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.     

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.     

Assays on Rhodnius for juvenile hormone activity

Contrary to expectation the effect of a given quantity of juvenile hormone (JH) topically applied to the abdomen of Rhodnius is greatly reduced if the cuticle is thin; either newly stretched by a large meal or wasted by prolonged starvation. The active substance apparently enters the body too rapidly and is eliminated in metabolism.The activity of the juvenile hormone and the most active JH-mimics is increased ten to fifteenfold if they are topically applied in dilute solution in peanut oil or triolein.Assays of the juvenile hormones of Cecropia at a concentration of 1 : 1000 in peanut oil (suitably diluted with octane) show that synthetic (racemic) C₁₈-JH of Röller (JH-1) is considerably less active than the natural enantiomorph; and the synthetic C₁₇-JH of Meyer (JH-2) has only about two-thirds of the activity of JH-1. Using this method of application, the natural juvenile hormone applied to the fifth instar larva of Rhodnius will cause a 50 per cent suppression of adult characters (grade 10 in the assay) at a dose of about 15 ng.     

Purification of the Drosophila Kc cell juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from the cytosol of Drosophila melanogaster Kc cells has been purified with the use of a juvenile hormone photoaffinity analog, 10,11-epoxy (2E, 6E) farnesyl diazoacetate (EFDA). The purification procedure consists of five chromatographic steps and the end product of the purification procedure showed homogeneity by means of both native and SDS polyacrylamide gel electrophoresis. Furthermore, using a racemic mixture of the natural hormone, [³H]juvenile hormone III (JH III), as the radioligand in this purification procedure, we demonstrate that the purified protein is likely the authentic intracellular JHBP.     

Bioassays of compounds with potential juvenoid activity on Drosophila melanogaster: Juvenile hormone III, bisepoxide juvenile hormone III and methyl farnesoates

Metabolites of the 6,7,10,11 bisepoxide juvenile hormone III (JHB₃), and other potential juvenoids, were tested for juvenile hormone activity using early instar or early stage pupae of Drosophila melanogaster. Importantly, methyl farnesoates were tested as they might have JH-like activity on Dipteran juveniles. Larvae were exposed to compounds in medium, or the compounds were applied to white puparia. In the assays employed in the present study, there was no indication for JH activity associated with the metabolites of JHB₃. The activity of methyl farnesoate (MF) was higher than that of JH III and far greater than bisepoxide JH III. As opposed to the two endogenous juvenile hormones, methyl farnesoate has weak activity in the white puparial bioassay. When fluorinated forms of methyl farnesoate, which is unlikely to be converted to JH, were applied to Drosophila medium to which fly eggs were introduced, there was a high degree of larval mortality, but no evidence of subsequent mortality at the pupal stage. One possible explanation for the results is that methyl farnesoate is active as a hormone in larval stages, but has little activity at the pupal stage where only juvenile hormone has a major effect.     

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.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Characterization of hemolymph juvenile hormone esterase from Lymantria dispar

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age

Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed.The synergistic effect of 1 μg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to >18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter's ability to synchronize epidermal cells.The significance of these observations in the analysis of time of action of juvenile hormone is discussed.     

Juvenile hormone induction of pheromone gland PBAN-responsiveness in Helicoverpa armigera females

The maturation of corpora allata (CA) and the competence of pheromone glands in the adult moth Helicoverpa armigera, are both age-related and appear to be correlated. Sex pheromone glands of pharate adults do not produce sex pheromone independently, nor do they respond to exogenous PBAN. Newly emerged moths produce significantly less pheromone than day one moths. JH (juvenile hormone) II was found to be the main JH form produced by CA in vitro. JH II primed pheromone glands of pharate adults to respond to PBAN. In addition, injection or topical application of JH II to newly-emerged females induced pheromone production in the presence of PBAN. Our findings suggest that JH is involved in the initiation of pheromone production of Helicoverpa armigera.     

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.     

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.     

Dependence of juvenile hormone activity on haemolymph factor in Tenebrio molitor pupae

Morphogenetic response of Tenebrio molitor pupae to juvenile hormone (JH) varies with age, it being low during the first 6 hr after pupal ecdysis and reaching a peak at about 18 hr after ecdysis. Parabiosis of 6-hr pupa to a 18-hr-old pupa causes a higher morphogenetic response in 6-hr pupa to JH. A similar increase in morphogenetic response was not noticeable when a 0·45 μm Millipore filter was placed between the parabiotic partners, suggesting that the factor(s) may be macromolecular. This view is further supported by the observation that the Millipore filter did not prevent transport of ecdysterone another agent that enhances morphogenetic response of pupae to JH.     

Farnesoic acid stimulation of C16 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata

Biosynthesis of C₁₆ juvenile hormone (C₁₆JH) by isolated corpora allata (CA) of the viviparous cockroach Diploptera punctata has been studied by a radiochemical assay. This assay uses the incorporation from methyl labelled l-methionine in the methyl ester moiety of C₁₆JH. The optimal concentration of l-methionine in the medium has been determined. When varying proportions of (methyl-³H)- and (methyl-¹⁴C)-labelled l-methionine are used in the assay, no isotope effect was observed indicating that labelled l-methionine can be used as a mass marker to quantify C₁₆JH synthesis in D. punctata. C₁₆JH synthesis was stimulated by addition of farnesoic acid (FA) to the medium (maximum at 30–40 μM). When stimulated with 30 μM FA, the C₁₆JH release rate was directly dependent on C₁₆JH synthesis rate, and no intraglandular accumulation of either C₁₆JH or its immediate precursor methyl farnesoate were observed. Spontaneous and FA-stimulated rates of C₁₆JH release were studied during the first reproductive cycle of the adult female. The two final steps of C₁₆JH biosynthesis are not physiologically rate-limiting, as the ratio of spontaneous to FA-stimulated C₁₆JH release (fractional endocrine activity ratio) is always lower than unity. There is a precise relationship between the length of the basal oöcytes and the rate of C₁₆JH release.     

Inhibitors of ester hydrolysis as synergists for biological activity of Cecropia juvenile hormone.

In Manduca sexta pupae the sensitivity to exogenous Cecropia C18 juvenile hormone (JH) is governed in large part by the rate at which the hormone is metabolized. By analysing the distribution of labelled JH and its metabolites, when dispensed by injection in light mineral oil, I have shown that the half-life of JH at concentrations ranging from 0·02 to 1 μg/g live weight of insect is approximately 24 hr.When the same doses of JH are dispensed in mineral oil containing endocrinologically inactive carboxylesterase inhibitors, organophosphate, and carbamate insecticides, the hormone's half-life in certain cases is prolonged significantly. The formulations containing those materials which most effectively prolong the hormone's half-life give higher assay scores than equivalent doses of hormone in mineral oil alone.From these results it is inferred that the chemicals tested synergize the Cecropia juvenile hormone's biological activity by serving presumably as inhibitors of the principal enzyme responsible for the rapid breakdown of exogenous C18-JH in haemolymph—a carboxylesterase. Preliminary in vitro experiments with carboxylesterase-rich Manduca pupal blood further support this inference.     

The effect of juvenile hormone and its antagonists on JH esterase activity in Tenebrio molitor

The hemolymph juvenile hormone esterase (JHE) and general carboxyl esterase activities in Tenebrio molitor show independent development-associated changes during larval-pupal and pupal-adult metamorphoses. JHE activity was high in pharate pupae and early pupae. Unlike in lepidopteran pupae that have been studied thus far, JH had no effect on JHE activity in pupae and pharate adults of Tenebrio. A JH antagonist, ethyl 4,2,tert butyl carboxy n benzoate (ETB), and 20-hydroxyecdysone had no effect on JHE activity. These observations suggest that although JHE activity in Tenebrio is precisely regulated during larval-pupal metamorphosis, JH and molting hormone do not appear to be involved in its regulation and that the proximate cues that influence JHE activity in Tenebrio pupae are different from that of lepidopterans.

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Résumé Les activités JHE (estérase juvénile hormone) de l'hémolymphe et carboxyl estérase générale de T. molitor changent indépendamment du développement pendant les métamorphoses larve-nymphe et nymphe-adulte. L'activité JHE est élevée chez les prénymphe et les jeunes nymphes. Contrairement aux chrysalides de papillons, JH n'a pas d'effet sur l'activité JHE chez les nymphes et les préadultes de Tenebrio. Un antagoniste de JH, l'éthyl 4,2,tert butyl carboxy n benzoate (ETB), 20-hydroxyecdysone et le précocène 2-agent anti-allate chez plusieurs insectes-, n'ont aucun effet sur l'activité JHE. Ces observations suggèrent que, bien que l'activité JHE chez T. molitor soit ajustée avec précision pendant la métamorphose larvo-nymphale, JH et hormone de mue ne paraissent pas être impliquées dans sa régulation et que les signaux immédiats, qui influent sur l'activité JHE de la nymphe de T. molitor, diffèrent de ceux des Lépidoptères.

     

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.     

Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.