Pcr-dgge Comparative Study On The Structure Of The Intestinal Flora Of Six Freshwater Fish

With the development of intensive and large-scale aquaculture and the increasing stressor in the modern time,the immunity of the aquatic animals has decreased,and the susceptibility to disease of the aquatic animals has increased greatly.In recent years,people use probiotics in aquaculture and aquatic food in order to improve the aquaculture ecological environment, improve the immunity of the breeding animal and inhibit the propagation of pathogenic micro-organisms,thereby reducing the occurrence of disease.Probiotics play a role by imposing on the intestinal flora and,therefore,It is a prerequisite to know the animal intestinal flora to study and develop probiotics.In this paper,16S rDNA PCR-DGGE is used to fingerprint to compare and analyze the structure of flora in the digestive tract of the health Aristichthys mobilis,Hypophthalmichthys molitrix,Ctenopharyngodonidellus,Megalobrama amblycephala,Pelteobagrus fulvidraco and Siniperca spp in the intensive aquaculture,and explore the differences of composition of intestinal flora,also lay the foundation of clarifying the intestinal flora fauna of freshwater fish and developing colonized probiotic.First of all,the intestinal tracts of six kinds of freshwater fishes were divided into three parts: foregut,midgut and hindgut according to the anatomy characteristics of the fish,and then used the revised Hoelzel's methods to extract total DNA of intestinal Microorganisms.Secondly,the landing PCR amplification using was done by the specific primers of bacterial 16S rDNA-V3 fragments for the total DNA amplification of intestinal gut,and then the denaturing gradient gel electrophoresis was done.Finally,the clear and advantaged marked bands of DGGE patterns were recovered,and then were cloned and sequenced.After using BLAST to compare the sequence,we used the DNAMAN software to build Phylogenetic trees.The diversity of intestinal bacteria in the six kinds of fresh fishes was investigated by denaturing gradient gel electrophoresis(DGGE) fingerprinting.The principal component analysis of DGGE profiles indicated that substantial inter subject variations existed in intestinal bacteria in freshwater fishes.The sequencing of 16S rDNA genes revealed that the similarity between in the sequencing gained and in the gene bank was 96%-100%From the analysis of the intestinal flora of Aristichthys mobilis,Hypophthalmichthys molitrix,Ctenopharyngodonidellus,Megalobrama amblycephala,Pelteobagrus fulvidraco and Siniperca spp,we found that:the distribution of bacteria in the foregut is generally less than that in the mid-gut and the hindgut in the same species of fish,while the total number of anaerobic bacteria is far greater than that of aerobic bacteria at the same intestinal gut;among the different species of fish,however,the distinction of the total number of the aerobic bacteria in the intestinal flora is much larger than that of the anaerobic bacteria.The species and the quantities of intestinal flora of six freshwater fish are all increasing gradually from the foregut to the hindgut and the law of the distribution of their types and quantities of anaerobic bacteria in intestinal flora is:the carnivorous Siniperca spp and Pelteobagrus fulvidraco are more than Aristichthys mobilis and Hypophthalmichthys molitrix that eat mainly planktonic plants and Ctenopharyngodonidellus,while the herbivorous Megalobrarna and amblycephala are the least. That is to say,the total number of anaerobic bacteria and bifidobacteria in gut of freshwater fish is increasing gradually with the changes from herbivorous fish to carnivorous fish.Lactobacillus is a significant component of the intestinal flora bacteria of fish,and the structure of which displays a distinct difference with the types of fish,feeding habits,and the environment of growth.The conclusion of the paper as following:1.The 16S rDNA PCR-DGGE technique was proved a fast and effective experimental technique which can be used to study ecology of intestinal flora in freshwater fish.2.The structure of intestinal flora in freshwater fish presents a difference in several of species,and the types and quantities are growing gradually from foregut to hindgut,while the total number of anaerobic bacteria is more than that of aerobic bacteria.3.The structure of intestinal flora in freshwater fish is related to the kinds of food.The total number of anaerobic bacteria and bifidobacteria in gut of freshwater fish is increasing gradually with the changes from herbivorous fish to carnivorous fish.4.Type A,B Bacteroides are mostly in the obligate anaerobic bacteria in the gut of the fish, the bacteria of the Aeromonas and Enterobacteriaceae are mainly types of the aerobic and facultative anaerobic bacteria.Aeromonas culicicola and Enterobacter are superior bacteria colonies in the gut of freshwater fish.