Eclosion hormone activity in developing embryos of the silkworm, Bombyx mori

The ultimate timing of hatching in the silkworm, Bombyx mori, is controlled by a circadian oscillator. The presence of eclosion hormone in developing embryos of the silkworm is demonstrated. Eclosion hormone activity first becomes detectable in embryos which have developed almost to the stage of the differentiation of the neuroendocrine system. Hormonal activity increases sharply to a maximum level 1 day before hatching and falls by about a half in the newly hatched larvae. Eclosion hormone was partially purified from the pharate first-instar larvae and approx, a 2100-fold purification was achieved. The molecular weight of the embryo eclosion hormone is estimated to be 7000 ～ 9000 Daltons by gel-filtration on Sephadex G-50 (superfine). The role of eclosion hormone on hatching behaviour of the silkworm, Bombyx mori, is discussed.     

Nutritive effects of d-amino acids on the silkworm, Bombyx mori

Nutritive effects of d-amino acids on the silkworm, Bombyx mori, were investigated by growth experiments using defined diets and also by analysis of free amino acids in the larval haemolymph. None of the d-forms of the usual ten essential amino acids could be utilized effectively, although d-methionine was utilized in lieu of the l-form only to a limited extent and d-histidine gave a positive but smaller effect than d-methionine. d-Proline, its l-form being semi-essential for the silkworm, was not utilized. d-Leucine, and to a lesser extent d-alanine and d-serine, were found to be somewhat toxic. Comparison of free amino acid patterns in the haemolymph of the fifth-instar larvae, which fed on diets either lacking l-forms of histidine, methionine and leucine singly or including the d-forms singly in place of these l-forms, supported the results of the growth experiments.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

Temporal organization of endocrine events underlying larval-larval ecdysis in the silkworm, Bombyx mori

The timing of ecdysis in the penultimate instar of Bombyx mori was demonstrated to be under the control of a circadian clock. The temporal organization of secretion of prothoracicotropic hormone (PTTH), ecdysone and juvenile hormone was studied with particular attention to the circadian control of the timing of hormone release. PTTH release occurs, at least, in the second and third night. The latter is responsible for evoking the larval ecdysis. Prothoracic gland initiates ecdysone secretion abruptly with a very short span after the second PTTH release and secrete enough amount of ecdysone for larval moulting, which takes place 11 h later. Juvenile hormone titer is relatively high before the second PTTH release and corpus allatum becomes dispensable for ensuring the larval moulting in 1.5 h. Based on these findings, interpretations for the endocrine system underlying precocious pupation and formation of intermediates, which are produced by neck ligation, are presented.     

Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth,haemolymph components and uric acid excretion in the silkworm, Bombyx mori

Effects of various kinds of dietary protein on growth of the silkworm, Bombyx mori, were determined using semi-synthetic diets. Also, the ingestion, digestion and utilization of dry matter and of nitrogen were measured. Nutritive effects of dietary proteins and supplementation of limiting amino acids on haemolymph protein and amino acids pattern were also investigated. Larval growth was largely dependent on the dietary proteins. When the larvae were reared on a diet containing weakly nutritive proteins such as gluten and zein, haemolymph protein was decreased and uric acid excretion was markedly accelerated. The free amino acid composition of the haemolymph manifested characteristic patterns according to the kinds of dietary protein.The supplementation of gluten and zein with their limiting amino acids resulted in a rise of haemolymph protein and a drop in uric acid excretion. The amino acid patterns in the haemolymph were greatly changed according to supplementation.     

Molecular and functional characterization of adipokinetic hormone receptor and its peptide ligands in Bombyx mori

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides, but its signaling pathways remain to be elucidated. In this study, we molecularly characterized the signaling of Bombyx AKH receptor (AKHR) and its peptide ligands in HEK293 cells. In HEK293 cells stably expressing AKHR, AKH1 stimulation not only led to a ligand concentration dependent mobilization of intracellular Ca²⁺ and cAMP accumulation, but also elicited transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We observed that AKH receptor was rapidly internalized after AKH1 stimulation. We further demonstrated that AKH2 exhibited high activities in cAMP accumulation and ERK1/2 activation on AKHR comparable to AKH1, whereas AKH3 was much less effective.     

Temporal organization of endocrine events underlying larval-pupal metamorphosis in the silkworm, Bombyx mori

The temporal organization of endocrine events underlying larval-pupal moult was examined. After the phagoperiod of 6–7 days, the last instar larvae of Bombyx mori purged the gut contents and then pupated 4–5 days thereafter. Juvenile hormone titer was considerably high on the first day of the fifth instar, but declined to an undetectable level on day 3. Release of prothoracicotropic hormone (PTTH) began after the decline in the junvile hormone titer and completed during the night of day 4. Prothoracic glands began to secrete ecdysone on day 5. Larvae could pupate on schedule if their brains were removed after the PTTH release. One time of PTTH release during the feeding stage could satisfy the requirement for pupation occuring on schedule. The phagoperiod could be shortened by allatectomy early in the fifth instar and prolonged by the injection of a certain dose of juvenile hormone analogue before the gated PTTH release, accounting for the role of juvenile hormone in the timing of PTTH release.     

In vitro diapause substance in the silkworm, Bombyx mori

By means of in vitro studies, in which isolated suboesophageal ganglions of the Bombyx silkworm were cultured, it was shown that at least two kinds of substances are biosynthesized and exert independent effects on determination of diapause or non-diapause in silkworm eggs. They are referred to as the diapause and non-diapause substance, respectively. Whether diapause or non-diapause eggs are laid may depend upon the different quality of these substances.     

The acceleration of apolysis by the brain hormone in the silkworm, Bombyx mori

The rôle of the brain hormone as an activation hormone of the prothoracic glands in silkworm larvae was examined using decapitated larva of the fourth instar. The gland was activated by the brain hormone injected and induced early larval or larval-pupal apolysis.     

In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria

Both adipokinetic hormone and octopamine have a stimulating effect on lipid release from locust fat body in vitro, when incubated in diluted haemolymph. The presence of adipokinetic hormone results in the formation of the flight-specific haemolymph lipoprotein A⁺ accepting the increased amount of lipids released into the incubation medium. In contrast, interconversions of lipoproteins do not occur when octopamine is added to the incubation medium, which is in line with the expectations: the lipid-mobilizing effect of octopamine is a limited and short-term effect. When fat body tissue is incubated with isolated haemolymph protein fractions, the lipid-mobilizing effect of adipokinetic hormone only occurs when the incubation medium contains both lipoprotein, A_y and protein fraction C, resulting in the formation of lipoprotein A⁺. In similar control incubations with the hormone omitted, some lipoprotein A⁺ is also formed (concomitant with a slight amount of lipid released), though significantly less than in incubations with hormone. Besides a stimulating function on lipolytic processes in the fat body, adipokinetic hormone is suggested to influence haemolymph lipoprotein rearrangement. A possible counteracting function of another factor in the haemolymph is discussed.     

Improved strength of silk fibers in Bombyx mori trimolters induced by an anti-juvenile hormone compound

Background

Bombyx mori silk fibers with thin diameters have advantages of lightness and crease-resistance. Many studies have used anti-juvenile hormones to induce trimolters in order to generate thin silk; however, there has been comparatively little analysis of the morphology, structure and mechanical properties of trimolter silk.

Changes in ecdysteroid and juvenile hormone levels in developing eggs of Bombyx mori

It is well-known that insect eggs can contain very high concentrations of ecdysteroids, which undergo drastic changes during embryogenesis. We found that this is equally valid for juvenile hormones. Three juvenile hormone-immunoreactive compounds were observed in developing Bombyx mori eggs. They were assumed to be juvenile hormones 1, 2 and 3 according to their retention time in HPLC. These hormones underwent drastic and sudden changes. In the space of one day their concentration was seen to rise rapidly from an undetectable level up to as high as 4 × 10^?6 micromoles per mg of eggs. Their presence was detected as early as the first day of embryonic development, as well as during the blastokinesis period (day 5 to day 9) and in late embryos (day 12 to day 14). Their relative concentrations varied greatly. On two occasions, day 1 and day 8, all three hormones were simultaneously present. Moreover, juvenile hormone 3 was present during the blastokinesis period, either alone or in combination with hormone 2. The latter was the only hormone present in late embryos, before hatching. Thus, with regard to both ecdysteroids (ecdysone and 20-hydroxyecdysone) and juvenile hormones, each day of embryonic development displayed a different hormonal pattern. These patterns undoubtedly constitute a “hormonal code” of embryogenesis control. While 20-hydroxyecdysone can be assumed to trigger cuticulogenesis in embryos as it does in larvae, the effects of the other hormones as well as their possible interactions are questionable.     

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca-corpora allata (CC-CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808-818]. The in situ hybridization analyses in the CC-CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC-CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize JH.     

Releasing signals of oviposition behaviour in Bombyx mori

The oviposition behaviour of Bombyx mori was studied by eliminating the sensory input received by the tactile setae of the anal papillae.It is shown that the removal of mechanical stimuli of the setae affects oviposition and its time course to some extent. In comparing this experimental result with the theory of Lorenz, summarized in the ‘hydromechanic model’, it is postulated that the sensory information from the mechanoreceptive setae surrounding the ovipositor is a key stimulus which releases the accumulated action-specific energy (‘drive’) and triggers and controls the organs involved in the behaviour.     

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.     

The control of chitinase activity by ecdysterone in larvae of Bombyx mori

The chitinase activity in the abdominal body walls of isolated abdomens of Bombyx mori larvae was examined.The activity in the abdomen just after ligation, decreased with the lapse of time, but increased by a fivefold factor whenever ecdysterone was injected into the abdomen at a sufficient dose to induce ecdysis. The increase in chitinase activity appeared some 9 hr after hormone injection. The increase in the enzyme activity was not blocked completely by actinomycin D and puromycin, and the rate of syntheses of RNA and protein in the body walls was not found to rise before the increase of chitinase activity.     

Vitellogenin titre in haemolymph of Locusta migratoria in normal adults,after ovariectomy,and in response to methoprene

Vitellogenin in the haemolymph of Locusta migratoria was assayed by rocket immunoelectrophoresis to elucidate aspects of its regulation. In many normal adult females, vitellogenin first appeared on days 5–9, rose quickly to peak levels, and declined before a second vitellogenic cycle; in others, it appeared later and built up more slowly. The timing of first appearance of vitellogenin, and proportions of early and late-developing individuals, differed markedly in groups from the same colony assayed in different years, suggesting effects of both genetic and environmental variation. Average peak levels of vitellogenin were 25–30 mg/ml. After ovariectomy, vitellogenin appeared near the normal time and increased for several weeks to about 300 mg/ml; haemolymph volume also increased greatly, so that the total haemolymph-vitellogenin pool reached about 300 mg/individual, or 100 times the normal amount. After ovariectomy, no cyclicity of vitellogenin accumulation was apparent. These results show that the ovary is not required for stimulation of vitellogenin synthesis, and suggest that normal cycling may depend on inhibition by the mature ovary. Females treated with ethoxyprecocene on day 1 of adult life to inactivate the corpora allata did not produce vitellogenin, but were induced to do so with the juvenile hormone analogue, methoprene. After injection of 150 μg of methoprene in mineral oil, there was one day lag, then vitellogenin increased in the haemolymph to the normal peak level and declined slowly to zero during 5 weeks; after a second injection of methoprene, vitellogenin re-appeared more rapidly, with less lag, reflecting accelerated secondary hormonal stimulation of vitellogenin synthesis in the fat body. Adult males showed no detectable haemolymph vitellogenin even after injection of large doses of methoprene.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Properties of flacherie virus of the silkworm, Bombyx mori

The flacherie virus of the silkworm, Bombyx mori, was isolated from infected larvae reared under aseptic conditions. Two types of infectious particles, tentatively designated FVS I and FVS II, were separated by density gradient centrifugation. Some properties of the separated particles were investigated. Electron micrographs showed that FVS I and FVS II were spherical particles with diameters of 27 ± 2 nm and 22 ± 2 nm, respectively. The sedimentation coefficients of FVS I and FVS II were 180 S and 134 S, respectively. It was concluded from experiments of incorporation of ³H-uracil inoculated into diseased larvae at late stage of flacherie disease that the nucleic acid of FVS II was RNA. The two types of particles were present in Sakaki and Wadayama strains of flacherie virus.