Enhancement by farnesol and farnesoic acid of juvenile-hormone biosynthesis in induced low-activity locust corpora allata

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose-dependent manner. Farnesol and FA also stimulate a dose-dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography-mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose-response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA-1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA-1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.     

Activity of the corpora allata of adult female Leucophaea maderae: effects of mating and feeding

Juvenile hormone III biosynthesis by corpora allata of adult female Leucophaea maderae was measured by an in vitro radiochemical assay. In fed females, JH III synthesis increases more than 20-fold after mating to a peak of 55 pmol/pair/h on day 9 and then rapidly declines. This increase in JH III synthesis concomitant with rapid oocyte growth in mated females is not observed in virgin females. The corpora allata from starved, virgin females appear to be inactive. The addition of 150 microM 2E,6E-farnesol (a) JH III precursor) to the incubation medium stimulates the corpora allata from starved, virgin females less than the corpora allata from starved, mated females. Both feeding and mating are necessary for the expression of a normal cycle of JH III synthesis in this cockroach.     

Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of 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.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.     

Control of C-16 juvenile hormone biosynthesis in active corpora allata of female African locusts

Summary

Corpora allata from 8-day-old female Locusta migratoria, during the phase of yolk deposition, exhibit high rates of C-16 juvenile hormone (JH) biosynthesis. The effect of different potential factors which may be involved in the regulation of corpora allata activity is reported. The biosynthetic activity of corpora allata was determined by radiochemical assay.

In maturing females, no changes in corpora allata activity are detected during one daily cycle. Starvation reduces JH biosynthesis only 3 days after the beginning of the food deprivation. Suppression of the median neurosecretory material by electrocoagulation of the internal cardiaca tract (TCC-I) does not disturb JH biosynthesis whereas the transection of the allata I nerve fibres (NCA-I) or the electrocoagulation of the lateral neurosecretory pericarya results in a rapid decline of JH biosynthesis. These data indicate that the median and lateral allatotropins are different, and that only the lateral neurosecretory material exerts an allatostimulating action on corpora allata at the time of vitellogenesis. The corpora allata response to the median allatotropin changes during oocyte growth. C-16 JH and/or 20-hydroxyecdysone treatments in vitro (addition in the culture medium) and in vivo (injection in female) do not influence JH production in our experimental conditions.     

Approaches to the biosynthesis of the cecropia C18-Juvenile Hormone

The juvenile hormone of the silkmoth Hyalophora cecropia—methyl 7-ethyl-3,11-dimethyl-cis-10,11-oxido-trans,trans-2,6-tridecadienoate—is a rare bis-homosesquiterpenoid whose biosynthesis has not been explained. In an attempt to do so, I have studied the incorporation of ¹⁴C- and ³H-labelled precursors into the carbon skeleton of C₁₈-Juvenile Hormone (JH) both in adult male insects and in short-term tissue cultures of corpora allata, the glands which are known to secrete the hormone. In these studies, also, novel combinations of microchemical derivatization procedures and radiochemical purification sequences, involving thin-layer, high-pressure liquid, and electron capture gas chromatography were used to assess the extent of isotope incorporation into sub-nanogram amounts of biosynthetic JH. Thus, in in vivo incubations I recovered label derived from acetate, glucose, methionine, and to a lesser extent from iso-leucine and valine. Bis-homofarnesol, farnesol, leucine, 3-methyl-2-pentenol, propionate, and mevalonate did not contribute significantly to the formation of the carbon skeleton. In parallel in vitro incubations I recovered label in the JH fraction when glucose, acetate, or methionine served as precursor.     

Activity of the corpora allata of adult female Aedes aegypti: effects of mating and feeding

The synthesis of juvenile hormone III (JH III) by the isolated corpora allata (CA) of Aedes aegypti adult female was studied using an in vitro radiochemical assay. We dissected the corpora allata-corpora cardiaca (CA-CC) complex attached to a piece of aorta. The complex was left connected to the intact head capsule to facilitate the visualization and transfer of the glands. A linear increase in the cumulative amount of biosynthesized JH III was found for at least the first 6 h of incubation; approximately 45% of the synthesized JH III was present in the medium. There was a dependence of JH III synthesis on exogenous methionine supply. Using reversed phase high performance liquid chromatography two major labeled products biosynthesized by the CA were separated. They co-migrated with JH III and methyl farnesoate (MF). The identity of the biosynthesized JH III was confirmed by gas chromatography-mass spectrometry. JH III synthesis was only 2.0 fmol/pair gland/h immediately after adult emergence, but increased to 32.6 fmol/ pair gland/h 18 h later in sugar-fed females. Two days after emergence, the CA biosynthetic activity slowly started to decrease, and reached values of around 5.3 fmol/pair gland/h by one week after emergence. Synthesis of JH was similar from either sugar-fed females mated or unmated. A blood meal resulted in a decrease of JH III synthesis in CA from mated females by 12 h after feeding and from virgin females by 24 h after feeding. JH III biosynthesis remained low for at least 96 h in mated females, but was back to higher levels 72 h after feeding in virgin females. Rates of JH III biosynthesis closely reflected the hemolymph levels of JH III both after emergence and after a blood meal described by Shapiro et al. (1986). The activity of the CA in Aedes aegypti females seems to be regulated by developmental changes and nutritional signals, and to be independent of mating stimulus.     

The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocdrca gregaria.

We have compared, on an individual basis, the volume of the corpora allata with their ability to synthesize and release juvenile hormone (JH) using glands taken at daily intervals throughout the period of sexual maturation and the first two ovarian cycles in $\text{Schistocerca gregaria}$. A standard $\text{in vitro}$ radiochemical assay was used to measure the rates of both spontaneous JH biosynthesis from [methyl-¹⁴C]-methionine, and of JH biosynthesis stimulated by optimal concentrations of [C-2 ³H]-farnesenic acid. Computation of results showed that there are, during this period, changes of up to 250-fold in the rate of spontaneous JH biosynthesis per unit volume corpora allata. It is concluded that the volume of the corpora allata is of no value as an indicator of the spontaneous synthetic activity of the glands in this species, and that the overall rate of JH synthesis is regulated by mechanisms that do not involve large changes in the volume of the gland cells. However, in the presence of farnesenic acid, there is a corelation between stimulated JH synthesis and glandular volume, suggesting that the volume of the gland reflects the maximum activity of the final two stages in JH biosynthesis.     

Juvenile hormone and reproduction in crickets, Gryllus bimaculatus De Geer: corpus allatum activity (in vitro) in females during adult life cycle

ABSTRACT. Incubation conditions were established for a short-term radiochemical assay of spontaneous juvenile hormone (JH) biosynthesis in vitro by corpora allata from adult female Gryllus bimaculatus. The only JH synthesized was shown by HPLC to be JH III. A further incubation product, predominantly extracted from the corpora allata, was thought to be the JH III precursor, methyl farnesoate. In adult females reared at a constant temperature of 27°C the synthetic activities of the corpora allata-corpora cardiaca complexes in vitro increase from almost zero to a high peak value 4 days after the imaginal moult. Thereafter the activity decreases to varying intermediate levels, but always lower than the first maximum. Two days after the first peak in corpus allatum activity, ovarian fresh weight increases dramatically and the first oviposition occurs 2 days later.
Topical application of JH III to females reared at 20°C, which usually have a low fecundity, causes a dose-dependent stimulation of egg production and oviposition.     

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.     

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.     

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.     

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.     

Inhibition of juvenile hormone III biosynthesis in cockroach corpora allata by interference with the S-adenosylmethionine-dependent transmethylation

The synthesis of juvenile hormone-III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S-adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl-³H]methionine or [³⁵S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S-adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′-deoxy-5′-methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH-III synthesis by intact glands can be inhibited by interfering with the S-adenosylmethionine-dependent transmethylation, and suggest that the product and inhibitor of that reaction, S-adenosyl-homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.     

Assay of HMG-CoA synthase in Diploptera punctata corpora allata

The conversion of acetyl-CoA to HMG-CoA was measured by a radioenzymatic assay in which the product is derivatized to its bis(p-nitrobenzyl)ester and separated from the substrate by TLC. The enzymatic assay was applied to post-mitochondrial supernatants of corpora allata homogenates from the cockroach, Diploptera punctata. The apparent K_M for the conversion of acetyl-CoA to HMG-CoA was 200 μM. The enzyme activity was measured during a cycle of JH synthesis in adult mated females. HMG-CoA synthase followed the pattern of juvenile hormone III synthesis closely with a peak on day 5 (1.89 ± 0.07 nmol/h/pair-eq.), and the decline of its activity occurred earlier than that of HMG-CoA reductase.