The molecular site of action of juvenile hormone and juvenile hormone insecticides during metamorphosis: how these compounds kill insects

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Journal of Insect Physiology 50 (2004) 111–121
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Review

The molecular site of action of juvenile hormone and juvenilehormone insecticides during metamorphosis: how these

compounds kill insects

Thomas G. Wilson �

Department of Entomology, 400 Aronoff Laboratory, Ohio State University, 318 West 12th Avenue, Columbus, OH 43210, USA

Received 11 September 2003; received in revised form 4 December 2003; accepted 5 December 2003

Abstract

Studies in a variety of insects during the past four decades has deepened our understanding of juvenile hormone (JH) physi-ology, but how this hormone works at the molecular level remains elusive. Similarly, the mechanism of toxicity of JH analogueinsecticides is still in question. There is much evidence from laboratory usage that JHAs act as JH agonists and generally showthe highest toxicity when applied at the onset of metamorphosis. A physiological basis for the toxicity and morphogenetic effectshas been suggested by recent work linking these effects with interference with the expression or action of certain genes, parti-cularly the Broad-Complex (BR-C) transcription factor gene, that direct metamorphic change. Misexpressed BR-C then leads toimproper expression of one or more downstream effector genes controlled by BR-C gene products, resulting in abnormal develop-mental and physiological changes that disrupt metamorphosis. Therefore, JH is a necessary molecule at certain times in insectdevelopment but becomes toxic when present during metamorphosis.# 2003 Elsevier Ltd. All rights reserved.

Keywords: Broad-Complex; Insect growth regulator; JH analogue; Insecticide toxicology; Methoprene

1. Introduction

The science of entomology, like other branches ofbiology, is being researched in considerable detailtoday. To unlock the secrets of insects, other dis-ciplines such as molecular biology and genetics havebeen called upon as never before. Some of us feel thatour focus has become hopelessly compartmentalized aswe strive to keep up with both our specialty and at thesame time, the field as a whole. Therefore, it is a happyoccasion when researchers from separate subdisciplinesin entomology realize that they are pursuing similargoals. In this article I review results from the intertwin-ing of efforts by insect physiologists to understand themolecular action of juvenile hormone (JH) with thoseby economic entomologists to understand the basis forthe toxicity of juvenile hormone analog (JHA) insecti-cides. Results from each effort have recently allowed

conclusions to be drawn that elucidate both final goals,although not to a level of satisfaction that permitseither group to rest on their laurels.

The intersection of these research goals owes its ori-gin to the methodology underlying insecticide develop-ment. An insecticide is toxic because it interacts with atarget macromolecule that is usually vital to the insect,and this interaction inactivates the macromolecule. Forexample, organophosphate or carbamate insecticidesbind acetylcholinesterase at the neuromuscular junctionof an insect, but unlike the substrate acetylcholine thatthese compounds mimic, they cause the enzyme to bephosphorylated or carbamylated, thereby rendering itinactive (Corbett, 1974; O’Brien, 1976). Death of theinsect can then ensue from faulty nerve impulse trans-mission. Novel insecticides are identified based on oneof several search strategies: (1) Numerous chemicals arescreened for toxicity by trial and error; a specific targetmacromolecule is not a component of the screen. Thiswas the usual methodology of earlier times when insec-ticides were discovered perhaps more by chance than

112 T.G. Wilson / Journal of Insect Physiology 50 (2004) 111–121

by systematic effort. If a particular chemical identifiedin this manner subsequently proves to be a successfulinsecticide, then identification of the target macromol-ecule for this insecticide becomes a later goal, oftendeceptively difficult, in the research program. Forexample, the insecticide cyromazine was discovered tobe effective against certain insect pests (Hart et al.,1982), but despite efforts, the mode of toxicity remainsunknown (Bel et al., 2000). (2) A previously identifiedinsecticide of proven success can be targeted in a searchfor more efficacious chemical analogs, building on suc-cess from the earlier compound. Synthetic pyrethroidshave been developed following the demonstrated insec-ticidal action of pyrethrum, a natural product, andpyrethroids have proven to be very effective. (3) Aphysiology found only in insects and closely relatedanimals, such as the presence of a chitinous exo-skeleton, can be used for the development of targetedinsecticides. This strategy is used currently in searchesfor novel, effective ‘‘biorational’’ insecticides. The lastsearch mode can result in the discovery of insecticidesthat are very nontoxic to vertebrates, such as chitinsynthesis inhibitors and JHAs (Retnakaran et al.,1985). The latter of course mimic JH, an insect hor-mone not found in vertebrates. This latter class ofinsecticides comprises a focus of this article.

2. Juvenile hormone

First, I will briefly summarize pertinent aspects ofJH endocrinology that have underscored a need for abetter understanding of JH action at the molecularlevel. A more comprehensive review, including histori-cal perspectives, has recently appeared (Gilbert et al.,2000). JH, a sesquiterpenoid molecule synthesized andsecreted by the corpus allatum, appears to be unique toinsects. It has not been detected in related arthropodssuch as crustaceans (Laufer et al., 1992) or ticks (Neeseet al., 2000). The existence of the hormone was hypo-thesized based on early physiological experiments(Wigglesworth, 1936), the chemical nature of the hor-mone was suggested following identification of JHactivity as farnesol derivatives in Tenebrio molitorexcrement (Schmialek, 1961), and in the late 1960sthe first of several JH homologs was chemically ident-ified (Roller et al., 1967; Meyer et al., 1970). Mostinsects have JHIII (epoxy farnesoic acid methyl ester,(Judy et al., 1973)) (Fig. 1), lepidopteran insects haveadditional similar molecules termed JHI, JHII, andJH0 that differ by ethyl instead of methyl sidechains(Schooley et al., 1984), and cyclorrhaphous dipteraninsect corpus allatal glands can synthesize JHIII bisep-oxide in culture (Richard et al., 1989). Recent work hasshown that JH acid, long thought to be merely aninactive JH metabolite, can act in a hormonal role in

certain tissues of Manduca sexta (Ismail et al., 1998).Even the basics of JH endocrinology are by no meanscompletely described.

The hormone is well known to insect physiologists tobe involved in a rich array of roles in insects, includingdevelopment, reproduction, behavior, pheromone pro-duction, adult diapause, and morph and caste determi-nation (Denlinger, 1985; Kerkut and Gilbert, 1985;Riddiford, 1985; Bownes, 1989; Nijhout and Wheeler,1982; Riddiford, 1994; Riddiford, 1996; Wyatt andDavey, 1996; Tillman et al., 1999; Sullivan et al., 2000;Tanaka, 2001; Moczek and Nijhout, 2002). The best-known and probably the most intriguing role is tomaintain the status of ecdysone-driven larval or nym-phal molts, thus preventing premature metamorphosis(Wigglesworth, 1934). During metamorphosis the JHtiter is low-to-absent; JH then returns to most, perhapsall, adult insects to control various aspects of repro-duction, especially oocyte development, vitellogeninproduction, and female receptivity (Wyatt and Davey,1996). JH involvement in a particular role can be veryinsect-specific. For example, vitellogenin biosynthesishas been demonstrated to be under JH control in theLocusta migratoria (Dhadialla et al., 1987) but not inthe Indian meal moth, Plodia interpunctella (Shirk et al.,1990). Hormone specificity can extend down to tissuespecificity in a particular species: in Drosophila melano-gaster vitellogenin biosynthesis appears to be stronglymediated by JH in ovarian follicle cells but weakly inadult fat body cells (Soller et al., 1999).

Many of these roles are intertwined with ecdysoneaction. The role of JH in modulating molts involves aneffect on ecdysone action, prompting a paradigm of

Fig. 1. Chemical structures of JH III and JHAs.

T.G. Wilson / Journal of Insect Physiology 50 (2004) 111–121 113

high JH titer modulating a larval-larval molt, and alow-to-absent titer allowing ecdysone to direct a moredevelopmentally advanced molt, including metamor-phosis. The interplay between these two hormones hasbeen verified at a molecular level as well (Berger et al.,1992); in this work, JH application inhibited expressionof a particular ecdysone-driven gene at the onset ofmetamorphosis. I will return to this action of JH onanother gene that is critical for initiating metamorphicchange.

Current efforts for understanding the endocrinologyof JH include isolating the responsible tissue wheneverpossible in order to explore the molecular level. Forexample, culture of fat body in the absence of JH, fol-lowed by hormone application, has shown JH involve-ment in L. migratoria (Abu-Hakima, 1981; Dhadiallaet al., 1987). In M. sexta (Riddiford, 1976; Riddifordet al., 1979), culture of larval or pupal epidermis hasbeen instrumental in providing a framework for deci-phering JH involvement during its interaction withecdysone. But the most promising approach may be theutilization of insect cell culture. Although the tissueorigin of primary cell cultures are often unknown, sev-eral lines are known to respond to JH; e.g. DrosophilaKc cells (Cherbas et al., 1989). Transfection of a cellculture with reporter gene constructs allows importantexperiments probing JH action at the molecular levelto be carried out (Berger et al., 1992). Such experi-ments are more difficult using tissue preparations.

3. Juvenile hormone analog insecticide development

Almost simultaneous with efforts to determine thestructure of JH was the chemical elucidation of a natu-ral product with JH activity from fir trees (Bowerset al., 1966). This compound, together with others sub-sequently synthesized (Bowers, 1969), was sufficientlydissimilar in structure to JH to encourage severalchemical companies, notably Zoecon Corporation, instudies synthesizing a variety of chemical analogs of JH(JHA) to test their efficacy as insecticides. This effortwas successful, validating the new paradigm of insecti-cide development based on the identification of mol-ecular targets that are specific to insects and the designof compounds to disrupt them. The compoundsdeveloped from these efforts were also termed ‘‘insectgrowth regulators’’ although they have been shownonly to disrupt development, not regulate it. Althoughit is clear that JHAs were patterned after JH and, as Iwill describe, act as JH agonists once inside an insect,how these molecules kill insects has not been easy todecipher. Much of the difficulty in solving this puzzlelies with our dismal knowledge of the mechanism ofaction of JH. There has not been a roadmap to guidethe research. This lack of knowledge of JH action is

not due to lack of effort, however, but more from theuniqueness of JH as a hormone. The steroid hormoneecdysone, with a huge literature of vertebrate steroidhormone endocrinology to guide the work, has beenmuch more amenable to problem solving. As a result,we understand much of how ecdysone works. JH hasremained more enigmatic.

The most successful early compound, methoprene(isopropyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadie-noate, Fig. 1) (Henrick et al., 1973), was found to bevirtually nontoxic to vertebrates (Siddall, 1976), con-firming the wisdom of the search mode that targeteda physiology unique to insects. Other JHAs weredeveloped by Zoecon that were generally similar instructure to methoprene (Staal, 1975; Retnakaran et al.,1985; Dhadialla et al., 1998), but none of these hasenjoyed the success of methoprene. Subsequently, agro-chemical companies, notably Sumitomo and Roche/Maag, developed aromatic non-terpenoidal compoundsthat also possessed the toxicity characteristics of meth-oprene but has a wider insect spectrum of effectiveness(Dhadialla et al., 1998). The chemical departure ofthese latter compounds from JH demonstrated thateffective JHAs need not be restricted to high chemicalsimilarity to JH to cause JH-like effects on insects.

Most insecticides have the property of being toxic toa wide range of insects; in contrast, JHAs are muchmore insect specific, and the predictability of the effi-cacy of a particular JHA against various targeted spe-cies is poor. For example, methoprene is very effectiveagainst dipteran insects but less so against lepidopteraninsects (Staal, 1975; Sehnal, 1976). In general, JHAsare not effective against major crop pests such as vari-ous species of noctuid moths. Since they lack the swiftkilling power of other insecticides such as pyrethroidsor organophosphates, they are not considered forinsect control when rapid insect population depletion isdesired. Among more closely related susceptible insects,however, the effects of these compounds can be verypredictable. For example, methoprene has been exam-ined in a variety of dipteran insects and found to havesimilar toxicity and morphogenetic effects against bothmosquitoes (Spielman and Williams, 1966; O’Donnelland Klowden, 1997) and cyclorrhaphous Diptera(Ashburner, 1970; Madhavan, 1973; Postlethwait, 1974;Sehnal and Zdarek, 1976; Wilson and Chaykin, 1985).Indeed, methoprene is very effective against mosquitoes(Mulla, 1995) and, because of its suitability for usenear humans, is used widely. Other consequences ofJHA application include failure of egg hatch for manyspecies (Riddiford, 1970; Staal, 1975), probably due todisruption of early embryonic development; sterility insome adult species, again possibly due to mortality ofthe embryos following fertilization of oviposited eggs,and most commonly, mortality during pupal develop-ment (Staal, 1975; Retnakaran et al., 1985).


Despite a slow beginning, efforts to understand howJHAs act to kill insects are now enjoying success. Itshould come as no surprise that much of the break-through has come from work with two model insects,M. sexta and D. melanogaster. Fortunately, these twospecies differ sufficiently both developmentally andphysiologically so that common aspects of JH actionand JHA toxicity, not aspects peculiar to one group ofinsects, can be identified.

4. JHAs act like JH on insects

When highly purified methoprene became availableto investigators at no charge from Zoecon scientists, itseffects on a variety of insects were examined. In most,perhaps all, of the studies methoprene behaved as a JHmimic: its effects were similar to those seen in labora-tory studies when these insects were treated with pur-ified JH (Beckage and Riddiford, 1982; Dean andMeola, 1997). The similar action of JHAs and JHfound by insect physiologists led them to conclude thatJHAs could be useful substitutes for the more labileand less available JH. Indeed, an early report showinga JHA to be more potent than the natural hormone(Bowers, 1969) has proven to be the rule rather thanthe exception. Studies examining methoprene metabolismhave concluded that this JHA, having an isopropyl ratherthan methyl ester, is not as readily metabolized and thuspersists in the treated insect (Hammock and Quistad,1981). The stability of JHAs within insects may explainmuch of their potency compared with JH homologs.

Two early reports suggested that methoprene wasexerting its effect not as a JH agonist but as aninhibitor of JH degradation (Brooks, 1973; Slade andWilkinson, 1973). Thus, the JH effects seen after meth-oprene treatment were postulated to result fromaccumulating endogenous JH instead of applied metho-prene. Since these earlier studies generally involvedapplication of methoprene to whole animals, the directvs indirect effect of methoprene was difficult to assess.More recent studies have assessed methoprene effects ininsect tissue culture or cell culture experiments, thuseliminating indirect-effect possibilities such as inhi-bition of JH degradation or stimulation of another sig-naling component, such as ecdysone, in the insect.Although initially plausible, the JH antagonist hypoth-esis of methoprene action is no longer supported: noreports of methoprene inhibition of JH metabolismhave appeared, and the results of numerous studies ofmethoprene effects are compatible with its action as aJH agonist. Studies with D. melanogaster have clearlyshown that methoprene acts as a JH agonist; it mimicsthe action of JH III on this insect very faithfully. Whenmethoprene is applied to flies at a time in developmentwhen the hormone is normally not present, it results in

disruption of development. Specifically, application towhite puparia (animals that have just entered metamor-phosis) results in metamorphic disruption as evidencedby morphogenetic effects such as abdominal bristle dis-ruption and malrotated male genitalia, as well as toxiceffects that can result in death during the pharate adultstage (Ashburner, 1970; Madhavan, 1973; Postlethwait,1974; Wilson and Fabian, 1986). JHIII application pro-duces identical results (Madhavan, 1973; Postlethwait,1974; Wilson and Fabian, 1986). Both methoprene andeach of three JH homologs were found to act in aqualitatively identical manner in Drosophila Kc cells(Cherbas et al., 1989).

Subsequent work examining more chemically diverseJHAs showed that these compounds also act as JHagonists. This has been especially true of the compoundpyriproxyfen, {2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxy]-pyridine}, Fig. 1). Although the chemistries of pyriproxfenand related compounds such as fenoxycarb stray fromthat of JH (Fig. 1), both are powerful JH agonists.Pyriproxyfen, for example, is more than an order ofmagnitude more efficacious than methoprene when appliedto D. melanogaster (Riddiford and Ashburner, 1991). It isalso more efficacious on the cat flea Ctenocephalides felis(Dean and Meola, 1997),M. sexta (Hatakoshi et al., 1988),Spodoptera litura (Hatakoshi et al., 1986), and acts as a JHduring L. migratoria reproduction (Edwards et al., 1993).

In addition to disrupting development in preadult D.melanogaster, methoprene can replace deficient JH inadults with fidelity. This was shown in experimentsapplying either methoprene or JHIII to JH-deficientadults (Postlethwait and Weiser, 1973; Wilson, 1982).Vitellogenic ovary development, proper male accessorygland protein synthesis, and larval fat body histolysis,all JH-dependent processes, could be restored by eithercompound in the JH-deficient adults. Furthermore,methoprene acts like JH in experiments at a molecularlevel in this insect; for example, the aforementionedability of methoprene to block transcription of ecdy-sone-driven heat-shock protein 23 in a dose-dependentmanner (Berger et al., 1992).

JHAs act like JH in other insects as well. As men-tioned earlier, toxic and morphogenetic effects similarto those seen in D. melanogaster have been seen in othercyclorhaphous insects (Sehnal and Zdarek, 1976) and inmosquitoes (Spielman and Williams, 1966; O’Donnelland Klowden, 1997). In lepidopteran insects, such asM. sexta, methoprene acts as a JH (Zhou et al., 1998).In allotectomized Locusta migratoria, synthesis of vitel-logenin is stimulated by treatment with methoprene(Dhadialla et al., 1987) and follicle cell patency is sup-ported by either JHIII or methoprene (Davey et al.,1993). In summary, the ability of methoprene and otherJHAs to mimic JH either in a disruptive role or in ahormonal replacement role in a variety of insects isstrong support that JHAs acts as JH agonists.


5. JH action at the molecular level

If JHAs act as JH agonists, then an understanding ofthe mechanism of action of JH should illuminate JHAtoxicity. Work done over a period of several decadeson the mechanism of action of JH has lead to certainconclusions. JH has been shown in a variety of insectsto affect gene transcription. Evidence for this rangesfrom earlier studies demonstrating that chromosomepuff morphology in D. melanogaster can be affected byJH (Richards, 1978) to more recent work demonstrat-ing specific gene transcriptional regulation by JH in avariety of insects (Jones, 1995; Hirai et al., 1998; Fenget al., 1999; Dubrovsky et al., 2000; Zhou et al., 2002;Venkataraman et al., 1994). Ecdysone also regulatesgene transcription (Thummel, 1996), but unlike ecdy-sone transcriptional control of many (several hundred)genes during metamorphosis (White et al., 1999), JHappears to affect a much smaller number. JH regulatestranscription of genes during both preadult and adultdevelopment (Jones, 1995).

JH also has effects in insects independent of regulat-ing gene transcription. The hormone has been shownto promote protein synthesis in male accessory glandsin a variety of insects (Wyatt and Davey, 1996). Somework in several insects has suggested JH control at thetarget cell membrane level, suggestive of second mess-enger signaling (Yamamoto et al., 1988; Sevala et al.,1995). Examples of vertebrate steroid hormones, prim-arily thought to act directly at the chromosome levelon gene transcription, have been reported recently toalso act at the plasma membrane (Zhu et al., 2003),and a recent argument has been made to include JH ina diverse group of lipid hormones that act at the mol-ecular level as signaling molecules not simply in theclassical steroid hormone role but at several levels(Wheeler and Nijhout, 2003). Therefore, results show-ing both male accessory gland and oocyte signal trans-duction at the membrane level may be unveilingmultiple actions of this hormone. It is interesting tonote in this regard that the vertebrate hormone thyrox-ine can interact with an oocyte membrane receptor forJH (Davey, 2000). Finally, JH effects have also beenassociated with mitochondria (reviewed in Wyatt andDavey, 1996).

However, at present most of the attention toward JHaction is centered on regulation of gene transcription.A major hurdle in deciphering this years-old con-undrum is our lack of understanding of a JH receptormolecule in any insect (Jones, 1995; Gilbert et al.,2000). Recent candidates for a JH receptor componentincludes the ultraspiracle (usp) gene product (Jones andSharp, 1997), which is an ecdysone receptor component(Henrich et al., 1990; Oro et al., 1992), and Metho-prene-tolerant (Met), a member of the bHLH-PAStranscription factor family (Ashok et al., 1998). USP

appears to bind JHIII in vitro (Jones and Sharp, 1997)and, as such, provides a neat explanation for ecdy-sone/JH interaction. Met mutants confer resistance toJH and JHAs (but not to other classes of insecticides)in vivo (Wilson and Fabian, 1986), and extracts fromMet mutants show reduced target-tissue intracellularJHIII binding (Shemshedini and Wilson, 1990). Per-haps the final elucidation of ‘‘the’’ JH receptor willinterweave both of these proteins into a common pic-ture. But, there is no doubt that a solution to this long-standing problem of the nature of the receptor willfacilitate work leading to an understanding of themechanism of action of this hormone.

6. Window of sensitivity of JHAs

When during development do insects show the high-est sensitivity to JHAs? The answer does more thansatisfy our biological curiosity: it provides a cluefor understanding the action of these molecules. Thisquestion has been well studied in D. melanogaster.Exposure of preadults to methoprene or pyriproxyfeneither topically applied or incorporated into thefood showed a sensitive period of around 12–15 hcentered at the onset of pupariation (Postlethwait, 1974;Riddiford and Ashburner, 1991). This sensitive perioddefines both the toxic and morphogenetic effects ofJHA (Postlethwait, 1974). A similar sensitive period hasbeen shown for topically applied JHIII and for theresistance phenotype of Met mutants (Wilson andFabian, 1986; Wilson, 1996). During this period indevelopment metamorphic change is accelerating(Thummel, 1996), and the endogenous JH titer is rap-idly decreasing (Bownes and Rembold, 1987; Sliteret al., 1987). One interpretation of this window of sensi-tivity is that JH (as JHA) is now present at exactly thetime in development when the insect does not want JHpresent, and its presence disrupts the hierarchy of ecdy-sone-mediated metamorphic change, resulting in theconsequences seen as developmental disruption andtoxicity.

The changes seen in insects treated with JHA duringthe sensitive period have suggested a mechanism bywhich these compounds act. In one study exploring thepathology of methoprene, serial sections of pharateadults that had been treated with methoprene duringthe puparial stage were examined for internal anatom-ical changes (Restifo and Wilson, 1998). The concen-tration of methoprene used was sufficient to ensuremortality in the pharate adults, but examination wascarried out shortly before death; therefore, the changesseen were methoprene-related, not death-associated.Most of the anatomical structures in these pharateadults appeared normal, suggesting that application ofmethoprene does not produce sweeping changes in


tissue development during metamorphosis. But, dose-dependent anatomical defects were present in the pha-rate adults. Importantly, many of these defects werenoted in the central nervous system, including defectsin brain structure that could certainly be responsiblefor the mortality that occurred. Other defects noted inthe methoprene-treated pharate adults included persist-ence of larval salivary glands and missing muscle struc-tures which, while dramatic changes, seem unlikely tobe life threatening.

What could be causing these changes during pupaldevelopment? One possibility is that methoprene actsas a toxin to directly affect the tissues involved. Whilepossible, the temporal separation of nearly the entirepupal developmental stage of 4 days between the meth-oprene-sensitive period and the appearance of thedefects in pharate adults argues against this expla-nation. A stronger hypothesis that is in accord with themethoprene-sensitive period is that of methopreneinterference, either direct or indirect, with early meta-morphic gene expression. This interference can thenlead to the defects seen in pharate adults. One (prob-ably the) major event occurring during pupariation isthe activation of gene expression (Thummel, 1996). Themagnitude of this event is illustrated by one of the firstgene microarray determinations that reported over 500genes in D. melanogaster to be transcriptionally regu-lated by ecdysone at a the onset of metamorphosis(White et al., 1999). These genes regulating metamor-phosis are activated in a hierarchical fashion: the earlygenes, many of which have been identified as encodingtranscription factors, are directly regulated by ecdy-sone, and the later ones, termed effector genes, are acti-vated (occasionally repressed) by the earlier geneproducts acting solely or in combination with otherproteins (Henrich and Brown, 1995; Segraves, 1994).Products from the effector gene activation carry out themetamorphic changes seen: apoptosis of most larvaltissues, growth and differentiation of cells that willform the adult tissues, and formation of the actualstructures. For example, activation of cell death in lar-val tissues, best understood for the larval salivaryglands (Buszczak and Segraves, 2000), is directed dur-ing pupariation by an ecdysone-regulated early genetermed E93 (Lee et al., 2000). One of the effector genesis the dronc caspase gene whose expression leads toapoptosis in larval tissues; however, ecdysone control isindirect, and dronc gene activation is directly controlledby the early gene product Broad-Complex (BR-C)(Cakouros et al., 2002). As might be predicted, certainBR-C mutants disrupt cell death in the larval salivaryglands, resulting in persistence of these tissues in pupae(Restifo and White, 1992).

Is there evidence that JH influences the expression ofany of these early genes? A well-studied gene whoseexpression appears to be affected by JH is in fact BR-C,

which encodes a small group of alternatively splicedtranscription factors (Bayer et al., 1996) controllingexpression of downstream effector genes (Karim et al.,1992; Crossgrove et al., 1996). A number of down-stream genes have been identified that are directlyregulated by BR-C proteins, including the aforemen-tioned dronc, Sgs4, Ddc, Fbp2, and Fork head (Bayeret al., 1997; Renault et al., 2001; Cakouros et al.,2002; Chen et al., 2002) to name a few from a listthat is likely to grow in future work. Studies inM. sexta suggest that methoprene can inhibit thetranscription of BR-C (Zhou et al., 1998), a conse-quence that would likely result in misexpression ofBR-C target effector genes and thus defects duringmetamorphic change. In D. melanogaster, BR-C alsois affected by methoprene application. Initially,BR-C came under suspicion when the pathology ofmethoprene-treated D. melanogaster mimicked certainaspects of the BR-C mutant phenotype, such asdeformities in the central nervous system, persistenceof the larval salivary glands, and musculature defectsmentioned earlier (Restifo and Wilson, 1998). Someof these defects also mimicked phenotypic defects ofanother early gene mutant, Deformed (Restifo andWilson, 1998), suggesting that BR-C is not the solegene affected by methoprene treatment. These defor-mities were not seen in Met mutants treated identi-cally with methoprene (Restifo and Wilson, 1998).This JH-induced phenocopy of BR-C mutants sug-gested that methoprene would act in D. melanogasteras in M. sexta to block BR-C transcription; however,a different result emerged when transcription wasexamined in pyriproxyfen-treated pupae: instead ofblocking transcription, this JHA induced anotherwave of BR-C expression in late pupae, presumablydisrupting expression of BR-C target genes thatdepend on precise temporal expression of this tran-scription factor, resulting in the observed defects inthe pharate adults (Zhou and Riddiford, 2002).Additionally, genetic studies show that BR-C andMet mutants interact: double mutants of each resultin phenotype enhancement (Wilson and Restifo,unpublished), widely interpreted as interaction of thegene products (Bradley, 1996; Chen et al., 1998).A hypothesis that can explain these results inD. melanogaster is that BR-C and MET act in tan-dem during normal development as a heterodimericcomplex to regulate expression of one or more down-stream genes. In wild-type flies if methoprene ispresent, it binds MET to result in a disruptedMET:BR-C heterodimer that subsequently is unableto correctly drive expression of effector genes, result-ing in the observed defects in pharate adults. In Metmutants, methoprene cannot interact as well with thealtered MET protein; therefore MET:BR-C-dependenteffector gene regulation is not derailed, and survival


and normal morphogenesis occurs (i.e. the flies are

resistant to methoprene).

7. A summary view/hypothesis of JHA action

The studies outlined above point to an action of

JHA insecticides to disrupt metamorphic change in sus-

ceptible insects, perhaps most readily seen in dipteran

insects. JH is present during larval development, when

it serves to modulate the action of the ecdysone surge

at each larval molt from triggering an undesired meta-

morphic molt. Thus, the hormone truly maintains the

‘‘status quo’’ of larval/nymphal development, and lar-

vae are refractory to JHA treatment since endogenous

JH is already present. At the onset of metamorphosis

JH is cleared from the insect and no longer is a barrier

to the prepuparial action of ecdysone that initiates the

sweeping metamorphic changes in the insect. These

changes are controlled by a flood of new gene products

resulting from this gene expression, primarily gene acti-

vation. The early genes activated by ecdysone encode

proteins that in turn regulate downstream effector

genes that are directly responsible for the morphologi-

cal and physiological changes that characterize meta-

morphosis. If JHA is applied to an insect during early

metamorphosis, it binds to MET or other JH-interacting

protein. This JH-protein complex then either alters

expression of one or more of the early ecdysone-

regulated genes, such as BR-C, at the top of the gene

expression hierarchy or interacts with BR-C protein

as a heterodimer. In either case the consequences are

not felt by the pupa until the pharate adult stage,

when misexpression of the downstream effector genes

results in some alteration of their gene products and

failure of proper developmental and physiological

changes normally controlled by them. Thus, the bris-

tle defects and failure of proper male genitalia

rotation seen even at sublethal doses of JHA, reflect

an expression failure of the proper downstream genes

that control these developmental processes instead of

a direct toxic effect of JHA on these tissues. Like-

wise, at slightly higher JHA doses more serious cen-

tral nervous system defects occur, presumably

resulting in lethality in the pharate adults. It is clear,

however, that JHA does not totally shut down BR-C

expression because a null allele for this gene results

in failure to enter metamorphosis (Kiss et al., 1988),

a far more severe effect on development than that

resulting from JHA application. Therefore, the effect

of JHA is subtle, affecting only a small subset of

metamorphic changes.

8. Questions that remain to be answered

In addition to providing more evidence for the pro-posed scenario of JHA action, future work needs toaddress several questions.

First, the basis for the effectiveness of the widechemical range of JHAs merits investigation. How cansuch different chemicals act similarly as JH agonists? Asimilar phenomenon is seen for the vertebrate hormoneestrogen, which is mimicked by a variety of environ-mental, industrial, and naturally occurring compoundstermed xenoestrogens (Mueller, 2003). CytochromeP450 investigators have also faced this problem withthe huge array of xenobiotic compounds that bind tothis cytochrome and result in the characteristic P450response. The solution to their problem, at least par-tially, became evident with genome sequencing and thediscovery that the genes encoding these proteins, theCyp genes, comprise a large family of genes (over 90in D. melanogaster) that result in an array ofCYP proteins with different protein binding affinities(Feyereisen, 1999). Previous work in M. sexta examin-ing this issue with JH binding proteins found that JHIand a photoaffinity analog of methoprene bound todifferent proteins in this insect (Osir and Riddiford,1988), suggesting a P450-similar story for JHAs, mul-tiple genes for multiple JHA-binding proteins. A simi-lar result was found for JH and methoprene-bindingproteins in follicle cell membranes of L. migratoria(Sevala et al., 1995). However, work in D. melanogasterhas argued against this explanation as widespread. Metmutants were found to be resistant to several JHAs aswell as to JHIII (Wilson and Fabian, 1986; Riddifordand Ashburner, 1991); thus, a defective single generesults in resistance to a variety of chemicals, workingperhaps through a promiscuous binding protein cap-able of binding a variety of different JHA chemicals.Recently, studies of multidrug resistance in a prokar-yote demonstrated that the membrane efflux pump pro-tein responsible for resistance to a variety of drugs hasjust this type of binding characteristic: a large bindingcavity that enables different ligands to bind to a slightlydifferent subset of amino acid residues (Yu et al.,2003). An approach to understanding this problem willinvolve studies of JHA binding and binding proteins ininsects, work that has previously proven challenging.

Second, the basis for the selectivity of insects in theirresponse to JHAs is an important question in need ofan answer. Although there is some insect specificity forother insecticide classes, we do not see JHA suscepti-bility widespread among insects as is commonly foundfor most broad-spectrum insecticides. Some of thisspecificity is undoubtably due to the ability of certaininsect pests to evade contact with JHAs, such as forestinsects living within trees. But, other insect speciesshow little or no metamorphic disruption following


direct exposure to JHA. It is clear that response tomethoprene differs between insect species at the mol-ecular level as well: BR-C expression is prevented in M.sexta but not in D. melanogaster following JHA appli-cation (Zhou et al., 1998; Zhou and Riddiford, 2002).Perhaps the very different developmental and physio-logical responses of lepidopteran vs dipteran insects toJHAs can be accounted for by different responses of asmall number of genes at the onset of metamorphosis.An understanding of the nature of the refractoriness ofsome insects to JHAs will not only tell us more aboutJH endocrinology in Insecta but also might lead tonovel JHA compounds having a wider insect targetspecies spectrum.

Third, the mechanism of action of JH needs a sol-ution or at least more than simply a modicum of pro-gress. Of the ones posed here, I believe that thisquestion is the closest to an answer. Work in D. mela-nogaster has established JH binding, both hemolymphand cytosolic components, and at least one geneinvolved in resistance to JH through a target-site mech-anism. A critical aspect of the work is the identificationof JH-responsible genes. While the identification of sev-eral seems clear (Dubrovsky et al., 2000), the avail-ability and power of microarray analysis should soonlead to identification of those that are transcriptionallyregulated, either activated or repressed, directly by JH.With target genes known, work can readily be carriedout to engineer a reporter gene system that, togetherwith well-studied D. melanogaster cell lines, can ident-ify components of the JH transcriptional machinery,which should help establish the precise mechanism ofaction. Our current understanding of the componentsof ecdysone action, including the receptor components,associated proteins, and many of the target genes, willthen be valuable for deciphering the nature of theinteraction of the ecdysone and JH signaling pathways.

Finally, the effect of JH and JHAs on vertebratephysiology must be settled. For many years the earlytoxicological data indicated that JHAs were very safechemicals in use around humans. But sporatic reportshave suggested effects of methoprene on vertebrateendocrine responses (Harmon et al., 1995). Methopreneentry into ponds due to mosquito spraying does notseem to be a causative agent of deformed frogs(Sessions et al., 1999), but the interaction of this chemi-cal with vertebrate cell culture gene expression requiresfurther attention. This work will allow not only thesafety of JHAs but also point to possible commonal-ities in vertebrate and invertebrate signaling pathways.

Acknowledgements

Dr. David Denlinger kindly invited me to write thisreview. I thank the National Science Foundation and

the National Institutes of Health for their support ofcurrent work on JH in my laboratory.

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