Comparison Of The Larval Intestinal Microbial Populations Of Three Different Frogs And The Effect Of Heavy Metals On The Changes Of Their Flora

The intestines of human and animal are an ecosystem consisting of a great number of commensal bacteria living in symbiosis with the host.The intestinal microbiota performs a number of important functions for their hosts,such as food digestion,metabolism,nutrient supply,stimulates immune system development and prevents colonization with pathogens.However,imbalance of intestinal microbiome is associated with an increased susceptibility to outside pathogens,resulting in developing inflammatory diseases and metabolic disorders.In recent years,with the widespread application of high-throughput sequencing technology in the study of intestinal microbiota,the majority of extant researches have focused on human and mammal intestinal microbiota,while little is known about the composition of amphibian intestinal microbiota.The present study,amphibian larva was selected as model organism to explore the composition of intestinal microbiota in amphibian by the method of high-throughput sequencing.Firstly,compared the composition and diversity of intestinal bacterial communities of Rana chensinensis(R.chensinensis),Pelophylax nigromaculatus(P.nigromaculatus),and Rana catesbeiana(R.catesbeiana)tadpoles.Secondly,we surveyed morphological alterations and the composition of intestinal bacteria community of R.chensinensis tadpoles during metamorphosis(G26,G32,G38,G42 and G46).And then we observed the R.chensinensis tadpoles intestinal microbial community changes associated with different concentrations of cadmium exposure(0,112 and 448 ? g Cd L-1).Our main results are as follows:1.The diversity of the intestinal microbial community was significantly different between the three species of tadpole.The tadpoles of R.catesbeiana had the largest diversity index and P.nigromaculatus tadpoles had the least diversity index.2.Venn diagram demonstrated that the 49 OTU(Operational taxonomic units,OTUs)shared in three tadpole species belong to three phyla,Proteobacteria(53.64%),Bacteroidetes(23.35%)and Fusobacteria(13.20%).Proteobacteria accounted for the largest proportion in the 49 OTU that shared in three tadpole species.Therefore,Proteobacteria is a core bacterial in intestinal microbiota of three tadpole species.3.The tadpoles of different species had significant difference in the composition of the intestinal microbiota both at the phylum and genus levels.At the phylum level,it is worth noting that Verrucomicrobia were not detected in R.chensinensis tadpoles.At the genus level,Cetobacterium and Plesiomonas were not appeared in R.chensinensis tadpoles,while these bacterium had higher quantity in R.catesbeiana tadpoles.Moreover,the proportion of Aeromonas were obviously richness in P.nigromaculatus tadpoles compared to the other two species.4.During metamorphosis,total body length,body weight,intestinal length and intestinal weight were reduced by 78%,51.9%,89%and 83.2%,respectively.5.The distinct OTU assignments and alpha-diversity of the intestinal microbiota among five groups revealed that bacterial diversity of R.chensinensis significantly increased from G26 to G38,peaked at G38,and then decreased to G46.6.Comparison of beta-diversities of the intestinal microbiota in five groups indicated that the composition of the tadpoles' intestinal microbiota varied throughout metamorphosis.The G26 and G32 tadpoles were largely colonized by Proteobacteria and Bacteroidetes,while the intestines of G38,G42 and G46 larvae were predominantly by members of Bacteroidetes,Proteobacteria,Fusobacteria and Firmicutes.Compared to aquatic larvae(G26,G32),terrestrial juveniles(G42,G46)showed major shifts in microbial composition,including reduction in Bacteroides,increases in Cetobacterium,Citrobacter,Shewanella and Fusobacterium.7.The alpha-diversity of the intestinal microbiota suggesting that the diversity of gut microbial community for the control group(0 p g Cd L-1)was higher than that of the 112 ? g Cd L-1 exposure group and significantly higher than that of the 448 ? g Cd L-1 exposure group.This result demonstrated that the diversity of intestinal microbial community declined with the increase of cadmium concentration.The beta-diversity indicate that Cd exposure results in restructuring of microbial communities.At the phylum level,it is worth noting that Fusobacteria and Spirochaetae were not detected in 448 ? g Cd L-1 exposure groups.Firmicutes rapidly decreased in 448 ? g Cd L-1 exposure group.At the genus level,Succinispira(Firmicutes),Desulfovibrio(Proteobacteria)and Fusobacterium(Fusobacteria)vanished in 448 ? g Cd L-1 exposure groups.In conclusion,our results indicated that the three tadpole species(R.chensinensis,P.nigromaculatus and R.catesbeiana)have significant difference in the composition and diversity of intestinal microbiota.And the diversity and composition of intestinal microbiota of R.chensinensis tadpoles have significantly changes during metamorphosis.In addition,the cadmium exposure caused R.chensinensis tadpoles intestinal microbial composition disorder,which affects the overall health of tadpoles.Our study may provide a new framework based on intestinal microbiota to understand the amphibian evolution and to evaluate the response of amphibians to environmental chemicals pollution.