Comparative Analysis of Bacterial Community Structures in Earthworm Skin, Gut, and Habitat Soil across Typical Temperate Forests

Abstract

Earthworms are essential components in temperate forest ecosystems, yet the patterns of change in earthworm-associated microbial communities across different temperate forests remain unclear. This study employed high-throughput sequencing technology to compare bacterial community composition and structure in three earthworm-associated microhabitats (skin, gut, and habitat soil) across three typical temperate forests in China, and investigated the influence of environmental factors on these differential patterns. The results indicate that: (1) From warm temperate forests to cold temperate forests, the soil pH of the habitat decreased significantly. In contrast, the physicochemical properties of earthworm skin mucus exhibited different trends compared to those of the habitat soil. (2) Alpha diversity analysis revealed a declining trend in Shannon indices across all three microhabitats. (3) Beta diversity analysis revealed that the transition from warm temperate deciduous broad-leaved forest to cold temperate coniferous forest exerted the most significant impact on the gut bacterial communities of earthworms, while its influence on the skin bacterial communities was comparatively less pronounced. (4) Actinobacteria and Proteobacteria were the predominant phyla in earthworm skin, gut, and habitat soil, but the trends in bacterial community composition differed among the three microhabitats. (5) Mantel tests revealed significant correlations between bacterial community structures and climatic factors, physicochemical properties of earthworm habitat soil, and physicochemical properties of earthworm skin mucus. The findings of this study offer novel perspectives on the interplay between earthworms, microorganisms, and the environment within forest ecosystems.

Keywords: bacteria; diversity analysis; earthworm; environmental factors; mantel test.

Figure 1

Distribution map of the study plot. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest.

Figure 2

Soil physicochemical properties in three temperate forest types. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest; TN, total nitrogen; TC, total carbon; TP, total phosphorus; TK, total potassium; NO₃⁻-N, nitrate nitrogen; NH₄⁺-N, ammonium nitrogen; C/N, carbon–nitrogen ratio. Significant differences are indicated by asterisks based on the nonparametric Kruskal–Wallis test: ** p < 0.01 and *** p < 0.001.

Figure 3

Physicochemical properties of earthworm epidermal mucus in three temperate forest types. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest; TN, total nitrogen; TC, total carbon; TP, total phosphorus; TK, total potassium; NO₃⁻-N, nitrate nitrogen; NH₄⁺-N, ammonium nitrogen; C/N, carbon–nitrogen ratio. Significant differences are indicated by asterisks based on the nonparametric Kruskal–Wallis test: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

MAT, MAP, and ST in three temperate forest types. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest; MAT, mean annual temperature; MAP, mean annual precipitation; ST, soil temperature. Significant differences are indicated by asterisks based on the nonparametric Kruskal–Wallis test: * p < 0.05 and *** p < 0.001.

Figure 5

Bacterial community α-diversity in earthworm habitat soil, skin, and gut across three temperate forest types. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest. Significant differences are indicated by asterisks based on the nonparametric Kruskal–Wallis test: * p < 0.05 and *** p < 0.001.

Figure 6

NMDS ranking plot based on Bray–Curtis dissimilarity in all samples. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest.

Figure 7

Bacterial community composition at the phylum level in three temperate forests. Taxa with relative abundances less than 1% were combined into “Others”. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest.

Figure 8

Variations in the relative abundance of dominant bacterial phyla across three temperate forests. DL, Dongling Mountains Warm temperate deciduous broad-leaved forest; CB, Changbai Mountains Temperate broad-leaved red pine forest; GH, Greater Hinggan Mountains Cold temperate coniferous forest. Significant differences are indicated by asterisks based on the nonparametric Kruskal–Wallis test: * p < 0.05 and ** p < 0.01.

Figure 9

Mantel tests revealing the correlations between bacterial community structure and climatic factors, soil physicochemical properties, and physicochemical properties of earthworm epidermal mucus. MAT, mean annual temperature; MAP, mean annual precipitation; ST, soil temperature; TN, total nitrogen; TC, total carbon; TP, total phosphorus; TK, total potassium; NO₃⁻-N, nitrate nitrogen; NH₄⁺-N, ammonium nitrogen; C/N, carbon–nitrogen ratio. Different asterisks indicate significant differences at * p < 0.05, ** p < 0.01, and *** p < 0.001.