Phylogenetic Analysis Of Intestinal Bacteria In Several Aquatic Animals

Molecular methods based on the recovery of microbial community DNA followed by PCR and 16S rRNA gene sequence analysis have proven useful for studying natural microbial populations and thus, are accepted methods in microbial ecology. Specifically, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes from total microbial DNA isolated from environmental samples provides a DNA fingerprint of each sample and permits subsequent identification of community members by sequence analysis. Such molecular techniques have been used to characterize the dominant microorganisms in several important aquatic species such as haddock larvae, salmon, rainbow trout and abalone etc..In this study, DGGE, 16S rRNA gene clone library analysis and real-time quantitative PCR (RTQ-PCR) were used to investigate the bacterial diversity in the intestinal tract of Chinese mitten crabs (Eriocheir sinensis) and anchovy (Coilia mystus) and to investigate the diversities of intestinal bacteria in eight kinds of fishes from the Changjiang river estuary and correlation between the intestinal bacteria in fish and their feed.Chinese mitten crab is an important product in the aquaculture. The aims of this study are to identify the dominant intestinal bacteria in the Chinese mitten crab and to investigate differences in intestinal bacteria between pond-raised and wild crabs. The principal component analysis (PCA) of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. Sequencing of 16S rRNA genes revealed that 90-95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs respectively, whereas Bacteroidetes was found to be common in all sampled crab groups. RTQ-PCR indicated that the abundance of Bacteroides and total bacterial load were approximately four-to-ten times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that a substantial part of the bacteria in the gut of Chinese mitten crabs have yet to be described. The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant populations in the gut of the Chinese mitten crab. This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for development of probiotic products for crab cultivation.Anchovy is a valuable marine protein resource in China and other countries. It usually lives in the coastal ocean and goes to estuary (in China, mainly the Changjiang River estuary) for reproduction in March or April and reaches the peak of reproduction in June or July. The intersubject variations were investigated by DGGE and principal component analysis (PCA) to reveal that the variations between two seasons are more obvious than intersubject variations in each season and intersubject variations among anchovy intestinal bacteria collected in July are not so obvious as that collected in March. 16S rRNA gene clone libraries of intestinal bacteria in anchovy in two seasons were constructed. Most clones were assigned to proteobacteria, but more clones in class Gammaproteobacteria were found in anchovy gut colleted in March and more clones in Betaproteobacteria and Alphaproteobacteria were found in July. This study demonstrated obvious differences in the intestinal bacterial composition of anchovy between two seasons. This knowledge will increase our understanding of the effects of water environment on bacterial community composition in the fish gut and provide necessary data for development of anchovy cultivation and reproduction.PCR-DGGE analysis and PCA were used to investigate the diversities of intestinal bacteria in eight kinds of fishes from the Changjiang river estuary. It was proved that there were abundant and various bacteria in the guts of eight kinds of fishes. The bacterial community structures in the guts of these fish were obviously different. Specifically, the gut bacteria of Glossogobius giuris that live in the understratum of the water environment was proved to be more obviouslly different to that in other seven fishes. The correlation between the intestinal microbiota in fish and their feed were obvious.In this study, intestinal bacteria of several important aquatic animals were analyzed with molecular techniques. The results of this work provides useful data and new insights for understanding the relationship between lifestyles, aqua-environments and gut bacteria which is the important basis for improving aquaculture.