PCR-DGGE FINGERPRINTING ANALYSIS ON INTESTINAL MICROBIAL COMMUNITY OF THREE INDOOR REARING FISHES

PCR-DGGE fingerprinting was explored to study intestinal microbial community of three indoor rearing ju-venile fishes (channel catfish, silver prussian carp and hybridized prussian carp). Each fishes with three repetitions were reared in a semi-recirculating system in Institute of Hydrobiology. During the experiment, water temperature was 23℃30℃, and fish were fed to satiation twice daily (9:00 and 15:00). After 30 days, 3 fishes of each treatment were randomly collected to extract microbial DNA. Gastrointestinal samples of all fishes were obtained by aseptically dis-secting the fish and carefully extracting the entire gastrointestinal tract. After dissecting, different segments of each gastrointestinal tracts from 3 fishes of the same treatment were pooled to analyse the bacterial communities. DNA from species samples were obtained according to the traditional Phenol-chloroform method. Bacterial domain-specific prim-ers F357 (5'-CCTACGGGAGGCAGCAG-3' with GC clamp in the 5' end) and R518 (5'-ATTACCGCGGCTGC TGG-3') were used to amplify the variable V3 region of 16S rRNA genes. PCR products were separated on a 9% polyacrylamide gel (ratio of acrylamide to bisacrylamide was 37.5∶1) in a 1TAE buffer and a denaturing gradient from 35% to 50% of urea and formamide. Unweighted pair-group method using arithmetic averages (UPGMA) and Multidimensional Scaling (MDS) were used to investigate community similarity of bacterial among different fish species. Moreover, rank abun-dance plots were used to determine differences in bacterial community structure based on taxa relative abundance. For each plot, a linear regression model was fitted, and it was represented by the equation, log10y = a + bx, where a was the intercept and b was the slope of the plot. The slope (b) was subsequently used as a descriptive statistic for changes in community structure. Linear regressions, coefficients of determination (r) and significance (P) were calculated by using SPSS 13.0 software. Special DNA bands on DGGE profile were sequenced by Shanghai Sunny Biotechnology Co. As showed in DGGE profile, various number of 16S rDNA bands were detected in different samples, and the average bands detected in channe1 catfish intestine (7.5) were less than that of silver prussian carp and hybridized prussian carp (15 and 14, respectively). In addition, Both UPGMA clustering and MDS ordination based on 1/0 matrix showed that the similarity of intestinal microbe of silver prussian carp and hybridized prussian carp was higher when comparing to the similarity with channel catfish. Also, rank-abundance plots and linear regression on the basis of relative abundance of DGGE bands revealed that the microbial community structure of the three fishes were significantly different. Bacterial community structures in silver prussian carp and hybridized prussian carp showed no significant difference (P=0.383), while they showed difference (P0.05) comparing with the intestinal bacteria in channe1 catfish. On the other hand, sequencing results showed that the bacteria in channel catfish intestine belonged to Proteobacteria, containing - Proteobacterium and -Proteobacterium, while the bacteria in silver prussian carp and hybridized prussian carp intes-tine contained Fusobacterium, Aeromonas and some unclassified-bacteria. In conclusion, the intestinal bacterial compo-sition is not identical in different kinds of juvenile fishes reared in the same condition.