| There were complex microbial communities in amphibian intestines.These intestinal microbiota were affected by many factors,such as genetic factors,phylogeny,food selection,environmental changes and behavior.They were closely related to the growth and development,nutritional metabolism,disease and immunity of the host.The dynamic relationship between intestinal microbiota and host may be conducive to various life activities of the host,but can also cause the imbalance of intestinal microbiota of the host and lead to the occurrence of diseases.Therefore,the study of intestinal microbiota was of great significance to understand the symbiotic relationship between intestinal microbiota and host.The growth and development of amphibians undergo abnormal development,the changes of morphological structure and physiological characteristics from larvae to adult,and the transformation of living environment from aquatic to terrestrial.These factors may cause the changes of intestinal microbiota.Therefore,the study of the characteristics of amphibian intestinal microbiota in different developmental stages was helpful to understand the adaptive strategies of intestinal microbiota in the process of metamorphosis and development,and was helpful to deepen the understanding of adaptive mechanism from aquatic to terrestrial of amphibian.In this study,16 S r RNA high-throughput sequencing was used to study the characteristics of intestinal microbiota of H.maoershanensis in three stages: forelimb bud stage,hindlimb bud stage and adult,as well as in different habitats,so as to explore how the intestinal microbiota of H.maoershanensis adapted to the changes of physiological structure and environment in different development stages,and mainly answer the following scientific questions:(1)What are the structural characteristics of intestinal microbiota in different developmental stages and habitats of H.maoershanensis?(2)How does the intestinal microbiota adapt to environmental changes during the development of H.maoershanensis?(3)Does habitat change affect intestinal microbiota of H.maoershanensis? The results as follows:(1)The results of Alpha analysis showed that:(1)There were significant difference in the Alpha diversity of intestinal microbiota between forelimb bud stage and hindlimb bud stage(Q < 0.05).Sobs index(108.67 ± 53.71),Ace index(137.41 ± 52.49)and Chao index(136.97 ± 58.86)of hindlimb bud stage were significantly higher than those of forelimb bud stage(Q < 0.05),but there were no significant difference between Shannon index(2.13 ± 0.96)and Simpson index(0.34 ± 0.25)(Q > 0.05).(2)There were no significant difference in the indices of Alpha diversity(Q < 0.05)between the intestinal microbiota at the forelimb bud stage and adult,means the similar diversity and richness of intestinal microbiota.(3)There were no significant differences in the Alpha diversity and richness of intestinal microbiota between the hindlimb bud stage and adult stage(Q > 0.05).(4)There were significant difference in the Alpha diversity of intestinal microbiota between different habitats(Q < 0.05),indicating that the diversity and richness of intestinal microbiota of H.maoershanensis in Zhuangyuanqiao were significantly higher than those in Shangdinghu.(5)There were no significant difference in the Alpha diversity index of intestinal microbiota of H.maoershanensis at hindlimb bud stage in different habitats(Q > 0.05).(6)There were no significant difference in the Alpha diversity index of the intestinal microbiota of adult in different habitats(P > 0.05).(2)The results of Beta analysis showed that:(1)Weighted and unweighted PCo A analysis showed that there were significant difference in intestinal microbiota structure between forelimb bud stage and hindlimb bud stage(P < 0.05).(2)There were significant difference in the structure of intestinal microbiota between forelimb bud stage and adults(P <0.05).(3)There were significant difference in the structure of intestinal microbiota between adult and hindlimb bud stage(P < 0.05).(4)There were significant difference in the structure of intestinal microbiota of H.maoershanensis at forelimb bud stage in Shandinghu and Zhuangyuanqiao(P < 0.05).(5)There were significant differences in the structure of intestinal microbiota of hindlimb bud stage in different habitats of H.maoershanensis(P <0.05).(6)There was no significant structural difference in the intestinal microbiota of adult H.maoershanensis between Shandinghu and Zhuangyuanqiao(P > 0.05).(3)Community composition analysis showed that:(1)In the two stages of tadpole,the composition of intestinal microbiota were very similar,and the dominant intestinal microbiota were Firmicutes(forelimb bud stage: 54.47% ± 31.23 %;hindlimb bud stage: 62.32 % ±27.92 %)and Proteobacteria(forelimb bud stage: 29.89 % ± 27.43 %;hindlimb bud stage:23.08 % ± 23.25 %).The dominant families of intestinal microbiota in the two periods were Peptostreptococcaceae(forelimb bud stage: 10.87 % ± 14.04 %;hindlimb bud stage: 37.1 %± 35.13 %),Clostridiaceae(forelimb bud stage: 21.60 % ± 18.32 %;hindlimb bud stage:12.05 % ± 14.75 %)and Enterobacteriaceae(forelimb bud stage: 9.52 % ± 15.77 %;hindlimb bud stage: 10.88 % ± 17.71 %).Among them,the relative abundance of Porphyromonadaceae,Desulfovibrionaceae and Micrococcaceae were significantly higher than that of forelimb bud stage(Q < 0.05).(2)Firmicutes and Proteobacteria were the dominant phylum of the forelimb bud stage.The relative abundance of Firmicutes and Fusobacteria were significantly higher in forelimb bud stage than in adult(Q < 0.05).The dominant families were Clostridiaceae,Enterobacteriaceae and Peptostreptococcaceae,and the relative abundance of Clostridiaceae and Peptostreptococcaceae were significantly higher than in adult.The dominant phylum in adult were Proteobacteria and Bacteroidetes,and the Bacteroidetes,Spirochaetes of adult were significantly higher than those in forelimb bud stage.The dominant families of adult were Flavobacteriaceae and Oxalobacteraceae,of which the relative abundance were significantly higher than forelimb bud stage(Q < 0.05).(3)From the level of phylum,we can see that the dominant phylum in these two stages were Firmicutes,Proteobacteria and Bacteroidetes.The relative abundance of Firmicutes,Fusobacteria in the hindlimb bud stage were significantly higher than that of adult.The relative abundance of Proteobacteria and Bacteroidetes,Chloroflexi of adult were significantly higher than that of hindlimb bud stage(Q < 0.05).From the family level,the dominant families of adult were Flavobacteriaceae and Oxalobacteraceae,and their relative abundance were significantly higher than that of the hindlimb bud stage.The intestinal microbiota of the hindlimb bud stage were dominated by Peptostreptococcaceae and Clostridiaceae,and the relative abundance were significantly higher than that of adult.(4)The dominant phylum of intestinal microbiota in different habitats of forelimb bud stage were Firmicutes and Proteobacteria,and the dominant families were Clostridaceae and Peptostreptococcaceae.(5)The dominant phylum of intestinal microbiota in the hindlimb bud stage in different habitats were Firmicutes and Proteobacteria,and the dominant families were Peptostreptococcaceae and Clostridaceae.(6)The dominant phylum of intestinal microbiota of adult in different habitats were Proteobacteria and Bacteroidetes,and the dominant families were Oxalobacteridae and Flavobacteriaceae.(4)The results of functional prediction analysis show that:(1)The function prediction of hindlimb bud stage were better than that in forelimb bud stage on the whole.At the level of Pathway level 1,genetic information processing in hindlimb bud stage was significantly better than that in forelimb bud stage(q < 0.05).At Pathway level 2,environmental adaptation,immune system,metabolic diseases,nervous system,nucleotide metabolism,replication and nucleotide metabolism,repair,transcription and translation were significantly higher in hindlimb bud stage than those in forelimb bud stage(q < 0.05).(2)Functional prediction results showed that there were significant differences between forelimb bud stage and adult in KEGG Pathway Level 1,cellular processes and environmental information processing of forelimb bud stage were significantly higher than adult(p < 0.05).The genetic information processing,human diseases,metabolism of adult were significantly higher than those of forelimb bud stage(p < 0.05).(3)There were significant difference in the functional prediction between hindlimb bud stage and adult.In KEGG Pathway Level 1,the functional abundance of environmental information processing,human diseases,metabolism and organismal systems of hindlimb bud stage were significantly higher than that of adult(q <0.001).The cellular processes and genetic information processing of intestinal microbiota in hindlimb bud stage were significantly higher than those in adult(q < 0.001).In conclusion,the intestinal microbiota of H.maoershanensis showed differences in richness,composition and structure between forelimb bud stage and hindlimb bud stage,while the intestinal microbiota of forelimb bud stage and adult,hindlimb bud stage and adult did not show differences in Alpha diversity,but the intestinal microbiota was significantly separated in structure.There were differences in functional abundance at different developmental stages of H.maoershanensis.This study also found that there were structural differences in the intestinal microbiota of H.maoershanensis in different habitats.These results showed that the differences in the structure and function of intestinal microbiota may be related to the physiological characteristics and food selection at different developmental stages,and habitats also had a certain impact on the intestinal microbiota. |
PDF Full Text Request