Ecdysteroid fluctuations in adult Drosophila melanogaster caused by elimination of pupal reserves and synthesis by early vitellogenic ovarian follicles

Radioimmunoassay (RIA) of whole body extracts of Drosophila melanogaster males and females demonstrates that at eclosion all individuals contain high levels of ecdysteroid. Highly polar ecdysteroids (presumably metabolites) in the meconium represent approximately half of the total ecdysteroid RIA-activity present at this time and are subsequently eliminated. Ecdysteroids remaining after the elimination of the meconium are also highly polar as shown by reverse-phase high pressure liquid chromatography (RP-HPLC). The amount of ecdysteroid RIA-activity found in whole body extracts declines in both sexes until 18 h post-eclosion when levels begin to increase in the female and drop to undetectable levels in the male. In the female the ovaries are the major source of ecdysteroid. The increase in whole body ecdysteroid in the female coincides with the initiation of ovarian ecdysteroid production and accumulation. Topical application of methoprene, a juvenile hormone (JH) analog, stimulates ovarian ecdysteroid synthesis in apterous-ts 78j (ap^{ts 78j}), a temperature-sensitive juvenile hormone-deficient mutant, corroborating previous results suggesting a role of juvenile hormone in ovarian ecdysteroid production. Stage 8–9 follicles, whose development is juvenile hormone dependent, are shown to be the most active in ecdysteroid production. The regulatory potential of these stages is discussed.     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.     

Juvenile hormone modifications of gene expression in the fat body and posterior silk glands of Bombyx mori

The patterns of protein synthetic activities were determined in the fat body and silk glands of Bombyx mori larvae during the fifth instar. Comparisons were made between control and juvenile hormone or methoprene—treated animals. In normal larvae, the relative synthetic activities of a few proteins increase up to the time of spinning. In treated larvae, the presence of high levels of juvenile hormone or methoprene never results in an arrest of the expression of differentiation at the molecular level. The syntheses of the most abundant markers of development in both organs are reduced by the hormone, but total inhibition has never been observed. The transfer of a large amount of proteins (28–29 kd) from the fat body to the haemolymph is decreased.     

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 μg/ml) and 20E (1 μg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.     

Ecdysteroid level and the change in sensitivity to a juvenile hormone analogue in Ephestia cautella larvae (Lepidoptera,Phycitidae)

A double-antibody ecdysone-specific radioimmunoassay was used to clarify whether the effects on metamorphosis of the juvenile hormone analogue methoprene are correlated with changes in ecdysteroids level. It appears that a small ecdysteroids peak, 5 days before pupation, is responsible for the transition from inhibition to defective metamorphosis. Study of the changes in ecdysteroid titer in last-instar larvae treated with the JHA 2 days prior to the appearance of the above small ecdysteroids' peak showed an immediate reduction in ecdysteroid level, followed by cyclic, successively reduced titer for about 20 days. After this period the larvae ceased to feed and entered to a diapauselike stage which ended in the death of the larvae. A similar effect on ecdysteroid titer and developmental arrest was exhibited by JHA-treated first-instar larvae. The mechanism of the interactions between JHA and ecdysteroid level deserves further investigation.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Interactions of ecdysteroid and juvenoid agonists in Plodia interpunctella (Hübner)

The influence of non-steroidal ecdysteroid agonists on Indianmeal moth larvae was assessed by rearing last instar larvae on diet treated with RH-5992 (tebufenozide) or RH-2485 (methoxyfenozide). Larvae were monitored for effects of the ecdysteroid agonists on weight, metamorphosis and mortality. Larvae treated with either of the ecdysteroid agonists at a concentration of 5 ppm or higher gained less weight and had greater mortality than did larvae reared on control diet. For example, the weights of control larvae increased approximately 400% by day 2, compared with only a 50% increase in weight when the larvae were treated with 25 ppm of RH-2485 or RH-5992. Similarly, mortality in control larvae was less than 10%, but was as much as 90–100% in larvae reared on diet treated with one of the ecdysteroid agonists. We also examined the effects of simultaneous treatment with a juvenile hormone (JH) mimic, either methoprene or fenoxycarb. The JH mimics prevented adult emergence, and the larvae continued to feed throughout the month-long observation period. However, larvae treated with a juvenile hormone mimic gained weight despite the presence of an ecdysteroid agonist in the diet. On diets treated with 0.1 ppm of RH-2485 or RH-5992, JH-treated larvae gained even more weight than did untreated controls. Interestingly, although the addition of a JH mimic to ecdysteroid-treated diet resulted in increased weight, it did not lead to reduced mortality. In fact, combinations of a JH mimic with 10 ppm RH 2485 or RH 5992 resulted in nearly 100% mortality compared with 40–70% mortality without the JH compounds. These results indicate that JH mimics overcome the inhibitory effects of ecdysteroid agonists on weight gain; however, they also resulted in increased mortality compared with moderate doses of ecdysteroid agonists alone. One specific action of these compounds at the cellular level was noted in that RH 5992 mimicked ecdysteroids by increasing uptake of ¹⁴C-GlcNAc in a Plodia interpunctella cell line, while fenoxycarb was inhibitory. Arch. Insect Biochem. Physiol. 38:91–99, 1998. Published 1998 Wiley-Liss, Inc.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

Quantal Division and a Postmitotic State in Myoblast Differentiation

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Quantal division and a postmitotic state in myoblast differentiation.

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Neurotropic effect of juvenile hormone III in larvae of the cockroach,Periplaneta americana

Juvenile hormone III and juvenile hormone analogs (methoprene, farnesyl methyl ether) cause a short-term depression of spike activity in last instar larvae of the cockroach Periplaneta americana, specifically in the prothoracic gland nerves, both in vivo and in vitro. The decreased nervous activity in the prothoracic gland nerves leads to decreased ecdysteroid secretion by the prothoracic gland.     

Effects of hormone agonists on Sf9 cells, proliferation and cell cycle arrest

Methoxyfenozide and methoprene are two insecticides that mimic the action of the main hormones involved in the control of insect growth and development, 20-hydroxyecdysone and juvenile hormone. We investigated their effect on the Spodoptera frugiperda Sf9 cell line. Methoxyfenozide was more toxic than methoprene in cell viability tests and more potent in the inhibition of cellular proliferation. Cell growth arrest occurred in the G2/M phase after a methoprene treatment and more modestly in G1 after methoxyfenozide treatment. Microarray experiments and real-time quantitative PCR to follow the expression of nuclear receptors ultraspiracle and ecdysone receptor were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment with no gene in common between the two treatments. Our results suggest two different signalling pathways in Sf9 cells.     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

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.     

HORMONAL CONTROL OF THE VITELLOGENESIS IN THE JAPANESE OAK SILKWORM, ANTHERAEA YAMAMAZ (LEPIDOPTERA: SATURNIIDAE)

Abstract Effects of ecdysteroid and juvenile hormone (JH) on vitellogenesis of the Japanese oak silkworm, Antheraea yamami are reported in this article. After topical treatment with 20-hydroxyecdysone alone or JH analog (i.e. methoprene) alone and combined treatment with these two chemicals, vitellogenin (Vg) titers in the fat body and haemolymph at the pupal stage were mostly higher than those of the control, indicating that both ecdysteroid and JH exerted a promoting effect on the synthesis of Vg. In contrast, the Vg uptake was markedly inhibited by JH while stimulating effect of the ecdysteroid could be shown that vitellin (Vt) titer in the ovary was lower after methoprene treatments, but higher after 20-hydroxyecdyson treatments. Meanwhile, effects of these two hormones on Vg synthesis in the fat body were also tested with the incubation in vitro with Grace medium containing H-leucine and the hormones. The results demonstrated that Vg synthesis was stimulated after treating with methoprene alone or 20-hydroxyecdysone alone and combined treating with these two chemicals, and particularly ecdysteroid had more marked positive effect. To comprehensively concluded our results, it could be regarded that ecdysteroid play the main role in the regulation of vitellogenesis for the Japanese oak silkworm.