In eukaryotes, genomic DNA is wrapped around an octamer of the core histone proteins H2A, H2B, H3 and H4. Silkworms have been known to bear holocentric chromosomes and their regulatory mechanisms of chromatin remodeling remain unclear. We have cloned the silkworm canonical core histones and their variants. The H2A variants, H2AX and H2AZ, were highly conserved in the silkworm, whereas the fly has only one H2A variant, H2Av, which is a chimera of H2AX and H2AZ with the function of both molecules. In the silkworm, all histones except for H2AX were ubiquitously expressed in all tested tissues, and H2AX was expressed only in the genital organs. A subcellular localization analysis of the cloned histones using an EGFP fusion construction demonstrated that the behaviors of the histones are the same as those of genomic DNA throughout the cell cycle. The fluorescence intensities of H2AX and H3.3 were weaker than those of the other histones. The incorporation of these histones into chromatin might be restricted due to their specific function in DNA repair and transcription activity. There were two H3 variants, H3.2 and H3.3, in the silkworm; H3.1 was not present. Lysine 9 on H3 was methylated and acetylated in silkworms bearing holocentric chromosomes.

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We investigated the cocoon repair behavior of silkworms after cutting off a part of the cocoons under construction. The results showed that the time elapsed from the start of spinning to the cut-off has a great impact on the cocoon repair behavior. When the cocoon part was cut off at a later cocooning stage, the silkworms spun the cut-off area for a longer time and more intensively for repair. However, when the cocoon part was cut off at an early cocooning stage, the silkworms did not engage in cocoon repair. The intensity of repair also depended on the size of the cut-off area. The silkworms restored their normal spinning behavior after completing the repair. They could complete cocoon construction even when the cocoon part was cut off 6 h after gut purge.

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Protein prenyl transferase, that is, farnesyltransferase (FT) and geranylgeranyltransferase (GGT), catalyze the farnesylation or geranylgeranylation of proteins including GTP-binding proteins (G proteins). The post-translational modifications by prenyl groups are important for proper signal transduction by G proteins. We report the cloning and mRNA expression analysis of genes encoding the α-and β-subunit of a putative FT (Bmftα and Bmftβ) and β-subunit of GGT type I (Bmggt-Iβ) in the silkworm, Bombyx mori. These proteins have important motifs for enzymatic activity, suggesting that they are functional as FT/GGT in the silkworm. A phylogenetic analysis revealed that each of these genes constituted the same clade with D. melanogaster homologue in the phylogenetic tree. Developmental expression analysis by a quantitative reverse transcription-polymerase chain reaction clarified that Bmftα, Bmftβ and Bmggt-Iβ were expressed strongly towards adult emergence. Furthermore, all of these genes were expressed strongly in the ovaries of female adults, and especially for Bmggt-Iβ a strong expression was also detected in the Malpighian tubules of the larval stage. Our results suggest that proper signal transduction with factors modified by these enzymes is essential for the development or physiological regulation of these tissues in the silkworm.

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Twenty-two strains of silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), were investigated to determine their susceptibilities to an isolate of entomopathogenic fungus, Beauveria brongniartii (Sacc.). Most strains showed resistance to B. brongniartii, but some were extremely susceptible. When second instar larvae were infected by dipping in a conidial suspension of B. brongniartii (1.9×109 c.f.u./ml), the differences in susceptibility between the resistant strains and susceptible strains were large: mortality rate of strain Akako was 100% and that of strain Japanese No.1 was 11.0±9.0%. When fifth instar larvae were infected by injection of the conidial suspension, the maximum difference in LD50 values among the strains was about 500-fold. F1 progenies from the cross of the susceptible strain and the resistant strain were resistant, a half of the backcrossed hybrids to the susceptible parent strain was resistant and a half of them was susceptible. Those results indicate that the susceptible strain possesses a recessive gene conferring high susceptibility to B. brongniartii isolate. Silkworm strains may be useful model insects to study the mechanisms and genes related to the resistance of lepidopteran pests against entomopathogenic fungi.

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We examined the Bombyx mori (silkworm) white gene, Bmwh3, for its possible utility as a transgenesis marker in the B. mori Aojuku white-egg translucent mutant (w-3^ol) strain. To express wild-type Bmwh3 in the w-3^ol strain, we used the GAL4/UAS system. The A3-GAL4 strain used drives expression of the transgene under the control of the UAS throughout all tissues at all stages. Whereas wild-type silkworms have dark brown eggs, opaque white larval integuments, and dark brown eyes, the w-3^ol mutants have white eggs, translucent larval integuments, and light brown eyes. Transgenic expression of Bmwh3 in w-3^ol mutant silkworms restored the morphology of the larval integument by increasing the uric acid content to near normal and partially restored the dark brown phenotype of the compound eye by slightly increasing the accumulation of ommochrome pigment. Eggs transgenically expressing Bmwh3 were brown/white mosaics. Thus, when linked to a strong promoter, Bmwh3 is likely to be a useful marker for transgenic screening of B. mori.

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In 2005, natural epizootics were observed during an outbreak of Setothosea asigna (Lepidoptera: Limacodidae) larvae in an oil palm plantation in South Sumatra, Indonesia. The causative agent was filterable, which implied it was a virus. Since preliminary testing using reverse PCR gave a positive result for the Thosea asigna virus (TaV), we experimented with the purification of the viral agent from the diseased larvae. Electron microscopy revealed nonenveloped virus-like particles that were spherical in shape and about 40nm in diameter. cDNA cloning followed by sequencing demonstrated that RNA purified from the particles contained two large open-reading-frames (ORFs) with a partly shared sequence and extensive homology (>98% identity at the nucleotide level) with ORFs of TaV encoding an RNA-dependent RNA polymerase and the capsid protein, respectively. 3’ RACE suggested that there is, like in TaV genomic RNA, no poly(A) tract at the 3’-terminus of the RNA. The pathogenicity of the purified particles against Limacodidae larvae in Japan was demonstrated to be very strong for Monema flavescens and Austrapoda dentata. These results indicated that the agent causing the epizootic disease among S. asigna larvae in the oil palm plantations was TaV which also has potential as a biological control agent for Limacodidae pests in Japan.

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Analysis of cDNA encoding the double-stranded ribonuclease (Bm-dsRNase) of the silkworm, Bombyx mori, elucidated a possible conclusion that the 41kDa mature dsRNase is produced from the 45kDa precursor protein in the middle midgut tissue by post-translational processing. To strengthen this speculation, the 41kDa protein was produced in vitro: the 43kDa-45kDa protein-rich fraction was partially purified by gel filtration and then treated with bovine trypsin. Specific conversion of the 43kDa and 45kDa proteins into the enzymically-active 41kDa protein was demonstrated by a Western blot analysis and dsRNase activity assay, strongly supporting that the 41kDa Bm-dsRNase is produced in vivo by the limited proteolysis that was induced by an endogenous trypsin-like enzyme, and immediately released into midgut lumen.

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