Study On The Adaptation Mechanism Of Rhesus Macaques(Macaca Mulatta) To High-altitude Environment Based On Fecal Metagenomes And Metabolomes

The extreme cold,low oxygen,and high ultraviolet radiation of the high-altitude environment force high energy demands on animals,but the relative scarcity and low nutritional quality of food resources pose a great challenge to their survival.Macaques（Macaca mulatta）are one of the few primates that are widely distributed at both high and low altitudes,and there have been many studies on the genetic mechanisms underlying their adaptation to high-altitude environments.The 16S rRNA sequencing revealed a significantly higher diversity of gut microbiota in high-altitude populations than in low-altitude populations,which is presumed to play an important role in animal adaptation to high-altitude environments.However,previous studies have only characterized taxonomic composition,and understanding of the mechanisms of animal adaptation to high altitude environments from the perspective of functional gene composition and metabolites of gut microbiota remains poorly understood.Here we collected fresh fecal samples from two high-altitude populations（Average altitude>3,000m）and two low-altitude populations（Average altitude<1,000m）of wild macaques.We screened a total of 40 samples（n=10 for each population）using gene amplification of six microsatellite loci for individual identification,and SRY and ZFX gene for sex identification.Fecal metagenomic and metabolomic analyses were performed on these samples to investigate the synergistic role of gut microbiota in the adaptation of macaques to high-altitude environments.Meanwhile,the similarity in the structure of gut microbiota composition between wild macaques,humans and dogs in the high-altitude environment was examined by reanalyzing the sequence of the 16S rRNA gene V4 region.The main results are as follows:（1）The diversity of gut microbial composition was significantly higher in high-altitude populations of wild macaques compared to low-altitude populations,and the gut microbial composition was more similar among individuals in high-altitude populations.Microbiota taxonomic annotation yielded 7471 species,with 37 microbiota species significantly higher in abundance in the high-altitude populations,16 of which was previously reported to be associated with host dietary digestion and energy metabolism.14 microbiota species were found to be significantly higher in low-altitude populations,but only 4 of these are associated with the host diet digestion and energy metabolism.The analysis of CAZyme composition revealed that genes encoding 22 glycoside hydrolases were significantly more abundant in high-altitude populations（P<0.05）,while low-altitude populations only had genes encoding12 glycoside hydrolases that were significantly more abundant（P<0.05）.（2）Non-targeted metabolomic techniques revealed fecal metabolic profiles in high-altitude macaques.In addition,metabolite traceability analysis revealed higher levels of steroid hormone synthesis and significantly higher metabolism of various amino acids including cysteine and methionine in high-altitude macaques compared to low-altitude macaques（P<0.05）.（3）Combined metagenomic and metabolite analyses revealed a significant enrichment of genes for the modules related to the synthesis of the energy substrate acetyl coenzyme A using CO₂ and pyruvate using oxaloacetate in the gut microbiota of high-altitude macaques,as well as a significant enrichment of genes for the conversion of acetyl coenzyme A to the energy substrate acetate（P<0.05）,and an enrichment of genes for the microbial consumption modules for the energy substrates acetyl coenzyme A,pyruvate and acetate produced.There were no significant differences between the high-low altitude groups and these energy substrates were not detected in feces.Fecal metabolomic analysis revealed that the abundance of the seven short-chain fatty acids and their conjugates detected were not significantly different between the high-and low-altitude groups（P>0.05）.（4）Met Origin-based traceability analysis of fecal metabolites revealed that among the metabolic pathways specific to the microbiota,all five metabolites corresponding to the four metabolic pathways of toluene degradation（ko00623）and polycyclic aromatic hydrocarbons（PAHs）degradation（ko00624）were significantly more abundant in the feces of the high-altitude group.BIO-Sankey network plots based on biological correlations showed that three microbiota were closely associated with three metabolites and four reactions in the toluene degradation pathway,with two microbiota positively associated with two metabolites.Six microbiota were closely associated with two metabolites and three reactions in the polycyclic aromatic hydrocarbons degradation pathway.Among these,five microbiota were positively associated with two metabolites.Enrichment by metagenomic functional modules also revealed a significant enrichment of R01633 reactive genes in the phthalate degradation（M00624）module of the PAHs degradation pathway.（5）A complementary study based on the composition of gut microbiota from 16S rRNA amplicon data revealed that the gut microbiota diversity was higher in the high-altitude group than in the low-altitude group among wild macaques,humans and dogs.Beta diversity analysis also revealed that the Uni Frac distance was significantly lower in the high-altitude environment than in the low-altitude environment for all three species（P<0.05）.The proportion and relative abundance of core bacterial types were significantly higher in the high-altitude environment than in the low-altitude environment（P<0.05）.Neutral community model analysis revealed that the gut microbiota of the three species had a greater dispersal coefficient at high altitudes than at low altitudes.In conclusion,this study reveals the synergistic mechanism of the gut microbiota in wild macaques adapting to high-altitude environments.The composition and functional structure of the gut microbiota in high-altitude macaques are adapted to the scarcity of high-quality food and the high energy demand in high-altitude environments.This adaptation can effectively improve the host food digestion,with a higher potential for producing energy substrates such as acetyl-Co A,pyruvate,and acetate,providing energy compensation for the host,and helping it adapt to the high-energy demand of high-altitude environments.It can also help the host to degrade exogenous toxic substances such as polycyclic aromatic hydrocarbons and toluene produced when consuming complex foods such as plant rhizomes.Meanwhile,this study also revealed the biological and statistical connections between gut microbiota and metabolites at the metabolic pathway level,providing basic data for understanding the role of gut microbiota in the host.Additionally,from the perspective of gut microbiota composition,high-altitude environments strongly drive convergent adaptation of wild macaques to high-altitude environments with human and dog gut microbiota.This further extends our understanding of the mechanisms of high-altitude adaptation in humans and other mammals.