Hajime Fugo

Basic pattern of fluctuation in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm, Bombyx mori

General features of the changes in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm Bombyx mori were analyzed by comparing the patterns of the titer changes between different races and between silkworms reared under different environmental conditions. In common to all types of the silkworms tested, we observed low PTTH titers during the phagoperiod of the final instar, a small rise in PTTH titer on the day before wandering, two middle-sized peaks of the titer at the wandering and prepupal stages, high PTTH titers during early pupal-adult development, and a gradual titer increase shortly before adult eclosion. Increases in hemolymph PTTH titer were closely correlated with increases in ecdysteroid titers and with subsequent occurrences of morphological and behavioral changes characteristic of the initiation or progression of metamorphosis. The timing of the increase in hemolymph PTTH titer on the day of wandering was photoperiodically controlled, but that timing at the later stages seemed not to be influenced by the light-dark cycle.

Identification of the Putative Protein Phosphatase Gene PTC1 as a Virulence-Related Gene Using a Silkworm Model of Candida albicans Infection

ABSTRACT Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.

Neurohormones in developing embryos of the silkworm, Bombyx mori: The presence and characteristics of prothoracicotropic hormone-S

Abstract The presence of prothoracicotropic hormone (PTTH) activity is shown in developing embryos of the silkworm, Bombyx mori. We isolated a form of PTTH which was effective in eliciting adult development of brainless pupae of Samia cynthia ricini, while it had no effect on brainless Bombyx pupae. This hormonal activity increased with age in non-diapause eggs, but was also present in diapause eggs. The hormone was partially purified from pharate first-instar larvae and approximately a 75-fold purification was achieved. The molecular weight of the hormone was estimated to be 4,000–5,000 Daltons by gel-filtration on Sephadex G-50. The hormone was stable to heat treatment (100°C for 10 min), but hormonal activity was completely destroyed by treatment with pronase, trypsin and α-chymotrypsin indicating the peptide nature of the material.

Eclosion hormone of the silkworm Bombyx mori. Expression in Escherichia coli and location of disulfide bonds.

A gene encoding eclosion hormone (EH) from the silkworm, Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH. Sequence analysis of cystine‐containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule. Evidence was also obtained that the 6 residues at the NH2‐terminal are dispensable but 4 residues at the COOH‐terminal play an important role in EH activity.

Eclosion hormone of the silkworm Bombyx mori: Purification and determination of the N-terminal amino acid sequence

Abstract Eclosion hormone (EH) was purified from 180,000 pupal heads of the silkworm, Bombyx mori , to a homogeneous state by a 16-step purification procedure, which consisted of heat treatment, fractional precipitations, gel-filtrations, ion-exchange chromatography, hydrophobic chromatography and high performance liquid chromatography. Approximately 2,000,000-fold purification was attained to obtain 1.2 nmol (10 μg) EH with an overall yield of 3.3%. EH activity was measured by its ability to elicit precocious eclosion behavior in a pharate adult Bombyx . The purified EH was active at a dose of 0.83 ng. By using an automated gas-phase sequencer, the sequence of the 13 N-terminal amino acid residues was determined to be: H-Ser-Pro-Ala-Ile-Ala-Ser-Ser-Tyr-Asp-Ala-Met-Glu-Ile.

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

Induction of dauer larvae by application of fenoxycarb early in the 5th instar of the silkworm, Bombyx mori

Fenoxycarb application from 0 h until 60 h of the 5th instar of Bombyx mori induced 100% dauer larvae. Between the 60 and 78 h, the ratio of fenoxycarb-induced dauer larvae decreased, and the ratio of supernumerary instar moulting larvae increased. After application of fenoxycarb at the 48 h of the 5th instar, the haemolymph prothoracicotropic hormone (PTTH) titer was higher in fenoxycarb-treated larvae than in control larvae. Furthermore, brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes from fenoxycarb-treated larvae released higher amounts of PTTH in vitro than the Br-CC-CA complexes of control larvae. Prothoracic glands (PGs) of fenoxycarb-treated larvae at the 48 h of the 5th instar exhibited basal and PTTH-stimulated secretory activities similar to that of control PGs until the 72 h of the 5th instar. After that time point, both basal and PTTH-stimulated secretory activity of PGs from fenoxycarb-treated larvae significantly decreased and remained low for the rest of the investigated period. The combined results suggest that the application of fenoxycarb affects the ability of the PGs to be stimulated by PTTH and the induction of dauer larvae in Bombyx mori is not due to inhibition of PTTH release from Br-CC-CA complexes.

Apoptosis and Adhesion of Hemocytes During Molting Stage of Silkworm, Bombyx mori

Abstract To clarify the regulatory mechanism of the rapid changes in the hemocyte density in the silkworm, Bombyx mori, during ecdysis, we evaluated the relationship between the hemocyte density and the incidence of apoptosis during this stage. We also evaluated the role of the sugar chains on the adhesion of hemocytes by analyzing the effects on the hemocyte density of the injection of enzymes that cut sugar chains and monosaccharides into the body cavity. The hemocyte density was increased in the molting stage and spinning, and then decreased after the ecdysis. During spinning, the diameter of the granulocytes markedly increased, in which fatty granules in the cytoplasm increased, becoming foamy. They were identified to be apoptotic hemocytes using the Hoechst staining and the Comet assay. The decrease in the hemocyte density during spinning was mainly caused by the apoptosis of granulocytes. Next, we focused on the fluctuation of hemocyte density during the molting stage. Examination of the changes in the hemocyte density induced by injecting glycoside hydrolases, neuraminidase, sialic acid, or monosaccharides into the body cavity during the fourth molt stage and the third day in fifth instar larva demonstrated that the alteration of hemocyte density was regulated by the attachment and detachment of hemocytes via a selectin ligand, sugar chains. As with the injection of glycoside hydrolase, neuraminidase, sialic acid and fucose raised the hemocyte detachment, and it was assumed that the selectin ligands include the sialyl Lewis x like sugar chains, the same as mammalian lymphocytes.

Involvement of Calcium, Inositol-1,4,5 Trisphosphate and Diacylglycerol in the Prothoracicotropic Hormone-Stimulated Ecdysteroid Synthesis and Secretion in the Prothoracic Glands of Bombyx mori

Abstract The objective of this study was to determine which intracellular second messenger systems are activated by prothoracicotropic hormone in the prothoracic glands (PGs) of Bombyx mori. Recombinant prothoracicotropic hormone (rPTTH) could stimulate ecdysteroid synthesis and secretion from day 6 PGs of the 5th instar of Bombyx mori within 30 min of in vitro incubation. However, rPTTH did not stimulate any increases in the glandular content of inositol 1,4,5-trisphosphate and cAMP during this short incubation period. Extracellular Ca2+ influenced the basal and rPTTH-stimulated ecdysteroid synthesis and release in a dose-dependent manner. The L-type Ca2+ channel antagonist, nitrendipine, inhibited the rPTTH-stimulated ecdysteroid synthesis and secretion (IC50 ∼28 μM). The phospholipase C inhibitor, 2-nitro-4-carboxyphenylN, N-diphenylcarbamate, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50 ∼19 μM). The protein kinase C inhibitor, chelerythrine chloride, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50∼14 μM). The protein kinase C activator, phorbol-12-myristate 13-acetate (PMA), could stimulate basal ecdysteroid synthesis and secretion (EC50∼1 μM) and its inactive α-isomer (4 α-PMA) was ineffective. The combined results suggest that the PTTH-stimulated ecdysteroid synthesis and release in the PGs of Bombyx is dependent on extracellular Ca2+ and the bifurcating second messenger signalling cascade of inositol 1,4,5-trisphosphate and diacylglycerol.

Disturbance of adult eclosion by fenoxycarb in the silkworm, Bombyx mori.

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.