Integrated Microbiome-Metabolome Analysis Reveals Intestine-Liver Metabolic Associations in the Moustache Toad

Abstract

The intestinal microbiota regulates host metabolic homeostasis through production of bioactive microbial metabolites. These microorganisms facilitate digestion, enhance immune function, maintain osmoregulation, and support physiological balance via these bioactive compounds, thereby enhancing environmental adaptation. Our study investigated intestinal microbiota-liver metabolic interactions in Leptobrachium liui using 16S rRNA gene sequencing and non-targeted liquid chromatography-tandem mass spectrometry metabolomics. Key findings include (1) comparable alpha diversity but distinct microbial community structures between the small intestine (SI) and large intestine (LI), with the SI dominated by Enterobacteriaceae (72.14%) and the LI by Chitinophagaceae (55.16%); (2) segment-specific microbe-metabolite correlations, with predominantly positive correlations in the SI and complex patterns in the LI involving fatty acids, amino acids, and energy metabolites; and (3) significant correlations between specific bacterial families (Aeromonadaceae, Enterobacteriaceae, Chitinophagaceae) and hepatic metabolites related to fatty acid metabolism, amino acid synthesis, and energy pathways, indicating potential gut-liver axis associations. These findings provide insights into amphibian intestinal microbiota-hepatic metabolite associations and may inform future studies of host-microbe interactions.

Keywords: Leptobrachium liui; amphibian; gut–liver axis; intestinal microbiota; microbiome–metabolome.

Figure 1

(a) Three-dimensional geographical distribution of the six sampling sites for adult male Leptobrachium liui in the Jiulongshan National Nature Reserve. The sampling sites are located at elevations between 720 and 939 m. Four specimens (ind01, ind02, ind05, ind11) were collected from streams in Waijiulong Valley, while two specimens (ind15, ind18) were collected from Zhongxinkeng Valley. (b) Habitat characteristics of the collection area. (c) Adult male Leptobrachium liui. Photographed by Guo-Hua Ding.

Figure 2

Bacterial community composition and differentially abundant taxa between the small intestine (SI) and large intestine (LI) of adult male Leptobrachium liui. Relative abundance of bacterial taxa at the (a) phylum, (b) order, and (c) family levels. Asterisks indicate significantly different taxa between intestinal segments (*: p < 0.05, **: p < 0.01). (d) Cladogram generated from the LEfSe analysis illustrating the phylogenetic distribution of bacterial lineages with significant differences between the SI and LI. (e) Linear discriminant analysis (LDA) scores of significantly enriched bacterial taxa in the SI (green bars) and LI (red bars). Only taxa meeting the threshold criterion of LDA score > 4.0 are shown.

Figure 3

Alpha and beta diversity of bacterial communities in the small intestine (SI) and large intestine (LI) of adult male Leptobrachium liui. Alpha diversity indices: (a) Sobs index, (b) Shannon index, (c) Simpson index, and (d) Pielou index. (e) Non−metric multidimensional scaling (NMDS) plot based on Bray−Curtis dissimilarity showing differences in bacterial community structure between the SI and LI, with a stress value of 0.053. (f) Analysis of similarities (ANOSIM) demonstrating significant differences in bacterial community composition between and within the SI and LI groups (R = 0.5204, p = 0.015).

Figure 4

Metabolic profiling of hepatic metabolites in adult male Leptobrachium liui. (a) Distribution of identified metabolites across KEGG metabolic pathways. Numbers indicate the counts of metabolites in each pathway. (b) Relative proportions of metabolite classes detected in negative (NEG) and positive (POS) ionization modes using LC−MS/MS. (c) Top 25 most abundant metabolites based on mean intensity values (log₁₀ scale), colored by their respective chemical classes.

Figure 5

Correlation analysis between bacterial families and metabolites in adult male Leptobrachium liui. Heatmaps of Spearman correlation between the relative abundances of predominant bacterial families and top metabolites in the (a) small and (b) large intestines. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Network-based visualization of significant correlations between bacterial families and metabolite classes in the (c) small and (d) large intestines. The line thickness in the network plots represents different significance thresholds, as indicated in the legend.