Journal of Insect Physiology 56 (2010) 1456–1464

Activities of natural methyl farnesoids on pupariation and metamorphosis ofDrosophila melanogaster

Grace Jones a,*, Davy Jones b, Xiaobo Li a, Lingfeng Tang a, Li Ye a, Peter Teal c, Lynn Riddiford d,e,Courtney Sandifer a, Dov Borovsky f, Jean-Rene Martin g

a Department of Biology, University of Kentucky, Lexington, KY 40506, United Statesb Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506, United Statesc U.S. Department of Agriculture, Agricultural Research Service, Chemistry Research Unit, Gainesville, FL 32608, United Statesd Department of Biology, University of Washington, Seattle, WA 98195-1800, United Statese Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, United Statesf University of Florida-IFAS, Florida Medical Entomology Laboratory, Vero Beach 32962, United Statesg Laboratoire de Neurobiologie Cellulaire et Moleculaire (NBCM) CNRS, UOR-9040, Gif-sur-Yvette Cedex, France

A R T I C L E I N F O

Article history:

Received 17 February 2010

Received in revised form 1 June 2010

Accepted 2 June 2010

Keywords:

Methyl farnesoate

Ultraspiracle

RXR

HMGCoA reductase

Juvenile hormone

Methyl epoxyfarnesoate

Bisepoxy JH III

JH esterase

A B S T R A C T

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP

and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila

larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were

similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation.

However, the order of activity was changed (JH III > bisJH III > MF) with respect to preventing prepupae

from eclosing as normal adults, whether administered in the larval diet or as topically applied at the

white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by

a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation.

Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as

a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of

pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three

hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval

attainment of pupariation. However, in this background dietary MF had increased activity in preventing

puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl

farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting

in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-

late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited

with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and

female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with

nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the

binding of secreted MF to USP.

� 2010 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Journal of Insect Physiology

journa l homepage: www.e lsev ier .com/ locate / j insphys

1. Introduction

The endocrine networks that regulate insect metamorphosisfrom the larva to the pupa, and then to the adult, are a popularmodel for discerning how hormonal signals are physiologicallyintegrated in the differentiation of body form (Emlen and Nijhout,

Abbreviations: MF, methyl farnesoate; JH III, methyl epoxyfarnesoate; JH, juvenile

hormone; bisJH III, methyl bisepoxyfarnesoate; HMGCR, 3-hydroxy-3-methyl-

glutaryl-CoA reductase; Drosophila, Drosophila melanogaster.

* Corresponding author. Tel.: +1 859 257 2105; fax: +1 859 257 7505.

E-mail addresses: gjones@pop.uky.edu, gjones@uky.edu (G. Jones).

0022-1910/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2010.06.001

2000; Riddiford et al., 2003). Among the endocrine actors in insectmetamorphosis is ‘‘juvenile hormone’’ (methyl epoxyfarnesoate,JH III). The ‘classic status quo model’ of endocrine coordination ofthe metamorphic transformation (Williams, 1953) is based on theinteraction of 20-hydroxy ecdysone (20E, which prompts eachmolt) with JH III or one of its homologs (JH I or JH II) (Truman andRiddiford, 2007). Under the current status quo model, a high level ofjuvenile hormone(s) at the larval molting surge of ecdysonemaintains the larval to larval molting pattern. Then, at sometimeduring the final larval instar the juvenile hormone level declines.That decline enables a small ecdysone surge to commit the larvaltissues for pupation at the next molt (Truman and Riddiford, 2002).It is also postulated that wandering behavior in preparation for

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–1464 1457

pupation by the final larval instar is delayed until the endogenousJH titer declines sufficiently. An evidence for the application of thatmodel to Drosophila is that inclusion of synthetic analogs of JH III,such as methoprene, into the larval diet prolongs the feeding stage,thereby delaying pupariation (Wilson and Fabian, 1986).

Insect cells express a farnesoid biosynthesis pathway in which acrucial enzymatic step is controlled by 3-hydroxy-3-methylglu-taryl-coenzyme A reductase (HMGCR). This pathway producesfarnesyl pyrophosphate (FPP). That product then branches intovarious pathways. In the known biochemical pathways involvingFPP: (1) proteins and heme are farnesylated directly from FPP,without production or utilization of free farnesol (Cui and Merz,2007; Caughey et al., 1975) and in a reaction specificity for whichfarnesol at even great excess cannot substitute for FPP (Saiki et al.,1993), (2) dolichol and ubiquinone are synthesized by condensa-tion of one or more isoprenyl units from isoprenyl phosphate ontoFPP, by a reaction mechanism that does not produce or utilize freefarnesol (Krag, 1998; Schenk et al., 2001), and (3) FPP incorporationinto squalene and cholesterol biosynthesis do not occur in insects(Belles et al., 2005). However, uniquely in insect corpora allatalcells, such as of the Drosophila larval ring gland, FPP is alsoconverted to farnesol, which is a precursor to biosynthesis of JH IIIor other methyl farnesoids (Fig. 1 in Belles et al., 2005). In the morethan 40 years of research on farnesoid biosynthesis in larval insectssince the determination of JH structure, there does not appear tohave been shown a single instance in which a function of farnesol isnecessary for larval life, without which the larva dies due tointernal dysfunction—except for the role of farnesol as a precursorfor methyl farnesoid biosynthesis.

For the past several decades, most models of insect metamor-phosis have focused on the biological activity of JH III (or JH I and JHII in Lepidoptera). Further, ring glands of higher Diptera in tissueculture also synthesize and release not only MF but also bisJH III(Richard et al., 1989a,b; Yin, 1994), raising questions as to whichhormone(s) are responsible for regulating developmental progres-sion. In addition, there is evidence in Drosophila that MF isproduced in a terminal biosynthetic route that is distinct from theterminal biosynthetic steps leading to production of JH III and bisJHIII (Moshitzky and Applebaum, 1995). Recently, MF and bisJH IIIhave been detected as being in the circulating hemolymph ofDrosophila larvae, along with JH III (Jones and Jones, 2007; Joneset al., 2010).

[(Fig._1)TD$FIG]

Fig. 1. Effect of dietary provision of natural methyl farnesoids on attainment of

pupariation by the indicated day since egg hatch. MF: methyl farnesoate; JH III:

juvenile hormone III; Bis: bisepoxy JH III; M/J/B: combined provision of MF, JH III

and bisJH III at the 6.2, 0.13, and 6.2 mmol doses, respectively, based on

approximate proportions of the hormones detected during late larval

development. MF and JH III exhibited similar effects (stronger than bisJH III) to

delay attainment of pupariation (n = 160 for each treatment).

Identifying targets of JH signaling is a goal that has consumedmuch experimental effort. A prepupal increase in JH has beenhypothesized to prevent some prepupal tissues, such as the visualsystem, from prematurely expressing the adult program (e.g.,Kiguchi and Riddiford, 1978). The gene encoding a JH-specificesterase (‘‘JHE’’) is expressed soon after that prepupal JH peak inLepidoptera (e.g., Roe et al., 1993; Sparks et al., 1983) and inDrosophila (Campbell et al., 1992, 1998; Kethidi et al., 2005; Klagesand Emmerich, 1979). In lepidopterans, the prepupal JHE peak isabolished by removal of the corpora allata. The JHE peak is restoredby exogenous JH (Jones and Hammock, 1985), and in somelepidopterans a lethal supernumerary molt to a second pupaoccurs if the prepupal JHE peak is inhibited (Jones and Hammock,1985). Hence, it has been hypothesized that this prepupal peak ofJH esterase is responsible for clearing methyl farnesoids that actedduring the early prepupal stage (DeKort and Granger, 1996).

In Drosophila, a teratogenic assay has been one approach totesting activities of natural methyl farnesoids, and syntheticanalogs. In that assay, exogenous application of the test compoundis made, to observe whether a disruption occurs at or prior topupariation, or at metamorphosis to the pupa, and/or at adulteclosion. Most typically, this approach has exposed the testanimals to a single compound at a time (Sehnal and Zdarek, 1976).Further, Harshman et al. (2010) in this symposium issue show thatin a teratogenic assay of inhibiting survival to pupariation, MF wasmore active than JH III.

Another approach to assessing methyl farnesoid activity is aremoval assay, in which endogenous methyl farnesoid produc-tion is suppressed, and observation made of the ensuingdevelopmental lesion(s). Surgical removal of the portion of thering glands producing methyl farnesoids is technically difficultfor Drosophila, and was mastered only by a few researchers(reviewed in Jones and Jones, 2007). More recently, genetictechniques have been fashioned for suppression the methylfarnesoid production, such as apoptotic ablation of the corporaallata cells of the ring gland (Liu et al., 2009; Riddiford et al.,2010), or RNAi-mediated suppression of a biosynthetic enzyme(Jones et al., 2010; Niwa et al., 2008). When the biosynthesis ofthe three methyl farnesoids is sufficiently suppressed, there is asevere disruption in larval–larval molting and larval pupariation(Jones et al., 2010). However, restoration of that biosyntheticpathway with farnesol rescued larval survival to the 3rd instar. Inview of the specialized role of farnesol in larval development,summarized above, a reasonable interpretation of this result isthat provision of dietary farnesol rescued larval developmentaldefects by way of rescuing the suppressed methyl farnesoidpathway in the corpora allatal cells. In contrast, replacementwith just exogenous JH III or just exogenous MF did not rescuelarval development. In fact, exogenous provision of JH III or MF inthe background of suppressed production of all three methylfarnesoids appeared more toxic than did the suppressed methylfarnesoid production alone (Jones et al., 2010). Also, Riddifordet al. (2010) reported that when endogenous production of allthree methyl farnesoids was ablated, provision of exogenous JHanalog to rescue of visual system development acted in partthrough a signaling system that did not include the putative JHreceptor (methoprene tolerant protein, MET; Wilson and Fabian,1986; Wilson, 2004).

The present paper provides the first analysis in Drosophila ofeffects in a teratogenic assay, of the combinatorial provision of allthree secreted methyl farnesoids. In addition, comparison is madeof the activities of these three Drosophila hormones, where theprovision is derived from internally produced versus exogenouslyapplied methyl farnesoids. Finally, we include data that evidencethe applicability of a methyl farnesoate–ultraspiracle hormone/receptor axis to a lower dipteran, the mosquito Aedes aegypti.

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–14641458

2. Methods and materials

2.1. Fly strains and mosquitoes

The fly strains used in these studies were maintained at 25 8C onstandard corn meal diet (cornmeal/malt/yeast/soy flour/cornsyrup/phosphoric acid–proprionic acid mix/agar/tegosept). Mos-quitoes were grown and maintained as described by Borovsky andCarlson (1992).

2.2. Dietary exposure to methyl farnesoids

In experiments in which larvae were reared in groups, the dietwas poured into 8 dram plastic vials, taking care to ensure the foodsurface was level and without bubbles. After solidification, thedose of the given experimental compound was applied to the foodsurface in 100 ml of ethanol (EtOH) carrier solvent. The ethanol wasallowed to evaporate overnight with gentle swirling. In the Di3.3promoter experiments (see below), forty eggs were manuallyplaced on food surface of each vial, with four vials per treatment.The developing larvae in each vial were categorized as to the day ofattainment of pupariation (if puparation was reached), and foradult emergence. Totals in each category were analyzed by chi-square analysis. In the Di11 promoter experiments, in which newlyhatched 1st instar larvae were reared individually, diet was placedinto individual 10 mm � 75 mm glass vials. The respective dose ofhormone in 6.25 ml of EtOH was applied to the surface, asdescribed previously (Jones et al., 2010). Totals in each scoringcategory were analyzed by chi-square as above.

Because all three compounds are secreted at the same timefrom ring glands explanted to culture from late last instar larvaejust prior to pupariation, we tested the effect of simultaneouslyapplying all three compounds to the diet, at a ratio approximatingthe late last instar endogenous levels. Our preliminary measure-ments of each compound during the feeding stage of the late lastinstar detected a ratio of MF to JH III of ca. 50:1, and the bisJH IIIlevel slightly lower than that of MF. In the present experiments, wethen applied the three compounds at a ratio of MF:JH III:bisJH III of49.5:1:49.5, and the total amount of methyl farnesoid appliedequaled 12.5 mmol in 100 ml of EtOH carrier (the same as themaximum dose applied for any individual methyl farnesoid).

2.3. Topical exposure to MF

The ‘‘white puparium assay’’ (Postlethwait, 1974) was used toassess the sensitivity of that stage to developmental disruption bytopical exposure to MF. The assay was performed on Canton Spuparia as described by Riddiford and Ashburner (1991).

2.4. Chemicals

JH III was obtained from Sigma–Aldrich (St. Louis, MO) and MFwas obtained from Echelon Inc. (Logan, Utah). bisJH III wasprepared by one of the authors (PT) as mixed isomers from JH III,following the procedure of Richard et al. (1989a). The crudebisepoxide in benzene was concentrated and dissolved in pentaneprior to separation by liquid chromatography. Liquid chromatog-raphy was performed using a Rheodyne 71251 injector, a KratosSpectraflow 4001 pump and Waters 4101 differential refractom-eter. The initial purification was performed using a 25 cm � 10 mm(id) Adsorbosil silica column (10 mm) (Alltech) eluted with 25%ethyl acetate in pentane at 5 ml/min. Peaks collected wereanalyzed by GC–MS for the presence of bisJH III (Teal et al.,2000). The fraction containing bisJH III was further purified using a25 cm � 2.1 mm (id) Adsorbosil silica column (5 mm) eluted with20% ethyl acetate in pentane at 0.5 ml/min. This resulted in partial

resolution (50% resolution) of four isomers of the bisepoxide. Thelast eluting peak had a retention time and chemical ionizationmass spectrum identical with bisJH III naturally produced by malesof the Caribbean fruit fly (Teal et al., 2000). Retention times andratios of diagnostic ions including m/e = 283 (M+1), 265(M+1�HOH), 251 (M+1�CH3OH), 233 (M+1�CH3OH�HOH), 205(M+1�CH3OH�HOH�CO) whereas the retention times for theother stereoisomers were different from the naturally producedbisepoxide.

2.5. Genetic interference with endogenous methyl farnesoid titers

2.5.1. Decrease in level of endogenous farnesoids

The genetic approach used was based on a binary Gal4/RNAisystem that we have reported previously, targeting 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) with an RNAi (Joneset al., 2010). When the RNAi is driven by a natural HMGCRpromoter fragment expressed in corpora allatal cells, the level ofthe enzyme HMGCR is strongly suppressed (Belgacem and Martin,2007), reducing the biosynthetic pathway of methyl farnesoids inthe corpora allatal cells. In one experimental design, a transgenicHMGCR promoter fragment was used to drive Gal4 expression(Di11-Gal4, maintained over a TM3 stubble-marked balancerchromosome that expresses actin5C-driven GFP) that we usedpreviously (Jones et al., 2010). After crossing to a homozygous linefor UAS-HMGCR-RNAi, the GFP marker was used to separate theDi11-Gal4; UAS-HMGCR-RNAi 1st instar larvae. Physiochemicalmeasurements have previously confirmed that expression of theHMGCR-RNAi in larvae reduced the circulating level of methylfarnesoids (Jones et al., 2010). In another experimental design, aweaker transgenic HMGCR promoter fragment was used (Di3.3-

Gal4, maintained as a homozygote; Belgacem and Martin, 2007) togenerate Di3.3-Gal4; UAS-HMGCR-RNAi larvae.

2.5.2. Increase in level of endogenous farnesoids

In these experiments, we used a binary Gal4/RNAi systemdesigned to inhibit the level of juvenile hormone esterase, anenzyme specialized to degrade methyl farnesoids. Homozygotesfor the construct UAS-JHEsterase-RNAi (Vienna RNAi Stock Center;5824), were crossed to a line with actin5C-Gal4/Cyo. Eclosing adultswere scored for +/� the Cyo marker; i.e., those without the Cyomarker express the JHEsterase-RNAi.

2.6. Normal expression of JH esterase gene

The normal (yw) expression of JH esterase during the prepupalstage was determined by extracting total RNA at hourly intervalsfrom 10 animals per replicate that were initially synchronized at thewhite puparium stage (hour 0). After synthesizing oligo-d(T)-primed cDNA, the abundance of the JHE transcript was determinedby qPCR using the following primers: forward primer – 50-AGGCCTTCATGGGCATTC; reverse primer – 50-TGGGCATTACCTTGG-GATTA. The abundance of the specific JHE product was normalized tothe abundance of ribosomal protein 49 (RP49) using the followingRP49 primers: forward primer – 50-TTCCTTGACGTGCCAAAA;reverse primer – 50-AATGATCTATAACAAAATCCCTGA.

2.7. Measurement of methyl farnesoid biosynthesis by Aedes aegypti

Methods used to culture exposed corpora allata (CA), oraccessory glands, of A. aegypti and to analyze for JH III and MFproduction were as described previously (Borovsky and Carlson,1992; Borovsky et al., 1992, 1994). Briefly, groups of exposed CA oraccessory glands (10 per group) were explanted from male orfemale adults at 1–5 days after adult emergence. Each group of 10CA or 10 accessory glands was incubated in tissue culture media

[(Fig._2)TD$FIG]

Fig. 2. Effect of dietary provision of natural methyl farnesoids on prepupal

development to adult eclosion. MF: methyl farnesoate; JH III: juvenile hormone III;

Bis: bisepoxy JH III; M/J/B: combined provision of MF, JH III and bisJH III at the 6.2,

0.13, and 6.2 mmol doses, respectively, based on approximate proportions of the

hormones detected during late larval development. JH III exhibited a much stronger

effect than either MF or bisJH III to prevent prepupae from attaining adult eclosion

(n = 160 for each treatment).

Fig. 3. White puparial (topical) assay of natural methyl farnesoids on development

of prepupae to adults. The data for MF were generated in the present study (N = 9–

18 per point, except for 52 at 1000 ng, 78 at 10,000 ng and 30 at 25,000 ng), while

those for JH III are adapted here from Wozniak et al., 2004. The datum point for bisJH

III is adapted here from Richard et al. (1989a). Adults that eclosed with abnormal

rotation of the genitalia (male) or defective bristles on the abdominal sternites

(females) were considered as abnormal. In this assay, JH III was much more active

than MF.

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–1464 1459

containing L-[methyl-H3]methionine for 4 h. Following incuba-tions, tissues and media were separately extracted with acetoni-trile and analyzed for biosynthesized JH III and methyl farnesoateusing HPLC and gas chromatography (Borovsky and Carlson, 1992;Borovsky et al., 1992). A portion of the studies described hereyielded data reported in Borovsky et al. (1994) as only total methylfarnesoid synthesis. Newly reported here are the data on amountsof MF and JH III recovered in the medium (indicating secretion)versus recovered with the cultured tissue. Results are expressed asthe mean of the indicated number of determinations � SEM.

2.8. Ultraspiracle ligand binding assay

The cDNA for A. aegypti nuclear receptor ultraspiracle, isoform A(aaUSPA) was cloned into the pET32 plasmid, expressed inbacteria, and purified by nickel resin column followed with aSuperdex-200 column, as described for Drosophila melanogaster

USP (dmUSP, Jones et al., 2001). aaUSPA possesses in its ligandbinding pocket a conserved tryptophan residue corresponding toW318 of dmUSP. As demonstrated previously, the presence of thatresidue in the ligand binding pocket enables a ligand binding assayfor farnesoids based on fluorescence quenching of the tryptophanresidue when ligand occupies the binding pocket (Jones et al.,2006). The purified aaUSPA was assayed using a ligand bindingassayed as used for dmUSP (Jones et al., 2006).

3. Results

3.1. Dietary exposure of normal larvae to methyl farnesoids

We first tested the activity of all three endogenously secretedmethyl farnesoid compounds to prolong the feeding stage. When12.5 mmol of respective compound were applied to the diet at theoutset of larval life, the larvae fed, grew, and molted to the 2ndinstar, and then to the 3rd instar, which then also continued to feedand grow. Daily observations did not discern any differencebetween the exposed and control animals, in their occurrence onthe food during the feeding periods between the molts. Dietary MFwas slightly more active than JH III in causing a delay in the 3rdinstar larvae reaching the post-feeding stage of pupariation (chi-square, p = 0.012), while bisJH III was less active than either MF orJH III (chi-square, p < 0.0001; Fig. 1). Our preliminary observationsdid not detect a change in the length of the wandering stage, hencethe delay in attainment of pupariation is interpreted here to resultfrom a delay in initiation of wandering behavior.

We then simultaneously exposed the animals to all threemethyl farnesoids in the diet throughout larval life, at a ratioapproximating the endogenous levels in the late last instar. As seenin Fig. 1, this application of all three compounds (total of 12.5 mmolmethyl farnesoid) was not more active in prolonging the feedingstage in normal larvae than was provision of 12.5 mmol of eitherMF or JH III alone (chi-square, p > 0.01).

3.2. Topical exposure to methyl farnesoids

It was of interest to ascertain the comparative effect of the threemethyl farnesoids on the metamorphic molt to the pupa, and thento the adult. As shown in Fig. 2, the dietary exposure to 12.5 mmolof total methyl farnesoid, at the natural late last instar ratio, was ofsimilar efficacy in preventing prepupal development to the adult,as was exposure to that dose of MF alone. However, JH III alone at12.5 mmol was much more active in preventing prepupaldevelopment to adult eclosion than was exposure to MF aloneor bisJH III alone (chi-square, p < 0.0001). Although larval deathduring the feeding studies occurred at or after the prepupal stage, itis possible that the death was caused by exogenous hormonal

action at an earlier stage in development. Therefore, the activity ofthe compounds was compared in the ‘‘white puparium assay’’. Inthis assay, the compounds were directly applied to the youngprepupal animals, thereby omitting potential effects of earlierdietary exposure on the subsequent prepupal and later stages. Asshown in Fig. 3, MF was about 100 times less active in the topicalwhite puparium assay than was JH III, in preventing normal adultdevelopment. Even at the higher doses of MF about 2/3 of thetreated flies eclosed but an increasing number of these showedabdominal defects of JH application at this time (Postlethwait,1974) and therefore were scored as abnormal adults.

3.3. Dietary exposure of larvae with partially suppressed

endogenous farnesoids

The results from the above experiments show that with respectto delaying attainment of pupariation, the activity of MF was moresimilar to that of JH III, than was its effect in comparison to JH III forpreventing prepupal animals from reaching adult eclosion. Weconsidered the possibility that the relatively weaker disruptiveeffect on normal development post-pupariation by MF than by JHIII (Figs. 2 and 3) might relate to the endogenous methyl farnesoidtiters, in that the exogenous-sourced compound is acting in thecontext of the endogenously produced titers. Hence, we took[(Fig._3)TD$FIG]

[(Fig._4)TD$FIG]

Fig. 4. Rescue by farnesol of a developmental block in larvae expressing Di11-driven

HMGCR-RNAi in their corpora allatal cells. The indicated dose of farnesol was

applied to the diet of individually reared larvae, which in a dose-dependent manner

rescued larval attainment of pupariation. Sibling control larvae containing the

HMGCR-RNAi transgene and a stubble-marked TM3 balancer (instead of the Di11

driver) did not exhibit blocked pupariation (n = 68–134 larvae per treatment).

[(Fig._5)TD$FIG]

Fig. 5. Activity of MF to prevent adult eclosion in genetic background of suppressed

methyl farnesoid production (HMGCR-RNAi). MF: methyl farnesoate; JH III: juvenile

hormone III; Bis: bisepoxy JH III; M/J/B: combined provision of MF, JH III and bisJH III

at the 6.2, 0.13, and 6.2 mmol doses, respectively, based on approximate

proportions of the hormones detected during late larval development. Larvae

expressing HMGCR-RNAi in the corpora allatal cells under the milder driver Di3.3

attain pupariation and then adult eclosion. However, their sensitivity to MF

blocking prepupal development to the eclosing adult is increased as compared to

MF activity on normal larvae (compare to Fig. 2) (chi-square, p < 0.0001) (n = 160

for each treatment).

[(Fig._6)TD$FIG]

Fig. 6. (A) Developmental phenotype of RNAi-inhibition of JH esterase. Animals

were genetically crossed to yield larvae expressing a JHesterase-RNAi driven by an

actin5C promoter (Gal4-UAS binary system). Siblings generated in that cross (that

carried the RNAi transgene but in the presence of a Cyo-marked balancer

chromosome, instead of a chromosome containing an actin5C-driven Gal4

construct) serve as positive controls for successful puparial development to

adult eclosion. Numbers emerging are shown, with percentages in parenthesis. The

pattern of suppressed emergence of both males and females expressing the RNAi-

JHesterase evidences a marked blockage in attainment of adult eclosion. (B) Normal

prepupal expression profile of JH esterase gene. Data for two independent replicates

are shown (except 8 h has a single point). Curve shown is drawn freehand. (C)

Example phenotype of failed adult eclosion in JHesterase-RNAi expressing animals,

which presumably experienced persistent levels of uncleared prepupal methyl

farnesoids.

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–14641460

advantage of the ability to genetically disrupt the endogenousproduction of all three methyl farnesoids. When the stronger Di11driver was used to drive the HMGCR-RNAi to suppress the methylfarnesoid production during larval development, very few larvaeattained pupariation (Fig. 4, first column bar). When dietaryfarnesol was provided from the outset of larval life, to rescue thelarval methyl farnesoid biosynthesis (that would produce all threeendogenous methyl farnesoids), the exposed animals developed tofeeding, growing 3rd instar larvae, which then in a dose-dependentmanner were rescued to attain pupariation (Fig. 4). However, weshowed previously that dietary provision instead of either MF or JHIII to larvae with suppressed methyl farnesoid production wasactually more toxic in preventing larval survival than thesuppression of methyl farnesoid production itself (Jones et al.,2010). In order to individually test MF against JH III in a less methylfarnesoid-suppressed background, we used in the present study aweaker driver (Di 3.3) that does not prevent most larvae fromsurviving to pupariation (unp. observations). Methyl farnesoidbiosynthetic activity in these larvae is presumably between that ofnormal larvae and the strongly suppressed (Di11) larvae. Theexposed larvae fed, grew, and molted to the 2nd instar, and then tothe 3rd instar, which then also continued to feed and grow. Dailyobservations did not discern any difference between the exposedand control animals, in their occurrence on the food during thefeeding periods between the molts. JH III in the diet exhibited againa strong effect and prevented the attainment of adult eclosion(p > 0.05 for 100% dose of JH III in Fig. 2 vs. Fig. 5). However, thetoxicity of dietary MF in preventing post-pupariation animals fromeclosing as normal adults was enhanced (Fig. 5, 30% eclosed),relative to the effect of MF on development of normal prepupae toadults (Fig. 2, 80% eclosed) (chi-square, p < 0.0001, comparing100% doses). These data, and the results above for effects of dietarymethyl farnesoids on normal larvae, strongly suggest that farnesol,methyl farnesoate, JH III and bisJH III in the diet do in fact penetratethe exposed larvae. If these compounds had not penetrated thefeeding, growing, and molting larvae, the positive or negativeeffects on development by the given compound would not havebeen observed.

3.4. Exposure to abnormally high, but endogenously sourced

methyl farnesoids

The above experiments exposed the prepupal animals toelevated methyl farnesoid levels by way of exogenous exposure.It could be argued that the results, though pharmacologicallyinteresting, only indirectly provide clues as to actions or potentialactions of endogenously produced levels of methyl farnesoids.Hence, we used a genetic means to deliver an RNAi topresumptively reduce the activity level of JH esterase (which

was not measured here), that specifically hydrolyzes the methylfarnesoids (Crone et al., 2007; Kamita et al., 2003). This enzyme isreported to markedly increase after puparium formation, however,because of some differences in the reported timing of this peak inDrosophila (Campbell et al., 1992; Kethidi et al., 2005; Klages andEmmerich, 1979; Niwa et al., 2008), we made here a higherresolution analysis of its prepupal expression. As shown in Fig. 6B,during the period following attainment of the white pupariumstage, expression is low until a marked increase at 6 h, whichpersists through 9 h. But then, the abundance of the mRNA rapidlydecreases at 11 h.

[(Fig._7)TD$FIG]

Fig. 7. Methyl farnesoate secretion and receptor binding in Aedes aegypti. (A) Exposed corpora allata (in removed head) or explanted accessory glands were cultured in vitro

and biosynthesized JH III or MF were measured by incorporation of tritiated methyl ester from L-[methyl-H3]methionine methyl donor. The culture medium was separately

measured for secreted JH III and MF, and the indicated cultured tissue was measured for biosynthesized JH III and MF contained within or adhering to the tissue. Measured

biosynthesis is expressed as fmol/CA (or accessory gland)/4 h � SEM. The male CA, female CA and male accessory glands were all detected to biosynthesize and release both

methyl farnesoate and JH III. (B) Recombinant nuclear receptor ultraspiracle from A. aegypti (isoform A) was detected in fluorescence quench binding assay to bind methyl farnesoate

as low as at 100 nM. Numbers shown above the histogram bar are the number of independent receptor preparations used to generate the shown average % fluorescence quenching

by the tested ligand.

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–1464 1461

We then used the RNAi system to inhibit appearance of JHesterase activity. A high degree of mortality was observed inanimals (of both sexes) expressing the JHesterase-RNAi under thecontrol of the actin5C-Gal4 driver, as compared to the sibling Cyo-balancer controls carrying only the unexpressed JHesterase-RNAi

transgene (Fig. 6A). As seen in Fig. 6C, the phenotype observedunder these conditions was failed emergence of apparentlypharate adults (possessing a bristled adult abdomen), which issimilar to a phenotype observed in animals exposed to exogenousdietary methyl farnesoids.

3.5. Applicability of concepts on methyl farnesoate to a lower dipteran

The above data, and those of Jones et al. (2006, 2010) support ahigher dipteran endocrine model in which methyl farnesoate issecreted by the corpora allata cells and binds to the nuclearreceptor ultraspiracle. These data prompt an experimentalconsideration of whether the model is also applicable to lowerdipterans, e.g., mosquitoes. As shown in Fig. 7A, when incubated inculture, the exposed corpora allata of both male and female A.

aegypti secreted not only JH III into the culture medium, but alsosecreted biosynthesized methyl farnesoate. In addition, the maleaccessory gland (previously only known as a source of epoxidizedmethyl farnesoid), also biosynthesized and secreted methylfarnesoate. Further, the A. aegypti ultraspiracle (isoform A) boundto methyl farnesoate, apparently with much higher affinity than toJH III (Fig. 7B).

4. Discussion

4.1. Targets of methyl farnesoid signaling

During the nearly 50 years since the discovery of the chemicalstructure of juvenile hormone, concepts on ‘how JH acts’ havebecome progressively more complex. The original concept that ‘JH’has ‘a’ receptor gave way to a view that JH may have more than onereceptor (e.g., Wheeler and Nijhout, 2003; Wang et al., 2009).However, additional studies during the past decade have raised theprospect that there actually exist in larval circulation multiplemethyl farnesoid hormones, each of which may have a receptor(s).In Drosophila, three methyl farnesoids are known to be secretedfrom the corpora allatal cells of the ring gland: MF, JH III and bisJH

III. In addition, there are three candidate methyl farnesoidreceptors: ‘‘ultraspiracle’’ (USP), that binds MF with nanomolaraffinity (Jones et al., 2006); ‘‘methoprene tolerant’’ (MET), thatbinds JH III with nanomolar affinity (Miura et al., 2005), and ‘‘germcell expressed’’ (GCE, Wilson et al., 2010), which apparently has notyet been tested for binding to bisJH III (or other methyl farnesoids).

The results of the studies reported here or contained in otherpapers relating to this symposium further raise the question as towhether one or more methyl farnesoid is targeting a differentaspect of an organ formation, or whether they are providingmultiple signal integration into the same aspect of the event, orwhether their signaling is redundant (e.g., Boerboom et al., 2000;Davis et al., 2007). For example, when differentiation ofproliferating eye discs is experimentally induced by the ecdy-sone-producing cells of wandering stage ring glands, thatdifferentiation is suppressed by cosecretions produced by theportion of the ring gland that produces the three methyl farnesoids(Vogt, 1943; Richard et al., 1989a,b). When differentiation of theprepupal visual system is precociously released by genetic ablationof the 3rd instar corpora allatal cells, that precocious differentia-tion can be suppressed by either exogenous JH III or exogenous JHanalog (Riddiford et al., 2010). However, that suppression ismediated only in part by MET, the putative JH III receptor(Riddiford et al., 2010). Those authors proposed that endogenousJH III correctly times the visual system maturation in part throughMET and in part through another signaling pathway yet to beidentified. A remaining possibility is that a different methylfarnesoid also provides signaling to an aspect of visual systemdevelopment, and that at the high exogenous dose JH III is alsocross-binding with the receptor (that is not MET) of that othermethyl farnesoid.

The model of different methyl farnesoids having nonredundanttargets finds support in the change in relative activities of MF:JHIII:bisJH III in the three teratogenic assays either used here orreported by Harshman et al. (2010). First, in a ‘delay in wanderingbehavior’ assay, our studies here found that both MF and JH IIIexhibited similar activities when delivered in the food to delaypupariation of otherwise normal larvae. Second, in a ‘larval death’assay, Harshman et al. (2010) reported that dietary MF was moreactive that JH III in causing larvae to die without reachingpupariation. Importantly, those authors found that trifluoroMF(fluorinated to inhibit epoxidation to JH III) was also more active in

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–14641462

that assay than was JH III, showing that the activity of theexogenous MF was not by way of its putative conversion to JH III.Third, Sehnal and Zdarek (1976) used a later time of treatment, of‘‘post-feeding’’ (i.e., wandering) larvae, before pupariation, andobserved that for Calliphora vomitora, MF was more active thanJH III in preventing adult eclosion from the puparium. Fourth, inan assay that instead treated animals yet later, at ‘whitepuparium’ stage, both our study here and that of Harshmanet al. (2010) found that JH III was much more active than MF inpreventing prepupae from developing to eclosed adults. Theactivity of the third compound, bisJH III, was also discordant witheither MF or JH III: it was the least active in the first and secondassay above, but was intermediate between JH III and MF in thefourth assay.

An additional consideration in interpreting these data is thatwhen the given methyl farnesoid compound is applied to normallarvae, it is merely adding to its respective endogenous titer (Staal,1977). If the endogenously produced compound is alreadysufficiently high to saturate its receptor(s), then adding more ofthe compound would have little effect on the signaling pathwaysmediated by that receptor. Instead, at sufficiently high pharmaco-logical doses, the compound might cross-activate other receptorsthat are not normally a target. For example, JH III has onlymicromolar affinity for USP, and published JH III titers forDrosophila indicate an average concentration of JH III across thebody in the low nanomolar range (Sliter et al., 1987; Bownes andRembold, 1987). However, if JH III is presented to USP at a sufficientconcentration, it will bind to the USP (Jones et al., 2001) and thereceptor then activates transcription of a susceptible promoter (Xuet al., 2002; Fang et al., 2005), or may potentiate 20-OH ecdysonetranscriptional activation (Henrich et al., 2003, 2009). Ourpreliminary data are that during the late final instar, MF is presentin circulation at a much higher concentration than is JH III.Unfortunately, the Kd of MF and bisJH III for MET (and GCE) are notyet known. Nevertheless, while acknowledging these caveats, thedata from our studies and those of Harshman et al. (2010) areparsimonious with a model in which MF and JH each can exert arespective activity in the presently assessed biological endpointsoccurring prior to pupariation.

4.2. Developmental consequence of absence of methyl farnesoids

Classical endocrine transplantation studies in higher dipterans,in which methyl farnesoid-producing corpora allata wereimplanted into otherwise intact feeding 3rd instar larvae, observeda delay in attainment of the wandering stage (Possompes, 1953).This result engendered the anticipation decades later that geneticremoval of methyl farnesoids from feeding 3rd instar larvae wouldcause a precocious attainment of the wandering stage. However,several recent studies that used genetic means to suppress 3rdinstar methyl farnesoid production each show instead that theexperimental larvae exhibited a delay in attainment of wandering(Liu et al., 2009; Jones et al., 2010; Riddiford et al., 2010).

In hindsight, that outcome was perhaps forecast by theadditional experiments of Possompes (1953), using the higherdipteran Calliphora erythrocephala. In his astutely designedendocrine studies, removal of the entire ring gland (containingboth edysone-producing cells and the methyl farnesoid-producingcorpora allata cells) from young 3rd instar larvae caused extensionof the feeding stage (delay of wandering). When he implanted intothose young 3rd instar larvae an entire ring gland from donors atthe very end of their feeding stage, the cessation of feeding andinitiating of wandering in the recipients was restored. If, instead,the implanted ring gland (from donors at the end of the feedingstage) did not include the corpora allatal region (therebymaintaining the recipient in a methyl farnesoid ablated status),

then rescue of faster attainment of wandering did not occur. Thatis, when the portion of the ring gland producing a dose of ecdysonesufficient to end the feeding stage was present, but in the context ofthe young 3rd instar being without any production of theendogenous three methyl farnesoids, the larva exhibited a delayin reaching of the wandering stage, rather than precociousattainment of the wandering stage.

4.3. Phenomenon of sensitized response to methyl farnesoid

imbalance

We have previously reported, and confirmed again here, thecurious occurrence of a sensitized response to methyl farnesoidexposure. When Drosophila larvae experience throughout larvaldevelopment a sufficiently suppressed production of all threemethyl farnesoids, most die at the 2nd to 3rd instar molt, and mostof the few attaining pupariation do not survive to adult eclosion(Jones et al., 2010). Farnesol is a dedicated precursor to all threemethyl farnesoids in the biosynthetic pathway in the corpora allata(Belles et al., 2005). When dietary farnesol was provided to those‘methyl farnesoid-suppressed’ larvae, such that presumablybalanced normal production of all three methyl farnesoids canbe resumed, the animals were rescued to pupariation in a dose-dependent manner. Yet, when JH III or MF were insteadindividually administered to these ‘methyl farnesoid-suppressed’larvae from the beginning of larval development, the deathphenotype was not rescued, even in part, and in fact the severity ofthe lethal phenotype was increased (Jones et al., 2010). In thepresent study, we used a milder suppression of all three methylfarnesoids throughout larval development, such that the methylfarnesoid-suppressed prepupae successfully reached adult eclo-sion. However, when these larvae were also at the same timeexogenously administered MF, it again caused a greater dose-dependent mortality to occur than was seen in normal animals thatwere administered the same exogenous MF treatment. This‘unbalanced methyl farnesoid rescue’, in the background of eithera strong or milder suppression of larval methyl farnesoidproduction, yields an increased toxicity, rather than rescue, bothin the context of larval–larval molting (Jones et al., 2010) and inprepupal to adult development (our studies here). So far as we areaware, this phenomenon of toxicity arising from unbalancedmethyl farnesoid rescue has not been previously described (Yinet al., 1995).

4.4. Functional role of Drosophila prepupal JH esterase peak

Recently, Riddiford et al. (2010) have provided evidence that, inDrosophila, circulating prepupal JH III, at least in part, has afunction to regulate the timing of development of the visualsystem, similar to the role previously described in Lepidoptera. Thefunctional role and regulation of the subsequent prepupal ‘‘JHesterase’’ peak that occurs in the prepupa of many holometabolousinsects has been postulated to be to remove the JH in prepupalcirculation, lest it disrupt subsequent pupal to adult metamorpho-sis (DeKort and Granger, 1996). In this model, the circulatingresidual JH (methyl farnesoids) directly induce the JH esterasepeak.

Most evidence for this role of the prepupal JH esterase has thusfar come from lepidopteran models. In vivo chemical inhibition ofJH esterase activity during the final instar feeding stage results inan elevation of the JH titer (Jones et al., 1990). Similar chemicalinhibition of the prepupal JH esterase peak can result in a pupal–pupal molt (Jones and Hammock, 1985). Hence, in at least somelepidopteran systems the prepupal JH esterase peak appears to benecessary to clear prepupal JH, so that pupal to adult developmentmay proceed.

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–1464 1463

Our fine-resolution determination of the timing of the ‘‘JHesterase’’ peak during the prepupal stage corresponds nicely to theend of the developmental window during which Riddiford et al.(2010) evidenced an active function of JH III to coordinate thetiming of visual system development. In addition, assuming thatthe JHesterase-RNAi expression did reduce the level of JH esteraseactivity, the lethal outcome shown here is the first experimentalevidence that methyl farnesoids are circulating at a level in earlyprepupa that, if not cleared by this enzyme, will then disruptnormal development to adult eclosion. We observed here a similarphenotype (failed adult eclosion) for exogenous application ofmethyl farnesoids (white puparium assay) and for persistentendogenous methyl farnesoids (RNAi-JHesterase). This provides thefirst experimental evidence that developmental phenotypesobserved by applying exogenous methyl farnesoids to prepupaeare informative about pathways of potential endogenous methylfarnesoid reception that remain accessible during pupal to adultmaturation in Drosophila.

5. Conclusions

The present study has provided additional evidence thatregulation of developmental programs involves methyl farnesoate,in addition to the classical JH III. We also provided evidence thatthat prepupal JH esterase is necessary to clear one or moreprepupal methyl farnesoids. Until now, it is the lepidopteran JHesterase promoters that have been utilized as models to investigatedirect JH regulation of gene activity (Harshman et al., 1994; Kethidiet al., 2005; Xu et al., 2002). Those investigations have generated amodel in which USP, the ecdysone receptor, MET and possibly GCE(Godlewski et al., 2006; Wilson et al., 2010) are all part of aregulatory complex that transduces the JH signaling to the JHesterase promoter (Bitra and Palli, 2009). Now, recent studies,including those presented in this symposium, are clarifying theparticipation of these receptors in transducing signaling by MF,20E, JH III (and bisJH III?) in Drosophila. Given the additionalindications in this present study of the functional necessity of thisenzyme for pupal development to the adult, the opportunities ofthe Drosophila genetic system are now available towards discern-ing mechanisms of MF, JH (and bisJH III) action in specific generegulation. Our data also indicate that the Drosophila model forbinding of secreted MF to the nuclear receptor USP is applicable tounderstanding MF/USP interaction in lower dipterans, such asmosquitoes.

Acknowledgements

Support for the research described here was provided, in part,by NIH grant GM075248 (to GJ); by NSF IBN0344933 (to LMR); bythe Florida Department of Agriculture and Consumers Servicesgrant 75900 and grants 97-00081 and 2007-037 from the UnitedStates–Israel Binational Science Foundation (BSF), Jerusalem (toDB). We express appreciation to Larry Harshman for providing ourearly access to the manuscript he submitted in connection withthis symposium issue. Dr. Alex Raikhel kindly provided the cDNAfor A. aegypti ultraspiracle isoform A. We want to acknowledgethe two reviewers selected by the journal that reviewed themanuscript.

References

Belgacem, Y.H., Martin, J.R., 2007. Hmgcr in the corpus allatum controls sexualdimorphism of locomotor activity and body size via the insulin pathway inDrosophila. PLoS ONE 2, e187.

Belles, X., Martın, D., Piulachs, M.D., 2005. The mevalonate pathway and thesynthesis of juvenile hormone in insects. Annual Review of Entomology 50,181–199.

Bitra, K., Palli, S.R., 2009. Interaction of proteins involved in ecdysone and juvenilehormone signal transduction. Archives of Insect Biochemistry and Physiology70, 90–105.

Boerboom, D., Pilon, N., Behdjani, R., Silversides, D.W., Sirois, J., 2000. Expressionand regulation of transcripts encoding two members of the NR5A nuclearreceptor subfamily of orphan nuclear receptors, steroidogenic factor-1 andNR5A2, in equine ovarian cells during the ovulatory process. Endocrinology141, 4647–4656.

Borovsky, D., Carlson, D.A., 1992. An in vitro assay for biosynthesis and metabolismof juvenile hormone by exposed corpora allata of Aedes aegypti. Journal ofMedical Entomology 29, 318–324.

Borovsky, D., Carlson, D.A., Ujvary, I., 1992. In vivo and in vitro biosynthesis andmetabolism of MF, juvenile hormone III, and juvenile hormone III acid in themosquito Aedes aegypti. Journal of Medical Entomology 29, 619–629.

Borovsky, D., Carlson, D.A., Hancock, R.G., Rembold, F., van Handel, E., 1994.De novo biosynthesis of juvenile hormone III and I by the accessory glandsof the male mosquito. Insect Biochemistry and Molecular Biology 24, 437–444.

Bownes, M., Rembold, H., 1987. The titre of juvenile hormone during the pupal andadult stages of the life cycle of Drosophila melanogaster. European Journal ofBiochemistry 164, 709–712.

Campbell, P.M., Healy, M.J., Oakeshott, J.G., 1992. Characterisation of juvenilehormone esterase in Drosophila melanogaster. Insect Biochemistry and Molec-ular Biology 22, 665–677.

Campbell, P.M., Oakeshott, J.G., Healy, M.J., 1998. Purification and kinetic charac-terisation of juvenile hormone esterase from Drosophila melanogaster. InsectBiochemistry and Molecular Biology 28, 501–515.

Caughey, W.S., Smythe, G.A., O’Keeffe, D.H., Maskasky, J.E., Smith, M.I., 1975.Heme A of cytochrome c oxicase. Structure and properties: comparisons withhemes B, C, and S and derivatives. Journal of Biological Chemistry 250, 7602–7622.

Crone, E.J., Sutherland, T.D., Campbell, P.M., Coppin, C.W., Russell, R.J., Oakeshott,J.G., 2007. Only one esterase of Drosophila melanogaster is likely to degradejuvenile hormone in vivo. Insect Biochemistry and Molecular Biology 37, 540–549.

Cui, G., Merz Jr., K.M., 2007. Computational studies on the farnesyltransferaseternary complex II: the conformational activation of farnesyldiphosphate.Biochemistry 46, 12375–12378.

Davis, M.M., Yang, P., Chen, L., O’Keefe, S.L., Hodgetts, R.B., 2007. The orphan nuclearreceptor DHR38 influences transcription of the DOPA decarboxylase gene inepidermal and neural tissues of Drosophila melanogaster. Genome 50, 1049–1060.

DeKort, C.A.D., Granger, N.A., 1996. Regulation of JH titers: the relevance ofdegradative enzymes and binding proteins. Archives of Insect Biochemistryand Physiology 33, 1–26.

Emlen, D.J., Nijhout, H.F., 2000. The development and evolution of exaggeratedmorphologies in insects. Annual Review of Entomology 45, 661–708.

Fang, F., Xu, Y., Jones, D., Jones, G., 2005. Interactions of ultraspiracle with ecdysonereceptor in the transduction of ecdysone- and juvenile hormone-signaling. FEBSJournal 272, 1577–1589.

Godlewski, J., Wang, S., Wilson, T.G., 2006. Interaction of bHLH-PAS proteinsinvolved in juvenile hormone reception in Drosophila. Biochemical and Bio-physical Research Communications 342, 1305–1311.

Harshman, L.G., Song, K.-D., Casas, J., Schuurmans, A., Kuwano, E., Riddiford, L.M.,Hammock, B.D., 2010. Bioassays of compounds with potential juvenoid activityon Drosophila melanogaster: bisepoxide juvenile hormone III metabolites andMFs. Journal of Insect Physiology 56, 1465–1470.

Harshman, L.G., Ward, V.K., Beetham, J.K., Grant, D.F., Grahan, L.J., Zraket, C.A.,Heckel, D.G., Hammock, B.D., 1994. Cloning, characterization, and genetics ofthe juvenile hormone esterase gene from Heliothis virescens. Insect Biochemis-try and Molecular Biology 24, 671–676.

Henrich, V.C., Beatty, J., Ruff, H., Callender, J., Grebe, M., Spindler Barth, M., 2009. In:Smagghe, G. (Ed.), The Multidimensional Partnership of EcR and USP IN:Ecdysones: Structures and Functions. Springer Science + Business Media B.V.,p. 361.

Henrich, V.C., Burns, E., Yelverton, D.P., Christensen, E., Weinberger, C., 2003.Juvenile hormone potentiates ecdysone receptor-dependent transcription ina mammalian cell culture system. Insect Biochemistry and Molecular Biology33, 1239–1247.

Jones, D., Jones, G., 2007. Farnesoid secretions of dipteran ring glands: what we doknow and what we can know. Insect Biochemistry and Molecular Biology 37,771–798.

Jones, G., Hammock, B.D., 1985. Critical roles of juvenile hormone and its esterase inthe prepupa of Trichoplusia ni. Archives of Insect Biochemistry and Physiology 2,397–404.

Jones, G., Hanzlik, T., Hammock, B.D., Schooley, D.A., Miller, C.A., Tsai, L.W., Baker,F.C., 1990. The juvenile hormone titre during the penultimate and ultimatelarval stadia of Trichoplusia ni. Journal of Insect Physiology 36, 77–83.

Jones, G., Wozniak, M., Chu, Y., Dhar, S., Jones, D., 2001. Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle. InsectBiochemistry and Molecular Biology 32, 33–49.

Jones, G., Jones, D., Teal, P., Sapa, A., Wozniak, M., 2006. The retinoid-X receptorortholog, ultraspiracle, binds with nanomolar affinity to an endogenous mor-phogenetic ligand. FEBS Journal 273, 4983–4996.

Jones, D., Jones, G., Teal, P., Hammac, C., Messmer, L., Osborne, K., Belgacem, Y.H.,Martin, J.R., 2010. Suppressed production of methyl farnesoid hormones yields

G. Jones et al. / Journal of Insect Physiology 56 (2010) 1456–14641464

developmental defects and lethality in Drosophila larvae. General and Compar-ative Endocrinology 165, 244–254.

Kamita, S.G., Hinton, A.C., Wheelock, C.E., Wogulis, M.D., Wilson, D.K., Wolf, N.M.,Stok, J.E., Hock, B., Hammock, B.D., 2003. Juvenile hormone (JH) esterase: whyare you so JH specific? Insect Biochemistry and Molecular Biology 33, 1261–1273.

Kethidi, D.R., Xi, Z., Palli, S.R., 2005. Developmental and hormonal regulation ofjuvenile hormone esterase gene in Drosophila melanogaster. Journal InsectPhysiology 51, 393–400.

Kiguchi, K., Riddiford, L.M., 1978. A role of juvenile hormone in pupa development ofthe tobacco hornworm, Manduca sexta. Journal of Insect Physiology 24, 673–680.

Klages, G., Emmerich, H., 1979. Juvenile hormone metabolism and juvenile hor-mone esterase titer in heamolymph and peripheral tissues of Drosophila. Journalof Comparative Physiology 132, 319–325.

Krag, S.S., 1998. The importance of being dolichol. Biochemical and BiophysicalResearch Communications 243, 1–5.

Liu, Y., Sheng, Z., Liu, H., Wen, D., He, Q., Wang, S., Shao, W., Jiang, R.J., An, S., Sun, Y.,Bendena, W.G., Wang, J., Gilbert, L.I., Wilson, T.G., Song, Q., Li, S., 2009. Juvenilehormone counteracts the bHLH-PAS transcription factors MET and GCE toprevent caspase-dependent programmed cell death in Drosophila. Development136, 2015–2025.

Miura, K., Oda, M., Makita, S., Chinzei, Y., 2005. Characterization of the Drosophilamethoprene-tolerant gene product. Juvenile hormone binding and ligand-de-pendent gene regulation. FEBS Journal 272, 1169–1178.

Moshitzky, P., Applebaum, S.W., 1995. Pathway and regulation of JHIII-bisepoxidebiosynthesis in adult Drosophila melanogaster corpus allatum. Archives of InsectBiochemistry and Physiology 30, 225–237.

Niwa, R., Niimi, T., Honda, N., Yoshiyama, M., Itoyama, K., Kataoka, H., Shinoda, T.,2008. Juvenile hormone acid O-methyltransferase in Drosophila melanogaster.Insect Biochemistry and Molecular Biololgy 38, 714–720.

Possompes, B., 1953. Recerches experimentales sur le determinisme de la meta-morphose de Calliphora erythrocephala meig. Archives de Zollogie Experimen-talr et Generale 89, 203–364.

Postlethwait, J.H., 1974. Juvenile hormone and the adult development of Drosophila.Biological Bulletin 147, 119–135.

Richard, D.S., Applebaum, S.W., Sliter, T.J., Baker, F.C., Schooley, D.A., Reuter, C.C.,Henrich, V.C., Gilbert, L.I., 1989a. Juvenile hormone bisepoxide biosynthesis invitro by the ring gland of Drosophila melanogaster: a putative juvenile hormonein the higher Diptera. Proceedings of the National Academy of Sciences of theUnited States of America 86, 1421–1425.

Richard, D.S., Applebaum, S.W., Gilbert, L.I., 1989b. Developmental regulation ofjuvenile hormone biosynthesis by the ring gland of Drosophila melanogaster.Journal of Comparative Physiology B 159, 383–387.

Riddiford, L.M., Ashburner, M., 1991. Effects of juvenile hormone mimics on larvaldevelopment and metamorphosis of Drosophila melanogaster. General andComparative Endocrinology 82, 172–183.

Riddiford, L.M., Hiruma, K., Zhou, X., Nelson, C.A., 2003. Insights into the molecularbasis of the hormonal control of molting and metamorphosis from Manducasexta and Drosophila melanogaster. Insect Biochemistry and Molecular Biology33, 1327–1338 Review.

Riddiford, L.M., Truman, J.W., Mirth, C.K., Shen, Y.C., 2010. A role for juvenilehormone in the prepupal development of Drosophila melanogaster. Develop-ment 127, 1117–1126.

Roe, R.M., Jesudason, P., Venkatesh, K., Kallapur, V.L., Anspaugh, D.D., Majumder, C.,1993. Developmental role of juvenile hormone metabolism in Lepidoptera.American Zoologist 33, 375–383.

Saiki, K., Mogi, T., Ogura, K., Anraku, Y., 1993. In vitro heme O synthesis by the cyoEgene product from Escherichia coli. Journal of Biological Chemistry 268, 26041–26045.

Sehnal, F., Zdarek, J., 1976. Action of juvenoids on the metamorphosis of cyclor-rhaphous Diptera. Journal of Insect Physiology 22, 674–682.

Sliter, T.J., Selakf, B.J., Baker, C., Schooley, D.A., 1987. Juvenile hormone in Drosophilamelanogaster: identification and titer determination during development. In-sect Biochemistry 17, 161–165.

Staal, G.B., 1977. Insect control with insect growth regulators based on insecthormones. Pontificiae Academiae Scientiarum Scripta Varia 41, 353–383.

Schenk, B., Fernandez, F., Waechter, C.J., 2001. The ins(ide) and outs(ide) of dolichylphosphate biosynthesis and recycling in the endoplasmic reticulum. Glycobiol-ogy 11 (5), 61R–70R.

Sparks, T.C., Hammock, B.D., Riddiford, L.M., 1983. The haemolymph juvenilehormone esterase of Manduca sexta (L.)-inhibition and regulation. Insect Bio-chemistry 13, 529–541.

Teal, P.E., Gomez-Simuta, Y., Proveaux, A.T., 2000. Mating experience and juvenilehormone enhance sexual signaling and mating in male Caribbean fruit flies.Proceedings of the National Academy of Sciences United States of America 97,3708–3712.

Truman, J.W., Riddiford, L.M., 2002. Endocrine insights into the evolution ofmetamorphosis in insects. Annual Review of Entomology 47, 467–500 (Review).

Truman, J.W., Riddiford, L.M., 2007. The morphostatic actions of juvenile hormone.Insect Biochemistry and Molecular Biology 37, 761–770 (Review).

Vogt, M., 1943. Zur Produktion und Bedeutung metamorphosefordernder Hormonewahrend der Larvaenentwicklung von Drosophila. Biologisches Zentralblatt 63,395–446.

Wheeler, D.E., Nijhout, H.F., 2003. A perspective for understanding the modes ofjuvenile hormone action as a lipid signaling system. Bioessays 25, 994–1001.

Wang, J., Lindholm, J.R., Willis, D.K., Orth, A., Goodman, W.G., 2009. Juvenilehormone regulation of Drosophila Epac—a guanine nucleotide exchange factor.Molecular and Cellular Endocrinology 305, 30–37.

Williams, C.M., 1953. Morphogenesis and the metamorphosis of insects. HarveyLectures 47, 126–155.

Wilson, T.G., Fabian, J.A., 1986. Drosophila melanogaster mutant resistant to achemical analog of juvenile hormone. Developmental Biology 118, 190–201.

Wilson, T.G., 2004. The molecular site of action of juvenile hormone and juvenilehormone insecticides during metamorphosis: how these compounds killinsects. Journal of Insect Physiology 50, 111–121.

Wilson, T.G., Fujiwara, Y., Baumann, A., 2010. Functional divergence of methoprenetolerant (Met) and germ cell expressed (gce), paralogs involved in juvenilehormone activity in the genus Drosophila. Journal of Insect Physiology 56, 1445–1455.

Wozniak, M.I., Chu, Y., Fang, F., Xu, Y., Riddiford, L.M., Jones, D., Jones, G., 2004.Alternative farnesoid structures induce different conformational outcomesupon the Drosophila ortholog of the retinoid X receptor, ultraspiracle. InsectBiochemistry and Molecular Biology 34, 1147–1162.

Xu, Y., Fang, F., Chu, Y.X., Jones, D., Jones, G., 2002. Activation of transcriptionthrough the ligand binding pocket of the orphan nuclear receptor ultraspiracle.European Journal of Biochemistry 269, 6026–6036.

Yin, C.M., 1994. Juvenile hormone III bisepoxide: new member of the insect juvenilehormone family. Zoological Studies 33, 237–245.

Yin, C.M., Zou, B.X., Jiang, M., Li, M.F., Qin, W., Potter, T.L., Stoffolano Jr., J.G., 1995.Identification of juvenile hormone III bisepoxide (JHB3), juvenile hormone IIIand MF secreted by the corpus allatum of Phormia regina (Meigen), in vitro andfunction of JHB3 either applied alone or as a part of a juvenoid blend. Journal ofInsect Physiology 41, 473–479.