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.     

Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (pI 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation by cyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.     

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.     

Critical roles for juvenile hormone and its esterase+ in the prepupa of Trichoplusia ni

In the caterpillar Trichoplusia ni (Lepidoptera: Noctuidae) it has been demonstrated by allatectomy that the appearance of juvenile hormone during the prepupal stage is crucial for the successful larval-pupal ecdysis of most larvae. Application of juvenile hormone or juvenile hormone esterase inhibitors at key times disrupted normal development as well. Thus the subsequent disappearance of juvenile hormone is regulated by degradation by juvenile hormone esterase in addition to a hypothetical reduction in biosynthesis. This reduction in juvenile hormone titer in the prepupa is just as critical for normal development as was its previous appearance. These observations on the critical role of juvenile hormone in the prepupa are in contrast to observations in some other species. For instance, in the case of Manduca sexta (Lepidoptera: Sphingidae), juvenile hormone is considered only supplementary to the action of prothoracicotropic hormone in the postwandering stage and primarily is required for normal pupal development. It thus appears that even within the Lepidoptera the role of juvenile hormone in prepupal development can vary dramatically.     

Characterization of two major isoforms of juvenile hormone esterase from Trichoplusia ni (Lepidoptera)

The two major isoforms of juvenile hormone (JH) esterase isolated from Trichoplusia ni were fragmented by cyanogen bromide and trypsin digestion. The resulting CNBr or CNBr/trypsin fragments were characterized and compared biochemically by SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis and HPLC. Similar and unique fragments were examined for sequence, antigenic determinants and carbohydrate moieties. The studies identified small regions of the proteins which possess either potentially different sequences or different post-translational modifications. The location of a glycosylated asparagine residue was determined, as well as a region containing an epitope probably composed of a linear sequence of residues. An N-terminal region was identified that contained charge variation between the two isoforms and the sequence was obtained for the only unique CNBr/trypsin fragment detected from that region. These are the first data on mapping of regions of charge variation, epitope location and glycosylation sites for this enzyme from any insect species.     

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.     

Approaches to the purification of the juvenile hormone esterase from the cabbage looper,Trichoplusia ni

Several approaches to the purification of juvenile hormone (JH) esterase from second-day last-instar larvae of Trichoplusia ni were taken, including: ammonium sulphate precipitation, polyethylene glycol precipitation, hydrophobic interaction chromatography, anion exchange chromatography, and gel filtration chromatography. The most successful procedure involved a combination of polyethylene glycol precipitation with anion exchange chromatography on DEAE Sephacel which yielded a 134-fold purification of juvenile hormone esterase. When this preparation was subjected to semi-preparative electrophoresis followed by isoelectric focusing on a polyacrylamide slab gel, a single band of apparently homogeneous enzyme was obtained. Juvenile hormone esterase activity was unstable after electrophoresis and isoelectric focusing. The stability of juvenile hormone esterase activity in a water solution is influenced by protein concentration and by agents protecting sulphydryl groups. The results of this study support the hypothesis that a single protein is responsible for the majority of the JH hydrolysis catalyzed by haemolymph from the larvae of T. ni used in this study.     

Juvenile hormone regulation of mRNA levels for a highly abundant hemolymph protein in larval Trichoplusia ni

Several hemolymph proteins ranging in size from 73 to 76 kDa increase to very high levels just prior to metamorphosis in Trichoplusia ni (Lepidoptera). One of these proteins (pI = 5.8, Mr = 76,000) was selected for a study of hormonal regulation. The appearance of this protein could be suppressed in vivo by topical treatment with the juvenile hormone analog fenoxycarb. An antiserum for this protein was prepared and shown to react selectively with the 76-kDa protein in whole hemolymph. Translation of poly(A)-containing RNA from untreated larvae yielded the 76-kDa protein, whose identity was verified with the antibody, whereas mRNA from juvenile hormone analog-treated larvae did not. These data indicate that juvenile hormone acts to regulate the level of the mRNA of this hemolymph protein.     

Characterization and the developmental role of plasma juvenile hormone esterase in the adult cabbage looper,Trichoplusia ni

Juvenile hormone (JH) esterase was characterized from the plasma of adult females of the cabbage looper, Trichoplusia ni, and compared with that present in 4th and 5th instar larvae. Ester hydrolysis was the principal route of JH metabolism. Gel filtration of plasma resolved a single peak of JH esterase which was distinct from that of the α-naphthyl acetate (α-NA) esterase activity. The JH esterase apparent molecular weight was 62,000 in prepupae and virgin, female adults and 69,000 in 2-day-old 4th instar larvae. Broad range isoelectric focusing of plasma of prepupae and adults resolved a major peak of activity at pH 5.5 with a minor peak of activity at pH 6.1 and in 4th instar larvae at pH 5.45 and 5.8, respectively. By this method JH esterase was resolved from the α-NA esterase activity. The plasma of prepupae and adults metabolized JH I at about twice the rate of JH III. JH esterase activity from adult plasma was more stable than the α-NA esterase activity. Adult JH esterase activity was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate in contrast to that of the α-NA esterase activity. Mated females oviposited 8 times more eggs than virgin females to 10 days after emergence. The total haemolymph protein content of virgin females remained high throughout the period of study whereas mated females showed a significant decline beginning on day 4. JH esterase activity remained unchanged in virgins whereas it declined drastically in mated females. The α-NA esterase activity declined to low levels shortly after emergence in both groups. JH and α-NA esterase activity was not affected by the application of the juvenoid, (RS)-methoprene. The present study provides evidence of a functional role for JH esterase in JH metabolism and reproduction in adult T. ni. JH esterases in the adult were identical to that of prepupae by the methods described above.     

Effects of the anti hormone-hormone mimic ETB on the induction of insect juvenile hormone esterase in Trichoplusia ni.

Juvenile hormone (JH) esterases can be artificially induced to appear in the hemolymph of last instar larvae of the lepidopterous insect $\text{Trichoplusia}$$\text{ni}$ (Noctuidae) by topical treatment with JH I, JH II, or dihomo branched juvenoids. ETB (ethyl-4-[2-(t-butylcarbonyloxy) butoxy] benzoate; ZR-2646) at high doses is a weak inducer of JH esterase (JHE). However, at doses of ETB that induce only low levels of JHE activity, ETB will block the JHE induction caused by the dihomo juvenoid epofenonane and at higher doses will reduce the induction caused by JH I or JH II. ETB is not a JHE inhibitor; rather, it appears to be acting as a JH agonist/antagonist in normal larvae and in isolated abdomens. These effects of ETB on JHE induction may illustrate a new mode of action of anti-JH's.     

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.     

In vivo and in vitro effects of epofenonane on juvenile hormone esterase activity in tissues of last stadium larvae of Trichoplusia ni

Topical application of the juvenoid, epofenonane, to last stadium postwandering larvae of Trichoplusia ni caused a precocious elevation of juvenile hormone esterase (JHE) activity that was tissue speific and time dependent. This increase in enzyme activity over controls was most dramatic in the hemolymph, whereas increases in the fat body were lower. Antibodies raised against JHE reacted on Western blots with a fat body and hemolymph protein present in epofenonane treated and untreated last stadium day 3 larvae. The abundance of this protein, which comigrated with JHE, closely coincided with the temporal increases in JHE catalytic activity that occurred in response to treatment in vivo with epofenonane.The presence of epofenonane (5–10,000 nM) in the medium at the start of fat body incubations failed to shift the temporal appearance of JHE activity or boost activity levels significantly over those of controls. If larvae were treated in vivo with epofenonane before fat body tissue was removed, only a small, but significant increase in JHE activity was found in vitro. The rate of enzyme secretion was insufficient to account for the rapid increases in enzyme activity that occur in the hemolymph in response to epofenonane, even though tissue held in vitro was deemed viable by monitoring lactate dehydrogenase activity in the medium, fat body intracellular ATP, and the incorporation of [³⁵S]methionine into fat body protein. Fat body tissue removed from various aged last stadium larvae released enzyme at different rates in vitro.     

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.     

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.     

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.     

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Developmental and behavioural responses of larval Trichoplusia ni to parasitization by an imported braconid parasite Chelonus sp

ABSTRACT. Larval Trichoplusia ni (Hübner) (Noctuidae) parasitized by Chelonus sp. (near curvimaculatus ) (Braconidae) precociously initiated pupation during the penultimate fourth instar. The temporal sequence of developmental markers exhibited by parasitized T. ni closely matched the temporal sequence in normal, pupating larvae. The parasitized larvae did not complete pupation, but consistently stopped development at a stage recognizable by a certain set of markers. This halt was observed in hosts from which parasites emerged and from hosts which had been stung but from which no parasites emerged. Weight gain and food consumption by parasitized hosts were significantly lower than normal, although most reached the fourth instar at the same time as normal larvae. Measurement of head capsule widths indicated that the width in precociously pupating larvae was less than the critical width associated with attainment of the pupation instar of normal larvae.     

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.