A Comparative Study On The Intestinal Flora Of Wild And Captive Musk Deer In Winter Based On High-Throughput Sequencing

As rare wildlife,musk deer is a class I protected species in China.Its wild population is becoming more and more endangered due to over-hunting and habitat reduction.On the other hand,high mortality and morbidity hinders the development of domesticated musk deer.As a ruminant,gut microbes is significant to the survival of musk deer.The formation of animal’s gut microbiota is the result of long-term evolution of species and is functionally indispensable of maintaining the health of hosts.Together,the host’s gut and gut microbiota constitute intestinal microbiome which is involved in the correct functioning in thehost’s immune and digestive systems.Due to the closed surrounding of musk enclosures,dense population and unreasonable feed formulation,it’s easy tocause the occurrence of infectious diseases between musk deer individuals.For the wild population,the ability to absorb nutrients from high-fiber foods is key to the survival of wild musk deer in winter.From the standpoint of the different intestinal microbes of wild and captive musk deer,we investigate the community structure,diversity and function of the intestinal microbes from the two groups of musk deer which live in different conditions.And excavate the dominant flora,significant differences flora and their related metabolic pathways.A comparative study of the intestinal microbes of two musk deer group can not only assesses the health status of the two populations,but also lays the foundation for the conservation of the rare wild animals.We used high-throughput sequencing techniques to performl6S rRNA sequencing and metagenomic DNA sequencing of intestinal flora.The results were summarized as follows:1.Analysis oftheintestinal microbial structureof wild and captive musk deer based on 16S rRNA sequencingBased on the 16S rRNA sequencing,the intestinal microbes were classified,we identified the highest abundance of 10 microbes’ phylum of the musk deer.Their relative abundances in the wild musk deer(W)and captive musk deer(C)are respectively:(Firmicutes:85.27%,59.80%),(Bacteroidetes:10.30%,29.23%),(Spirochaetes:0.11%,2.98%),(Proteobacteria:1.17%,3.14%),(Tenericutes:1.26%,1.20%),(Cyanobacteria:0.49%,0.49%),(Fibrobacteres:0.03%,0.30%),(Verrucomicrobia:0.14%,0.11%),(Actinobacteria:0.61%,0.42%)and(Euryarchaeota:0.06%,0.32%).Through comparison,it was found that the intestinal flora structure of the two groups of musk deer were similar in overall,Firmicutes and Bacteroidetes were the dominant bacteria,but their proportion was significant different.As one of the important indexes of nutrient absorption and energy metabolism,the ratio of Firmicutes and Bacteroidetes(F/B)in the W group was significantly higher than that in the C group(W:8.28,C:2.05).High ratio of Firmicutes can help the body absorb more nutrients from food and cause fat deposits.This is an important physiological mechanism of wild musk deer to adapt to the barren food in winter.The top 10 highest abundance of intestinal microbes were:5-7N15,Ruminococcus,Clostridium,Treponema,Roseburia,Bacteroides,Oscillospira,Anaerostipes,Coprococcusand Dorea.The biggest difference of genus between the two groups was Treponema.The core bacteria which were present in all the musk deer stool samples were Roseburia,Coprococcus,Clostridium,Oscillospira,Blautia,rc4-4,Ruminococcusand5-7N15.All the core bacterias belonged to the Firmicutes except for the 5-7N15,and most of the core bacteria had the function ofproducing short chain fatty acid(SCFA).2.Analysis of intestinal microbial diversity and the bacteria with significant difference from the wild and captive musk deerWe analyzed the similarity,the number of bacteria,the abundance,the difference in flora structure and the diversity of the samples within and between the two groups by analyzing a diversity and β diversity of the wild and captivemusk deer.Generally speaking,there was a significant separation between the two groups,the difference was significant between the groups than difference of samples within group.The differences in microbial diversity among the samples in the W group were greater,but were slightly lower than those in the C group.It could be seen that,thirty years’ domestication process of musk deer did not significantly reduce the diversity of thei rintestinal microbes.Microbes with significant differences between the two groups were excavated by T-test and LEfSe analysis.Within those microbes,Clostridium,Roseburia,Oscillospira,Anaerostipes,Enterococcus,Dorea,Blautia,BifidobacteriumandFaecalibacteriumshowed higher abundance in W group,and they all belonged to Firmicutes.While most of those microbes showed higher abundance in C group which belong toBacteroidetes and Spirochaetes,like Treponema,5-7N15,Ruminococcus,Bacteroides,Paludibacter,Methanobrevibacter,Phascolarctobacterium,Prevotella,Paraprevotella and Shewanella.3.Analysis of the different function of intestinal microflora from wild and captive musk deer based on metagenomic DNA sequencingBased on the high-throughput sequencing method,we analyzed the function of the intestinal flora of the musk deerfrom three aspects:the metabolic pathway,cluster of orthologous groups and carbohydrate enzyme.Based on KEGG database,we counted the unique enzyme reaction of each group.The unique enzyme reaction of intestinal flora from wild musk deer was represented by K06045(squalene-hopene/tetraprenyl-beta-curcumene cyclase),K01047(secretory phospholipase A2),K00036(glucose-6-phosphate 1-dehydrogenase),K00832(aromatic-amino-acid transaminase),K00022(3-hydroxyacyl-CoA dehydrogenase)and K01692(enoyl-CoA hydratase).These enzymes were mainly involved in biological processes including metabolism of terpenoids and polyketone compounds,sesquiterpenoids and triterpenoid biosynthesis,synthesis and metabolism of SCFA,Phenylalanine,Tyrosine and Tryptophan biosynthesis.We speculated that intestinal flora of wild musk deer contained high abundance of bacteria associated with SCFAproduce.SCFA were not only the important energy source to the body,but also helped to resist the occurrence of some intestinal diseases.A total of 4 883 genes were annotated to 37 glycosyltransferase families(GTs),and the involved major enzymes were cellulase synthase,chitinase,phosphodiester D-mannose aminotransferase,glucosyltransferase,hyaluronan synthase and chitosan oligosaccharide synthase.In all carbohydrate esterase(CEs),the highest abundance gene of the musk deer’sintestinal flora was CE4,accounting for 32.9%of the total number of family annotated genes.CE4 and CE9 showed higher abundance of acetyl xylan esterase,chitin deacetylase,chitooligosaccharide deacetylase,peptidoglycan GlcNAc deacetylase,peptidoglycan N-acetylmuramic acid deacetylase.Acetyl xylan esterase and chitin deacetylase had the function of digesting the cell wall of fungi and plant,chitin of mold cell wall,dermatoplasm of bacterial cell wall.We hypothesized that the high abundance of chitinase in the wild muskdeer intestinal flora was related to their fungi recipe,it also confirmed the correlation between the function of intestinal flora and food.In general,the intestinal flora formation of musk deer was the result of food,growth environment and its own physiological characteristics.Similarly,intestinal flora also played an important role in the nutrient absorption and energy metabolism of the host.The characteristic of high relative abundance of Firmicutes in wild musk deer intestinal florahelped the body to draw more nutrients from food,at the same time,SCFA produced by Firmicutes could help to resist inflammation.In addition,some of the secondary metabolites from plant metabolism also had an important protective effect such as terpenoids.