Study on karyotype and genome size of entomopathogenic microsporidians

微孢子虫的鉴定是基于光镜和电镜观察的形态特征。近年来，脉冲场凝胶电泳不仅为小单细胞生物的染色体研究提供了一种替代方法，而且还为小孢子虫的鉴定提供了一种选择。另一方面，在这些研究中使用的微孢子虫不是克隆的。从患病的家蚕幼虫中已经发现了微孢子虫的混合感染。本研究首先研究了混合侵染条件下寄主与昆虫细胞培养物的相互作用，并利用光镜对克隆的小孢子虫核型进行了估计。然后，我们对克隆的小孢子虫进行了脉冲场凝胶电泳，并讨论了虫食性小孢子虫的种内和种间变异。在家蚕和frugiperda Spodoptera SF21AEII细胞培养中，bombycis NIS . More 001在参与混合感染时，对其他小孢子虫Nosema sp. NIS M11和Vairimorha sp. NIS M12具有优势，但优势并不通过竞争消除小孢子虫，并且小孢子虫可以在同一寄主中共存。因此，在讨论微孢子虫的特性和鉴定时，强烈建议克隆微孢子虫是重要的。用极限稀释法在昆虫细胞培养中建立了几个小孢子虫的克隆体，并用Axioplan 2通用显微镜对克隆的小孢子虫的营养细胞染色体数进行了估计。微孢子虫Vairimorpha sp. NIS M12有13条染色体。在小孢子虫基因组DNA分析中，明确了从巴西分离株提取基因组需要碱法EDTA溶液处理孢子。脉冲场凝胶电泳显示不同种类微孢子虫的条带模式不同，提示该方法是鉴别微孢子虫的有效工具。本文所建立的方案适用于所有分离株，这一结果证明了克隆的重要性。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