一株家蚕异型微孢子虫感染特征和分类地位

Infection characteristics and classification of a microsporidian isolated from silkworm

摘要:

【背景】家蚕微粒子病是一种对蚕业生产危害巨大的蚕病，该病病原是蚕种生产唯一检疫对象，而家蚕病原性微孢子虫种类多、来源复杂，给蚕种生产微粒子病的防控增加了难度。【目的】研究一株从蚕种检疫样品中分离的微孢子虫(命名为GXM15)的致病性和分类地位，鉴定并分析其来源，完善家蚕病原性微孢子虫分类和数据库，为蚕种生产控制家蚕微粒子病提供参考依据。【方法】采用生物试验方法测定GXM15微孢子虫对家蚕的半数感染浓度(IC50)和胚种传染率；显微镜观察GXM15微孢子虫孢子形态，利用透射电子显微镜观察GXM15微孢子虫的超微结构；采用PCR扩增、T克隆和测序获得GXM15微孢子虫的SSU rRNA基因和ITS片段DNA序列，并利用MEGA 5.0和DNAstar软件构建GXM15微孢子虫的系统发育树和遗传距离分析。【结果】GXM15微孢子虫对家蚕的IC50为8.29×104个/mL，是家蚕微孢子虫(Nosema bombycis，Nb)的2.28倍；GXM15微孢子虫对家蚕的胚种传染率为3.6%，明显低于Nb；GXM15微孢子虫形态呈短卵圆形，大小为(2.05±0.20)× (3.25±0.30) μm，GXM15微孢子虫体积是Nb微孢子虫的2.19倍；GXM15微孢子虫超微结构具双核，极丝13圈，极丝倾斜角约45°，符合Nosema属的特征；GXM15微孢子虫SSU rRNA基因在系统发育树中位于Nosema属分支中，遗传距离分析表明GXM15微孢子虫与Nb同属异种，是一株新微孢子虫。【结论】GXM15微孢子虫是一株家蚕病原性微孢子虫，根据GXM15微孢子虫致病性和分类地位研究，可以为蚕种生产防控家蚕微粒子病提供参考依据。

Abstract:

[Background] Silkworm pebrine is a serious disease that has caused great damage to the silkworm production and its pathogen is the unique quarantine object that for silkworm egg production. The sources of pathogenic microsporidan of silkworm B. mori are variety and complex, which increases the difficulty of controlling pebrine in the silkworm egg production. [Objective] The present study were conducted to investigate the pathogenicity and classification feature of a microsporidan which was isolated from the silkworm mother moths (name GXM15), and its source was analysed, which will improve the classification and database of silkworm pathogenic microsporidian and will be provide reference basis for the control of silkworm pebrine in silkworm egg production. [Methods] The 50% infectious concentration (IC50) and the germinative infection rate of GXM15 were investigated with biological test methods. The spore morphology of GXM15 was observed with optical microscope, its internal structure was observed under transmission electron microscope, the SSU rRNA gene and ITS sequence of GXM15 were obtained by PCR amplification, clone and sequencing, the phylogenetic tree based on SSU rRNA sequences was constructed with the software of MEGA 5.0, and the genetic distance of SSU rRNA and ITS sequence were analyzed with software of DNAstar. [Results] The IC50 of GXM15 is 8.29×104 spores/mL, which is 2.28 times to that of Nosema bombycis (Nb) and the germinative infection rate of GXM15 is 3.60%, that is significantly lower than that of Nb. The spore of GXM15 is short oval in shape and (2.05±0.20)× (3.25±0.30) μm in size, and the volume of GXM15 is 2.19 times to that of Nb. There are binuclear and has 13 polar filament coils in the spores of GXM15, and its angles of polar filament is 45°, that is conform to the taxonomic features of Nosema. The SSU rRNA of GXM15 is grouped in the Nosema clade, and the genetic distance analysis of SSU rRNA and ITS show that GXM15 and other Nosema microsporidian are belong to different species of the same genus, GXM15 is a new microsporidian. [Conclusion] GXM15 is a new pathogenic microsporidan of Bombyx mori, and study on the pathogenicity and classification feature of GXM15 microsporidan can provide basis for prevention and control of the silkworm pebrine.