Bacterial diversity in various coastal mariculture ponds in Southeast China and in diseased eels as revealed by culture and culture-independent molecular techniques

Mariculture ponds are widely distributed in Chinese coasts and have become a threat to the health of coastal ecosystems. In order to improve our understanding on the microbial composition in mariculture environments, we sampled a variety of ponds farming different animals or plants around the Dongshan Island and Xiamen Island in Southeast China and isolated cultures from the tissues of diseased eels. Analysis by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE), clone library and direct culturing methods revealed highly diverse bacterial communities in these samples. Bacterial communities in the Dongshan samples were dominated by Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. The Gracilaria verrucosa pond harbours the most abundant species (20 DGGE bands), followed by Epinephelus diacanthus pond (18 bands), Haliotis diversicolor supertexta pond I (18 bands) and Penaeus vannamei pond (11 bands). In comparison with surface waters, Penacus orientalis pond sediment showed a much more complex bacterial community, from which only sequences affiliated with Deltaproteobacteria, Firmicutes, Acidobacteria and candidate phylum TM6 were found. Bacterial cultures in diseased eels were closely related to two pathogenic genera, Aeromonas in Gammaproteobacteria and Bacillus, in Firmicutes. Clones affiliated with another two genera, Escherichia and Vibrio, that have pathogenic potentials were also identified. Phylogenetic analysis of a total of 131 sequences showed that 48.9% of the sequences were clustered into Gammaproteobacteria and formed the most abundant group, followed by Alphaproteobacteria (19.1%), Firmicutes (7.6%), Bacteroidetes (5.3%), Deltaproteobacteria (5.3%), Actinobacteria (4.6%), Chloroplast (3.8%), Acidobacteria (2.3%), Cyanobacteria (1.5%), Betaproteobacteria (0.7%) and TM6 (0.7%). 43.7% (28/64) of the phylogenetic clusters cannot be classified into any known genus and 44.3% (58/131) of the sequences show < 95% similarity to public database records, suggesting that abundant novel species exist in mariculture ponds. Gathering bacterial diversity data in mariculture ponds and diseased fish is meaningful for the prevention and control of fish diseases and for the improvement of our understanding of microbial ecology in a pond environment.