Diversity and Composition of Gut Microbiota in Different Developmental Stages of the Tibetan Toad (Bufo tibetanus)

Abstract

The intestinal microbiota is vital for host immunity and metabolism, and its changes are associated with the development stage of hosts. However, little is known regarding how growth and development of anurans affect the diversity of their microbiota, which has a complex life cycle. The Tibetan toad (Bufo tibetanus) is a wild population in the high-altitude area of southwest China, which has special adaptability to the environment. Here, the microbial community of the Tibetan toad at six developmental stages (from the tadpole at Gosner stage 18 to the 8-year-old adult) was assessed using high-throughput 16S rRNA sequencing. The alpha diversity index analysis showed that the Chao, Ace, and Shannon indices were highest at Gosner stage 32 and decreased as development progressed, and their alpha diversity remained unchanged over time in adult stages. Beta diversity revealed that the gut microbiota structure differed significantly from Gosner stages 18 to 31, and it became similar to adult toads from Gosner stages 45 to 46 and in juvenile groups. At the phylum level, Firmicutes, Proteobacteria, and Actinobacteria were dominant phyla in tadpoles and adults. The relative abundance of Firmicutes and Proteobacteria in the adult group was significantly higher and lower than that of tadpoles, respectively. The linear discriminant analysis effect size (LEfSe) analysis identified seven phyla exhibiting significant differences during life stages: Verrucomicrobiota, Bacteroidota, and Proteobacteria (Gosner 18 to 31), Cyanobateria and Chloroflexi (Gosner 32 to 41), Actinobacteriota (Gosner 45 to 46), Desulfobacterota (juvenile group), and Firmicutes (adult group). A pathway enrichment analysis revealed that the metabolism and biosynthesis of secondary metabolites were significantly enriched across all developmental stages. This research unveiled variations in the intestinal microbiota composition during development in anurans. Factors such as developmental stage, habitat type and feeding habit jointly affected the gut microbial diversity and community composition in the Tibetan toad. The findings of this study can provide information for understanding the influence of historical developments on the intestinal microbiota and provide protection information for anurans.

Keywords: Bufo tibetanus; Illumina MiSeq sequencing; development stages; intestinal microbiota.

Figure 1

Rarefaction curves of samples. (A): Sobs index; (B): Shannon index. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult.

Figure 2

Comparison of alpha diversity of intestinal microbiota at different growth stages of B. tibetanus. (A): Chao index; (B): Ace index; (C): Shannon index. Different letters indicate significant differences among groups. Capital letters represent extremely significant differences (p < 0.01), and lowercase letters represent significant differences (p < 0.05).

Figure 3

PCoA plot based on Bray–Curtis distances (A) and hierarchical clustering tree (B) of gut microbes at different growth stages. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult.

Figure 4

Community barplot analysis showing the relative abundance at the phylum (A) and genus (B) level at different growth stages. Abundances of phyla or genera less than 1% were classified as “others”. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult.

Figure 5

Cladogram of LEfSe analysis showing differentially abundant bacterial taxa in the gut microbiota among groups. The yellow node represents no significant difference between the sample groups. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult.

Figure 6

LEfSe analysis of the top 10 gut microbiota in all groups. LDA scores of differentially abundant taxa in the gut microbiota of the six groups from the LEfSe analysis. The top 10 dominant taxa in each group are shown. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult.

Figure 7

Predicted functions of the intestinal microbiota on KEGG pathways. T1: Gosner 18–31; T2: Gosner 32–41; T3: Gosner 42–44; T4: Gosner 45–46; J: juvenile; A: adult. Different letters indicate significant differences among groups. Capital letters represent extremely significant differences (p < 0.01), and lowercase letters represent significant differences (p < 0.05).