Maintaining silkworm races can probe to be labor intensive because stock races must be reared annually. The most effective way of reducing labor is to extend the period of preservation. A method for rearing silkworms bi-annually was recently proposed; however, it required several complicated procedures for egg preservation. In this study, we developed a simpler method for the preservation of silkworm diapause eggs, involving minimum care, refrigeration, and without terminating diapause. We first optimized storage conditions by investigating the relationship between the long-term storage of diapause eggs and the number of days needed to achieve diapause at 25°C, as well as the number of days required for termination at 5°C. We then developed a very simple 2-year egg storage method, using a high-humidity refrigerator for egg storage and only one intermediate care procedure. Using this new method, the period from one generation to the next is now 2years.

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The silkworm, Bombyx mori, is closely related to the wild silkworm, B. mandarina, although process of their divergence is not fully elucidated. To evaluate the evolutionary relationship of the domesticated silkworm strains, alkaline phosphatase gene (Alp) cluster in B. mandarina was identified and characterized. Two Alp genes, Alp-m (man) and Alp-s (man), were shown to be duplicated in tandem in the genome of B. mandarina inhabiting Japan. Gene arrangement and exon-intron structure of each gene were highly identical to those in B. mori. Localization of the two ALP proteins in the epithelial cells of the B. mandarina’s midgut were similar to those of B. mori. These results consistently suggest that Alp-m (man) and Alp-s (man) are B. mandarina orthologs of Alp-m and Alp-s of B. mori, respectively, and that the duplication of Alp genes was prior to the speciation. By comparing the structure of the intervening sequences of two Alp genes, a possible phylogenetic relationship among the polymorphism in B. mori was discussed. The nucleotide sequence reported in this paper has been submitted to DDBJ/GeneBank (accession number: AB270698).

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The EcoRI site is located between the 5.8S and 28S rRNA genes in Bombyx mandarina (Japan)(Japanese type rDNA) but is absent in the rDNA of B. mandarina (China)(B. mori type rDNA) including the domesticated B. mori. Digestion of PCR products amplified by primers of both flanking sides of this EcoRI site with EcoRI should only be successful for amplification products of B. mandarina (Japan) containing Japanese type rDNA. The distribution of Japanese type rDNA in Japan, Korea, and China was determined using this rapid detection method. Japanese type was not detected in China or in Korea despite having 27 chromosomes. These findings suggest that the choromosome fusion and the rDNA type separation were independent evolutionary events. Invasion of B. mori chromosome #11 into Japanese B. mandarina (Japan) was examined.

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We have explored the gene expression and localization of an aquaporin (AQP) in the silk gland of Bombyx mori. Whole mount in situ hybridization studies with digoxigenin-labelled RNA probes derived from an open reading frame of B. mori AQP (Bommo AQP) showed its mRNA distribution strictly at the anterior silk gland (ASG) from vigorously feeding larva. Its expression disappeared from actively spinning larva. Northern hybridization analysis of Bommo AQP in the ASG demonstrated that a single transcript of 2.3kb was abundantly present during the feeding phase of fifth instar stadium and its expression decreased after spinning. Immunocytochemical studies using an antipeptide antibody against the Bommo AQP molecules revealed that the positive reaction was localized at the apical surface of ASG cells from feeding larva and disappeared from spinning larva. Further the apical surface localization of Bommo AQP was found in the posterior division of the middle silk gland (posterior MSG) and the strong immunoreaction was also observed at the apically-opened vacuoles, cavities and pits predominantly existing in the most posterior MSG. The immunoreaction at the posterior MSG almost disappeared in the spinning phase. The occurrence and disappearance of AQP at the limited region of the silk gland was evidently coincided with that found in the H⁺-translocating vacuolar-type ATPase. An osmoregulatory work along the length of the silk gland enables a silkworm larva to stabilize the liquid silk in a native state with entrained water during growth and development.

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Flow cytometry is a useful technique for studying plant genome size and ploidy and for analyzing the degree of polysomaty of various organs. Polysomaty is a common biological phenomenon in higher plants and is detectable as multiple peaks in the histogram by flow cytometric analysis. However, if polysomaty occurs in the target tissue, the multiple peaks may cause mis-estimates of genome size and ploidy obtained by flow cytometric analysis. In this study, we used flow cytometry to determine whether polysomaty occurred in various organs of several mulberry species. We did not find markedly polysomatic organs in any of the above-ground vegetative organs, whereas anthers exhibited a polysomatic state. Based on these results, we tried to ascertain the possibility distinguishing between two types of periclinal cytochimeras by flow cytometric analysis of mulberry leaves. One type of the cytochimera was distinguishable from its progenitor and the other type of cytochimera by flow cytometry.

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The integument of the Eri silkworm, Samia ricini (Lepidoptera, Saturniidae) was observed using fluorescence microscopy by staining with the fluorescent dye DAPI. Intact integument usually consists of a single cell layer of epidermal cells and non-cellular cuticle layers. Partial folding of the epidermis is sometimes observed with cell debris-like structures between the cuticular layers. In order to determine how such structures arise, a part of the integument around the scoli was injured artificially by pinching with forceps so that the epidermal cell layer was disrupted without breaking the cuticle layer. After the injury, some parts of the disrupted epidermal cells had embedded in newly secreted cuticle. These observations suggest that epidermal cell debris is cleared from the wound site by embedding in the cuticle.

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To estimate the contribution of phospholipase C (PLC) in the virulence of Pseudomonas aeruginosa against Bombyx mori, PlcB, PlcH, and PlcN mutants were created by the sacB-based counterselection method. Insertion of Ωaac cassette (selection marker, Gm^R) in plcB, plcH, and plcN was confirmed by Southern hybridization and the mutants were designated as PAO1plcB, PAO1plcH, and PAO1plcN, respectively. Inactivation of PLC genes did not cause growth deficient in vitro. The culture filtrates of PAO1, PAO1plcB, and PAO1plcN had hemolytic activity, but not PAO1plcH. When the PLC mutants were inoculated into the fourth instar of Bombyx mori larvae, all mutants maintained high virulence, indicating that at least inactivation of a single plc gene in P. aeruginosa did not affect the virulence against B. mori. It was different from the results obtained in another Lepidopteran host model, Galleria mellonella.

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The vit (scanty vitellin) eggs of Bombyx mori deposited by the females homozygous for the mutation were investigated for the manifestation of inherited characteristics. Mutant eggs produced by vit/vit ovaries were white in color due to the lack of yolk proteins. These eggs could be fertilized and grown to the final stage of embryogenesis but did not hatch and died, irrespective of whether the genotype of embryo was homozygous or heterozygous for the vit gene. Mating experiments showed that the trait of yolk color was transmitted in a manner of pseudomaternal inheritance, whereas the character of embryonic lethality was manifested following a mode of maternal inheritance.

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