2013 AdvInsectSci-Smagghe Et Al

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This chapter was originally published in the book Advances in Insect Physiology, Vol. 43, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institutions administrator.
All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institutions website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: Guy Smagghe, Luis E. Gomez, Tarlochan S. Dhadialla, Bisacylhydrazine Insecticides for Selective Pest Control. In Tarlochan S. Dhadialla, editor: Advances in Insect Physiology, Vol. 43, Burlington: Academic Press, 2012, pp. 163-249. ISBN: 978-0-12-391500-9 Copyright 2012 Elsevier Ltd. Academic Press
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CHAPTER TWO
Bisacylhydrazine Insecticides for Selective Pest Control

Guy Smagghe*, Luis E. Gomez, Tarlochan S. Dhadialla
*Department of Crop Protection, Ghent University, Ghent, Belgium Dow AgroSciences LLC, Indianapolis, Indiana, USA
Contents
1. Introduction 2. Chemical Structures of Ecdysteroids and Non-Steroidal Ecdysone Agonists 3. Ecdysteroid-Specific Mode of Action 3.1 Bioassays for tissue and cellular effects 3.2 Bioassays for whole organism effects 4. Methoxyfenozide Global Uses 4.1 Introduction 4.2 Bulb vegetables 4.3 Cereals 4.4 Citrus 4.5 Cole crops (Brassica vegetables) 4.6 Cucurbits 4.7 Forages 4.8 Forestry 4.9 Fruiting vegetables 4.10 Leafy vegetables and legumes 4.11 Oilseeds 4.12 Ornamentals 4.13 Pome fruits 4.14 Small fruits 4.15 Stone fruits 4.16 Tree nuts 4.17 Tropical fruits 5. Methoxyfenozide Formulation 5.1 Formulation types and commercial products 6. Environmental Fate, Metabolism, and Residue Analysis of Methoxyfenozide 6.1 Introduction 6.2 Metabolism and environmental fate studies 6.3 Environmental fate and characteristics of methoxyfenozide 6.4 Hydrolysis of methoxyfenozide under aqueous conditions 6.5 Metabolism of methoxyfenozide in animals 6.6 Metabolic fate of methoxyfenozide in plants
Advances in Insect Physiology, Volume 43 ISBN 978-0-12-391500-9 http://dx.doi.org/10.1016/B978-0-12-391500-9.00002-4
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164 7. Toxicological Profile of Methoxyfenozide 7.1 Mammalian 7.2 Avian 7.3 Aquatic 7.4 Fish 7.5 Terrestrial 8. Sublethal and Ovicidal Effects 8.1 Sublethal effects 8.2 Ovicidal effects 8.3 Population effects by sublethal and ovicidal effects 9. Resistance and Resistance Management 10. Conclusions and Future Prospects Acknowledgements References
Guy Smagghe et al.
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Abstract
In this chapter, we review five members of a novel class of chemistry, the non-steroidal bisacylhydrazine (BAH) compounds that are true agonists of the steroidal insect moulting hormone, 20-hydroxyecdysone. Also referred to as ecdysone agonists (EAs), the five BAH compounds have been commercialized for the control of lepidopteran and coleopteran larvae. Of these, four compounds (methoxyfenozide, tebufenozide, chromafenozide, and fufenozide) are predominantly toxic to lepidopteran larvae, while the fifth compound, halofenozide, is active on both lepidopteran and coleopteran larval pests in turf. The evidence for the basis of this insect selective toxicity is reviewed. The nonsteroidal EA BAH insecticidal compounds are important tools in integrated pest management and insect resistance management programmes because of their selective insect toxicity, novel mode of action, and reduced risk for eco- and mammalian toxicology. In reviewing these BAH insecticides, there is greater emphasis on methoxyfenozide, the most widely used insecticide in this class of chemistry.
1. INTRODUCTION
The two principle hormones that regulate insect growth, development, and reproduction are the steroidal moulting hormone, 20hydroxyecdysone (20E), and the sesquiterpenoid juvenile hormone (JH). Any interference with the titres of these hormones, or the mechanisms by which they manifest their actions, results in abnormal or detrimental growth, development, and reproduction. Ecdysteroids, which are widespread in the animal and plant kingdoms (http:/ /ecdybase.org; Lafont et al., 2002), are signalling molecules that fulfil

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diverse functions in the life of an insect by virtue of their roles as hormones, pheromones, or insect deterrents (Nijhout, 1994). Interestingly, they do not occur naturally in vertebrates, which is a feature that makes mimics of this important hormone suitable as ligand-dependent gene-switch ligands in different applications in agriculture and medicine due to the reduced likelihood of pleiotropic effects. The first attempts to synthesize insecticides with 20E activity were made in the 1970s (Watkinson and Clarke, 1973). This concept was inspired by Carroll Williams in 1967 to introduce the potential use of insect hormones as environmentally benign third-generation insecticides for which the pest insects would not be able to develop resistance because the hormones are required to complete development. The rationale was that if the pest insect is treated with a chemical that mimics the action of ecdysteroids or JH at an inappropriate life stage, the treated insect would go through abnormal development, leading to mortality. Unfortunately, early attempts to test this hypothesis using steroidal molecules with a cholesterol backbone mimicking the structure and function of ecdysteroids failed because of the chemical and metabolic instability of the steroid in the insect (Watkinson and Clarke, 1973). After the early attempts of Watkinson and Clarke (1973), there was a long lag period in the discovery of additional analogues of ecdysteroids. It was nearly two decades after the hypothesis of Williams (1967) that researchers at Rohm and Haas Company (Spring House, PA) synthesized the first non-steroidal ecdysone agonist (EA), belonging to the chemical class of BAHs (Hsu, 1991; Wing, 1988; Wing et al., 1988). Structureactivity optimization of the first such compound during the subsequent few years at Rohm and Haas Company led to the synthesis of three highly effective compounds that have since been commercialized. It is notable that the BAH insecticides have been the subject of over 2000 refereed journal articles and other research reports since their discovery. It is beyond the scope of this chapter to review all published works, especially those published before 2006. For overview of the topic, the reader is referred to fairly extensive reviews published before 2006 (Dhadialla and Ross, 2012; Dhadialla et al., 1998, 2005, 2010; Oberlander et al., 1995; Palli et al., 2005a,b,c). In the current insecticide marketplace, there are five registered BAH insecticides. Three were discovered by Rohm and Haas Company (tebufenozide coded as RH-5992, methoxyfenozide coded as RH-2485, and halofenozide coded as RH-0345), one by Nippon Kayaku/Sankyo Companies (chromafenozide coded as ANS-118, CM-001), and one by Jiangsu Pesticide Research Institute Company Limited (fufenozide coded as JS-118). Of the

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three BAH insecticides discovered and originally commercialized by Rohm and Haas Company, two (methoxyfenozide and halofenozide) are currently owned by Dow AgroSciences LLC and tebufenozide was purchased by Nippon Soda Co., Ltd. from Dow AgroSciences LLC, in 2010. Four of the BAH compounds (methoxyfenozide, tebufenozide, chromafenozide, and fufenozide) have a spectrum of control largely specific for lepidopteran larvae, while halofenozide has a broader spectrum of control that includes coleopteran and lepidopteran larvae, showing special utility as a soil insecticide and marketed for control of turf pests. To date, methoxyfenozide is the most widely registered and used BAH insecticide, with registrations in more than 50 countries for use on a variety of crops ranging from vegetables to specialty uses such as in forestry and tea production. This chapter focuses on methoxyfenozide, with greater emphasis on its spectrum of activity for control of lepidopteran pests and its toxicological profile. Where appropriate, other EA structures are mentioned throughout the chapter to exemplify the utility of these compounds for insect control. Previous reviews of this non-steroidal class of chemical insecticides have focused on their mode of action as agonists of 20E, their selectivity to target pests, and their use in non-insecticidal applications such as gene switches in plant and animal systems (Dhadialla and Ross, 2012; Dhadialla et al., 1998, 2005, 2010; Oberlander et al., 1995; Palli et al., 2003, 2005a,b,c). Gomez et al. (2011) presented a review of the BAH insecticides and their utility as green chemistry compounds. The BAH insecticides novel mode of action as non-steroidal EAs, their selective insect toxicity, and other characteristics make these insecticides important tools for insecticide resistance management (IRM) and integrated pest management (IPM) programmes. For an extensive review of structureactivity relationships (SARs) of BAH EAs and ecdysteroids and their action and interaction at the molecular level, the reader is referred to Chapters 3 and 4.
2. CHEMICAL STRUCTURES OF ECDYSTEROIDS AND NON-STEROIDAL ECDYSONE AGONISTS

The chemical structure of the first non-steroidal ecdysteroid agonist with potent insecticidal activity was published in 1988 (Hsu, 1991; Wing, 1988; Wing et al., 1988). This novel compound with a non-substituted BAH structure was coded as RH-5849. In the decade that followed the discovery of RH-5849, extensive work at Rohm and Haas Company with

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this new class of chemistry involved the synthesis of additional compounds guided by ligandreceptor binding and whole-insect mortality SAR assays for over 4000 BAH analogues. These efforts led to the discovery of the three insecticidal compounds, tebufenozide, methoxyfenozide, and halofenozide, which were commercialized by Rohm and Haas Company. Further efforts by scientists in industries in Japan and China to find additional chemistries with an ecdysone mode of action led to the discovery of two new compounds, chromafenozide and fufenozide, both of which are members of the BAH chemical class. The main difference between the three BAH insecticides discovered at Rohm and Haas Company and chromafenozide is that, in chromafenozide, the A-ring (for nomenclature of phenyl rings in BAH compounds see Fig. 2.1) is fused to a dihydropyran ring. For fufenozide, a dihydrofuran ring was fused with the A-ring. The structures of 20E, the first BAH insecticidal prototype (RH-5849), and the five commercial BAH insecticidal compounds are shown in Fig. 2.1. In parallel with these developments, SAR studies on compounds from the BAH chemical class have been done by different groups and reviewed (Dinan and Hormann, 2005; Minakuchi et al., 2003; Nakagawa, 2005; Nakagawa et al., 1998, 1999, 2000, 2001, 2005; Sawada et al., 2003; Smagghe et al., 1999b, Wheelock et al., 2006). Dinan and Hormann (2005) reported that six-membered aryl groups on either side of the two negative centres favoured activity against lepidopteran and coleopteran insects. Activity against lepidopterans could be further enhanced by modifying the substitutions on the A-ring at the 4-position with 1-2carbon-containing lipophilic groups, or with 2,3- or 2,(3,4)-ring patterns. Substitutions on the B-ring were also found to be less specific, though 2-, 2,5-, 3,5-, or 3,4,5-positions were favourable. In this context, the higher activity of methoxyfenozide with a methoxy group on the A-ring and two methyl groups on 3,5-positions of the B-ring can be explained. Using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), Hormann et al. (2008) reported on the effect of substituents at the two aromatic moieties of BAHs. The insecticidal activities of a panel of 77 BAH compounds were determined against three economically important pest insect species: beet armyworm, Spodoptera exigua (Hu bner), Asiatic rice borer, Chilo suppressalis (Walker), and Colorado potato beetle, Leptinotarsa decemlineata Say. Comparison of the two lepidopteran CoMFA models revealed a similarity in the steric contours of the B-ring and two important electrostatic contours near each of the A-carbonyl groups and the B-ring.

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Ecdysteroid
X
OH H Y
Gene switch ligand

O
HO H HO H OH
O X = H, Y = OH X = OH, Y = OH X = OH, Y = H
N H
O RG-102240 (GS-S)
O Ecdysone (E) 20-Hydroxyecdysone (20E) Ponasterone A (PonA)
New chemistries
F O N H O F N O O F HN
Insecticides
O N H N O O
O
N H N O
B
Cl
a-Acylaminoketone Halofenozide (RH-0345)

O O
Tebufenozide (RH-5992)
O N H O N O O
Tetrahydroquinoline
N H
N O O O
N H
N Cl N Cl N
CN
Methoxyfenozide (RH-2485)
Chromafenozide (ANS-118)
Oxadiazoline
Cl
O N H N O O Cl
g-Methylene-g-lactam
Fufenozide (JS-118)
Figure 2.1 Chemical structures of the steroidal ecdysteroids (ecdysone, 20-hydroxyecdysone, ponasterone A (Pon A)), non-steroidal ecdysone agonists commercialized as insecticides, and a series of new chemistries that have not been commercialized but have been shown to act as ecdysone agonists in ecdysone recptor (EcR) ligand-binding assays. The BAH insecticides, tebufenozide, methoxyfenozide, and halofenozide (RH-5992, RH-2485, and RH-0345, respectively, all developed by Rohm and Haas Company, USA), chromafenozide (ANS-118, CM-001; jointly by Nippon Kayaku and Sankyo, Japan), and fufenozide (JS-118, by Jiangsu Institute, China), tetrahydroquinoline (THQ) (by FMC, USA), a-acylaminoketone and oxadiazoline (both by Intrexon, USA), and g-methylene-g-lactams (by CSIRO, Australia). The BAH ligand RG-102240 (GS-S) (by RheoGene, now Intrexon, USA) is used as a gene-switch activator ligand in the RheoSwitch system. The terminology of labelling the two phenyl rings as Aand B-rings of BAH compounds is indicated for tebufenozide as an example.
The disposition of a steric-enhancing CoMFA contour and a negative charge-enhancing contour near the A-ring is also consistent with the high lepidopteran potency found for chromafenozide, which possesses an A-fused chroman ring in the negative charge-enhancing region in the Lepidoptera model. These contours are absent for the coleopteran tested, in which chromafenozide shows only low toxicity. In contrast to the

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prominent A-ring steric-diminishing and negative-charge-enhancing contours of the lepidopteran CoMFA models, numerous steric-enhancing regions occur around both A- and B-positions in the coleopteran model. The geometric disposition of hydrogen-bond-donor-enhancing regions differed as well. Additionally, certain hydrogen-bond-acceptor regions that are present in Lepidoptera are absent in the coleopteran model. Overall, the SAR analysis for each type of insect toxicity could be partially, but not entirely, rationalized in view of the ecdysone receptor ligand-binding domain (EcRLBD) sequence alignments and putative contact residues relative to the known crystallized Heliothis virescens (Fabricius) EcR-LBD/BYI06830 holoreceptor (Billas et al., 2003). For more details on SARs between ecdysteroids and non-steroidal agonists, the reader is referred to Chapter 3. With the successful development of the BAH insecticides, the search continued for novel chemistries that would control non-lepidopteran pests, particularly sucking insects such as mosquitoes, aphids, leafhoppers, and whiteflies. To achieve this, in addition to traditional approaches, insect EcR sequences were obtained by cloning and genome projects and were used to design highthroughput screening (HTS) assays in cell lines and yeast in industry and academia (Dhadialla et al., 2005; Palli et al., 2005a; Swevers et al., 2004; Tice et al., 2003). Interestingly, these large HTS efforts, with conventional new lead chemistry optimization, resulted in the identification of new EcRbinding compounds belonging to chemistries different from the BAH chemistry: 3,5-di-tert-butyl-4- hydroxy-N-isobutyl-benzamide (DTBHIB), tetrahydroquinoline (THQ), a-acylamidoketone, oxadiazoline chemistries, and g-methylene g-lactams (Fig. 2.1) (reviewed in Birru et al., 2010; Dhadialla et al., 1998, 2005; Palli et al., 2005a; Tice et al., 2003, Chapter 3). Unlike the predominant insecticidal activity of the BAH for lepidopteran insects, some compounds of the THQ group demonstrated a higher potency for dipterans than lepidopterans (Palli et al., 2005b), and the g-methylene g-lactams targeted the EcRs from the sheep biting louse, Bovicola ovis (Schrank), and the Australian sheep blowfly, Lucilia cuprina (Wiedemann), with high affinity (Birru et al., 2010). While these new chemistries demonstrate the potential for discovery of new non-steroidal EAs with a broader spectrum of activity different from the BAH insecticides, none of the new chemistries discovered outside the BAH chemical class have led to newly registered insecticides. The insecticidal activity of the benzamide compound discovered at Sumitomo Company, Japan, was not described, and attempts to replicate the results of Mikitani (1996) using DTBHIB and analogues failed to

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demonstrate that these compounds were competitive inhibitors of tritiatedponasterone A (3H-Pon A) binding to the fruit fly, Drosophila melanogaster (Meigem), EcR, and ultraspiracle (DmEcR/DmUSP) heterodimer proteins produced by in vitro transcription and translation (Dhadialla et al., 2005). In addition, after Elbrecht et al. (1996) at Merck Research Laboratories, West Point, PA, reported the isolation of an iridoid glycoside, 8-Oacetylharpagide, from a plant, the common bugle, Ajuga reptans (Aiure), as having ecdysteroidal activity, Dinan et al. (2001) demonstrated that the ecdysteroidal activity claimed by Elbrecht et al. (1996) was due to copurification of ecdysteroids in their 8-O-acetylharpagide preparation. In addition to control of agricultural pests with the BAH insecticides, there were successes to control gene expression in artificial systems with these insecticides and other BAH chemistries. Scientists at RheoGene (now Intrexon Ltd.) developed an EcR-based gene switch, RheoSwitch, in animal cells that could be transactivated using a BAH ligand, RG102240 (GS-S) (Fig. 2.1). Continuing with this effort, Lapenna et al. (2009) reported the discovery of novel semi-synthetic ecdysteroids with high potency as ligands in a gene-switch system, possibly reflecting their good permeability and metabolic stability. We believe that the latter efforts constitute valuable progress towards the goal of identifying new EcR-based gene-switch activator molecules suitable for drug development and for ligand-dependent transgene activation in plants.
3. ECDYSTEROID-SPECIFIC MODE OF ACTION

In the earlier reviews on the BAH insecticides with ecdysteroid activity (Dhadialla et al., 1998, 2005; Nakagawa, 2005; Oberlander et al., 1995), the focus on mode of action was from studies using RH-5849, tebufenozide (RH-5992), and halofenozide (RH-0345). The unsubstituted BAH, RH5849, which was not commercialized, had a broader spectrum of insect toxicity than the five that have been commercialized since then. In this section, the focus will be on work published since 2005 on the five commercialized BAH insecticides. References will be made to earlier publications where relevant.
3.1. Bioassays for tissue and cellular effects

A number of in vitro and in vivo assays have been used to study the effects and mode of action of the BAH insecticides. For these assays, larvae of susceptible and non-susceptible insects and a number of insect cell lines and dissected tissues were used.

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Wing et al. (1988) were the first to use D. melanogaster embryonic Kc cells to demonstrate that like 20E, RH-5849 also induced aggregation and clumping of otherwise confluent cultures of Kc cells. Similar morphological effects of four of the commercialized BAH compounds, tebufenozide, methoxyfenozide, halofenozide, and chromafenozide, have also been demonstrated for cell lines derived from embryos or tissues of D. melanogaster (Clement et al., 1993; Mosallanejad et al., 2008a,b; Nakagawa, 2005); the mosquitoes, Aedes sp., Anopheles sp., and Culex sp. (Beckage et al., 2004; Smagghe et al., 2002); the midge, Chironomus tentans Fabricius (Smagghe et al., 2003a,b; Spindler-Barth et al., 1991); the forest tent caterpillar, Malacosoma disstria Hu bner; the spruce budworm, Choristoneura fumiferana (Clemens) (Sohi et al., 1995); the Indian meal moth, Plodia interpunctella Hu bner (Oberlander et al., 1995); the cotton boll weevil, Anthonomus grandis (Boheman) (Dhadialla and Tzertzinis, 1997; Soin et al., 2009); the beet armyworm, S. exigua, and the silk moth, Bombyx mori (Linnaeus) (Mosallanejad et al., 2008a,b; Swevers and Iatrou, 2003). There are no data available for the mode of action of fufenozide, but the chemical structure and its selective efficacy on lepidopteran pests suggest that this compound also would act as a non-steroidal EA. Some of these cell lines, imaginal wing discs, and larval claspers from susceptible insects also have been used to study the relative binding affinities, and biochemical and molecular effects of tebufenozide (Mikitani, 1996), methoxyfenozide, halofenozide, or chromafenozide and other BAH EAs (Dhadialla et al., 2005; Farkas and Slama, 1999; Mosallanejad et al., 2008a,b; Nakagawa, 2005; Nakagawa et al., 2002a,b,c; Smagghe and Swevers, 2013; Smagghe et al., 1996, 2000, 2002; Soin et al., 2009, 2010a,b). Cytosolic and/or nuclear extracts from 20E-responsive cells and tissues containing functional EcRs, and bacterially expressed EcRs and USPs from different insects, also have been used to determine the relative binding affinities of BAH insecticidal compounds or to screen for new chemistries with a similar mode of action in radiometric competitive receptor-binding assays (Table 2.1). In all these studies, it was evident that the BAH insecticidal compounds manifest their activity by binding specifically to the ecdysone receptor complex (EcR/USP) of the insect moulting hormone or ecdysteroids. Although all arthropods have EcR complex proteins, and the respective moulting hormones (ecdysteroids) manifest their action via interaction with the EcR/ USP receptor protein, four of the five commercialized BAH insecticidal compounds, tebufenozide, methoxyfenozide, chromafenozide, and fufenozide are predominantly toxic only to lepidopteran larvae while
Table 2.1 Relative affinities of 20-hydroxyecdysone (20E), ponasterone A (Pon A), and the BAH compounds on engineered cells, nuclear receptor cellular extracts, or in vitro expressed ecdysone receptor and ultraspiracle (EcR and USP, respectively) proteins from insects of different orders, demonstrating their high insect order specificity Insect species 20E Pon A RH-5849 Tebufenozide Methoxyfenozide Halofenozide Chromafenozide References
Insect order: Lepidoptera Plodia interpunctella 210 nM (extracts, IAL-PID2 cells) Spodoptera exigua (imaginal discs, Se4 cells) 90 nM 3 nM 230 nM 3 nM 0.5 nM 129 nM Dhadialla et al. (1998, 2005) 9.0 nM Smagghe and Degheele (1995), Mosallanejad et al. (2008a,b) Smagghe et al. (2000), Soin et al. (2009a) Nakagawa et al. (2002a), Toya et al. (2002), Ogura et al. (2005b) Swevers et al. (2003), Soin et al. (2009a)
2.6 nM
870 nM
12 nM
9.6 nM
Spodoptera littoralis (imaginal discs, Sl2b cells) Spodoptera frugiperda (Sf9 cells)
158 nM
1.3 mM
2.287 nM
6.024 nM
500 nM
2.6 nM
166 nM
8.9 nM
363 nM
1.5 nM
3.5 nM
ca 1 nM
Bombyx mori (Bm5)
36 nM
10 nM
400 nM
0.91.1 nM
0.13.4 nM
78 nM
0.11 nM
Insect order: Diptera 54145 nM Drosophila melanogaster (extracts, Kc, S2 cells) 0.9 nM 2 mM 192 nM 6.6 mM 124987 nM 0.57.8 mM 1.1 mM Cherbas et al. (1988), Dhadialla et al. (1998, 2005), Nakagawa et al. (2002b, 2005), Soin et al. (2010a,b) Kapitskaya et al. (1996), Dhadialla et al. (2005) 230 nM Grebe et al. (2000), Smagghe et al. (2002) 2 mM
Aedes aegypti
28 nM
2.8 nM
30 nM
Chironomus tentans
278 nM
0.356.5 nM 27 nM
7.3 nM
Insect order: Coleoptera Leptinotarsa decemlineata 247425 nM 6 nM 740 nM 1.3117 mM 1.1 mM 26 mM Smagghe et al. (1996a), Dhadialla et al. (1998), Ogura et al. (2005a) Dhadialla et al. (2005)
Continued
Tenebrio molitor (extracts)
> 10 mM > 10 mM
> 10 mM
> 10 mM
Table 2.1 Relative affinities of 20-hydroxyecdysone (20E), ponasterone A (Pon A), and the BAH compounds on engineered cells, nuclear receptor cellular extracts, or in vitro expressed ecdysone receptor and ultraspiracle (EcR and USP, respectively) proteins from insects of different orders, demonstrating their high insect order specificitycont'd Insect species 20E Pon A RH-5849 Tebufenozide Methoxyfenozide Halofenozide Chromafenozide References
Anthonomus grandis (AG-3C cells)
247 nM
4.46.1 nM
45 mM
7.113 nM
5 mM
15117 mM
Dhadialla and Tzertzinis (1997), Soin et al. (2009b)
Insect order: Orthoptera Locusta migratoria 10,000 nM 1.18 nM > 10 mM > 10 mM > 10 mM > 10 mM Hayward et al. (2003)
Insect order: Hemiptera Bemisia argentifolia Bemisia tabaci Myzus persicae Nezara viridula 34.5 mM 0.3 mM 8 nM > 10 mM > 10 mM > 400 mM > 100 mM 2028 mM > 10 mM > 10 mM Dhadialla et al. (2005) Carmichael et al. (2005) Carmichael et al. (2005) TohidiEsfahani et al. (2011)

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halofenozide is toxic to lepidopteran and coleopteran larvae. Additionally, methoxyfenozide and tebufenozide have ovicidal and sublethal effects on adult Lepidoptera (see Sections 4 and 8). The main reason for this selective toxicity is their extremely high binding affinity to lepidopteran EcRs. As exemplified in Table 2.1, the insect steroid moulting hormone 20E and the potent phytoecdysteroid Pon A bind to EcRs from different insect orders with similar affinities. Methoxyfenozide, tebufenozide, and chromafenozide bind to the lepidopteran EcRs with affinities that are equivalent to that of Pon A (approximate Kd values of 110 nM), which is about 100-fold more active than 20E. But intriguingly, this was not the case for the binding of these compounds to EcRs from coleopteran (Colorado potato beetle, L. decemlineata, the yellow mealworm, Tenebrio molitor (Linnaeus), and cotton bollworm, A. grandis), orthopteran (the migratory locust, Locusta migratoria (Linnaeus)), and hemipteran insects (whiteflies, Bemisia tabaci (Gennadius), the green peach aphid, Myzus persicae (Sulzer), and the southern green stink bug, Nezara viridula (Linnaeus)) even when tested at high concentrations of greater than 110 mM (Carmichael et al., 2005; Dhadialla and Tzertzinis, 1997; Dhadialla et al., 2005; Hayward et al., 2003; Smagghe et al., 2006; Tohidi-Esfahani et al., 2011). Insect cells, like vertebrate or plant cells, can be transformed with constructs containing a reporter gene encoding the green fluorescent protein or the firefly luciferase protein driven by a ligand-dependent transcriptional factor and appropriate DNA response element. For understanding the mode of action of the BAH insecticidal compounds, insect cells have been transformed with one of the aforementioned reported genes under the control of either an endogenous EcR complex or a heterologous EcR complex and ecdysone response elements. Such transformed insect cell lines have been used to screen new chemistries with EA activity, for SAR purposes to optimize the structure of the putative chemistries, or to understand the SAR of the EcR complex proteins and their interactions (as reviewed in Dhadialla and Ross, 2007; Dhadialla et al., 2005, 2010; Palli et al., 2005a; Palli and Retnakaran, 2000; Smagghe and Swevers, 2013). Upon exposure to an EA compound, there is a concentration-dependent fluorescent or luminescent signal by the transgenic cells which can be read in an HTS set-up. Depending upon which EcR complex was tested in the transformed insect cell, the activity of test compounds in in vitro cell-based assays correlated with insecticidal activity of the test compounds in in vivo whole larval mortality assays (Smagghe and Swevers, 2013; Soin et al., 2009, 2010a,b; Swevers et al., 2004). In contrast, while the BAH insecticides also bind to

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the EcRs from the dipteran fruit flies, D. melanogaster and the yellow fever mosquito, Aedes aegypti (Linnaeus), the binding affinities are relatively weak and of no toxicological consequence. D. melanogaster cells deficient in the DmEcR have been used with success to investigate the interaction of BAH insecticides with the non-target crustacean, Crangon crangon (Linnaeus), EcR/USP (Verhaegen et al., 2010). Similarly, vertebrate CHO cells transformed with different insect EcR and/or USP have been used in HTS mode to screen for novel EA chemistries (Beatty et al., 2009). Despite the extensive understanding of the molecular basis of the action of the BAH insecticidal compounds and analogous chemistries, and the availability of a variety of receptorligand binding and cell-based assays, no new commercial chemistries have been found for non-lepidopteran insect pests. While the reader is referred to extensive reviews on the mode of action of the BAH insecticides by Dhadialla et al. (1998, 2005, 2010) and Dhadialla and Ross (2012), subsequent chapters in this volume provide in-depth reviews on the SAR of ecdysteroids and synthetic agonists for interaction with the EcR and biological activity (Chapter 3), and the molecular basis of interaction of ecdysteroids and non-steroidal EA compounds with the LBD residues of EcR proteins from several insects (Chapter 4).
3.2. Bioassays for whole organism effects

The commercialized BAH insecticides are toxic to susceptible insects mainly via ingestion. Toxicity via topical application is expressed only when very high doses are applied. The effects of BAH compounds have been studied in many different insects (Dhadialla et al., 1998, 2005; Nakagawa, 2005; Palli and Retnakaran, 2001; Retnakaran et al., 1997; Slama, 1995; Smagghe and Degheele, 1994a,b; Smagghe et al., 1996b,c, 2002). In general, because the BAH insecticides are more metabolically stable in vivo than ecdysteroids and because they are true EAs at the receptor level, ingestion of these insecticidal compounds creates hyperecdysonism, a term coined by Williams (1967). Hyperecdysonism represents a state of excess ecdysteroidal activity in susceptible insects, which results in effects and symptomatology of a moult event. In insects, a moult from one developmental stage to another is triggered at the appropriate time within a developmental stage by increasing titres of 20E, the insect moulting hormone. Although the toxicity and the pest control spectrum of the five leading BAH EAs vary, their toxic symptomatology is very similar.

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A B
Figure 2.2 Scanning electron micrographs of third instar larva of Cydia pomonella at 24 h after treatment with tebufenozide showing (A) clear premature head capsule apolysis (small white arrow heads) without shedding of the old head capsule (large black arrows), leading to the presence of a double head capsule ( 60). (B) The inset (from white arrow heads in (A)) indicates the presence of the new capsule that was not sclerotized ( 200). (C) Light microscope photomicrograph showing the abnormal attachment of thoracic muscles to the cuticle (arrows) ( 40) (adapted from Smagghe et al., 2004).
One of the first effects of ingestion of a BAH EA by susceptible larvae is inhibition of feeding within a few hours of treatment (314 h) (Dhadialla et al., 1998, 2005; Retnakaran et al., 1997; Slama, 1995; Smagghe, 1995; Smagghe et al., 1996a), which prevents further plant damage. Morphologically, the susceptible intoxicated larva becomes moribund and has a slipped head capsule, which is indicative of the ongoing moulting process, but terminates as a precocious lethal moult. In Fig. 2.2, the morphological effects are shown and the new cuticle is not tanned or sclerotized. One resulting consequence is that the mouthparts under the slipped head capsule remain soft and mushy, preventing any crop damage even if the head capsule came off by mechanical or physical force (Dhadialla et al., 2005; Smagghe et al., 2004). The physiological and biochemical basis for the action of BAH insecticides to disrupt larval development manifested by a lethal moult have been studied, but not as extensively as the molecular basis of their action. Normal insect moults initiated by 20E are very tightly regulated in concert with required gene expression and repression as well as the interplay with hormones (20E, eclosion hormone, and bursicon) for normal growth and development of insects. Timely release of eclosion hormone for successful moulting behaviour is necessary and happens when 20E titres decline to basal levels (Truman et al., 1983), but with potent BAH insecticidal compounds, these

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normal processes are disrupted. The BAH insecticides trick the intoxicated susceptible larva to initiate an untimely moult and, by virtue of their metabolic stability, these compounds inhibit repression of certain genes and release of eclosion hormone, which is normally dependent upon declining titres or basal levels of 20E. Hence, the susceptible intoxicated larva is unable to successfully complete the initiated moult. Examination of the newly cuticularized head and mouth parts under the slipped head capsule of the BAH insecticide-intoxicated larva reveals a lack of sclerotization and tanning of the new cuticle (Figs. 2.2 and 2.3). Other symptoms of intoxicated susceptible larvae include extrusion of the hindgut and loss of haemolymph and moulting fluid from haemorrhage of the deformed new
LM-24 h A NC SHC
TEM-12 h B1
oPC
24 h D
72 h
oEC oPC EM
B2
ES mv mv ES
C2 C1
ES mv mv N fC nEC
ES
nEC nPC mv
N N
BL
BL BL
Figure 2.3 Treatment of a susceptible fifth-instar larva of Spodoptera exigua with a BAH insecticide (exemplified here with tebufenozide), inducing a lethal premature moult. (A) Photomicrograph showing the induction of premature head capsule apolysis. The black arrow indicates the newly synthesized untanned white capsule (NC) under

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cuticle, all of which result in desiccation and ultimate death. These effects have been demonstrated in a number of treated lepidopteran larvae (reviewed in Dhadialla et al., 2005), in sensitive Coleoptera (L. decemlineata; T. molitor; A. grandis; white grubs; Japanese beetle, Popillia japonica (Newman); multicoloured Asian lady beetle, Harmonia axyridis (Pallas)) treated with halofenozide (Carton et al., 2003; Dhadialla et al., 1998, 2005; Farinos et al., 1999; Smagghe and Degheele, 1994; Smagghe et al., 1996a), and in Diptera (A. aegypti; Anopheles gambiae (Giles; southern house mosquito, Culex quinquefasciatus (Say); northern house mosquito, Culex pipiens (Linnaeus); C. tentans) (Beckage et al., 2004; Boudjelida et al., 2005; Smagghe et al., 2003a). The reasons for the lethal precocious moult effects of BAH insecticides also have been investigated at the ultrastructural level in different susceptible insects, such as S. exigua (Smagghe et al., 1996c), C. fumiferana (Palli and Retnakaran, 2001; Retnakaran et al., 2003; Retnakaran et al., 1997), the golden twin-spot moth, Chrysodeixis chalcites (Esper) (Smagghe et al.,
the old brown sclerotized head (white arrow), demonstrating slipped head capsule (SHC), and severe growth inhibitory effects. (BD) Transmission electron microscope (TEM) micrographs of the integument 12 h (B), 24 h (C), and 72 h (D) after treatment with tebufenozide. The photomicrographs represent the induction of premature moulting with forced apolysis at 12 h, a new epicuticle layer at 24 h, and inhibition of ecdysis, leading to death at 72 h. (B1) Fine structure of the integument showing the endocuticular lamellae of the old cuticle (oPC), which are digested by the moulting fluid, morphologically resemble the initial formation of an ecdysial membrane (EM). Ecdysial space (ES) formation and detached epidermal microvilli (mv) are clearly visible beneath the old cuticle. The apical border of the epidermal cell is vacuolated and the nuclei of epidermal cells (N) contain dense chromatin masses and many vacuoles and vesicles. (B2) Inset of microvilli (mv) with clear start of deposition of new epicuticular material with secretion of new cuticulin patches, plasma membrane plaques, at the tips of the epidermal microvilli (small black arrows). Bars for A1 2.2 mm and A2 0.6 mm. (C1) Formation of a double cuticle. The epidermal cells contain numerous vacuoles and vesicles and irregularly shaped nuclei (N) with dense chromatin masses. BL, basal lamina; ES, ecdysial space. (C2) Inset of the apical epidermis, demonstrating the production of fibrous cuticle (fC) under the newly secreted epicuticular layer (nEC) (large open white arrows) at the tips of the epidermal microvilli (mv) (small black arrows). Bars for B1 1.1 mm, B2 0.6 mm. (D) The integument shows the conspicuous absence of a high number of procuticular lamellae underneath the new epicuticle and signs of epidermal cell degeneration. BL, basal lamina; EM, ecdysial membrane; ES, ecdysial space; mv, epidermal microvilli; nEC, epicuticle of the newly secreted cuticle; nPC, procuticle of the new cuticle; oEC, the old epicuticle partially digested; BL, basal lamina. Bar 2.5 mm (adapted from Smagghe, 1995; Smagghe et al., 1996c, unpublished data).

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1997), L. decemlineata (Dhadialla et al., 2005; Smagghe et al., 1999d), and cultured abdominal sternites of T. molitor (Soltani et al., 2002). Examination of the cuticle following intoxication with one of the BAH compounds revealed that the larvae synthesize a new cuticle that is malformed. Figure 2.3 shows effects on S. exigua fifth instar larva and its new cuticle formation after intoxication with tebufenozide. Unlike normal cuticle synthesis, the lamellate endocuticle deposition in tebufenozideintoxicated larvae is disrupted and incomplete. Within 12 h of treatment, the epidermal cells in intoxicated larvae have fewer microvilli, show hypertrophied Golgi complexes and an increased number of vesicles compared to normal epidermal cells active in cuticle synthesis. The visual observations of precocious production of new cuticle have been demonstrated by in vivo and in vitro experiments to show the inductive effects of tebufenozide and 20E on the amount of chitin in S. exigua larval cuticle and chitin synthesis in cultured integument claspers of European corn borer, Ostrinia nubilalis (Hu bner), respectively (Smagghe et al., 1999c). At the physiological level, the state of hyperecdysonism, manifested by BAH insecticides in intoxicated susceptible larvae, is achieved by various mechanisms. Blackford and Dinan (1997) demonstrated that while larvae of the tomato moth, Lacanobia oleracea (Linnaeus), detoxified ingested 20E as expected, they remained susceptible to BAH EAs. This suggested that the metabolic stability of the EAs induced the hyperecdysonism state in L. oleracea. In another study, RH-5849 was shown to repress ecdysteroidogenesis in the larvae of the blowfly, Calliphora vicina (Robineau-Desvoidy), as a result of its action on the ring gland (Jiang and Koolman, 1999). In BAH-treated larvae of S. exigua and L. decemlineata, Smagghe et al. (1995) reported that the induction of premature apolysis happened without a surge in the titre of free ecdysteroids. Similarly, Gu et al. (2008) observed that when larvae of B. mori were treated with tebufenozide, the ecdysteroid levels were initially inhibited, but 24 h after treatment, the ecdysteroid levels increased. When B. mori prothoracic glands were cultured in vitro, treatment with tebufenozide showed no interference with the stimulatory effect of the glands with prothoracicotrophic hormone for ecdysteroidogenesis (Gu et al., 2008). In contrast, the titres of ecdysteroids in primary cultures of abdominal sternites as collected from pupae of T. molitor were increased upon exposure to halofenozide as compared to that in control abdominal sternites cultures (Soltani et al., 2002). The amounts of ecdysteroids increased with increasing incubation times and concentrations of 110 mM halofenozide in vitro. Topical application of 10 mg halofenozide in solvent

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solution to newly ecdysed pupae of T. molitor in vivo also caused a significant increase in the haemolymph ecdysteroid titre as compared to that in control pupal haemolymph. However, there was no effect in the timing of the normal ecdysteroid release in the haemolymph. In contrast to these in vitro and in vivo effects observed in the above T. molitor study, Williams et al. (1997) observed that injection of RH-5849 into larvae of the tobacco hornworm, Manduca sexta (Linnaeus), induced production of midgut cytosolic ecdysone oxidase and ecdysteroid phosphotransferase activities, which are involved in the inactivation of 20E. Moreover, both 20E and RH-5849 caused induction of ecdysteroid 26-hydroxylase activity in the midgut mitochondria and microsomes. Subsequent results by Williams et al. (2002) suggested that induction of ecdysteroid 26-hydroxylase by RH-5849, tebufenozide, and halofenozide may be a universal action of BAH insecticidal compounds in susceptible lepidopteran larvae. In the same study, these effects were not observed in non-susceptible larvae of the wax moth, Galleria mellonella (Linnaeus), although its EcR is capable of binding tebufenozide. The binding in G. mellonella may not be sufficient for transactivation of genes that are induced or repressed by 20E. The fact that effective binding of tebufenozide or potent ecdysteroids to the LBD of EcR proteins results in a conformational change in the EcR protein, which is a prerequisite for transactivation of 20E-dependent genes, was demonstrated using limited proteolysis of radiolabelled EcR proteins from susceptible and nonsusceptible insects in the absence and presence of muristerone A and RH-5849 (Cherbas et al., 1998) and tebufenozide (Dhadialla et al., 2005, 2007). The diagram in Fig. 2.4 illustrates the relative metabolic stabilities of 20E and tebufenozide (or other BAH insecticidal compounds) and their titres during a larval stage of susceptible control in untreated or susceptible tebufenozide-intoxicated insects, respectively. Several hours following ingestion of BAH insecticidal compounds by a susceptible larva, genes that are dependent upon increasing titres of 20E are activated. However, genes that are normally activated or repressed for expression by decreasing titres or absence of 20E are not expressed in the presence of ingested BAH molecules in the haemolymph. During a normal moult, the release of eclosion hormone to initiate the eclosion behaviour in the moulting larvae depends upon clearance of 20E titres in the haemolymph (Truman et al., 1983). The presence of BAH molecules in the insects haemolymph inhibits the release of eclosion hormone, resulting in lack of eclosion behaviour, which leads to an unsuccessful lethal moult. The expression and repression of several genes during a moult cycle in M. sexta and C. fumiferana larvae treated with BAH insecticide has been investigated by different authors (Dhadialla et al., 2005; Palli

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Schematic of 20E, tebufenozide, and eclosion hormone liters in haemolymph of normal and intoxicated susceptible lepidopteran larva
Normal moult Larval instar (N) MI 5 Larval instar (N+1) MC EH 4 6 4 8
MI
3 2
2 1 Ingestion of tebufenozide Normal feeding Time (h) 1
10
Figure 2.4 Schematic representation of titres of the insect moulting hormone, 20E (thin line), and release of eclosion hormone (EH; dotted line), which triggers the ecdysis of the larva to complete a normal moult. The solid bold line represents relative titres of ingested BAH insecticide (exemplified here with tebufenozide) in a susceptible lepidopteran larva (adapted from Dhadialla et al., 2005). Owing to the metabolic stability and amount of BAH (tebufenozide in this case) in the insect haemolymph, eclosion hormone, the release of which is normally dependent upon the complete decline of 20E, is not released and the intoxicated insect is not able to complete the moult, which leads to premature death. Methoxyfenozide, halofenozide, and chromafenozide most likely undergo a similar metabolic fate, which is detrimental to intoxicated insect stage. The ecdysone agonists trigger a moult attempt any time during the feeding stage of a susceptible larval instar. Events that take place during the moult and are dependent upon the increasing or decreasing titres of 20E are also shown. The numbers in bold and regular font represent different events triggered by a BAH insecticide (exemplified here with tebufenozide) and 20E, respectively. 1, inhibition of feeding; 2, initiation of new cuticle synthesis; 3, apolysis of old cuticle from new cuticle resulting in an ecdysial space filled with moulting fluid; 4, head capsule slippage; MI, moult initiated; MC, moult completed; 5, derailment of the moulting process; 6, in the case of BAH insecticide treatment, eclosion hormone is not released, and the larva stays trapped in its old cuticle with its slipped head capsule covering the mouth parts (see Figs. 2.2 and 2.3); 7, moult attempt is lethal and the ecdysone agonist intoxicated larvae dies of starvation, haemorrhage, and desiccation; 8, cuticle formation continues and moulting fluid starts to be resorbed; 9, moult attempt is completed after release of EH (in case of a normal 20E-regulated moult) and larva ecdyses into the next larval stage; 10, new cuticle hardens and the mouth parts are sclerotized so that the larva may continue its growth and development into the next stage (N 1) from Dhadialla et al., 2005; (with permission from Elsevier).

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et al., 1995, 1996, 1999, 2005a,b,c; Retnakaran et al., 1995, 2001, 2003). Essentially, in untreated larvae, titres of 20E increase when apolysis occurs, but in larvae intoxicated with BAH insecticidal compounds (here exemplified with tebufenozide), early 20E-activated genes such as MHR3 (in M. sexta), CHR3 (in C. fumiferana), and E75 (in D. melanogaster) are expressed. As 20E titres decline during normal development, dopadecarboxylase (DDC), which requires a transient exposure to 20E followed by its clearance, is expressed. In contrast, in tebufenozide-intoxicated larvae, genes like DDC are not expressed, which prevents tanning and hardening of the already malformed new cuticle. During the intermoult period, when 20E is absent, genes like those for the 14-kDa larval cuticle protein (LCP14) that are normally repressed in the presence of 20E, are expressed. Once again, the prolonged presence of BAH EA in the haemolymph prevents the expression of LCP14, and perhaps other genes that would normally be inactive in the absence of 20E. These results, with the EcR/USP ligandbinding data provided compelling evidence that the BAHs act as true mimetics of the insect moulting hormone, 20E. However, their ability to induce lethal moults in susceptible intoxicated larvae is due to a combination of their ecdysone agonistic activity and much longer metabolic stability in the intoxicated larval haemolymph and tissues. For more details on the molecular interaction of ecdysteroids, BAHs, and other EA chemistries with the receptor complex of 20E in susceptible insects and non-sensitive species, we refer the reader to Chapter 4.
4. METHOXYFENOZIDE GLOBAL USES 4.1. Introduction

In this chapter, our emphasis is predominantly on the registrations and uses of methoxyfenozide because the other BAH insecticides (chromafenozide, fufenozide, and halofenozide) have limited geographic and use registrations (Table 2.2). The first global registrations of methoxyfenozide by Rohm and Haas Company occurred in 1998 in Brazil, Colombia, Indonesia, Israel, and Mexico. Its first registered uses included pome fruits (apples and pears), stone fruits (peaches, plums, etc.), and feed and fodder crops (grasses and others). First registrations of methoxyfenozide in the United States occurred in 2000 for pome fruits and cotton. Other significant methoxyfenozide registrations occurred, after the product was acquired by Dow AgroSciences LLC, in China and Japan (2001), Australia (2002), and Annex I inclusion (European
Table 2.2 Commercial bisacylhydrazine (BAH) insecticides Methoxyfenozide Tebufenozide Halofenozide
Chromafenozide
Fufenozide
Trade name
Falcon
Confirm Mimic
Mach 2
Cyclone Kanpai Killat Matric
JS118 Fufenozide
Integro
Intrepid Pacer
Romdan
Prodigy
Phares Podex Lepidoptera 47 1993 Lepidoptera and grubs Lepidoptera Lepidoptera China N/A
Runner Pests spectrum Lepidoptera Countries of registration Year of first registration Manufacturer
a
60 1998
Puerto Rico Japan (United States) 2002 Dow AgroSciences 1999
Dow AgroSciences Nippon Soda Co., Ltd.
Nippon JPRIb Sankyo Agro Co.
The trade names for methoxyfenozide, tebufenozide, and halofenozide are registered trademarks of Dow AgroSciences LLC. Jiangsu Pesticide Research Institute.

185
Union) in 2005. Currently, methoxyfenozide is registered in more than 50 countries (Table 2.2). The pest specificity for lepidopteran larvae has played a major role in its wide acceptance as an IPM tool where it can be sprayed in the presence of pollinators including the large earth bumblebee, Bombus terrestris (Linnaeus) (Mommaerts et al., 2006) and it has been documented to have good compatibility with natural enemies in many taxonomic groups including mites (Acari: Anystidae; Acari: Phytoseiidae) (Bostanian et al., 2010; Colomer et al., 2011; Laurin and Bostanian, 2007), nematodes (Nematoda: Steinernematidae) , 2010), and several families of predatory and parasitic insects (Radova (Hemiptera: Anthocoridae, Hemiptera: Pentatomidae, Hymenoptera: Ichneumonidae, Neuroptera: Chrysopidae; and Dermaptera: Forficulidae) (Amor et al., 2012; Bengochea et al., 2012; Colomer et al., 2011; Mahdian et al., 2007; Rimoldi et al., 2008; Schneider et al., 2006, 2007; Zotti et al., 2012a,b). Zhao et al. (2012) documented similar levels of selectivity to Trichogramma japonicum (Ahmead) (Hymenoptera: Trichogrammatidae), a common egg parasitoid, by tebufenozide and fufenozide. Its effects on adults at sublethal doses and its documented ovicidal activity discussed in Section 8 has played an important role in its adoption in several crops including pome and stone fruits, and more recently, in extensive crops such as soybeans in Argentina, Brazil, and the United States. A global review of methoxyfenozides use in different countries is presented in this section; additionally, a global summary of crops, rates, and pests for methoxyfenozide is found in Table 2.3. For specific information regarding use rates and pre-harvest intervals for specific countries for methoxyfenozide, the reader is referred to product label(s) for the country of interest (http:/ /www.dowagro.com/products/label/index.htm).
4.2. Bulb vegetables

Bulb vegetables (garlic, onions, and others) are generally considered specialty crops because they are often grown in relatively small areas compared to other crops. The first registration for methoxyfenozide in bulb vegetables was granted in Israel in 1999. Major pests controlled with methoxyfenozide in bulb vegetables include several species of cutworms (Agrotis spp.) and armyworms (Spodoptera spp.).
4.3. Cereals
Methoxyfenozide was first registered in Colombia in 1998 for lepidopteran control in rice and corn. The product is now registered in more than 20 countries for control of pests such as cutworms, Agrotis spp., C. suppressalis,

Table 2.3 Global summary of crops, rates, and pests for methoxyfenozide as an insect control agent Rates Latin name Common name Cropsa
Bulb vegetables Florence Fennel Garlic Green onion Leek Onion 2.312 g a.i./hl H2O 70280 g a.i./ha e) Achyra rantalis (Guene Agrotis spp. Mythimna unipuncta (Haworth) Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) e) Spodoptera ornithogalli (Guene Trichoplusia ni (Hu bner) Cereals Corn Milo/sorghum Rice Seed corn Wheat 48 g a.i./hl H2O 19.2240 g a.i./ha Agrotis spp. Chilo suppressalis (Walker) Diatraea grandiosella Dyar Mythimna separata (Walker) Rupella albinella (Cramer) Scirpophaga incertulas (Walker) Spodoptera frugiperda (J.E. Smith) Tryporyza incertulas (Walker) Cutworms Asiatic rice borer Southwestern corn borer Rice ear-cutting caterpillar South American white stem borer Yellow stem borer Fall armyworm Rice stem borer Garden webworm Cutworms Armyworm Beet armyworm Southern armyworm Fall armyworm Yellow-striped armyworm Cabbage looper

Citrus Clementine Grapefruit Mandarin Orange Tangerine Citrus hybrids 7.236 g a.i./hl H2O 72280 g a.i./ha Archips argyrospila (Walker) Egira curialis Grote n and Davis Marmara gulosa Guille Papilio cresphontes Cramer Peridroma saucia (Hu bner) Phyllocnistis citrella Stainton Platynota stultana Walsingham Cole crops 2.312 g a.i./hl H2O Bok choy 70280 g a.i./ha Broccoli Broccoli raab Brussels sprouts Cabbage Cauliflower Cavalo Chinese broccoli Chinese mustard cabbage Collard Cress Kohlrabi Mizuna Mustard greens Napa Petsai Rape greens e) Achyra rantalis (Guene Agrotis spp. e) Evergestis rimosalis (Guene Hellula undalis (F.) Pieris brassicae (L.) Pieris rapae (L.) Plutella xylostella (L.) Pseudaletia unipuncta (Haworth) Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) e) Spodoptera ornithogalli (Guene Trichoplusia ni (Hu bner) Garden webworm Cutworms Cross-striped cabbageworm Cabbage webworm Cabbage worm Imported cabbageworm Diamondback moth True armyworm Beet armyworm Southern armyworm Fall armyworm Yellow-striped armyworm Cabbage looper
Continued
Fruittree leafroller Citrus cutworm Citrus peelminer Orangedog Variegated cutworm Citrus leafminer Omnivorous leafroller

Table 2.3 Global summary of crops, rates, and pests for methoxyfenozide as an insect control agentcont'd Crops Rates Latin name Common name
Cucurbits Cucumber Melon Vegetable sponge Watermelon 48280 g a.i./ha Diaphania hyalinata (L.) Diaphania nitidalis (Stoll) Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) Forages Alfalfa Clover Grasses Lupin Mixed forage stand 14.4600 g a.i./ha Agrotis spp. Autographa californica (Speyer) Colias eurytheme Boisduval Fissicrambus mutabilis (Clemens) Parapediasia teterrella (Zincken) Pediasia trisecta (Walker) Mythimna unipuncta (Haworth) Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) e) Spodoptera ornithogalli (Guene Spodoptera praefica (Grote) Cutworms Alfalfa looper Alfalfa caterpillar Striped sod webworm Bluegrass webworm Larger sod webworm Armyworm Beet armyworm Southern armyworm Fall armyworm Yellow-striped armyworm Western yellow-striped armyworm Melonworm Pickleworm Beet armyworm Southern armyworm Fall armyworm
Forestry Silviculture Nursery trees Christmas trees 9.6 g a.i./hl 36120 g a.i./ha Alsophila pometaria (Harris) Choristoneura fumiferana (Clemens) Ennomos subsignaria (Hu bner) Epiphyas postvittana (Walker) Euproctis chrysorrhoea (L.) Hyphantria cunea (Drury) Lymantria dispar (L.) Malacosoma americanum (F.) Opodiphthera eucalypti (Scott) Orgyia leucostigma (J.E. Smith) Spodoptera spp. Thaumetopoea pityocampa (Schiff) Thyridopteryx ephemeraeformis (Haworth) Fruiting vegetables Chilli pepper Eggplant Paprika Pimento Sweet pepper Tomato Tomatillo 8.428.8 g a.i./hl H2O Agrotis spp. 48600 g a.i./ha Helicoverpa armigera (Hu bner) Helicoverpa zea (Boddie) Heliothis virescens (Fabricius) e) Neoleucinodes elegantalis (Guene Cutworms Corn earworm Tomato fruitworm Tobacco budworm Small tomato borer
Continued
Fall cankerworm Spruce budworm Elm spanworm Light brown apple moth Browntail moth Fall webworm Gypsy moth Eastern tent caterpillar Emperor gum moth White-marked tussock moth Armyworms Pine processionary caterpillar Bagworm

e) Rachiplusia nu (Guene Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Tuta absoluta (Meyrick) Leafy vegetables Arugula Chard Chervil Lettuce Orach Purslane Spinach 2.312 g a.i./hl H2O 70280 g a.i./ha e) Achyra rantalis (Guene Agrotis spp. Mythimna unipuncta (Haworth) Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) e) Spodoptera ornithogalli (Guene Trichoplusia ni (Hu bner) Legumes Bean pods Dry beans Green bean 2.312 g a.i./hl H2O 70280 g a.i./ha e) Achyra rantalis (Guene Agrotis spp. Mythimna unipuncta (Haworth)
Beet armyworm Southern armyworm South American tomato moth
Garden webworm Cutworms Armyworm Beet armyworm Southern armyworm Fall armyworm Yellow-striped armyworm Cabbage looper
Garden webworm Cutworms Armyworm
Spodoptera exigua (Hu bner) Spodoptera eridania (Stoll) Spodoptera frugiperda (J.E. Smith) e) Spodoptera ornithogalli (Guene Trichoplusia ni (Hu ber) Oil seeds Cotton Peanut Soybean Sunflower 14.4600 g a.i./ha Alabama argillacea (Hu bner) Anticarsia gemmatalis Hu bner Heliothis virescens (F.) Helicoverpa gelotopoeon (Dyar) Helicoverpa zea (Boddie) e) Rachiplusia nu (Guene Spodoptera frugiperda (J.E. Smith) Trichoplusia ni (Hu bner) Ornamentals Greens Roses Flowers Perennials 9.6 g a.i./hl 36120 g a.i./ha Alsophila pometaria (Harris) e) Callopistria floridensis (Guene Choristoneura fumiferana (Clemens) Ennomos subsignaria (Hu bner)
Beet armyworm Southern armyworm Fall armyworm Yellow-striped armyworm Cabbage looper
Cotton leafworm Velvetbean caterpillar Tobacco budworm
Bollworm Fall armyworm Cabbage looper
Fall cankerworm Florida fern caterpillar Spruce budworm Elm spanworm

Continued

Outdoor areas Residential areas
Epiphyas postvittana (Walker) Euproctis chrysorrhoea (L.) Hyphantria cunea (Drury) Lymantria dispar (L.) Malacosoma americanum (F.) Opodiphthera eucalypti (Scott) Orgyia leucostigma (J.E. Smith) Spodoptera spp. Thyridopteryx ephemeraeformis (Haworth)
Light brown apple moth Browntail moth Fall webworm Gypsy moth Eastern tent caterpillar Emperor gum moth White-marked tussock moth Armyworms Bagworm
Pome fruits Apple Crab apple Loquat Pear Quince (Asian pear) 3.615 g a.i./hl H2O 72173 g a.i./ha Archips argyrospila (Walker) Archips rosanus (L.) Choristoneura rosaceana (Harris) Cydia pomonella (L.) Epiphyas postvittana (Walker) Grapholita molesta (Busck) Grapholita prunivora (Walsh) Apple leaf roller Rose leaf folder Spruce budworm Codling moth Light brown apple moth Oriental fruit moth Lesser apple worm
Lacanobia subjuncta (Grote and Robinson) Apple pandemis Pandemis pyrusana Kearfott Platynota idaeusalis (Walker) Phyllonorycter blancardella (F.) Phyllonorycter crataegella (Clemens) Small fruits Aronia berry Blueberry Bushberry Chilean guava Cranberry Currant Elderberry European barberry Grapes Gooseberry Honeysuckle Huckleberry Jostaberry Juneberry Lingonberry Mayhaw Salal Sea buckthorn Spanish lime Strawberry 3.69.6 g a.i./hl H2O 36275 g a.i./ha Acrobasis vaccinii Riley Agrotis spp. Archips argyrospilus (Walker) Archips rosanus (L.) Clysia ambiguella (Hubner) Choristoneura rosaceana (Harris) Endopiza viteana Clemens Grapholita packardi Zeller Helicoverpa zea (Boddie) Itame argillaceari (Packard) Lobesia botrana (Dennis & Schiffermu ller) Pandemis pyrusana Kearfott Spodoptera spp.
Continued
Tufted apple bud moth Spotted tentiform leafminer Apple blotch leafminer
Cranberry fruitworm Cutworms Apple leaf roller Rose leaf folder Grape berry moth Obliquebanded leafroller Cherry fruitworm Bollworm Blueberry spanworm European grapevine moth Apple pandemis Armyworms

Stone fruits Apricot cherry Nectarine Peach Plum Prune 3.612 g a.i./hl H2O 72240 g a.i./ha Anarsia lineatella Zeller Archips argyrospila (Walker) Argyrotaenia velutinana (Walker) Choristoneura rosaceana (Harris) Cydia pomonella (L.) Grapholita molesta (Busck) Grapholita packardi Zeller Grapholita prunivora (Walsh) Lithophane antennata (Walker) Pandemis limitata (Robinson) Pandemis pyrusana Kearfott Platynota idaeusalis (Walker) Platynota stultana Walsingham Schizura concinna (J.E. Smith) Peach twig borer Fruittree leafroller Redbanded leafroller Obliquebanded leafroller Codling moth Oriental fruit moth Cherry fruitworm Lesser appleworm Green fruitworm Three-lined leafroller Apple pandemis Tufted apple bud moth Omnivorous leafroller Redhumped caterpillar
Spilonota ocellana (Denis & Schiffermu ller) Eye-spotted bud moth Tea 9601920 g a.i./ha Adoxophyes honmai (Yasuda) Ascotis selenaria Denis and Schiffermu ller Caloptilia theivora (Wals.) Smaller tea tortrix Giant looper Tea leaf roller

Homona magnanima Diakonoff Spodoptera litura F. Tree nuts Almond Beech nut Butter nut Chestnut Chinquapin Hazelnut Macadamia Pecan Pistachio Walnut 3.64.8 g a.i./hl H2O 67426 g a.i./ha Acrobasis nuxvorella Neunzig Amyelois transitella (Walker) Anarsia lineatella Zeller Choristoneura rosaceana (Harris) Cnephasia longana (Haworth) Cydia caryana (Fitch) Cydia latiferreana (Walsingham) Cydia pomonella (L.) Hyphantria cunea (Drury) Schizura concinna (J.E. Smith) Tropical fruits Acerola Avocado Black sapote Canistel Carambola Coffee Feijoa Guava Jaboticaba 3.66 g a.i./hl H2O 175280 g a.i./ha Argyrotaenia citrana (Fernald) Conopomorpha cramerella (Snellen) Conopomorpha sinensis (Bradley) Deanolis albizonalis Hampson Deudorix epijarbas (Moore) Homona spargotis Meyrick Orange tortrix Cocoa pod borerh Lychee fruit borer Mango seed borer Oriental cornelian Avocado leaf roller
Continued
Oriental tea tortrix Cluster caterpillar
Pecan nut casebearer Navel orangeworm Peach twig borer Spruce budworm Omnivorous leaftier Hickory shuckworm Filbertworm Codling moth Fall webworm Redhumped caterpillar

Kiwi Lychee Longan Mamey (white sapote) Mango Papaya Passion fruit (granadilla) Pomegranate Pulasan Spanish lime Rambutan Pulasan Sapodilla Star apple Wax jambu
a
Marmara salictella Clemens Marmara spp. Noorda albiizonaliis Hampson Oiketicus kirbyi (Guilding) Stenoma catenifer Walsingham
Citrus peel miner Miners Bag worm Avocado seed moth
Refer to local methoxyfenozide labels for specific recommendations for crops, pests, and rates in the country of interest.

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rice ear-cutting caterpillar, Mythimna separata (Walker), South American white borer, Rupella albinella (Cramer), fall armyworm, Spodoptera frugiperda (J.E. Smith) and stem borers in sweet corn, rice, sorghum, and wheat. Methoxyfenozide represents a new mode of action for IRM in cereal crops, which with its relatively long residual effect makes it very attractive for growers to use. Ready-Jones et al. (2007) reported that a single application of methoxyfenozide at 29 g a.i./ha significantly reduced insect injury in rice. Furthermore, when an application of methoxyfenozide at 29 g a.i./ha was followed by an application of gamma-cyhalothrin at 17 g a.i./ha, a significant reduction in insect injury was observed with a significantly greater yield.
4.4. Citrus
Methoxyfenozide is registered for use in several citrus crops including grapefruits, lemons, oranges, and several tangerine cultivars and hybrids in several countries. The citrus leafminer represents one of the most important pests where methoxyfenozide has shown utility for IPM programmes. Since it was first reported as an invasive species in Florida in 1993 (http:/ /www.freshfromflorida.com/pi/pest-alerts/phyllocnistis-citrella.html) and in California in the year 2000 (http://www.ipm.ucdavis.edu/PMG/r107303211.html), the citrus leafminer has become a major pest in the United States because it not only affects the trees by directly feeding on the flush (young leaves), but also spreads the bacterium, Xanthomonas axonopodis pv. citri, that causes citrus canker. Research for citrus leafminer, Phyllocnistis citrella (Stainton), control in the United States has been conducted with methoxyfenozide for several years. Figure 2.5 shows research done by Dow AgroSciences LLC (unpublished data)
0.4 0.3 0.2 0.1 0
Intrepid 140 g a.i./ha + Dimethoate 1006 g a.i./ha + Oil Malathion 2760 g a.i./ha
Figure 2.5 Comparison of formulated methoxyfenozide (Intrepid) mixed with Dimethoate and oil applied by air blast versus applications of malathion for citrus leafminer control in Florida. The Y-axis shows number of live larvae in 10 flush, where one flush is a plant terminal with newly emerged leaves (unpublished results, Dow AgroSciences LLC).

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comparing methoxyfenozide in mixture with dimethoate as a tool for controlling citrus leafminer. Other pests that are controlled by metn hoxyfenozide in citrus include citrus peelminer, Marmara gulosa (Guille and Davis), orangedogs, Papilio spp., and cutworms.
4.5. Cole crops (Brassica vegetables)

Methoxyfenozide was first registered in Indonesia for control of lepidopteran pests in 1998. Key pests controlled by methoxyfenozide in broccoli, cauliflower, cabbage, and other cole crops include cabbage looper, Trichoplusia ni (Hu bner), several webworm species, cabbageworms, Pieris spp., and diamond back moth, Plutella xylostella (L.).
4.6. Cucurbits
The first registration for methoxyfenozide use on cucurbit crops was granted in South Korea in 1999. Cucurbits are highly specialized crops that require pollinators (i.e. bees and bumble bees) to produce commercial harvest. Because methoxyfenozide is compatible with pollinators and it has no restriction for application time, it has become a major insecticide for control of several species of Lepidoptera including melonworm, Diaphania hyalinata (L.), pickleworm, Diaphania nitidalis (Stoll), and several species of armyworms, Spodoptera spp. Figure 2.6 shows an example of one of the many studies done by Dow AgroSciences LLC (unpublished results) during the development of methoxyfenozide for use in cucurbits.
5 4 3 2 1 0
Intrepid 40 g a.i./ha Xentari 600 g product/ha Lufenuron 20 g a.i./ha Untreated
Figure 2.6 Evaluation of methoxyfenozide (Intrepid) for control of melon worm, Diaphania hyalinata and pickleworm, Diaphania nitidalis as compared to other insecticides (Lufenuron and Xentari) on the market. The Y-axis shows the number of live larvae/ 5 terminals/10 fruits per plot (unpublished results, Dow AgroSciences LLC).

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4.7. Forages
Methoxyfenozide is currently registered for control of cutworms, Agrotis spp., alfalfa looper, Autographa californica (Speyer), and several species of armyworms, Spodoptera spp. among other important pests in forage crops. Its relative long residual control is an important reason for its adoption in these crops.
4.8. Forestry
Methoxyfenozide was registered after tebufenozide for use in forest areas due to its higher potency and widely studied effects on eggs and sublethal effects on adult insects in those plant species. C. fumiferana, the elm spanworm, Ennomos subsignaria (Hu bner), webworms, gypsy moth, Lymantria dispar (L.), and, recently introduced to the United States, the light brown apple moth, Epiphyas postvittana (Walker), are among the lepidopteran species controlled with methoxyfenzoide in forestry agroecosystems.
4.9. Fruiting vegetables

Chile, Colombia, and Indonesia first registered methoxyfenozide for lepidopteran control in fruiting vegetables in 1998. The fruiting vegetable crop group is where methoxyfenozide is registered in most countries because of its flexibility of application, ease to tank mix with other chemicals (as stated in Section 5), and its compatibility with IPM and IRM programmes Spodoptera spp., Agrotis spp., tomato fruit worm, Helicoverpa zea (Boddie), and South American tomato moth, Tuta absoluta (Meyrick) are among the pests controlled in fruiting vegetables. Figure 2.7 shows studies done by Dow AgroSciences LLC (unpublished) during the development of the product to determine the effect of methoxyfenozide on fruit damage control in tomatoes.
4.10. Leafy vegetables and legumes

The first registration of methoxyfenozide on leafy vegetables (lettuce, spinach, etc.) was granted in Indonesia in 1998, while the first registrations in legumes occurred a year later in Israel. Methoxyfenozide is used in these crops to control Spodoptera spp., Agrotis spp., and webworms.
4.11. Oilseeds
The first registration of methoxyfenozide in oilseeds occurred in 1998 in Colombia and Indonesia for control of S. frugiperda; cotton leafworm, Alabama argillacea (Hu bner); velvetbean caterpillar, Anticarsia gemmatalis

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22 20 18 16 14 12 10 8 6 4 2 0
Methoxyfenozide 48 g a.i./ha Spinosad 36 g a.i./ha Chlorfenapyr 29 g a.i./ha Untreated
Figure 2.7 Evaluation of methoxyfenozide efficacy for beet armyworm, Spodoptera exigua control in tomatoes in Guatemala as compared to insecticides (spinosad and chlorfenapyr) with different mode of action. The Y-axis shows per cent of fruit damaged (unpublished results, Dow AgroSciences LLC).
(Hu bner); and cotton bollworm, Helicoverpa zea (Boddie). In the United States, methoxyfenozide was first registered to control lepidopteran larvae in cotton where it was successfully used to control Spodoptera spp., soybean looper, Chrysodeixis includens (Walker), and cabbage looper, T. ni (Hu bner) until transgenic Bacillus thuringiensis (Bt) cotton was registered, which caused a general decline in uses of insecticides in this crop. Hardee and Burris (2003) reported that in Tennessee, methoxyfenozide was successfully used to control beet armyworm infestations with a single application during the season. Methoxyfenozide has also proven to be a very effective insecticide for common soybean pests including C. includens and A. gemmatalis (Fitzpatrick et al., 1999, 2000; Willrich et al., 2002). Willrich et al. (2002) demonstrated that control of A. gemmatalis and C. includens with methoxyfenozide was achieved either when used as a preventive measure or when applied at the economic threshold with 70 and 140 g a.i./ha, respectively.
4.12. Ornamentals
Because of its compatibility with IPM programmes and its good performance compared to other insecticides, methoxyfenozide is widely used in ornamentals for control of several species of Lepidoptera including some invasive species such as L. dispar and E. postvittana. Other lepidopteran species controlled with methoxyfenozide in ornamentals include fall cankerworm,

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Alsophila pometaria (Harris), Florida fern caterpillar, Callopistria floridensis e), and C. fumiferana. (Guene
4.13. Pome fruits

Methoxyfenozide is currently registered in more than 25 countries for control of lepidopteran pests in pome fruits including codling moth, Cydia pomonella (L.), oriental fruit moth, Grapholita molesta (Busck), E. postvittana, and several other species of tortrix leaf rollers. Because of its adverse impact on fecundity and fertility of the target insect species (discussed in Section 8), methoxyfenozide is commonly used at the petal fall stage of growth to manage early infestations of C. pomonella, C. rosaceana, and apple pandemic, Lacanobia subjuncta (Grote and Robinson) in U.S. apples as a true ovicidal treatment as proposed by Brunner et al. (2007). Studies on tank mixes with methoxyfenozide showed that when mixed with the neonicotinoid insecticides acetamiprid, clothianidin, or thiacloprid, and applied at 350 and 1350 degree days (DD) once per generation, it consistently reduced C. pomonella fruit injury in apples and pears (Granger et al., 2005). The tankmix application was applied once per codling moth generation (350/ 1350 DD) and was preceded by the application of a crop oil at 1%.
4.14. Small fruits

Methoxyfenozide is used in management programmes for lepidopteran pests of small fruits in numerous countries to control several pests including leafrollers and fruitworms in bush berries, cane berries, and grapes. Methoxyfenozide and tebufenozide were evaluated by Isaacs et al. (2005) for control of grape berry moth, Clysia ambiguella (Hu bner), comparing them to the broad-spectrum insecticides azinphosmethyl, carbaryl, and fenpropathrin. When applications of methoxyfenozide or tebufenozide were timed to egg hatch, both compounds were very successful in controlling the pest. In another study, application of methoxyfenozide at 120 and 240 g a.i./ha successfully controlled the blueberry spanworm, Itame argillaceari (Packard), in wild blueberries (Ramanaidu, 2010). Additionally, methoxyfenozide has proven to be a very successful product for invasive lepidopteran species in small fruits. In 2009, the European grapevine moth (EGVM), Lobesia botrana (Dennis & Schiffermu ller), was first reported threatening the wine industry in Napa County, California. Methoxyfenozide and other green chemistry products were tested for control of EGVM. Currently, methoxyfenozide is considered among the most

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efficacious insecticides to control this invasive species for both egg and larval stages (http://westernfarmpress.com/management/second-generationegvm-trapped-sonoma-county).
4.15. Stone fruits

Methoxyfenozide was first registered in Argentina for lepidopteran control in stone fruits in 1999. Since then, additional registrations have been granted in the European Union and the United States. Target pests include G. molesta, C. pomonella, fruittree leafroller, Archips argyrospila (Walker), omnivorous leafroller, Schizura concinna (J.E. Smith), redbanded leaf roller, Argyrotaenia velutinana (Walker), and peach twig borer, Anarsia lineatella (Zeller). Because of its specificity and low impact on beneficial arthropods, some of which are needed for pollination of the crop, methoxyfenozide has found wide adoption in IPM programmes for stone fruits where outbreaks of other pests such as mites and aphids, by use of non-selective insecticides, have the potential to cause significant economic impact.
4.16. Tree nuts

Methoxyfenozide is registered in several countries to control lepidopteran pests including pecan nut casebearer, Acrobasis nuxvorella (Neunzig), navel orangeworm, Amyelois transitella (Walker), A. lineatella, and C. pomonella. Tree nut crops include almond, beech nut, butter nut, chestnut, chinquapin, hazelnut, macadamia, pecan, pistachio, and walnut. Since its launch in the United States, it was quickly adopted by almond growers for control of A. transitella because of its compatibility within IPM programmes. Applications are made at hull-split to target eggs and neonate larvae before entering the shell.
4.17. Tropical fruits

The tropical fruit group includes a variety of highly appealing fruits for fresh consumption including kiwi, carambola, lychee, mango, papaya, pomegranate, and rambutan among a long list of crops. Methoxyfenozide was first registered in 1999 for use in pomegranate in Israel. Currently, methoxyfenozide is used to control several key lepidopteran pests including orange tortrix, Argyrotaenia citrana (Fernald), lychee fruit borer, Conopomorpha sinensis (Bradley), and mango seed borer, Deanolis albizonalis (Hampson). Because of its characteristics as a green chemistry, methoxyfenozide is a compound widely used in this group and more registrations are achieved every year in

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different countries. For instance, Lewis and Gorton (2007) published research done in Australia to support registration of methoxyfenozide in Australia for control of the avocado leaf roller, Homona spargotis (Meyrick). In their research, they compared methoxyfenozide to tebufenozide and spinosad finding that tebufenozide was slower acting than methoxyfenozide and spinosad, but untreated controls for all three compounds were similar at 8 days after treatment. When assessing residual control, only methoxyfenozide was able to provide a residual effect all the way to 15 days after treatment, suggesting a greater period of residual activity.
5. METHOXYFENOZIDE FORMULATION
The formulation of an active ingredient (a.i.) into a pesticide is directly related to its chemical and physical properties. Methoxyfenozide is a nonreactive solid with a high melting point (MP 204205 C). It has very low solubility in water and non-polar solvents, but the molecule will degrade by exposure to temperatures at or above the MP, or exposure to hot concentrated acids (Carlson et al. 2001). Methoxyfenozide is exceptionally suited for mixtures with other insecticides because its low reactivity and low solubility allow it to be combined with many other a.is (Gomez et al., 2011).
5.1. Formulation types and commercial products

Commercial products containing methoxyfenozide as the a.i. are formulated as dustable powders, suspension concentrates (SCs, flowable concentrates), and wettable powders (WPs) (see Table 2.4). Different commercial formulation types are chosen depending on several factors with physical properties of the a.i. among the most important factors to consider because physical properties will dictate what is feasible with a given a.i. in terms of formulation. Market preferences are also considered when choosing a commercial formulation. For instance, markets where large area applications are done usually prefer higher loading formulations that require less storage area versus a more diluted formulation. For those markets, a high-loading WP formulation of methoxyfenozide (i.e. Intrepid or Runner 80 WP) could be used, but those formulations may require applicators and product handlers to use more protective equipment versus a liquid (SC) formulation due to respiratory concerns with fine powders. Suspension concentrates are generally easier to use because they can be measured more accurately during an application (i.e. they do not require having a scale to weigh them) versus a

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Table 2.4 Global trade names and formulation types for methoxyfenozide Registered names Formulation Methoxyfenozide concentration
Falcon Falcon Integro
SCa WPc SC SC WP
f d
20% (w/w)b 4% (w/w) 240 g/le 240 g/l 80% (w/w) 0.5% (w/w) 1.5% pencycuron 0.25 imidacloprid 0.5% (w/w) 1.5% pencycuron 100 g/l 240 g/l 100 g/l 0.5% (w/w) 240 g/l 80% (w/w)
Intrepid Intrepid Monceren AD Monceren Runner Pacer Prodigy Prodigy

DP (DL ) DP (DL) SC SC SC DP (DL) SC WP
Runner Runner Runner

a b c
Suspension concentrate. Percentage by weight. Wettable powder. d Suspension concentrate. e Grams per litre. f Japan code for driftless formulation. Falcon , Integro , Intrepid , Pacer , Prodigy , and Runner are trademarks of Dow AgroSciences LLC. Monceren is a trademark of Bayer.
solid product; for that reason, the SC formulations of methoxyfenozide are more commonly used in a greater number of markets around the globe.
6. ENVIRONMENTAL FATE, METABOLISM, AND RESIDUE ANALYSIS OF METHOXYFENOZIDE 6.1. Introduction

After discovery of an insecticidal compound (or other pesticide) with desirable efficacy, the largest costs in bringing it to market occur in studies that establish limits of safety of the a.i. and its metabolites to mammalian, avian, and aquatic animal species, as well as the metabolism and residue fate of the

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metabolites and the a.i. in plants, fruits and vegetables, soils, and groundwater. Unlike pharmaceutical drugs, which are directly delivered to the patient (notwithstanding the toxicological effects within the ingesting human or animal patient), agricultural pesticides are sprayed on the crops infested with the pest (insect, fungus, or weed). This application method poses a risk to the environment, non-pest arthropods, and animal species. Therefore, every registration of each such new pest-controlling agent requires extensive testing of the molecule as an a.i. Additionally, registration of a pesticide in each country where it will be used will have its own registration requirements, which have to be met. This dictates if additional tests must be done. Due to the nature of pesticide application, safety to the environment, animals (mammalian, avian, aquatic), and beneficial insects must be established. However, as absolute safety to all non-target organisms may not always be achievable, the registration agencies in different countries will set limits of safety for use of the pesticide. In 1995, the U.S. Government introduced the Presidential Green Chemistry Challenge Award to any pesticide that met the set criterion. In 1998, this award was given to Rohm and Haas Company for three commercial insecticides (tebufenozide, methoxyfenozide, and halofenozide) belonging to the BAH chemical class. By granting this award, the U.S. Government recognized the outstanding chemical processes for BAH insecticides that reduce negative impact on human health and the environment. Both tebufenozide and methoxyfenozide were registered by the Environment Protection Agency (EPA) under its Reduced Risk Pesticide Program. Methoxyfenozide, like other members of its class (tebufenozide, chromafenozide, and fufenozide; Fig. 2.1), is largely toxic to lepidopteran larvae (Dhadialla and Ross, 2012; Dhadialla et al., 2005; Gomez et al., 2011; Toya et al., 2002; Yanagi et al., 2006; Zhang et al., 2003). Given the molecular basis of action of methoxyfenozide (Section 3), it is a paradox that these insecticide members of the BAH chemical class are selectively lethal to lepidopteran larvae, with no lethal activity to adult lepidopterans, and of little to no consequence to non-lepidopteran insect pests, which includes beneficial arthropods. Detailed decision documents containing large databases of information, including a global evaluation of toxicology, metabolism, and residues, produced for methoxyfenozide by the joint meeting on Pesticide Residues by the Food and Agriculture Organization/World Health Organization (FAO/ WHO), and other reports are in public domain (Federal Register, 2006, 71 (109), pp. 3284932853).

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6.2. Metabolism and environmental fate studies

To understand and follow the metabolism of an a.i. of a pesticide, traditionally the active molecule is radiolabelled using 14C. Often the molecule is uniformly labelled and/or at specific substitutions to fully understand the metabolic fate of the compound in the test animal, plant, or in the environment. The parent radiolabelled compound and its metabolites are extracted from the test organism, tissue, or soil and analysed using high-pressure liquid chromatography for fractionation of extracts, radio detectors (for detection of fractions containing radiolabelled parent or its metabolites), and mass spectrometry to characterize by mass the metabolites in a particular fraction using instruments that are most often coupled (Mao et al., 2006; Wang and Wotherspoon, 2007). For studies on the metabolic fate of methoxyfenozide in animals, plants, and the environment, methoxyfenozide with either one or both of the phenyl rings uniformly labelled or with the t-butyl group labelled with C14 have been used.
6.3. Environmental fate and characteristics of methoxyfenozide

Methoxyfenozide has a log Pow of 3.72 (http://www.fao.org/ag/AGP/ AGPP/Pesticid/JMPR/Download/2003_eva/methoxyfenozide%202003. pdf) that suggests potential for bioaccumulation. However, actual studies with fish and clams have shown that methoxyfenozide is rapidly eliminated with a 50% depuration estimate of about 0.3 days (http://www.unece.org/ trans/danger/publi/ghs/ghs_rev03/English/04e_part4.pdf). As part of registration, studies are quite often required to determine the stability and degradation of the molecule to be registered under both aerobic and anaerobic conditions. In such laboratory studies where 14Cmethoxyfenozide was incubated in soil under aerobic conditions in the dark at 25 C for 365 days, methoxyfenozide was shown to degrade slowly, with a value of greater than 365 days for reaching 50% degradation (DT50) (http://www.apvma.gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf). However, under field conditions, degradation was significantly more rapid with DT50 values of 92327 days. These differences between lab and field studies suggest that in the lab studies, it is possible that conditions for aerobic metabolism were not maintained. Laboratory studies under anaerobic conditions again revealed very low levels of metabolism of radiolabelled methoxyfenozide (http:/ /ec.europa.eu/food/plant/protection/evaluation/newactive/methoxyfenozide_review_report.pdf). In both

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O O O O O N N O OH O N N O
OH
OH
CH2OH O OH OH OH
O O N N O
RH-152068 (g)
CH2OH O O N N O CH2OH
HO
CH2OH O OH OH OH
O O N OH N O
RH-131154 (g,h)
RH-131157 (r)
O O N N O O OH N N O
Meatabolite H (2 isomers) (g,h)

CH2OH O OH OH OH
O O N N O
Methoxyfenozide (g,h,r)
RH-141518 (g,h,r)
CH2OH
RH-117236 (g,h,r)
R O
CH2OH O OH OH OH
O N O N O OH HO O N N O O OH N N O
CH2OH
CH2OH O OH OH OH
O O N N O
RH-151065 (g,h)
RH-141508 (r)
CH2OH O
RH-141511 (g,h,r)
CH2OH O OH N O
Natural products (lactose, lipids triglycerides)
OH HO
N O
CH2OH
RH141512 (r)
RH-141513 (r)
Figure 2.8 Proposed metabolic fate of methoxyfenozide derived from studies in animals using C14-methoxyfenozide labelled uniformly at one or both the phenyl rings, or at the t-butyl group. The RH-# under each compound is the Rohm and Haas Company code number for each of the metabolites identified. Letters in brackets following the coded compound numbers indicate the animal species in which the metabolite was identified: g, goat; h, hen; r, rat.
aerobic and anaerobic studies, the first metabolites were the alcohol and acid derivatives, RH-117236 and RH-131154, respectively (Fig. 2.8). Finally, laboratory soil photolysis studies revealed that methoxyfenozide has a minor route of degradation via exposure to sunlight with no major derivative products of methoxyfenozide (http://ec.europa.eu/food/plant/ protection/ evaluation/newactive/methoxyfenozide_review_report.pdf).
6.4. Hydrolysis of methoxyfenozide under aqueous conditions

Incubation of 14C-methoxyfenozide in sterile buffers at pH 5, 7, and 9 at 25 C in the dark, to separate from photolytic degradative effects, indicated that methoxyfenozide is stable to hydrolysis (http://www.apvma.gov.au/ registration/assessment/docs/prs_methoxyfenozide.pdf). Studies reported in this reference also revealed that methoxyfenozide is stable to aqueous photolysis in sterile deionized water buffered at pH 7. On the other hand,

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when methoxyfenozide is incubated in irradiated natural pond water at 25 C, with 12:12 h (photophase:scotophase) using a lamp simulating natural sunlight, minor degradation of the radiolabelled methoxyfenozide was observed with DT50 of 866 days in darkness and DT50 of 77 days in light. In water-sediment systems, minimum mineralization of methoxyfenozide was observed under aerobic conditions in the dark, with no major metabolite in the water column and the main route of dissipation was through transfer to the sediment phase (http://ec.europa.eu/food/plant/protection/evaluation/ newactive/methoxyfenozide_review_report.pdf). In groundwater leaching studies, methoxyfenozide was shown to have minimal leaching potential from surface-applied material. Most of the surface-applied methoxyfenozide was retained in the top 010 cm soil. However, in U.S. field studies, < 5% of applied methoxyfenozide was found to move down to 7691 cm below after 30 days in four sites (http://www. apvma.gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf). The most rapid movement of methoxyfenozide was observed in deep sand, where $ 90% of it had moved down to 91 cm (http://www.apvma.gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf).
6.5. Metabolism of methoxyfenozide in animals

Extensive studies to access the toxicological risk and registrability of methoxyfenozide have been done. In most studies, the primary end chemical entities of methoxyfenozide have been methoxyfenozide itself and a glucuronide metabolite in animals. Metabolism studies have been carried out in a number of animals by feeding known quantities of 14C-methoxyfenozide (radiolabelled uniformly at each of the two phenyl rings or at the t-butyl group) in diets and analysing tissues, excreta, and urine for methoxyfenozide and its metabolites. The main outcome of these studies has been that methoxyfenozide residues are low in tissues, milk, and eggs. Proposed metabolic pathways of methoxyfenozide from the various animal studies are shown in Fig. 2.8. When lactating goats were fed $ 50 ppm dietary equivalents of 14Cmethoxyfenozide (uniformly labelled at the phenyl rings or at the t-butyl position) in diet for seven consecutive days, analysis of the excrements and tissues revealed that 7484% of the ingested dose of the residue was excreted in the faeces and an additional 57% in the urine (http://www.fao.org/ ag/AGP/AGPP/Pesticid/JMPR/Download/2003_eva/methoxyfenozide% 202003.pdf). Analysis of the edible tissues like liver, kidney, fat, and muscle

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revealed detection of 0.261.2, 0.0450.2, 0.0180.053, and < 0.025 mg/kg, respectively. The major metabolite in liver and kidney was RH-141518 with trace amounts of other glucuronides of methoxyfenozide. In contrast, the major residue in fat, muscle, and milk was methoxyfenozide itself, reaching a plateau of very low levels ( 0.018 ppm) within 2436 h of ingested dose. In similar studies in hens, which were fed $ 60 ppm 14C-methoxyfenozide incorporated in diet for seven consecutive days, a majority of the parentingested compound was recovered unmetabolized in the excreta. The other metabolite recovered in the eggs and tissues in addition to methoxyfenozide was RH-141518, which constituted < 0.03% of the ingested dose. When rats were administered a single oral dose of 14C-methoxyfenozide, it rapidly reached peak blood or plasma levels within 1530 min and 6270% of the dose was systemically absorbed (http://whqlibdoc.who.int/ publications/2004/924166519X_methoxyfenozide.pdf; http:/ /www.apvma. gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf). Analysis of the various tissues and excrement showed that methoxyfenozide was metabolized into its demethylated, hydroxylated, and glucuronated end points and eliminated in the bile, faeces, and urine with the ingested methoxyfenozide found only in faeces and not in urine or bile. Results of the above studies indicated that metabolism of methoxyfenozide in rats is qualitatively the same as in poultry and ruminants.
6.6. Metabolic fate of methoxyfenozide in plants

Similar to the metabolic fate studies of methoxyfenozide in animals, 14 C-methoxyfenozide (labelled at each phenyl ring or at the t-butyl position) has been used to study its metabolic fate in apples, grapes, cotton, and rice (http://www.fao.org/ag/AGP/AGPP/Pesticid/JMPR/Download/2003_eva /methoxyfenozide%202003.pdf). The metabolites of methoxyfenozide identified in these studies are shown in Fig. 2.9. The first metabolites formed are the alcohols of the parent compound at the methyl (dimethylphenyl B-ring) or methoxy (methoxyphenyl A-ring) substitutions on the bis-phenyls (RH-131364 and RH-117236), which, depending on the plant species tested, may be further metabolized to additional modification of the second methyl substitution on the B-ring (RH-131157). The final metabolites detected have been a bis-alcohol at the B-ring, an acid at one of the methyl positions in B-ring, or a glucoside conjugate of the parent compound. In studies on apples and grapes, the half-life of methoxyfenozide was < 30 days with the decline primarily due to fruit growth as there was very

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O O N N O O OH N N O
Methoxyfenozide (1)
RH-117236 (9)
CH2OH O O N N O OH O N N O
CH2OH O OH OH
CH2OH
RH-131364 (2)
O O O N N O O O CH2OH O O N N O CH2OH O O O N N O N OH
RH-141511 (7)
O O NN O
OH
OH
RH-151065 (8)
RH-131154 (3)
N O
OH
RH-152068 (6)
CH2OH OH OH
O OH
RH-131157 (4)
RH-152073 (5)
Figure 2.9 Metabolic pathway of methoxyfenozide derived from residue fate and metabolism studies of radiolabelled methoxyfenozide in crops and soil. The numbers in brackets represent the crop or soil in which the metabolites were identified: (1) C14-methoxyfenozide labelled uniformly at one or both the phenyl rings, or at the t-butyl group; (2) apples, grapes, rice, and radish roots from confined rotational crop (CRC) study; (3) grapes (tentative identification, tID), soil; (4) apples, rice, CRC-mustard, radish roots and tops, wheat forage and straw; (5) grapestID, CRC-mustard leaves, radish leaves, wheat straw; (6) CRC-mustard, radish roots and tops; (7) rice, CRC-wheat straw; (8) grapes, rice, CRC-mustard, radish roots and tops, wheat forage and straw; (9) rice, grapestID, CRC-wheat straw and grain, soil.
little metabolism of methoxyfenozide in apples and grapes. In apples, little over 2% of the total residue present consisted of two metabolites, RH131364 and RH-131157 (Fig. 2.9). In grapes, there were two minor metabolites of methoxyfenozide at < 4% and identified as glucose conjugates of RH-117236 and RH-131364 with a majority of the sample consisting of unchanged methoxyfenozide in both the fruit and the leaves.

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In a rice study, where the treatment of radiolabelled methoxyfenozide was at the pre-flag leaf and postflowering stages, the major component of the residues analysed was methoxyfenozide itself, with 5059% of it in grain and 6569% in straw. The other minor metabolites detected only comprised about 35% of the total residues in straw and grain. In confined crop rotational studies on mustard, radish, and wheat crops planted at 30, 90, and 365 days (nominal) in a sandy loam soil previously treated with radiolabelled methoxyfenozide at a rate of 2.2 kg a.i./ha, the parent methoxyfenozide was the major residue analysed in immature and mature crop samples. In general, total radioactive residue levels decreased significantly with increasing plant-back intervals (http://www.fao.org/ag/ AGP/AGPP/Pesticid/JMPR/Download/2003_eva/methoxyfenozide% 202003.pdf). The presence of methoxyfenozide and its metabolites in crop tissues grown in sandy loam soil treated with radiolabelled methoxyfenozide indicated the translocation of methoxyfenozide and/or its metabolites into the plants. While the radish roots contained the highest levels of unmetabolized methoxyfenozide, in the other crops the major metabolites were the A-ring phenol and B-ring alcohol and glucose conjugates of the phenyl rings (Fig. 2.9).
7. TOXICOLOGICAL PROFILE OF METHOXYFENOZIDE 7.1. Mammalian

Methoxyfenozide has been registered as a reduced risk insecticide by the U. S. EPA due to its novel mode of action, green chemistry, and mammalian and ecotoxicological profiles. To be classified as a reduced risk insecticide, like all other insecticides, extensive studies to establish the limits of toxicological safety to mammals, avian, terrestrial, and aquatic organisms, and the environmental fate of the parent a.i. and its metabolites are required by the registration authorities in geographical regions where insecticide uses must be registered. Studies on the components that are mixed to formulate the a.i. to establish their safety are also required. The endpoints and study results for mammalian toxicology of methoxyfenozide, tebufenozide, and chromafenozide are summarized in Tables 2.5 and 2.6. In addition to the short-term studies summarized in the Table 2.5, several long-term studies to understand any effects due to chronic exposure of methoxyfenozide were also conducted (http://whqlibdoc. who.int/publications/2004/924166519X_methoxyfenozide.pdf; http://

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Table 2.5 Mammalian risk profiles of registered bisacylhydrazine (BAH) insecticides Methoxyfenozide Tebufenozide Chromafenozide
Acute oral LD50 (rat, mouse) Acute dermal LD50 Eye irritation (rabbit) Dermal sensitization (guinea pig) Ames test Acute inhalation Reproduction (rat)
> 5000 mg/kg > 2000 mg/kg Non-irritating Negative Negative > 4.3 mg/l No effect
> 5000 mg/kg > 5000 mg/kg > 5000 mg/kg > 2000 mg/kg Non-irritating Slightly irritating Non-sensitizer Mildly sensitizing Negative > 4.3 mg/l No effect No effect Negative
Table 2.6 Acute (short-term) mammalian toxicological study results for methoxyfenozide Study duration Animal Effects NOEL (mg/kg/day)
28 days
Rat dermal No skin irritation or treatmentrelated systemic effects There was complete recovery of changes in haematological parameters observed after treatment during 4-week recovery period There was increase in liver weight and liver hypertrophy at midhigh doses. There were minimal changes in haematological parameters at high dose Decrease in body weight gain in males and females dosed at high doses No adverse effects
1000 Not applicable
4-week Dog recovery
90 days
Rat
69male 72female
90 days
Mouse
428male 589female 198male 209female
90 days
Dog
1 year
Dog
Mid- to high doses: slight to moderate haematological effects High dose: increased liver and thyroid weights; histopathological
10male 13female

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Table 2.6 Acute (short-term) mammalian toxicological study results for methoxyfenozidecont'd Study duration Animal Effects NOEL (mg/kg/day)
changes in liver, spleen, and bone marrow were considered secondary to mild met-haemoglobinaemia. The effects at mid-dose were not considered adverse by U.S. EPA and EU EU/EPA: 106 male 111female
NOEL, no observable effect level. Results of these studies are from http://whqlibdoc.who. int/publications/2004/924166519X_methoxyfenozide.pdf, http://www.apvma.gov.au/registration/ assessment/docs/prs_methoxyfenozide.pdf, http://ec.europa.eu/food/plant/protection/evaluation/ newactive/methoxyfenozide_review_report.pdf, http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi? dbname2002_register&docid02-23996-filed.pdf.
www.apvma.gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf; http://ec.europa.eu/food/plant/protection/evaluation/newactive/methoxyfe nozide_review_report.pdf; http://frwebgate.access.gpo.gov/cgi-bin/getdoc. cgi?dbname2002_register&docid02-23996-filed.pdf). The no observable effect levels (NOELs) in 18-month mouse studies were 1020 and 1354 mg/ kg for males and females, respectively. In this study, no treatment-related effects were observed. In another 24-month study with rats exposed to chronic doses of methoxyfenozide, the NOEL for males and females were 10 and 12 mg/kg, respectively. In this study, haematologic effects as increase in liver weight, hepatocyte hypertrophy, and follicular cell hypertrophy of the thyroid, at mid- and high doses were observed. In male rats, there was decreased survival, while in females there was decreased body weight, increased adrenal weights, chronic progressive glomerulonephropathy of kidneys, hyperplasia of renal pelvic epithelium, and uremic changes of multiple organs at the high dose were observed. Further, two-generation reproductive studies using rat and rabbit to evaluate the toxicological effects of methoxyfenozide on reproduction and development revealed that methoxyfenozide had no adverse effects on the reproductive performance of rat parent or offspring (NOEL of 1552 and 1956 mg/kg for male and female, respectively, parent or offspring) and no maternal or developmental effects in rat or rabbit at high dose (NOEL of 1000 mg/kg in both species) (http://whqlibdoc.who.int/publications/ 2004/924166519X_methoxyfenozide.pdf; http://www.apvma.gov.au/ registration/assessment/docs/prs_methoxyfenozide.pdf; http://ec.europa.eu/

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food/plant/protection/evaluation/newactive/methoxyfenozide_review _report.pdf; http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname 2002_register&docid02-23996-filed.pdf). In the two-generational rat study, increased liver weight and hepatocyte hypertrophy at mid- and high dose were observed in the parent rat. However, in EU, the effects at the mid-dose were not considered adverse. In genotoxicity (three in vitro and one in vivo) and neurotoxicity (acute and chronic), there were no effects observed and the results were negative for both tests (http://whqlibdoc.who.int/publications/2004/92416 6519X_methoxyfenozide.pdf; http://www.apvma.gov.au/registration/ass essment /docs/prs_methoxyfenozide.pdf; http://ec.europa.eu/food/plant /protection/evaluation/newactive/methoxyfenozide_review_report.pdf; http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname2002_regis ter&docid02-23996-filed.pdf). In the neurotoxicity studies, the NOEL for acute studies was 2000 mg/kg, and 1318 and 1577 mg/kg for male and female, respectively, in sub-chronic studies (http://whqlibdoc.who. int/publications/2004/924166519X_methoxyfenozide.pdf; http://www.ap vma.gov.au/registration/assessment/docs/prs_methoxyfenozide.pdf; http:// ec.europa.eu/food/plant/protection/evaluation/newactive/methoxyfenozid e_review_report.pdf; http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi? dbname2002_register&docid 02-23996-filed.pdf). The NOEL for Acceptable Daily Intake (ADI) from the rat and/or dog chronic studies was 10 mg/kg/day.
7.2. Avian
Methoxyfenozide was found to be practically nontoxic to both bobwhite quail, Colinus virginianus (Linnaeus), and mallard duck, Anas platyrhynchos (Sibley and Monroe), in 14-day acute toxicity and 8-day dietary toxicity studies, respectively, carried out according to U.S. EPA guidelines (Table 2.7). The LD50 values for both bobwhite quail and mallard duck were > 2250 mg a.i./kg body weight and > 5620 ppm of a.i., respectively. In 21- or 22-week reproductive toxicity tests using methoxyfenozide, the no observed effective concentration (NOEC) for both mallard duck and bobwhite quail was 1000 ppm.
7.3. Aquatic
Both acute and chronic studies have been carried out to observe effects on aquatic arthropods. In chronic studies, methoxyfenozide was slightly toxic to the water flea, Daphnia magna (Straus), and moderately toxic to the mysid

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Table 2.7 Summary of end point for birds, Daphnia magna, honey bees, and earthworms for methoxyfenozide, tebufenozide, and chromafenozide Methoxyfenozide Tebufenozide Chromafenozide
Avian: Mallard duck (LC50 8-day dietary) Bobwhite quail (LC50 8-day dietary) Aquatic: Bluegill sunfish, acute (LC50 96 h) Daphnia magna, acute (EC50 48 h) Honey bee (oral and contact: acute LC50) Earthworm (LC50 14 days)
> 5620 mg/g > 5620 mg/g
> 5000 mg/g > 5000 mg/g > 5000 mg/g (Japanese quail, 14 days)
> 4.3 mg/l
> 3.0 mg/l
3.7 mg/l 100 mg/bee 1213 mg/kg
3.8 mg/l 234 mg/bee 1000 mg/kg
> 189 mg/l (3 h)
> 1000 mg/kg
shrimp, Mysidopsis bahia (Molenock). The most sensitive species that incurred the greatest toxicity was the midge, Chironomus riparius (Meigen). The larvae were the most sensitive with an NOEC for adult emergence of 18 mg a.i./l based on initial overlying water concentration. The sensitivities to methoxyfenozide that were observed in microcosm studies (http:// www.apvma.gov.au/registration/assessment/docs/ prs_methoxyfenozide.pdf) were similar to those observed in lab studies. Interestingly, the results of the microcosm study also indicated that if the concentrations of methoxyfenozide in water fell sufficiently, the C. riparius population could recover. As pointed out in the mode of action for methoxyfenozide (Section 3), the sensitivity of Chironomus species to methoxyfenozide is correlated to its high EcR-binding affinity (Smagghe et al., 2002). This correlation of methoxyfenozide binding to EcR with very high affinity is very similar to the reasons for selective toxicity of methoxyfenozide primarily to lepidopteran larvae. As mentioned in Section 3 on the mode of action, methoxyfenozide manifests its action via interaction with the EcR complexes of susceptible insects. The insect moulting steroidal hormone, 20E, also manifests its actions via interaction with the EcR proteins. The presence of EcR complexes is very unique to all insects and many arthropods. No vertebrates or

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plants are known to have the EcR gene or protein, even though all vertebrates have multiple forms of steroid hormones and specific hormone receptors (e.g. estrogen and estrogen receptor, testosterone and testosterone receptor, retinoic acid and retinoic acid receptor, etc.). Based on this, it would be safe to assume that methoxyfenozide would not have any effects on aquatic or terrestrial plants. No effects were found in a U.S. EPA study on the green alga, Pseudokirchneriella subcapita (Korshikov), in which technical grade methoxyfenozide a.i. (120 h exposure) and the 240 SC formulation (240 g methoxyfenozide per litre of SC) (96 h exposure) were found not to be toxic up to the solubility limits of 3.4 mg/kg for technical product and 107 mg/l for formulated product (http://ec.europa.eu/food/plant/ protection/evaluation/newactive/methoxyfenozide_review_report.pdf).
7.4. Fish
Methoxyfenozide has also been tested to determine the LC50 values in acute and chronic studies on several species of fish (Cyprinodon variegates, Lepomis macrochirus, Onocorhynchus mykiss, Pimephales promaleas) (http://ec.europa.eu/ food/plant/protection/evaluation/newactive/methoxyfenozide_review_ report.pdf). It must be pointed out that, due to the very low solubility of methoxyfenozide in water, the maximum concentrations at which it can be tested are limited by its solubility. In 96-h acute toxicity studies on several species of fish (http://ec.europa. eu/food/plant/protection/evaluation/newactive/methoxyfenozide_review_ report.pdf) using either the technical methoxyfenozide or the 240 SC formulated methoxyfenozide, the LC50 values ranged from > 2.8 mg a.i. per liter to > 130 mg 240 SC product per liter. The higher LC50 values for the formulated product reflect its greater solubility than just for the technical grade of the unformulated product.
7.5. Terrestrial
In several sections, especially Section 3 on mode of action, reference to published data and explanations have been given to explain why methoxyfenozide and several other BAH insecticides are predominantly Lepidoptera specific. This is despite the fact that methoxyfenozide and other BAH insecticides manifest their action in susceptible insects via interaction with the same target site (EcR) that is used for the manifestation of the hormonal effects of 20E and other ecdysteroids in all insects. (Dhadialla and Ross, 2012; Dhadialla et al., 2005).

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Methoxyfenozide has been shown to have no impact on most beneficial insects including bees, predators, and parasitoids. The outcome of a number of bee toxicity studies indicate that methoxyfenozide is practically nontoxic to adult honey bee, Apis mellifera L. (LD50 > 100 mg a.i. per bee for acute contact and oral toxicity) (http://ec.europa.eu/food/plant/protection/ evaluation/newactive/methoxyfenozide_review_report.pdf). In two other studies, honey bee brood development was not adversely effected when A. mellifera colonies were fed syrup containing methoxyfenozide (http:// www.apvma.gov.au/registration/assessment/docs/prs_methoxyfenozide. pdf) or when bees were exposed to a seasonal rate of 480 g/ha in a semi-field study (http://dsp-psd.pwgsc.gc.ca/collection_2008/pmra-arla/H113-92008-15E.pdf). When B. terrestris (L.) were exposed to methoxyfenozide via oral route by incorporating methoxyfenozide in drinking sugar water or the pollen, or dermal route via topical contact, it resulted in no acute toxicity to the worker bees, or any adverse effects on larval development (Mommaerts et al., 2006). In the same study, exposure of B. terrestris to JH analogues, pyriproxifen and kinoprene, resulted in high number of dead larvae in the JH analogueexposed nests. A conclusion drawn by the authors from this study was that both the EAs (tebufenozide and methoxyfenozide) at their recommended concentrations are safe to be used in combination with B. terrestris. In another laboratory study, Kim et al. (2006) tested two different rates (full field rate or 10% of the field rate) of methoxyfenozide and several other insecticides to investigate the lethal and sublethal effects on Deraeocoris brevis (Uhler), an important generalist predator in pome fruits in the western United States. Both methoxyfenozide and spinosad had no acute toxicity to D. brevis nymphs and adults at both the tested rates, and no effect on egg hatch and nymphal survival just after hatch. While methoxyfenozide also had no sublethal effects on adults at the full rate tested, it slowed development of fourth instars following treatment of second instar nymphs, and lowered fecundity by 30% in the subsequent generation compared with the untreated check. Using a cabbage leaf dip method and doseresponse effects of various insecticides, including methoxyfenozide, labelled for use on crucifer crops, Cordero et al. (2007) conducted insecticide toxicity bioassays to determine the effects on two hymenopteran parasitoids, Diadegma insulare (Cresson) and Oomyzus sokolowskii (Kurdjumov) of P. xylostella, in the southeastern United States. In this study, all tested insecticides (spinosad, indoxocarb, esfenvalerate, methoyl, acetamiprid, acephate, emamectin benzoate, and

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methoxyfenozide) were found to have toxic effects on the adult stages of D. insulare or O. sokolowskii, or both. However, relative to the other insecticides tested, which resulted in 100% mortality to both the parasitoids 72 h after exposure, methoxyfenozide was considerably less toxic with 62% mortality of O. sokolowski adults. In several other studies on the safety of methoxyfenozide and other insecticides on beneficial insects, methoxyfenozide was shown to have no detrimental effects on the parasitoid, Encarsia smithi (Silvestri) of the camellia spiny whitefly, Alearocanthus camelliae (Kanmiya & Kasai) (Hemiptera) infesting tea plants (Yamashita and Yakahi, 2011); the insect egg parasitoid, Telenomus remus (Nixon) (Carmo et al., 2010); the ichnemonid adult, Hyposoter didymator (Thunberg) (Schneider et al., 2008); adults of egg parasitoids, Trichogramma atopovirilia (Oatman & Platner), by exposing the adult parasitoids to fresh dry formulated methoxyfenozide (Intrepid 240 SC) film applied on glass plates and further evaluation of the capacity of parasitism per adult fe pli-geti) of the male (Giolo et al., 2007); the parasitic wasp Psyttalia concolor (Sze olive fruit fly Bactrocera oleae (Rossi) (Bengochea et al., 2012); the predatory stinkbug, Picromerus bidens (Linnaeus), which has been considered as a potential biocontrol agent of several defoliator pests in various agricultural and forest ecosystems (Mahdian et al., 2007); predators of Chrysoperla carnea (Stephens) and Forficula auricularia (Linnaeus) (Zotti et al., 2012a,b); Neoseiulus fallacies (Garman), a key predator of tetranchid mites in IPM programmes across Canada (Bostanian et al., 2010); Orius laevigatus (Fieber) and Amblyseius swirskii (Athias-Henriot) as predators of Frankliniella occidentalis (Pergannde) and Bemisia tabacci (Gennadius) in commercial pepper greenhouses (Amor et al., 2012; Colomer et al., 2011); and the generalist predator, Labidura riparia (Pallas) (Kohno et al., 2007). In the same study, Kohno et al. (2007) also demonstrated the absence of detrimental effects by chromafenozide. Moving on to another important terrestrial organism, the earthworm, Eisenia foetida (Savigny), an Australian study was carried out according to OECD guidelines (http://www.apvma.gov.au/registration/assessment/ docs/prs_methoxyfenozide.pdf). Methoxyfenozide was found practically nontoxic to E. foetida with 14-day NOEC values of 1213 mg a.i. and 1250 mg of formulated SC per kilogram dry soil in each case, respectively. At concentrations of $ 2.1 mg a.i. in 240 SC per kilogram dry soil in a 56-day E. foetida chronic study, no adult mortality or reproductive effects on progeny production were observed. However, a slight increase in the body weight of methoxyfenozide exposed E. foetida at the highest rate was observed.

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8. SUBLETHAL AND OVICIDAL EFFECTS

The BAH insecticides can affect insect population not only by direct action on lepidopteran larvae but also by sublethal effects on larvae and adults. Direct effect on lepidopteran eggs has been observed in several insect species, thus having the potential to affect all life-cycle stages of the target pests resulting in an overall population effect over time. Sublethal effects and ovicidal activity have been observed since early development of the BAH insecticides by Rohm and Haas Company. These effects became more obvious during field characterization of these compounds by Rohm and Haas Company, Dow AgroSciences LLC, and a number of researchers around the world. These effects may be more notorious when large crop areas are sprayed exposing a significant percentage of the overall insect population to the effects of these insecticides, which also minimizes the immigration of new non-treated adults into the fields. Most of the early characterization of the sublethal and ovicidal effects by the BAH insecticides was focused on members of the Tortricidae that affect tree fruits (i.e. leaf rollers and C. pomonella). Because methoxyfenozide is developed and registered in so many countries, more researchers have been able to demonstrate those effects on a larger number of lepidopteran species. In recent years, more publications have been published about these effects on other pests of economic importance in the Tortricidae and Noctuidae. For instance, research from Argentina (D. Igarzabal et al., unpublished data) and Uruguay (Castiglioni et al., 2004, 2011) has demonstrated the ovicidal and sublethal effects of methoxyfenozide on A. gemmatalis and the bean shoot borer, Crocidosema ( Epinotia) aporema Wals., two of the most important pests of soybean in South America. This section will focus on reviewing the available data on sublethal effects on larvae, adults, and the ovicidal potency of methoxyfenozide. Also, tebufenozide will be discussed where there are applicable data.
8.1. Sublethal effects

Sublethal effects of methoxyfenozide and tebufenozide have been reported for many pests of economic importance. These effects have been studied at different developmental stages through ingestion, injection, or contact of sublethal doses to larvae or adults of Bombycidae, Crambidae, Noctuidae, and Tortricidae.

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8.1.1 Sublethal effects on larvae Direct effects on larvae as a result of exposures to sublethal doses of tebufenozide were studied on E. postvittana. Third instars exposed to 2 and 3 ppm of the chemical were more susceptible at high temperatures compared to untreated larvae, but no effect was observed with other instars tested (Whiting et al., 1999). Pineda et al. (2009a,b) recorded lower pupal weight, higher pupal mortality, increase in deformed pupae and adults of S. frugiperda when larvae were fed diet with concentrations equivalent to LC10 and LC25 for the species. Additionally, larvae exposed to those sublethal concentrations had a prolonged life in the larval stage. In a study on L. dispar larvae, it was found that LC10 and LC30 doses of methoxyfenozide caused abnormality in larval development and lower efficiency of conversion of ingested food. Survival rate, sixth instar larval weight, pupal ratio, and female pupal weight were also reduced by the sublethal concentrations of methoxyfenozide. Additionally, several enzyme levels were also affected by ingestion of sublethal concentrations of methoxyfenozide (YueZhi et al., 2009). 8.1.2 Effects on pupae and adults reared from larvae exposed to sublethal doses Another series of parameters studied include the effects on pupae and adults developing from larvae treated with sublethal doses of methoxyfenozide and tebufenozide. These parameters have been studied on multiple members of the Crambidae, Noctuidae, and Tortricidae. Rodriguez et al. (2001) observed that fecundity (number of eggs laid by females) and fertility (number of eggs that hatched) were not affected in the sugarcane borer, Diatraea saccaralis (F.), in the first generation of adults resulting from tebufenozidetreated larvae, but subsequent exposure of following generations to sublethal doses of the a.i. resulted in detrimental effects on fecundity and fertility of later generations. A similar effect was observed for beet armyworm, S. exigua, where reduction in fertility was recorded after continuous treatment of subsequent generations with tebufenozide (Smagghe et al., 1998). Cao and Han (2006) also observed reproductive disadvantages when developing a resistant strain of P. xylostella. A life table of the population showed that subsequent generations exposed to tebufenozide expressed decreased copulation rate and fecundity by adults and reduction in egg hatch. Codogan et al. (2002) did not observe any effects on adult C. fumiferana reared from tebufenozide-treated larvae, but significantly less oviposition was observed from those females when tebufenozide was present on the surfaces offered

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for oviposition. In contrast, Dallaire et al. (2004) found reductions in adult weight, delayed ovarian development, which resulted in lower female fecundity, and lower male mating success of C. fumiferana and C. rosaceana (Harris) when adults were reared from tebufenozide-treated larvae. Ovarian development studies focusing on the role of methoxyfenozide and tebufenozide in delaying the transition from vitellogenesis to choriogenesis have also been carried out in other Lepidopteran species including C. pomonella and B. mori (Sun et al., 2003; Swevers and Iatrou, 2003). Similarly, several adult reproductive and pupal parameters, including fertility and fecundity of Platynota idaeusalis (Walker) and L. botrana (Den & Schiff ), were affected when larvae were exposed to sublethal doses of methoxyfenozide and enztebufenozide (Biddinger and Hull, 1999; Biddinger et al., 2006; Sa n Irrigaray et al., 2005). Studies on S. exigua (Rodr guez de-Cabezo quez et al., 2010), Spodoptera littoralis (Boisduval) (Adel and Sehnal, Enr 2000; Pineda et al., 2004), and Spodoptera litura (F.) (Seth et al., 2004; Shahout et al., 2011) have shown pupal and adult deformities, female fertility and fecundity reduction, and male reduction in sperm production and sperm transfer resulting from larval exposure to sublethal concentrations of tebufenozide and methoxyfenozide. In contrast, Carpenter and Chandler (1994) and Zarate et al. (2011) only reported reduction in male fertility in H. zea exposed to tebufenozide and lower pupal weight, pupal deformities, and pupal mortality as well as longer larval development. 8.1.3 Sublethal effects when pupae or adults are exposed to BAH insecticides
8.1.3.1 Sublethal effects on pupae
Sundaram et al. (2002) investigated the effect of exposures to sublethal doses of tebufenozide injected in C. fumiferana pupae. Adults emerging from such treated pupae expressed wing deformities that affected their ability to mate and reproduce.
8.1.3.2 Sublethal effects on adults
Several studies have investigated the sublethal effects on adults by exposing them to methoxyfenozide or tebufenozide. The route of exposure by these studies includes oral, direct exposure to an insecticidal spray, or by exposure to dry residues. Reinke and Barrett (2007a) evaluated the effect of methoxyfenozide or tebufenozide on recently emerged G. molesta adults (unmated) by topical exposure to treated surfaces paired with untreated

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adults of the opposite sex. In their study, they found that significant reduction of fecundity and adult longevity was observed only when females were exposed to methoxyfenozide. Similar results for C. aporema were observed by Castiglioni et al. (2011) when recently emerged females exposed to methoxyfenozide-treated surfaces expressed reduction in fertility and fecundity. However, these effects were not expressed by females exposed to the insecticide once they had started oviposition. These results differ from studies done on C. pomonella 48- to 72-h-old moths exposed to residue-treated surfaces with label rates of tebufenozide that resulted in significant reduction of fecundity and fertility (Knight, 2000). Similarly, exposure of C. pomonella to sublethal doses of methoxyfenozide or tebufenozide during their whole adult stage resulted in a significant reduction in number of eggs laid and number of eggs hatched (Sun and Barrett, 1999). In similar studies, adult G. molesta exposed to methoxyfenozide or tebufenozide by contact with treated surfaces or ingestion during their adult stage expressed reduced fecundity and fertility (Batista Neto et al., 2011; Reinke and Barrett, 2007a), but reduction in female longevity was only observed by Reinke and Barrett (2007a). Interspecific differences among members of the Tortricidae were observed in recently emerged adult C. pomonella, Argyrotaenia velutiana (Walker), C. rosaceana, and P. idaeusalis exposed to treated surfaces with methoxyfenozide or tebufenozide. Reduction in fertility and fecundity of both sexes by both compounds were observed for C. rosaceana and P. idaeusalis, while for A. velutiana and C. pomonella only methoxyfenozide affected both sexes and tebufenozide affected only female moths (Myers and Hull, 2003; Sun et al. 2000; Sun and Barrett, 2003). In contrast, Smagghe et al. (2004) observed that, when recently emerged adult C. pomonella were sprayed with a sublethal dose of tebufenozide, the adult moths were sterilized by the application. Similar studies for S. littoralis exposed to methoxyfenozide showed that the compound affected fertility and fecundity regardless of the adult sex (Pineda et al., 2006, 2007, 2009a,b). A separate study on a related species, S. exigua, showed that effect on number of eggs laid by female moths decreased as the time of exposure to methoxyfenozide increased from 48 to 72 h, and the adult longevity was reduced only in males exposed to the insecticide (Luna et al., 2011). 8.1.4 Effects of methoxyfenozide on moth mating and egg laying behaviours The effect of methoxyfenozide on the calling ability by females (attractiveness) and response to pheromones by the males (responsiveness) along with selection of the egg-laying site are other sublethal effects that have been

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documented in members of the Tortricidae. Lab bioassays and field trials conducted on C. pomonella showed that exposure of the adults to treated surfaces with methoxyfenozide only affected male responsiveness but did not affect female attractiveness (Barrett, 2008; Hoelscher and Barrett, 2003a). Similar studies conducted on Argyrotaenia vetutinana (Walker) and C. rosaceana resulted in similar findings where male responsiveness was affected by exposure to methoxyfenozide while female attractiveness remained unaffected (Hoelscher and Barrett, 2003b). A study on G. molesta exposed to surfaces treated with methoxyfenozide showed similar results in terms of male responsiveness, but in contrast, it also showed a decrease in female attractiveness (Reinke and Barrett, 2007b). Studies done on C. aporema Wals. and C. fumiferana showed that females expressed selective oviposition behaviour when methoxyfenozide- and tebufenozide-treated surfaces were offered resulting in a reduction or no oviposition by adult females (Castiglioni et al., 2004; Codogan et al., 2002). When male C. pomonella were treated with an electrostatic powder containing methoxyfenozide, the male responsiveness and ability of the males to find the calling females were not affected (Huang et al., 2009), which could infer a difference in the effect by the delivery method. Sublethal effects can vary depending on the specific BAH insecticide used, insect species, gender exposed, and duration of exposure. In the studies reviewed in this section, methoxyfenozide consistently had stronger effects than tebufenozide regardless of the pest species and gender. In addition to the effects on fecundity and fertility, exposure of adults to methoxyfenozidetreated surfaces appears to negatively affect the sexual behaviour of adults (responsiveness and attractiveness) in some species. In general, it appears that male responsiveness may be impacted much more than female attractiveness by methoxyfenozide.
8.2. Ovicidal effects

The ovicidal effects of tebufenozide and methoxyfenozide have been reported in several studies for key insect pests in the Crambidae, Noctuidae, and Tortricidae. Overall, methoxyfenozide is more active than tebufenozide across all species where effects were compared. Results among and within species vary depending on the evaluation methods and the target stage of eggs. In general, either eggs treated within the first 24 h after oviposition or that were laid on surfaces treated with methoxyfenozide or tebufenozide had increased mortality versus eggs exposed to these compounds closer to hatch. For C. pomonella, Pons et al. (1999) reported that tebufenozide was significantly

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more effective in reducing egg hatch activity when eggs were laid on tebufenozide pre-treated surfaces, than when application was done after eggs were laid and residual effect on fruit was low. Methoxyfenozide, on the other hand, had greater residual activity than tebufenozide on eggs treated either before or after oviposition on fruit (Borchert et al., 2004). In contrast, Charmillot et al. (2001) reported that methoxyfenozide had both larvicidal and ovicidal effects while tebufenozide was exclusively larvicidal when apples were dipped on solutions of the products. They also reported that preventative treatment (before egg lay) was more effective than curative treatment (after egg lay). Borchert et al. (2004, 2005) reported ovicidal effects on G. molesta by methoxyfenozide. In field applications, they reported that the ovicidal effect of methoxyfenozide would produce significant effects on overall G. molesta and C. pomonella control. Brunner et al. (2007) proposed that, under commercial conditions, an early application of methoxyfenozide as an ovicide for C. pomonella control would allow growers to transition to a delayed larvicidal application moving from 250 to 300 DD for their first larvicidal treatment. Ovicidal effect by either direct spray on eggs after laying or contact with residues have also been reported on O. nubilalis (Trisyono and Chippendale, 1997), D. grandiosella (Trisyono and Chippendale, 1998), D. saccharalis (Rodriguez et al., 2001), C. fumiferana (Codogan et al., 2002), Endopiza viteana (Clemens) (Isaacs et al., 2005), C. oponema (Castiglioni et al. 2004, 2011), and A. gemmatalis (Fig. 2.10) (Igarzabal et al., unpublished data). Pineda et al. (2004) reported an ovicidal effect of methoxyfenozide on S. littoralis only when the solution contained acetone, but not when it was diluted in water, which may be due to the low solubility of methoxyfenozide in water. Charmillot et al. (2007) reported no ovicidal effect on Grapholita lobarzewskii (Nowicki) when fruits
Figure 2.10 The three photographs in this figure show the effects on Anticarsia gemmatalis trial with methoxyfenozide as ovicidal treatment from Igarzabal et al., unpublished data. (A) Non-hatched eggs after application. (B) Dead neonate larva that hatched from treated egg. (C) Larvae that hatched from untreated eggs.

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dipped on methoxyfenozide or tebufenozide solutions were exposed to mated females for oviposition. Zamora et al. (2008) did not observe egg mortality on S. frugiperda eggs treated after oviposition with solutions of methoxyfenozide at concentrations up to 1000 ppm. These results differ from F. Haile and M. Kempe (2009, unpublished data) that tested the effect of methoxyfenozide on S. frugiperda eggs either by residue contact (oviposition on pre-treated corn plants) or by direct application on eggs laid within the previous 24 h. In their study, egg laying was reduced > 82% at all rates and egg mortality before hatching was observed for eggs laid on pretreated surfaces (Table 2.8). Also, a reduction in egg hatching was observed when eggs were sprayed with methoxyfenozide solutions (Table 2.9).
8.3. Population effects by sublethal and ovicidal effects

Population reduction over time by the sublethal and ovicidal effects of the BAH insecticides has been proposed by several authors in the previous sections, but because of the complexity of evaluating those long-term effects, quantitative data has not been published for any species. L.A. Hull (unpublished data) recorded adult P. idaeusalis captured in sex pheromone-monitoring traps in large orchard plots at the Penn State University FRECArendtsville Farm from 1991 to 2011 as an indirect way to measure overall population magnitude in his research plots. The effect of tebufenozide applications, and more dramatically methoxyfenozide, on the reduction in number of adults captured over time after the two products were first applied in those fields can be observed in Fig. 2.11.
Table 2.8 Effect of methoxyfenozide on Spodoptera frugiperda eggs laid on corn plants pre-treated with the insecticide at different concentrations 4 days 2 days post-treatment post-treatment Treatment Rate (ppm) Avg. # eggs per plant % Reduction in egg oviposition % Larval hatching
Intrepid Intrepid Intrepid Intrepid
125 250 500 1000
2.6 0.2 0.0 0.0 14.4
81.9 98.6 100.0 100.0
0.0 0.0 83.3
Untreated
Intrepid is a registered trademark of Dow AgroSciences LLC for methoxyfenozide. From Haile and Kempe (2009, unpublished data).

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Table 2.9 Effect of methoxyfenozide on Spodoptera frugiperda eggs directly sprayed after oviposition Pre-treatment 2 days post-treatment Treatment Rate (ppm) Avg. # eggs per plant Avg. # larvae hatching per plant % Larval hatching
Intrepid Intrepid Intrepid Intrepid
125 250 500 1000
18.0 14.0 13.0 12.8 20.6
3.4 4.8 1.0 0.0 14.0
18.9 34.3 7.7 0.0 67.9
Untreated
Intrepid is a registered trademark of Dow AgroSciences LLC for methoxyfenozide. From Haile and Kempe (2009, unpublished data).
Tebufenozide* Methoxyfenozide Rynaxypyr Spinetoram

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 0 200
18 36 9 18 36 18 27 9 9
TABM (1 trap/orchard) TABM (2 traps/orchard) 400 600 800 1000
18 18 9 18 9 18 18 36 55 9 27 27 9 9 9 18
18 36 36 55 36 64
18 27 36 91 91 55
* % of insecticide trmts (11 total trmts) treated with designated compounds.
Mean number of months captured/season
Figure 2.11 Tufted apple bud moth (TABM), Platynota idaeusalis, adults captured in pheromone traps in orchards treated with various insecticides from 1991 to 2011 at Pennsylvania State University FRECArendtsville Farm (L.A. Hull, Pennsylvania State University, 2011, unpublished data).

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9. RESISTANCE AND RESISTANCE MANAGEMENT

Tebufenozide was the first non-steroidal EA with commercial application and has been used in Western Europe since the mid-1990s. In the United States, the first uses occurred concurrently in 1994 in Alabama and Mississippi under Section 18 exemptions (Dhadialla et al., 1998; Walton et al., 1995). For the newer EAs, methoxyfenozide was first used in Brazil, Colombia, Indonesia, Israel, and Mexico in 1998 and now in many crops in more than 50 countries. The commercial use of halofenozide in turf and golf courses started in the late nineties in the U.S. turf market, and the use of chromafenozide to control pest Lepidoptera in Japan began in the late nineties. To date, the BAH insecticides form a separate group 18 in the IRAC mode of action classification, being a key to IRM (www.irac.org). Resistance to tebufenozide was documented for the first time in C. pomonella around Avignon in southern France (Sauphanor and Bouvier, 1995; Sauphanor et al., 1998) and in the green-headed leafroller, Planotortrix octo (Dugdale), in New Zealand (Wearing, 1998). The latter has been well described before in detail by Dhadialla et al. (1998). Shortly after these first incidences, more data were collected for an important agricultural pest insect, S. exigua, of vegetables in open field and greenhouse. Indeed, many populations of this noctuid pest are resistant to nearly all insecticide groups and it is well known for its high potential to rapidly develop insecticide resistance (Arthropod Pesticide Resistance Database; http://www.pesticideresistance.com/). A survey for baseline resistance to tebufenozide of S. exigua collected in different greenhouses in Western Europe (Spain, Belgium, and the Netherlands) was performed and the data showed a lack of resistance development (Smagghe et al., 2003a,b). At the same time, Smagghe et al. (1998) maintained a laboratory strain of S. exigua under continuous pressure with sublethal concentrations of tebufenozide for over 12 generations, and in the sixth generation a significantly lower toxicity (about fivefold) was observed (Fig. 2.12). In addition, retention and metabolism studies showed the importance of oxidative metabolism, leading to a rapid clearance from the insect body (Smagghe and Degheele, 1997; Smagghe et al., 1998, 1999a). Similarly, Moulton et al. (2002) reported on development of resistance to tebufenozide in third instars of S. exigua from Bangbuathong (Thailand), which reached levels of 150-fold compared to a laboratory-

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A
100
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y = -75.89 69.21 2 R = 0.79
80 60 40 20 0 -0.4
%mortality
50
0 0 3 6 9 12 15
0.1
0.6
Log LC50 (mg/l)
Conc RH-5992 (mg/l)
Figure 2.12 Selection assay in a susceptible strain of the beet armyworm (Spodoptera exigua). (A) Induction of tolerance via continuous treatment of sublethal doses of tebufenozide ( RH-5992 ca LC25) over subsequent generations via administration to all five larval stages of a susceptible laboratory strain. The green lines represent the dosetoxicity curves for larvae of generations 0, 1, and 4 (susceptible); orange, generations 4, 5, and 6 (slightly tolerant); and red, generations 712 (tolerant). (B) Fitness cost associated with tolerance for tebufenozide. The linear regression plot shows a negative relationship between the mean fecundity, expressed as percentage of the mean number of eggs per female of the Gx generation as compared with G0 moths, and the logarithm of for LC50 tebufenozide (adapted from Smagghe et al., 1998).
inbred reference strain not previously exposed to tebufenozide. In this region of Thailand, it is typical that many insecticides, including organophosphates (OPs), pyrethroids, benzoylphenyl ureas, Bt formulations, and even new insect growth disruptors (IGDs, formerly called IGRs) have been rendered ineffective in a rapid manner due to illadvised agricultural practices, most notably dilution of insecticide residues on leaves by overhead drench irrigation (Moulton et al., 2002). This practice is likely to be responsible for the high incidence of insecticide multi-resistance in this area and the highly accelerated rate of resistance development. When this Thailand strain was dosed with methoxyfenozide, a 120-fold lower toxicity was observed. These selection assays with tebufenozide and methoxyfenozide showed a reduction in toxicity for both compounds, suggesting instability of resistance and at least some commonality of resistance mechanism (Moulton et al., 2002).

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Interestingly during the selection of S. exigua for resistance to tebufenozide in the laboratory by Smagghe et al. (1998), as exemplified in Fig. 2.12, a decrease in oviposition was noted with reduced toxicity, indicating that there was a fitness cost associated with development of resistance. Similar results were obtained by Cao and Han (2006) who reported that a resistance level of 94-fold to tebufenozide in P. xylostella was achieved over 35 generations accompanied with fitness cost. The life table tests indicated that the resistant strain showed reproductive disadvantages, including decreased copulation rate, reproductive productivity, and egg hatchability. The resistant insects had a relative fitness that is only 30% of the net reproductive rate of the susceptible strain. More recently, similar data were obtained for the newer fufenozide (Sun et al., 2012). These data indicated that selection of resistance to tebufenozide and fufenozide had considerable fitness costs, and therefore, rotational use of insecticides without cross-resistance is recommended to delay development of resistance. A greenhouse-raised strain of S. exigua from southern Spain was also found to be resistant to tebufenozide and methoxyfenozide. Although the level of resistance was lower in this second strain, it was high enough to allow studies on the mechanism(s) of resistance. In general, a higher breakdown metabolism leads to lower levels of the parent toxophore. For tebufenozide and methoxyfenozide, the major first phase route of detoxification was through oxidation (Smagghe et al., 1998, 2003a,b). In these experiments, it was of interest that addition of the P450 inhibitor, piperonyl butoxide, and also metyrapone, could significantly synergize the toxicity of tebufenozide and methoxyfenozide, whereas S,S,S-tributyl phosphorotrithioate (DEF), an esterase inhibitor, was ineffective (Smagghe, 2004; Smagghe et al., 1998, 1999a), indicating that a lower toxicity was more likely from an increase in oxidative activity, rather than in esterase activity. It is inevitable that even though the BAH insecticides possess a new mode of action via binding to the EcR of susceptible insects, parameters such as uptake, metabolism and excretion can play a major role in the risks for resistance development to the BAH insecticides and cross-resistance to other insecticide groups designated by IRAC. Indeed, more recent, strains of S. exigua collected in different regions in Pakistan between 2008 and 2010 showed relatively low (3- to 41-fold) levels of resistance to methoxyfenozide, while levels for pyrethroids and organophosphates (OPs) were very high, reaching up to more than 130-fold (Ishtiaq et al., 2012). Similar data were obtained by Zhou et al. (2011) with different field strains from regions in China in 20082010. Recently, Liu et al. (2011a,b) reported absence of cross-resistance with a

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methoxyfenozide mid-resistant population (30-fold) of the old world bollworm, Helicoverpa armigera (Hu bner) and 12 different OPs, carbamates, and pyrethroids. Synergism assays with piperonyl butoxide, phosphate defoliant and diethyl maleate in vivo, and enzyme activity assays for mixed function oxidases, general esterases, and glutathione-S-transferases in vitro confirmed the enhancement of activity of the three enzymes. In a study to determine the insecticide resistance or cross-resistance status of > 30 populations in Greece, Voudouris et al. (2011) reported reduced susceptibility to all tested compounds (azinphosmethyl, phosalone, deltamethrin, thiacloprid, fenoxycarb, tebufenozide, methoxyfenozide, and diflubenzuron). In addition, elevated cytochrome P450 activity, followed by elevated glutathione-S-transferase activity and reduced carboxyl esterases activity were reported; but no sodium channel or acetylcholine esterase (AChE) resistance mutations were found in any of the about 1000 individuals of each larval instar screened with diagnostic polymerase chain reaction (PCR). In conclusion, all the above reports pointed that several reasons account for varying degrees of resistance, including selection pressure, cropping structure, and migration. On the genetics behind the selection for resistance to tebufenozide in S. exigua by dietary exposure in the laboratory, Jia et al. (2007) estimated the heritability (h2) of resistance. After selection with tebufenozide 62 times during 75 generations, a resistant strain was achieved with a resistance ratio of 39. The h2 in the early, middle, and latter selection stages was 0.1075 (F0F25), 0.2780 (F26F50), and 0.0538 (F51F75), respectively. The h2 for the entire selection experiment was 0.1556. The susceptibility to tebufenozide increased 3 fold when the culture was kept for 21 generations without exposure to the pressure with tebufenozide after 43 rounds of selection. These data suggest that S. exigua has the capability of developing resistance to tebufenozide, and it is difficult for it to recover the sensitivity to tebufenozide over a short time period. In addition, it was of interest that bioassay by these authors also revealed that this resistant strain had high crossresistance to methoxyfenozide (RR 71), moderate cross-resistance to abamectin (RR 13), low cross-resistance to emamectin benzoate (RR 7), indoxacarb (RR 8), and fufenozide (RR 5). This suggested that when tebufenozide is applied by interruption and/or alternation with other insecticides possessing a different mode of action, the resistance development rate in S. exigua would be delayed. Two years later, Liu et al. (2009) performed a somewhat similar experiment to select H. armigera for resistance to methoxyfenozide. After 29 times

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of selection during 33 generations, the population developed 33-fold resistance to methoxyfenozide. The h2 for the entire selection experiment was 0.0830; the respective h2 in the early, middle, and latter selection stages was 0.0715, 0.0768, and 0.0947. Therefore, it requires 34 and 27 generations of laboratory reared H. armigera kept under selection pressures that result in 80% and 90% mortality, respectively, at each generation to develop a 10-fold increase in methoxyfenozide LC50. Thus, the number of generations required for a 10-fold increase would be even more in the field due to the changes in allele frequency and environmental variation or both. Interestingly, at the cellular level, Retnakaran et al. (2001) reported that tebufenozide accumulated selectively in the spruce budworm midgut CF203 (C. fumiferana-203) cells in contrast to dipteran Dm-2 (D. melanogaster) cells which actively excluded the compound. It is possible that such exclusion systems may also account for the fact that older instars of the white-marked tussock moth, Orgyia leucostigma, are resistant to tebufenozide (Retnakaran et al., 2001). The characterization of all such possible resistance processes is essential to provide information that can be helpful to prevent resistance from developing towards this valuable group of IGDs. However, more information is needed with additional strains collected from the field, especially where growers have severe pest control problems, before a general interpretation can be formulated on resistance risks for this new type of IGDs in the field. While the available data indicate oxidative metabolism as the primary reason for development of resistance to tebufenozide and methoxyfenozide, there is no evidence so far that suggests that target site modification could be involved as another route to resistance development in field or laboratory insect populations. However, at the cellular level, there is evidence that there could be alterations in the target site(s). Using insect cell lines, Cherbas and co-workers (P. Cherbas, personal communication) were the first to report that in vitro cultured Kc cells of D. melanogaster did not respond to 20E after continuous exposure. Similarly, Spindler-Barth and Spindler (1998) reported that cells of another dipteran, the midge, C. tentans, maintained in the continuous presence of increasing concentrations of 20E or tebufenozide over a period of 2 years, developed resistance to both compounds. In these resistant subclones, all 20E-regulated responses that are known to occur in sensitive cells were no longer detectable, suggesting that the hormone signalling pathway itself was interrupted (Grebe et al., 2000; Spindler-Barth and Spindler, 1998). Further ligand-binding experiments with extracts containing EcR from susceptible and resistant cells indicated that ligand binding to EcR from resistant clones was significantly

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decreased. Moreover, an increase in 20E metabolism and a reduction in receptor concentration were noted in some clones. Similar effects were observed in another study using imaginal discs of S. littoralis selected for resistance to tebufenozide (Smagghe et al., 2001). To further understand the mechanisms of resistance to BAH insecticidal compounds, Mosallanejad et al. (2008a,b) selected ecdysteroid-responsive cell lines of S. exigua (Se4) for resistance by continuous exposure to 20E and methoxyfenozide in order to obtain 20E- and methoxyfenozideresistant mutant cell lines. As shown in Fig. 2.13, the use of insect cell cultures allowed a rapid selection of very high levels of resistance (1,000,000-fold) such that these embryonic lepidopteran Se4 cells lost their sensitivity from 0.1 nM at the start of resistance selection to 100 mM methoxyfenozide at the end of the experiment over a relatively short period of about 50 passages. These authors showed that resistance in these cells was not due to a differential metabolism and uptake of methoxyfenozide and 20E compared to the sensitive cells. Because crossresistance existed between the 20E- and methoxyfenozide-selected cells, it was hypothesized that the resistance mechanism may be at the level of the ecdysone signalling pathway, which is the common effector pathway for both compounds (Dhadialla et al., 1998; Nakagawa, 2005). Previously, this was also hypothesized for the prototype compound RH5849 using D. melanogaster Kc cells (Wing, 1988). Based on these results, Swevers et al. (2008) investigated the various steps in the ecdysteroid signalling pathway by measuring the activity of selected transcription factors known to be involved in this cascade in susceptible and resistant cell lines. The early gene in the ecdysone signalling pathway, HR3, was constitutively expressed in the resistant cell lines grown in the presence of 20E and methoxyfenozide. In addition, the gene FTZ-F1 was constitutively expressed in both resistant and sensitive Se4 cells, suggesting that its expression was not regulated by the addition of methoxyfenozide and 20E. Further analysis of the functionality of the EcR/USP complex in the resistant Se4 cells revealed the existence of a normally functioning EcR/USP complex in these cells. Very similar data were obtained for resistant cell lines derived from other Lepidoptera cell lines such as ovarian Bm5 (derived from B. mori) and midgut C. fumiferana CF-203 cells (Mosallanejad, 2009). It is proposed that the resistance mechanism exists at the junction between the conserved ecdysone regulatory cascade and the differentiation programme in the cell line. Indeed, RNAi studies in IAL-PID2 cells have confirmed the

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110-4
110-5
Concentration (M)
110-6
110-7
110-8
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Se4-RH-2485-R4 Se4-20E-R4
110-10 0 20 40 60
Number of passages B
70 60 Number of genes Number of genes 50 40 30 20 10 Induced Repressed 0 70 60 50 40 30 20 10 0 Absence of methoxyfenozide Presence of methoxyfenozide
Figure 2.13 Use of insect cell lines to screen and study mechanisms of insecticide resistance. (A) Development of resistance towards methoxyfenozide and 20-hydroxyecdysone (20E) in lepidopteran Se4 (Spodoptera exigua) cells following successive selection with increasing concentrations over different passages for the
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involvement of the ecdysone regulatory cascade in cell cycle arrest and morphological transformation (Siaussat et al., 2007a,b, 2008). Continuing with this line of research to understand the molecular basis of resistance to BAH insecticidal compounds and 20E in insect cells, Mosallanejad et al. (2010) selected a dipteran cell line, Schneider 2 (S2) cells from D. melanogaster, for resistance towards methoxyfenozide. Although methoxyfenozide is an insecticide displaying high specificity against lepidopteran insects (Dhadialla et al., 1998), it also has considerable activity in EcR reporter assays in S2 cells (EC50 16.6 mM; Soin et al., 2010b) and is one of the most active compounds from a library of BAH chemotypes. According to Nakagawa et al. (2002b,c), the IC50 of methoxyfenozide is < 1 mM in the binding assay using D. melanogaster Kc cells, while the IC50 of Pon A is about 1 nM. The advantage of the S2 insect cell line is that it represents a useful model for researchers because of the availability of the D. melanogaster genomic sequence, extensive database, and corresponding commercially available microarray slides. Hence, Mosallanejad et al. (2010) investigated the functionality of the EcR/USP complex and performed a microarray study to determine transcript expression profiles of genes involved in methoxyfenozide resistance. The information provided insights in altered functioning of the ecdysone signalling pathway as a contributing factor to the resistance mechanism to methoxyfenozide, resulting in a total loss of susceptibility, and in the functional link between ecdysone signalling and cell proliferation. The most striking observation was that most differentially expressed transcripts increased or decreased in expression in the presence or absence, respectively, of methoxyfenozide (Fig. 2.13). In both conditions, the EcR complex is not acting as a liganddependent transcriptional activator, as continuous presence of methoxyfenozide inactivates EcR, while in the other condition, the activating ligand is absent. As in the absence of ligand, EcR can act as a repressor (Tsai et al., 1999), it was proposed that the preferential decrease in
Se4-RH-2485-R4 and the Se4-20E-R4 subclone, respectively. (A) The passage numbers at which growth was observed for different concentrations of compounds during the selection period. (B) Classification of genes differentially expressed between the methoxyfenozide-resistant dipteran S2 (D. melanogaster) cells and the sensitive cells maintained in the absence of methoxyfenozide. The two graphs on the right display numbers of differentially expressed genes for different classes of genes in the presence or absence of methoxyfenozide for the resistant cells. Genes that are induced and repressed are separated in different bars (redrafted from Mosallanejad et al., 2007, 2010).

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expression after removal of the selection pressure restores the repressor function of EcR in the resistant cells and that the observed deficiency of the EcR complex in continuous presence of an EA involves both activation and repressor functions. As a consequence, this microarray study provided a significant list of leads in the search for possible mechanisms of resistance against EAs and uncovered more complex levels of EcR signalling than were previously conceived. In summary, although it is inevitable that insects will ultimately develop resistance to any new insecticide, particularly when it is misused, resistance to the BAH insecticidal EAs should be prevented from occurring too soon with good monitoring programmes and by implementation of resistance management tactics. The high level of safety of both tebufenozide and methoxyfenozide to predators and parasitoids allows growers to profit from the biological control approaches in current IPM programmes, reducing the pressure for resistance development. In addition, different resistance prevention tactics such as rotation with insecticides with different modes of action and enzyme synergists are available as a result of recent fundamental and applied research. According to IRACs classification, the BAH insecticides belong to group 18. To date, some instances of resistance or cross-resistance to tebufenozide and methoxyfenozide have been reported in different pests and no information is available in the literature for resistance development to chromafenozide and halofenozide.
10. CONCLUSIONS AND FUTURE PROSPECTS

The discovery of the non-steroidal EA BAH insecticides with a novel mode of action via the receptors for the insect moulting hormone, 20E, and their selective spectrum of insect activity have become very useful insecticides for incorporation into IPM and IRM programmes. Additionally, the BAH insecticidal compounds have been instrumental in enhancing our understanding of the structure and function of EcRs and the physiological, biochemical, and molecular basis of ecdysteroid action. Their utility is evidenced by the extensive literature around this class of chemistry, specific insecticidal compounds and their use for selective control of lepidopteran pests. The selective insect toxicity of BAH insecticides prompted the need and the potential for the discovery of newer BAH compounds or members of very different chemistries that would have a different or broader spectrum of insect toxicity than the current BAH insecticides. These efforts led to discovery of new chemotypes, not belonging to the BAH chemistry, with some

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like the THQ and g-methylene g-lactams that have a spectrum of activity extending to non-lepidopteran arthropods. While these new chemistries have not materialized for commercialization, given how much more we know of the target site (EcR-LBD) of these and BAH insecticides, there is the potential for discovery of non-BAH compounds with commercial value as insecticides for non-lepidopteran insect pests. Unlike the JH analogues (see Chapter 5) that were discovered long before the non-steroidal EA BAH insecticidal compounds, the mode of action of the BAH insecticides is much better understood at the biochemical and molecular level. The 3D structure activity of some of the BAH compounds in comparison with an ecdysteroid with residues in the LBD of EcR from susceptible and non-susceptible insects is very well understood at the crystalline structure level. This great advance opens the possibility to rationally design chemotypes that would be toxic to non-lepidopteran insects. This possibility is described in Chapter 4. Finally, with an increased understanding of the ecdysteroid signalling pathway and genes involved, it is possible to disrupt the growth and development of insect pests by finding new insecticidal chemotypes or approaches, such as RNAi (Barchuk et al., 2008; Pollard et al., 2008; Siaussat et al., 2007a,b) to knock down or inactivate key targets involved in the ecdysone signalling pathway, or another hormone signalling pathway. A topic not discussed in this review, and which has been discussed in earlier reviews of the BAH IGDs (Dhadialla and Ross, 2005; Dhadialla et al., 1998, 2005, 2010; Palli et al., 2005a,b,c), is the use of the BAH insecticidal compounds or others with the same mode of action as ligands for regulated gene expression in plants and animals, which do not normally have the EcR genes (Martinez et al., 1999; Padidam et al., 2003; Palli et al., 2005a,b,c; Unger et al., 2002). This research involves transforming plant or animal systems with genes encoding the EcR and USP proteins or their LBDs fused to appropriate DNA-binding domains and activation factors under the influence of constitutive or regulated promoters and appropriate DNA response elements fused to the gene of interest needed to be transactivated with a ligand. The ligand in this case would be one of the BAH insecticidal compound or member of another chemistry that binds with transactivational activity to the EcR-LBD. Many advances have been made in this area of research, though the results have not been transformed into application in the field.
ACKNOWLEDGEMENTS
We (L. E. G. and T. S. D.) would like to thank Dow AgroSciences LLC for their support and encouragement to undertake writing this review and the following colleagues for their input

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and technical reviews to increase the quality of this document: Kirk Brewster, Jon Babcock, Carl Corvin, Don Kelley, Kari Lukasik, and Mike Shaw. The authors especially thank Dr. Rayda Krell for assisting in editing the chapter. G. S. acknowledges past and present financial support from the Special Research Council of Ghent University and the Fund for Scientific Research (FWO-Vlaanderen, Belgium).
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Bisacylhydrazine Insecticides for Selective Pest Control

2012 Elsevier Ltd. All rights reserved.

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2. CHEMICAL STRUCTURES OF ECDYSTEROIDS AND NON-STEROIDAL ECDYSONE AGONISTS

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Gene switch ligand

O Ecdysone (E) 20-Hydroxyecdysone (20E) Ponasterone A (PonA)

a-Acylaminoketone Halofenozide (RH-0345)

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3. ECDYSTEROID-SPECIFIC MODE OF ACTION

3.1. Bioassays for tissue and cellular effects

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Bombyx mori (Bm5)

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Tenebrio molitor (extracts)

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Anthonomus grandis (AG-3C cells)

Dhadialla and Tzertzinis (1997), Soin et al. (2009b)

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3.2. Bioassays for whole organism effects

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Normal moult Larval instar (N) MI 5 Larval instar (N+1) MC EH 4 6 4 8

2 1 Ingestion of tebufenozide Normal feeding Time (h) 1

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4. METHOXYFENOZIDE GLOBAL USES 4.1. Introduction

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Table 2.2 Commercial bisacylhydrazine (BAH) insecticides Methoxyfenozide Tebufenozide Halofenozide

Cyclone Kanpai Killat Matric

Puerto Rico Japan (United States) 2002 Dow AgroSciences 1999

Dow AgroSciences Nippon Soda Co., Ltd.

Nippon JPRIb Sankyo Agro Co.

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4.2. Bulb vegetables

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Beet armyworm Southern armyworm South American tomato moth

Garden webworm Cutworms Armyworm

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Cotton leafworm Velvetbean caterpillar Tobacco budworm

Bollworm Fall armyworm Cabbage looper

Fall cankerworm Florida fern caterpillar Spruce budworm Elm spanworm

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Outdoor areas Residential areas

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Oriental tea tortrix Cluster caterpillar

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