Degradation of juvenile hormone and methylation of juvenile hormone acid by corpora cardiaca–corpora allata of the cockroach,Nauphoeta cinerea: I. Biochemical aspects

Corpora cardiaca-corpora allata (CC-CA) from vitellogenic females of Nauphoeta cinerea degraded, in vitro, racemic and (10R)-juvenile hormone III (JH III) at a rate of 249 pmol/CC-CA/h and 786 pmol/CC-CA/h, respectively. The major metabolite formed was JH III acid, together with some highly polar products. CC-CA homogenates degraded racemic JH III to a small extent, whereas (10R)-JH III was degraded efficiently to JH III acid. No highly polar products were formed by CC-CA homogenates. When CC-CA were incubated with racemic JH III acid, some of this substance was degraded to highly polar products, and a minor part was methylated to JH III. CC degraded very little JH III acid and did not methylate it to JH III. CC-CA homogenates methylated JH III acid very efficiently; we measured an apparent K_max of 37.8 μM and a V_max of 1,260 pmol/4 h/ CC-CA equivalent. The addition of JH III acid to CC-CA in vitro increased the rate of biosynthesis of JH III, as determined by measuring incorporation of methyl[¹⁴C]methionine into JH III. These data indicate that the metabolite JH III acid can enter the CA and be methylated to JH III.     

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 hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.

     

Changes in the size of corpora allata,in the juvenile hormone III titer in the hemolymph,and in the protein content of terminal oocytes throughout the first gonadotropic cycle in Aiolopus thalassinus (Insecta: Orthoptera: Acrididae)

In Aiolopus thalassinus (Fabr.), the changes in the volume of the corpora allata (CA), in the concentration of juvenile hormone III (JH III) in the hemolymph, and in protein content in the terminal (t) oocytes were studied during the first gonadotropic cycle. These parameters could be better related to the volume of t-oocytes than to age after emergence. The JH III titer curve was maximum (2.9 pmol/10 μl) at an oocyte volume of 1.2 mm³. Before oviposition (days 10–16) the JH III titer decreased to 0.65 pmol/10 μl hemolyph. The increase in JH III titer reflected a period of high protein storage in the t-oocytes. The largest volume of the CA was reached at the beginning of yolk storage in the t-oocytes. The highest JH III titer did not correspond with the largest volume of CA, which occurred much earlier. © 1993 Wiley-Liss, Inc.     

Juvenile hormone biosynthesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae)

The in vitro production of juvenile hormones (JH) was investigated by using corpora allata (CA) of larvae and corpora cardiaca-corpora allata (CC-CA) complexes of adult females of the fall armyworm Spodoptera frugiperda. In female moths, JH release was high compared to that in 5th and 6th instar larvae. Concentrations of 0.11-0.12 mM methionine, 180-200 mM Na(+), 5.8-8.3 mM K(+), 10-50 mM Ca(2+) and a pH range of 5.7-6.3 yielded optimal incorporation of L-[methyl-(3)H] methionine in vitro by CC-CA complexes. The highest hourly incorporation occurred during a 9-h incubation period following a 1.5-h lag-phase. JH release from CC-CA complexes of adult females was shown to be age-dependent with a peak value on day 2 (approx. 4 pmol h(-1) CA(-1)). By a combination of reversed phase (RP)- and normal phase (NP)-high performance liquid chromatography (HPLC), two major labelled products released by the complex were separated. One compound co-migrated with chemically synthesized JH II diol, the second compound with JH III diol. Only traces of JH II and III could be detected in some samples. Gland extracts also contained both the major radiolabelled products. Double labelling experiments using [3H]methionine and [14C]acetate confirmed their de novo synthesis in CC-CA complexes of female moths. The nature of chemically synthesized reference JH III diol was proved by LC-MS (ESI mass spectrometry) and 1H-NMR (nuclear magnetic resonance spectroscopy).     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

Parasitism-induced effects of Glyptapanteles liparidis (Hym., Braconidae) on the juvenile hormone titer of its host, Lymantria dispar: the role of the parasitoid larvae

Parasitization by the gregarious larval endoparasitoid Glyptapantles liparidis induces a dramatic increase in the hemolymph juvenile hormone (JH) titer (especially JH III) of its host larva, Lymantria dispar. Here, we investigated the role of the parasitoid larvae in JH synthesis and release by in vitro and in vivo experiments. GC-MS analyses confirmed that the rising hemolymph JH titer coincided with the time at which the parasitoids molt to the second larval instar. Peak values in host hemolymph titers were observed prior to parasitoid emergence, and titers dropped to negligible levels within 24 h after parasitoid emergence. Whole body extracts from excised second instar parasitoids yielded JH III and trace amounts of JH II. The in vitro secretory activity of the corpora allata (CA) of L. dispar larvae was not enhanced by parasitization. When the host's CA were separated by neck ligation, we found elevated JH III titers, but no JH II in the hemolymph of the posterior section, which contained the parasitoids. Parasitoids that were kept in in vitro culture produced and released only JH III. The parasitoids’ ability to secrete JH and to molt independently from their host's molting cycles indicates that at least second instar parasitoids are hormonally self-reliant.     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

20-hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. 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 plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or −24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the −24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the −24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.     

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm,Bombyx mori

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain-corpora cardiaca-corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.     

Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH-I and 10, 11-epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH-III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [³H]JH-III and to a lesser extent JH-I. Other proteins from the hemolymph do not bind JH-III. Binding of JH-III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH-III. It is concluded that lipophorin is the JH-III-specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH-III-specific binding site is probably located on apolipophorin I.     

Fenoxycarb,a potent contaminant of the silkworm,Bombyx mori L., does not influence its juvenile hormone titer

Fenoxycarb (FC) effects were studied on juvenile hormone (JH) titers and JH‐esterase activities in the silkworm, B. Mori. In the literature, FC was observed to induce high JH titers but also to act without corpora allata (CA). These contradictory results did not permit us to conclude whether FC was a potent JH mimic or it was acting through the enhancement of JH titers in the hemolymph. Analysis of hemolymph JH‐esterase activities during the last larval instar reveals that FC was not a JH‐esterase inhibitor. Considering JHs, only JH II was detected in the European hybrid 200×300. Furthermore, JH titer was exactly identical in control and FC‐treated larvae, i.e., it dropped during the first 2 days of the last larval instar and became undetectable after day 2. This result is important since it contradicts the generally admitted concept that FC was acting by increasing the titer of JH. On the contrary, it was found that, despite its non‐terpenoid chemical structure, FC might be a JH mimic. In addition, FC suspected contamination of mulberry leaves was analyzed from a physiological viewpoint. We observed that "contaminated" mulberry leaves‐fed larvae became permanent larvae through the inhibition of their prothoracic glands (PG) activity and without any modification of their JH titers, i.e., exactly as for FC‐treated larvae. This last point adds information concerning the suspected implication of FC in the induction of the non‐spinning syndrome. Arch. Insect Biochem. Physiol. 40:141–149, 1999. © 1999 Wiley‐Liss, Inc.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during 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 once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml^?1 on day 1 to 0.27 U ml^?1 on day 3. When larvae were treated with (7S)-hydroprene (a JH analog), the titer remained elevated (2.09 U ml^?1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml^?1 on day 5 to 1.62 U ml^?1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head-ligated day 5 larvae were treated with either (7S)-hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml^?1 and 2.05 U ml^?1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml^?1). The combined results indicate the titer of the hemolymph factor is regulated by JH.     

Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina

The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a K_d for racemic JH III of 33 ± 6 nM. The density of the binding sites is 212 ± 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [³H]JH III with JH III > JH II > JH I > JH III > JH III diol > JHB₃ = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells.     

Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps

Last instar larvae of the tobacco budworm, Heliothis virescens F., fail to pupate and have little 20-hydroxyecdysone when parasitized by Toxoneuron nigriceps (Viereck). In this paper, we extend these observations to juvenile hormone (JH) to determine if parasitism by this wasp affects other endocrine systems. To this end, we compared the production of JH by corpora cardiaca-corpora allata complexes (CC-CA), the metabolism of JH by haemolymph enzymes, and the haemolymph titre of JH in parasitized and non-parasitized control larvae of H. virescens during the last larval instar. CC-CA from parasitized and control larvae had similar peaks of JH synthesis on day 1 of the fifth instar, with JH II accounting for more than 90% of total JH in both groups. On subsequent days, JH synthesis dropped to undetectable levels more quickly in non-parasitized controls than in parasitized larvae. JH metabolism by haemolymph of parasitized and control animals increased from low levels on day 1 of the fifth instar to high levels on days 2 and 3 of the instar. JH metabolism was significantly higher in control larvae than in parasitized larvae. After day 3, JH metabolism decreased in both groups, but was significantly higher in parasitized larvae. The major metabolite of JH in both groups was JH acid, though traces of JH diol and JH acid diol were also detected. The haemolymph titre of JH in both groups peaked on day 1 of the fifth instar and, similar to the synthesis of JH by CC-CA, decreased more rapidly in control larvae. As a result, non-parasitized animals had significantly lower JH titres on day 2. The higher JH titres observed in parasitized larvae during the early fifth instar may contribute to their developmental arrest. The possible role of these JH alterations in the host developmental and metabolic redirection is discussed and a more comprehensive physiological model accounting for host-parasitoid interactions is proposed.     

Identification and action of juvenile hormone III from sexually mature alate females of the red imported fire ant,Solenopsis invicta

Analysis of extracts of hemolymph obtained from sexually mature alate females of Solenopsis invicta from monogyne colonies resulted in identification of juvenile hormone III (JH III). The average amount of JH III was 0.32±0.04 pmol/μmolof hemolymph. Topical application of 0.038 pmol of JH III was sufficient to stimulate alates to shed their wings in the presence of the queen. The time in which alates were induced to dealate decreased linearly with increasing concentrations of JH III from 0.038 to 3.8 pmol. However, higher JH III concentrations deviated from linearity and did not reach dealation times comparable with those that occur after mating flights. Thus, it appears that the mechanism of dealation that occurs when female alates are out of the influence of their queen is different from the one associated with mating flights. Application of 0.42 μmol of precocene II inhibited dealation of alates in queenless colonies. However, this inhibition was reversed after applying 38 pmol JH III to precocene-treated alates. The sizes of corpora allata (CA) from sexuals treated with JH III did not differ from those of controls. However, the sizes of CA were reduced in alates treated with precocene II. The results indicated that JH was important to dealation.     

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca-corpora allata (CC-CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808-818]. The in situ hybridization analyses in the CC-CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC-CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize 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.     

Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly 14C-labeled BCAA, together with [methyl-3H]methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of 14C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans.