Earthworm gut virome

The work published in The ISME Journal called “Benzo[a]pyrene stress impacts adaptive strategies and ecological functions of earthworm intestinal viromes”

Earthworm is known as “ecosystem engineer”. These muscular dwellers major contributed to soil structure amendment, nutrition improvement, and soil microbiome adaptability impacted by the voracious feeding habits. Moreover, these little creatures may improve bioremediation of contaminated soils, such as organic pollutants and microplastics. And they are often used as biological indicators for the ecological risk assessment of soil pollutants.

For earthworms, the main members of intestinal microorganisms are Proteobacteria, Firmicutes, Bacteroidetes and Actinomycetes, and many ecological functions are mediated by symbiotic microorganisms. For example, the gut microbes of earthworms are involved in the decomposition of organic matter, nutrient fixation, pollutant degradation, and other biogeochemical cycles. 

Bacteriophages (i.e. bacterial viruses) have lysogenic and lytic life cycle, and they can directly regulate the bacterial community structure by lysing the host through predation. On the other hand, the auxiliary metabolic genes carried by the virus can be used to increase or redirect the resource acquisition, central carbon and energy metabolism in the host cell.

In previous studies, high pollution Cr stress would promote synergistic interaction between phage and bacteria to adapt to extreme environment. However, our understanding of the viriome in the earthworm gut is still lacking. The earthworm intestine is a unique anaerobic environment with rich nutrition and relatively stable physical and chemical properties, which is quite different from the soil environment.

In order to further deepen the understanding of the virome of earthworms and explore the unique interaction between bacteria-phage in the earthworm gut in polluted habitats,
BaP, a typical high molecular weight polycyclic aromatic hydrocarbons with high toxicity, was selected as a typical pollutant in this study, and the pollution gradients of different concentrations were set for the culture of earthworms. 

The research focuses on the following three questions: 
1.    How does BaP affect the structure of bacteria-phage community in earthworm gut?
2.    How does the relationship of intestinal bacteria-phage interaction change under different levels of BaP stress?
3.    To what extent do intestinal bacteria-phage assist earthworms in adapting to environmental stress?

We hypothesize that within a certain concentration range, bacteriophage and bacteria have a cooperative relationship. The metabolic function of bacteriophage helps intestinal bacteria to cope with pollution stress. However, the cooperative relationship disappeared with the increase of contamination concentration.

After a series of pretreatment, earthworms were cultured for 28 days under 5 concentrations of pollutants added soil.

After culture, earthworms were dissected to obtain intestinal contents, then DNA and RNA were extracted, library construction and illumina sequencing were performed.

The result show that BaP significantly induced ROS production in the earthworm gut and the intestinal microbial profile altered significantly under BaP-induced stress.

BaP stress also significantly changed the functional characteristics of microorganisms. Low level BaP exposure enriched the expression of genes coding for bacterial energy production pathways (e.g., carbohydrate, lipid, and amino acid metabolism)

Through KEGG enrichment analysis, we found that low-level BaP exposure significantly enriched genes the pathways closely related to microbial energy production. However, enriched genes in the high-level BaP group were distributed in the pathways related to microorganisms adaptation and survival.

BaP exposure also affected the composition and lifestyle of phage community. Phages adopted a lysogenic adaptive strategy at low BaP levels and converted toward a lytic lifestyle at high BaP levels.

And we find that phage-carried AMGs could enhance cell membrane resistance, increase antioxidant capacity and enhance metabolic degradation ability of BaP.

And then transcriptomic analysis further confirmed the functional activity of phage-carried AMGs

BaP contamination affected phage-bacterium interactions and phage adaptive strategies.High-level BaP exposure resulted in more phage-host associations relative to low-level BaP exposure. The bacteria were subjected to greater phage predation pressure at high BaP concentrations. Phages in the earthworm gut exposure to BaP were more inclined to adopt the Piggyback-the-Winner (PtW) life strategy.

In summary, low-level BaP stress can stimulate microbial metabolism, which enhanced the antiphage defense system and cooperative interaction between phages and bacteria. In contrast, high-level BaP exposure damaged bacterial metabolism and the antiphage systems, resulting in the shift from cooperative to antagonistic relationship between phages and bacteria.

This work was financially supported by National Natural Science Foundation of China 
, The Key R&D Project of Jiangsu Province, and NSF ERC on Nanotechnology-Enabled Water Treatment. 

Mingming Sun and Pingfeng Yu are the corresponding authors.

We would like to thank all of you at the College of Resources and Environmental Sciences, Nanjing Agricultural University.

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