Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta

A new sensitive method for determining juvenile hormone (JH) hydrolysis has been developed which measures the release of tritiated methanol from JH labelled in the methyl ester group. Using this assay we investigated the interaction of JH with haemolymph esterases and haemolymph JH-binding protein. Haemolymph from fifth instar larvae of Manduca sexta contains two families of esterases which can be distinguished by their reactivity with diisopropylphosphorofluoridate (DFP). One group consists of general esterases which are capable of hydrolysing free JH but not JH complexed to the binding protein and are completely inhibited by low concentrations of DFP (10⁻⁴ M). The other group (JH-specific esterases), relatively DFP resistant, has little detectable general esterase activity but can hydrolyse JH bound to the binding protein as well as free JH. The major JH-esterase has a sedimentation coefficient of 4·98 S and a diffusion coefficient of 6·4 × 10⁻⁷ cm² sec⁻¹. The molecular weight calculated from these values is 6·7 × 10⁴. The general esterases are present throughout the larval stage, but the JH-specific esterases are barely detectable until the fourth day of the fifth instar when they suddenly appear at a high concentration. Since the general esterases cannot hydrolyse bound JH, one function of the binding protein is to protect JH during transport in the early instars, thus confirming that the binding protein is a true carrier of JH. In the late fifth instar prior to metamorphosis, however, JH-specific esterases appear in the haemolymph resulting in the hydrolysis of JH complexed to the carrier protein. Thus, by lowering JH titre, the JH-esterases play an important rôle in development in M. sexta.     

Juvenile hormone metabolism in the plasma,integument, midgut,fat body,and brain during the last instar of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) III esterase and JH III epoxide hydrolase activity was found in the integument, midgut, fat body, and brain during last instar development of the tobacco hornworm, Manduca sexta. JH esterase activity was primarily located in the cytosol in these tissues while the majority of the JH epoxide hydrolase activity was found in the microsomes. A prewandering (on day 3) and postwandering (on day 8) peak in plasma JH III esterase activity occurs in the last instar of gate I M. sexta. The JH esterase activity profile in integument, midgut, fat body, and brain followed a similar pattern to that of the plasma. The only exception to this was the absence of the postwandering, prepupal (on day 8) JH esterase peak in the fat body. The topical application of the juvenoid, (RS)-methoprene, failed to induce fat body JH esterase activity but increased activity in the plasma, integument, midgut, and brain in M. sexta prepupae. These results indicate that the source of plasma JH esterase activity is not always the fat body as previously hypothesized. The developmental profile of tissue JH epoxide hydrolase activity was also similar to that of JH esterase suggesting that both enzymes may be regulated partly by the same factors and that JH epoxide hydrolase may also have an important, previously unrecognized functional role in JH regulation and insect metamorphosis. Multiple isoelectric forms of tissue-specific JH esterases and JH epoxide hydrolases were found in integument, midgut, fat body, and brain. The JH esterases in these tissues had isoelectric points more acidic than that for plasma. Tissue α-naphthyl acetate esterase, developmental profiles, and inhibitor sensitivity to 3-(octylthio)-1,1,1-trifluoropropan-2-one differed significantly from that for JH esterase, suggesting that they represent different enzymes. ©1992 Wiley-Liss, Inc.     

Release of a juvenile hormone binding protein by fat body of the Indian meal moth, Plodia interpunctella,in vitro

When fat body of fifth instar larvae of Plodia interpunctella was cultured in vitro in a chemically defined medium, the tissue released a low mol. wt protein (FBBP) that binds juvenile hormone (JH). This FBBP has the same mol. wt, estimated by gel permeation chromatography, as the haemolymph JH binding protein. Furthermore, the FBBP protected JH from degradation by general esterases isolated from the haemolymph. Treatment of fat body with cycloheximide inhibited incorporation of [¹⁴C] leucine into the FBBP protein fraction and reduced the amount of FBBP released into the medium. We conclude that one source of the JH binding protein found in the haemolymph is the fat body.     

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.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

Hydrolysis of juvenile hormone in diapausing and non-diapausing larvae of the southwestern corn borer,Diatraea grandiosella

Summary The role of juvenile hormone (JH) esterases in relation to the diapause state of the southwestern corn borer,Diatraea grandiosella, was examined. The facultative larval diapause of this insect is dependent upon the presence of JH. Plasma, fat body, midgut, and body wall extracts metabolized [³H]JH I and [³H]JH III to JH-acid in vitro. JH-diol, JH-acid-diol, or conjugated polar metabolites were not detected. A longer half life of [³H]JH I was found in vitro in the plasma of diapausing larvae than in that of non-diapausing larvae. Although JH hydrolytic activity was relatively low in the plasma of pre-diapausing and diapausing larvae, systematic changes were observed suggesting that JH esterases may be involved in regulating the JH titer during this period. The JH hydrolytic activity found in the plasma of diapausing larvae was 3 to 5 times lower than that found in the plasma of mid-last instar non-diapausing larvae. Gel filtration profiles obtained from the plasma of diapausing and non-diapausing larvae suggested that JH esterases and -naphthyl-acetate esterases are different enzymes. Multiple overlapping peaks of JH hydrolytic activity with an apparent molecular weight range of 43,000 to 75,000 were detected, whereas 2 separate peaks of -naphthyl-acetate hydrolytic activity (apparent mol. wt. ca. 54,000, and 120,000) were detected. Gel filtration of supernatants of fat body indicated that JH was hydrolyzed at a lower rate by the fat body of pre-diapausing larvae than by that of non-diapausing larvae.     

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.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

An effect of the microsporidian Vairimorpha ephestiae (Microsporidia: Burenellidae) on activity and spectrum of nonspecific esterases in different tissues of the greater wax moth galleria mellonella (Lepidoptera: Pyralidae) larvae

The effect of the microsporidian Vairimorpha ephestiae Matted (Microsporidia: Burenellidae) on nonspecific esterases was studied in hemolymph, fat body and midgut of the larvae of Galleria mellonella L. (Lepidoptera: Pyralidae). Esterase patterns were analyzed by the polyacrylamide gel electrophoresis, the total esterase activity was detected spectrophotometrically. The increase of total esterase activity was registered in hemolymph of inflected larvae. An overexpression of esterase isozyme in hemolymph was already detected at the 3rd day post infection. No changes in esterases pattern were observed in the fat body's homogenates of the G. mellonella larvae possessing the symptoms of microsporidiosis. The degradation of esterase isozymes and the decrease of total esterase activity in the pattern of the midgut homogenates of infected larvae were registered during parasite sporogony. The greatest esterase activity in hemolymph and midgut tissues was registered during vegetative reproduction of parasite, but the least level of esterase activity was observed during mass sporogony of microsporidia.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae)

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

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.