凡纳滨对虾繁育系统菌群结构及优势菌遗传多样性分析

Microbial community structure and genetic diversity of dominant bacteria in Litopenaeus vannamei breeding system

【目的】探讨凡纳滨对虾(Litopenaeus vannamei)工厂化繁育系统内发生细菌性玻化症(shrimp postlarva bacterial vitrified syndrome,BVS)时期可培养微生物的菌群特征以及优势病原菌的遗传多样性。【方法】采用细菌体外培养方法结合基因测序技术对不同育苗阶段的亲虾、受精卵、无节幼体、蚤状幼体、糠虾幼体、仔虾,及其育苗池水和饵料内可培养细菌菌群的组成与结构特征进行研究,并通过多位点序列分析(multilocus sequence analysis,MLSA)方法解析病原菌的遗传多样性。【结果】系统内分离纯化的526株具有典型形态差异和群落优势的细菌分属于4门5纲16目24科38属113种。在纲水平上γ-变形菌纲(Gammaproteobacteria)丰度最高,共453株,占总分离株的86.1%;在属水平上弧菌属(Vibrio)丰度最高,共369株,占总分离株的70.2%;在种水平上,溶藻弧菌(Vibrio alginolyticus)为最优势种,共112株,占总分离株的21.3%,并且分布于整个繁育系统,在饵料中具有最高丰度。多元关联分析表明,随着对虾幼体的发育,饵料对幼体体内可培养细菌的菌群结构影响逐渐增加。对112株潜在溶藻弧菌的MLSA分析表明,其中100株菌株进一步确认为溶藻弧菌。进一步利用MLSA构建系统发育树分析其遗传多样性发现,100株溶藻弧菌分为9个簇,分离自同类样品的菌株广泛分布在不同的簇中。【结论】在BVS发生时期,凡纳滨对虾工厂化繁育系统中具有丰富的可培养微生物种类。对虾幼体发育过程中,饵料对幼体体内可培养细菌的菌群结构具有重要影响。溶藻弧菌为凡纳滨对虾工厂化繁育系统中的优势弧菌,分布于整个繁育系统,且具有丰富的遗传多样性。本研究为解析对虾繁育系统可培养微生物演替规律提供了数据支撑,也为对虾苗期病原防控和健康养殖提供了科学依据。

[Objective]To investigate the characteristics of culturable microorganisms and the genetic diversity of dominant pathogens during shrimp postlarva bacterial vitrified syndrome(BVS)of Litopenaeus vannamei in the factory breeding system.[Methods]The composition and structural characteristics of culturable bacterial communities in shrimp(parent shrimp,fertilized egg,nauplius,zoea,mysis,and postlarva),water,and bait samples at different breeding stages were studied by in vitro bacterial culture method combined with gene sequencing technology,and the genetic diversity of the pathogenic bacteria was studied by multilocus sequence analysis(MLSA).[Results]The 526 isolates with typical morphological differences and community dominance belonged to 113 species,38 genera,24 families,16 orders,5 classes,and 4 phyla.At the class level,Gammaproteobacteria had the highest abundance,with 453 isolates(86.1%of the total isolates).At the genus level,Vibrio had the highest abundance,with 369 isolates(70.2%).At the species level,V.alginolyticus was the most dominant species,with 112 isolates(21.3%).V.alginolyticus was distributed in the whole breeding system and had the highest abundance in the baits.The multiple correlation analysis showed that the influence of baits on the structure of culturable bacterial community increased gradually with the development of larvae.The MLSA confirmed 100 out of the 112 potential isolates as V.alginolyticus.MLSA was further employed to construct a phylogenetic tree for revealing the genetic diversity of the isolates.The 100 V.alginolyticus isolates were classified into 9 clusters,and the isolates from the same type of samples showed dispersed distribution in different clusters.[Conclusion]During the occurrence of BVS,abundant culturable microorganisms existed in the factory breeding system of L.vannamei.The baits exerted effect on the structure of culturable bacterial community in the larvae.V.alginolyticus was the dominant species of Vibrio in the factory breeding system of L.vannamei,with wide distribution and high genetic diversity in the breeding system.The results of this study provide data support for analyzing the succession rules of culturable microorganisms in shrimp breeding system and lays a foundation for pathogen prevention and control and healthy breeding of shrimp.