Anaerobic fungi (AF) play an essential role in feed conversion due to their potent fiber degrading enzymes and invasive growth. Much has been learned about this unusual fungal phylum since the paradigm shifting work of Colin Orpin in the 1970s, when he characterized the first AF. Molecular approaches targeting specific phylogenetic marker genes have facilitated taxonomic classification of AF, which had been previously been complicated by the complex life cycles and associated morphologies. Although we now have a much better understanding of their diversity, it is believed that there are still numerous genera of AF that remain to be described in gut ecosystems. Recent marker-gene based studies have shown that fungal diversity in the herbivore gut is much like the bacterial population, driven by host phylogeny, host genetics and diet. Since AF are major contributors to the degradation of plant material ingested by the host animal, it is understandable that there has been great interest in exploring the enzymatic repertoire of these microorganisms in order to establish a better understanding of how AF, and their enzymes, can be used to improve host health and performance, while simultaneously reducing the ecological footprint of the livestock industry. A detailed understanding of AF and their interaction with other gut microbes as well as the host animal is essential, especially when production of affordable high-quality protein and other animal-based products needs to meet the demands of an increasing human population. Such a mechanistic understanding, leading to more sustainable livestock practices, will be possible with recently developed -omics technologies that have already provided first insights into the different contributions of the fungal and bacterial population in the rumen during plant cell wall hydrolysis.
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.
The rumen contains a multi-kingdom, commensal microbiome, including protozoa, bacteria, archaea, fungi and viruses, which enables ruminant herbivores to ferment and utilize plant feedstuffs that would be otherwise indigestible. Within the rumen, virus populations are diverse and highly abundant, often out-numbering the microbial populations that they both predate on and co-exist with. To date the research effort devoted to understanding rumen-associated viral populations has been considerably less than that given to the other microbial populations, yet their contribution to maintaining microbial population balance, intra-ruminal microbial lysis, fiber breakdown, nutrient cycling and genetic transfer may be highly significant. This review follows the technological advances which have contributed to our current understanding of rumen viruses and drawing on knowledge from other environmental and animal-associated microbiomes, describes the known and potential roles and impacts viruses have on rumen function and speculates on the future directions of rumen viral research.