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.     

Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae

The prothoracic glands of the early last-instar larva of Mamestra brassicae (day 0–3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-1, day-2 and day-3) were given juvenile hormone 5–10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-1, day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain, presumably PTTH, and ecdysone released from the prothoracic glands themselves.     

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

Summary Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupalManduca sexta (Lepidoptera) and larvalSarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (ED₅₀) was 0.20 forManduca and 0.15 forSarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, whenM. sexta larval prothoracic glands were challenged byS. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.     

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.     

The role of juvenile hormone during larval-pupal transformation of Spodoptera littoralis: Switchover in the sensitivity of the prothoracic gland to juvenile hormone

Application of JHA to the final instar of Spodoptera littoralis larvae before they attain maximal body weight causes delay to or completely blocks the onset of metamorphosis, as indicated by the dorsal vessel exposure. When the same dose of JHA is administered to the larvae just after their attaining maximal body weight, at the time of the onset of gut-emptying, metamorphosis is substantially accelerated.By means of ligature experiments, it is shown that JHA has a direct effect on the prothoracic glands. It inhibits them at the beginning of the final instar and then stimulates them shortly before pupation. It seems that under normal conditions JH is necessary for pupal cuticle formation; those larvae which pupate without JH show adult characteristics.     

Inhibition of insect development by simultaneous action of prothoracic gland hormone and juvenile hormone

Injections of large amounts (around 20 μg) of ecdysterone and of Cecropia C₁₇-juvenile hormone or its bio-analogue 6,7-epoxygeranyl 3,4-methylendioxyphenyl ether into a pupa of Tenebrio molitor may inhibit the apolysis and cuticle secretion. Some of the treated insects secrete a new cuticle only locally, others do not even undergo apolysis although they survive until the controls ecdyse as adults. The development of internal organs is also hindered. These results are tentatively explained in terms of a mutually antagonistic action of the two hormones on nucleic acid synthesis.     

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.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects

Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone (rPTTH) from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or are inconsistent with, previous studies over the past 20years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of cAMP (adenosine 3':5' cyclic monophosphate) synthesis; requirement for extracellular Ca(2+) in in vitro studies; ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells in each brain hemisphere as the source of PTTH (the prothoracicotropes); the ability of antibodies to rPTTH to inhibit ecdysteroidogenesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.     

The effect of juvenile hormone on pupal pigmentation of Pieris brassicae L.

The role of juvenile hormone (JH) in the morphological colour adaptation of pupae of Pieris brassicae controlled by environmental factor was analyzed. First the effects of JH I and its analogue, Farnesyl-Methyl-Ether (FME) were tested. Secondly the JH-titres of the last instar larvae were measured under various light conditions which influence the future pigmentation of the pupae.During the sensitive period, which occurs before pupation, blue light (410 nm) produces the strongest, darkness medium, and yellow light (570 nm) the lightest pigmentation of the pupae.JH I as well as FME has an inhibiting effect on the formation of the black spots in the cuticle. However, this effect only becomes apparent (a) if the insects are kept under blue light during the sensitive period (which normally leads to a strong black pigmentation) and (b) only when these animals were treated with JH I or FME either at the beginning, or 10 hr after the beginning of the sensitive period. In the last larvae instar, JH could be found only during the sensitive period. Fourteen hours after the beginning of the sensitive period the JH concentration reaches a maximum of 30–100 pg JH per insect. The JH-titre resulting from blue light conditions is significantly different from those of the larvae kept under white or yellow light. An additional maximum of 60 pg JH per animal was found 8 hr/after the beginning of the sensitive period. Obviously, JH affects the process of pigmentation of the pupae, but the described results are not sufficient to explain thoroughly the regulation of pigmentation modified by environmental factors. The effects of further factors are discussed.     

Both prothoracicotropic hormone and an autocrine factor are involved in control of prothoracic gland ecdysteroidogenesis in Locusta migratoria and Schistocerca gregaria

In Bombyx mori, ecdysteroidogenesis by the prothoracic glands (PGs) is controlled by both prothoracicotropic hormone (PTTH) and a factor secreted by the glands themselves. This factor, which is active both in vitro and in vivo, has been named 'autocrine factor' (AF). To find out whether or not this dual control also exists in other species, in particular in hemimetabolous ones, we applied similar methods as were used to discover AF in Bombyx to the locusts Locusta migratoria and Schistocerca gregaria. Our results unequivocally show that locust PGs also secrete an as yet unidentified autocrine factor. Possible roles of AF are discussed.     

Mass fragmentographic analysis of juvenile hormone 1 levels during the last larval instar of Pieris brassicae

A method is presented here for routine analyses of juvenile hormone levels using coupled gas-liquid chromatography-mass fragmentography with chemical ionization. This method is sensitive and highly specific, but needs complex equipment. It has been used for an analysis of Pieris brassicae haemolymph during the last larval instar. Only juvenile hormone I was detected at significant levels (between less than 100 pg/ml and 6 ng/ml). Juvenile hormone I variations are far more complex than expected and show a discrete peak (1 ng/ml) at the time when pupal programming takes place. This finding is consistent with the ‘classical scheme’, where pupal moult occurs in the presence of reduced juvenile hormone levels.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

Identification of two nuclear polyhedrosis viruses from the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae)

Two nuclear polyhedrosis viruses from the cabbage moth Mamestra brassicae found in two geographical areas in Europe have been characterized and compared. These two virus isolates have similar biological activities and have the same host range. The two M. brassicae nuclear polyhedrosis viruses can be distinguished by restriction endonuclease analysis of their DNA. They appear to be distinct but related virus strains.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.     

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.     

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.