海洋蛭弧菌生物防治对虾苗期细菌性病害的分子生态学研究

Bdellovibrio-like organisms (BLO) are small-sized, parasitic bacteria which proliferate by predation on other Gram-negative bacteria，and have important application value for biocontrol of deleterious bacteria. However, there is little information available concerning the response relationship between community structures of BLO and bacteria, let alone the mechanism of action. This situation severely limits the reasonable application of BLO. As is well known, great economical loss owing to the high mortality rate of larval shrimp during hatchery phase frequently occurs, and this is mainly due to the outbreak of bacterial diseases. In this project, the diversity and dynamics of marine BLO and bacteria in larviculture water, from three hatcheries of shrimp (Litopenaeus vannamei), will be firstly explored and compared based on Illumina high-throughput sequencing analysis of 16S rRNA gene. The variation of BLO and bacteria contents in water will be examined by quantitative PCR and other methods. Then predominant BLO will be isolated and identified, and physiological and ecological characteristics of them will be further studied. Subsequently, three kinds of dominant BLO will be selected, and their therapeutic effect on shrimp larvae infected with pathogenic bacteria will be evaluated. Furthermore, the selected BLO will be sent back to larviculture water to investigate the effects of them on community structure of bacteria, as well as on growth and survival of larvae. The results would reveal the spatial and temporal changes in community structures of BLO and bacteria in water during shrimp larviculture. The response relationship between BLO and bacterial structures, including the action mechanism will be elucidated. And the ecological function of dominant BLO in larviculture water would be clarified. These findings can provide scientific foundation for applying marine BLO on biocontrol of bacterial diseases in shrimp larviculture and other mariculture environments.

蛭弧菌类生物(Bdellovibrio-like organisms)，简称蛭弧菌，是一类以裂解其它细菌为生的小型寄生性细菌，在生物防治有害细菌上具有重要应用价值。目前对蛭弧菌与细菌群落结构间响应关系和机制还不清楚，限制了蛭弧菌合理应用。对虾育苗期极易暴发细菌病害，经常造成幼体大量死亡，损失巨大。本项目以16S rRNA基因为靶标，采用Illumina高通量测序技术，分析和比较3个凡纳滨对虾苗场育苗过程中海洋蛭弧菌和细菌多样性变化；定量PCR等检测菌含量；分离鉴定并选择3种优势蛭弧菌，掌握其生理生态特性；评价它们对幼体感染病原菌的治疗效果，并进一步研究它们回接正常育苗水体后，对细菌群落更替和幼体生长存活影响。揭示对虾育苗水体蛭弧菌和细菌群落结构的时空演替特征、响应关系和作用机制，阐明优势蛭弧菌在育苗水体中的生态学功能，为海洋蛭弧菌在对虾育苗及其他海水养殖环境中防治细菌性病害提供科学依据。

蛭弧菌类生物(Bdellovibrio-like organisms)，简称蛭弧菌，是一类以裂解其它细菌为生的小型寄生性细菌，在防治有害细菌上具有重要应用价值。对虾育苗期极易暴发细菌尤其是弧菌病害，经常造成幼体大量死亡。(1)本项目通过16S 宏基因组测序等技术对广东湛江三个凡纳滨对虾(Litopenaeus vannamei)苗场育苗期水体菌群多样性研究显示，苗池菌群随幼体发育呈明显演替，不同苗场菌群在育苗后期总体上趋向一致，并以变形菌门、拟杆菌门和放线菌门占绝对优势，科水平上则以红杆菌科、黄杆菌科和微杆菌科最为优势，优势科丰度分布与水体理化因子有着明显相关性。(2)建立了基于Illumina MiSeq平台的海洋蛭弧菌16S 宏基因组高通量测序方法，发现育苗后期苗池水体海洋蛭弧菌种群结构总体上也趋向一致，但其多样性与总的细菌多样性间无明显相关性；苗池海洋蛭弧菌与弧菌含量间也无明显相关性。高通量测序和分离培养鉴定都表明对虾苗池海洋蛭弧菌以嗜盐噬菌弧菌科(Halobacteriovoracaceae)为主，并以类群(cluster) XIII为绝对优势种群。(3)通过海水共培养研究了三株不同种群海洋蛭弧菌对对虾病原弧菌的控制效果，发现蛭弧菌和病原弧菌的密度均显著影响捕食效率。(4)进一步评估其中两株蛭弧菌对对虾幼体感染荧光弧菌后的保护作用，结果显示单一海洋蛭弧菌很难控制急性暴发性的荧光弧菌病。(5)高浓度海洋蛭弧菌投放正常育苗水体后，一定时间内对水体菌群多样性和种群结构有着明显影响。综上所述，海洋蛭弧菌在调控对虾苗池细菌含量和种群多样性上起重要作用，利用海洋蛭弧菌防控苗期弧菌病害有一定效果，但单一菌株的作用有其局限性。蛭弧菌类型、投菌量、作用时间、病原类型以及苗池本底蛭弧菌种群结构都是影响防控效果的重要因素。本研究丰富和深化了对虾育苗细菌生态学内容，对海洋蛭弧菌与宿主菌的互作有了新的认识，从菌群演替角度探讨了海洋蛭弧菌生物防控苗期细菌病害的分子生态学机制，为海洋蛭弧菌在海水养殖特别是对虾育苗中的合理应用提供了科学依据。