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PROCEEDINGS ISC Congress Japan.Pdf Contents Preface……………………………………………………………………………………………… 4 Keynote Lecture…………………………………………………………………………………… 7 Section 1: Mulberry………………………………………………………………………………… 13 Section 2: Bombyx mori……………………………………………………………………… 41 Section 3: Non-mulberry silkworms……………………………………………………………… 75 Section 4: Bacology of silkworms………………………………………………………………… 93 Section 5: Post-cocoon technology………………………………………………………………115 Section 6: Economy…………………………………………………………………………………123 Section 7: Sericulture in non-textile industries and new silk applications…………………161 Section 8: Silk processing, trading and marketing……………………………………………189 Contents Preface………………………………………………………………………………………………000 Keynote Lecture……………………………………………………………………………………000 Section 1: Mulberry…………………………………………………………………………………000 Section 2: Bombyx mori………………………………………………………………………000 Section 3: Non-mulberry silkworms………………………………………………………………000 Section 4: Bacology of silkworms…………………………………………………………………000 Section 5: Post-cocoon technology………………………………………………………………000 Section 6: Economy…………………………………………………………………………………000 Section 7: Sericulture in non-textile industries and new silk applications…………………000 Section 8: Silk processing, trading and marketing……………………………………………000 Keynote Lecture PROCEEDINGS KEYNOTE LECTURE Keynote Lecture Use of silkworms as an experimental animal for evaluation of food and medicine Kazuhisa Sekimizu Mulberry Teikyo University Institute of Medical Mycology Genome Pharmaceuticals Institute Use of a large number of mammalian animals for evaluation of therapeutic effects of drug candidates becomes to be difficult due to high cost and ethical issues from the view of animal welfare. To solve the problems, we are proposing use of silkworms(1). In Japan, a long history of silk industry has established maintenance of silkworm strains Bombyx mori and methods for taking care of silkworms. Thus, silkworm has a large number of advantages as an experimental animal. However, little attention has been paid for use of silkworms as experimental animals for evaluating functions of food and medicine. Non-mulberry silkworms Bacology of the silkworms Post-cocoon technology Economy To evaluate the system of food and medicine by using silkworms, we tried to establish disease models with silkworms including infectious diseases. When pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa etc.) or true fungi (Candida albicans etc.) are injected into blood of silkworms, they die within several days(2). Antibiotics clinically used for human patients showed therapeutic effects in silkworms. Furthermore, their ED50 values, which show the amount of drugs needed to cure 50% of animals, of antibiotics in silkworms infected with Staphylococcus aureus Sericulture in non-textile (3) were consistent to the values in mammalian animals . This means that we can evaluate industries and new silk applications therapeutic effects of antibiotics by silkworms without sacrificing mammalian animals. － 7 － Silk processing, trading and marketing The 25th International Congress on Sericulture and Silk Industry By searching samples obtained from culture supernatant of soil bacteria, we discovered novel antibiotics, lysocin E (4). Lysocin E showed therapeutic effects in mouse models. Development of lysocin E for human patients is an important subject in future. We established hyperglycemia model with silkworms. When we added glucose or sucrose in to the artificial diet of silkworms, we observed rapid increase in glucose concentration in the blood of silkworms. Silkworms with high concentrations of glucose in the blood showed termination of their developments. We found that insulin decreased in the concentration of glucose in the hyperglycemic silkworms, and that insulin recovered the silkworms from the suppression of the developments. By using the hyperglycemia model of silkworm, we demonstrated that a lactic acid bacteria, Enterococcus faecalis YM0831, was effective to suppress the blood glucose level in silkworms and that this strain also showed suppressive effects in sucrose tolerance test with healthy humans(5). When we injected peptidoglycans beta-glucans, bacterial and fungal cell wall components, into the blood of silkworms, paralytic peptide, a cytokine of silkworm, was activated resulting in muscle contraction(6). By using this phenomenon, we can easily evaluate immuno-activating substances in food samples. We found that a strain of Lactococcus lactis 11/19-B1, has high activity of immuno-stimulation. － 8 － PROCEEDINGS Keynote Lecture Mulberry Bombyx mori Non-mulberry silkworms In summary, silkworms are highly valuable as experimental animals for evaluating functions of food and therapeutic effects of drug candidates. We can reduce number of mammalian animals needed to sacrificing for evaluation of these purposes. Bacology of the silkworms References (1) Sekimizu, K., Hamamoto, H. Drug Discov. Ther. (2016) 10, 1-2. (2) Kaito, C., Akimitsu, N., Watanabe, H., Sekimizu, K. Microb. Pathog. (2002) 32, 183- 190. (3) Hamamoto, H., Kurokawa, K., Kaito, C., Kamura, K., Manitra Razanajatovo, I., Kusuhara, H., Santa, T., Sekimizu, K. Antimicrob. Agents Chemother. (2004) 48, 774- Post-cocoon technology 779. (4) Hamamoto, H., Urai, M., Ishii, K., Yasukawa, J., Paudel, A., Murai, M., Kaji, T., Kuranaga, T., Hamase, K., Katsu, T., Su, J., Adachi, T., Uchida, R., Tomoda, H., Yamada, M., Souma, M., Kurihara, H., Inoue, M., Sekimizu, K. Nat. Chem. Biol. (2015) 11, 127-133. Economy (5) Matsumoto, Y., Ishii, M., Hasegawa, S., Sekimzu, K. Comm. Biol. (2019) 2, 157 (6) Ishii, K., Hamamoto, H., Kamimura, M., Sekimizu, K. J. Biol. Chem. (2008) 283, 2185-2191. Sericulture in non-textile industries and new silk applications － 9 － Silk processing, trading and marketing Section 1 Mulberry PROCEEDINGS ML-LP: Lead Paper Keynote Lecture Variation of mulberries (Morus spp.) Hiroaki Yamanouchi Radiation Breeding Division, Institute of Crop Science, NARO, National National Mulberry Agriculture and Food Research Organization, Hitachiomiya 2425, Ibaraki, Japan E-mail: [email protected] Mulberry is a plant belonging to the genus Morus that possesses several variations. Mulberry (Morus) consists of various species; one variation of the genus Morus is ploidy, and it comprises diploid and polyploid species. The examples of the reported ploidies are Bombyx mori diploid (2n = 2x = 28; e.g., M. alba and M. bombycis), tetraploid (2n = 4x = 6; e.g., M. boninensis), hexaploid (2n = 6x = 84; e.g., M. tiliifolia), and docosaploid (2n = 22x = 308; e.g., M. nigra) (Machii et al. 2002). In addition, a haploid species M. notabilis has been reported (He et al., 2013). Polyploidization generally occurs in angiosperms and plays an important role in evolution. Morus may be just in evolution through polyploidization. For sericulture, some diploid species derived from the east side of Eurasia have been primarily used. Morus species are naturally distributed in almost all continents, except in Non-mulberry silkworms Australia and Antarctica. Each species is originally distributed in only certain continents and its neighboring islands or in more restricted regions. Some species are artificially distributed for use as silkworm food (leaf) and as fruits for human consumption. Especially, diploid species derived from East Asia, represented by M. alba, are artificially distributed around the world. For example, there is an indigenous cultivar from Africa, ‘Enbu’, in the Japanese NARO Genebank. ‘Enbu’ is not a cultivar of M. mesozygia, which Bacology of the silkworms is only an African native species; however, it is probably derived from some diploid species introduced into Africa perhaps from Asia. Therefore, it is important to analyze mulberries using various methods such as follows. 1) Flow cytometry analysis Flow cytometry is a technique performed using a flow cytometer. “A flow cytometer is Post-cocoon technology an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination” (Givan, 2004). In the case of plants, protoplast and subcellular organelles, especially the nucleus, are primarily analyzed by flow cytometry (Vrána et al., 2014), whereas cells are not generally analyzed unlike in the case of animals. Economy Flow cytometry is often used to analyze the nuclear DNA content and the related parameters, e.g., genome size, ploidy, endoreduplication, and cell cycle. In general, the suitable plant tissue is chopped using a razor blade in a buffer solution, and the nuclei isolated in the buffer are stained using a fluorescent dye, followed by application into the flow cytometer. In general, two fluorescent dyes are used for staining nuclear DNA in plants, i.e., 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). PI (an intercalator) binds to DNA and is not affected by base composition, whereas DAPI (a Sericulture in non-textile DNA-specific dye) binds preferentially to adenine–thymine (A–T)-rich regions of the industries and new silk applications DNA helix (Doležel et al., 1992). In the analysis of ploidy and endoreduplication, both PI － 13 － Silk processing, trading and marketing The 25th International Congress on Sericulture and Silk Industry and DAPI can be used for staining nuclear DNA. The presence of endoreduplication is represented by multiple peaks on a flow cytometry histogram. Only a single peak was observed when rice leaves were analyzed, whereas remarkable multiple peaks were observed when the leaves
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