Developmental Role of Juvenile Hormone Metabolism in Lepidoptera

SYNOPSIS. Lepidopteran juvenile hormone (JH) esterase appearsto have a functional role in the regulation of embryogenesis,larval growth and development, and adult reproduction. In preovipositionaland newly laid eggs of the tobacco hornworm, Manduca sexta,JH esterase activity was elevated presumably to metabolize maternalJHs, and then declined after blastoderm formation. Also, a singlepeak in hemolymph JH esterase activity was found prior to ecdysisin the second through the fourth instar of M. sexta, the functionof which is unclear. However, in the last instar, elevated hemolymphJH esterase activity was noted prior to wandering and againprior to ecdysis to scavenge the last traces of JH necessaryfor normal development. The hemolymph JH esterase is likelyof multiple tissue origin for the prewandering peak with thefat body excluded as a source for the prepupal peak; an inhibitoryfactor from the brain and JH regulate JH esterase biosynthesis.In adult cabbage loopers, Trichoplusia ni, elevated hemolymphJH esterase activity appeared to be important in reducing theJH titer and preventing egg maturation. Structure/activity datawith trifluoromethylketones were incorporated into the designof a novel, JH esterase inhibitor, the sulfone and hydrate ofoctylthio-1,1,1- trifluoropropan-2-one, with selective and persistent,in vivo inhibitory activity. The topical application of thiscompound to last instar larvae and virgin adults of T. ni producedjuvenizing effects (delayed pupation and induced egg maturation/oviposition,respectively) providing direct evidence of a functional rolefor JH esterase in lepidopteran development.     

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.     

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.     

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.     

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.     

Larval esterases of the southwestern corn borer, Diatraea grandiosella: Temporal changes and specificity

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella. Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis.The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.     

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 partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time- and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.     

Characterization of hemolymph juvenile hormone esterase from Lymantria dispar

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.     

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.     

Effects of juvenile hormone I and the anti-juvenile hormone fluoromevalono-lactone on development and juvenile hormone esterase activity in post-feeding last-stadium larvae of Trichoplusia ni (Hübner)

Treatment of post-feeding (early day 3; wandering phase) last-stadium larvae of the cabbage looper, Trichoplusia ni, with the anti-juvenile hormone, fluoromevalonolactone, prevented the normal ecdysis to the pupa. It caused the formation of larval-pupal intermediates, a dose-dependent delay in the time of tanning, and a decrease in juvenile hormone esterase activity at the time of the prepupal juvenile hormone esterase peak. Fluoromevalonolactone was inactive as juvenile hormone esterase inhibitor in vitro. Conversely, juvenile hormone I accelerated the time of tanning, induced the early appearance of juvenile hormone esterase activity, and prevented adult eclosion. Although most of the larvae that were treated with fluoromevalonolactone immediately after the prepupal burst of juvenile hormone (late on day 3; post-spinning phase) still became larval-pupal intermediates, the time of tanning and juvenile hormone esterase activity were close to normal. Topical treatment of day-3 larvae with radiolabelled juvenile hormone I resulted in the rapid appearance and decline of radiolabelled juvenile hormone I in the haemolymph which was associated with the increased production of juvenile hormone I acid and the induced appearance of juvenile hormone esterase activity. Thus, in post-feeding last-stadium larvae of T. ni, juvenile hormone seems to be necessary for the proper formation of the pupa. Juvenile hormone is also involved in determining the time of pupation, and it appears to induce its own degradation.     

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.     

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.     

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.     

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.     

An in vitro system for studying juvenile hormone induction of juvenile hormone esterase from the fat body of Trichoplusia ni (Hübner)

An in vitro fat body culture was used to study juvenile hormone esterase (JHE) regulation. The present study shows juvenile hormone can directly induce JHE activity to appear in the culture medium in a dose-dependent manner at physiological concentrations of JH. This induced appearance of JHE can be blocked with actinomycin D. Biochemical characterization of the in vitro produced JHE demonstrated that it had the same isoelectric points as that of the in vivo JHE activity. The JHE inhibitor 1-1-1 trifluoro-tetradecan-2-one gave the same inhibition profile and I₅₀ toward both in vivo and in vitro produced JHE activities. Finally, the JHE activity induced in vitro was immunologically similar to that occurring in vivo. The system should be useful for high resolution studies on the regulation of JHE.     

Interactions between a non-terpenoid carbamate with juvenile hormone activity and esterases in the last larval instar of Adoxophyes reticulana Huebn

The summer fruit tortrix moth is very susceptible to compounds with juvenile hormone activity. Ro 13-5223, a non-terpenoid carbamate, is 3–4 orders of magnitude more active in inhibiting metamorphosis in the last-instar larvae than juvenile hormone I. Larvae reared in permanent contact with this substance are characterised by higher juvenile hormone esterase activity but lower α-naphthyl esterase activity when compared to the untreated controls. In vitro Ro 13-5223 inhibits juvenile hormone hydrolysis but only in dosages which are far above the concentrations found in haemolymph of larvae exposed to the ¹⁴C-labelled compound. It does not serve as a substrate for juvenile hormone esterase in vitro even though it induces the enzyme activity in vivo. All these characteristics may account for the very high biological activity of Ro 13-5223 which disrupts humoral coordination of insect development.     

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.     

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.