基于生态科赫法则的对虾白便综合症病因学机理研究

凡纳滨对虾(Litopenaeus vannamei)白便综合症(white feces syndrome, WFS)因致病机理不明仅以典型病症命名。相关性分析认为WFS是多病原菌协同作用，肠道菌群失衡造成的，但缺乏因果关系证据。本项目拟结合Spike-in质粒绝对定量，扩增子和深度宏基因组监测对虾养殖和WFS过程中肠道菌群和毒力基因组成。结构方程模型明确WFS发生环境诱因。毒力基因-表型（WFS严重程度）三角测量确定导致病害的毒力基因，组装指示毒力基因所在contigs，或/和FishTaco耦合潜在多病原菌与指示毒力基因。选择性分离，生态科赫法则模拟WFS暴发条件（如环境诱因、多病原菌侵染时序及比例），因果关系确定多致病菌。解析病原菌如何协同突破对虾肠道定殖拮抗和免疫反馈，潜在的互作通路，揭示WFS的病因学机理。同时，建立qPCR检测的指示毒力基因丰度为自变量，定量预测WFS技术。

The etiological mechanism of shrimp (Litopenaeus vannamei) white feces syndrome (WFS) is unknown, thus is named by its typical symptoms. Microbiome-wide association studies have inferred that WFS is attributed to polymicrobial pathogens and dysbiosis of the gut microbiota, while there is no causal evidence. This proposal will integrate spike-in plasmid, amplicons and deep metagenomics sequencing to track the profiles of gut bacterial community and their coded virulence genes as shrimp ages and over WFS progression. A structural equation model is applied to identify the driving factors of WFS. Virulence genes-WFS severity triangulation model is used to screen virulence genes that cause WFS. The hosts of these virulence genes are inferred by assembling their carrying contigs, and/or by FishTaco for coupling pathogens and virulence genes. Accordingly, we could simulate the emergent conditions of WFS (i.e. driving factors, infection order and proportion of polymicrobial pathogens) to causally determine the pathogens of WFS by the ecological Koch postulates. We further uncover how polymicrobial pathogens overcome the shrimp colonization resistance and immunity, and the underlying interactive pathways. Additionally, we develop a technique that quantitatively predicts WFS incidences by using qPCR assayed abundances of WFS-discriminatory virulence genes as independent variables.