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.     

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.     

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.     

Analysis of ecdysteroidogenic activity of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The cellular mechanism underlying ecdysteroidogenesis throughout the last larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid secretory activity of the prothoracic glands and cAMP accumulation of gland cells, as well as changes in responsiveness to stimulation by prothoracicotropic hormone (PTTH) and 1-methyl-3-isobutylxanthine (MIX). It was found that the prothoracic glands during the first 3 days of the last instar cannot produce detectable ecdysteroid and showed no response to stimulation by PTTH or 1-methyl-3-isobutylxanthine (MIX). However, artificial elevation of cellular cAMP levels by in vitro dibutyryl cAMP treatment stimulated the glands to secrete detectable ecdysteroid, implying the presence of a cAMP-dependent ecdysteroidogenic apparatus during this stage. From days 3 to 8, basal gland activities fluctuated, but the glands showed activation responses to PTTH and to the chemicals that increase cellular cAMP levels. After the occurrence of the peak in basal gland activity on day 9, glands on day 10 showed no response to PTTH, implying a refractory state of the glands to PTTH stimulation. For cAMP accumulation, it was found that glands on day 2 began to show increased cAMP accumulation to PTTH, implying that the acquisition of gland competency for elevation of cAMP levels after stimulation by PTTH precedes that of ecdysteroid production. Moreover, during most parts of the last larval instar (between days 3 and 8) and at the pupation stage, greatly increased cAMP accumulation upon stimulation by PTTH was observed only in the presence of MIX, indicating that cAMP phosphodiesterase levels may be high during these stages. From these results, we concluded that development-specific PTTH signal transduction during the last larval instar, which shows a different pattern from that of the penultimate larval instar, may play an important role in regulating changes in prothoracic gland activity and in leading to larval-pupal metamorphosis.     

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: a cytophysiological analysis of the ring gland

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.     

Daily changes in neuroendocrine control of moulting hormone secretion in the prothoracic gland of the cockroach Periplaneta americana (L.)

Time of day related changes in ecdysteroid secretion by the prothoracic gland of last instar nymphs were studied using in vitro coincubations of prothoracic glands and brains under a 12-h light:12-h dark cycle. The experiments reveal that the cells of the prothoracic gland of the cockroach nymphs do not have an endogeneous circadian oscillator determining rhythmicity of ecdysteroid secretion. PTTH release in the scotophase is responsible for the peak of ecdysteroid production during the photophase.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

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.     

NEUROANATOMICAL STUDIES OF PROTHORACIC GLANDS OF COTTON BOLLWORM HELZCOVERPA ARMZCERA (LEPIDOPTERA: NOCTUIDAE)*

Abstract Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T-shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15^:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar. Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent. Freeze-fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.     

Physiological investigation on the role of the innervation in the regulation of the prothoracic gland in Periplaneta americana

Under in vitro conditions the prothoracic gland nerve of the last larval instar of Periplaneta americana shows the same efferent nervous activity as under in situ conditions–ie, low activity at the 9th day and high activity at the 20th day of the molting interval. Isolation of the prothoracic ganglion from the subesophageal ganglion provokes an increase in this nerve activity, suggesting an inhibitory effect of the subesophageal ganglion on prothoracic gland nerve activity in vivo. Only in 20-day-old larvae does electrical stimulation of isolated prothoracic glands in vitro via the gland nerve result in a slightly increased release of ecdysteroids from the gland. This effect could not be influenced by different lengths of stimulation periods. Denervation of the prothoracic gland by transection of the gland nerve on the 13th day of the molting interval results in a complete abolition of the first peak of ecdysteroid production in the gland but has no influence on the occurrence and the amount of the main ecdysteroid peak just before the molt. The results suggest the participation of nervous activity in special periods of prothoracic gland regulation in the cockroach.     

Steroid transport through the surface of the prothoracic gland cells in Galleria mellonella L.

Steroid transport through the cell surface of the giant polyploid prothoracic gland cells of Galleria mellonella L. was studied by an ultracytochemical method. The alkaloid digitonin, known to form a complex with all sterols having a free-OH radical in position 3, proved to be suitable for studying the interiorisation of moulting hormone precursors and the release of synthesized hormones. The results suggest that cholesterol uptake in the last larval instar occurs by macropinocytosis during the feeding period, while the release of the steroids produced by the gland occurs by reverse micropinocytosis mostly on days 5-7 of the instar. The two processes are not simultaneous. The intracytoplasmic localisation of the reaction product confirms the steroidogenic role of the prothoracic gland.     

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.     

Growth and respiratory enzyme activity in the prothoracic glands of Galleria

In the penultimate-larval instar, the total volume of the prothoracic gland and the activities of some oxidative mitochondrial enzymes (cytochrome oxidase, NADH: cytochrome c oxidoreductase, succinate: cytochrome c oxidoreductase) undergo cyclic variations associated with larval growth. These specifically larval-larval growth cycles are absent in the prothoracic glands of normal last-instar larvae. Here the cycles can be induced artificially by implantation of brain or corpora cardiaca-allata complexes or, by exogenous application of juvenile hormone. The smallest size of the prothoracic gland in relation to the size of the body, as well as the minimal activity of all the three mitochondrial enzymes in the gland, have been found exactly at the moment of the pre-pupal peak of ecdysteroid in the body. The possibility that the prothoracic glands alone can synthetize ecdysteroid during the peak is questioned.     

Nachweis von häutungsaktiven Stoffen in isolierten Prothorakaldrüsen und Oenocyten beiBombyx mori während des 5. Larvenstadiums

Summary The content of moulting hormones has been determined in homogenates of isolated prothoracic glands and oenocytes during the 5th instar of the silkworm,Bombyx mori by means of the Calliphora bioassay.Prothoracic glands show variable activity in the production of moulting hormones, reaching a maximum near the end of the larval period. Comparable activities, but at higher levels, could be demonstrated in oenocytes. Controls with doubled quantities of tissue produced in a proportionate reaction in the bioassay. Fat bodies were inactive.Prothoracic glands and oenocytes incubated together resulted in a slower pupation index than would be expected from the sum of single determinations of oenocytes and prothoracic glands. This is explained by the ability of prothoracic glands to build conjugates of ecdysones.Die Arbeit wurde mit Unterstützung der Deutschen Forschungsgemeinschaft durchgeführt     

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Temporal changes in DNA synthesis of prothoracic gland cells during larval development and their correlation with ecdysteroidogenic activity in the silkworm, Bombyx mori

DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the 3rd, 4th, and last larval instars were examined. During the early stages of both the 3rd and 4th larval instars, a dramatic increase in the number of DNA-synthesizing cells of the prothoracic glands was detected. However, during the latter stages of each instar, the number of DNA-synthesizing cells greatly decreased. The determination of glandular protein content showed that dramatic increases occurred during the latter stages of each larval instar. Comparison of changes in prothoracic gland cell DNA synthesis with ecdysteroidogenic activity showed that the increase in DNA synthesis precedes ecdysteroidogenesis. The cellular mechanism underlying changes in prothoracic gland cell DNA synthesis during the last two larval instars was further analyzed by determining the in vitro DNA synthesis of the glands, their responsiveness to hemolymph growth factors, and changes in the growth-promoting activity of hemolymph during development. It was found that both growth factors and the responsiveness of the prothoracic gland cells to growth factors from hemolymph may play roles in regulating DNA synthesis of gland cells.     

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.     

Autocrine activation of DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori

Autocrine activation of DNA synthesis in prothoracic gland cells in last instar larvae of the silkworm, Bombyx mori, was studied using both a long-term in vitro organ culture system and immunocytochemical labeling with 5-bromo-2'-deoxyuridine (BrdU). When prothoracic glands were incubated in a small volume of culture medium (10 microl/gland), the numbers of DNA-synthesizing cells per gland increased significantly, and DNA synthesis was stimulated less by hemolymph, as compared with glands incubated in a large volume (50 microl/gland). Moreover, glands cultured in groups (6 glands per group in a 50-microl drop) also resulted in much higher levels of DNA synthesis than those cultured individually in a 50-microl drop. The mechanism by which alternation of the volume of the incubation medium results in changes in the levels of DNA synthesis was further examined. When prothoracic glands were incubated in medium (50-microl drop per gland) that was preconditioned with glands (in a 10-microl drop individually), a dramatic increase in DNA synthesis activity was also observed, indicating that prothoracic glands may release a factor that stimulates their own DNA synthesis. The growth-promoting factor was further characterized and it was found that the factor is heat stable, and its molecular weight was estimated to be between 1,000 and 3,000 Da. Moreover, the factor also stimulated corpus allatum cell DNA synthesis in vitro. Injection of concentrated putative growth-promoting factor into day 4 last instar-ligated larvae greatly increased cell DNA synthesis of the prothoracic glands, indicating the in vivo function of the present autocrine factor.     

Effects of the corpora allata on sexual receptivity and completion of oocyte development in females of the cricket, Gryllus bimaculatus

Abstract Allatectomy of young penultimate nymphs of Gryllus bimaculatus De Geer (Gryllidae) resulted in prothetelic creatures which exhibited reproductive competence. The same operation performed on young last instar nymphs resulted in moulting to morphologically normal adults. Allatectomized morphologically normal adult females, as well as prothetelic ones, showed the same level of sexual receptivity as untreated control females. Allatectomized morphologically normal and prothetelic females laid viable eggs, but rate of egg laying and number of eggs produced by these females were much reduced in comparison with the controls. Administration of methoprene (a Juvenile Hormone analogue) to allatectomized females restored egg production to a more or less normal rate. Removal of the spermatophore within 10 min of copulation had no effect on subsequent sexual receptivity of the females, nor on the reduced rate of egg laying by the allatectomized females, but did affect the rate of egg laying by control females.
It is suggested that the corpora allata (CA) and the Juvenile Hormone (JH) play no major role in controlling basic sexual receptivity of G.bimaculatus females, and do not have an all-encompassing control on egg production, though they do exert a marked quantitative effect on the rate of egg production.     

Control of prothoracic gland activity in larvae of Galleria mellonella

Prothoracic glands of last instar wax moth larvae maintain spontaneous secretory activity both in decapitated larvae and in isolated abdomens into which they have been transplanted, as judged by their ability to induce secretion of a new cuticle. Their activity is hormonally stimulated by the brain and inhibited by the prothoracic and mesothoracic ganglia. The subesophageal ganglion seems to suppress the inhibitory influence of the thoracic ganglia. The prothoracic glands of larvae decapitated at different times during the last instar all respond to brain implantation, and this response does not change when brains are implanted at increasing intervals after decapitation. The prothoracotropic activity of the isolated brain is highest in brains of pupae and adults but is relatively and consistently low in brains of last instar larvae. The results demonstrate that the control of prothoracic glands is a complex process governed by the nervous integration of various stimuli.