Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits

Abstract

Background: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined.

Results: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions.

Conclusions: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.

Keywords: Microbiome; Network analysis; Rumen; Strain diversity; Virome.

Fig. 1

Prophages identified from the rumen microbial genomes. a, Number of prophages identified from 8,902 rumen metagenome-assembled genomes (MAGs) including 1,726 RUG2 MAGs [46] and 7,176 MAG assembled in this study (see supplementary information for details). b, The taxonomy of the identified prophage vOTUs. c, The prophage genome encoding one antimicrobial resistance gene (ARG) identified from an Agathobacter sp900546625 genome. d, A prophage gene (second from the 5’ end) under positive selection. This prophage was carried by the genome of one Prevotella sp900317685 strain coexisting with other strains of this species in 46 of the 240 RUG2 samples. Inset figure panel (Upper right corner): distribution of host species that carry various numbers of prophages among the 240 RUG2 samples. See also Supplementary Table 2 for the comparison between the vOTUs taxonomic classification based on the old and new International Committee on Taxonomy of Viruses (ICTV) taxonomy and Supplementary Table 3 for the full list of ARG-carrying prophages and their annotations

Fig. 2

Strain level host specificity of rumen viruses. a, Inter- and intra-species host specificity of the rumen viruses exemplified with one of the RUG2 samples [46], ERR3275126. Each circle represents one microbial genome and is color-coded based on species, while each square represents one vOTU. Connected vOTUs and microbial genomes have matches between the vOTU protospacer sequences and the corresponding microbial spacer sequences. Unconnected circles represent coexisting microbial genomes whose spacer sequences did not match any protospacer sequences. b, The percentage of vOTUs in each of the RUG2 samples (240 in total) infecting a single genome (or MAG) of bacteria, multiple genomes (or MAGs) of different bacterial species, or one of the multiple genomes (MAGs) of the same species (i.e., co-existing bacterial strains of the same species that lack a spacer that matches a protospacer sequence)

Fig. 3

Co-occurrence networks showing the modular organization of rumen microbiome and microbe-virus interactions. a, Rumen microbe-only network. b, Microbe-virus network. Both networks were built with the microbes and viruses identified with a prevalence greater than 50% in the 240 RUG2 samples [46]. Microbial nodes are denoted as circles, and viral nodes are denoted as squares. The microbial species of the same phylum or predicted hosts of the viruses are displayed with one distinct color. Large circles represent core bacterial species ubiquitous in the 975 metagenomes used in developing the RVD [11], while small circles represent non-core microbial species. The colors of the edges designate different connections between different nodes. The three largest modules in each network are highlighted in red, green, and blue. See also Supplementary Fig. 4 for the largest three modules of the microbe-virus network

Fig. 4

Rumen viral richness is associated with both dietary composition and animal production traits. a, The effect of dietary composition on viral richness. b, Viral richness variations between animals with differing production traits. Box plots indicate the median (middle line), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers) as well as individual observations (dots). Statistical significance was tested using the two-sided non-parametric Wilcoxon signed-rank test. p values below 0.05, 0.01, and 0.001 are indicated as “*”, “**”, and “***”, respectively. See also Supplementary Table 1 for detailed information about the studies included. TMR: total mixed ration, primarily consisting of corn silage and concentrate (grain); LLS: low lipid starch diet; HLS: high lipid starch diet; basal: a basal diet with 9.6% crude protein (CP), 14.1% nonfiber carbohydrates (NFC), and 67.3% neutral detergent fiber (NDF); NFC: a diet with 10% CP, 28.3% NFC, and 53.6% NDF; Protein: a diet with 15.6% CP, 16.3% NFC, and 59.3% NDF

Fig. 5

Principal coordinates analysis comparing rumen virome compositions between dietary compositions and between animal production traits. a, Comparison of rumen viromes between dietary compositions. b, Comparison of rumen viromes between animal production traits. Permutational multivariate analysis of variance (PERMANOVA) was used to compare the overall viromes. p values below 0.05, 0.01, and 0.001 are indicated as “*”, “**”, and “***”, respectively. See also Supplementary Table 1 for detailed information about the studies included. TMR: total mixed ration, primarily consisting of corn silage and concentrate (grain); LLS: low lipid starch diet; HLS: high lipid starch diet; basal: a basal diet with 9.6% crude protein (CP), 14.1% non-fiber carbohydrates (NFC), and 67.3% neutral detergent fiber (NDF); NFC: a diet with 10% CP, 28.3% NFC, and 53.6% NDF; Protein: a diet with 15.6% CP, 16.3% NFC, and 59.3% NDF

Fig. 6

Phages infected bacteria have varying abundant in ruminants fed different diets or with different efficiency. Differential abundance analysis identified several vOTUs (blue) that were differentially abundant in ruminants fed different diets (a) and animals differing in feed efficiencies or methane emissions (b). The log2-fold changes of their predicted hosts are denoted in red, and those also significantly differentially abundant between animal cohorts are indicated by red arrows. See also Supplementary Table 2 for detailed information about the studies included. TMR: total mixed ration, primarily consisting of corn silage and concentrate (grain)