基于高通量测序分析杂色鲍肠道菌群动态变化及其与病害相关性

Haliotis diversicolor in China has experienced mass mortality with diverse symptom and pathogens in grow-out stage of pond small abalone. Disease has been regarded as a bottleneck in developing the aquaculture industry. Intestinal microflora balance has a profound impact on the host health and disease through their function in the development and maintenance of host physiology, immunology and nutrition.Therefore, it was speculated that the susceptibility of small abalone to disease may due to the alteration of intestina microflora along with the change of seasons. By ribosomal tag pryosequencing of DNA and RNA, we are going to observe the dynamic changes of gut flora in the development of disease. And we would investigate the key point in developing process of disease and aquire the core bacterial flora in controlling intestinal microbial balance of abalone.Combining the data of the bacterial flora and environmental factors in abalone farmed pond water, and immune factors of host, we will further probe into the relationship between the intestinal flora and disease. All informations could help to understand the disease triggering mechanism and explore the protecting policy of the mariculture shellfish in coast of South of China. The results of this study would also help to rich invertebrate micro-ecological theory and bacterial etiology theory, and promote sustainable development of Blue Agriculture in China.

杂色鲍养成阶段病害频发已成为制约产业可持续发展的"瓶颈"，而季节转换期肠道菌群失调可能是病害爆发的潜在原因。本项目立足于动物微生态理论和肠道菌群失调理论，采用高通量测序技术，从DNA和RNA水平上对杂色鲍病害发生过程中肠道细菌类群结构及其变化进行动态监测；获得与病害发生相关的肠道菌群变化的节点及核心微生物类群；综合分析养殖水体中菌群结构，环境参数和宿主血细胞免疫因子的变化趋势，揭示肠道菌群与病害间的关联性，探讨杂色鲍病害发生机理和探索生态防治途径。研究结果将有助于丰富无脊椎动物肠道微生态学和鲍细菌性病因学理论，为促进"蓝色农业"的可持续发展奠定基础。

鲍是我国重要的增养殖经济贝类。自然资源锐减和市场需求，推动了鲍养殖业的迅猛发展，已达到集约化、产业化、工厂化的规模。养殖鲍产量2013年达110,380吨，占全世界产量的88.8%。但自2006年以来，季节转换期鲍病害爆发、并发、多发已成为制约产业发展的“瓶颈”。尽管对各种病原微生物已进行了较为深入的研究，但相对于鲍病害爆发症状的多样性，难以确认单一病原体是病害大规模爆发的主因，推测其共附生菌群失调可能是病害爆发的潜在诱因。立足于此，本项目分别选取了病害高发期（5月及12月）养殖鲍稚鲍和幼鲍，采用分子生态学技术与高通量测序技术，对其共附生菌群动态变化与病害相关性开展研究，从DNA 和RNA 水平上对杂色鲍病害发生过程中其共附生细菌类群结构及其变化进行动态监测；同时结合养殖水体中菌群结构，环境参数和宿主免疫指标的变化趋势，揭示鲍微生物群落与病害发生、发展、爆发间的关联性。.研究结果表明，鲍病害的发展是一个复杂的过程，微生物群落在这一过程中此消彼长，与养殖鲍健康状态息息相关：（1）鲍微生物群落结构的变化趋势与鲍病害进程维持着高度一致性。健康鲍与濒死鲍具有高丰度、高多样性和高均匀度，但其组成及结构完全不同的微生物群落结构；而处于病害发生过程中的病鲍却拥有优势菌群显著，极为失衡的微生物群落结构。（2）除肠道微生物类群外，肝胰腺也是检测微生物群落结构从而预测鲍病害发生重要器官；（3）弧菌菌群和支原体分别是稚鲍和幼鲍病害爆发的核心微生物类群；而玫瑰杆菌和嗜冷菌属可能是维系宿主健康的有益菌群；（4）相较于溶解氧、酸碱度，Toc等未发生明显变化的环境参数，环境温度月际变化较为明显，结合微生物群落变化数据，推测温度是影响菌群结构和组成的主要因素。（5）4月份温度波动，病害发生前期，鲍免疫活性突然增加以抵抗环境压力（如温度变化），5月份，温度升高，某些微生物类群丰度迅速增加，引起微生物群落结构整体失衡，此时鲍相关免疫指标下降。本研究结果改变了“单一的病原微生物是引发养殖鲍病害爆发主要原因”常规论断，在微生物群落结构变化与病害间有了新的认识，从微生物群落结构整体性变迁的节点了解病害发生的进程，并获得了与宿主健康状态相关的核心微生物类群，为深度解析宿主病害机制奠定基础；同时，为预知病害爆发，实时了解病害发生进程，进而在适宜阶段采取有效措施控制病害发展提供数据支持。