Haemolymph juvenile hormone III esterase activity during adult life of female and male crickets,Gryllus bimaculatus de Geer (Ensifera,Gryllidae)

Using an in vitro method, juvenile hormone III degradation was studied in the plasma of adult female and male crickets, Gryllus bimaculatus. The primary route of juvenile hormone III metabolism in cricket haemolymph is ester hydrolysis to juvenile hormone III acid by juvenile hormone III esterase. Juvenile hormone III esterase activity in females' haemolymph is low just after imaginal moulting. A sharp peak of enzyme activity is observed on day 3 after emergence, and two subsequent peak values occur in older animals. Plasma juvenile hormone III esterase activity in freshly ecdysed males is also low, but increases rapidly thereafter. Another increase in enzyme activity is observed in older males. The fluctuations in juvenile hormone III esterase activity are discussed in correlation with changes in haemolymph volume, haemolymph protein content, haemolymph juvenile hormone III titer, and the rates of juvenile hormone III biosynthesis in vitro of the corpora allata.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

Effects of juvenile hormone I and the anti-juvenile hormone fluoromevalono-lactone on development and juvenile hormone esterase activity in post-feeding last-stadium larvae of Trichoplusia ni (Hübner)

Treatment of post-feeding (early day 3; wandering phase) last-stadium larvae of the cabbage looper, Trichoplusia ni, with the anti-juvenile hormone, fluoromevalonolactone, prevented the normal ecdysis to the pupa. It caused the formation of larval-pupal intermediates, a dose-dependent delay in the time of tanning, and a decrease in juvenile hormone esterase activity at the time of the prepupal juvenile hormone esterase peak. Fluoromevalonolactone was inactive as juvenile hormone esterase inhibitor in vitro. Conversely, juvenile hormone I accelerated the time of tanning, induced the early appearance of juvenile hormone esterase activity, and prevented adult eclosion. Although most of the larvae that were treated with fluoromevalonolactone immediately after the prepupal burst of juvenile hormone (late on day 3; post-spinning phase) still became larval-pupal intermediates, the time of tanning and juvenile hormone esterase activity were close to normal. Topical treatment of day-3 larvae with radiolabelled juvenile hormone I resulted in the rapid appearance and decline of radiolabelled juvenile hormone I in the haemolymph which was associated with the increased production of juvenile hormone I acid and the induced appearance of juvenile hormone esterase activity. Thus, in post-feeding last-stadium larvae of T. ni, juvenile hormone seems to be necessary for the proper formation of the pupa. Juvenile hormone is also involved in determining the time of pupation, and it appears to induce its own degradation.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Induction and regulation of juvenile hormone esterases during the last larval instar of the cabbage looper, Trichoplusia ni

Juvenile hormone esterase titres were monitored in gate I and gate II last instar larvae of Trichoplusia ni using JH III as substrate. Two peaks of activity were observed for both gate I and gate II larvae, although the first and second juvenile hormone esterase peaks for the gate II larvae are extended and delayed one day, respectively. Head or thoracic ligations before the prepupal stage lower or block the appearance of both esterase peaks. Juvenile hormone I and II, as well as homo and dihomo juvenoids can induce the second juvenile hormone esterase peak in both normal and ligated larvae, and increase the esterase titre during the first peak in nonligated larvae. Induction of the juvenile hormone esterases is possible in non-ligated larvae as soon as the moult to the last instar has occurred and in ligated larvae as soon as the first esterase peak has started to decline. Distinct mechanisms of regulation are present for the first and second juvenile hormone esterase peaks. Juvenile hormone does not appear to be involved in regulating its own metabolism by directly inducing the first esterase peak; however, evidence is consistent with a brief burst of juvenile hormone which occurs prior to pupation inducing the production of the second peak of juvenile hormone esterase activity.     

Interactions between a non-terpenoid carbamate with juvenile hormone activity and esterases in the last larval instar of Adoxophyes reticulana Huebn

The summer fruit tortrix moth is very susceptible to compounds with juvenile hormone activity. Ro 13-5223, a non-terpenoid carbamate, is 3–4 orders of magnitude more active in inhibiting metamorphosis in the last-instar larvae than juvenile hormone I. Larvae reared in permanent contact with this substance are characterised by higher juvenile hormone esterase activity but lower α-naphthyl esterase activity when compared to the untreated controls. In vitro Ro 13-5223 inhibits juvenile hormone hydrolysis but only in dosages which are far above the concentrations found in haemolymph of larvae exposed to the ¹⁴C-labelled compound. It does not serve as a substrate for juvenile hormone esterase in vitro even though it induces the enzyme activity in vivo. All these characteristics may account for the very high biological activity of Ro 13-5223 which disrupts humoral coordination of insect development.     

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels.Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture.Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT (S-benzyl-O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male).This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.     

Juvenile hormone esterase is a biochemical anti-juvenile hormone agent

Juvenile hormone esterase, purified by affinity chromatography from the larval hemolymph of Manduca sexta in the fifth stadium, was injected into larvae of the same species in the earlier stadia resulting in a blackening of the cuticle following ecdysis to the next larval stadium. This anti-juvenile hormone response was dose-dependent for an injection in the second, third or fourth stadium. Cuticular blackening was prevented by treating larvae with the juvenoid epofenonane. Larval response to injected juvenile hormone esterase also varied with the time of injection within a single stadium, having a maximum effect for injections at the time of head capsule slippage. Juvenile hormone esterase activity measured from the hemolymph after injection of larvae in the second stadium decreased over an 11 h time-course. Because the anti-juvenile hormone effects resulting from a single injection of juvenile hormone esterase were dependent on the time of injection, it appears that when juvenile hormone biosynthesis is active in the insect, the duration of enzyme activity limits the anti-juvenile effects that can be induced.     

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.     

Effect of lighting conditions on endocrine events in Galleria mellonella

Galleria mellonella larvae reared in a light:dark (LD) 12:12 regime terminate feeding and pupate 1 day earlier than insects kept in constant darkness. Rearing conditions have no influence on the body weight attained at pupation. In both rearing conditions body weights attained by females were greater on the average by 50% than the body weights of males.In LD 12:12 all distinctive changes in the juvenile hormone titre and juvenile hormone esterase activity occurred 1 day earlier than in constant darkness. The peak value of juvenile hormone esterase activity was by 22% higher in insects reared under light conditions as compared with animals kept in constant darkness.Last-instar G. mellonella larvae reared in constant darkness were more sensitive to cooling and juvenile hormone analogue application. The chilling-induced elevation of juvenile hormone titre was also higher in constant darkness. The chilling-induced elevation of the brain allatotropic activity was not influenced by rearing conditions (brain activity measured as the number of extra-larval moults produced by hosts implanted with chilled brains). However, rearing in an LD 12:12 regime decreases the host sensitivity to implanted brains.     

A novel protein that binds juvenile hormone esterase in fat body tissue and pericardial cells of the tobacco hornworm Manduca sexta L

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm moth Manduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and native M. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.     

Improved assay conditions for measurement of corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata

Assay conditions for the short-term, radiochemical, in vitro determination of the spontaneous rate of juvenile biosynthesis by isolated corpora allata from Leptinotarsa decemlineata have been further improved, permitting the measurement of juvenile hormone biosynthesis by individual pairs of corpora allata. The final incubation product has been identified as juvenile hormone III with the aid of High-performance liquid chromatography (HPLC) and juvenile hormone esterase degradation. Using the new assay conditions, the activities of adult corpora allata during maturation were found to be significantly higher in reproductive, long-day animals than in pre-diapause, short-day beetles. During diapause no activity was detectable, whereas corpora allata from post-diapause beetles were reactivated totally after 5 days. Simultaneous determination of the in vitro rates of juvenile hormone biosynthesis and corpus allatum volumes revealed no clear correlation although the results suggest that the volume may be indicative of the maximal capacity for juvenile hormone production. Corpora allata from a population of beetles did not display any synchronous diurnal rhythmicity.     

Critical roles for juvenile hormone and its esterase+ in the prepupa of Trichoplusia ni

In the caterpillar Trichoplusia ni (Lepidoptera: Noctuidae) it has been demonstrated by allatectomy that the appearance of juvenile hormone during the prepupal stage is crucial for the successful larval-pupal ecdysis of most larvae. Application of juvenile hormone or juvenile hormone esterase inhibitors at key times disrupted normal development as well. Thus the subsequent disappearance of juvenile hormone is regulated by degradation by juvenile hormone esterase in addition to a hypothetical reduction in biosynthesis. This reduction in juvenile hormone titer in the prepupa is just as critical for normal development as was its previous appearance. These observations on the critical role of juvenile hormone in the prepupa are in contrast to observations in some other species. For instance, in the case of Manduca sexta (Lepidoptera: Sphingidae), juvenile hormone is considered only supplementary to the action of prothoracicotropic hormone in the postwandering stage and primarily is required for normal pupal development. It thus appears that even within the Lepidoptera the role of juvenile hormone in prepupal development can vary dramatically.     

Prepupal regulation of juvenile hormone esterase through direct induction by juvenile hormone

The regulation of the prepupal peak of juvenile hormorne esterase activity was investigated and found to be directly induced by juvenile hormone. Allatectomy and reimplanation as well as juvenile hormone application experiments all indicated that the appearance of prepupal juvenile hormone esterase activity was in response to a prepupal burst of juvenile hormone. Implantation experiments indicated that the effect of juvenile hormone is not mediated through the isolated brain or subesophageal ganglion.     

Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (pI 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation by cyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.     

Developmental regulation of juvenile hormone esterase in Trichoplusia ni: Its multiple electrophoretic forms occur during each larval ecdysis

Juvenile hormone esterase activity has been found during the intramoult period of each larval stadium in Trichoplusia ni. The activity is indistinguishable from that occurring during the final larval stadium, on the basis of its four isoelectric forms and kinetic data. The 4th- to 5th-instar intramoult peak in activity also occurs in other Lepidoptera (Heliothis virescens, Spodoptera exigua, Manduca sexta and Hyphantria cunea). Further, the final species also possessed a peak of activity during the intramoult period to the penultimate larval instar. The findings have important implications for the current concept that the function of juvenile hormone esterase is, by reason of its anti-juvenile hormone action, an enzyme of the last larval instar which enables metamorphosis to begin.     

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.