Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role?

Burying beetles, Nicrophorus orbicollis, depend on the location of an unpredictable resource, a small vertebrate carcass, for reproduction. When they discover a carcass, they undergo a correlated rapid rise in titers of juvenile hormone (JH) in the hemolymph and ovarian development. This study investigates the regulation of the changes in JH during breeding in both male and female burying beetles and the role of JH in ovarian development. JH biosynthesis by the corpora allata (CA), measured in vitro, increased in females within an hour of their discovery of a carcass and increased later in males. After returning to low rates as oviposition began, JH biosynthesis rose again 3 days later in females but not in males. Neither the ovaries nor testes synthesized JH. There was a concomitant fall in JH esterase activity within 12 h of discovery of the carcass in both males and females. Although the rise in JH titers and biosynthesis and the fall in JH esterase is correlated with ovarian development, application of methoprene or JH III in the absence of a carcass did not result in vitellogenin uptake by the oocytes. Therefore, we conclude that, in spite of the rapid rise in JH before oviposition, it is not sufficient to regulate vitellogenin synthesis and/or its uptake by the ovaries. We suggest that its role has been preempted to organize social behavior and coordinate parental behavior between mates.     

JH biosynthesis by reproductive tissues and corpora allata in adult longhorned beetles, Apriona germari

We report on juvenile hormone (JH) biosynthesis from long‐chain intermediates by specific reproductive tissues and the corpora allata (CA) prepared from adult longhorned beetles, Apriona germari. The testes, male accessory glands (MAGs), ovaries, and CA contained the long‐chain intermediates in the JH biosynthetic pathway, farnesoic acid (FA), methyl farnesoate (MF), and JH III. The testes and ovaries, but not CA, produced radioactive JH III after the addition of ³H‐methionine and, separately, unlabeled methionine, to the incubation medium. We inferred that endogenous FA is methylated to MF in the testes and ovaries. Addition of farnesol led to increased amounts of FA in the testes, MAGs, ovaries, and CA, indicating oxidation of farnesol to FA. Addition of FA to incubation medium yielded increased JH III, again indicating methylation of FA to MF in the testes, MAGs, ovaries, but not CA. Addition of MF to incubation medium also led to JH III, from which we inferred the epoxidation of MF to JH III. JH biosynthesis from farnesol in the testes, MAGs, and ovaries of A. germari proceeds via oxidation to FA, methylation to MF, and epoxidation to JH III. This is a well‐known pathway to JH III, described here for the first time in reproductive tissues of longhorned beetles. © 2010 Wiley Periodicals, Inc.     

Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex

The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y-organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ovaries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph.     

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.     

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Rates of Juvenile Hormone biosynthesis and degradation during reproductive development and diapause in the boll weevil, Anthonomus grandis

Abstract. The role of Juvenile Hormone (JH) during reproductive development and diapause was investigated in the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae). JH sythesized by corpora allata (CA) in vitro of A.grandis was identified as JH-UJ by high-performance liquid chromatography and by conversion to the methoxyhydrin. Optimal conditions for the use of a short-term assay in vitro were established to examine profiles of CA activity. In addition, rates of JH degradation by JH-specific esterase were determined. Patterns of CA and JH-esterase activity during reproductive development and the diapause state were established with laboratory-reared reproductive weevils and diapausing weevils collected as larvae and pupae in the field after the cotton-growing season. The results indicate that JH production is elevated in reproductive females whereas males and winter field-collected females show no CA activity. Vitellogenin concentrations in haemolymph and rates of oviposition were studied in relation to CA activity and JH degradation. An attempt to induce diapause in the laboratory failed.     

Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea

The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed.     

Methyl farnesoate binding proteins in tissues of the spider crab, Libinia emarginata

Methyl farnesoate (MF) binding proteins (MFBPs) were found in the ovaries, testes, accessory glands, and hemolymph of the spider crab Libinia emarginata, by photoaffinity labeling the tissues in vitro with tritiated farnesyl diazomethyl ketone ([3H]-FDK). Specificity was demonstrated by competitive displacement of [3H]-FDK with MF. SDS-PAGE followed by fluorography revealed several labeled proteins in the hemolymph and testes with molecular masses ranging from 29 to 116 kDa, and two in the ovary that were 97 and 70 kDa. Tissues from reproductive animals bound twice as much label per gram weight compared to those that were from non-reproductive crabs.     

Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce.     

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.     

Seasonal variation of energy metabolism in ghost crab Ocypode quadrata at Siriú Beach (Brazil)

The aim of this study was to analyze the seasonal variations of carbohydrate and lipid metabolism of the ghost crab Ocypode quadrata (Fabricius, 1787) on a sandy beach in the southern region of Brazil. Crabs and hemolymph samples were collected monthly in the field. Hepatopancreas, gills, gonads and claw muscles were used for glycogen determination. In males, blood glucose levels increased in the summer and in the winter. The glycogen values increased significantly in the hepatopancreas in the winter, but remained constant in the muscle, gonads and gills. In females, hemolymph glucose levels, glycogen values in the hepatopancreas and in the gills remained constant throughout the year; however, muscular glycogen increased in the spring and gonad glycogen decreased in the summer. Hemolymph triglyceride levels of males and females and total cholesterol of males decreased significantly in the spring. In females, a significant increase of total cholesterol levels was found in the winter. The findings suggest that in O. quadrata lipids seem to be an important reserve of energy used during reproduction, both in males and females, while glycogen may be used during periods of intense activity or fasting.     

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.     

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.     

Stage-specific production and release of juvenile hormone esterase from the ovary of Diploptera punctata

Juvenile hormone esterase (JHE) is a catabolic enzyme that specifically degrades juvenile hormone (JH) and has been identified in hemolymph and tissues in both larvae and adults of numerous insect species. This study investigates the presence of JHE in ovaries of the viviparous cockroach, Diploptera punctata, and the in vitro release of JHE from these ovaries during the first gonadotrophic cycle. JHE is released in vitro from maturing basal (most posterior) follicles and from follicle cells isolated from oocytes during the short period of time between spermatophore release and chorion formation. Enzyme release is dependent upon the presence of calcium in the medium. This released ovarian JHE appears to be larger than and to display ionic characteristics that are different from the isolated hemolymph and fat body JHEs. In addition, JHE activity measured in homogenates of whole ovaries and subsequently oviposited basal oocytes increases dramatically following spermatophore release, coincident with a previously described decline in JH titer in the ovary. A likely role for ovarian JHE is the site-specific degradation of JH in and around the oocyte prior to fertilization and embryonic development.     

Seasonal variation of energy metabolism in ghost crab Ocypode quadrata at Siriú Beach (Brazil)

The aim of this study was to analyze the seasonal variations of carbohydrate and lipid metabolism of the ghost crab Ocypode quadrata (Fabricius, 1787) on a sandy beach in the southern region of Brazil. Crabs and hemolymph samples were collected monthly in the field. Hepatopancreas, gills, gonads and claw muscles were used for glycogen determination. In males, blood glucose levels increased in the summer and in the winter. The glycogen values increased significantly in the hepatopancreas in the winter, but remained constant in the muscle, gonads and gills. In females, hemolymph glucose levels, glycogen values in the hepatopancreas and in the gills remained constant throughout the year; however, muscular glycogen increased in the spring and gonad glycogen decreased in the summer. Hemolymph triglyceride levels of males and females and total cholesterol of males decreased significantly in the spring. In females, a significant increase of total cholesterol levels was found in the winter. The findings suggest that in O. quadrata lipids seem to be an important reserve of energy used during reproduction, both in males and females, while glycogen may be used during periods of intense activity or fasting.     

Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation

Methyl farnesoate (MF), a crustacean juvenile hormone (JH) analog, plays important roles in the regulation of a number of physiological processes such as molting, metamorphosis, and reproduction. Understanding its metabolic pathway is a key for various potential applications in crustacean aquaculture, including artificial seed production and enhancement of growth. Although the synthetic pathway of MF is well established, little is known about its degradation and recycling in crustaceans. In insects, juvenile hormone esterase (JHE), a carboxylesterase, is responsible for JH inactivation. Two cDNAs, encoding JHE-like carboxylesterases (CXEs) from the hepatopancreas and ovary of Pandalopsis japonica, were isolated by using a combination of in-silico data mining from an expressed sequence tag (EST) database and traditional PCR-based cloning. The full length Pj-CXE1 (2084bp) and Pj-CXE2 (1985bp) cDNAs encoded proteins composed of 584 and 581 amino acids, respectively. The active site sequence and domain organization of the Pj-CXEs were highly conserved, including the catalytic triad and other motifs, which suggested that both Pj-CXEs are biologically active carboxylesterases. Phylogenetic analysis of the deduced sequences of Pj-CXEs showed that both were most closely related to the JHEs from non-lepidopteran insects. End-point RT-PCR showed that Pj-CXE1 was expressed primarily in the gonad, whereas Pj-CXE2 was expressed in both the hepatopancreas and hindgut. Quantitative PCR showed that Pj-CXE1 was upregulated in the gonads by eyestalk ablation (ESA). In contrast, ESA had no significant effect on Pj-CXE2 expression in hepatopancreas or gonad. This is the first report of the cloning of two JHE-like CXE cDNAs in decapods and the upregulation of Pj-CXE1 by acute withdrawal of eyestalk neuropeptides. Further study is needed to understand the function of CXEs in MF metabolism and its regulation by eyestalk neuropeptides.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

Juvenile hormone III produced in male accessory glands of the longhorned beetle,Apriona germari,is transferred to female ovaries during copulation

We report on juvenile hormone (JH) biosynthesis in vitro by male accessory glands (MAGs) in the longhorned beetle, Aprionona germari, accompanied by the transfer of JH from males to females during copulation. JH was extracted from the MAGs and separated by reversed‐phase high‐performance liquid chromatography. JH III was identified as the major JH by gas chromatography–mass spectrometry. A radiochemical assay and a non‐radioactive method were used to measure the in vitro rate of JH biosynthesis by the MAGs. After 4 h of incubation with ³H‐methionine in the medium, the radioactivity in the MAGs substantially increased. In a separate assay, incubation of the MAGs with non‐radioactive methionine for 4 h resulted in a 39% increase in JH III. Seven‐day‐old males were injected with medium 199 containing ³H–methionine and 24 h later they were mated with virgin females. Hemolymph and the MAGs were collected from the mated males and hemolymph, ovaries and eggs were collected from the mated females for assaying radioactive JH. The radioactivity incorporated into JH in the MAGs was transferred to the females during copulation and later transferred into their eggs. Assayed 1 h after copulation, JH III level in the MAGs decreased 42% and the content of JH III in the male hemolymph did not change, whereas the content of JH III in the female hemolymph and ovaries both increased. © 2010 Wiley Periodicals, Inc.