高温浸酸防治家蚕微粒子病胚种垂直传播的机理研究

Pebrine caused by Nosema bombycis is the only statutory quarantine object in sericulture. Transovarial transmission (TT) of the disease leads to huge damages to the silkworm eggs production. The preliminary experiment of the research group has proved that acid treatment with high temperature (47.5℃, 6-7min) has obvious effect on the TT of pebrine disease. The real-time PCR indicated that the pathogens in the newly hatched silkworms from the eggs with high temperature acid treatment reduce more than 97%. Previous researches thought that the high temperature acid treatment could kill or inhibit the meronts. However, there is still lack of strong evidence about the hypothesis. Furthermore, How does it affect the basal metabolism of the microsporidia? Are the silkworm eggs involved in the defense against the pathogen? The questions are still unknown. In the project, real-time PCR、paraffin and RNA-Seq methods will be used to clarify the function of the treatment and its mechanism. The results will provide scientific basis for its popularization and application in sericulture.

家蚕微粒子病是家蚕微孢子虫引起的唯一法定检疫蚕病，其胚种垂直传播对养蚕业尤其是蚕种生产造成严重危害。项目组前期实验已经证明，在产卵后20h高温浸酸（47.5℃，6-7min）处理带毒蚕卵对家蚕微粒子病胚种传染有明显的防治效果。实时荧光定量PCR检测也表明，与常规浸酸（46℃，5min）相比，高温浸酸处理后孵化的蚁蚕中家蚕微孢子虫数量减少97%以上。前人认为高温浸汤或浸酸处理可以杀死家蚕微孢子虫裂殖体，从而抑制病原的增殖。然而，该假说尚缺乏直接可靠的证据。而且，高温浸酸如何影响家蚕微孢子虫基础代谢从而致其死亡？家蚕胚胎是否参与对微孢子虫的抑制？这些问题都还有待阐明。本项目拟采用实时荧光定量PCR、石蜡切片荧光染色、高通量转录组测序等工具，阐明高温浸酸在家蚕微粒子病胚种垂直传播中的防治作用及其机理，为生产上应用高温浸酸防治家蚕微粒子病的胚种传播提供科学依据。

家蚕微粒子病是家蚕微孢子虫引起的唯一法定检疫蚕病，其胚种垂直传播对养蚕业尤其是蚕种生产造成严重危害。项目组前期工作发现，蚕种高温即时浸酸对家蚕微粒子病胚种传染有明显的防治效果。本研究利用荧光定量 PCR对卵龄 24～240 h蚕卵中的家蚕微孢子虫进行定量检测，发现高温即浸处理后每日取样蚕卵中的病原量显著低于阳性对照组，且明显低于常规即浸组，表明蚕种高温即浸处理对家蚕微孢子虫具有显著的灭活作用。对蚕卵孵化后饲养至3龄眠和5龄起的幼虫逐条进行显微镜镜检，结果显示高温即浸处理对家蚕微粒子病的相对防治效果分别为92.30±2.95%和81.80±0.06%，结茧率达83.33%～88.29%，确证高温即时浸酸对家蚕微粒子病的胚种垂直传播有显著的防治作用；通过吉姆萨染色法对高温浸酸适期（卵龄18～22 h）带毒蚕卵中家蚕微孢子虫的发育状态进行观察，发现带毒蚕卵中的病原为双核、四核裂殖体。浸酸处理48h后，阳性对照组和常规即浸处理组蚕卵中均能观察到裂殖体，高温处理组则未能发现病原，推测高温即浸处理的作用有效靶点为家蚕微孢子虫的裂殖体阶段。通过RNA-Seq技术分析了高温即浸处理后家蚕胚胎和病原微孢子虫的基因表达变化，对差异表达基因进行GO功能分析和KEGG pathway分析，发现高温即浸处理能够刺激蚕卵免疫应答增强对病原的抗性，同时影响家蚕微孢子虫的基础代谢导致病原裂殖体死亡。研究结果为生产上应用高温即浸防治家蚕微粒子病提供了科学依据。