Comparative analysis of gut microbial composition and functions of forest musk deer in different breeding centres

doi:10.16829/j.slxb.150701

Abstract

Abstract:

Intestinal diseases are common in the breeding of forest musk deer (Moschus berezovskii, FMD). Gut microbiota has evolved with the host and formed a complex microecosystem with the gastrointestinal tract of animals. To compare the effects of different breeding environments on gut microbial composition and functions of captive FMD, a total of 215 fresh feces samples were collected from 5 different breeding centres in China, and a 16S rRNA gene high-throughput sequencing was performed. The differences in gut microbial composition, diversity, and functions of FMD from different breeding centres were analyzed. The results showed that Firmicutes and Bacteroides were the absolute dominant bacteria phyla in the gut microbiota of FMD fed without compound probiotics from Qilian County, while Firmicutes and Proteobacteria were the absolute dominant bacteria in the gut microbiota of FMD fed with compound probiotics from Liangdang County and Feng County. The composition, dominant bacteria phyla, dominant bacteria genera, potential pathogenic bacteria, metabolism-related, and disease-related functions of gut microbiota in FMD from different breeding centres were significantly different. The α diversity and disease-related function enrichment of gut microbiota in FMD from Qilian were significantly lower than those from other breeding centres, and enterotype 2 was dominant, with Firmicutes, UCG-005,and Bacteroides as the marker bacteria. The relative abundance of potential pathogenic bacteria of gut microbiota in FMD from Liangdang and Feng Counties was low. We speculated that the difference in food composition might be the main factor leading to the difference in the gut microbiota of FMD from different breeding centres, and the use of compound probiotics might be an important factor leading to the decrease of α diversity and potential pathogenic bacteria. The results of this study can provide a scientific basis for the artificial breeding management of FMD, and also have certain significance for the assessment of the artificial breeding environment and the plan of reintroduction in the future.

Key words: Forest musk deer, Gut microbiota, Functions, Diversity, 16S rRNA gene sequencing

摘要：

肠道疾病是养殖林麝 (Moschus berezovskii) 常见疾病。动物肠道微生物伴随宿主进化并与胃肠道构成了复杂的微生态系统。为探究不同饲养环境对圈养林麝肠道微生物组成和功能的影响，本研究对采自国内5个不同养殖场的215份粪便样品进行了16S rRNA基因高通量测序，对比分析不同养殖场林麝肠道微生物组成、多样性和功能的差异。结果显示，厚壁菌门和拟杆菌门是未喂食复合益生菌的祁连县养殖场林麝肠道菌群的绝对优势菌门，而喂食复合益生菌的甘肃两当县和陕西凤县的4家养殖场林麝肠道菌群的绝对优势菌门为厚壁菌门和变形菌门。不同养殖场林麝肠道菌群组成、优势菌门、优势菌属、潜在致病菌、代谢及疾病相关功能均有显著差异。祁连县养殖场林麝肠道微生物的α多样性和疾病相关功能表达量显著低于其他养殖场，并以肠型2为主，其主导菌为厚壁菌门、UCG-005和拟杆菌属；两当县和凤县的4家养殖场林麝肠道菌群潜在致病菌相对丰度较低。本研究推测食物组成差异可能是导致不同养殖场林麝肠道微生物差异的主要因素，复合益生菌的使用可能是导致α多样性和潜在致病菌下降的重要因素。该结果可为林麝的人工养殖和有效管理提供科学依据，也对人工饲养环境评估和未来的再引入计划具有一定指导意义。

关键词: 林麝, 肠道微生物, 功能, 多样性, 16S rRNA基因测序

CLC Number:

Q938

Feng JIANG, Pengfei SONG, Jingjie ZHANG, Hongmei GAO, Haijing WANG, Zhenyuan CAI, Daoxin LIU, Tongzuo ZHANG. Comparative analysis of gut microbial composition and functions of forest musk deer in different breeding centres[J]. ACTA THERIOLOGICA SINICA, 2023, 43(2): 129-140.

江峰, 宋鹏飞, 张婧捷, 高红梅, 汪海静, 蔡振媛, 刘道鑫, 张同作. 不同养殖场林麝肠道微生物组成和功能的差异[J]. 兽类学报, 2023, 43(2): 129-140.

Figures/Tables 6

Fig. 1 Analyses of gut microbial composition of forest musk deers (FMD) in different breeding centres. A: Rarefaction curves based on Sobs; B: Relative abundance of bacterial phyla. The dominant phyla were shown in red; C: Cluster heat map analysis of identifiable bacterial genera (Top 70), the red, blue, black, orange and green text indicated Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Planctomycetes, respectively. D: Venn diagrams at phylum and genus level. QL: Farm in Qilian County; LD: Farm in Liangdang County; FX: Farm in Feng County; FR:Free-range farm. Same notes in the following figure

Fig. 2 Analyses of gut microbial diversity of forest musk deers in different breeding centres. Analysis of differences in α diversity between groups based on Sobs (A), Shannon (B), Chao1 (C) and PD (D) indices. PCoA analysis (E) and NMDS analysis (F) among different farms. *P < 0.05 (Wilcoxon rank-sum test), **P < 0.01, ***P < 0.001

Fig. 3 Differences of dominant bacteria in gut microbiota of forest musk deers in different breeding centres. Analysis of the differences between the dominant phyla (A); Analysis of the differences between the dominant genera belonging to Firmicutes (B), Proteobacteria (C), Bacteroidetes (D), Actinobacteria (E). ***P < 0.001 (Kruskal-Wallis test)

Fig. 4 Functional annotation based on KEGG database (A) and eggNOG database (B), and analysis of the differences of metabolic functions between groups at the level-2 level. **P < 0.01, ***P < 0.001. ns, not significant

Fig. 5 Analysis of the differences of potential pathogenic bacteria (A) and disease related functions (B) of forest musk deers in different breeding centres. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant

Fig. 6 Analysis of different enterotype in forest musk deers from different breeding centres. A: Analysis of optimal cluster number and enterotype map; B: Distribution of enterotype in different farms; C: Analysis of α diversity in different enterotypes; D: LEfSe analysis of different enterotypes at phylum and genus levels; E: Difference analysis of biomarkers of different enterotypes

References 0

	Bäckhed F, Ley R E, Sonnenburg J L, Peterson D A, Gordon J I. 2005. Host-bacterial mutualism in the human intestine. Science, 307 (5717): 1915-1920.
	Bolger A M, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics, 30: 2114-2120.
	Chen L, Liu M, Zhu J, Gao Y, Sha W L, Ding H X, Jiang W J, Wu S P. 2020. Age, gender, and feeding environment influence fecal microbial diversity in Spotted Hyenas (Crocuta crocuta). Current Microbiology, 77 (7): 1139-1149.
	Costello E K, Lauber C L, Hamady M, Fierer N, Gordon J I, Knight R. 2009. Bacterial community variation in human body habitats across space and time. Science, 326 (5960): 1694-1697.
	Cui X X, Wang Z F, Yan T H, Chang S H, Wang H, Hou F J. 2019. Rumen bacterial diversity of Tibetan sheep (Ovis aries) associated with different forage types on the Qinghai-Tibetan Plateau. Canadian Journal of Microbiology, 65 (12): 859-869.
	David L A, Maurice C F, Carmody R N, Gootenberg D B, Button J E, Wolfe B E, Ling A V, Devlin A S, Varma Y, Fischbach M A, Biddinger S B, Dutton R J, Turnbaugh P J. 2014. Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505 (7484): 559-563.
	De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet J B, Massart S, Collini S, Pieraccini G, Lionetti P. 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proceedings of The National Academy of Sciences of The United States of America, 107 (33): 14691-14696.
	Edgar R C. 2013. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods, 10 (10): 996-998.
	Ezenwa V O, Gerardo N M, Inouye D W, Medina M, Xavier J B. 2012. Microbiology. Animal behavior and the microbiome. Science, 338 (6104): 198-199.
	Fan J M, Zheng X L, Wang H Y, Qi H, Jiang B M, Qiao M P, Zhou J W, Bu S H. 2019. Analysis of genetic diversity and population structure in three forest musk deer captive populations with different origins. G3-Genes Genomes Genetics, 9 (4): 1037-1044.
	Fan Z X, Li W J, Jin J Z, Cui K, Yan C C, Peng C J, Jian Z Y, Bu P, Price M, Zhang X Y, Shen Y M, Li J, Qi W H, Yue B S. 2018. The draft genome sequence of forest musk deer (Moschus berezovskii). Gigascience, 7 (4): 1-6.
	Fountain‑Jones N M, Clark N J, Kinsley A C, Carstensen M, Forester J, Johnson T J, Miller E A, Moore S, Wolf T M, Craft M E. 2020. Microbial associations and spatial proximity predict North American moose (Alces alces) gastrointestinal community composition. Journal of Animal Ecology, 89 (3): 817-828.
	Heinritz S N, Weiss E, Eklund M, Aumiller T, Louis S, Rings A, Messner S, Camarinha‑Silva A, Seifert J, Bischoff S C, Mosenthin R. 2016. Intestinal microbiota and microbial metabolites are changed in a pig model fed a High-Fat/Low-Fiber or a Low-Fat/High-Fiber Diet. PLoS ONE, 11 (4): 0154329.
	Hu X L, Liu G, Shafer A B A, Wei Y T, Zhou J, Lin S, Wu H, Zhou M, Hu D F, Liu S Q. 2017. Comparative analysis of the gut microbial communities in forest and alpine musk deer using high‑throughput sequencing. Frontiers in Microbiology, 8: 572.
	Hu X L. 2017. The quantitative studies on the dynamics of gastrointestinal parasites and microbiota in forest musk deer and the indicative function on health. Ph.D thesis. Harbin: Northeast Forestry University. (in Chinese)
	Jewell K A, McCormick C A, Odt C L, Weimer P J, Suen G. 2015. Ruminal bacterial community composition in dairy cows is dynamic over the course of two lactations and correlates with feed efficiency. Applied and Environmental Microbiology, 81 (14): 4697-4710.
	Jiang F, Gao H M, Qin W, Song P F, Wang H J, Zhang J J, Liu D X, Wang D, Zhang T Z. 2021. Marked seasonal variation in structure and function of gut microbiota in forest and alpine musk deer. Frontiers in Microbiology, 12: 699797.
	La Reau A J, Meier‑Kolthoff J P, Suen G. 2016. Sequence-based analysis of the genus Ruminococcus resolves its phylogeny and reveals strong host association. Microbial Genomics, 2 (12): 1-13.
	La Reau A J, Suen G. 2018.The ruminococci: key symbionts of the gut ecosystem. Journal of Microbiology, 56 (3): 199-208.
	Lang J M, Pan C, Cantor R M, Tang W H W, Garcia‑Garcia J C, Kurtz I, Hazen S L, Bergeron N, Krauss R M, Lusis A J. 2018. Impact of individual traits, saturated fat, and protein source on the gut microbiome. mBio, 9 (6): e01604-18.
	Ley R E, Peterson D A, Gordon J I. 2006. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell, 124 (4): 837-848.
	Li Y M, Hu X L, Yang S, Zhou J T, Zhang T X, Qi L, Sun X N, Fan M Y, Xu S H, Cha M H, Zhang M S, Lin S B, Liu S Q, Hu D F. 2017. Comparative analysis of the gut microbiota composition between captive and wild forest musk deer. Frontiers in Microbiology, 8: 1705.
	Li Y M, Hu X L, Yang S, Zhou J T, Qi L, Sun X M, Fan M Y, Xu S H, Cha M H, Zhang M S, Lin S B, Liu S Q, Hu D F. 2018. Comparison between the fecal bacterial microbiota of healthy and diarrheic captive musk deer. Frontiers in Microbiology, 9: 300.
	Liu X, Zhao W, Yu D, Cheng J G, Luo Y, Wang Y, Yang Z X, Yao X P, Wu S S, Wang W Y, Yang W, Li D Q, Wu Y M. 2019. Effects of compound probiotics on the weight, immunity performance and fecal microbiota of forest musk deer. Scientific Reports, 9 (1): 19146.
	Lv X H, Qiao J Y, Wu X M, Su L N. 2009. A review of mainly affected on musk‑deer diseases: purulent, respiratory system and parasitic diseases. Journal of Economic Animal, 13 (2): 104-107. (in Chinese)
	Magoč T, Salzberg S L. 2021. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 27 (21): 2957-2963.
	Matulova M, Nouaille R, Capek P, Péan M, Delort A M, Forano E. 2008. NMR study of cellulose and wheat straw degradation by Ruminococcus albus 20. FEBS Journal, 275 (13): 3503-3511.
	Nicholson J K, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W, Pettersson S. 2012. Host-Gut microbiota metabolic interactions. Science, 336 (6086): 1262-1267.
	O’Toole P W, Jeffery I B. 2015. Gut microbiota and aging. Science, 350 (6265): 1214-1215.
	Shanahan F. 2010. Probiotics in perspective. Gastroenterology, 139 (6): 1808-1812.
	Sun X N, Cai R B, Jin X L, Shafer A B A, Hu X L, Yang S, Li Y M, Qi L, Liu S Q, Hu D F. 2018. Blood transcriptomics of captive forest musk deer (Moschus berezovskii) and possible associations with the immune response to abscesses. Scientific Reports, 8 (1): 599.
	Wan Y, Wang F L, Yuan J H, Li J, Jiang D D, Zhang J J, Li H, Wang R Y, Tang J, Huang T, Zheng J S, Sinclair A J, Mann J, Li D. 2019. Effects of dietary fat on gut microbiota and faecal metabolites, and their relationship with cardiometabolic risk factors: a 6-month randomised controlled-feeding trial. Gut, 68 (8): 1417-1429.
	Wang Q, Garrity G M, Tiedje J M, Cole J R. 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 73 (16): 5261-5267.
	Wang Y Y, Cao P H, Wang L, Zhao Z Y, Chen Y L, Yang Y X. 2017. Bacterial community diversity associated with different levels of dietary nutrition in the rumen of sheep. Applied Microbiology and Biotechnology, 101 (9): 3717-3728.
	Waters J L, Ley R E. 2019. The human gut bacteria Christensenellaceae are widespread, heritable, and associated with health. BMC Biology, 17 (1): 83.
	Wu S R, Cui Z H, Chen X D, Zheng L X, Ren H, Wang D D, Yao J H. 2021. Diet-ruminal microbiome-host crosstalk contributes to differential effects of calf starter and alfalfa hay on rumen epithelial development and pancreatic α-amylase activity in yak calves - ScienceDirect. Journal of Dairy Science, 104 (4): 4326-4340.
	Yang Q S, Meng X X, Xia L, Feng Z J. 2003. Conservation status and causes of decline of musk deer (Moschus spp.) in China. Biological Conservation, 109 (3): 333-342.
	Yin J, Li Y Y, Han H, Chen S, Gao J, Liu G, Wu X, Deng J P, Yu Q F, Huang X G, Fang R J, Li T J, Reiter R J, Zhang D, Zhu C R, Zhu G Q, Ren W K, Yin Y L. 2018. Melatonin reprogramming of gut microbiota improves lipid dysmetabolism in high-fat diet-fed mice. Journal of Pineal Research, 65 (4): e12524.
	Yoo J Y, Groer M, Dutra S V O, Sarkar A, McSkimming D I. 2020. Gut microbiota and immune system interactions. Microorganisms, 8 (10): 1587.
	Zhang C H, Zhang M H, Wang S Y, Han R J, Cao Y F, Hua W Y, Mao Y J, Zhang X J, Pang X Y, Wei C C, Zhao G P, Chen Y, Zhao L P. 2010. Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice. ISME Journal, 4 (2): 232-241.
	Zhang Z G, Xu D M, Wang L, Hao J J, Wang J F, Zhou X, Wang W W, Qiu Q, Huang X D, Zhou J W, Long R F, Zhao F Q, Shi P. 2016. Convergent evolution of rumen microbiomes in high‑altitude mammals. Current Biology, 26 (14): 1873-1879.
	Zhao X F, Zhang X Y, Chen Z S, Wang Z, Lu Y Y, Cheng D F. 2018. The divergence in bacterial components associated with Bactrocera dorsalis across developmental stages. Frontiers in Microbiology, 9: 114.
	Zhou C, Zhang W B, Wen Q C, Bu P, Gao J, Wang G N, Jin J Z, Song Y J, Sun X H, Zhang Y F, Jiang X, Yu H R, Peng C J, Shen Y M, Price M, Li J, Zhang X Y, Fan Z X, Yue B S. 2019. Comparative genomics reveals the genetic mechanisms of musk secretion and adaptive immunity in Chinese forest musk deer. Genome Biology and Evolution, 11 (4): 1019-1032.
	Zhou L Y, Xiao X H, Zhang Q, Zheng J, Li M, Yu M, Wang X J, Deng M Q, Zhai X, Li R R. 2018. Improved glucose and lipid metabolism in the early life of female offspring by maternal dietary genistein is associated with alterations in the gut microbiota. Frontiers in Endocrinology, 9: 516.
	吕向辉, 乔继英, 吴晓民, 苏丽娜. 2009. 主要危害麝类的化脓性、呼吸系统疾病及寄生虫病研究. 经济动物学报, 13 (2): 104-107.
	吴家炎, 王伟. 2006. 中国麝类. 北京: 中国林业出版社.
	国家林业局. 2009. 中国重点陆生野生动物资源调查. 北京: 中国林业出版社.
	胡晓龙. 2017. 林麝肠道寄生虫和菌群动态的量化研究及其健康指示作用. 哈尔滨: 东北林业大学博士学位论文.

[1]	ZHANG Tongzuo, JIANG Feng, ZHANG Jingjie, CAI Zhenyuan, GAO Hongmei, GU Haifeng, SONG Pengfei. A review of wildlife conservation and management strategies of Sanjiangyuan National Park [J]. ACTA THERIOLOGICA SINICA, 2023, 43(2): 193-205.
[2]	XIE Bo, NONG Xiuping, HUANG Guoli, HUANG Rong, YAO Wei, LIN Jianzhong, ZHOU Qihai. Preliminary survey of mammals and birds diversity by using camera traps in the Guangxi Encheng National Nature Reserve [J]. ACTA THERIOLOGICA SINICA, 2023, 43(2): 215-223.
[3]	ZHANG Qinghao, YAO Song, XU Kai, LIU Tong, XIAO Wenhong, BAI Bingyong, HUANG Xiaoqun, XIAO Zhishu. Bird and mammal diversity inventory by camera trapping in the Neixiang Baotianman National Nature Reserve, Henan Province [J]. ACTA THERIOLOGICA SINICA, 2023, 43(2): 206-214.
[4]	TIAN Xinmin, ZHANG Minghai. Genetic diversity of wapiti in northeast China based on fecal DNA [J]. ACTA THERIOLOGICA SINICA, 2023, 43(1): 41-49.
[5]	Qi ZHAO, Qi ZHANG, Haoling LI, Yue LAN, Xingan YAN, Guijun ZHAO, Wenhua QI. Distinct patterns of microsatellite and functional analysis of forest musk deer and its closely related species [J]. ACTA THERIOLOGICA SINICA, 2022, 42(6): 705-715.
[6]	Ruotong CHENG, Yibo CHEN, Xiangqiong MENG, Jiarui CHEN, Qing WEI. Genetic diversity of male specific region of Y chromosome in Tibetan antelope [J]. ACTA THERIOLOGICA SINICA, 2022, 42(5): 609-614.
[7]	Xinmin TIAN, Mingdong LIAN, Yaqi SONG, Xiaohui LIU, Mengping YANG, Hong CHEN. Genetic diversity and demographic history of Siberian flying squirrel (Pteromys volans) population in northern Zhangguangcai Mountains, Heilongjiang, China [J]. ACTA THERIOLOGICA SINICA, 2022, 42(4): 398-409.
[8]	Min MAO, Ming YANG, Xinyu LIU. Effects of hibernation on cecal microbiota in Daurian ground squirrel [J]. ACTA THERIOLOGICA SINICA, 2022, 42(4): 420-431.
[9]	Shigang GU, Yanhui ZHAI, Chao XU, Dawei YU, Zhiqiang HAN, Quanmin ZHAO, Xiangpeng DAI. Research progress of interspecies somatic cell nuclear transfer in wild animals [J]. ACTA THERIOLOGICA SINICA, 2022, 42(4): 442-450.
[10]	WANG Donghui, LIU Yuliang, SHEN Fujun, CAI Zhigang, AN Junhui, HOU Rong. The effect of frozen semen of captive giant pandas on their population genetic diversity [J]. ACTA THERIOLOGICA SINICA, 2022, 42(3): 261-269.
[11]	SONG Wenyu, LI Xueyou, ONDITI Kenneth Otieno, JIANG Xuelong. Methodological advances in explaining community assembly with nichebased theory [J]. ACTA THERIOLOGICA SINICA, 2022, 42(3): 312-324.
[12]	JI Yunrui, YU Zhucheng, YU Jie, CHEN Zhuo, TONG Zhe, LI Diqiang, LIU Fang. Using camera traps to survey mammals and birds in Zhejiang Jiangshan Xianxialing Provincial Nature Reserve [J]. ACTA THERIOLOGICA SINICA, 2022, 42(2): 211-218.
[13]	WANG Junhua, WONG Kai-Chin, CHEK Si-Nga, VU Ka-Man, CHAN Hoi-Hou, LIANG Jie, HE Xiangyang, ZHANG Libiao. New records of bat species and their conservation status in Macao, China [J]. ACTA THERIOLOGICA SINICA, 2022, 42(1): 125-130.
[14]	FAN Chao, ZHANG Liangzhi, FU Haibo, LIU Chuanfa, LI Wenjing, ZHANG He, TANG Xianjiang, CHENG Qi, SHEN Wenjuan, ZHANG Yanming. Seasonality of abundant and rare taxa in gut microbiota of plateau pikas [J]. ACTA THERIOLOGICA SINICA, 2021, 41(6): 617-630.
[15]	SHAO Ruiqing, LI Yankuo, ZHONG Yifeng, WU Heping, LUO Xiaomin, XIONG Yu, CAO Kaiqiang. Preliminary survey on mammal and bird species diversity using infrared camera traps in the Matoushan National Nature Reserve, Jiangxi Province [J]. ACTA THERIOLOGICA SINICA, 2021, 41(6): 706-713.