Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Larval-pupal transformation of the prothoracic glands of Mamestra brassicae

The sensitivity of the prothoracic glands to juvenile hormone and prothoracicotropic hormone (PTTH) of penultimate (5th)-instar larvae of Mamestra brassicae was compared with that of the same-instar larvae destined for pupal ecdysis by allatectomy. The activity of the prothoracic glands was assessed using either moulting of isolated abdomens or ecdysone radioimmunoassay. Juvenile hormone application immediately after neck-ligation (which removes brain-corpora cardiaca-corpora allata complex) prevented prothoracic gland function in larvae at all stages. When larvae were allatectomized 12 hr after ecdysis, followed by neck-ligation at different times and given juvenile hormone immediately, the hormone inhibited the prothoracic glands of young larvae, but activated the prothoracic glands from day-5 or older larvae. Juvenile hormone I, juvenile hormone II and methoprene activated the prothoracic glands, but juvenile hormone III was relatively ineffective. Brain implantation instead of juvenile hormone application led to activation of the prothoracic glands at all stages.Allatectomy thus caused changes leading to metamorphosis including a transformation of the prothoracic glands from ‘larval’ to ‘pupal’ type. After this change these prothoracic glands were able to respond not only to PTTH but also to juvenile hormone just as in last-instar larvae.     

Cellular events in the prothoracic glands and ecdysteroid titres during the last-larval instar of Spodoptera littoralis

Changes in prothoracic gland morphology were correlated to developmental events and ecdysteroid titres (20-hydroxyecdysone equivalents) during the last-larval instar in Spodoptera littoralis. After ecdysis to the last-larval instar the haemolymph ecdysteroid titre remained at about 45 ng/ml, when the prothoracic glands appeared quiescent. The first signs of distinct gland activity, indicated by increased cell size and radial channel formation, were observed at about 12 h prior to the cessation of feeding (36 h after the last-larval moult), accompanied by a gradual increase in ecdysteroid titre to 110 ng/ml haemolymph, at the onset of metamorphosis. During this phase ecdysteroid titres remained at a constant level (140–210 ng/ml haemolymph) and prothoracic gland cellular activity was absent for a short period. The construction of pupation cells occurred when haemolymph ecdysteroids titres increased to 700 ng/ml. A rapid increase in ecdysteroids began on the fourth night (1600 ng/ml haemolymph) reaching a maximal level (4000 ng/ml haemolymph) at the beginning of the fourth day. In freshly moulted pupae a relatively high ecdysteroid titre (1100 ng/ml haemolymph) was still observed, although during a decrease to almost negligible levels. The increase in ecdysteroid level during the third and the fourth nights of the last-larval instar was correlated with the period when almost all the prothoracic gland cells showed signs of high activity. Neck-ligation experiments indicated the necessity of head factors for normal metamorphosis up to the second to third day of the instar. The possibility that the prothoracic glands are under prothoracicotropic hormone regulation at these times is discussed.     

Evaluation of Serangium n. sp. (Col., Coccinellidae), a predator of Bemisia tabaci (Hom., Aleyrodidae) on cassava

Abstract  The potential of a new, previously unidentified Serangium species (Col., Coccinellidae) to control the high Bemisia tabaci (Gennadius) (Hom., Aleyrodidae) populations on cassava was evaluated. Field and laboratory studies were carried out to determine the abundance and feeding capacity of this Serangium species feeding on B. tabaci on cassava. Serangium nymphs and adults were most abundant in cassava fields late in the season, rising sharply from 5 months after planting (MAP) to a peak at 7–8 MAP. Pre-imaginal development averaged 21.2 days and was longest in eggs and shortest in the L₁ instar. Mean total prey consumption of immature Serangium increased with the stage of development with the lowest consumption in the L₁ instar and highest in the L₄ instar. Mean daily consumption was lowest on the first day after hatching in the L₁ instar and rose to a peak on the 13th day after hatching in the L₄ instar. Each Serangium larva consumed a mean of over 1000 nymphs during its entire development. These results have demonstrated the potential of this Serangium species to control B. tabaci populations on cassava.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Control of larval-pupal-adult molt in the moth Sesamia nonagrioides by juvenile hormone and ecdysteroids

Sesamia nonagrioides (Lepidoptera: Noctuidae) larvae reared under long day (LD; 16L:8D) conditions pupate after 5 or 6 larval instars, whereas under short day (SD; 12L:12D) conditions they undergo up to 12 additional molts before pupating. This extended period of repeated molting is maintained by high levels of juvenile hormone (JH). Previous work demonstrated that both LD and SD larvae decapitated in the 6th instar pupate but further development is halted. By contrast, about one-third of SD larvae from which only the brain has been removed, undergo first a larval molt, then pupate and subsequently developed to the adult stage. Debrained LD larvae molt to larvae exceptionally but regularly pupate and produce adults. Implanted brains may induce several larval molts in debrained recipient larvae irrespectively of the photoperiodic conditions. The results of present work demonstrate that the prothoracic glands (PGs) and the corpora allata (CA) of debrained larvae continue to produce ecdysteroids and JHs, respectively. PGs are active also in the decapitated larvae that lack JH, consistent with the paradigm that CA, which are absent in the decapitated larvae, are the only source of this hormone. Completion of the pupal-adult transformation in both LD and SD debrained insects demonstrates that brain is not crucial for the development of S. nonagrioides but is required for diapause maintenance. Application of JH to headless pupae induces molting, presumably by activating their PGs. It is likely that JH plays this role also in the induction of pupal-adult transformation in debrained insects. Application of the ecdysteroid agonist RH 2485 (methoxyfenozide) to headless pupae also elicits molting: newly secreted cuticle is in some cases thin and indifferent, in other cases it bears distinct pupal or adult features.     

Effects of ultralow doses of fenoxycarb on juvenile hormone–regulated physiological parameters in the silkworm,Bombyx mori L.

Effects of fenoxycarb at ultralow doses were investigated on juvenile hormone (JH)–regulated parameters in the silkworm, B. mori. Like JH, this non-terpenoid carbamate is able to induce permanent larvae in the last larval instar. However, whereas micrograms of JH are needed to produce this effect, only a few picograms of fenoxycarb are necessary to induce the same effect. The effects of fenoxycarb observed in this study were only visible from day 4 of the last larval instar—that is, when the JH titer has dropped to undetectable levels and JH-repressed physiological parameters would naturally be expressed. We observed that the permanent larvae induced with low doses of fenoxycarb (100 pg/larva) had no 20-hydroxyecdysone (20E) peak. Their prothoracic glands (Pgs) were completely inactive and very weakly sensitive to prothoracicotropic hormone (PTTH). Fenoxycarb at doses of 1 ng/larva also significantly inhibited silk gland growth and coloration, whereas carotenoid content of the hemolymph was maintained at high levels, which could reflect an inhibition of its uptake by the silk glands. Total hemolymph protein levels in last instar larvae were also depressed at these doses. So, it seems that low doses of fenoxycarb are sufficient to maintain in a juvenilized status the physiological parameters that are normally expressed when JH titer has declined. Moreover, from an endocrinological viewpoint, we demonstrated that the corpora allata (CA) are not necessary for fenoxycarb to induce those effects and discussed its possible mode of action. Arch. Insect Biochem. Physiol. 37:178–189, 1998. © 1998 Wiley-Liss, Inc.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

The metabolism of 3H-moulting hormone in Calliphora erythrocephala during larval development

The activity in whole insects for converting ³H-α-ecdysone to ³H-β-ecdysone after injection is low (half-maximal) in young last instar larvae, maximal in mature larvae, and minimal (fourth-maximal) at the white puparial stage. Because moulting hormone titre is low throughout the last larval instar and increases at the formation of the puparium it appears that hydroxylation at C-20 is not a key step in regulating β-ecdysone biosynthesis during larval development.The activity for catabolizing ³H-β-ecdysone is maximal in second instar larvae, about thirdmaximal throughout most of the third instar, and minimal at pupariation (thirtieth-maximal). Thus inactivation may play a rôle in regulating moulting hormone titre during larval development.     

Larval diapause of the European corn borer, Ostrinia nubilalis: Further experiments examining its hormonal control

The possible involvement of juvenile hormone (JH) in controlling the mature larval diapause of the European corn borer, Ostrinia nubilalis, was examined using biological and chemical assays for JH titres, topical applications of JH mimic, and injections of 20-hydroxy-ecdysone. Bioassays of extracts of larval haemolymph showed that (1) 4th instar pre-diapausing larvae had a higher JH titre (ca. 1450 Galleria Units (GU)/ml) than equivalent non-diapausing larvae (ca. 340 GU/ml), and that (2) 5th instar pre-diapausing larvae contained a JH titre of ca. 320 GU/ml, which declined to ca. 90 GU/ml in newly-diapaused larvae. Chemical assasys carried out on extracts of whole larvae showed that early diapausing larvae contained an extremely low titre of JH. In addition, the application of JH mimic or 20-hydroxy-ecdysone or both agents to diapausing larvae failed to reveal the presence of a functional JH titre during diapause. The application of JH mimic to early 5th instar non-diapausing larvae produced moribund larval-pupal intermediates rather than supernumerary larvae. Our results, therefore, suggest that although JH may control some phases of diapause induction, it is not involved in maintaining diapause.     

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.     

Juvenile hormone coordinates the regulation of the hemolymph ecdysteroid titer during pupal commitment in Manduca sexta

The timing and magnitude of the pupal commitment peak in the hemolymph ecdysteroid titer of fifth instar Manduca sexta larvae are controlled by the combined effects of prothoracicotropic hormone (PTTH), a prothoracic gland-stimulating factor present in the hemolymph, and the biosynthetic competence of the prothoracic glands themselves. The present data indicate those individual effects are coordinated by juvenile hormone (JH): (1) Treatment of larvae with the JH analog (7S)-hydroprene prevents the normal precommitment drop in the titer of the stimulatory factor; (2) treatment of larvae with (7S)-hydroprene suppresses in a dose- and time-dependent manner the biosynthetic competence of the prothoracic glands; and (3) (7S)-hydroprene acts directly on the brain to inhibit the release of PTTH in vitro. Thus, during Manduca development, a drop in the JH titer early in the fifth instar results in a rapid drop in the titer of the stimulatory factor, the gradual acquisition by prothoracic glands of biosynthetic competence, and lastly, the gated release of PTTH into the hemolymph. The resulting increase in ecdysone synthesis by the prothoracic glands gives rise to the small peak in the ecdysteroid titer that drives pupal commitment.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.