Study On The Mechanism Of Early Ossification Of Chinese Giant Salamander And The Diversity Of Intestinal Microflora In Metamorphosis

Anuran metamorphosis is accompanied by a complex set of changes in morphology and physiology that transform an aquatic larva to a terrestrial juvenile.These changes include organ resorption(tail,gills),de novo development(limbs,lung)and organs and tissues remodeling(liver,intestine and skeleton).To learn more about changes in intestinal microbiota,characteristics of endochondral ossification and molecular mechanism mediating endochondral ossification throughout amphibian metamorphosis,we first studied the morphological alterations of typical B.gargarizans and its intestine during metamorphosis and then inventoried the intestinal microbial communities of B.gargarizans at pre-metamorphosis(G30),pro-metamorphosis(G38),metamorphosis climax(G42)and complete metamorphosis(G46)stages via 16S rDNA gene sequencing.In addition,regarding to skeletal development,we performed a procedure for staining cartilage and bone in hind limbs and vertebral columns to visualize skeletal development in B.gargarizans through metamorphosis.Then we used RNA-sequencing to characterize the transcriptome of B.gargarizans and investigated its developmentalexpression profiles at varying developmental stages(G30,G37,G42 and G46)and verified the sequencing results using Real-time quantitative PCR.Our main results are as follows:1.Morphological data indicated that during metamorphosis,both B.gargarizans and its intestine at G26,G31 and G38 stages increasingly developed,but were vestigial at G42 and G46 stages.Snout vent length,total body weight,intestinal weight and intestinal length of B.gargarizans from the climax to the end were reduced by 56.8%,29.8%,91.8%and 91.8%,respectively.2.Through 16S rDNA sequencing,267 OTUs were obtained from the intestines of B.gargarizans at four developmental stages.The distinct OTU assignments and alpha-diversity of the intestinal microbiota among four groups revealed that bacterial diversity of B.gargarizans decreased as metamorphosis progressed.3.Comparison of beta-diversities of the intestinal microbiota in four groups indicated that the composition of the tadpoles’ intestinal microbiota varied throughout metamorphosis.G30 and G38 groups shared similar bacterial composition and they were largely predominantly colonized by Proteobacteria and Firmicutes,while G42 and G46 groups shared similar bacterial composition and they were predominantly colonized by members of Bacteroidetes,Proteobacteria,Fusobacteria and Firmicutes.Compared to aquatic larvae(G38),terrestrial juveniles(G46)showed major shifts in microbial composition,including reduction in Proteobacteria,the remodeling of Firmicutes,increases in Bacteroidetes and Fusobacteria,the disappearance of Actinobacteria,the appearance of Verrucomicrobia.4.Correlations using the heatmap showed potential links between changes in the gut microbiota composition and altered morphological parameters.Total body weight,total body length,snout vent length,intestinal weight and intestinal length were positively associated with the abundance of genera Enterobacter and Flavobacterium.Through metamorphosis,changes in abundance of genera Akkermansia,Enterobacter,Bifidobacterium and Alpinimonas in B.gargarizans were found to be correlated with loss of body tissue.5.Double staining of bone and cartilage in B.gargarizans showed that limb bones and vertebral columns were well developed following metamorphosis development.Femur ossification began at stage 38,and its proportions increased from 29%to 84%at G43.6.Through transcriptome sequencing,we obtained 118,385,689 clean reads from B.gargarizans at four developmental stages,and 137,264 unigenes were assembled,of which 32.08%matched known protein databases.We further identified 10,352 differentially expressed genes(DEGs)by constructing DGE libraries among four key developmental stages during B.gargarizans metamorphosis.Then eight expression patterns were characterized.7.Based on comparative analyses of differentially expressed genes and Nr database,28 genes involved in endochondral ossification were identified.These genes were responsible for differences in the developmental effects of thyroid hormones in cartilage and bone among the four stages in B.gargarizans.And of 28 selected genes,10 were validated via q-PCR and the result produced values consistent with the expression changes observed by seqnencing analysis.In conclusion,B.gargarizans intestinal microbial community diversity decreased and its composition shifted during metamorphosis.We hypothesized that multiple factors,such as diet,living environment,intestinal morphology and body loss shaped the composition of these symbiotic host-associated communities through amphibian metamorphosis.During metamorphosis,B.gargarizans skeletons were well developed and the analysis of their differential expression performed by RNA-sequencing provides an invaluable resource to identify genes involved in endochondral ossification.It could provide experimental references for further researchs on characteristics of microbial diversity and molecular mechanism mediating endochondral development throughout amphibian metamorphosis.