For detection and identification of pathogenic microorganisms which were infected with silkworm, Bombyx mori using PCR, amplification of DNA of generally assumed pathogenic microorganisms has been performed using specific primers. Thus, detection of unknown pathogens with the possibility to infect silkworms requires designing new specific primers, which takes time before detection. To develop a method of detecting silkworm infected with all pathogenic microorganisms by an identical procedure under identical conditions, we designed Hyper Detection of infectious disease based on Genome Profiling (HDGP). HDGP is a new technique utilizing the concept of genome profiling (GP), and it is capable of simultaneously detecting infected silkworm and identifying the cause of infection without specific primers. We detected entomopathogenic protozoa that infected silkworm and virus DNA-contaminated silkworms DNA to investigate the usefulness of HDGP, and confirmed significant differences in the profile image between the pathogenic microorganisms-contaminated silkworm and healthy silkworm. We also obtained similar results in a comparative experiment of assumptive infected tomatoes and pathogenic fungus. These findings suggest that HDGP may detect infections regardless of the species of host and infectious pathogen.

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The silk gland of Bombyx mori and Samia cynthia ricini produces vast amounts of silk proteins and stores them in the glandular lumen as a liquid silk during the larval growth and development. We have explored the system regulating pH in the silk gland, because the gelation of fibroin is pH-dependent. By injecting the pH-sensitive dye (phenol red) into silkworm larvae, we have estimated the pH in the glandular lumen. Although the entry of dye was unsuccessful in the anterior silk gland (ASG) of Bombyx, the lumen of the middle silk gland (MSG: major reservoir for fibroin) and that of the posterior silk gland (PSG: the fibroin factory) were colored with phenol red. The coloration by phenol red indicated that the MSG was acidic (pH 5~6) in the vigorously feeding larvae leading to gelation of silk proteins at the MSG and that the PSG was neutral (pH 7~8). When the larvae started spinning, the lumen in the MSG became neutral. A similar pattern in the luminal pH shift was obtained in the silk gland of Samia cynthia ricini (Eri-silkworm) with a dye-injection experiment. In Samia, the H⁺-translocating vacuolar-type ATPase (V-ATPase) locates at the apical surface of PSG, where the fibroin is produced, secreted and temporarily stored. The V-ATPase was also distributed at the apical surface of the anterior MSG as well as that in ASG. These V-ATPases became undetectable after the onset of spinning. The V-ATPase at the plasma membrane of silk gland cells regulates the physico-chemical state of liquid silk in the glandular lumen, in particular at the MSG of Bombyx and at the PSG of Samia, respectively.

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The frequency of single strand breaks (SSBs) occurring on both strands of the pBR322 plasmid DNA region flanked by a pair of primers used for polymerase chain reaction (PCR) amplifications was determined after irradiation with ¹³⁷Cs γ rays. We verified that real time PCR is suitable for the detection and quantitative analysis of SSBs caused by γ ray irradiation. The utility of this approach was also supported by the comparison of the practical experimental data with the Monte Carlo simulation. The potential application of this PCR method for the detection of genomic DNA damage was also confirmed.

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A total of five new cell lines were established from Helicoverpa armigera; including KU-HaEmb1 (HaEmb1) and KU-HaEmb2 (HaEmb2) from embryos, KU-HaPO1 (HaPO1) and KU-HaPO2 (HaPO2) from pupal ovaries, and KU-HaAO1 (HaAO1) from adult ovaries. These cell lines were examined for their permissiveness for homologous H. armigera nucleopolyhedrovirus (HearNPV) and seven heterologous NPVs. Upon infection with HearNPV, the cell line HaAO1 yielded the highest productivity in terms of amounts of polyhedra, budded virus (BV), polyhedrin and viral DNA among the six cell lines, including the five newly established cell lines and the cell line from Helicoverpa zea (BCIRL-Hz-AM1), which is commonly used for in vitro studies and production of HearNPV. The cell lines HaPO1, HaPO2 and HaEmb2 were also permissive for HearNPV but they yielded lower productivity than that of the cell line HaAO1. Only the cell line HaEmb1 was nonpermissive for HearNPV infection among the cell lines established. Examination with seven heterologous NPVs showed that all the new cell lines continued to proliferate without any cytopathic effects (CPE), following infection with five NPVs from Bombyx mori, Hyphantria cunea, Lymantria dispar, Orgyia pseudotsugata and Spodoptera litura. Upon infection with NPVs from Autographa californica (AcMNPV) and Spodoptera exigua (SeMNPV), all the new cell lines, with the exception of HaAO1, manifested CPE to varying degrees, synthesizing significant amount of viral DNA. These cell lines, with the exception of AcMNPV-infected HaPO1 that produced significant amount of BVs, yielded negligible or very low level of BVs and polyhedrin following infection with AcMNPV and SeMNPV, indicating that they were semipermissive for these two NPVs. These results indicate that the newly established H. armigera cell line HaAO1 provide a promising candidate for in vitro production of HearNPV biopesticide, as well as a useful system for the analysis of molecular mechanisms of NPV-cell interactions.

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We purified the major yolk protein, vitellin, from Saturnia japonica, by column chromatographies. SDS-PAGE and immunoblot analysis of the S. japonica vitellin (SjVn) showed that SjVn consisted of only a large subunit with a molecular size of approximately 200kDa. We then cloned and sequenced cDNA of the S. japonica vitellogenin (SjVg), a SjVn precursor. The SjVg cDNA was 5731 nucleotides long and encoded 1776 amino acids for the entire subunit. The molecular weight of the predicted polypeptide was 200,000. Consensus motifs, such as GL/ICG (at the amino acid position 1592) and DGGR (located 17 residues upstream from the GL/ICG motif) were found in the deduced amino acid sequence. There is no RXRR motif, which is a cleavage site between the small and large subunits. Two polyserine regions were found in the deduced amino acid sequence.

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A complex type marner-like element (MLE) was isolated by PCR with genomic DNA of Bombyx mandarina. The clones of this MLE, homologous to the ‘Cecropia-ITR-MLE’ in BmTNML locus that we have isolated previously from B. mori, were roughly classified into four groups according to the state of inserted elements: (1) Containing a retrotransposon (L1Bm) and another element, (2) containing L1Bm alone, (3) containing another element alone and (4) without insertions. This inserted element was named BmamaT1, as it was homologous to the B. mori MLE called Bmmar6, which was previously reported to be similar to a maT-family member, Bmmar1. Many of the current Cecropia-ITR-MLE clones had, as the BmamaT1 insertion site, the sequence of TA/TATA, which may be a transposition target site. Moreover, the BmamaT1 elements that inserted into the Cecropia-ITR-MLE region were highly homogeneous, making one group when a phylogenetic tree was made together with BmamaT1 members isolated directly from the B. mandarina genome. Many of the inserted type BmamaT1 elements had complete ORF which possessed the so-called DDD catalytic triad. All these findings were taken to indicate that a large fraction of the BmamaT1 elements are still highly active.

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The surface and internal structures of the glossy and small sized imaginal eyes in the varnished-eye mutation (ve) of Bombyx mori were observed in comparison with the normal compound eyes by scanning electron microscopy and light microscopy. The ve mutant eyes were found to have apart and irregular facets (surfaces of corneas) and degenerated internal structures at the crystalline cones and retinular pigment cells covering the rhabdoms. We infer that the ve mutant gene causes differentiation disorders in the ommatidium during eye morphogenesis.

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In the silkworm, non-susceptibility to B. mori densonucleosis virus type-1 (DNV-1) is controlled by a dominant gene, Nid-1. In this report, simple and effective methods for linkage analysis and for mapping were improved. Three strains were used: No. 908, a virus resistant strain carrying Nid-1, and RF02 and RF50, which were used as standard wild type strains in our previous linkage analyses and mapping. Linkage analysis of Nid-1 was carried out by using survivors of two kinds of backcrosses, (RF50×908) females crossed to RF02 males and (RF02 × 908) females crossed to an RF50 male, after oral inoculation with DNV-1. The ordered DNA patterns from two families of BF₁ segregants was enough to determine linkage group by analysis of RFLP patterns of previous mapped cDNA clones, specific for each of the twenty-eight linkage groups (RFLGs). The results indicated that Nid-1 was linked to linkage group seventeen (RFLG17). Individual genomic DNA samples of 64 survivors from the backcross of RF02×(RF50×908) were used to locate five clones and Nid-1. The relative distances between them were determined by repeated three-point analysis and showed that Nid-1 was closely linked to two genes (m025 and m237), at a distance of 4.7cM from them.

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