1

1 Insect endocrinology and hormone-based pest

2 control products in IPM

3

4 Daniel Doucet1, Michel Cusson2 and Arthur Retnakaran3

6 1) Canadian Forest Service, Sault Ste. Marie, Ontario, Canada

7 2) Canadian Forest Service, Quebec City, Canada

8 3) Brock University, Ste Catharines, Ontario, Canada

10

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12 1 Introduction

13 Integrated pest management (IPM) methods were developed largely as a

14 response to the negative consequences of the intensive use of broad-spectrum,

15 highly potent chemical insecticides in the early to mid- 20th century (Kogan 1998).

16 These insecticides, belonging to the carbamate, organophosphate and

17 organochlorine families, display many drawbacks, including environmental

18 persistence, bioaccumulation, development of resistance among target pests,

19 toxicity to non-target species (especially natural enemies) and human health

20 risks. While IPM focuses mainly on preventative tactics (crop rotation, etc.) rather

21 than remedial ones, synthetic chemical insecticides are still very much needed to

22 achieve effective control (Kogan 1998).

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23

24 The study of insect physiology has been driven, in no small part, by the need for

25 safe alternatives to broad-spectrum insecticides. Theoretically at least, digestion,

26 excretion, neuronal communication, metabolism and other physiological

27 processes all comprise “insect-specific” components that are vulnerable and

28 targetable by synthetic molecules. To this day, however, IPM-compatible pest-

29 control products that target the insect endocrine system far outnumber those

30 targeting other systems. In particular, hormone mimics that control development

31 have enjoyed not only wide appeal but also many commercial successes, and

32 additional control products targeting hormone production and function are

33 currently under development. In this chapter we endeavour to provide an

34 overview of (i) insect endocrinology, (ii) existing control products that mimic

35 ecdysone and juvenile hormone (JH) action, and (iii) possible development of

36 disruption control strategies based on novel endocrine functions that are likely to

37 generate new IPM tools in the future.

38

39 2 Basics of hormone biochemistry and biology

40 The term “hormone” was first coined by Ernest Starling a hundred years ago to

41 define any chemical messenger, secreted by an organ, which travels through the

42 bloodstream to affect the physiology of another, distant organ (reviewed in

43 Henderson 2005). At that time, hormones were known to be important mediators

44 of vertebrate physiology, but Kopec (1917) soon demonstrated that similar

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45 secretions exist in insects, with his isolation of a brain factor promoting moulting

46 in a lepidopteran larva.

47

48 Insect hormones fall into four classes based on their chemical structures: i)

49 peptide and protein hormones, ii) biogenic amines, iii) prostaglandins, and iv)

50 terpenoid lipids. Peptide hormones are chains of amino acids usually shorter

51 than 20-30 residues. Longer chains are traditionally referred to as proteins. This

52 is by far the most abundant class of insect hormones, with several hundred

53 different peptides isolated to date, from various species. Biogenic amines are

54 derivatives of amino acids and are involved in signal transduction. In insects,

55 octopamine and tyramine are two such compounds that regulate important

56 aspects of locomotor and non-locomotor behavior, circadian rythms and stress

57 response (Gole and Downer 1979; Fussnecker et al. 2006). Prostaglandins are

58 oxygenated metabolites of arachidonic acid and play diverse roles including

59 mediation of cellular immunity and release of egg-laying behaviour (Stanley

60 2006). Terpenoid hormones are lipid molecules constructed from the basic

61 hydrocarbon unit 2-methyl-1,3-butadiene, also called isoprene. In insects, two

62 important subclasses exist: the juvenile hormones, which are derivatives of linear

63 chains of three isoprene units (i.e., sesquiterpenes) and the ecdysteroids, which

64 consist of a tetracyclic cholestane ring system derived from cholesterol, which is

65 elaborated from smaller isoprenoids. Both hormones act on the timing and nature

66 of molting in all insects. Most successful hormone-based insecticides mimic or

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67 inhibit the activity of these hormones; the principal groups are presented in

68 greater detail in subsequent sections.

69

70 3 Overview of the insect endocrine system

71 Hormone secretion in insects is accomplished by a limited number of glands,

72 organs and tissues, and, as observed in other animal groups, the central nervous

73 system (CNS) plays an overarching role. The CNS integrates sensory

74 information and translates it into nervous or hormonal outputs that bring about

75 the physiological, behavioral and developmental processes necessary for

76 survival (Nijhout 1994). The endocrine control of these processes by the CNS

77 can be either direct or indirect. A good example of direct control is the induction

78 of diapause in Bombyx mori by the diapause hormone (DH), released by

79 secretory neurons from the suboesophageal ganglia (Hasegawa 1957, Sato et al.

80 1993). More frequently, however, the CNS modulates the secretions of other

81 endocrine glands by releasing inhibitory or stimulatory (tropic) neurohormones.

82 This hierarchical organization between hormones and neurohormones can be

83 highly complex and include endocrine feedback loops. Such is the case for insect

84 ecdysis, where the proper unfolding of this innate behavior is ensured by a tightly

85 controlled spatial and temporal release of several peptidic hormones (more than

86 6, Kim et al. 2006). While a thorough review of insect hormones (and their sites

87 of production) would be beyond the scope of this chapter, a survey of some of

88 the most important ones is presented in Table 1.

89
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90 3.1 CNS and associated neurohemal organs

91 The CNS is comprised of two major endocrine centers: the brain-retrocerebral

92 complex (RC) and the perisympathetic organs of the ventral nerve cord. The RC

93 is a bipartite structure, posterior to the brain, composed of the corpora allata (CA)

94 and corpora cardiaca (CC). Clusters of neurosecretory cells are located in

95 various parts of the insect brain (e.g. medial, lateral and ventral) and the majority

96 release their secretions via the CC (Nijhout 1994). Some brain neurosecretory

97 cells instead use the CA as a neurohemal organ (e.g. secretion of

98 prothoracicotropic hormone, PTTH), while fewer still release their secretions

99 distally, for example in the vicinity of the proctodeum (hindgut) (e.g. the

100 proctodeal nerves in Manduca sexta, which synthesize the eclosion hormone

101 (EH), Truman and Copenhaver 1989). The glandular portions of the CA and the

102 CC also secrete their own hormones besides those from neurosecretory cells:

103 the CA secrete JH (see section 5) while the CC contain endocrine cells that

104 produce peptides such as the adipokinetic hormone (AKH).

105

106 The ganglia of the ventral nerve cord also deploy segmentally distributed

107 neurohemal organs that are functionally close to the CC. These perisympathetic

108 organs (PSOs) can emerge either from the ganglion itself, anteriorly or

109 posteriorly to the ganglion (Nijhout 1994). Although anatomically and functionally

110 related to the CC, the PSOs release a different collection of neuropeptides

111 (Predel et al. 1999; Predel et al. 2000).

112
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113 3.2 Other endocrine glands

114 While the CNS is the most structurally and functionally complex insect endocrine

115 tissue, peripheral organs and tissues also contribute in important ways to

116 hormonal control of physiological processes. This is particularly true for the

117 regulation of developmental events. The prothoracic glands (PGs), located in the

118 thoracic segments of immature insects are devoted to the secretion of the

119 hormone ecdysone. Ecdysone is responsible for triggering molting in insects and,

120 along with JH, has been a target of choice for the development of insecticidal

121 hormone analogs (see sections 4 and 5 below). The Inka cells come into play

122 later during the molting cycle, at the time of ecdysis. These cells are specialized

123 in the secretion of two peptides regulating ecdysis-related behaviors: the

124 preecdysis triggering hormone (PETH) and the ecdysis triggering hormone (ETH)

125 (Žitňan et al. 2003). The number, morphology and distribution of Inka cells within

126 the body can be quite variable between insects of different orders, but they are

127 always in close association with the epitracheal glands, near the spiracles of the

128 tracheal system. Finally, the epiproctodeal glands (EPGs) are another example

129 of endocrine structures specialized in the secretion of peptides associated with

130 molting. In Manduca, these glands are present as a pair of single, multinucleated

131 cells, at the junction of the hindgut and the rectum, in close contact with the

132 proctodeal nerve (Davis et al. 2003). EPGs synthesize a myoinhibitory-like

133 peptide (MIP-like I) that is speculated to shut off ecdysone production by the

134 PGs, at the end of each molt (Davis et al. 2003).

135
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136 Some insect tissues have endocrine functions beside their more obvious primary

137 physiological role(s). For instance, endocrine cells have been detected in the

138 midgut of several insect species by using antibodies recognizing peptidic

139 hormones. Midgut endocrine cells have features of typical secretory cells (e.g.

140 abundant secretory granules, clear cytoplasm; Brown et al. 1985, Neves et al.

141 2003) and have been found to release peptides primarily involved in midgut

142 contraction and other aspects of digestive physiology. Hormones secreted by

143 these cells include allatostatins and allatostatin-like peptides (AS-like,

144 helicostatins in Helicoverpa armigera, (Davey et al. 2005)) and crustacean

145 cardioactive peptide (CCAP, Sakai et al. 2004). Interestingly, both AS and CCAP

146 were originally discovered in functions unrelated to invertebrate midgut

147 physiology: AS as an inhibitor of JH synthesis by the CA (Stay and Tobe 2007)

148 and CCAP as a peptide regulating heartbeat in the crab and ecdysis in insects

149 (Stangier et al. 1987, Žitňan and Adams 2005).

150

151 Evidence also exists to the effect that a few peptidic hormones are produced by

152 the fat body. Claeys et al. (2003) were able to detect messenger RNAs encoding

153 four neuroparsins (NPP1-4) in fat body of the locust Schistocerca gregaria. Very

154 little is known about the function of these neuroparsins, but their expression is

155 stage- and sex-dependent, and is regulated by JH and 20-hydroxyecdysone

156 (Claeys et al. 2006). Rachinsky et al. (2006) also detected low levels of

157 allatotropin, a JH biosynthesis stimulating peptide, in the fat body of Manduca

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158 sexta. In addition, the fat body is the source of uncharacterized growth factors

159 that stimulate the proliferation of gut stem cells (Smagghe et al. 2003).

160

161 The ovaries of many insects constitute a source of ecdysteroids that play

162 important roles in reproductive physiology. In the mosquito Aedes aegypti,

163 ovarian-secreted ecdysone triggers the transcription, in the fat body, of yolk

164 protein precursor genes (e.g. vitellogenin, lipophorin) whose products are then

165 transported back to the ovaries and incorporated into developing eggs (Raikhel

166 et al. 2005). In other species, such as the locust Locusta migratoria, the bulk of

167 ovarian ecdysteroids are stored in the oocyte, where they are believed to play a

168 role in cuticle formation during embryonic development (Lagueux et al. 1984).

169

170 4 Ecdysone and Ecdysone agonists

171 4.1 Ecdysone functions

172 The elucidation of the structure of ecdysone by Butenandt and Karlson in 1954

173 was the major catalyst for research on the physiology of the molting hormone

174 and its role in insect metamorphosis. It soon became apparent that the

175 ecdysone-induced puffs on the giant polytene chromosomes of Chironomus

176 tentans were produced by induction of gene transcription and thus led the way to

177 molecular studies on the mode of action of ecdysone at the genetic level (Clever

178 and Karlson 1960; Karlson 1956; Ashburner et al. 1974). We now know that the

179 biologically active form of ecdysone is 20-hydroxy ecdysone (20E), which acts as

180 the ligand of a heterodimeric receptor system consisting of two nuclear receptors,
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181 the ecdysone receptor (EcR) and ultraspiracle (USP). USP is an allosteric

182 effector for ligand binding by the EcR. 20E binds to the ligand-binding domain of

183 the EcR subunit of the EcR-USP dimer (EcR complex). In turn, this complex

184 binds to specific sequence on the DNA, designated as the “ecdysone response

185 element” (EcRE), located upstream from the gene that is to be activated or

186 repressed (Nordeen et al. 1998). Binding of the 20E-liganded EcR complex to an

187 EcRE causes transactivation of gene transcription. Conversely, the unliganded

188 complex causes repression. The degree of binding affinity appears to correlate

189 with activity. The bipolar transcriptional ability, both repression and activation, is

190 probably aided by corepressors and coactivators, as in vertebrate steroid

191 hormone receptor systems, but details are lacking at present (King-Jones and

192 Thummel 2005).

193

194 Upon reaching a critical body size, the prothoracic glands, in response to the

195 neuropeptide prothoracicotropic hormone (PTTH), produces the prohormone α-

196 ecdysone. α-ecdysone is rapidly converted into the active 20E in tissues such as

197 the fat body, by ecdysone monooxygenase. This hormone plays a central role in

198 the regulation of growth, metamorphosis and reproduction, and participates in a

199 panoply of physiological activities (Palli et al. 2005; Riddiford et al. 2003). The

200 major role of 20E is the regulation of the molting process, which consists of three

201 phases during each instar: (i) cell division and cuticle elaboration during the

202 intermolt phase in the absence of 20E, (ii) digestion of the old cuticle with

203 concomitant synthesis of a new cuticle, triggered by high levels of 20E, and (iii)
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204 ecdysis or shedding of the remnants of the old cuticle after 20E has been cleared

205 from the hemolymph (Retnakaran et al. 2003; Riddiford et al. 2003; Truman

206 2005; Žitňan and Adams 2004).

207

208 In addition to molting, 20E is involved in the metamorphic remodeling required for

209 the larval–pupal and pupal-adult transformation; this process inter alia involves

210 formation of new tissues after the old ones have degenerated as a result of

211 programmed cell death. There is complete replacement of larval fat body and

212 midgut, midgut stem cell differentiation, elimination of larval silk glands and

213 thorough reorganization of the nervous system (Brown et al. 2006; Yin et al.

214 2005; Parthasarathy and Palli 2007; Sekimoto et al. 2006). In adults of some

215 species, 20E takes on a reproductive function in ovarian development and

216 vitellogenesis (Raikhel et al. 2005). In addition, this hormone regulates several

217 unique functions such as muscle reorganization (Lovato et al. 2005), diapause

218 termination (Denlinger et al. 2005), neuropeptide release (Hossain et al. 2006),

219 immune response (Franssens et al. 2006), chromatin remodeling (Zraly et al.

220 2006), DNA amplification for increased protein production for pupation (Foulk et

221 al. 2006), non-genomic effects such as electrochemical changes in membranes

222 (Schlattner et al. 2006), epithelial morphogenesis in imaginal discs (Hammonds

223 and Fristrom 2006), and many others illustrating the pleiotropic nature of 20E.

224

225 Having described the various functions of 20E and the ways in which it interacts

226 with a receptor complex to modulate gene expression, we can examine how the
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227 various synthetic ecdysone agonists function and how they differ from the natural

228 hormone to permit exploitation of the analogs as narrow spectrum control

229 products.

230

231 4.2 20E regulation of molting

232 The major function of 20E is the regulation of the molting process in insects. In

233 the Lepidoptera, this hormone appears as a single peak during the middle of

234 each larval stadium, except for the last one where there are two peaks; the

235 earlier, smaller peak is the commitment peak which triggers the reprogramming

236 towards pupal development, followed by the larger molt peak. The presence of

237 high titers of JH during the larval 20E peak defines the molt as larval whereas the

238 absence of JH during the commitment peak results in pupal transformation,

239 which involves the turning-off of larval genes and turning-on of pupal genes. The

240 physiological and molecular basis of the switch has been elegantly elucidated by

241 Riddiford and her associates showing for instance that the Broad Complex gene

242 (BR-C) is the transcription factor that specifies the pupal cuticle (Riddiford et al.

243 2003). Thus, 20E can both repress and activate genes and its effects can be

244 modulated by JH.

245

246 4.3 Steroidal and non-steroidal analogs of ecdysone

247 Various plant ecdysteroids (phytoecdysones) and synthetic steroids have been

248 assessed for their ability to act as agonists or antagonists of ecdysone and for

249 their insecticidal activity, but with limited success. Besides being difficult to
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250 synthesize, steroids are large molecules lacking contact activity and are prone to

251 oxidative breakdown. Over 200 plant ecdysteroids have been studied and none

252 of them have shown high potential as control products (Dinan 2001). Similarly,

253 several synthetic steroids were tested for biological activity, but none proved very

254 effective (Robbins et al. 1970). A few steroid analogs such as cucurbitacins and

255 brassinosteroids were tested for antagonistic activity but, again, the effects were

256 weak (Dinan et al. 1997; Charrois et al. 1996). More recently, however, non-

257 steroidal ecdysone agonists such as tetrahydroquinoline compounds showed

258 some activity, especially against mosquitoes (Palli et al. 2005). By far the most

259 effective compounds among the non-steroidal ecdysone agonists are the

260 diacylhydrazines, serendipitously discovered in the Rohm and Haas (RH)

261 laboratories at Spring House, PA, USA (Hsu 1991). Initial optimization following

262 discovery of the chemistry led to a first promising candidate, RH-5849, but soon

263 three other compounds displaying greater activity were synthesized: (i) RH-5992

264 (tebufenozide) was active against several lepidopterans and was registered

265 under the commercial names Mimic®, Confirm®, and Romdan®; (ii) RH-2485

266 (Methoxyfenoxide) was several times more active than RH-5992 on

267 lepidopterans, and was marketed under the names Intrepid®, Runner®, Prodigy®

268 and Falcon®; (iii) RH-0345 or Halofenozide was active against Coleopterans in

269 addition to Lepidopterans and was registered under the name Mach 2®. Rohm

270 and Haas has since sold all these compounds to Dow Agrosciences,

271 Indianapolis, IN. More recently Nippon Kayaku, Saitane and Sankyo, Ibaraki,

272 from Japan, have come up with a new diacylhydrazine which they have named
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273 ANS-118 / CM-001 or chromafenozide, which is active against lepidopterans and

274 is registered under the names Matric® and Killat® (Nakagawa 2005) (Fig. 1).

275

276 4.4 Mode of action of diacylhydrazines

277 Diacylhydrazines are functionally similar to 20E and bind to the EcR receptor

278 complex as ligands (Nakagawa 2005). While they mimic the natural hormone in

279 triggering the molting process, the latter is never completed. The treated larva

280 shows all the initial signs of molting, such as feeding cessation, head-capsule

281 slippage (with the new head capsule remaining untanned), and loosening of the

282 cuticle due to apolysis. However, the similarity between 20E action and

283 diacylhydrazines stops at this juncture. There is no ecdysis, resumption of

284 feeding, or sclerotization and darkening of the new head capsule. Rather, the

285 larva is in a state of developmental arrest or suspended animation and eventually

286 dies of starvation and desiccation.

287

288 The effects of the diacylhydrazines on the external morphology, histological

289 changes (macro and ultra) and gene expression have been well studied in

290 several insect species, such as the spruce budworm, Choristoneura fumiferana

291 (Retnakaran et al. 1997b). When sixth instar spruce budworm larvae are fed 70

292 µg of RH-5992, they stop feeding within 6 h and remain quiescent. At 24 h post

293 feeding, the head capsule slips, revealing a white, untanned, fragile new head

294 capsule, and the body wall appears loose, suggesting that apolysis has occurred.

295 The new head capsule is very thin and wrinkled, and is buckled inward. A time-
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296 course study of ultrastructural changes induced by RH-5992 revealed

297 hypertrophy of the Golgi and large ecdysial droplets in the cuticle 3 h post

298 feeding. By 12 h, the old cuticle was partially digested by the ecdysial fluid, while

299 synthesis of a new cuticle had begun, with a clear ecdysial space in between the

300 old and the new cuticle. Between 24 h and 48 h, development had come to a

301 stand still, with the larva bearing remnants of the old cuticle and a thin new

302 cuticle with no endocuticular lamellaea, and ecdysis did not take place.

303

304 The physiological effects of diacylhydrazines are reflected at the molecular level,

305 where they derail the fine order of genes expressed and repressed by ecdysone

306 in the molting cycle. The three phases of the ecdysone peak have distinct gene

307 expression patterns (Fig. 2). For instance, during the first phase or growth phase,

308 genes such as LCP-14 (encoding a larval cuticular protein of 14 kDa in size) are

309 expressed in the absence of 20E, and constitute growth genes (Hiruma et al.

310 1997). During the second or molt initiation phase, genes such as MHR3

311 (Manduca hormone receptor 3) are expressed in the presence of 20E (Langelan

312 et al. 2000). During the third phase, a set of genes such as DDC (encoding the

313 dopadecarboxylase enzyme) are expressed after the transient exposure to, and

314 subsequent decline of, 20E (Hiruma et al. 1995). Using an in vitro assay with

315 preparations of the integument, the effect of RH-5992 on the expression of LCP-

316 14, MHR3 and DDC was investigated. In a manner similar to that observed with

317 20E, this agonist induced the expression of MHR3 and repressed the expression

318 of LCP-14 and DDC and the effect persisted over an extended period of time
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319 (Retnakaran et al. 1995). Unlike 20E, RH-5992 persists in the epidermis and is

320 not cleared from the system; as a result, DDC expression is persistently

321 suppressed, and so are ecdysis, sclerotization and resumption of feeding.

322

323 In spruce budworm, the high biological stability of RH-5992 also implies that the

324 timing of its application can be flexible. For instance, when 3rd -5th instars are

325 exposed to RH-5992 before the 20E peak, the characteristic signs of molt

326 induction are observed. If larvae are treated with the agonist after the 20E peak,

327 there are no effects on that instar. However, owing to its stability, the material is

328 carried over to the next instar, wherein it manifests its effect (Palli et al. 1995).

329 This carry-over effect was also dramatically shown in the diapausing second

330 instar. When the first instar larvae are allowed to graze on a RH-5992-coated

331 surface, they consume small amounts of the material, mostly after the rise of the

332 20E titer, and are therefore unaffected. They spin hibernacula and molt into 2nd

333 instars, and are normally poised to go into diapause, as a result of a lack of 20E.

334 However, since the RH-5992 has been carried over from the 1st instar,

335 precocious molting is initiated in the second instar larvae, which die in this state

336 within the hibernacula (Doucet et al. 2007). The latter examples illustrate the

337 fundamental difference in the mode of action of molt-inducing insecticides and

338 classical neurotoxic insecticides: while diacylhydrazines may be slow acting, they

339 can in some circumstances target instars that might be difficult to control by

340 contact or systemic, fast-acting compounds.

341
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342 4.5 Diacylhydrazine effects on insects

343 As one would expect, diacylhydrazine agonists interfere with many of the

344 pleiotropic actions of 20E, with adverse consequences for most of them. Only a

345 few studies on the effects of diacylhydrazines on functions other than molting

346 have been conducted, primarily on adult reproduction and diapause. Ovarian

347 development and vitellogenesis are regulated by 20E in some insects, and

348 treatment with diacylhydrazines often results in reduced fecundity and impaired

349 fertility (Farinas et al. 1999). The ecdysone agonists appear to affect sperm

350 counts as well as their movement through the reproductive tract (Seth et al.

351 2004). Diapause in immature stages seems to result from a lack of 20E but can

352 be prevented or broken by nonsteroidal ecdysone agonists (Doucet et al. 2007;

353 Denlinger et al. 2005).

354

355 4.6 Diacylhydrazines as pest-control products

356 The non-steroidal ecdysone agonists have been tested on a variety of insects

357 with varying degrees of success. The activity of different diacylhydrazines on a

358 representative list of insects is shown in Table 2. The Mimic® formulation of RH-

359 5992 (tebufenozide) works very well against the spruce budworm, a serious pest

360 of the balsam fir (Abies balsamea) and white spruce (Picea glauca) in the boreal

361 forests of North America (Retnakaran et al. 1997a; Cadogan et al. 1998).

362 Laboratory studies show that RH- 2485 (methoxyfenozide) is about 10 times

363 more active than tebufenozide (Sundaram et al. 1998a). The grape berrymoth,

364 Lobesia botrana, is very sensitive to methoxyfenoxide, which shows excellent

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365 potential for control of this pest. Older larvae are more susceptible than younger

366 ones, and treated adults display reduced fecundity and fertility (Sáenz-de-

367 Cabezón Irigaray et al. 2005).

368

369 In some instances the molt-inducing activity of diacylhydrazines is very weak,

370 and thus this class of compounds cannot be retained as an effective control

371 solution. The obliquebanded leafroller, C. rosaceana, shows relatively low

372 susceptibility to tebufenozide (RH-5992) and methoxyfenozide (RH-2485)

373 (Ahmad 2002). In the case of the white-marked tussock moth, treatment with RH-

374 5992 induces head capsule slippage but the larva, after remaining quiescent for

375 a week to 10 days, becomes active and molts into the next instar (Retnakaran et

376 al. 2003). In vitro and in vivo data tend to indicate that the potency of

377 diacylhydrazines is related to absorption and excretion rates in a given species

378 (Smagghe et al. 2001, Retnakaran et al. 2001). Other cases of low susceptibility

379 include the coddling moth, Cydia pomonella (Sauphanor and Bouvier 1995) and

380 the green headed leafroller, Planotortrix octo, from New Zealand (Wearing 1998),

381 to name a few. This only goes to show that these compounds, while very

382 effective on some species, have their own limitations and are not a general cure-

383 all.

384

385 4.8 Ecdysone agonists as candidates for integrated pest management (IPM)

386 Over the years pest management methods have progressively evolved towards

387 an ecologically based systems approach, combining biological, cultural, physical

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388 and chemical tools in a way that minimizes economic, health and environmental

389 risks. Such an IPM approach is a dynamic process constantly aiming at

390 maximizing target specificity and minimizing environmental side effects.

391 Ecdysone agonists have so far been shown to be insect-specific and, among the

392 various agonists, some are far more active on one group of insects than others.

393 In general, their mammalian toxicity is very low; the acute oral toxicity for rat and

394 mouse is >5000 mg/kg for tebufenozide, methoxyfenozide and chromafenozide,

395 and >2850 mg/kg for halofenozide. These compounds have no detectable effects

396 on reproduction, and are negative in Ames mutation assay (Dhadialla et al.

397 2005). These nonsteroidal ecdysone agonists are also relatively safe for the

398 environment and do not have any negative impact on forest-litter-dwelling

399 organisms such as earthworms and Collembola (Addison 1996).

400 Macroinvertebrates in freshwater ponds are unaffected by tebufenozide

401 (Kreutwizer et al.1994). Tebufenozide and methoxyfenozide have been shown to

402 have little effect on bumblebees (Mommaerts et al. 2006). A summary of safety

403 to parasites, predators and pollinators is provided by the National Registration

404 Authority of Australia (Anonymous 2002). Tebufenozide was also found to have

405 little impact on a generalist predator, the lacewing Chrysoperla carnea (Medina et

406 al. 2003). The persistence, breakdown and catabolism of tebufenozide has been

407 well studied. In conifer needles and forest litter some persistence was observed,

408 but well within tolerance levels (Sundaram et al. 1996). Thus the environmental

409 safety and narrow spectrum of activity of these nonsteroidal ecdysone agonists

410 make them a valuable addition to the arsenal of candidates for IPM.
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411

412 5 Juvenile Hormones

413 5.1 Chemical nature, biosynthesis and functions

414 The juvenile hormones (JHs) form a family of lipophilic, sesquiterenoid molecules

415 with epoxide and methyl-ester functionalities. All are derived from the mevalonate

416 pathway intermediate, farnesyl diphosphate (FPP), or from one of its ethyl-

417 branched homologs. The majority of insects produce only one chemical form of

418 JH, JH III (C-16), but the Lepidoptera produce four additional, ethyl-substituted

419 JHs, [JH 0 (C-19), JH I (C-18), JH II (C-17) and 4-methyl JH I (C-19)] and the

420 Diptera produce a bis-epoxy form of JH III (JH-B3; Fig. 3). These hormones are

421 all produced de novo by the CA. JH biosynthesis begins with the condensation of

422 three units of acetyl-CoA (for JH III and JH-B3) or two units of acetyl-CoA and

423 one of propionyl CoA (lepidopteran JHs), leading to the formation of the isoprene

424 (C-5) and homo-isoprene (C-6) building blocks of FPP and ethyl-substituted

425 FPPs. Unlike the enzymatic steps that are responsible for FPP formation, which

426 are common to most living organisms, those that convert FPP into JH are

427 specific to insects.

428

429 Although JH plays multiple roles in insects, it owes its name to its juvenilizing

430 effects during larval moults: the presence of elevated JH titres during ecdysone

431 secretion represses the expression of metamorphic genes, thus maintaining the

432 insect in a juvenile (larval) state. Shortly after the final larval moult, JH

433 biosynthesis ceases and a JH-specific catabolic enzyme known as JH esterase

 20

434 (JHE) is secreted, which leads to a rapid decline in the JH titre. Under these

435 conditions, ecdysone triggers the metamorphic program leading to the pupal

436 moult. In adult insects, JH is a gonadotropin and is best known for its stimulatory

437 effects on vitellogenesis, inducing the production of vitellogenin by the fat body

438 and/or its uptake by developing oocytes. In males, JH has been implicated in the

439 production of accessory sex gland secretions and in the control of courtship

440 behaviour. JH has also been shown to be involved in the regulation of various

441 other processes, including embryogenesis, migration, cast differentiation,

442 polyphenism and reproductive diapause (Cusson 2004). Although many of the

443 roles played by JH have been well characterized, its mode of action at the

444 molecular level remains unclear. Several proteins have been tentatively identified

445 as JH receptors, but conclusive evidence regarding their role as receptors is still

446 lacking (see Palli and Cusson 2007, for a more complete account of recent work

447 in this area). The presence of a JH response element (JHRE) in the promoter

448 region of the JH-responsive JHE gene suggests that JH can act through a

449 nuclear receptor. Some nuclear proteins do, indeed, bind to this JHRE, but

450 binding requires that the proteins be dephosphorylated, a process that appears

451 to be induced by JH (Kethidi et al. 2006).

452

453 5.2 JH analogs and IPM

454 Following the initial isolation of JH, Caroll Williams (1956) predicted the dawn of

455 JH-based insecticides. It was surmised that synthetic JH-like molecules would

456 fatally interfere with JH functions and that insects would not likely develop
 21

457 resistance against such compounds (Williams 1967). Many JH analogs

458 (molecules with JH effects, with or without a JH-like terpenoid structure) were

459 subsequently designed, synthesized and assayed for insecticidal activity (Sláma

460 et al. 1974). Some of them (e.g., methoprene, hydroprene, fenoxycarb,

461 pyriproxifen, diofenolan; Fig. 4) were found to be effective against certain pests

462 and have since enjoyed commercial success, particularly for the control of

463 insects that are injurious in the adult stage (e.g., mosquitoes, fleas, whiteflies,

464 etc.), but their efficacy against phytophagous larval insects (e.g., caterpillars) has

465 often proven to be limited, largely because the analogs interfere with

466 metamorphosis, once larval feeding has ended (see Dhahialla et al. 1998; 2005

467 for reviews).

468

469 5.3 Anti-JHs

470 It has long been recognized that a strategy involving the induction of precocious

471 metamorphosis through the inhibition of JH biosynthesis would be better suited to

472 the control of immature phytophagous insects than one involving the disruption of

473 metamorphosis with JH analogs (Cusson and Palli 2000). Although a number of

474 naturally occurring and synthetic inhibitors of JH biosynthesis have been

475 evaluated for their ability to trigger precocious metamorphosis, none have yet

476 been developed commercially. The “precocenes”, isolated from the bedding plant

477 Ageratum houstonianum, originally generated much hope and enthusiasm as

478 they caused premature metamorphosis in the heteropteran species Oncopeltus

479 fasciatus. However, these compounds proved to be ineffective against

 22

480 holometabolous insects. Inhibitors of mevalonate pathway enzymes known to

481 have hypocholesterolemic activity in mammals were also tested on insects;

482 examples include the fungal metabolite compactin, an inhibitor of HMG-CoA

483 reductase (Monger et al. 1982), and the fluorinated mevalonate analog,

484 fluoromevalonate, an inhibitor of enzymes involved in the processing of

485 mevalonate (Quistad et al. 1981), both of which were found to induce precocious

486 metamorphosis in lepidopteran larvae, but required high doses and/or repeated

487 applications. Allylic alcohol derivatives of dimethylallyl diphosphate, the C-5 chain

488 initiator for FPP production by FPP synthase (FPPS), provided similar results

489 (Quistad et al. 1985). Inhibitors of later, insect-specific steps of JH biosynthesis,

490 such as formation of the methyl ester moiety and epoxidation, were also

491 examined for their ability to block JH biosynthesis and induce precocious

492 metamorphosis. Brevioxime, a compound with a sesquiterpene-like structure

493 isolated from the fungus Penecillium brevicompactum, was shown to inhibit in

494 vitro JH biosynthesis by the CA of Locusta migratoria (Castillo et al. 1998).

495 Similarly, the synthetic 1,5-disubstituted imidazole, KK-42, an inhibitor of the P-

496 450-linked enzyme that epoxidizes the JH precursor methyl farnesoate (MF),

497 inhibited in vitro JH biosynthesis by the CA of L. migratoria (Castillo et al. 1998)

498 and the cockroach Diploptera punctata (Pratt et al. 1990). Again, concentrations

499 of the compounds required to achieve inhibition were relatively high.

500

501 Recent developments in the area of insect genomics have led to the cloning and

502 characterization of enzymes specific to JH biosynthesis, including JH acid methyl

 23

503 transferase from B. mori (Shinoda and Itoyama 2003) and MF epoxydase from D.

504 punctata (Helvig et al. 2004). The ability to produce these enzymes in

505 heterologous expression systems should lead to substantial improvements in our

506 capacity to assess their three-dimentional structures and design potent and

507 highly specific inhibitors that could be used as anti-JH insecticides. In addition, to

508 the above proteins, several mevalonate pathway-specific enzymes have been

509 cloned from various species of insects. Although these may not provide the most

510 suitable target sites for insecticide development, given that they are found in

511 most living organisms, the lepidopteran homolog of at least one of them, FPPS,

512 appears to display significant structural singularities believed to be instrumental

513 in the biosynthesis of the ethyl-substituted JHs (Cusson et al. 2006; Sen et al.

514 2006); this enzyme may thus prove to be a suitable target for the design of

515 Lepidoptera-specific inhibitiors (see Palli and Cusson 2007 for a more in-depth

516 review).

517

518 Identification of a JH receptor is perhaps the most promising avenue for the

519 development of highly effective anti-JH compounds. As indicated above,

520 however, this receptor has so far remained elusive, despite sustained and

521 continuing research efforts. Once a receptor has been isolated, it should become

522 possible to design cell-based high-throughput assays for the screening of

523 potential JH antagonists that are effective at the target tissue level (Minakuchi

524 and Riddiford 2006; Palli and Cusson 2007).

525
 24

526

527 6 Future directions and conclusion

528 Insecticides targeting endocrine functions have a young history, and it remains to

529 be seen at what rate new products will be successfully brought on the market.

530 Agonists and antagonists of peptidic hormones are currently being investigated,

531 such as cyclic backbone peptides inhibiting the action of PBAN (Altstein 2004).

532 Other suitable targets for peptidomimetics include receptors for PTTH and

533 ecdysis-related peptides (Palli and Cusson 2007). An important factor in the

534 development of new insecticidal compounds will be the identification of new

535 target sites ranging from hormone receptors to hormone biosynthetic and

536 degradative enzymes. Target site identification has been greatly helped by the

537 complete sequencing of insect genomes, including species of economic

538 importance [the honeybee (Honeybee Genome Sequencing Consortium (2006));

539 Bombyx mori (Xia et al. 2004)] as well as vectors of human diseases (Anopheles

540 gambiae, Holt et al. 2002). Thus the full repertoire of peptidic hormones, and

541 receptors for both peptidic and non-peptidic hormones, can eventually be known

542 for a given sequenced insect genome by mining for genes with endocrine

543 functions. This is exemplified by the discovery of 56 G-protein-coupled receptors

544 (GPCRs) for neurohormones and biogenic amines in the honeybee genome

545 (Hauser et al. 2006). Similarly, Drosophila genomics has made possible the

546 identification of the suite of enzymes controlling ecdysone biosynthesis in this

547 species (Gilbert and Warren 2005). With large-scale DNA sequencing becoming
 25

548 more affordable, additional fully-sequenced insect genomes should become

549 available in the near future, including those of agricultural pests.

550

551 This large-scale gene identification effort will feed into the strategies currently

552 used in agrochemistry to synthesize and screen compounds against target sites

553 (i.e., proteins). High-throughput instrumentation now allows the screening of 500

554 to 1000 compounds per day in in vitro assays on a given target (Allenza &

555 Eldridge 2007). In vitro assays, while allowing a higher throughput than whole-

556 insect assays, are still trial-and-error operations. A target protein needs to be

557 expressed in sufficient quantities and in the proper conformation to retain its in

558 vivo functions in an in vitro context. Furthermore, some hormone receptors

559 require two or more subunits to be functional (e.g., ecdysone receptor), thus

560 increasing the complexity and cost of some in vitro assays (Allenza & Eldridge

561 2007). To avoid these pitfalls, the discovery of novel hormone-based insecticidal

562 compounds will require an intimate knowledge of the target at the molecular,

563 cellular and whole-organism levels.

564

565 IPM specialists will likely witness the introduction of additional, endocrine-based

566 control products in the foreseeable future. New approaches in the way endocrine

567 disruption is delivered are also in development, for instance by using genetically

568 modified plants or microorganisms that interfere with hormone action (Palli and

569 Cusson 2007). By exploiting the diversity and specificity of insect hormone
 26

570 systems, these new applications methods should bring even more

571 environmentally attractive control options for IPM.

572

573

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1107

1108 Žitňan, D., and Adams M.E. (2005). Neuroendocrine regulation of insect ecdysis.

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1119

1120

1121

1122

1123

1124

1125

1126

1127

1128

1129
 51

1130 Figure captions

1131

1132 Fig 1. Chemical structures of Insect molting hormones, a phytoecdysteroid and

1133 five of the most promising diacylhydrazine ecdysone agonists.

1134

1135 Fig 2. Mode of action of 20E and Tebufenozide (RH-5992). The 20E peak in the

1136 penultimate lepidopteran larval instar is schematically represented delineating

1137 the different phases of the normal molting process (A) and contrasting it with the

1138 effect of RH-5992 (B).

1139

1140 Fig 3. Structures of Juvenile Hormones (JHs) produced by insects.

1141

1142 Fig 4. Structures of four JH analogs commercially available.

1143

1144

1145

1146

1147

1148

1149

1150

1151

1152
 Table 1: Survey of selected insect hormones, their functions, structures, sites of synthesis, target tissues and receptors.
Hormone
Amino acid sequence length
Juvenile hormone
(e.g. JH III)
Ecdysone
Octopamine
Prostaglandins
(e.g. PGE2)
Diapause hormone (DH)
Prothoracicotropic
104-125 aa protein
hormone (PTTH)
Eclosion hormone (EH)
Adipokinetic hormone
(AKH)
Pre-ecdysis triggering
hormone (PETH)
Ecdysis-triggering
hormone (ETH)
Allatostatins (including
myoinhibitory peptides
(MIPs) and helicostatins)
Crustacean cardioactive
peptide (CCAP)
Neuroparsins 78 or 83 aa proteins
Pheromone biosynthesis
activating neuropeptide
(PBAN)
Structure/
Primary role Site of synthesis Target tissue(s) Receptor References
Maintenance of immature Epidermis, fat body,
Corpora allata (CA) Unknown Goodman & Granger 2005
characters many others
Ecdysone Receptor-
Prothoracic glands, Epidermis, Henrich 2005
Ultraspircale
Initiation of the molting cycle ovaries in some many others during Lafont et al. 2005
heterodimer (EcR-
species metamorphosis
USP)
Regulation of locomotor and
Central nervous system, GTP-binding protein
non-locomotor behaviors, Peripheral and central Balfanz et al. 2005
fat body, female coupled receptors
circadian rhythm and stress nervous systems Fussnecker et al. 2006
reproductive system (GPCRs)
response
Hemocytes,
Modulation of insect immunity, Fat body, hemocytes, Uncharacterized
terminal abdominal Stanley 2006
release of egg-laying behavior midgut GPCR(s)
ganglion
Suboesophagial DHR Sato et al. 1993
24-25 aa peptide Promotion of diapause entry Ovaries
ganglion (GPCR) Homma et al. 2006
Stimulation of ecdysone Brain
Prothoracic gland Unknown Rybczynski 2005
synthesis neurosecretory cells
CCAP-producing neurons
of the ventral nerve cord,
Brain ventral medial
61-73 aa protein Activation of ecdysis behavior Inka cells, non-neuronal Unknown Žitňan & Adams 2005
neurosecretory cells
cells of abdominal
transverse nerves
Mobilization of lipid stores for
8-11 aa peptide Corpus cardiacum Fat body AKHR (GPCR) Schooley et al. 2005
insect flight
Activation of preecdysis I Žitňan & Adams 2005
11 aa peptide Inka cells Central nervous system Unknown
behavior Kim et al. 2006
Activation of preecdysis II and ETHR Žitňan & Adams 2005
15-26 aa peptide Inka cells Central nervous system
ecdysis behaviors (GPCR) Kim et al. 2006
Pleiotropic: Inhibition of
ecdysone production, inhibition Central nervous system, Prothoracic glands,
DAR-1, DAR-2, and
8-18 aa peptides of JH production and/or epiproctodeal glands, corpora allata, Stay & Tobe 2007
others (GPCRs)
inhibition of visceral muscle gut gut
contraction
Activation of motor pattern CG6111 (GPCR,
Heart,
9 aa peptide during ecdysis, stimulation of Central nervous system isolated from Žitňan & Adams 2005
Central nervous system
heart contractions Drosophila)
Pleiotropic: anti-JH effects, Central nervous system,
Fat body, Raikhel et al. 2005
antidiuretic, hypertrehalosemic fat body, male Unknown
others tissues (?) Claeys et al. 2003
and hyperlipemic effects accessory glands, testis
Stimulation of pheromone Suboesophagial
30-35 aa peptide Pheromone gland PBANR (GPCR) Blomquist et al. 2005
synthesis ganglion
Table 2. A representative list of insect pests that have been tested with nonsteroidal ecdysone
agonists (diacylhydrazines) for control.

Common name Scientific name Ecdysone agonist Control Reference

Cotton leaf worm Spodoptera littoralis Methoxyfenozide Very effective Carton et al. 2000

Corn ear worm Helicoverpa zea Tebufenozide Very effective Chandler et al. 1992

Spodoptera
Fall army worm Tebufenozide Very effective Chandler et al. 1992
frugiperda

Leaf skeletonizer Uraba lugens Tebufenozide Poor control Mansfield et al. 2006

Tebufenozide/
Codling moth Cydia pomonella Resistant strains Loriatti et al. 2005
Methoxyfenozide
Choristoneura
Spruce budworm Tebufenozide Very effective Cadogan et al. 2005
fumiferana
Nantucket pine tip
Rhyacionia frustrana Tebufenozide Excellent control Philip et al. 2005
moth
Tebufenozide/ Excellent control at egg
Grape berry moth Endopiza viteana Isaacs et al. 2005
Methoxyfenozide hatch
European corn Safest control, protects
Ostrinia nubilalis Tebufenozide Ebaid 2004
borer predators
Wawrzyniak 2004
Cabbage butterfly Pieris brassicae Methoxyfenozide Excellent control on L3

Oriental fruit moth Grapholita molesta Methoxyfenozide Good control Arioliet et al. 2004

Tebufenozide/
Grape wine moth Lobesia botrana Good control Charmillot et al. 2003
Methoxyfenozide

Autumn gum moth Mnesampala privata Tebufenozide Good control Elek et al. 2003

Cotton bollworm Helicoverpa armigera Methoxyfenozide Good potential Zhao-Xiao et al. 2003

Common cutworm Spodoptera litura Chromafenozide Good activity Yanagi et al. 2006

Cabbage army
Mamestra brassicae Chromafenozide Good activity Yanagi et al. 2006
worm
Mediterranean flour
Ephestia kuhniella Tebufenozide Very effective Hami et al. 2005
moth
Formosan
Coptotermes
subterranean Halofenozide Significant reduction Raina et al. 2003.
formosanus
termite

Japanese beetle Popillia japonica Halofenozide As good as imidacloprid Mannion et al. 2001

Colorado potato Leptinotarsa

Halofenozide Very effective Carton et al. 2000
beetle decemlineata
Figure 1

A) OH OH B) OH

20
OH OH
HO HO
OH OH
HO HO
O O
20-hydroxyecdysone α-ecdysone
(active molting hormone) (prohormone)

OH OH
C) D)
H
C N N C
HO
OH O O
HO
O
RH-5849
Ponasterone
(orginial diacylhydrazine)
(phytoecdysteroid)

E) F)
CH3 CH3 CH3 OCH3
H H
C N N C CH2CH3 C N N C

O O O O
CH3 CH3

Tebufenozide Methoxyfenozide
(RH-5992; Mimic®; (RH-2485; Intrepid®; Runner®;
Confirm®; Romdan®) Prodigy®; Falcon®)

G) H)
CH3
H H
C N N C Cl C N N C O

O O O O
CH3

Halofenozide Chromafenozide
(RH-0345; Mach 2®) (ANS-118; Matric®; Killat®)
Figure 2
Figure 3
Figure 4