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.     

Regulation of ecdysone hydroxylation in Locusta migratoria: Rôle of the moulting hormone level

After repetitive injections of moderate doses of ecdysone, ecdysterone or phenobarbital to young Vth (last) instar larvae of Locusta migratoria, the conversion rate of ecdysone to ecdysterone in vivo is significantly higher than in control insects. Similarly, 5 hr after injection of a low dose of ecdysone or ecdysterone, a strong ‘induction’ of ecdysone 20-monooxygenase activity occurs. This ‘inductive’ effect is blocked by cycloheximide.Simultaneous injections of ecdysone and ecdysterone show that hydroxylation of ecdysone is inhibited by the product of the reaction, ecdysterone. Removal of the prothoracic glands and X-ray treatment of the hemocytopoietic tissue do not affect ecdysone hydroxylation. The mechanism of induction and inhibition of ecdysone 20-monooxygenase shown in this study is probably responsible for the important variations of this key enzyme which have been reported from several insect species.     

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.     

Moulting and ecdysone release in response to electrical stimulation of protocerebral neurosecretory cells in Locusta migratoria

Locusta migratoria larvae were submitted to electrical stimulation of the protocerebral neurosecretory cells (median neurosecretory cells of the pars intercerebralis and lateral neurosecretory cells), during the last larval instar. The effects of the treatment were observed both on the duration of the stage and on the variations in haemolymph ecdysone levels. In untreated larvae, there was an initial ecdysone peak at the beginning of day 5, which was followed by 4 larger peaks between days 6 and 8. Stimulation of the median neurosecretory cells at the beginning of the instar resulted in the formation of one very large hormonal peak at the end of day 3: a day and a half earlier than in the control groups. Moulting was likewise accelerated. Stimulation also increased the size of the peaks, as compared with the controls. Stimulation of the lateral neurosecretory cells had a weaker ecdysiotropic effect; neither the number nor the size of the peaks were changed, though, like ecdysis, they occurred earlier. Stimulation of the deutocerebrum had no effect on either ecdysone titres or moulting. Electrical stimulation of the median neurosecretory cells at the end of day 5, that is after the occurrence of the first ecdysone peak, shortened the larval stage while having no significant effect on ecdysone levels in the haemolymph. The neuroendocrine control of ecdysis in Locusta is discussed.     

The control of prepupal puffing patterns in vitro; Implications for prepupal ecdysone titres in Drosophilia melanogaster

In late third instar larvae and prepupae of Drosophila melanogaster there is a complex change in puffing patterns in the salivary gland chromosomes. There are two peaks of activity in this period. The first, in larvae, is known to be under the control of the moulting hormone ecdysone. The second, in prepupae, is now shown by the in vitro culture of prepupal glands to be under the specific control of β-ecdysone in a manner similar to the first. A new class of puffs, active between these two peaks, whose induction is inhibited by ecdysone in vitro, is described. The behaviour of these puffs, exemplified by 75CD and 63E, suggests a period of very low ecdysone titre in vivo. The developmental significance of the role of ecdysone during prepupal development is discussed.     

Ecdysone titre and metabolism in relation to cuticulogenesis in embryos of Locusta migratoria

Eggs of Locusta migratoria contain remarkably high concentrations of ecdysone and several other ecdysteroids. During the time-span of embryonic development (11 days) 4 distinct peaks of ecdysone concentration (up to 8 μM) are observed in the egg, demonstrating the ecdysiosynthetic capacity of the embryo. Only during postblastokinetic development, is ecdysone efficiently hydroxylated to 20-hydroxyachieved through conjugation. On the basis of optical and electron microscopic observations, we have been able to correlate precisely each of the four peaks of ecdysone concentration in the egg with the time of deposition of a cuticle by the embryonic tissues (peak 1: serosal cuticle; peak 2: first embryonic cuticle; peak 3: second embryonic cuticle; peak 4: third embryonic cuticle).     

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.     

Moulting hormone level during the last instar of Galleria mellonella larva

Using radioimmunoassay the moulting hormone titres of the greater wax moth were determined during the last larval instar. Two peaks were observed, one when the larvae start to spin and another just before the pupation. The second peak exhibits the higher MH level, equivalent to 3600 ng/g ecdysterone. By TLC-RIA analysis three compounds were detected: ecdysone, ecdysterone and a very polar metabolite (VPM). The pattern of MHs during the last larval instar is described and the possible changes in the activity of enzymes of MH metabolism and ecdysone-ecdysterone conversion is discussed.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

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.     

Moulting of insect tracheae captured by light and electron-microscopy in the metathoracic femur of a third instar locust Locusta migratoria

The insect tracheal system is an air-filled branching network of internal tubing that functions to exchange respiratory gases between the tissues and the environment. The light and electron-micrographs presented in this study show tracheae in the process of moulting, captured from the metathoracic hopping femur of a juvenile third instar locust (Locusta migratoria). The images provide evidence for the detachment of the cuticular intima from the tracheal epithelial cells, the presence of moulting fluid between the new and old cuticle layers, and the withdrawal of the shed cuticular lining through larger upstream regions of the tracheal system during moulting. The micrographs also reveal that the cuticular intima of the fine terminal branches of the tracheal system is cast at ecdysis. Therefore, the hypothesis that tracheoles retain their cuticle lining at each moult may not apply to all insect species or developmental stages.     

Ultrastructural and immunocytochemical investigation of ecdysteroid secretion by the prothoracic gland of the waxmoth Galleria mellonella

Summary The formation and secretion of ecdysteroid by the prothoracic gland cells of Galleria mellonella (Insecta, Lepidoptera) were investigated electron microscopically and immunocytochemically. The moulting hormone ecdysone becomes first evident in vesicles and tubules of the smooth endoplasmic reticulum (SER). The SER forms secretory granules in which ecdysone was shown immunocytochemically. The Golgi apparatus seems not to be directly involved in ecdysone secretion. The secretory granules are released from the cells by exocytosis.Supported by the Sächsische Akademie der Wissenschaften zu LeipzigThe author is grateful to Mrs. Angelika Schmidt for her excellent assistance     

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.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

Activité,in vivo, d''inhibiteurs de la biosynthèse de l''ecdysone chezLocusta migratoria

Summary Among about 50 compounds synthesized to inhibit enzymes involved in the biosynthesis pathway of ecdysone we selected seven molecules which showed a strong effect on ecdysone production byLocusta migratoria prothoracic glands incubatedin vitro. These molecules mostly possess a specific activity on ecdysone biosynthesis which is irreversible. The compounds were administered in one or several injections of aqueous or oily solutions at different times in the course of the two last larval instars. Three inhibitors led in a 10% ratio to a prolongation (sometimes more than 3 times the standard length) of the instar, pointing out a decrease in the ecdysone biosynthesis. Two other inhibitors induced some morphogenetic modifications in the adults, as size reduction or wing alterations, and metamorphosis difficulties. Thein vivo low activity compared with the strong onein vitro could be due to difficulties for the compounds to reach the prothoracic glands without degradation. The variation of inhibitory activity which appearsin vivo between the seven compounds studied is not linked either with the chemical structures of the molecules (which are very near one another) or with theirin vitro activity.

     

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.     

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.     

The secretion and metabolism of alpha-ecdysone by cockroach (Leucophaea maderae) tissues in vitro

Hemolymph β-ecdysone levels are high (～1.6 μg/ml) in late last instar cockroach ($\text{Leucophaea}$$\text{maderae}$) nymphs; the level of α-ecdysone (～0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.