In many animals, as well as in insects, diapause or growth arrest is an essential feature of the life cycle. However, diapause features and their mechanisms are surely diversified, thus it may be difficult to achieve an integrated understanding. We found that an insect pentapeptide called Yamamarin and conjugated to palmitic acid (C16-DILRG-NH₂) causes reversible growth arrest in the rat hepatoma cells and inhibits embryonic development in the silkworm, Bombyx mori. The latter effect resembles action of the structurally unrelated diapause hormone, a tetraeicosapeptide released under certain environmental conditions from the central nervous system of B. mori. Despite different incidence in mammalian cells and insects, we here propose that C16-DILRG-NH₂ may open up new vistas in the development of biophamaceuticals and pest management.

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The White-sided egg mutation (Se) of Bombyx mori produces eggs which are opaque with wrinkles in homozygotes, whereas they have gray bands on the anterior, posterior and ventral regions in heterozygotes. Here, the surface and internal chorion structures of these eggs were observed morphologically. No peculiarity was exhibited in the mutation with respect to apparent egg dimensions. When cross-sectional chorion specimens were observed using a scanning electron microscope, the internal features, mainly in the middle layer of the Se homozygous, were found to be deformed. Also the middle layer of eggs of the Se heterozygous was disordered in opaque areas. It can therefore be concluded that the Se alleles take part in the formation of the middle-layer architecture during choriogenesis.

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Mariner-like elements (MLEs) were isolated from Chinese, Korean and Japanese individuals of Bombyx mandarina by the long PCR methods using the inverted terminal repeat of the Hyalophora cecropia MLE as a primer. The amplified clones of about 1.3 kbp in size were regarded to be full-length MLEs and termed Cecropia-ITR-MLEs. The sequences of the elements were determined, trimmed to eliminate any insertions and used to construct a phylogenetic tree. As a result, the Cecropia-ITR-MLE sequences of B. mandarina could be divided into major groups here called the Continental type and the Japanese type. Chinese and Korean specimens mostly belonged to the former group, which is very diverse and may well consist of subgroups. A majority of Honshu (the main island of Japan) specimens belonged to the latter, which is a much more closely related group of sequences. Interestingly, Tsushima (south of Korea) and Hokkaido (close to Russia) specimens mainly fell into the former type, whereas Fukuoka (south of Tsushima) individuals included both.

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It is known that the GacS/GacA two-component system is important for the virulence of Pseudomonas aeruginosa in invertebrate and vertebrate hosts and in plants (Rahme et al., 1995; Tan et al., 1999; Jander et al., 2000). In this study, the gacA nonpolar mutant C2 and polar mutant C3 were generated and inoculated into Bombyx mori. The polar mutants showed reduced virulence, whereas no significant difference was observed between the gacA nonpolar mutant and wild type strain with respect to the ability to kill B. mori larvae. This suggests that the reduced virulence was due to a polar effect of the insertions, rather than to the lack of the gacA gene product.

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Long-term preservation of the ovaries and spermatozoa of the silkworm (Bombyx mori) has been established in our current studies. Female moths yielded eggs which were artificially inseminated with semen of cryopreserved and stored in liquid nitrogen for 971 days. The fertilization rate and the number of eggs laid were almost equivalent to those obtained by normally mated moths. Second, ovaries from 4th instar larvae were removed and cryopreserved in liquid nitrogen for 1,451 days. The thawed ovaries were transplanted to surgically castrated 3rd instar female larvae. The recipient female moth yielded fertilized eggs with natural mating. Third, the female moths that had been transplanted with the cryopreserved ovaries at 3rd instar were artificially inseminated with the semen cryopreserved for 971 days. The recipient females also yielded fertilized eggs in this experiment. These results may contribute to the development of long-term preservation procedures of genetic resources of B. mori.

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The baculovirus expression system (BES) using Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for the high-level expression of sufficient quantities of recombinant proteins in a broad taxonomic range of insect cell lines and insect larvae. In the case of the silkworm, Bombyx mori, however, the BES using AcNPV tends to be much less exploited because AcNPV is believed to be inefficient in the replication of B. mori and cell lines derived from it. In this study, we have searched for high-permissive silkworm strains with higher production levels of a recombinant protein, by screening the susceptibility of 163 silkworm strains to the recombinant AcNPV, which expresses firefly luciferase. Based on their relative luciferase expression levels in larval hemocytes, the silkworm strains tested were divided into 3 groups: 5 high-permissive strains, 74 middle-permissive strains, and 84 low-permissive strains. Among the 5 high-permissive strains (c11, d17, f10, f38, and l312), d17 was the most productive, showing the luciferase activity of 3.5×10⁴±1,764 relative light units (RLU) per microgram of cell proteins. This remarkable susceptibility indicated that the silkworm d17 strain is very useful for large-scale protein production by the BES using AcNPV.

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In general, the domesticated silkworm, Bombyx mori, is an oligophagous insect that feeds on mulberry leaves. However, one mutant strain, Sawa-J, can feed on other plants and a low cost artificial diet (LP-1) on which ordinary silkworms do not feed. The food preference of Sawa-J was reported to be controlled by the polyphagous gene (pph) belonging to the third linkage group. To determine the pph locus, we performed a three-point test by using Ze and lem genes as markers. We obtained recombination values of 8.64% between Ze and pph, 14.16% between pph and lem, and 20.24% between Ze and lem. Using the value 20.8 between lem and Ze in the silkworm linkage map, the pph locus was mapped to position 12.9 on the third linkage group.

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