【作者】 孙苏燕；

【导师】 张德民；

【作者基本信息】 宁波大学 ， 海洋生物， 2011， 硕士

【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。更多还原

【Abstract】 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.更多还原