Comparative Analysis of the Symbiotic Microbiota in the Chinese Mitten Crab (Eriocheir sinensis): Microbial Structure, Co-Occurrence Patterns, and Predictive Functions

Abstract

Symbiotic microorganisms in the digestive and circulatory systems are found in various crustaceans, and their essential roles in crustacean health, nutrition, and disease have attracted considerable interest. Although the intestinal microbiota of the Chinese mitten crab (Eriocheir sinensis) has been extensively studied, information on the symbiotic microbiota at various sites of this aquatic economic species, particularly the hepatopancreas and hemolymph, is lacking. This study aimed to comprehensively characterize the hemolymph, hepatopancreas, and intestinal microbiota of Chinese mitten crab through the high-throughput sequencing of the 16S rRNA gene. Results showed no significant difference in microbial diversity between the hemolymph and hepatopancreas (Welch t-test; p > 0.05), but their microbial diversity was significantly higher than that in the intestine (p < 0.05). Distinct differences were found in the structure, composition, and predicted function of the symbiotic microbiota at these sites. At the phylum level, the hemolymph and hepatopancreas microbiota were dominated by Proteobacteria, Firmicutes, and Acidobacteriota, followed by Bacteroidota and Actinobacteriota, whereas the gut microbiota was mainly composed of Firmicutes, Proteobacteria, and Bacteroidota. At the genus level, Candidatus Hepatoplasma, Shewanella, and Aeromonas were dominant in the hepatopancreas; Candidatus Bacilloplasma, Roseimarinus, and Vibrio were dominant in the intestine; Enterobacter, norank_Vicinamibacterales, and Pseudomonas were relatively high-abundance genera in the hemolymph. The composition and abundance of symbiotic microbiota in the hemolymph and hepatopancreas were extremely similar (p > 0.05), and no significant difference in functional prediction was found (p > 0.05). Comparing the hemolymph in the intestine and hepatopancreas, the hemolymph had lower variation in bacterial composition among individuals, having a more uniform abundance of major bacterial taxa, a smaller coefficient of variation, and the highest proportion of shared genera. Network complexity varied greatly among the three sites. The hepatopancreas microbiota was the most complex, followed by the hemolymph microbiota, and the intestinal microbiota had the simplest network. This study revealed the taxonomic and functional characteristics of the hemolymph, hepatopancreas, and gut microbiota in Chinese mitten crab. The results expanded our understanding of the symbiotic microbiota in crustaceans, providing potential indicators for assessing the health status of Chinese mitten crab.

Keywords: Chinese mitten crab; hemolymph; hepatopancreas; high-throughput sequencing; intestine; symbiotic microbiota.

Figure 1

Comparison of alpha diversity indices of the bacterial communities at different sites. (A) Comparison of the Sobs index of the bacterial communities at different sites, (B) comparison of the Shannon index of the bacterial communities at different sites, (C) comparison of the Simpson index of the bacterial communities at different sites, and (D) comparison of the Chao index of the bacterial communities at different sites. Higher Sobs and Chao values indicate a higher richness; higher Shannon and lower Simpson values indicate a higher diversity. Statistical significances between the two sites were considered at ** p < 0.01, and *** p < 0.001 by Welch t-test. B: hemolymph (B1–B12); H: hepatopancreas (H1–H12); I: intestine (I1–I12).

Figure 2

Analysis of the bacterial community structure at different sites. (A) Principal coordinate analysis based on the Bray-Curtis metric of the bacterial communities. The percentages indicate the relative contribution of the principal components. (B) The taxonomic clustering tree based on Bray-Curtis metric of the bacterial communities. B: hemolymph (B1–B12); H: hepatopancreas (H1–H12); I: intestine (I1–I12).

Figure 3

Distribution of the bacterial communities in all thirty-six samples at the phylum level. B: hemolymph (B1–B12); H: hepatopancreas (H1–H12); I: intestine (I1–I12).

Figure 4

(A) The petalogram shows the number of genera that were shared at different sites of twelve crabs. (B) Network of co-occurring bacterial OTUs across three sites. Only Spearman’s correlation coefficient (r > 0.7 or r < −0.7 significant at p < 0.05) is shown. The nodes are colored according to phyla. Green edges represent positive correlations and red edges represent negative correlations. Node size is proportional to the betweenness centrality of each OTU, and edge thickness is proportional to the weight of each correlation.

Figure 5

(A) LEfSe showing differences in the bacterial communities at the OTU level for three sites. (B) LEfSe showing differences in the predicted functions of different sites as predicted by Tax4Fun analysis. B: hemolymph (B1–B12); H: hepatopancreas (H1–H12); I: intestine (I1–I12).

Figure 6

Heatmap profiles showing the functional categories (Kyoto Encyclopedia of Genes and Genomes (KEGG) level 2) of the bacterial communities, as predicted by Tax4Fun analysis. The functional clustering analysis is based on the unweighted pair-group method with arithmetic means (UPGMA). Rows represent the KEGG Orthology (KO) functions, columns represent the 36 samples, and the color intensity in the heatmap represents the relative abundance of the functional categories. B: hemolymph (B1–B12); H: hepatopancreas (H1–H12); I: intestine (I1–I12).