Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

Abstract

bThe development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. Ciliate protozoa have been found to colonize the intestinal tracts of a wide range of ruminant and nonruminant herbivores (1). In the rumen ecosystem, ciliates can account for up to 50% of the total microbial nitrogen, reaching densities of 10 5 to 10 6 cells/ml rumen fluid (2, 3). Although ciliates are not essential for feed degradation and survival of the host (4), it is believed that they contribute to overall gut function, for example, by adding degradative complexity (5), by their ability to scavenge oxygen (6), or by their grazing behavior, which helps to shape and regulate prokaryotic populations (4,7,8). During fermentation of ingested plant material, some of the known rumen ciliates release large amounts of hydrogen produced in their hydrogenosomes (9-11), thereby providing ideal conditions for commensal hydrogenotrophic methanogens. As a consequence, the total number of rumen ciliate protozoa as well as ciliate community composition appears to influence the amount of methane emitted by the host (12-14). However, studies so far have used limited numbers of animals to analyze the impact of ciliate community structure or the roles of individual members of ciliate commun...

Metatranscriptomic Profiling Reveals the Effect of Breed on Active Rumen Eukaryotic Composition in Beef Cattle With Varied Feed Efficiency

“…This inconsistency might be due to the difference on the fraction of data being analyzed and on the focus of these studies. Earlier studies were mainly focused on rumen ciliates, and the taxonomic assignment was made based on sequence data generated using ciliate-specific marker gene (Kittelmann et al, 2013;Henderson et al, 2015;Kittelmann et al, 2015;Comtet-Marre et al, 2017). Although the rumen ciliate reference database used in these studies was based on a full-length 18S rRNA gene, it was built for the analysis of Trichostomatia and thus does not contain reference sequences of flagellated protozoa.…”

“…A recent survey based on 18S rRNA gene amplicon sequencing revealed that almost all protozoal sequencing data from 742 rumen content samples worldwide were assigned to 12 genus-equivalent protozoal groups, namely, Anoplodinium-Diplodinium, Enoploplastron, Entodinium, Epidinium, Eremoplastron-Diploplastron, Eudiplodinium, Metadinium, Ophryoscolex, Ostracodinium, Polyplastron, Dasytricha, and Isotricha (Henderson et al, 2015). Similar rumen ciliate community structure has also been observed in cattle using high-throughput sequencing and microscopic methods (Kittelmann and Janssen, 2011;Kittelmann et al, 2015). The differences between metatranscriptomic and traditional identification methods further emphasize that more isolated and pure culture-based studies are needed to comprehensively characterize rumen eukaryotic microorganisms.…”

Buccal Swabbing as a Noninvasive Method To Determine Bacterial, Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

“…Ciliate protozoal sequences were clustered using the prefix-suffix method (22). Representative OTUs were subjected to BLAST searches against a newly assembled BLAST database composed of the following databases: a modified Greengenes gg_13_5 database, which was depleted of all sequences with taxonomy assignments beginning with "k__Archaea" (26); RIM-DB (25); and databases for intestinal ciliate protozoa (27) and anaerobic fungi (28). Relative abundance tables were generated at the species level (bacteria and archaea) or genus level (ciliate protozoa).…”

An adhesin from hydrogen‐utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen‐producing microorganisms

“…Several studies have shown that endosymbiotic methanogens in the genus Methanobrevibacter reside in protozoa species belonging to the family Isotrichidae (Chagan et al ., ; Irbis and Ushida, ), which suggests that different adhesins may be responsible for interactions with this protozoal family. Eudiplodinium maggii appeared to be the dominant protozoal species in the protozoa sample before reverse panning; however, it is possible that this species is overrepresented in the data, as larger protozoa tend to have more 18S rRNA gene copies per genome (Kittelmann et al ., ). Although quantification of protozoa by 18S rRNA gene copies may introduce a bias towards larger protozoa, this does not affect our interpretation of the pyrosequencing data as we compared the relative abundance of protozoal species in two samples that were processed by the same method.…”