Juvenile hormone esterase activity in the pupating and diapausing larvae of Sesamia nonagrioides

The development of the Mediterranean corn borer, Sesamia nonagrioides, under long-day (LD) photoperiod is associated with juvenile hormone (JH) decline and pupation in the 5th or 6th larval instar. The larvae grown under short-day (SD) conditions maintain a moderate JH titer and enter diapause during which they undergo several extra larval molts. Both types of larvae exhibit similar levels of juvenile hormone esterase (JHE) activity that increases in each instar during the period of low ecdysteroid titer and drops when the titer rises to a molt-inducing peak. A suppression of JHE activity within 24h after application of an ecdysteroid agonist suggests that the drop of activity is a rapid and possibly direct response to ecdysteroids or their agonist. Esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) suppressed more than 98% of the JHE activity without affecting pupation timing and adult development. The data indicate that JHE is not crucial for the switch between larval development, diapause, and metamorphosis in S. nonagrioides.     

Juvenile hormone esterase activity and ecdysteroid titer in Heliothis virescens larvae injected with Microplitis croceipes teratocytes

Juvenile hormone esterase (JHE) activity in the hemolymph of 5th-instar Heliothis virescens larvae injected with Microplitis croceipes teratocytes was inversely related to the number of teratocytes injected. JHE activity in the hemolymph of larvae injected with 750 3-day-old teratocytes (the approximate number from one parasitoid embryo) was depressed to less than 5% of those levels found in control larvae. During the latter portion of the digging stage and in the burrowing-digging (BD) stage JHE activity in larvae treated with 350 teratocytes was approximately 40% of control values. However, injection of 180 teratocytes did not significantly affect JHE titers. Two-day-old teratocytes caused the greatest reduction in JHE titer with decreasing effects observed with injections of 3- to 6-day-old teratocytes. Nevertheless, because 2-day-old teratocytes were difficult to separate from host hemocytes, 3-day-old teratocytes were used in most of these studies. Injections of nonparasitized H. virescens hemolymph plasma, Micrococcus luteus bacterial cell walls, washed M. croceipes eggs, or teratocytes from Cotesia congregata did not depress JHE titers. Teratocyte injections also significantly reduced growth of host fat body. Ecdysteroid titers in cell formation, day 2 (CF2) larvae injected as new 5th instars with 350 3-day-old teratocytes failed to increase, as compared to noninjected and saline-injected controls. An injection of 1 μg/larva of 20-hydroxyecdysone at the BD stage permitted normal pupation in 50% of the teratocyte-treated larvae as compared to 0% pupation for teratocyte-treated control larvae not treated with 20-hydroxyecdysone. Teratocytes seem to be responsible for the inhibition of JHE release and thus indirectly impact on ecdysteroid titers. © 1992 Wiley-Liss, Inc.     

Reduced activity of juvenile hormone esterase in microsporidia-infected Lymantria dispar larvae

Infection of the fat body of Lymantria dispar (Lep.: Lymantriinae) larvae with the microsporidium Vairimorpha disparis has severe effects on juvenile hormone (JH) metabolism of the host. Beginning 8 days postinfection, activity of the JH degrading enzyme JH-esterase was significantly lower in the hemolymph of infected than uninfected larvae. Activity remained low as microsporidiosis progressed. JH titers were slightly elevated in infected larvae; the difference was not significant in most cases. This disturbance of JH metabolism may be due to generally impaired fat body functions and high demand for resources by the developing pathogen.     

Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA-JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA-JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak on the first day of the pupal phase (P0). This pattern of activity is distinct from that of hemolymph JHE activity, which peaks just prior to wandering on V4 and again just prior to pupation (V9). Although the CA-JHE and hemolymph-JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA-JHE and hemolymph-JHE activities are similar, but not identical, in nature. Exposure of the V6 CA in vitro to JH II (0.1 μM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA-JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA-JHE may also be integrally related to this positive feedback mechanism.     

Juvenile hormone activity in Dysdercus cingulatus Fabr by juvenile hormone esterase inhibitor, OTFP

Application of juvenile hormone esterase inhibitor 3-octylthio-1,1,1- trifluropropan-2-one (OTFP) to 5th instar nymphs and virgin females of D. cingulatus revealed the profound role played by juvenile hormone esterase (JHE) in metamorphosis and reproduction. The ability of OTFP to cause delay and the formation of malformed nymphs, suggests that inhibition of JHE in vivo maintains a higher than normal hemolymph JH titer. It is obvious that OTFP does inhibit in vivo JHE activity in late instar nymphs. Further, the application of JHE inhibitor, OTFP to virgin females demonstrates that substituted trifluropropanones can indirectly stimulate egg development by inhibiting JHE activity in virgin females.     

Juvenile hormone modulation of insect cold hardening: Ice-nucleating activity

Data are presented offering the first evidence for probable endocrine involvement in the control of cold hardening in Eurosta solidaginis. Juvenile hormone (JH) deprivation experiments in which the corpora allata were removed by head ligation resulted in a loss of supercooling (SC) capacity in larvae collected over 2 years. This loss of supercooling capacity is indicative of synthesis of organismal pools of ice-nucleating agents (INA). Larval sensitivity to JH removal (ligation) on SC is seasonally dependent. For example, in 1983, larvae were most sensitive in October, secondarily so in September, and relatively insensitive in December regardless of acclimation temperature. While in 1984, larvae acclimated to +5 °C were most sensitive in November, secondarily so in October, and relatively insensitive in December, and larvae acclimated to +15 °C were most sensitive in October and December and least so in November. Supercooling point elevations as great as 7 °C over controls were observed with maximal responses occurring within 1 day following ligation. In 1983, juvenile hormone replacement following ligation generally resulted in an expansion of supercooling capacity when compared to controls. As with ligation, the sensitivity to JH replacement on SC was seasonally dependent: September and October larvae being the most sensitive with December larvae being insensitive. Larvae collected in 1984 were given a greater dose of JH than those in the previous year and showed no significant change in SC over ligated-acetone controls. Hormone analog potentiation experiments in which unligated larvae collected in 1983 and acclimated to +5 °C were given methoprene resulted in depression of supercooling points for September and October larvae. JH titres appear to play an important role in the regulation of SC capacity in E. solidaginis larvae.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Proteomic analysis of parasitized Plutella xylostella larvae plasma

Insects use their innate immunity to defend themselves against foreign invaders, such as microorganisms, nematodes and parasites. Cotesia plutellae, an endoparasitoid wasp that parasitizes the diamondback moth Plutella xylostella, uses several strategies to attack the host immune system, such as injection of viruses, venom, and serosal membrane-derived cells denoted teratocytes. However, the proteome profiles related to these immune deficiency systems have yet to be clearly defined. In this study, we investigate differences in protein expression patterns in parasitized P. xylostella larvae, with a view to identifying parasitism-specific factors. Using 2D polyacrylamide gel electrophoresis, proteins in the host plasma were assessed every 48 h after parasitism by C. plutellae. A large number of protein spots (350 in total) were detected, and approximately 50 spots were differentially expressed in the parasitized P. xylostella larvae every 48 h. In total, 26 potential candidates, including P. xylostella Serpin 2 (pxSerpin 2), translationally controlled tumor protein, signal transduction histidine kinase, apolipophorin-III, and fatty-acid binding protein were identified through quadrupole time-of-flight tandem mass spectrometry and sequence homology analysis. These proteins were classified into the following functional groups: immunity, signaling, lipid metabolism, energy metabolism, amino acid/nucleotide metabolism, and others. The pxSerpin 2 gene was cloned, and its expression profile investigated during the course of parasitism. Real-time PCR analysis of pxSerpin 2 revealed a poor correlation between the mRNA level and protein abundance. Our results clearly suggest that parasitism-specific proteins participate in suppression of the host immune response.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

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

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

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.     

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 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.     

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.     

Juvenile hormone-binding protein and juvenile hormone esterase activity in hemolymph from last-instar nymphs of Leucophaea maderae

The concentration of the juvenile hormone-binding protein (JHB) in hemolymph was determined throughout the last nymphal instar. It was found to be 3.9 μM at the molt to the instar, rising to 13 μM by mid-instar, and dropping to 6.7μM the day before emergence. Endocrine control of its production during the last nymphal instar could not be established. The apparent juvenile hormone esterase (JHF) activity was low at the molt to the last instar, but rose about fivefold by mid-instar, and then modestly declined. On the day of emergence, JHF activity rose to the highest level observed. A four- to fivefold increase in absolute JHF activity was determined during the first half of the last nymphal instar. This increase is not regulated by JH. Removal of the JHB from hemolymph samples by precipitation with a polyclonal specific antibody increased the JHF activity up to 1,000-fold. Thus, changes in the concentrations of JHB can affect the apparent activity of JHE, which is unrelated to the production or degradation of the JHF.     

Juvenile hormone activity in larvae and adult females of the locust, Schistocerca gregaria

The isolation and purification of fractions with juvenile hormone activity from whole animal extracts of larvae, and from extracts of haemolymph from larvae and adults is described. Using the Galleria bioassay three such fractions were isolated from both third and fourth instar hoppers and from adult females. The chromatographic behaviour of these fractions indicates that one contains JH III, but the other two contain unknown juvenilizing compounds.     

昆虫神经毒性酯酶活性的测定

首次报道了针对昆虫建立的神经毒性酯酶(NTE)活性检测方法以及据此法所测得的棉铃虫幼虫的NTE活性。将测定脊椎动物NTE活性的方法改进并微量化以适用于无脊椎动物昆虫体内NTE活性测定。对于棉铃虫幼虫,该法测得其头部、中肠和脂肪体等3个部位的NTE活性分别为5.30,1.40和14.50nmolminmgprotein。     

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.