The role of juvenile hormone esterase in terminating larval feeding and initiating metamorphic development in Trichoplusia ni

This study shows, first, that when JH degradation by JHE is blocked with an inhibitor (EPPAT, O-ethyl-S-phenyl-phosphoramidothiolate), prothoracicotropic/ecdysone release/effects are postponed in the cabbage looper Trichoplusia ni (Hübner) (Noctuidae). Thus, JHE is an important component of JH degradation, implying that without normal degradation the JH titer will become abnormally high. Second, this accumulation of endogenous JH in EPPAT treated larvae results in an extra larval molt. Therefore, JHE is also important in the control of the nature of the molt, by controlling the JH titer. Third, this study demonstrates that EPPAT at proper doses is a viable probe for studying enzyme and hormone action in vivo without pharmacological artifacts.

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Résumé Cette étude indique d'abord que, lorsque la dégradation de l'hormone juvénile (JH) par la JHE est bloquée par un inhibiteur (EPPAT, O-éthyl-S-phényl-phosphoramidothiolate) les effets prothoracicotropiques—libération d'ecdysone—sont retardés chez Trichoplusia ni Hübner. Ainsi, la JHE est un élément important de la dégradation de JH, impliquant que sans une dégradation régulière, la teneur en JH deviendra anormalement élevée. Cette accumulation d'hormone juvénile endogène chez les larves traitées à l'EPPAT provoque de plus une mue larvaire supplémentaire. Par conséquent, JHE est importante aussi dans le contrôle de la nature de la mue, en déterminant la teneur en JH. Enfin, cette étude a montré que l'EPPAT à des doses appropriées est un moyen efficace pur étudier l'action hormonale in vivo sans artéfacts pharmaceutiques.

     

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.     

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.     

Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis.

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.     

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 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.     

Characterization of affinity-purified juvenile hormone esterase from Trichoplusia ni

Juvenile hormone (JH) esterase was purified greater than 1000-fold in one step from hemolymph and whole larval homogenates from the last larval instar of Trichoplusia ni to give a single diffuse band that migrates at Mr = 64,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was based on an affinity chromatography procedure that employs trifluoromethyl ketone ligands. Isoelectric focusing of the purified preparations resulted in multiple bands that coincided to all significant hydrolysis of juvenile hormone detected in this manner. Kinetic experiments using optically pure enantiomers of JH II as substrates showed the two main electromorphs of JH esterase from the hemolymph to have apparently identical kinetic parameters as well as a similar capability to distinguish between substrates that differ in the orientation of the epoxide moiety of JH. However, the enzyme could hydrolyze esters lacking the JH structure. The proteins were shown to be monomers and to have asparagine-linked oligosaccharides, most likely of hybrid structure. Immunochemical and other evidence showed that the affinity-purified proteins were responsible for all significant JH esterase activity during periods of rapid esterolysis in vivo.     

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.     

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.     

Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta

The mechanisms of degradation of juvenile hormone esterase (JHE) were investigated in larvae of the tobacco hornworm, Manduca sexta. JHE is removed from the hemolymph by the pericardial cells by receptor-mediated endocytosis and is ultimately degraded in the lysosomes. Immunoprecipitation experiments and native PAGE followed by Western blotting showed that JHE associates with a putative heat shock cognate protein (Hsp). Approximately 25% of the active JHE in the pericardial cell complex is associated with the putative Hsp 1 h postinjection of affinity purified JHE. Electron microscope analysis revealed that the putative Hsp is located in the trans-Golgi network of pericardial cells, where it is hypothesized to be involved in sorting of proteins destined for the lysosomes, from those destined for the cell membrane. Data acquired from immunoprecipitation and Western blotting experiments argue against the involvement of ubiquitin in the degradation of JHE. Injection of radiolabeled JHE into larvae of M. sexta followed by SDS-PAGE of pericardial cell homogenates revealed covalent binding of an unidentified protein to JHE in the pericardial cell complex. Arch. Insect Biochem. Physiol. 34:275–286, 1997. © 1997 Wiley-Liss, Inc.     

粉纹夜蛾颗粒体病毒重组增效蛋白的增效作用

采用时间 剂量 死亡率模型 ,分析了粉纹夜蛾 (Trichoplusiani)颗粒体病毒重组增效蛋白P96对棉铃虫 (Helicoverpaarmigera)核型多角体病毒 (HaNPV)感染棉铃虫幼虫的增效作用。结果显示 :感染后 11d ,HaNPV P96组的LC50 值为 3.4 7× 10 3 多角体 /mL ,比HaNPV组 ( 3.89× 10 4 多角体 /mL)降低了 91.0 8% ;在 1.6× 10 4 ～ 1.6× 10 6多角体 /mL浓度范围内 ,HaNPV P96组的LT50 值较HaNPV组缩短 0 .3～ 1.8d。P96显著提高了HaNPV对棉铃虫幼虫的毒力     

Endocrine interrelationship between the parasitoid Chelonus sp. and its host Trichoplusia ni

The egg-larval parasitoid Chelonus sp. induces the precocious onset of metamorphosis in the 4th (penultimate) stadium of its host Trichoplusia ni, emerges from the prepupa, and then feeds on it. Qualitative and quantitative changes in ecdysteroids and juvenile hormone were measured. Hemolymph of 3rd-to 4th-instar host larvae and the parasitoids they contained, as well as nonparasitized and parasitized eggs, were analyzed. In the host hemolymph a broad peak of ecdysteroids during molting into the 4th stadium and a continuous increase from day 2 (onset of precocious wandering) until day 4 (emergence of parasitoid) were observed; 20-hydroxyecdysone and 20,26-dihydroxyecdysone were predominant. The juvenile hormone titer fluctuated in the 3rd and early 4th stadium and fell to undetectable levels shortly before the precocious onset of wandering. The parasitoid's ecdysteroids started to increase on the molt to the 2nd instar (= early 4th instar of the host) and thereafter fluctuated on a high level, 20-hydroxyecdysone, 20,26-dihydroxy-ecdysone, and ecdysone being predominant. The juvenile hormone titer was high in late 1st-instar parasitoids, decreased to low levels at ecdysis into the 2nd instar, and increased again to high levels in the 2nd-instar larvae at the time when their shape changed from flat to cylindrical. After ecdysis to the 3rd instar the juvenile hormone titer fell. A comparison revealed that both ecdysteroids and juvenile hormone fluctuate independently in parasitoid and host at most stages, suggesting that the parasitoid produces its own hormones. The first data on ecdysteroids and juvenile hormones in the egg stage of a parasitoid/host system are reported. At the stage of eye pigmentation parasitized eggs contained more immunoreactive midpolar ecdysteroids than non-parasitized ones. 20-Hydroxyecdysone and 20,26-dihydroxyecdysone were the predominant ecdysteroids in both nonparasitized and parasitized eggs, but the latter contained several additional ecdysteroids which were not seen in nonparasitized eggs. The titer of juvenile hormone was similar in both. Shortly before hatching the ecdysteroids were low in parasitized and nonparasitized eggs, but the content of juvenile hormone was much higher in the former. At this stage the majority of parasitoids have already eclosed and teratocytes are released. The results of HPLC analysis indicated the presence of juvenile hormone III together with juvenile hormones I and II in parasitized eggs, but only juvenile hormones I and II in nonparasitized eggs.     

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.     

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。     

The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata

Following unilateral allatectomy in the viviparous cockroach, Diploptera punctata, the remaining corpus allatum (CA) can synthesize juvenile hormone (JH) at rates equal to that of a pair of control CA. A single CA undergoes changes in volume and cell number similar in magnitude to those occurring during the normal cycle of JH synthesis. There is no hyptertrophy of the CA associated with compensation in JH synthesis. Therefore, the rate of JH synthesis per cell or per unit volume of single CA following unilateral allatectomy is twice that of a single control CA.     

重组保幼激素酯酶的纯化和生物学效应

培养昆虫细胞生产重组昆虫保幼激素酯酶时细胞培养液的蛋白质浓度为153.2～188.0 μg/mL。批量处理纯化重组保幼激素酯酶时酶蛋白活力回收率33%,效果与梯度分离方法相当,但简便快速,可作为大量分离纯化的第一步。重组保幼激素酯酶对烟草天蛾Manduca sexta幼虫的生物学活性测定结果验证了重组保幼激素酯酶对烟草天蛾幼虫和自身天然酶有相似的生物学活性。     

Circadian changes in melatonin in the nervous system and hemolymph of the cabbage looper moth,Trichoplusia ni

Quantitative levels of melatonin and 5-hydroxytryptamine were measured over the scotophase in the protocerebrum, subesophageal ganglion, optic lobes, thoracic ganglia, and hemolymph of adult male cabbage looper moths, Trichoplusia ni, using HPLC with electrochemical detection. Melatonin levels were very low (s < 1 pmol) or undetectable during the photophase, but increased in all tissues during the dark. Lowest mean levels in the dark were observed in the optic lobes (0.3 to 0.7 pmols). Maximal mean levels in the protocerebrum (5.2 pmols) occurred in the early part of the scotophase, but in all other tissues (2.8 in the subesophageal ganglion; 9.5 in thoracic ganglia) and hemolymph (18 pg/l) maximal mean levels were observed later in the dark. Levels of 5-hydroxytryptamine in each tissue, in contrast, were higher than melatonin levels in the photophase, and in the protocerebrum and thoracic ganglia decreased during the dark, but in the optic lobes and subesophageal ganglion remained unchanged. Further, decreases in 5-hydroxytryptamine during the dark were significantly lower than the increased levels of melatonin, suggesting that active synthesis of 5-hydroxytryptamine was occurring. In a second experiment, when measured from individuals in three different photoperiods (618, 1212, 186 lightdark) maximum mean melatonin levels in the brain (protocerebral and subesophageal ganglia) peaked within the first 1.5 h of the dark and remained at measurable levels for the duration of the dark. In a third experiment, levels of melatonin in the brain and thoracic ganglia displayed rhythmicity in continuous dark conditions but not in continuous light, when compared with profiles obtained in a normal light dark regime.Abbreviations CNS Central nervous system - HPLC-ECD high performance liquid chromatography with electrochemical detection - MEL melatonin - 5HT 5-hydroxytryptamine - N-ac-SHT N-acetyl-5-hydroxytryptamine