Carbohydrates, proteins and lipids play an important role in the overall physiology of insects. Here, we describe the lipid distribution in internal organs by using fluorescence lipid, which shows that more lipids were accumulated in the ovary than in the fat body and cuticle. On the other hand, protein accumulation in pupal developing ovary was resolved by SDS-PAGE, showing that vital proteins like vitellin, 30kDa, egg specific protein and lipophorin were gradually accumulated in the ovary. Furthermore, well-differentiated ovariole was observed in day 4 pupal ovary, and we examined the lipophorin uptake by using the fluorescence immunolocalization technique. Lipophorin was present in nurse cells and follicle cells and marginally with perioocyte space in immature ovariole. In the mature ovariole, nurse cell was narcotized and lipophorin appeared in follicular cell. Altogether, present findings illustrate that lipids and proteins gradually accumulated in the developing ovary. But lipophorin was endocytic uptake in immature ovariole, after the maturation uptake halted.

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Nucleopolyhedroviruses (NPVs) of saturniid wild silkworms are important in both insect pathology and biotechnology, because they cause severe damage to silk production in Asian countries and are also utilized as expression vectors for the production of valuable proteins in wild silkworms with larger body sizes than the domesticated silkworm, Bombyx mori. In this study, partial genomic DNA sequences of the Antheraea yamamai NPV (AnyaNPV) were determined and compared with the corresponding sequences of the A. pernyi NPV (AnpeNPV) and Samia ricini NPV (SariNPV). Their sequences were highly homologous, suggesting that they might be variants derived from a common ancestral NPV. They also shared large deletions in the coding region of the ecdysteroid UDP-glucosyltransferase gene (egt), although a clone of SariNPV possessed a full-length egt. The 4th instar larvae of S. ricini infected with egt-deleted NPVs died earlier than those infected with a cloned SariNPV carrying the full-length egt, suggesting that the egt deletions reduce production of polyhedral occlusion bodies (POBs) in the infected insects. In addition, SariNPV possessed large deletions in the contiguous cathepsin and chitinase gene region (v-cath~chiA), and its infection to S. ricini larvae did not cause obvious liquefaction of the larval bodies as well as burst release of POBs, suggesting that the v-cath~chiA deletions reduce horizontal transmission in the insect population. These results seem to indicate that the deletions detected in the egt and/or v-cath~chiA regions of the saturniid NPVs may not be significantly disadvantageous and have some good reasons, at least, in their infection cycles under the rearing conditions of their hosts.

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We analyzed 7708 714-bp COI nucleotide sequences from 101 populations of Japanese Bombyx mandarina, and identified three highly variable single nucleotide polymorphism (SNP) sites, SNPs156, 325, and 666, to enable the classification of these populations. At SNP325, two alleles, SNP325-C and -T, were exclusively distributed; C was nearly fixed in the eastern part of the Japanese archipelago, whereas T was fixed in the western part of the archipelago; no SNP325-C was observed in this region. The northern part of Kyoto and Hyogo Prefectures were in the boundary between these two regions, that is, SNP325-C and -T segregated in populations distributed in this region. SNP156 divided the population in the Tohoku district into two, one in the north and the other in the south; the population in the region spanning the north to the middle of Yamagata and Miyagi Prefectures carried exclusively SNP156-T, which was nearly fixed in this region. This trend was also observed in the southwestern part of Hokkaido. In contrast, SNP156-C was nearly fixed in other areas. SNP666-A was nearly fixed in the two Sanin populations, Daisen and GotsuIzumo. Some other populations in the western area showed segregation of SNP666-G and A. Consequently, Japanese B. mandarina populations were virtually divided into four divisions by COI haplotypes. These results strongly suggest the phylogeographic history of B. mandarina in the Japanese archipelago during and after the last glacial maximum.

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The Bombyx mori nucleopolyhedrovirus (BmNPV) La strain is a variant isolated in Laos (Iwashita, 1993). Although DNA fingerprinting analysis revealed nucleotide sequence variations in the genomes of BmNPV La and T3 (the type strain of BmNPV), other features of La, including virulence and growth properties, are unknown. Here we compared the pathogenicity and replication of La and T3 in B. mori larvae and cultured cells. Larval bioassays revealed that the median lethal dose of La BV was approximately 2-fold lower compared with that of T3. We found that La induced earlier host liquefaction compared with T3, and the median lethality time of La was approximately 6h shorter compared with that of T3. Further, La-infected B. mori larvae released more occlusion bodies (OBs) into the hemolymph compared with those infected with T3. These results show that the virulence of La is higher compared with that of T3. Moreover, La may be suitable for use in a BmNPV-based expression vector system, because La produced approximately 4-fold and 1.75-fold higher levels of polyhedrin mRNA and protein, respectively, compared with T3. Moreover, we found that multicapsid occlusion-derived viruses (ODVs) were more numerous in La-infected cells compared with those in T3-infected cells. However, there was no significant difference in the median lethal oral dose. Together, these results show that La exhibits unique features that mediate pathogenesis, production of the Polyhedrin protein, and the formation of multicapsid ODVs.

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Ishigameyoh (gap) was identified as a spontaneous mutant of Bombyx mori with an apterous and sterile phenotype. Phenotypic observations revealed that the gap mutation affected the development of imaginal discs and the primordia of adult organs, including the wings, legs, compound eyes, and reproductive organs. Therefore, Ishigameyoh (gap) is a metamorphosis-defective mutant. We found that oogenesis depends on the gap gene, whereas spermatogenesis is independent. Ovary transplantation experiments suggest a cell-autonomous requirement for the gap gene in the process of oogenesis. The gap mutation has been maintained by crossing +^gap/gap moths as the u90 strain. However, the u90 strain is difficult to maintain because +^gap/+^gap and +^gap/gap moths cannot be distinguished phenotypically. For efficacious maintenance, we established a molecular marker that is closely linked to the gap locus. First, we localized the gap locus to chromosome 5 using phenotypic markers. Second, the gap locus was narrowed down to within a 1-Mb region using PCR-based markers. Within this 1-Mb region, we finally established a PCR-based marker that amplified different-sized products from +^gap/+^gap and gap/gap offspring of the u90 strain. This marker allows the distinction of +^gap/+^gap and +^gap/gap moths with an error rate of <1% and therefore reduces the labor needed to maintain the u90 strain. Moreover, this marker helps to identify +^gap and gap individuals before pupation, which is necessary for larvae-based genetic and physiological analyses of the gap gene.

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Fibroin, a major component of silk fiber, is composed of light (L) chains, heavy chains, and fhx/P25 in the silkworm Bombyx mori. Sericin cocoon (Nd-s^D) is a silkworm strain expressing a mutant fibroin L chain that is accumulated in the endoplasmic reticulum (ER) of posterior silk glands (PSGs). However, little is known about the effects of accumulation in PSGs in Nd-s^D strain. We compared the PSG gene expression profiles of 5th-instar larvae of Nd-s^D strain and the fibroin-producing normal strain. cDNA representational difference analysis identified candidate genes whose expression levels were higher in Nd-s^D strain than in normal strain. We focused on heat shock proteins and cathepsin B, a major lysosomal protease. We confirmed upregulation of BiP(GRP78), Hsp20.8, and cathepsin B at the transcriptional and/or translational levels. These results suggest that the accumulation of mutant L chain induces the unfolded protein response in PSGs of Nd-s^D strain, which will be a valuable tool for protein quality-control studies of silk grand.

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Bombyx mori silk fibroin is a natural protein material characterized by mechanical toughness and biocompatibility, which makes it a promising candidate for the development of biomaterials. To adapt silk fibroin to a diverse range of applications, versatile methods for tuning its properties are desirable. Here, we report the in vivo incorporation of an alkyne-bearing unnatural amino acid, 4-ethynylphenylalanine (EthPhe), into B. mori silk fibroin and demonstrate that EthPhe can be used as a selective chemical handle for conjugation with functional molecules by click chemistry. EthPhe-incorporated silk fibroin (AlkyneSilk) is produced by oral administration of EthPhe to transgenic B. mori larvae expressing a mutant of the B. mori phenylalanyl-tRNA synthetase α-subunit in posterior silk glands. We verified that ethynyl groups in AlkyneSilk selectively react with azide-bearing molecules, demonstrating the utility of AlkyneSilk as a “clickable” silk material which can be easily decorated with desired functional molecules.

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