Abstract

The rumen protozoa, alongside fungi, comprise the eukaryotic portion of the rumen microbiome. Rumen protozoa may account for up to 50% of biomass, yet their role in this ecosystem remains unclear. Early experiments inferred a role in carbohydrate and protein metabolism, but due to their close association with bacteria, definitively attributing these functions to the protozoa was challenging. The advent of ‘omic technologies has created opportunities to broaden our understanding of the rumen protozoa. This study aimed to utilize these methods to further our understanding of the role that protozoa play in the rumen in terms of their metabolic capacities, and in doing so, contribute valuable sequence data to reduce the chance of mis or under-representation of the rumen protozoa in meta’omic datasets. Rumen protozoa were isolated and purified using glucose-based sedimentation and differential centrifugation, extracted RNA was Poly(A) fraction enriched and DNase treated before use in a phage-based, cDNA metatranscriptomic library. Biochemical activity testing of the phage library showed 6 putatively positive plaques in response to carboxymethyl cellulose agar (indicative of cellulose activity), and no positive results for tributyrin (indicative of esterase/lipase activity) or egg yolk agar (indicative of proteolysis). Direct sequencing of the cDNA was also conducted using the Illumina HiSeq 2500. The metatranscriptome identified a wealth of carbohydrate-active enzymes which accounted for 8% of total reads. The most highly expressed carbohydrate-active enzymes were glycosyl hydrolases 5 and 11, polysaccharide lyases and deacetylases, xylanases and enzymes active against pectin, mannan and chitin; the latter likely used to digest rumen fungi which contain a chitin-rich cell membrane. Codon usage analysis of expressed genes also showed evidence of horizontal gene transfer, suggesting that many of these enzymes were acquired from the rumen bacteria in an evolutionary response to the carbohydrate-rich environment of the rumen. This study provides evidence of the significant contribution that the protozoa make to carbohydrate breakdown in the rumen, potentially using horizontally acquired genes, and highlights their predatory capacity.

Highlights

  • Rumen protozoa are classified into 2 groups, namely the entodinomorphs and holotrichs

  • With the view to developing the current understanding of the role and functions of the rumen protozoa, this study created a phage-based metatranscriptomic library from purified protozoal cDNA that was functionally screened for lipase, protease and cellulase activity

  • Direct sequencing data confirmed the functional screening results by demonstrating that carbohydrate metabolism is a primary function of the rumen protozoa, especially when compared with lipolytic and/or proteolytic activity

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Summary

Introduction

Rumen protozoa are classified into 2 groups, namely the entodinomorphs and holotrichs. These protozoa are distinctive in terms of their phenotypic and behavioral adaptations, which allow survival in this harsh anaerobic environment. In addition to a high A-T bias, rumen protozoa are further complicated by the presence of two types of nuclei: the macro and micronucleus. This lack of reference sequences for rumen protozoa often results in their mis- or under-representation in meta ‘omic datasets as well as poor coverage during annotation (Comtet-Marre et al, 2017). Recent developments in the field of ‘omics, allow researchers to delve further than ever before into otherwise challenging microbiomes

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