An ultrastructural analysis of the ecdysoneless ((l(3)ecd 1ts) ring gland during the third larval instar of Drosophila melanogaster

Summary In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd ^1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.     

An ultrastructural and developmental analysis of the corpus allatum of juvenile hormone deficient mutants ofDrosophila melanogaster

Summary The ultrastructure of the corpus allatum of theapterous mutantsap ⁴ andap ^56f ofDrosophila melanogaster during larval-pupal-adult metamorphosis and adult life was correlated with the gland's ability to synthesize juvenile hormone in vitro. During the early wandering period of the third instar of both mutants, a high concentration of smooth endoplasmic reticulum, mitochondria and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. Juvenile hormone biosynthesis by the glands is high at that time and, in fact, only slightly lower than that of wild type glands. In contrast to the wild type gland, the cells of the pupal and pharate adult corpus allatum of both mutants contains highly electron dense mitochondria with tubular cristae but no whorls of smooth endoplasmic reticulum nor glycogen clusters. The frequency and size of the lipid droplets, putatives depots of the juvenile hormone precursors, in cells of theap ^56f gland is a function of the insect's age, but both are lower than in wild type gland cells. Juvenile hormone biosynthesis by both mutant glands remains at the basal level when compared to increased synthesis by the wild type gland. The frequency and density of lipid droplets in cells of theap ⁴ corpus allatum are much lower than in theap ^56f glands. During adult life, the ultrastructural profile of theap ^56f corpus allatum is similar to that of the wild type gland although the in vitro production of juvenile hormone by the former is much lower than that of the wild type gland. The ultrastructural features of the adult corpus allatum ofap ⁴ homozygotes reveal precocious degeneration and support the view that this non-vitellogenic mutant is a juvenile hormone deficient mutation.     

An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.     

The effects of azadirachtin A on the morphology of the ring complex of Lucilia cuprina (Wied) larvae (Diptera: Insecta)

This study investigated the effect of azadirachtin A (AZA) on the ultrastructure of the endocrine glands (the prothoracic gland, the corpus allatum, and the corpus cardiacum) responsible for controlling moulting and ecdysis in Lucilia cuprina larvae. All constituent endocrine glands within the ring complex exhibited ultrastructural change after exposure of larvae to AZ A in the diet. The effects on the nucleus were the most noticeable and included: (1) crenulation of nuclear shape, (2) clumping of heterochromatin, and (3) pyknosis. The degenerative changes to the nuclei appeared to precede changes within the cytoplasm. It was concluded that the degeneration of all three endocrine glands within the ring complex would contribute to a generalised disruption of neuroendocrine function.     

Switchover in the sensitivity of the prothoracic glands to juvenile hormone in the cotton leafworm, Spodoptera littoralis

Switchover in the sensitivity of the prothoracic glands to juvenile hormone analogue during the last-larval instar of Spodoptera littoralis occurs in the middle of the third scotophase i.e. at the end of phagoperiod when the body weight is maximal and the ecdysteroid is increasing in the haemolymph. Application of the analogue to larvae neck-ligated before the switchover completely inhibits or delays metamorphosis due to an inhibitory effect on the prothoracic gland cells and is not mediated by the nervous system. This inhibition by the analogue is dose-dependent, and when complete inhibition of metamorphosis occurs, the prothoracic glands cells degenerate. Treatment of neck-ligated larvae with the analogue after the switchover stimulates metamorphosis by accelerating the appearance of an ecdysteroids peak in the haemolymph. The stimulatory effect of the analogue to the prothoracic glands in neck-ligated larvae is not direct one, and some unknown factors seem to play a role therein.

The probale role of prothoracicotropic hormone as a synchronizing factor in the switchover in the sensitivity of the prothoracic glands to juvenile hormone is discussed.     

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.     

Effect of allatectomy on ecdysteroid-dependent development of Rhodnius prolixus larvae

the regulation of haemolymph titres of ecdysteroids during larval development of the bloodsucking bug, Rhodnius prolixus was studied. Corpus allatum ablation in 4th-instar larvae 1 day after feeding was reflected in an increase of the intermoult period and in a high level of ecdysial arrest. These effects could be corrected by juvenile hormone and ecdysone therapies. Comparison of the ecdysteroid titres in haemolymph determined in control and allatectomized larvae, at different intervals after feeding, showed that allatectomy drastically depressed the ecdysteroid levels. Juvenile hormone treatment reestablished ecdysteroid titres in the haemolymph of allatectomized insects. Isolated prothoracic glands from allatectomized larvae had a very low production of ecdysteroid-RIA-activity when compared with prothoracic glands from control or allatectomized larvae which received in vivo juvenile hormone treatment. The complexity of the corpus allatum-prothoracic glands interaction in Rhodnius post-embryonic development is discussed.     

Degeneration of moulting glands in male crickets

The degeneration of the prothoracic glands of the male cricket, Gryllus bimaculatus, was analyzed by using an in vitro assay for ecdysteroid release from the moulting glands in last instar nymphs as well as in adult animals, and correlated with light and transmission electron microscopy. Apoptosis was examined by the TUNEL-reaction. The ability to synthesize ecdysteroids reached a peak at the 8th day of the last larval instar, identified as the moulting peak. After adult ecdysis it decreased to barely measurable values. Prothoracic gland degeneration was initiated at the time of the moulting peak, characterized by TUNEL positive reactions, nuclear and cytoplasmatic condensation, a striking abundance of residual basal laminae; besides a great amount of autophagic vacuoles are observed. The results reveal that apoptosis and autophagy are the basic mechanisms for programmed cell death in the prothoracic gland of Gryllus bimaculatus.     

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.     

The architecture of the accessory reproductive glands of the male desert locust. 5: ultrastructure during maturation

The accessory reproductive glands of the male desert locust were studied with the electron microscope from the time of adult emergence until full sexual maturity was attained (10 days). Observations on the changes in the nucleus and the cytoplasm of the glandular epithelium, particularly those organelles involved in the elaboration of cell secretions, were made during the maturation of the accessory glands. Cells of gland 16 (the functional seminal vesicle) exhibit numerous ribosomal aggregates, have a concentration of mitochondria along their luminal surface, and show some secretory activity, even at the time of adult emergence. On the other hand, all other accessory glands show very little development of the secretory apparatus (rough-surfaced endoplasmic reticulum, polyribosomes, Golgi elements, elaborate mitochondria, etc.) until after the fifth adult day. These changes parallel changes in the secretory activity of the adult corpus allatum; and it is suggested that the corpus allatum hormone regulates the maturation of the truly glandular accessory glands of the locust, but not that of the functional vesicle.     

Histophysiological studies on the corpus allatum during prepupal diapause in Monema flavescens (Lepidoptera)

The appearance of the corpus allatum, the central endocrine gland of diapause, was examined histologically in the slug moth prepupae, Monema flavescens (Lepidoptera) Before beginning of diapause, the secretory cells of the corpus allatum increase in size with the enrichment of the cytoplasm in quantity and in number of organelles, while RNA synthesis also starts. As diapause progresses, the secretory cells have many large unstained vacuoles in the cytoplasm, which were demonstrated to contain some substances of lipoidal nature. It is not clear that the substance is juvenile hormone itself or a material related to juvenile hormone. Agranular ER is the most characteristic organelle connected with mitochondria and situated around the vacuoles during diapause. Electron micrographs show that agranular ER and mitochondria have an essential role for the production of juvenile hormone. The function of the corpus allatum during diapause and the relationship between juvenile hormone and diapause are discussed.     

Die Bildung spezifischer Proteine während der Metamorphose der Speicheldrüsen von Wilhelmia lineata (Simuliidae)

Histolysis of pupal salivary glands and their further development during metamorphosis of Wilhelmia lineata were investigated by light microscopy. After pupal-adult apolysis the distal parts of the glands degenerate. The adult gland develops from a ring of tightly packed very small cells with small nuclei at the anterior end of the degenerating pupal gland. The gland of the female pharate adult consists of a spherical accessory gland with small cells and oval nuclei and a little U-shaped main gland with larger cells and round nuclei. The two U-shaped lobes are orientated horizontally. The gland of the male pharate adult has the same shape and cellular structure as the accessory gland of the female. During further development the main gland becomes greatly elongated as the cells increase in size, the cells become flattened, and the secretion of saliva into the gland lumen can be seen. The glands are fully differentiated at the time of pupal-adult ecdysis. Soluble proteins from the one-day-old female pharate adult show a very similar pattern to those of pupal and male glands. With growth of the female main gland and secretion of saliva into the gland lumen increasing amounts of proteins, which have been characterized earlier (Poehling et al., 1976) occur in the gland. This could suggest a specific rôle of these proteins during bloodsucking in the adult females.     

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.     

Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori

The insect brain regulates the activity of the prothoracic glands to secrete ecdysteroids, which affect growth, molting, and metamorphosis. Here we report the identification of a novel prothoracicostatic factor and its receptor in the silkworm Bombyx mori. The prothoracicostatic factor purified from pupal brains of B. mori is a decapeptide with the conserved structure of an insect myosuppressin and thus named Bommo-myosuppressin. Bommo-myosuppressin dose dependently suppressed the cAMP level and inhibited ecdysteroidogenesis in the larval prothoracic glands at much lower concentrations than the prothoracicostatic peptide, the other prothoracicostatic factor reported previously. In vitro analyses using a prothoracic gland incubation method revealed that Bommo-myosuppressin and prothoracicostatic peptide regulate the prothoracic gland activity via different receptors. In situ hybridization and immunohistochemistry revealed the existence of Bommo-myosuppressin in the brain neurosecretory cells projecting to neurohemal organs in which it is stored. We also identified and functionally characterized a specific receptor for Bommo-myosuppressin and showed its high expression in the prothoracic glands. All these results suggest that Bommo-myosuppressin functions as a prothoracicostatic hormone and plays an important role in controlling insect development.     

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.     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

Cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach, Diploptera punctata

The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.     

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.