The practical conditions of an enzyme-linked immunosorbent assay (ELISA) for ecdysteroids were optimized. A complex of ecdysone and horseradish peroxidase (HRP) was prepared as an enzyme tracer, and a competition between the tracer and free ecdysteroids was employed for the ELISA system. The binding of rabbit antiserum against 20-hydroxyecdysone (20E) was better with the direct method than indirect method that used anti-rabbit IgG. Optimal blocking using 1% casein before the competition assay had lower background compared to no blocking or blocking using 1% BSA. The optimized method showed a determination range of 0.06 - 6ng equivalent of 20E. The system made it possible to determine ecdysteroids in both in vitro and in vivo samples. Changes in ecdysteroid titer in hemolymph of Agrius convolvuli between 4th molting and eclosion determined by the ELISA optimized in this study was similar to that reported concentrations in Manduca sexta.

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A cDNA linkage map in the domesticated silkworm, Bombyx mori, based entirely on backcross segregant analysis is reported. One hundred segregants from a single pair mating derived from a backcross of a RF02 female and a F₁ (RF02×RF50) male were used as the mapping population. More than 194 randomly isolated cDNA clones whose linkage groups had been determined previously were analyzed out of which 189 were unambiguously placed on the linkage map by repeated three-point analysis. Allelic differences in cDNA markers were based on RFLP analysis by Southern blot hybridization. Every recombinant value for each clone on the same chromosome was calculated directly. The majority of the mapped clones corresponded to single loci dispersed on every 28 chromosomes. The map covers about 66% of the genome and provides important landmarks for Bombyx genome analysis.

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A short antibacterial peptide was designed and synthesized on the basis of the active sites of insect defensins. The 9-mer peptide, ALYLAIRRR-NH₂, was tested to determine whether it can suppress the proliferation of methicillin-resistant Staphylococcus aureus (MRSA) in vivo and in vitro. In vivo evaluation of the peptide against MRSA was carried out histopathologically. Silk sutures pretreated or non-treated with the peptide were embedded in the back skin of mice for 24h. Sutures treated with the peptide showed strong inhibition of the proliferation of MRSA judging from examination by light microscopy of biopsy samples. On the contrary, non-treated sutures showed numerous Gram-positive loci. For in vitro experiments, silk fibroin films containing different concentrations of the 9-mer peptides were prepared. MRSA seeded on the culture plates was covered with a transparent fibroin film and incubated at 37°C for 24h. No MRSA colonies were detected under the films containing the 9-mer peptide, whereas many colonies appeared under the control film without the peptide. These results suggest this synthetic antibacterial peptide is a useful lead peptide for development of novel therapeutic agents against infection with antibiotic-resistant bacterial pathogens.

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An enhanced gene delivery system, monitored by the expression of a luciferase gene, was devised for an insect cell line BmN4, by altering the ratios of several liposome components. First, three kinds of commercially available polyethylenimine (PEI) reagents with different sizes were tested; one (PEI reagent with an average molecular weight of 1,800) gave a much better transfection efficiency than others. Using this particular reagent (PEI-1800), we newly formulated a liposome composed of three polycationic lipids: dioleoylphosphatidylchorine (DOTAP), dioleoylphosphatidylethanolamine (DOPE) and PEI-1800 in a ratio of 1:1:1. The current liposome gave a 2.5-fold efficiency compared to a commercially available liposome (CellFECTIN), a 2:3 mixture of TM-TPS (tetramethyl-tetrapalmitylspermine) and DOPE. The effects of divalent cations on the transfection with the newly devised liposome were investigated in detail. It is concluded that a mixture containing the PEI-1800 reagent provides a low cost and stable transfection procedure for the cultured silkworm cell line.

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A recessive mutation affecting the body color trait of newly hatched larvae has been found spontaneously in the experimental strain possessing a small egg mutant gene. The current mutation is characterized by a whitish color in newly hatched larvae, instead of the normal body color of black. Also this mutation was found to cause retarded larval growth, abnormal feature of pupal wings and undeveloped gonads. Linkage analysis showed that the mutant gene controlling these characters is linked to the melanism gene (mln), which belongs to the 18th linkage group. The novel mutant gene was named “Hakugi” in Japanese and “undeveloped gonads” in English with the gene symbol gon.

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