文蛤浮游期发病幼虫细菌群落分析和致病菌的分离鉴定  

Analysis of bacterial community and identification of pathogenic bacteria in the diseased clam larvae of Meretrix petechialis

作　　者：隋炜金 王鸿霞[1] 刘保忠[1,2] SUI Weijin;WANG Hongxia;LIU Baozhong(Key Laboratory of Experimental Marine Biology,Institute of Oceanology,Chinese Academy of Sciences,Qingdao 266071,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院海洋研究所,实验海洋生物学重点实验室,山东青岛266071 [2]中国科学院大学,北京100049

出　　处：《水产学报》2023年第6期154-162,共9页Journal of Fisheries of China

基　　金：现代农业产业技术体系(CARS-49);山东省重点研发计划(农业良种工程)项目(2021LZGC029)。

摘　　要：为研究文蛤育苗过程中幼虫病害及其主要致病菌,实验通过构建细菌16S rDNA克隆文库、病原菌分离纯化、人工感染和药敏实验等方法对育苗场发病的浮游期文蛤壳顶幼虫样品进行了系统分析。结果显示,发病的文蛤壳顶幼虫细菌群落多样性低,地中海弧菌占比高达75%以上,推断其可能为引发此次幼虫发病的主要致病菌。从发病幼虫的匀浆组织中分离获得该优势菌株,测序及系统发育鉴定为地中海弧菌。人工感染实验确定了其致病性,菌株US2-01在1.0×10^(6) CFU/mL的菌液浓度下浸泡感染文蛤浮游幼虫,96 h累计死亡率为84%。药敏实验表明,地中海弧菌菌株US2-01在12种抗生素的测试中对常用的青霉素、氨苄西林、红霉素等抗生素具有一定的耐药性,对四环素和多西环素中度敏感,对头孢他啶、庆大霉素、卡那霉素等其余7种抗生素呈现高度敏感。本研究首次报道了地中海弧菌为文蛤浮游期幼虫致病的一种潜在病原,研究结果可为文蛤幼虫疾病研究及贝类苗种培育过程中的病害防控提供科学依据。The clam Meretrix petechialis is an important commercial bivalve mollusk widely distributed in China,Japan and Korea.The development of clam culture depends on the plenty of seed supply.However,mass mortalities of M.petechialis larvae often occur during hatchery rearing.Outbreak of disease is the main bottleneck for successful spat production.So,the characterization of pathogenic bacterial strains causing epizootics in larval culture is essential for developing efficient management strategies to prevent and control disease outbreaks in clam hatchery.In present study,the moribund umbo larvae of clam sampled from hatchery were analyzed by constructing bacterial 16S rDNA clone library.The results showed that the bacterial community diversity of moribund larvae was low,and the Vibrio mediterranei clones accounted for more than 75%in the library,indicating they may be the causative bacteria of clam larvae mortality.Furthermore,the dominant strain was isolated from the moribund larvae,and identified as V.mediterranei by 16S rDNA sequencing and phylogenetic analysis.The virulence of the obtained strain US2-01 was determined by artificial infection test,a dose effect was observed for the pathogenicity of US2-01 on clam larvae.The onset time of US2-01 infection was related to its concentration,and the onset time was delayed in the low concentration group(1.0×10^(4) CFU/mL).The cumulative mortality rate of infected clam larvae was 84%when they were immersed in final concentration 1.0×10^(6) CFU/mL US2-01 for 96 hours.The clinical signs were observed during the artificial infection experiment,the treated larvae gradually lost their swim ability,showing symptoms such as the shedding of veliger cilia,partial tissue dissociation,and the empty stomach,which were consistent with the symptoms of the naturally diseased larvae in hatchery.Finally,the drug sensitivity test was performed and the results showed that V.mediterranei strain US2-01 was resistant to commonly used antibiotics such as penicillin,ampicillin and erythromycin

关 键 词：文蛤幼虫 地中海弧菌 16S rDNA克隆文库 药敏实验 

分 类 号：S944.3[农业科学—水产养殖]