中华蟾蜍蝌蚪肠道菌群的结构及其影响因素研究

Amphibians possess a unique evolutionary status, and gut microbiota structure of amphibians is important for us to understand the symbiotic relationships between vertebrates and their associated gut microbiota. However, the contributions of various environmental and biological factors, e.g. host genetics, habitat conditions and food types, to the the variations of gut microbial structure of amphibians remain elusive. In the proposed project, we will collect Asiatic toad (Bufo gargarizans) tadpoles in Huaihe River Basin-Dabie Mountain area, and investigate the the gut microbial structure of Asiatic toad tadpoles using 16S rDNA amplicon sequencing. In addition, we will utilize mitochondrial and microsatellite molecular markers to explore the effects of host genetics on the gut microbiota structure. Furthermore, the influences of host genetics, habitat conditions and food types on the gut microbial structure will be demonstrated in transplantation experiments of Asiatic toad eggs by controlling food types and water conditions. This project will provide strong insights into the symbiotic relationships between amphibians and gut microbiota, and scientific evidences on gut microbiota succession in the metamorphosis development of Asiatic toads.

两栖动物进化地位特殊，其肠道菌群结构对于解析脊椎动物与肠道菌群的共生机制意义重大。然而宿主的遗传背景、生境条件和食物类型等影响因素与两栖动物肠道菌群结构之间的关系目前尚不清楚。本项目拟在淮河流域-大别山区采集中华蟾蜍（Bufo gargarizans）蝌蚪，运用16S rDNA扩增子高通量测序技术，检测该物种在不同生态环境中的肠道菌群结构，并利用线粒体和微卫星分子标记，探究肠道菌群结构的宿主遗传效应。通过进一步开展中华蟾蜍卵带的移植实验，控制食物类型和水环境，阐明宿主的遗传背景、生境条件和食物类型等因素对肠道菌群结构的影响。本项目将为揭示两栖动物与肠道菌群的共生机制提供强力支撑，并为研究肠道菌群在中华蟾蜍变态发育过程中的演替奠定基础。

健康的肠道菌群对于维持宿主营养代谢、免疫调节等生命活动至关重要。肠道菌群结构与功能受到诸多内源性（如遗传背景）和外源性（如食物类型）因子的影响。基于16S rRNA基因扩增子测序技术，本项目系统探究了宿主性别、遗传背景、生长发育阶段、饲养条件等内外源因子对中华蟾蜍（Bufo gargarizans）肠道菌群结构的影响作用。首先，基于两个地理种群的冬眠期中华蟾蜍肠道菌群研究，发现生长发育阶段决定了肠道菌群的结构差异，而性别和遗传背景却没有显著效应。然后，通过中华蟾蜍的卵带移植和人工饲养实验，不仅验证了饲养条件（如食物类型和投喂模式）对中华蟾蜍蝌蚪生长发育的重要性，而且揭示了不同饲养条件对中华蟾蜍蝌蚪肠道菌群结构的影响作用，为进一步阐明肠道菌群的功能奠定基础。最后，我们检验了基于粪便无创检测两栖动物蝌蚪肠道菌群结构的有效性，同时还发现不同DNA提取方法和不同测序批次会掩盖肠道菌群的生物学差异。本项目不仅能够深化我们对两栖动物肠道菌群结构及其影响因素的了解，而且有利于更科学地驯养中华蟾蜍，从而确保该物种开发利用的可持续性。