凡纳滨对虾幼体肠道菌群演替特征及其与苗期病害相关性

The intestinal microbiota plays important roles in immunity, nutrient processing and disease resistance for aquatic animals. The water column, associated with larviculture of the Pacific shrimp, Litopenaeus vannamei, affect the colonization and succession of bacterial community in larval intestinal tract since water act as surrounding environment contacted both by larvae and microorganisms. During the early development phase, shrimp larvae experience extensive metamorphosis which may lead to poor physical fitness. Subsequently, pathogens and/or opportunistic pathogens invade easily into the intestinal tract and disrupt the balance of intestinal microbiota. Thus shrimp larvae frequently suffer mass mortality due to bacterial diseases associated with intestinal tract. To date, there is little information available about the intestinal microbiota in shrimp larvae, let alone their succession and relevance with bacterial diseases. In this project, Illumina MiSeq high-throughput sequencing, targeted the 16S rRNA gene, is taken to investigate the initial colonization and dynamics of the aquatic and intestinal microbiota for healthy and diseased larvae during the nursery phase. Following that, the dominant bacteria are isolated from intestines of healthy larvae, and identified with potential to become probiotics. Then the ability of the top two beneficial bacteria, inoculated to initial larviculture water, will be evaluated about their colonization in larval intestinal tract, their effects on larval survival and growth, as well as their prophylaxis effect on typical larval vibriosis. The results of this project would reveal the structure and dynamics of intestinal microbiota of the shrimp larvae, particularly with respect to their relationships to occurrence of bacterial diseases. The mechanisms of action of the two autochthonous probiotics on modulating structure of bacterial community, on improving larval performance as well as on preventing colonization by pathogens would be elucidated. These findings would provide effective paradigm to manipulate structure of intestinal microbiota of shrimp larvae which are increasingly interested using as an alternative means for disease prevention in larviculture.

肠道微生物对水产动物营养、免疫与抗病起着重要作用。育苗水体作为幼体与微生物的共同介质深刻影响幼体肠道菌群定植与演替。对虾幼体阶段频繁变态而体质脆弱，机会病原菌极易入侵幼体肠道而导致菌群失衡，造成幼体细菌病害频发而大量死亡。目前对对虾幼体变态过程中肠道菌群结构演替及其与病害相关性缺乏深入认识。本项目以16S rRNA基因为靶标，采用Illumina MiSeq高通量测序研究比较凡纳滨对虾(Litopenaeus vannamei)正常和发病苗池幼体肠道菌群结构差异；从健康幼体肠道分离鉴定两种优势有益菌，评价它们接入水体后在水体和幼体肠道定植能力，对幼体生长存活影响及对弧菌病预防效果。揭示对虾苗期幼体肠道菌群结构时空演替特征及其与病害发生关联，阐明两种土著有益菌对幼体肠道菌群结构调控效应和预防弧菌病可能机制，为应用土著有益菌微生态调控对虾幼体肠道菌群结构以预防苗期病害提供科学依据。

肠道菌群对水产动物营养、免疫与抗病起着重要作用，深入了解对虾幼体阶段肠道菌群演替特征及其与病害发生相关性，有助于调控幼体肠道菌群以预防苗期病害和促进健康育苗。(1)本项目首先通过高通量测序评估了4种DNA提取试剂盒对凡纳滨对虾幼体肠道菌群的提取效果，发现不同试剂盒对幼体肠道菌群提取存在明显偏差，其中细菌试剂盒因具有重复性最好等优点建议优先使用。(2)比较了两个苗场凡纳滨对虾幼体肠道菌群演替特征，解析了健康和发病幼体肠道菌群差异及其与病害发生相关性。结果表明凡纳滨幼体肠道菌群多样性随幼体发育总体上先降后升，在溞状后期显著偏低；肠道菌群以变形菌门占绝对优势，其次为拟杆菌门、厚壁菌门和放线菌门；平行苗池间幼体肠道菌群结构异质性与随机性随时间而增加；幼体肠道菌群结构随着投饵起始及饵料转换明显更替，并以溞状至糠虾阶段演替最为显著；CX苗场发病幼体在大量死亡前其肠道菌群结构已与正常幼体差异显著，尤其是弧菌科等丰度显著升高。(3)从对虾幼体肠道和水体分离到多株潜在益生菌株，包括一个已发表细菌新种；对育苗环境弧菌多样性和抗药性研究发现，溶藻弧菌最为普遍存在，且多数弧菌的耐药性较为严重。(4)评估了4株土著菌和3株芽孢杆菌对凡纳滨对虾幼体感染荧光弧菌的保护效应，其中能较明显减缓荧光病的两株益生菌都难以持续定植于水体和幼体肠道。本研究揭示了凡纳滨对虾育苗期幼体肠道菌群多样性及其演替特征，明确了饵料类型是影响菌群演替的关键因素，发现幼体肠道菌群结构偏离正常状态和特定类群如弧菌的急剧扩增极易导致疾病暴发，探索了有益菌对幼体感染病原弧菌的保护效应及其对肠道菌群影响，为通过调节肠道菌群预防对虾苗期病害提供了科学依据。