Benzo[a]pyrene stress impacts adaptive strategies and ecological functions of earthworm intestinal viromes

Abstract

The earthworm gut virome influences the structure and function of the gut microbiome, which in turn influences worm health and ecological functions. However, despite its ecological and soil quality implications, it remains elusive how earthworm intestinal phages respond to different environmental stress, such as soil pollution. Here we used metagenomics and metatranscriptomics to investigate interactions between the worm intestinal phages and their bacteria under different benzo[a]pyrene (BaP) concentrations. Low-level BaP (0.1 mg kg^-1) stress stimulated microbial metabolism (1.74-fold to control), and enhanced the antiphage defense system (n = 75) against infection (8 phage-host pairs). Low-level BaP exposure resulted in the highest proportion of lysogenic phages (88%), and prophages expressed auxiliary metabolic genes (AMGs) associated with nutrient transformation (e.g., amino acid metabolism). In contrast, high-level BaP exposure (200 mg kg^-1) disrupted microbial metabolism and suppressed the antiphage systems (n = 29), leading to the increase in phage-bacterium association (37 phage-host pairs) and conversion of lysogenic to lytic phages (lysogenic ratio declined to 43%). Despite fluctuating phage-bacterium interactions, phage-encoded AMGs related to microbial antioxidant and pollutant degradation were enriched, apparently to alleviate pollution stress. Overall, these findings expand our knowledge of complex phage-bacterium interactions in pollution-stressed worm guts, and deepen our understanding of the ecological and evolutionary roles of phages.

Fig. 1. Toxicity of Benzo[a]pyrene (BaP) to earthworm intestines and gut bacterial community.

Reactive oxygen species (ROS) (A) and superoxide dismutase (SOD) activity (B) of earthworm gut exposed to BaP at 0 (CK), 0.1 (B1), 2.0 (B2), 20 (B3), and 200 (B4) mg per kg soil. C Relative abundance of the top 10 abundant intestinal bacteria genus. D Alpha diversity (ACE and Chao1 indexes for richness, and Shannon, Simpson, Pielou indexes for evenness) of the bacterial community in the earthworm intestines. The radar map is drawn according to the data in Table S2 after normalization. The error bars in this study mean standard deviation of three replicates. *p < 0.05 indicates significant differences between the BaP exposure treatment and control, based on the t-test.

Fig. 2. Functional profile and metatranscriptome validation of prokaryotic community in earthworm intestines.

A Heatmap shows the relative abundance of KEGG pathway (Level 2) of earthworm intestinal prokaryotic community. Abundance is normalized among different treatments based on Z-score. Red stars represent pathways with significant differences in expression levels in metatranscriptome. The regulation of genes related to energy and metabolism (B), stress adaptation (C) and BaP degradation (D) in metatranscriptomes relative to the control group. The histogram shows the top 15 enriched KEGG orthology (KO) pathways of enriched genes under low- (E) and high- (F) level BaP exposure. The number of enriched genes in each pathway is labeled in front of the histogram, and the bars are ranked by p values. “Relative abundance” represents the proportion of significantly enriched genes to all genes in the KEGG pathway. The dark red single stranded nucleic acid shown in the diagram represents data obtained from the metatranscriptome.

Fig. 3. Composition and lifestyle of earthworm gut virome.

A Gene-sharing network associates viral contig in earthworm gut (red nodes) with datasets of viral genomes that includes viral sequence from bee gut (yellow nodes), human gut (blue nodes), soil (green nodes), and RefSeq (gray nodes). The edges indicate similarity based on shared protein clusters. B The matrix layout shows the number of VCs that are exclusive (one circle) or shared (multiple circles) between the five different datasets used for clustering. C Venn diagram of shared VC among the five different datasets. D Community compositions (family level) of phages in earthworm gut. E Transmission electron microscopy (TEM) image of typical phage in earthworm intestines. The scale bars shown in the figures are 100 nm. F Proportions of lytic phage and lysogenic phage in earthworm gut. G Abundance (Log10 RPKM) of prophage.

Fig. 4. Phage-encoded auxiliary metabolic genes (AMGs) and their transcriptome profiles under BaP stress.

A Heatmap shows the classification of AMG in KEGG level 2 pathways and their relative abundance. B Number of AMGs in lysogenic and lytic viral contig respectively. “Occurred number of contig” means the number of contigs carrying a certain class of AMGs in different lifestyles. C Genomic context and protein structure of six phage encoded AMGs associated with BaP resistance and degradation. D Significantly up-regulated or down-regulated gene abundance in different samples and its log2 fold change (fc) compared with the control. When log2 (fc) >0 represents that the gene in the sample is significantly up-regulated compared with control, and vice versa. E Rank the differentially expressed genes according to the log2 fold change. Each circle represents a gene, and the color of the circle represents the significance of the difference in gene expression. The genes annotated to the same function were distinguished by different symbols with detailed information listed in Table S8.

Fig. 5. Analysis of phage-bacterium interactions.

A Predicted phage-host associations. Left: the viral contigs that can match to host. Different colors present different taxonomic classification at the family level. Middle: the number of phage-host pairings in different samples. Right: the distribution of bacterial hosts at the phylum level. The bar chart below counts the number of pairings in each sample, and the classification of the different hosts in the bar is consistent with the information of the hosts on the right. B Identification of antiviral defense systems in bacteria. Left: taxonomic classification of defense systems carrying bacteria at the phylum level. Middle: the number of defense systems in different samples. Right: the subtype and the defense mechanisms of the defense system. The bar charts below show the number of antiviral defence systems. C The phylogenetic tree of the phage host and the bacteria that carry the antiviral defense system. D The expression abundance of defense system genes in metatranscriptomes are presented by the abundance relative to control. E Phage-to-bacterium ratio in earthworm intestines at different concentrations of BaP.

Fig. 6. Conceptual depiction of metabolic functions of bacteria and phages under different BaP stress.

The pathways “glycolysis/gluconeogenesis” and “nucleotide excision repair” were respectively taken as examples to emphasize the differences in physiological metabolic states of bacteria under low (A) and high (B) BaP concentration conditions. The yellow fonts represent genes that are significantly enriched in the transcriptome, and the color squares below the genes represent the log2 fold change compared to the control group. The blue fonts represent phage-encoded AMGs that are prominently expressed.