Understanding the biodiversity and genetics of the gut microbiome has important implications for host physiology. One underexplored and elusive group is ciliated protozoa, which play crucial roles in regulating gut microbial interactions. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies for all major abundant clades. With these genomes, we firstly resolved the taxonomic and phylogenetic framework that reclassified them into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Via extensive horizontal gene transfer and gene family expansion, rumen ciliates possess a broad array of enzymes to synergistically degrade plant and microbial carbohydrates. In particular, ~80% of the degrading enzymes in Diplodiniinae and Ophryoscolecinae act on plant cell wall, and the high activities of their cellulase, xylanase and lysozyme reflect the potential of ciliate enzymes for biomassconversion. Additionally, the new ciliate dataset greatly facilitated the rumen metagenomic analyses by allowing ~12% of reads to be classified.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Understanding the biodiversity and genetics of the gut microbiome has important implications for host physiology. One underexplored and elusive group is ciliated protozoa, which play crucial roles in regulating gut microbial interactions. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies for all major abundant clades. With these genomes, we firstly resolved the taxonomic and phylogenetic framework that reclassified them into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Via extensive horizontal gene transfer and gene family expansion, rumen ciliates possess a broad array of enzymes to synergistically degrade plant and microbial carbohydrates. In particular, ~80% of the degrading enzymes in Diplodiniinae and Ophryoscolecinae act on plant cell wall, and the high activities of their cellulase, xylanase and lysozyme reflect the potential of ciliate enzymes for biomass-conversion. Additionally, the new ciliate dataset greatly facilitated the rumen metagenomic analyses by allowing ~12% of reads to be classified.