健康和患病凡纳滨对虾幼虾消化道菌群结构的比较
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摘要
凡纳滨对虾养殖过程中,早期阶段是病害易感阶段,而消化道菌群结构与对虾健康关系密切。因此,探讨幼虾的消化道菌群尤其是弧菌类细菌与对虾发病的关系对病害有效防治具有重要意义。本实验采用Illumina测序研究了凡纳滨对虾健康和患病幼虾的消化道细菌群落结构,并基于纯培养和16S r DNA,rec A和pyr H基因序列比对,分析了幼虾消化道中弧菌的主要种类组成。结果发现,健康幼虾消化道中α-变形菌纲和厚壁菌门丰度较高,而患病幼虾中γ-变形菌纲、拟杆菌门、放线菌门和β-变形菌纲较高,其中放线菌门丰度差异显著。基于科水平的响应比分析,发现患病幼虾消化道中动性球菌科和噬菌弧菌科的丰度显著降低,而弧菌科的丰度显著升高。相似度分析发现,驱动群落变异的OTU主要来源于弧菌属、海洋杆状菌属、冷杆菌属、假交替单胞菌属以及未分类至属的红杆菌科和微杆菌科。健康幼虾消化道弧菌组成以锡那罗亚州弧菌为主,而患病幼虾消化道弧菌组成以坎氏弧菌为主。尽管健康和患病幼虾消化道内细菌群落结构整体差异不显著,但一些重要类群丰度变化显著,其特征与已知的生态功能一致。
The early stage of shrimp aquaculture is susceptible to bacterial infection and the bacterial community structure in digestive tract is highly associated with shrimp health. Therefore, it is of great significance to explore the relation between shrimp bacterial community of digestive tract and juvenile shrimp disease. We investigated the intestinal bacterial community composition of healthy and diseased juvenile shrimp(Litopenaeus vannamei) by using Illumina sequencing, and explored Vibrio diverstiy based on 16 S r DNA, rec A and pyr H gene sequences of Vibrio isolates. The results showed that the average relative abundances of Alphaproteobacteria and Firmicutes are higher in healthy juvenile shrimp digestive tract than those in diseased one, while these of Gammaproteobacteria,Bacteridetes, Actinobacteria and Betaproteobacteria exhibited an opposite pattern and the variation in Actinobacteria is significant. In addition, response ratio analysis at the bacterial family level shows that the relative abundances of Planococcaceae and Bacteriovoracaceae families significantly decreased, while that of Vibrionaceae remarkably increased in diseased juvenile shrimp digestive tract in relation to healthy one. Similarity percentage(SIMPER) analysis revealed that the main contribution to the variation of intestinal bacterial community structure between healthy and diseased shrimp is attributed to 8 OTUs affiliated with Vibrio, unclassified Rhodobacteraceae,Marivita, Cryobacterium, unclassified Microbacteriaceae and Pseudoalteromonas. As to Vibrio assamblege composition, we found that culturable V. sinaloensis strains were the dominant species in healthy juvenile shrimp digestive tract, while V. campbellii is dominant in diseased juvenile shrimp.
The early stage of shrimp aquaculture is susceptible to bacterial infection and the bacterial community structure in digestive tract is highly associated with shrimp health. Therefore, it is of great significance to explore the relation between shrimp bacterial community of digestive tract and juvenile shrimp disease. We investigated the intestinal bacterial community composition of healthy and diseased juvenile shrimp(Litopenaeus vannamei) by using Illumina sequencing, and explored Vibrio diverstiy based on 16 S r DNA, rec A and pyr H gene sequences of Vibrio isolates. The results showed that the average relative abundances of Alphaproteobacteria and Firmicutes are higher in healthy juvenile shrimp digestive tract than those in diseased one, while these of Gammaproteobacteria,Bacteridetes, Actinobacteria and Betaproteobacteria exhibited an opposite pattern and the variation in Actinobacteria is significant. In addition, response ratio analysis at the bacterial family level shows that the relative abundances of Planococcaceae and Bacteriovoracaceae families significantly decreased, while that of Vibrionaceae remarkably increased in diseased juvenile shrimp digestive tract in relation to healthy one. Similarity percentage(SIMPER) analysis revealed that the main contribution to the variation of intestinal bacterial community structure between healthy and diseased shrimp is attributed to 8 OTUs affiliated with Vibrio, unclassified Rhodobacteraceae,Marivita, Cryobacterium, unclassified Microbacteriaceae and Pseudoalteromonas. As to Vibrio assamblege composition, we found that culturable V. sinaloensis strains were the dominant species in healthy juvenile shrimp digestive tract, while V. campbellii is dominant in diseased juvenile shrimp.
引文
[1]杨少丽,王印庚,董树刚.海水养殖鱼类弧菌病的研究进展[J].海洋水产研究,2005,26(4):75-83.YANG S L,WANG Y G,DONG S G.Progress o research on Vibriosis in marine cultured fish[J].Marin Fish Research,2005,26(4):75-83(in Chinese).
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[13]宛立,王吉桥,高峰,等.南美白对虾肠道细菌菌群分析[J].水产科学,2006,25(1):13-15.WAN L,WANG J Q,GAO F,et al.Bacterial flora in intestines of white leg shrimp(Penaeus vanname Booen)[J].Fisheries Science,2006,25(1):13-15(in Chinese).
[14]Mardis E R.Next-generation DNA sequencing methods[J].Annual Review of Genomics and Human Genetics,2008,9:387-402.
[15]Caporaso J G,Lauber C L,Walters W A,et al.Ultra high-throughput microbial community analysis on th Illumina Hi Seq and Mi Seq platforms[J].The ISMEJournal,2012,6(8):1621-1624.
[16]李筠,吕艳,李军,等.苗期中国对虾幼体异养细菌区系及其变化与病害发生的关系[J].中国海洋大学学报(自然科学版),2004,34(6):1003-1007.LI Y,LU Y,LI J,et al.Study of heterotrophic bacteria flora,dominant vibrios composition of larvae and pos larvae of Penaeus chinensis and its relationship with disease[J].Journal of Ocean University of Qingdao2004,34(6):1003-1007(in Chinese).
[17]Zheng Y,Yu M,Liu Y,et al.Comparison of cultivabl bacterial communities associated with Pacific whit shrimp(Litopenaeus vannamei)larvae at different health statuses and growth stages[J].Aquaculture,2015.
[18]Thompson F L.,Gevers D.,Thompson C C.,et al Phylogeny and molecular identification of vibrios on th basis of multilocus sequence analysis[J].Applied an Environmental Microbiology,2005,71:5107-5115.
[19]Caporaso J G,Kuczynski J,Stombaugh J,et al.QIIMEallows analysis of high-throughput communit sequencing data[J].Nature Methods,2010,7(5):335336.
[20]Edgar R C.Search and clustering orders of magnitud faster than BLAST[J].Bioinformatics,2010,26(19)2460-2461.
[21]De Santis T Z,Hugenholtz P,Keller K,et al.NAST:multiple sequence alignment server for comparativ analysis of 16S r RNA genes[J].Nucleic Acids Research2006,34(2):W394-W399.
[22]Garland J L,Mills A L.Classification and charac terization of heterotrophic microbial communities on th basis of patterns of community-level sole-carbon-sourc utilization[J].Applied and Environmental Microbiology1991,57(8):2351-2359.
[23]Bending G D,Turner M K,Jones J E.Interaction between crop residue and soil organic matter quality an the functional diversity of soil microbial communities[J]Soil Biology and Biochemistry,2002,34(8):1073-1082.
[24]Sullam K E,Essinger S D,Lozupone C A,et al Environmental and ecological factors that shape the gu bacterial communities of fish:a meta-analysis[J]Molecular Ecology,2012,21(13):3363-3378.
[25]王春忠,林国荣,严涛,等.长毛对虾海水养殖环境以及虾肠道微生物群落结构研究[J].水产学报,2014,3(5):706-712.WANG C Z,LIN G R,YAN T,et al.Microbia community in the shrimp(Penaeus penicillatus)intestin and its culture environment[J].Journal of Fisheries o China,2014,38(5):706-712(in Chinese).
[26]丁贤,李卓佳,陈永青,等.芽孢杆菌对凡纳对虾生长和消化酶活性的影响[J].中国水产科学,2005,11(6)580-584.Ding X,Li Z J,Chen Y Q,et al.Effects of probiotics o growth and activities of digestive enzymes of Pennau vannamei[J].Journal of Fishery Sciences of China,200511(6):580-584.
[27]Boopathy R,Kern C,Corbin A.Use of bacillu consortium in waste digestion and pathogen control in shrimp aquaculture[J].International Biodeterioration and Biodegradation,2015.
[28]王丽娜,吴联熙.蛭弧菌的噬菌特性及其在水污染检测和控制应用中的研究进展[J].微生物学通报,199421(1):54-57.WANG L N,WU L X.Phagocytosis characteristics o the Bdellovibrio and its research progress in wate pollution detection and control applications[J]Microbiology,1994,21(1):54-57(in Chinese).
[29]储卫华,朱卫,康春涛.海洋蛭弧菌的分离鉴定及其对副溶血弧菌的作用[J].微生物学通报,2009,36(1):2024.Chu W H,Zhu W,Kang C T.Isolation,identification o marine Bdellovibrios and its effect on Vibrio parahaemolyticus[J].Microbiology,2009,36(1):2024(in Chinese).
[30]宋志萍,蔡俊鹏,王志,等.蛭弧菌的分离及其生长条件和裂解能力的研究[J].微生物学报,2005,45(4)571-575.Song Z P,Cai J P,Wang Z,et al.Study of bacteria-lysi abilities and growth conditions of Bdellovibrio[J].Act Microbiologica Sinica,2005,45(4):571-575(i Chinese).
[31]Gladney L M,Tarr C L.Molecular and phenotypi characterization of Vibrio navarrensis isolates associate with human illness[J].Journal of Clinical Microbiology2014:01544-14.
[32]Janda J M,Newton A E,Bopp C A.Vibriosis[J].Clinic in laboratory medicine,2015,35(2):273-288.
[33]Gomez-Gil B,Fajer-Avila E,Pascual J,et al.Vibri sinaloensis sp.nov.,isolated from the spotted ros snapper,Lutjanus guttatus Steindachner,1869[J]International Journal of Systematic and Evolutionar Microbiology,2008,58(7):1621-1624.
[34]Tzuc J T,Escalante D R,Herrera R R,et al.Microbiot from Litopenaeus vannamei:digestive tract microbia community of Pacific white shrimp(Litopenaeu vannamei)[J].Springer Plus,2014,3(1):280.
[2]Zhou J,Fang W,Yang X,et al.A nonluminescent an highly virulent Vibrio harveyi strain is associated wit"bacterial white tail disease"of Litopenaeus vanname shrimp[J].Plo S ONE,2012,7(2):e29961.
[3]Boirivant M,Amendola A,Butera A.Intestina microflora and immunoregulation[J].Mucosa Immunology,2008,1:S47-S49.
[4]Xiong J,Wang K,Wu J,et al.Changes in intestina bacterial communities are closely associated with shrim disease severity[J].Applied Microbiology an Biotechnology,2015:6911-6919.
[5]Yasuda K,Kitao T.Bacterial flora in the digestive trac of prawns,Penaeus japonicus Bate[J].Aquaculture1980,19(3):229-234.
[6]Rungrassamee W,Klanchui A,Chaiyapechara S,et al Bacterial population in intestines of the black tige shrimp(Penaeus monodon)under different growt stages[J].Plo S One,2013,8(4).
[7]马甡,于明超,李卓佳.虾类消化道菌群研究进展[J]中国海洋大学学报(自然科学版),2008,37(6):889-893MA S,YU M C,LI Z J.A review of studies on th effects and influencing factors of microflora in th digestive tract of shrimp[J].Periodical of Ocea University of China:Natural Science Edition,200837(6):889-893(in Chinese).
[8]Xiong J,Zhu J,Wang K et al.The temporal scaling o bacterioplankton composition:high turnover and predictability during shrimp cultivation[J].Microbia Ecology,2014,67(2):256-264.
[9]Zhang D,Wang X,Xing J,et al.Bacterioplankton assemblages as biological indicators of shrimp health status[J].Ecological Indicators,2014,38:218-224.
[10]Xiong J,Zhu J and Zhang D.The application of bacteria indicator phylotypes to predict shrimp health status[J]Applied Microbiology and Biotechnology,2014,98(14):8291-8299.
[11]Sung H H,Hsu S F,Chen C K,et al.Relationship between disease outbreak in cultured tiger shrimp(Penaeus monodon)and the composition of Vibrio communities in pond water and shrimp hepatopancrea during cultivation[J].Aquaculture,2001,192(2):101110.
[12]Ruwandeepika D,Arachchige H,Sanjeewa Prasad Jayaweera T,et al.Pathogenesis,virulence factors and virulence regulation of Vibrios belonging to the Harvey clade[J].Reviews in Aquaculture,2012,4(2):59-74.
[13]宛立,王吉桥,高峰,等.南美白对虾肠道细菌菌群分析[J].水产科学,2006,25(1):13-15.WAN L,WANG J Q,GAO F,et al.Bacterial flora in intestines of white leg shrimp(Penaeus vanname Booen)[J].Fisheries Science,2006,25(1):13-15(in Chinese).
[14]Mardis E R.Next-generation DNA sequencing methods[J].Annual Review of Genomics and Human Genetics,2008,9:387-402.
[15]Caporaso J G,Lauber C L,Walters W A,et al.Ultra high-throughput microbial community analysis on th Illumina Hi Seq and Mi Seq platforms[J].The ISMEJournal,2012,6(8):1621-1624.
[16]李筠,吕艳,李军,等.苗期中国对虾幼体异养细菌区系及其变化与病害发生的关系[J].中国海洋大学学报(自然科学版),2004,34(6):1003-1007.LI Y,LU Y,LI J,et al.Study of heterotrophic bacteria flora,dominant vibrios composition of larvae and pos larvae of Penaeus chinensis and its relationship with disease[J].Journal of Ocean University of Qingdao2004,34(6):1003-1007(in Chinese).
[17]Zheng Y,Yu M,Liu Y,et al.Comparison of cultivabl bacterial communities associated with Pacific whit shrimp(Litopenaeus vannamei)larvae at different health statuses and growth stages[J].Aquaculture,2015.
[18]Thompson F L.,Gevers D.,Thompson C C.,et al Phylogeny and molecular identification of vibrios on th basis of multilocus sequence analysis[J].Applied an Environmental Microbiology,2005,71:5107-5115.
[19]Caporaso J G,Kuczynski J,Stombaugh J,et al.QIIMEallows analysis of high-throughput communit sequencing data[J].Nature Methods,2010,7(5):335336.
[20]Edgar R C.Search and clustering orders of magnitud faster than BLAST[J].Bioinformatics,2010,26(19)2460-2461.
[21]De Santis T Z,Hugenholtz P,Keller K,et al.NAST:multiple sequence alignment server for comparativ analysis of 16S r RNA genes[J].Nucleic Acids Research2006,34(2):W394-W399.
[22]Garland J L,Mills A L.Classification and charac terization of heterotrophic microbial communities on th basis of patterns of community-level sole-carbon-sourc utilization[J].Applied and Environmental Microbiology1991,57(8):2351-2359.
[23]Bending G D,Turner M K,Jones J E.Interaction between crop residue and soil organic matter quality an the functional diversity of soil microbial communities[J]Soil Biology and Biochemistry,2002,34(8):1073-1082.
[24]Sullam K E,Essinger S D,Lozupone C A,et al Environmental and ecological factors that shape the gu bacterial communities of fish:a meta-analysis[J]Molecular Ecology,2012,21(13):3363-3378.
[25]王春忠,林国荣,严涛,等.长毛对虾海水养殖环境以及虾肠道微生物群落结构研究[J].水产学报,2014,3(5):706-712.WANG C Z,LIN G R,YAN T,et al.Microbia community in the shrimp(Penaeus penicillatus)intestin and its culture environment[J].Journal of Fisheries o China,2014,38(5):706-712(in Chinese).
[26]丁贤,李卓佳,陈永青,等.芽孢杆菌对凡纳对虾生长和消化酶活性的影响[J].中国水产科学,2005,11(6)580-584.Ding X,Li Z J,Chen Y Q,et al.Effects of probiotics o growth and activities of digestive enzymes of Pennau vannamei[J].Journal of Fishery Sciences of China,200511(6):580-584.
[27]Boopathy R,Kern C,Corbin A.Use of bacillu consortium in waste digestion and pathogen control in shrimp aquaculture[J].International Biodeterioration and Biodegradation,2015.
[28]王丽娜,吴联熙.蛭弧菌的噬菌特性及其在水污染检测和控制应用中的研究进展[J].微生物学通报,199421(1):54-57.WANG L N,WU L X.Phagocytosis characteristics o the Bdellovibrio and its research progress in wate pollution detection and control applications[J]Microbiology,1994,21(1):54-57(in Chinese).
[29]储卫华,朱卫,康春涛.海洋蛭弧菌的分离鉴定及其对副溶血弧菌的作用[J].微生物学通报,2009,36(1):2024.Chu W H,Zhu W,Kang C T.Isolation,identification o marine Bdellovibrios and its effect on Vibrio parahaemolyticus[J].Microbiology,2009,36(1):2024(in Chinese).
[30]宋志萍,蔡俊鹏,王志,等.蛭弧菌的分离及其生长条件和裂解能力的研究[J].微生物学报,2005,45(4)571-575.Song Z P,Cai J P,Wang Z,et al.Study of bacteria-lysi abilities and growth conditions of Bdellovibrio[J].Act Microbiologica Sinica,2005,45(4):571-575(i Chinese).
[31]Gladney L M,Tarr C L.Molecular and phenotypi characterization of Vibrio navarrensis isolates associate with human illness[J].Journal of Clinical Microbiology2014:01544-14.
[32]Janda J M,Newton A E,Bopp C A.Vibriosis[J].Clinic in laboratory medicine,2015,35(2):273-288.
[33]Gomez-Gil B,Fajer-Avila E,Pascual J,et al.Vibri sinaloensis sp.nov.,isolated from the spotted ros snapper,Lutjanus guttatus Steindachner,1869[J]International Journal of Systematic and Evolutionar Microbiology,2008,58(7):1621-1624.
[34]Tzuc J T,Escalante D R,Herrera R R,et al.Microbiot from Litopenaeus vannamei:digestive tract microbia community of Pacific white shrimp(Litopenaeu vannamei)[J].Springer Plus,2014,3(1):280.