Bacterial Keystone Taxa Regulate Carbon Metabolism in the Earthworm Gut

Abstract

As important ecosystem engineers in soils, earthworms strongly influence carbon cycling through their burrowing and feeding activities. Earthworms do not perform these roles in isolation, because their intestines create a special habitat favorable for complex bacterial communities. However, how the ecological functioning of these earthworm-microbe interactions regulates carbon cycling remains largely unknown. To fill this knowledge gap, we investigated the bacterial community structure and carbon metabolic activities in the intestinal contents of earthworms and compared them to those of the adjacent soils in a long-term fertilization experiment. We discovered that earthworms harbored distinct bacterial communities compared to the surrounding soil under different fertilization conditions. The bacterial diversity was significantly larger in the adjacent soils than that in the earthworm gut. Three statistically identified keystone taxa in the bacterial networks, namely, Solirubrobacterales, Ktedonobacteraceae, and Jatrophihabitans, were shared across the earthworm gut and adjacent soil. Environmental factors (pH and organic matter) and keystone taxa were important determinants of the bacterial community composition in the earthworm gut. Both PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) and FAPROTAX (Functional Annotation of Prokaryotic Taxa) predicted that carbon metabolism was significantly higher in adjacent soil than in the earthworm gut, which was consistent with the average well color development obtained by the Biolog assay. Structural equation modeling combined with correlation analysis suggested that pH, organic matter, and potential keystone taxa exhibited significant relationships with carbon metabolism. This study deepens our understanding of the mechanisms underlying keystone taxa regulating carbon cycling in the earthworm gut. IMPORTANCE The intestinal microbiome of earthworms is a crucial component of the soil microbial community and nutrient cycling processes. If we could elucidate the role of this microbiome in regulating soil carbon metabolism, we would make a crucial contribution to understanding the ecological role of these gut bacterial taxa and to promoting sustainable agricultural development. However, the ecological functioning of these earthworm-microbe interactions in regulating carbon cycling has so far not been fully investigated. In this study, we revealed, first, that the bacterial groups of Solirubrobacterales, Ktedonobacteraceae, and Jatrophihabitans were core keystone taxa across the earthworm gut and adjacent soil and, second, that the environmental factors (pH and organic carbon) and keystone taxa strongly affected the bacterial community composition and exhibited close correlations with microbial carbon metabolism. Our results provide new insights into the community assembly of the earthworm gut microbiome and the ecological importance of potential keystone taxa in regulating carbon cycling dynamics.

Keywords: bacterial community; bacterial community structure; bacterial diversity; earthworm gut; keystone taxa; microbial carbon metabolism.

FIG 1

Diversity of the bacterial community in the earthworm gut and adjacent soil. (a) Shannon’s index; (b) Chao1 index; (c) richness; (d) evenness. Different lowercase letters indicate significant differences based on Tukey’s honestly significant difference (HSD) test (P < 0.05). HMs, adjacent soil under high-manure treatment; HMe, earthworm gut under high-manure treatment; HMLs, adjacent soil under high-manure and -lime treatment; HMLe, earthworm gut under high-manure and -lime treatment.

FIG 2

Taxonomic composition of the bacterial community in the earthworm gut and adjacent soil at the phylum (a) and genus (b) levels. Different lowercase letters indicate significant differences based on Tukey’s HSD test (P < 0.05). HMs, adjacent soil under high-manure treatment; HMe, earthworm gut under high-manure treatment; HMLs, adjacent soil under high-manure and -lime treatment; HMLe, earthworm gut under high-manure and -lime treatment.

FIG 3

Common and endemic bacterial taxa in the earthworm gut and adjacent soil. (a) Venn diagram showing the number of unique and shared OTUs in the earthworm gut and adjacent soil under manure treatments; (b) composition and abundance of 520 shared OTUs in soil and earthworm gut; (c) taxonomic trees of the shared OTUs. Different colors represent the classification levels of kingdom, phylum, class, order, family, genus, and species. HMs, adjacent soil under high-manure treatment; HMe, earthworm gut under high-manure treatment; HMLs, adjacent soil under high-manure and -lime treatment; HMLe, earthworm gut under high-manure and -lime treatment.

FIG 4

Carbon metabolism of the microbial community. (a) Carbon source metabolic activity indicated by the average well color development (AWCD) in the earthworm gut and adjacent soil under manure treatments; (b) relative abundance of carbon source-related metabolic pathways in the prediction of functional pathways using PICRUSt2 analysis; (c) AWCD values of individual carbon sources determined by Biolog assay. Different lowercase letters indicate significant differences based on Tukey’s HSD test (P < 0.05). HMs, adjacent soil under high-manure treatment; HMe, earthworm gut under high-manure treatment; HMLs, adjacent soil under high-manure and -lime treatment; HMLe, earthworm gut under high-manure and -lime treatment.

FIG 5

Structural composition of keystone taxa. (a) Venn diagram of unique and shared keystone taxa in the earthworm gut and adjacent soil; (b) systematic classification tree of keystone taxa. Different colors represent the classification levels of kingdom, phylum, class, order, family, genus, and species. Bright red nodes represent the shared keystone taxa in the earthworm gut and adjacent soil. (c) Chord diagram indicating the composition and relative abundance of keystone taxa in the earthworm gut and adjacent soil under manure treatments. HMs, adjacent soil under high-manure treatment; HMe, earthworm gut under high-manure treatment; HMLs, adjacent soil under high-manure and -lime treatment; HMLe, earthworm gut under high-manure and -lime treatment.

FIG 6

Effects of keystone taxa on microbial carbon metabolism. (a) Correlation analysis between keystone taxa and carbon metabolism indicated by PICRUSt2, FAPROTAX, and Biolog analysis. Carbon (C) cycling is predicted by PICRUSt2 analysis, and the C metabolic pathway is predicted by FAPROTAX analysis. C sources are indicated by the average well color development (AWCD), which was measured by Biolog assay. The shapes of nodes represent different units, and the size indicates the relative abundance of carbon metabolism or degree of connectivity of bacterial OTUs. Red edges represent negative correlations, while blue edges represent positive correlations. The thickness of each connection is proportional to the value of Spearman’s correlation coefficient. (b) Contributions of environmental factors (pH and OM) and the bacterial community to microbial carbon metabolism determined using structural equation modeling. The bacterial community is indicated by diversity (Shannon index), composition (first axis in canonical correspondence analysis [CCA1]), and keystone taxa (the sum of relative abundance). *, P < 0.05; **, P < 0.01; ***, P < 0.001.