Japanese reference strains of silkworm microsporidians were established initially as laboratory cultures in the Pebrine Research Laboratory, Division of Silkworm Pathology, Sericultural Experiment Station in Tokyo. These are Nosema bombycis NIS-001, Nosema sp. NIS-M11, Nosema sp. NIS-M14, Vairimorpha sp. NIS-M12, Microsporidium sp. NIS-M25, Pleistophora sp. NIS-M27 and Thelohania sp. NIS-M32. N. bombycis NIS-001 has been maintained as the major reference strain in this laboratory for more than 80 years. Other silkworm microsporidians were derived from spore samples subjected to mass pebrine inspection during the silkworm egg production. Spore samples of these microsporidians were fed to silkworm larvae, and diseased ones were inspected for isolation of microsporidians after identification by spore morphology under a microscope. Later, microsporidian isolates resulting from the mass pebrine inspection were discriminated from N. bombycis by fluorescent antibody techniques, ELISA and latex adhesion tests. Recently, analyses of the small subunit ribosomal RNA genes, by sequencing and PCR, have been recognized to be highly useful and reliable for the identification of silkworm microsporidians. Results of these molecular identification methods are in good agreement with those from the latex adhesion tests of environmental spores with monoclonal antibody sensitized latex.
N. bombycis spores were commonly shown to infect Lepidoptera when tested in Central Honshu and their isolates exhibited marked differences in spore size and pathogenicity to the silkworm. By mass pebrine inspection, N. sp. NIS-M11 was frequently detected in B. mori and in the cabbage white, Pieris rapae. The latex adhesion test of environmental spores indicates that a spore surface antigen of N. sp. NIS-M11 is identical to that of N. mesnili isolates from P. rapae. Epizootiological study of V. sp. NIS-M12 suggests that its natural host is a pyralid, Lepidogma kiiensis(Lepidoptera: Pyralidae), and that this parasite causes only light chronic infection in the original host.
A highly synchronous infection method of insect cell cultures by artificial germination of environmental spores was developed and revealed the basic mode of the life cycle of silkworm microsporidians in insect cell cultures. Whereas octosporous sporogony of N. sp. NIS-M11 and V. sp. NIS-M12 could be induced in insect cell lines, disporous sporogony appear to be the basic mode of sporogony in Nosema and Vairimorpha species. Primary spore formation and the extrusion of a secondary infective form from primary spores were directly demonstrated in insect cell cultures infected with N. bombycis, and this evidence changed the concept of the sporogonial sequence in microsporidia. Cloning of N. bombycis NIS-001 was accomplished by a limiting dilution method and several clones of N. bombycis NIS-001 have been obtained. Generally, insect cell lines from Lepidoptera are permissive to silkworm microsporidians, and several insect cell cultures infected persistently with silkworm microsporidians have continued for more than 100 passages. However, some cultures infected persistently with silkworm microsporidians last only less than five passages.
Accumulation of molecular data about microsporidia has changed remarkably present notions about their phylogeny, as well as the definition of new species, strongly indicating that unnamed silkworm microsporidians should be described in the modern way.

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A cDNA stretch encoding one of the silkworm HSC70 genes with its upstream sequence has been identified, based on partial cDNA sequences registered in a Bombyx mori EST database. The deduced amino acid sequence with 649 residues was 89 and 96% identical to those from Drosophila melanogaster hsc4 and Manduca sexta Hsc70-4, respectively. The expression analysis by the reverse transcription polymerase chain reaction demonstrated that the mRNA transcription occurred in all tissues examined and was not stimulated by heat shock. These results suggested that the current structure corresponds to the silkworm heat shock cognate 70-4 gene and named BmHSC70-4. Cloning and sequencing of the genomic 5′-flanking, putative promoter region of BmHSC70-4 revealed the presence of several canonical transcription elements such as the GATA box, CCAAT motif and HSE (heat shock element), although without a TATA box element. The deletion analysis of the 5′ flanking region of the BmHSC70-4 gene, that fused to the firefly luciferase coding sequence as a reporter, indicated that the longest construct (−1, 639/+26) showed the highest promoter activity, and the constructs −111 to −59 and −1, 110 to −941 contained the basal transcription enhancer and the repressor elements, respectively.

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The suboesophageal body (SB) of the silkworm, Bombyx mori, has been known for a long time, but its function has not been clarified. This study demonstrates that SB produces lysozyme, a tissue-specific chymotrypsin inhibitor (SCI-SB) and a P27K ortholog of a glycoprotein known from another moth. The nucleotide sequences of SCI-SB and P27K cDNAs were newly determined and that of the lysozyme was confirmed to be identical to the previous data. The amount of lysozyme in SB is enhanced during molting and rises profoundly in larvae challenged with a bacterial injection. Lysozyme was shown to be released into haemolymph but could not be detected in other tissues. By contrast, the P27K protein, which is 56.5% identical with a haemolymph glycoprotein of Manduca sexta, occurs in B. mori in all analyzed tissues except the silk glands. Its function is unknown. SCI-SB belongs to the Kunitz-type proteinase inhibitors and exhibits 87.1, 85.5 and 85.7% homologies with chymotrypsin inhibitors SCI-I, SCI-II and SCI-III, respectively, which are known from silkworm haemolymph. However, SCI-SB is expressed exclusively in SB. The nature and expression patterns of identified proteins indicate that SB is an immunity-associated organ.

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The infectivity of Bombyx mori cypovirus 1 (BmCPV-1) in four different lepidopteran cell lines (BmN4, SES-Bm-130, TUAT-Spli-221 and IPLB-Sf-21) was analyzed. The B. mori derived BmN cell line showed the highest susceptibility to a BmCPV-1 inoculum that was prepared by dissolving polyhedra in alkaline solution and removing polyhedrin after neutralization with HCl. Incubation of the BmN cells at 30°C in TC-100 medium containing 10% FBS was optimal for the formation of BmCPV-1 polyhedra. The formation of polyhedra was virus-dose dependent. In BmN cells, polyhedra were detected at 3 days after inoculation at 25°C and the number of cells containing polyhedra increased up to 8 days post infection. There remained many apparently healthy cells in the BmN culture even at 13 days after inoculation, indicating that the transmission of BmCPV-1 from infected to healthy cells was rare or did not occur. Adsorption time of the virus to the cell was estimated to be more than 16 hours suggesting that low affinity between the virus and cell was one reason for low infectivity of BmCPV-1 to cultured cells. The development of an infection system for BmCPV-1 in cultured cells is also discussed.

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Thirty clones were derived by plaque assay on FRI-Splm 1229 cells from a wild isolate of Hyphantria cunea nucleopolyhedrovirus (HycuNPV). Analysis with restriction endonucleases (RENs) revealed that 29 clones out of 30 were distinct in their REN patterns, suggesting a very high degree of naturally occurring genotypic variation. Budded virion (BV) yields of 10 clones were examined in FRI-Splm 1229 cells at 48 and 72 h postinfection (pi), showing that productivity of BV and polyhedrin in the cell culture was different among clones. Two clones with higher BV productivity and two clones with lower BV productivity were each selected and examined for the virulence in per os inoculated H. cunea larvae as well as for the production of BV and polyhedrin in the cell culture. The virulence was different among selected clones, although no clear correlation was made between the virulence for H. cunea larvae and the productivity of BVs and/or polyhedrin in the cell culture. These results indicate that the wild isolate of HycuNPV consists of diverged variants that are distinct in genotype and biological activity.

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To study the application of Bombyx mori fibroin to wound care, the effect of fibroin on human skin fibroblasts was examined. The fibroin obtained from silk glands enhanced the proliferation of cultured fibroblast cells by up to 150% of the no fibroin control. Native molecular fibroin prepared from cocoons elicited an enhancement comparable to the above, whereas a degraded fibroin preparation from degummed silk was inactive. Alkali treatment of cocoons caused degradation of fibroin with a decrease in the biological activity. Furthermore, fibroin from cocoons that underwent prolonged alkali treatment exhibited an inhibitory action on fibroblast growth.

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In a few genetic strains of the silkworm, Bombyx mori, two mature larvae jointly spin a large cocoon called a double cocoon with a high frequency. We measured the behaviour of the silkworm to spin double cocoons and analysed the data to examine the relationships between the two silkworms concerning the sharing of the cocoon construction, the relative positions of the two silkworms, the moving properties and the spinning speed. As a result, we were able to visually show the spinning positions and the sharing of the two silkworms for the construction of the cocoon. The results showed that they fixed their bodies in the same or opposite direction with a high frequency. Furthermore, in one race of double cocoons, pseudo-periodicity existed in the serial change of the angle between the two silkworm bodies. There was a difference between the transition probabilities of the two different kinds of double cocoons. The behaviour of one silkworm affected the movement of the other silkworm in one of the double cocoons, though the two silkworms behaved more independently in the other double cocoon. They moved the spinneret significantly faster when the hind parts of the body were being fixed than when they were moving, although there was no definite difference between the spinning speed of the two silkworms.

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