The larvae of most Lepidoptera secrete fibrous proteins from their labial glands, collectively known as silk. Silk is used to construct shelters for the larvae and cocoons in which the larvae pupate. The water insoluble core of the silk filament is produced in the posterior gland section. Its crucial component, the heavy chain fibroin (H-fibroin), is characterized by hierarchical arrangement of species specific amino acid repeats. The H-fibroin genes of different Lepidoptera are homologous but their repetitive region has diversified. Amino acid composition, complexity of basic repetitive units, and arrangement of these units into iterated higher order domains determine physical properties of the silk fiber. In most Lepidoptera, the fiber includes two other peptides, light chain fibroin (L-fibroin) and P25 that occurs as two proteins differing by the degree of glycosylation. For the silkworm it has been shown that six H-fibroin, six L-fibroin, and one P25 molecules are assembled into an elementary silk unit. The fiber of tussah silk is different because it lacks the L-fibroin and P25 components; it seems to be composed of H-fibroin dimers. The fiber core in all species is enveloped by several sericins that are derived from two genes expressed in the middle section of the gland. Differential splicing and probably also different extent of glycosylation provide for the diversity of sericins that serve as a glue for fiber adherence. The silk further contains several proteins of smaller size that are not essential for silk function but may provide protection against predators, molds, and microbes. Greatly diversified proteins called seroins, to emphasize their production both in the middle, sericin producing and the posterior, fibroin producing gland sections, were identified as products of one or two genes, depending on the species. Two other genes specifically expressed in the silk glands encode a Kunitz and a Kazal type protease inhibitors that are active on certain fungal and bacterial proteases. The inhibitors are produced before cocoon spinning and constitute very stable cocoon components. Structures of several other small silk proteins await elucidation. Silk produced by a handful of species has been woven into textiles for several millennia, and used as additive into cosmetics and occasionally also remedies. Modern technologies offer the potential to produce for practical use specific silk components such as the protease inhibitors. Creation of recombinant silkworms secreting silk fibers of new quality has become feasible. Industrial spinning of a silk dope prepared under in vitro conditions is a challenge for current research.

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The sorbitol dehydrogenase (SDH) gene of Bombyx mori begins to be expressed in diapause eggs after 5°C-chilling for 40-50 days. Because three elements similar to consensus sequences bound by members of the steroid hormone receptor superfamily such as Seven-up (SVP)/COUP-TF, were found in the 5′-upstream region of the SDH gene, we tried to isolate cDNAs for SVP from Bombyx eggs, as a step to understand the mechanism of how 5°C controls the SDH gene expression. Two cDNAs for SVP were cloned and named Bm svp-α and β, respectively. From the deduced amino-acid sequences, Bm svp-α protein was shown to have partial A/B region and C, D, E and F regions, whereas Bm svp-β protein lacked E (corresponding to the ligand-binding domain) and F regions. In non-diapause eggs, the temporal profile in the amount of Bm svp-α mRNA showed a peak in the middle of embryogenesis, while mRNA amounts of svp-β were much lower than those of svp-α. On the other hand, in diapause eggs, 5°C-chilling for 40-60 days enhanced levels of mRNA and protein for Bm svp-α, suggesting that Bm svp-α protein is a strong candidate of transcription factors that control the SDH gene expression induced by the cold.

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Homologues of proliferating cell nuclear antigen gene (pcna) were identified and characterized in Hyphantria cunea nucleopolyhedrovirus (HycuNPV), and two cell lines, NSC-HyCu15B and Sf9, from the fall Webworm, H. cunea, and the fall armyworm, Spodoptera frugiperda, respectively. The structure of PCNAs from NPVs and their host cells was comparatively analyzed. The HycuNPV pcna homologue had an open reading frame (ORF) encoding a polypeptide of 250 amino acid residues with a predicted molecular weight (MW) of 27, 080, and shared 34 and 64% amino acid sequence identities with PCNAs of Autographa californica MNPV and Orgyia pseudotsugata MNPV, respectively, suggesting a high degree of divergence within NPV PCNAs. Analyses of pcna cDNAs from NSC-HyCu15B and Sf9 cells showed that both cDNAs encoded polypeptides of 260 amino acid residues with MWs of 28, 968 and 29, 001, respectively. The NSC-HyCu15B and Sf9 cell PCNAs shared a very high degree of amino acid sequence identities not only within lepidopteran PCNAs (96-98%), but also between lepidopteran and dipteran PCNAs (78-80%). Multiple alignments of PCNAs from representative organisms revealed that NPV PCNAs shared higher identities with PCNAs from insects (30-45%) and other eukaryotes (26-42%), and lower identities with PCNAs from archaea (13-23%) and Paramecium bursaria chlorella virus 1 (15-25%). No significant closer relationship was observed in the amino acid sequence identities between PCNAs from HycuNPV and H. cunea from which HycuNPV was primarily derived. In addition, multiple alignment revealed that similarly to archaea PCNAs, NPV PCNAs possessed a unique gap around the region that was important for eukaryotic PCNAs to interact with DNA polymerase ε. Circumstantial evidence suggested that baculovirus pcna homologues had descended from a common ancestor that was derived by a certain NPV after the split to groups I and II.

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A series of 1-substituted and 1, 5-disubstituted imidazoles have been shown to terminate diapause in pharate first instar larvae of the wild silkmoth, Antheraea yamamai, and the gypsy moth, Lymantria dispar. To understand the mode of action for the imidazole compounds, we analyzed imidazole compound (KK-42)-binding proteins by affinity chromatography using a synthetic resin-coupled KK-42 analog. Two binding proteins (KK-42BPs) of 40 and 45 kDa were isolated in soluble fractions prepared from diapausing pharate first instar larvae. The N-terminal amino acid sequence from the first 20 residues was determined for the 45 kDa protein. This 45 kDa protein appeared throughout the periods of pre-diapause and diapause, and it disappeared after the KK-42 application and long period of chilling. It was further demonstrated through specific positive staining with antiserum that the 45 kDa KK-42BP was localized in yolk cells. These results are the first report of a protein associated with artificial hatching in insects.

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Antisense and double-stranded RNA (dsRNA) interference (RNAi) was investigated in a silkworm ovarian cell line, BmN, using the firefly luciferase as a reporter. The expression of the luciferase from the reporter plasmids was suppressed in the transfected cells when the amount of antisense RNA was in excess of the mRNA amount. The size of the antisense RNA was also found to be an important factor in the interfering efficiency. On the other hand, a small amount of dsRNA induced an efficient RNAi. The sequence-dependency of the dsRNA-derived RNAi was verified in transient expression experiments using the Renillia luciferase, emphasizing the usefulness of dsRNA as a template for gene silencing in the biotechnology of the silkworm.

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Morphological aspects of the micropylar apparatus in Bombyx mori chorion of mature eggs were observed by scanning electron microscopy, with special reference to the external orifice or opening, and to the internal tubes that were thought to offer the channels of sperm entry. The opening located on the outside surface varied in shape among eggs from the same ovary; the shape fell into 5 categories tentatively named bobbin, triangle, cross, starfish and asterisk, whose apparent apex number is from 2 to 6, respectively. The tubular channels located on the inside surface area close to the opening existed also in 5 types, differing in number from 2 to 6. The most abundant were the triangular opening and the 3-tubed internal apparatus in the normal strain p22. Analysis of incidence distribution using the eggs from p22 indicated that the shape of the external opening was highly correlated with the number of internal channels. The channel number tended to be lower in the miniature egg mutation, emi, and higher in the giant egg mutation Ge compared to the normal, implying that the expression of gene systems producing the micropylar apparatus is affected by the egg size.

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The nucleotide sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), including 5.8S rDNA, were determined for 5 isolates of Metarhizium anisopliae from Japan and Korea in order to analyze their phylogenetic relationships. These sequences were aligned with published sequences of the Metarhizium isolates from different countries. The bootstrapped neighbor-joining tree showed that isolates of Metarhizium species were divided into two major groups (groups I and II). Isolates belonging to M. anisopllae formed a sister group (group I), separate from M. flavoviride and M. album (group II). Isolates of group I were divided into at least 4 subgroups (subgroups I-A, B, C, and D), and Japanese isolates tested belonged to the subgroup I-A.

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To develop techniques to produce stromata of Cordyceps militaris (L.) Link on a large scale, the silkworm, Bombyx mori, was infected and the growth of stroma was investigated. The 5th instar larvae and the pupae of the silkworm reared in a sterilized indoor environment on an artificial diet and were inoculated with C. militaris by spraying, dipping, or by hypodermic injection. The rate of infection differed among the three methods; the highest being in the hypodermic injection. The rate of infection was also higher in the pupae than in the larvae. The formation and growth of the stromata were better in the silkworm pupae. A conidial suspension was injected at different sites on the pupal body. Infection rates, however, did not differ significantly among the sites. The formation and growth of the stromata were not affected by the injection site either.

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The effect of carbonic anhydrase (CA), which specifically binds δ-endotoxin in an in vitro binding assay, on the insecticidal activity of Bacillus thuringiensis (Bt) was examined in the larvae of the silkworm, Bombyx mori. The results showed that the mortality from Bt was significantly lower in the larvae administrated with 50μg/g CA diet than in the larvae administrated with no CA. No significant difference was observed in the diet consumption and weight gain of the test larvae. These results indicate that CA interacts with δ-endotoxin not only in vitro but also in vivo to lower the insecticidal activity of Bt.

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