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.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Sex-specific developmental profiles of juvenile hormone synthesis in honey bee larvae

Summary Juvenile hormone synthesis in drone larvae of the honey bee was measured by an in vitro radiochemical assay. The developmental profile of corpora allata activity in male larvae showed considerable differences from queen larvae, the presumptive reproductive females, and was comparable to workers, the sterile female morph. Drone and worker larvae, however, differed drastically in the regulation of juvenile hormone biosynthesis, as revealed by the addition of farnesoic acid to the culture medium. This precursor stimulated juvenile hormone synthesis of drone glands nearly eightfold, whereas in worker larvae it is known to lead to an accumulation of methyl farnesoate. The sex-specific differences in endocrine activity indicate a role for juvenile hormone in the expression of genetically determined sexually dimorphic characters during metamorphosis, a role not currently accounted for in models describing endocrine regulation of insect development. Correspondence to: K. Hartfelder     

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robb's tissue culture medium incorporate ³H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of ³H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

Effects of parasitization of Trichoplusia ni by Chelonus sp.

ABSTRACT. Parasitization of Trichoplusia ni (Huebner) (Lepidoptera: Noctuidae) by Chelonus sp. (Hymenoptera: Braconidae), an egg-larval parasitoid, leads to precocious cocoon spinning of the host in the fourth (penultimate) stadium followed by parasitoid emergence from the prepupa. We have investigated the mechanism by which Chelonus sp. disrupts host development. The developing larva and fluids injected by the adult female separately from the egg, are not the source of these effects, but it remains a possibility that the teratocytes, originating from the trophamnion of the parasitoid egg, are responsible. The titre of the juvenile hormone esterase activity in the haemolymph of the parasitized fourth instar host is similar to that in the initial period of the final instar of normal T. ni, but lacks the postwandering peak of activity. The increased JHE activity leads to a reduced JH titre early in the fourth stadia. This indicates that disruption of host development occurs within 12h after apolysis to the fourth stadium, if not before. Anti-juvenile hormone activity is not detected in extracts of parasitized T. ni. The morphological and behavioural changes associated with precocious development of the T. ni host are prevented by applications of juvenile hormone I, juvenile hormone II and the juvenoid, Ro 10–3108, but not juvenile hormone III and the juvenoid R 20458. However, these applications fail to prevent the onset of juvenile hormone esterase activity, another marker of precocious development. These observations indicate that simple anti-juvenile hormone activity may not be the mechanism of disruption of host development. Development of the parasitoid is disrupted by application of Ro 10–3108 and juvenile hormones I, II and III, but timing of eclosion is only affected by application of juvenile hormone I, juvenile hormone II and Ro 10–3108. This observation may indicate a discrimination by the parasitoid between its own juvenile hormone III and the host's juvenile hormone II.     

Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite

A radiochemical assay measuring juvenile hormone synthesis by corpora allata incubated in vitro was adapted for use with the termite Zootermopsis angusticollis. Corpora allata from 3–4-day old virgin female neotenic reproductives were used in these studies because this caste showed the highest rates of juvenile hormone synthesis (0.6 pmol h^?1 per pair corpora allata). Juvenile hormone-III synthesis was linear for up to 6 h over the range of concentrations of labelled l-methionine from 27–280 μM. Rates of juvenile hormone synthesis were stimulated up to 10-fold in a dose-dependent manner by the addition of farnesoic acid to the incubation medium. However, the relatively high concentration of 120 μM farnesoic acid reduced the rates of juvenile hormone synthesis. The radiochemical assay was used to determine rates of juvenile hormone synthesis in vitro by corpora allata from larvae with a queen and king vs orphaned larvae. The presence of reproductives resulted in a suppression of larval corpus allatum activity relative to orphaned controls.     

Precocene I and II inhibition of vitellogenic oöcyte development in Drosophila melanogaster

Adult female Drosophila melanogaster were exposed to precocene I and II, antiallatropin compounds which result in juvenile hormone deficiency in many insects. The presence of juvenile hormone in Drosophila adults was evaluated by examining vitellogenic oöcyte development, a process regulated by juvenile hormone in these flies. Both precocenes reduced the number of vitellogenic oöcytes present 43 hr after exposure in a dose-dependent manner. Precocene I was effective when applied to either newly eclosed females prior to vitellogenic oöcyte development or to gravid females. Precocene I was also effective in decapitated females, indicating that the action of the compound is not mediated by the brain. Corpus allatum volume, presumably a reflection of secretory activity, increased between 0 and 24 hr after eclosion in control females but not in precocene-treated females even after 48 hr. However, when females were removed from precocene medium, gland volumes increased within 48 hr to approximately those of control flies. This result is consistent with the reversibility of the precocene effect on Drosophila adults. These results suggest that precocene acts on the corpus allatum of Drosophila adult females to produce juvenile hormone deficiency.     

Effects of juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

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.     

Effects of beta-ecdysone and juvenile hormone on the Na+/K+-dependent ATPase in imaginal disks of Drosophila melanogaster.

The evagination of imaginal disks of Drosophila melanogaster is induced in vitro by β-ecdysone and inhibited by juvenile hormone. The possibility that these hormones act by changing intracellular Na⁺ and K⁺ levels was investigated by studying their effects on the sodium-potassium dependent adenosinetriphosphatase (NaK ATPase), an enzyme with a major rôle in regulating Na⁺ and K⁺ levels in cells. We find that β-ecdysone has no effect on this enzyme and can induce evagination even when intracellular Na⁺ concentrations are increased 2 to 3 fold by ouabain. Juvenile hormone stimulates the enzyme, but still acts to inhibit evagination when NaK ATPase activity is inhibited by ouabain. We conclude that the actions of β-ecdysone and juvenile hormone on imaginal disk evagination do not directly involve the NaK ATPase or require specific changes in Na⁺ and K⁺ concentrations.     

Control of juvenile hormone production: the relationship between precursor supply and hormone synthesis in the tobacco hornworm,Manduca sexta

Tissue culture conditions for the production of juvenile hormone by excised corpora allata from the tobacco hornworm, Manduca sexta, were optimized and hormone output was monitored by incorporation of [¹⁴C-methyl]-methionine. Production was moderate in early fifth instar but undetectable in late fifth instar larvae. It was low in pupae, unmeasurable in adult males and high in adult females. Hormone production by glands of adult females was unaffected by the presence in the medium of juvenile hormone III, the hormone analogue, Methoprene, dibutyrylcyclic AMP, dibutyryl cyclic GMP and 25-hydroxycholesterol, an inhibitor of cholesterol synthesis in mammals.The assay of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in homogenates of corpora allata from M. sexta was optimized and activity in homogenates from animals at various life stages was determined. The correlation between the activity of the enzyme and hormone production by intact glands was excellent in all cases examined except for adult males, which have very high HMG-CoA reductase levels, but produce no hormone.Unlike the HMG-CoA reductase of mammalian tissue, the activity of corpus allatum enzyme is unaffected by Mg²⁺ ATP or fluoride ion, an inhibitor of phosphoprotein phosphatase. The activity is also unaffected by the presence of cAMP, cGMP, IBMX or 25-hydroxycholesterol.     

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 esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male).This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.     

The roles of juvenile hormone and 20-hydroxy-ecdysone during vitellogenesis in isolated abdomens of Drosophila melanogaster

Both juvenile hormone and 20-hydroxy-ecdysone seem to be involved in the regulation of vitellogenesis in Drosophila melanogaster. It is the purpose of this paper to begin to define the functions of these two hormones. Although vitellogenin synthesis does not occur at a high rate in 1-day-old female abdomens isolated from the head and thorax before 0.75 hr after eclosion, both ZR515 (a juvenile hormone analogue) and 20-hydroxy-ecdysone can cause in these preparations vitellogenin synthesis and secretion into the haemolymph. The synthesis and secretion into the haemolymph of all three vitellogenins which are detectable by electrophoresis in sodium dodecyl sulphate-containing gels of polyacrylamide is promoted by both hormones. That result excludes the hypothesis that these two hormones regulate the synthesis of different vitellogenins. A dose-response curve showed that an injection of 0.2 μl of a 10^?6 M 20-hydroxy-ecdysone solution was sufficient to promote vitellogenin synthesis and secretion in isolated abdomens. Ovaries from isolated female abdomens treated with juvenile hormone analogue showed nearly normal amounts of all three vitellogenins and morphologically normal advanced vitellogenic follicles, whereas ovaries from isolated abdomens treated with 20-hydroxy-ecdysone contained little vitellogenin and no vitellogenic follicles. We conclude that under the conditions used, juvenile hormone permits vitellogenin uptake into the oöcyte much more readily than does 20-hydroxy-ecdysone.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

Influence of temperature and carbon dioxide concentration on juvenile hormone titre and dependent parameters of adult worker honey bees (Apis mellifera L.)

It is known that juvenile hormone plays an important role in the regulation of labour division and of the different life spans, and that the microclimate of the bee hive is characterized by its high CO₂ concentration and its varying temperature depending on the presence of brood.We have investigated the influence of microclimates characteristic of breeding and broodless areas on the juvenile hormone titre in the haemolymph and whole body extracts, on the corpora allata in vitro activity, on the degradation of juvenile hormone and on the dry weight of the hypopharyngeal glands using bees of known ages. A microclimate of 35°C and 1.5% CO₂, as observed in the breeding area, induces a rapid and pronounced increase in the juvenile hormone titre. On the other hand, this titre remains low in bees kept at 27°C and 1.5% CO₂, a microclimate associated with broodless combs. Rates of juvenile hormone synthesis by corpora allata in vitro were found to be extremely low, even in the presence of farnesenic acid, and not related to the juvenile hormone titre. In vitro incubation of juvenile hormone in haemolymph revealed no degradation while injected juvenile hormone was found to be degraded and taken up by the gut at rates only weakly correlated with the juvenile hormone titre.We propose a hypothetical model for the regulation of the juvenile hormone titre as well as the course of labour division by the varying microclimates observed in the bee hive.     

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.