Study Of Biodiversity Of Rumen Ciliates

Rumen ciliates are unicellular eukaryotic microorganisms that live in mutualistic symbiosis with their hosts in the rumen of ruminants,and belong to Protozoa,Ciliophora,which is characterized by complex morphology and rich species diversity.Rumen ciliates are an important part of rumen microorganisms and play an important role in rumen microecosystem,especially in stabilizing rumen environment and influencing host substance metabolism.Due to various factors,there is a lack of basic research on the biodiversity,taxonomy and phylogeny of rumen ciliates in China,which has seriously hindered the in-depth research on their physiological,biochemical and ecological functions.Objectives The biodiversity of rumen ciliates was systematically studied by combining morphological and molecular biology methods.Based on 18S r RNA gene sequences of rumen ciliate species,we investigated the phylogenetic relationships of rumen ciliates.Methods Rumen ciliates of Holstein cattle in China were studied for their biodiversity based on morphological and molecular biological methods.Firstly,we used the microscopic observation method to analyze the overall diversity by density counting and genus composition ratio.Then we used the four morphological study method of ciliates to characterize the typical features of the common genus representative species of rumen ciliates in terms of live morphology,infraciliature pattern,macronucleus morphology and ultrastructure.Finally we obtained the 18S r RNA gene sequences of the common genus representative species of rumen ciliates based on the single cell amplification sequencing technology,and explored the phylogenetic relationships of rumen ciliates through phylogenetic analysis based on the sequences of newly sequenced species in this study and the18S r RNA gene sequences of rumen ciliates available in Genbank database.Results and Conclusions Based on the above methods,the experimental results and findings obtained in this study were as follows:(1)The mean density of rumen ciliates in Chinese Holstein cattle was 3.2±3.4×10~8ind/L.A total of 13 genera of rumen ciliates were detected,including Entodinium,Diplodinium,Eudiplodinium,Eremoplastron,Diploplastron,Ostracodinium,Polyplastron,Elytroplastron,Metadinium,Ophryoscolex,Epidinium,Isotricha,Dasytricha.The five most frequency of appearance genera of rumen ciliates were Entodinium,Eremoplastron,Diplodinium,Isotricha,and Ostracodinium in that order.The composition percentage of Entodinium was as high as84.0±10.2%,while the composition percentage of the other 12 genera were less than 5%,in particular,the lowest composition ratios of 0.01±0.05%,0.1±0.2%,and 0.1±0.2%were found for the genera Elytroplastron,Metadinium,and Polyplastron,respectively.(2)In vivo morphological observations describe the morphological characteristics of 22species of rumen ciliates in ten genera.The results of infraciliature pattern of 28 species of rumen ciliates from 12 genera broadly grouped into the following three types:Entodiniinae-type consisting of"C"AP and thread-like VP;Diplodiniinae-type consisting of"C"AP and semi-annular DP and thread-like VP at the same level;Ophryoscolecinae-type consisting of spiral folded AP at the anterior end of the body and semi-annular large DP and fine thread-like VP at the more posterior end.The macronucleus morphology of 25 species of rumen ciliates in 13 genera has the following types:oval or short rod-shaped(Isotricha,Dasytricha);straight rod-shaped(Entodinium,Ostracodinium,Diploplastron);curved rod-shaped(or thick at the anterior end with a special shape)(Diplodinium,Polyplastron,Elytroplastron,Metadinium,Eudiplodinium);and poke-shaped(thin at both ends and wide in the middle)(Epidinium,Ophryoscolex).The results of infraciliature pattern and macronucleus morphology of the subfamily Diplodiniinae rumen ciliates have intergeneric diversity,reflecting that their internal phylum taxonomic status may be more complex.We obtained the ultrastructure of the striations of 18 species of rumen ciliates in 11 genera,which can be broadly classified as follows:longitudinal,neat,wide bars(Entodinium exiguum,Eremoplastron dilobum,Epidinium,Diploplastron);twisted,discontinuous lines(Diplodinium,Ostracodinium,Polyplastron,Eudiplodinium);irregular and uniformly distributed in sunken circular pores(Ophryoscolex).Scanning electron microscopic ultrastructural observations showed that the striations of the surface membrane were similar and different among genera,indicating that they may have some systematic taxonomic value.(3)In this study,the 18S r RNA gene sequences of 17 representative species of nine common genera(including Entodinium,Diplodinium,Eudiplodinium,Ostracodinium,Polyplastron,Ophryoscolex,Epidinium,Isotricha,Dasytricha)rumen ciliates were obtained by single-cell amplification technique,and the 18S r RNA gene sequences of Entodinium rostratum were obtained for the first time.The phylogenetic relationships of rumen ciliates were explored based on the newly sequenced 18S r RNA gene sequences of Entodinium rostratum and the 18S r RNA gene sequences of 13 genera and 29 species of Ophryoscolecidae rumen ciliates in the public database.(4)The phylogenetic tree based on 18S r RNA gene sequences showed that all three subfamilies in the Ophryoscolecidae were monophyletic,and this result was in full agreement with the taxonomic results of the infraciliature in this study,indicating that the molecular phylogenetic analysis based on 18S r RNA gene sequences can accurately reflect the phylogenetic relationships of rumen ciliates at the level of the subfamily Ophryoscolecidae.The phylogenetic status of each genus within the subfamily Diplodiniinae was partially inconsistent between the morphological results and the molecular phylogenetic results based on 18S r RNA gene sequences,and more morphological results and molecular data of multiple genera are needed to clarify the taxonomic relationships within this subfamily.Significance In this study,we obtained the density and genus composition of ciliates in the rumen of dairy cows by using ciliate morphology,and characterized the typical features of representative species of common genera of rumen ciliates to improve the morphological data base of common taxa of rumen ciliates and provide basic information for teaching and research in related fields;the morphological features and the results of phylogenetic analysis based on single-cell 18S r RNA gene sequencing were complemented and corroborated with each other to explore the phylogenetic relationships of rumen ciliates,and the 18S r RNA gene sequences of Entodinium rostratum were sequenced for the first time,which further increased the diversity of the gene pool of rumen protozoa.This study provides basic information for domestic and even international studies on the biodiversity of rumen ciliates,and also further provides basic information for studying the biological functions and discovering genetic resources of rumen ciliates.In addition,this study can also provide theoretical information for the optimization technology of rumen microbial community in dairy cows to improve livestock production performance and reduce enteric methane emissions from ruminants,which is important for mitigating the global greenhouse effect and preventing and treating human diseases.