Characterization and analysis of novel carboxyl/cholinesterase genes possessing the Thr-316 motif in the silkworm, Bombyx mori

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.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Only one esterase of Drosophila melanogaster is likely to degrade juvenile hormone in vivo

Previously we identified juvenile hormone esterase (JHE) from Drosophila melanogaster by the criteria that it showed both appropriate developmental expression and kinetics for juvenile hormone (JH). We also noted three further esterases of D. melanogaster with some JHE-like characteristics, such as a GQSAG active site motif, a particular amphipathic helix, or close phylogenetic relationship with other JHEs. In this study, these JHE-like enzymes were expressed in vitro and their kinetic parameters compared with those of the previously identified JHE. Despite considerable phylogenetic distance between some of the esterases, they could all hydrolyse racemic JHIII. However, only the previously identified JHE had kinetic parameters (K(M) and k(cat)) towards various forms of JH (racemic or individual isomers of JHIII, JHII, JHI, and methyl farnesoate) consistent with a physiological role in JH regulation. Furthermore, only this JHE showed a preference for artificial substrates with acyl chain lengths similar to that of JH. This suggests that there is probably only one physiologically functional JHE in D. melanogaster but multiple esterases with JH esterase activity. Genomic comparisons of the selective JHE across 11 other Drosophila species showed a single orthologue in 10 of them but Drosophila willistoni has 16 full-length copies, five of them with the GQSAG motif and amphipathic helix.     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

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.

---

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.

     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

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.     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.     

EVOLUTIONARY ENDOCRINOLOGY OF JUVENILE HORMONE ESTERASE: FUNCTIONAL RELATIONSHIP WITH WING POLYMORPHISM IN THE CRICKET,GRYLLUS FIRMUS

The existence, nature, and physiological consequences of genetic variation for juvenile hormone esterase (JHE) activity was studied in the wing-polymorphic cricket, Gryllus firmus. Hemolymph (blood) JHE activity was sixfold lower in nascent short-winged (SW) females, relative to nascent long-winged (LW) females during the last juvenile stadium (stage). Morph-associated genetic variation for JHE activity had two causes, variation in loci: (1) regulating whole-organism enzyme activity; and (2) controlling the degree to which JHE is secreted into the blood Reduced JHE activity in nascent SW-selected individuals was associated with reduced in vivo juvenile hormone catabolism. This suggests that variation in JHE activity during juvenile development may have important physiological consequences with respect to the regulation of blood levels of juvenile hormone and consequent specification of wing morph. This is the first definitive demonstration of genetic variation for hormonal metabolism in any insect and a genetic association between hormone metabolism and the subsequent expression of morphological variation (wing morph). However, we have not yet firmly established whether these associations represent causal relationships In contrast to the clear association between JHE activity and wing morph development, we observed no evidence indicating that variation in JHE activity plays any direct or indirect role in causing the dramatic differences in ovarian growth between adult wing morphs. Variation in JHE activity also does not appear to be important in coordinating the development of wing morph with the subsequent expression of reproductive differences between adult morphs. Finally genetic variation for the developmental profiles of JHE activity during juvenile and adult stages are remarkably similar in three Gryllus species. This suggests that genetic correlations between JHE activities during different periods of development, which underlie these activity profiles, have been conserved since the divergence of the three Gryllus species.     

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.     

The effect of juvenile hormone and its antagonists on JH esterase activity in Tenebrio molitor

The hemolymph juvenile hormone esterase (JHE) and general carboxyl esterase activities in Tenebrio molitor show independent development-associated changes during larval-pupal and pupal-adult metamorphoses. JHE activity was high in pharate pupae and early pupae. Unlike in lepidopteran pupae that have been studied thus far, JH had no effect on JHE activity in pupae and pharate adults of Tenebrio. A JH antagonist, ethyl 4,2,tert butyl carboxy n benzoate (ETB), and 20-hydroxyecdysone had no effect on JHE activity. These observations suggest that although JHE activity in Tenebrio is precisely regulated during larval-pupal metamorphosis, JH and molting hormone do not appear to be involved in its regulation and that the proximate cues that influence JHE activity in Tenebrio pupae are different from that of lepidopterans.

---

Résumé Les activités JHE (estérase juvénile hormone) de l'hémolymphe et carboxyl estérase générale de T. molitor changent indépendamment du développement pendant les métamorphoses larve-nymphe et nymphe-adulte. L'activité JHE est élevée chez les prénymphe et les jeunes nymphes. Contrairement aux chrysalides de papillons, JH n'a pas d'effet sur l'activité JHE chez les nymphes et les préadultes de Tenebrio. Un antagoniste de JH, l'éthyl 4,2,tert butyl carboxy n benzoate (ETB), 20-hydroxyecdysone et le précocène 2-agent anti-allate chez plusieurs insectes-, n'ont aucun effet sur l'activité JHE. Ces observations suggèrent que, bien que l'activité JHE chez T. molitor soit ajustée avec précision pendant la métamorphose larvo-nymphale, JH et hormone de mue ne paraissent pas être impliquées dans sa régulation et que les signaux immédiats, qui influent sur l'activité JHE de la nymphe de T. molitor, diffèrent de ceux des Lépidoptères.

     

A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth,Plutella xylostella

Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI = 5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.     

Genome-wide comparison of genes involved in the biosynthesis,metabolism, and signaling of juvenile hormone between silkworm and other insects

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.     

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.