Editorial: Advances in the Understanding of the Commensal Eukaryota and Viruses of the Herbivore Gut

Herbivores play an important role in the survival of humanity, contributing food and textiles, as well as social and economic value. For decades, optimizing the productivity, health, welfare, and environmental footprint of herbivorous animals, particularly ruminant livestock, has been the subject of an extensive, global research effort. Much of this research effort has focused on the herbivore gut. The specialized nature of the herbivore digestive tract and its resident microbes enables the breakdown of highly fibrous plant materials, which are unable to be utilized by omnivores and carnivores. In recent years, the bacteria and methanogenic archaea have been the major focus of research efforts, with the other gut microbes being understudied in comparison

Assessment of the Accuracy of High-Throughput Sequencing of the ITS1 Region of Neocallimastigomycota for Community Composition Analysis

Anaerobic fungi (Neocallimastigomycota) are common inhabitants of the digestive tract of large mammalian herbivores, where they make an important contribution to plant biomass degradation. The internal transcribed spacer 1 (ITS1) region is currently the molecular marker of choice for anaerobic fungal community analysis, despite its known size polymorphism and heterogeneity. The aim of this study was to assess the accuracy of high-throughput sequencing of the ITS1 region of anaerobic fungi for community composition analysis. To this end, full-length ITS1 clone libraries from five pure cultures, representing the ITS1 region size range, were Sanger sequenced to generate a reference dataset. Barcoded amplicons of the same five pure cultures, and four different mock communities derived from them, were then sequenced using Illumina HiSeq. The resulting sequences were then assessed in relation to either the reference dataset (for the pure cultures) or the corresponding theoretical mock communities. Annotation of sequences obtained from individual pure cultures was not always consistent at the clade or genus level, irrespective of whether data from clone libraries or high-throughput sequencing were analyzed. The detection limit of the high-throughput sequencing method appeared to be influenced by factors other than the parameters used during data processing, as some taxa with theoretical values >0.6% were not detected in the mock communities. The high number of PCR cycles used was considered to be a potential explanation for this observation. Accuracy of two of the four mock communities was limited, and this was speculated to be due to preferential amplification of smaller sized ITS1 regions. If this is true, then this is predicted to be an issue with only six of the 32 named anaerobic fungal clades. Whilst high-throughput sequencing of the ITS1 region from anaerobic fungi can be used for environmental sample analysis, we conclude that the accuracy of the method is influenced by sample community composition. Furthermore, ambiguity in the annotation of sequences within pure cultures due to ITS1 heterogeneity reinforces the limitations of the ITS1 region for the taxonomic assignment of anaerobic fungi. In order to overcome these issues, there is a need to develop an alternative taxonomic marker for anaerobic fungi.</p

Simultaneous amplicon sequencing to explore co- occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats

Anaerobic fungal communities differ along the horse digestive tract

Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation.</p

PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi:Insights, Challenges, and Opportunities

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit

A Proposed Taxonomy of Anaerobic Fungi (Class Neocallimastigomycetes) Suitable for Large-Scale Sequence-Based Community Structure Analysis

Anaerobic fungi are key players in the breakdown of fibrous plant material in the rumen, but not much is known about the composition and stability of fungal communities in ruminants. We analyzed anaerobic fungi in 53 rumen samples from farmed sheep (4 different flocks), cattle, and deer feeding on a variety of diets. Denaturing gradient gel electrophoresis fingerprinting of the internal transcribed spacer 1 (ITS1) region of the rrn operon revealed a high diversity of anaerobic fungal phylotypes across all samples. Clone libraries of the ITS1 region were constructed from DNA from 11 rumen samples that had distinctly different fungal communities. A total of 417 new sequences were generated to expand the number and diversity of ITS1 sequences available. Major phylogenetic groups of anaerobic fungi in New Zealand ruminants belonged to the genera Piromyces, Neocallimastix, Caecomyces and Orpinomyces. In addition, sequences forming four novel clades were obtained, which may represent so far undetected genera or species of anaerobic fungi. We propose a revised phylogeny and pragmatic taxonomy for anaerobic fungi, which was tested and proved suitable for analysis of datasets stemming from high-throughput next-generation sequencing methods. Comparing our revised taxonomy to the taxonomic assignment of sequences deposited in the GenBank database, we believe that >29% of ITS1 sequences derived from anaerobic fungal isolates or clones are misnamed at the genus level

RIM-DB: a taxonomic framework for community structure analysis of methanogenic archaea from the rumen and other intestinal environments

Methane is formed by methanogenic archaea in the rumen as one of the end products of feed fermentation in the ruminant digestive tract. To develop strategies to mitigate anthropogenic methane emissions due to ruminant farming, and to understand rumen microbial differences in animal feed conversion efficiency, it is essential that methanogens can be identified and taxonomically classified with high accuracy. Currently available taxonomic frameworks offer only limited resolution beyond the genus level for taxonomic assignments of sequence data stemming from high throughput sequencing technologies. Therefore, we have developed a QIIME-compatible database (DB) designed for species-level taxonomic assignment of 16S rRNA gene amplicon data targeting methanogenic archaea from the rumen, and from animal and human intestinal tracts. Called RIM-DB (Rumen and Intestinal Methanogen-DB), it contains a set of 2,379 almost full-length chimera-checked 16S rRNA gene sequences, including 20 previously unpublished sequences from isolates from three different orders. The taxonomy encompasses the recently-proposed seventh order of methanogens, the Methanomassiliicoccales, and allows differentiation between defined groups within this order. Sequence reads from rumen contents from a range of ruminant-diet combinations were taxonomically assigned using RIM-DB, Greengenes and SILVA. This comparison clearly showed that taxonomic assignments with RIM-DB resulted in the most detailed assignment, and only RIM-DB taxonomic assignments allowed methanogens to be distinguished taxonomically at the species level. RIM-DB complements the use of comprehensive databases such as Greengenes and SILVA for community structure analysis of methanogens from the rumen and other intestinal environments, and allows identification of target species for methane mitigation strategies