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.     

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.     

An in vitro study on regulation of prothoracic gland activity in the early last-larval instar of the silkworm Bombyx mori

The endocrine mechanisms that regulate prothoracic gland (PG) activity in early stages of final larval instar of the silkworm Bombyx mori were investigated using a newly developed long-term cultivation system of the gland. The PGs dissected from day-0 fifth instar larvae did not secrete detectable amounts of ecdysone for the first 24 h in culture but started secretion within the next 2 days. The amount of secreted ecdysone increased day by day. When day-0 PGs were co-cultivated with corpora allata, however, they remained inactive for at least 8 days. PGs dissected from 1-day younger larvae (day-3 fourth instar larvae) secreted ecdysone for the first 24 h but stopped secretion for the next 24 h, followed by recovery of ecdysone secretory activity. By contrast, PGs from day-1 fourth instar larvae remained active throughout a cultivation period without any sign of inactivation. However, when the same glands were exposed to a high titer of 20-hydroxyecdysone for the second 24h in culture, they gradually lost their activity. These results indicate that PGs of fourth instar larvae are inactivated by ecdysteroid through a negative feedback mechanism and that thus inactivated PGs spontaneously recover ecdysone secretory activity in the early fifth instar unless inhibited by juvenile hormone.     

Involvement of PI3K/Akt signaling in PTTH-stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx mori

The prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by prothoracic gland in larval insects. Previous studies showed that Ca²⁺, cAMP, extracellular signal-regulated kinase (ERK), and tyrosine kinase are involved in PTTH-stimulated ecdysteroidogenesis by the prothoracic glands of both Bombyx mori and Manduca sexta. In the present study, the involvement of phosphoinositide 3-kinase (PI3K)/Akt signaling in PTTH-stimulated ecdysteroidogenesis by B. mori prothoracic glands was further investigated. The results showed that PTTH-stimulated ecdysteroidogenesis was partially blocked by LY294002 and wortmannin, indicating that PI3K is involved in PTTH-stimulated ecdysteroidogenesis. Akt phosphorylation in the prothoracic glands appeared to be moderately stimulated by PTTH in vitro. PTTH-stimulated Akt phosphorylation was inhibited by LY294002. An in vivo PTTH injection into day 6 last instar larvae also increased Akt phosphorylation of the prothoracic glands. In addition, PTTH-stimulated ERK phosphorylation of the prothoracic glands was not inhibited by either LY294002 or wortmannin, indicating that PI3K is not involved in PTTH-stimulated ERK signaling. A23187 and thapsigargin, which stimulated B. mori prothoracic gland ERK phosphorylation and ecdysteroidogenesis, could not activate Akt phosphorylation. PTTH-stimulated ecdysteroidogenesis was not further activated by insulin, indicating the absence of an additive action of insulin and PTTH on the prothoracic glands. The present study, together with the previous demonstration that insulin stimulates B. mori ecdysteroidogenesis through PI3K/Akt signaling, suggests that crosstalk exists in B. mori prothoracic glands between insulin and PTTH signaling, which may play a critical role in precisely regulated ecdysteroidogenesis during development.     

Activity of the prothoracic gland and its sensitivity to prothoracicotropic hormone in the penultimate and last-larval instar of Bombyx mori

The time course of secretion of ecdysone in vitro by the prothoracic glands of Bombyx mori was studied through the penultimate and last-larval instars. Ecdysone was produced by the glands in high amounts by the penultimate instar at 72 and 84 h while the glands in the last instar exhibited a high activity over 4 days around the time of gut purge and thereafter. The glands in the penultimate instar produced ecdysone at a low level throughout the instar before the sharp peak of activity, when they became inactive and remained so for the first 3 days of the last instar after when they regained secretory activity. Sensitivity of the glands to prothoracicotropic hormone varied in accord with the changes in their secretory activity. Inactive glands were not stimulated by 22K-prothoracicotropic hormone. In addition, glands with maximal activity in the penultimate instar were insensitive to 22K-prothoracicotropic hormone. These results suggest that the prothoracic glands in the penultimate and last-instar larvae are physiologically different.     

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.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

昆虫成虫蜕皮激素研究进展

绝大多数成体昆虫羽化后,幼虫期间负责蜕皮激素合成的前胸腺即发生退化,但在一些内部生理及外部环境因子的调控下,某些成体组织(如生殖腺)可扮演类似前胸腺的角色合成与分泌蜕皮激素。蜕皮激素的功能发挥是经受体介导的,包括核受体(如EcR/USP)和膜受体(如DopEcR),它们广泛表达于成体许多组织,参与成虫行为、生殖、寿命、滞育及免疫应答等众多方面的调节,对维持基本的生理功能具有重要作用。就成虫蜕皮激素的产生组织及影响其滴度的因素、成虫蜕皮激素受体概述与组织分布、成虫蜕皮激素信号通路的功能发挥等研究进展方面加以综述。     

Prothoracic glands in the regulation of ecdysone titres and metabolic fate of injected labelled ecdysone in Locusta migratoria

In the absence of the prothoracic glands, fifth instar larvae of Locusta migratoria contain no demonstrable quantities of ecdysone and ecdysterone (assayed together in the Calliphora bioassay), whereas normal larvae show a high peak of ecdysone activity. The metabolic fate of injected radiolabelled ecdysone is found to be very similar in prothoracectomized larvae to that of normal larvae (hydroxylation rate, dehydrogenation of ecdysone and ecdysterone, inactivation rate). However, in the absence of the prothoracic glands, the larvae excrete radiolabelled ecdysone in their faecal material at a rate which is considerably higher than that of normal insects of the same age. These results are discussed in view of the regulation of the ecdysone titres by the prothoracic glands in L. migratoria.     

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.     

Ecdysteroid release by the prothoracic gland of Gryllus bimaculatus (Ensifera: Gryllidae) during larval-adult development

The in vitro secretion of ecdysteroids from the prothoracic glands of larvae of Gryllus bimaculatus was analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (65-93%), with lesser amounts of ecdysone (7-35%). Production and release of ecdysteroids from the prothoracic glands are calcium-dependent. The rate of ecdysteroid release was low during the beginning and the end of the last two larval stages and high in between. Prothoracic glands from young adult females produced only minor amounts of ecdysteroids and ceased hormone production around day 4 after the moult.     

PTTH-stimulated ecdysone secretion is dependent upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta

PTTH stimulates ecdysteroid secretion by the insect prothoracic glands. The peptide activates cAMP synthesis in a calcium-dependent manner, ultimately enhancing ecdysteroid synthesis. We have found that PTTH stimulates a rapid increase in tyrosine phosphorylation of at least four proteins in the prothoracic glands of larval Manduca sexta, as seen on Western blots of glandular lysates probed with antibody directed against phosphotyrosine. PTTH-stimulated tyrosine phosphorylation is blocked by an inhibitor of Src family tyrosine kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1). The inhibitor also blocks PTTH-stimulated ecdysone secretion, as well as PTTH-stimulated cAMP synthesis. Direct activation of the catalytic subunit of adenylyl cyclase by forskolin is not affected by PP1. In addition, ecdysteroid secretion stimulated by the cAMP analog dbcAMP is not blocked by PP1. These findings point to an important role for a Src-family tyrosine kinase at a very early step in the PTTH signaling pathway, prior to the activation of adenylyl cyclase.     

Involvement of calcium in prothoracicotropic stimulation of ecdysone synthesis in Galleria mellonella

The cytosolic free calcium was measured with Fura-2 in single prothoracic gland cells of Galleria larvae. During the last two larval instars calcium concentration correlated with ecdysone secretion by the glands. Addition of prothoracicotropic hormone (PTTH) from brains of Galleria larvae to prothoracic glands in vitro induced a significant increase in calcium in the gland cells. This effect of PTTH was abolished by removal of extracellular calcium, or by the addition of lanthanum or of the calcium channel antagonists nicardipine and verapamil. The calcium channel agonist Bay K 8644 evoked an increase in intracellular calcium. TMB-8, an inhibitor of intracellular calcium mobilization, did not block the PTTH-stimulated rise in calcium concentration or ecdysone production, indicating that intracellular calcium stores are not involved in the calcium-mediated ecdysone synthesis. Moreover, PTTH seems to exert its action by influencing dihydropyridine-sensitive calcium channels in the plasma membrane. © 1996 Wiley-Liss, Inc.     

Steroidal regulation of hydrolyzing activity of the dietary carbohydrates in the silkworm, Bombyx mori

Blood sugar is an essential energy source for growth and development and is maintained at a constant level through precise regulation of formation and utilization. Sugars are produced from dietary carbohydrates by enzymatic hydrolysis in the digestive tract, which are under the homeostatic control of paracrine and prandial mechanisms in mammals. Here, we show that dietary carbohydrates hydrolyzing activity of the digestive tract is developmentally regulated by the steroid hormone ecdysone in the silkworm, Bombyx mori. The dietary carbohydrates hydrolyzing activity remained high throughout the last larval period and then decreased to negligible levels until the pupal period. However, dietary carbohydrates digestive activities were constitutively high when the steroidogenic organ, prothoracic glands were ablated. The prothoracic glands produced and released a large amount of ecdysone at the end of the larval period, suggesting that ecdysone is responsible for the decrease in dietary carbohydrates hydrolyzing activity. In fact, ecdysone decreased the activity to negligible levels in silkworms lacking the prothoracic glands. The present results indicate that the dietary carbohydrates hydrolyzing activity is regulated by ecdysone and that an increase in ecdysone titer decreases that activity at the end of the larval period, suggesting that ecdysone is essential for metabolic coordination during development.     

Size assessment and growth control: how adult size is determined in insects

Size control depends on both the regulation of growth rate and the control over when to stop growing. Studies of Drosophila melanogaster have shown that insulin and Target of Rapamycin (TOR) pathways play principal roles in controlling nutrition-dependent growth rates. A TOR-mediated nutrient sensor in the fat body detects nutrient availability, and regulates insulin signaling in peripheral tissues, which in turn controls larval growth rates. After larvae initiate metamorphosis, growth stops. For growth to stop at the correct time, larvae need to surpass a critical weight. Recently, it was found that the insulin-dependent growth of the prothoracic gland is involved in assessing when critical weight has been reached. Furthermore, mutations in DHR4, a repressor of ecdysone signaling, reduce critical weight and adult size. Thus, the mechanisms that control growth rates converge on those assessing size to ensure that the larvae attain the appropriate size at metamorphosis.     

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.     

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.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.