虎斑颈槽蛇肠道微生物组成和分布特征
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摘要
蛇类是一种研究能量平衡的模型动物,而肠道微生物则帮助宿主获取能量和营养。大多数肠道微生物的研究集中在圈养动物上,而对于野生蛇类的研究很少。本研究从3只野生虎斑颈槽蛇(Rhabdophistigrinus)的大肠、小肠和泄殖腔内容物提取微生物总DNA,进行16SrRNA基因V4区域扩增子测序,分析虎斑颈槽蛇肠道核心微生物组成和分布特征。结果表明,阿尔法指数在不同肠道区域的多样性不存在显著性差异。大肠、小肠和泄殖腔3个肠道部位共享534个操作分类单元(OTUs),大肠包含最多特有OTUs(388个)。门水平,梭杆菌门(Fusobacteria)在小肠(52.87%±14.49%)、大肠(41.12%±22.60%)和泄殖腔(65.70%±10.44%)均为优势菌。属水平,鲸杆菌属(Cetobacterium)在小肠(46.36%±13.86%)、大肠(21.95%±9.82%)和泄殖腔(58.18%±14.29%)为优势菌,柠檬酸杆菌属(Citrobacter)在小肠和泄殖腔中有显著性差异。此外,在其肠道检测到很多潜在的致病菌,例如,柠檬酸杆菌属、明串珠菌属(Trichococcus)和丹毒丝菌属(Erysipelothrix),了解了潜在致病菌在虎斑颈槽蛇肠道中的分布情况。在聚类分析中发现泄殖腔的数据重复性较好,将本研究泄殖腔数据与前人研究过的食鱼蝮(Agkistrodonpiscivorus)泄殖腔高通量测序数据进行联合分析,为更广泛开展不同蛇类肠道微生物组成的差异性分析提供理论借鉴依据。
Snakes are model animals for studying vertebrate energy balance. The gut microbes help the host get energy and nutrients. Currently, researches focused on the captive animals, but little is known about the gut microbes in the wild snakes. We described intestinal microflora in large intestine, small intestine and cloaca in 3 wild Rhabdophis tigrinus by using a V4 region of 16 S rRNA gene amplicon sequencing and analyzed the core composition and distribution characteristics of the intestinal microflora. The results showed no significant difference in the diversity of alpha index in different intestinal regions(Table 1). The three intestinal regions shared 534 Operational Taxonomic Units(OTUs), among which the large intestine contained388 more unique OTUs(Fig. 3 c). At the phylum level, Fusobacteria were found to be dominant in the small intestine(52.87% ± 14.49%), large intestine(41.12% ± 22.60%) and cloaca(65.70% ± 10.44%)(Fig. 1). At the genus level, we indicated that the genus of Cetobacterium had an advantage in all intestinal segments,including the small intestine(46.36% ± 13.86%), large intestine(21.95% ± 9.82%) and cloaca(58.18% ±14.29%)(Fig. 2), and that Citrobacter showed a significant difference between small intestine and cloaca. In addition, many potentially pathogenic bacteria, for example, Citrobacter, Trichococcus and Erysipelothrix,were detected in the gut, which is helpful for. understanding the distribution of potential pathogens in the intestines of Rhabdophis tigrinus. In cluster analysis, the data repeatability of cloaca samples were well(Fig.3 d) and combined analysis with the Agkistrodon piscivorus data provided the theoretical basis for further comparison analysis of intestinal microflora among different snake species(Fig. 4).
Snakes are model animals for studying vertebrate energy balance. The gut microbes help the host get energy and nutrients. Currently, researches focused on the captive animals, but little is known about the gut microbes in the wild snakes. We described intestinal microflora in large intestine, small intestine and cloaca in 3 wild Rhabdophis tigrinus by using a V4 region of 16 S rRNA gene amplicon sequencing and analyzed the core composition and distribution characteristics of the intestinal microflora. The results showed no significant difference in the diversity of alpha index in different intestinal regions(Table 1). The three intestinal regions shared 534 Operational Taxonomic Units(OTUs), among which the large intestine contained388 more unique OTUs(Fig. 3 c). At the phylum level, Fusobacteria were found to be dominant in the small intestine(52.87% ± 14.49%), large intestine(41.12% ± 22.60%) and cloaca(65.70% ± 10.44%)(Fig. 1). At the genus level, we indicated that the genus of Cetobacterium had an advantage in all intestinal segments,including the small intestine(46.36% ± 13.86%), large intestine(21.95% ± 9.82%) and cloaca(58.18% ±14.29%)(Fig. 2), and that Citrobacter showed a significant difference between small intestine and cloaca. In addition, many potentially pathogenic bacteria, for example, Citrobacter, Trichococcus and Erysipelothrix,were detected in the gut, which is helpful for. understanding the distribution of potential pathogens in the intestines of Rhabdophis tigrinus. In cluster analysis, the data repeatability of cloaca samples were well(Fig.3 d) and combined analysis with the Agkistrodon piscivorus data provided the theoretical basis for further comparison analysis of intestinal microflora among different snake species(Fig. 4).
引文
Akins L N,Ayayee P,Leff L G.2018.Composition and diversity of cyanobacteria-associated and free-living bacterial communities during cyanobacterial blooms.Annals of Microbiology,68:493-503.
Beaupre S J.2002.Biology of Vipers.Utah:Eagle Mountain Publishing,463-480.
Cani P D,Vos W M D.2017.Next-generation beneficial microbes:the case of Akkermansia muciniphila.Frontiers in Microbiology,8:1765
Caporaso J G,Kuczynski J,Stombaugh J,et al.2010.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,7(5):335-336.
Colston T J,Noonan B P,Jackson C R.2015.Phylogenetic analysis of bacterial communities in different regions of the gastrointestinal tract of Agkistrodon piscivorus,the Cottonmouth Snake.PLoSOne,10(6):e0128793.
Costello E K,Gordon J I,Secor S M,et al.2010.Postprandial remodeling of the gut microbiota in Burmese pythons.Isme Journal,4(11):1375-1385.
Dao M C,Everard A,Aron-Wisnewsky J,et al.2016.Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity:relationship with gut microbiome richness and ecology.Gut,65(3):426-436.
Diaz-Heijtz R,Wang S,Anuar F,et al.2011.Normal gut microbiota modulates brain development and behavior.Proceedings of the National Academy of Sciences,108(7):3047-3052.
Ezenwa V O,Gerardo N M,Inouye D W,et al.2012.Animal behavior and the microbiome.Science,338(6104):198-199.
Flint H J,Scott K P,Louis P,et al.2012.The role of the gut microbiota in nutrition and health.Nature Reviews Gastroenterology Hepatology,9(10):577-589.
Foster G,Ross H M,Naylor R D,et al.2010.Cetobacterium ceti gen.nov.,sp.nov.,a new gram-negative obligate anaerobe from sea mammals.Letters in Applied Microbiology,21(3):202-206.
Hong S W,Lee J S,Chung K S.2011.Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines,and feces of earthworms.Journal of Microbiology,49(4):544-550.
Huang W,Chen X,Wang K,et al.2019.Comparison among the microbial communities in the lake,lake wetland,and estuary sediments of a plain river network.Microbiology Open,8(2):e00644.
Iii J G H,Hanning I,Beaupre S J,et al.2008.Denaturing gradient gel electrophoresis for the determination of bacterial species diversity in the gastrointestinal tracts of two crotaline snakes.Herpetological Review,39(4):433-438.
Jho Y S,Park D H,Lee J H,et al.2011.Aerobic bacteria from oral cavities and cloaca of snakes in a petting zoo.Korean Journal of Veterinary Research,51(3):243-247.
Keenan S W,Engel A S,Elsey R M.2013.The alligator gut microbiome and implications for archosaur symbioses.Scientific Reports,3(3):2877.
Kohl K D,Brun A,Magallanes M,et al.2017.Gut microbial ecology of lizards:insights into diversity in the wild,effects of captivity,variation across gut regions,and transmission.Molecular Ecology,26(4):1175-1189.
Kohl K D,Cary T L,Karasov W H,et al.2013.Restructuring of the amphibian gut microbiota through metamorphosis.Environmental Microbiology Reports,5(6):899-903.
Kope?nyJ,Mrázek J,Killer J.2010.The presence of bifidobacteria in social insects,fish and reptiles.Folia Microbiologica,55(4):336-339.
Ley R E,Hamady M,Lozupone C,et al.2008.Evolution of mammals and their gut microbes.Science,320(5883):1647.
Mc Coy A N,Araújo-pérez F,Azcárate-Peril A,et al.2013.Fusobacterium is associated with colorectal adenomas.PLoSOne,8(1):e53653.
Mc Laughlin R W,Cochran P A,Dowd S E.2015.Metagenomic analysis of the gut microbiota of the Timber Rattlesnake,Crotalus horridus.Molecular Biology Reports,42(7):1187-1195.
Mundy R,Macdonald T T,Dougan G,2010.Citrobacter rodentium of mice and man.Cellular Microbiology,7(12):1697-1706.
Reboli A C,Farrar W E 1989.Erysipelothrix rhusiopathiae:an occupational pathogen.Clinical Microbiology Reviews,2(4):354-359.
Roggenbuck M,B?rholm S I,Blom N,et al.2015.The microbiome of new world vultures.Scientific Reports,5:5498.
Rooks M G,Garrett W S.2016.Gut microbiota,metabolites and host immunity.Nature Reviews Immunology,16(6):341-352.
Rubinstein,M R,Wang X W,Liu W,et al.2013.Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-Cadherin/β-Catenin signaling via its FadA adhesin.Cell Host Microbe 14(2):195-206.
Saleem M.2015.Microbiom Community Ecology.New York:Springer International Publishing,89-113.
Segata N,Izard J,Waldron L,et al.2011.Metagenomic biomarker discovery and explanation.Genome Biology,12(6):R60.
Sharon G,Segal D,Ringo J M,et al.2010.Commensal bacteria play a role in mating preference of Drosophila melanogaster.Proceedings of the National Academy of Sciences,107(46):20051-20056.
Sullam K E,Essinger S D,Lozupone C A,et al.2012.Environmental and ecological factors that shape the gut bacterial communities of fish:a meta-analysis.Molecular Ecology,21(13):3363-3378.
Suzuki T A,Nachman M W.2016.Spatial heterogeneity of gut microbial composition along the gastrointestinal tract in natural populations of house mice.PLoS One,11(9):e0163720.
Tsuchiya C,Sakata T,Suqita H.2008.Novel ecological niche of Cetobacterium somerae,an anaerobic bacterium in the intestinal tracts of freshwater fish.Letters in Applied Microbiology,46(1):43-48.
Turnbaugh P J,Ley R E,Mahowald M A,et al.2006.An obesity-associated gut microbiome with increased capacity for energy harvest.Nature,444(7122):1027-1031.
Vences M,Lyra M L,Kueneman J G,et al.2016.Gut bacterial communities across tadpole ecomorphs in two diverse tropical anuran faunas.Science of Nature,103(3/4):25.
Waite D W,Taylor M W.2014.Characterizing the avian gut microbiota:membership,driving influences,and potential function.Frontiers in Microbiology,5(4):223.
姜雅风.2002.虎斑颈槽蛇行为的观察.四川动物,21(1):29-31.
时云朵,孙豪.2017.黑眉锦蛇肠道菌群结构分析.湖南农业大学学报:自然科学版,43(3):292-297.
王莉民,楚文瑛.2013.6株明串珠菌属细菌的分离鉴定.国际检验医学杂志,34(13):1775-1776.
赵尔宓.1998.中国动物志:爬行纲第三卷有鳞目蛇亚目.北京:科学出版社,282-295.
Beaupre S J.2002.Biology of Vipers.Utah:Eagle Mountain Publishing,463-480.
Cani P D,Vos W M D.2017.Next-generation beneficial microbes:the case of Akkermansia muciniphila.Frontiers in Microbiology,8:1765
Caporaso J G,Kuczynski J,Stombaugh J,et al.2010.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,7(5):335-336.
Colston T J,Noonan B P,Jackson C R.2015.Phylogenetic analysis of bacterial communities in different regions of the gastrointestinal tract of Agkistrodon piscivorus,the Cottonmouth Snake.PLoSOne,10(6):e0128793.
Costello E K,Gordon J I,Secor S M,et al.2010.Postprandial remodeling of the gut microbiota in Burmese pythons.Isme Journal,4(11):1375-1385.
Dao M C,Everard A,Aron-Wisnewsky J,et al.2016.Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity:relationship with gut microbiome richness and ecology.Gut,65(3):426-436.
Diaz-Heijtz R,Wang S,Anuar F,et al.2011.Normal gut microbiota modulates brain development and behavior.Proceedings of the National Academy of Sciences,108(7):3047-3052.
Ezenwa V O,Gerardo N M,Inouye D W,et al.2012.Animal behavior and the microbiome.Science,338(6104):198-199.
Flint H J,Scott K P,Louis P,et al.2012.The role of the gut microbiota in nutrition and health.Nature Reviews Gastroenterology Hepatology,9(10):577-589.
Foster G,Ross H M,Naylor R D,et al.2010.Cetobacterium ceti gen.nov.,sp.nov.,a new gram-negative obligate anaerobe from sea mammals.Letters in Applied Microbiology,21(3):202-206.
Hong S W,Lee J S,Chung K S.2011.Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines,and feces of earthworms.Journal of Microbiology,49(4):544-550.
Huang W,Chen X,Wang K,et al.2019.Comparison among the microbial communities in the lake,lake wetland,and estuary sediments of a plain river network.Microbiology Open,8(2):e00644.
Iii J G H,Hanning I,Beaupre S J,et al.2008.Denaturing gradient gel electrophoresis for the determination of bacterial species diversity in the gastrointestinal tracts of two crotaline snakes.Herpetological Review,39(4):433-438.
Jho Y S,Park D H,Lee J H,et al.2011.Aerobic bacteria from oral cavities and cloaca of snakes in a petting zoo.Korean Journal of Veterinary Research,51(3):243-247.
Keenan S W,Engel A S,Elsey R M.2013.The alligator gut microbiome and implications for archosaur symbioses.Scientific Reports,3(3):2877.
Kohl K D,Brun A,Magallanes M,et al.2017.Gut microbial ecology of lizards:insights into diversity in the wild,effects of captivity,variation across gut regions,and transmission.Molecular Ecology,26(4):1175-1189.
Kohl K D,Cary T L,Karasov W H,et al.2013.Restructuring of the amphibian gut microbiota through metamorphosis.Environmental Microbiology Reports,5(6):899-903.
Kope?nyJ,Mrázek J,Killer J.2010.The presence of bifidobacteria in social insects,fish and reptiles.Folia Microbiologica,55(4):336-339.
Ley R E,Hamady M,Lozupone C,et al.2008.Evolution of mammals and their gut microbes.Science,320(5883):1647.
Mc Coy A N,Araújo-pérez F,Azcárate-Peril A,et al.2013.Fusobacterium is associated with colorectal adenomas.PLoSOne,8(1):e53653.
Mc Laughlin R W,Cochran P A,Dowd S E.2015.Metagenomic analysis of the gut microbiota of the Timber Rattlesnake,Crotalus horridus.Molecular Biology Reports,42(7):1187-1195.
Mundy R,Macdonald T T,Dougan G,2010.Citrobacter rodentium of mice and man.Cellular Microbiology,7(12):1697-1706.
Reboli A C,Farrar W E 1989.Erysipelothrix rhusiopathiae:an occupational pathogen.Clinical Microbiology Reviews,2(4):354-359.
Roggenbuck M,B?rholm S I,Blom N,et al.2015.The microbiome of new world vultures.Scientific Reports,5:5498.
Rooks M G,Garrett W S.2016.Gut microbiota,metabolites and host immunity.Nature Reviews Immunology,16(6):341-352.
Rubinstein,M R,Wang X W,Liu W,et al.2013.Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-Cadherin/β-Catenin signaling via its FadA adhesin.Cell Host Microbe 14(2):195-206.
Saleem M.2015.Microbiom Community Ecology.New York:Springer International Publishing,89-113.
Segata N,Izard J,Waldron L,et al.2011.Metagenomic biomarker discovery and explanation.Genome Biology,12(6):R60.
Sharon G,Segal D,Ringo J M,et al.2010.Commensal bacteria play a role in mating preference of Drosophila melanogaster.Proceedings of the National Academy of Sciences,107(46):20051-20056.
Sullam K E,Essinger S D,Lozupone C A,et al.2012.Environmental and ecological factors that shape the gut bacterial communities of fish:a meta-analysis.Molecular Ecology,21(13):3363-3378.
Suzuki T A,Nachman M W.2016.Spatial heterogeneity of gut microbial composition along the gastrointestinal tract in natural populations of house mice.PLoS One,11(9):e0163720.
Tsuchiya C,Sakata T,Suqita H.2008.Novel ecological niche of Cetobacterium somerae,an anaerobic bacterium in the intestinal tracts of freshwater fish.Letters in Applied Microbiology,46(1):43-48.
Turnbaugh P J,Ley R E,Mahowald M A,et al.2006.An obesity-associated gut microbiome with increased capacity for energy harvest.Nature,444(7122):1027-1031.
Vences M,Lyra M L,Kueneman J G,et al.2016.Gut bacterial communities across tadpole ecomorphs in two diverse tropical anuran faunas.Science of Nature,103(3/4):25.
Waite D W,Taylor M W.2014.Characterizing the avian gut microbiota:membership,driving influences,and potential function.Frontiers in Microbiology,5(4):223.
姜雅风.2002.虎斑颈槽蛇行为的观察.四川动物,21(1):29-31.
时云朵,孙豪.2017.黑眉锦蛇肠道菌群结构分析.湖南农业大学学报:自然科学版,43(3):292-297.
王莉民,楚文瑛.2013.6株明串珠菌属细菌的分离鉴定.国际检验医学杂志,34(13):1775-1776.
赵尔宓.1998.中国动物志:爬行纲第三卷有鳞目蛇亚目.北京:科学出版社,282-295.