Detection of juvenile hormone I,II and III in adult monarch butterflies (Danaus plexippus)

Hemolymph of Danaus plexippus (monarch butterfly) was analyzed for juvenile hormone titer by gas liquid chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. Laboratory reared, reproductively active, adult males and females contained JH I, II and III. JH II predominated with titers ranging from 2.3 to 11 ng/ml hemolymph. Titers of JH I and JH III were approx. 1 order of magnitude lower than those of JH II. JH I, II and III acids were also detected, but at levels generally lower than the corresponding JHs. JH titers in field collected reproductively inactive, gregarious adult monarchs, were 1–2 orders of magnitude lower than those of laboratory reared reproductively active monarchs. JH II was again the predominant JH in these animals.     

Disturbance of Adult Eclosion by Fenoxycarb,a Juvenile Hormone Mimic,in the Beet Armyworm,Spodoptera exigua

Effect of exogenous juvenile hormones (JHs) on pupal development was assayed in the beet armyworm, Spodoptera exigua. Fenoxycarb, a potent JH mimic, was applied topically to different ages of the pupae, and showed significant inhibition of normal adult eclosion even at 0.1 μg dose when it was applied at the early pupal stage (day 0). As the pupal development underwent, the susceptibility of the pupae fenoxycarb decreased. RH5992, a potent ecdysteroid mimic, did not, however, any similar inhibitory effect on the pupae. Natural JH types (JH I, JH II, and JH III) were applied on day 0 pupae to compare their inhibitory effects on adult eclosion. Both JH I and JH II significantly inhibited adult eclosion at 1.0 μg dose, but JH III did not even at 10.0 μg dose. It was noted that fenoxycarb-treated pupae showed little rectum development. Fenoxycarb did not, however, show any negative effect on the development of compound eye and wing imaginal discs, and on the pupal hemolymph protein pattern. These results suggest that there should be a commitment period requiring an absence of JH for a normal adult metamorphosis during early pupal development and that the endogenous type of JH in S. exigua is JH I or JH II or both JHs like other lepidopteran species.     

Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nauphoetacinerea

By the combined use of high-pressure liquid chromatography, $\text{Galleria}$ bioassay and gas chromatography/ chemical ionization/mass spectrometry we were able to isolate and identify the three known natural juvenile hormones (JHs) from haemolymph extracts of larval and adult females of the cockroach $\text{Nauphoeta}$$\text{cinerea}$. This is the first demonstration of the simultaneous occurrence of the three JHs in the same insect and the first time JH I and II have been identified in a hemimetabolous insect. Quantitative investigations show that the composition of the three JHs is different at different developmental stages. The haemolymph of larvae contains a high percentage of JH I and II, whereas the haemolymph of adult females in the oocyte maturation stage contains mostly JH III. This suggests more juvenilizing functions for JH I and II and more gonadotropic functions for JH III.     

Influence of juvenile hormone and mating on oogenesis and oviposition in the codling moth,Cydia pomonella

Oogenesis in the codling moth, Cydia pomonella, and the role of juvenile hormones (JHs) were addressed. Rudimentary ovarian structures were recognisable in day 3–4 pupae, when haemolymph JH was still undetectable by coupled gas chromatography‐mass spectrometry in the selected ion mode (GC‐MS/SIM). The presence of developing oocytes was observed by light microscopy on day 8, coincident with very low JH titres (0.74 ± 0.05 ng/ml JH II). Chorionation was only evident upon emergence, following an increase in JH in the pharate adult (0h old: 4.71 ± 0.34 ng/ml JH II). Analysis of haemolymph from virgin and mated females indicated that JH II was predominant, with approximately equal and lower quantities of JHs I and III (3.3‐ to 5.0‐fold less). When pupae or newly emerged adults were treated with JH homologues, no alteration in ovarian protein content was apparent, but the JH mimetic, fenoxycarb, depressed the number of oocytes filling ≥ 50% follicular volume. Chorion deposition was stimulated by JHs I, II, or III (10 μg), but not by fenoxycarb (0.05 μg, 10 μg). Mating provided correct stimuli for enhanced choriogenesis and egg laying, and, since haemolymph JH titres were concomitantly elevated (approximately 2‐fold), it was postulated that the rise in JH elicited both these events. Application of JHs to virgin females, however, could not mimic mating; only increases in choriogenesis were induced: JH‐treatment of virgins (or mated insects) significantly decreased oviposition rates over 24 and 48 h and markedly reduced the life‐time total number of eggs. Arch. Insect Biochem. Physiol. 41:186–200, 1999. © 1999 Wiley‐Liss, Inc.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Detection of only JH III in several life-stages of Nauphoeta cinerea and Thermobia domestica

We have conducted a reinvestigation into both the identification and quantification of juvenile hormone (JH) from several developmental stages of the cockroach, Nauphoeta cinerea, and the firebrat, Thermobia domestica, using a gas chromatography-mass spectrometric (GC/MS) method. We detected only JH III in these animals in contrast to prior studies in which JH I, II, and/or III had been reported using a different scheme relying on HPLC purification and subsequent GC/MS analysis under chemical ionization (CI) conditions. Very high levels (approximately 800 ng/g) of JH III were found in N. cinerea embryos at stages after dorsal closure whereas first stadium nymphs and female penultimate stadium nymphs contained only low levels (approximately 1 ng/g and approximately 7 ng/ml respectively); in adult females at the stage of rapid oocyte growth approximately 150 ng JH III per ml of hemolymph was measured. T. domestica nymphs and egg laying adults contained only low levels (approximately 1 ng/g) of JH III. The results emphasize the caution which must be used in interpreting results of procedures for analysis of JH at parts-per-billion levels, and also enforce prior observations that the higher JH homologs are not present except in the Lepidoptera.     

Juvenile hormone in Drosophila melanogaster: Identification and titer determination during development

Juvenile hormone (JH) III was identified in whole-body extracts of eggs, larvae, pupae, pharate adults and adults of Drosophila melanogaster. Titers of the hormone varied according to stage of development. Highest levels were found in post-feeding (wandering) larvae and adults; only low levels were found in feeding last-stadium larvae and in pupae. None of the other known JHs were detected (limit of detection ≈0.01 ng/g). These results are discussed in the light of the known physiological roles of JH in the development of Drosophila and other insects.     

Parasitoid-host endocrine relations: self-reliance or co-optation?

High titers of juvenile hormone (JH) maintain developmental arrest in Manduca sexta larvae parasitized by Cotesia congregata. Parasitized hosts exhibit up to 9.5 times greater amounts of total hemolymph JH (from 0.6±0.09 to 2.51±0.43 ng/ml) compared to non-parasitized controls. Elevated titers are observed throughout the fifth instar, even beyond egression of the parasitoids on day 5. GC–MS analysis revealed that in hemolymph of unparasitized control larvae, JH I is the major homolog and levels of JH III are negligible; in parasitized individuals the amounts of JH I, II, and III rise, and JH III predominates. Neck ligation ensured separation of M. sexta’s corpora allata from the posterior section, which contained most of the parasitoids in the infected insects. When the posterior region was sampled, JHs were not detected in the non-parasitzed larvae, but in those parasitized, JH III was found (1.98±0.29 ng/ml, 24 h post-ligation). JH III was the only homolog produced and secreted by the parasitoid in in vitro culture. This is the first report stating that a parasitoid secretes JH III and may contribute, at least in part, to the circulating titer in the host hemocoel, concurrently promoting host production of JH I and II.     

Ecdysteroids and juvenile hormones of whiteflies, important insect vectors for plant viruses

Ecdysteroids and juvenile hormones (JHs) regulate many physiological events throughout the insect life cycle, including molting, metamorphosis, ecdysis, diapause, reproduction, and behavior. Fluctuation of whitefly ecdysteroid levels and the identity of the whitefly molting hormone (20-hydroxyecdysone) have only been reported within the last few years. An ecdysteroid commitment peak that is associated with the reprogramming of tissues for a metamorphic molt in many holometabolous and some hemimetabolous insect species was not observed in last nymphal instars of either the sweet potato whitefly, Bemisia tabaci (Biotype B), or the greenhouse whitefly, Trialeurodes vaporariorum. Ecdysteroids reach peak levels 1-2 days prior to the initiation of the nymphal-adult metamorphic molt. Adult eye and wing differentiation which signal the onset of this molt begin earlier in 4th instar T. vaporariorum (Stages 4 and 5, respectively) than in B. tabaci (Stage 6), and the premolt peak is 3-4 times greater in B. tabaci ( approximately 400 fg/microg protein) than in T. vaporariorum ( approximately 120 fg/microg protein). The JH of B. tabaci nymphs and eggs was found to be JH III, supporting the view that JHs I and II are, with rare exception, only present in lepidopteran insects. In B. tabaci eggs, JH levels were approximately 10 times greater on day 2/3 (0.44 fg/egg or 0.54 ng/g) than on day 5 (0.04 fg/egg or 0.054 ng/g) post-oviposition. Approximately, 1.4 fg/2nd-3rd instar nymph (0.36 ng/g) was detected. It is probable that the relatively high level of JH in day 2/3 eggs is associated with the differentiation of various whitefly tissues during embryonic development.     

Simultaneous preparation of both enantiomers of juvenile hormones labeled at C-10 with tritium at high specific activity

We report an improved method for the synthesis of high specific activity insect [10-(3)H]juvenile hormones (JH) I, II, and III which affords both enantiomers of each in high optical purity. A synthetic route for JH I was modified to give higher yields and purity. We increased the specific activity of the synthetic [10-(3)H]JHs using normal phase liquid chromatography optimized to give near baseline resolution of [10-(3)H]JHs and unlabeled JHs. Racemic [10-(3)H]JHs and their corresponding diol metabolites were enantiomerically separated using a chiral column eluted with 2-propanol:hexane. Acidic hydration of the unnatural antipode of the [10-(3)H]JHs gives the diol antipode with the same stereochemistry as that from epoxide hydrolase action on the natural JH antipode. The [10-(3)H]JH diol enantiomers can also be resolved with the same chiral column using a more polar solvent. The synthesis of high specific activity chiral ethyl ester analogs of JH I and II can also be accomplished using this synthetic route.     

Titres of juvenile hormone I,II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.     

Enzyme immunoassays of JH III and JH III diol using acetylcholinesterase as tracer: An alternative to radioimmunoassay

Immunogens were prepared by coupling JH III and JH III diol to human serum albumin. Specific and sensitive antibodies were obtained by immunizing rabbits. Pure acetylcholinesterase (EC 3.1.1.7) from electric eel (Electrophorus electricus) was covalently coupled to the acids from hydrolyzing JH III or JH III diol. The enzyme immunoassays (EIA) were performed in 96-well microtiter plates coated with second antibody (pig anti-rabbit).The JH III EIA performance was equivalent to or better than radioimmunoassay using an iodinated tracer: the sensitivity was 0.91 ng/ml at 50% B/B₀, the detection limit was 0.2 ng/ml; cross-reactivity was less than 1% for the diols of JH I, JH II and JH III, and 30% for JH III acid. For the JH III diol assay, the EIA sensitivity was 1.9 ng/ml at 50% B/B₀ and the detection limit was 0.2 ng/ml; cross-reactivity was less than 1% for JH III and JH III acid, and 14 and 10% for JH I diol and JH II diol. Finally, use of semi-automatic apparatus allowed rapid and easy EIA analyses in which the enzyme label is a long-lived reagent and handling of radioactive compounds is avoided.     

The identification of an aphid juvenile hormone, and its titre in relation to photoperiod

ABSTRACT. Whole body extractions from larval and adult apterous forms of Megoura viciae , and from adult Aphis fabae , were analysed for the known insect juvenile hormones (JHs) by a gas chromatography-mass spectrometric method. Low levels of JH III were detected in both aphid species, the first identification of a juvenile hormone from an homopteran insect. Although the mean titre in adult M. viciae is higher in long-day than in short-day reared insects (0.12±0.03 v. 0.04±0.01 ng/g), titres were variable and measurements overlapped. The results are discussed in the context of the hormonal control of aphid polymorphism and the question of identity of homopteran and hemipteran JH.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Juvenile hormone and ovarian maturation in the diptera: A review of recent results

The roles of the JHs and ecdysteroids in ovarian maturation of adult Aedes aegypti, A. atropalpus, Musca domestica and Drosophila melanogaster have been characterized by comparing the effects of surgical (allatectomy, ovariectomy, decapitation, abdominal ligation), genetic, and nutritional (sugar feeding) manipulations. The results show that in all species JHs, or their mimics, and 20-hydroxyecdysone act in combination to stimulate ovarian maturation and vitellogenin (Vg) synthesis and that high doses of exogenous JHs, or their mimics, stimulate ovarian ecdysteroid synthesis, at least in A. aegypti, A. atropalpus, and D. melanogaster. Thus the gonadotropic regulatory mechanisms that exist in various dipteran species are more similar than originally suspected. In M. domestica, and possibly D. melanogaster, 20-hydroxyecdysone is present in the hemolymph at vitellogenic levels in newly emerged females and may persist in ovariectomized adults. If true for D. melanogaster, this could explain why topical application of JH, or its mimics, to ovariectomized, isolated abdomens is effective in stimulating Vg synthesis in the absence of the ovaries.     

A quantitative assay for the juvenile hormones and their precursors using fluorescent tags

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.

Methods

A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).

Conclusions

The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.

Significance

The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.     

Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male. Arch. Insect Biochem. Physiol. 38:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Hydrolytic degradation of juvenile hormones in haemolymph and corpora allata of Galleria mellonella (L).

Hydrolytic rates of juvenile hormones (JHs) I, II and III by the corpora cardiaca-corpora allata complex (CC-CA) and by the haemolymph of Galleria mellonella remain in the same order (III greater than I greater than II in CC-CA and I greater than III greater than II in haemolymph) throughout the last larval instar. Haemolymph hydrolytic activity shows peak at the end of feeding when 80 pmol JH I versus 15 pmol JH II is degraded per 1 microliter and minute; hydrolysis rapidly declines in the apolysing insects. Hydrolytic rates in CC-CA reach a maximum of 240 fmol/pair per min for JH III and 85 fmol/pair per min for JH II in pharate pupae. Brain implantations or chilling of freshly ecdysed last instar larvae, which are known to elevate JH titer and induce supernumerary larval molt, do not affect JH hydrolysis. The results indicate that the dominance of JH II in Galleria may be at least partly controlled by preferential hydrolysis of homologs I and III.     

Juvenile hormones and their titer regulation in Galleria mellonella

Juvenile hormones I, II and III occur in Galleria but JH II is dominant. Its concentration reaches a peak of 3 pmol/g body wt in the penultimate instar, drops to zero in the last larval instar and, except for a small peak in prepupae (0.2 pmol/g), remains undetectable until pharate adults. After emergence the titer reaches over 5 pmol/g in both sexes. Presence of JH II is associated with JH II acid; JH III acid occurs even more often, including stages lacking JH III. Brain implantation into freshly ecdysed last instar larvae effects a similar JH peak as in the penultimate instar and causes an extra larval molt. The opposite treatment, i.e. decerebration of fresh last instar larvae, elicits a continuous rise of JH II to 10 pmol/g and an increase of otherwise rare JH I to 3 pmol/g. Sham operations of these larvae or decerebration of old larvae elevate practically only JH II titer to 1–1.5 pmol/g. Implanted brain-corpora cardiaca-corpora allata complexes maintain in various hosts 0.14–1.6 pmol/g of JH II. The significance and regulation of these fluctuations in JH titer are discussed.     

The activity of juvenile hormone mimics for the eggs of Rhodnius prolixus

Twenty four juvenile hormone (JH) mimics of several different chemical classes were applied to freshly laid eggs of Rhodnius prolixus. Activity of the JH mimics was assessed in terms of their ability to inhibit eclosion. All chemical classes tested except one of the natural JHs and two very closely related long chain compounds contained members which inhibited eclosion; the most active chemicals were derivatives of geranyl para-aminobenzoic acid esters and aryl methylene dioxyphenyl ethers. In general, those chemicals of a particular class which had highest morphogenetic activity when applied to last instar larvae, were also most active in inhibiting eclosion. However, not all structure/activity relationships of JH mimics for last instar larvae were applicable to eggs; in particular the presence of a terminal epoxide group increased activity greatly for eggs but only slightly for last instar larvae. The dose per unit weight to inhibit eclosion is far greater than the equivalent dose to disrupt normal metamorphosis.