Study on karyotype and genome size of entomopathogenic microsporidians

根据光镜和电子显微镜观察的形态特征，对微孢子虫进行了鉴定。近年来，脉冲场凝胶电泳法不仅为小单细胞生物的染色体研究提供了新的选择，也为微孢子虫的鉴定提供了新的选择。另一方面，这些研究中使用的微孢子虫不是克隆的。微孢子虫的混合感染已从病蚕幼虫中发现。本研究首先研究了微孢子虫在混合感染下寄主与细胞的相互作用，并利用光学显微镜对克隆的微孢子虫进行了核型鉴定。然后，我们给出了克隆的微孢子虫的脉冲场凝胶电泳数据，并讨论了昆虫性微孢子虫的种内和种间变异。无论是在家蚕中还是在果蝇SF21AEII细胞培养中，家蚕微孢子虫都是…另一种微孢子虫Nosema sp.NIS M11和Vairimorha sp.NIS M12参与混合感染时，优势微孢子虫不能通过竞争消除次要微孢子虫，微孢子虫可在同一宿主中共存。因此，在讨论微孢子虫的特性和鉴定时，克隆微孢子虫是非常重要的。用有限稀释法在昆虫细胞培养中建立了几个微孢子虫克隆，并用Axioplan 2万能显微镜对克隆的微孢子虫营养细胞的染色体数目进行了估计。观察到微孢子虫Vairimora sp.的13条染色体。NIS M12。在微孢子虫基因组DNA分析中，明确了巴西微孢子虫基因组提取需要用EDTA碱性溶液处理。在脉冲场凝胶电泳法中，不同种的微孢子虫的带型不同，提示该方法是鉴定微孢子虫的有用工具。本报告中建立的方案可以适用于所有使用的分离物，这一结果表明了克隆的重要性。较少

The identification of microsporidian parasites has been based on morphological characters observed with light and electron microscopy. Pulsed-field gel electrophoresis has recently provided an alternative for not only the investigation of chromosomes in small single-celled organisms but also the identification of microsporidians. On the other hand, microsporidians used in those studies were not cloned ones. Mixed infection of microsporidians has been known from the diseased silkworm larvae. In this study, the interaction of the parasites in insect hosts and insect cell cultures under mixed infection was investigated first and estimate of the karyotype of the cloned microsporidians was made by light microscopy. Then, we presented data from pulsed-field gel electrophoresis of cloned microsporidians and the intra- and inter-specific variations of entomophathogenic microsporidians was discussed.Both in the silkworm and in the Spodoptera frugiperda SF21AEII cell culture, Nosema bombycis NIS … More 001 dominated on the other microsporidians, Nosema sp. NIS M11 and Vairimorha sp. NIS M12, when it was involved in the mixed infections, However, the dominant did not eliminate the minor ones by competition and microsporidians could co-exist in the same host. It was, therefore, strongly suggested that cloning of microsporidians would be important when the characterization and identification of the parasites were discussed.Several clones of those microsporidians in insect cell cultures were established by a limiting dilution method and attempt to estimate the number of chromosomes for the vegetative cells of cloned microsporidians was made by an Axioplan 2 universal microscope. Thirteen chromosomes were visualizied for the microsporidian, Vairimorpha sp. NIS M12. In genomic DNA analysis of microsporidia, it was clarified that alkali EDTA solution treatment of spore was needed for genome extraction from Brazilian isolates. The banding patterns were different among the species of microsporidia in pulse-field gel electrophoresis, suggesting that this method useful tool for identification of microsporidia. Established protocol in this report could apply for all isolates used, and this restult demnonstrated the importance of cloning. Less