Ultrastructural study of the regressing prothoracic glands of blattarian insects

Summary The prothoracic glands, source of the molting hormone ecdysone, regress within a few days after the final molt, a process which was analyzed with electron microscopic methods in the cockroaches Leucophaea and Blaberus. This strictly timed event is accompanied by drastic alterations in cellular fine structure. Early signs of breakdown appear in groups of nuclei whose substance becomes segregated into patches of contrasting electron density characteristic of pyknosis.The most conspicuous change in the cytoplasm of parenchymal cells concerns the appearance of large, heterogeneous inclusion bodies in which various cellular elements become segregated. These compartments seem to represent autophagic vacuoles within which the gradual degradation of much of their contents takes place, presumably under the influence of lysosomal enzymes. Undigested swirls of membranous character may remain sequestered within these packets for some time.At advanced stages of cellular atrophy, plasma membranes and nuclear envelopes have gradually disappeared, and masses of protoplasm undergoing autolysis become invaded by a greater number of hemocytes than are present in nymphal glands. These phagocytic elements appear to engulf debris of parenchymal cells as well as some degenerating connective tissue elements. After the completion of the regressive process, the axial band of musculature characteristic of the nymphal gland persists on its own. Whether or not some parenchymal cells (or possibly their precursors) capable of reactivation persist in the proximity of this muscle is unknown.The resorption of the prothoracic gland in the newly emerged insect is the result of physiological autolysis and seems to be aided by the activity of phagocytic hemocytes.Dedicated to Professor W. Bargmann on his 60th birthday in friendship and admiration.This study was supported by Research Grants AM-03984, NB-02145 and NB-05219 from the U.S.P.H.S.I wish to express my thanks to Mrs. S. Wurzelmann, Mrs. C. Jones, Mrs. C. Grubman, and Mr. S. Brown for their excellent technical assistance.     

Autocrine activation of ecdysteroidogenesis in the prothoracic glands of the silkworm, Bombyx mori

Ecdysteroidogenesis in the prothoracic glands is activated by the neuropeptide, prothoracicotropic hormone (PTTH). The present study demonstrates autocrine activation of ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori. Using both a long-term in vitro organ culture system and an ecdysteroid radioimmunoassay, it was found that either decreasing the incubation volume, from 100 to 5 microl, or increasing the number of glands incubated per drop (50 microl) from 1 to 5 significantly increased ecdysteroid secretion. Prothoracic gland-conditioned medium was used to clarify the autocrine factor. The results showed that activation of ecdysteroidogenesis by the prothoracic gland-conditioned medium appeared to be dose dependent and a dramatic increase in ecdysteroid secretion was observed after 6h of incubation in the conditioned medium. Moreover, it appeared that autocrine activation occurred when glands were incubated in large volumes of incubation medium and during a short incubation period, indicating that the factor may exert its action in situ at some specific developmental stages. This tropic factor was further characterized, and it was found that the factor seemed to be heat-stable, with a molecular weight estimated to be between 1000 and 3000 Da. Injection of the concentrated putative autocrine factor into day 5 last instar larvae greatly increased ecdysteroidogenic activity of the prothoracic glands compared to those injected with saline, indicating the possible in vivo function of the present factor.     

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

The fine structure of blattarian prothoracic glands

Summary The ultrastructural characterization of the prothoracic glands derives interest from the fact that they are the only known source of a steroid hormone (ecdysone) among invertebrates. The present material includes nymphal glands of two blattarian species studied at graded intervals over a period of several intermolt cycles.Among the prominent cytological features of this organ are long and intricately interwoven cellular processes that may be separated from each other by extracellular channels with an average width of 0.5 . The plasma membranes of the cells facing these spaces are coated by an external lamina (boundary membrane) representing extensions of the same extracellular material that provides the connective tissue sheath of this and other insect organs. Micropinocytotic caveolae and vesicles present along this large cellular surface area may facilitate the transport of material between glandular cells and hemolymph, or vice versa.The cytoplasm contains a fairly moderate amount of membranous structures. Golgi units are distinctive but never spectacular; ergastoplasmic elements are sparse in most cells. This sparsity as well as the absence of membrane-bounded secretory granules is in line with the known steroidal nature of the secretory product involved. Some cells show a striking array of microtubules.Certain structural attributes of nucleoli, mitochondria, and lysosome-like bodies seem to vary in conjunction with distinct phases of the intermolt cycle.Dedicated to an inspiring teacher and loyal friend, Professor F. Wassermann on the occasion of his 80th birthday, August 13, 1964. — Supported by Research Grants AM-03984 and NB-02145 from the U.S.P.H.S. — I wish to thank Mrs. Cynthia Jones and Mr. Stanley Brown for their skillful assistance.     

Ultrastructure of active and inhibited prothoracic glands.

Changes in the cytoplasm of prothoracic gland cells were compared in pharate adults, dauer pupae, and aminophylline inhibited pupae of H. cecropia. For the first 3 to 4 days after transfer from 4 to 22°C, a similar sequence of changes in the cytoplasmic elements was observed. At day 4 the cytoplasm of pharate adults exhibited further differentiation which was consistent with the initiation of secretion, while dauer and inhibited pupae remained at the stage achieved at day 4 and did not advance further even after a substantial lapse of time. These results are interpreted as indicating that the early changes represent a response by the cells to the temperature change, while the initiation of secretion requires the intervention of brain hormone.     

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.     

Activation of prothoracic glands by brains in vitro

The effects of brains from both diapausing and non-diapausing Mamestra brassicae pupae on the prothoracic glands from pupae of the same condition were studied by observations of the morphological changes and bioassay of the prothoracic glands in vitro.It was ascertained that the active brains intensified the hormonal activity of prothoracic glands from younger diapausing pupae more than those from older pupae. Further, these results coincided with the fact that the prothoracic glands from brainless pupae were more difficult to activate by active brains the longer the time after the glands had been extirpated.The brains from both younger and older diapausing M. brassicae pupae were able to activate co-cultured inactive prothoracic glands in vitro. These results suggest that even the brain from diapausing pupae of M. brassicae can synthesize and release the prothoracic gland activating hormone in vitro.     

Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.     

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.     

Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands

Abstract The ichneumonid endoparasitoid Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) injects a polydnavirus when it oviposits into a host. We compared the development of Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae parasitized in the penultimate (fourth) stadium with those parasitized in the last (fifth) stadium by C.sonorensis and show that hosts stung in the fifth stadium exhibited arrested or delayed development compared to the controls. Parasitoids developed normally to the point of emergence in larvae stung in the fifth stadium but most did not successfully emerge from the host. The prothoracic glands in all successfully parasitized fifth stadium hosts and most unsuccessfully parasitized fifth stadium hosts showed some degree of virally-induced degeneration. Larvae stung in the fourth stadium developed more slowly than controls and either did not moult or developed to a fifth and sometimes a supernumerary sixth stadium before parasitoid emergence. Unsuccessfully parasitized hosts were delayed in their development but eventually moulted to the fifth and, in some cases, a supernumerary sixth stadium before pupating. Hosts stung in the fourth stadium showed no signs of prothoracic gland degeneration whether successfully parasitized or not. In addition, calyx fluid injections into early fourth stadium hosts did not cause prothoracic gland degeneration even after these hosts moulted to the fifth stadium, suggesting that degeneration induced by polydnavirus is specific to the last stadium of the host.     

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.     

Symbiont-mediated RNA interference in insects

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.     

Presence of sialic acid in prothoracic glands of Galleria mellonella (Lepidoptera)

The presence of sialic acid (SA) in prothoracic glands (PGs) of Galleria mellonella was determined by the methods of electron microscopy (EM), histochemistry, spectrophotometry (SP) and electronic ionization (EI)-mass spectroscopy. Histochemical observations were carried out by the cationic dye ruthenium red (RR), staining with and without neuraminidase digestion in the larval stage. Neuraminidase-sensitive SA was demonstrated by the decrease in the amount of RR-binding following neuraminidase digestion. The total amount of SA was found to be 0.09016 mg g(-1) in dry tissue by spectrophotometric determination. EI-mass spectroscopy results confirmed the EM and SP observations. The fragmentation scheme derived from EI-mass analysis exhibited the presence of the lactonized form of Neu5Gc7, 9Ac(2). On the basis of the various pieces of evidence described above, it was firmly concluded that Neu5Gc7, 9Ac(2) molecules were present in PGs of G. mellonella.     

Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro

Isolated prothoracic glands from Tenebrio larvae synthesize in vitro α-ecdysone, but not β-ecdysone from 4-¹⁴C-cholesterol. Isolated abdominal oenocytes from the larvae synthesize mainly β-ecdysone, but only little α-ecdysone. When prothoracic glands and oenocytes are cultured together, the α-ecdysone derived from the prothoracic glands is oxidized by the oenocytes to β-ecdysone. The newly synthesized hormones are not stored in the cells, but are secreted into the medium if sufficient amounts of non-labelled hormones are present. If no unlabelled hormones are added to the culture medium, the newly formed hormones are converted to a large extent into polar conjugates.