Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.     

Juvenile hormone metabolism in the plasma,integument, midgut,fat body,and brain during the last instar of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) III esterase and JH III epoxide hydrolase activity was found in the integument, midgut, fat body, and brain during last instar development of the tobacco hornworm, Manduca sexta. JH esterase activity was primarily located in the cytosol in these tissues while the majority of the JH epoxide hydrolase activity was found in the microsomes. A prewandering (on day 3) and postwandering (on day 8) peak in plasma JH III esterase activity occurs in the last instar of gate I M. sexta. The JH esterase activity profile in integument, midgut, fat body, and brain followed a similar pattern to that of the plasma. The only exception to this was the absence of the postwandering, prepupal (on day 8) JH esterase peak in the fat body. The topical application of the juvenoid, (RS)-methoprene, failed to induce fat body JH esterase activity but increased activity in the plasma, integument, midgut, and brain in M. sexta prepupae. These results indicate that the source of plasma JH esterase activity is not always the fat body as previously hypothesized. The developmental profile of tissue JH epoxide hydrolase activity was also similar to that of JH esterase suggesting that both enzymes may be regulated partly by the same factors and that JH epoxide hydrolase may also have an important, previously unrecognized functional role in JH regulation and insect metamorphosis. Multiple isoelectric forms of tissue-specific JH esterases and JH epoxide hydrolases were found in integument, midgut, fat body, and brain. The JH esterases in these tissues had isoelectric points more acidic than that for plasma. Tissue α-naphthyl acetate esterase, developmental profiles, and inhibitor sensitivity to 3-(octylthio)-1,1,1-trifluoropropan-2-one differed significantly from that for JH esterase, suggesting that they represent different enzymes. ©1992 Wiley-Liss, Inc.     

A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time- and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.     

Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis

Changes in activity of the corpora allata (CA) during larval-pupal-adult development of the tobacco hornworm Manduca sexta were studied by transplantation assays, measurements of in vitro juvenile hormone (JH) and JH acid synthesis, and determination of JH acid methyltransferase (JHAMT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities. The data from these assays demonstrate that the CA cease to secrete JH by day 4 of the last larval instar (wandering stage). With regard to JH synthesis, they remain inactive throughout the prepupal, pupal, and most of the pharate adult periods. CA of females, but not of males, resume JH synthesis shortly before eclosion. The biochemical basis of the inactivation process is the loss of JHAMT activity. However, prepupal CA produce JH acids, as shown by enzyme and in vitro assays. Pupal and pharate adult CA do not synthesize JH acids although levels of HMG-CoA reductase activity seem to remain relatively high. Radiolabeled JH was recovered from hemolymph of allatectomized prepupae that had been injected with radiolabeled JH acid. These results provide further evidence that certain peripheral tissues (eg, imaginal discs) convert JH acid secreted by the prepupal CA to JH and, thus, that JH acid is a prohormone in the prepupal period. The CA change from hormone secretion to prohormone secretion during larval-prepupal transformation, a unique functional alteration in an endocrine gland.     

Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation

The structure of human Janus kinase 2 (JAK2) comprising the two C-terminal domains (JH1 and JH2) was predicted by application of homology modelling techniques. JH1 and JH2 represent the tyrosine kinase and tyrosine kinase-like domains, respectively, and are crucial for function and regulation of the protein. A comparison between the structures of the two domains is made and structural differences are highlighted. Prediction of the relative orientation of JH1 and JH2 was aided by a newly developed method for the detection of correlated amino acid mutations. Analysis of the interactions between the two domains led to a model for the regulatory effect of JH2 on JH1. The predictions are consistent with available experimental data on JAK2 or related proteins and provide an explanation for inhibition of JH1 tyrosine kinase activity by the adjacent JH2 domain.     

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.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

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.     

Comparison of effects of juvenile hormone mimic and synthetic juvenile hormone I on the bug Lygaeus kalmii

ABSTRACT. The possibility that JH mimics cause artifactual responses was investigated by comparing one such mimic (JH-M) and a 'true' JH (JH I) in the effects of each upon L.kalmii larval colouring and development. Post-blastokinesis treatment with synthetic JH I affected larval colour pattern in the same way as JH-M, but higher hormone concentrations were required. JH I also caused higher larval mortality. JH-M and JH I were equally effective in producing supernumerary instars.     

Juvenile hormone (JH) esterase of the mosquito Culex quinquefasciatus is not a target of the JH analog insecticide methoprene

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.     

The role of juvenile hormone in the control of reproductive function in Drosophila virilis under nutritional stress

To investigate the role of juvenile hormone (JH) in the control of Drosophila reproduction under stress, JH degradation and reproduction were studied under nutritional stress and JH treatment in Drosophila virilis females of wild type (wt) and a heat stress (hs) mutant: this mutant does not respond to heat stress by alterations in JH metabolism and has decreased JH level and fertility under normal conditions. One day of starvation results in a decrease of JH degradation, a delay in oocyte maturation, degradation of early vitellogenic egg chambers, accumulation of mature oocytes and a 24 h oviposition arrest in both wt and hs females. A fertility decrease was observed in both wt and hs females 24 h following the end of starvation. JH treatment leads to a decrease of JH degradation and an arrest of oviposition for 24 h in fed females. JH treatment prior to starvation seems to protect some oocytes from resorption: in JH-treated wt females, fertility increases rapidly following the end of starvation. The dynamics of JH degradation and fertility are similar following starvation and JH treatment. The role of JH in the accumulation of mature oocytes and the delay of oviposition under stress are discussed.     

Role of DopR in the molecular mechanism of the dopamine control of juvenile hormone metabolism in female Drosophila

The effect of a decreased availability of the D1-like dopamine receptor (DopR) in Drosophila (caused by DopR antagonist added into food) on the juvenile hormone (JH) synthesis rate in young female D. melanogaster has been studied. The JH degradation rate and the alkaline phosphatase (ALP) and tyrosine decarboxylase (TDC) activities were used as indicators of the JH synthesis rate. Treatment of the flies with butaclamol, a specific DopR antagonist, has been demonstrated to increase the JH degradation rate, and the stress reactivity of the system of JH metabolism and to decrease the ALP activity and stress reactivity, and to increase the TDC activity and stress reactivity. As shown earlier, all this indicates a decrease in the JH synthesis rate in young female drosophila with a decreased DopR availability. It is concluded that the activating effect of dopamine on JH synthesis in Drosophila is mediated by D1-like receptors.     

Effects of endogenous esterases and an allatostatin on the products of Manduca sexta larval corpora allata in vitro

Abstract A rapid and simple method has been developed for the simultaneous measurement of juvenile hormone (JH) and JH acid synthesized in vitro by larval corpora allata (CA) of the tobacco hornworm, Manduca sexta. An organic solvent partition of incubation medium efficiently separates JH acid from JH, and a radioimmunoassay which recognizes the two moieties equivalently is then employed to quantify each. The change in the biosynthetic product of the CA from JH to JH acid appears to begin slowly at the time of ecdysis to the last (fifth) larval stadium and is not complete until just prior to wandering (day 4). The inclusion of the JH esterase inhibitor S-benzoyl-O-ethyl phosphoramidothiolate in incubations of corpora allata revealed that the activity of JH esterases from the gland parallels gland activity and that significant hydrolysis of newly synthesized JH by these esterases occurs in incubations of glands taken at the beginnings of the fourth and fifth larval stadia. An allatostatin, which is proposed to inhibit the corpus allatum during the time of the change in its product, inhibits both JH I and JH I acid synthesis.     

Self-transferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecalis var. zymogenes.

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Ben-), and those from strain JH3 had also lost resistance to the bacteriocin (Bnr-). The majority of Hly-, Ben- variants from JH1 retained bacteriocin resistance (Bnrplus). Strains JH1 and JH3 contained a plasmid deoxyribonucleic acid species of molecular weight 38 times 10-6 (plasmids pJH2 and pJH3, respectively), and strain JH1 also contained a 50 times 10-6 molecular weight plasmid (pJH1) which has previously been shown to carry the genes determining resistance to the antibiotics kanamycin, neomycin, streptomycin, erythromycin, and tetracycline. Hly-, Bcn-, Bnr- variants of strain JH3 had completely lost plasmid pJH3. Hly-, Bcn-, Bnr- variants of strain JH1 had completely lost plasmid pJH2 and retained plasmid pJH1, but Hly-, Bcn-, Bnrplus variants had retained both plasmids pJH2 and pJH1. The Hlyplus, Bcnplus, Bnrplus traits from both parental strains were transferable to nonhemolytic S. faecalis strains during mixed incubation in broth at 37 C, and hemolytic recipient strains were found to have received plasmid pJH2 from strain JH1 and pJH3 from JH3. We conclude that the Hlyplus, Bnrplus traits are borne on plasmid pJH2 in strain JH1 and pJH3 in strain JH3 and that, in Hly-, Bcn-, Bnrplus variants of strain JH1, plasmic pJH2 has suffered a mutation affecting hemolysin and bacteriocin expression. We infer that the plasmids transfer by conjugation. Beta-hemolytic activity is the only property distinguishing the zymogenes variety from S. faecalis. Since we have shown that this activity is plasmid borne in strains JH1 and JH3, we endorse the view that the varietal status of zymogenes should be dropped.     

Juvenile hormone titers in virgin and mated Choristoneura fumiferana and C. rosaceana females: assessment of the capacity of males to produce and transfer JH to the female during copulation

We used a radioimmunoassay (RIA) to assess the effect of mating on juvenile hormone (JH) titer in females of the tortricid moths Choristoneura fumiferana and C. rosaceana. Virgins had undetectable levels of JH in their hemolymph on the 5th day of the pupal stage but titers rose to 1-4 and 0.2-0.5 ng JH II eq./ml, respectively, after emergence. On days 1, 3 and 5 following copulation, females of both species had higher JH titers than virgins of the same ages, with the greatest difference between virgin and mated females observed on day 3 for C. fumiferana and on day 5 for C. rosaceana. This increase was apparently not the result of a male-to-female transfer of JH during copulation since: (i) the accessory sex glands (ASGs) of males of both species displayed a very limited ability to convert JH acid into JH, (ii) ASGs produced no JH when incubated in vitro in the presence of L-[methyl-(3)H]-methionine, (iii) ASGs of males injected with L-[methyl-(3)H]-methionine 24 h prior to dissection contained no JH-associated radioactivity, and (iv) freshly formed spermatophores dissected out of females mated to similarly injected males contained no trace of radioactive JH. In addition, the JH content of ASGs and spermatophores, as measured by RIA, was not higher than that of virgin-female hemolymph, on a per-mg basis. However, in contrast with earlier findings in other species of moths, the CA of male C. fumiferana and C. rosaceana maintained in vitro in the presence of tritiated methionine produced and released JH I, JH II and JH III in quantities and proportions similar to those reported for female glands.     

The role of juvenile hormone in the larval diapause of the European corn borer,Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.     

Effects of the anti hormone-hormone mimic ETB on the induction of insect juvenile hormone esterase in Trichoplusia ni.

Juvenile hormone (JH) esterases can be artificially induced to appear in the hemolymph of last instar larvae of the lepidopterous insect $\text{Trichoplusia}$$\text{ni}$ (Noctuidae) by topical treatment with JH I, JH II, or dihomo branched juvenoids. ETB (ethyl-4-[2-(t-butylcarbonyloxy) butoxy] benzoate; ZR-2646) at high doses is a weak inducer of JH esterase (JHE). However, at doses of ETB that induce only low levels of JHE activity, ETB will block the JHE induction caused by the dihomo juvenoid epofenonane and at higher doses will reduce the induction caused by JH I or JH II. ETB is not a JHE inhibitor; rather, it appears to be acting as a JH agonist/antagonist in normal larvae and in isolated abdomens. These effects of ETB on JHE induction may illustrate a new mode of action of anti-JH's.