Differences in Microbial Community Structure Determine the Functional Specialization of Gut Segments of Ligia exotica

Abstract

Ligia feed on seashore algae and remove organic debris from the coastal zone, thereby playing an important role in the intertidal ecosystem. Nevertheless, the specific roles of distinct gut segments in the gut transit remain unclear. We collected and identified Ligia exotica specimens in the coast of Aoshanwei, Qingdao, Shandong Province, and analyzed their foreguts and hindguts for 16S rRNA, metagenomics, metabolomics, and proteomics. The concentrations of common metabolites, NO₃^--N and NH₄⁺-N, and the contents of C and N were measured. The gut transit decreased the abundances of the dominant phyla Cyanobacteria but increased Proteobacteria, Firmicutes, and Actinobacteria, and Planctomycetes and Bacteroidetes remained relatively constant. The foregut gut microbiota is involved in the carbohydrates and amino acids metabolism, as well as the decomposition of polysaccharides. The hindgut gut microbiota performs a variety of functions, including carbohydrate and amino acid metabolism, fermentation, cell motility, intracellular transport, secretion, and vesicular translocation, and the decomposition of polysaccharides, disaccharides, and oligosaccharides. The results of omics analyses and molecular experiments demonstrated that the metabolic processes involving amino acids and carbohydrates are more active in the foregut, whereas the fermentation, absorption, and assimilation processes are more active in the hindgut. Taken together, the differences in microbial community structure determine the functional specialization of different gut segments, i.e., the foregut appears to be the primary site for digesting food, while the hindgut further processes and absorbs nutrients and then excretes them.

Keywords: 16S rRNA gene sequencing; gut transit; ligia exotica; metabolome; metagenome; proteome.

Figure 1

Collection and identification of Ligia exotica in the coast of Aoshanwei, Qingdao, Shandong province. The red dot represents sample collection site (A); the morphology of L. exotica dorsal (B); and abdomen (C). Phylogenetic tree of isopoda based on the COI sequences; the “Species” represent the species we studied (D).

Figure 2

The 16S rRNA gene sequencing analysis in the foregut and hindgut of L. exotica. Venn diagram of gut bacteria in the foregut and hindgut samples of L. exotica (A). Histogram of relative abundance of species at the phylum (B) and genus (C) level in the foregut and hindgut of L. exotica. The abundance clustering heatmap of bacteria at the genus level in the foregut and hindgut of L. exotica (D). The presence and absence of gut microbiota between L. exotica in South Korea and L. exotica in Qingdao, Shandong Province. Absence or presence of a certain gut microbes is marked in yellow or blue (E). The β diversity of gut microbiota in the foregut and hindgut of L. exotica based on the PLS-DA method (F).

Figure 3

KEGG and EggNOG analysis in metagenomic sequencing of the foregut and hindgut of L. exotica. Number of genes in metabolic pathways annotated to KEGG level 2 by the gut microbiota of L. exotica (A). Relative abundance on the first (B) and second (C) levels of KEGG in the foregut and hindgut of L. exotica. The functional proteins contained in the L. exotica gut microbiota (D). Distribution of functional LDAs based on EggNOG level 1 differences in the foregut and hindgut of L. exotica (E). Heatmap of horizontal distribution based on EggNOG level 2 (F).

Figure 4

CAZy and CARD analysis in metagenomic sequencing of the foregut and hindgut of L. exotica. Relative abundance of the six functions of CAZy in the foregut and hindgut of L. exotica (A) and relative abundance of the level 2 of CAZy in the foregut and hindgut of L. exotica (B). Differential functional LDA distribution based on CAZy level 1 in the foregut and hindgut of L. exotica (C). Attribution analysis of resistance genes in the foregut (D) and hindgut (E) at the phylum. Heatmap of top 30 antibiotic resistance gene abundance clustering in the foregut and hindgut of L. exotica (F).

Figure 5

Metabolome analysis in the foregut and hindgut of L. exotica. Amounts of different metabolites in the foregut and hindgut of L. exotica (A). KEGG enrichment analysis of differential metabolites (B). Protein digestion and absorption pathway involved in differential metabolites in the foregut and hindgut of L. exotica, where red represents metabolites that represent significant upregulation of abundance in the foregut, while green represents metabolites that represent significant upregulation of abundance in the hindgut (C).

Figure 6

Proteome analysis in the foregut and hindgut of L. exotica. The bar chart of protein quantitative differences (A). Red color refers to the number of proteins significantly upregulated in the hindgut, and blue color refers to the number of proteins significantly upregulated in the foregut. KEGG diagram of significantly upregulated and downregulated differentially expressed proteins (B). Heatmap illustrating digestive enzyme expression among different gut segments, where red boxes indicate high expression patterns of proteins in different gut segments, and blue boxes indicate low expression patterns of proteins in different gut segments (C).

Figure 7

The determination of physico-chemical properties in the foregut and hindgut. The content of amino acids commonly found in the foregut and hindgut (A). The content of carbohydrate commonly found in the foregut and hindgut (B). The content of metabolites significantly more abundant in the hindgut than in the foregut and the content of metabolites present only in the hindgut (C). The concentrations of NO₃⁻-N and NH₄⁺-N and the elemental percentages of C and N in the foregut and hindgut (D–G).

Figure 8

The diagram of the process of ingested food digestion by L. exotica.