Noriaki Agui

Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombyx mori

Abstract In the silkworm, Bombyx mori, apolysis of the spiracle is the first visible sign of the initiation of a larval moult. After spiracular apolysis, the characteristic sequence of new spiracle formation can be recognized easily through the cuticle around the old spiracle. This sequence, called the spiracle index, was used as the basis for a precise developmental chronology of the larval moulting period. At certain spiracle-index stages, histological changes in the larval ventral abdominal integument were also examined. By allatectomy and abdominal ligations, the critical periods for the secretion of juvenile hormone and ecdysone respectively were found to be at the initiation of spiracular apolysis and at the time when general apolysis was complete. The haemolymph-ecdysteroid level as measured by radioimmunoassay was about 30–50 ng/ml during the first 2 days of the 4th instar, 60–70 ng/ml at the time of initiation of spiracular apolysis, and 200 ng/ml at the time of completion of apolysis of the general body surface. The maximal level of the hormone was about 290 ng/ml at the mid-moulting period. The relationship between the ecdysteroid titre and morphological events occurring during the larval moult was discussed.

The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release

When the parasitoid Apanteles kariyai laid eggs into host Pseudaletia separata larvae, before prothoracicotropic hormone (PTTH) was released in the last instar preparatory to metamorphosis, the host did not pupate and the larvae of the wasps emerged. The ecdysteroid titer of unparasitized intact larvae increased up to 1 microgram/ml 1 day before pupation, whereas the titer of parasitized larvae was maintained at a low level without the surge. Isolated prothoracic glands from intact larvae synthesized much more ecdysone than those of parasitized larvae both in vivo and in vitro. Administration of exogenous PTTH caused the activation of the prothoracic glands seen during parasitization. Injection of 20-hydroxyecdysone (20-HE) into the parasitized larvae caused by hosts pupation, but did not affect the development of the wasp larvae. However, the sensitivity of the integument to 20-HE was lower in parasitized than in unparasitized larvae. Injection of a mixture of adult wasp calyx and venom fluids into last instar unparasitized larvae delayed their pupation, suggesting that calyx and venom fluids are factors contributing to disturbance of the normal function of brain-prothoracic gland system. These results show that parasitization inhibits secretion and/or synthesis of PTTH and also delays the larval-pupal commitment of the integument by keeping the ecdysteroid level low.

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Abstract Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.

In vitro activation of neurosecretory brain cells in Mamestra brassicae by β-ecdysone

The effect of ecdysone or of the prothoracic gland on the neurosecretory cells of brains taken from diapausing pupae of the cabbage armyworm, Mamestra brassicae L., was investigated histologically and functionally in vitro. When the diapausing brain was cultured in a medium containing β-ecdysone, the neurosecretory cells of the median group I in the pars intercerebralis increased staining intensity more than those of brains cultured in medium alone. Furthermore, when the prothoracotropic activity of a brain treated with β-ecdysone was compared with that of an untreated brain in vitro, the brain treated with hormone showed higher activity than the untreated one. These results suggest that β-ecdysone acts on the neurosecretory cells of pupal brain in M. brassicae as a positive feedback regulator.

Larval-pupal transformation of the prothoracic glands of Mamestra brassicae

Abstract 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.

Activation of prothoracic glands by brains in vitro

Abstract 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 relationship between nutrition, vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

Abstract The effect of adult nutrition on oogenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.

Ecdysone as a feedback regulator for the neurosecretory brain cells in Mamestra brassicae

Abstract The effect of β-ecdysone injection or prothoracic gland removal on the neurosecretory brain cells in the cabbage armyworm, Mamestra brassicae was investigated. The prothoracic gland removal of the 6-day-old last instar larvae induced the release of stainable neurosecretory material from type II cells in the pars intercerebralis 24 hr after treatment. The injection of β-ecdysone into the prothoracic gland-extirpared larva stimulated both the synthesis of stainable material in the type II cells and transportation of stainable material from neurosecretory cells to axon tracts. These results suggest that ecdysone may have a role as a positive feedback regulator for the synthesis and release of prothoracicotropic hormone in M. brassicae brain.

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

Abstract The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.

Hormonal control of vitellogenin mRNA levels in the male and female housefly, Musca domestica

Abstract The temporal changes and hormonal control of vitellogenin gene transcript (vitellogenin mRNA) levels in the female and male housefly, Musca domestica, were investigated during the first cycle of oogenesis by Northern blot analysis using housefly vitellogenin cDNA as a probe. The vitellogenin mRNA in the ovary and fat body in female flies accumulated in a stage-dependent fashion in parallel with the change of vitellogenin concentration in the haemolymph during egg formation. No vitellogenin mRNA was detected in the fat body of male flies. The accumulation of vitellogenin mRNA in the decapitated females of autogenous strain (in which vitellogenesis had been prevented by decapitation at the pre-vitellogenic stage) was accelerated by administration of 20-hydroxyecdysone in a dose-dependent manner. The male flies of the same strain, however, were about 100 times less sensitive to 20-hydroxyecdysone than the female flies. The time at which 20-hydroxyecdysone stimulates the vitellogenin gene was slower in males than in females. Furthermore, 20-hydroxyecdysone activated the vitellogenin gene not only in the fat body of males and females but also in the ovary of females. The vitellogenin gene expression in male flies was not affected by any dose of juvenile hormone analogue (methoprene). The female flies, however, responded to juvenile hormone analogue, though its efficacy on the induction of vitellogenin gene expression was lower and the lag time for induction was longer than those observed with the 20-hydroxyecdysone-treated female flies. The response of the ovary to juvenile hormone analogue was much stronger than that of the fat body. These results clearly indicate that the accumulation of vitellogenin mRNA in the housefly takes place in a sex-, stage- and tissue-specific fashion during the normal gonotropic cycle, and that the effect of ecdysteroid is more pronounced than that of juvenile hormone on the induction of vitellogenin gene transcription.