Investigation on Bacterial Diversity in Portunus Trituberculatus Rearing Pond by Cultivation-dependent and Independent Approaches
|Keywords||Portunus trituberculatus Aquaculture pond Microbial community Vibrio 16S rDNA gene|
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.