16S RRNA-base Analysis Of Bacterial Diversity In The Microbial Flora Of The Red-eared Slider And Four Eye-spotted Turtle Intestinal Tract

The vertebrate intestine is a complex ecosystem of microenvironment and colonizesmany microbial communities. The intestinal microflora plays an important role in the host,including the food decomposition, the metabolism, nutrient supply, improve immunity anddefense against invading pathogens. Nowadays the research on animal intestinal microbes hasbecome the hottest topic in the micro-ecology. In recent years, with the continuousdevelopment of sequencing technology and mature, a lot of animals intestinal microflorastructure has been known, but for the turtle which has on earth more than200million yearslife history, there has not been reported.Therefore, we selected red eared turtle (exotic specie) and four eye-spotted turtle (localspecie) as the research objects, depended on the metagenomic method and454sequencingtechnology to analysis the diversity and the abundance of the intestinal bacterial community.In this way, we can better understand structure of the freshwater turtle intestinal microbes,provide a theoretical basis for the symbiotic system construction between the host (red earedturtle, four eye-spotted turtle) and intestinal microbes, and for the construction of theintestinal flora monitoring platform.In this study, we extracted the microbial metagenomic DNA and used the Roche454sequencing technology to analysis the fecal microflora of3adult red eared turtles and3adultfour eye-spotted turtle individuals. Fecal bacteria were characterized by amplifying theV3-V5region of bacterial16S rRNA gene. Sequence analysis showed that red-eared turtlesand four eye-spotted turtle have a rich diversity of intestinal microbes. A total of the163785effective sequences and1766OTUs (similarity to sequences are classified under97%) weobtained. The result of the sequences annotation show that the groups up to16phylum,respectively for Firmicutes, Bacteroidetes, Spirochaetes, Fusobacteria, Proteobacteria,Tenericutes, Deferribacteres, Synergistetes, Actinobacteria, Planctomycetes, Elusimicrobia,Verrucomicrobia, Fibrobacteres, Lentisphaerae, Deinococcus-Thermus and Euryarchaeota(Archaea). In the intestinal microflora, the Firmicutes and Bacteroidetes as the absolutedominant bacteria widely exist in each sample. In addition, sequence annotation results showthat in the intestinal contains a huge number of unknown bacterial (8014sequences),accounting for the total sequence of4.89%, and those unclassified bacteria sequence may becan provide an important basic data for cognition, development and utilization in the future. For different species of the intestinal microflora reflects host specificity. According to theBeta diversity and the analysis of the intestinal flora, for the different species, red eared turtlesand four eye-spotted turtles shows great difference, and the difference in different speciesmuch greater than the intraspecific. Red eared turtle samples were obtained71368sequencesand1061OTUs, four eye-spotted turtle obtained92417sequences and705OTUs, and dependon the alpha diversity analysis, the result show that the diversity of red eared turtle intestinalmicrobial is more abundant than the four eye-spotted turtle. In the classification, red earedturtle got a total of15categories, four eye-spotted turtle got12categories, the two groups ofsamples in the dominant bacteria ratio also showed difference, red eared turtle intestinalpredominant flora as Firmicutes, Bacteroidetes and Spirochaetes, but the four eye-spottedturtle is more concentrated on Firmicutes and Bacteroidetes. So we can presumably thatSpirochaetes may play a certain special role in the red eared turtle intestinal. To the bacterialphylotype, two groups of sample differences also exist, for example the Bacteroidetes andSynergistetes. While in the unknown strain, red eared turtle also shows more abundantpotential resources for development, a large number of unclassified sequences presented2239.67±716.59in the red eared turtle, the proportion was9.67±2.34%, relatively fewnumbers of unclassified sequences presented in four eye-spotted turtle (431.67±114.29), theproportion was only1.43±0.45%.In addition, sequence annotation results show that the intestinal of the red eared turtleand four eye-spotted turtle contains a variety of pathogens, including Brevinema,Edwardsiella, Shigella and Salmonella etc.. As a pet, the red eared turtle has a long timecontact with humans, so these pathogens is likely to pose a potential threat to cause zoonoticdisease. And according to the further statistical results showed, intestinal pathogenic bacteriaexists difference in two groups of samples, mainly reflected in the Treponema, Edwardsiella,Campylobacter, Brevinema and Klebsiella, and only genera Salmonella and Aeromonaso existin two groups of samples. Therefore, a comprehensive survey of intestinal pathogenic bacteriaspecie, is critical to the etiology and prevention of diseases in humans and animals.