Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives

Abstract

Bombyx mori, the domesticated silkworm, is of great importance as a silk producer and as a powerful experimental model for the basic and applied research. Similar to other animals, abundant microorganisms live inside the silkworm gut; however, surprisingly, the microbiota of this model insect has not been well characterized to date. Here, we comprehensively characterized the gut microbiota of the domesticated silkworm and its wild relatives. Comparative analyses with the mulberry-feeding moths Acronicta major and Diaphania pyloalis revealed a highly diverse but distinctive silkworm gut microbiota despite thousands of years of domestication, and stage-specific signatures in both total (DNA-based) and active (RNA-based) bacterial populations, dominated by the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Most fungal sequences were assigned to the phyla Ascomycota and Basidiomycota. Environmental factors, including diet and human manipulation (egg production), likely influence the silkworm gut composition. Despite a lack of spatial variation along the gut, microbial community shifts were apparent between early instars and late instars, in concert with host developmental changes. Our results demonstrate that the gut microbiota of silkworms assembles into increasingly identical community throughout development, which differs greatly from those of other mulberry-feeding lepidopterans from the same niche, highlighting host-specific effects on microbial associations and the potential roles these communities play in host biology.

Fig. 1

Gut bacterial community dynamics among mulberry-feeding lepidopterans. a Boxplot of species richness (number of OTUs) and community diversity measured by Shannon index. Letters above each host species indicate significant differences (one-way ANOVA, LSD post-hoc test, P < 0.05, see Supplementary Table S7) in the mean values. b Relative abundance of bacterial phyla in different samples. c PCoA plot based on community structure. Each symbol represents a sample, colored by host phylogeny. Variation in communities segregated strongly according to host phylogeny, with B. mori, A. major and D. pyloalis forming discrete groups (PERMANOVA test with 999 permutations, P ≤ 0.05, see Supplementary Table S8). d Heatmap showing the relative abundance of dominant taxa. Hierarchical cluster analysis was based on the Bray–Curtis distance with complete-linkage method. Each bar or column represents an individual insect

Fig. 2

Gut fungal community dynamics among mulberry-feeding lepidopterans. a Relative abundances of fungal phyla in different samples. b Heatmap showing the relative abundance of dominant taxa in each species and mulberry leaves. Cluster analysis used the Bray–Curtis distance and complete-linkage method. Each bar or column corresponds to a species (three individuals for every species)

Fig. 3

Spatial structuring of the B. mori gut microbiota. a Spatial variation of bacterial species richness and diversity. Inset: a schematic of the silkworm gut. n.s. not significant (one-way ANOVA, LSD post-hoc test, P > 0.05, see Supplementary Table S9). b PCoA plot showing variation in community structure among gut regions (PERMANOVA test with 999 permutations, P > 0.05, see Supplementary Table S8). Each point represents an individual insect

Fig. 4

Gut bacteria dynamics across successive life stages of B. mori. a Boxplot of species richness and community diversity throughout development. Different letters indicate statistical significance (one-way ANOVA, LSD post-hoc test, P < 0.05, see Supplementary Table S10). b Phylum-level taxonomic succession. c Overlap of OTUs at the larval stage. d PCoA showing that gut microbiotas segregated strongly according to host developmental stage, with early instars and late instars forming discrete groups (PERMANOVA test with 999 permutations, P < 0.01, see Supplementary Table S8). e Heatmap of major taxa over the life cycle. Cluster analysis used the Bray–Curtis distance and complete-linkage method. Each point, bar or column represents a single individual

Fig. 5

Metabolically active gut bacteria across life stages of B. mori and in A. major, and D. pyloalis. a Phylum-level community composition of B. mori and b other insects. c PCoA showing communities correlated with host developmental stage and d phylogeny (PERMANOVA test with 999 permutations, P ≤ 0.01, see Supplementary Table S8). e Heatmap of major taxa found in B. mori and f A. major and D. pyloalis. Cluster analysis used the Bray–Curtis distance and complete-linkage method. Each point, bar, or column represents a single individual