Fungal Metabolism of trans-2-Octenoic Acid

Juvenile hormone esterases (JHEs) function in juvenile hormone (JH) degradation. In the silkworm, Bombyx mori, we have characterized authentic JHE (Bmjhe) and five other carboxyl/cholinesterase (CCE) genes (Bmcce-1 to -5) with GQSAG, a motif sequence of JHE. But none of the genes appeared to function in vivo as a JHE, except for Bmjhe. Recently it was reported that the GQSAG motif might be dispensable, and that the Thr-316 residue has functional significance for JHE activity. On the basis of these findings, we identified two novel JHE candidates, Bmcce-6 and Bmcce-7, that lack GQSAG but possess Thr-316. In the CCE phylogenetic tree, BmCCE-6 was close to the lepidopteran JHE cluster, while BmCCE-7 constituted the same cluster as pheromone-degrading esterases. The developmental expression profiles were different among Bmjhe, Bmcce-6, and Bmcce-7. None of the proteins hydrolyzed JH in vitro. Our results suggest that only one CCE (BmJHE) functions as JHE in the silkworm.     

Improved insecticidal efficacy of a recombinant baculovirus expressing mutated JH esterase from Manduca sexta

Juvenile hormone esterase (JHE), a member of the carboxylesterase family (EC 3.1.1.1), metabolizes JH that is found in juvenile insects. A highly conserved amphipathic alpha helix is found on the surface of known JHEs. This helix is implicated in receptor-mediated binding and endocytosis of JHE by the pericardial cells resulting in the clearance of JHE activity from the hemolymph. In this study, Lys-204 and Arg-208 of the amphipathic alpha helix of the JHE of Manduca sexta (MsJHE) were mutated to histidine residues generating MsJHE-HH. Pharmacokinetic studies following the injection of MsJHE-HH into the hemocoel of larval M. sexta, Heliothis virescens, and Agrotis ipsilon indicated that MsJHE-HH and wild type MsJHE are cleared at similar rates. The infectivity (lethal concentration and lethal time) of a recombinant baculovirus, AcMsJHE-HH, expressing MsJHE-HH was not significantly different than that of a recombinant baculovirus, AcMsJHE, expressing MsJHE in first instars of H. virescens and A. ipsilon. However, the mass of AcMsJHE-HH-infected larvae was 40–50% lower than that of larvae infected with AcMsJHE, and 70–90% lower than that of wild type AcMNPV-infected larvae.     

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.     

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.     

Novel assay for determining the metabolic fate of juvenile hormone III: A study with Drosophila melanogaster

A thin-layer chromatographic assay was developed for the resolution of hydrolytic and conjugative catabolites of juvenile hormone (JH). A single-dimension, dual-development thin-layer system allowed complete resolution of the catabolites. Thus, this system provided a means for the rapid and economic analysis of JH hydrolysis even when different hydrolytic activities were present concurrently. Purified hydrolytic enzymes were found to be superior to chemical methods for the generation of small amounts of standards of JH catabolites. The relative levels of activities of an epoxide hydrolase and an esterase toward JH III were found to be similar in microsomal preparations from three lines of adult Drosophila melanogaster isolated from a field population. However, selection of flies by exposure to cut orange resulted in the elevation of levels of epoxide hydrolase activities, whereas esterase levels were not affected to the same extent. The formation of the JH acid-diol was not detected under the conditions of this study, suggesting that the JH acid and diol were not good substrates for epoxide hydrolase and juvenile hormone esterase, respectively.     

Regulation of JH titers: The relevance of degradative enzymes and binding proteins

Juvenile hormones play a crucial role in development, metamorphosis, and reproduction of insects. This mini-review discusses the nature of the juvenile hormones identified in insects and their changes in concentration in the hemolymph during development and reproduction. The hemolymph titer is largely determined by the rate at which juvenile hormones are synthesized and released by the corpora allata, but other factors are also involved in titer regulation, such as the affinity and concentration of juvenile hormone binding proteins in the hemolymph and the rate of juvenile hormone degradation in hemolymph and tissues. Juvenile hormone specific esterases occur in hemolymph and tissues, whereas epoxide hydrolases, which may degrade the hormone, are exclusively tissue bound. The activities of these degradative enzymes and the concentration of binding proteins change during the insect life cycle and these changes are related to fluctuations in hormone titer. However, we are still a long way from understanding the subtle interactions between these components in regulation of juvenile hormone titers. In particular, our knowledge is hampered by lack of information about the types, concentrations, and affinities of intracellular juvenile hormone receptors. © 1996 Wiley-Liss, Inc.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of JH epoxide hydrolase versus JH esterase activity in the cabbage looper, Trichoplusia ni, by juvenile hormone and xenobiotics

JH III esterase and JH III epoxide hydrolase (EH) in vitro activity was compared in whole body Trichoplusia ni homogenates at each stage of development (egg, larva, pupa and adult). While activity of both enzymes was detected at all ages tested, JH esterase was significantly higher than EH activity except for day three of the fifth (last) stadium (L5D3). For both enzymes, activity was highest in eggs. Adult virgin females had 4.6- and 4.0-fold higher JH esterase and EH activities, respectively, than adult virgin males. JH III metabolic activity also was measured in whole body homogenates of fifth stadium T. ni that were fed a nutritive diet (control) or starved on a non-nutritive diet of alphacel, agar and water. With larvae that were starved for 6, 28 and 52 h, EH activity per insect equivalent was 48%, 5% and 1%, respectively, of the control insects. At the same time points, JH esterase activity levels in starved T. ni were 29%, 4% and 3% of that of insects fed the nutritive diet. Selected insect hormones and xenobiotics were administered topically or orally to fifth stadium larvae for up to 52 h, and the effects on whole body EH and JH esterase activity analyzed. JH III increased the JH III esterase activity as high as 2.2-fold, but not the JH III EH activity. The JH analog, methoprene, increased both JH esterase and EH activity as high as 2.5-fold. The JH esterase inhibitor, 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), had no impact on EH activity. The epoxides trans- and cis-stilbene oxide (TSO and CSO) in separate experiments increased the EH activity approximately 2.0-fold. TSO did not alter JH esterase levels when topically applied, but oral administration reduced activity to 70% of the control at 28 h, and then increased the activity 1.8-fold at 52 h after the beginning of treatment. CSO had no effect on JH esterase activity. Phenobarbital increased EH activity by 1.9-fold, but did not change JH esterase levels. Clofibrate and cholesterol 5alpha,6alpha-epoxide had no effect on EH. JH esterase activity also was not affected by clofibrate, but cholesterol 5alpha,6alpha-epoxide reduced the JH esterase activity to 60-80% of the control. The biological significance of these results is discussed.     

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.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.     

Growth parameters associated with endocrine events in larval Trichoplusia ni (Hübner) and timing of these events with developmental markers

Numerous behavioural and morphological markers were found during the last instar of Trichoplusia ni which permit selection of highly synchronous groups of larvae for physiological or biochemical experiments. Growth parameters were also examined and it was found that the occurrence of a 6th instar was associated with a head-capsule width below the critical threshold of 1.66 mm. Starvation experiments indicated that the critical body weight triggering the first release of prothoracicotropic hormone (PTTH) was not the same for all larvae and was associated closely with a critical ratio of body size: head-capsule size and/or body size: initial body size at the beginning of the instar. The time of prothoracicotropic hormone-ecdysone release in preultimate instars was also associated with a similarly calculated ratio. The ratio was very similar from instar to instar. Neck or thoracic-abdominal ligation of larvae attaining various markers provided indications of times of release of critical amounts of prothoracicotropic hormone, ecdysone and juvenile hormone. The time of peak juvenile-hormone-esterase (JHE) activity in the haemolymph during the prepupal stage was determined with these markers.     

保幼激素的代谢

保幼激素的代谢由保幼激素酯酶、保幼激素环氧水解酶和保幼激素二醇激酶等共同催化完成。在这些代谢酶的作用下,保幼激素代谢成保幼激素酸、保幼激素二醇、保幼激素酸二醇和保幼激素二醇磷酸。作者总结了保幼激素代谢的研究方法;按实验室和昆虫种类为线索,归纳和概括了每一种保幼激素代谢酶的研究进程;对保幼激素酯酶和保幼激素环氧水解酶作了序列分析;最后对保幼激素的代谢研究进行了展望。