Table_6_Effects of Captivity and Season on the Gut Microbiota of the Brown Frog (Rana dybowskii).XLSX

The gut microbiota of amphibians is affected by exogenous and endogenous factors. We performed a comprehensive analysis using high-throughput sequencing technology and functional predictions and observed general changes in the gut microbiota of frogs in different growth stages, seasons, and growth environments. There were no significant differences in microbial richness and diversity between juvenile and adult wild frogs, between the summer and autumn groups of captive frogs, or between wild and captive frogs. There were significant differences in the gut microbiota community structure of Rana dybowskii between the summer and autumn groups of captive frogs and between wild and captive R. dybowskii, whereas the differences between juvenile and adult wild frogs were not significant. The dominant gut bacterial phyla in frogs from both captive and wild environments included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Linear discriminant effect size (LEfSe) analysis showed that Bacteroidetes and Firmicutes were significantly enriched in captive and wild R. dybowskii, respectively linear discriminant analysis (LDA > 4). The core operational taxonomical units (OTUs) that were found in >90% of all frogs tested encompassed 15 core OTUs. The captive frogs exhibited 15 core OTUs in addition to the above overall core microbiota, whereas the wild frogs exhibited 19 core OTUs in addition to the above overall core microbiota. Predictions made using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) suggested that eleven KEGG pathways, such as infectious diseases, immune system diseases, metabolism, metabolism of other amino acids, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, neurodegenerative diseases, and transport and catabolism, were enriched in captive frogs. The relative abundance of several red-leg-syndrome-related pathogens increased significantly in captive frogs compared with that in wild frogs. To our knowledge, this is the first study on the effects of individual seasons and captivity on the gut microbiota of frogs.

两栖动物肠道菌群（gut microbiota）受外源与内源因素的影响。本研究采用高通量测序技术与功能预测方法开展系统性分析，观察了不同生长阶段、季节及生长环境下蛙类肠道菌群的整体变化特征。野生蛙类的幼体与成体、圈养蛙类的夏季组与秋季组，以及野生与圈养蛙类之间，其菌群丰富度与多样性均无显著差异。圈养中国林蛙（Rana dybowskii）的夏季组与秋季组，以及野生与圈养中国林蛙之间，其肠道菌群群落结构存在显著差异；而野生幼体与成体中国林蛙的菌群群落结构差异并不显著。圈养与野生环境下蛙类的肠道优势细菌门包括厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）、变形菌门（Proteobacteria）以及放线菌门（Actinobacteria）。线性判别分析效应量（Linear discriminant effect size, LEfSe）分析显示，拟杆菌门（Bacteroidetes）与厚壁菌门（Firmicutes）分别在圈养与野生中国林蛙中显著富集（线性判别分析（LDA）得分>4）。在所有受试蛙类中检出率超过90%的核心操作分类单元（operational taxonomical units, OTUs）共计15个。圈养蛙类除上述整体核心菌群外，还拥有15个专属核心OTUs；而野生蛙类除上述整体核心菌群外，另有19个专属核心OTUs。采用未观测状态重建群落系统发育分析（Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, PICRUSt）进行的功能预测结果显示，圈养蛙类的11条KEGG通路显著富集，包括传染病、免疫系统疾病、代谢、其他氨基酸代谢、辅酶与维生素代谢、萜类与聚酮类化合物代谢、神经退行性疾病相关通路以及转运与分解代谢通路等。与野生蛙类相比，圈养蛙体内数种与红腿综合征（red-leg syndrome）相关的病原菌相对丰度显著升高。据我们所知，本研究是首个探讨单一季节与圈养环境对蛙类肠道菌群影响的相关研究。