摘要
三疣梭子蟹(Portunus trituberculatus)是我国海产经济蟹类,近年来浙江省沿海的三疣梭子蟹养殖业发展非常迅速,但是病害发生频繁,成为其稳产、高产的主要限制瓶颈。养殖水体中微生物的多样性特别是异养细菌的菌群结构及其演替规律对于探明梭子蟹发病原因及制定病害防治措施具有重要意义,本文采用培养法和免陪养法对三疣梭子蟹养殖池塘细菌的群落组成进行了研究。
首先,对宁海县明港东坝三疣梭子蟹养殖塘水体中的可培养细菌进行了为期3个月(7、8、9月份)的跟踪调查,每个月采一次样,共分离纯化198株异养细菌,根据其菌落形态特征分为15个类群;16S rDNA基因的变性梯度胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)带型和基因序列分析表明,198株异养细菌分属于4个门,5个纲,16个科,26个属,56个种,81个发育型,主要属于γ-变形杆菌纲( Gammaproteobacteria, 130株, 63.1%)和芽孢杆菌纲(Bacilli,47株,22.8%)。通过比较7、8、9月份的异养细菌群落组成发现水体中的异养细菌群落类群演替规律如下:(1)81个发育型中没有一个种或发育型同时出现在3个月份;(2)同个种或发育型在2个月份中同时出现的有7个科8个属,其余9个科18个属仅在一个月份中出现。
对7-11月份三疣梭子蟹养殖水体中的弧菌数量及其群落演替进行了调查,结果表明,V. natriegens的大量出现不仅与病害无关,还可能具有重要的生态功能,而V. harveyi的大量出现可能与病害发生相关。
其次,应用不依赖于培养的分子生物学技术——构建16S rDNA克隆文库的方法调查了同一三疣梭子蟹养殖塘水体中7月份和8月份细菌的群落结构。通过2个16S rDNA克隆文库的构建,共获得了131条有效序列。相对于培养法而言,2个文库中细菌的多样性更丰富,可归属为5个门(变形菌门(Proteobacteria)、蓝细菌门(Cyanobacteria)、放线菌门(Actinobacteria)、CFB类群(Cytophaga-Flavobacterium-Bacteroides)以及厚壁菌门(Firmicutes)),优势类群为蓝细菌纲和γ-变形杆菌纲。
最后,我们对健康和发病的三疣梭子蟹(Portunus trituberculatus)养殖水体及其体液的弧菌数量和群落结构进行了比较研究。共分离得到72株弧菌可分为7个菌落类群,7个种,11个发育型。病害塘养殖水体的弧菌数量与健康塘相比,没有较大变化,但是蟹体体液中的弧菌群落结构变化明显,病害蟹弧菌平均数量较健康蟹增加了2个数量级,但弧菌种属多样性明显减少,健康蟹体液主要是4个种5个发育型(V.alginolyticus、V.campbellii、V.natriegens和V. harveyi(Ⅱ、Ⅲ)),而病害塘主要是1个种2个发育型(V.harveyi(Ⅳ)和(Ⅴ)),且V. harveyi(Ⅰ)的数量大大增加,成为优势发育型。这些结果似乎表明三疣梭子蟹发病与养殖水体中的弧菌数量及群落结构关系不大,而与蟹体中的弧菌数量及群落结构关系密切,可能与弧菌的个别种甚至同个种的个别发育型的优势生长有关。
Portunus trituberculatus is a kind of marine crabs of economic value in china. Aquaculture of Portunus trituberculatus in coast of Zhejiang province has developed rapidly. However, frequent occurrence of disease has already become the bottleneck of the high yield of Portunus trituberculatus. The investigation on diversity of microorganism in aquaculture water (especially the community structure and succession of heterotrophic bacteria) is of importance for find out the causation of disease and to frame the disease control measures.
First, a follow-up survey about heterotrophic bacteria in the water of Dongba Portunus trituberculatus rearing pond at Minggang of Ninghai county had been done from July to September. A total of 198 strains of heterotrophic bacteria were roughly clustered into 15 colony-based groups. DGGE finger-printing and 16S rDNA gene sequencing analysis showed that 198 strains of heterotrophic bacteria belongs to 4 phylums, which including 5 classes, 16 families, 26 genuses, 56 species and about 81 phylogenetic types. The dominant classes isγ-Proteobacteria (130 strains, accounted for 63.1%) and Bacilli (47 strains, accounted for 22.8%). By comparing the community composition of heterotrophic bacteria in pond water from July to September, it was found that the community succession rule of heterotrophic bacteria was as follows: (1) There was no species or phylogenetic type could been found in the three months simultaneously; (2) There were 7 families, 8 genera appeared in two months at the same time while the remaining 9 families and 18 genera appeared only in one month.
In addition, the amount and community succession of Vibrio were investigated in the pond water for Portunus trituberculatus rearing pond from July to November by means of the plate count and 16S rDNA sequences analysis. The survey results showed that: 1). The emergence of large amount of V. natriegens have no relation to the disease, and may have important ecological role instead. 2). The dominance of V. harveyi in the pond may be associated with disease.
Secondly, culture-independed method--16S rDNA clone library construction, was also used to investigate bacterial community structure in the same water of Portunus trituberculatus rearing pond in July and August. One hundred and thirty one effective sequences from two 16S rDNA clone libraries had been obtained. The diversity of bacteria in two clone library was richer than that revealed by culture-depended method. All of the bacteria could be attributed to five phylums (Proteobacteria, Cyanobacteria, Actinobacteria, CFB group (Cytophaga-Flavobacterium-Bacteroides) and Firmicutes). The dominant groups of bacteria were cyanobacteria andγ-Proteobacteria.
Finally, the amount and community succession of Vibrio were comparativey investigated in the pond water and body fluid in healthy and diseased Portunus trituberculatus rearing pond. The total 72 strains of Vibrio could be divided into 7 groups, 7 species and 11 phylogenetic types. The number of Vibrio had no significant difference in the water of both ponds. But the community structure of Vibrio in body fluid of the crab changed obviously. The average number of diseased crab increased by two orders of magnitude compared to the healthy one, and the diversity of Vibrio species decreased significantly, it was changed from 4 species (V.alginolyticus, V.campbellii, V.natriegens and V. harveyi (Ⅱ,Ⅲ) into 1 species (V.harveyi (Ⅳ,Ⅴ)). In addition, V. harveyi (Ⅰ) became the dominant phylogenetic types. These results seems to indicate that the disease occurrence in Portunus trituberculatus has no direct relation with the amount and community composition of Vibrio in the pond water, but closely related to the amount and community composition of Vibrio in crab, and mainly related to the diversity of Vibrio, individual species of Vibrio or even the individual development patterns of the same species.
引文
陈萍,李吉涛,李健,刘淇,刘萍.溶藻弧菌对三疣梭子蟹抗氧化酶系统的影响[J], 海洋科学. 2009 , 33(5): 59-63.
国家海洋局.海洋调查规范[M].北京:海洋出版社, 1975.
高尚德,陈旭仁,吴以平.中国对虾养成后期间虾池水体和底泥中细菌含量的变化[J].水产学报, 1994, 18(2):138-142.
郭平,许美美.对虾养殖池水域环境细菌的动态变化[J].海洋与湖沼, 1994, 25(6):625-630.
韩一凡,莫照兰,李杰等.溶藻弧菌的PCR快速检测方法[J].中国海洋大学学报. 2009, 39(6): 1237-1240.
何伟贤.三疣梭子蟹养殖常见病及防治办法[J].水产科学. 2004, 25(5): 29-31.
金珊,王国良,薛良义等.海水网箱养殖水域异养细菌和弧菌的数量动态[J].海洋渔业, 1999, 21(4):154-156.
李秋芬,曲克明,陈碧鹃等.老化虾池生态系中几类主要细菌的季节变化特征[J].海洋水产研究, 2002, 23(2):12-28.
林克文.三疣梭子蟹养殖及病害防治[J].科学养鱼, 2002,(3): 24-25.
李筠,吕艳,李军等.苗期中国对虾幼体异养细菌区系及其变化与病害发生的关系[J].中国海洋大学学报, 2004, 34(6): 1003-1007.
柳承璋,宋林生,吴青.分子生物学技术在海洋微生物多样性研究中的应用[J].Marine Sciences, 2002, 26(8):27-30.
刘淇,李海燕,王群,刘萍,戴芳钰,李健.梭子蟹牙膏病病原菌--溶藻弧菌的鉴定及其系统发育分析[J].海洋水产研究, 2007, .28(4):9-13.
刘真,邵宗泽.南海深海沉积物烷烃降解菌的富集分离与多样性初步分析[J].微生物学报, 2007, 47(5):869-873.
刘晶晶,陈全震,曾江宁等.海水养殖区微生物生态研究.浙江海洋学院学报(自然科学版) [J], 2006, 25(1):72-77.
马绍赛,周诗赉,陈聚法,等.滩涂养殖菲律宾蛤仔死亡及生态环境效应调查研究[J].海洋水产研究, 1997, 18(2): 1-8.
倪纯治,林燕顺等.海水养虾池的几种致病弧菌生态[J].台湾海峡. 1995, 14(1): 73-79.
潘晓艺,沈锦玉,余旭平等.水产养殖中枯草芽孢杆菌的分子鉴定.水生生物学报[J], 2007, 31(1):139-141.
钱丽君,张德民,徐小红.应用DGGE分析三疣梭子蟹养殖塘底泥细菌的多样性[J].水产学报, 2007, 31(2): 204-210.
孙苏燕,张德民,钱丽君,等.三疣梭子蟹养殖塘表层底泥异养细菌群落比较研究[J].水产学报, 2010, 34(5): 820-828.
施慧,许文军.徐汉祥.梭子蟹酵母菌人工感染实验和组织病理学初步研究[J].海洋水产研究. 2005, 26(2): 48-52.
温丹,张德民,初航.网箱养殖海区底泥产芽孢细菌多样性[J].海洋与湖沼, 2009, 40(5): 615-621.
王国良,金珊,李政,等.三疣梭子蟹(Portunus trituberculatus)乳化病的组织病理和超微病理研究[J].海洋与湖沼,2006a, 37(4): 297-304.
王国良,金珊,陈寅儿,李政,三疣梭子蟹肌肉乳化病的病原及其致病性研究,海洋科学进展[J],2006b, 24(4): 526-531.
王建平;余晓巍;周志强;刘长军.宁波市三疣梭子蟹主要病害流行特征及应对措施[J]. 河北渔业,2008, (9): 49-53.
王晓颖,席峰,袁建军等.虾池沉积环境中若干功能菌及弧菌的时空变化[J].厦门大学学报(自然科学版) [J], 2006, 45,增刊:250-256.
王文兴.青岛太平角和即墨丰城沿海对虾养殖场异养菌群和条件致病菌的研究.黄渤海海洋[J], 1983, 1(2):133-137
吴后波,潘金培.弧菌属细菌及其所致海水养殖动物疾病[J].中国水产科学, 2001, 8(1): 89-93.
许文军,金海卫,丁跃平等.秋冬季节三疣梭子蟹Portunus trituberculatus(Mies)暂养常见疾病及防治措施[J].现代渔业信息, 2004, 19(7): 23-25.
许文军,徐汉祥,金海卫,张学舒,罗海忠,徐国辉.梭子蟹乳化病病原的研究.浙江海洋学院学报(自然科学版) [J]. 2003, 22(3): 209-213.
谢文阳,邱秀慧,海洋弧菌多样性[J],世界科技研究与发展. 2005, 27(2): 4-41.
叶亚新,黄勇,王金虎.分子生物学技术在环境微生物多相分类中的应用[J].苏州科技学院学报(自然科学版). 2004, 21(4):46-53.
赵青松,徐镇,陈寅儿等.梭子蟹乳化病的间接ELISA诊断[J].上海水产大学学报, 2005, 14(3): 248-252.
张志南,田胜艳.异养细菌在海洋生态系统中的作用[J].青岛海洋大学学报, 2003, 33(3): 375-383.
张宝涛,王立群,伍宁丰等. PCR-DGGE技术及其在微生物生态学中的应用.生物信息学[J], 2006, 3:132-134.
张英珊,郑凤英.海洋环境基因组学在海洋生态研究中的应用[J].生态环境, 2006, 15(1):179-183.
张恒庆,初航,温丹,等.三疣梭子蟹养殖池塘水体中异养细菌菌群特征的研究[J].辽宁师范大学学报(自然科学版), 2008, 31(2): 221-224.
张德民,黄志勇,杨惠芳等.几株红假单胞菌属细菌的表观特征及其遗传多样性研究[J].微生物学报, 2000, 40(3):229-236.
张德民,黄志勇等.紫色非硫细菌Rhodocista属一新分离株的鉴定及其系统学研究[J].微生物学报,2000,4(1): 14-20.
张晓君,房海,陈翠珍等.致病性哈氏弧菌生物学及分子特征[J].中国兽医学报. 2009, 29(9): 1120-1124.
郑天凌,庄铁城,蔡立哲等.微生物在海洋污染环境中的生物修复作用.厦门大学学报(自然科学版)[J], 2001, 40(2): 524-533.
郑小宏,温彩霞.祝立.罗源湾欧鳗海水网箱养殖病害调查研究[J].福建畜牧兽医, 2000, 22(5): 3.
Burford M A, Costanzo S D, Dennison W C, et al. A synthesis of dominant ecological processes in intensive shrimp ponds and adjacent coastal environments in NE Australia[J]. Mar Pollut Bull, 2003, 46: 1456-1469.
Caruso G, Genovese L, Mancuso M, et al. Effects of fish farming on microbial enzyme activities and densities:comparison between three Mediterranean sites[J]. Lett Appl Microbiol,2003,37(4): 324-328.
Colwell R K,Xuano Mao C,Chang J. Interpolating, extrapolating, and comparing incidence-based species accumulation curves[J].Ecology, 2004, 85:2717-2727.
Dunbar J,Takala S,Barns M S et al. Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning.Appl[J]. Environ Microbio, 1999, 65:1662-1669.
Erin A G,William F,Paul R J et al. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments[J].Appl Environ Microbiol, 2007, 5:3272-3282.
Fuhrman J A,McCallum T B,Davis A A. Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans[J].Appl Environ Microbiol, 1993, 59:1294-1302.
Giovannoni S J,Britschgi T B,Moyer C L et al. Genetic diversity in Sargasso Sea bacterioplankton.Nature[J], 1990, 34(5): 60-63.
Gowen R J, Bradbury N B. The ecological impact of samonid farming in coastal waters: a review [J].Oceanogr Mar Biol Ann Rev,1987, (25): 563-575.
Good I L. The population frequencies of species and the estimation of population parameters.Biometrika[J], 1953, 40:237-264.
Hill TCJ,Walsh KA,Harris JA et al. Using ecological diversity measures with bacterial communities[J].FEMS Microbiology Ecology, 2003, 43:1-11.
Hugenhdtz P,Goebel B M,Pace N R. Impact of cuture independent studies on the emerging phylogenetic view of bacterial diversity[J]. J Bacteriology, 1998, 180(18): 4765-4774.
Klapponbach J A,Saxman P R,Cole J R et al. The ribosomal RNAoperon copy number database[J]. Nucleic Acids Res, 2001, 29:181-184.
Morris C E,Bardin M,Berge O et al. Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999[J]. Microb. Mol. Biol,Rev. 2002, 66: 592-616.
Muyzer G,Waal E C,Uittrlinden A G. Profiling of complex microbial populations bydenaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Appl Environ Microbiol, 1993, 59:695-700.
Mattew T Cottrell,David L. Kirchman. Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization.Appl[J]. Environ Microbiol, 2000,66(12): 5116-5122.
Matthew S Payne,Mike R Hall,Raymond Bannister et al. Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus) [J].Aquaculture, 2006, 258:80-90.
Olivier Nercessian,Yves Fouquet,Catherine Pierre et al. Diversity of Bacteria and Archaea associated with a carbonate-rich metalliferous sediment sample from the Rainbow vent field on the Mid-Atlantic Ridge[J].Environmental Microbiology, 2005,7(5):698-714.
Singleton D R,Furlong M R,Rathbun S L et al. Quantitative comparison of 16S rRNA gene sequence libraries from environmental samples.Appl. Environ[J]. Microbiol, 2001, 67:4374-4376.
Solbrig O T. From genes to ecosystems:a research agenda for biodiversity.Report of a IUBS—SCOPE—UNESCO workshop.The International Union of Biological Sciences, 1991, 51 Boulevardole Montmorenny Paris France.
Torsvik V,Goksoyr J,Daae F L. High diversity in DNA of soil bacteria.Appl[J]. Environ Microbiol. 1990, 56:782-787.
Toranzo A E,Magarinos B,Romalde J L. A review of the main bacterial fish diseases in mariculture systems [J].Aquaculture, 2005, 24(6):37-61.
Ursula Dorigoa,Laurence Volatierb,Jean-Franc-ois Humberta. Molecular approaches to the assessment of biodiversity in aquatic microbial communities[J].Water Research, 2005, 39:2207-2218.
Vezzulli L, Chelossi E, Ricardi G, et al. Bacterial community structure and activity in fish farm sediments of the Ligurian sea (Western Mediterranean)[J]. Aqu Int, 2002, 10 (2): 123-141.
Watve M G, Gangal R M. Problems in measuring bacterial diversity and a possible solution[J]. Applied and Environmental Microbiology, 1996, 62(11): 4299-4301.