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.     

Characterization and affinity purification of juvenile hormone esterase from Bombyx mori

Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.     

Identification and partial characterization of juvenile hormone esterase from cotton pest Dysdercus cingulatus

Juvenile hormone esterase (JHE), a selective enzyme that hydrolyzes the methyl ester of insect juvenile hormone plays an important role in regulating metamorphosis in nymphs as well as reproduction in adults. Studies on JH degradation provide insight into the possibilities of physiological disruption in the insects. In the present study, the JH degrading enzyme, JHE from the cotton pest Dysdercus cingulatus (Heteroptera) is characterized. Electrophoretic analysis of haemolymph during various developmental stages showed the JHE bands prominent only on the final day of 5th instar nymph, and the esterase substrate specificity confirmed the presence of JHE isoforms. In an attempt to clone cDNA of JHE gene from the final instar nymphs, mRNA isolated from fat bodies was coupled with JHE gene-specific primers and the cDNA was synthesized using RT-PCR. The PCR amplified cDNA showed the presence of JHE isoforms in D. cingulatus.     

Recombinant juvenile hormone esterase as a biochemical anti-juvenile hormone agent: Effects on ovarian development in Acheta domesticus

By investigating the effects of recombinant juvenile hormone esterase (JHE) on the stimulation of ovarian development and egg laying in the house cricket Acheta domesticus L., we have tested the hypothesis that recombinant JHE (derived from the tobacco budworm Heliothis virescens) can be used as a biochemical anti-juvenile hormone (JH) agent. Recombinant JHE, produced by a genetically engineered baculovirus, was affinity-purified and injected into females of A. domesticus. JHE was cleared rapidly from the hemolymph of the crickets. However, upon repeated injection, significant reductions were seen in the extent of development of the ovaries and in the numbers of eggs laid. The effects of JHE could be rescued by topical application of the JHE inhibitor, OTFP. Thus, we have demonstrated an anti-JH effect on reproduction and that the recombinant JHE derived from a lepidopteran is active in an orthopteran insect. Arch. Insect Biochem. Physiol. 34:359–368, 1997. © 1997 Wiley-Liss, Inc.     

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.     

Biochemical characterization of juvenile hormone esterases from lines selected for high or low enzyme activity inGryllus assimilis

In a previous study, activity of the insect endocrine regulator juvenile hormone esterase (JHE), in the cricketGryllus assimilis, was subjected to bidirectional selection. This resulted in three pairs of high- and low-selected lines, each of which differed by 3.5-fold in JHE activity. In the present study, juvenile hormone esterases from these lines were characterized with respect to the Michaelis constant (K _m), thermostability, and inhibition. None of three high-selected JHEs differed from its respective low-selected JHE in the Michaelis constant (K _m) for juvenile hormone. Similarly, the high-selected JHEs did not differ from the low selected JHEs in thermostability or inhibition by either of two general esterase inhibitors (DFP, eserine) or a “JHE-specific” inhibitor (OTFP). Thus no evidence was obtained to suggest that the response to selection was due to allozymes or isozymes with altered kinetic or stability properties. Kinetic and stability properties were also very similar for the JHEs from the three high-selected or the three low-selected lines. Finally, none of the thermostability or inhibition profiles for any of the six JHEs exhibited sharp discontinuities, thus providing no evidence for the existence of multiple isozymes. The available evidence points to genetically variable regulators which affect the synthesis, degradation, or tissue distribution of JHE as being responsible for the divergence in JHE activity between the selected lines.     

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.     

Potential ligands of DmP29, a putative juvenile hormone esterase binding protein of Drosophila melanogaster

We previously reported the identification of a putative juvenile hormone esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155-163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant. Given the high abundance, and "sticky" nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.     

Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea

The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.     

Coordinate induction of electrophoretic variants of juvenile hormone esterase

Major and minor electrophoretic variants of juvenile hormone esterase (JHE) were found in the hemolymph of last instar larvae of Trichoplusia ni, both before and after metamorphic commitment. The average ratios of activity of the two major forms were similar during both last stadium peaks in activity. Immunological analysis showed that the hemolymph concentration of JHE during this stadium paralleled the level of enzymatic activity, and no putative higher molecular weight, inactive forms were detected. Immunological analysis provided the first evidence of relatedness of major and minor forms. After hormonal stimulation, the concentration of the two major forms increased concomitantly and by a similar proportion, suggesting that charge variation, at least for these two major forms, is not a point of hormonal or developmental regulation of JHE.     

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.     

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

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.