Hemolymph Juvenile Hormone Esterase Activity Research Articles

In Vitro and in Vivo Inhibition of Haemolymph Juvenile Hormone Esterase Activity by the Ethanol Extract of Clerodendrum inerme in Fifth Instar Larva of Castor Semilooper, Achaea janata (L.)

Juvenile hormone (JH) is a unique hormone in insects that controls growth, development, metamorphosis and reproduction. It has been well established that JH esterase is an enzyme involved in JH regulation. Results of both in vitro and in vivo studies revealed that ethanol leaf extract of Clerodendrum inerme significantly inhibits haemolymph JH esterase activity affecting growth and normal development of the test insect, castor semilooper ( Achaea janata ), a serious pest of oil seed crops like castor and groundnut.

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

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

Synthesis and biological activity of novel anti-juvenile hormone agents

Ethyl 4-[2-(tert-butylcarbonyloxy)butoxy]benzoate (ETB) is a partial juvenile hormone (JH) antagonist which targets larval epidermis. We converted ETB to ethyl 4-(2-substituted alkyloxy)benzoates. These compounds induced precocious metamorphosis in the silkworm, Bombyx mori, which is a clear sign of JH deficiency, and their activity was completely counteracted by the simultaneous application of a JH agonist, methoprene. Among these novel compounds, ethyl 4-(2-benzylhexyloxy)benzoate (KF-13) showed high precocious metamorphosis-inducing activity at low doses, but its activity decreased with increasing dose, probably due to their JH-like activity. KF-13 is an enantiomeric compound. The (S)-enatiomer of KF-13 was more active than the (R)-enantiomer at low doses, but at high doses the activity was reversed (R>S). Hemolymph JH esterase activity, which is indispensable for the initiation of pupation in normal last-instar larvae, was induced in 4th-instar larvae by treatment with KF-13. 2-(6-Methyl-3-pyridyloxy)hexyl and 2-phenoxyhexyl analogs showed JH activity when topically applied to allatechtomized 4th-instar larvae.

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.

Synthesis and Anti-Juvenile Hormone Activity of Ethyl 4-[2-(6-Methyl-3-pyridyloxy)alkyloxy]benzoates

A series of ethyl 4-[2-(6-methyl-3-pyridyloxy)alkyloxy]benzoates was prepared and tested for activity to induce precocious metamorphosis in larvae of the silkworm, Bombyx mori. Of the compounds tested, ethyl 4-[4-methyl-2-(6-methyl-3-pyridyloxy)pentyloxy]benzoate (5) was the most effective when applied to 24 hr-old 3rd instar larvae. There was no significant difference in precocious metamorphosis-inducing activity between 5R(+)- and 5S(−)-enatiomers. The activity of compound 5 could be fully counteracted by methoprene, a juvenile hormone (JH) agonist, but not by the dietary administration of 20-hydroxyecdysone. When 3rd instar larvae were treated with compound 5, hemolymph JH esterase activity, which is indispensable for the initiation of pupation in normal last instar larvae, was induced during the 4th instar. © Pesticide Science Society of Japan

A morph-specific daily cycle in the rate of JH biosynthesis underlies a morph-specific daily cycle in the hemolymph JH titer in a wing-polymorphic cricket

A previous study documented a high amplitude, morph-specific daily cycle in the hemolymph JH titer in the wing-polymorphic cricket, Gryllus firmus. The JH titer rose and fell 10–20 fold in the flight-capable [LW(f), long-winged] morph during the late-photophase-early scotophase, while it was relatively constant during that time in the flightless (SW, short-winged) morph. In the present study we documented a dramatic morph-specific daily cycle in the in vitro rate of juvenile hormone (JH) biosynthesis that was tightly correlated with the hemolymph JH titer on days 5–7 of adulthood. Biosynthetic rates rose and fell 1–2 fold between the late photophase-early scotophase on each of days 5–6 and 6–7 of adulthood in the LW(f) morph, while biosynthetic rates were relatively constant during this period in the flightless, short-winged morph (SW), except for a slight dip in the rate of biosynthesis late in the photophase on these days. Similar morph-specific patterns of JH biosynthesis were observed whether rates were measured on corpora allata attached to corpora cardiaca in males or females, or on corpora allata alone. Hemolymph juvenile hormone esterase activity was significantly higher in the LW(f) vs. the SW morph during the beginning of scotophase, when the JH titer is decreasing rapidly in the LW(f) morph. Results indicate that the morph-specific daily cycle in the JH titer in G. firmus is primarily regulated by a morph-specific daily cycle in the rate of JH biosynthesis and to a lesser degree by hemolymph JH esterase activity. This is the first documentation of a diurnal cycle in the rate of JH biosynthesis in any insect, or a daily cycle in the rate of JH biosynthesis that is correlated with a specific morph in a polymorphic species. Results have important implications for the endocrine regulation of dispersal polymorphism, circadian rhythms of insect hormone titers and their regulators, and general studies of the JH titer and its regulation in insects.

Fenoxycarb levels and their effects on general and juvenile hormone esterase activity in the hemolymph of the silkworm, Bombyx mori

We studied the relationship between fenoxycarb levels and general esterase (GE) activity, juvenile hormone esterase (JHE) activity, and induction of permanent fifth instar (dauer) larvae in Bombyx mori. Fenoxycarb (1 μg/animal) was topically applied to B. mori at 0 h of the fifth instar. Hemolymph fenoxycarb levels, as determined by an ELISA, reached a peak within 1 h, and then decreased to undetectably low at 42 h. After 48 h, the feces of these larvae contained 99% of the applied fenoxycarb dose. The above fenoxycarb treatment induces dauer larvae with persistently increased hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb later in the fifth instar (48 h), when it still could induce dauer larvae, resulted in a similar pattern of elimination from the hemolymph and produced steadily elevated hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb late in the fifth instar (132 h) did not prevent pupal ecdysis; it increased hemolymph JHE activity, but not GE activity. The latter treatment prevented the JHE activity decrease observed in control larvae before pupal ecdysis. In vivo, in control animals, the highest hemolymph JHE activity was observed 12 h after pupal ecdysis. At that developmental time, fenoxycarb inhibited this enzyme in vitro ( IC 50≈2 μM) . The combined results suggest: (1) fenoxycarb is rapidly excreted from the larval body; (2) induction of dauer larvae and increased hemolymph GE and JHE activities are not directly associated with its presence in the larval body of B. mori.

CDNA cloning and characterization of Bombyx mori juvenile hormone esterase: an inducible gene by the imidazole insect growth regulator KK-42

The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3 H -JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)☆

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides ( M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only ∼ 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent (∼100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.

Characterization and temporal aspects of haemolymph juvenile hormone esterase in adult cockroach, Periplaneta americana

Daily variations in the in vitro haemolymph juvenile hormone esterase activity (hJHE) of adult male and female Periplaneta americana were monitored over a 2 week period from the time of adult emergence and throughout the first reproductive cycle of the adult female. Kinetic analysis of hJHE from females indicated an apparent K m JH III of 5.59±1.75 μ m ( V max=140 pmol JH III hydrolysed h −1 per μl haemolymph). In females the mean rate of JH III metabolism in diluted haemolymph shortly after emergence was 27.5±1.5 pmol h −1 μl −1 ( n=16) and remained relatively low (16–32 pmol h −1 μl −1) over much of early adult development. Activity remained at this level during the first two days of the 4 day reproductive cycle, but showed a much increased broad peak of activity (74–93 pmol h −1 μl −1) at 60–72 h post-extrusion. This peak lags behind the whole body JH titre peak, which could suggest that elevated levels of JH III may bring about the induction of JH esterase(s). A different pattern of JHE activity was observed in adult males. hJHE rates in males at emergence were almost twice as high (81.5±15.8 pmol h −1 μl −1, n=16) as those found in females at this time, but thereafter showed a downturn to moderate levels (44–68 pmol h −1 μl −1) that were maintained for the remainder of the study. Rapid (FPLC) DEAE–sepharose ion exchange chromatography, ultrafiltration and fast-flow superose gel filtration chromatography were employed to achieve an efficient partial purification (166-fold) of the hJHE from cell-free plasma of reproductively active female P. americana 48–72 h post-ootheca extrusion. Gel filtration and SDS–polyacrylamide gel electrophoresis (PAGE) revealed an enzyme having apparent molecular mass of between 60 and 70 kDa, whilst non-denaturing PAGE and iso-electrofocusing resolved a single acidic enzyme peak with a pI of 4.9.

Inhibitory effects of parasitism by the gregarious endoparasitoidCotesia congregata on host testicular development

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasp's polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997. © 1997 Wiley-Liss, Inc.

Role of the corpora allata and juvenile hormone in the control of imaginal diapause and reproduction in three species of locusts

To understand the hormonal regulation of imaginal diapause and ovarian development in three species of locusts, Nomadacris succincta, Nomadacris japonica and Locusta migratoria, juvenile hormone (JH) biosynthesis by the corpora allata (CA) in vitro and JH esterase activity in the hemolymph were measured. The two species of Nomadacris exhibited a positive correlation between oocyte growth and JH biosynthesis by CA in vitro, although the correlation was significant only in N. succincta. In contrast, L. migratoria showed a negative correlation, indicating a higher rate of JH synthesis associated with more suppressed ovarian development. In this species, however, the hemolymph JH titer was higher in reproductively active females than in sexually immature females, indicating a positive correlation between the hemolymph JH titer and ovarian development. The levels and temporal pattern of variation in mean hemolymph JH esterase activity were rather similar between reproductively active and inactive females in L. migratoria. It is thus unlikely that the hemolymph JH esterase plays an important role in the regulation of the hemolymph JH titers in this species. Topical application of 100 μg of methoprene stimulated ovarian development significantly in L. migratoria, whereas 450 μg failed to induce ovarian development or vitellogenesis in sexually immature females of N. succincta and N. japonica. Besides JH, there appear to be other factors present that are involved in the control of the termination of diapause in the two Nomadacris species.

Parasitism by Microplitis demolitor induces alterations in the juvenile hormone titers and juvenile hormone esterase activity of its host, Pseudoplusia includens

Microplitis demolitor is a polydnavirus-carrying parasitoid that attacks the larval stage of Pseudoplusia includens and other noctuids. Parasitism or injection of wasp components like polydnavirus and teratocytes has a juvenilizing effect on P. includens development. Here we measured hemolymph juvenile hormone (JH) titers and juvenile hormone esterase activity in P. includens larvae after parasitism or injection of wasp components. Results were compared to nonparasitized larvae. During the fifth stadium, JH titers of nonparasitized larvae fluctuated between 0.08 and 0.50 ng/ml, whereas titers in parasitized larvae never fell below 2 ng/ml. P. includens larvae injected with calyx fluid plus venom or calyx fluid plus venom and teratocytes exhibited JH titers intermediate between parasitized and nonparasitized larvae. Nonparasitized larvae exhibited two peaks of JH esterase activity. The first occurred between 12 and 48 h in association with cessation of larval feeding, while the second occurred immediately before pupation. In contrast, levels of JH metabolism remained at low levels in parasitized larvae and larvae injected with calyx fluid plus venom or calyx fluid, venom and teratocytes. Only trace amounts of JH were detected in in vitro assays of teratocytes or M. demolitor larvae suggesting neither released JH into the hemolymph of parasitized larvae. Although calyx fluid plus venom and teratocytes did not elevate JH titers to the levels measured in parasitized larvae, their effects on formation of larval-pupal intermediates by P. includens were similar to those generated by exogenous application of methoprene or JH.

Artificial selection on JHE activity inGryllus assimilis: Nature of activity differences between lines and effect on JH binding and metabolism

Genetic lines of the cricket Gryllus assimilis selected for elevated vs. decreased activity of hemolymph juvenile hormone esterase (JHE) exhibited a 6.8-fold difference in enzyme activity by the seventh generation of selection. This documents that hemolymph JHE activity has the capability for rapid evolutionary change. This is the only insect endocrine trait for which such data are currently available. The difference in hemolymph JHE activity between the lines was due, to an equivalent degree, to variation in whole-cricket enzyme activity and allocation of JHE activity to the hemolymph compartment. No differences in kinetic or thermostability characteristics were observed between JHEs from the high- vs. low-selected lines. This suggests that the variation in JHE activity between the lines resulted from selection of variable regulatory genetic factors affecting the synthesis, degradation, or activation of enzyme activity. JHE and JH-binding activity in the hemolymph are genetically correlated, possibly due to co-regulation by the same genetic factors. Finally, in vivo JH degradation was significantly elevated in lines selected for elevated JHE activity. This documents that evolutionary changes in JHE activity alter in vivo JH metabolism. © 1996 Wiley-Liss, Inc.

Artificial selection on JHE activity in Gryllus assimilis: Nature of activity differences between lines and effect on JH binding and metabolism

Genetic lines of the cricket Gryllus assimilis selected for elevated vs. decreased activity of hemolymph juvenile hormone esterase (JHE) exhibited a 6.8-fold difference in enzyme activity by the seventh generation of selection. This documents that hemolymph JHE activity has the capability for rapid evolutionary change. This is the only insect endocrine trait for which such data are currently available. The difference in hemolymph JHE activity between the lines was due, to an equivalent degree, to variation in whole-cricket enzyme activity and allocation of JHE activity to the hemolymph compartment. No differences in kinetic or thermostability characteristics were observed between JHEs from the high- vs. low-selected lines. This suggests that the variation in JHE activity between the lines resulted from selection of variable regulatory genetic factors affecting the synthesis, degradation, or activation of enzyme activity. JHE and JH-binding activity in the hemolymph are genetically correlated, possibly due to co-regulation by the same genetic factors. Finally, in vivo JH degradation was significantly elevated in lines selected for elevated JHE activity. This documents that evolutionary changes in JHE activity alter in vivo JH metabolism. © 1996 Wiley-Liss, Inc.

Juvenile hormone degradation in adult wing morphs of the cricket, Gryllus rubens

Juvenile hormone—III metabolism was compared between adult wing morphs of the cricket Gryllus rubens to determine if variation in hormone degradation could be responsible for the dramatic fecundity differences between female morphs. As in other insects, hormone was degraded almost exclusively to juvenile hormone acid in dilute hemolymph in vitro, while significant quantities of juvenile hormone acid, diol and acid-diol as well as more polar metabolites were produced in vivo within 1 h after injection of racemic hormone. Activities of the degradative enzyme juvenile hormone esterase in hemolymph ranged from 4 to 14 nmol/min/ml serum during the first 10 days of adulthood in each morph of each sex. Activities were similar to values reported in other adult orthopterans and were substantially lower than those in last-stadium G. rubens. The 70 min in vivo half-life of racemic hormone injected into day 5–6 short-winged females was significantly higher than that of mid-last-stadium G. rubens and was similar to values reported in adult Diploptera punctata, Locusta migratoria and Manduca sexta. The amount of (10-R)- or racemic hormone degraded in vivo within 1 h after injection was strongly correlated with hemolymph juvenile hormone esterase activity in individual day 5–7 but not day 2–3 adults. This indicates that the hemolymph esterase contributes significantly to in vivo metabolism of exogenous hormone during some periods in adults. Midgut, fat body and ovaries exhibited significant activities of both juvenile hormone esterase and epoxide hydrolase, indicating that the hydrolase may contribute to tissue-specific and to whole cricket hormone metabolism. Importantly, no major differences were observed between adult wing morphs with respect to (1) percentage racemic or (10-R)-juvenile hormone—III degraded in vivo, (2) developmental profiles of hemolymph juvenile hormone esterase activity or (3) activities of juvenile hormone esterase or epoxide hydrolase in various non-hemolymph tissues. These data (1) provide no evidence that hormone metabolism differs significantly between adult morphs and (2) contrast with the importance of differential hormone degradation in morph induction during the juvenile stage.

Developmental Role of Juvenile Hormone Metabolism in Lepidoptera

SYNOPSIS. Lepidopteran juvenile hormone (JH) esterase appears to have a functional role in the regulation of embryogenesis, larval growth and development, and adult reproduction. In preovipositional and newly laid eggs of the tobacco hornworm, Manduca sexta , JH esterase activity was elevated presumably to metabolize maternal JHs, and then declined after blastoderm formation. Also, a single peak in hemolymph JH esterase activity was found prior to ecdysis in the second through the fourth instar of M. sexta , the function of which is unclear. However, in the last instar, elevated hemolymph JH esterase activity was noted prior to wandering and again prior to ecdysis to scavenge the last traces of JH necessary for normal development. The hemolymph JH esterase is likely of multiple tissue origin for the prewandering peak with the fat body excluded as a source for the prepupal peak; an inhibitory factor from the brain and JH regulate JH esterase biosynthesis. In adult cabbage loopers, Trichoplusia ni , elevated hemolymph JH esterase activity appeared to be important in reducing the JH titer and preventing egg maturation. Structure/activity data with trifluoromethylketones were incorporated into the design of a novel, JH esterase inhibitor, the sulfone and hydrate of octylthio-1,1,1- trifluoropropan-2-one, with selective and persistent, in vivo inhibitory activity. The topical application of this compound to last instar larvae and virgin adults of T. ni produced juvenizing effects (delayed pupation and induced egg maturation/oviposition, respectively) providing direct evidence of a functional role for JH esterase in lepidopteran development.

In vivo and in vitro degradation of juvenile hormone-III in presumptive long-winged and short-winged Gryllus rubens

Degradation of exogenous juvenile hormone-III was compared between long-winged and short-winged morphs of the cricket Gryllus rubens during the last stadium. This was done to determine if morph-specific differences in haemolymph juvenile hormone esterase activity, which are thought to be important in morph determination, result in corresponding differences in in vivo hormone degradation. Percentage degradation of racemic or (10-R)-juvenile hormone-III was significantly higher in plasma from the long-winged vs the short-winged morph during the mid-stadium while no differences between the morphs were observed early in the stadium. These results paralleled morph-specific differences in plasma juvenile hormone esterase activity. Similar patterns were obtained in vivo, except for early-stadium, short-winged crickets where hormone degradation was much higher than expected based on haemolymph juvenile hormone esterase activity. When esterase activity in long-winged individuals was inhibited in vivo by a trifluoropropanone derivative, there was a corresponding decrease in in vivo hormone degradation. No morph-specific differences were observed in degree of hormone binding by haemolymph components during either the early or mid-stadium, indicating that variation in hormone binders cannot account for differences between morphs in degree of hormone degradation. Importantly, differences between morphs in hormone degradation were much less than differences in haemolymph juvenile hormone esterase activity. These results collectively document morph-specific covariation in haemolymph juvenile hormone esterase activity and in vivo hormone degradation. However, because differences between morphs in hormone degradation are not large, their functional significance with respect to morph determination remains uncertain.

Regulation of juvenile hormone esterase gene expression in the tobacco budworm ( Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A) +RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.

The regulation of the first peak of haemolymph juvenile hormone esterase activity during the last stadium of the cabbage looper, Trichoplusia ni

The role of the head and a juvenoid, epofenonane, in the regulation of the first peak of haemolymph juvenile hormone esterase activity was examined during the fifth (last) stadium of the cabbage looper, Trichoplusia ni. The topical application of epofenonane to unligated day-1 larvae brought about an average 36% increase in the haemolymph juvenile hormone esterase activity. Epofenonane treatment also resulted in a 2-fold increase in the juvenile hormone and α-naphthyl acetate esterase activity of the whole body of unligated larvae suggesting that the juvenoid causes an increase in esterase biosynthesis or activation. Ligation and starvation prevented the appearance of the first peak of haemolymph juvenile hormone esterase activity. Application of epofenonane to newly ligated larvae had no effect whereas it significantly increased the juvenile hormone and α-naphthyl acetate esterase activity of the haemolymph in larvae ligated for 24 h prior to the treatment. In newly starved larvae, epofenonane increased the haemolymph juvenile hormone and α-naphthyl acetate esterase activity above respective controls. In 24 h starved larvae, juvenile hormone esterase activity of the haemolymph increased in response to epofenonane, head extract, or a combination of head extract injection and epofenonane application whereas α-naphthyl acetate esterase activity increased only to head extract or to a combination of head extract injection and epofenonane application. Injection of head extracts of day-1, fifth-instar larvae into the same aged ligated larvae increased both the juvenile hormone and α-naphthyl acetate esterase activity in the haemolymph by about 3-fold 5 h later compared to the respective controls. This increase, however, was not reflected in a similar increase in the whole body esterase levels suggesting that head extract may have caused a release of esterases from tissues into the haemolymph. Injection of head extract from day-1 larvae into the same aged unligated larvae inhibited the normal increase in the haemolymph juvenile hormone esterase activity seen between day 1 and 2 and no inhibition was noted with injections of body extracts or bovine serum albumin. Head extract injection significantly decreased the juvenile hormone esterase activity in the whole body, and haemolymph of unligated and 24 h ligated larvae treated with epofenonane. The head-extract-induced inhibition of haemolymph juvenile hormone esterase activity was dose dependent and the esterase activity in the haemolymph was unaffected when incubated in vitro with head extract for a period of 4 days at 30°C.