Comparative Analysis Of Gut Microbiota In Five Herbivores

Complex ecosystems have been forged by co-adaptation over millennia between hosts and gut microbiota to create stable and specific microbiomes.It is believed that the coevolution of animals and microbes led to the development of mutualistic relationships between hosts and their microbial colonizer.The main aim of this study was to explore the relationship between gut microbiota and herbivore evolution.Fecal microbiota in Axis porcinus,Gervus albirostris,Budorcas taxicolor tibetana and Equus hemionus were compared to reveal the coadaptation between microbiota and host phylogeny in different evolutionary stages.The effects of diet,environment and host physiology on the gut microbiota were examined by comparing microbiotas of the same species in different captivity,ages and health state respectively.Transplantations of the fecal microbiotas from five animals into germ-free zebrafish recipients were performed to reveal how host responses to these microbial community and the effect of microbiota on host physiology and metabolism.The details are as follows:1.Coevolution of host and gut microbiota in five herbivores as revealed by 16 S r RNA high throughput sequencingFecal microbiota in captive Axis porcinus(n=12),Gervus albirostris(n=15),Equus hemionus(n=14),Budorcas taxicolor tibetana(n=13)and Rhinopithecus roxellana(n=37)were compared by the Illumina Mi Seq platform targeting the V4 region of the 16 S r RNA.The community composition of Axis porcinus,Gervus albirostris and Budorcas taxicolor resembled each other,while the community composition of Equus hemionus were distinct from those of the three ruminants.Although phylum Firmicutes and phylum Bacteroidetes were two most dominant taxa in Equus hemionus,phylum Firmicutes contributed less to the microbiota of Equus hemionus(40.80%)than to captive Budorcas taxicolor(60.21%),Gervus albirostris(67.36%)or Axis porcinus(59.67%)The phylum Bacteroidetes contributed more to the gut microbiota of Equus hemionus(31.49%)than to captive Budorcas taxicolor(28.70%),Gervus albirostris(23.17%)or Axis porcinus(22.03%).Compared with the three ruminant,the microbiota of Equus hemionus was featured by relative high abundance of phylum Verrucomicrobia(15.11%),which only comprised 1.8%,1.3% and 1.0% of the microbiota of captive Budorcas taxicolor,Gervus albirostris and Axis porcinus,respectively.Meanwhile,phylum Spirochaetes and Fibrobacteres contributed significantly more to the gut microbiota of Equus hemionus than the three ruminants.Compared to these herbivores,Rhinopithecus roxellana had a similar community composition on Phylum level,while a distinctive community composition was observed in Rhinopithecus roxellana on genus level.For community richness comparison,ruminants with larger body size(Budorcas taxicolor,Gervus albirostris)had significantly higher microbial richness than ruminants with small body size(Axis porcinus).Microbial richness was significantly higher for ruminants(Budorcas taxicolor,Gervus albirostris and Axis porcinus)compared with Equus hemionus.On the PCo A plot,microbial communities from ruminants clustered together and microbiotas of Equus hemionus were distinct from those of the ruminants.At the same time,the microbiotas of Rhinopithecus roxellana formed a single cluster.bipartite network and the dendrogram based on databases of microbiotas also showed that the phylogenetic relationships among the microbiota from the four herbivores mirror the relationships of their host species.By analyzing specific OTUs that were differentially distributed among three ruminants,we found that relative abundance of those OTUs showed a descending trend as body size increases.Meanwhile,PICRUSt prediction indicated that the abundance of most microbial activity genes,especially metabolism related genes appeared in the pattern “Budorcas taxicolor< Gervus albirostris< Axis porcinus” The following fecal SCFAs concentrations analysis also confirmed that all concentrations of SCFAs conformed to the law.The results showed that there were obvious coevolution trends in the gut microbiotas and host of these animals.2.Comparative analysis of fecal microbiota in wild and captive takins16S r RNA Illumina Miseq sequencing and denaturing gradient gel electrophoresis(DGGE)were employed to characterize microbial community and diversity in the feces of wild(n=6)and captive takins(n=6).The takin exhibited an intestinal microbiota dominated by three phyla: Firmicutes(57.4%),Bacteroidetes(24.2%)and Proteobacteria(12.3%).At family/genus level,Ruminococcaceae,Bacteroidaceae,Acinetobacter,Clostridium,Lachnospiraceae,Rikenellaceae,Bacillus,Comamonas and Spirochaetaceae were dominant.Distinctive microbiotas between wild and captive takins were observed based on microbial community composition and captive takins had significantly higher community diversity.The results of PCR-DGGE were generally consistent with the results of high-throughput sequencing,while PCR-DGGE showed that wild and captive takins shared a similar microbial community structure.Quantitative real-time PCR(q PCR)were also utilized to monitor predominant bacteria in three Sichuan takin individuals housed in Chengdu zoo over a half-year period,which showed that microbial communities of the three takins were relatively similar to each other and stable during our study period.Our results suggested that diet was a major driver for shaping microbial community composition.3.Diarrhea associated fecal microbiota in captive Sichuan golden snub-nosed monkeys as revealed by 16 S r RNA high throughput sequencingDiarrhea are often correlated with profound alterations in the intestinal microbiota termed dysbiosis.Herein,we characterized the fecal microbiota in diarrhea(n=15)and healthy monkeys(n=37)by the Illumina Mi Seq platform.Concentrations of fecal short-chain fatty acids(SCFAs)and copy numbers of virulence factor genes were also assessed using gas chromatography and Quantitative PCR(q PCR),respectively.The results showed that diarrhea monkeys harbored distinctive microbial composition compared to healthy monkeys and had 45% fewer Bacteroidetes,although diarrhea monkeys had a similar microbial structure to healthy monkeys.Among healthy subjects,old monkeys also had the lowest relative abundance of Bacteroidetes.Linear discriminant analysis effect size(LEf Se)and canonical correlation analysis(CCA)identified significant differences in microbial taxa between diarrhea and healthy monkeys.PICRUSt analysis revealed that several pathogenic genes were enriched in diarrhea monkeys,while glycans-metabolism genes were overrepresented in healthy monkeys.Meanwhile,there was a positive correlation between the abundance of nutrition-metabolism related genes and individual digestive capacity of healthy monkeys.Consequently,the abundance of genes encoding heat stable enterotoxin were significantly higher in diarrhea monkeys than healthy monkeys(P<0.05).In healthy subjects,adult monkeys had significant higher concentrations of butyrate and total SCFAs than old monkeys(P<0.05).The results suggested that pathogens and malabsorption were two main causes of diarrhea,which were closely related to the microbial composition and functions.In conclusion,the present study demonstrated that diarrhea had a microbial component and alterations in microbial composition caused significant changes in systemic metabolic states.4.Transcriptome analysis of germ-free zebrafish recipients transplanted with the microbiotas from five animals We performed transplantations of the fecal microbiotas from Axis porcinus,Gervus albirostris,Equus hemionus,Budorcas taxicolor and Rhinopithecus roxellana into germ-free zebrafish recipients.RNA-seq was employed to analysis the transcriptome of germ-free zebrafish larvae after exposure to the microbiotas from the five animals.In hierarchical clustering analysis based on DEGs FPKM values(expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced)of each sample/group,AP-zebrafish(zebrafish transplanted with Axis porcinus microbiota)had the most similar expression profiles to GA-zebrafish and they had closest phylogenetic relationship.Phylogenetic relationships from near to far,it followed by BT-zebrafish,RR-zebrafish and EH-zebrafish,which was generally consistent with the phylogenic relationship of the five animals.According to gene clustering in heatmap and H-cluster?K-means and SOM analysis,most DEGs were consistent with the pattern “Axis porcinus>Gervus albirostris>Budorcas taxicolor tibetana” or “Axis porcinus<Gervus albirostris<Budorcas taxicolor tibetana”.Quantitative real-time RT-PCR(q RT-PCR)of biomarkers of host metabolism and innate immune responses were also conducted.Compared with conventional raised zebrafish,m RNA expression of innate immune and metabolism genes in germ-free zebrafish were in the state of suppression/ low expression.Transplantations of different microbiotas were able to stimulate and recapitulate the expression of innate immune and metabolism genes to varying degree.In general,highest expression levels of innate immune genes were induced by Rhinopithecus roxellanae and Equus hemionus microbiota.When comparing the influence of microbiotas from Budorcas taxicolor,Gervus albirostris and Axis porcinus on these genes,we also found the pattern“Budorcas taxicolor> Gervus albirostris> Axis porcinus”.These results indicated that the transcriptome responses of Germ-free zebrafish to gut microbiota from the five animals also reflected the phylogenic relationship of the five animals,which would help us understand how host responses to these microbial community and the effect of microbiota on host physiology and metabolism in coevolutionary history.