Differences and correlation analysis of feeding habits and intestinal microbiome in Schizopygopsis microcephalus and Ptychobarbus kaznakovi in the upper reaches of Yangtze River

Abstract

Background: The intestinal microbiota has co-evolved with the host to establish a stable and adaptive microbial community that is essential for maintaining host health and facilitating food digestion. Food selection is a critical factor influencing variations in gut microbial composition, shaping gut microbiome communities, and determining the ecological niches of fish.

Methods: In this study, high-throughput amplicon sequencing of 16S rRNA and 18S rRNA was utilized to compare the dietary and gut microbial differences between Schizopygopsis microcephalus and Ptychobarbus kaznakovi, both collected from the same sites in the Tuotuo River and Tongtian River, which are tributaries of the Yangtze River. We compared the microbial community structure, diet composition, and diversity between the two fish species using various analytical methods, including LefSe, α-diversity and β-diversity analyses. Additionally, we constructed co-occurrence networks to determine their correlations.

Results and discussion: The alpha diversity results indicated that S. microcephalus exhibited higher intestinal microbiota and feeding diversity compared to P. kaznakovi. Furthermore, the beta diversity results revealed significant differences in both intestinal microbiota and eukaryotic communities between the two species. The dominant bacterial phyla in both S. microcephalus and P. kaznakovi included Proteobacteria, Firmicutes, Actinobacteriota, Chloroflexi, and Verrucomicrobiota; however, Firmicutes was significantly more abundant in P. kaznakovi (P = 0.006), while Actinobacteriota was significantly higher (P = 0.019) in S. microcephalus at the phylum level. The primary food sources for S. microcephalus and P. kaznakovi were identified as Streptophyta (54.41%, 77.50%) and Cercozoa (8.67%, 1.94%), with Bacillariophyta (25.65%) was also the main food of constituting a major component of the diet for S. microcephalus. These differences suggested that S. microcephalus and P. kaznakovi occupy distinct dietary niches. To further explore the relationship between gut microbiota and feeding habits, we identified significant correlations between various food components and the gut microbial community through co-occurrence networks. This study enhances our understanding of the co-evolution and co-adaptation between host gut microbiota and feeding behaviors in sympatric fish species.

Keywords: DIETS; Ptychobarbus kaznakovi; Schizopygopsis microcephalus; correlation analysis; ecological niche; intestinal microbiota.

Figure 1

Overview of intestinal bacterial and diet composition of S. microcephalus and P. kaznakovi. (A, B) Venn diagram displaying the OTUs overlap of intestinal bacteria between S. microcephalus and P. kaznakovi, and the composition of the top five phyla. (C, D) Venn diagram displaying the OTUs overlap of diet between S. microcephalus and P. kaznakovi, and the composition of the top five phyla.

Figure 2

Comparison of alpha diversity between S. microcephalus and P. kaznakovi. The alpha diversity of bacteria (A, B) and eukaryote (C, D) were estimated by the Ace index and Shannon index. Asterisks indicate significant differences between S. microcephalus and P. kaznakovi according to a Wilcoxon rank-sum test: ^*p < 0.05.

Figure 3

Comparison of beta diversity between S. microcephalus and P. kaznakovi. Bacteria (A, B) and eukaryotes (C, D) used PCoA and cluster analysis to assess the distribution differences among the profiles.

Figure 4

Differences in the composition between S. microcephalus and P. kaznakovi. (A, B) Differences in composition of intestinal bacteria between S. microcephalus and P. kaznakovi at phylum and genus levels. (C) The significant differences in bacterial abundance between S. microcephalus and P. kaznakovi. (D, E) Differences in composition of eukaryotes between S. microcephalus and P. kaznakovi at phylum and genus levels. (F) The significant differences in eukaryotic abundance between S. microcephalus and P. kaznakovi. The percentage accumulative histogram displays only the taxonomically annotated species. The cladograms generated by LEfSe by non-parametric factorial Kruskal-Wallis rank sum tests and Wilcoxon rank sum tests. Significance at the phylum, class, and genus levels were indicated, with different letters representing significance at the genus level.

Figure 5

Differences in KEGG pathways between S. microcephalus and P. kaznakovi. Functional abundance distribution of S. microcephalus (A) and P. kaznakovi (B); (C) Results of significance differences in functional abundance between S. microcephalus and P. kaznakovi based on the Wilcoxon rank-sum test.

Figure 6

Correlation analysis between gut bacteria and diet in S. microcephalus and P. kaznakovi based on Spearman's correlation analysis between OTUs. Co-occurrence networks of bacteria in S. microcephalus (A) and P. kaznakovi (E); (B, F) are respective modularity class. Co-occurrence networks of eukaryotes in S. microcephalus (C) and P. kaznakovi (G); (D, H) are respective modularity class. Each network displays with the top 60 abundances, a correlation coefficient >|0.7|, and a P value < 0.05. The nodes were colored by taxonomy at phyla levels. The size of each node is proportional to the number of connections. Each modularity class marks the modules that have a proportion greater than 10%, with M1 having the highest proportion.

Figure 7

Correlation analysis of the gut bacteria and diet combination in S. microcephalus and P. kaznakovi. Co-occurrence analysis of the gut microbes and diet in S. microcephalus and P. kaznakovi based on Spearman's correlation analysis between OTUs. Co-occurrence networks of bacteria and eukaryotes in S. microcephalus (A) and P. kaznakovi (D) for the combined analysis; (B, E) are respective modularity class. Each network displays with the top 60 abundances, a correlation coefficient >|0.7|, and a P value < 0.05. The nodes were colored by taxonomy at phyla levels. The size of each node is proportional to the number of connections. Each modularity class marks the modules that have a proportion greater than 10%, with M1 having the highest proportion. The Mantel test analysis of S. microcephalus (C) and P. kaznakovi (F) are based on Spearman's analysis and demonstrates the top 15 abundant phyla with differences.