter focuses on the current knowledge regarding the primary structure and mechanisms of vitellogenin (Vg)
biosynthesis in insects. So far, Vg sequences have been reported from 20 insect species; seven of them belong to
Hemimetabola and thirteen belong to Holometabola. Comparison of the coding sequences revealed that some features
like GLI/CG motif, cystein residues, and a DGXR motif upstream of the GLI/CG motif, were highly conserved near the
carboxy terminal of all insect Vgs. Moreover, a consensus (R/K)XX(R/K) or RXXR cleavage sequence motif existed at
the amino-terminal of all sequences outside the Apocrita except for Lymantria dispar where it existed on the C–terminal.
Furthermore, this chapter shows how Vg post-transcriptional processing exhibits a complicated pattern in insects
belonging to different orders. We also provide a brief account of hormonal regulation of Vgs and their uptake by the
oocyte. The Vg genes are large and specify a single transcript of 6-7 kb encoding a primary Vg precursor of ~200-kD,
which undergoes proteolytic processing to generate subunits ranging from ~50-180-kD. Recent molecular studies have,
however, shown that the proteolytic cleavage of Vg is more complicated in hemimetabolous insects (cleaved into several
subunits: small, medium and large) than in those of holometabolous insects, where Vg molecule is cleaved into two
subunits (one large and one small). Exceptions are Vgs of the Suborder Apocrita (higher Hymenoptera) where the Vg
primary precursor remains uncleaved. Nevertheless, the yolk proteins of higher Diptera (such as Drosophila melanogaster)
form a different family of proteins and are also not cleaved.
Keywords: insect vitellogenins; strucutre; biosynthesis; processing; regulation; uptake
* For Correspondence (email: mtufail@kobe-u.ac.jp)
Overview
1. Introduction (7b). Post-transcriptional cleavage of

2. Characteristics of vitellogenins P. americana Vgs
3. Copy number of Vg genes in insects (7c). Post-transcriptional cleavage of
4. Structural features of insect vitellogenins A. aegypti Vg
5. Vitellogenins as a molecular marker 8. Glycosylation, phosphorylation and sulphation
6. Biosynthesis and post-transcriptional cleavage of 9. Hormonal control of Vg biosynthesis
insect vitellogenins (9a). Dictyoptera
7. Examples of post-transcriptional cleavage pathway (9b). Orthoptera
of insect Vgs (9c). Heteroptera
(7a).Post-transcriptional cleavage of 10. Uptake of vitellogenins by the oocytes
L. maderae Vgs 11. Conclution
12. References
This article has been scientifically edited by Chandrasekar R.
69
1. Introduction
Vitellogenins (Vgs) are precursors of the major egg yolk protein, vitellins (Vns), in many oviparous
animals. Vg of most insect species is synthesized extraovarially in fat body cells in a tissue-, sex-, and
stage-specific manner, released into the hemolymph and then incorporated into the developing oocytes
via recepter-mediated endocytosis (see reviews: Byrne et al., 1989; Raikhel and Dhadialla, 1992).
During this course, the Vg molecules are modified through cleavage, glycosylation, lipidation, and
phosphorylation (see reviews: Raikhel and Dhadialla, 1992; Hagedorn et al., 1998; Giorgi et al., 1999;
Tufail et al., 2005). After incorporation into eggs, the Vgs represent a major component of egg yolk
proteins and serve as storage proteins to provide a source of amino acids for the developing embryo. The
synthesis of Vg in most, if not all, insect species is regulated at the transcriptional level. The sex- and
tissue-associated, and hormone-mediated developmental specificities of Vg transcription have been
reported for many insect species (Bownes, 1986; Belles, 1998, Tufail et al., 2000; Tufail and Takeda,
2002).
In insects, a sex-linked “female-protein” was first found in the hemolymph of the silkmoth
Hyalophora cecropia and was shown to participate in yolk formation by Telfer (1954). The major source
of this protein was identified as the fat body and it was named “vitellogenin” as the precursor of Vn or
yolk protein (YP) by Pan et al. (1969). Later studies, however, have shown that this protein is not always
female specific because it can be synthesized, although in smaller amounts, in the males of some species
(both from Hemimetabola and Holometabola), and also that there are vitellogenic tissues other than the
fat body as in the higher Diptera (for detailed account see: Belles, 1998; Raikhel and Snigirevskaya,
1998; Giorgi et al., 2005).
The YPs, in insects, may be divided into four categories: 1) Vn of most insect species, which is
derived from Vg, the precursor which is synthesized in the fat body cells in a female specific manner,
secreted into the hemolymph and then ultimately taken up by the developing oocytes; 2) YPs of higher
Diptera, such as Drosophila melanogaster, which are the products of the genes expressing both in the
female fat body cells and ovarian follicle cells and are incorporated into developing oocytes; 3) Proteins
such as the egg specific protein (ESP) of Bombyx mori produced in ovarian follicle cells and
incorporated into the developing oocytes; and 4) Proteins such as the 30-kD protein of B. mori produced
in the fat body cells, but in a sex non-specific manner, which is secreted into the hemolymph and
sequestered into the developing oocytes.
2. Characteristics of vitellogenins
Insect Vgs are synthesized as a primary Vg gene product of ~200 kD, which is cleaved by dibasic
endoproteases into large (140-190 kD) and small (40-60 kD) polypeptides within the fat body before
being secreted into the hemolymph (Trewitt et al., 1992; Chen et al., 1994; Yano et al., 1994b;
Kageyama et al., 1994 ； Hiremath and Lehtoma, 1997a; Hirai et al., 1998; Lee et al., 2000a, b; Comas et
al., 2000; Tufail et al., 2000, 2001, 2007; Tufail and Takeda, 2002). Female-specific Vg mRNAs of 6-7
kb have been seen only in the fat body cells (see above references). In insects such as cockroaches,
however, the primary Vg gene product is also cleaved into polypeptides of ~90-110 kD (medium
polypeptides) in addition to the large and small subunits (Tufail et al., 2001; Tufail and Takeda, 2002,
and unpublished data) (Fig. 1). The recent molecular studies have shown that the Vgs from
hemimetabolous insects are more strick in cleavage and is even more complicated cleaved into several
polypeptides than in those from holometabolous insects where Vgs are either not cleaved as in those of
Apocrita (Nose et al., 1997) or cleaved only into large and small polypeptides as in that of the mosquito,
Aedes aegypti (Chen et al., 1994). The Vgs of Cyclorrhapha, appropriately called yolk proteins, are even
quite different from those of other insects and also are not processed. Vgs generally exist as dimers, but
monomeric molecules of about 300 kD are known from Nauphoeta cinerea (Imboden et al., 1987).
70
(A)
Periplaneta americana Periplaneta australiasae Blatta lateralis

M ♂ ♀ E ♂ ♀ E ♂ ♀ E
(kD) (kD) (kD)
200 175 175
170
155 155
150
116
100 100 100
97 90
66
60
50 50 50
45
30
SDS-PAGE (7%) SDS-PAGE (7%) SDS-PAGE (7%)
(B)
Leucophaea maderae Asiablatta kyotensis
M ♂ ♀ E ♂ ♀ E
(kD) (kD)
200
170
155
116 112
100 116
97 110
92
90 95
66 87
60
55 50
45
30
SDS-PAGE (7%) SDS-PAGE (7%)
Fig. 1. Biosynthesis and proteolytic cleavage of Vg precursor in five cockroach species, three of them
belonging to Blattoidea (A) and two to Blaberoidea (B). The primary translation product is cleaved
soon after its synthesis into small (~30-60-kD), medium (~80-115-kD) and large (~150-180 kD) subunit
polypeptides which are secreted and deposited in the egg (E) as respective vitellin (Vn) polypeptides.
The identified Vgs/Vns subunit polypeptides are indicated by lines on the right. M and arrows indicate
the molecular weight markers (kD).
Vg genes and/or complete cDNAs have been cloned from 20 insect species (Table 1). The
comparison of deduced amino acid sequences of Vgs from different insect species has shown that their
structures are highly conserved (Chen et al., 1997) and form a gene superfamily. The immunochemical
cross-reactivity in insects, however, is generally confined to the family level of taxonomic relationship
(Shirk, 1987). Recently, the similarity in Vn-antigenicity among 10 cockroach species belonging to two
superfamilies has been reported (Tufail et al., 2000) (Fig. 2). The similarity in Vn antigenicity was
limited to within the superfamily in cockroaches (Tufail et al., 2000) except for that of Leucophaea
71
maderae (Blaberoidea), wherea a 90 kD Vn-polypetide was antigenically related to that of P. americana

(Blattoidea), and thus the similarity extended in this case beyond the superfamily level (Fig. 2).
The insect Vgs have been shown to be homologous Blattoidea Blaberoidea

with the Vgs of other invertebrates such as nematodes M 1 2 3 4 5 6 7 8 9 10
and those of oviparous vertebrates such as amphibia and 200
birds (Spieth et al., 1985; Blumenthal and
Zucker-Aprison, 1987; Nardelli et al., 1987; Wahli, 116
97
1988; Byrne et al., 1989; Trewitt et al., 1992; Chen et
al., 1994). Similarity of proteins has also suggested 66
having homology with Vgs of other invertebrates, such
as ticks (Chinzei et al., 1983; Rosell and Coons, 1991), 45
sea urchins (Shyu et al., 1986), millipedes (Prasath and
Subramoniam, 1991) and Crustacea (Chen and Chen, SDS-PAGE (7%)
1993).
1 2 3 4 5 6 7 8 9 10
3. Copy number of Vg genes in insects
The Vg genes/cDNAs are reported from several

insect species belonging to different taxonomic groups
(Table 1). The insect Vgs are coded by one to several
(depending on the species) gene copies (see review:
Tufail et al., 2005). It has long been reported that
Leucophaea mederae Vg is coded by only a single Vg Anti-P. am.100 - kD Vn immunoblot
gene (Masler and Offengand, 1982; Storella et al., 1985;
della-Cioppa and Engelmann, 1987; Don-Wheeler and 1 2 3 4 5 6 7 8 9 10
Engelmann, 1991, 1997). We have recently cloned two
Vg-related cDNAs from L. maderae and provided
evidence that Vg of this cockroach is a product of two
different Vg genes (Tufail et al., 2007). Both genes (Vg1
and Vg2) are transcribed since they could be amplified,
through RACE- and RT-PCR, on mRNA from fat bodies
of different individual females (Tufail et al., 2007). The
Vg1 and Vg2 cDNAs encode mature proteins of 1,898
and 1,896 residues, respectively, and share 96% over all Anti-P. am. 50 - kD Vn immunoblot
identity in their structures (Tufail et al., 2007). Both Vg
primary products share common RXXR consensus
cleavage sites and are processed into smaller subunits as Fig. 2. Similarities in Vn antigenicity
repoted previously (Tufail and Takeda, 2002; Tufail et among ten cockroach species. Egg exracts
al., 2005, 2007) and shown in Fig. 1 (B). We have also were separated by SDS-PAGE (7%) and
cloned and characterized two Vg molecules from either Coomassie blue stained (top) or
blotted to nitrocellulose membrane and
Periplaneta americana (Tufail et al., 2000, 2001, 2005). probed with anti- P. americana 100 - kD Vn
Multiple copies of Vg genes/cDNAs have also been antiserum (middle) or anti- P. americana 50
sequenced from other insect species incuding Locusta - kD Vn antiserum (bottom). Lanes 1-5
migratoria (Wyatt et al., 1984), R. clavatus (Hirai et al., represent species of Blattoidea: Periplaneta
1998); P. stali (Lee et al., 2000a) and Aedes aegypti americana (Lanes 1), Periplaneta
(Romans et al., 1995) (see for more detail in review by australasiae (Lanes 2), Periplaneta japonica
Tufail et al., 2005). The physiological role of multiple (Lanes 3), Blatta lateralis (Lanes 4),
copies of Vg genes in some insect species is, however, Periplaneta fulginosa (Lanes 5). Lanes 6-10
represent species of Blaberoidea: Blabarus
unclear. We speculate that different Vg genes may have sp. (Lanes 6), Pycnoscelus surinamensis
different functions. For instance, the plasma clotting (Lanes 7), Leucophaea maderae (Lanes 8),
protein of crayfish shares structural characteristics with Nauphoeta cinerea (Lanes 9), Asiablatta
Vgs but has unique function (Hall et al., 1999). Our kyotensis (Lanes 10). M indicate the
hypothesis is also supported by the fact that the Vg gene molecular weight markers (kD).
is expressed in male honey bees (Trenczek and Engels,
72
1986; Trenczek et al., 1989; Valle, 1993; Piulachs et al., 2003) suggesting that Vg may have also some
additional function than as a yolk precursor. Recently, it has been reported that Vg gene is involved in
regulation of hormonal dynamics, and has multiple coordinating effects on social organization of worker
honey bees (Guidugli et al., 2005; Nelson et al., 2007). Delimiting of the general and specific
physiological roles of multiple Vgs/isoforms should prove to be import.
Table 1. Reference information of cloned insect Vgs
Species Order Accession # Authors
a). Hemimetabola
Blattella germanica Dictyoptera AJ005115 Martin et al., 1999; Comas et al., 2000
Periplaneta americana-Vg1 Dictyoptera AB034804 Tufail et al., 2000
Periplaneta americana-Vg2 Dictyoptera AB047401 Tufail et al., 2001
Leucophaea maderae -Vg1 Dictyoptera AB052640 Tufail & Takeda, 2002
Leucophaea maderae -Vg2 Dictyoptera AB194976 Tufail et al., 2007
Riptortus clavatus Hemiptera U97277 Hirai et al., 1998
Plautia stali-Vg1 Hemiptera AB033498 Lee et al., 2000a
Plautia stali-Vg2 Hemiptera AB033499 --------------------
Plautia stali-Vg3 Hemiptera AB033500 --------------------
Graptopsaltria nigrofuscata Hemiptera AB026848 Lee et al., 2000b
Nilaparvata lugens Hemiptera AB353856 Naeemullah et al, unpublished
b). Holometabola
Anthonomus grandis Coleoptera M72980 Trewitt et al., 1992
Aedes aegypti Diptera U02548 Chen et al., 1994, Romans et al., 1995
Anopheles gambiae Diptera AF281078
Bombyx mori Lepidoptera D13160 Yano et al., 1994a, b
Lymantria dispar Lepidoptera U90756 Hiremath & Lehtoma, 1997a, b
Antheraea pernyi Lepidotera AB049631
Antheraea yamamai Lepidoptera AB055843
Samia cynthia Lepidotera AB055844
Athalia rosae Hymenoptera AB007850 Kageyama et al., 1994, Nose et al., 1997
Pimpla nipponica Hymenoptera AF026789 Nose et al., 1997
Apis mellifera Hymenoptera AJ517411 Piulachs et al., 2003
Encarsia formosa Hymenoptera AY553878 Donnell, 2004
Solenopsis invicta Hymenoptera AF512520
The accession numbers refer to database information linked to DDBJ
4. Structural features of insect vitellogenins
The primary structures of Vgs are now available from 20 insect species, seven of them that are
hemimetabolous and thirteen that are holometabolous (Table 1). The most striking characteristic of all
insect Vgs is the existence of polyserine domains, which are at least conserved at the N-termini of all
(except Lymatria dispar and Anthonomus grandis) sequenced Vgs outside the apocitan Hymenoptera
(Fig. 3). The cockroach and mosquito Vgs are still unique in harboring these domains also at the
C-termini (Chen et al., 1994; Tufail et al., 2000, 2001, 2005; Tufail and Takeda, 2002; Comas et al.,
2000) (Fig. 3). The role of polyserine domains is yet unclear, but their presence might serve as good
substrates for kinases. In P. americana and L. maderae, the polyserine domains were found to be highly
phosphorylated (Tufail et al., 2001; 2005 Tufail and Takeda, 2002). In vertebrates, a component of Vg,
phosvitin, also contains a polyserine region, and most of the serine residues are phosphorylated.
Phosphoserine tract represents extreme concentrations of negative charge (Goulas et al., 1996), which
may promote solubility of the Vg (Gerber-Huber et al., 1987) or provide a region for chelating essential
metal ions such as Ca2+ and Fe3+ (Nardelli et al., 1987; Taborsky, 1991). There is evidence that
dephosphorylation of Vg reduces its uptake by oocytes (Miller et al., 1982; Dhadialla et al., 1992) which
73
suggests that phosphorylated residues may contribute to the interaction between Vg and its receptor on
the oocyte surface.
Another characteristic of insect Vgs for which the sequences are available is that in all of them
(except apocritan Hymenoptera) the primary Vg gene product is cleaved in the fat body before being
secreted into the hemolymph. However, the cleavage in Vgs from hemimetabolous insects is more
complicated (cleaved into several subunits) than in those of holometabolous insects, where the Vg
primary product is cleaved into only one small and one large polypeptide (Fig. 3) (see review: Tufail et
al., 2005). The cleavage is accomplished by the subtilisin-like endoproteases, proprotein convertases
(Barr, 1991; Rouille et al., 1995), which recognize a consensus (R/K)XX(R/K) sequence motif
immediately preceding the cleavage site. This consensus cleavage site sequence motif is found in the
Vgs of all known hemimetabolous insects, with its position particularly conserved near the N-terminus in
a region of relatively low conservation and is flanked by polyserine domains (Fig. 3). In L. maderae, the
cleavage site was also determined near the C-termini of both Vg precursors (Tufail and Takeda, 2002;
Tufail et al., 2007). In the case of R. clavatus Vg, a cleavage site near the center was found in addition to
those at the N- and C-termini (Hirai et al., 1998) (Fig. 3), but the cleavage site sequence (KFKKAN) at
the latter position is not a consensus for the 1896
protease. It was speculated that the dibasic L. maderae-Vg2

1898
endoprotease might cut next to the KFKK L. maderae-Vg1
Dictyoptera
(which fits the consensus), and then two P. americana-Vg1

1880
additional amino acids (Ala and Asn) are

Hemimetabola
1860
P. americana-Vg2
removed by some other protease (Hirai et al., B. germanica

1845
1998). 1971
G. nigrofuscata
Hemiptera
1890
The other common features emerged in P. Stali Vg-1
insect Vgs include the existence of a GL/ICG R. clavatus

1860
motif, and up to nine cysteine residues that N. lugens

2045
follow at conserved locations near the 1856

A. rosae
C-termini (Romans et al., 1995; Nose et al.,
1791
1997; Lee et al., 2000b; Tufail et al., 2000)
Hymenoptera
P. nipponica
(Fig. 4). In addition, a DGXR motif has also E. formosa

1797
been observed 17-19 residues upstream of the S. invicta

1623
GL/ICG in all insect Vgs except for that of L. A. mellifera 1754

Diptera Coleoptera
maderae Vgs (Tufail et al., 2001, 2007;

Holometabola
1772
Tufail and Takeda, 2002, and references A. grandis
therein) (Fig. 4). Similar sequences were also A. aegypti

2132
found in human von Willebrand factors A. gambiae

2035
(vWf) (Baker, 1988) and mucin 2 L. dispar

1732
glycoprotein (Muc 2) (Gum et al., 1994), 1767

Lepidoptera
B. mori
both of which are members of the Vg gene
superfamily. The GL/ICG motif and cysteine
1763
A. pernyi
residues were necessary for oligomerization A. yamamai

1760
of vertebrate Vns (Mayadas and Wagner, S. cynthia

1764
1992; Mouchel et al., 1996). It was also

demonstrated that the DG residues located
near the cysteine-rich repeat contributed to Fig. 3. Diagramatic representation of the cleavage
establishing appropriate folding of the LDL sites and polyserine stretches in Vgs from 20 insect
receptor protein (Djordjevic et al., 1996). One species (see Table 1 for references). The arrows and
possible function of insect Vns oligomerized white lines indicate the putative or determined
cleavage sites following the consensus RXXR
in eggs would be binding lipids, in which
cleavage site sequence. The black segments show
inactive ecdysteroids are enclosed. The the polyserine domains. Numbers indicate the
ecdysteroids are then released as Vns and amino acid residues deduced from the N-terminua
undergo proteolysis during embryogenesis (excluding the signal peptides). In the case of
(Hagedorn et al., 1998; Giorgi et al., 1999). It P. stali, isoform 1 has been shown.
has been proposed that the DG residues
74
together with the GL/ICG motif and the following cysteine residues might participate in forming the
structure necessary for insect Vns to function properly during embryogenesis (Tufail et al., 2000).
The comparison of deduced amino acid sequences of Vgs from different insects has shown that they
can be aligned confidently along their entire lengths, with five particular subdomains (data not shown)
where the sequences are well conserved (Chen et al., 1997; Sappington and Raikhel, 1998; Lee et al.,
2000b). Furthermore, a comparison of signal peptides of all known insect Vgs shows a high similarity
(Fig. 5), suggesting that they are orthologous and point to a common ancestor signal peptide for insect
Vgs. The comparison of overall amino acid identity ratio in Vg amino acid sequences indicates that the
identity ratio is, in general, directly proportional to the phylogenetic relationship; that is, the closer the
relationship the higher the identity ratio and vice versa (Tufail et al., 2001, 2007; Tufail and Takeda,
2002), as shown in Fig. 6.
AgVg --------------------------------------------------------------- LmVg1 1606 ENQRFLLQASNIYRNKTR-GLCGNMDGEEITDLLTPNECYELDYKKFFEAYTNGNQH
LdVg RSRS----------------------------------------------------------- LmVg2 1604 ENQRFLLQASNIYRNKTR-GLCGNMDGEEITDLLTPNECYELDYKKFFEAYTNGNQH
BmVg KSRS----------------------------------------------------------- PaVg1 1631 DGKRVLLQASNGYRDEVR-GLCGTFDGEPTTDFTTPSNCILNDPKAFINSYRLGGDR
AaVg SSSDSSSSSSSSEFSSLSRSGS----------------------------------------- PaVg2 1647 DGERVMLHAGNHYRNQVR-GLCGTFDGEPSTDFKAPQNCHVRDVEDLILAYTLVRDL
AgaVg SSSSSDESDDSN--------------------------------------------------- BgVg 1580 DGSRIMIQASNMYRNFTK-GLCGNMDGEFVNDVLTPWGCYAKDMALFVASYADNSNS
ApVg RSRS----------------------------------------------------------- GnVg 1746 DGSRVKLQADSGYMSKTH-GLCGPYNGEYIDDFTTPQDCVVDDPVVFAKTYAFKEDY
AyVg RSRS----------------------------------------------------------- PsVg1 1692 DGKRLVVSVSNHHRDRVR-GICGTMDGEPSYDFTTPANCVVKDPQQFASSYALPEAS
AmVg --------------------------------------------------------------- PsVg2 1637 DGHRAAITMPNSYRNKLR-GLCGTFDGEPFNDFTTSANCVVKDYQQFISSYAIADES
EfVg --------------------------------------------------------------- PsVg3 1685 DGQRVQIQIPNSYRNMVR-GLCGTFDGEPVNDYTSPRNCVMRSRQQFVGSYAVADST
ScVg RSRS----------------------------------------------------------- RcVg 1658 DGARVQLSASSSYKGNVR-GLAGTFDGQEANDFTTPNNRVLRNPLQFAATYALLDSE
SiVg --------------------------------------------------------------- NlVg 1856 DGANFIIDADSYHRGEVR-GLCGTYSGDKYSPLHPEKNKCIMREAIPFAATYALPGS
ArVg 1655 DGERIKLILNNEYRDQIR-GLCGTFNGEPATDFTAPQNCILKNPEHFAASYALTKDQ
LmVg1 DSESRERTTTQNPMDNSIRPNIHRKQNMVVITRVVRRDTDVCFSAEPLKTCIDNSRAADTRIQ PnVg 1586 DGQRVQIKASGKYRSDIR-GLCGNFDGEPDNDFTSPKDCVLLKPEEFAASYALTTKD
AgVg 1598 DGQRVKVTATGNKLRDSVYGLCGRFSQDKHEDFTVPSNCVTRDTRKFVESYQVEKGQ
LmVg2 DSESRERTTTQNPMDNSIRPNIHRKQNMVVITRVVRRDTDVCFSAEPLKTCIDNSRAADTRIQ
LdVg 1534 DGKRGIFTTSQ-YRNITR-GICGQNSGDPLDDYKTPLGIVD-HSQHFGAAFTLDLEK
PaVg1 --SSSSSESAEGAPEKSSRKTSPPQTSTLHRTMVVEEVTEICFSMRPLPECAPRFKPADTLKK
BmVg 1568 DGQRFVVFTQD-YRNSTR-GICGRMSGEQRDDYLTPEGLVD-KPELYAAAYSLNEEN
PaVg2 ----------RGENKLHRAQQPSRNC-HMHLHRIVTHNGKQCISKLALTECAPLCREESHTTK
AaVg 1876 DGYRARFFSDYSFYNNFV-GLCGTNNGEYFDEFVTPDQCYMRKPEFFAASYAITGQN
BgVg ----NQIGPKPTLIPSQSPMTSDDKC-MTQQPRHTYYENQFCVSEKPLDTCMPLICHATESYT
AgaVg 1799 DGYRARFFADQSYFNNFV-GLCGTNNGEGEDDFITPDQCVMRKPEYFAASYALTGMN
GnVg --------LPSGNKPSDSRRHGPQSC-TNMRIMIQKRGSRTCFSAQPQNVCSSKCQATEIIQK
ApVg 1562 DGQRFVVLASE-NRQSAR-GICGSMSGEPRDDYLTPEGLVD-KPEHYAASYALNDEN
PsVg1 ----------------------MPSC-STYKIQIVKEGGRSCFSLRPQVSCNPNCKPTKNIQK AyVg 1559 DGQRFVVLASE-NRQSAR-GICGSMSGEPRDDYLIPEGLVD-KPEYYAASYALNDEN
PsVg2 ----------------------QSGC-TVFQPQIVQDGQNTCFSTEPQAVCGSNCKPSRKVER AmVg 1562 DGERVMLKASEDYRYSVR-GLCGNFDHDSTNDFVGPKNCLFRKPEHFVASYALISNQ
PsVg3 ----------------------QEGC-TNHKIRIAEVDGKTCFSIKPQVTCNPNCQAYGSLEK EfVg 1605 DGSRTKVKVGNRYRDSVR-GLCGNNDGESVDDQQTPQGYLIQNPLEFAATYALTNEE
RcVg ----------------------GSSC-SKLMTQIVEENDKSCFSTTPQPACASHCRPTGKIQK ScVg 1563 DGQRLVVLASE-NRESAR-GICGSMSGEPRDDYLTPEGLVD-KPEYYAASYALNDEN
NlVg --------------RSNNNSSERLANPTKLIQDVKNNGDQVCISIRPVPKCQKGFSPAGSSEK SiVg 1522 DGEHILLMISDNYLNAVR-GLCGNYDTQPNNDFIIPENCILTKAEEFAATYAMTQES
ArVg -----------------------GSC-TKFATATIEENGKTCFSLRPVATCAAGCKATRKQET
PnVg ------------------DSSDHKRC-NTLRTKVIEEEDQICFSLRPLPTCAEGCTANRTKPK LmVg1 YMDKTCIRYFPIDDMNYFPKQQRQRNPAYPSDLSDVLSKSISGTSSQTSSASSNENKQNRHSH
AgVg ---------------------------TVMKHRYIEENGEICFTIRPLPVCN--TSVKQVVTK LmVg2 YMDKTCIRYFPIDDMNYFPKQQRQRNPAYPSDLSDVLSKSISGTSSQTSSASS--MKISKPSF
LdVg ------------------YQKQRGPCQVQNQVQYHENHGEICITTTPISACQSHCHSSNYQVQ PaVg1 EDNAWMLNYRAQPCVSRNFTSTDIIGKHMPRNPASRGFELPHPVNDTSSSSSSSSESSSSSSS
BmVg ------------------YDKHKGACEVRQQVQFYENHGDICITTSRVPSCQSHCRAGDYKIQ PaVg2 DRSRLRDENICVREDVQLVNLTNHRHAEKSGIRPYDIDDDSSSSSSSSSSSSSSSSSSKSNST
AaVg -SSSSSSSEEQKEFHPHKQEHSMKECPVQHQHQFFEQGDRICFSLRPLPVCHSKCAATEKISK BgVg EVRKIKATQNEQTCVPQFHQPLVSHQMRLSQVIKLADTSSSSESSSSSESHENNSSPSSESQV
AgaVg -SSSSEERKPNREHFFEKQQYTEKECPVKYQAQYVEQGDKICFTSRPLPTCASQCKATEKAPK GnVg STRPK--PVSQYSTWSASKIAENITCVKKNDKHHNIIPSFHNDHSKQSHNNRTKPSPEDVCQY
ApVg ------------------YDKQRGPCAIKQQVQYYENHGEICITTTQLPACQSHCRGEDYKIQ PsVg1 LRG---------PTKPHKKQGQSGACYPREVVFADVVSDTDAGRGRRLQTMPGND---IEGKL
AyVg ------------------YDKQRGPCAIKQQVQYYENHGEICITTTQLPACQSHCRGEDYKIQ PsVg2 -CKG--------PSKEYLSQPKNEECYPKEVVYADVISDAEAGRRVRLSTPRNNT---IGGKK
AmVg ---------------------VNKHCTIHRTQVKETDDK-ICFTMRPVVSCASGCTAVETKSK PsVg3 -CQG--------ERKEINQQAQNAPCYPKRVIFADVVSDYDAGR--SHGTGTLNS---TRSKP
EfVg ---------------------SEKQCMKHRTMVQRNKDQ-ICFSKRPVPSCSSRCSEAETRNK RcVg -CQG--------PAVQRQQQAQQSPSYERRVILGDVVNELEAGRQQQQNNKSKKNKANGSEER
ScVg ------------------YDKQKGPCAVKQQVQYYENHGEICITTTQLPACQSHCRGEEYKIQ NlVg SNSN---------VEQLKRQADQMTCFRRRHIFANVITSNDYDRSSSSSSSNRNNNRNNNKNN
SiVg --------------------------------------------------------------- ArVg -CEG--------PAKENARRAQQAHCYREAVLFGNVVSDQDAGRSKNKDSKWENNKSRKSGSG
PnVg -CHG--------SALEHARKASQAVCSQKSPRPGNVVSDRDAGRKYSENSNWGYHSRQNSDDE
LmVg1 QQQFI-CLPDS-PAFEHYLKLIKKGINPDFTRKKNFVQLEVKIPTK-CIKSQ---~~~~COOH AgVg Q-WRN--------------SPSEQ-CIKKVLPLYTNVISNQNGSQMRTKLASG----------
LmVg2 QQQFI-CLPDS-PASEHYLKLIKKGINPDFTRKKNFVQLEVKIPTK-CIKSH---~~~~COOH LdVg TN----------SQIQQWKKIAQETAYQPKLTHTVILRFDEEWKIAGEQ---KGLEWGSQKVY
PaVg1 KIKFH-CLTKG-PTASHWLKMVKKGVNPDFSKKREHKQLEVDIPAK-CVRH----~~~~COOH BmVg SD----------PKTQELKALATQQAYYPEYKYTSILRSDPTWQEESQS--CGEDQWQSETVY
PaVg2 TEAFV-CFPPG-PTADHYTKLVRKGVSPDFSRKTDIVNLRVTIPSR-CVSKI---~~~~COOH AaVg -CTGP-----AKAFNYAYQQKAKQECVKREVYYGDIIYNQEYYHPRYRYYNHNVEESSSSSSS
BgVg IDVNFYCVPLGPAANHYMKLVKKGILPDLSRNRNGKRVVLPVEIPIQCEPVLN--~~~~COOH AgaVg -CSGP-----AQAYFTEYHQKAQQHCVKPQYYFGNVISEQEAGRQRYNYYYKDFDLSDSSSSE
GnVg EVPFR-CVENE-KARDFIQPVMRGRVPPKLNNYQPSEKLVIGVPQS-CIFSY---~~~~COOH ApVg -SD---------PKTQELKTKAKQEAYQPENKYTSVLRSDSQWQQEMTASSSSEEDWGSETVY
PsVg1 HIEFH-CRPDNDNLTGHWLRMVQKGANPDLSQKSSNSKMSVMLPEG-CTSIH---~~~~COOH AyVg -SD---------PKTQELKNKAKQEAYQPENKYTTVLRSDSQWQQEMTASSSSEEDWGSETVY
PsVg2 SIEFH-CAPSTDSLTVHWLNLVKKGANPDFSQKSTTKRMNVLVPET-CKPKEYN-~~~~COOH AmVg -CEGD-------SLNVAKSLQDHD-CIRQERTQQRNVISDSESGRLDTEMSTWGYHHN-----
PsVg3 TIEFH-CLNES-SATRHWVTMVKKGANPDFSQKKSNYQMSVGLPEK-CVPNSYQM~~~~COOH EfVg QCEG--------PARQNAEKAKKSPRVSLSARPGNVISDREAGRKNEEDSSERKGNE------
RcVg IVDFH-CVQAS-SSSRNWAQMIKNGANPDFSAKEKHRQILLQLPTG-CVPKA---~~~~COOH ScVg SD----------PKTQELKTKAKQQAYQPQNKYTSVLRSDSQWQQEMTATLSSEEDWGSETIY
NlVg EVDYV-CMSHGKNAQFWINQIFQGGYVLLEQKQ-HNATFMKNIPQR-CVRDN---~~~~COOH SiVg -CQG--------PAPENKRKAEQSTCMSRSYRPSDVISDREAGRSSTKNRGWGYH--------
ArVg TVKMH-CVQSS-SSSQQLVHRIKQGANPDFSQKSHDKSSRIEVPES-CVAQQ---~~~~COOH
PnVg VMPMH-CMPKN-IAAERMADRIKQGANPDFSQKSYTKKNGFDIPVG-CHAA----~~~~COOH LmVg1 STSSSHSSHSHSHSHSKSHSHSSSSQSHSRPKHSRPEQSRSSSSASRSRHSASRASRASSQNS
AgVg NVPVH-CIQGT-KTAYYYKSLIDQGGNPDFSRKSETRTARMEVAAQ-CN------~~~~COOH LmVg2 SLNFVIHSSHSHSIPILIPSHSSSSQSHSRPKHSRPEQSRSSSSASRSRHSASRASRASSQNS
LdVg AVQAV-CKGKKDPEFRMYKDQIHQGQNPQVTG--VPKVEQYRVPTT-CTE-----~~~~COOH PaVg1 SSSESASNSDLHNNSTSSSSSS-----------------------------------------
BmVg HVQVT-CKSKLDHDFRMYKEQIKKGQNPEVSG--IPSVKQFKVPVT-CQP-----~~~~COOH PaVg2 SSSSSESNESALP--------------------------------------------------
BgVg NKSKRQPNSRPRSSSSSSSSSSSESNESVLAKKIIN---------------------------
AaVg YFDVH-CFEKDSTQAKKYKSEIGRGYTPDFKSFAPHKTYKFNYPKS-CVYKAY--~~~~COOH
GnVg NDDSAG---------------------------------------------------------
AgaVg YVDVH-CRDATDSVAQLYKQQIRKGVNPDMSNKSVTKTVKFFLPKK-CVHVY---~~~~COOH
PsVg1 PR-------------------------------------------------------------
ApVg AAQVT-CRPKIDQQFRSHRDHIKQGQNPEVTG--VPKVKQFKVPTA-CNA-----~~~~COOH
PsVg2 GTKG-----------------------------------------------------------
AyVg AAQVT-CRPKIDQQFRSHRDHIKQGQNPEVTG--VPKVIQFKVPTA-CNA-----~~~~COOH
PsVg3 SIRN-----------------------------------------------------------
AmVg PYKFH-CMEKN-EAAMKLKKRIEKGANPDLSQKPVSTTEELTVPFV-CKA-----~~~~COOH
RcVg MKVQ-----------------------------------------------------------
EfVg KIEFH-CVPKS-DASEKVADRVEKGANPDLTQKNTSKTEFQKIPVS-CKA-----~~~~COOH NlVg ---------------------------------------------------------------
ScVg AAQVT-CRPKLDQQFRSHRDHIKQGQNPVVSG--VPKVKQFKVPTS-CNA-----~~~~COOH ArVg GKKS-----------------------------------------------------------
SiVg -------------------------------------------------------~~~~COOH PnVg QGKN-----------------------------------------------------------
Fig. 4. Comparison of the C-terminal part of 24 Vg amino acid sequences from 20 insect species. Dashes
represent spaces introduced for maximum alignment. The DGXR and GL/ICG motifs and
cysteine-residues are shown with shaded frames. Numbers indicate the amino acid positions excluding
the signal peptides.
75
5. Vitellogenins as a molecular marker
Various authors have considered the phylogenetic relationships between Vgs of invertebrates and
vertebrates (Romans et al., 1995; Chen et al., 1997; Sappington and Raikhel, 1998). Previously, Nose et
al. (1997) constructed a phylogenetic tree of insects using the deduced amino acid sequences of Vgs
Leucophaea maderae Vg1 -MWYTLLCCLVVGGVA-----

Leucophaea maderae Vg2 -MWYTLLCCLVVGGVA-----
Periplaneta americana Vg1 -MWKGFLCCLLVAGVTS----
Periplaneta americana Vg2 MMWKTLLCCLLAV---SAA--
Blattella germanica MTWNALLCCLLV----SAASA
Graptopsaltria nigrofuscata -MWAPLYMCLLVAGAIA----
Plautia stali-Vg1 MLWSSAL--LLAFACLAAA--
Plautia stali-Vg2 MNWTLVA--LLTFVGLAAA--
Plautia stali-Vg3 -MWAPFTLFVVAFLTLAS---
Riptortus clavatus -MWSPVIICLLV-GLASA---
Nilaparvata lugens -MNG-LTLILCVIAVAGVSA-
Athalia rosae -MWSPLLLCLLV-GIASA---
Pimpla nipponica -MWCPLFLVLLA-GAATA---
Anthonomus grandis -MWSTVALCLLV-GL-SYVSS
Lymantria dispar -MRLLLISAFIAVV-SS----
Bombyx mori -MKLFVLAAIIAAV-SS----
Aedes aegypti -MLAKLLLLALA-GLTAA---
Anopheles gambiae -MIAKLLLLTLV-GLCTA---
Antheraea pernyi -MKLLFLAVVIAAV-SS----
Antheraea yamamai -MKLLFLAVVIAAV-SSHRA-
Apis mellifera -MLLLLT-LLLFAGTVAA---
Encarsia formosa -MILSHALLLLLVGAACA---
Samia cynthia -MKLFVLAVLIAAV-SS----
Solenopsis invicta -MWYLAF-LLII-GAYAADHA
Fig. 5. Alignment of the signal peptide of 24 Vgs from 20 insect species (see Table 1 for references).
from 6 insect species. The tree was in agreement with those based on morphological characteristics and
ribosomal DNAs, suggesting that Vg can be used as a molecular marker to indicate phylogenetic
relationships. Later on, Lee et al. (2000b) constructed a phylogenetic tree based on amino acid sequences
of 9 insects (3 hemimetabolous and 6 holometabolous); the results were not different from those reported
by Nose et al. (1997). The difference in phylogenetic trees based on Vg amino acid sequences is reported
only by Comas et al. (2000), where the cicada, G. nigrofuscata forms a cluster, although with only 57%
76
bootstrap value, with cockroaches (P. americana and B. germanica). Tufail and Takeda (2002) have
reported a phylogenetic (neighbour-joining) tree based on 4 Vg amino acid sequences from three
cockroach species (P. americana, L. maderae and B. germanica). The tree was in agreement with
another molecular tree constructed based on the DNA sequences of mitochondrial 12S rRNA genes
(Tufail and Takeda, 2002) and with the most widely accepted phylogeny of cockroaches constructed by
McKittrick (1964). A recent phylogentic analysis using 23 Vg sequences from 19 insect species by
Tufail et al. (2007) and the present phylogenetic (neighbour-joining) tree constructed based on 24 Vg
sequences from 20 insects in this article (Fig. 6) reflect, in general, the current phylogenetic
classification of insects, suggesting that Vgs are still phylogenetically bound, although there exists a
divergence among them that can be used as a molecular marker.
n dis
A. g ra
A. ga A.
a m
eg bi ica
L. d yp ae n
ispa ti po
r ip o sa
n
Holometabola
P. fo rm
E.
B. m a
o ri er
llif
me
A.
S. cyn
thia ta
S. invi
Coleoptera
A. yamam
ai Diptera
Leptdoptera
yi Hymenoptera
rn
pe
A. A. rosae
Hemiptera
Dictyoptera P. stali-Vg1
L. m a
derae
-Vg1
Hemimetabola
P. stali-V
g 2 g2
ae-V
ader
L. m R. P. stali-Vg
cla 3
va
i ca tu
s
ma n N.
ger g2
ta
B. a -V 1 lu ge
sca
an Vg ns
ric a-
u
e an
rof
am i c
P.
nig
er
am
G.
0.1 P.
Fig. 6. Phylogenetic analysis of 24 Vgs corresponding to 20 insect species. A distance analysis of amino acid
sequences was performed using CLUSTAL W program (Thompson et al., 1994) and used as input for
a neighbor-joining tree construction program (MEGA2, Kumar et al., 2001). Scale indicates distance
(number of amino acid substitutions per site). Source of amino acid sequences is given in Table 1.
6. Biosynthesis and post-transcriptional cleavage of insect vitellogenins
Vg is synthesized along the ribosomes that are associated with the rough endoplasmic reticulum and
subsequently transferred to the Golgi apparatus and eventually packaged into secretary granules
emerging from the trans-Golgi network (Mazzimi et al., 1989; Snigirevskaya et al., 1997; Giorgi et al.,
1989, 1998, 1999). During this process, the Vg is known to be modified post-translationally by
glycosylation, phosphorylation, lipidation and proteolytic cleavage (Kunkel et al., 1980; Brennan and
Mahowald, 1982; Osir et al., 1986b; Takahashi, 1987; Della-Cioppa and Engelmann, 1987;
Don-Wheeler and Engelmann, 1997; Sappington and Raikhel, 1998; Giorgi et al., 1999; Tufail et al.,
2005). Studies on Vg from many insect species show that most insect Vgs are synthesized as one or
more large precursors which are cleaved within the fat body into two or more (depending on the species)
subunits as discussed above. These polypeptides are assembled together and are subsequently secreted
into the hemolymph as large oligomeric proteins (Della-Cioppa and Engelmann, 1987).
77
A 1 2 3 4 5 6 7 8 9 10
Vg subunits
200 170 kD
150 kD Fig. 7. The content fluctuations of P. americana Vgs. The
116 100 kD content fluctuations of P. americana Vgs were determined
97
through SDS-PAGE by comparing the hemolymph from 1-10
66 day-old adult females. The same amount of hemolymph samples
50 kD
45 (0.05 ml equivalent per lane) was loaded on each lane. Numbers
on the left indicate the molecular weight markers (kD). The
SDS-PAGE (7%)
content level of Vg polypeptides (indicated on the right) after
B first appearing in the hemolymph increased continuously until
100
100 kD day 8 (B), which then decreased as was taken up by the
80
developing oocytes. The 170- and 150-kD Vg polypeptide bands
(% )
levels (%)
50 kD
60
were barely visible on SDS-PAGE gels. (B) Quantification of the
40
Vg levels from the SDS-PAGE at different time points. The day
Vg
20
8 Vg levels are expressed as 100% (based on Tufail et al., 2005).
0
1 2 3 4 5 6 7 8 9 10
Adult stage (days)
In hemimetabolous insects, the primary Vg gene product is cleaved into several polypeptides ranging
from 50-180 kD and unlike holometabolous insects where the primary Vg product is cleaved into two
polypeptides (one large and one small). In hemimetabolous insects, the Vg biosynthesis and processing
have been studied in detail in the cockroaches P. americana and L. maderae (Tufail et al., 2000, 2001,
2005, 2007; Tufail and Takeda 2002). In P. americana, the synthesis of Vg is accomplished by two Vg
genes (Vg1 and Vg2), and their products are cleaved soon after their synthesis into three major Vg
polypeptides [170-, 100- (multisubunits), and a 50-kD] and a minor Vg polypeptide (150-kD) (Fig. 1)
(Tufail et al., 2000, 2001, 2005). The Vg gene starts being expressed at two days after emergence in the
female fat body cells, whereas Vg is first detected in the hemolymph by immunobloting in 4 days old
adult females, 2 days after the mRNA of Vg gene first appeared (Tufail et al., 2000), suggesting that the
synthesis of Vg is regulated at the transcriptional level in this species under these conditions. A study on
the fluctuation of Vg content in the hemolymph of P. americana showed that its level in the hemolymph
increased until day 8, and then decreased as it was sequestered by the developing oocytes (Fig. 7) (Tufail
et al., 2005).
In L. maderae, five Vg subunit polypeptides are synthesized (112-, 100-, 92, 87- and 50-kD) (Tufail
and Takeda, 2002; Tufail et al., 2005, 2007), which are coded by two Vg genes (Vg1 and Vg2) (Tufail et
al., 2007). The subunit polypeptides are assembled into the protein backbone of the secreted Vg dimer
(Della-Cioppa and Engelmann, 1987). It has been reported that biosynthesis of Vg in L. maderae occurs
in a stepwise fashion: (1) the co-translational glycosylation of the primary translation product (190-kD)
produces a pro-Vg of 203-kD, (2) the post-translational phosphorylation of 203-kD Vg to 220-kD
pro-Vg, (3) the proteolysis processing of 220 kD pro-Vg to form the constituent polypeptides
(Della-Cioppa and Engelmann, 1987; Tufail and Takeda, 2002; Tufail et al., 2007).
In holometabolus insects, the Vg biosynthesis and processing has been studied in detail in the
mosquito, Aedes aegypti. Native Vg in this insect has a molecular weight of 337-kD and an isoelectric
point of 6.3. It is comprised of two polypeptide subunits of 200- and 66-kD. Bose and Raikhel (1988)
have shown, using an in vitro translation of the vitellogenic fat body mRNA, that both Vg subunits are
derived from a common precursor of 220 kDa, thus originating from the same gene.
7. Examples of post-transcriptional cleavage pathway of insect Vgs
As mentioned earlier, the Vg cleavage is accomplished by the subtilin-like endoproteases,

proprotein covertases (Barr, 1991; Rouille et al., 1995), which recognize a consensus (R/K)XX(R/K) or
RXXR sequence motif immediately preceding the cleavage site. This tetra-residue motif exists at
N-termini of all Vg sequences outside the Apocrita, except that for L. dispar where it is located on the
C-terminus (Fig. 3). The following discussion provides examples of some insect Vgs where
post-transcriptional cleavge patterns have been thoroughly characterized.
78
(7a). Post-transcriptional cleavage of L. maderae Vgs
The Vg of L. maderae was reported to consist of four Vg subunit popypeptides (112, 100, 92 and 50
kD) (Tufail and Takeda, 2002; Tufail et al., 2005) and were believed to be coded by a single Vg gene
(Maseler and Offengand, 1982; Storella et al., 1985; della-Cioppa and Engelmann, 1987; Don-Wheeler
and Engelmann, 1991, 1997). Recently, Tufail et al. (2007) have reported that L. maderae Vg is a
product originating from two precursor polypeptides (pro-Vg1 and proVg2), and that 112-kD
polypeptides are further processed. The proposed processing pathway of L. maderae Vg precursor
molecules is summarized in Fig. 8.
A Lm L (T w o L m V g -g e n e s: L m V g 1 a n d L m V g 2 )
Vg m Vg
1 2
T r a n sc rip tio n
V g1 V g2 (T w o m R N A s)
T r a n sla tio n
Vg1 Vg2 (T w o p r e -p r o -V g s)
V g1 V g2 (T w o p r o -V g s)
P o st-tr a n slatio n a l
p r o te o ly tic p r o ce ssin g
LmVg-polypeptides
V g1 V g2 V g1 V g2
(fat-body)
1 5 5 -k D s 1 1 2 -k D s
LmVn-polypeptides
(oocyte)
V g1 V g2 V g1 Vg2 Vg1 V g2 V g1 Vg2
5 5 -k D s 1 0 0 -k D s 9 2 -k D s 8 7 -k D s
Vg1 Vg2
9 0 -k D s
B
Lm V g1 RRLR RNPR RRTR
? ? 1 913
N - SP 5 5 -kD 1 0 0 -k D 9 2 -, 9 0 -kD 1 1 2 -, 8 7 -kD -C
C
S1 C P S2 P C S3 S4
P
N - SP 5 5 -kD 1 0 0 -kD 9 2 -, 9 0 -kD 1 1 2 -, 8 7 -kD -C
? ? 1 911
Lm V g2 RRLR RNPR RRTR
Fig. 8. Summary of the Vg biosynthesis and post-transcriptional processing pathway of L. maderae Vgs
(Pro-Vg1 and ProVg2). The precursor polypeptides are cleaved into five subunit polypeptides (55-,
100-, 92-, 112- and 87-kD) in the fat body before being secreted into the hemolymph (see text and
Tufail et al., 2007). The 87-kD polypeptide is a minor component originated from Vg1- and Vg2-112
kD polypeptides. The 92-kD Vn polypeptide in the oocyte is further processed and results in the
production of an additional 90-kD Vn polypeptide. The origin and position of all Vg/Vn subunits
compared to their ladder products is shown in B. Post-tranlational cleavage sites (complete, C or
partial, P) following RXXR consensus cleavage sequences (shown with arrows) for both L. maderae
primary products are indicated with dotted lines. Possible but unknown cleavage sites at the
C-termini of 92- and 112-kD polypeptides (to generate the 90- and 87-kD subunits, respectively) are
marked with question marks (see Tufail et al., 2007). S1-S4 indicate the stretches (shown with black
boxes) different in amino acid sequences in both L. maderae Vg precursors. SP is the signal peptide.
Numbers on the right indicate the amino acids deduced from the translation initiation methionine
(based on Tufail et al., 2007).
79
Two Vg genes exist in L. maderae that are transcribed and then translated into two precursor
polypeptides (Pro-Vg1 and -Vg2). It is noteworthy that both Vg precursors share common cleavage sites
and are processed as shown in Fig. 8, and as described previously (Tufail and Takeda, 2002; Tufail et al.,
2005, 2007). Our immunoblot analysis using antibodies generated against peptides unique to each of
Vg1 and Vg2 reveals that the 112-kD polypeptides are processed into 87-kD polypeptides (Fig. 8)
(Tufail et al., 2007). Based on the position of peptides used for antibodies, the 87-kD polypeptides must
come from the cleavage of 112-kD polypeptides at the C-termini. However, nothing is clear about the
cleaved C-terminal ~25 kD polypeptide. Furthermore, the Vg1-112-kD polypeptide is cleaved
completely, as no antigen was detected at the 112-kD position. In contrast, the Vg2-112-kD polypeptide
is cleaved incompletely, and most of the 112-kD polypeptide remains uncleaved (Tufail et al., 2007).
The incomplete cleavage is a common characteristic of cockroach Vgs (Tufail et al., 2001, 2005).
A Pa P (Two PaVg-genes: PaVg1 and PaVg2)

Vg a Vg
1 2
Transcription
Vg1 Vg2 (Two mRNAs)
Translation
Vg1 Vg2 (Two pre-pro-Vgs)
Vg1 Vg2 (Two pro-Vgs)

Post-translational
proteolytic processing
PaVg1-polypeptides
Vg1 Vg1 Vg2

PaVg2-polypeptides
Vg2
(fat-body/oocyte)
(fat-body/oocyte)
100-kD 170-kD 150-kD 100-kD
Vg1 Vg1 Vg2 Vg2
100-kD 70-kD 50-kD 100-kD
B
PaVg1 RTRR
C 1896
N- SP 100-kD 170-kD 100-kD 70-kD -C
P
Polyserine domains Polyserine domains
P C 1876
N- SP 50-kD 150-kD 100-kD -C
PaVg2 Polyserine domains Polyserine domain
Fig. 9. Biosynthesis and post-transcriptional processing patterns of both P. americana Vg precursors

(Pro-Vg1 and Pro-Vg2). Both Vg precursors are synthesized and then cleaved in the fat body before
being secreted into the hemolymph (see text). The origin and position of all Vg/Vn subunits
compared to their ladder products is shown in B. Post-tranlational cleavage sites (complete, C or
partial, P) are indicated with dotted lines. RTRR is the determined cleavage site sequence (shown
with arrow). SP is the signal peptide. Numbers on the right indicate the amino acids deduced from
the translation initiation methionine (based on Tufail et al., 2001, 2005).
80
(7b). Post-transcriptional cleavage of P. americana Vgs
P. americana Vg is coded by two Vg genes (Vg1 and Vg2) (Tufail et al., 2000, 2001, 2005). The
primary precursors are cleaved into three major [(170, 100 (multisubunits) and 50 kD)] and a minor (150
kD) polypeptide (Fig. 1). The post-transcriptional processing pattern of both Vgs (Vg1 and Vg2) of P.
americana is summarized in Fig. 9, as described previously (Tufail et al., 2000, 2001, 2005). The
pro-Vg1 is first processed completely to 100- and 170-kD polypeptides (Tufail et al., 2001, 2005). The
N-terminal amino acid sequence of 170-kD polypeptide matched the deduced amino acid sequence of
Vg1 following the RTRR cleavage site sequence and thus formed the C-terminus of this molecule
(Tufail et al., 2000, 2001). The 170-kD polypeptide is then be cleaved incompletely into 100- and 70-kD
polypeptides in the fat body before being secreted into the hemolymph (Storella et al., 1985).
In the case of Vg2, the primary precursor is first cleaved completely (at C) to produce 150- and
100-kD polypeptides. The 150-kD is then further processed, resulting in the production of 50- and
100-kD polypeptides in the fat body. The determined N-terminal amino acid sequences of 150- and
50-kD polypeptide demonstrated that they share the same sequences and are thus the N-terminus of the
Vg2 (Fig. 9, B) (Tufail et al., 2001, 2005). It is to be noted that the cleavage of 150-kD polypeptide is
incomplete as the 150-kD polypeptide is deteced in the hemolymph and egg extracts (Fig. 1, A) (Tufail
et al., 2001, 2005).
6.5-kb
A Vg mRNA
Translation
Pre- Fig.10 The synthesis of Aedes aegypti

Pro-Vg Vg from 6.5 kb Vg mRNA, and its
224-kD
post-transcriptional processing in the
fat body. Pro-Vg is synthesized and
then cleaved into two subunits in the
fat body before being secreted into the
Pro-Vg
250-kD hemolymph. The origin and position
of the cleaved Vg subunits compared
Post-translational to their ladder product is shown in B.
proteolytic processing
RYRR is the cleavage site (indicated
by arrow and a dotted line), which is
recognized by the cleavage enzyme,
S-Su L-Su
Vg convertase (reproduced from
Sappington and Raikhel, 1998).
62-kD 190-kD
S-Su L-Su
66-kD 200-kD
B
AaVg
RYRR
2123
N- SP 66-kD 200-kD -C
Polyserine domains Polyserine domain
81
(7c). Post-transcriptional cleavage of A. aegypti Vg
The post-translational cleavage process of A. aegypti Vg is shown in Fig. 10. The precursor
polypeptide of 250-kD is coded by a 6.5 kb Vg mRNA. In vitro pulse-chase experiments with
radioactive amino acids revealed that the primary precursor is cleaved in a rate-limiting and regulated
manner to give rise to two Vg polypeptides of 190- and 62-kD, respectively (Dhadialla and Raikhel,
1990). These subunit polypeptides then undergo post-translational modifications leading to mature
subunits of 220- and 66-kD, respectively (Dhadialla and Raikhel, 1990) (Fig.10). Using monoclonal Vg
subunit-specific antibodies, the small polypeptide was shown to be located at the amino-terminal of the
Vg precursor, while the large polypeptide exists at the C-terminal of the precursor (Fig. 10B). The
cleavage site between the two polypeptide subunits in the Vg precursor molecule consists of a consensus
sequence (RYRR) specific for subtilisin-processing endoproteases conserved among insects (Chen et al.,
1994).
8. Glycosylation, phosphorylation and sulphation
Although the primary function of Vg is to provide a bulk of amino acids to the embryo, it also
carries with it carbohydrates, phosphates and sulphates (Kunkel et al., 1980; Brennan and Mahowald,
1982; Osir et al., 1986b; Takahashi, 1987; Diolla-Cioppa and Engelmann, 1987, 1997; Dhadialla and
Raikhel, 1990; Sappington and Raikhel, 1998; Giorgi et al., 1999). These conjugates result from
post-translational modifications (glycosylation, phosphorylation, lipidation) of Vg that not only support
the embryo but are also involved in the internalization of the carrier into the oocyte. The glycosylation,
phosphorylation and sulphation shall be considered here only briefly. For a detailed account see Tufail et
al. (2005).
All insect Vgs characterized until now were glycosylated (Kunkel and Nordin, 1985; Osir et al.,
1986b; Della-Cioppa and Engelmann, 1987; Don-Wheeler and Engelmann, 1997; Giorgi et al., 1998). In
L. maderae, all mature Vg polypeptides are glycosylated (Don-Wheeler and Engelmann, 1997), and
attached carbohydrates are exclusively N-linked mannose oligosaccharides (Konig et al., 1988). These
findings were confirmed by the existence of 14 putative asparagine-linked glycosylation sites (having a
consensus sequence: NXS/T) in both L. maderae Vg cDNAs (Tufail and Takeda, 2002; Tufail et al.,
2007). Such putative Asn-linked glycosylation sites were also found in both Vgs (Vg1 and Vg2) of
P. americana (Tufail et al., 2000; 2001), which were 20 and 17, respectively. Studying the role of
glycosylation on Vg synthesis, processing and secretion from L. maderae, Della-Cioppa and Engelmann
(1997) have demonstrated that blocking of glycosylation with tunicamycin produced an aglycosylated
Vg precursor of 190-KD which was accumulated within the fat body, as reported also from Locusta,
Aedes, and Blattella (Wyatt et al., 1984; Wojchowski et al., 1986; Dhadialla and Raikhel, 1990),
suggesting that glycosylation is an important step in subsequent secretion of Vg by the fat body. This led
to the conclusion that although glycosylation does not play any role in Vg synthesis, its involvement in
the subsequent processing steps (like phosphorylation, proteolysis) is speculated.
Moreover, studying Vg binding to follicle membrane preparations from B. germanica, Konig et al.
(1988) have suggested a complex role for covalently bound carbohydrate. In that, glycopeptide prepared
by protease digestion of vitellogenin inhibited binding of the intact protein, but after α−mannosidase
treatment, which removes most of the mannose residues, these glycopeptides stimulated binding of the
intact protein. The authors concluded that high-mannose oligosaccharides were necessary, but not
sufficient, for binding of Vg to the receptor. However, a different situation was reported by Osir et al.
(1986a, b) from Manduca sexta, where Vg deglycosylated with endoglycosidase-H competed effectively
with the native protein, and was taken up with the latter by the follicles.
Insect Vgs are also heavily phosphorylated, and many at serine residues (Engelmann and Friedel,
1974; Takahashi, 1987; Della-Cioppa and Engelmann, 1987; Don-Wheeler and Engelmann, 1997). L.
maderae Vgs also have high levels of phosphorylation (Tufail and Takeda, 2002, Tufail et al., 2007).
The predicted phosphorylation sites were serine (S), threonine (T), and tyrosine (Y) residues, and once
82
again the phosphorylation at S-residues was particularly high, and this was primarily due to the fact that
the polyserine domains exist at both of the termini (Fig. 3) (Tufail and Takeda, 2002; Tufail et al., 2007).
These polyserine stretches were also found in both Vg molecules (Vg1 and Vg2) of P. americana and
were found to be highly phosphorylated (Tufail et al., 2000, 2001). Della-Cioppa and Engelmann, 1987,
reported that the phosphorylation of L. maderae Vg occurs in fat-body endoplasmic reticulum, just prior
to secretion of the protein into the hemolymph. A possible role of phosphorylation has already been
discussed above.
In the mosquito, A. aegypti, the Vg is glycosylated, phosphorylated and sulphated (Raikhel and
Bose, 1988; Dhadialla and Raikhel, 1990; Chen et al., 1994). Protein sulphation was first demonstrated
in D. melanogaster. Following in vivo radiolabeling with inorganic [35S]-sulfate, radioactive label was
found in all three yolk polypeptides (Baeuerle and Huttner, 1985). Pulse-labeling experiments with
[35S]-sulfate of mouse fibroblasts expressing Drosophila YP2 demonstrated that the intracellular site of
tyrosine sulfation is the trans-Golgi network (Friederich et al., 1988). This observation is in line with the
finding that tyrosylprotein sulfotransferase, the enzyme that catalyzes protein sulfation at tyrosine
residues, is specifically oriented towards the lumen in the cisternae of the trans-Golgi region (Baeuerle
and Huttner, 1985; Huttner, 1988). In A. aegypti, both Vg subunit polypeptides are reported to be
sulphated (Dhadialla and Raikhel, 1990). For more detail see a recent review by Tufail et al. (2005).
9. Hormonal control of Vg biosynthesis
The biosynthesis of Vg is regulated hormonally (Hagedorn, 1985; Raikhel and Dadhialla, 1992;
Wyatt and Davey, 1996). The identified hormones involved in Vg biosynthesis are juvenile hormone
(JH), ecdysone, and neuropeptides. Among them, JH is the best-known for transcription of Vg genes and
the consequent control of Vg production (Engelmann, 1983; Wyatt and Davy, 1996). JHs were initially
identified from their action in metamorphosis to maintain the larval stage status. However, at adult stage
it works as gonadotropin. Several forms of the JH (JH 0, JH I, JH II, JH III), differing in the length of the
side chains, have been identified. JH III, methyl (2E, 6E)-10, 11-epoxy-3, 7, 11 –trimethy l-2,
6-dodecadienoate, is the most frequently involved in vitellogenesis, and is present in a vast majority of
insect orders. It has been reported in a number of insects (L. maderae: Della-Cioppa and Engelmann,
1980; L. migratoria: Couble et al., 1979; Nair et al., 1981; A. aegypti: Raikhel and Lea, 1990) that fat
body cells increase in ploidy and undergo a phase of intensive proliferation of the rough endoplasmic
reticulum after the adult molt, and that these processes, in addition to being a prerequisite for Vg
synthesis, are JH-dependent.
The action of JH or JH analogues upon Vg synthesis has been described in many insect species, and
especially in locusts and cockroaches (see Wyatt and Davey, 1996; Belles, 1998) (also see the section of
“hormonal regulation of vitellogenesis”). Recently, the transcription of Vg gene has been shown to start
as early as 2 hours after the primary stimulation of JH III to allatectomized females of B. germanica
(Comas et al., 1999). The situation was different in L. migratoria (Edwards et al., 1993), where the
administration in vivo of cycloheximide simultaneously with JH analogue delayed the appearance of Vg
mRNA by about 24 hours with respect to the animals treated with the analogue only, suggesting that the
action of JH on Vg transcription in this species depends on a prior process involving the JH-induced
synthesis of another protein or proteins. This should not be surprising, given the relatively slow response
of Vg gene of L. migratoria, which requires a lag time of some 24 hours after induction of the JH (see
Wyatt and Davey, 1996).
The insects can be divided into three types on the basis of the mechanism of hormonal stimulation
in the synthesis of Vg. Type I includes insects which use only JH for Vg biosynthesis. In majority of
insect species, JH is the best-known gonadotropic hormone (Engelmann, 1983; Wyatt and Davy, 1996).
Type II includes insects such as dipterans: mosquitoes and Drosophila melanogaster which require JH
and ecdysone (produced by the ovaries) (Hagedorn et al., 1975). Type III includes lepidopterans that
require JH, ecdysteroids and additional hormones to regulate their reproductive biology.
83
In this chapter we shall review only the key findings together with more recent ones that
demonstrate the role of hormones in vitellogenesis, for insects belonging to the hemimetabola. For more
detail, many comprehensive reviews are available that provide a bulk of information and the historical
background on the subject (see, for example, Engelmann, 1983; Hagedorn, 1985; Davey, 1993; Wyatt
and Davey, 1996; Davey, 1997; Strambi et al., 1997; Belles, 1998, 2005).
(9a). Dictyoptera
The cockroaches were the first insects in which the effect of the CA on production of Vg was
demonstrated. It appears that JH is the only hormone involved in vitellogenesis in cockroaches. Most of
the information derives from P. americana, L. maderae, B. germanica, N. cinerea and D. punctata
(Engelmann, 1983). In P. americana, an oviparous cockroach, the ovarian gonotrophic cycles, including
both the synthesis and uptake of Vgs are regulated by the CA (see reviews Engelmann, 1983; Wyatt and
Davey, 1996). After inhibition or removal of CA, ootheca production was stimulated by administration
of the JH analogues, hydroprene or fenoxycarb (Edwards et al., 1985; Weaver and Edwards, 1990). In B.
germanica, the growth of terminal oocytes responds to different doses of JH I and analogues (Kunkel,
1973). Mating, ingestion of high quality food, social interactions and the presence of vitellogenic
ovaries, influence JH synthesis by the CA in B. germanica (Schal et al., 1997). One or more additional
factors may be involved in the decline of Vg synthesis in this cockroach species late in the gonadotropic
cycle because JH levels remain high while Vg production is declining (Martin et al., 1995). In B.
germanica, as in all other cockroaches studied to date, vitellogenesis and cyclic maturation of oocytes
depends upon JH III (see Schal et al., 1997; Belles, 1998, and references therein).
In L. maderae, Vg synthesis by the fat body has been reported to be strictly controlled by JH or its
analogues. The synthesis of Vg controlled by JH or JH analogues has been demonstrated even in the
males and nymphs of some cockroach species including N. cineraea, D. punctata, B. germanica, and L.
maderae (Don-Wheeler and Engelmann, 1991, and references therein). In these nymphs and males,
usually only a fraction of the Vg is made compared to normal vitellogenic adult females. Thus, there are
stage- and sex-specific differences in the response of the fat body to JH. Recently, a JH analogue,
methoprene, was used to induce the synthesis of Vg in adult females and males of L. maderae, and it was
reported that the synthesis of Vg precursor in the sexually dimorphic fat bodies occurred in the same
stepwise fashion (Don-Wheeler and Engelmann, 1997), suggesting again the sole and strong role of JH
in Vg biosynthesis.
Engelmann (2002) has provided information regarding JH and ecdysone interactions in immature
and adult females of L. maderae which is related to the control of the CA and stimulation of Vg
synthesis in the target tissues and subsequent vitellogenesis. He has shown that the topical application of
400 µg of the JH analogue, methoprene, to females of penultimate instar failed to induce Vg synthesis.
However, last instar females showed an increasing production of Vg as they aged during the first half of
the instar, which then declined to zero in the second half of the last instar when the prothoracic glands
had become highly active. A few days before the metamorphic molt, the responsiveness to methoprene
reached maximal levels, suggesting that the fat body developed competency to produce Vg during the
last nymphal instar, but increasing titers of ecdysone then interfered with the action of methoprene and
consequently the production of Vg was curtailed. Methoprene applied to allatectomized adult females
induced Vg synthesis in a dose dependent manner. This induction was, however, quantitatively reduced
by implanted active prothoracic glands, particularly when low doses of methoprene had been applied.
The effect of the prothoracic glands (i.e., ecdysone) appears to signal an interference with the action of
methoprene at the target tissues, the fat body. The author demonstrates that ecdysone inhibits
vitellogenesis by two independent mechanisms: 1) inhibition of the CA resulting in the inhibition of JH
and 2) inhibition of Vg synthesis by the fat body, and both of these mechanisms appear to be operative in
immature and mature animals (Engelmann, 2002).
84
(9b). Orthoptera
JH is essential for vitellogenesis in several species of grasshoppers and locusts (Engelmann, 1983;
Wyatt and Davey, 1996). The fat bodies of Melanoplus bivittatus and Locusta migratoria have been
used as models for the study of JH action (Roberts and Jefferies, 1986). JH is involved in the synthesis of
Vg in the female fat body cells (Dhadialla and Wyatt, 1983; Dhadialla et al., 1987). The Vg mRNAs
were detected in the fat body in response to JH or JHA (Chinzei et al., 1982; Chinzei and Wyatt, 1985;
Zhang et al., 1993; Glinka and Wyatt, 1996). JH also regulates the uptake of Vgs by the developing
oocytes, and the mature ovary appears to have feedback to the brain and/or the CA, which then down
regulates the production of JH until the primary set of eggs is laid. An appreciable delay has been
observed between the time of administration of a JH analogue and the appearance of Vg mRNA in L.
migratoria. This time lag increased when cycloheximide was co-administered, suggesting the possibility
that activation of Vg gene expression requires JH to first stimulate the synthesis of a transcription factor
(Edwards et al., 1993; Wyatt et al., 1996).
It has also been reported that Vg synthesis can be induced in last stage larvae and adults by the
ovary maturating parsin (Lom OMP), a neuropeptide from the brain, but ovariectomized larvae fail to
respond, suggesting the participation of a factor from the ovaries, possibly 20HE, as in the Diptera
(Girardie and Girardie, 1996). Moreover, it has been shown that Lom OMP has two distinct but
complementary gonadotropic effects: an ecdysteroidogenic effect triggered by its C-terminal domain
with the ovary as the target tissue and a protecting effect on Vg mRNA probably triggered by its other
gonadotropic domain, the N-terminal, with the fat body as the target tissue (Girardie et al., 1998). Thus,
it is clear now that Vg synthesis and vitellogenesis in L. migratoria require not only JH, but also 20 HE
and an ecdysteroidogenic neurohormone as in dipteran insects (Girardie and Girardie, 1996; Girardie et
al., 1992, 1996, 1998). However, JH is the principal hormone in locusts, since in the adult, it is the only
hormone that induces the expression of Vg genes.
In the cricket, Acheta domesticus, JH controls Vg synthesis in the fat body, stimulating transcription
of Vg genes and synthesis of Vg polypeptides, which are exported into the hemolymph (Bradley and
Edwards, 1978; Benford and Bradley, 1986; Strambi et al., 1997). At the same time, JH also induces
morphological changes in the follicular epithelium, including the enlargement of interfollicular spaces
for the uptake of Vg by the developing oocytes. In Gryllus bimaculatus, the time of the first appearance
of Vg in the hemolymph is correlated with the onset of JH synthesis by the CA (Kempa-Tomm et al.,
1989, 1990), suggesting the role of JH in Vg synthesis. JH III is the major JH in many gryllids like A.
domesticus, Teleogryllus commodus and G. bimaculatus (see Strambi et al., 1997). The allatectomy
completely abolishes oocyte growth in A. domesticus, but it did not totally suppress the ovarian
development and egg-laying in T. commodus, G. bimaculatus and G. campestris (see Strambi et al.,
1997) as in Rhodnius prolixus (Pratt and Davey, 1972; Wang and Davey, 1993).
In contrast to the stimulatory effect of ecdysteroids on vitellogenesis in Locusta (Girardie and

Girardie, 1996; Girardie et al., 1992, 1996, 1998) and Gryllus, (Behrens and Hoffmann, 1983) as in the
cockroach Blabarus (Perriere et al., 1993), ecdysteroids have been reported to inhibit vitellogenesis in
other insects, notably the cockroaches Diploptera (Friedel et al., 1980) and Leucophaea (Engelmann,
1971, 2002). Recently, Hatle et al. (2003) has presented data from the lubber grasshopper, Romalea
microptera, where ecdysteroids are shown to have no effect on vitellogenesis, and is different from those
of other orthopterans. Thus the role of ecdysteroids in reproduction of non-dipteran insects is not yet
clearly defined (Gaede et al., 1997; Tawfik et al., 1997).
85
(9c). Heteroptera
JH is the principal hormone governing vitellogenesis in Heteroptera (Davey, 1997). Most of the
data available is from the blood-sucking bug, Rhodnius prolixus (Davey, 1993) in which JH is
responsible for Vg production in the fat body. Similar data have been reported from other Heteropteran
species including both haematophagous, such as Triatoma protracta, and phytophagous like Oncopeltus
fasciatus and Pyrrhocoris apterus (Engelmann, 1983). In R. prolixus, the synthesis of Vg is reported to
be stimulated by a low dose of methoprene (Chinzei et al., 1994). Allatectomy halts or severely retards
egg development in O. fasciatus, Dysdercus cigulatus, Triatoma infestans, Dindymus versicolor,
Dysdercus intermedius, and Dysdercus koenigii (see Davey, 1997, and references therein). There are
discrepancies, however, in the requirement for JH for vitellogenesis in R. prolixus and O. fasciatus,
where allatectomy did not lead to a complete cessation of egg production (Pratt and Davey, 1972; Wang
and Davey, 1993; Kelly and Hunt, 1982). In many heteropteran species, JH is involved not only in Vg
synthesis in the fat body (Coles, 1965; Mundall and Engelmann, 1977), but also controls access of Vg to
the oocyte (Davey, 1993), and uptake of Vg by the oocytes (Wang and Davey, 1992). Topical
application of JHs or their analogues to a variety of Heteroptera terminated the adult reproductive
diapause. Riptartus clavatus females developed vitellogenic oocytes after being treated with Altosid®
(Numata and Hidaka, 1984), JH I or JH II (Shinoda, 1996). However, fewer vitellogenic follicles were
produced when females were treated with JH III. This is in contrast to the two-spotted stinkbug, Perillus
bioculatus, where JH III is involved in ovarian maturation and Vg synthesis (Adams et al., 2002).
Thus, all of the species of Heteroptera examined to date show stimulation of vitellogenesis by JH.
In most of the cases, the synthesis of Vg seems to be dependent only on JH, except in R. prolixus and O.
fasciatus, where Vg synthesis can take place in the apparent absence of JH.
10. Uptake of vitellogenins by the oocytes
Following secretion into the hemolymph, Vgs are taken up by developing oocytes. The uptake of
Vg by developing oocytes during oogenesis is a characteristic phenomenon of all oviparous animals
(Stifani et al., 1990; Raikhel and Dhadialla, 1992) and is achieved through receptor-mediated
endocytosis, a ubiquitous mechnism for the selective intake of macromolecules by animal cells
(Goldstein et al., 1979; Anderson and Kaplan, 1983). Vg is taken up by the developing oocytes through
channels between the follicular cells (Telfer, 1961, 1965; Raikhel and Dhadialla, 1992). JH acts with the
membrane receptor to promote Vg uptake by promoting widening of the intercellular spaces, referred to
aspatency, in the follicular epithelium. This occurs as a result of cell shrinkage from Na+/K+-ATPase
activation, and phosphatidylinositol and protein kinase C are involved (Ilenchuk and Davey, 1987; Davy,
1993). Receptors (VgR) where Vg then anchors are localized in coated pits on the surface of growth
competent oocytes which are engulfed with the yolk protein precursor into cells by receptor mediated
endocytosis (Telfer et al., 1982; Byrne et al., 1989; Raikhel and Dhadialla, 1992). For a complete
account of VgR proteins and their functioning see Tufail and Takeda (this volume).
Although several types of yolk protein precursors are accumulated by insect oocytes (see reviews:
Raikhel and Dhadialla, 1992, Izumi et al., 1994; Rajaratnam, 1996, Sappington and Raikhel, 1998), Vg,
with few exceptions, is by far the most abundant in all insect species. The proteins accumulated other
than Vg in the oocytes include lipophorin (Machado et al., 1996), arylphorin (Telfer et al., 1983),
microvitellogenin (Kawooya et al., 1986) and several other follicle specific proteins (Sato and
Yamashita, 1991). Telfer and Pan (1988) have estimated the rate at which these proteins are incorporated
into the oocytes and the ultimate amplification factor they may attain upon storage in the oocyte.
However, the receptor specificity for these additional proteins is not yet clear. The competition
experiments using Vg and other hemolymph proteins have shown that some of them may utilize the
same receptor system with Vg, while others are apparently taken up by an independent endocytotic
pathway (Machado et al., 1996). The cytoplasm of the oocyte also contains a number of lipid droplets
together with triglycerides, phospholipids and granules of glycogen in them. In addition, some proteases
are also taken up by the oocytes during vitellogenesis.
86
Near the end of vitellogenesis, a thin protein sheet, the vitelline membrane, an acellular coat that
surrounds the oocyte, is secreted at the inner surface of the follicle cells (Dhadialla and Raikhel, 1992).
There is evidence that 20-hydroxyecdysone regulates its formation. The vitellin membrane in D.
melanogaster is composed of numerous proteins with molecular mass ranging from 14-130 kD (Fargnoli
and Waring, 1982). Wyatt, (1991) has repoted that they are encoded by a family of genes. The last
synthetic act of follicle cells is the formation of chorion which is composed of a number of sclerotized
proteins secreted under the control of gene families (small or large) (Waring and Mahowald, 1979;
Kafatos et al., 1987). The chorion is placed on the egg while it is still present in the ovary, and before
fertilization. Once the chorion is secreted, this layer forms an impermeable covering that restricts the
trafficking of substances in or out with the exception of sperm that enters through a specialized opening,
the micropyle. The number of micropyles varies (depending on the species) from one to several. When
the secretion of the chorion is completed, the old follicle cells degenerate and leave the chorion as the
outer surface of the egg which then passes into the median oviduct.
11. Conclusion
The sequencing of Vg genes/cDNAs from several insect species has not only led to an
understanding of the Vg primary structures, but also has made it clear that how Vg biosynthesis is
regulated and how this molecule undergoes a complicated pattern of post-transcriptional processing. The
comparison of coding sequences of all known insect Vgs has shown that their structures are highly
conserved and form a gene superfamily. This is, perhaps, because of the nutritional demands of the
insect embryos that certain amino acid residues and motifs (like cystein residues, and GLI/CG and
DGXR motifs at the carboxy-terminal) of Vg primary product remain conserved. Overall, the picture
that has emerged is that the Vg genes are large and each specifies a single transcript of 6-7 kb encoding
Vg precursor protein of ~200 kD, which undergoes proteolytic cleavage to generate subunits ranging
from 50-180 kD. These subunits are then post-translationally modified by glycosylation, phosphorylation
and sulphation to be functionally mature Vg polypeptides. Recent molecular studies have revealed that
the proteolytic cleavage of Vg is more complicated in hemimetabolous insects (cleaved into several
subunits: small, medium and large) than in those of holometabolous insects, where Vg molecule is
cleaved into two subunits (one large and one small). The cleavage is accomplished by the subtilisin-like
proprotein convertases, which recognize a consensus (R/K)XX(R/K) sequence motif preceding the
cleavage site. This tetra-residue moif exists at N-termini of all Vg sequences outside the Apocrita, except
that for L. dispar where it is located on the C-terminus.
Although the recent molecular cloning of Vgs has provided a bulk of information about the protein
primary structure and the post-transcriptional processing patterns of this molecule, it has also raised new
questions. For example, the importance of the differential cleavage of the Vg precursor in different
insects; the regulation of the extent of cleavage at each site, both in the fat body and the oocyte; and the
nutritional and physiological role of the cleaved Vgs for the developing embryo offer promising areas for
future research.
Moreover, another issue still waiting to be resolved that why Vg of some insects is coded by
multiple Vg genes. We speculate that different Vg genes may have different functions. For instance, the
plasma clotting protein of crayfish shares structural characteristic with Vgs but has a unique function
(Hall et al., 1999). Our hypothesis is also supported by the fact that Vg gene is expressed in honey bee
males (Trenczek and Engels, 1986; Trenczek et al., 1989; Valle, 1993; Piulachs et al., 2003) suggesting
that Vg may have also some additional function than as a yolk. Delimiting of the general and specific
physiological roles of multiple Vgs/isoforms should prove to be important.
87
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Chapter – 5
Molecular mechanisms of insect vitellogenin/ lipophorin receptors

Muhammad Tufail* and Makio Takeda
Graduate School of Science and Technology, Kobe University,
Nada, Kobe 657-8501, Japan.
Abstract
Insect vitellogenin and lipophorin receptors (VgRs/LpRs) are members of the low density lipoprotein receptor (LDLR)
gene superfamily, and play a critical role in oocyte development by mediating endocytosis of the major yolk protein
precursors, Vg and Lp, respectively. All LDLR family receptors share a group of five structural modules: cysteine-rich
repeats constituting the ligand-binding domain (LBD), epidermal growth factor (EGF)-like repeats, repeats containing a
YWTD motif, a transmembrane domain, and a cytoplasmic domain. Each structural domain is specified for a distinct and
important functions. The analysis of the protein primary structures reveals that insect VgRs harbor two LBDs with 5
repeats in the first and 8 repeats in the second domain as compared to LpRs which have a single 8-repeat LBD. Moreover,
the cytoplasmic tail of all insect VgRs contains a LI internalization signal instead of the NPXY motif found in LpRs and in
majority of other LDLR family receptors. The exception is that of the fire ant VgR which also contains NPXY motif in
addition to LI signal. The cockroach VgRs still harbor another motif (NPTF) which is also believed to be a functional
internalization signal. The expression studies demonstrate that insect VgRs are ovary-specific members of the LDLR
family as compared to LpRs which are transcribed in a wide range of tissues including ovary, fat body, midgut, brain,
testis, Malpighian tubules and muscles. The functional importance of individual receptors may lie in their tissue-specific
expression. This chapter provides a brief review of the current knowledge pertaining to molecular structures and
physiological functions of VgRs and LpRs in insects. Moreover, this chapter also compares insect VgRs/LpRs with other
family receptors reported both from vertebrates and invertebrates to provide information on the evolutionary aspects
among these proteins.
Keywords: insect vitellogenin receptors; lipophorin receptors; LDLRs; structure; expression; regulation;
* For Correspondence (email: mtufail@kobe-u.ac.jp)
Overview
8. Developmental profile and cellular distribution of the
1. Introduction ovarian LpR mRNA
2. Insect VgRs/LpRs are LDLR family members 9. Structural organization of insect VgRs/LpR and other
3. Characteristics of LDLR family receptors family receptors
4. Insect Vg/Lp receptors in context of other LDLR family (9.A). Ligand-binding domain
members (9.B). EGF-precursor homology domain
5. VgRs are ovary-specific receptors of the LDLR family (9.C). O-linked sugar domain
6. LpRs are universally expressed receptors of the LDLR (9.D). Transmembrane domain
family (9.E). Cytoplasmic domain
7. Developmental profile and cellular distribution of VgR 10. Concluding remarks
mRNA and the protein 11. References
95
1. Introduction
All oviparous species provision their eggs with yolk to ensure ample supply of amino acids, proteins,
lipids and carbohydrates for the future embryo. In insects, most of the yolk protein components are
synthesized extraovarially in the fat body and are internalized by the competent oocytes. The
accumulation of protein reserves in insect oocytes is achieved by membrane-bound receptors through
receptor-mediated endocytosis (reviewed in Raikhel and Dhadialla, 1992, Sappington and Raikhel, 1998;
Izumi et al., 1994; Rajaratnam, 1996; Van Antwerpen et al., 2005; Meenakshi et al., 2007). Although
several types of yolk protein precursors (YPPs) are accumulated by insect oocytes, vitellogen (Vg), with
few exceptions, is by far the most abundant in all insect species. The uptake of Vg in insect oocytes is
achieved by membrane-bound receptors (VgRs) (reviewed in Raikhel and Dhadialla, 1992, Sappington
and Raikhel, 1998; Van Antwerpen et al., 2005). VgR genes and/or cDNAs have been investigated in a
wide group of animals, both vertebrates and invertebrates including insects. In insects, VgRs have been
sequenced from six insect species: Aedes aegypti (yellow fever mosquito) (Sappington et al., 1996),
Solenopsis invicta (fire ant) (Chen et al., 2004), Periplaneta americana (American cockroach) (Tufail
and Takeda, 2005), Blattella germanica (German cockroach) (Ciudad et al., 2006), Leucophaea maderae
(Madeira cockroach) (Tufail and Takeda, 2007) and a yolk protein receptor (YPR) of Drosophila
melanogaster (Schonbaum et al., 1995) (We will use VgR instead of the convential terminology (YPR)
in the text). Moreover, a genome sequence (accession no. EAA06264) coding for VgR from malaria
mosquito, Anopheles gambiae is also available. The insect VgR gene transcript of ~7.5 kb encodes a
large ovary-specific protein of ~180-215 kDa, roughly twice the size of vertebrate VgRs (95-115 kDa)
(Ferenz, 1993; Sappington and Raikhel, 1995; Tufail and Takeda, 2005, 2007).
The proteins accumulated other than Vg in the oocytes include lipophorin (Lp) (Machado et al.,
1996), arylphorin (Telfer et al., 1983), microvitellogenin (Kawooya et al., 1986) and several other
follicle specific proteins (Sato and Yamashita, 1991). Lp serves as a reusable shuttle for transporting
lipids from the fat body to oocytes (in lepidopteran) and, in some insects, it is endocytosed as an intact
molecule by Lp receptor (LpR) (Telfer et al., 1991). However, the receptor specificity for other
additional proteins is not yet clear. Lps are also reported to be the carrier of hydrocarbon from
hemolymph to the oocyte (Sevala et al., 1999; Fan et al., 2002). Recent studies have shown that high
density Lp (HDLp) is the one endocytosed as an intact particle by the cell surface Lp receptor (Van Hoof
et al., 2003, 2005). LpRs have been cloned from several insect species such as Locusta migratoria
(Dantuma et al., 1999), A. aegypti (Cheon et al., 2001; Seo et al., 2003), and Galleria mellonella (Lee
et al., 2003), Bombyx mori (Gopalapillai et al., 2006) and B. germanica (Ciudad et al., 2007) and L.
maderae (Tufail et al., 2009). However, it remains unclear whether or not the LpR plays any part in lipid
shuttling mechanism (Ryan et al., 2000; Arrese et al., 2001).
2. Insect VgRs/LpRs are LDLR family members
Insect VgRs and LpRs are members of the low density lipoprotein receptor (LDLR) gene
superfamily. Other members of this receptor family include the LDLR, a prototype of the LDLR family
(Yamamoto et al., 1984; Willnow, 1999), very LDLR (VLDLR) (Sakai et al., 1994), apolipoprotein E
receptor 2 (Kim et al., 1996), LDLR-related protein (LRP) (Herz et al., 1988) and megalin (Saito et al.,
1994). These receptors can be divided in receptors with a single ligand binding domain (LBD) and EGF-
precursor domain (EGFD), such as the classical LDLRs (Yamamoto et al., 1986), vertebrate VgRs (Bujo
et al., 1994; Okabayashi et al., 1996), Nematode VgR (Grant and Hirsh, 1999 and VLDLRs (Takahashi,
et al., 1992; Sakai et al., 1994), and receptors possessing multiple (more than two) clusters of these two
domains, such as the LRPs (human and chicken, Herz, et al., 1988; Nimpf et al., 1994) and megalins
(Saito et al., 1994; Hjalm et al., 1996). The insect VgRs have two clusters and make a unique subclass of
LDLR family receptors (see Tufail and Takeda, 2007 and references therein), whereas LpRs belong to a
group having a single cluster (Fig. 1). The differences in number and arrangement of these repeated
sequences are thought to be responsible for the diversity of ligands which bind to this family of receptors
(Hobbs et al., 1990).
96
N N
ligand-binding
domain-1
Ligand-binding repeat
EGF repeat
YWTD repeat
EGF-precursor
domain-1
N N
N
ligand-binding
domain-2
EGF-precursor
domain-2
O-linked sugars
Transmembrane
NPTF
NPTF
NPTY
NPTY
NPTY
Cytoplasmic
LI
LI
C C C C C
PaVgR LmVgR LmLpR GgVgR HsLDLR
(American cockroach) (Madeira cockroach) (Chicken) (Human)
Fig.1. Schematic comparison of the LDLR family receptors. All LDLR family members have the identical
structural organization. Insect VgRs have two ligand binding/EGF-precursor domains as compared to
insect LpRs, vertebrate VgRs and LDLRs. PaVgR: P. americana VgR, LmVgR: L. maderae VgR,
LmLpR: L. maderae LpR, GgVgR: G. gallus VgR, HsLDLR: H. sapiens LDLR.
3. Characteristics of LDLR family receptors
The LDLR family receptors are membrane-bound proteins that can be found in species ranging
from nematodes to insects and mammals. The genes are originated from a common evolutionary
ancestor. These receptors share a characteristic set of five structural modules (Fig. 1): i) cystein-rich
ligand binding repeats (LBRs) (Class A module), ii) epidermal growth factor (EGF)-like repeats (Class B
module), iii) repeats containing a YWTD motif that are proposed to form a β-propeller domain
(Springer, 1998), iv) a transmembrane domain anchoring the receptor to the plasma membrane, and v) a
cytoplasmic domain containing at least one copy of the tight-turn-tyrosine motif (NPXY) [LI/LL and
NPXF motif in insect VgRs, see Sappington and Raikhel, 1998, Tufail and Takeda, 2005, 2007] which is
involved in receptor internalization via coated pits (Goldstein et al., 1985; Chen et al., 1990).
Despite structural homology of LDLR family members, these receptors are involved in a diverse
array of biological functions, including lipoprotein transport into cells (Goldstein and Brown, 1974),
97
clearance of protein complexes from plasma (Herz et al., 1992) as well as vitamin carriers. More recent
findings, however, show that some members are also involved in signaling events that play a role in
neuronal migration, synaptic transmission or embryonic development (Hiesberger et al., 1999;
Trommsdorff et al., 1999) (for review see: Schneider and Nimpf, 2003). The VgRs/LpRs, as indicated
above, are particularly involved in reproduction. The chicken VgR, for example, has been shown to
import very low-density lipoprotein (VLDL), riboflavin-binding protein, and alpha2-macroglobulin into
growing oocytes (Stifani et al., 1990; Mac Lachlan et al., 1994). Indeed, mutations that abrogate
expression of the VgR result in non-egg laying hens. In D. melanogaster, female sterility occurs in
insects having a genetic deficiency of yl (Schonbaum et al., 1995). Analysis of oocytes produced by yl-
females shows a drastic reduction in numbers of coated pits and coated vesicles and very little
proteinaceous yolk (DiMario and Mahowald, 1987). The LpR is involved in the transfer of lipids by
internalizing the HDLp through receptor-mediated endocytosis (Dantuma et al., 1999). Transfection
experiments showed that HDLp is recycled upon LpR-mediated endocytosis in mammalian (CHO) cells
(Van-Hoof et al., 2002) but not in insect (S2) cells. This suggests that the recycling mechanisms is cell-
type specific (Van-Hoof et al., 2005), adding complexicity to the receptor-mediated endocytosis of the
Lp.
LDLR family members have the ability to bind multiple ligands. The chicken VgR, for example,
recognizes at least eight different ligands (Hiesberger et al., 1995; Jacobsen et al., 1995). LDLR-related
protein binds more than 20 different ligands (Strickland et al., 1995; Kounnas et al., 1996). The
mammalian VLDLR has been shown to bind apoE specifically, whereas apoB, a ligand of the LDL
receptor, does not interact with this receptor (Takahashi et al., 1992). In insects, the structurally very
similar receptors for Vg/YP from the mosquito (Sappington et al., 1996) and Drosophila (Schonbaum et
al., 1995) recognize quite unrelated ligands suggesting that insect VgRs may also have possibility to
recognize more than one ligand. The LDLR binds the ligand at the cell surface at neutral pH,
transporting that to endosomes where the low-pH environment induces ligand release. The two activities
of the receptor, ligand binding and pH-triggered release, are attributed to distinct domains, referred to as
the LBD and the EGF precursor homology domain, respectively (Brown and Goldstein, 1986). Electron
microscopy studies have indicated that the receptor adopts an extended arrangement at neutral pH,
presenting the tandemly repeated LBRs for ligand binding (Jeon and Shipley, 2000). The 3.7 A° X-ray
structure revealed that at endosomal pH the receptor closes by forming intramolecular long-range
contacts between the β-propeller domain and the central LA repeats of the ligand-binding domain
(Rudenko et al., 2002).
4. Insect Vg/Lp receptors in context of other LDLR family members
LDLR family receptors represent a variety of protein molecules, all of which are characterized by
various arrangements of five structural modules. To review the phylogenetic relationship of insect Vg/Lp
receptors with other LDLR family, we performed a phylogenetic analysis of 32 lipoprotein receptors
available in the literature and GenBank database. Using a neighbour-joining tree construction program
based on distances of amino acid sequences, we obtained the tree shown in Fig. 2. The tree places the
insect Vg/Lp receptors in separate groups. The insect LpRs appear as a sister group of vertebrate
VLDLRs and VgRs. Interestingly, the insect VgRs appear as the sister group of other receptors (Fig. 2).
Moreover, the branch lengths among the LpRs are shorter among the LDLR members used for
phylogenetic analysis, suggesting that the divergence among LpRs is not rapid. Our phylogenetic
analysis also reveals that the insect LpRs are more closely related to the vertebrate VLDLRs/VgRs and
LDLRs than to insect VgRs and that the split of a single LBD receptors and multiple LBD receptors
occurred at an early stage in evolution.
98
ia LpR
B om
LPR
R
LP
byx
Ae
ro
ea
Galler
usta
ha
de
op
LpR
op
s
hi
uc
Lp
Loc
la
Le
P
Lp
Bos LDLR
R
pR
Blattella L
sL
like)
lu
Su
alin-
al
sL
(Meg
Hom Insect
G
P
Homo LR LRP
DL
o LDL LpRs
R bditis
R
LR
Ps orha
Mus LDLR Caen
LDLRs
alin
s LDLR Megalins Gallus meg
Xenopu
B os L
Oncorhynchus VgR Vertebrate RP2
VgRs/VLDLRs
mega
lin
R
us Vg
Xenop gR R M Insect Solen
us opsis
sV VL VgRs
llu LDL VgR
Ra
DLR
G a DL
sV
ttu
R Leuc
Bo
s
opha
L
VL
e a Vg
Homo V
R
DL
Bl
at
R
te
lla
Pe
Vg
rip
R
l an
Aed
eta
Anopheles VgR
Dr
es V
Vg
oso
R
p
gR
hil
aY
Caenorhabditis VgR
PR
0.1
Fig. 2. Phylogenetic (neighbor-joining) analysis of 32 LDLR family receptors. A distance analysis of amino
acid sequences was performed using CLUSTAL W program (Thompson et al., 1994) and used as input
for a neighbor-joining tree construction (MEGA Ver3.1, Kumar et al., 2001). The RME-2 (VgR) of the
nematode (Caenorhabditis elegans) was used as an out group. The scale indicates distance (number of
amino acid substitutions per site). Sequences used from the data base were: Leucophaea maderae VgR
(accession number: AB255883), Blattella germanica VgR (accession no: AM050637), Periplaneta
americana VgR (accession no: AB077047), Aedes aegypti VgR (ov) (accession no. L77800), Anopheles
gambiae VgR (accession no: EAA06264), Drosophila melanogaster yolk protein receptor (YPR)
(accession no: U13637), Solenopsis invicta VgR (accession no: AY262832), L. maderae LpR (accession
no: AB218869), B. germanica LpR (L) (accession no: AM403063), Locusta migratoria LpR (accession
no: AJ000010); A. aegypti LpR (accession no: AF355595), Bombyx mori LpR(1) (accession no:
AB201471), Galleria mellonella LpR (accession no: DQ482581), D. melanogaster LpR (accession no:
NM_733119), Gallus gallus VgR (accession no: X80207), Xenopus laevis VgR (accession no: AB006906),
Oncorhynchus mykiss VgR (accession no: AJ003117), Rattus norvegicus VLDLR (accession number:
NP_776914), Mus musculus VLDLR (accession no: AAH13622), Bos taurus VLDLR (accession no:
NP_776914), Homo sapiens VLDLR (accession no: NP_003374), Mus musculus LDLR (accession no:
AF425607), H. sapiens LDLR (accession no: L00352), X. laevis LDLR (accession no: Q99088), Sus
scrofa LDLR (accession no: AAC17444), B. taurus LDLR (accession no: XP_874020), C. elegans LRP
(megalin-like) (accession no: Q04833), B. taurus (LRP2) megalin (accession no: XP_592673), G. gallus
megalin (accession no: XP_967944), G. gallus LRP (accession no: X74904), H. sapiens LRP (accession
no: X13916) and C. elegans VgR (RME-2) (accession no: AF185706).
99
5. VgRs are ovary-specific receptors of the LDLR family
VgRs are ovary-specific members of the LDLR gene family (Schneider, 1996). Recently, we have
cloned and characterized two VgRs from two cockroach species, P. americana (Tufail and Takeda,
2005) and L. maderae (Tufail and Takeda, 2007). An ovary-specific VgR transcript of ~7.3 kb was
observed throughout the period of ovarian development at high levels especially in previtellogenic
ovaries of both species. RNA in situ hybridization and immunocytochemistry localized VgR mRNA and
the protein only in germ line-derived cells, the oocytes, and revealed that VgR gene transcription and
translation begin very early during oocyte differentiation in the germarium (Tufail and Takeda, 2005).
The immunoblot analyses probed ovary-specific VgR proteins of ~210 and ~215 kD in P. americana and
L. maderae, respectively on the day of female emergence. The expression of VgR protein during very
early periods of oocyte development clearly demonstrates that VgR is not the limiting factor for Vg
uptake. We demonstrated that VgR-endocytotic machinery starts functioning soon after the ligand (Vg)
becomes available (Tufail and Takeda, 2005) (Fig. 3). The VgR signals were high during vitellogenic
periods (Tufail and Takeda, 2005, 2007). Our investigations along with those reported from other insects
(Schonbaum et al., 1995; Sappington et al., 1996; Chen et al., 2004, Tufail and Takeda, 2005;
Meenakshi et al., 2007), annelids (Hafer, 1992) and vertebrate species (Bujo et al., 1994; Okabayashi et
al., 1996 and Davail et al., 1998) indicates that VgRs are ovary-specific members of the LDLR gene
superfamily, and this is consistent with the VgR function.
Endocytosed by
VgR
VgR Ovary
VgR Vg
Vg
gene gene
1 2 3 4 5
Last nymphal Adult stage (days)
stage
Fig. 3. Diagrammatic representation of the stage-specific expression of VgR/Vg genes and their products, and
the onset of Vg uptake by the ovary of P. americana. The VgR gene starts expressing in ovaries before
adult emergence whereas the VgR protein is detected in previtellogenic ovaries on the day of female
emergence. The Vg gene transcript was detected in two-day old adult female fat body cells, whereas the
Vg primay product was detected in the hemolymph of four-day-old adult females, two days after the
Vg gene expression. The receptor-endocytotic machinery starts fuctioning soon after the ligand
becomes available. The Vg uptake was observed in 5-day old ovaries. (based on Tufail et al., 2000, 2001,
2005; Tufail and Takeda, 2005).
6. LpRs are universally expressed receptors of the LDLR family
In contrast to VgRs, the insect LpRs are expressed in in a wide range of tissues including ovary, fat
body, midgut, brain and testis (Tsuchida and Wells, 1990; Dantuma et al., 1999; Gondim and Wells,
2000; Cheon et al., 2001; Seo et al., 2003; Lee et al., 2003; Ciudad et al., 2007). In B. mori, four LpR
isoforms (LpR1-LpR4) are reported and one (LpR4) is cloned from the central nervous system
(Gopalapillai et al., 2006). In A. aegypti, two variants of LpR gene product specific to the fat body and
100
ovary have been sequenced (Cheon et al., 2001; Seo et al., 2003). Recently, we performed Northern blot
analysis with total RNA extracted from the adult female ovaries, fat body, muscles, Malpighian tubules,
midgut and head. The LpR mRNA was detected in all selected tissues (Tufail et al., 2009). In ovaries, a
transcript of around 4.0 kb was observed and matched the size of the cDNA we cloned (3362 bp).The
presence of LpR transcript(s) in different tissues suggests the tissue-specific usage of these proteins.
7. Developmental profile and cellular distribution of VgR mRNA and the protein
Recently, we have observed the developmental profile of VgR gene transcription in two cockroach
species, P. americana and L. maderae (Tufail and Takeda, 2005, 2007). The VgR mRNA was produced
at all stages of ovarian development especially with its level high during early previtellogenic periods.
Almost a similar pattern of VgR gene transcription was also observed in adult B. germanica (Ciudad
et al., 2006). In S. invicta, the VgR mRNA levels were higher in virgin alate females than in actively
reproductive queens (Chen et al., 2004). Among insects, the most diverging pattern of VgR gene
transcription was observed in the mosquito, A. aegypti, where mRNA levels started to increase rapidly
after one day of adult emergence, and continued to increase dramatically during the vitellogenic periods,
reaching its peak at 24 h post blood meal (Cho and Raikhel, 2001). In non insect species like chickens
and rainbow trout also high levels of VgR mRNA in early vitellogenic periods, and low or no VgR
mRNA levels in fully vitellogenic periods were observed (Bujo et al., 1995; Davail et al., 1998),
suggesting that VgR gene is particularly expressed at high levels long before vitellogenesis starts. The
expression pattern of VgR protein, in cockroaches, is almost complementary to that of the VgR gene
transcript; a high transcriptional level of VgR mRNA during early adult (previtellogenic) periods is
followed by increased protein level during late adult periods, the vitellogenic phase as seems to be
functionally required (Tufail and Takeda, 2005, 2007; Ciudad et al., 2006).
The studies using RNA in situ hybridization and immunocytochemistry localized VgR mRNA and
the protein only in germ line-derived cells, the oocytes, in P. american, and revealed that VgR gene
transcription and translation starts very early during oocyte differentiation in the germarium (Tufail and
Takeda, 2005). The VgR transcript was abundant and found to be evenly distributed throughout the
oocyte. No VgR transcript was detected in any somatic cells of the ovary (see Tufail and Takeda, 2005)
In dipteran ovaries, the VgR gene transcript was localized in the nurse cells in addition to the oocyte of
each follicle (Sappington et al., 1996; Schonbaum et al., 2000). Transgenic studies of the yl gene
encoding Drosophila YPR and yl-LacZ reporter analysis have revealed that yl mRNA is transcribed only
in the germ line nurse cells and that the yl transcript is accumulated in developing oocytes (Schonbaum
et al., 2000). The cockroach oocyte, however, lacks nurse cells, suggesting VgR transcript is likely to be
transcribed by the oocyte nucleus. In P. americana, the VgR protein were evenly distributed throughout
the oocyte in early previtellogenic ovaries whereas it was accumulated mainly in the oocyte cortex
adjacent to the follicular epithelium in late previtellogenic periods and before the vitellogenesis was
initiated (Tufail and Takeda, 2005). Also, in Drosophila, the Yl protein was reported to be present at the
cortex of vitellogenic stage oocytes, and was detected by immunogold labeling in coated vesicular and
tubular structures (Schonbaum et al., 2000). In previtellogenic stage oocytes, however, Yl was uniformly
distributed throughout the oocyte (Schonbaum et al., 2000).
8. Developmental profile and cellular distribution of the ovarian LpR mRNA
The developmental expression pattern of L. maderae LpR mRNA revealed that LpR gene is
expressed during all stages of the ovarian development (Tufail et al., 2009). The transcript level was low
in early previtellogenic periods (until day 2) which was increased to its maximum during the late
previtellogenic periods on day 5, and remained high during vitellogenic periods. A similar phenomenon
has also been observed in A. aegypti LpR (Cheon et al., 2001) where the transcript was detected
throughout the ovarian development but increased after the onset of vitellogenesis, peaking by 24 h post
blood meal. Recently, Ciudad et al. (2007) have isolated two LpR isoforlms (L and S) in B. germanica
and reported a high level of L-isoform during previtellogenic stage and low during later stages
101
(vitellogenic) whereas the S-isoform is expressed at very low level during the first days, increases
slightly in the mid-vitellogenesis and decreases during later periods (days 6 and 7). In A. aegypti ovary,
the LpR transcript is localized exclusively in the germ-line cells, nurse cells and oocytes throughout the
ovarian development. The LpR mRNA was detected in the germarium (Cheon et al., 2003) similar to
VgR from the same species (A. aegypti) (Cho et al., 2001) and from other species including P.
americana (Tufail and Takeda, 2005), and D. melanogaster (Schonbaum et al., 2000). Thus, it appears
that the genes involved in the receptor-endocytotic machinery are specifically expressed long before
functionally required.
9. Structural organization of insect VgRs/LpR and other family receptors
The VgR/LpR sequences are now available from several insect species (Table 1). The consensus
emerged from the sequence analysis is that these proteins along with other LDLR family members
including VLDLRs, LDLRs, LRPs and megalins have the same global structure and share a group of 5
structural domains (Fig. 1), which are discussed in detail below.
(9.A). Ligand-binding domain
The ligand-binding domain is composed of multiple cysteine-rich repeats involved in binding of

the ligand. The family of LDLR members is composed of a large variety of receptors that differ in
number of LBDs and number of cysteine-rich repeats in each domain. The sequence analysis shows that
the insect VgRs harbor 13 ligand-binding reprats LBRs (class A repeats or A modules), which are
divided into two domains with 5 repeats in the first and 8 in the second domain (Fig. 1). This contrasts to
other related family members like classical LDLRs (Yamamoto et al., 1986) which have a single
7-repeat domain, vertebrate VgRs (Bujo et al., 1994; Okabayashi et al., 1996) and VLDLRs (Takahashi,
et al., 1992; Sakai et al., 1994) which have a single 8-repeat domain (Fig. 1), and LRPs (human and
chicken, Herz, et al., 1988; Nimpf et al., 1994) and megalins (Saito et al., 1994; Hjalm et al., 1996)
which have four domains of 2-7, 8, 10 and 11 repeats. The exception is S. invicta VgR which has
4 repeats in the first LBD (Chen et al., 2004). On the other hand, the insect LpRs so far cloned (Dantuma
et al., 1999; Cheon et al., 2001; Seo et al., 2003; Lee et al., 2003; Gopalapillai et al., 2006; Ciudad et al.,
2007) (see Table 1) have shown a single 8-repeat domain and thus structurally resemble to vertebrate
VLDLRs/VgRs. A splice variant of LpR sequenced recently from the mosquito fat body, however,
differs from other insect LpRs in having only 7 ligand-binding repeats (Seo et al., 2003) Also,
Drosophila and B. germanica have a receptor with 7-reapeat LBD.
Each LBR or A module is of ~40 residues long and habour 6 conserved cysteine residues (Fig. 4).
Six cysteine residues form three disulfide bonds (C1-C3, C2-C5 and C4-C6) (Fig. 4), constituting a
scaffold which stabilizes the module structure in conjunction with the calcium-binding site (Blacklow
and Kim, 1996; Fass et al., 1997). Mutations of the calcium-coordinating residues result in familial
hypercholesterolemia, consistent with the known calcium requirement for receptor function (Goldstein
and Brown, 1974) and folding (Blacklow and Kim, 1996). Structural comparison of Class A module
among a wide range of LDLR-family receptors including Class A module in insect VgRs/LpRs reveals a
number of highly conserved residues (Ullman et al., 1995; Sappington and Raikhel, 1998, 2005) (Fig. 4).
In addition, a conserved sequence of acidic amino acid residues (bold letters) (CDxxxDCxDGSDE) is
present in the C-terminal part of each LBR. Originally, these acidic residues were proposed to mediate
the interaction of LDLR (Lalazar et al., 1988) with basic amino acid residues of apolipoprotein E (ApoE)
(Wilson et al., 1991). Confusion arose when Fass et al. (1997) using crystallographic analysis
demonstrated that many acidic residues were involved in coordination of Ca2+. In calcium-bound
condition, however, not all negative charges are bound, still allowing their involvement in ligand binding
(North and Blacklow, 2000). The incorporation of a calcium ion in the structure explains the calcium
requirement for correct folding and disulfide bond formation of LBR (Blacklow and Kim, 1996; Atkins
et al., 1998) and for binding of lipoproteins to LDLR (Daniel et al., 1983).
102
Module Ligand-binding (Class A) repeats Linker residues
VgRs-LBD-I
LemVgR R1 CPENAFK------CHSDGT----CISGYHH-CDGHVDCNDGSDEIN--C 6
BlgVgR R1 CQGQGTFE-----CHNGA-----CISETKH-CDGHVDCTDGSDEVD--C 4
PeaVgR R1 CP-SGFFT-----CHNGE-----CINDDKH-CDGTSDCKDGSDEFD--C 4
AeaVgR R1 CAENEYR------CDNGA-----CIPDVNH-CNGAKDCTDGSDEVG--C 4
AngVgR R1 CGAHEFQ------CENGA-----CIPAAGH-CNDIQDCADGSDESG--C 4
DrmYPR R1 CDAGQFQ------CRDGG-----CILQAKM-CDGRGDCKDSSDELD--C 4
SoiVgR R1 CEDGYFQ------CNSGE-----CIPVDKK-CDYIDHCIDGSDEDFE-C 10
LemVgR R2 CLEPRWFR-----CRTGR-----CISSSLR-CDTDDDCGDWSDEED--C 9
BlgVgR R2 CKEPDWFR-----CRNGR-----CISSGMR-CDDDDDCGDWSDEDD--C 8
PeaVgR R2 CKEPHWFR-----CHNGR-----CTSKSFH-CDGVDDCGGWSDEED--C 9
AeaVgR R2 CKKPMWYR-----CKHDKS----CISATFL-CDKHDDCPLGDDEEN--C 11
AngVgR R2 CRAPFWYR-----CRHEST----CISGSSR-CDGQRDCLGGDDEEN--C 11
DrmYPR R2 CRPPHWFP-----CAQPHGA---CLAAELM-CNGIDNCPGGEDELN--C 17
SoiVgR R2 ------------------------------------------------
LemVgR R3 C-KKDEWH-----CINDNN----CIPTDWV-CDGKQDCMDGTDELQG-C 4
BlgVgR R3 CTDSEWR------CM-DNN----CIIIDWV-CDGRQDCMDGSDELQG-C 5
PeaVgR R3 CTADEWR------CV-DNN----CIFMDWV-CDGKQDCMDGSDELQG-C 5
AeaVgR R3 CSKFEFT------CT-DKM----CIPLDLV-CDGVSHCLDGSDETIG-C 6
AngVgR R3 CSKAEFT------CT-DRA----CIPADLV-CDGVQHCLDGSDETIG-C 6
DrmYPR R3 CSKYEFM------CQQDRT----CIPIDFM-CDGRPDCTDKSDEVAG-C 6
SoiVgR R3 CAKDQFK------CK-NQE----CIPAAKY-CDMVNDCLDESDEHDG-C 5
LemVgR R4 CSDGFV-------CNNHH-----CIP-VTFHCDGSDDCGDGSDERN--C 10
BlgVgR R4 CHDGFM-------CKNGH-----CLP-ITFHCDGSDDCGDNSDEDY--C 10
PeaVgR R4 CEDGFV-------CGNYH-----CIP-NSFLCDGFDDCGDNSDEKL--C 10
AeaVgR R4 CKG FV-------CKNKR-----CINSHDWVCDGIDDCGDGSDEEN--C 2
AngVgR R4 CKG—FL-------CRNKH-----CLQSHHWVCDGLDDCGDGSDEEH--C 3
DrmYPR R4 CPGEGHL------CANGR-----CLRRKQWVCDGVDDCGDGSDERG--C 3
SoiVgR R4 CTNKF-L------CTDGH-----CIN-KEWVCDGRNDCPDGNDEWN--C 10
LemVgR R5 CTHERNFHL----CRDNRT----CISLDEL-CDGVRNCPDYSDEGIK-C 6
BlgVgR R5 CTTDKNLHL----CHDGRT----CISLNEL-CDEVQHCPDHSDEGPG-C 6
PeaVgR R5 CKLEKNLFL----CADRQE----CVEVREL-CDGTPHCYDGSDEGPA-C 6
AeaVgR R5 CDLEHGKFE----CADNST----CVDLKLV-CDGKDDCGDHSDEGGS-C 4
AngVgR R5 CTLEHGKYE----CANNHT----CVDVTQV-CNGADDCGDGSDEGPG-C 6
DrmYPR R5 CEPQKGKFL----CRNRET----CLTLSEV-CDGHSDCSDGSDETDL-C 5
SoiVgR R5 CKTENYQYM----CAN-HR----CISLKVV-CDKKDDCGDGSDEGPG-C 4
VgRs-LBD-II
LemVgR R1 CSEDKFK------CKEDNL----CIPRDFR-CNGRRDCPSG-EDELD-C 4
BlgVgR R1 CSEDKFK------CKSDNL----CIPRNFR-CNGRKDCQSG-EDELD-C 4
PeaVgR R1 CSGEMFK------CKTDNF----CIPGRMR-CDGKIDCPNGGEDELN-C 4
AeaVgR R1 C---EFK------CTSG-E----CLTISKR-CNGNKDCADGSDEKG--C 10
AngVgR R1 C---AFR------CASG-E----CLARGLR-CNGRVDCMDQSDEQG--C 12
DrmYPR R1 C---EFR------CHSG-E----CLTMNHR-CNGRRDCVDNSDEMN--C 11
SoiVgR R1 CSKNEYK------CSEHNI----CIQRNQL-CDGIENCPNGEDETSE-C 5
LemVgR R2 CSDTEFS------CKNG-Q----CIPGDKL-CDDEKDCIDGSDEKN--C 2
BlgVgR R2 CLDSQFT------CKNG-Q----CISIEKL-CNGERDCLDGSDEKN--C 2
PeaVgR R2 CRDDQFV------CHNG-Q----CISITKK-CDGDSDCRDGSDEYY--C 4
AeaVgR R2 CQYDEFM------CADKSK----CIDQTRR-CDEHVDCGDGSDEMK--C 8
AngVgR R2 CRWNEFR------CADGSR----CIAATSR-CDSRPDCADRSDEAN--C 8
DrmYPR R2 CSPSQFA------CHSGEQ----CVDKERR-CDNRKDCHDHSDEQH--C 8
SoiVgR R2 CKENQFM------CKNG-D----CIRLKDR-CNSRYDCTDQSDEQN--C 4
LemVgR R3 CSEETDFQ-----CRTGE-----CIDILDR-CDLVPDCRDASDEEN--C 4
BlgVgR R3 CEEAIQFK-----CSSGE-----CVDIHDR-CDHYPDCTDGSDESN--C 4
PeaVgR R3 CNEDLQFK-----CRTGD-----CIVKSWY-CDGSKDCEDGSDEEN--C 4
AeaVgR R3 CHEHQHA------CPDGM-----CIDVNTL-CDGFPDCLDGSDEVG--C 12
AngVgR R3 CTRYQFS------CADGF-----CVDATAR-CDQVPDCPDGSDEQE--C 13
DrmYPR R3 CHVHQHG------CDNGK-----CVDSSLV-CDGTNDCGDNSDELL--C 5
SoiVgR R3 CKSDEFQ------CKFTET----CIPKTKM-CDSNPDCDDLSDEED--C 4
103
LemVgR R4 CAEGHYR------CVVGV-----CIPYTWV-CDGQSDCVDGSDEKD--C 4
BlgVgR R4 CPPTDFK------CHIGV-----CVPKYWV-CDGEPDCIDGTDELN--C 4
PeaVgR R4 CEPSAFK------CALGQ-----CIPEEWV-CDGQSDCVDDTDEQN--C 4
AeaVgR R4 CGPLMFR------CNMGQ-----CIPKWWE-CDGNPDCTDGSDEHDK-C 5
AngVgR R4 CAAGMFR------CNSGQ-----CVPGSWE-CDGSPDCHDASDEHES-C 8
DrmYPR R4 CEPGMFQ------CGSGS-----CIAGSWE-CDGRIDCSDGSDEHDK-C 4
SoiVgR R4 CTSNEFK------CNNGK-----CIPNTFV-CDNDNDCEDGEDEAAEKC 5
LemVgR R5 CDAG-SFS-----CNNGR-----CIDRHLL-CNGDNDCGDYSDEME--C 7
BlgVgR R5 CGPD-LFS-----CNNGR-----CVDKKLV-CNHNDDCGDSSDEIT--C 7
PeaVgR R5 CGPG-AFS-----CGNGR-----CIDQTLL-CNNVDDCGDRSDEDP--C 15
AeaVgR R5 CGAGFTK------CALGH-----CIEDRLL-CDGNNDCGDNSDELN--C 6
AngVgR R5 CGVGF--------CISSALV---CDGNDD--CGDGTDEEH--------C 10
DrmYPR R5 CPP-DMHR-----CLSGQ-----CLDRSLV-CDGHNDCGDKSDELN--C 10
SoiVgR R5 CKMPKMFK-----CPNGD-----CISDSLL-CNGINDCNDGSDEVH--C 10
LemVgR R6 CQPTEIP------CLSHNKTVISCVPSSAR-CNDVAECPLGDDERA--C 2
BlgVgR R6 CQTTEIT------CTSHNKSVISCVPMSAR-CNDIPDCPLGDDERG--C 2
PeaVgR R6 CKEGEYT------CHPHGKNVTICLPSSGR-CNGTAECPLGDDERG--C 1
AeaVgR R6 CVGLEDDNPTKYLCPRSGK----CLDIAVR-CNGTAECPDGEDEAG--C 2
AngVgR R6 CSEQAIANGTAYRCARSGA----CLPAAAR-CNGTAECPHGEDETG--C 2
DrmYPR R6 CAEDQYQ------CTSNLKI---CLPSAVR-CNGTTECPRGEDEAD--C 3
SoiVgR R6 CSLNEYR------CLGTDI----CLPKNVR-CDGKNDCPQSDDEQN--C 2
LemVgR R7 CLDFQFR------CSNGR-----CIPQEWT-CDKTDDCDDGSDEDPVLC 12
BlgVgR R7 CMDFQFK------CNDGR-----CIPFEWT-CDGTKDCADGSDENQMHC 12
PeaVgR R7 CQDFQFT------CYNGK-----CIPSEWV-CDGINDCGDGSDENNARC 11
AeaVgR R7 CGLQEFQ------CKSGK-----CIRKEWR-CDKEVDCDDGSDEVD--C 14
AngVgR R7 CGLREFQ------CSDGQ-----CIRQEWR-CDHDQDCDDGSDERN--C 15
DrmYPR R7 CSIYEFK------CRSGRE----CIRREFR-CDGQKDCGDGSDELS--C 21
SoiVgR R7 CFENEFA------CDNKR-----CIPELWV-CDKANDCGDNSDEKN--C 12
LemVgR R8 CREYS--------CKNGD-----CISMSFV-CDGRKDCSDGSDEGGL-C 3
BlgVgR R8 CTEYS--------CDNGA-----CVSLSLV-CNGRQDCSDSSDEGGF-C 3
PeaVgR R8 CTDYA--------CNDGQ-----CISLSLA-CNNKRNCEDGSDEGGQ-C 3
AeaVgR R8 CGEGTFE------CKPGV-----CIEMSQV-CNGKKDCDDGKDEGKG-C 3
AngVgR R8 CGRDTFE------CGPGE-----CIPVAKL-CDGRRDCTNGHDEEGA-C 3
DrmYPR R8 CRPHLFD------CQDGE-----CVDLSRV-CNNFPDCTNGHDEGPK-C 3
SoiVgR R8 CDEFK--------CSVGT-----CLPYSKV-CDGNRDCPDGSDETGK-C 3
LpRs-LBD
LemLpR R1 CSLRQFR------CNNGR-----CIPLTWT-CEGDDDCGDNSDETSPEC 5
BlgLpR R1 -------------------------------------------------
LomLpR R1 CTLRQFQ------CANGH-----CIPLTWM-CEGEDDCGDNSDETNAVC 5
AeaLpR R1 CSERQFR------CNDGH-----CIHVSFV-CDGEADCSDGSDEHSREC 6
BomLpR R1 CPMKQFQ------CANGK-----CIPMTWV-CEGDDDCGDNSDESIEEC 5
GamLpR R1 CSLKQFQ------CANGK-----CIPLSWV-CEGENDCGDNSDENIDEC 6
DrmLpR R1 -------------------------------------------------
LemLpR R2 CSETEFK------CNNGR-----CIPVHWQ-CDNEKDCSDGSDEIPSVC 4
BlgLpR R2 CSSSEFK------CNNGR-----CIPVHWQ-CDNEKDCSDGSDEVPSVC 4
LomLpR R2 CTDQEFR------CNNGR-----CIPSHWQ-CDNEKDCADGSDEIPQVC 4
AeaLpR R2 CSDDKFR------CKSGR-----CIPKHWQ-CDGENDCSDGSDEDSEKC 4
BomLpR R2 CTSSEFR------CKTGR-----CIPLSWK-CDNEKDCSDSSDEDPTVC 4
GamLpR R2 CTSSEFR------CKTGR-----CIPLSWR-CDNEKDCSDGSDEEPGTC 4
DrmLpR R2 CSSDQFR------CGNGN-----CIPNKWR-CDQESDCADGSDEANELC 4
LemLpR R3 CSPEEFT------CRAMPGE---CVSLTWM-CDDNQDCSDGSDEKA--C 3
BlgLpR R3 CSPEEFT------CRAMPGE---CVPLTWM-CDDNPDCSDGSDEKA--C 3
LomLpR R3 CASDEFT------CRTAPGE---CVPLAWM-CDDNPDCSDGSDEKA--C 3
AeaLpR R3 CSSEEFT------CRSGTGT---CIPLAWM-CDQNRDCPDGSDEMS--C 3
BomLpR R3 CGPEEFT------CRGKPGE---CVPLTWM-CDDNPDCSDGSDEKA--C 3
GamLpR R3 CDPEEFT------CRGKHGE---CVPLTWM-CDDNPDCSDGSDEKA--C 3
DrmLpR R3 CSPDEYA------CKSGEGQ---CVPLAWM-CDQSKDCSDGSDEHN--C 3
LemLpR R4 CRSDEFT------CGNGK-----CIQKRWV-CDRDDDCGDGSDERD--C 4
BlgLpR R4 CRSDEFT------CGNGK-----CIQRRWV-CDRDDDCGDGSDEQD--C 4
LomLpR R4 CRSDEFT------CANSK-----CIQQRWV-CDRDDDCGDGSDEKD--C 4
AeaLpR R4 CRSDEFT------CANGR-----CIQKRWQ-CDRDDDCGDNSDEKG--C 4
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BomLpR R4 CRSDEFT------CGNGK-----CIQMRWV-CDGDDDCGDDSDEVK--C 4
GamLpR R4 CRSDEFT------CGNGK-----CIQQRWV-CDGDDDCGDDSDEVK--C 4
DrmLpR R4 CRADEFT------CGNGR-----CIQKRWK-CDHDDDCGDGSDEKE--C 4
LemLpR R5 CNPETEFN-----CSEN-F----CITSRWR-CDGDADCHDGSDEIG--C 9
BlgLpR R5 CNAETEFN-----CSEN-F----CITARWR-CDGDADCPDGSDEVN--C 12
LomLpR R5 CAPETEFN-----CSDNNM----CITARWQ-CDGDLDCQDGSDEQG--C 8
AeaLpR R5 CDPLKQFA-----CSEN-Y----CITSKWR-CDGEPDCPDGSDERG--C 10
BomLpR R5 CQPQSHFS-----CTDG-Q----CISAWWH-CDGDVDCADGSDEVG--C 9
GamLpR R5 CQPHTHFS-----CADG-H----CISARWR-CDGDIDCPDGSDEME--C 9
DrmLpR R5 CDSVAEHT-----CTNG-A----CIAKRWV-CDGDPDCSDGSDERS--C 9
LemLpR R6 CLPREFE------CEDRLT----CVHQSWV-CDGDKDCPGGSDESITRC 4
BlgLpR R6 CLPREFE------CEDRLT----CIHQSWV-CDGDKDCPGGSDESASRC 4
LomLpR R6 CLPREFE------CLDRMT----CIHQSWV-CDGDRDCPDGSDEDVSRC 4
AeaLpR R6 CLSLEYQ------CSDRIT----CIHKSWI-CDGEKDCPQGDDEMPPIC 4
BomLpR R6 CISTEFE------CNDRIT----CVHRAWV-CDGDHDCPDGGDEALELC 5
GamLpR R6 CLSAEFE------CRDRLT----CVHRAWV-CDGDRDCPGGDDEAPELC 5
DrmLpR R6 CLSHEYQ------CKDRIT----CLHHSWL-CDGDRDCPDGDDEHTANC 4
LemLpR R7 CRPDQFQ------CR-NRA----CIPGHLH-CSGHAECPDESDEEN--C 6
BlgLpR R7 CRPDQFQ------CR-NRA----CIPGHLH-CSGAPECPDESDEEN--C 6
LomLpR R7 CRPDQFQ------CR-NRI----CIPGHLH-CSGHADCSDGSDEEN--C 6
AeaLpR R7 CRPDQFQ------CKKDKT----CINGHFH-CNGKPECSDGSDEVD--C 6
BomLpR R7 CRLDQFQ------CK-DHS----CIPGALY-CNGVKDCPDGSDEYN--C 6
GamLpR R7 CRLDQFQ------CK-DHS----CIPGALY-CNGDKDCPDGSDEFN--C 6
DrmLpR R7 CRADQFQ------CG-DRS----CIPGHLT-CNGDKDCADGSDERD--C 12
LemLpR R8 CDQRTEFE-----CGGGM-----CIPLSKV-CDKKPDCPNWEDEPGDKC 4
BlgLpR R8 CDPRTEFE-----CGGGM-----CIPIAKV-CDKKPDCPNWEDEPTDKC 4
LomLpR R8 CDPKTEFE-----CGGGM-----CIPLSSV-CDKKPDCPNWEDEPQEKC 4
AeaLpR R8 CNPKTEFD-----CGGGM-----CIPLSKV-CDKKPDCPEFQDEPNDKC 4
BomLpR R8 CDKRTEFD-----CGGGM-----CIPLSKV-CDKHVDCPNFEDEPRDRC 4
GamLpR R8 CDKKTEFD-----CGGGM-----CIPLSKS-CDKKPDCPDFEDEPRDKC 4
DrmLpR R8 CNATSEFD-----CGGGQ-----CVPLSKV-CDKRKDCPDGEDEPAGKC 4
Consensus CxxxxxxxxxxxxCxxxxxxxxxCIxxxxxxCDxxxDCxDGSDExxxxC
S-bond S-bond
S-bond
Fig. 4. Comparison of Class A module sequences from know insect VgRs and LpRs. The VgR sequences were
form L. maderae VgR (LemVgR.), B. germanica VgR (BlgVgR), P. americana VgR (PeaVgR), A. aegypti
VgR (AeaVgR), A. gambiae VgR (AngVgR), D. melanogaster YPR (DrmYPR), S. invicta VgR (SoiVgR),
whereas LpR sequences were from L. maderae LpR (LemLpR), B. germanica LpR (BlgLpR), L.
migratoria LpR (LomLpR), A. aegypti LpR (AeaLpR), A. gambiae LpR (AngLpR), B. mori LpR(1)
(BomLpR), G. mellonella LpR (GamLpR.), D. melanogaster LpR (DrmLpR). Hyphens indicate gaps
inserted to maximize the alignment. Modules are numbered from amino-terminal to carboxy-terminal
position in modular domains. The bold hyphens show the missing Class A modules. Consensus
sequence indicates residues crucial for maintaining the global tertiary structure, whereas x is any
residue. The brackets connecting cysteine residues reveal the probable di-sulfide bonds.
The structural analysis of insect VgRs/LpRs reveals that the Class A modules of LBDs are
connected by a short linker sequences (Fig. 4). Solution NMR studies of the tandem repeats LBR1-LBR2
and LBR5-LBR6 have demonstrated that the linkers in both module pairs are flexible, permitting
essentially unrestricted relative motion of each module with respect to its partner in the pair (Kurniawan
et al., 2000; Beglova et al., 2001). The absence of contacts between adjacent ligand-binding modules in
the X-ray structure suggests that the interdomain flexibility directly observed in these two cases applies
generally to the linkers connecting all of the LBR in the ligand binding domain. Such flexible
intermodule connections allow the LDLR to bind a variety of heterogeneous lipoprotein particles
different in size, shape and curvature.
105
(9.B). EGF-precursor homology domain
The second region of LDLR extracellular domain contains EGF- like repeats (Class B repeats) of
~40 amino acids, which are present either singly or in a group of two (Fig. 1, 5). The insect VgRs have
seven EGF-like repeats with 4 in the first and 3 in the second domain, whereas the LpRs have three
repeats similar to the LDLRs and Vg/VLDLRs (Fig. 1, 5). Although these repeats harbor six conserved
cysteine residues similar to LBRs, but differ in pattern of bonding, which occurs between CI and CIII,
CII and CIV, and CV and CVI (Fig. 5) (for detail see review: Sappinton and Raikhel, 2005). The repeats
surround a ~260-amino acid domain containing six YWXD motifs that are proposed to form a beta-
propeller structure (Springer, 1998). Insect VgRs have three groups of six YWTD motifs while LpRs
have only a single cluster of six YWTD motif repeats (Fig. 1). The structural analysis of insect
VgRs/LpRs reveals that these receptors strictly follow the LDLR family rules in positioning the
signatures of these sequence motifs (Fig. 1). Insect VgRs possess two clusters of EGF-precursor like
domin (Tufail and Takeda, 2005, 2007) unlike their LpRs cognates that contain only one cluster (Cheon
et al., 2001; Seo et al., 2003; Tufail et al., 2009) (Fig. 1). The YWXD motif appears to be involved in
hydrogen bond and serve to orient adjacent β-sheets (Springer, 1998). Unlike the LBRs and EGF-like
repeats, the repeats containing YWXD motif are not themselves modules; rather the whole domain of six
repeats is the unit involved in evolutionary shuffling (Springer, 1998).
Previous studies have shown that LDLR with deleted EGF-precursor domain still binds VLDL;
however, the acid-dependent dissociation of ligand is prevented in endosomes (Davis et al., 1987).
Recently, Rudenko and colleagues (2002) have suggested a plausible molecular mechanism for the acid-
dependent release of ligand. Their crystallographic analysis of the LDLR extracellular region at
endosomal pH revealed that the LBD folds back onto the β-propeller. This conformation is stabilized by
interactions between repeats R4/R5 and the β-propeller and involves the β-propeller in displacement of
the ligand from the LBD (Rodenko et al., 2002).
(9.C). O-linked sugar domain
The third region of LDLR ectodomain is enriched in serine (S) and threonine (T) residues and many
of them are, putatively, O-linked glycosylated (Willnow, 1999) (Fig. 1). Many LDLRs have an O-linked
sugar domain (OLSD) at this position but the presence of this domain is not universal (Bujo et al., 1995).
The VgR gene, for example, produces an ovary-specific VgR with O-linked sugar domain in L. maderae
(Tufail and Takeda, 2007), P. americana (Tufail and Takeda, 2005), B. germanica (Ciudad et al., 2006) and
A. aegypti (Sappington et al., 1996) but without this domain in D. melanogaster (Schonbaum et al., 1995)
and S. invicta (Chen et al., 2004). On the other hand all insect LpRs (see for reference Table 1) possess
O-linked sugar domain. The OLSD in insect VgRs is ~30 residues long and there exist 6 S/T residues in
each P. americana and L. maderae, whereas 8 and 9 S/T residues in B. germanica and A. aegypti,
respectively. In contrast, the OLSD in insect LpRs is much longer. In L. maderae, for example, this region
is over 70 residues long and 30% of the residues are S/T (Tufail et al., 2009), whereas A. aegypti LpR
contains an OLSD of over 250 residues of which 26% is contributed by the S/T residues (Cheon et al.,
2001). Also, insect LpRs have revealed insertion/deletion in this region. Recently, two LpR splice variants
differening in 24 amino acids have been reported from B. germanica. Splice variants in the OLSD have also
been described in LpRs of A. aegypti (Cheon et al., 2001; Seo et al., 2003), G. mellonella (Lee et al., 2003)
and B. mori (Gopalapillai et al., 2006). The function of O-linked sugar domain is not yet resolved. There is
possibly that OLSD provides a rigid stalk allowing the ectodomain to extend into the extracellular space
(Jentoft, 1990). Alternatively, glycosylation may protect the receptor from denaturation during recycling
through the slightly acidic endosomal compartments (Tyko and Maxfield, 1982). As a third function, O-
glycosylation may modulate the rate of proteolytic cleavage of the ectodomain by membrane-bound
proteases (Kingsley et al., 1986; Kozarsky et al., 1988; Mullberg et al., 1997). However, LDLR molecules
lacking OLSD have no affect on ligand binding, endocytosis and degradation (Davis et al., 1986). Also, the
absence of this domain in vertebrate VgRs and in some insect VgRs argues for some other physiological
function of this region.
106
Module EGF precursor (Class B) like repeats

VgRs- EGFD-I
LemVgR RA CKAIK----CDHL--CVPTPRG-AI-----CVCKVGYTMAKN-KT-C
BlgVgR RA CRTAW----CNHR--CIPTPQG-PQ-----CVCQTGYTMAVN-NT-C
PeaVgR RA CPTVG----CSHQ--CIPSPQG-PL-----CVCQVGYKTVDN-KT-C
AeaVgR RA CDSMR----CPEG--CKATPHG-AV-----CLCKPGFRFNKKSKV-C
AngVgR RA CKALH----CAPQT-CKVLPDGKPV-----CLCGVGFRFEPSAGR-C
DrmYPR RA CDAKK----CALGAKCHMMPASGAE-----CFCPKGFRLAKFEDK-C
SoiVgR RA CSSAG----CQSN--CHQTPKG-SV-----CTCKPGYKLQKDNRT-C
LemVgR RB CLKYG-I--CDQK--CKNLPGSYS------CYCDEGYFLADDNHS-C
BlgVgR RB CLEYG-I--CDQK--CLNMPGKYD------CYCDEGYELAEDKRT-C
PeaVgR RB CMEYG-I--CDQR--CRNLQGSYS------CYCDEGYEVGSDKRS-C
AeaVgR RB CERYG-L--CSQG--CENTPGSFK------CTCVDKFKLKDDSRT-C
AngVgR RB CARYG-L--CSQG--CINTPGSFR------CTCIDQFHLMRDGRT-C
DrmYPR RB CKEQDDL--CSQG--CENTSGGYR------CVCDAGYLLDKDNRT-C
SoiVgR RB CQAYG-I--CDQD--CMNVPGSYA------CTCQREYYLENDKRT-C
LemVgR RC CEISG----CSDI--CLLAP—NQTYT----CGCPEHKVRGLDKHS-C
BlgVgR RC CKKAD----CSDL--CLLAP--NRTYT---CACPEHKVRGADMHS-C
PeaVgR RC CHNNW----CSDI--CLLAPNKTYTYT---CACPENKQLGADKHT-C
AeaVgR RC CLGTF----CSHL--CLLAP--NDSYS---CACPYGMSLKADKHS-C
AngVgR RC CANHT----CTHL--CLLTS--NATYA---CACPQGMELSRDKHS-C
DrmYPR RC CENAT----CSHL--CLLAEPEIGGHS---CACPDGMRLAPDHRR-C
SoiVgR RC CYSNP----CSQL--CMLNQ--NKGYT---CGCTLDKKLNADKHT-C
LemVgR RD CQIDNGH--CSHI--CALS-LKRTV-----CLCPIGMELKHD-GHTC
BlgVgR RD CQKENGG--CSHI--CALS-QKRMV-----CLCPMGMQLKKN-EKTC
PeaVgR RD CHKNNGG--CSHI--CALA-LKHTV-----CLCPVGMVLNRD-NKTC
AeaVgR RD CMKQNGG--CSHI--CVPAGMYSSA-----CICPTGMIFSSPKNTTC
AngVgR RD CTRSNGG--CSHI--CVAGGLYTSA-----CVCPTGMVFNTTLARVC
DrmYPR RD CQQQNGG--CSHI--CVGEGPYHSI-----CLCPAGFVYRDAGNRTC
SoiVgR RD CQKNNGN--CSHV--CLPSLITSFI-----CACPPGMELSND-NRTC
VgRs-EGFD-II
LemVgR RA CLGKDP---CEDI--CLKTPRGPR------CKCSHGFALLSDGSR-C
BlgVgR RA CKEGYP---CQQV--CMKTPRGPQ------CGCSKGFKLLNNGAK-C
PeaVgR RA CNAKSP---CDQI--CQPTLAAQD------ATVHKGYVLSSDGAK-C
AeaVgR RA CAKSP----CEHK--CIKTPTGAI------CECREGFTLAPNKKS-C
AngVgR RA CTGGLGP--CAQI--CQKSPAGSI------CACLEGYELAGDRKT-C
DrmYPR RA CRSASGRQVCQHK--CRATPAGAV------CSCFDGYRLDADQKS-C
SoiVgR RA CTVN---NFCKGM--CYKTPAGAV------CGCQSGYRLAVDMIS-C
LemVgR RB CDMQA----CAQV--CHNKPGSFS------CACDPGFELRSDRIS-C
BlgVgR RB CESQV----CAQV--CHNTPGSFS------CICDAGFELRSDRIS-C
PeaVgR RB CEIGGA---CAQV--CHNTRGSFS------CSCHPGFQLRSDHVS-C
AeaVgR RB CAEGRP---CAQQ--CRNTFGSYR------CSCNPGFMLRSDKIS-C
AngVgR RB CARGQP---CAQQ--CANVHGSYR------CACHDGFMLRPDKAS-C
DrmYPR RB CQEQQP---CAQL--CENTLGGYQ------CQCHADFMLRQDRVS-C
SoiVgR RB CEL-DI---CSQM--CRNTIGSYE------CFCKDEFIIRNDKTS-C
107
LemVgR RC CANHT----CSEM--CVLSPGRNPS-----CLCSDSTTRSMGEP--C
BlgVgR RC CANHT----CDEM--CVLSPSGYPV-----CLCSDGTIVEMGKS--C
PeaVgR RC CANHS----CSEL--CVMNPGGTPS-----CLCSGGQVVEMGEL--C
AeaVgR RC CDTKQIN--CSHV--CVPGADGHGV-----CICHNGERIHGTDI--C
AngVgR RC CDRAN----CTHL--CVAVGP-VAR-----CICENGLEIAEGDR--C
DrmYPR RC CAHAH----CHGL--CLQADYGYE------CMCGN-RLVAEGER--C
SoiVgR RC CDAEY----CDYM--CVLKKE-NAT-----CICSDGESIESNST--C
LpRs-EGFD
LemLpR RA CLENNGG--CSQK--CADTPGGYY------CDCLHGYKLMDN-KT-C
BlgLpR RA CKENNGG--CSQL--CVDTPGGYY------CDCRRGFKLMDN-RT-C
LomLpR RA CAVNNGG--CSQK--CVDTPAGYY------CDCLPGYKLVDN-HT-C
AeaLpR RA CLENNGG--CSHL--CVDTPAGFY------CDCKPGYKLVNN-RT-C
BomLpR RA CEKNNGG--CSQR--CVDTPIGYY------CDCEKGYKLIDN-RT-C
GamLpR RA CAKNNGG--CTQR--CVDTPVGYY------CDCDKGYKLIDN-RT-C
DrmLpR RA CASKNGG--CMHQ--CIDLKVGHH------CECHEGYKLSPDKRN-C
LemLpR RB CDSPGA---CSQT--CINEKGTFK------CQCVEGYLRDPRNHTRC
BlgLpR RB CEVPGT---CSQS--CINEKGTFK------CQCVEGYLRDPRDHTRC
LomLpR RB CEIPGA---CSQE--CINEKGTFK------CQCVEGYLRDPRDPTRC
AeaLpR RB CAEAGS---CSQK--CTNEIGTSK------CECMPGYLRDPRDHTKC
BomLpR RB CADPGS---CSQI--CINEKGTFK------CECHTGYARDPRDRTRC
GamLpR RB CADPGA---CSQM--CINEKGTFK------CECHAGYARDPRDRTRC
DrmLpR RB CEVPGK---CSQI--CVNEIGGFK------CECEAGYMRDPKNHTRC
LemLpR RC CQAVNGH--CSHL--CLPAPQINSRSPKISCACPDGLRLMADGLM-C
BlgLpR RC CQAVNGH--CSHL--CLPAPQINSRSPKISCACPDGLRLMEDRLM-C
LomLpR RC CQAVNGH--CSHL--CLPAAQINAHSPKISCACPDGLQLMQDGLM-C
AeaLpR RC CQAVNGH--CSHL--CLPAPQINSRSPKISCACPTGLKLMDDGLM-C
BomLpR RC CAAVNGH--CSHL--CLPAPRFGPNSPRVSCACPNGLKLLPDDQM-C
GamLpR RC CAAVNGH--CSHL--CLPAPRIGTHAPRVSCACPNGLRLLPDNQM-C
DrmLpR RC CQSVNGH--CSHL--CLPAPRINERSPRISCACPTGLKLMVDGLM-C
Consensus CxxxxxxxxCxxxxxCxxxxxxxxxxxxxxCxCxxxxxxxxxxxxxC
S. bond S. bond
S. bond
Fig. 5. Comparison of Class B module sequences from know insect VgRs and LpRs. Sequence information is
given in Figs. 2, 4 and Table 1. Hyphens indicate gaps inserted to maximize the alignment. Modules are
numbered from amino-terminal to carboxy-terminal position in modular domains. Consensus sequence
indicates residues crucial for maintaining the global tertiary structure, whereas brackets connecting
cysteine residues reveal the probable di-sulfide bonds.
(9.D). Transmembrane domain
The transmembrane domain (TMD) is a short stretch of ~24 amino acid residues long that holds
the LDLR in the lipid bilayer. It plays an important role in in receptor functioning and several recycling
passes between plasma membrane and various endocytic vesicle system (see Maxfield and McGraw,
2004). The TMD forms a transmembrane α-helix and functions as a membrane anchor (Willnow, 1999;
Herz and Bock, 2002). The structural analysis reveals that the sequence of this domain is enriched in the
hydrophobic residues. In insect VgRs/LpRs, the hydrophobic residues found in TMD are alanine (A),
glycine (G), isoleucine (I), leucine (L), phenylalanine (F) or valine (V). The sequence comparison shows
that this domain is conserved within members of the same group than other groups of the LDLR family.
108
(9.E). Cytoplasmic domain
This region is exposed to the cytoplasm and plays a crucial role in endocytosis and intracellular
transport of the LDLR through clathrin-coated pit internalization signal, FDNPVY (Davis et al., 1987)
(Fig. 6). The NPXY sequence motif adopts a tight hairpin conformation that serves as a binding site for a
variety of adaptor proteins and signaling molecules. A single copy of this motif has also been identified
VgRs-Intracellular domain
LemVgR * * *
KYGFNKDSKFGFSMHFSNPTFGVQSPESHLDTPSHSLVP--GQHQYV
BlgVgR KYRGKKNSKFGFSMHFQNPTFGVRNSTENPSTPPQGLVP--GQHQYT
PeaVgR KRFGYKGPKLNFSLHFKNPTFGIKESDV---AVPQVLVP--GQHQYT
AeaVgR IYRRRYQHKFDIGMHFHNPELSTADAAEVKMFQKVPRLN--QTTTHN
DrmYPR QYRQRGHTDLNINMHFQNPLATLGGTKAFLEHERAEAGV--GFTTET
SoiVgR LKSKPASNLSCSSIHFQNPSYDRSDEIEVMLDSMASSELSPGQHEYI
LemVgR *
NPFDSAGVREHPDGKVVMIPHEKISPAKLETPTHTAEADEG--AEME
BlgVgR NPFDSSGVLEHPDGKVLILSQQEKPPVIIRIPSQSSERTDAADFEME
PeaVgR *
NPFDNAEALKQLEGSVIQESRLKKLADHIQLE---------------
AeaVgR ELTLETPPHRPPCQGDPNSSESTGQNNVTSTALELENMSDVDSMEDA
DrmYPR GTVSSRGSNDTFTTTSASSSFAAQQFSVPNALQRLLRPRQSAS----
SoiVgR INPNNKGMKAAENNAKKSNQCSEGKNI--------------------
LemVgR DDTDQGFITDTDSMKVKLIP---------------------------
BlgVgR DDTSQEFVSDNNDMKAPLIS---------------------------
PeaVgR -DEDAEDYAPDGSDKAPLIH---------------------------
AeaVgR YDC-------RDDPLQRLIL---------------------------
DrmYPR ----------GDPMAQELLLESPSRESKLHALDGGGAGGDGDGGCGV
SoiVgR ----------EEEKQDALIYFVHNSK---------------------
LemVgR --------------------------------------~~~~~COOH
BlgVgR --------------------------------------~~~~~COOH
PeaVgR --------------------------------------~~~~~COOH
AeaVgR --------------------------------------~~~~~COOH
DrmYPR GRQVPDILVADMDDDAAKSAGQFGGNYAGNDANARFVS~~~~~COOH
SoiVgR --------------------------------------~~~~~COOH
LpRs-Intracellular domain
LemLpR * *
RHYLHRNVTSMNFDNPVYRKTP--EDQFSLEKNQYQP-TKIYPSTVG
BlgLpR RHYLHRNVTSMNFDNPVYRKTT--EDQFSLEKNQYQP-QRIYPATVG
LomLpR RHYLHRNVTSMNFDNPVYRKTT--EDQFSLEKNQYQP-QRIYPATVG
AeaLpR KHHVHRNSTSMNFDNPVYRKTT--EDQFSLEKNLPNR---MYPSTVG
BomLpR RHYVHHNVTSMNFDNPVYRKTT--EDQFALEKNGYAPGSKLYPSTVG
GamLpR RHYVHRNVTSMNFDNPVYRKTT--EDHFALEKNGYAPGSKLYPSTVG
DrmLpR RYCSKRRINSMNFENPIYRKTTTTEDHFSLRKNLPAR--IYDHTSVM
LemLpR *
EEAQEPLTSPGTNDYV~~~~~COOH
BlgLpR EEAQEPLTSPGTNDYV~~~~~COOH
LomLpR EEAHEPLTSPGTNDYV~~~~~COOH
AeaLpR EEAQEPLNRPGTNDFV~~~~~COOH
BomLpR EEAQEPLNKPNTEF-V~~~~~COOH
GamLpR EEAQEPLNTSGTNDFV~~~~~COOH
DrmLpR DEEYSPVIGISSY---~~~~~COOH
Fig. 6. Interacellular domain of insect VgRs/LpRs showing various sequence motifs. The cytoplasmic domain
of insect VgRs harbor LI/LL and NPTF/NPTY internalization signal motifs (shown with dark-shaded
frames), whereas the LpRs are strictly bound to only tight-turn tyrosine (NPTY) signal and is indicated
also with dark-shaded frame. Possible casein kinase II site (S/TXXD/E) and a cGMP-dependent pritein
kinase site (RXS/T) in L. maderae LpR (LemLpR) are shown with light-shaded frames. Possible serine
(S) phosphorylation sites predicted with Netphos 2.0 server (Blom et al., 1999) are indicated with
asterisks. Possible transduction sequence motifs are boxed. For sequence information see Fig. 4 and
Table 1.
109
in VLDLR, the ApoER2 and LR11/SorLA-1, whereas LRP1, LRP1B and Megalin each harbors two
copies. In addition to the two NPXY motifs, LRP1 possesses a YXXL motif which serves as the
dominant internalization motif (Li et al., 2000). Interestingly, LRP5, LRP6, ST7 and LRP3 lack the
characteristic NPXY motif. Instead, they contain YXXL and/or dileucine-based endocytosis motifs. The
insect VgRs are unique in having a LI internalization signal (LL in Drosophila YPR) (Fig. 6) instead of
the NPXY motif found in majority of LDLR family receptors (see Sappington and Raikhel, 1998).
Among insects, only the fire ant VgR contains NPXY signal in addition to LI (Chen et al., 2004)
(Fig. 6). The cockroach VgRs (Tufail and Takeda, 2005, 2007; Ciudad et al., 2006) still harbor another
motif (NPTF) which is also believed to be a functional internalization signal (Davis et al., 1987). In
contrast, the insect LpRs all contain only NPXY internalization motif (Fig. 6). There are also observed
some other motifs in the cytoplasmic region of insect VgRs that might be involved in internalization. For
example, the intracellular domain in three cockroach VgRs contains a NPFD motif similar to the
NPFX(1,2)D motif in yeast that can direct efficient uptake of the Kex2p receptor (Tan et al., 1996; Dunn
and Hicke, 2001; Howard et al., 2002). Moreover, cytoplasmic region of A. aegypti VgR contains a Src-
homology 3 (SH3) binding sequence motif PXXP, which is also present in the same region of some other
LDLR family members (Hjalm et al., 1996; Sun and Souter, 1999). Drosophila yl receptor tail harbour a
AKSAGQF motif similar to that of the mannose 6-phosphate receptor motif (AKGMEQF) that functions
together with LL and YXXØ (where X is any amino acid and Ø is an amino acid with a bulky
hydrophobic side chain) motif as an internalization signal (Denzer et al., 1997).
Moreover, recent studies have shown that phosphorylation of S residue in the intracellular region
regulates the receptor-mediated endocytosis of LRP1 (Bu et al., 1998; Li et al., 2001a). We have
identified one potential phosphorylated S residues in cytoplasmic domain of P. americana VgR (Tufail
and Takeda, 2005) and four in that of L. maderae VgR (Tufail and Takeda, 2007) (Fig. 6). Also, three
poteintial phosphorylated S residues were predicted in addition to a casein kinase II (CKII) (S/TXXD/E)
phosphorylation site and a cGMP-dependent protein kinase (RXS/T) site in the intracellular domain of
the L. maderae LpR (Fig. 6, Tufail et al., 2009). The potential protein Kinase C (PKC) and CKII
phosphorylation cites were predicted in the interacellular loops that are involved in regulation of the
functional activity in all GPCRs (Witt-Enderby et al., 2003). The involvement of putative CKII and
cGMP-dependent protein kinase sites in functional regulation of L. maderae LpR is not excluded. The
potential CKII sites have been identified in cytoplasmic region of several LDLRs (see review:
Rodenburg et al., 2006) including the locust LpR (Dantuma et al., 1999). Phosphorylation at this
location is believed to be involved in several functions especially in signal transduction. However, it has
also been assumed that it is involved in ubiquitination and subsequent degradation of the cytoplasmic
proteins (Djordjevic et al., 2000). SH3 (PXXP) binding motif is also involved in signal transduction
cascades (Herz and stickland, 2001; Li et al., 2001b) and PDZ-binding consensus sequence motif
(S/TXV) has been identified in megalin and may be involved in signal transduction cascades (Gotthardt
et al., 2000; Takeda et al., 2003). The endodomain of P. americana VgR has a SDV motif similar to
PDZ-binding motif and might be involved in the signal transduction cascades.
10. Concluding remarks
The recent development described in this chapter provide evidence that insect Vg/Lp receptors,
irrespective of their origin and recognition of quite unrelated ligands, are highly conserved in their
modular structure. It is, however, not clear why these receptors differ in number of LBDs. What is the
role of two LBDs in insect VgRs and how is ligand specificity determined by these receptors? Also, the
mechanism of endocytic vesicle trafficking, the regulatory role of Rab proteins (a family of small
GTPases) in vesicular traffic are other intriguing questions. Moreover, the LpRs sequenced from
different tissues bear a very high structural homology. The exact mechanism of LpR-endocytotic
machinery and the fate of the HDLp should be clarified.
110
LDLR family members have the ability to bind multiple ligands. The chicken VgR, for example,
recognizes at least eight different ligands (Hiesberger et al., 1995; Jacobsen et al., 1995). LDLR-related
protein binds more than 20 different ligands (Strickland et al., 1995; Kounnas et al., 1996). The
mammalian VLDL receptor has been shown to bind apoE specifically, whereas apoB, a ligand of the
LDL receptor, does not interact with this receptor (Takahashi et al., 1992). A very high structural
similarity of insect VgRs with Drosophila YPR (Schonbaum et al., 1995) contrasting to quite different
ligand suggests that the insect VgRs may endocytose more than one ligand similar to their vertebrate
mates. Moreover, recent investigations have proved that LDLR members are also involved in signal
transduction in addition to endocytosis of the macromolecules. Several fingerprints of the putative signal
transduction motifs in the intracellur domain of insect VgRs/LpRs suggests that these receptors might
also be involved in the signal transduction cascades. These mysteries should be clarified in the future
studies.
Table 1. Sequence information of the insect vitellogenin receptors (VgRs) and lipophorin receptors (LpRs)
Species Order Accession # Authors
VgRs
Drosophila melanogaster Diptera U13637 Schonbaum et al.., 1995
Aedes aegypti Diptera L77800 Sappington et al.., 1996
Solenopsis invicta Hymenoptera AY262832 Chen et al.., 2004
Periplaneta americana Dictyoptera AB077047 Tufail and Takeda, 2005
Blattella germanica Dictyoptera AM050637 Ciudad et al., 2006
Leucophaea maderae Dictyoptera AB255883 Tufail & Takeda, 2007
Anopheles gambiae Diptera EAA06264
LpRs
Locusta migratoria (fb) Orthoptera AJ000010 Dantuma et al., 1999
Aedes aegypti (ov) Diptera AF355595 Chen et al., 2001
Aedes aegypti (fb) Diptera AY348869 Seo et al., 2003
Galleria mellonella (fb) Lepidoptera DQ482581 Lee et al., 2003
Bombyx mori LpR1 Lepidoptera AB201471 Gopalapillai et al., 2006
Bombyx mori LpR2 Lepidoptera AB201472
Blattella germanica L Dictyoptera AM403063 Ciudad et al., 2006
Blattella germanica S Dictyoptera AM403064
Leucophaea maderae Dictyoptera AB218869 Tufail et al., 2009
Drosophila melanogaster Diptera NM_733119
The accession numbers refer to database information linked to DDBJ/NCBI
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NAL B OO
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Views on Insect Molecular Biology, Vol.(1), 2009 Invited
InvitedReview
Review
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 Vol. (1). Page No. XX, 2009
SIONË
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 Vol. (1), 119 – 130, 2009
Chapter – 6
Nutritional Signaling in Anautogenous Mosquitos
Immo A. Hansen#* and Geoffrey M. Attardo$

# Department of Biology & Institute of Applied Biosciences, New Mexico State University,
PO Box 30001, MSC 3A, Las Cruces NM 88003-8001, USA.
$
Yale School of Public Health and Epidemiology,
Rm 606 LEPH, 60 College Street, New Haven, CT 06520, U.S.A.
Abstract
Mosquitoes are vectors of diseases that cause tremendous human mortality and morbidity. The drive behind the
vector capacity of mosquitoes is the requirement for blood to initiate their reproductive process. This chapter will
describe the molecular mechanisms linking the nutritional stimuli generated from a blood meal to the production
of yolk protein precursors. Research on the physiology of autogenous (not requiring a blood meal for egg
production) versus anautogenous (requiring a blood meal for egg production) showed that mosquitoes capable of
producing eggs without a blood meal had higher levels of nutrients and specifically a higher concentration of
certain amino acids in their hemolymph. Analysis of the effect of amino acids on hormonal stimulation of yolk
protein precursor genes by 20-hydroxyecdysone showed that specific amino acids are essential for hormonal
activation of these genes. The mechanism mediating the amino acid signal to regulate gene transcription is
mediated by the TOR (target of rapamycin) pathway. TOR is a conserved pathway throughout eukaryotic
organisms that has been adapted to regulate reproduction in mosquitoes. The entire pathway has not been
elucidated, however the downstream effector was determined to be a GATA type transcription factor the
translation of which is regulated by the TOR pathway. Another important mechanism regulating expression of
yolk protein precursors are insulin like peptides. The insulin pathway is conserved in multicellular organisms and
undergoes significant crosstalk with the TOR pathway. Knockdown analyses on components of this pathway
impact yolk protein precursor expression.
Keywords: Ades aegypti; insulin growth factor , nutritional; TOR pathway; vitellogenesis
* For Corespondence (email: immoh@nmsu.edu )
1. Introduction Females of anautogenous mosquito species
2. Biology of autogenous and anautogenous take vertebrate blood in order to gain nutrients
mosquitoes that are used for reproduction. As a consequence
3. Mosquito reproductive physiology and molecular
biology
of this unique life style species of the genera
4. Amino acid signaling Anopheles, Aedes, and Culex have become
5. Insulin signaling highly effective vectors for parasitic protozoans
6. Conclusion and viruses that cause several severe tropical
7. References diseases. Mosquito-borne diseases infect and kill
millions of people in underdeveloped countries
and are therefore a global health threat.
The article has been scientifically edited by Chandrasekar R.
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Many preceding and recent research projects aimed at the understanding of the phenomenon of
anautogeny and tried to elucidate mosquito nutrition and nutritional signaling in reproductive tissues. In
this review we will discus work on the molecular biology of mosquito reproduction. The conversion of
blood to yolk, termed vitellogenesis, starts with the digestion of blood proteins by a set of trypsin-like
proteases in the gut. This process is followed by synthesis of the yolk proteins in the fat body and their
secretion into the hemolymph of the mosquito. Subsequently the yolk proteins are taken up by the
developing oocytes. Big gains of knowledge have been made in identifying nutritional signal
transduction events within female mosquitoes. Especially insulin signaling and amino acid-induced
signaling via the target of rapamycin-signaling pathway in different reproductive organs/tissues have
been reported. These findings pave the way for the development of novel, unconventional mosquito
control strategies.
2. Biology of autogenous and anautogenous mosquitoes
Many species of blood feeding insects have evolved to regulate their reproductive cycle around the
acquisition of a blood meal. The requirement for blood to initiate reproduction is termed anautogeny.
Consequentially, many of these insects are potent disease vectors. Mosquitoes are the most significant
vectors on the list in terms of the number of diseases that they transmit and the number of people that
those diseases affect. Different species of mosquitoes have developed variations on the requirement of
blood for oogenesis. These variations range from mosquitoes that do not require blood at all, to
mosquitoes that must have a blood meal to begin oogenesis. Mosquitoes that do not require blood for
reproduction are termed autogenous and can be either obligatory or facultative autogenous. Mosquitoes
that do require blood are considered anautogenous; this condition can also be obligatory or facultative
depending upon the species and conditions. The terms autogeny and anautogeny were first defined in
1929 (Roubaud, 1929). Since then many species of mosquitoes have been characterized as to the type of
reproductive strategy they undertake (Rioux et al., 1975;Vinogradova, 1965).
Mosquitoes show a range of phenotypes in their autogenic or anautogenic tendencies. Strains of

the pitcher plant mosquito Wyeomyia smithii in the northern region of North America (>40° N) do not
blood feed at all (O'Meara and Lounibos, 1981). Some species of mosquitoes begin their adult life
autogenously and switch to anautogeny after their first reproductive cycle. Many strains of Aedes
atropalpus, only blood feed after having laid their first batch of eggs (O'Meara and Craig, Jr., 1969).
Other facultative autogenous mosquitoes will feed immediately if given the opportunity, but can develop
a first batch of eggs without feeding if blood is not available, as is the case with the black marsh
mosquito Aedes taeniorhynchus. There is a cost to autogenous egg development in these mosquitoes as
egg numbers are significantly reduced in non-blood fed mosquitoes when compared to blood fed
mosquitoes (O'Meara and Evans, 1973; O'Meara and Edman, 1975). Some facultative autogenous
mosquitoes require specific environmental and nutritional conditions during larval development to allow
them to develop eggs autogenously (O'Meara, 1979). Finally, obligatory anautogenous mosquitoes under
no circumstances develop eggs without a blood meal. Some species that for the most part are strictly
anautogenous, have strains which are autogenous. An example of this is found in Ae. aegypti, which is
usually obligatory anautogenous. Autogenous “feral” strains of A. aegypti have been identified in Africa
that live away from their normal human hosts in the jungle. These mosquitoes appear to have developed
autogenous reproduction in response to a reduction in host density (Trpis, 1977). Typically, the most
effective disease vectors are obligatory anautogenous for example: A. aegypti, the vector of dengue fever
and Anopheles gambiae, the primary vector of malaria.
To understand how expression of autogenous and anautogenous reproductive modes in mosquitoes

are regulated, a number of studies have been conducted comparing the differences between these two
reproductive types. Environmental factors are an important determinant of the expression of autogeny or
anautogeny in some facultatively autogenous mosquito species. A study done on different populations
of A. taeniorhynchus in Florida shows that the expression of autogenous egg development is very
different from site to site. These differences are a result of a genetic factors, but it is hypothesized that
host abundance at the individual sites has shaped these predispositions in different populations (O'Meara
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and Evans, 1973). Photoperiod and temperature are important regulators of autogenic expression as well.
In the mosquito Culex tarsalis, longer photoperiods result in a large increase in the expression of
autogeny while shorter photoperiods prompt anautogeny (Harwood, 1966). Temperature also plays an
important role in the expression of autogeny in these mosquitoes. At temperatures below 21°C
expression of autogeny is severely reduced (Brust, 1991). The sensitivity to photoperiod and temperature
by these mosquitoes is most likely an adaptation that promotes survival and reproductive success during
the fall and winter months when conditions are harsh and resources are scarce.
Another environmental factor shown to affect expression of autogeny is poor larval nutrition and
stress. C. tarsalis larvae that were fed on a reduced diet and or exposed to crowding during development,
developed into adults expressing much lower levels of autogenic egg development (Reisen et al., 1986).
Finally, the mating status of the female mosquitoes has also been shown to have an effect on the
ability to develop eggs without a blood meal. In Ae. taeniorhynchus, autogenous egg maturation will not
begin until the female has mated with a male (O'Meara and Evans, 1976). It is hypothesized that a
substance from the male accessory gland included with the sperm is responsible for this stimulus. In
addition to environmental stimuli, genetics are a critical determinant of the reproductive capabilities of a
mosquito.
Data from extensive crossbreeding experiments between autogenous and anautogenous strains of
Culex pipiens suggests that in these mosquitoes there are most likely multiple genes responsible for
conferring the autogenous phenotype. Of the three chromosomes, these genes are linked to chromosome
one (the sex chromosome) and chromosome three, creating a partial sex linkage. Expression of autogeny
appears to be gene dosage dependant (Spielman, 1957). In A. atropalpus, which has both autogenous and
anautogenous strains, a similar genetic analysis was performed. The genetics of autogeny in this
mosquito appear to be significantly different, as the trait of autogeny appears to be conferred by a single
dominant autosomal gene (O'Meara and Craig, Jr., 1969). Further research demonstrated that while the
gene conferring autogeny is dominant in this species, there are other modifier/enhancer genes that
optimize the level of fecundity in mosquitoes carrying the autogeny gene. When these genes are removed
through crosses with anautogenous strains, efficiency of autogenic egg development decreases (O'Meara,
1972). The genes responsible for conferring or supporting autogenic or anautogenic status have not yet
been identified. There are many factors regulating the expression of autogeny and anautogeny in
mosquitoes. What are the physiological differences and changes that are happening in the mosquito as a
result of these genetic and environmental stimuli and how do they result in one or the other form of
reproductive strategy? Comparative physiological analyses between autogenous and anautogenous
mosquitoes show significant differences between mosquitoes expressing these two reproductive types.
Nutritional analysis of autogenous and anautogenous strains of C. tarsalis shows the autogenous
mosquitoes having greater amounts of total lipids, total carbohydrates and total proteins. Interestingly,
male mosquitoes from the autogenous strain were also found to have higher levels of nutrients than
males from the anautogenous strain (Su and Mulla, 1997). A similar analysis was performed in
autogenous and anautogenous strains of Aedes albopictus. This analysis also found that the autogenous
strains contained significantly higher energy reserves in the form of metabolizable protein and lipid
stores (Chambers and Klowden, 1994). A more specific nutritional analysis determined the levels of free
AAs in the hemolymph of these autogenous strains immediately after eclosion. Specifically, arginine,
glycine, isoleucine, leucine, lysine, phenylalanine, serine, threonine and valine showed the greatest
difference in concentration between the two strains (Su and Mulla, 1997).
Life cycle analysis of autogenic and anautogenic C. tarsalis shows that the autogenic strains take a
day longer to complete larval development, but are able to lay eggs one to two days earlier than their
anautogenous counterparts. However, the first batch of eggs laid by the autogenous mosquitoes contains
significantly fewer eggs than those laid by the anautogenous strains (Reisen and Milby, 1987). A
disparity between nutritional reserves is a common theme that arises upon comparison of autogenous
with anautogenous mosquitoes. It appears that the environmental and genetic factors that facilitate
121
autogeny to take place in some mosquitoes result in an accumulation of nutrients in the larval stage,
which is then carried into the adult stage. This abundance of nutrients somehow triggers the activation of
egg development in these mosquitoes. This information is important because it highlights the fact that
anautogenous mosquitoes maintain relatively low levels of systemic nutrients and this most likely
prevents egg development. Furthermore, it indicates that nutritional cues are playing a critical role in this
system in mosquitoes. The nutritional deprivation in anautogenous mosquitoes is lifted when these
mosquitoes feed on nutrient rich blood, which consequentially marks the beginning of egg development.
Better understanding of the role of nutrition in egg development, has come about through study of
reproduction in A. aegypti.
3. Mosquito reproductive physiology and molecular biology
A blood meal triggers a series of events in different tissues of mosquito females. One of the best
understood blood meal-activated processes is the activation of a digestive protease called ‘early trypsin’
in the midgut of Aedes aegypti, in the first hours after a blood meal. Early trypsin mRNA is present at
high levels in the midgut before a blood meal and is rapidly translated afterwards under the control of
Juvenile hormone. The activity of early trypsin in turn activates the transcription of late trypsin (Noriega
et al., 1994; Noriega et al., 1996; Noriega et al., 1999). Blood feeding initiates the process of
vitellogenesis, a key physiological event in mosquito reproduction (Fig.1). Vitellogenesis is the
utilization of nutrients from a blood meal for the large-scale synthesis and secretion of yolk protein
precursors (YPPs) in a tissue called the fat body. The fat body is analogous to vertebrate liver and fat
tissues. Fat body cells are specifically responsible for the synthesis of YPPs in mosquitoes (Hagedorn et
al., 1973; Hagedorn and Judson, 1972). The main YPPs genes activated during vitellogenesis are
vitellogenin (vg), vitellogenic carboxypeptidase (vcp), vitellogenic cathepsin B (vcb) and lipophorin (lp).
Out of these genes vg is the most highly expressed. Vitellogenesis occurs exclusively in the fat body and
is divided into three main stages: previtellogenesis, vitellogenesis and post vitellogenesis (Raikhel et al.,
2002).
The mosquito fat body is a functionally diverse tissue. Its functions range from storage and
metabolism to protein synthesis during the mosquito’s life cycle. These changes in function are regulated
in a hormonally dependent manner (Raikhel, 1987). Upon eclosion from pupae to adult, the fat body
undergoes hormonally regulated changes that allow it to become responsive to signals that induce
vitellogenesis. A transient peak in juvenile hormone III (JH) occurring immediately after eclosion is
associated with these changes (Flanagan and Hagedorn, 1977; Raikhel and Lea, 1990; Hagedorn, 1994).
These changes make the fat body competent to respond to the steroid hormone 20-hydroxyecdysone
(20E) and to synthesize the massive amounts of protein required for egg maturation (Raikhel and Lea,
1983; Dittman et al., 1989). The orphan nuclear receptor AaFTZ-F1 has recently been shown to be
essential for attainment of competence by the fat body to respond to 20E. Furthermore, the translation of
AaFTZ-F1 is regulated by the presence of JH (Li et al., 2000; Zhu et al., 2003). Once remodeling of the
fat body has been completed and hormonal competence has been attained, the fat body enters a
reproductive state of arrest during which it becomes dormant and YPP gene expression is repressed.
The state of arrest is relieved upon blood feeding by the female mosquito and vitellogenesis
begins. Activation of vitellogenesis causes the fat body to undergo further structural changes that
facilitate protein synthesis (Snigirevskaya et al., 1997). Blood feeding also stimulates an increase in JH
esterase activity and inhibition of JH production causing a rapid decrease in the basal hemolymph JH
titer (Readio et al., 1998). Another response to blood feeding is the secretion of a neurosecretory
hormone releasing factor from the ovaries (Lea, 1967). This factor in turn causes the release of ovarian
ecdysteroidogenic hormone (OEH) from the brain, which in turn stimulates the production of the steroid
hormone, ecdysone, by the ovaries (Hagedorn et al., 1975). Ecdysone travels to the fat body and is
hydroxylated to 20E. 20E titers increase and peak at 24 hours post blood meal (PBM), and then rapidly
decline back to basal levels by 36 hours (Hagedorn, 1985; Hagedorn, 1989).
122
Molting, metamorphosis and reproduction are regulated in part by 20E in D. melanogaster and
other insects (Hagedorn, 1989; Thummel, 1996; Segraves, 1994). 20E is the primary stimulus that up
regulates YPP gene expression. YPP gene expression and protein synthesis positively correlate with 20E
titers. When 20E titers decline, YPP synthesis declines as well (Deitsch et al., 1995; Hagedorn, 1985;
Raikhel, 1992). After activation by 20E YPP genes are transcribed and translated specifically in the fat
body. The YPP proteins are then processed in the fat body and secreted into the hemolymph where they
travel to the ovaries and are endocytosed by the developing oocytes. At around 36 hours PBM the fat
body converts back to a nutrient storage and metabolism function until the next vitellogenic cycle is
initiated (Fig.1) (Raikhel, 1992).
Fig. 1 Nutritional and hormonal regulation of mosquito vitellogenesis.
20E is a main regulator of vitellogenesis (Spielman et al., 1971), but there were results that implied
that 20E by itself is not sufficient to activate vitellogenesis. Observation of YPP secretion indicates that
these genes are expressed before hemolymph levels of 20E begin to increase (Hagedorn et al., 1975).
Other experimental results showed that injection of physiological levels of 20E into previtellogenic
mosquitoes were not sufficient to induce YPP synthesis and consequent egg development. Only very
large (non-physiological) doses of 20E were able to activate low levels of YPP expression (Borovsky D.
and Van Handel, 1979; Lea, 1982; Fuchs and Kang, 1981). However, 20E injections into autogenous
mosquitoes successfully activate YPP synthesis (Fuchs and Kang, 1981). These data suggested that a
second control mechanism maintains the state of arrest in anautogenous mosquitoes. In contrast to these
results, in vitro cultured fat bodies from 3-5 day previtellogenic mosquitoes treated with physiological
levels of 20E successfully initiated vitellogenesis. The media in which the fat bodies were cultured in
contained AAs (Raikhel et al., 1997). This suggested that the availability of nutrients acts as a signal to
the fat body, which then deactivates the previtellogenic state of arrest, allowing 20E to activate YPP
genes and activate vitellogenesis. Further support for this hypothesis came from experiments in which
solutions containing AAs and physiological levels of 20E have been infused into the hemolymph of
previtellogenic anautogenous mosquitoes; these mosquitoes successfully underwent egg development
(Uchida K, 1998). These data together with the physiological analyses of autogenic and anautogenic
mosquitoes further supported the idea of nutritional requirements for initiation of vitellogenesis.
123
4. Amino acid signaling
Nutrients are a key stimulus in alleviating the vitellogenic state of arrest in anautogenous
mosquitoes. However, the question of how they were doing this remained standing. Higher amino acid
(AA) titers in the hemolymph of autogenous mosquitoes suggested AAs could act as a signal activate
vitellogenesis (Uchida et al., 1998; Su and Mulla, 1997). AAs would make an ideal signaling mechanism
as hemolymph titers of AAs increase dramatically after a blood meal (Uchida et al., 2003; Uchida et al.,
1990). A more detailed examination of the role of AAs in stimulating egg development in mosquitoes
shows that when the anautogenous mosquito C. pipiens is infused with a balanced solution of AAs, it is
capable of initiating egg development. This experiment was also performed with seven other species of
anautogenous mosquitoes. Five of the seven underwent successful egg development (Uchida et al.,
2001). Furthermore, specific AAs are essential for egg development as when they are omitted from the
infusion mixture, egg development fails to occur. These essential AAs are leucine, isoleucine, lysine,
phenylalanine, threonine, tryptophan, valine, cysteine, arginine and asparagine (Uchida K, 1998). This
study agrees with previous work in which adult A. aegypti were fed artificial blood meals from which
individual AAs were omitted and egg development was recorded. With the exception of asparagine the
same AAs were found to be essential for egg development (Dimond et al., 1956). AAs essential for egg
development are also essential for larval growth in A. aegypti and in general for growth in mammals
(Rose, 1938; Singh and Brown, 1957).
These experiments clearly establish the link between AAs and reproduction in mosquitoes. They
also reveal parallels between the AAs essential for egg development and those required for immature
development in insects as well as in mammals. A well-characterized nutritional signaling system that fit
with the data presented above was the TOR kinase nutritional signaling pathway. The TOR kinase
(Target of Rapamycin) is a serine/threonine kinase that is ubiquitously expressed in eukaryotes (Raught
et al., 2001). It has been well characterized in its role as a nutrient sensor in multiple systems. TOR
functions in concert with the insulin-signaling pathway. Crosstalk occurs between these pathways
through TOR interactions with a small GTPase called RHEB (Saucedo et al., 2003) which functions
downstream of the TSC proteins and the AKT and PI3 kinases (Hafen, 2004). The primary effect this
pathway mediates is the control of cellular growth via regulation of transcription and translation in
response to environmental nutrients, specifically AAs.
In S. cerevisiae AA and nitrogen source deprivation results in transcriptional activation of nitrogen

catabolite repression genes via the TOR pathway (Cooper, 2002). In D. melanogaster AAs act directly
upon the fat body via the TOR pathway to regulate the growth of the whole organism by the secretion of
an insulin binding protein originating from the fat body (Colombani et al., 2003b). AAs and TOR are
also involved in regulation of blastocyst trophoectoderm differentiation and embryonic implantation in
mammals (Martin and Sutherland, 2001). The exact mechanism of how TOR senses the presence of AAs
is not known. However, some of the AAs that stimulate TOR activity are known. Branched chain AAs
and leucine in particular have been identified as activators of TOR in mammalian adipocyte tissue.
Stimulation with leucine results in increased protein synthesis, hyperplastic growth and tissue
morphogenesis in rat adipocytes (Lynch et al., 2003). In addition, limitation of the cationic AA arginine
inhibits global D. melanogaster growth, which is regulated by TOR (Colombani et al., 2003).
The mechanisms by which TOR transduces its response to AA stimulation are better understood.
TOR is involved in the regulation of both transcription and translation. TOR regulates translation
through the maintenance of the phosphorylation state of two translational regulatory proteins. TOR
phosphorylates and activates the p70 S6 kinase in response to nutritional stimuli (Chung et al., 1992;
Fox et al., 1998; Brown et al., 1995). The S6 kinase phosphorylates the ribosomal protein S6 that
specifically facilitates the translation of mRNAs containing a 5’-polypyrimidine tract. These types of
mRNAs typically encode ribosomal proteins, translation elongation factors, and growth control proteins
(Jefferies et al., 1997). TOR also regulates the phosphorylation state of the PHAS-I/4E-BP1 factor. This
factor in a dephosphorylated state binds to and inhibits the eIF-4E translational initiation factor which
124
functions in cap recognition and recruitment of ribosomes to the mRNA. Phosphorylation of 4E-BP1
inactivates it and derepresses eIF-4E allowing translation to proceed (Brunn et al., 1997).
In yeast, TOR has also been well documented in its ability to regulate transcription. TOR regulates
the nuclear localization of a GATA type transcription factor named Gln3 via maintenance of its
phosphorylation state in yeast (Beck and Hall, 1999; Bertram et al., 2000; Cooper, 2002). In mammals
the CHOP (C/EBP homologus protein) and AS (Asparagine synthetase) genes are both negatively
regulated in the presence of AAs. Specifically, both are upregulated during leucine starvation. Control
of these genes has been localized to cis elements found in the 5’ regulatory region. The CHOP gene
contains an element called the AARE (AA response element) while the AS gene contains two elements
called NSRE –1 and –2 (Nutrient sensitive response element). Transfer of the AARE element alone to a
basal promoter sequence confers AA responsiveness to it, while both NSRE’s are necessary to confer
nutrient sensitivity. The AARE and NSRE-1 elements both show homology to the binding sites for the
C/EBP and ATF/CREB families of transcription factors (Bruhat and Fafournoux, 2001). CHOP
expression was linked to TOR in a study showing that insulin like growth factor 1 (IGF 1) is required for
CHOP activation and that treatment with the TOR inhibitor rapamycin as well as inhibitors of the insulin
pathway prevented activation (Entingh et al., 2001).
The TOR signaling pathway is indeed the key pathway transducing AA signals in mosquitos. As
mentioned above, it has been demonstrated that AAs act directly upon the fat body to activate basal
expression of the Vg gene and that 20E is incapable of activating Vg without them. The nutritionally
regulated TOR (Target of Rapamycin) kinase signal transduction pathway mediates the AA signal
(Hansen et al., 2004). AA/TOR-signaling results in phosphorylation and activation of S6 kinase (S6K)
(Hansen et al., 2005). Two cationic AA transporters are essential for AA/TOR signaling in the mosquito
fat body (Attardo et al., 2006). One final downstream step in this signaling cascade is the translation of a
GATA factor, which is the specific transcriptional activator of the Vg gene (Park et al., 2006). Forkhead
box (FOX) transcription factors are also very likely targets since it has been shown that they also play a
prominent role in mosquito reproduction (Hansen et al., 2007). The results of these studies clearly show
that processing of nutritional information via AA/TOR and S6K signaling is a central step in the
regulation of egg development in mosquitoes. However, blood-meal-induced activation of the TOR-
signaling pathway and subsequent egg maturation depends on the accumulation of adequate nutritional
reserves during larval development of the mosquito (Shiao et al., 2001). Small, malnourished mosquitoes
do not posses the TOR molecular machinery and are therefore not competent to start vitellogenesis
directly after a blood meal. Competence can be regained by treatment with JH.
5. Insulin signaling
Nutrient- and insulin signaling coordinate protein synthesis to regulate cell growth, development,
fecundity, metabolic homeostasis, and lifespan in multicellular organisms. Two major signaling
pathways have been identified which transduce nutritional information: the insulin/PI3K-signaling
pathway and the AA/TOR signaling pathway. TOR kinase represents a convergence point for both
pathways (Colombani et al., 2003a). At the top of the insulin/PI3K signaling cascade are the insulin-like
peptides (ILPs). Seven ILPs have been identified in Anopheles gambiae (Krieger et al., 2004) and eight
genes encoding ILPs in the yellow fever mosquito, Aedes aegypti (Riehle and Brown, 1999). Transcripts
for five of the ILPs occurred predominantly in brain of larval, pupal, and adult mosquitoes. Transcripts
of two other ILP genes were present in the head, thorax and abdomens of all stages. The eighth ILP was
predominantly expressed in abdomen.
ILPs play an important role in insect reproduction since it was shown, that ablation of median
neurosecretory cells in the Drosophila brain, which produce ILPs leads to reduced fecundity and
increased lifespan (Broughton et al., 2005). In addition, transplantation experiments in Drosophila
reveal, that insulin/PI3K signaling is necessary for vitellogenesis. Ovaries deficient with the insulin
receptor substrate CHICO did not undergo vitellogenesis when transplanted into wild-type females
(Richard et al., 2005). A key transcriptional regulator of the insulin/PI3K signaling is a forkhead
125
transcription factor, FOXO, that is inactivated by active insulin signaling (Puig et al., 2003). Induced
expression of dFOXO in the fat body from the onset of adulthood increased life-span and reduced
fecundity in female Drosophila significantly while no effect was seen in male flies (Giannakou et al.,
2004).
Exposure to human insulin influences the susceptibility of anophiline mosquitoes to Plasmodium

infection. Insulin-fed mosquitoes showed significantly higher oocyst infection rates than controls and
increased oocyst densities in both A. gambiae and A. stephensi (Beier et al., 1994). In turn, molecules
from the invading parasite also elicit functional responses in female mosquitoes that are regulated
through the insulin signaling pathway or by cross-talk with interacting pathways (Luckhart and Riehle,
2007). In addition to the effect of insulin on Plasmodium development several studies show that
application of vertebrate insulin influences mosquito reproduction. Porcine insulin activates
ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. (Graf et al., 1997).
Bovine insulin in combination with 20-hydroxyecdysone activated transcription of the YPP gene
vitellogenin (Vg), and this process was sensitive to the phosphoinositide-3 kinase (PI-3k) inhibitor
LY294002 as well as the TOR inhibitor rapamycin (Roy et al., 2007).
Mosquitoes possess an insulin receptor (IR) that is expressed before a blood meal mainly in the
nurse cells of ovaries (Helbling and Graf, 1998). Immunocytochemistry showed that IR was localized in
the cell membranes of follicle cells surrounding the oocyte and nurse cells (Riehle and Brown, 2002). A
key enzyme of the insulin signaling cascade, the protein kinase Akt, was identified and cloned as a
cDNA from ovaries of the mosquito A. aegypti. When A. aegypti ovaries were treated with bovine
insulin in vitro, Akt was threonine-phosphorylated (Riehle and Brown, 2003). RNAi-mediated
knockdown of the mosquito InR, Akt, and TOR inhibited insulin-induced Vg gene expression as well as
S6 Kinase phosphorylation in in vitro fat body culture assays (Roy et al., 2007). The results of these
experiments prove that the insulin receptor pathway is present in several reproductive tissues of
mosquitoes. Further work is necessary to elucidate the connections between nutrition, reproduction, and
disease transmission.
6. Conclusions
Research on nutritional signaling in mosquitoes has provided us with fundamental insights into its
biology and function, but many open questions remain. It is now clear that TOR signaling plays a central
role in the regulation of mosquito reproduction and several of the involved mechanisms have already
been elucidated. It remains to be learned if and how ILPs and insulin receptor-signaling are involved in
the regulation of reproduction and in disease transmission. Importantly, the results of these studies will
be valuable for the development of transgenic mosquitoes that can be used for fighting mosquito-borne
diseases like malaria. It appears that mosquito signal transduction will continue to be a fascinating and
rewarding research subject in the future.
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the hemolymph of the female Culex pipiens pallens (Diptera: Culicidae), after a blood meal. J.
Med. Entomol. 27, 302-308.
Vinogradova EB. (1965). Autogenous development of ovaries in blood-sucking mosquitoes. Zoologische
Zhurnal. 210-219.
Zhu J., Chen L., and Raikhel AS. (2003). Posttranscriptional control of the competence factor betaFTZ-
F1 by juvenile hormone in the mosquito Aedes aegypti. Proc. Natl. Acad. Sci. USA, 100, 13338-
13343.
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 Vol. (1). Page No. XX, 2009
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Short Views onShort
Insect Molecular
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on Insect Vol.(1),
Molecular 2009
Biology Invited
Invited Review
Review
TERNA
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ËIN
 Vol. (1), 131 – 145, 2009
Chapter – 7
Pheromone Biosynthesis Activating Neuropeptide (PBAN) and its

G-protein coupled receptor
Rachel Bober1,2 & Ada Rafaeli2‡

1
Department of Entomology, Hebrew University, P.O. Box 12, Rehovot 76100, Israel
2
Institute for Technology & Storage of Agricultural Products, Agriculture Research Organization,
Volcani Center, Bet Dagan 50250, Israel.
Abstract
In many moth species, pheromone production, correlated with female receptivity, is under neuroendocrine control due to
circadian release of a neuropeptide, Pheromone-Biosynthesis-Activating Neuropeptide (PBAN), produced in the
subesophageal ganglion, located near the brain. PBAN is responsible for the activation of pheromone production by adult
female moths during the scotophase through its binding to a G-protein coupled receptor (PBAN-R). This short review
describes the discovery of this GPCR through homologies with GPCRs of similar ligands in model genome data banks
and a novel method for detection of this GPCR at the protein level. A phylogenetic study provides an overall review of
known GPCRs for this neuropeptide family. We describe the molecular biology approaches that have been used to study
functionality and distribution of this GPCR. We discuss approaches for the design of antagonists and include data on
steric hindrence effects. Studies on mutagenesis and molecular modelling, focussing on binding domains, enlighten the
interaction between the ligand and its receptor. Finally, we discuss physiological studies that reveal the developmental
regulation of this GPCR. Future studies incorporating such multidisciplinary approaches to insect physiology can impact
on effective designs of biological agents for manipulating insect behaviour.
Keywords: PBAN, GPCR, moths; pyrokinin; neuropeptide; receptor-ligand interactions; photoaffinity-

biotin label; gene expression; mutagenesis; molecular modeling
‡
For Correspondence (email: vtada@volcani.agri.gov.il)
Overview
1. Introduction: Reproductive behavior in moth (c) Quantitative Reverse-Transcription Real Time

species Polymerase Chain Reaction (RT-Real-Time-
2. PBAN and FXPRLamide neuropeptides QPCR)
3. Discovery of the G-protein coupled PBAN- 6. Drug (insecticide) design
receptor (a) Mutagenesis
(a) G-protein coupled Receptor Gene (b) Molecular modeling and in silico experiments
(b) Protein Level 7. Physiological studies - Developmental Regulation
4. Phylogeny of the PBAN receptor Gene 8. Concluding Remarks
5. Approaches in Molecular Biology 9. Acknowledgements
(a) Functional expression 10. References
(b) Reverse-Transcription Polymerase Chain
Reaction (RT-PCR) and Sequencing
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1. Introduction: Reproductive behavior in moth species
Adult, sexually receptive female noctuid (night-flying) moths signal their receptivity to mate by
producing and releasing a volatile, species-specific sex-pheromone blend from a pheromone gland
situated between the ultimate and penultimate terminal segments of the abdomen (Rafaeli and Gileadi,
1995). Sexual communication is thus dependent on the synchronous production of sex pheromones that
will attract the male and broadcast receptivity of the mature female.
By definition, pheromones are substances secreted into the environment by one individual and
received by a conspecific in which they elicit a specific response (Karlson and Lüscher, 1959). Factors
such as age and photoperiod can influence the timing and success of sex-pheromone production and
release and thereby the successful meeting of the sexes for reproduction (Fig. 1). In most female moths
Juvenile Hormone (JH), produced and released by the corpora allata, acts as a major endocrine effector
for vitellogenesis (yolk accumulation) and egg development (Ramaswamy et al., 1997).
Pheromone production and release by

mature virgin females is strictly gated within a
time-window during the scotophase (night-time).
In H. armigera, for example, pheromone
production is gated within a window of
receptivity during the latter part of the
scotophase. Photoperiod influences the release of
a neuropeptide: Pheromone Biosynthesis-
Activating Neuropeptide, termed PBAN, which is
produced by a set of neurosecretory cells in the
subesophageal ganglion (SOG) situated just
below the brain. PBAN is transferred through
nerve connections and stored in the corpora
cardiaca (CC) from where it is photoperiodically
released into the hemolymph (Ramaswamy et al.,
1995; Iglesias et al., 1999), usually during the
scotophase. PBAN reaches its target tissue, which
is the pheromone gland situated at the tip of the
abdomen (Rafaeli and Gileadi, 1995) and
stimulates the pheromone biosynthetic pathway
resulting in the production of the species-specific
pheromone blend. At this same time neural Fig.1 A schematic diagram showing effect of age
messages are also sent down to induce a typical and photoperiod on reproductive behavior
behavior known as "calling" behavior. in the moth Helicoverpa armigera.
Moth sex-pheromone blends may be categorized into two types (Ando et al., 2004). PBAN does
not influence the biosynthesis of the type 2 class of sex pheromones, which are composed of polyene
hydrocarbons and their epoxides. These are biosynthesized from diet-derived linoleic or linolenic acids
by chain elongation, desaturation, and decarboxylation. PBAN has been shown to stimulate only fatty
acid derived sex-pheromone components belonging to the type 1 sex-pheromones. These are linear fatty
acid-derived compounds, 10–18 carbons in chain length, containing an oxygenated functional group and
one to three double bonds. They are biosynthesized de novo from acetate by specific enzymes in the
pheromone gland and released by calling females.
The site of action of PBAN is not completely resolved: it may either up-regulate ACCase (Eliyahu
et al., 2003; Tsfadia et al., 2008); or activate the reduction of fatty acids by the fatty acyl reductase
(Martinez et al., 1990; Moto et al., 2003); or perhaps, by a two-step action involving both enzymes
(Eltahlawy et al., 2007). Readers interested in a more comprehensive coverage of the pheromone
biosynthetic pathways are encouraged to see other recent reviews (Tillman et al., 1999; Jurenka, 2003).
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2. PBAN and FXPRLamide neuropeptides
PBAN was first identified in H. zea as a 33-amino acid C-terminal amidated peptide (Raina et al.,
1989) shortly followed by the identification of a homolog in Bombyx mori (Kitamura et al., 1989).
Subsequent studies revealed that PBAN is a member of the pyrokinin/PBAN family of peptides with a
common C-terminal FXPRLamide motif (X=G/S/T/V), which represents the minimum sequence
necessary in eliciting activity, and with diverse functions in several species of insects (see reviews:
Rafaeli, 2002; Rafaeli and Jurenka, 2003). PBAN-like peptides are found in other insects where they
have other functions such as contraction of hindgut muscles in Leucophaea maderae (Holman et al.,
1986); melanization in Lepidoptera larvae (Matsumoto et al., 1990); induction of embryonic diapause in
B. mori (Imai et al., 1991); acceleration of puparium formation in several flies (Zdarek et al., 1997) and
ecdysone biosynthesis in prothoracic glands of B. mori (Xu et al., 2004; Watanabe et al., 2007).
Moreover, the gene encoding PBAN has post-translational processing sites that could produce
four additional PBAN-gene neuropeptides: PGN-24; PGN-18 (β-SNP); PGN-8 (γ-SNP); PGN-7 (α-SNP)
(see review Rafaeli and Jurenka, 2003) all having the FXPRLamide motif (Ma et al., 1996). The
homolog of PGN-24 in B. mori, termed SGNP I (which has 23 amino acids) has been shown to act as a
diapause hormone (Imai et al., 1991; Sato et al., 1993) and has also been implicated in the induction of
ecdysone synthesis in larval prothoracic glands of the same species (Xu et al., 2004; Watanabe et al.,
2007). PGN-18 bears close similarity to a PBAN-like peptide isolated from larval Pseudaletia unipuncta
(Pss-PT) (Matsumoto et al., 1992). Cap2b-3 from Drosophila melanogaster (pyrokinin-1) has a similar C
terminal to PGN-24. Ecdysis triggering hormone (ETH), involved in ecdysis also bears a PRXamide C-
terminal, as does a vertebrate neuropeptide, Neuromedin U, which is involved in food intake control.
Neuromedin U has been shown to mimic PBAN action with a pheromonotropic activity at higher ligand
concentrations (Choi et al., 2003). It can therefore be concluded that not all PBAN-like peptides have
pheromonotropic functions (although they may mimic this function at higher ligand concentrations) and
those found in other organisms, therefore may represent different functions. These findings emphasize
the ubiquity and multifunctional nature of these PRXamide peptides.
Pharmacological studies led us to the conclusion that PBAN is recognized by a G-protein

coupled receptor (GPCR) (Rafaeli and Gileadi, 1996a; b) since NaF, an activator of G-protein, was able
to mimic downstream events and activate the pheromone gland to produce pheromone in the absence of
PBAN. On ligand-receptor binding this G-protein induces the action of a calcium sensitive adenylate
cyclase as a result of the influx of calcium into the cell to bind to calmodulin (Jurenka et al., 1991,
Jurenka, 1996). The action of a calcium-calmodulin sensitive adenylate cyclase results in the increase of
intracellular cyclic-AMP levels (Rafaeli and Soroker, 1989), leading to a stimulation of pheromone
biosynthesis. In B. mori, however, it has been demonstrated that cyclic-AMP is not involved in these
downstream events (Hull et al., 2007).
3. Discovery of the G-protein coupled PBAN-receptor
(a) G-protein coupled Receptor Gene
Identification of peptide receptors in insects was enhanced with the complete sequencing and
annotation of the D. melanogaster genome (Adams et al., 2000). From the genomic annotation of
D. melanogaster, it was determined that approximately 44 genes code for peptide GPCRs (Hewes and
Taghert, 2001). Four of the GPCRs in the genome of Drosophila were similar to neuromedin U receptors
in vertebrates and at least one receptor was characterized as binding FXPRLamide peptides (Park et al.,
2002). We were the first to identify a PBAN-receptor from moths (Choi et al., 2003). The seven
transmembrane domains, which are indicative of GPCRs, were deduced by hydropathy analysis. The
ERY variant of the DRY domain is located just downstream of transmembrane domain 3. The DRY
domain is highly conserved among GPCRs and is considered to be crucial for the coupling and
activation of G proteins. Several serine and threonine residues are present in the C-terminal tail, which
133
are likely to be responsible for phosphorylation signals and two putative N-glycosylation sites are
present in the N-terminal region and between transmembrane domains 4 and 5.
(b) Protein Level
Since the PBAN-R is an insoluble membrane protein, present at low relative protein concentrations
in the pheromone gland, it was difficult to sequence the protein using the available biochemical
strategies and in the absence of an insect model genome. To overcome this difficulty an exclusive
approach to study PBAN-PBAN-R interactions was developed in our laboratory. By creating a
photoaffinity biotinylated HezPBAN analog we showed specific binding to a putative membrane
receptor (Rafaeli and Gileadi, 1997; 1999; Rafaeli et al., 2003). Membranes of pheromone gland cells
were prepared from pheromone glands and incubated in the presence of this analog and in the additional
presence or absence of unlabeled analog for competitive displacement. After the reaction time,
irradiation using UV enabled the covalent binding of the photoaffinity group to the surrounding
molecules. The membranes were then run on SDS-PAGE, transferred to nitrocellulose and the blots were
treated with avidin-HRP. Avidin was used to bind to all biotinylated groups and HRP was used for
detection by chemoilluminescence. This method allowed visualization of bound PBAN to its receptor
whilst the competitive displacement enabled the determination of specific binding (Rafaeli et al., 2003).
In adult female pheromone glands a specific band was detected in the region of ~50kD.
These binding studies were subsequently verified using Sf9 cells expressing the HezPBAN-R,
showing identical binding characteristics as were found in H. armigera adult female pheromone glands.
One can deduce that the 50kDa protein represents the actual PBAN-R protein. Moreover, Northern blot
evidence using a DIG-PBAN-R probe showed the expected product size for both Sf9 cells expressing the
HezPBAN-R and H. armigera pheromone glands (Rafaeli et al., 2007).
The deduced amino acid sequences from the heliothine moths H. zea and H. armigera have a 99%
homology in the 346 amino acid sequence indicating a size of 38.6kDa. The estimation of a molecular
weight of 50kDa for the complex PBAN-R-photoaffinity-biotinylated-PBAN-analog using SDS-PAGE
was in contradiction to the deduced amino acid sequence. After accounting for the attached biotin-
labeled PBAN, the apparent size of the receptor would be reduced to about 45 kDa. The apparent
differences in size between deduced sequences and gel-migration estimates could be explained by
unpredictable migration on SDS-PAGE due to glycosylations or other modifications.
Using this novel method we demonstrated the spatial (Rafaeli et al., 2007) and temporal (Rafaeli
and Bober, 2005) distribution of the PBAN-R protein in membranes of H. armigera brain, thoracic
ganglion and ventral nerve cord as compared to adult pheromone glands.
4. Phylogeny of the PBAN receptor Gene
Since the first identification, a number of additional receptors have been sequenced based on
homology to the HezPBAN-R or identified from genome sequencing projects (Table 1). A high degree
of homology is evident in the transmembrane domains with many amino acids being conserved across all
receptors but very little homology is evident in the N- and C-terminal domains (Stern et al., 2007). This
is the reason for the 70-75% homology found in the nucleotide sequence of the D. melanogaster
pyrokinin receptors (Table 1). The B. mori receptor differed in the C-terminal, which extends 67 amino
acids compared to the heliothine receptor. This extension has been implicated in desensitization and
internalization of the GPCR (Hull et al., 2005; see review Matsumoto et al., 2007). In addition, a
diapause hormone receptor was identified in ovaries of B. mori, which differed from the B. mori PBAN-
R in pheromone glands (Table 1).
From the phylogenetic tree (Fig. 3) the close similarities of the various PBAN-R's is apparent
(Moth PBAN-R cluster). These are more closely related to the D. melanogaster group of pyrokinin-like
receptors (Fly PK-like cluster) than to the moth diapause hormone-like receptors (DH-like cluster).
134
Amongst the three D. melanogaster pyrokinin-like receptors, CG9918 (AF368273) is the most distantly
removed from the PBAN-R and was therefore used in further mutagenesis experiments (see below).
Similar conclusions regarding the separate cluster of the pyrokinin-like receptors were drawn by a recent
phylogenetic tree analysis with respect to Anopheles gambia (Olsen et al., 2007).
5. Approaches in Molecular Biology
Much of the research concerning the physiology and behavior of sex-pheromone production has
focused in the past on traditional behavioral and biochemical methods. Molecular biology techniques
have opened new avenues for in depth research into mechanisms that might otherwise be missed by
biochemical approaches alone. Moreover, these techniques serve to validate our biochemical knowledge
and together provide information on the physiological regulation of sex-pheromone production.
(a) Functional expression
With our extensive pharmacological studies concerning downstream events of the PBAN-PBAN-R
interaction it was possible to exploit the influx of calcium as indicator of ligand-receptor interactions in
an expression system in order to verify the functionality of the cloned receptor. The full-length PBAN-R
was cloned and expressed in Sf9 insect cells and was shown to mobilise calcium, using fluo-4/AM as
calcium indicator, in response to PBAN in a dose-dependent manner (Choi et al., 2003). The EC50 for
PBAN was found to be 25 nM, a lower value when compared to the activation of the expressed CG8795
(PK-R-2) Drosophila receptor and much lower when compared to the activation of the expressed
CG9918 (PK-R-1) (Choi et al., 2007). On the other hand PK-1 activated the CG9918 receptor at lower
EC50 than the activation of the PBAN-R, verifying its identity as a PK-1 receptor (Cazzamali et al.,
2005). Clearly there is a differential response with the various ligands and the various PBAN-like
receptors and these affinity differences can be utilized in the categorization of the various receptors.
However, when comparing the EC50 values of the endogenous PBAN-R in pheromone glands
challenged with various PRX-amide peptides and PBAN with the cellular activity (calcium influx in Sf9
cells) of the expressed PBAN-R, differences are observed (Stern et al., 2007). Whilst EC50 values of
the expressed PBAN-R reach similar levels when challenged with either the full sequence or the shorter
gene derived neuropeptides (25-200 nM), this is not the case when pheromonotropic activity of the
endogenous receptor is examined. Here the EC50 values of the shorter peptides increase by several
orders of magnitude (0.54 nM-9.73 mM). In addition, when observing the maximal absolute levels of
pheromone produced by these glands on challenge with the PRX-amide peptides, not all the peptides
activate to the same extent and, for example, PGN-8 and PGN-24 induce significantly lower levels of
pheromone production than PGN-18, PGN-7 or PBAN. Neuromedin U too, never reaches the activation
levels obtained by PBAN.
This point emphasizes the importance of comparing the endogenous physiological responses to the
responses of cloned receptors. In vivo, a very low concentration (180 pM) (Ramaswamy et al., 1995) of
circulating PBAN could stimulate pheromone biosynthesis. Calcium assays indicate that once activated
the receptor can remain activated for a period of time (approximately 5 min) (Choi and Jurenka, 2006).
This finding could explain discrepancies in the amount of PBAN required for activating the receptor in
the heterologous expression system (25 nM) (Choi et al., 2003) and in an in vitro pheromone gland assay
(0.54 nM) in heliothines (Stern et al., 2007). A lower concentration is required in vivo due to the receptor
remaining activated over a period of time. The heliothine PBAN-receptor does not have a C-terminal
extension compared to the B. mori receptor and this extension is important for internalization of the
receptor after activation (Hull et al., 2004). Calcium assay data indicates that the heliothine receptor
remains activated and is not internalized after ligand binding (Choi and Jurenka, 2006).
These results also indicate a degree of selectivity of the endogenous PBAN-R to the entire
sequence of PBAN, which can only be observed when pheromone production is assayed. This point is
emphasized when dose-response relationships are studied using PBAN and a non-active, free-acid
135
PBAN ligand containing a C-terminal free acid. Free-acid PBAN analog does not in itself stimulate
pheromone production (Matsumoto et al., 1992) (Fig. 2a). However, in the presence of the amidated
form the free-acid PBAN significantly inhibits the normal stimulatory levels of sex-pheromone by ~50%
(Fig. 2b). This is a similar phenomenon observed in normal dose-response curves with increasing levels
(non-physiological) of PBAN (Rafaeli 1994). This phenomenon may be due to steric hindrance by the
full sequence of PBAN interfering in the PBAN-PBAN-R interaction (Fig. 2a).
Fig.2. Dose-response relationships of PBAN (PBAN-NH2) and the effect of the free-acid C-terminal PBAN
analog (PBAN-OH) in isolated gland incubations of 2-4 day-old adult female H. armigera. Glands were
incubated for two hours in 10 µl drops of physiological saline (as reported in Rafaeli, 1994) in the
presence or absence of PBAN-NH2 or PBAN-OH. Data are means ± SEM of 4-18 individual replicates
(a) Effect of increasing doses on pheromone production in response to PBAN-NH2 or PBAN-OH. (b)
Effect of various concentrations of PBAN-OH when added to maximal pheromone production
stimulation by 2.5 pmole/gland of PBAN-NH2. Different letters depict a statistically significant difference
at P<0.05 (Oneway ANOVA comparisons for all pairs using Tukey Kramer HSD).
136
(b) Reverse-Transcription Polymerase Chain Reaction (RT-PCR) and Sequencing
In order to identify the spatial distribution of the PBAN-R gene and to reveal possible new
members of the pyrokinin/PBAN family we used RT–PCR analysis together with partial sequencing of
the PCR products. When using this method it is extremely important to include controls such as: no
template controls and no reverse transcriptase controls, since the polymerase chain reaction multiplies
amplicons exponentially and therefore will multiply any contaminations (primer-dimer; secondary
structure, primers contamination). We exploited this approach to confirm the presence of the PBAN-R
gene in neural tissues (Rafaeli et al., 2007).
Since, the protein is undetectable in the aedeagus, the male tissue which is homologous to the
female pheromone gland, and, since PBAN did not induce a physiological (pheromone production) or a
cellular (cyclic-AMP production) response in this tissue (Rafaeli et al., 2003) we chose the male
aedeagus as a negative control in these PCR reactions. Surprisingly, the gene for the PBAN receptor was
also detected in this tissue (Rafaeli et al., 2007). Moreover, the presence of the gene in the aedeagus was
unexpected in the light of previous reports showing Northern blot analyses of various tissues in B. mori
(Hull et al., 2004) where they demonstrated the presence of the PBAN-R solely in pheromone glands of
adult females compared to adult testes, ovaries, fat body and flight muscle. However, in this study the
male aedeagus or neural tissues were not included. Indeed, our results showed the absence of the PBAN-
R in the ovaries and larvae confirming the B. mori Northern blot data (Rafaeli et al., 2007).
(c) Quantitative Reverse-Transcription Real Time Polymerase Chain Reaction (RT-Real-Time-QPCR)
RT-Real-Time-QPCR is a powerful and ultra-modern molecular biology tool which can specify
temporary and spatial relative gene expression levels and can reveal gene regulatory mechanisms.
Whereas RT-PCR results in endpoint detection, RT-Real-Time-QPCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon production at each PCR cycle (in real time). This method
first requires the selection of a reference gene in order to compensate for the differences in loading and
RT efficiency. It is recommended to choose more then one gene and proper reference genes may not
necessarily be "house-keeping" genes such as actin.
In our studies (Rafaeli et al., 2007) we chose GTP-BPα as a reference gene. This gene was
selected from several genes (actin A3a, GAPDH, Ribosomal protein 49) since its relative standard
curves were generated with serial 4 x dilution of the pheromone gland cDNA, providing at least 4 points
for comparison with the PBAN-R gene. Analysis of the dissociation curves for the target and reference
genes showed a single melt peak. Relative expression of the real time-PCR products was determined
using the ∆∆CT method (Livak and Schmittgen, 2001). A plot of the log cDNA dilution versus ∆CT
resulted in a slope close to zero. This provided validation for the use of the 2-[∆∆CT] method (Livak and
Schmittgen, 2001) indicating a similar efficiency for both the target and internal control genes. The
average CT was calculated for the PBAN-R and GTP-BPα transcripts and the ∆CT was determined for
each cDNA dilution which demonstrated the method's validation. It should be noted that standard curves
using H. armigera actin A3a resulted in invalid data indicating that this gene undergoes changes in its
expression in the pheromone gland and cannot be used as a reference. Two negative control reactions (a)
no template control and (b) no reverse transcriptase were prepared and analyzed in parallel with the
experimental cDNA samples during all RT-Real-Time-QPCR assays.
RT-Real-Time-QPCR showed that the PBAN-R gene is expressed at lower levels in the other
tissues relative to the pheromone gland in H. armigera (Rafaeli et al., 2007). These findings confirmed
that PBAN or PBAN-like receptors are also present in the neural tissues as well as the male aedeagus. Its
presence in neural tissues may represent a neurotransmitter/neuromodulator-like function for PBAN-like
peptides. The appearance of the gene in the male homologous tissue poses several intriguing questions.
Is this a different gene representing a PBAN-like-receptor which, at the protein level, binds with high
affinity to another PBAN-like ligand and is therefore missed by our binding assay? Is this a pseudogene,
137
a remnant of an evolutionary forgotten function? Or is it a silenced gene that does not translate into a
protein?
6. Drug (insecticide) design
The understanding of the molecular mechanism(s) responsible for pheromone production is crucial
in order to devise ideal strategies for mating disruption specifically for the modeling of effective and
selective antagonists that will block pheromone-mediated communication and thereby prevent successful
mating. In order to rationally design antagonists for the PBAN-R, information about the natural binding
domains is required.
Several approaches could be taken to identify compounds that would antagonize the activation of
the PBAN-R. These approaches are utilized by the pharmaceutical industry to identify drug candidates
that could be effective in treating a variety of diseases. One approach is to utilize a combinatorial
chemical library and screen thousands to millions of compounds. This is a high-throughput approach that
relies on the varied chemical composition of the library to obtain a lead compound that could be further
explored for antagonistic activity. This approach was pursued to identify lead antagonists for PBAN
activity (Ben Aziz et al., 2005; 2006; Altstein et al., 2007) using a sequential D-Phe replacement library.
The antagonistic properties of a few linear and backbone cyclic conformational constrained peptide
libraries and their analogs, were tested for the ability to inhibit pyrokinin/PBAN mediated functions.
However, very high concentrations were necessary for inhibitory activity in the latter studies thereby
encountering possible problems associated with steric hindrance. Clearly, there is a need for the design
of additional, improved antagonists that are more potent. A second approach is to utilize a phage display
library to identify short peptide sequences that could bind to the receptor. These peptide sequences could
then be studied to identify properties of the binding interaction to aid in the identification of antagonists.
A third approach is to experimentally identify the binding domains of the receptor with the natural
ligand. Once the binding pocket is identified then the rational design of a small molecule antagonist
could be undertaken. In fact the pharmaceutical industry utilizes all of these approaches in their search
for drug candidates. We have chosen to identify the natural binding domains of the receptor with natural
ligands in order to determine likely chemistries that could inactivate the receptor.
(a) Mutagenesis
In a phylogenetic (Fig. 3) and peptide activation comparison (Stern et al., 2007), the Drosophila
PK-R-1 (CG9918) is not as closely related to the HezPBAN-R as the GPCRs encoded by the Drosophila
PK-R-2 genes CG8784 and CG8795. In addition, the D. melanogaster pyrokinin-1 and PGN-24 are
identical in the last eight amino acids of the C-terminal ending (GLWFGPRLamide) and the Drosophila
gene, CG9918 has been shown to code for a pyrokinin-1 receptor (Cazzamali et al., 2005). PGN-24 is
relatively poor at stimulating pheromontropic activity by isolated pheromone glands and it exhibits lower
potency of the expressed PBAN-R. Thus, because of the more distant relationship and differences in
peptide activation we utilized sequence differences between PBAN-R and PK-R-1 to create chimera
receptors to determine the active extracellular domains using both in silico (see (b)) and biochemical
mutagenesis studies (Choi et al., 2007). The N-terminal and extracellular loops (ECL1, ECL2 and ECL3)
of the HezPBAN-R were replaced by the corresponding N-terminal and extracellular loops of the PK1-R.
Using the chimeras were challenged with the various PBAN-like peptides. In all the chimeras lower
activation by PBAN was observed and in the case of chimera 2 (ECL2 exchanged), PBAN activation
was lost compared to the other chimeras. The expression of chimera 2 receptor was shown to be
unaffected using enhanced green fluorescent protein- (EGFP-) labeled receptor and thus the decreased
activity of this chimera was not the result of poor receptor expression. Chimera studies exchanging
ECL3 demonstrated that it is directly involved in peptide ligand recognition (Choi et al., 2007).
138
Fig.3. Unrooted neighbor-joining tree based on the deduced amino acid sequences of PRX-amide GPCRs in
eukaryotes. The phylogenetic tree was reconstructed using MEGA 4.0. Numbers along branches indicate
bootstrap support from 1,000 replicates. The scale bar indicates 0.01 change per unit length. PBAN-R,
pheromone biosynthesis activating neuropeptide receptor: Hea, Helicoverpa armigera (AY792036); Hez,
Helicoverpa zea (AY319852); Hev, Heliothis virescens (EU000525/6); Spl, Spodoptera littoralis
(DQ407742); Bom, Bombyx mori (AB181298); Plx, Plutella xylostella (AY974334); PK-R, pyrokinin
receptor: Nav, Nasonia vitripennis (XM001600537), Drm, Drosophila melanogaster (AF522189 (CG8784);
AF522190 (CG8795); AF368273 (CG9918)); Ang, Anopheles gambiae (AY900218/9); Aea, Aedes aegypti
(XM001657160); DH-R, diapause hormone receptor: A. gambiae (AB23041); B. mori (AB164390,
AB164387, NM001043448); Trc, Tribolium castaneum (XM963408,XM963710); Apm, Apis mellifera
(XM623963); N. vitripennis (XM001606151); CAPA-like-R, CAPA-like receptor: A. mellifera
(XM623963); A. gambiae (AY900217); Ghrelin-R, Ghrelin receptor: Caj, Callithrix jacchus (EF526306);
neuromedin-like –R, neuromedin-like –receptor: Dar, Danio rerio (XM680313).
139
(b) Molecular modeling and in silico experiments
We studied the receptor-ligand interactions using molecular modeling techniques. The seven
transmembrane helices of HezPBAN-R were identified, built, packed and oriented correctly after
multiple sequence alignment of the HezPBAN-R and several other GPCRs using the X-ray structure of
rhodopsin as a template (Stern et al., 2007). Molecular dynamics simulations were run on three different
β-turn types of the C-terminal hexapeptide of PBAN (Nachman et al., 1991; Wang et al., 1994; Clark
and Prestwich, 1996) and the results were clustered into 12 structurally distinct groups. The lowest
energy conformation from each group was used for computer simulated docking with the model of the
HezPBAN-R. Highest scoring complexes were examined and putative binding sites were identified
(Stern et al., 2007).
Using the chimera receptors and our PBAN-R model, in silico docking experiments showed that
the cysteine disulfide bridge, which occurs between the ECL1 and ECL2 is broken. In addition, the
number of hot points (i.e. contact points in a residue in the receptor with the YFSPRLamide C-terminal),
reduced significantly in relation to the PBAN-R with chimera 2 showing the greatest reduction. This
agreement with the experimental results is evidence to support the validity of the predicted PBAN-R
model and that the docking solutions are not far from the actual binding conformations of the PBAN
ligand with the PBAN-R. This project has highlighted the usefulness of molecular modeling in
determining ligand-binding domains and in simulating docking of the ligand to this important GPCR
(Stern et al., 2007).
7. Physiological studies - Developmental Regulation
As mentioned previously pheromone production is dependent on the age and maturation of female
moths. In some moth species (Bombyx mori) (Chatani and Ohni, 1976) the induction of vitellogenesis
occurs during pharate development (before eclosion) whereas in other moth species (eg. Helicoverpa
armigera) (Fan et al., 1999a) adult maturity is reached after emergence and so, in this latter species, on
emergence, the female does not possess fully developed eggs. Only on the second night the eggs have
fully developed. In the latter species, the production of JH is induced during the 6th hour after emergence
(Fan et al., 1999a).
The role of JH in the regulation of pheromone biosynthesis has been demonstrated in a few species
of moths that exhibit migratory behavior (Cusson and McNeil, 1989; Gadenne, 1993; Picimbon et al.,
1995) but has been questioned in other moth species. Treatment with fenoxycarb (FX), a JH analog,
induced earlier calling, mating and oviposition by females of A. ipsilon and caused recovery of ovarian
maturation, calling and mating in allatectomized females (Gadenne, 1993). We have previously
demonstrated a clear correlation between the activity of corpora allata (CA), reflecting JH biosynthesis,
and the competence of pheromone glands to respond to PBAN (Fan et al., 1999a) in pharate females of
H. armigera. We have demonstrated that JH triggered pharate pheromone glands to respond to PBAN
and induced the appearance of the PBAN-R protein (Rafaeli et al., 2003). It is feasible that up-regulation
of other, female pheromone-specific proteins, which are required for the biosynthetic pathway in the
production of sex pheromones will also occur. Indeed, the production of several female-typical 35S-
labelled proteins were observed in JH treated female pupae, which were not present in untreated female
pupae (Rafaeli et al., 2003). Thus, it would be of interest to test whether JH also has an influence on the
expression of specific enzymes typically involved in the biosynthetic pathway of moth sex pheromones.
Since JH affects nuclear receptors leading to up-regulation of transcription factors, we can exploit
physiological studies in elucidating JH's role in the up-regulation of the PBAN-R, thus providing another
approach that could lead to the development of a strategy to inhibit the expression of the PBAN-R at
certain times during development. On the other hand, in contrast to its up-regulatory role in pharate
females, JH caused down-regulation in the receptor-protein and pheromone levels of adult females
(Rafaeli and Bober, 2005). These results showed that JH treatment advances the normal age-dependent
program and acts as an “ageing (maturation) hormone”.
140
Mating induces several changes in the behavior of the female: pheromone production and release
(calling) is inhibited (Fan et al., 1999b; 2000; Nagalakshmi et al., 2004) and the levels of JH increase in
the hemolymph due to an allatotropic effect (Fan et al., 1999b). The inhibition of PBAN release is one
of the contributing factors that cause the inhibition of pheromone production (Nagalakshmi et al., 2007).
Most of these changes are attributed to the transfer of seminal peptides by the male from the accessory
glands (Nagalakshmi et al., 2004; 2007). For detailed information in this field readers are encouraged
to consult recent reviews on the subject (Kubli, 2003; Rafaeli, 2005; Ram and Wolfner, 2007)
Table 1: Sequence identities of structurally related moth PBAN-Rs and other insect PRXamide receptors.
Neuropeptide Moths Sequence Accession numbers Reference

Receptor identities (%)
PBAN-R Helicoverpa zea 100 AY319852 Choi et al., 2003

PBAN-R Helicoverpa armigera 98 AY792036 Rafaeli et al., 2007
PBAN-R Heliothis virescens 95 EU000526 Kim et al., 2007
PBAN-R Heliothis virescens 94 EU000525 Kim et al., 2007
PBAN-R Spodoptera littoralis 87 DQ407742 Zheng et al., 2007
PBAN-R Bombyx mori 77 AB181298 Hull et al., 2004
PBAN-R Plutella xyllostella 73 AY974334 Lee and Boo, 2006
Neuropeptide Insects Sequence Accession numbers Reference
Receptor identities (%)
PK-1-R (CG9918) Drosophila elanogaster 75 AF368273 Cazzamali et al., 2005

PK-2-R (CG8784) Drosophila elanogaster 74 AF522189 Rosenkilde et al., 2003
PK-2-R (CG8795) Drosophila elanogaster 70 AF522190 Rosenkilde et al., 2003
DH-1-R Bombyx mori 76 AB164390 Homma et al., 2006
DH-2-R Bombyx mori 66 AB164387 Homma et al., 2006
8. Concluding Remarks
Progress has been made in the understanding of the physiological mechanisms underlying
pheromone biosynthesis but many fundamental questions remain unanswered. We still have little
understanding of the binding domains involved in ligand-receptor interaction; the transcriptional control
of this GPCR and the physiological significance of this GPCR in neural tissues as well as in the male
aedaegus. Gene knockdown techniques are now available and may be utilized in providing answers
concerning the regulation of enzymes involved in the biosynthetic pathway (Ohnishi et al., 2006). Some
encouraging progress has been made with the introduction of computational biology and molecular
modeling studies but clearly other approaches are needed to complement the study. Differences in
functional assays of expressed receptors and endogenous tissues have emphasized the need to validate
functional expression studies. Such studies can impact on the effective design of future biological agents
for insect control. Needless to say, this multidisciplinary approach will require future standardization of
experimental protocols (for example biological assays), which will lead to improved reproducibility,
increased sensitivity and the ability to unify the data obtained from the different approaches.
9. Acknowledgements
The studies conducted in our laboratory were supported by a research fund Grant No. IS-3634-04C
from BARD, the US-Israel Binational Agricultural Research and Development Fund in collaboration
with Prof. Russell Jurenka and Dr. Man-Yeon Choi, Department of Entomology, Iowa State University,
Ames, IA 50011-3222, USA. The molecular modeling studies were performed by Dr. Peter Stern,
Chemical Physics Department, Weizmann Institute of Science, 76100 Rehovot, Israel and Dr. Lian Yu,
Department of Mathematics, Beijing Normal University, Beijing 100875, PR CHINA. We thank Mr. Avi
Azrielli for dedicated care of the insect culture and gas-chromatographic analyses. This is a part of the
Ph.D. Dissertation by R.B. and contribution No.507/08 from the Agricultural Research Organization,
Volcani Center, Bet Dagan, Israel.
141
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NAL B OO
Short
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Views on Insect Molecular Biology, Vol.(1), 2009 Invited Review
T
TERNA
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SIONË
ËIN
 Vol. (1), 147 – 158, 2009
Chapter – 8
Molecular mechanisms of sex pheromone reception in Lepidoptera

Emmanuelle Jacquin-Joly1* and Martine Maïbèche-Coisné2
UMR 1272 INRA-UPMC PISC Physiologie de l’Insecte : Signalisation et Communication,
1
Route de Saint-Cyr, F-78000 Versailles, France, 2 7 Quai Saint Bernard, F-75000 Paris, France.
Abstract
Sex pheromones play a key role in animal mating success and particularly in insects. The largest background on the
molecular mechanisms of their reception in insect antennae has been accumulated in moths, which offer a very sensitive
and specific system to define new concepts in signal recognition. Pheromone reception takes place in morpho-functional
units, the pheromone-sensitive sensilla, and depends on the intervention of proteins from different families involved in
specific steps. A global scheme of events has emerged: first, volatile pheromones are bound by Pheromone-Binding
Proteins to cross the aqueous medium that embeds the pheromone-sensitive neurons. Then, pheromones interact with
specific receptors located in the dendritic membrane of these neurons and the chemical signal is transformed in an electric
signal that will be transmitted to the brain. Signal termination may then be ensured by specific enzymes, the Pheromone-
Degrading Enzymes. Although we still lack a consensus view on the exact function of each protein family, the occurrence
of a diversity of members in each family accounts for their participation in the specificity of pheromone recognition. The
combinatorial expression of these proteins within a sensillum may ensure the specificity, the sensitivity and the dynamic of
the olfactory reception, defining the functional phenotypes of pheromone-sensitive neurons.
Key-words: Olfaction; Pheromone; Pheromone-Binding Protein; Pheromone Receptor; Pheromone-

Degrading Enzyme; Sensory Neuron Membrane Protein; Lepidoptera.
* For Correspondence (email: jacquin@versailles.inra.fr )
1. Introduction
2. The moth antennae and the pheromone reception
More than half a century ago, Karlson and
structures Lüscher (Karlson and Luscher, 1959), while
3. The Pheromone-Binding Proteins (PBPs) and their studying insect responses to invisible signals,
potential role in the first discrimination of pheromone introduced the concept of pheromone as
molecules
4. The Pheromone Receptors (PRs) and their contribution to “substances which are secreted to the outside by
the specific response of ORNs an individual and received by a second individual
(A) PR discovery of the same species, in which they release a
(B) Functional studies
(C) Co-receptors: OR83b orthologs and SNMPs
specific reaction, for example, a definite
5. The Pheromone-Degrading enzymes (PDEs) and their role behaviour or a developmental process”. The
in signal termination same year, the first pheromone, the sex
6. Conclusion pheromone bombykol, was purified and
7. Acknowledgements
8. References identified in a moth, the silkmoth Bombyx mori
(Butenandt et al., 1959).
147
Since then, pheromones have been identified in several thousands of insect species, mainly due to
their potential as attractants or repellents in crop protection strategies. Among the different pheromone
communication systems used by animals, the moth sex-pheromone communication appears as an ideal
system to study the underlying molecular mechanisms and in general to study the principles of olfaction.
Indeed, it offers a very sensitive and simple binary system by which a female when sexually ready emits
a species-specific pheromone blend to attract receptive males. To the specificity of the female message
corresponds a highly sensitive and specific perception system in male. Pheromone perception consists of
both chemical signal reception at the peripheral level, the level of the antennae, and the corresponding
electrical signal integration by brain centres, leading to the appropriate behaviour. Our view of the
mechanisms of pheromone reception was strongly influenced by the discovery of the olfactory receptor
gene super family, first in mammals and a decade later in insects, together with the development of
biochemical, molecular and electrophysiological approaches. In this minireview, after a brief
presentation of the pheromone reception structures carried by moth antennae, we will present recent
advances gained in the understanding of the molecular mechanisms underlying the specificity of sex-
pheromone reception in moth antennae, which may be ensured by the contribution of different protein
families involved in specific steps.
2. The moth antennae and the pheromone reception structures
Insect antennae carry thousands of innervated olfactory structures, the sensilla, each functioning
independently (Fig.1). The sensilla are classified in different morphological types according to their
shape and in different functional types according to the odorants they respond to. In moths, pheromone
detection takes place in sensilla called trichoid sensilla (from the Greek trichos = hair) because of their
long hair shaft (30 to 600 µm). In some species, antennae have a sophisticated shape (such as the
pectinated antennae of B. mori, Fig.1A) that increases their surface to accommodate a larger number of
pheromone-sensitive sensilla. Male moths often have enlarged antennae with numerous pheromone-
sensitive sensilla. In contrast, most female moths are unable to smell their pheromone and their antennae
are usually narrower and carry less sensilla. Like other olfactory sensilla, trichoid sensilla are perforated
by numerous tiny pores that allow the entrance of pheromone molecules (Fig.1B). These sensilla usually
house one or two olfactory receptor neurons (ORNs). These ORNs are bipolar cells that ensure the
transformation of the chemical message into an electrical message that will be transmitted to the brain.
ORN cell bodies are enclosed in enveloping accessory cells (Fig. 1B): the thecogen cell, the trichogen
cell, and the tormogen cell (Steinbrecht, 1997). The ORN dendrites extend in the sensillum lumen filled
with the sensillum lymph whose ionic and proteinic composition is regulated by the accessory cells. The
ORN axons group together in antennal nerves that enter the primary processing centre in the central
nervous system, the antennal lobes, where they synapse with central neurons in glomerular structures.
Axons of pheromone-sensitive neurons project to a sexually dimorphic macroglomerular complex.
Pheromone information is relayed via antennal lobe projection neurons to both the mushroom bodies
and the lateral protocerebrum.
Due to the compartmentalisation of the insect peripheral olfactory system into different kinds of
sensilla, distinct pheromone-sensitive neurons can then be surrounded by different molecular
environments that could interfere with their reception properties. Deeply studied by electrophysiology,
pheromone-sensitive neurons usually respond to a single pheromone component, rarely to two (Renou
and Lucas, 1994). This situation is clearly different from the responses of “generalist” ORNs that are
usually activated by a family of related chemicals (Hallem et al., 2004). The following paragraphs will
give the current views of the underlying molecular mechanisms that lead to such as specific response.
148
A B
0.5 cm
10 µm
Fig.1. Organization of moth antennae. A : A pair of pectinated antennae on the head of a male silkmoth,
Bombyx mori (Photography: E. Jacquin-Joly). The antennal branches are carrying numerous cuticular
expansions, the sensilla. B: detailed organization of an olfactory sensillum. The sensillum houses
olfactory receptor neurons (ORNs) whose dendrites expend in the sensillum lymph and whose axons
converge to the brain. The ORN cell bodies are enclosed in enveloping accessory cells.
3. The Pheromone-Binding Proteins (PBPs) and their potential role in the first discrimination of
pheromone molecules
Crossing the aqueous sensillum lymph in order to interact with the receptors located in the ORN
dendritic membrane is a challenge for hydrophobic pheromone molecules. Specific classes of proteins
able to bind odorant molecules, named Odorant-Binding Proteins (OBPs), were found in high
concentration in the insect sensillum lymph (up to 10 µM, (Klein, 1987), where they are supposed to
solubilise and transport the chemicals to the receptors. Among OBPs, Pheromone-Binding Proteins
(PBPs) were proposed to bind pheromones (Fig.2). The first PBP was indeed discovered in the moth
Antheraea polyphemus using a tritium-labelled pheromone as a probe (Vogt and Riddiford, 1981). Since
then, OBPs and PBPs have been found in a great number of insect species from various orders (review in
Vogt, 2003; Pelosi et al., 2006). PBPs are small extracellular proteins (15 to 16 kDa) with an acidic
isoelectric point and 3D-structures revealed several α-helices stabilized by 3 disulfide bridges (Sandler
et al., 2000; Mohanty et al., 2004). These helices contribute to form a more or less hydrophobic cavity
where the ligand is bound. However, the exact function of PBPs is still intensely debated. Their potential
role include the solubilisation, the concentration, the removal or inactivation of pheromone (review in
Kaissling, 2004). It has been also proposed that PBPs could participate in the first step of pheromone
discrimination by specific binding with pheromonal molecules (reviewed in Leal, 2003). This hypothesis
is supported by the observation that, in most moth species, the pheromone signal consists of a blend of
several molecules and, in accordance, several PBPs (usually 3 in moths), have been identified within a
species, some of them being even co-expressed within the same sensilla (Jacquin-Joly et al., 2000;
Forstner et al., 2006). However, this function as a first filter is still under debate, as illustrated below for
the best characterized PBP, the BmorPBP1 from B. mori. This PBP is enriched in male antennae
(Krieger et al., 1996) where it is expressed in long trichoid sensilla (Steinbrecht et al., 1992; Maida et
al., 1993) that housed two ORNs, one sensitive to the pheromone bombykol and the other to its oxidized
form bombykal (Kaissing et al., 1978). This PBP clearly binds bombykol, as demonstrated in vitro
(Wojtasek and Leal, 1999a). In addition, when the B. mori bombykol receptor BmorOR1 (see III) is
149
heterologously expressed in Drosophila melanogaster antennae, its response to bombykol is greatly

enhanced when co-expressed with BmorPBP1 (Syed et al., 2006). Recombinant BmorPBP1 has been
produced, and its structure resolved by both X ray (Sandler et al., 2000) and NMR (Horst et al., 2001)
approaches, leading to a complete scheme of bombykol/PBP interaction (Wojtasek and Leal, 1999a).
However, its specific binding with bombykol is still under debate. In one hand, using in vitro
competitive binding assays, Gräter et al. (2006) showed that both bombykol and bombykal were bound
by BmorPBP1 with nearly the same high affinity. This PBP may then ensure the unspecific travelling of
both bombykol and bombykal within the same sensillum. On the other hand, other studies suggest that
BmorPBP1 could preferentially bind to bombykol. When applied directly to the receptor neurons via tip-
opened sensilla during electrophysiological recordings, pheromone components have to be solubilized,
either by DMSO or by a PBP. In such an in vivo study, bombykal in combination with BmorPBP1 failed
to activate the bombykal-responding neurons, whereas bombykol in combination with BmorPBP1
elicited significantly stronger responses of the bombykol-responding neurons than bombykol combined
with any other solubilizers (Pophof, 2002, 2004). In addition, in stable HEK cell lines expressing
BmorOR1, BmorPBP1 has been shown to selectively mediate a response to bombykol but not to
bombykal, whereas BmorOR1 expressed alone responds to both bombykol and bombykal when these
compounds are dissolved by DMSO (Grosse-Wilde et al., 2006). These results strongly support a
BmorPBP1-filter function through a preferential binding with bombykol over bombykal, as recently
supported by computational analyses (Charlier et al., 2008). Thus, another still unidentified PBP might
ensure the bombykal transport in B. mori pheromone-sensitive sensilla. In a similar manner, the
pheromone receptor HvirOR13 from Heliothis virescens (see III) responded to several DMSO-dissolved
pheromonal components when expressed in HEK cells, indicating a limited specificity of this receptor.
However, when DMSO is replaced by a PBP, HvirPBP2, cells expressing HvirOR13 specifically
responded to the main component of the sex pheromone blend (Grosse-Wilde et al., 2007). Altogether,
these data provide evidence that a combination of distinct receptor and PBP underlies the specific
response observed in the detection of a pheromone component in vivo. However, it has to be noticed that
in other experiments, using in vivo expression in D. melanogaster antennae (Syed et al., 2006) or in vitro
expression in Sf9 cell (Kiely et al., 2007), the functional properties of a given receptor resemble that of
the natural receptor although the natural PBP/OBP repertoire is not present.
As an additional role, it has been proposed that moth PBPs may also contribute to the activation of
ORNs (Pophof, 2002, 2004). Rather than the pheromone alone, the complex PBP-pheromone could
dock with the receptor. Interestingly, a very recent study demonstrated that it is the case in
D. melanogaster. LUSH, a D. melanogaster OBP involved in the binding of the aggregation pheromone
11-cis-vaccenyl acetate (CVA) encounters a conformational change while binding with CVA. This
LUSH/CVA conformation would be the active form that interacts with the receptor (Laughlin et al.,
2008). Thus, more than 20 years after the first discovery of PBPs, their exact contribution to the specific
response of moth pheromone-sensitive ORNs still await further investigations.
4. The Pheromone Receptors (PRs) and their contribution to the specific response of ORNs
(A). PR discovery
Since moth pheromones consist of volatile components, pheromone receptors (PRs) belong to the
family of insect olfactory receptors (ORs). A great amount of insect OR sequences are now available,
thanks to rapid progress in insect genome sequencing and analyses, leading to functional studies and
high throughput de-orphanization. Much less is known on PRs and only a few were recently identified
among ORs, using the following criteria: 1) candidate moth PRs should be expressed only (or
preferentially) in male antennae, 2) they should be more conserved than other ORs (20% identities in
average), since pheromone detection must be under different kinds of selective pressure than detection of
food or host odours (Krieger et al., 2004), 3) their expressing cells should be surrounded by PBP-
expressing cells. Among different OR sequences retrieved from genome analyses of the noctuid
Heliothis virescens, Krieger et al. (2004) were able to identify a small group of 4 receptors sharing more
than 40% identity, expressed exclusively in the antennae of male moths, in cells located beneath
150
pheromone-sensitive sensilla and surrounded by PBP-expressing cells. In B. mori, a differential

screening strategy associated with the analyses of the available genome led to the identification of 6
potential PRs expressed specifically or predominantly in male antennae (Sakurai et al., 2004; Nakagawa
et al., 2005). Interestingly, among the B. mori OR repertoire, 2 ORs were found to be widely enriched in
the female antennae (Wanner et al., 2007), leading to the hypothesis that they may detect odours critical
to female behaviour, such as possible male-produced courtship pheromones (Anderson et al., 2009).
Very recently, based on the observation that moth PRs exhibit around 40% identities, homology cloning-
based strategies led to successful identification of additional PRs in species for which no genomic data
are available (Mitsuno et al., 2008, Jacquin-Joly, personal data). All together, these PR sequences form a
distinct sub-family within the insect OR family. Like ORs, they present a seven-transmembrane domain
(7TM) structure (Fig.2) and topology predictions let suggest that they present an inverted orientation
within the membrane compared to classical 7TM receptors, experimentally confirmed for some insect
ORs (Benton et al., 2006, Lundin et al., 2007; Jordan et al., 2009). Their expression patterns follow the
same rule as for ORs, that is one ORN expresses only one type of PR. For instance, two of the identified
B. mori PRs (BmorOR1 and BmorOR3) are mutually exclusively expressed in a pair of adjacent
pheromone-sensitive neurons located in the same sensilla, one specifically tuned to bombykol and the
other to bombykal (Nakagawa et al., 2005).
(B). Functional studies
Only a few PRs have been functionally characterized, using heterologous in vitro expression
system such as HEK, Sf9 cell lines (both coupled to calcium imaging), Xenopus oocytes (coupled to
voltage-clamp recordings) and in vivo expression in D. melanogaster antennae, either in the “empty
neuron” (Hallem et al., 2004) or the T1 sensilla (Kurtovic et al., 2007), coupled to electrophysiology.
Pheromonal ligands have been assigned to HvirOR13 (Grosse-Wilde et al., 2007; Kurtovic et al., 2007),
BmorOR1 and BmorOR3, (Sakurai et al., 2004; Nagakawa et al., 2005; Syed et al., 2006; Kurtovic et
al., 2007) and to 3 PRs identified by homology cloning (Mitsuno et al., 2008). The general observation
is that PRs appeared to be very selective compared to insect “general” ORs that usually recognize a
variety of ligands. However, this notion has been challenged by the discovery that the presence of PBPs
can affect the response profile of PRs expressed in HEK cells, as demonstrated for BmorOR1 and
HvirHR13 (Grosse-Wilde et al., 2006, 2007, see II).
(C). Co-receptors: OR83b orthologs and SNMPs
In situ, PRs appeared to be co-expressed (Mitsuno et al., 2008) with an atypical OR that belongs
to a subfamily of ORs unusually highly conserved across insect orders (Krieger et al., 2003), the
D. melanogaster OR83b ortholog family. This subfamily consists of receptors expressed in many (if not
all) ORNs in addition to the specific OR typically expressed in each neuron. In D. melanogaster, OR83b
is necessary for OR addressing to the neuron membrane and it acts as a dimerization partner for OR
function (Larsson et al., 2004; Neuhaus et al., 2005; Benton et al., 2006) (Fig. 2). Interestingly, a
D. melanogaster OR83b mutant can be rescued by orthologs from different insect orders, including
Lepidoptera (Jones et al., 2005), suggesting that the OR83b function has been conserved throughout
evolution. As a proof, co-expression of the B. mori OR83b ortholog with BmorOR1 in Xenopus oocytes
increased the percentage of bombykol-responsive oocytes from 10-15% to over 95% (Nakagawa et al.,
2005) suggesting that moth PRs could require an OR83b ortholog to be fully functional in vivo (Fig.2).
Although it is now admit that insect ORs function as heterodimers with an OR83b ortholog, the way they
transduce the signal has been recently challenged by quite contradictory studies. Insect ORs have been
for long proposed to interact with a G protein (as vertebrate ORs), activating upon ligand binding an IP3
transduction pathway and leading to ion channel opening and neuron depolarization. Such a pathway has
been recently confirmed by in vivo studies of several D. melanogaster mutants lacking key elements of
the IP3 pathway (Kain et al., 2008) that showed reduced odor responses. On the contrary, Sato et al.
(2008) recently demonstrated in vitro that some insect OR heteromeric complexes, including the one
formed by BmorOR1 and the B. mori OR83b ortholog, function directly as ligand-activated non-
selective cation channels. Another study from Wicher et al. (2008) proposed that insect ORs could form
151
ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated

non-selective cation channels. Thereby, ORs could provide a rapid and transient as well as a sensitive
and prolonged odorant signalling via both an ionotropic and a metabotropic pathway.
In addition to this complex scheme, other co-receptor proteins, the Sensory Neuron Membrane
Proteins (SNMPs), are also thought to play a role in pheromone detection (Fig.2). First identified in
pheromone-sensitive ORNs of A. polyphemus (ApolSNMP1) (Rogers et al., 1997), SNMP homologs
have been found in other insect species (Rogers et al., 2001, Nichols and Vogt, 2008, Forstner et al.,
2008). These membrane bound proteins are homologous to the vertebrate CD36 receptors (which mainly
recognize proteinaceous ligands, cholesterol and fatty acids), and can be classified in three groups
(Nichols and Vogt, 2008). The third group includes two subgroups referred to as SNMP1 and SNMP2
expressed in association with chemosensory organs. In moths, they have been proposed to be involved in
the recognition of lipophilic pheromone components (Rogers et al., 2001; Vogt, 2003). In H. virescens,
these two SNMPs are differentially expressed in cells of pheromone-sensitive sensilla (Forstner et al.,
2008): SNMP1 appeared to be co-expressed in the same cell with the pheromone receptor HvirOR13,
whereas SNMP2 appeared to be expressed in supporting cells, suggesting distinct functions for the 2
SNMP subtypes. The role of SNMP in pheromone detection has been demonstrated using a D.
melanogaster SNMP-null mutant (Benton et al., 2007). In D. melanogaster, one SNMP is expressed in a
population of ORNs implicated in pheromone detection. Electrophysiological studies of a SNMP-mutant
revealed that this SNMP is essential for the responses of ORNs expressing the CVA receptor OR67d.
SNMP, however, is not necessary for the responses of the conventional odorant receptor OR22a to its
short hydrocarbon fruit ester ligands. Interestingly, when HvirOR13 is ectopically expressed in D.
melanogaster OR67d-expressing neurons, its activation by H. virescens pheromone ligand is dependant
on the presence of SNMP (Benton et al., 2007). Thus, SNMP function seems to be conserved and
specific to pheromone reception. It has been proposed that SNMP may acts in concert with PRs to
capture pheromone molecules on the surface of olfactory dendrites (Benton et al., 2007).
5. The Pheromone-Degrading enzymes (PDEs) and their role in signal termination
After pheromone/PR interaction, an important step in sensory detection is the cessation of

signalling. Indeed, pheromone molecules need to be removed from the immediate vicinity of PRs so that
fresh plume of sex pheromone can be detected. This function is supposed to be carried out by Odorant-
Degrading Enzymes (ODEs), called Pheromone-Degrading Enzymes (PDEs) in the case of pheromonal
substrates (Vogt and Riddiford, 1981) (Fig.2).
Different enzymatic activities have been characterized in the past in the antennae of various
lepidopteran species. Several antennal esterases were first characterized in A. polyphemus, and in
particular, a male specific sensillar enzyme has been studied in great detail (Vogt and Riddiford, 1981;
Vogt et al., 1985; Klein, 1987). This sensillar esterase was shown to have a good affinity for the
pheromone and the pheromone half life in vivo was estimated at 15ms in presence of this enzyme (Vogt
et al., 1985). Beside esterases, other enzymatic activities were identified in moths such as aldehyde-
oxidases/dehydrogenases (Rybczynski et al., 1989, 1990; Tasayco and Prestwich, 1990a, 1990b, 1990c),
alcohol-oxidases/dehydrogenases (Kasang et al., 1989), epoxide-hydrolase (Prestwich et al., 1989). Only
few candidate enzymes have been molecularly identified in insects, mostly in Lepidoptera. Several
esterases have been cloned from the antennae of A. polyphemus (Ishida and Leal, 2002, 2005), Mamestra
brassicae (Maïbèche-Coisné et al., 2004a), Spodoptera littoralis and Sesamia nonagrioides (Merlin et
al., 2007). Aldehyde oxidase genes have been identified in M. brassicae (Merlin et al., 2005) and in
B. mori (Pelletier et al., 2007). Some of these genes are specifically expressed in the antennae, relevant
with a function in olfaction, and in particular, the esterases from noctuids and one aldehyde oxidase from
B. mori are clearly expressed at the base of pheromone-sensitive sensilla, suggesting a function as PDE
(Maïbeche-Coisné et al., 2004a; Pelletier et al., 2007).
152
Pheromone
PBP
PDE
SNMP OR83b Sensillum lymph

Dendritic
membrane
ORN cytoplasm
PR
Fig.2 Hypothetical view of the different steps of pheromone reception in a moth pheromone-sensitive
sensillum. After entering the sensillum lymph through cuticular pores, pheromone molecules are
supposed to be carried by Pheromone-Binding Proteins (PBPs) to cross the lymph. Then, they reach the
ORN dendritic membrane where either the pheromone alone or the complex pheromone/PBP interacts
with a heteromeric receptor formed by a pheromone receptor (PR) and its partner OR83b. The
interaction with the receptor may be mediated by the intervention of another membrane protein, the
Sensory Neuron Membrane Protein (SNMP). After receptor activation, the pheromone molecules are
supposed to be degradated by Pheromone-Degrading Enzymes (PDEs).
The correlation between the catabolic properties of the antennal enzymes and the chemical nature
of the sex pheromone components has led to the hypothesis that insects possess specific PDEs able to
efficiently degrade incoming conspecific sex pheromone components. However, to play a significant
role, enzymatic inactivation of pheromones should be extremely fast. Indeed, while flying to a sex
pheromone source, male moths encounter small pockets of pheromone molecules separated by clean air
spaces and it was shown that they can react to the loss of the odorant trail in less than 0.5 second (Baker
and Vogt, 1988). This means that they are able to reset their sensory system on a millisecond timescale.
The few functional data available in vitro on native ODEs are controversial. Indeed, some authors using
antennal extracts have calculated the half life of moth pheromone to be approximately 4 min (Kasang et
al., 1989) whereas others using moth sensillar preparations have found only few millisecond half-life
(Vogt et al., 1985; Rybczynski et al., 1989). Interestingly, two recent in vitro studies on recombinant
antennal esterases from A. polyphemus and Popillia japonica, a beetle, demonstrated that each
recombinant PDE degrades the respective pheromone of the corresponding species with an estimated
half-life of milliseconds (Ishida and Leal 2005, 2008). These recent kinetic data strongly suggest that
enzymatic inactivation could play a significant role in the dynamic of signal termination. However, the
role of these enzymes in the dynamic of pheromone signal termination in vivo has not been demonstrated
yet and their involvement in pheromone-guided behaviour is still unknown.
153
In addition to extracellular enzymes supposed to break down chemicals near PRs, intracellular
enzymes have been found in insect chemosensory appendages, such as cytochrome P450, UDP-
glucosyltransferases (UGT) or glutathione-S-transferases (GST) (Rogers et al., 1999; Wang et al., 1999;
Wojtasek and Leal, 1999b; Vogt, 2005). Well studied in mammals, these enzymes are known to always
act in two sequential steps: the products of P450 activity (phase 1) are metabolized by UGTs or GSTs
(phase 2). Their combined action results in the production of hydrophilic metabolites that can be easily
excreted. Their supposed function is to protect the sensory neurons, which are directly exposed to the
external environment, against volatile xenobiotics (Heydel et al., 2001; Lazard et al., 1990; Minn et al.,
2005; Zhang et al., 2005). A P450 specific to the male antennae has been characterized as a potential
PDE in the scarab beetle Phyllopertha diversa (Wojtasek and Leal, 1999b; Maïbèche-Coisné et al.,
2004b). In moths, several antennal P450s have been identified, among them the first insect olfactory
specific P450 (Maïbèche-Coisné et al., 2002). However, no functional data are yet available in moths on
these enzymes.
From all these data, one can postulate that sequential enzymatic steps occur in the chemosensory
sensilla: an extracellular step in the vicinity of the receptors that allows to quickly metabolize
pheromones into chemical forms that can no more activate sensory neurons, as suggested for moth
antennal esterases, followed by several intracellular steps leading to the final excretion.
6. Conclusion
As illustrated in this review, pheromone recognition in moth antennae relay on the intervention of
different protein families. The different properties of members of each family together with their
combinatorial expression in different functional types of pheromone-sensitive sensilla may contribute to
the specific ORN responses. However, despite numerous studies, the exact contribution of each family is
still intensively debated. Anyway, the elucidation of pheromone reception mechanisms has already
defined new concepts in signalling research and will continue to do so. Also, it opens new perspectives
for insect control, each of the PBP, PR, SNMP and PDE families offering relevant specific targets to
perturb one of the most efficient insect behaviours.
7. Acknowledgements
We would like to thank our former and current PhD students (Christine Merlin, Julien Pelletier,
Isabelle Brigaud, Nicolas Durand), post-doctoral fellow (Sébastien Malpel), technicians (Marie-Christine
François, Françoise Bozzolan) and scientists at UMR PISC (Versailles-Paris, France) that contributed to
many of the cited references. Our work on moth pheromone reception at UMR PISC is supported by
INRA, University Paris VI (BQR 2005), Fonds National de la Science (ACI JC5249), Génoscope (CNS,
Evry, France) and ANR “Neurosciences” (ANR-07-NEUR-037-01).
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NAL B OO
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T
Short Views on Insect Molecular
Short Views Biology,
on Insect Vol.(1),Biology
Molecular 2009 Research
ResearchArticle
Article
TERNA
MIS
SIONË
ËIN
 Vol. (1), 159 – 173, 2009
Chapter – 9
Autophagic programmed cell death in the peripheral fat body

tissues of the silkworm, Bombyx mori L.
Prosothaman Sumithra#, Raman Chandrasekar # and Muthukalingan Krishnan*#
* Biomedical Medical Diagnostic Laboratory, Department of Environmental
Biotechnology, Bharathidasan University, Tiruchirappalli – 620 024, Tamilnadu, India.
#
Insect Molecular Biology Laboratory, Department of Environmental Biotechnology,
School of Environmental Sciences, Bharathidasan University, Tiruchirappalli- 620 024,
Tamilnadu, India.
Abstract
Programmed cell death (PCD) generally occurs within a developmental context in response to physiological stimuli such
as hormones and is dependent on de novo gene expression. Despite the ubiquity of this phenomenon, little is known
about what tells a cell to die, and less still about the physiological and molecular mechanisms that bring about death. One
system that has proven to be very amenable for the study of PCD is the larval peripheral fat body (PF) tissues of the
silkworm, Bombyx mori. Here we assess the two major pathways of PCD via apoptosis and autophagic cell death both by
morphological and molecular methods in the disintegrated larval PF tissues during the larval – pupal transformation.
Larval PF tissue began to disintegrate by the process of PCD on day of spinning (S0) and its structural integrity was
completely lost by day one of pupa (P1). PCD in PF was well characterized by chromatin condensation by acridine orange
staining, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and
intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies by morphological
examination and DNA fragmentation by agarose gel electrophoresis. Western blot analysis of proteasomal subunits
proved that the involvement of proteolytic activity during the PCD of peripheral tissues. Lysosomal participation during
the PCD of PF tissues was investigated by elevated level of marker enzyme acid phosphatase distinctly on the day 1 of
pupal period. Further to investigate the effective role of 20-hydroxyecdysone (20E) on these events, we cultured the larval
PF tissues with or without 20E and analyzed by chromatin condensation via acridine orange staining and DNA laddering
studies.
Key Words : Bombyx mori; peripheral fat body; PCD; 20E; 26S Proteasome; Acid phosphatase
* For correspondence (email: krish14156@yahoo.com)
Overview 3. Result
(a) Chormatin Condensation
1. Introduction
(b) DNA laddering studies
2. Materials and Methods
(c) Lysosomal Acid Phosphatase – a marker enzyme
(a) Experimental animals
(d) 26S & 20S Proteasome
(b) Detection of chromatin condensation
(e) Morphological Examination
(c) Tissue preparation for Electron Microscopy
(f) Role of 20E on PCD
(d) DNA extraction and Electrophoresis
4. Discussion
(e) Gel Electrophoresis and Immunoblotting
5. Acknowledgement
(f) ACP assay
6. Reference
(g) In vitro culture of fat body tissues
159
Short Views on Insect Molecular Biology, Vol.(1), 2009 Research Article
1. Introduction
In contrast to vertebrate cells where time’s arrow tends to lead irreversibly to differentiation, death
and whole-tissue regeneration, insect cells follow a path of make do and mend, with cells regenerating
and changing their function through life. Some cells are irreplaceable, some cells complete their
functions and are then sacrificed, and some cells live a finite lifetime to be replaced by yet another
generation by the process of PCD. Genetic dissection of cell death programs has identified as conserved
elements of core cell-death pathway, as well as an understanding of how these proteins bring about the
destruction of a cell (Buszczak and Segraves 2000). Cells use different pathways for active self-
destruction as reflected by different morphology: while in apoptosis (or type I) nuclear fragmentation
associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic
degradation of cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of
PCD (Bursch, 2004). The most extensively studied category, apoptosis (type I) is characterized by cell
rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation and fragmentation,
nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-bound apoptotic
bodies that are rapidly digested by macrophages or neighboring cells (Kerr et al., 1972; Wyllie et al.,
1980; Clarke 1990; Takayama et al., 2003; Boyce et al., 2004; Danial and Korsmeyer 2004; Festjens et
al., 2004; Lorenzo and Susin 2004; Chandrasekar et al., 2008a & 2008b). Rather, cells may use different
pathways to commit suicide, including autophagic PCD or type II cell death. Its main features are the
appearance of abundant autophagic vacuoles in the cytoplasm, which is accompanied by mitochondrial
dilation and enlargement of the ER and the Golgi apparatus. However, apoptosis and autophagic PCD
are not mutually exclusive phenomena. PCD generally takes place in four steps: the decision to die,
death, engulfment and degeneration (Steller 1995). Indeed, Zakeri et al. (1995) detailed non-apoptotic
types of PCD with diverging molecular symptoms such as a lack of endonuclease activation and changes
in sub-cellular hydrolase traffic, and Bowen et al. (1998) described a number of examples of non-
apoptotic vacuolar PCD in a range of invertebrates including insects. More recently, a number of
reviews covering morphological and functional features of autophagic PCD throughout the living world
were published (Clarke 1990; Zakeri et al., 1995; Bursch 2001; Bursch et al., 2004; Chandrasekar et al.,
2008a). The genetic and molecular background of the type II (autophagic) mechanism of PCD is not
well known, although, the morphological and biochemical features of autophagocytosis are characterized
in detail. Cells undergoing autophagic PCD the first changes visible at the electron microscope level
comprise formation of autophagic vacuoles (AV), which gradually degrade cytoplasmic structures.
These observations with dying cells meet well with the current concept of macroautophagy (herein
referred to as autophagy), namely that it ensues through a sequence of events highly conserved from
yeast to humans including : the sequestration of cytoplasmic constituents or certain organelles in a
random manner by so called isolatory membranes and the autophagosomes thus formed, subsequently
fuse with lysosomes forming autophagic vacuoles (AV), inside which the macromolecules of the
engulfed organelles are digested at acidic pH by the lysosomal enzymes (Klionsky and Emr 2000;
Reggiori and Klionskz 2002; Bursch 2004; Chandrasekar 2006; Chandrasekar et al., 2008a).
Ecdysteroids play a central role in the cell death of numerous in insect tissues (Chinzei 1975;
Truman and Schwartz 1984, Dai and Gilbert 1999; Terashima et al., 2000; Tsuzuki et al., 2001; Halaby
et al., 2003). During metamorphosis, ecdysteroids display different effects on cell death, i.e., either an
increasing or decreasing ecdysteroid titer can elicit PCD, depending on the developmental stage (Truman
and Schwartz 1984; Kimura and Truman 1990; Truman et al., 1992). Evidence is now mounting that
intracellular proteolysis is involved in PCD (Martin and Green, 1995). One of the major difficulties in
examining the biochemical pathways that regulate this process is that in most cases, condemned cells are
interspersed among healthy ones, making the observed biochemical changes difficult to interpret.
Ubiquitin – mediated proteolysis is required not only for protein turnover during homeostasis and stress,
but also during the PCD of some cells (Muller and Schwartz 1995). In the muscle cells of Manduca sexta
committed to die an increase in the expression of polyubiquitin was described which has been shown as
an essential mediator of proteolysis (Myer and Schwartz 1996). Ubiquitin functions as a post-
translational adduct to proteins, where its best characterized role is to target proteins for proteolysis via
an ATP/ubiquitin-dependent proteinase, which contains the multicatalytic proteinase (MCP; proteasome)
160
as an integral component. This complex degrades the target substrate and returns intact ubiquitin protein
to the cytoplasm (Rechsteiner et al., 1993).
The autophagic degradation of larval organs during metamorphosis can be regarded as a form of
PCD. The developmental process of different larval tissues during metamorphic transformation showed
that tissues such as the silk gland and gut are completely histolyzed (Shiba et al., 2001; Lee et al., 2002;
Uhlirova et al., 2003), while other tissues such as fat body undergo reorganization with histolysis (Rizki
1978; Rabossi et al., 2004) and predetermined imaginal tissues differentiate and grow into organs and
external structures (Fristrom and Chihara 1978; Uhlirova et al., 2003). Typically, PCD occurs in a
temporal window of opportunity following a molt. The intersegmental muscles that facilitate adult
eclosion degenerate in the first few days of adult life (Lockshin and Williams 1965; Schwartz and
Truman 1982; Schwartz, 1992). The motoneurons innervating these muscles undergo PCD concurrently
(Truman 1983). Similarly, the prothoracic gland degenerates following production of the ecdysteroids
that will stimulate adult differentiation (Ozeki 1966; Dai and Gilbert 1997).
Fat body in insects is one of the eukaryotic systems which epitomizes the complexity of gene
control as displays an orchestral expression of genes in response to intra and extra molecular cues. Our
previous studies on B. mori (Vanishree 1998) and Amsacta albistriga (Chandrasekar 2006;
Chandrasekar, et al., 2008a, 2008b) reported that PF tissues located near to the cuticle region reaches
high abundance during mid-larval period and begins to be disintegrated during the larval – pupal
transformation while the ecdysone titer is high. This protrudes the amenable way at first time to
investigate the molecular studies on PCD in the larval PF tissues of the silkworm, B. mori, which has
spawned and provided an opportunity to address a number of intriguing questions surrounding apoptosis
in insects.
a. Experimental animals
Eggs of a hybrid of a multivoltine strain of B. mori (local Tamil Nadu White) and a bivoltine
strain, NB2D4, were purchased from the local Government Grainage, Tiruchirappalli, India, and
maintained at a temperature of 27±2˚C and 75±5% relative humidity. Larvae that hatched out were fed
with freshly chopped, tender leaves of mulberry, MR2 variety, until the third instar and were given
coarse leaves until the end of the fifth stadium. After the fourth molt, larvae were sexed and those
weighing 0.8g were selected for the experiments as described previsouly by Chandrasekar et al. (2007).
The life span of the silkworms under these conditions is 30-32 days, with the final fifth stadium, lasting
for 6 days. Fifth stadium larvae cease feeding to begin cocoon spinning on day 7 and the larval – pupal
ecdysis occurs on day 8. For convenience, the day of spinning was termed by S0 and the unsclerotized
soft pupal stage day P0. The pre-pupal period lasts for 3 days (S0-S2) and the pupal period for 7 days (P1-
P7).
b. Detection of chromatin condensation
Acridine orange (Sigma-Aldrich) stain that is commonly used to reveal the condensed chromatin
that is characteristic of apoptosis (Hopwood et al., 2001). Using this method of detection, when viewed
under appropriate filters, apoptotic cells are highly fluorescent making them easily identifiable from non-
apoptotic cells. The larvae were chilled on ice for 2-3 min before dissection in insect ringer solution.
The larval PF tissues were collected throughout the V instar, spinning stage and the pupal stage, placed
on a glass slide and tapped gently with a brass rod to disperse the tissue. Acridine orange (50µM in
insect ringer solution) stain was added and the tissue was viewed immediately under a fluorescence
microscope.
161
c. Tissue preparation for Electron Microscopy
According to Muller et al. (2004) insects were inflated with 4% paraformaldehyde, 0.5%
glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for 5 min at room temperature. Larval PF lobes (S2,
P0, P1) were dissected fixed under the fixative solutions for 1 h at 4˚C. After rinsing with 0.1M
cacodylate buffer, the fat body pieces were post-fixed for 1 h in 0.5% osmium tetroxide. After
dehydration in a graded series of ethanol (50% ethanol, 15 min; 70% ethanol, 25 min; 90% ethanol, 30
min; 100% ethanol two times each for 30 min) and then in propylene dioxide (10 min) at room
temperature, the tissue was embedded in Araldite (Araldite: Propyele oxide, 1:1, overnight). The
specimens were thermally cured at 50˚C for two days. Thin sections of 60nm-90nm were cut with a
diamond knife. Sections were picked in copper grids and stained with uranyl acetate for 8 min, lead
citrate for 2 min and then blotted dry. Preparations were analyzed with the Philips Electron Microscope
(Philips 410 LS, Austria) at an acceleration voltage of 75 kV.
d. DNA extraction and Electrophoresis
Genomic DNA was extracted from larval PF tissues throughout the V instar larval, spinning and
pupal stages by adopting the Sambrook et al. (1989) methodology with slight modification. PF tissues of
B. mori were collected and homogenized with 400 - 800µl of TEN 9 buffer (50mM TrisCl, 100mM
EDTA, 200mM NaCl) and the homogenate was centrifuged at 12000 rpm at 4˚C for 10-15 min. Then,
equal volume of 20% SDS was added and the tubes were inverted few times and incubated at room
temperature for 30 min. The lysate was treated with 5µl proteinase K (10mg/ml) for 4 h at 60˚C. At end
of the incubation, phenol and chloroform in the ratio of 1:1 was added and the tubes were inverted
several times and refrigerated for 15 -30 min. Then, the tubes were centrifuged at 15,000 rpm at 4˚C for
10-15 min and the aqueous phase was collected. To this added equal volume of 0.3M sodium acetate
and 3 volume of 70% ice cold ethanol. The tubes were inverted gently and kept at -20˚C over night.
The tubes were centrifuges at 15,000 rpm for 15 min at 4˚C and the pellet was washed with 70% ice cold
ethanol. Dried pellets were dissolved in 200µl of TE buffer (10mM Tris HCl, 1mM EDTA). To this 5µl
of DNase-free RNase (2mg/ml) was added to the samples and digestion proceeded for 30 min at 37˚C.
After washing with 70% ethanol the pellet were dissolved in 50µl of TE buffer and stored at -85˚C.
DNA was subjected to electrophoresis in 1.2% agarose gel and was visualized by ethidium bromide
staining.
e. Gel Electrophoresis and Immunoblotting
PF tissues were homogenized in ice cold 20mM Tris HCl, 50mM NaCl, pH 7.5 and centrifuged at
12,000 rpm at 4˚C for 15 min. The supernatant was mixed with loading buffer and boiled for 5 min.
20µg of total protein was subjected to electrophoresis on 12% sodium dodecyl sulphate-polyacrylamide
gels. Proteins were transferred to nitrocellulose paper for 30 min at 30 V according to the procedure of
Towbin et al. (1979). Following transfer, membranes were blocked in transfer buffer saline (20mM Tris-
HCl, 0.5M NaCl, pH 7.5) containing 3% non-fat dried milk for 1 h, at 20˚C. The blocked membranes
were then incubated in the presence of rabbit anti- ATPase subunit (47kDa) and 20S – regulatory subunit
(29kDA) primary antibodies in TTBS (20mM Tris-HCl, 0.5M NaCl, 0.05% Tween 20, pH 7.5)
containing 2% non fat dried milk overnight at 4˚C. Antibody binding was visualized by 4-chloro-1-
naphthol/ hydrogen peroxide liquid chromogenic substrate using anti-rabbit secondary antibodies labeled
with hydrogen peroxidase .
f. ACP assay
The enzyme assay was carried out according to the method of Henrickson and Clever (1972). The
reaction mixture contained 150mM sodium acetate buffer (pH 5.0) and 20µg of tissue homogenate
proteins. It was incubated at 37˚C for 10 min to exclude glucose-6-phosphatase activity (Beaufay et al.,
1954). The reaction was initiated by the addition of 5µmol of substrate, p-nitrophenyl bisodium
phosphate (Sigma) to the assay mixture followed by incubation for 1 h at 37˚C. The reaction was
162
terminated by the addition of 0.5ml of 0.1N NaOH, and the color developed was measured at 420nm
against substrate blank. The p-nitrophenol was used for the preparation of standard curve. The activity
of the enzyme was expressed as nmol of p-nitrophenol released /h/µg of fat body protein.
g. In vitro culture of fat body tissues
To study the effect of 20-hydroxyecdysone on PCD of the PF tissues, (at S2 stage); different
concentration (1, 2, 3, 4 and 5µg) of 20E was added to 1ml of Grace’s insect cell culture medium and
incubated for desired timing at 25˚C. Ethanol equivalents were also added to the control medium. The
effects of 20E on the PCD of the cultured tissues were determined by Acridine orange staining and DNA
laddering studies between 6 h intervals.
3. Results
Traditionally, peripheral and perivisceral fat body tissues were considered as one multifunctional
organ and frequently compared to mammalian liver and adipose tissue (Keeley 1985; Locke 2003). Our
previous studies on B. mori (Vanishree et al., 2005) and A. albistriga (Chandrasekar et al., 2008b)
clearly indicate the existence of functional differences between peripheral and perivisceral fat body
tissues, while PF tissue is predominantly responsible of biosynthetic activity, whereas the perivisceral fat
body is specialized as a storage organ. After the synthesis and sequestration of storage protein,
respective fat body cells undergo PCD (Chandrasekar et al., 2008a).
a. S0 : day zero of spinning stage

b. S1 : day one of spinning stage
c. S2 : day two of spinning stage
d. P0 : day zero of pupal stage
e. P1: day one of pupal stage
Fig.1. Fluorescent microscopic images of larval peripheral tissues stained with acridine orange showing the
onset of programmed cell death by the pattern of chromatin condensation in dying tissues. The live
tissues emit bright green fluorescence whereas the dying tissues emit bright orange fluorescence showing
the chromatin condensation.
(a). Chromatin Condensation
Acridine orange is one of the several nuclei acid stains routinely used to reveal the condensed
chromatin characteristic of apoptotic cells (Mc Gahon et al., 1995; Longthorne and Williams 1997;
Hopwood et al., 200l). Using this method of detection, and when viewed under blue filters, apoptotic
cells are highly fluorescent making them easily identifiable from non-apoptotic cells. The live cells emit
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an intense green fluorescence whereas the dead cells display an intense dark orange fluorescence. The
larval PF from day one larval (V1) to day one pupal (P1) tissues were freshly collected, stained with
acridine orange and viewed under fluorescence microscope using appropriate filter. An important
outcome of this study is that the larval peripheral tissues underwent PCD during the day one of pupal
period by emitting dark red fluorescence showing the presence of chromatin condensation (Fig. 1f) while
larval – pupal transformation stages (V6, S0, S1, S2, P0) (Fig. 1a-e) emitted a bright green fluorescence
indicating the presence of active live cells.
Fig.2. Electrophoretic detection of DNA fragmentation in the total

genomic DNA isolated from larval peripheral fat body tissues of
the last larval instar. Strong DNA fragmentation with appearance
of low – molecular weight DNA fragments can be observed during
the larval – pupal transformation prominently on day one of
pupal period.
V2 – V6 : day two to day six of last larval instar

S0 – S 2 : day zero to day two of spinning stage
P0 – P 1 : day zero to day one of pupal stage
(b). DNA laddering studies
To examine the DNA fragmentation, genomic DNA was extracted and electrophoresed on 2%
agarose and examined under UV light. Clear DNA laddering pattern of low-molecular weight fragments
<1kb were observed in the disintegrated larval PF tissues during larval – pupal transformation
significantly on day one of the pupal stage (Fig. 2). It is important to note that the larval peripheral
tissues lost its synthetic activity and underwent cell death in a programmed pattern.
(c). Lysosomal Acid Phosphatase – a marker enzyme
Lysosomal activation is an integral part 0.06

released /h/mg of fat body protein)
Enzyme activities (nmoles of PNP
of apoptosis in some systems, including insect

tissues during metamorphosis and degenerative 0.05
mammalian tissues (Halaby et al., 2003). 0.04

Lysosomal acid phosphatase is the ideal
0.03
enzyme for PCD and was estimated by
adopting the Hendrickson and Clever (1972) 0.02
methodology in peripheral tissues (V1 to P1) as 0.01

reflection of the lysosomal activity which in
0
turn indicates the status of the cell. The
V1 V2 V3 V4 V5 V6 S0 S1 S2 P0 P1
present work lays the ground work for the Stages
involvement of acid phosphatase during
autophagic degradation of disintegrated
peripheral tissues. Our current observation Fig. 3 Graphical representation of lysosomal acid
phosphatase activity in the larval peripheral fat body
demonstrates that the PF lysosomal acid
tissues in the B. mori showing the elevated level of
phosphatase activity increased gradually and enzyme activity in the day one of pupal stage. The
reached maximum on day one of the pupal enzyme activity is expressed in nmole of p-
stage (Figure 3 and 4). On the other hand, the nitrophenol phosphate released per hour per mg of
enzyme activity was also analyzed in fat body protein. The standard deviation and
polyacrylamide gels, which showed a standard error are from 5 experimental replicates.
significant elevation of enzyme activity exactly V1 – V6 : day one to day six of last larval instar
on the day one of pupal stage (P1) (Fig. 4) S0 – S2 : day zero to day 2 of spinning stage
P0 – P1 : day zero to day one of pupal stage
where the complete disintegration of larval
peripheral fat tissues takes place.
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Fig. 4 Polyacrylamide Gel Electrophoretic pattern of

lysosomal acid phosphatase activity in the larval
peripheral fat body tissues of B. mori showing the
elevated level of enzyme activity on day one of pupal
stage. After electrophoresis the gel was incubated
with substrate solution containing acetate buffer (pH
– 5.0), sodium chloride, 10% Magnesium chloride,
sodium α – napthyl phosphate, fast blue RR salt stain
for half – an – hour in dark Purple color bands were
visualized by adopting the method of Vallejos, (1983).
V3 – V6 : day three to day six of last larval instar

S0 – S2 : day zero to day 2 of spinning stage
(d). 26S & 20S Proteasome
Total protein homogenates prepared from the PF of the larvae of B. mori were collected from each
day of the 5th larval stages and subjected to 12% SDS- PAGE (Fig. 5 a). The presence of the 26S
proteasome in the larval peripheral fat tissues throughout the V instar stages was analyzed by Western
blot technique using MS73 subunit of 26S proteasome antibody which recognizes an ATPase subunit of
the 26S proteasome regulatory cap. One distinct band with an apparent molecular mass of 47kDa was
identified in the Western blot (Fig. 5b) analysis. This protein was not detected at the last larval instar
(V4-V6) but increasing amounts of MS73 was started appearing on day of the spinning stage (S0) and
reached maximum on day one of pupal stage, indicating involvement of this proteolytic activity during
the PCD of larval PF tissues which occurs on day one of pupal stage (Fig.5a)
Fig.5. Protein profile (a) and immunodetection of proteasome subunits in the larval peripheral fat body
tissues of B. mori subjected to 12% SDS – PAGE showing the cross reactivity of 47kDa (S6/MS73) - ATPase
subunit of the 26S proteasome (b) and 28kDa – regulatory subunit of 20S proteasome (c).
V4 – V6 : day four to day six of last larval instar

S0 – S2 : day zero to day 2 of spinning stage
165
The presence of 20S proteasome activity in the larval fat tissues was analyzed by immunoblotting
using the anti MCP antibody (Thanks to Prof. Martin Rechsteiner) which recognizes the regulatory
complex (19S) of 20S proteasome complex. Three distinct bands with apparent molecular mass of
28kDa region were labeled in Western blot. Western blot profile of the samples demonstrated the lack of
regulatory complex of 20S proteasome throughout the feeding period, but the appearance of 19S protein
was revealed in cells of the PF during commencement of the 1st day of pupal stage (Fig. 5c).
(e). Morphological Examination
Morphology of the larval PF tissues undergoing PCD was examined by electron microscopic
techniques. Apoptotic morphology (eg. shrinkage of cells and nuclei, cytoplasmic blebbing, ruptured
mitochondria, or detached cells which lost their intercellular connections) was not observed by electron
microscopic analysis and the followings were the prominent changes observed during the disintegration
of larval peripheral fat tissues; The first step in the
formation of the autophagic vacuoles is the appearance of
the so-called autophagosomes (Ag) or segregosomes. On
day two of spinning larva great number of mitochondria
were seen which indicates the active lively stage whereas
on the same day it structured for its disintegration by the
formation of autophagosomes (Ag) (fig. 6a). The
appearance of autophagosomes is in great numbers by the
end of the feeding period (S2) indicating the
commencement of the PCD process in these cells (Fig.
6a). In the day zero of pupa the formed autophagosomes
Fig.6 Electron micrographs of larval

peripheral fat body tissues undergoing
programmed cell death during larval –
pupal transformation in the silkworm,
B. mori. Formation of autophagosome
on day two of spinning larva (a);
aggregation of autophagosome on day
zero of pupa (b, c); after 3 h on day one
of pupa the autophagosomes fuses with
lysosomes to form autophagic vacuoles
(d); after 10 hrs on day one pupa lytic
enzyme in the autophagic vacuoles
digested the aggregated materials and
forms undigested one as apoptotic
bodies (e).
(Scale bar = 1µm).
AB – Apoptotic Bodies;
Ag – Autophagosomes;
Av – Autophagic Vacuoles;
L – Lipid;
M – Mitochondria;
N - Nucleus
166
aggregates together (Fig.6b, c) but after 6h the number of mitochondria are less (Fig. 6b) which indicates
that the larval peripheral fat body get ready for the disintegration. On the same day but after 12h, the PF
tissues prearranged themselves by complete disappearance of mitochondria (Fig. 6c). Later on the
aggregated autophagosomes fuse with lysosomes in which the digestion of the macromolecules begins
inside these organelles known as autophagic vacuoles after 3 hrs of day one of pupal stage in PF cells
(Fig. 6d). Autophagic vacuoles fuse very often with each other where degradation of the sequestered
organelles and materials began. Finally most of the cytoplasmic organelles were wrapped by autophagic
vacuoles (AV) and the undigested residues turned into electron-dense material after 10hrs in day one of
the PF cells. These undigested residues appear on the electron micrographs as dark, electron-dense spots
as apoptotic bodies (AB) or smears inside them (Fig. 6e). At the end of the larval (P1) stage, the
structure of the segregated organelles is totally destroyed. Only the undigested, homogenously electron-
dense material fills the inside of the large autophagic vacuoles (Fig. 6e).
(f). Role of 20E on PCD
Investigation of cell death during insect metamorphosis has provided new insights into the
mechanism by which a hormone, in which the steroid hormone 20-hydroxyecdysone, induces death in
some tissues while simultaneously promoting the further development of others. Changes in
responsiveness of the PF tissues to 20E, an effective inducer of PCD were examined by culturing the fat
body tissues in insect Grace’s medium supplemented with different concentrations of 20E and were
determined by chromatin condensation and DNA laddering studies.
Fig.7. In vitro studies on the effect of 20E on

chromatin condensation on the day two of spinning
stage peripheral fat body tissues of the silkworm,
B. mori after 12 h incubation at 25˚C. (Live cell
emits green fluorescence; dead cell emits dark
brown fluorescence)
Control treated with ethanol (a); 1µg/ml of 20E

(b); 2µg/ml of 20E (c); 3µg/ml of 20E (d); 4µg/ml of
20E (e); 5µg/ml of 20E (f); 3µg/ml of 20E (24h
incubation at 25˚C) (g); 4µg/ml of 20E (24h
incubation at 25˚C) (h)
Fig. 8. In vitro studies on the

effect of 20E on DNA
fragmentation by 1.2% agarose
gel electrophoresis in the day
two of spinning stage peripheral
fat body tissues treated with 4
and 5µg/ml of 20E after 12 h
incubation at 25˚C.
To test this hypothesis stating that ecdysteroids induce PF tissues PCD directly; S2 (spinning day
2) PF tissues were incubated in supplemented Insect Grace’s medium with or with out 20E (1 to 5
µg/ml) for up to 24 h. Chromatin condensation appeared to be 20E dose – and time – dependent and was
examined regularly during 6 hours intervals. Early signs of chromatin condensation occurred by
increasingly after 12 h incubation with maximum concentration (5µg/ml) of 20E (Fig. 7). After 24h,
167
almost every cell contained a highly condensed and fragmented nucleus. Among the five doses of 20E
tested, 5µg/ml was the most effective inducer of 100% chromatin condensation after 12 h incubation
(Fig. 7f). A dose of 3 and 4µg/ml causes only partial nuclear condensation (Fig. 7g, h), whereas 1 and 2
had no effect even after 24 h incubation (data not shown). This approach led to the unexpected finding
that 20E (5µg/ml) had direct effect on the PCD of larval PF tissues showing that the induction crop up
before 48h than the naturally occurring PCD of these tissues. induces PCD of peripheral tissues Control
experiments i.e. culture of PF tissues in 20E – free Insect Grace’s medium were also examined for
chromatin condensation in order to study the effect of 20E as an effective inducer of PCD. In control
experiments there was no sign of chromatin condensation were observed by acridine orange staining
even after 24 h incubation.
Total genomic DNA was extracted from the PF tissues cultured in Insect Grace’s medium treated
with 3, 4 and 5µg/ml after 12h incubation and subjected to agarose gel electrophoresis. Clear nuclear
fragmentation was noted in 5µg/ml, which strongly support the hypothesis that 20E – an effective
inducer of PCD in the PF tissue (Fig. 8).
4. Discussion
In the last decade a tremendous progress has been achieved in understanding the control of
apoptosis by survival and death factors as well as the molecular mechanisms of preparation and
execution of the cells suicide. Autophagic PCD appears to be a phylogenetically old phenomenon, it may
occur in physiological or disease status (Bursch 2001). However, it is an open question whether these
pathways exist and /or coexist during the physiological, developmental processes of insects. To approach
this problem, we studied the events of PCD in the larval fat body cells (PF tissues) during the last stage
of larval development of silkworm, B. mori. This organ can be regarded as a practically homogenous
tissue, consisting of a single cell type, the trophocytes, which die via autophagocytosis (Locke and
Collins 1966; Sass and Kovacs 1975; 1977; Chandrasekar et al., 2008a). Hence we made an attempt to
study the hallmarks of autophagic PCD in the larval PF tissues.
There are two major morphological categories of PCD: type I, apoptotic cell death, and type II,
autophagic cell death (Pilar and Landmesser 1976; Chu-Wang and Oppenheim 1978; Clarke 1990;
Lockshin and Zakeri 1996). Each type displays different specific morphological and biochemical
features. Based on our fluorescent studies, ultrastructural analysis, and DNA laddering studies, cell death
of larval PF tissues appears to display characteristics of both types, i.e. condensed chromatin under the
nuclear membrane, DNA fragmentation, apoptotic body formation (type I), as well as numerous
autophagic vacuoles, and intense membrane endocytosis (type II). Disintegrated, fragmented or
shrinkage cells of PF tissues were not registered through electron microscopic analysis and did not reveal
the characteristic nuclear morphology, or the blebbing. On other hand, extensive autophagy – indicated
by the extremely quick and extensive increase in the lysosomal compartment of the cells showing the
elevated level of lysosomal marker enzyme, acid phosphatase (stage P1) which was observed in the PF
tissues from the beginning of the spinning period confirming the results of earlier studies made on other
lepidopteron insects (Locke and Collins 1966; Sass and Kovas 1975; 1977; Muller et al., 2004;
Chandrasekar et al., 2008a). These events definitely pave the way for DNA fragmentation exactly on the
day one of pupal stage in the PF tissues. These combined features have also been observed in other
cases, such as the dying cells in the posterior necrotic zone of the forelimbs of chick embryos that
display all the characteristics of type I cell death but contain numerous autophagic vacuoles as (Mottet
and Hammer 1972; Hurle and Hinchliffe 1978; Clarke, 1990). Our observations are in good agreement
with earlier descriptions (Schwartz 1992; Zakeri et al., 1993; Muller et al., 2004; Chandrasekar et al.,
2008a,b) showing that in the dying intersegmental muscles during the postembryonic development of
insects and the larval fat body cells of M. sexta, the classical apoptotic morphology can be recognized.
However, some other data suggest that the autophagic and apoptotic pathways may coexist in certain
degrading larval organs (salivary and prothoracic glands) of insects (Dai and Gilbert 1997).
168
DNA fragmentation is an early hallmark for apoptosis and therefore an important criterion for
identifying the type of PCD. One of the aims of the present work was to see whether DNA fragmentation
occurs in dying insects PF cells showing a typical morphologic signs of autophagocytotic cell death.
Although this distinction appears very clear, the technique used to identify DNA fragmentation, such as
the DNA laddering technique, is quite critical. Using this technique, laddering was detected during the
final stage of the disintegration of PF tissues of B. mori, strongly indicating that the process is
autophagic phenotype. The DNA fragmentation, as an integral part of the PCD in natural developmental
processes of insects was described in those organs where the apoptotic and autophagic features were
simultaneously observed but the performance of the classic DNA ladder failed (Zakeri et al., 1993) with
the only exception of a highly synchronized in vitro cell culture triggered to death (Dai and Gilbert
1999). According to our best knowledge, it is the second report on the occurrence of DNA fragmentation
in larval PF tissues of B. mori with morphological pure autophagic phenotype where as the first report
was observed in larval fat body cells of M. sexta (Muller et al., 2004).
Lysosomes are normally associated with cytoplasmic turnover, whereas, in some cells undergoing
cell death, autophagic vacuoles that are derived from lysosomes play a significant role in cellular
breakdown (Beaulaton and Lockshin 1977; 1982; Clarke, 1990). Zahrebelski et al. (1995) studies
suggested that the release of hydrolytic enzymes from lysosomes might be the final event causing lysis
of the membrane and irreversible loss of viability. Acid phosphatase has been used as the marker
enzyme for lysosomes and a marker for apoptosis. It plays a critical role in the degradation of insect
tissues (salivary gland, intersegmental muscle, etc.) and mammalian tissues (mammary gland, prostate
gland, etc.) (Halaby et al., 2003). In silkworm, B. mori, the elevated level of lysosomal acid phosphatase
during the histolysis of the PF tissues may play a vital role in formation of autophagic vacuoles, which
may “cleanse the system” by engulfing apoptotic bodies or cell debris during the late stages of cell death
as reported for prothoracic gland cell death of Drosophila and M. sexta (Dai and Gilbert 1991). Based
on studies of professional phagocytes by Vieira et al. (2002) stated that in Caenorhabditis elegans
phagosomes likely fuse with endosomes /lysosomes, resulting in the formation of degradative
phagolysosomes. The trigger for cell death and the dramatic increase in polyubiquitin expression in the
degenerating intersegmental muscles (ISM) of M. sexta is the decline of the level of circulating molting
hormone. It is well known that ubiquitin may serve as a macromolecular tag attached to certain proteins
to mark them for degradation by the proteasome (Pickart 2001). The ubiquitin-protein conjugates may
also be decomposed in the lysosomal compartment of the cells (as it was described in various cell types)
(Laszlo et al., 1990; Mayer et al., 1991). Therefore, the regulatory ATPase subunit of the 26S
proteasome (47kDa- MS73) and the 28kDa - regulatory subunit of 20S proteasome may have a role in
the PCD of the larval PF tissues during metamorphosis. In M. sexta MS73 is found at high level only in
body wall muscles that are undergoing or are destined to undergo PCD (Low et al., 1997). MS73
immunoreactivity has now been found to be present in two other muscle types that undergo
developmentally PCD, the intersegmental muscles ISM1-2 and ISM7-8, which die just after pupal
ecdysis, and the proleg retractor muscle (PRM), which dies just before pupal ecdysis. In all three muscle
types, the level of MS73 more than doubles just before death occurs (Low et al., 1997). Our immunoblot
analysis was in good agreement with that of Low et al. (1997) showing in the dying PF cells that the
expression of 47 kDa – ATPase subunit of 26S and 28 kDa – regulatory subunit of 20S proteasome were
elevated just before the death occurs. The nuclear location and accumulation of ubiquitin and
proteasomes which coincide with the start of autophagy suggest that this highly conserved system may
be involved in the regulation of differential gene activation necessary to accomplish the process of
autophagocytosis in the larval fat body cells of M. sexta (Muller et al., 2004).
During an insect’s metamorphosis from larval to adult form, different tissue types respond to a
series of transient increases in the titer of a steroid hormone, 20E. The present data confirmed that 20E
act directly on larval PF tissue to trigger PCD. The effective dose 5µg/ml trigger the PCD of peripheral
tissues 2 days before, that exactly on day 2 of spinning stage after 12h incubation, whereas in the
developmental PCD of these tissues occurs prominently on day 1 of pupal period. In previous studies,
the involvement of 20E in the onset of PCD of insect tissues was demonstrated in tissues such as
intersegmental muscles and motoneurons by in vivo observations (Lockshin and Williams 1965;
169
Schwartz and Truman 1982) or by in vitro culture with 20E (Streichert et al., 1997). Although these
observations and in vitro manipulations provide strong evidence for the direct action of 20E on tissues,
the tissues in these studies consisted of several types of cells. This left open the possibility of
intercellular signaling that triggered PCD. Anterior silk glands are stimulated to full PCD by the rise of
hemolymph ecdysteroids in the photophase of day 2. The titer of approximately 1.5µg/ml in the late day
2 (Kiguchi 1983) may trigger PCD. This indicates that the degeneration of tissues requires stronger
steroid stimulation than that needed for tissue differentiation (Terashima et al., 2000). The present study
provides an in vitro system for further investigations of the molecular and cellular mechanisms of PCD
in the larval PF tissues.
The pace in cell death research is frantic and, sadly, this has resulted in considerable confusion
caused by a lot of contradictory publications. In conclusion, the present study indicates that the PCD
seen in PF tissues during larval – pupal transformation of B. mori lies between classic apoptosis and
autophagy, since its exhibits some characteristics of both phenomena. We have provided an overview of
state of the physiological PCD field in PF tissues of B. mori to highlight some of the unanswered
questions, as well as the genetic screening tools available to further this work.
5. Acknowledgement s
The authors thank Prof. Martin Rechsteiner, Department of Biochemistry, University of Utah
School of Medicine, Salt Lake City, UT, for the generous gift of Proteasome antibodies and
Dr. Lawrence I. Gilbert and Dr. James T. Warren, Department of Biology The University of North
Carolina at Chapel Hill, NC, for the generous gift of 20E. The financial support of the CSIR and DST,
New Delhi for gratefully acknowledged.
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174
NAL B OO
Short
IO
Views on Insect Molecular Biology, Vol.(1), 2009 Invited
T
TERNA
MIS
Review
SIONË
ËIN
 Vol. (1), 175 - 190, 2009
Chapter – 10
Apoptosis Regulation in Mosquito and its importance to

Malaria Infection.
Sylvester Sindakira Leonard Lyantagaye*.
Department of Molecular Biology and Biotechnology, University of Dar es Salaam
P.O. Box 35179 - Dar es Salaam, Tanzania.
Abstract
Apoptosis or Programmed cell death (PCD) is a biochemically regulated and active cell death program conserved
from worms to humans. Deregulation of apoptosis has been associated with many human diseases. In malaria,
PCD is observed following the invasion of the malaria parasite Plasmodium ookinete in the mosquito midgut
epithelial cells, an essential step in the transmission life cycle of the malaria parasite. Malaria parasite initiates the
inhibition of mosquito oogenesis by indusing apoptosis in the follicular epitherial cells, affecting the process of egg
production in mosquito. It is, therefore, hypothesized that targeted regulation of apoptosis process in mosquito
could be used in search for new ways to fight against the malaria epidemic.
Keywords: Apoptosis; Mosquito; Plasmodium ookinete; Malaria epidemic; Caspase; Bcl-2; IAP; Michelob_x;
midgut epitherial cells; programmed cell death
*For correspondence (email: lyantagaye@amu.udsm.ac.tz)
Overview
1. Introduction
1. Introduction
2. Historical meaning of the world apoptosis Apoptosis or Programmed cell death
3. The process of apoptosis (PCD) is a biochemically regulated and active
(3a) Apoptosis in development process cell death program conserved from worms to
(3b) Apoptosis in the immune system
(3c) Apoptosis in diseases humans. Apoptosis plays a critical role in many
4. Molecular mechanisms of apoptosis processes such as development, immune
(4a) Caspases responses, elimination of cancerous or infected
(4b) The regulation of apoptosis by the Bcl-2 protein
family cells and aging. Studies on apoptosis have been
(4c) Signalling pathways of apoptosis done more extensively in higher vertebrates than
(4d) The extrinsic or death receptor pathway in invertebrates. Deregulation of apoptosis has
(4e) The intrinsic or mitochondrial pathway
(4f) Caspase independent apoptosis pathways
been associated with many human diseases e.g.,
5. The role of apoptosis in mosquito during Plasmodium cancer in the case of excessive down regulation
invasion and HIV/AIDS or Alzheimer’s disease in the
6. Conclusion case of excessive up regulation. Most of the
7. Acknowledgments
8. References literature available on mosquito apoptosis is on
the occurance of apoptosis in the midgut
epithelial cells during oogenesis or the early
stages of Plasmodium invasion.
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Although there have been several interesting findings from studies done on the role of apoptosis in
the process of malaria infections, such findings have not had significant impact towards the efforts to
control the disease. One of the reasons for such insuficient impact is the lack of sound strategies to fit
together the informations from the fragmented findings to form a harmonious or credible whole. What is
needed would be to combine the informations as much as possible available on different aspects of the
disease pandemic and make such combined informations public so that researchers will not have to
duplicate studies. This chapter reviews studies on the molecular mechanisms of programmed cell or
apoptosis and its role during the malaria parasite invasion of the mosquito midgut. The knowledge of the
molecular mechanisms behind the apoptosis during mosquito-parasite interactions could help in efforts
to search for new effective ways to prevent transmission of the malaria parasite to human.
2. Historical meaning of the world apoptosis
The word apoptosis has been introduced into modern scientific writing by Kerr et al. (1972) to
describe a form of cell death distinct from necrosis. However, the word apoptosis was originally used in
medical and philosophical writings of the classical Greek and Roman times, ca. 460 – 370 BC, to
describe structural changes related to tissue and cell death (Degli Esposti, 1998). Later on, from 129 AD
onwards, the Roman medical writers extended the medical meaning of apoptosis to “dropping of the
scabs”. Besides its origin in medical texts, apoptosis has been used also in philosophical writings of the
classical world, particularly, the application to political and social contexts as a synonymous of failure,
ruin, decline and decadence (Degli Esposti, 1998). It seems, however, that Kerr et al. (1972) chose the
word apoptosis for its meaning of “falling of leaves from trees or dropping of petals from flowers” in
ancient Greek and this has remained the accepted etymology in biomedical sciences. This analogy
emphasizes that the death of living matter is an integral and necessary part of the life cycle of organisms.
3. The process of apoptosis
Apoptosis is a normal component of the development and health of multicellular organisms. It is

estimated that in the human body about 100, 000 cells are produced every second by mitosis and a
similar number die by apoptosis (Vaux and Korsmeyer, 1999). Cells die in response to a variety of
stimuli and during apoptosis they do so in a controlled, regulated fashion (Leist and Jaattela, 2001), and
sometimes apoptotic cell death is known as programmed cell death (Ameisen, 1996). Apoptosis is a
process in which cells play an active role in their own death, which is why apoptosis is often referred to
as cell suicide (Kumar and Cakouros, 2004; Olney et al., 2004; Kim et al., 2004). This makes apoptosis
distinct from necrosis in which uncontrolled cell death leads to lysis of cells, inflammatory responses
and, potentially, to serious health problems (Kroemer et al., 1995; Saikumar et al., 1999).
Upon receiving specific signals instructing the cell to undergo apoptosis a number of distinctive
biochemical and morphological changes occur in the cell. A family of proteins known as caspases are
typically activated in the early stages of apoptosis. These proteins cleave key cellular substrates that are
required for normal cellular function including structural proteins in the cytoskeleton and nuclear
proteins such as DNA repair enzymes. The caspases can also activate other degradative enzymes such as
DNases, which begin to cleave the DNA in the nucleus (Schwartzman and Cidlowski, 1993). These
stereotypical changes are manifestations of intrinsic suicide machinery that has been conserved through
evolution (Vernooy, 2000). The result of these biochemical changes is the appearance of the
characteristic morphological changes in the cell.
The apoptotic characteristic changes include the cell shrinkage, nuclear condensation, and collapse
of the cell into small intact membrane bound fragments known as apoptotic bodies (Fig.1), a form that
allows for easy clearance e.g., by macrophages (Saikumar, et al. 1999). These phagocytic cells (the
macrophages) are responsible for removing apoptotic cells from tissues, avoiding many of the problems
associated with necrotic cell death.
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Fig.1. Hallmarks of the apoptotic cell death process. Adopted from

http://www.microbiologybytes.com/virology/index.html.
In order to promote their phagocytosis by macrophages, apoptotic cells often undergo plasma
membrane changes that trigger the macrophage response (Dai et al., 1995; Vitkovic, 1997; Balanco et
al., 2001). One such change is the translocation of the plasma membrane phospholipid
phosphatidylserine from the inner leaflet of the cell to the outer surface. Membrane changes can often be
observed morphologically using microscope through the appearance of membrane blebs or blisters (Kerr
et al., 1972; Savil and Fadok, 2000). In vitro, the apoptotic bodies are also sometimes observed (Kerr et
al., 1972; Savil and Fadok, 2000).
Apoptosis plays a critical role in many processes such as development, immune responses,
elimination of cancerous or infected cells and aging (Fleisher, 1997; Reed, 2000). Apoptosis is also
important in stress response; cells that have been damaged by stress e.g. oxidative stress (Mollace et al.,
2002) are induced to die by apoptosis so that they do not become necrotic and so release toxic
components onto surrounding cells and tissues.
(3a) Apoptosis in developmental process
In the embryonic stages in particular, but also after birth, cells that have served a function in
development and are no longer needed are removed by apoptosis. Apoptosis is important for the
formation of body cavities and is involved in homeostatic regulation of cell number, whereby excess
cells are deleted (Jacobson et al., 1997). For example, resorption of the tadpole tail at the time of
metamorphosis into a frog (Jacobson et al., 1997; Hanada et al., 2003), the formation of the fingers and
toes of the mammalian fetus, which requires the removal of the tissue between them (Jacobson et al.,
1997; Dünker et al., 2002) require apoptosis (Fig. 2). Exposure to noxious stimuli such as heat,
irradiation, or toxins leads to injury. If injury is too severe to repair, cells die by apoptosis (Jacobson et
al., 1997). Apoptosis is also required for the sloughing off of the inner lining of the uterus (the
endometrium) at the start of menstruation (Vinatier et al., 1996), it is also required for the formation of
the proper connections (synapses) between neurons in the central nervous system (CNS) (Yachnis et al.,
1998).
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Fig. 2. Examples of apoptosis in physiology and pathology showing the physiological role of apoptosis in
embryonic development and morphogenesis. A, the formation of body cavities, B, Morphogenesis of
fingers and toes, C, removal of tadpole tail during metamorphosis. D, irreparable damaged cells die by
apoptosis. From Jacobson et al. (1997).
During developmental process, cells whose genome (DNA) is damaged can disrupted embryonic
development leading to birth defects (Hook and Lee, 2004), or become cancerous (Motoyama and Naka,
2004). Cells respond to such DNA damage by increasing their production of p53, a potent inducer of
apoptosis (Burmeister et al., 2004). Mutations in the p53 gene for instance are common in cancer cells
(Giaccia, 1996), suggesting that the suppression of p53 gene may leads to cancer development.
(3b). Apoptosis in the immune system
Apoptosis is needed to destroy cells that are threat to the integrity of the organism. For example,
one way by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis
(Banda et al., 1992). CTLs may also induce apoptosis in each other and even in themselves (Trapani and
Sutton, 2003). T-cells can bind to the surface of other cells that display the antigen and trigger a
response. The response may involve other lymphocytes and any of the other leukocytes (von Herrath and
Oldstone, 1996; Takahashi et al., 2001). This is important for the deletion of self-reactive cells, non-
reactive cells, and no-longer required cells. Defects in the apoptotic machinery have been associated with
autoimmune diseases such as lupus erythematosus (Santiago-Raber et al., 2004) and rheumatoid arthritis
(Pap et al., 2003).
(3c). Apoptosis in diseases
Dysregulation of apoptotic signalling can play a primary or secondary role in various diseases with
insufficient apoptosis leading to e.g. cancer (cell accumulation, resistance to therapy, defective tumour
surveillance by the immune system), autoimmunity (failure to eliminate autoreactive lymphocytes),
persistent infections (failure to eradicate infected cells), whereas excessive apoptosis contributes to e.g.
neurodegeneration (Alzheimers’ disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral
sclerosis), autoimmunity (uncontrolled apoptosis induction in specific organs), AIDS (depletion of T
lymphocytes), and ischaemia (stroke, myocardial infarction) (Thompson, 1995; Reed, 2002).
Malfunction of the death machinery results from the mutation of genes that code for factors directly or
indirectly involved in the initiation, mediation, or execution of apoptosis, and several mutations in
apoptosis genes have been identified as a causing or contributing factor in human diseases (Mullauer et
al., 2001), making apoptosis an important therapeutic target.
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4. Molecular mechanisms of apoptosis
The decision by a cell whether to commit suicide or not, depends on the balance between the
survival signals and the death signals received. The continued survival of most cells requires that they
receive continuous stimulation from other cells and, for many, continued adhesion to the surface on
which they are growing (Comoglio et al., 2003). Some examples of positive signals are growth factors
for neurons and interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes (Araujoa and
Cotmana, 1995). The withdrawal of such signals will lead to apoptosis. Receipt of negative signals leads
the cell to apoptosis program. Examples of negative signals are: increased levels of oxidants within the
cell (Dahle and Kvam, 2004), damage to DNA by these oxidants or other agents like ultraviolet light and
X-ray radiation, and chemotherapeutic drugs (Brantley-Finley et al., 2003; Szymczyk et al., 2004; Zaidi
et al., 2004), and binding of ligands to the cell surface receptors, such as tumor necrosis factor-alpha
(TNF-α) to the TNF and Fas ligand (FasL) to Fas (Smith et al., 1994; Brojatsch et al., 1996; Nagata,
1997; Screaton et al., 1997; Pan et al., 1997; MacFarlane et al., 2003).
The core component of the apoptosis machinery is a proteolytic system involving a family of
proteases known as ced (cell death defective) in C. elegans, and caspases in mammals (Steller, 1995;
Nicholson, 1999; Chang and Yang, 2000). The activated proteases cleave various cellular proteins,
leading to the programmed cell death (apoptosis) (Thornberry and Lazebnik, 1998, Budihardjo et al.,
1999). The programmed cell death machinery has also been characterised in the fly, Drosophila
melanogaster (Abrahams, 1999; Colussi et al., 2000; Gaumer et al., 2002).
(4a). Caspases
The term caspases is derived from cysteine-dependent aspastate-specific proteases to signify that
they are proteolytic enzymes (proteases) that specifically cleave at aspartate residues in target proteins
within the cell during apoptosis (Fiers, 1999). According to unified nomenclature, the caspases are
referred to in the order of their publications. At least fourteen vertebrate caspases have been reported,
caspase-1 to –14 (Yuan et al., 1993; Cohen, 1997; Van de Craen et al., 1998; Creagh and Martin, 2001;
Kuechle et al., 2001). Inactive caspases are synthesised and exist in cells as zymogens (pro-caspases),
they are converted to active caspases through a regulated proteolytic process. The proteolytic processing
occurs at critical aspartic acid residues that conform to the substrate recognition consensus (Earnshaw
et al., 1999; Chang and Yang, 2000). There are three basic domains in the immature form: pro-domain,
large and small subunits (Fig. 3). The variable size (23 to 216 amino acids) NH2-terminal domain (pro-
domain) is used to interact with other proteins. The large (20 kD) and small (10 kD) subunits, each of
which contributes amino acids to the active sites, make up the catalytic domain (Thornberry and
Lazebnik, 1998; Earnshaw et al., 1999). The pro-apoptotic caspases can be divided into the group of
initiator caspases including procaspase-2, -8, -9 and –10, and into the group of effector caspases
including pro-caspase-3, -6 and –7. Whereas the effector caspases possess only short pro-domains, the
initiator caspases possess long prodomains, containing death effector domains (DED) in the case of
procaspase-8 and –10 or caspase recruitment domains (CARD) as in the case of procaspase-9 and
procaspase-2.
The effector caspases are the executioners in the cell; they cleave the proteins that actually lead to
the morphological features related to apoptosis (Raff, 1998; Budihardjo et al., 1999). Many structural
and regulatory proteins are inactivated by caspases, while other substrates can be activated. Several
caspases substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell
fate (Fischer and Häcker, 2003; Fischer et al., 2004)
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Fig. 3: Cleavage of an inactive pro-caspase

precursor into an active caspase (from Raff,
1998).
Caspases play two roles in bringing about the death of the cell. They transduce death signals that
are generated in specific cellular compartments, and they cleave a number of cellular proteins, resulting
in the activation of some and the inactivation of others. The cleavage events are thought to lead, through
a number of mechanisms, to many of the biochemical and morphological changes associated with
apoptosis (Thornberry and Lazebnik, 1998; Earnshaw et al., 1999). It is important to note that, in
mammals and flies, mutant phenotypes suggest caspases can also play important non-apoptotic roles, and
the functions of a number of caspases are still unclear (Alnemri et al., 1996; Vernooy, 2000).
Phylogenetic analysis classifies caspases into two groups illustrated in Fig. 4, the ICE (interleukin-1ß-
converting enzyme) subfamily and CED (cell death defective) subfamily (Thornberry and Lazebnik,
1998; Nicholson, 1999) based on substrate specificity, function and activation.
It has been suggested that about 200 proteins are targeted by caspases during apoptosis (Nicholson,
1999). Some examples have been extensively reported on. Active caspase can activate other pro-
caspases to overcome the inhibitory effects of apoptosis inhibitors.
Caspase-3 cleaves the anti-apoptotic Bcl-

2 and Bcl-xL, the ICAD/DFF45, the DNA
repair enzyme, PARP (poly-ADP-ribose
polymerase), and gelsolin. The cleavage of the
Bcl-2 and Bcl-xL results in the inhibition of
their activities, whereas the cleavage of the
ICAD/DFF45 results in the translocation of
CAD (caspase-activated deoxyribonuclease) to
the nucleus with consequent degradation of the
nuclear DNA (Sakahira et al., 1998). The
cleavage of PARP leads to its de-activation
(Takahashi et al., 1996) and the fragmented
DNA cannot be repaired. The activation of
gelsolin promotes the apoptotic features in the
nuclear and cytoplasmic membrane. Lamins is
activated through cleavage by caspase-6, and it
too promotes the apoptotic features in the
nuclear and cytoplasmic membrane (Kothakota
Fig. 4. The family of identified mammalian caspases et al., 1997). Caspase-8 cleaves and activates
(from Thornberry and Lazebnik, 1998). The murine Bid, which results in the translocation of tBid
caspase homologue, caspase-14, a keratinocyte (truncated Bid) to the mitochondria and
specific caspase (Van de Craen et al., 1998; Kuechle consequent release of cytochrome c (Li et al.,
et al., 2001), is not shown. 1998).
180
(4b). The regulation of apoptosis by the Bcl-2 protein family
The Bcl-2 gene was initially cloned and characterized as a candidate proto-oncogene involved in
the t(14:18) translocation that is characteristic of the human B-cell malignancy follicular lymphoma
(Tsujimoto et al., 1984; Vaux et al., 1988) and is the vertebrate homologue of the C. elegans ced-9
gene. Bcl-2 facilitates survival of cells in which it is expressed and possesses a hydrophobic tail that
allows it to associate with various cellular membranes, including those of the mitochondrial outer
membrane, the endoplasmic reticulum and the nuclear envelope (Adams and Corry, 1998).
A number of Bcl-2 family members have been identified. Some of which, like Bcl-2, protect cells
from apoptosis while others actually promote apoptosis. The members of the Bcl-2 family are
characterized by conserved amino acid sequence motifs (Figure 1.5). These are called Bcl-2 homology
(BH) domains and there are at least 4 different BH domains (BH 1-4) some of which confer pro-
apoptotic activity, while others confer anti-apoptotic activity to the proteins in which they are present
(Adams and Cory, 1998). Bcl-XL, Bcl-w, Mcl-1, A1, BHRF1, LMW5-HL, E1B-19K and CED-9 protect
cells from apoptosis. The pro-apoptotic group of Bcl-2 members can be devided into subgroups: the Bax-
subfamily which consists of Bax, Bak, and Bok that all possess the domains BH1, BH2, and BH3, Bcl-
XS which possesses of BH3 and BH4, and the BH3-only proteins (e.g., Bik, Hrk, Bim, Bad, Bid, Egl-1
and Noxa) which have only the short BH3 motif, an interaction domain that is both necessary and
sufficient for their killing action (Saikumar et al., 1999; Cory and Adams, 2002; Mund et al., 2003).
In a viable cell, the proapoptotic Bcl-2 family members Bax, Bak, and BH3-only proteins are
antagonized by anti-apoptotic members such as Bcl-2. In response to an apoptotic stimulus, BH3-only
members are activated by transcriptional upregulation (Bax, Noxa), subcellular relocalization (Bim),
dephosphorylation (Bad), or proteolysis (Bid). Activated BH3-only proteins prevent anti-apoptotic Bcl-2
members from inhibiting pro-apoptotic members. In addition, they might directly induce a
conformational change of Bax and Bak, which subsequently oligomerize and insert into the
mitochondrial membrane where they form pores either by themselves or by associating with the
permeability transition pore complex. In consequence, pro-apoptotic factors are released from the inner
mitochondrial membrane into the cytosol, such as cytochrome c, which contributes to the formation of
the apoptosome and the subsequent activation of the caspase cascade (Cory and Adams, 2002; Mund
et al., 2003).
(4c). Signalling pathways of apoptosis
Two main pathways leading to apoptosis have been described (Fig. 5). One involves the release of
cytochrome c from mitochondria due to the signals arising within the cell, this pathway is known as the
intrinsic or mitochondrial pathway. The other involves plasma membrane receptors of TNF family
triggered by the binding of the death activators, e.g., tumor necrosis factor (TNF), lymphotoxin and Fas
ligand (FasL). This pathway is also known as extrinsic pathway. (Ashkenazi, 1997; Ashkenazi and Dixit,
1998; Green, 1998; Saikumar et al., 1999; Couzinet et al., 2002). Both two mechanisms depend on
proteolytic activation of caspases (Fig. 5). Caspase independent mechanisms also exist, for instance the
mechanism that involves the apoptosis-inducing factor (AIF).
(4d). The extrinsic or death receptor pathway
The death receptor pathways involve at least six transmembrane receptors belonging to the tumour
necrosis factor (TNF) receptor superfamily: TNFR1 (p55), Fas/CD95/Apo1, CAR1, DR3 (Apo3,
TRAIL), DR4 and DR5 (Smith et al., 1994; Brojatsch et al., 1996; Nagata, 1997; Pan et al., 1997;
Screaton et al., 1997; MacFarlane et al., 2003). These are integral membrane proteins with their receptor
domains exposed to the surface of the cell. Binding of the complementary death activators, e.g. FasL,
TNF, oligomerise and recruit adapter proteins (FADD, TRADD) to form death-inducing signaling
complexes (DISC) (Saikumar et al., 1999). FADD then binds to the clustered death domains of the
181
receptor and the pro-domain of caspase-8 (FLICE or MACH), which leads to the oligomerisation and
activation of initiator caspase-8, which sequentially activates effector caspases e.g., caspase-3 (Strasser
and Newton, 1999). FLASH inhibitory proteins (FLIP) or other proteins involved in survival pathways
as well as death mechanisms, e.g., RIP, may participate in complex, but tightly regulated mechanisms
that determine life or death (Malinin et al., 1997).
Alternatively, the DISC formation is reduced or blocked completely, so the caspase cascade cannot
be propagated directly but has to be amplified via the mitochondria (Scaffidi et al., 1998). Caspase-8
cleaves Bid (a pro-apoptotic member of the Bcl-2 family proteins) in the cytosol. The cleaved truncated
Bid (tBid) relocates to the mitochondria surface where it forms a complex with Bax (another pro-
apoptotic protein) leading to the activation of mitochondrial apoptosis pathway.
Fig. 5 Summary of executive caspases activation during apoptosis.
(4e). The intrinsic or mitochondrial pathway
This pathway is usually activated in response to other lethal stimuli such as DNA damage,
oxidative stress, and hypoxia (Rich et al., 2000). Apart from their role in energy metabolism and Ca2+
homeostasis, mitochondria play a crucial role in the regulation of cell death. Critical to this role is the
mitochondrial permeability transition pore (MPTP), whose opening uncouples mitochondria, preventing
them from providing for the energy needs of the cell leading to cell death (Nagahara et al., 2000;
Halestrap, 2003). Opening of the MPTP is tightly regulated by the mitochondria membrane potential and
matrix pH, and it helps regulate matrix Ca2+ concentrations. The transmembrane potential results from
the pumping of protons from the inner mitochondrial membrane by the electron transport chain,
generating ATP through the phosphorylation of ADP (Kroemer and Reed, 2000).
The mitochondrial pathway is essential for oncogene- and chemotherapy-induced apoptosis

(Cecconi et al., 1998; Hakem et al., 1998; Pirinia et al., 2002; Arimura et al., 2003). The MPTP opening
leads to the release of pro-apoptotic proteins contained in the mitochondrial intermembrane space.
These include cytochrome c that activates the caspase cascade responsible for the changes to the cell
structure and function that occur in apoptosis, and the caspase independent apoptosis-inducing factor
(AIF) (Cande et al., 2002; Arimura et al., 2003).
In a viable cell, Bcl-2 is found on the outer mitochondrial membrane and is bound to a molecule of
the protein Apaf-1 (Saikumar et al., 1999; Rippo et al., 2000). Internal damage to the cell due to reactive
oxygen species (ROS), for example, causes Bcl-2 to release Apaf-1 (Garcia-Ruiz et al., 2000). Cessation
of survival stimuli is thought to generate apoptotic signals through ill-defined mechanisms involving
182
MAP kinase and PI-3 kinases, which lead to translocation of other pro-apoptotic proteins such as Bax
from the cytosol to the outer mitochondrial membrane (Saikumar et al., 1999).
Transcription, in some cases mediated by p53, may be required to induce proteins such as Bax.
The activated Bax penetrates mitochondrial membranes, causing cytochrome c to leak out (Xia et al.,
2002). The released cytochrome c and Apaf-1 bind to molecules of pro-caspase-9. The resulting complex
of cytochrome c, Apaf-1, pro-caspase-9 and ATP, (apoptosome) forms in the cytosol. Caspase 9 cleaves
and, in so doing, activates effector caspases such as caspase-3, and others (Garcia-Ruiz et al., 2000). The
action of caspases, endonuclease(s), and possibly other enzymes leads to cleavage of structural proteins
in the cytoplasm, degradation of chromosomal DNA, and membrane changes which mark cells for
phagocytosis. For instance, an effector caspase cleaves the inhibitor of the caspase activated DNAse
(ICAD) leading to the activation of the endonuclease caspase activated DNAse (CAD) and DNA
degradation (Saikumar et al., 1999).
Apoptotic actions of stimuli such as drugs and radiation are mediated in most cases by p53-
dependent mechanisms including the transcription of pro-apoptotic proteins such as Bax (Newton and
Stresser, 2000). As a transactivator of transcription, p53 can induce apoptosis by upregulating the
expression of pro-apoptotic Bax, or Fas/FasL interactions (Mullauer et al., 2001). However, the
expression of Mdm2 protein can cause p53 degradation thus inhibiting its accumulation. The gene
encoding Mdm2 is itself activated by p53, thereby providing a negative autoregulatory loop. Some
agents cause cellular alterations such as ATP depletion that act directly on Bax. Anti-apoptotic proteins
such as Bcl-2 and Bcl-xL inhibit the membrane permeabilizing effects of Bax and other pro-apoptotic
proteins; however, they may also bind directly to Apaf-1 and inactivate this adapter protein, thereby
preventing the activation of caspase-9 (Saikumar et al., 1999).
Kumar and Vaux (2002) proposed a new model of the intrinsic pathway (Fig. 6). In the new
model, intrinsic pathways, such as those initiated by cell stress, induce activation of caspase-2, which is
required for permeabilization of mitochondria, release of cytochrome c, and apoptosis. According to this
model, mitochondria may act as amplifiers rather than initiators of caspase activity.
The intrinsic (mitochondrial) and extrinsic (receptor) pathways converge at the activation of
effector caspases e.g. caspase-3 (Saikumar et al., 1999). The activated caspase-3 then cleaves various
death substrates (cellular proteins), including poly ADP ribose polymerase (PARP), which is a 116 kD
DNA repair enzyme, to trigger apoptosis (Nicholson, 1999). It is also known that effector caspases can
induce a feedback mechanism and activate caspase-8 and –9. A feedback role of the effector caspases is
to cleave inhibitory Bcl-2 family proteins in turn the pro-apoptotic Bcl-2 family proteins and apoptosis
inducing molecules are activated. A second feedback mechanism of the effector caspase is to help
stimulate the release of caspase-activating proteins from mitochondria (Pirnia et al., 2002).
Effector caspases as well as initiator caspases can also be activated by granzyme B, an Asp-
specific serine protease. Granzyme B is an effector for cytotoxic T lymphocytes (CTL) and natural killer
cells and is delivered into virally infected cells during CTL- and natural killer cell-mediated apoptosis.
Granzyme B-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner, but
Granzyme B can also initiate caspase 3 processing and apoptosis in the absence of mitochondria
(Andrade et. al., 1998; Saikumar et al., 1999; Goping et al., 2003). This leads to the destruction of the
targeted infected cell.
Activated caspase-8 may also lead to the activation of the enzyme sphingomyelinase from
sphingomyelin that lead to the production of ceramide. Production of ceramide is a common event for
most of the inducers of apoptosis (Perry and Hannun, 1998). Alteration of the sphingomylin/ceramide
pathway is associated with resistance of human breast carcinoma MCF7 cell to tumour necrosis factor-
alpha-mediated cytotoxicity (Cai et al., 1997), suggesting the involvement of sphingomylin/ceramide
pathway in apoptosis.
183
(4f). Caspase independent apoptosis pathways
Although caspase specific inhibitors delay

apoptosis (Monney et al., 1998; Perfettini et al., 2002),
they are sometimes incapable of preventing it. There is
now evidence that caspase-independent apoptosis
pathways exist. They probably operate in conjunction
with the caspase-dependent pathways.
Following the mitochondrial outer membrane

permeabilisation, the cell might be committed to die
even if caspases are not activated (or are inhibited), as a
result of a loss of mitochondrial function and/or owing
to other apoptogenic proteins released from the
mitochondria. For instance, Smac (Diablo) when
released from mitochondria interferes with IAP
(inhibitor of apoptosis proteins) thereby releasing the
inhibitory effects of IAP thus lowering the threshold for
caspase activation, hence allowing apoptosis to occur
(Yang et al., 2000; Ekert et al., 2001; Halestrap, 2003).
Apoptosis-inducing factor (AIF) is a protein that

is normally located in the intermembrane space of
mitochondria. When the cell receives a death signal and
Fig. 6: A new model of apoptosis showing
that the intrinsic pathways in response to cell
the MPTP opens, AIF is released from the
stress; caspase-2 activation is required before mitochondria, migrates into the nucleus, and binds to
mitochondrial permeabilization and DNA. A mitochondrion-specific nuclease
apoptosis can take place. From Kumar and (endonuclease G) also translocates to the nucleus during
Vaux (2002). apoptosis. Once translocated, together AIF and
endonuclease G cleave chromatin DNA into
nucleosomal fragments leading to apoptosis independently of caspases. (Susin et al., 1999; Cande et al.,
2002, Arimura et al., 2003). The release of the DNAse activity from mitochondria can be induced by
factors that can lead to the MPTP opening such as the truncated Bid (tBid) (Van Loo et al., 2001).
5. The role of apoptosis in mosquito during Plasmodium invasion
A mosquito is a slender long-legged fly insect with aquatic larvae, and is important vector of
disease. The bite of the bloodsucking female mosquito can transmit a number of serious diseases
including malaria, dengue fever, west Nile fever, and viral encephalitis result in millions of human
deaths each year worldwide and significant agricultural losses. During metamorphosis, apoptotic cell
death is readily apparent in insects, where the larval tissues must atrophy to give way for those of the
adult (Schwartz et al., 1990).
Activation of caspases by proteolytic processing is a critical step during apoptosis in insects, like
in mammals (Manji and Friesen, 2001). Cells from the fruit fly Drosophila melanogaster and the
nocturnal moth Spodoptera frugiperda have been used extensively for studies on apoptosis. On the bases
of sequence similarity and biochemical activity, seven caspases have been identified in Drosophila
melanogaster (Vernooy, 2000). Drosophila DRONC and DCP-2/DREDD, which contain long pre-
domains, are initiator caspaces, whereas DCP-1, drICE, and DECAY contain short predomains and
therefore are likely effector caspases (Manji and Friesen, 2001). Caspases have also been identified from
lepidopteran insects (moths and butterflies). In particular, Sf-caspase-1 is the principal effector caspase
of SF21 cells, an established cell line from from S. frugiperda (Manji and Friesen, 2001). Studies on
D. melanogaster, have also shown that IAPs and IAP antagonists such as reaper, hid and grim have a
184
principal role in regulating cell death in insects. IAPs inhibit the activity of caspase. The IAP antagonist
removes this inhibition after binding to IAP (Palaga and Osborne, 2002). A reaper orthologue has also
been identified in the bowlfly (Chen et al., 2004). In mosquitos, michelob_x (mx) has been identified as a
missing IAP antagonist. Mx has a highly conserved amino-terminal IAP binding motif, and its expression
induces rapid cell death in insect cell lines and is a potent cell tissue abrator in vivo. And, like reaper in
Drosophila, mx is transcriptionally induced by ultraviolet irradiation to mediate cell death (Zhou et al.,
2005) suggesting that the mx is a role player in the regulation of apoptosis in mosquito.
Many host-parasite interactions are regulated in part by the programmed cell death of host cells or
the parasite. In mosquitoes, apoptosis of midgut epithelial cells mediated by activation of caspases is
observed after cell invasion by Plasmodium ookinete, an essential step in the transmission life cycle of
the malaria parasite (Al-Olayan et al., 2002, Abraham and Jacobs-Lorena, 2004). Also, Vaidyanathan
and Scott (2006) have demonstrated the apoptosis of midgut epithelial cells associated with West Nile
virus infection. This implies that one other important role of apoptosis in mosquito is as the host defence
against parasites and other pathogens. On the other hand, it has been demonstrated by Hopwood et al.
(2001) that malaria parasite initiates the inhibition of mosquito oogenesis by inducing apoptosis in the
follicular epitherial cells. This suggests that apoptosis could be used as a mechanism to control the
malaria vector egg production hence reducing the population of malaria parasite-carrying mosquitos
from the envirnment. In addition, following the parasite invasion, apoptosis in the mosquito midgut
epithelial cells is often accompanied by the induction of nitric oxide synthase and peroxidase (Barillas-
Mury and Kumar, 2005) whose activation leads to the production of nitric oxide, reactive nitrogen
intermediates and oxygen radicals (Hurd and Carter, 2004). These putative signal molecules are toxic
and induce apoptosis of the parasites cells through the activation of caspases, hence limiting the survival
of the malaria parasite (Hurd and Carter, 2004, Barillas-Mury and Kumar, 2005).
6. Conclusion
Dysregulation of apoptotic signalling can play a primary or secondary role in various diseases.
Malfunction of the death machinery results from several mutations in apoptosis genes have been
identified as a causing or contributing factor in human diseases, making apoptosis an important
therapeutic target. In mosquitoes, apoptosis of midgut epithelial cells mediated by activation of caspases
is observed after cell invasion by Plasmodium ookinete or associated with West Nile virus infection.
This implies that one other important role of apoptosis in mosquito is as the host defence against
parasites and other pathogens. On the other hand, malaria parasite initiates the inhibition of mosquito
oogenesis by inducing apoptosis in the follicular epitherial cells. This suggests that apoptosis could be
used as a mechanism to control the malaria vector egg production hence reducing the population of the
malaria transmission vector from the envirnment. In addition, following the parasite invasion, apoptosis
in the mosquito midgut epithelial cells is often accompanied by the induction of apoptotic cell death of
the Plasmodium cells, hence limiting the survival of the malaria parasite This suggests that if
scientifically manipulated, apoptosis could be used as a mechanism to reduce or control global malaria
infections.
7. Acknowledments
I would like to express my sincere thanks to Prof. Jasper Rees and Dr. Mervin Meyer not only for
their intellectual and moral support, but also for reading my entire manuscript, correcting my errors with
patience and good will.
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NAL B OO
IO
T
Short Views onShort
Insect Molecular
Views Biology,
on Insect Vol.(1),
Molecular 2009
Biology Research Article
TERNA
MIS
SIONË
ËIN
 Vol. (1), 191 – 201, 2009
Chapter – 11
Molecular mechanisms of cold hardiness in the European corn

borer (Ostrinia nubilalis, Hubn.)
Duško P. Blagojević*1, Mihajlo B. Spasić1 and Gordana N. Grubor – Lajšić2
1
Department of Physiology, Institute for Biological Research,
Bulevar despota Stefana 142, 11060 Belgrade, Serbia
2
Department of Biology and Ecology, Faculty of Sciences,
University of Novi Sad, D.Obradovica 2, 21000 Novi Sad, Serbia
Abstract
Here we review the results of the molecular and physiological responses to cold stress in the freeze-tolerant European
corn borer Ostrinia nubilalis indicative of molecular mechanisms of cold hardiness. Low temperatures induce phase
changes in energy production and subsequent reactive oxygen species (ROS) production, provoking physiological
responses to activate the antioxidative defense system (ADS) towards the regulation of cellular ROS concentration (H2O2,
superoxide and lipid peroxides), and redox equivalents glutathione, ascorbate and NADPH. The role of the ADS after
cold exposure in diapausing Ostrinia is twofold i.e. in early diapause ADS acts towards protection and promotes increased
activity of the pentose phosphate pathway while in late diapause ADS seems to be involved in the preservation of reduced
ascorbate and protection of membranes. Our results in Ostrinia larvae transferred to cold indicate that hydrogen peroxide
could be a regulatory molecule that mediates different physiological processes including melanogenesis. Several other
possible mechanisms could exist such as nitric oxide based determination of cell adaptation. Because manganese
superoxide dismutase (MnSOD) finally tunes the amount of hydrogen peroxide and nitric oxide in the mitochondria, this
enzyme may possibly be a trigger that determines physiological process of cells.
Keywords: Ascorbate; catalase; glutathione; hydrogen peroxide; melanin; reactive oxygen species; superoxide dismutase.
*For correspondence (email: dblagoje@ibiss.bg.ac.yu)
1. Introduction Insect cold hardiness is the sum of

2. Materials and Methods molecular and physiological mechanisms, as well
(a) European corn borer (Ostrinia nubilalis, Hubn;
Lepidoptera: Pyralidae)
as behavioral strategies that enable cold survival
(b) Antioxidant defense system (ADS) adaptations. Cold survival depends on both the
3. Results and Discussion length of exposure and the degree of coldness.
(a) ADS in diapause Other intrinsic factors such as the cooling rate
(b) ADS in exposure to cold
4. Conclusion and the presence of water also influence cold
5. Acknowledgements hardiness. Freeze-tolerant insects are
6. References characterized by relatively high supercooling
points which initiate freezing in extra cellular
spaces instead within the cell, therefore
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preventing injures of cell membranes caused by encroaching ice crystals. Cold tolerance and winter
diapause in insects that live in temperate climates occur at the same time while their survival mainly
depends on physiological and biochemical changes in response to low ambient temperatures.
Although there is considerable variation in the response of insects to cold (Bale, 1987, Sinclair,
1999) two main strategies are well known and a third has recently been recognized (Zachariassen, 1985,
Worland et al., 1998, Holmstrup et al., 2002). Insects either keep their body fluids liquid below their
melting point (freeze avoidance), survive the formation of ice in their tissues (freeze tolerance), or
depress their melting point by keeping their body fluids in vapor pressure equilibrium with the
surrounding ice (cryoprotective dehydration). Molecular and physiological mechanisms of these
strategies include increased synthesis of a limited number of low-molecular weight organic solutes like
polyols (glycerol and sorbitol), sugars (trehalose) and amino acids (alanine and proline) (Goto et al.,
2001, Michaud and Denlinger, 2007, Koštal et al., 2007). These compounds colligatively protect the
entire body from low temperature damage by increasing the osmolarity of body fluids and reducing the
freezing point (Lee, 1991, Storey, 1997). Desiccation and freezing are closely related processes and non-
colligative effects of these compounds include stabilization of membranes, proteins and other molecular
structures of cells (the water replacement hypothesis) (Quinn, 1985, Crowe et al., 1987, Meng, 2001).
The increase in polyol concentration relies on season and is initiated by short day length and
reduced temperature over a period of days and weeks (Lee, 1991, Hoffman, 2003). The elevation of
glycerol content is due to temperature-dependent activation of glycogen phosphorylase, which increases
the amount of free glucose molecules to be further metabolized to glycerol (Chen and Denlinger, 1990).
Higher production of glycerol is the result of the inhibition of fructose 1,6-bisphosphate kinase and
pyruvate kinase and activation of glycogen phosphorylase, glyceraldehyde-3-phosphatase and polyol
dehydrogenase. There are data showing that regulation operates during seasonal acclimation of insects
(Li et al., 2002, Koštal et al., 2004a,b). In addition, low temperature appears to favour the catabolism of
sugars via the pentose phosphate pathway (PPP) rather than the glycolytic pathway, consequently
generating the reducing equivalents (NADPH) for polyol synthesis (Kageyama, 1976, Wood and Nordin,
1980). There are also a few well-known proteins that have a role in mechanisms of cold hardiness
underlying adaptation to low temperatures. Some insects produce antifreeze proteins (AFPs), ice-
nucleating agents (INAs) and heat shock proteins (Michaoud and Denlinger 2004). Other possible
proteinous candidates are the aquaporins, the heat-shock (stress) proteins and late embryogenesis
abundant (LEA) proteins. LEA proteins occur in desiccation-tolerant organisms and protect other
proteins from aggregation (Wise and Tunnacliffe, 2004)
Antifreeze proteins (AFPs) and ice-nucleating agents (INAs) prevent injury of insect cells from ice
crystal formation. INAs elevate the crystallization temperature of the heamolymph, that results in an
increased possibility of ice crystals formation in the extracellular spaces at higher subzero temperatures.
As a consequence, water is drawn from the cells increasing their osmolarity (Zacchariassen, 1985).
There is evidence that AFPs are synthesized during the preparation for overwintering (Duman, 2004).
AFPs adhere to the surface of the ice crystals, hindering their growth and lowering the freezing point of
insect haemolymph via the process of thermal hysteresis (Duman and Serianni, 2002). The freezing point
is further reduced by the effect of polyols, organic anions (Li et al., 1998) and protein enhancers
(Duman, 2002). The level of 23 kDa small heat shock protein (HSPs) transcript increases in
nondiapausing flesh fly, Sarcophaga crassipalpis, after cold exposure. The transcript was highly
expressed beginning at the onset of diapause and continuing throughout diapause (Yocum et al., 1998).
Insect cold hardiness is closely linked to overwintering and insects predict the onset of winter by
real-time detection and monitoring of day length and temperature changes. In overwintering insects in
temperate climates, cold tolerance and diapause occur at the same time while their survival mainly
depends on physiological and biochemical adaptations to environmental changes. Diapause is suggested
to be an inherited factor conferring cold hardiness (Michaud and Denlinger 2004). Diapause is a dynamic
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process under hormonal control being associated with low metabolic rate (Lefevere and De Kort, 1989).
Diapause proceeds in a species-specific manner, usually in response to stimuli that anticipate
unfavorable environmental conditions, in genetically determined stage of metamorphosis. During this
time insects are sensitive to environmental stimuli, which may affect the intensity and length of
diapause. However, metabolic activity in diapause is suppressed even during periods favourable for
development. During diapause insects do not feed, there are inactive, with a lowered respiration rate
while their metabolism is “tuned” to the slow utilization of existing energetic reserves. The reduced
respiration rate is a consequence of reduced levels of mitochondrial cytochromes, (Storey, 1990,
Wipking et al., 1995) some of which may be degraded by fat body mitochondria, being subsequently re-
synthesized at the end of the quiescent period (Gruetzmacher and Keeley, 1982).
Respiration and oxygen consumption in aerobic cells is accompanied by generation of reactive

oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion radical (O2.-), lipid
peroxides, and hydroxyl radicals. The main cellular site of ROS generation is in the mitochondria where
superoxide anion and hydrogen peroxide are produced during respiration (Fig. 1). If not eliminated, ROS
propagates further oxidative processes leading to damage of cellular molecules and resulting in disturbed
homeostasis and cellular death. On the other hand, ROS are involved in cellular signaling, including
gene expression, signal transduction, and physiological regulation (Dalton et al., 1999, Suzuki et al.,
1997, Halliwell and Gutteridge, 2007). ROS involvement in cellular signaling is part of a complex redox
based physiological regulation (Schafer and Buettner, 2001) while unbalanced ROS levels influence the
concentrations of important cellular redox molecules such as glutathione and ascorbic acid. A shift of
redox status towards a more oxidative state could result in impaired development (Kirlin et al., 1999).
ROS concentration in organisms is regulated by the actions of complex antioxidant defense system
(ADS). The overall ADS in insects is NADPH consuming and is composed of enzymatic (superoxide
dismutases - SOD, catalase – CAT, glutathione and ascorbate peroxidase – GPx and APx and glutathione
and dehydroascorbate reductase – GR and DHAR) and low molecular weight antioxidants of different
classes. In insects ROS are generated by different metabolic pathways and controlled by antioxidants
leading to the regulation of cellular redox concentration. Investigations of the role of antioxidant systems
in supporting environmental and programmed adaptations to low temperatures suggest that ADS reduces
the risk of oxidative damages. ADS optimizes the concentration of ROS which is also involved in signal
transduction and the cellular redox state (Blagojević, 2007). In addition, freeze-tolerant species elevate
their ADS in preparation for recovery from freezing and continued development.
It has been suggested that biochemical mechanisms of cold hardiness and freezing injury are
connected with ADS (Rojas and Leopold, 1996, Grubor-Lajšić et al., 1997, Joanisse and Storey, 1998,
Jovanović-Galović et al., 2004, 2007). Evidence that the biosynthesis of polyol cryoprotectants is closely
connected with antioxidative enzymes is supported by the requirement for reduced equivalents
(NADPH), which are generated through the pentose phosphate pathway (PPP) (Stanić et al., 2004). On
the other hand, glycerol, ethylene glycol and trehalose, quoted as potential free radical scavengers are
accumulated during the winter period in freeze-tolerant insect species. These compounds act as both
anhydroprotectors and anti-freeze compounds (Block, 1990, Duman et al., 1991). However, it is
questionable whether their antioxidant action has an additional physiological role, simply represents a
consequence of their chemical properties and their relatively high concentration under certain
physiological conditions or developmental stage of insects (Grubor-Lajšić et al., 1992, Worland et al.,
1998, 2000). Furthermore, a close correlation between diapause and the accumulation of cryoprotectants,
such as polyhydroxy alcohols and trehalose, is reported for several insect species. Additional studies
have shown that major changes occur during winter diapause both in total metabolic flux and in relative
activities of different metabolic pathways (Macrae, 2005). The contribution of PPP to glycolysis in
diapausing larvae of Eurosta solidaginis increases at low temperatures (Tsumuki et al., 1987).
Furthermore, PPP is activated during sorbitol and glycerol synthesis in diapausing eggs of Bombyx mori
(Kageyama, 1976) and during cold induced glycerol synthesis in Protophormia terranovae (Wood and
Nordin, 1980). In cold-hardy insects flux through PPP is critically important for generating the reducing
equivalents (NADPH) needed for the synthesis of polyol cryoprotectants. It has been shown that for the
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synthesis of glycerol from glycogen, 86% of the total carbon flow must be routed through the PPP to
generate the required reducing power (Storey and Storey, 1988). On the other hand, as described above,
ADS are NADPH consuming processes. In summary, it appears that ADS is associated with cold-
hardiness metabolic pathways and physiological processes. This review summarizes our research on the
role of ADS and possible molecular mechanisms operating in cold hardiness, using the freezing tolerant
European corn borer (Ostrinia nubilalis, Hubn; Lepidoptera: Pyralidae) as a model.
(a). European corn borer (Ostrinia nubilalis, Hubn; Lepidoptera: Pyralidae)
The European corn borer is a pest of maize. It overwinters in corn stalks as diapausing fifth instar
larvae. O. nubilalis possesses sufficient supercooling ability to avoid freezing over their normal
environmental temperature ranges. Being freeze tolerant, O. nubilalis larvae are capable of surviving ice
formation in the extracellular body compartment (supercooling point c.– 20°C, lower lethal temperature
c – 28°C). During winter larvae accumulate high levels of glycerol and trehalose in response to cold
exposure and become freeze tolerant, i.e. they are capable of surviving extracellular ice formation.
(Grubor-Lajšić et al., 1992). Larvae and pupae of the European corn borer were collected from maize
plants in fields in Vojvodina, Serbia. They were kept overnight under natural conditions in ventilated
plastic containers between the sheets of lightly moisturized filter paper and then analyzed. A summery of
the experimental protocols is presented in Table 1.
Table 1. Experimental protocols used in research on European corn borer
physiological state and seasonal period tissue treatment measured reference

parameters
o
Eggs, larval instars, nondiapause haemolymph Cold 5 C glycerol Nordin et al.
and diapause J. Insect Physiol. 30: 563
(1984)
Diapausing 5th instar larvae haemolymph Ambient glycerol, trehalose Grubor-Lajšić et al.
(from November to March) conditions CryoLetters 12: 177
(1991)
Diapausing 5th instar larvae (January) whole body, Progressive glycerol, Grubor-Lajšić et al.
haemolymph, chilling from 15 trehalose, Physiol. Entomol. 17:
o
fat body C to -5 oC glycogen 148 (1992)
Diapausing 5th instar larvae (December) haemolymph Cold -3oC -12oC ADS enzymes Grubor-Lajšić et al.
fat body Arch Insect Biochem
Physiol 36: 1 (1997)
Diapausing 5th instar larvae whole body Cold -12oC ADS enzymes Stanić et al.
(November and February) PPP enzymes Eur J Entomol 101: 459
(2004)
5th instar diapausing larvae early whole body Ambient ADS enzymes Jovanović-Galović et al.
(October), mid- (December), and conditions Arch Insect Biochem
late diapause (February) Physiol 55: 79 (2004)
nondiapausing 5th instar larvae (July)
pupae emerged from nondiapausing larvae
(August).
pupae emerged from diapausing larvae
(May)
5th instar larvae mid diapause (December); mitochondria Ambient ADS enzymes Jovanović-Galović et al.
nondiapausing 5th instar larvae (July); conditions Arch Insect Biochem
pupae from nondiapausing larvae (August); Physiol 64: 111 (2007)
pupae from the diapausing larvae (May)
Glycerol is the major cryoprotectant in diapausing larvae of O. nubilalis. In the laboratory,

temperatures triggering glycerol synthesis range from 0 to 5°C (Storey and Storey, 1991). Nordin and
collaborators (1984) showed that the ability of fifth instar O. nubilalis larvae to accumulate glycerol
depends upon chilling. Eggs, second, third and fourth instar larvae do not have this ability, so a specific
developmental programme is required for insects to respond to cold stress. As expected, diapausing fifth
instar larvae of O. nubilalis accumulated glycerol in substantial amounts upon exposure to winter
194
conditions. However, glycerol content was significantly higher in larvae at the beginning of diapause. O.
nubilalis larvae accumulate glycerol in the haemolymph at the beginning of September (diapause onset)
and reach its highest levels in mid diapause (Grubor-Lajšić et al., 1991). In contrast to nature condition
results, exposure of larvae to low temperatures in the laboratory resulted in the expected elevation in
glycerol content in mid-diapause. Relatively high levels of glycerol in November (about 2% of the total
body weight) could provide adequate cryoprotection, even when exposed to sub-zero temperatures.
These results are in agreement with general conclusion that insects at different stages of diapause
respond differently to low temperature (Pio and Baust, 1988). The antioxidative status was different at
these two periods in diapause, suggesting a relationship between cold hardiness mechanisms and the
antioxidative defense system.
(b). Antioxidant defense system (ADS)
ADS in European corn borer includes enzymes, superoxide dismutases (SOD), catalase (CAT),
ascorbate and glutathione peroxidase (APx, GPx, respectively), dehydroascorbat reductase (DHAR),
glutathione reductase (GR) and cellular antioxidants and cofactors such as glutathione (GSH) and
ascorbic acid (AsA). In O. nubilalis, the decomposition of peroxides is performed by glutathione-S
transferases isoforms (GST) with peroxidase activity. These isoforms of GST are members of a large
family of GST based detoxifying system. In insects, GST peroxidase activity has been detected from the
Delta, Epsilon and Sigma classes (Singh et al., 2001, Vontas et al., 2001, Ortelli et al., 2003).
Resulting ADS reactions oxidase cellular GSH and ASA to its oxidized forms (oxidized
glutathione - GSSG and dehydroascorbate - DAsA). However, they can be reduced by GR and DHAR
respectively, using NADPH as reducing cofactor. Thus, the ADS action requires redox equivalents and,
therefore, antioxidant processses in cells can be viewed as GSH and/or NADPH consuming (Fig.1).
However, there are many molecules with antioxidant and ROS scavanger properties, but with other
regulatory (peroxiredoxin, thioredoxin, metallothionein, glucose-6-phosphate dehydrogenase) or
physiological functions (detoxification of secondary oxidative stress, xenobiotic detoxification, DNA
repair).
3. Results and Discussion
(a). ADS in diapause
Our experiments on the diapausing larvae of the European corn borer (Ostrinia nubilalis) showed
ADS changes during diapause, additionally supporting diapause as a dynamic process. Negative
correlation between DHAR and GR activity, together with changes in GSH and ASA content during
diapause led us to conclude that an active and enzymatically controlled establishment of optimal and
equilibrated concentration of cellular redox molecules is operative, and that ADS is a part of the redox
regulatory components. Results also suggest that use of redox equivalents (AsA and GSH) have a
strategic and economic character: (1) more AsA, less GSH, and vice versa; and (2) accelerated AsA
regeneration, slower GSH reduction, and vice versa. Their usage is balanced according to availability,
additional functions, and production of temporary dominant ROS molecules.
Furthermore, elimination of H2O2 in insects, including O. nubilalis, can be achieved by (1) CAT
activity and GSH recycling; and (2) an ascorbate recycling antioxidative mechanism (see Fig. 1).
Correlation analysis in diapausing larvae and pupae showed a negative correlation between GR and
DHAR activities in diapausing larvae, as well as between CAT and DHAR in nondiapausing ones
suggesting that if H2O2 elimination by CAT and GSH recycling mechanism is well balanced it may
prevent oxidative damage. The second mechanism could be less active, and vice versa. Since negative
correlation between GR and DHAR activity was found in both diapausing larvae and pupae emerged
from diapausing larvae, the mechanism of H2O2 regulation proposed above is, therefore, a characteristic
of insect diapause including development. This points also to a precise and very important coordinated
195
regulation of the concentration of hydrogen peroxide and redox active molecules by antioxidative
enzymes in insect diapause.
Fig.1. Antioxidant defense system (ADS), pentose phosphate pathway (PPP) and melanogenesis. Abbreviations:
MnSOD - manganese superoxide dismutase; CAT - catalase, APx - ascorbate peroxidase, DHAR -
dehydroascorbate reductase, GR - glutathione reductase, GPx - glutathione peroxidase, GST –
glutathione-S-transferase, AsA - ascorbate, DAsA - dehydroascorbate, GSH - reduced glutathione,
GSSG - oxidised glutathione, LOH - lipid alcohol, LOOH - lipid peroxide, LOSG - lipid glutathione,
RNS – reactive nitrogen species.
In mitochondria of diapausing O. nubilalis larvae the pattern of ADS enzymes seems to parallel
changes in energy production and O2 consumption and protect against oxidative stress (Jovanović-
Galović et al., 2007). However, in mitochondria, lower metabolic activity and respiratory rate in
diapause were accompanied by lower ADS levels of CAT, DHAR and GST, suggesting changes in H2O2
and ascorbate turnover. At the same time a positive correlation between GPx and GR suggests the
coordinated elevation of their activity towards the protection of lipid molecules and the reduction of the
oxidized form of glutathione to achieve optimal level for mitochondrial redox state in the terminal phase
of diapause and metamorphosis.
Another interesting point regarding ADS in diapause is the increase of GST mediated lipid
peroxidase activity at the end of the diapause. This could be a mode of preparing the organism for the
active high metabolic phase after diapause and the possible elevation of lipid peroxides due to free
radical mediated lipid peroxidation as an unavoidable consequence of elevated ROS production. In this
way the integrity and functionality of the membranes are preserved. The fact that peroxidase activity was
increased while overall GST activity (including peroxidase activity isoform) decreases, suggests also that
the rearrangement of separate activities of individual GST isoforms takes place during diapause. This is
important since the GST super family is the main detoxification enzymatic system, but also involved in
the metabolism of regulatory compounds (Udomsinprasert et al., 2004).
(b). ADS in exposure to cold
Previous comparisons of the freeze-tolerant Ostrinia nubilalis and the freeze-intolerant

Mediterranean borer Sesamia cretica exposed to low temperature (-3oC) revealed that O. nubilalis larvae
196
elevated ADS components. On the other hand, Sesamia response to cold at the level of ADS is different,
since further decrease in temperature exeed stress resistance capacities resulting in death (Grubor-Lajšić
et al., 1997). These imply different metabolic and regulatory characteristics in different insect cold
resistance strategies. The above-mentioned results agree with the available evidence suggesting that
freeze-tolerant species respond to cold by elevating the level of antioxidant defence. This could be the
mechanism for preparing the body for free radical attack during the thawing and re-warming process.
However, it seems that ADS during exposure to cold in diapausing Ostrinia has also a few other very
important physiological tasks additionally to the antioxidative. These tasks include preservation of redox
homeostasis, regulation of physiological concentration of redox molecules such as ascorbate,
glutathione, NADPH, hydrogen peroxide and NO and promotion of the pentose phosphate pathway.
This is achieved by overlapping of individual ADS components and recomposition of ADS toward
specified physiological goals. The degree of these processes depends on the period of diapause when
cold exposure was performed.
Our studies on the level of ADS enzymes in diapausing O. nubilalis larvae exposed to low
temperature (8oC and -12oC) revealed a shifted role of the ADS in early and mid- diapause. In early
diapause the ADS is directed towards protection and promotes increased activity of the pentose
phosphate pathway. In mid diapause the role of the ADS seems to be focussed on the preservation of
reduced ascorbate underlining the importance of reduced ascorbate in the physiology of this insect
species.
At the beginning of diapuse, superoxide is more rapidly removed by increased SOD activity
resulting in elevated hydrogen peroxide concentration. Furthermore, in larvae exposed to cold in early
diapause, peroxidative activity is also enhanced, obtaining increased protection to cellular lipids, and
integrity of cellular membranes. Exposure of larvae to low temperatures resulted in the elevation of PPP
enzymes activities, especially pronounced in early diapause. Glucose-6-phosphate dehydrogenase from
cold hardy insects has high substrate affinity, even at low temperatures, which aids the production of
NADPH needed to synthesize polyhydroxy alcohols from sugars (Storey et al., 1991). In O. nubilalis the
level of ascorbate was significantly lower after cold acclimation in both periods of diapause, suggesting
its utilization. Taking into account the enhanced PPP activity during cellular cryoprotection (synthesis of
glycerol) under freezing conditions, we hypothesized that dehydroascorbate contributes to the overall
activity of this metabolic pathway. Since dehydroascorbate at a physiological pH is unstable, with a half-
life of a few minutes, it may be rapidly metabolized through diketogulonate to 5-carbon intermediates,
which could enter the pentose phosphate pathway. In addition, the increased DHAR activity observed in
O. nubilalis larvae exposed to cold in February could reflect an intense reduction of an oxidized form of
ascorbate and the important role of this redox active molecule in numerous redox regulated processes
within the cells (Schafer and Buettner, 2001). Hence, the ADS is an important and linked metabolic and
physiological mechanism involved in redox homeostasis (Stanić et al., 2004).
The insect ADS is very flexible, dynamically organized and operates not only to eliminate ROS
but also to remodel particular ROS (H2O2 and NO.) to produce direct physiological effects. Our results
from ADS monitoring upon cold exposure of O. nubilalis emphasize the importance of superoxide
removal and the regulation of appropriate concentrations of H2O2 suggesting its dual role in cold
hardiness.
Free radical damage will occur if H2O2 levels exceed the concentration which can be tolerated by
cells. However, hydrogen peroxide also has a role in cellular signaling, and in the presence of cellular
antioxidants, mediates physiological processes. The presence of adequate amount of H2O2 is the first and
key step of melanogenesis. The first and key step in melanogenesis, tyrosine hydroxylation to DOPA,
can be accomplished with high yields in the presence of hydrogen peroxide and one of the biogenic
intracellular antioxidants such as ascorbic acid, NADH or glutathione (Kasraee et al., 2003). The
subsequent formation of dopachrome, and the production of melanin pigments were shown in the
presence of a known DOPA oxidising factor as peroxidase. This new concept can readily explain the
melanin synthesizing property of extracutaneous cells and the high association between intracellular
197
hydrogen peroxide formation and melanin synthesis by the cells. Cell culture methods showed that the
decomposition of intracellular hydrogen peroxide in melanocytes causes these cells to significantly loose
their melanin synthesizing ability, indicating a role for hydrogen peroxide in addition to tyrosinase, in
the rate limiting step of melanin synthesis in melanocytes. In summary, it can be proposed that melanin
production serves as a defense mechanism utilized by the cells to protect themselves against oxidative
stress by consuming intracellular hydrogen peroxide.
Mitochondrial SOD (manganese containing form of SOD located at the inner mitochondrial
membrane) enzyme regulates the level of mitochondrial superoxide elimination and hydrogen peroxide
production. However, MnSOD is also the subject of NO metabolic pathway (Filipović et al., 2007,
Stojanović et al., 2005), and is a part of several other possible mechanisms that could exist such as nitric
oxide based determination of cell adaptation or death. Because manganese superoxide dismutase
(MnSOD) finally tunes the amount of hydrogen peroxide and nitric oxide in the mitochondria, this
enzyme may possibly be a switch that determines the fate of cells.
4. Conclusion
In conclusion, ADS is very flexible, dynamically organized system and operates not only to
eliminate ROS but also to remodel particular ROS (H2O2 and NO.) to produce direct physiological
effects. ADS is involved in the regulation of the concentrations of redox active antioxidants glutathione
and ascorbic acid which have regulatory and constitutive physiological functions depending on the
availability and production of temporary dominant free radical molecules. In O. nubilalis low
temperature favours the catabolism of sugars via the pentose phosphate pathway and generates the
reducing power (NADPH) for polyol synthesis. Elevation in the activity of the pentose phosphate
pathway enzymes is especially pronounced in early diapause. Glycerol content and the activity of key
enzymes of the pentose phosphate pathway support the connection between this metabolic pathway and
the antioxidative system and the notion that the antioxidative defense system in larvae of Ostrinia
nubilalis is closely connected with metabolic changes characteristic for diapause and cold hardiness. The
changes in the activity of antioxidative enzymes during diapause suggest that diapause is a dynamic
process, which can be divided into phases. Insects in the preadult state may modulate phenotype
expression according to stage before diapause induction. Further investigation of the precise regulatory
roles of individual ADS components in cold hardiness will picture in more detail involvement of
hydrogen peroxide, nitric oxide and other redox active and antioxidant molecules in general
physiological mechanism.
5. Acknowledgments
This work was funded by Ministry of Science of Republic of Serbia, Grant No. 143034B “The role
of redox-active substances in the maintenance of homeostasis”.
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Chapter – 12
Molecular characteristics of the W chromosome, Z chromosome and

retrotransposable elements of the silkworm, Bombyx mori
Hiroaki Abe1,* and Tsuguru Fujii2

1
Department of Biological Production, Faculty of Agriculture,
Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, 183-8509, Fuchu, Tokyo, Japan;
2
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Yayoi 1-1-1, 113-8657, Bunkyo-ku, Tokyo, Japan.
Abstract
Sex chromosomes of the silkworm, Bombyx mori, are designated as ZW (XY) for females and ZZ (XX) for males. The
female mode of development is determined by the presence of a single W chromosome. Therefore, it is presumed that the
female-determining gene (Fem) is present on the W chromosome. Until date, 12 W-specific random amplified
polymorphic DNA (RAPD) markers have been identified. One of the W-translocation chromosomes, the sex-limited
Zebra-W strain or T(W;3)Ze, is thought to be the product of reciprocal translocation between W and chromosome 3. We
have compared the T(W;3)Ze chromosome to the parental W chromosome using the 12 W-specific RAPD markers. The
T(W;3)Ze chromosome lacked 2 W-specific RAPD markers, strongly indicating that the region containing these two
markers has been deleted. Additionally, we have investigated T(W;10)+w-2 (the sex-limited Black-egg-W strain) and
T(W;2)Y (the sex-limited Yellow-cocoon-W strain). The T(W;10)+w-2 chromosome lacked 1 W-specific RAPD marker,
while the T(W;2)Y chromosome lacked 11 such markers. These results indicate that the W chromosomes of the sex-
limited strains are the products of reciprocal translocation accompanied by deletion, and that only an extremely limited
region is required to determine femaleness. Furthermore, the W chromosome does not recombine with either the Z
chromosome or autosomes, and no genes for morphological traits have been mapped to the W chromosome. Many long
terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons, and their derivatives, had
accumulated as strata on the W chromosome. These elements on the W chromosome were expected to be largely intact.
On the other hand, the retrotransposable elements on the Z chromosome were altered by recombination or
rearrangement. In particular, the LTR-type retrotransposons on the Z chromosome were excluded by unequal crossing
over or intra-element homologous recombination between LTRs.
Keywords: Bombyx mori; silkworm; W chromosome; Z chromosome; sex chromosome; retrotransposon
* For correspondence. (email: wfem@cc.tuat.ac.jp )
1. Introduction ancestral species, Bombyx mandarina, can still

Sericulture of the silkworm, Bombyx mori be found in nature. In Japan, silkworms have
dates back about 4,500 years. It is generally been reared for more than 1,700 years. Since the
thought that sericulture began on the Asian latter half of the 19th century, raw silk has been
continent. B. mori, belongs to the family of one of the most important exports from Japan.
Bombycidae in the order Insecta. The silkworm The silkworm has been used in genetic studies
has been completely domesticated and is no since the birth of genetics in the early 1900s,
longer found in the wild. However, a presumed primarily in Japan.
203
Sericulture in Japan has at present Overview

decreased to its lowest level. However, 1. Introduction
silkworms are convenient experimental model 2. Historical background of isolation of translocations
organisms for several reasons. For example, they between W and autosomes
do not escape from the rearing bed even in the (2A) pSa +p W chromosome (T(W;2) pSa +p
chromosome) (sex-limited sable)
absence of bait as they have lost the ability to (2B) pSa +p W +od chromosome
look for bait by themselves; this makes the act of (2C) Female-killing chromosome (Df(pSa +p W +od)Fem
rearing silkworms an easy task. The adult moths chromosome)
(2D) T(W;3)Ze chromosome (sex-limited zebra)
cannot fly, and controlling pairings between (2E) Male-killing factor derived from the T(W;3)Ze
females and males is easy. Secondly, many chromosome
genetic marker strains exist, with almost all traits (2F) T(W;10)+w-2 chromosome (sex-limited black egg)
being evident in larvae. Variant traits include egg (2G) T(W;2)Y chromosome (sex-limited yellow cocoon)
3. Why did we analyze the W chromosome?
color, larval markings, body color, body shape, (3A) W-specific RAPD markers
blood color and cocoon color (Fujii et al., 1998; (3F) DNA sequence of the W-specific RAPD markers
Goldsmith et al., 2005). Finally, a single female (3C) W-specific lambda phage and BAC clones
4. Presence of W-specific RAPD markers in silkworm
moth can lay a large number of eggs (from 200 - strains maintained in Japan
500 in her lifetime). The chromosomes of the 5. Presence of W-specific RAPD markers in W chromosome
silkworm are small, holocentric, and numerous variants
(2n=56). Thus, cytogenetic analysis of the (5A) [T(W;3)Ze chromosome]
(5B) [T(W;10)+w-2 chromosome]
silkworm is difficult in comparison with (5C) [T(W;2)+p chromosome]
Drosophila. However, 400 or more visible (5D) [T(W;2)Y chromosome]
mutations have been placed on linkage maps for (5E) [Df(pSa +p W +od)Fem chromosome (DfZ-DfW
chromosome)]
B. mori (Goldschmidt and Katsuki, 1931; (5F) [W(B-YL-YS)Ze chromosome]
Doira,1987, 1992; Goldsmith, 1995; Goldsmith (5G) [Z1 and ZeWZ2 chromosomes]
et al., 2005). 6. Order of W-specific markers and position of the putative
Fem gene on the W chromosome
7. Relationship between sex-heterochromatin body and W
This chapter focuses on the present state of chromosome
molecular analyses of the W and Z chromosomes 8. Retrotransposable elements on the W and Z chromosomes
of B. mori. It begins with an overview of the of B. mori
(8A). Kabuki
history of studies conducted on the W (8B). Yokozuna
chromosome. This is followed by an explanation (8C). BMC1
of why we have chosen to analyses the W and Z (8D). Pao
chromosomes. Finally, we have focused on the (8E). Kamikaze
(8F). Kendo
nucleotide sequences of retrotransposable 9. Transposable elements on the Z chromosome
elements on the W chromosome. We have 10. Identification of the W chromosome using W-derived
discussed why many retrotransposable elements BAC probes
11. Conclusion
accumulated on the W chromosome in contrast to 12. Acknowledgements
the Z chromosome. 13. References
2. Historical background of isolation of translocations between W and autosomes
In this section, as a foundation for understanding W chromosome translocations, we have

discussed the history of research conducted on the W chromosome of B. mori. In general, Japanese
sericulture is stable in the spring season in Japan. However, it becomes unstable in summer and autumn,
because the quality of mulberry leaves deteriorates after spring and because of unfavorable weather
conditions, namely high temperature and humidity. Therefore, isolation of more vigorous silkworm
strains during the summer and autumn seasons is desired. In 1906, Toyama, one of the founders of
silkworm genetics, rediscovered Mendel’s laws in a study of inheritance of cocoon color. He recognized
heterosis in hybrids between Japanese and Thai strains (Toyama, 1906 cited in Tazima, 1964). Following
his advice, the Japanese government decided to encourage the rearing of hybrid silkworms and began
distributing hybrid eggs in 1914. Thereafter, almost all the silkworm strains reared at Japanese
sericultural farms have been F1 hybrids. At the same time, for the production of F1 eggs, males and
females must be separated at or before the cocoon stage. In the absence of separation, sibling moths
204
copulate mutually and females cannot be used for the production of F1 hybrid eggs. Therefore,
determining the sex of silkworms for hybridization became critical to silkworm egg producers.
Traditionally, separation was accomplished by inspecting Ishiwata’s germal discs in the female and
Herald’s gland in the male on the post-ventral surface of the 5th (final) instar larvae. This method was
generally performed by a professional “discriminator”, but it was inconvenient. Therefore, development
of a convenient method of discriminating males from females that even an amateur was capable
desiring.
Tanaka (1916) determined the chromosomal madeup of the silkworm in terms of sex linked (Z-
linked) inheritance namely that females were ZW (equivalent to XY) and males were ZZ (XX).
However, it was not clear which of the two chromosomes played the principal role in sex determination.
Subsequently, Hashimoto (1933) obtained a very interesting result related to the W chromosome. He
found that the polyploid individuals such as ZZW and ZZWW were phenotypically normal females, and
postulated that in B. mori, the female mode of development was determined by the presence of a single
W chromosome regardless of the number of autosomes or Z chromosomes present. Based on these
studies, the existence and function of the W chromosome was almost certain. However, there were no
methods at that time to identify the W chromosome cytologically. Moreover, it was very difficult to
prove the existence of W by genetic experimentation, and despite the fact that many morphological gene
loci had been found on the Z chromosome, none had been found on the W chromosome. Furthermore,
recombination or crossing over is restricted to males in B. mori.
Tazima proposed, “If we can translocate any one autosome that carries dominant genes for
noticeable traits to the W chromosome, we will be able to discriminate females and males based on these
traits easily”. In that way, the effort to isolate translocations between the W chromosome and autosomes
that was, isolation of the “sex-limited strains” was initiated.
(2A). pSa +p W chromosome (T(W;2) pSa +p chromosome) (sex-limited sable)
In the course of investigating chromosomal aberrations induced by X-rays, Tazima obtained a

strain in which two copies of chromosome 2 were attached (marked by +p and pSa where +p = normal
larval marking pSa = sable marking). Surprisingly, in 1941, he discovered a translocation between W and
this attached chromosome 2. In only one of the batches isolated in the study the sex ratio was distorted
with all females displaying +p pSa traits and all males displaying p (plain). There were no +p pSa males in
this batch. He concluded that the attached chromosome in the sex-segregating strain (marked by +p and
pSa) had attached to the W chromosome. He further hypothesized that this W chromosome could be used
as a marker for sex distinction in silkworm larvae. Tazima tested his assertion by crossing this female
with a p male and observing the pattern of segregation in the next generation. He named the strain with
this chromosome, which we now refer to technically as (T(W;2)pSa +p ), the W-PSa strain (Tazima,
1941, 1944).
Subsequently, Tazima tested if it was appropriate to use the T(W;2)pSa +p for commercial breeding
by determining the growth curves for T(W;2)pSa +p larvae in the final instar. In normal strains, the
growth curve for females was 20% higher than that for males. However, in the W-PSa strain, the growth
curve for females was lower than that for males. He thought that this handicap was due to the extra
chromosome 2 translocated to the W chromosome (partial triploid status). Therefore, he planned to
remove the excess part of the translocated chromosome by irradiation with X-rays. In the course of the
experiment, several new larval marking females (e. g., W-P (T(W;2)+p), W-Sa (T(W;2)pSa and W-B
(T(W;2)pB) were isolated as byproducts (Fig. 1B, C, and D). He also verified Hashimoto’s observations
and concluded that the femaleness of B. mori is determined by the presence or absence of the W
chromosome. These processes were described in detail by Tazima (2001), who had worked toward the
practical use of the W-translocation strain for many years. In 1976, selection of a strain appropriate for
commercial breeding was finally completed (Tazima, 2001).
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(2B). pSa +p W +od chromosome
Tazima continued to induce chromosomal aberrations. After exposing female pupae to 40 ºC for
24 h, he obtained a complex translocation, pSa +p W +od, which was presumably the product of a
connection between the short fragment of the Z chromosome that included the +od gene (od; distinct
translucent, 1-49.6) and the pSa +p W chromosome (Fig. 1A) (Tazima, 1948). From the results of a
dissociation experiment with the pSa +p W +od chromosome, Tazima concluded that the chromosome 2
fragment containing pSa +p was translocated to the left end of the W chromosome, and the Z chromosome
fragment containing the +od gene was translocated to the right end via an exceptional crossover between
the Z and pSa +p W chromosomes (the right-end model) (Tazima, 1948, 1964). However, as will be
mentioned later, our recent results appear to support the left-end model (Fujii et al., 2006).
Fig. 1. Visible markers translocated from autosomes to the W chromosome. These markers are very useful to
distinguish B. mori males from females in larval stages. (A) Fifth instar larvae of the sex-limited strain
carrying the pSa +p W +od chromosome. The female has normal and sable skin due to the genotype od/ pSa
+p W +od, p/p, whereas the male has translucent and plain skin due to the genotype od/od, p/p. (B) Sex-
limited W-P strain (Chinese 137). The female has normal larval marking (Z/T(W;2)+p, p/p), whereas the
male has white, plain skin (Z/Z, p/p). (C) Mosaic larvae. The female region has brown skin
(Z/T(W;2)pSa, +p/+p) and the male region has white skin (Z/Z, +p/+p). (D) Sex-limited W-B strain. The
female has black skin (Z/T(W;2)pB, +p/+p), whereas the male has white normal larval marking skin (Z/Z,
+p/+p). (E) Fifth-instar larvae of the Z101 strain. (a): Translucent and zebra marking female (Z(od)/
T(W;3)Ze, p/p). (b): Translucent and plain skin male (Z(od)/Z(od), p/p). (c): Normal skin and sable male
(Z (od) /Df( pSa +p W +od)Fem, p/p). (F) Sex-limited Zebra-W strain. The female has zebra marking
(Z/T(W;3)Ze, p/p) whereas the male lacks markings (Z/Z, p/p).
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(2C). Female-killing chromosome (Df(pSa +p W +od)Fem chromosome)
During the course of dissociation experiments using the pSa +p W +od chromosome, Tazima (1948)
found an exceptional individual that was phenotypically male and marked with +p, pSa and +od among the
progeny of an X-ray irradiated Z/pSa +p W +od female. In a subsequent study, he presumed that this
exceptional male had a mutant W chromosome that was generated by the deletion of the female-
determining region of the pSa +p W +od chromosome (Tazima, 1952). For convenience, the mutant W
chromosome was designated Df(pSa +p W +od)Fem (Fujii et al.,2006). Tazima (1952) proved that a
female with Df(pSa +p W +od)Fem dies during embryogenesis, whereas a male with Df(pSa +p W +od)Fem
exhibits normal viability. Based on genetic analyses, it was presumed that Df(pSa +p W +od)Fem was a
female-killing chromosome and that female killing was independent of the Z chromosome (Tazima, 1952).
Fig. 2 Visible markers translocated from autosomes to the W chromosome. These markers are very useful to
distinguish males from females and identify mosaic individuals in embryonic stages. (A) Mosaic eggs
laid by a Z/T(W;10)+w-2, mo/mo, w-2/w-2 female crossed with a w-2/w-2 male. Black eggs and black
regions in the egg are female, whereas white eggs and white regions are male. (B) Mosaic moths with the
egg (eye) color marker gene (+w-2/w-2 and w-2/w-2). Black eyes are due to +w-2/w-2, whereas white eye are
due to w-2/w-2. (C) Bilateral mosaic larva with “multilunar” larval marking. (D) Bilateral mosaic larva
with “striped” larval marking. (E) Several cocoon colors. (F) Cocoons spun by the sex-limited yellow
cocoon strain. The yellow cocoon is female (Z/T(W;2)Y), whereas the white cocoon is male (Z/Z).
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Recently, Fujii et al. (2006) analyzed the genetic behavior of the Df(pSa +p W +od)Fem
chromosome during male meiosis using phenotypic and molecular markers (Z101 strain) (Fig. 1E). In
that analysis, it was revealed that Df(pSa +p W +od)Fem was connected to the deleted Z chromosome and
that this fused chromosome behaves as a Z chromosome during male meiosis (Fig. 3). Furthermore, a
ZZW-type triploid female with Df(pSa +p W +od)Fem is viable, whereas a ZW-type diploid female with
Df(pSa +p W +od)Fem dies during embryogenesis (Fujii et al., 2006). Therefore, we concluded that the
Df(pSa +p W +od)Fem chromosome does not have a female-killing factor, but rather partial deletion of the
Z chromosome causes death of ZW-type diploid females with Df(pSa +p W +od)Fem (Fujii et al., 2006).
Based on these results, it was assumed that the fused chromosome composed of the deleted Z and
the Df(pSa +p W +od)Fem chromosome was generated by a single translocation event between pSa +p W
+od and Z. This complicated chromosome was designated DfZ-DfW (Fujii et al., 2006) (Fig. 3). This
assumption strongly supports the left-end model for the structure of the pSa +p W +od chromosome. We
have described the W chromosomal region of the Df(pSa +p W +od)Fem chromosome in the following
section.
od Sa
Fig. 3. Schematic representation of a chromosomal translocation between the + p +p and Z chromosomes.
(A) The +od pSa +p W chromosome. The positions of W-specific RAPD markers are indicated to the right
of the W chromosome. Fem; putative female-determining gene. (B) Schematic representation of
chromosomal translocation between the +od pSa +p W chromosome and the Z chromosome, and
generation of the DfZ-DfW chromosome.
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(2D). T(W;3)Ze chromosome (sex-limited zebra)
Several researchers undertook the challenge of inducing new translocations between the W
chromosome and an autosome carrying dominant alleles of genes with noticeable traits (egg color, larval
marking, and cocoon or blood color). Hashimoto (1948) was able to isolate a translocation of a
chromosome 3 fragment bearing the Ze (zebra) marker to the W chromosome (T(W;3)Ze) after γ-ray
irradiation. In the resulting strain with the T(W;3)Ze chromosome, female larvae had zebra markings,
whereas male larvae had whitish skin (Fig. 1F).
(2E). Male-killing factor derived from the T(W;3)Ze chromosome
As described above, Hashimoto (1948) isolated a translocation between the W chromosome and
chromosome 3 bearing the Ze gene (Fig. 1F). As a result, T(W;3)Ze and Df(3)Ze chromosomes were
generated. Hashimoto (1953) showed that Z/T(W;3)Ze, 3/Df(3)Ze females were viable, whereas Z/Z,
3/Df(3)Ze males die during embryogenesis. Based on these observations, he concluded that T(W;3)Ze
was a product of reciprocal translocation between W and chromosome 3. Furthermore, he suggested that
T(W;3)Ze lacks the part of W that normally contains the putative male-killing factor, which according to
him was instead connected to the Df(3)Ze chromosome. Unfortunately, the Df(3)Ze chromosome was
lost before detailed analyses could be conducted.
(2F). T(W;10)+w-2 chromosome (sex-limited black egg)
Egg color results from pigments produced in cells of the serosa, a single-layer membrane that
covers the embryo. The cells of the serosa are derived from cleavage nuclei such that the genotype of
serosa cells is identical to that of the embryo. The abundance of egg color (serosa color) is a unique
feature of the silkworm. Normal eggs have dark brown (black) pigmentation, whereas one of the
mutants, w-2 (white 2 gene), has yellowish-white pigmentation. Tazima et al. (1951) were able to isolate
a translocation of a chromosome 10 fragment bearing the +w-2 gene to the W chromosome (T(W;10)+w-2)
after irradiation with X-rays. In this strain (T(W;10)+w-2), white eggs were males of the genotype Z/Z, w-
2/w-2 and black eggs were females of the genotype Z/T(W;10)+w-2, w-2/w-2. In addition to being helpful
for determining the sex at the egg stage, the T(W;10)+w-2 chromosome was also useful to detect mosaic
eggs (Fig. 2A).
(2G). T(W;2)Y chromosome (sex-limited yellow cocoon)
Many cocoon color mutants have been maintained in Japan. A white (colorless) cocoon is
conventionally considered to be normal, as most silkworms reared in Japan for industrial purposes have
white cocoons. However, during the long history of sericulture, strains with other cocoon colors have
been observed and maintained, including yellow, golden yellow, pinkish, or green (Fig. 2E). Yellow and
pinkish colors are attributed to the presence of carotenoids, carotenes, and xanthophylls derived from
mulberry leaves. Green color is due to flavonoids (Doira, 1978). However, the expression of cocoon
color is complicated, as there appear to be many genes related to permeability of pigments in the
alimentary canal and silk glands. In general, yellow-blooded larvae are also yellow cocoon spinners
(Doira, 1978). Y is an allele of the yellow blood gene (Y, 2-25.6). In a larva with the Y allele, the
hemolymph is deep yellow in color because carotenoids from mulberry leaves pass through the digestive
organs.
To establish a sex-limited yellow cocoon strain, Kimura et al. (1971) used γ-ray or X-rays to
irradiate a total of 707 female pupae of a strain with a normal W chromosome and with the Y allele from
1961 to 1969 (nine years). After irradiation of the female pupae, the researchers crossed the emerged
female moths with white cocoon males (white blood, +Y/+Y), reared female individuals, crossed them in
the same manner, and investigated the subsequent generations. A total of 4,502 batches of eggs were
analyzed over the 9-year period; only 1 batch was observed in which all the females made yellow
cocoons and all the males made white cocoons. Thus, Kimura et al. (1971) were able to isolate a
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translocation of a chromosome 2 fragment bearing the Y allele to the W chromosome, and obtained a
sex-limited yellow cocoon strain (T(W;2)Y ). In that strain, females had yellow cocoons (Z/T(W;2)Y,
+Y/+Y,) and males had white cocoons (Z/Z, +Y/+Y) (Fig. 2F). This sex-limited yellow cocoon strain did
not have evident physiological defects (Kimura et al., 1971). However, during a subsequent breeding
process aimed at the commercial use of this strain, several physiological defects (less healthy, a lighter
weight cocoon shell and proctocele-larvae) were recognized in females containing the T(W;2)Y
chromosome (Niino et al., 1987). To eliminate these defects, Niino et al. (1988) irradiated γ-ray
irradiated this strain and isolated a derivative in which the non-Y portions of the translocated
chromosome 2 fragment were deleted.
3. Why did we analyze the W chromosome?
The W chromosome was interesting from the point of view of sex determination. One example of a
successful sex determination analysis in B. mori came from studying a heritable mosaic strain with an
interesting allele of the mo gene (Goldschmidt and Katsuki, 1931). In the normal strain (+mo/+mo), the
eggs contain one nucleus. However, in the mosaic strain, the eggs laid by a female (mo/mo) contain two
nuclei due to a failure to eliminate one of the polar bodies, and both nuclei are fertilized. This mosaic
strain has been used to study topics such as developmental biology and genetic pathology (Abe et al.,
1990, Shimada and Kobayashi, 1992). Using a mosaic strain with the T(W;2)pSa, T(W;2)pB or
T(W;10)+w-2 chromosome, sexual mosaics such as gynandromorphs that contain distinct boundaries
between female and male tissues (cells) can easily be obtained at the embryo, larva, and moth stages
(Figs. 1C, 2A, 2B, and 2C). These facts strongly indicate that no diffusible substances such as sex
hormones found in mammals influence sex determination or sexual development in B. mori. Moreover,
the data suggest that a female or male is genetically determined only by the presence or absence of the W
chromosome.
Additionally, an interesting feature of the W chromosome is that it does not recombine with the Z
chromosome or autosomes. Without recombination, the W chromosome remains static, undergoing no
change during transmission from mother to daughter. Even assuming that there is a gradual accumulation
of nucleotide changes over time, it seems unlikely that more extensive restructuring would occur.
Therefore, the structure of W should be unusually stable. Another interesting feature of the sex
chromosomes in B. mori is that although there is evidence for the presence of many genes on Z (Fujii et
al., 1998; Koike et al., 2003), the W chromosome is, by contrast, devoid of functional genes except for
the putative Fem gene. Therefore, we wished to analyze the nucleotide sequence of the W chromosome
with the idea that it might reveal aspects of chromosome function and sex determination.
As mentioned above, the main focus of study of the B. mori W chromosome was for the practical
use of the resulting strains. As a consequence, a molecular biological study of the W chromosome was
not performed for many years even after it was used in practice. Promboon et al. (1995) constructed a
linkage map of random amplified polymorphic DNAs (RAPDs). Subsequently, Yasukochi (1998)
created a dense genetic map based on 1018 molecular markers. Still later, an amplified fragment length
polymorphism (AFLP) map (Tan et al., 2001), a simple sequence repeat-based consensus linkage map
(SSR) (Miao et al., 2005), and a single nucleotide polymorphism linkage map (SNP) (Yamamoto et al.,
2006) were constructed. However, no W-specific markers of B. mori were reported in these studies.
Recently, a draft sequence of the genome of the p50 strain of B. mori was constructed by 3-fold whole-
genome shotgun (WGS) sequencing by Mita et al. (2004) in Japan and, in addition, a draft sequence of
the p50 strain was constructed by 5.9-fold WGS sequencing in China (Xia et al., 2004). However, these
nucleotide sequence data were obtained using only the male (ZZ) genome. Until date, systematic
molecular analysis of the W chromosome of B. mori has not been reported.
To analyze the W chromosome molecular biologically, Abe et al. (1995) felt that the acquisition of
a W-specific nucleotide sequence was very important as a first step. They attempted to obtain a RAPD
marker on the W chromosome using a large set of arbitrary 10-mer primers. Unexpectedly, the W-
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translocation strains have also proved very useful for molecular biological analysis of the W
chromosome (see below).
(3A). W-specific RAPD markers
Initially, we used the T(W;2)+p chromosome and not the normal W chromosome as template DNA
for polymerase chain reaction (PCR) aimed at detection of W-specific RAPD markers for two reasons.
First, if there are polymorphisms in the normal W chromosome at the molecular level, we cannot
discriminate the differences based on larval appearance. Therefore, it is necessary to determine the
standard type of W chromosome. Second, even if a given W-specific RAPD marker is derived from the
chromosome 2 fragment and not the W chromosome region, it should be possible to access the W
chromosome region via a chromosome walk. For these reasons, we used genomic DNA from males and
females of the Chinese137 strain (Fig. 1B) with the T(W;2)+p chromosome as a PCR template and
screened approximately 400 arbitrary 10-mer primers for their ability to serve as RAPD markers.
Fortunately, we obtained one female-specific RAPD marker, Female-218 (Abe et al., 1995, 1996). The
Female-218 RAPD marker resulted in the amplification of a product from the T(W;2)pSa and T(W;2)pB
chromosomes, but not from the normal W chromosome (Ohbayashi et al., 1996). At that time, whether
the Female-218 RAPD marker was derived from the W region or the chromosome 2 fragment region
was not known. Recently, it was clarified that the Female-218 RAPD marker is localized to the
chromosome 2 fragment region of the translocated W chromosomes (T(W;2)+p, T(W;2)pSa and
T(W;2)pB) according to the results of detachment experiments (Yokoyama et al., 2003). More recently,
we found another chromosome 2 fragment-specific RAPD marker, W-Maji, while screening another set
of arbitrary 10-mer primers (Fujii et al., 2006).
Thus, it is possible to find female-specific RAPD markers using genomic DNA from the
Chinese137 strain (with T(W;2)+p) as a PCR template. However, obtaining a RAPD marker for the W
chromosome region remained a goal for our group, as this would allow study of the normal W
chromosome. In subsequent analyses, we used genomic DNA from several strains with normal W
chromosomes as templates. Fortunately, we obtained 12 W-specific RAPD markers from the normal W
chromosome (Abe et al., 1998a, 2005b).
(3B). DNA sequence of the W-specific RAPD markers
The deduced amino acid sequences of 11 of the 12 W-specific RAPD markers show similarity to
the amino acid sequences of retrotransposons from various organisms reported previously (exception:
W-Musashi; Abe et al., 1998a, 2005b). Moreover, almost all amino acid sequences of W-specific RAPD
markers contain boundaries of two or three retrotransposable elements (Abe et al., 1998a, 2005b).
(3C). W-specific lambda phage and BAC clones
To analyze the W chromosome in detail, we have constructed genomic DNA lambda phage
libraries of B. mori. From these libraries, we obtained two lambda phage clones containing the W-
Kabuki and W-Samurai RAPD sequences. The DNA sequences of these two phage clones comprise the
nested structure of several retrotransposable elements (Ohbayashi et al., 1998; Abe et al., 2000, 2005b).
A B. mori bacterial artificial chromosome (BAC) library, the RPCI-96 library, has been
constructed in Japan using genomic DNA extracted from female and male mixed population of the p50
silkworm strain (Mita, unpublished data). In addition, two other B. mori BAC libraries, p50 and C108,
were also constructed using genomic DNA of the p50 and C108 strains, respectively (Wu et al., 1999).
We used the W-specific RAPD marker sequence to identify W-specific BAC clones and subjected these
clones to shotgun sequencing. However, due to the presence of many repetitive DNA elements, in
particular the non-long terminal repeat (LTR) retrotransposons BMC1 (Abe et al., 1998b) and Kendo
(Abe et al., 2002), assembly of the shotgun clone sequence did not result in a single contig. However,
although assembly of the shotgun sequence proved to be difficult, sub-sections of the clone sequences
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could be assembled into contigs and had been analyzed in detail (Abe et al., 2005a). As shown in Fig. 4,
the structural features of the W chromosome of B. mori are quite different from structures typical of
other chromosomes. Many LTR and non-LTR retrotransposons, retroposons (Bm1), DNA transposons
and their derivatives have accumulated on the W chromosome.
Fig. 4. The presence of transposable elements on the W chromosome sequence. Nested transposable elements
in the BAC clone that includes the W-Kabuki RAPD marker sequence. This figure is based on the
recent DNA sequence information. See Abe et al (2005a) for details.
4. Presence of W-specific RAPD markers in silkworm strains maintained in Japan
Abe et al. (2005b) determined the presence or absence of W-specific RAPD markers in the
silkworm strains maintained in Japan. All strains with normal W chromosomes used in the study were
positive for all the W-specific RAPD markers, with the exception of the W-Kamikaze marker. These
results strongly indicate that the level of diversity in the W chromosome is low in the silkworm strains
maintained in Japan.
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5. Presence of W-specific RAPD markers in W chromosome variants
(5A). [T(W;3)Ze chromosome]
As mentioned above, Hashimoto (1953) hypothesized that T(W;3)Ze chromosome was a product
of reciprocal translocation between W and chromosome 3. If reciprocal translocation occurred, then the
W region in the T(W;3)Ze chromosome should be shorter than that in the original W chromosome.
Moreover, if the W region was shortened, the fact that it still functioned as a W, (i.e., a female-
determining) chromosome, suggests that the putative Fem was still present. Thus, we concluded that it
was important to determine whether or not the W region of T(W;3)Ze is indeed shorter than that in the
original W chromosome. However, deletions of the W chromosome could not be detected using
conventional genetic methods. To confirm this deletion, a molecular biological comparison between the
T(W;3)Ze chromosome and the original W chromosome used in the translocation experiment would be
useful. However, the original strain used in the translocation experiment is not available for comparison
because there is no precise rearing record. Fortunately, however, the normal W chromosomes of strains
maintained in Japan (the Japanese-W-Eve type) are almost identical, suggesting that they provide a
suitable point of comparison to the putative W deletion strain T(W;3)Ze (Abe et al., 2005b). Hence,
partial deletion of the W in T(W;3)Ze can be detected by the disappearance of W-specific RAPD
markers isolated on the basis of recognition of the normal W. The W chromosome region of the
T(W;3)Ze chromosome lacks 2 of the 12 W-specific RAPD markers (W-Mikan and W-Samurai) (Abe et
al., 2005b). These results strongly indicate that the W chromosome region containing the W-Samurai
and W-Mikan markers was deleted in the T(W;3)Ze chromosome due to reciprocal translocation (Fig.5).
Fig.5 Schematic representation of a

reciprocal chromosomal translocation
between W and chromosome 3.
Reciprocal translocation between W
and chromosome 3 (Zebra), and
generation of a male-killing
chromosome and the T(W;3)Ze
chromosome of the sex-limited Zebra-
W strain.
(5B). [T(W;10)+w-2 chromosome]
As discussed above, we thought that the W chromosomes of other sex-limited strains were not the
result of simple fusion of an autosomal fragment to the end of an unaltered W chromosome but, rather
are the products of reciprocal translocation accompanied by the breakage (deletion) of the W
chromosome. Therefore, we next looked at the presence or absence of the W-specific RAPD markers on
the T(W;10)+w-2, T(W;2)+p, and T(W;2)Y chromosomes. The W chromosome region of the T(W;10)+w-2
chromosome was observed to lack 1 of the 12 W-specific RAPD markers (W-Mikan) (Abe et al.,
2005b). This result strongly indicates that the region containing the W-Mikan RAPD marker was deleted
in the T(W;10)+w-2 chromosome (Fig. 6).
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Fig..6 Schematic representation of

reciprocal chromosomal translocation
between W and chromosome 10.
Reciprocal translocation between W (+w-2)
and chromosome 10, and the generation of
the T(W;10)+w-2 chromosome of the sex-
limited Black-egg-W strain.
(5C). [T(W;2)+p chromosome]
We did not detect deletion of the W chromosome region in the T(W;2)+p chromosome using W-
specific RAPD markers (Abe et al., 2005b). We belived that the breakpoint of this T(W;2)+p
chromosome was close to the telomere of the W chromosome.
(5D). [T(W;2)Y chromosome]
We next analyzed the strains with each of three T(W;2)Y chromosomes (T(W;2)Y-Abe, T(W;2)Y-
Chu, and W(W;2)Y-Ban) maintained by three independent research groups. We determined the presence
or absence of the W-specific RAPD markers on the three T(W;2)Y chromosomes. T(W;2)Y-Chu
contained 6 of the 12 W-specific RAPD markers (W-Rikishi, W-Yukemuri-L, W-Yukemuri-S, W-
Bonsai, W-Samurai, and W-Mikan). However, both T(W;2)Y-Abe and T(W;2)Y-Ban contained only 1 of
the 12 W-specific RAPD markers (W-Rikishi) (Abe et al., 2008). Based on these results, we suspected
that the original W chromosome of T(W;2)Y-Abe and -Ban differed from the Japanese-W-Eve type.
However, the original W chromosome of the T(W;2)Y was the W chromosome of the C125 strain
(Kimura et al., 1971), which has the normal Japanese-W-Eve type (Abe et al., 2008). Moreover, the
original T(W;2)Y chromosome was clearly produced by only one event (translocation) in a single female
isolated from the C125 strain (Kimura et al., 1971). Therefore, we examined another possibility namely
that all the marker-positive regions were deleted from the T(W;2)Y-Abe and -Ban chromosomes except a
region containing the W-Rikishi RAPD marker and the putative Fem gene.
To determine the presence or absence of regions around the W-Rikishi RAPD marker sequence in
the T(W;2)Y-Abe and -Ban chromosomes, we obtained a BAC clone (1C7C) positive for the W-Rikishi
RAPD marker. Following shotgun sequencing of the 1C7C BAC clone, no single contig could be
constructed due to the presence of repetitive DNA elements. However, we identified many regions
containing boundaries between retrotransposable elements, which were used to design 7 new W-specific
PCR markers. The T(W;2)Y-Abe and -Ban types were positive for all 7 W-specific PCR markers (Abe et
al., 2008). These results indicate that almost all regions of the W chromosome were deleted from the
T(W;2)Y-Abe and -Ban chromosomes, while the region containing the sequence from the 1C7C BAC
clone and the putative Fem gene remained.
The results of the marker analysis help us learn the origins of these translocations. The T(W;2)Y-
Chu, -Abe, and -Ban chromosomes were thought to be produced in the following manner. First, the
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region of the W chromosome containing the 6 W-specific RAPD markers, W-Kabuki, W-Kamikaze, W-
Musashi, W-Sasuke and W-BMC1-Kabuki, was deleted by X-ray irradiation. Then, the chromosome 2
fragment containing the Y gene was translocated to a region that contains the putative Fem gene and is
positive for a different set of 6 W-specific RAPD markers (W-Mikan, W-Samurai, W-Bonsai, W-
Yukemuri-L, W-Yukemuri-S, and W-Rikishi). The resulting chromosome likely represented the
T(W;2)Y-Chu chromosome. The region of the W chromosome containing the 5 W-specific RAPD
markers, W-Mikan, W-Samurai, W-Bonsai, W-Yukemuri-L, and W-Yukemuri-S, was further deleted
following irradiation during the breeding process (Fig.7). The resulting changes are thought to be present
in the T(W;2)Y-Abe and -Ban chromosomes (Abe et al., 2008).
Fig. 7. Schematic representation of generation of sex-limited Yellow-cocoon-W strains. Generation of the

T(W;2)Y-Chu and T(W;2)Y-Abe chromosomes. Y, the yellow blood gene on chromosome 2.
(5E). [Df(pSa +p W +od)Fem chromosome (DfZ-DfW chromosome)]
Fujii et al. (2006) revealed that Df(pSa +p W +od)Fem (DfZ-DfW) contains the left side of W that
includes 3 of the 12 W-specific RAPD markers (W-Mikan, W-Samurai, and W-Bonsai) (Fig. 3). These
results indicate that the W-specific RAPD markers are arranged in the order W-Mikan, W-Samurai, and
W-Bonsai (starting from the left end of the W chromosome), and that the male-killing factor
(Hashimoto, 1953) is located between the left end and the W-Bonsai marker region. However, we
believe that a true male-killing factor between the left end and the W-Bonsai RAPD marker does not
exist because males with the DfZ-DfW chromosome have normal viability. The DfZ-DfW chromosome
includes a fragment of the left side of the W chromosome that includes the above 3 W-specific RAPD
markers. If we assume that a male-killing factor does not exist on the W chromosome of B. mori, then
why would the Z/Z, 3/Df(3)Ze males die before completing embryogenesis? It is well established that
some genes are haploinsufficient such that the presence of only one normal copy of the gene is not
sufficient for normal function. Therefore, one possible explanation of the embryonic death observed in
the Z/Z, 3/Df(3)Ze males is that a haploinsufficient gene is required during embryogenesis on the
chromosome 3 around the Ze locus.
215
(5F). [W(B-YL-YS)Ze chromosome]
We have reasoned that if more W

chromosome variants could be isolated,
then we could map W-specific RAPD
markers and the putative Fem gene on the
W chromosome to a smaller region.
Therefore, X-ray irradiated female pupae
of the sex-limited Zebra-W strain,
containing the T(W;3)Ze chromosome
and isolated an exceptional zebra male
larva. After the male was allowed to
copulate with the females, genomic DNA
was extracted from the legs of the male
and used as a template for PCR. Three W-
specific RAPD markers (W-Bonsai, W-
Yukemuri-L, and W-Yukemuri-S) were
detected in this male. These results
indicate that breakage occurred on the
T(W;3)Ze chromosome and that the
resulting derivative W;3 translocated
fragment contains the detected RAPD Fig. 8 Schematic representation of X-ray induced
markers as shown in Fig. 8. We generation of the W(B-YL-YS)Ze chromosome from the
designated this chromosome W(B-YL- T(W;3)Ze chromosome. Shown, generationof the W(B-YL-
YS)Ze chromosome by X-ray irradiation. See Abe et al.
YS)Ze (Abe et al., 2008). (2008) for details.
(5G). [Z1 and ZeWZ2 chromosomes]
When we have applied X-ray irradiation to break the T(W;3)Ze chromosome, we also obtained a
fragment of the W chromosome, which we designated ZeW. We determined that the ZeW fragment was
positive for 3 of the 12 W-specific RAPD markers (W-Bonsai, W-Yukemuri-L, and W-Yukemuri-S).
Unexpectedly, it revealed that the Z chromosome was also broken into a large fragment (Z1) with +sch (1-
21.5) and a small fragment (Z2) with +od (1-46.9). Moreover, we found that the ZeW fragment was
connected to the Z2 fragment. We have designated this joined chromosomal fragment ZeWZ2 (Fig. 9)
(Fujii et al., 2007).
We have next analyzed the genetic behavior of the Z1 and ZeWZ2 fragments during meiosis in males
(Z1 ZeWZ2/Z) and females (Z1 ZeWZ2/W). The Z1 fragment and the Z or W chromosome segregated
properly but non-disjunction was observed between the ZeWZ2 fragment and the Z chromosome, and
between the ZeWZ2 fragment and the W chromosome. Furthermore, the 2A:Z/Z1males, which were the
products of non-disjunction between ZeWZ2 and W, had observable phenotypic defects. Z/Z1 male moths
could walk and copulate with females but were incapable of flapping their wings. To analyze why the
Z/Z1 males could not flap their wings, we looked at the indirect flight muscles in these males. In the
“flapless” males, the indirect flight muscles were found to be broken or decayed (Fujii et al., 2007). Z1
was not considered as the cause of the flapless phenotype, because the Z/Z1 ZeWZ2 males could flap their
wings vigorously. The Z/Z1 ZeWZ2 status is thought to be a “balanced translocation” in B. mori (Fujii
et al., 2007). In humans, balanced translocation carriers have no clinical symptoms (reviewed in
Emanuel and Shaikh, 2001).
216
Fig. 9. Isolation of changes to the T(W;3)Ze chromosome induced by X-ray irradiation. Males of the genotype
sch +od/sch +od were crossed to X-ray-irradiated females. A chromosomal fragment dissociated from the
T(W;3)Ze chromosome is designated as the ZeW fragment. The ZeW fragment is positive for three of 12
W-specific RAPD markers (W-Yukemuri-L, W-Yukemuri-S, and W-Bonsai). The long fragment of the
Z chromosome bearing the +sch locus (1-21.5) is designated Z1, and the short fragment of Z bearing the
+od locus (1-49.6) is designated Z2. The chromosome composed of the ZeW fragment and the Z2 fragment
is designated ZeWZ2. See Fujii et al (2007) for details.
Moreover, in Drosophila melanogaster, the genes that encode major myofibrillar proteins such as
actin, myosin, and tropomyosin are haploinsufficient for flight. For example, haploidy for the myosin
heavy chain (MHC) gene results in its reduction in adult thoracic, leg, and larval muscles of the fly. This
affects the function of the flight muscles in haploid animals, although the leg and larval muscles function
normally (Bernstein et al., 1983; Mogami et al., 1986). In B. mori, the absence of dosage compensation
has been suggested based on gene expression patterns. Twelve of 15 genes on the Z chromosome are
expressed more abundantly in males (ZZ) than in females (ZW) (Suzuki et al., 1998, 1999; Koike et al.,
2003). However, the mRNA levels of 3 of 15 genes are equivalent in males and females, or are higher in
females (Koike et al., 2003). Thus, it is likely that expression is regulated on a gene-by-gene basis.
Therefore, we presume that haploinsufficient gene(s) is/are involved in indirect flight muscle
development that is present on the Z2 region of the Z chromosome in B. mori. However, if there are
haploinsufficient genes on the Z chromosome, then the expression of the gene(s) must necessarily be up-
regulated during female development in order to compensate for the presence of only one copy of the
gene(s) in females (Z/W).
217
6. Order of W-specific markers and position of the putative Fem gene on the W chromosome
The presence or absence of molecular markers in W chromosome variants makes it possible to

map the positions of the markers relative to the putative Fem gene (Fig. 10). The T(W;10)+w-2
chromosome does not contain the W-Mikan RAPD marker and the T(W;3)Ze chromosome does not
contain the W-Samurai and W-Mikan RAPD markers (Abe et al., 2005b). The female-killing DfZ-DfW
chromosome contains the W-Bonsai, W-Samurai, and W-Mikan RAPD markers (Fujii et al., 2006).
Moreover, the W(B-YL-YS)Ze chromosome contains the W-Yukemuri-L, W-Yukemuri-S, and W-
Bonsai RAPD markers (Abe et al., 2008). From these results, although the order of W-Yukemuri-L and
W-Yukemuri-S could not be determined, that of 5 W-specific RAPD markers could be determined:
W-Mikan, W-Samurai, W-Bonsai, and W-Yukemuri-S or W-Yukemuri-L (from the left end of the
W chromosome) (Fig. 10). Moreover, the evidence suggests that the regions containing these 5 W-
specific RAPD markers do not contain the putative Fem gene, as their loss has no effect on sex
determination. The T(W;2)Y-Abe and -Chu chromosomes do not contain the W-Kabuki, W-Kamikaze,
W-Musashi, W-Sasuke, W-Sakura, and BMC1-Kabuki RAPD markers. However, the T(W;2)Y-Abe
chromosome contains the W-Rikishi RAPD marker. From these results, the order of W-Kabuki,
W-Kamikaze, W-Musashi, W-Sasuke, W-Sakura, and BMC1-Kabuki RAPD markers could not be
determined, but we can concluded that the W-Rikishi RAPD marker is distal to the right end of the W
chromosome (Fig. 10). This positional information should be of significant help towards the goal of
cloning the putative Fem gene.
Fig. 10. Higher-resolution mapping of W-specific RAPD markers and the putative Fem gene on the W
chromosome using W chromosome variants. The order of six W-specific RAPD markers
(W-Kabuki, W-Kamikaze, W-Musashi, W-Sasuke, W-Sakura and BMC1-Kabuki) and two W-
specific RAPD markers (W-Yukemuri-L and W-Yukemuri-S) could not be determined. Mapping
of the other markers is shown. For the T(W;3)Ze, T(W;10)+w-2 and DfZ-DfW chromosomes, see
Abe et al. (2005b) and Fujii et al. (2006).
218
7. Relationship between sex-heterochromatin body and W chromosome
Cytologically, a sex-heterochromatin body (SB), which was observed in the nuclei of lepidopteran
females, had been deduced to be composed of condensed W chromosomes (Ennis, 1976; Traut and
Marec, 1996). The W chromosome of B. mori had been assumed to be composed almost entirely of
heterochromatin because the number of SBs in the nucleus of somatic cells in polyploid females
corresponds to the number of W chromosomes (Ito, 1977). However, Traut et al. (1986) showed that
deletion of approximately half of the W chromosome in Ephestia results in a drastic size reduction of the
SB. Similarly, in B. mori, we observed several smaller SBs in a single nucleus of a cell having the
T(W;2)Y-Chu type chromosome. Moreover, no SB was detected in the nuclei of female moths having the
T(W;2)Y-Abe type chromosome. These results strongly indicate that the W chromosome region of the
T(W;2)Y-Abe type chromosome was too short to form an SB detectable by light microscopy (Abe et al.,
2008).
8. Retrotransposable elements on the W and Z chromosomes of B. mori
Although we have not yet analyzed all the W-specific BAC clones completely, several parts of the
W-specific clones, especially 45J23S containing W-Kabuki RAPD marker have been analyzed in detail.
As shown in Fig. 4, one BMC1(a), an LTR retrotransposable element-like sequence FUI, an LTR
retrotransposon Yokozuna, and a retroposon Bm1 are inserted into LTR retrotransposon Kabuki.
Moreover, Kabuki is inserted into retrotransposon Kendo. Furthermore, Kendo is inserted into an
unknown sequence. The major focus of this chapter is the retrotransposable elements on the
W chromosome. A similar retrotransposable elements was observed in B. mori, by Takahashi and
Fujiwara (2002) and others (Eickbush 1995, 2002; Eickbush and Malik 2002; Matsumoto et al., 2004;
Osanai et al., 2004; and Wickter et al., 2007).
(8A). Kabuki
The Kabuki element was reconstructed by eliminating the inserted BMC1, Bm1 FUI, and
Yokozuna sequences (Fig. 4) (GenBank Accession No. AB032718). Kabuki is flanked by a 5-bp target
site duplication, 5'-CCCTT-3'. We determined the Kabuki consensus sequence. The Kabuki element is
5342 bp long and flanked by identical 182 bp sequences at both ends, which are thought to be 5’ and 3’
LTRs. The Kabuki consensus sequence contains two open reading frames (ORFs). However, Kabuki
ORF1 did not contain any cysteine and histidine (Cys) motifs found in retroviral gag genes. Kabuki
ORF2 contained four regions with sequence similarity to the functional domains —Protease (Pro),
reverse transcriptase (RT), RNase H (RH), and integrase (Int)— found in the pol region of the gypsy-Ty3
group of retrotransposons. The order of the four domains in the ORF2 of Kabuki, Pro-RT-RH-Int, (from
5' to 3') is identical to that within the retrotransposons of the gypsy-Ty3 group. Therefore, Kabuki
belongs to the gypsy-Ty3 group (Abe et al., 2000) (Fig. 11).
(8B). Yokozuna
Yokozuna is 4738 bp long, including a 208-bp LTR and a 183-bp LTR on either side. It is flanked
by a 5-bp target site duplication, 5'-TAATT-3'. Yokozuna contains a single ORF. The first domain of
ORF contains one putative Cys motif. The entire deduced ORF amino acid sequence bears strong
similarity to copia of Drosophila. Therefore, Yokozuna belongs to the Ty1-copia group (Ohbayashi
et al., 1998) (GenBank Accession No. AB014676) (Fig. 11).
(8C). BMC1
In B. mori, the BMC1 is considered a LINE-like element because it is dispersed throughout the
genome and the number of BMC1 elements is estimated to be approximately 3500 copies per haploid
genome (Ogura et al., 1994). Although the full-length BMC1 elements are dispersed throughout the
219
genome, most members are preferentially truncated to varying extents at their 5' ends. Moreover, long
ORFs could not be detected within the BMC1 full unit (Ogura et al., 1994). However, the BMC1(a)
inserted into Kabuki is a complete full-length element (GenBank Accession No. AB018558). This
BMC1 is 5091 bp long and has a 5' untranslated region (5'-UTR), two intact ORFs, and a 3'-UTR
sequence, which terminates in a poly(A) tail. The ORF1 of BMC1 encodes three putative Cys motifs,
while ORF2 encodes a protein containing an endonuclease (EN) domain and an RT domain (Abe et al.,
1998b) (Fig. 11).
(8D). Pao
The Pao element was detected in one of the spaces between the repeated codes for ribosomal
RNA. The original Pao element was 4791 bp long (including two LTRs of 634 bp), but had no codes for
RH and Int (Xiong et al., 1993) (GenBank Accession No. L09635). However, Abe et al. (2001) found
the sequence for RH and Int in this element by PCR, which was deleted from the isolate of Xiong et al.
(1993). Pao-like elements share common features that distinguish them from the other groups of LTR
retrotransposons. While the elements of Ty1-copia group encode only one Cys motif in their gag-like
region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the Int
domain of the retrotransposon polyprotein seems to be conserved in the Pao-like elements, but the
number of amino acid residues between D and E varies and is greater than 35 (Abe et al., 2001). Based
on these structural features and a comparison of the deduced amino acid sequences of the RT domain,
Abe et al. (2001) agreed with the suggestion of Xiong et al. (1993) that the Pao-like elements are
members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, the LTR retrotransposons
should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like
groups (Fig. 11).
Fig.11 Structure of six retrotransposons

of Bombyx mori. Each box with an arrow
indicates one LTR (long terminal
repeat). PBS, primer-binding site; PPT,
polypurine tract; Pro, protease domain;
RT, reverse transcriptase domain; RH,
RNase H domain; Int, integrase domain;
EN, endonuclease domain; 5’-UTR,
5’untranslated region; 3’-UTR,
3’untranslated region; poly(A), poly(A)
tail. Cys, cysteine and histidine motif are
indicated solid bar in the box of ORF.
(8E). Kamikaze
Abe et al. (1998a) identified a W-specific RAPD marker, W-Kamikaze, from B. mori, which
contains a part of a novel Pao-like retrotransposon Kamikaze. To determine the precise sequence of the
Kamikaze element, Abe et al. (2001) obtained the lambda phage clone, which contains the W-Kamikaze
RAPD sequence. The Kamikaze element was found near the left end of this phage clone. This element
220
was thought to be about 7100 bp long (GenBank Accession No. AB042120). However, based on our
recent analysis, it is now thought to be about 7300 bp long. The Kamikaze consensus sequence contains
two ORFs. ORF1 contained three putative Cys motifs. ORF2 contained Pro, RT, RH, and Int domains.
The order of five domains in the ORFs of Kamikaze, Cys-Pro-RT-RH-Int, from 5' to 3', is common to
that in the Pao-like elements (Fig. 11).
(8F). Kendo
Kendo is the most abundant non-LTR retrotransposon next to the BMC1 in B. mori genome (Abe
et al., 2002). Despite numerous sequences of B. mori, a complete full-length Kendo had not been found.
As mentioned below, the longest Kendo has been found in the Z-specific BAC clone (Koike et al.,
2003). However, in the WGS sequence data (Mita et al., 2004), we could not find longer Kendo than that
in the Z chromosome. Therefore, we think that the Kendo in the Z chromosome (Koike et al., 2003) is
the complete “full-length” element. The full-length Kendo is about 3670 bp long and contains a single
ORF encoding EN and RT domains (Fig. 11).
9. Transposable elements on the Z chromosome
Koike et al. (2003) analyzed a 320-kb sequence of the Z chromosome of B. mori. This 320-kb
sequence contains many transposable element-like sequences, 47 non-LTR retrotransposons, 50
retroposons, 10 DNA-type transposons, and other uncharacterized repetitive sequences. The non-LTR
retrotransposons BMC1, the DNA-type transposon mariner (Robertson and Asplund, 1996; Tomita et
al., 1997), and the retroposon Bm1 (Adams et al., 1986) are dispersed throughout the region of 320-kb
on the Z chromosome. However, these retrotransposons and DNA-type transposons were found to be
incomplete. As mentioned above, we think that the Kendo in this region (AB090307, nucleotide
positions: 89135–85464 from 5' to 3'; Koike et al., 2003) is the complete full-length element. It should
be noted that at that time, no LTR retrotransposons had been found in this 320-kb region. However, we
found a part of LTR retrotransposon in this region (AB090307, nucleotide positions: 34714–34358 from
5' to 3'). We determined that this sequence was a solo LTR of the LTR retrotransposon Suzuka.
Moreover, this LTR is flanked by a direct repeat (5'-GCTT-3'). This direct repeat seems to be a target
site duplication of Suzuka. Many solo LTRs of LTR retrotransposons have been reported in barley
(Shirasu et al., 2000). These solo LTRs may result from removal of the internal domain of LTR
retrotransposons by unequal crossing over or from intra-element recombination between LTRs (Fig. 12).
Similarly, in the Z chromosome of B. mori, it seems likely that recombination plays an important role in
the exclusion of the LTR type retrotransposons. We could not conclude whether the solo LTR of Suzuka
in a 320-kb region had been generated by unequal crossing over or from intra-element homologous
recombination between LTRs. Nonetheless, unequal crossing over and intra-element recombination
between LTRs are likely to be mechanisms for deletions, mutations, and decreases in the Z chromosome
size.
10. Identification of the W chromosome using W-derived BAC probes
Until recently, cytogenetic identification of the W chromosome had been considered very difficult
because the chromosomes of B. mori are small and numerous (2n = 56). Even in the pachytene
chromosomes from oocytes, it was impossible to distinguish the W chromosome from other
chromosomes. However, Sahara et al. (2003) used four W chromosome-derived BAC clones as probes
for fluorescence in situ hybridization (FISH) on chromosome preparations from female larvae of
B. mori. Although scattered hybridization signals were observed in all chromosomes, surprisingly, all
four W-BAC probes highlighted the whole of “one chromosome” in a paint-like manner. These results
strongly indicate that the chromosome painted by these W-BAC probes is the W chromosome. The type
of nucleotide sequence participating in this “painting” is unknown. However, it is likely that these four
W-BAC clones contain many repetitive sequences that are dispersed through the entire length of the W
chromosome.
221
Fig.12 Formation process of a solo-LTR in the genome. Homologous recombination between two LTRs from
the same LTR retrotransposon copy. The yellow box indicates a target site duplication. The green line
indicates an ORF of LTR retrotransposon. The target site duplication is conserved after the recombination at
the both ends of solo-LTR. The ORF and another solo-LTR generated by recombination are removed.
11. Conclusion
The W chromosome of B. mori has been artificially modified for practical use (Tazima, 1941,
1944, 1948, 1964; Tazima et al., 1951; Hashimoto, 1948; Kimura et al., 1971). In the past, the intension
was to isolate translocations of autosomal fragments to the W chromosome, rather than to delete the W
chromosome. However, in the process of generating these strains, deletions of the W chromosome were
unconsciously selected. From these strains, we can conclude that even large deletions of the W
chromosome or large translocations to W do not affect female sex determination. Therefore, we think
that a full-length, “normal” W chromosome is not essential for femaleness. Instead, we conclude from
test of the translocation and deletion strains that putative Fem genes are not distributed evenly over the
W chromosome, no gene governing femaleness is located in the regions deleted from W in T(W;2)
Y-Abe, and that although the exact length of the region can not be determined, only what appears to be
an extremely limited region around the W-Rikishi RAPD marker is required to determine the female
mode of development.
We have proposed that the W chromosome evolves more slowly than other chromosomes, as
crossing-over is restricted to males in B. mori and W is recombinationally isolated from the Z
chromosome and autosomes. Furthermore, a higher mutation rate is predicted for Z or autosomes relative
to W because of the greater number of cell divisions that occur in spermatogenesis than in oogenesis.
Indeed, many transposable elements, which have undergone little to no changes, are found on the W
chromosome (Abe et al., 2005a). However, it is likely that large-scale changes such as insertions of
transposable elements, large deletions or translocations occur more frequently on the W chromosome
than on Z or on the autosomes as these rearrangements do not affect female sex determination.
222
Chromosome fragments that have translocated to W lose the opportunity to undergo chromosomal
recombination and behave like part of the W chromosome. Conversely, chromosome fragments
translocated from W to the Z chromosome gain the opportunity to undergo chromosomal recombination.
Interestingly, lack of recombination and genetic degeneration of the W chromosome are closely
connected in the evolution of heteromorphic sex chromosomes (reviewed in Charlesworth et al., 2005).
In Diptera, the relationship between transposable elements and generation of a non-recombining Y
chromosome has been studied extensively (Charlesworth et al., 1994; Charlesworth 2001). In
Drosophila miranda, for example, chromosome degeneration is driven by accumulation of transposable
elements (Steinemann and Steinemann 1992, 1997, 1998, 2005; Steinemann et al., 1993). Apart from
these well-studied examples from Drosophila, the fate of chromosome fragments translocated onto sex
chromosomes is not well understood (reviewed in Charlesworth et al., 2005). Therefore, B. mori strains
with autosomal or Z chromosome fragments translocated to the W chromosome (such as pSa +p W +od,
T(W;2)pSa+p, T(W;10)+w-2, T(W;2)Y and T(W;3)Ze) and with W chromosome fragments translocated to
the Z chromosome (such as DfZ-DfW and ZeWZ2) will be useful for molecular biological analysis aimed
at understanding how the W chromosome has degenerated and/or evolved over time.
In the course of the Drosophila genome projects, researchers were not able to assemble Y
chromosome sequence data into a single contig due to the presence of repetitive sequences (Adams
et al., 2000). To identify new genes on the Y chromosome of D. melanogaster, another strategy
(TBLASTN search) was developed (Carvalho et al., 2001; Carvalho and Clark 2005). Recently,
numerous Y-linked repetitive sequences in the Y chromosome of the African malaria mosquito
Anopheles gambiae have been obtained (Krzywinski et al., 2004, 2005). However, no Y chromosome
genes were identified based on a TBLASTN search (Krzywinski et al., 2006). Thus, both in Drosophila
and Anopheles, molecular biological analyses of the Y chromosome are made very difficult by the
presence of unusual chromosomal features.
Many transposable elements have accumulated on the W chromosome as strata. On the other hand,
we found that the nucleotide sequences do not show the characteristics of typical transposable elements
(Abe et al., 2005a). WGS sequencing of B. mori using male genome DNA and the continuing effort to
annotate the sequence provide valuable biological information about the autosomes and the Z
chromosome (Mita et al., 2004; Xia et al., 2004). Although it has been predicted that almost all
transposable elements on the autosomes and Z chromosome have degenerated because of recombination
or rearrangement, genome studies in B. mori using male genome DNA will allow us to look at many
fragments of transposable elements and repetitive sequences. Therefore, we are using the WGS data to
analyze the uncharacterized nucleotide sequences of the W chromosome. The results indicate that these
sequences are transposable elements that do not fit into the conventional classification (Abe et al.,
manuscript submitted). The retrotransposable elements of the W chromosome of B. mori provide an
excellent model for tracing the development of the organization of the non-recombinant sex
chromosome.
12. Acknowledgements
The authors are grateful to Dr. Raman Chandrasekar, for a critical reading of this article. This work
was supported by grant from MEXT (No. 19040008 to H. A.) in Japan.
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NAL B OO
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Short Views onShort
Insect Molecular
Views Biology,
on Insect Vol.(1),
Molecular 2009
Biology
TERNA
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ËIN
 Vol. (1), 2009
APPENDIX - A
GLOSSARY OF TERMS
Accession Number: A unique identifier that is Autoradiography: A process in which radioactive

assigned to single database entry for a DNA or protein materials are incorporated into cells structures, which
sequence. are then placed next to a film or photographic
emulsion, thus forming a pattern on the film
Allele: One different variants of a gene. corresponding to the location of the radioactive
compounds within the cell.
Alignment: Refers to the procedure of comparing two
or more sequences by looking for a series of Autophagic vacuole (AV): A 2º lysosome derived
individuals characters or character pattern that are in from isolation bodies and 1º lysosomes Synonyms:
the same order in the sequences. Of the two types of cytoloysome, cytosergresome.
alignment, local and global, a local alignment is
generally the most useful. Bacterial artificial chromosome (BAC): A high-
capacity and very stable cloning vector based on the F
Amplification: An increase in the number of copies of plasmid of Escherichia coli.
a specific DNA fragment.
Bioinformatics: the science of informatics as applied
Antibody: Soluble serum proteins (immunoglobulins) to biological research. Informatics is the management
which are synthesized by a special set of cells of and analysis of data using advanced computing
immune system, the B-lymphocytes, in responses to techniques. Bioinformatics is particularly important as
antigens. an adjunct to genomics research, because of the large
amount of complex data this research generates.
Antigen: Specific chemical structure, for example a
particular sequence of a protein, which induces the Biomarker: observable change (not necessarily
production of antibodies in an organism and pathological) in the function of an organism, related to
specifically binds of antibodies. a specific exposure or event.
Annetocin: A neuropeptide hormone with high Calcium imaging: a technique designed to show the
homology to members of the Vasopressin /oxytocin calcium (Ca2+) status of a tissue or a medium, taking
superfamily of neurohypopysial hormones. Evidence advantage of calcium indicators (molecules that can
suggests that annetocin elicits stereotypical egg-laying respond to the binding of Ca2+ ions by changing their
behaviours in some invertebrates including ovulation spectral properties).
and oviposition.
Caspases: derived from cysteine-dependent aspartate-
Annotation: The prediction of genes in a genome, specific proteases to signify that they are proteolytic
including the location of protein-encoding genes, the enzymes (proteases) that specifically cleave at aspartate
sequence of the encoded proteins, any significant residues in target proteins within the cell during
matches to other proteins of known function, and the apoptosis.
location of RNA-encoding genes. Predictions are based
on gene models; e.g., hidden Markov models of introns Consensus: A single sequence that represents, at each
and exons in proteins encoding genes, and models of subsequent position, the variation found within
secondary structure in RNA. corresponding columns of a multiple sequence
alignment.
Apoptosis: The most common form of physiological
(as opposed to pathological) cell death. Apoptosis is an Chorion: Protective membrane around the eggs of
active process requiring metabolic activity by the insects.
dying cell. Often called programmed cell death,
although this is not strictly accurate. Chromosome: The DNA in a cell is divided into
structures called chromosomes. Chromosomes are
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large enough to be seen under a microscope. In Coding regions: those parts of the DNA that contain
humans, all cells other than germ cells usually contain the information needed to form proteins. Other parts
46 chromosomes: 22 pairs of autosomes and either a of the DNA may have non-coding functions (e.g. start-
pair of X chromosomes (in females) or an X stop, pointing or timer functions) or as yet unresolved
chromosome and a Y chromosome (in males). In each functions or maybe even 'noise'.
pair of chromosomes, one chromosome is inherited
from an individual's father and one from his or her Codon: a set of three nucleotide bases in a DNA or
mother. RNA sequence, which together code for a unique
amino acid. For example, the set AUG (adenine,
Chemiluminescence: In this method, a biotinylated uracil, guanine) codes for the amino acid methionine.
primer is used dideoxy sequencing reactions, and the Complementary DNA (cDNA): Viral reverse
biotinylated products are transferred from the transcriptase can be used to synthesize DNA that is
sequencing gel to a nylon membrane and detected complementary to RNA (e.g. an isolated mRNA). The
using a three-step procedure. The multivalent cDNA can be used, for example, as a probe to locate
streptavidin cross-links the biotinylated sequencing the gene or can be cloned in the double-stranded form.
product to biotinylated alkaline phosphatase. The
immobilized enzyme dephosphorylates a dioxetane Corpora allata: Ductless glands, usually found in pairs
substrate, which emits light and is detected by (singular corpus allatum), that synthesize and release
autoradiography. Chemiluminescence is comparable in juvenile hormone.
sensitivity to traditional radiolabeling and can also be
used with chemical sequencing. Database: A computerized storehouse of data the
provides a standardized way for locating, adding,
Circadian clock: the biological timer or clock removing, and changing data.
mechanism that is regulated by endogenous oscillation
with ca 24 hours periodicity. Deletion: in the process of DNA replication, a
deletion occurs if a nucleotide or series of nucleotides
Circadian pacemaker: the central (driving) circadian is not copied. Such deletions may be harmless, may
oscillator that regulates subordinate or peripheral result in disease, or may in rare cases be beneficial.
(driven) oscillator in the circadian system.
DNA (Deoxyribonucleic Acid): the molecule that
Clone: A term which is applied to genes, cells, or encodes genetic information. DNA is a double-
entire organisms which are derived from - and are stranded helix held together by bonds between pairs of
genetically identical to - a single common ancestor nucleotides.
gene, cell, or organism, respectively. Cloning of genes
and cells to create many copies in the laboratory is a DNA Adducts: Chemicals (alkylating agents) may
common procedure essential for biomedical research. form covalent bonds with deoxyribonucleic acid
Note that several processes which are commonly (DNA) to form an adduct in which a methyl or ethyl
described as cell 'cloning' give rise to cells which are group is added. DNA adducts are believed to play a
almost but not completely genetically identical to the major role in mutagenesis and clastogenesis, as well as
ancestor cell. 'Cloning' of organisms from embryonic in carcinogenesis.
cells occurs naturally in nature (e.g. with the
occurrence of identical twins). The laboratory cloning DNA annealing: The sticking together (renaturing) of
of a sheep ('Dolly') using the genetic material from a complementary single strands of DNA to make a
cell of an adult animal has recently been reported. double-stranded DNA after the strands have first been
pulled apart (denatured). This involves forming
Cloning: The process of producing a genetically hydrogen bonds between the base pairs.
identical copy (clone).
DNA probe: a piece of single-stranded DNA, typically
Cloning vector: DNA molecule originating from a labelled so that it can be detected (for example, a
virus, a plasmid, or the cell of a higher organism into radioactive or fluorescent label can be used), which can
which another DNA fragment of appropriate size can single out and bind with (and only with) another
be integrated without loss of the vectors capacity for specific piece of DNA. DNA probes can be used to
self-replication; vectors introduce foreign DNA into determine which sequences are present in a given
host cells, where it can be reproduced in large length of DNA or which genes are present in a sample
quantities. Examples are plasmids, cosmids, and yeast of DNA.
artificial chromosomes; vectors are often recombinant
molecules containing DNA sequences from several DNA repair genes: genes which code for proteins
sources. which correct 'mistakes' in DNA sequences. When
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these genes are altered, mutations may be able to necrosis factor (TNF), lymphotoxin and Fas ligand
accumulate in the genome, ultimately resulting in (FasL). The other one is known as intrinsic apoptosis
disease. See genetic mutation, p53 and suppressor gene. pathway, both two mechanisms depend on proteolytic
activation of caspases. Caspase independent
DNA replication: the process of making copies of mechanisms also exist, for instance the mechanism that
strands of DNA. Existing DNA is used as a template involves the apoptosis-inducing factor (AIF).
for synthesising the new strands.
Ecdysone: The steroid hormone secreted by the
Dot blot: Method for detecting a specific message. A prothoracic gland that is converted to 20-
spot of solution is dotted onto nitrocellulose paper, a hydroxyecdysone, which stimulates moulting fluid
specific radiolabeled complementary probe is allowed excretion.
to bind.
Full gene sequence: the complete order of bases in a
Electrophoresis: A method of separating large gene. This order determines which protein a gene will
molecules (such as DNA fragments or proteins) from a produce.
mixture of similar molecules. An electric current is
passed through a medium containing the mixture, and Functional genomics: Assessment of the function of
each kind of molecule travels through the medium at a genes idetntified by between-genome comparisons.
different rate, depending on its electrical charge and The function of a newly identified gene is tested by
size. Separation is based on these differences. Agarose introducing mutations into the gene and then
and acrylamide gels are the media commonly used for examining the resultant mutant organism for an
electrophoresis of proteins and nucleic acids. altered phenotype.
Endocrine Disrupting Chemical: An exogenous Follicular epitherial cell: A cell lining a follicle such
substance that causes adverse health effects in an intact as that of the thyroid gland.
organism, or its progeny, consequent to changes in
endocrine function. Gene: a length of DNA which codes for a particular
protein, or in certain cases a functional or structural
Endocrine glands: Groups of cells specialized to RNA molecule.
synthesize hormones and secrete them into the blood
to regulate other types of cells. Examples are pituitary, Gene expression: The process by which a gene's coded
thyroid, parathyroid, adrenal glands, ovary and testis, information is converted into the structures present
placenta and B cells of pancreas. and operating in the cell. Expressed genes include those
that are transcribed into mRNA and then translated
Endonuclease: An enzyme that cleaves its nucleic acid into protein and those that are transcribed into RNA
substrate at internal sites in the nucleotide sequence. but not translated into protein (e.g., transfer and
ribosomal RNAs).
Exogenous DNA: DNA which has been introduced
into an organism but which originated outside that Gene Families: Groups of closely related genes that
organism (e.g. material inserted into a cell by a virus). make similar products.
Exon: exons are those portions of a gene which code Gene Library: A collection of cloned DNA fragments
for proteins. which, taken together, represent the entire genome of
a specific organism. Such libraries or 'gene banks' are
Exonuclease: An enzyme that digests the ends of a assembled so as to allow the isolation and study of
piece of DNA. Expressed sequence tag (EST): a short individual genes. Gene libraries are produced by first
strand of DNA (approximately 200 base pairs long) breaking up or 'fractionating' an entire genome. This
which is part of a cDNA. Because an EST is usually fractionation can be accomplished either by physical
unique to a particular cDNA, and because cDNAs methods or by use of restriction enzymes. The genome
correspond to a particular gene in the genome, ESTs fragments are then cloned (multiplied in number) and
can be used to help identify unknown genes and to stored for later use.
map their position in the genome.
Gene Product: the protein produced by a gene.

Extrinsic apoptosis pathway: one of the two main
mechanisms leading to apoptosis which involves Gene sequence: The order of nucleotide bases in a
plasma membrane receptors of TNF family triggered DNA molecule that constitute a gene.
by the binding of the death activators, e.g., tumor
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Genetic Code: the set of codons in DNA or mRNA. Genome: all the genetic material in the chromosomes
Each codon is made up of three nucleotides which call of a particular organism; its size is generally given as its
for a unique amino acid. For example, the set AUG total number of base pairs.
(adenine, uracil, guanine) calls for the amino acid
methionine. The sequence of codons along an mRNA Genomic Library: A collection of clones made from a
molecule specifies the sequence of amino acids in a set of randomly generated overlapping DNA
particular protein. fragments representing the entire genome of an
organism.
Genetic engineering (gene manipulation, genetic
manipulation): The manipulation of an organism's Genomics: the study of genes and their function.
genetic endowment by introducing or eliminating Recent advances in genomics are bringing about a
specific genes through modern molecular biology revolution in our understanding of the molecular
techniques. A broad definition of genetic engineering mechanisms of disease, including thecomplex interplay
also includes selective breeding and other means of of genetic and environmental factors. Genomics is also
artificial selection. stimulating the discovery of breakthrough healthcare
products by revealing thousands of new biological
Genetic Map: a map of a genome which shows the targets for the development of drugs, and by giving
relative positions of the genes and/or markers on the scientists innovative ways to design new drugs,
chromosomes. vaccines and DNA diagnostics. Genomics-based
therapeutics include 'traditional' small chemical drugs,
Genetic Mutation: a change in the nucleotide protein drugs, and potentially gene therapy.
sequence of a DNA molecule. Genetic mutations are a
kind of genetic polymorphism. The term 'mutation', Genotoxin: A toxin (poisonous substance) which
as opposed to 'polymorphism', is generally used to harms the body by damaging DNA molecules causing,
refer to changes in DNA sequence which are not for example, mutations or tumours.
present in most individuals of a species and either have
been associated with disease (or risk of disease) or have Genotype: the particular genetic pattern seen in the
resulted from damage inflicted by external agents (such DNA of an individual. 'Genotype' is usually used to
as viruses or radiation). refer to the particular pair of alleles that an individual
possesses at a certain location in the genome. Compare
Genetic Polymorphism: a difference in DNA this with phenotype.
sequence among individuals, groups, or populations
(e.g. a genetic polymorphism might give rise to blue Germarium: The structure within an ovariole in
eyes versus brown eyes, or straight hair versus curly which the oogonia give rise to oocytes.
hair). Genetic polymorphisms may be the result of
chance processes, or may have been induced by Housekeeping genes: The genes which are expressed
external agents (such as viruses or radiation). If a in all cells and which code for molecules that are
difference in DNA sequence among individuals has necessary for basic maintenance and essential cellular
been shown to be associated with disease, it will functions.
usually be called a genetic mutation. Changes in DNA
sequence which have been confirmed to be caused by Heterologous Expression Systems: systems that
external agents are also generally called 'mutations' allow expression of a gene in a different organism.
rather than 'polymorphisms'.
Hemimetabolous: The insects that have a gradual or
Genetically Modified Organism (GMO): The incomplete metamophosis and the development of
modification of the genetic characteristics of a wings on the outside of the body wall.
microorganism, plant or animal by inserting a
modified gene or a gene from another variety or Holometabolous: The type of metamorphosis in
species. Genetically modified organisms (GMOs) may which a complex change occurs between larvae and
be micro-organisms designed for use as biopesticides or adults, involving a pupal stage. Wings develop on the
seeds that have been altered genetically to give a plant outside of the body wall.
better disease resistance or growth.
Hybridization: The process of joining two
Genomic DNA: The basic chromosome set consisting complementary strands of DNA or one each of DNA
of a species-specific number of linkage groups and the and RNA to form a double-stranded molecule.
genes contained therein.
232
Immunohistochemistry:histochemical visualization to discover the associations between genetic mutations

technique of an antigen by using antibody raised and disease.
against the antigen.
Messenger RNA (mRNA): the DNA of a gene is
In Situ Hybridization (ISH): Use of a DNA or RNA transcribed (see transcription) into mRNA molecules,
probe to detect the presence of the complementary which then serve as a template for the synthesis of
DNA sequence in cloned bacterial or cultured proteins.
eukaryotic cells.
Metabonome/Metabolome: constituent metabolites
Intron: a length of DNA which is interspersed among in a biological sample.
the protein-coding sequences (exons) in a gene. Introns
are transcribed (see transcription) into mRNA but are Metabonomics/Metabolomics: techniques available to
then cut out of the mRNA sequence before protein identify the presence and concentrations of metabolites
synthesis occurs. in a biological sample.
Intrinsic apoptosis pathway: also known as Meristic: A type of of ovariole in which nurse cells
‘mitochondrial pathway’, is one of the two main accompany the oocytes within the follicle and supply
mechanisms leading to apoptosis; it involves the release it with nutrients.
of cytochrome c from mitochondria due to the signals
arising within the cell. The other one is known as Metallothioneins (MTs): Ubiquitous low molecular
extrinsic apoptosis pathway, both two mechanisms weight proteins and polypeptides of extremely high
depend on proteolytic activation of caspases. Caspase metal and sulfur content. They are thought to play
independent mechanisms also exist, for instance the roles both in the intracellular fixation of the essential
mechanism that involves the apoptosis-inducing factor trace elements zinc and copper, in controlling the
(AIF). concentrations of the free ions of these elements, in
regulating their flow to their cellular destinations, in
Juvenile hormone: A hormone that is released by the neutralising the harmful influences of exposure to
corpora allata into the hemolymph, and involved in toxic elements such as cadmium and mercury and in
many physiological functions including the protection from a variety of stress conditions.
metamorphosis and reproduction.
Microarray: a component of a device for screening
Library: a set of clones of DNA sequences from an genomic or cDNA for mutations, polymorphisms or
organism's genome. A particular library might include, gene expression. The array is a small glass slide or
for example, clones of all of the DNA sequences other solid surface on which thousands of immobilized
expressed in a certain kind of cell, or in a certain organ oligodeoxynucleotide probes have been synthesized or
of the body. robotically deposited in a predetermined array, so that
automated recording of fluorescence from each of the
Ligand-receptor interaction: The interactions spots may score successful hybridizations. An array
between a molecules (usually of an extracellular origin) may be designed for the detection of all known genes
and a protein on or within a target cell. One type of of a species or selected specific sequences. The array
ligand-receptor interaction can be between steroid may also consist of different antibodies or proteins.
hormones and their cytoplasmic or nuclear receptors.
Another can be between secreted pholypeptide ligands Mutation: A change, deletion, or rearrangement in the
and transmembrane receptors. DNA sequence that may lead to the synthesis of an
altered inactive protein the loss of the ability to
Macroarray: A low-density array of DNA molecules produce the protein. If a mutation occurs in a germ
used for parallel hybridisation analysis (see cell, then it is a heritable change in that it can be
microarray). transmitted from generation to generation. Mutations
may also be in somatic cells and are not heritable in
Marker: a sequence of bases at a unique physical the traditional sense of the word, but are transmitted
location in the genome, which varies sufficiently to all daughter cells.
between individuals that its pattern of inheritance can
be tracked through families and/or it can be used to Multivesicular body (MVB): A structure bound by a
distinguish among cell types. A marker may or may single unit membrane containing material carried to it
not be part of a gene. Markers are essential for use in by pinocytosis vesicles and 1º lysosomes. MVBs have
linkage studies and genetic maps to help scientists to inner vesicles arising by invagination at their surface
narrow down the possible location of new genes, and and are concerned in membrane turnover and protein
digestion.
233
Molecular clock hypothesis: The hypothesis that Organelle: Sub-cellular structures that perform a role
sequences change at the same rate in the branches of an within each cell. These vary widely from the nucleus,
evolution ary tree. containing all our genetic information, to the golgi
apparatus which processes protein and secretes it in
Mosquitoes: common flying insects in the family vesicles on demand by the cell.
Culicidae that are found around the world. There are
about 3,500 species. The females of most mosquito Orthologs: Homologous genes in different species that
species suck blood (hematophagy) from other animals, evolved from a common ancestral gene by speciation
which has made them the most deadly disease vector (as opposed to paralogs, which result from duplication
known, killing millions of people over thousands of within a genome and subsequent divergence).
years and continuing to kill millions per year by the
spread of infectious diseases. Mosquitoes are a vector Organogenesis: The production of organ systems
agent that carries disease-causing viruses and parasites during animal embryogenesis.
from person to person without catching the disease
themselves. The principal mosquito borne diseases are Oogenesis: the process of meiosis in female organisms
the viral diseases yellow fever and dengue fever, from an oogonium to a primary oocyte, to a secondary
transmitted mostly by the Aedes aegypti, and malaria oocyte, and then to an ovum. Spermatogenesis is a
carried by the genus Anopheles. similar process of forming sperm by meiosis in males.
Nitrocellulose membrane (NC): A membrane with a Ovariole: The tubes that comprise the ovary and
high nonspecific absorbing power for biological contain the oocyte as they develop into eggs.
macromolecules. Very important as a receptor in blot-
transfer methods. Oocyte: An immature egg cell formed from the
oogonium within the ovariole.
Northern blot: An electroblotting method in which
RNA is transferred to a filter and detected by Oogonium: The first stage in the development in the
hybridization to32 P-labelled RNA or DNA. germarium of an egg from a female germ cell.
Nurse Cells : The sister cells of the oocytes in insects. Pinocytic vesicle: Microvesicle arising at the plasma
The nurse cells produce the bulk of the cytoplasmic membrane surface that is involved in conveying
content of the mature oocyte. membrane and protein into the cell.
Nucleic Acid: one of the family of molecules which Protein granule (PG): Membrane-bound vesicle
includes the DNA and RNA molecules. Nucleic acids storing protein that has been pinocytosed from the
were so named because they were originally discovered haemolymph. The protein is often crystalline. PGs are
within the nucleus of cells, but they have since been often composite structures, having fused with AVs.
found to exist outside the nucleus as well.
Panoistic: An ovariole that lacks nurse cell and
Nucleotide (= base): the 'building block' of nucleic nourishes the oocyte through the follicular epithelium.
acids, such as the DNA molecule. A nucleotide
consists of one of four bases - adenine, guanine, Pharmacogenomics: The science of understanding the
cytosine, or thymine – attached to a phosphate-sugar correlation between an individual patient's genetic
group. In DNA the sugar group is deoxyribose, while make-up (genotype) and their response to drug
in RNA (a DNA related molecule which helps to treatment. Some drugs work well in some patient
translate genetic information into proteins), the sugar populations and not as well in others. Studying the
group is ribose, and the base uracil substitutes for genetic basis of patient response to therapeutics allows
thymine. Each group of three nucleotides in a gene is drug developers to more effectively design therapeutic
known as a codon. A nucleic acid is a long chain of treatments.
nucleotides joined together, and therefore is sometimes
referred to as a 'polynucleotide'. Phenotype: a set of observable physical characteristics
of an individual organism. A single characteristic can
Nucleus: the membrane bound structure containing a be referred to as a 'trait', although a single trait is
cell's central DNA found within all eukaryotic cells. sometimes also called a phenotype. For example, blond
hair could be called a trait or a phenotype, as could
Oligonucleotide: A molecule made up of a small obesity. A phenotype can be the result of many
number of nucleotides, typically fewer than 25. These factors, including an individual's genotype,
are frequently used as DNA synthesis primers. environment, and lifestyle, and the interactions among
234
these factors. The observed manifestation of a Probe: Single-stranded DNA or RNA molecules of
genotype. The phenotype may be expressed physically, specific base sequence, labelled either radioactively or
biochemically, or physiologically. immunologically, that are used to detect the
complementary base sequence by hybridisation.
Phylogeny: (phylogenesis, phylogenetic, phylogenic):
The evolutionary history of a particular taxonomic Promoter: a segment of DNA located at the 'front'
group, usually a species. end of a gene, which provides a site where the enzymes
involved in the transcription process can bind on to a
Pheromone :a substance which is secreted to the DNA molecule, and initiate transcription. Promoters
outside by an individual and received by a second are critically involved in the regulation of gene
individual of the same species, in which it releases a expression.
specific reaction, for example, a definite behaviour or a
developmental process. Proteome: total protein complement expressed by a
cell, tissue or organism.
Plasmid: A structure composed of DNA that is
separate from the cell's genome. In bacteria, plasmids Proteomics: study of protein properties on a large
confer a variety of traits and can be exchanged between scale to obtain a global, integrated view of cellular
individuals - even those of different species. Plasmids processes including expression levels, post translational
can be manipulated in the laboratory to deliver specific modifications, interactions and location.
genetic sequences into a cell.
Programmed cell-death (PCD): is death of a cell in
Plasmodium: a genus of parasitic protozoa. Infection any form, mediated by an intracellular program. In
with these parasites is known as malaria. The genus contrast to necrosis, which is a form of cell-death that
Plasmodium was created in 1885 by Marchiafava and results from acute tissue injury and provokes an
Celli. Currently over 200 species in this genus are inflammatory response, PCD is carried out in a
recognized and new species continue to be described. regulated process which generally confers advantage
during an organism's life-cycle. PCD serves
Polymerase chain reaction (PCR): The first practical fundamental functions during both plant and metazoa
system for in vitro amplification of DNA, and as such (multicellular animals) tissue development.
one of the most important recent developments in
molecular biology. Two synthetic oligonucleotide Randomly Amplified Polymorphic DNA (RAPD):
primers, which are complementary to two regions of Variation of the polymerase chain reaction used to
the target DNA (one for each strand) to be amplified, identify differentially expressed genes. mRNA from
are added to the target DNA (that need not be pure), two different tissue samples is reverse transcribed, then
in the presence of excess deoxynucleotides and Taq amplified using short, intentionally nonspecific
polymerase, a heat-stable DNA polymerase. In a series primers. The array of bands obtained from a series of
(typically 30) of temperature cycles, the target DNA is such amplifications is run on a high resolution gel and
repeatedly denatured (around 90°C), annealed to the compared with analogous arrays from different
primers (typically at 50-60°C) and a daughter strand samples. Any bands unique to single samples are
extended from the primers (72°C). As the daughter considered to be differentially expressed; they can be
strands themselves act as templates for subsequent purified from the gel, and sequenced and used to clone
cycles, DNA fragments matching both primers are the full-length cDNA.
amplified exponentially, rather than linearly. The
original DNA need thus be neither pure nor abundant, Recombinant DNA: DNA molecules that have been
and the PCR reaction has accordingly become widely created by combining DNA more than one source.
used not only in research, but in clinical diagnostics
and forensic science. Recombinant protein: a protein derived from
recombinant DNA.
Polymorphism: in this context, the existence of inter-
individual differences in DNA sequences coding for Regulatory Gene: a gene which controls the protein-
one specific gene. The effects of such differences may synthesising activity of other genes. Restriction
vary dramatically, ranging from no effect at all to the enzymes (restriction endonucleases): a class of bacterial
building of inactive proteins. enzymes that cut DNA at specific sites.
Primer: Short pre-existing polynucleotide chain to Reverse Transcriptase: An enzyme used by

which new deoxyribonucleotides can be added by retroviruses to form a complementary DNA sequence
DNA polymerase. (cDNA) from an RNA template -usually the genome
of the retrovirus. The enzyme then performs a
235
complimentary template of the cDNA strand such that chromosomal difference, that region can be used as a
a double stranded DNA molecule is formed. This marker in chromosome mapping.
double stranded DNA molecule is then inserted into
Sequencing: determining the order of nucleotides in a
the chromosome of the host cell which has been
DNA or RNA molecule, or determining the order of
infected by the retrovirus. Reverse transcriptase is one
amino acids in a protein.
of the key components that HIV uses to mount its
attack. Sex chromosome: A chromosome which is involved
in sex determination.
Retrotransposon: A type of transposable element
with a sequence similar to an integrated retroviral Sex-limited strain: The silkworm, Bombyx mori,
genome that transposes by means of an RNA strains in which some noticeable traits express only in
intermediate. These elements ca be divided into sub- females because the dominant genes (chromosome
group (mainly LTR retrotransposons and non-LTR fragments) for noticeable traits were translocated to
retrotransposons) based on whether or not they have the W chromosome artificially.
long terminal repeats (LTRs).
Single Nucleotide Polymorphism (SNP): Inter-
Ribosome: Small particles that are found in all cells individual variations in the genetic code at the level of
both freely in the cytoplasm and more commonly one nucleotide.
found on endoplasmic reticulum. The ribosome Southern Blotting: Transfer by absorption of DNA
particle consists of protein and ribosomal RNA, where fragments separated in electrophoretic gels to
the RNA is translated from the mRNA transcribed membrane filters for detection of specific base
from nucleus DNA. This effectively is the point of sequences by radiolabeled complementary probes.
creation of a protein, where each nucleotide in the
polynucleotide is translated into part of the nucleotide Species: Groups of populations (which are groups of
sequence for the completed protein. individuals living together that are separated from
other such groups) which can potentially interbreed or
RNA (ribonucleic acid): a molecule similar to DNA, are actually interbreeding, that can successfully
which helps in the process of decoding the genetic produce viable, fertile offspring (without the help of
information carried by DNA. human technology).
RNAse (= ribonuclease, RNAase): Widely distributed Splicing: the removal of introns from the sequence of
type of enzyme that cleaves RNA. May act as mRNA. When an mRNA molecule is synthesized
endonucleases or exonucleases depending upon the from a DNA template, introns are transcribed (see
type of enzyme. transcription) along with exons. In the splicing
process, this material is cut out and the exons are
RT-PCR (= reverse transcriptase polymerase chain
joined together to form a continuous coding sequence.
reaction; reverse transcription PCR): PCR in which
the starting template is RNA, implying the need for an Slot blot: A dot blot in which samples are placed on a
initial reverse transcriptase step to make a DNA membrane through a series of rectangular slots in a
template. Some thermostable polymerases have template. This is slightly advantageous because
appreciable reverse transcriptase activity; however, it is hybridization artifacts are usually circular.
more common to perform an explicit reverse
transcription, inactivate the reverse transcriptase or Structural genomics: The effort to determine the 3D
purify the product, and proceed to a separate structures of large numbers of proteins using both
conventional PCR. experimental techniques and computer simulation.
RAPD: Randomly amplified polymorphic DNA. A Stadium: The period from one ecdysis to the next,
set of several genomics fragments amplified by a single during which the fat body changes in the characteristic
PCR primer; some what variable from individual to intermolt/molt sequence of development.
individual; +/- heterozygotes for individual fragments
can act as markers in genome mapping. Storage protein: a common means of these reserving
amino acids in insects is called “storage proteins.
RFLP: Restriction fragment length polymorphism. At storage proteins (SP) appear to be a special adaptation
some chromosomal locations a probe sometimes in insect molting, metamorphosis, and reproduction.
detects different sizes or different numbers of
Subspecies: A group of organisms that is
restriction fragments (often as a result of presence and
geographically isolated from and may display some
absence of restriction sties), and this situation is an
morphological differences from other populations of a
RFLP. If an individual is heterozygous for such a
species, but is nevertheless able to interbreed with
236
other such groups within the species where their other individuals of the same species or from wholly
ranges overlap. different species. Genetic material may also be of an
artificial nature. Foreign genetic information can be
Suppressor Gene: a gene which helps to reverse the added to the organism during its early development
effects of damage to an individual's genetic material, and incorporated in cells of the entire organism. As an
typically effects which might lead to uncontrolled cell example, mice embryos have been given the gene for
growth (as would occur in cancer). A suppressor gene rat growth hormone allowing mice to grow into large
may, for example, code for a protein which checks adults. Genetic information can also be added later in
genes for misspellings, and/or which triggers a cell's development to selected portions of the organism. As
self-destruction if too many genetic mutations have an example, experimental genetic therapy to treat
accumulated. cystic fibrosis involves selective addition of genes
responsible for lung function and is administered
Taq polymerase: A heat-stable DNA polymerase that directly to the lung tissue of children and adults.
is normally used in the polymerase chain reaction. It Transgenic organisms have been produced that provide
was isolated from Thermus aquaticus. enhanced agricultural and pharmaceutical products.
Insect resistant crops and cows that produce human
Toxicogenomics: a new scientific subdiscipline that hormones in their milk are just two examples.
combines the emerging technologies of genomics and
bioinformatics to identify and characterize Transgenic Organism: an organism whose genome
mechanisms of action of known and suspected has been altered by the incorporation of foreign, or
toxicants. Currently, the premier toxicogenomic tools exogenous DNA.
are the DNA microarray and the DNA chip, which
are used for the simultaneous monitoring of expression Translation: the process during which the information
levels of hundreds to thousands of genes. in mRNA molecules is used to construct proteins.
Tissue-specific gene expression: The expression of a Translocation: A type of chromosomal aberrations in

gene in a specific and reproducible subject of tissues which a fragment of a chromosome breaks off and
and cells during the development of a higher connects to a non-homologous chromosome.
eukaryote.
URF : Unassigned reading frame. An open reading
tRNA: (transfer RNA): A class of small RNA frame (ORF) whose function has not yet been
molecules that bear specific amino acids to the determined.
ribosome during translation; the amino acid is inserted
into the growing polypeptide chain when the Vitellin: The vitellogenins that have been deposited in
anitcoden of the tRNA paris with a coden on the the oocyte cytoplasm after processing.
mRNA being translated.
Vitellogenin: A protein, precursor of several yolk
Transcription: the process during which the proteins, especially phosvitin and lipovitellin in the
information in a length of DNA is used to construct eggs of various vertebrates, synthesized in the liver
an mRNA molecule. cells after oestrogen stimulation. Also found in
Transcriptomics: techniques available to identify invertebrates.
mRNA from actively transcribed genes.
Vitellogenesis: The process by which yolk produced
Transcriptome: mRNA from actively transcribed by the fat body (in insects) is taken up by the oocyte
genes Transcript Profiling: see transcriptomics and deposited in its cytoplasm.
Transfer RNA (tRNA): RNA molecules which bond
Western blot: Membrane carrying an imprint of
with amino acids and transfer them to ribosomes,
proteins separated by electrophoresis; can be probed
where protein synthesis is completed.
with a labeled antibody to detect a specific protein.
Transformation: A process by which the genetic X- and Y- linkage: The inheritance pattern of genes
material carried by an individual cell is altered by found on both the X and the Y chromosomes (rare).
incorporation of exogenous DNA into its genome or Xenobiotic(s): substances not normally present in the
Transfer of purified DNA, rDNA, for example, to reference organism.
bacteria or higher cells.
Z and W chromosomes: A pair of the sex
Transgenic: An organism whose genome has been chromosomes found in heterogametic female insects
altered by the inclusion of foreign genetic material. (ZZ for male and ZW for female).
This foreign genetic material may be derived from
237
NAL B OO
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Short Views on Insect Molecular Biology

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 Vol. (1), 2009
APPENDIX - B
AUTHOR INDEX
A
Blacklow, SC. 102
Anathakrishnan, TN. 1
Bu, G. 110
Adams, TS. 86
Brust, RA. 121
Abraham, EG. 179,185
Bernstein, AI. 217
Abe, H. 203,212,213,219, 218,223
Beier, MS. 126
Adms, JM. 181
Broughton, SJ. 125
Adms, MD. 142
Blau, J. 30
Akerman, MEW. 12
Bloch, G. 37
Atkinsen, H. 15
Buys, KS. 50
Achoff, J. 21
Byrne, BM 72
Allada, R. 22,23,24
Butenandt, A. 147
Ashmore, LJ. 24
Beulaton, J. 169
Atkionson, H 15
Bowen, ID. 160
Abdelalam 35
Boyce, M. 160
Anand, AN 52
Bursch, W. 160
Attardo, GM. 59
Brantely Finley, C. 179
Ashfaq, M. 62
Burmester, T. 50,52,56,58,59,61
Anderson, SO. 86
Buchner, E. 38
Arrese, EL. 96
Blagojevic, D. 193
Ando, T. 132
Block, W. 193
Altstein, M. 138
Bower, M. 51
Al-Olayan, EM. 185
Brown, MS 97,124
Alnemri, ES. 180
Bujo, H. 7,100
Arimura, T. 182,184
Ashkenazi, A. 181
C
B
Cashmore, AR. 29
Babu, SPB 1 Carvalho, AB. 223
Bae, K. 23 Cai, Z. 183
Balanco, JMdF. 175 Chandrasekar, R. 4,5,16,49,54,58,61,159
Bale, JS. 190 160,168
Baker, ME. 88 Chandrakant, L. 13
Barillas-Mury, C. 185 Chalier, L. 150
Barr, PJ. 74 Chamber, GM. 150
Bean, DW. 50 Chrysanthis, G. 53
Bembenek, J. 33,34 Chu, J. 14
Benes, H. 59 Chiang, CS. 22,31
Ben-Aziz, O. 138 Chiba, Y. 36,38
Benton, R. 152 Cheon, HM. 53,100
Belles, X. 70,83 Chang, DC. 22,31
Beglova, N. 105 Charlesworth, D. 223
Bertram, PG 125 Chu-Wang, IW. 168
Beck, T. 125 Choi, MY. 133,135,138
Beresford, PJ. 56 Cho 62,102
Butterworth, FM. 58 Chen, KH. 70,97,102
Bishop, GP. 58 Chen, P. 183
Bownes, M. 59 Chang, HY. 179
Beintema, JJ. 62 Chen, CP. 192
Corpuz, LM. 59
238
Colombani, J. 124 Giannakou, ME. 126

Cooper, TG. 125 Giaccia, AJ. 178
Coles, GC. 86 Glossop, NRJ. 24
Colussi, PA. 179 Gotter, AL. 31
Cecconi, F. 182 Gordadze, AV. 59
Clark, BA. 142 Goldstein, JC. 97,102
Clarke, PGH. 160 Gondim, KC. 100
Cyran, SA. 23 Goto, M. 192
Cymborowski, B. 33,36 Goldschmidt, R. 204
Goldsmith, MR. 204
Gopalapillai, K. 96,100,111,102,106,
E Gordana Grubor-Lajsic 191
Grabherr, R. 11
Grant, B. 7,96
Earnshaw, WC. 180
Grima, B. 24,28
Edwards, JP. 83,84
Graft, R. 126
Emery, P. 24,29,32,37
Grater, F. 150
Engelmann, F. 77,79,83,84
Grosse-Wilde, E. 150
Ekert, PG. 184
Green, DR. 181
Entingh, AJ. 125
Graccia-Ruizc, C. 182,183
Eickbush, TH. 219
Gruber-Lajsic, G. 194,195,193
Eliyahu, D. 132
Gruetzmacher, MC. 193
Emanuel, BS. 216
Gulduzli, KR. 73
F H
Fadok, V. 177
Harshman, GL. 11
Fang, Y. 23
Haunerland, NH. 4,50,53,55
Fagotto, F. 6,7
Hayshi, I. 12
Fargnoli, J. 87
Hassan, J. 29
Fass, D. 102
Hahn, DA. 52
Fan, Y. 140
Hansen, IA. 58,119,125
Federici, BA. 7
Hagedorn, NH. 74,84,122
Feldman, JR. 22
Hall, M. 72,81
Festjens, N. 160
Hafen, 124
Fiers, W. 179
Hallem, EA. 148,151
Fischer, SF, 179
Halliwell, B. 193
Filipovic, M. 198
Hashimoto, H. 222
Fleisher, TA. 177
Heydel, J. 154
Flanagan, TR. 122
Helfrich-Forster, C. 38
Forstner, M. 149
Hewes, RS. 133
Fox, HL. 124
Herz, J. 102
Frisch, B. 37
Hirai, J. 70,72
Friederich, E. 83
Hiremath, S. 70
Friedel, T. 82,85
Hiramatsu, N. 7
Fristrom, D. 159
Hjalam, G. 102
Fukada, H. 6
Hiesberger, T. 98
Fujiwara 58
Hobbs, HH. 96
Fujii, T. 58,203,204
Howard 110
Holman, GM. 133
Hopwood, JA. 163
G Hoffmann, AA. 192
Holmstrup, M. 192
Homberg, U. 36,37
Gao, XY. 12
Hull, JJ. 133,134,135,141
Gadnne, C. 140
Hurle, J. 168
Geong, HG. 13
Hunter-Ensor, M. 29
Gerber-Huber, S. 73
Hwang, UW. 60
Giribet, G. 60
Giorgi, F. 70,74,77,82
239
I Krieger, MJ. 125,150,151

Kiely, A. 151
Kitamura, A. 133
Iwai, S. 31,34
Klein, V. 152
Itoh, MT. 34
Kiguchi, K. 170
Indrasith, LS. 55
Klionsky, DJ. 160
Indokuchi, T. 53
Kroemer, G. 176,182
Izumi, S. 55,96
Kirlin, WG. 193
Immo Hansen, 63,119
Krishnan, M. 53,159
Imboden, H. 70
Krishnan, B. 24
Ilenchuk, TT. 86
Krzywinski, J. 223
Iglesias, F. 132
Kimura, K. 209,210
Imai, K. 133
Koike, Y. 210,216
Ishida, Y. 150,151
Kostal, V. 192
Ito, S. 216
Kothakota, S. 180
Kotaki, T. 6
Koeppe, JK. 6
J Koing, R. 7,82
Kost 11
Jacobsen, MD. 175 Ko, HW. 24,28
Jacobsen, L. 111 Kounnas, MZ. 98,111
Jacquin-Joly, E. 147,149,151 Konopka, RJ. 23
Jamorz 61 Kumar, S. 176,185
Jean-Antoine, 13 Kuechle, MK. 179
Jeon, H. 98 Kurtovic, A. 151
Jentoft, N. 106 Kunkel, JG. 82
Jefferies, HB. 124 Kubli, E. 141
Jurenka, RA. 132,133,135
Joanisse, DR. 193
Jones, G. 53,56,62 L
Jovanovic-Galvoic, A. 193,196
Lam, SK. 13
Laras, K. 13
K Lampel, J. 33
Lauverijaj, S. 51
Kageyama, T. 193 Lalzar, A. 102
Kassang, G. 152,153 Larsson, MC. 151
Kadener, S. 23 Lazard, D. 154
Kasraee, B. 197 Levst, M. 176
Kawasaki, 53 Lee, JW. 14
Kafatos, FC. 87 Lee, JM. 6,0,72,73,76
Kawooya, 55,86 Lee, C. 28
Kain, P. 151 Lee, CS. 100,111,106
Karlson, P. 132,149 Lee, DW. 141
Kerr, JFR. 160,176 Lee, RE. 192
Kelly, TJ. 86 Lea, 122,123
Kempa-Tomm, S. 85 Levine, JD. 30
Keeley, L. 4,163,193 Levenbook, L. 52
Kim, EY. 24,28,51 Leal, WS. 152,154
Kim, DH. 96,102 Leck, B. 159
Kim, JK. 176 Lefevere, KS. 191
Kim, JK. 176 Lim, C. 23
Klarsfeld, A. 37 Lin, JM. 23,24
Kloss, B. 24,28,31 Linn, CE. 34
Kinnear, LE. 55 Li, Y. 110
Kiran Kumar, N. 58 Li, C. 122
Kingsely, DM. 106 Livak, KJ. 137
Kumar, S. 181 Li, H. 180
Kurniawan, ND. 105 Li, N. 192
Kozarsky, 106 Li, YP. 192
240
Loher, JD. 38 Mouchel, N. 74

Lorenz, MW. 52 Mundall, E. 86
Locke, M. 165 Mullberg, J. 106
Lockshin, RA. 168 Mitsuno, H. 151
Low, P. 169 Mohanty, S. 149
Luckhart, S. 126 Muller F. 162,168,169
Luiz Paulo, MA. 49 Mullacer, L. 183
Lynch, CJ. 124 Monney, L. 184
Lundin, C. 151 Motoyama, N. 178
Mund, T. 181
Mogami, K. 216
M Myers, MP. 29
Masuda, K. 12
Marion, S. 13 N
Margaret, K. 14
Malpel, S. 38 Nagata, M. 5
Markova, EP. 24,31 Nagata, S. 177,179
Martinek, S. 28 Nakajima, M. 22
Martin, PM. 124, 128 Nakamura, K. 38
Martinez, T. 56,132 Nassel, DR. 37
Martin, D. 84 Nawathean, P. 28,29
Martin, SJ. 179 Naumann, V. 61
Martin Rechsteiner, 168 Nair, KK. 83
Magee, GP. 30 Nardelli, D. 72,73
Markl, J. 59 Nachman, RJ. 140
Maruta, K. 56 Nagalakshmi, VK. 141
Massey, HC. 59 Nakagawa, T. 149
Mayadas, TN. 74 Nagahara Y. 180
Machado, EA. 86 Newton, K. 180
Max Field FR. 106,108 Nelson, CM. 73
Matsumoto, S. 133,136 Newby, 30
Ma, PWK. 133 Niino, T. 208
Maibech-coisne, M. 152,154 Nimpf, J. 96,98
Maida, R. 147 Nishiitsutsuji-Uwo, J. 36,38
Mayer, RJ. 160,169 Nordin, JH. 191
Manji, GA. 184 Noriega, PM. 122
Mac Farlane, M. 177 Noriega, FG. 122
Malinin, NL. 180 Nose, Y. 70,74,76
Macrae, TH. 193,218
Matsumoto, T. 219
Merg, FG. 192
Mc Gahon, AJ. 163 O
Merlin, C. 152
Mc Laren, JS. 7 Okamura, Y. 12
Mc Gauphey, GH. 8 Ohnish, A. 141
Mc Donald, MJ. 30 Oker-Blom, C. 11
Mc Neil, GP. 30 Okmamoto, A. 36
Meenakshi, M. 51,96,100 Ogawa, K. 53,55,57
Memmel, NA. 61 Okabayashi, K. 96
Miller, MS. 73 O’Meara, GF. 120,121
Miller, SG. 50,56 Ogura, T. 219
Mikhailov, VS. 11 Osanni, M. 219
Michand, MR. 190 Osir, EO. 7,77
Miao, XX. 210 Osborne, B. 185
Mita, K. 209,210 Olney, JW. 176
Mihajlo Spasic 191 Olsen, SS. 135
Motohashi, T. 12 Ozeki, K. 161
Mouchet, J. 13 Ortelli, F. 195
Moreira, CK. 59
Muller, AC. 55
241
P Rieger, D. 34
Riddiford, LM. 53,58,149,152
Rizki, TM. 51,161
Pan, M. 5,52,53,55
Rich, T. 182
Pan, G. 178
Rojas, RR. 193
Park, EY. 11
Robertson, NH. 221
Park, JH. 30
Rothenfluh, A. 28
Park, SK. 30
Roberts, PE. 85
Park, Y. 133
Roberts, SK. 36
Palaga, T. 185
Roberts, DB. 55
Pap, T. 178
Rosll, R. 6,72
Page, TL. 36,38
Rosso, MN. 15
Pau, R. 55
Rouille, Y. 78
Parola, P. 13
Romans, P. 74,76
Patino, R. 7
Rodenburg, KW. 110
Peralta, A. 11
Rogers, ME. 152
Pedigo, LP. 2
Rubin, EB. 32
Petri, B. 38
Rudenko, G. 98, 106
Pittendrigh, CS. 21,36
Rybczynski, R. 151
Price, JL. 28
Ryan, MF. 9
Perfettini, J. 184
Ryan, RO. 5,53,96
Pelletier, J. 152
Persaud, DR. 58
Perriece, C. 85
Phadtare, S. 12 Q
Pickart, CM. 169
Pilar, G. 168 Quinn, PJ. 192
Pirinia, F. 182, 183 Queyriaue, 13
Pio, CJ. 195 Qi-Miao Shao, 21
Piulachs, MD. 73,87
Prasath, EB. 6,72
Pratt, GE. 85 S
Puig, O. 126
Pophof, B. 150
Sass, M. 168
Powers, AM. 13
Saitoh, R. 11
Prestwich, GD. 152
Sappington, TW. 6,55,76,77,81,86,96,98,
Promboon, A. 210
100,106,110
Sharma, HC. 15
Salerno, AP. 6
R Samuelson, P. 12
Sauman, I. 22
Ravi, V. 13 Saunders, DS. 21
Ray, A. 55 Sandler, BH. 149,150
Ram, KR. 141 Sambrook, J. 162
Raff, M. 179 Saez, L. 22
Raikhel, AS. 55,70,75,81,82,86, Sakamoto, K. 23
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Rajaratnam, 86,96 Sathyanarayanan, S. 23
Rafaeli, A. 131,132,133,136,137, Sato, Y. 133
140,141 Sato, K. 149
Raina, AK. 133 Saito, A. 102
Rachel Bober 131 Saucedo, LJ. 124
Ramesawamy,SB. 132,135 Saikumar, P. 176,181,182,183
Rabossi, A. 161 Sakahira, H. 180
Reiter, RJ. 30 Sahara, K. 222
Renn, SC. 30 Savill, J. 175
Reum, L. 57 Sakurai, HT. 58,151
Rechsteiner, M. 161,166,170 Sakai, J. 96,102
Reed, JC. 177,178,182 Schenider, WJ. 98,100
Reppert, SM. 22,28,30,31,34 Scheller, K. 53,55,58
242
Schenkel, H. 56 Tanaka, F. 22
Schal, C. 84,85 Tazima, Y. 205,206,207,222
Schwartz, LM. 160,161,168,170 Takahashi, H. 219
Schin, K. 51 Takahashi, S. 82,96
Schonbaum, CP. 7,98,100,101 Tanaka, Y. 205
Scaffidi, C. 182 Tasayco, M. 152
Schafer, FQ. 193,197 Takayama, S. 160
Segraves, WA. 123,160 Tawtik, AI. 85
Sehadova, H. 31,37 Terashima, J. 170
Seema 14 Telang, A. 50
Seo, SJ. 53,100,101,105,106,11 Thomson, JA. 50,55
Shiao, SH. 125 Thummel, CS. 123
Shirk, PD. 50,51 Trommsdorff, M. 98
Shinbo, H. 53 Trpis, M. 120
Shafer, DT. 24,28 Tillman, JA. 132
Shao, QM. 36 Tsfadia, O. 132
Shimizu, I. 23,31.33 Towbin, H. 162
Shiba, H. 161 Tomita, S. 221
Shimada, T. 210 Tomita, J. 22
Siwicki 37 Truman, JW. 160,161
Sinclair, BJ. 192 Trang, LTD. 21,33,31
Singh, SP. 195 Truman, JW. 33,34,
Stanewsky, R. 22,24,29,33,37 Tufail, M. 69,73,74,75,71,81,95
Streichert, LC. 170 100,106
Steller, H. 160,179 Tsumuki, H. 191
Strasser 183 Trenczek, TA. 73,87
Stanic, B. 197 Traut, W. 216
Storey, KB. 192,193,194,197 Tysell, B. 53
Steinemann, M. 220
Stifani, S. 7,86,98
Strambi, A. 84 U
Storella JR. 72,79,81
Strickland 98,110,111
Ueno, K. 53
Steinbrecht, RA. 146
Ullman, CG. 102
Springer, TA. 97,106
Ushirogawa, H. 36
Specker, JZ. 6
Uchida, K. 123,124
Spielman, A. 123
Uhliova, M. 161
Smith, CK. 11
Udomsinprasert, R. 196
Smith, CA. 179,181
Snigirevskaya 77
Suzuki, MG. 216
Sumithra, P. 159 V
Suh, J. 37
Suri, V. 28,29 Valle, D. 87
SumioKa, H. 53 Van Loo, G. 184
Subramoniam, T. 72 Vaux, D. 176
Sun, XM. 110 Vaux, DL. 181,183
Su, T. 121,124 Van Hoof, D. 98
Suzuki, MG. 216 Van Antwerpen, R. 96
Susin, SA. 184 Van de Craen M. 179
So, WW. 24,30 Van Engelen, FA. 15
Syed, Z. 150,151 Vanishree, V. 4,161,163
Sylvester Lyantagaya, 175 Vaidyanthan, R. 185
Szymczk, K. 179 Vivien-Roels, B. 34
Vitkovic, L. 177
Vinogradova, EB. 120
T Von Frisch, K. 31
Vogt, RG. 152,154
Vernooy, SY. 176,180,184
Telfer, WH. 5,52,53,55,86,96
Von Herrath, G. 178
Takeda, M. 21,31,36,69,70, 76,77,81
Vontas, JG. 195
81,83,95,96,100,106,110
243
W
Zheng, H. 29
Wang, Z. 7,51,53,85,86
Wang, VS. 142
Wang, Q. 154
Watanabe, K. 133
Wanner, KW. 151
Warner, E. 13
Waddel, B. 38
Walker, PA. 51
Waring, GL. 87
Weber, F. 28
Webb, BA. 59
Weaver, RJ. 84
Wheeler, DE. 5,52,55
Wimmer, AE. 2
Williams, CM. 30,33,161
Wipking, W. 193
Wise, S. 37
Wise, MJ. 192
Wilson, C. 102
Wicker, T. 219
Willnow, TE. 106
Withyachumnarnkul, B. 34
Wiedenmann, G. 36
Wigglesworth, VB. 50
Willott, E. 57,58
Wojchowski, DM. 82
Wojtasek, H. 150,154
Wood, Jr. 193
Worland, R. 192
Wu, C. 211
Wyllie, AH. 160
Wyatt, GR. 72,83,84,85,87
X
Xia, T. 183
Xia, Q. 210,223
Xiong, Y. 219,220
Xu, J. 133
Z
Zakeri, ZF. 168,169
Zachariassen, KE. 192
Zakharkin, SO. 59
Zavodska, R. 37
Zhong, BX. 55
Zhang, J. 30
Zhu, J. 33,123
Zheng, L. 141
Zhang, X. 154
Zhou, L. 185
Zhao, J. 37
244
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tM

Edited b Volurn+ {1}, April 2009

Editor: f)r. Raman Chandrasekar

First Published April Z00g

You must not circulate this book in any other

Publication press: Naehiyar silk and printing Hquse, south India.

S ubscriptions und Distri bution

International Book lulission @

Entomology as a science of interdepended branches like molecular entomology, insect

Raman Chandrasekar, Ph.D.,

1. Perspective of Molecular Approaches to Entomology 1

2. Molecular structure of insect clocks 21

3. Insect Hexamerin Storage Proteins: Biosynthesis, Utlization

4. Structural characteristics/ biosynthesis mechanisms of insect

5. Molecular mechanisms of insect Vitellogenin /lipophorin receptor 95

6. Nutritional signaling in adult mosquitoes 119

7. Pheromone Biosynthesis Activating Neuropetide (PBAN) and

8. Molecular mechanisms of sex pheromone reception in Lepidoptera 147

10. Apoptosis Regulation in Mosquito and its Importance to 175

12. Molecular characteristics of the W chromosome, Z chromosome and

Appendix - A (Glossary and Terms) 229

Prof. Makio TAKEDA Prof. Ada Rafaeli

Short Views on Insect Molecular Biology, Vol.(1), 2009

Prof. Marc J. Klowden Prof. Randolf Menzel

Short Views on Insect Molecular Biology, Vol. (1), 2009

Perspective of Molecular Approaches to Entomology

Keywords: molecular biology, sensillomic, baculoviruses, display techniques, pheromone

*For Correspondence (email: koreanchandru@yahoo.com)

*Present Address: College of Agriculture Entomology,

2. Molecular and Biotechnology approaches

Biochemistry, genetics and

Fig.2 The Molecular basis of life.

DNA (structural genomics)

3. Physiological vital Proteins

The appearance of storage

Peripheral fat body tissues

Fig.5 Schematic diagram showing the synthesis

(3b) Vitellogenin (Vg)

Vitellogenin (Vg) is a multifaceted protein and so,

The Vgs of most insects are members of a conserved

Fig.7 A pair of pectinated antennae

7. Baculoviruses as Expression Vectors

Research on the silkworm Bombyx mori

Insect offer many advantages for gene

The display of proteins on the surface of

communicable diseases, including parasitic (malaria, trypanosomosis, leishmaniosis, filariosis, etc.),

Arthropods—which are responsible for transmitting a broad range of diseases—have infinitely

Chikungunya virus is geographically distributed in

The genome of CHIKV consists of a linear, positive-sense, single-stranded RNA of approximately

10. Integrated pest management

integrated and used in a system that Indian Council of Agricl. Research

ecologically sound and economically

interacts with the environment, natural DBT, New Delhi

11. Concluding remarks

The ideal transgenic technology should be commercially feasible, environmentally benign

Publishers, Inc. Enfield, USA – Plymouth, UK, pp. 69-101.

Molecular Structure of Insect Circadian Clocks

*For Correspondence (email: mtakeda@kobe-u.ac.jp)

Fig.1 Three functional subunits of the circadian clock system.

2. Molecular structure of Drosophila circadian clock

(2a). Molecular components of circadian pacemaker

(2b). Molecular circuitry of circadian oscillator and roles of post-translational mediators