Journal of Insect Biotechnology and Sericology
Online ISSN : 1884-7978
Print ISSN : 1346-8073
ISSN-L : 1346-8073
89 巻, 3 号
選択された号の論文の3件中1~3を表示しています
Regular Articles
  • Mari Sekiguchi, Sayuri Ishikawa, Shin-ichiro Asano, Hisanori Bando, Ma ...
    2020 年 89 巻 3 号 p. 3_045-3_053
    発行日: 2020年
    公開日: 2021/02/18
    ジャーナル フリー

     In the infection process of BmNPV, GP64, the major membrane protein of budded viruses, is required for binding to host cell receptors and for pH-dependent membrane fusion and is, therefore, essential for viral infection. Based on previous studies, we predicted that tyrosine at position 172 of T3 GP64 was an important amino acid residue for membrane fusion function. In this study, we examined the effects of replacement of the amino acid in GP64 on the proliferation of BmNPV T3 in BmN cells and silkworm larvae as well as its membrane fusion activity. Y172T3H increased both membrane fusion activity and early viral proliferation in BmN cells. On the other hand, viral proliferation in silkworm larvae was significantly decreased, and the survival time of infected larvae was longer by 24 h. These results show that Y172T3 in GP64 is implicated in efficient BmNPV proliferation in the silkworm larvae, possibly through membrane fusion activity.

  • Nozomi Fujiwara, Masaaki Azuma, Masanobu Itoh, Jun Kobayashi
    2020 年 89 巻 3 号 p. 3_055-3_062
    発行日: 2020年
    公開日: 2021/02/18
    ジャーナル フリー

     The process of molting is essential for the growth of lepidopteran larvae and their metamorphosis into adults. However, the investigation of cellular renewal and regeneration is limited by the lack of efficient methods for the isolation and characterisation of midgut stem cells in vitro. Here, we demonstrate an easy and secure protocol for midgut stem cell isolation from molting larvae and pharate pupae of the silkworm, Bombyx mori. This meth- od is based on the physiology of stem cells, which lie loosely associated with the base of epithelial cells and multiply during the molting or metamorphosing phase. They finally intercalate or replace existing epithelial cells as they elongate and differentiate into mature forms. Using approximately 10 insects, it was possible to obtain >500,000 cells with high homogeneity and >80% viability. Stem cells prepared from the fourth instar molting larvae and pharate pupae were spherical and could be preserved at 4°C for 1-2 weeks with no viability loss. However, the number of living cells showed a steady decline when cultured in Grace’s insect culture medium for one week. Supplements such as fetal bovine serum, lipid-rich bovine serum albumin, or heat-treated native B. mori plasma did not support stem cell proliferation or suppress cell death. Although continuous stem cell culture is still difficult, the described method for midgut investigation could potentially utilized for biochemical studies of stem cells and pathological assays in vitro, which may lead to novel strategies for agricultural pest management.

  • Yuji Imura, Haruka Nakamura, Mitsuyoshi Nozawa, Yoshinori Hatakeyama
    2020 年 89 巻 3 号 p. 3_063-3_071
    発行日: 2020年
    公開日: 2021/02/18
    ジャーナル フリー

     The microsporidium Nosema bombycis is a causal pathogen of pébrine disease in the silkworm, Bombyx mori. In the sericultural industry, pébrine inspection is intensively performed to eliminate microsporidia from B. mori to maintain strains and produce eggs commercially for various purposes. However, detailed properties of microsporidia isolated via pébrine inspection have not been mentioned. In this study, we analyzed microsporidian strains isolated from B. mori specimens during mass pébrine inspection by spore size comparison and phylogenetic analysis of small subunit ribosomal RNA gene sequencing. Next, we elucidated the microsporidian fauna in the rearing room in five different years. A total of 310 samples were detected via pébrine inspection, 47.4% of which were detected in the late autumn rearing season. Isolated microsporidian strains were classified into two groups based on spore size: Nosema or Vairimorpha and Pleistophora. Phylogenetic analysis suggested classification into four genera: Nosema, Vairimorpha, Cystosporogenes, and Endoreticulatus. Some strains classified into genera Nosema and Vairimorpha exhibited high genetic homology with N. bombycis and Vairimorpha sp. NIS-M11, which cause pébrine disease. Thus, conventional pébrine inspection facilitates the elimination of microsporidia, thereby preventing the spread of this disease. The genus Cystosporogenes was first detected from insects in Japan. However, classification by spore size is misunderstood in the genus Pleistophora, which infects B. mori larvae. Our present results suggest that the recent status of the microsporidian fauna in the silkworm rearing room is slightly different from that observed via conventional recognition of spore shapes under microscopy.

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