Effects of Captivity and Season on the Gut Microbiota of the Brown Frog (Rana dybowskii)

Abstract

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.

Keywords: 16S rRNA gene; amphibian; intestinal microbiota; microbial diversity; ontogenetic; red-leg syndrome.

FIGURE 1

(A) Veen diagrams show the number of shared and unique core Operational taxonomic units (OTUs) among the core OTUs of all samples (>90%), the core OTUs of wild samples (100%), and the core OTUs of captive samples (100%). (B) Veen diagrams show the number of shared and unique OTUs among CS, CA, and W groups. (C) Community bar plot analysis of bacterial at the phylum level.

FIGURE 2

Non-metric multidimensional scaling (NMDS) plot based on the weighted UniFrac distance matrix between the gut bacterial communities of (A, left) the W1 (juvenile) and W2 (adult) groups of wild frogs, (B, left) the CS (summer) and CA (autumn) groups of captive frogs, and (C, left) the C (captive frogs) and W (wild frogs) groups. Composition (at the family level) of the gut bacterial communities of (A, right) juvenile and adult wild frogs, (B, right) the summer and autumn groups of captive frogs and (C, right) captive and wild frogs.

FIGURE 3

Relative abundances of predicted genes in the metagenome related to level-1 and level-2 KEGG pathways; (A) red boxes, CS (summer) group; green boxes, CA (autumn) group; (B) yellow boxes, W (wild frogs) group; blue boxes, C (captive frogs) group. Asterisks indicate significant differences between groups.

FIGURE 4

Family, order, class and phyla that were differentially represented between captive groups and wild groups, as indicated by relative abundances based on linear discriminant analysis effect size (LEfSe) analysis linear discriminant analysis (LDA > 4, P < 0.05). Red boxes and green boxes represent enrichment in the W (wild) and C (captive) groups, respectively. Each circle’s diameter is proportional to the taxon’s abundance. The strategy of multiclass analysis is non-strict (at least one class differential). Circles represent taxonomic ranks from domain to family, from the inside to the outside. Circles represent taxonomic ranks from phylum to genus, from the inside to the outside. Labels are shown at the class, order and family levels. The scores are shown for all taxa with an LDA score > 4.