养殖大菱鲆肠道微生物多样性及功能分析
|
摘要
本课题是利用分子手段和传统培养手段对养殖大菱鲆肠道菌群结构及功能特征进行研究。首先，利用宏基因组测序技术及16S rRNA分析技术对大菱鲆肠道菌群结构进行研究，共得到33,998条拼接序列（contigs）和95条操作分类单元序列（OTUs）。研究结果显示在大菱鲆肠道中变形菌门与厚壁菌门所占的比例最高（95%），且存在于肠道粘膜和内含物中。进一步将大菱鲆肠道菌群按照属来划分可知，弧菌属所占的比例最大，且存在于大菱鲆肠道的各个部分中，尤其在直肠内含物中所占比例最大。弧菌属中的四个主要类群，包括哈氏弧菌、霍乱弧菌、创伤弧菌以及副溶血弧菌，均被认为是海水鱼类的潜在致病菌。与其他物种肠道菌群结构相比，大菱鲆肠道中含有较高比例的-变形菌纲和柔膜菌纲，弧菌属和支原体属分别在这两个纲中占优势地位。16S rRNA基因序列分析结果显示，大菱鲆肠道各部分菌群的多样性是逐渐降低的（从胃到直肠），且大菱鲆肠道中的某些菌群可能来源于周围海水环境。
其次，为了能够提高培养效率，本课题选择了18种固体培养基对大菱鲆肠道菌群进行培养，包括8种基础培养基，8种1%肠道上清培养基以及2%、4%肠道上清培养基。结果共得到1,711个菌落和24个OTUs，其中Zobell2216E/Zobell2216E+固体培养基中分离的菌落数和菌群多样性最高，而MRS/MRS+固体培养基中的培养结果最不理想。含有肠道上清的培养基（1%、2%、4%）中的菌群结构与基础培养基中的菌群结构存在差异，且肠道上清中可能存在某些物质能够提高大菱鲆肠道菌群的培养效率。对培养得到的菌落进行分类分析可知，-变形菌门所占的比例最大（82%），其次是厚壁菌门和放线菌门（分别为15.6%和2.4%）。进一步按照属来划分可知，弧菌属所占比例最大（49.4%）。此外，还检测到许多潜在的致病菌和益生菌。致病菌包括假单胞菌属、发光菌属以及肠杆菌属。益生菌包括芽孢杆菌属、类芽孢杆菌属、假单胞菌属以及希瓦氏菌属。另外，培养实验所得到的大多数OTUs均是首次在大菱鲆肠道中报道，这些细菌在大菱鲆肠道中的作用还需进一步研究。
最后，本课题对大菱鲆肠道宏基因组拼接数据进行功能分析。结果显示，Clustering-based subsystems所占比例最大，约占大菱鲆肠道宏基因组拼接数据的15.9%，此外，与代谢相关的功能也占有较高比例。利用比较宏基因组学方法分析不同物种肠道宏基因组可知，在大菱鲆肠道宏基因组数据中与群体感应及生物膜形成系统相关的基因所占的比例较大，且这些基因均来源于弧菌属，包括创伤弧菌、霍乱弧菌以及副溶血弧菌。在养殖鱼类肠道宏基因组中，与压力应答以及蛋白折叠系统相关的基因所占比例较高，此外还检测到一些抗生素抗性以及重金属抗性基因。这些数据证明大菱鲆肠道微生物可能受到了养殖过程中某些人为因素的影响，从而产生相应的特殊功能。另外，与淡水鱼（杂交条纹鲈鱼）肠道宏基因组相比，大菱鲆肠道宏基因组中铁元素吸收及代谢系统所占的比例较大，这证明在海洋动物中可能存在着特有的代谢功能。
Culture-independent methods and culture-dependent technologies were appliedto unveil the taxonomic composition and functional diversity of the farmed turbot(Scophthalmus maximus) gastrointestinal (GI) microbiome. First, metagenomicscombined with16S rRNA sequence analysis was used to analyze the taxonomicdistribution and a total of33,998contigs and95operational taxonomic units (OTUs)were identified, respectively. Proteobacteria and Firmicutes which existed in both GIcontent and mucus were dominated in the turbot GI microbiome. At the genus level,Vibrio was observed in all parts of turbot GI tract, especially in rectum content.Within Vibrio, the four main species, V. vulnificus, V. cholerae, V. parahaemolyticusand V. harveyi were recognized as potential pathogens which were responsible formany fish diseases. As compared taxonomic distribution between hosts,Gammaproteobacteria and Mollicutes were overabundant in turbot GI microbiomes.Among them, members of Vibrio and Mycoplasma were dominant.16S rRNAsequence analysis also indicated that the bacterial community diversity decreasedalong the turbot GI tract (from stomach to rectum) and the turbot GI tract may harborsome bacteria which originate from associated seawater.
Second, to enhance the cultivation efficiency, eighteen agar media were tested forcultivating fish-gut-associated bacteria in farmed adult turbot, including agar mediawith or without1%gastrointestinal (GI) supernatant, and2%and4%GI supernatantagar media. A total of1,711colonies were analyzed, and24operational taxonomicunits (OTUs) were identified. Most colonies and OTUs were obtained from Zobell2216E agar media, while a low diversity was identified from MRS/MRS+agar media.Agar media with GI supernatant (1%,2%, and4%) yielded different profiles of OTUsfrom those of the corresponding original media and provided some substances thatenhanced the cultivation efficiency of bacteria in turbot GI tract.Gammaproteobacteria represented the large majority of the colonies (82%). Firmicutes and Actinobacteria represented15.6%and2.4%colonies, respectively. Atthe genus level,49.4%of all colonies belonged to Vibrio. Other potential pathogensincluding Pseudomonas, Photobacterium, and Enterobacter and potential probioticsincluding Bacillus, Paenibacillus, Pseudomonas and Shewanella were also obtainedfrom agar media. Additionally, most of OTUs identified in this study had highhomology with the species that were first described in turbot GI tract. The impact ofthese species on the turbot physiology and health should be further investigated.
Last, functional analyses indicated that the clustering-based subsystem and manymetabolic subsystems were dominated in the turbot GI metagenome, accounting for15.9%of turbot GI metagenome. Compared to other gut metagenomes, quorumsensing and biofilm formation was overabundant in the turbot GI metagenomes.Genes associated with quorum sensing and biofilm formation was found in specieswithin Vibrio, including V. vulnificus, V. cholerae and V. parahaemolyticus. In farmedfish gut metagenomes, the stress response and protein folding subsystems wereover-represented and several genes regarding antibiotic and heavy metal resistancewere also detected. These data suggested that the turbot GI microbiome may beaffected by human factors in aquaculture. Additionally, iron acquisition andmetabolism subsystem was more abundant in the turbot GI metagenome thanfreshwater fish (hybrid striped bass) gut metagenome suggesting that uniquemetabolic potential may be observed in marine animals.
其次，为了能够提高培养效率，本课题选择了18种固体培养基对大菱鲆肠道菌群进行培养，包括8种基础培养基，8种1%肠道上清培养基以及2%、4%肠道上清培养基。结果共得到1,711个菌落和24个OTUs，其中Zobell2216E/Zobell2216E+固体培养基中分离的菌落数和菌群多样性最高，而MRS/MRS+固体培养基中的培养结果最不理想。含有肠道上清的培养基（1%、2%、4%）中的菌群结构与基础培养基中的菌群结构存在差异，且肠道上清中可能存在某些物质能够提高大菱鲆肠道菌群的培养效率。对培养得到的菌落进行分类分析可知，-变形菌门所占的比例最大（82%），其次是厚壁菌门和放线菌门（分别为15.6%和2.4%）。进一步按照属来划分可知，弧菌属所占比例最大（49.4%）。此外，还检测到许多潜在的致病菌和益生菌。致病菌包括假单胞菌属、发光菌属以及肠杆菌属。益生菌包括芽孢杆菌属、类芽孢杆菌属、假单胞菌属以及希瓦氏菌属。另外，培养实验所得到的大多数OTUs均是首次在大菱鲆肠道中报道，这些细菌在大菱鲆肠道中的作用还需进一步研究。
最后，本课题对大菱鲆肠道宏基因组拼接数据进行功能分析。结果显示，Clustering-based subsystems所占比例最大，约占大菱鲆肠道宏基因组拼接数据的15.9%，此外，与代谢相关的功能也占有较高比例。利用比较宏基因组学方法分析不同物种肠道宏基因组可知，在大菱鲆肠道宏基因组数据中与群体感应及生物膜形成系统相关的基因所占的比例较大，且这些基因均来源于弧菌属，包括创伤弧菌、霍乱弧菌以及副溶血弧菌。在养殖鱼类肠道宏基因组中，与压力应答以及蛋白折叠系统相关的基因所占比例较高，此外还检测到一些抗生素抗性以及重金属抗性基因。这些数据证明大菱鲆肠道微生物可能受到了养殖过程中某些人为因素的影响，从而产生相应的特殊功能。另外，与淡水鱼（杂交条纹鲈鱼）肠道宏基因组相比，大菱鲆肠道宏基因组中铁元素吸收及代谢系统所占的比例较大，这证明在海洋动物中可能存在着特有的代谢功能。
Culture-independent methods and culture-dependent technologies were appliedto unveil the taxonomic composition and functional diversity of the farmed turbot(Scophthalmus maximus) gastrointestinal (GI) microbiome. First, metagenomicscombined with16S rRNA sequence analysis was used to analyze the taxonomicdistribution and a total of33,998contigs and95operational taxonomic units (OTUs)were identified, respectively. Proteobacteria and Firmicutes which existed in both GIcontent and mucus were dominated in the turbot GI microbiome. At the genus level,Vibrio was observed in all parts of turbot GI tract, especially in rectum content.Within Vibrio, the four main species, V. vulnificus, V. cholerae, V. parahaemolyticusand V. harveyi were recognized as potential pathogens which were responsible formany fish diseases. As compared taxonomic distribution between hosts,Gammaproteobacteria and Mollicutes were overabundant in turbot GI microbiomes.Among them, members of Vibrio and Mycoplasma were dominant.16S rRNAsequence analysis also indicated that the bacterial community diversity decreasedalong the turbot GI tract (from stomach to rectum) and the turbot GI tract may harborsome bacteria which originate from associated seawater.
Second, to enhance the cultivation efficiency, eighteen agar media were tested forcultivating fish-gut-associated bacteria in farmed adult turbot, including agar mediawith or without1%gastrointestinal (GI) supernatant, and2%and4%GI supernatantagar media. A total of1,711colonies were analyzed, and24operational taxonomicunits (OTUs) were identified. Most colonies and OTUs were obtained from Zobell2216E agar media, while a low diversity was identified from MRS/MRS+agar media.Agar media with GI supernatant (1%,2%, and4%) yielded different profiles of OTUsfrom those of the corresponding original media and provided some substances thatenhanced the cultivation efficiency of bacteria in turbot GI tract.Gammaproteobacteria represented the large majority of the colonies (82%). Firmicutes and Actinobacteria represented15.6%and2.4%colonies, respectively. Atthe genus level,49.4%of all colonies belonged to Vibrio. Other potential pathogensincluding Pseudomonas, Photobacterium, and Enterobacter and potential probioticsincluding Bacillus, Paenibacillus, Pseudomonas and Shewanella were also obtainedfrom agar media. Additionally, most of OTUs identified in this study had highhomology with the species that were first described in turbot GI tract. The impact ofthese species on the turbot physiology and health should be further investigated.
Last, functional analyses indicated that the clustering-based subsystem and manymetabolic subsystems were dominated in the turbot GI metagenome, accounting for15.9%of turbot GI metagenome. Compared to other gut metagenomes, quorumsensing and biofilm formation was overabundant in the turbot GI metagenomes.Genes associated with quorum sensing and biofilm formation was found in specieswithin Vibrio, including V. vulnificus, V. cholerae and V. parahaemolyticus. In farmedfish gut metagenomes, the stress response and protein folding subsystems wereover-represented and several genes regarding antibiotic and heavy metal resistancewere also detected. These data suggested that the turbot GI microbiome may beaffected by human factors in aquaculture. Additionally, iron acquisition andmetabolism subsystem was more abundant in the turbot GI metagenome thanfreshwater fish (hybrid striped bass) gut metagenome suggesting that uniquemetabolic potential may be observed in marine animals.
引文
1. Acheson DWK&Luccioli S (2004) Mucosal immune responses. Best Pract ResClin Gastroenterol18:387–404.
2. Adams DO&Hamilton TA (1984) The cell biology of macrophage activation.Ann Rev Immunol2:283–318.
3. Agulleiro MJ, Anguis V, Can avate JP, Mart′nez-Rodr′guez G, Mylonas CC,Cerda`J (2006) Induction of spawning of captive-reared Senegal sole (Soleasenegalensis) using different delivery systems for gonadotropin-releasinghormone agonist. Aquaculture257(1–4):511–524.
4. Aly SM, Ahmed YAG, Ghareeb AAA&Mohamed MF (2008) Studies onBacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on theimmune response and resistance of Tilapia nilotica (Oerochromis niloticus) tochallenge infections. Fish Shellfish Immunol25:128–136.
5. Amann RI, Ludwig W, Schleifer KH, Amann RI, Ludwig W&Schleifer KH(1995) Phylogenetic identification and in situ detection of individual microbialcells without cultivation. Microbiol Rev59:143–169.
6. Austin B&Austin DA (1999) Bacterial fish pathogens: disease of farmed andwild fish,3rd ed. Springer-Verlag KG, Berlin, Germany.
7. Austin B (2006) The bacterial microflora of fish, revised. ScientificWorldJournal6:931-945.
8. Bae JW, Rhee SK, Park JR, Kim BC&Park YH (2005) Isolation of uncultivatedanaerobic thermophiles from compost by supplementing cell extract ofGeobacillus toebii in enrichment culture medium. Extremophiles9:477–485.
9. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Múzquiz JL(2006) The role of probiotics in aquaculture. Vet Microbiol114(3-4):173-186.
10. Balcázar JL (2007) Probiotics in health maintenance: do they really work? Br JInfect Control8:26–29.
11. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Gironés O&Muzquiz JL(2007a) Sequencing of variable regions of the16S rRNA gene for identificationof lactic acid bacteria isolated from the intestinal microbiota of healthysalmonids. Comp Immunol Microbiol Infect Dis30:111–118.
12. Balcázar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Gironés O&Múzquiz JL(2007b) In vitro competitive adhesion and production of antagonistic compoundsby lactic acid bacteria against fi sh pathogens. Vet Microbiol122:373–380.
13. Balcázar JL et al.(2007c) Changes in intestinal microbiota and humoral immuneresponse following probiotic administration in brown trout (Salmo trutta). Br JNutr97:522–527.
14. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Gironés O&Muzquiz JL(2007d) Enhancement of the immune response and protection induced byprobiotic lactic acid bacteria against furunculosis in rainbow trout(Oncorhynchus mykiss). FEMS Immunol Med Microbiol51:185–193.
15. Bano N, DeRae Smith A, Bennett W, Vasquez L&Hollibaugh JT (2007)Dominance of Mycoplasma in the guts of the Long-Jawed Mudsucker,Gillichthys mirabilis, from five California salt marshes. Environ Microbiol9:2636-2641.
16. Bates JM, Akerlund J, Mittge E&Guillemin K (2007) Intestinal alkalinephosphatase detoxifies lipopolysaccharide and prevents inflammation inzebrafish in response to the gut microbiota. Cell Host Microbe2:371–382.
17. Ben-Oov E, Kramarsky-Winter E&Kushmaro A (2009) An in situ method forcultivating microorganisms using a double encapsulation technique. FEMSMicrobiol Ecol68:363-371.
18. Berg RD (1992) Translocation and the indigenous gut flora. In: Fuller R, ed.Probiotics: The scientific basis London; Chapman and Hall, London. pp55–85.
19. Bollmann A, Lewis K&Epstein SS (2007) Incubation of environmental samplesin a diffusion chamber increases the diversity of recovered isolates. Appl EnvironMicrob73:6386–6390.
20. Bosch TC&McFall-Ngai MJ (2011) Metaorganisms as the new frontier. Zoology114:185–189.
21. Bromage N, Mazorra C, Bruce M, Brown N, Shields R (2000) Halibut culture. In:Stickney RR (ed) Encyclopedia of aquaculture. Wiley, New York, pp425–432.
22. Bruns A, Cypionka H&Overmann J (2002) Cyclic AMP and acyl homoserinelactones increase the cultivation efficiency of heterotrophic bacteria from thecentral Baltic Sea. Appl Environ Microb68:3978–3987.
23. Bury N&Grosell M (2003a) Iron acquisition by teleost fish. Comp BiochemPhysiol Part C Toxicol Pharmacol135:97-105.
24. Bury NR&Grosell M (2003b) Waterborne iron acquisition by a freshwaterteleost fish, zebrafish Danio rerio. J Exp Bio206:3529-3535.
25. Bury NR, Walker PA&Glover CN (2003) Nutritive metal uptake in teleost fish. JExp Biol206:11-23.
26. Capkin E&Altinok I (2009) Effects of dietary probiotic supplementations onprevention/treatment of yersiniosis disease. J Appl Microbiol106:1147–1153.
27. Carpenter S&O’Neill LAJ (2007) How important are Toll-like receptors forantimicrobial responses? Cell Microbiol9:1891–1901.
28. Cerdà-Cuéllar M&Blanch AR (2002) Detection and identification of Vibrioscophthalmi in the intestinal microbiota of fish and evaluation of host specificity.J Appl Microbiol93(2):261-268.
29. Cerdà-Cuéllar M&Blanch AR (2004) Determination of Vibrio scophthalmi andits phenotypic diversity in turbot larvae. Environ Microbiol6:209-217.
30. Cerda`J, Douglas S, Reith M (2010) Genomic resources for flatfish research andtheir applications. J Fish Biol77(5):1045–1070.
31. Chabrillón M, Rico R M, Balebona M C, Mori igo M A (2005) Adhesion to sole,Solea senegalensis Kaup, mucus of microorganisms isolated from farmed fish,and their interaction with Photobacterium damselae subsp. piscicida. J Fish Dis28:229-237.
32. Cheesman SE&Guillemin K (2007) We know you are in there: Conversing withthe indigenous gut microbiota. Res Microbiol158:2–9.
33. Chen G&Brown A (1985) Bacterial growth and the concentrations of cyclicnucleotides in Legionella pneumophila cultures. Curr Microbiol12:23–26.
34. Clapper WE&Meade GH (1963) Normal flora of the nose, throat, and lowerintestine of dogs. J Bacteriol85:643–648.
35. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM,Bandela AM, Cardenas E, Garrity GM&Tiedje JM (2007) The ribosomaldatabase project (RDP-II): introducing myRDP space and quality controlledpublic data. Nucleic Acids Res35:169-172.
36. Colwell RR, Kaper JB, Joseph SW (1977) Vibrio cholerae, Vibrioparahaemolyticus and other Vibrios: occurrence and distribution in ChesapeakeBay. Science198:394–396.
37. Colwell RR, Huq A (2001) Marine ecosystems and cholera. Hydrobiologia460:141–145.
38. Conceic a o LCE, Ribeiro L, Engrola S, Araga o C, Morais S, Lacuisse M,Soares F, Dinis MT (2007) Nutritional physiology during development ofSenegalese sole (Solea senegalensis). Aquaculture268(1–4):64–81.
39. Condon C (2009) RNA processing. In: Moselio S (ed) Encyclopedia ofmicrobiology,3rd edn. Elsevier, Oxford, vol5, pp395–408.
40. Connon SA&Giovannoni SJ (2002) High-throughput methods for culturingmicroorganisms in very-low-nutrient media yield diverse new marine isolates.Appl Environ Microb68:3878–3885.
41. Coppola S, Zoina A&Marino P (1976) Interactions of N6-(delta2-isopentenyl)adenine with cyclic AMP on the regulation of growth and beta-galactosidasesynthesis in Escherichia coli. J Gen Microbiol94:436–438.
42. Corthésy B, Gaskins HR&Mercenier A (2007) Cross-talk between probioticbacteria and the host immune system. J Nutr137:781S–790S.
43. Dandekar T, Snel B, Schmidt S, Lathe W, Suyama M, Huynen M&Bork P (2002)Comparative genome analysis of Mollicutes. In Molecular Biology andPathogenicity of Mycoplasmas. New York, USA: Kluwer Academic/Plenum, pp.255–278.
44. Davis KE, Joseph SJ&Janssen PH (2005) Effects of growth medium, inoculumsize, and incubation time on culturability and isolation of soil bacteria. ApplEnviron Microb71:826–834.
45. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T,Dalevi D, Hu P&Andersen GL (2006) Greengenes, a chimera-checked16SrRNA gene database and workbench compatible with ARB. Appl EnvironMicrobiol72:5069-5072.
46. Dethlefsen L, McFall-Ngai M&Relman DA (2007) An ecological andevolutinary perspective on human-microbe mutualism and disease. Nature449:811–818.
47. Dickschat JS (2010) Quorum sensing and bacterial biofilms. Nat Prod Rep27:343-369.
48. Divya KR, Isamma A, Ramasubramanian V, Sureshkumar S&Arunjith TS (2012)Colonization of probiotic bacteria and its impact on ornamental fish Puntiusconchonius. J Environ Biol33(3):551-555.
49. Fasano A&Shea-Donohue T (2005) Mechanisms of disease: the role ofintestinal barrier function in the pathogenesis of gastrointestinal autoimmunediseases. Nat Clin Pract Gastroenterol Hepatol2:416–422.
50. Ferrari BC, Winsley T, Gillings M&Binnerup S (2008) Cultivating previouslyuncultured soil bacteria using a soil substrate membrane system. Nat Protoc3:1261–1269.
51. Ferrari BC&Gillings M (2009) Cultivating fastidious bacteria: viability stainingand micromanipulation from a soil substrate membrane system. Appl EnvironMicrob75:3352–3354.
52. Forne′I, Abia′n J, Cerda`J (2010) Fish proteome analysis: model organisms andnon-sequenced species. Proteomics10(4):858–872.
53. Fraser CM, Gocayne JD, White O, Adams MD., Clayton RA., Fleischmann RD,et al.(1995) The minimal gene complement of Mycoplasma genitalium. Science270:397–404.
54. García-Aljaro C, Eberl L, Riedel K&Blanch AR (2008) Detection ofquorum-sensing-related molecules in Vibrio scophthalmi. BMC Microbiol8:138.
55. Gatesoupe FJ (2008) Updating the importance of lactic acid bacteria in fishfarming: natural occurrence and probiotic treatments. J Mol MicrobiolBiotechnol14:107–114.
56. Gómez GD&Balcázar JL (2008) A review on the interactions between gutmicrobiota and innate immunity of fish. FEMS Immunol Med Microbiol52:145–154.
57. Gonzalez A, Stombaugh J, Lozupone C, Turnbaugh PJ, Gordon JI&Knight R(2011) The mind-body-microbial continuum. Dia Clin Neurosci13:55–62.
58. Gonza′lez C, Lo′pez-D′az JTM, Garc′a-Lo′pez ML, Prieto M&Otero A (1999)Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius),and aquacultured rainbow trout (Oncorhynchus mykiss). J Food Prot62:1270–1277.
59. Good IJ (1953) The population frequencies of species and the estimation ofpopulation parameters. Biometrika40:237-264.
60. Goodman SE&Gordon JI (2010) Our unindicated coconspirators: Humanmetabolism from a microbial perspective. Cell Metab12:111–116.
61. Graber JR&Breznak JA (2005) Folate cross-feeding supports symbiotichomoacetogenic spirochetes. Appl Environ Microb71:1883–1889.
62. Guardabassi L, Schwarz S&Lloyd DH (2004) Pet animals as reservoirs ofantimicrobial-resistant bacteria. J Antimicrob Chemother54:321-332.
63. Hackstadt T&Williams JC (1981) Biochemical stratagem for obligate parasitismof eukaryotic cells by Coxiella burnetii. P Natl Acad Sci USA78:3240–3244.
64. Hansen GH&Olafsen JA (1999) Bacterial interactions in early life stages ofmarine cold water fish. Microb Ecol38:1–26.
65. Hazen TH, Pan L, Gu JD&Sobecky PA (2010) The contribution of mobilegenetic elements to the evolution and ecology of Vibrios. FEMS Microbiol Ecol74:485-499.
66. He X, Nie X, Yang Y, Liu X, Pan D, Cheng Z&Liang X (2012) Multi-biomarkerresponses in fishes from two typical marine aquaculture regions of South China.Mar Pollut Bull doi:10.1016/j.marpolbul.2012.09.002.
67. Hoarau G, Boon E, Jongma DN, Ferber S, Palsson J, Van der Veer HW,RijnsdorpAD, StamWT,Olsen JL (2005) Loweffective population size andevidence for inbreeding in an overexploited flatfish, plaice (Pleuronectesplatessa L.). Proc R Soc B272:497–503.
68. Holland MCH&Lambris JD (2002) The complement system in teleosts. FishShellfish Immunol12:399–420.
69. Honda T&Iida T (1993) The pathogenicity of Vibrio parahaemolyticus and therole of the thermostable direct haemolysin and related haemolysins. Rev MedMicrobiol4:106–113.
70. H rmannsperger G&Haller D (2010) Molecular crosstalk of probiotic bacteriawith the intestinal immune system: clinical relevance in the context ofinflammatory bowel disease. Int J Med Microbiol300:63–73.
71. Horsley RW (1977) A review of bacterial flora of teleosts and elasmobranches,including methods for its analysis. J Fish Biol10:529–553.
72. Hosoi T, Ametani A, Kiuchi K&Kaminogawa S (2000) Improved growth andviability of lactobacilii in the presence of Bacillus subtilis, catalase, or subtilisin.Can J Microbiol46:892–897.
73. Huber T, Faulkner G&Hugenholtz P (2004) Bellerophon: A program to detectchimeric sequences in multiple sequence alignments. Bioinformatics20:2317-2319.
74. Hugenholtz P, Hooper SD&Kyrpides NC (2009) Focus: synergistetes. EnvironMicrobiol11:1327–1329.
75. Imsland AK, Foss A, Conceic a o LEC, Dinis MT, Delbare D, Schram E,Kamstra A, Rema P, White P (2003) A review of the culture potential of Soleasolea and S. senegalensis. Rev Fish Biol Fish13(4):379–407.
76. Irianto A&Austin B (2002) Probiotics in aquaculture. J Fish Dis25:633–642.
77. Irwin JA (2010) Extremophiles and their application to veterinary medicine.Environ Technol31(8-9):857-869.
78. Janssen PH (2003) Selective enrichment and purification of cultures ofMethanosaeta spp. J Microbiol Methods52:239–244.
79. Kaeberlein T, Lewis K&Epstein SS (2002) Isolating ‘uncultivable’microorganisms in pure culture in a simulated natural environment. Science296:1127–1129.
80. Kennedy J, Marchesi JR&Dobson ADW (2008) Marine metagenomics:strategies for the discovery of novel enzymes with biotechnological applicationsfrom marine environments. Microb Cell Fact7:27.
81. Kennedy J, Baker P, Piper C, Cotter PD, Walsh M, Mooij MJ, Bourke MB, ReaMC, O'Connor PM, Ross RP, Hill C, O'Gara F, Marchesi JR&Dobson AD(2009) Isolation and analysis of bacteria with antimicrobial activities from themarine sponge Haliclona simulans collected from Irish waters. Mar Biotechnol11(3):384-396.
82. Kim BS, Kim JN&Cerniglia CE (2011) In Vitro culture conditions formaintaining a complex population of human gastrointestinal tract microbiota. JBiomed Biotechnol2011:838040.
83. Kim DH&Austin B (2006) Innate immune responses in rainbow trout(Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish Shellfish Immunol21:513–524.
84. Kim JJ, Kim HN, Masui R, Kuramitsu S, Seo JH, Kim K&Sung MH (2008)Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceaerequiring growth-supporting factors. J Microbiol Biotechn18:611–615.
85. Kuda T, Kyoi D, Takahashi H, Obama K&Kimura B (2011) Detection andisolation of p-nitrophenol-lowering bacteria from intestine of marine fishescaught in Japanese waters. Mar Pollut Bull62(8):1622-1627.
86. Kwak JK, Park SW, Koo JG, Cho MG, Buchholz R&Goetz P (2003)Enhancement of the non-specific defense activities in carp (Cyprinus carpio) andflounder (Paralichthys olivaces) by oral administration of Schizophyllan. ActaBiotechnol23:359–371.
87. Lamendella R, Domingo JW, Ghosh S, Martinson J&Oerther DB (2011)Comparative fecal metagenomics unveils unique fuctional capacity of the swinegut. BMC Microbiol11:103.
88. Lavery TJ, Roudnew B, Seymour J, Mitchell JG&Jeffries T (2012) Highnutrient transport and cycling potential revealed in the microbial metagenome ofAustralian sea lion. PLoS One7: e36478.
89. Lazado CC, Caipang CMA, Rajan B, Brinchmann MF, Kiron V (2010)Characterization of GP21and GP12: two protential probiotic bacteria isolatedfrom the gastrointestinal tract of Atlantic cod. Probiotics&Antimicro Prot2:126-134.
90. Li CQ, Liu WC, Zhu P, Yang JL&Cheng KD (2011) Phylogenetic diversity ofbacteria associated with the marine sponge Gelliodes carnosa collected from theHainan island coastal waters of the South China Sea. Microb Ecol62(4):800-812.
91. Lunder T, S rum H, Holstad G, Steigerwalt AG, Mowinckel P&Brenner DJ(2000) Phenotypic and genotypic characterization of Vibrio viscosus sp. nov. andVibrio wodanis sp. nov. isolated from Atlantic salmon (Salmo salar) with ‘winterulcer’. Int J Syst Evol Microbiol50:427-450.
92. Markowitz VM, Chen IM, Chu K et al.(2012) IMG/M: the integratedmetagenome data management and comparative analysis system. Nucleic AcidsRes40:123-129.
93. McClelland DBL (1976) Peyer’s-patch-associated synthesis of immunoglobulinin germ-free, specifi c-pathogen-free, and conventional mice. Scand J Immunol5:909–915.
94. McDonald R, Schreier HJ&Watts JE (2012) Phylogenetic analysis of microbialcommunities in different regions of the gastrointestinal tract in Panaquenigrolineatus, a wood-eating fish. PLoS One7: e48018.
95. Metchnikoff E (1901) Surlaflore du corps. Human Member Protocol ofManchester Literature and Philosophical Society.45:1–38.
96. Meyer F, Paarmann D, D'Souza M et al.(2008) The metagenomics RAST sever-a public resource for the automatic phylogenetic and functional analysis ofmetagenomes. BMC Bioinformatics9:386.
97. Milla′n A, Gomez-Tato A, Fernandez C, Pardo BG, Alvarez-Dios JA, Calaza M,Bouza C, Va′zquez M, Cabaleiro S, Martinez P (2010) Design and performanceof a turbot (Scophthalmus maximus) oligo-microarray based on ESTs fromimmune tissues. Mar Biotechnol12(4):452–465.
98. Milla′n A, Go′mez-Tato A, Pardo BG, Ferna′ndez C, Bouza C, Vera M,Alvarez-Dios JA, Cabaleiro S, Lamas J, Lemos ML, Mart′nez P (2011) Geneexpression profiles of the spleen, liver, and head kidney in turbot (Scophthalmusmaximus) along the infection process with Aeromonas salmonicida using animmune-enriched oligo-microarray. Mar Biotechnol13(6):1099–1114.
99. Millner R, Walsh SJ, D′az de Astarloa JM (2005) Atlantic flatfish fisheries. In:Gibson RN (ed) Flatfishes: biology and exploitation. Blackwell Publishing,Oxford, pp240–271.
100. Mitra S, Rupek P, Richter DC, Urich T, Gilbert JA, Meyer F, Wilke A&HusonDH (2011) Functional analysis of metagenomes and metatranscriptomes usingSEED and KEGG.. BMC Bioinformatics12(Suppl1): S21.
101. Mollet FM, Kraak SBM, Rijnsdorp AD (2007) Fisheries-induced evolutionarychanges in maturation reaction norms in North Sea sole Solea solea. Mar EcolProg Ser351:189–199.
102. Montalto M, D’Onofrio F, Gallo A, Cazzato A&Gasbarrini G (2009) Intestinalmicrobiota and its functions. Dig Liver Dis Suppl3:30–34.
103. Montes M, Farto R, Pérez MJ, Nieto TP, Larsen JL&Christensen H (2003)Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus)culture by phenotypic analysis. J Appl Microbiol95:693-703.
104. Montes M, Farto R, Pérez MJ, Armada SP&Nieto TP (2006) Genotypicdiversity of Vibrio isolated associated with turbot (Scophthalmus maximus)culture. Res Microbiol157:487-495.
105. Moran D, Turner SJ&Clements KD (2005) Ontogenetic development of thegastrointestinal microbiota in the marine herbivorous fish Kyphosus sydneyanus.Microb Ecol49:590-597.
106. Mulder IE, Wadsworth S&Secombes CJ (2007) Cytokine expression in theintestine of rainbow trout (Oncorhynchus mykiss) during infection withAeromonas salmonicida. Fish Shellfish Immunol23:747–759.
107. Munroe TA (2005) Systematic diversity of the Pleuronectiformes. In: Gibson RN(ed) Flatfishes: biology and exploitation. Blackwell Publishing, Oxford, pp10–41.
108. Navarrete P, Espejo RT&Romero J (2009) Molecular analysis of microbiotaalong the digestive tract of juvenile Atlantic salmon (Salmo salar L.). MicrobEcol57:550-561.
109. Naylor R, Burke M (2005) Aquaculture and ocean resources: raising tigers of thesea. Annu Rev Environ Resour30:185–218.
110. Nayak SK (2010) Probiotics and immunity: a fish perspective. Fish ShellfishImmunol.,29(1):2-14.
111. Newaj-Fyzul A, Adesiyun AA, Mutani A, Ramsubhag A, Brunt J&Austin B(2007) Bacillus subtilis AB1controls Aeromonas infection in rainbow trout(Oncorhynchus mykiss, Walbaum). J Appl Microbiol103:1699-1706.
112. Nichols D, Lewis K, Orjala J et al.(2008) Short peptide induces an‘uncultivable’ microorganism to grow in vitro. Appl Environ Microb74:4889–4897.
113. Nishibuchi M&Kaper JB (1995) Thermostable direct hemolysin gene of Vibrioparahaemolyticus: a virulence gene acquired by a marine bacterium. InfectImmun63:2093–2099.
114. Oakey HJ, Cullen BR&OwensL (2002) The compete nucleotide sequence of theVibrio harveyi bacteriophage VHML. J Appl Microbiol93:1089–1098.
115. O’Hara AM&Shanahan F (2006) The gut flora as a forgotten organ. EMBO Rep7:688–693.
116. Ohno M, Shiratori H, Park MJ et al.(2000) Symbiobacterium thermophilum gen.nov., sp. nov., a symbiotic thermophile that depends on co-culture with aBacillus strain for growth. Int J Syst Evol Micr50:1829–1832.
117. Omsland A, Cockrell DC, Fischer ER&Heinzen RA (2008) Sustained axenicmetabolic activity by the obligate intracellular bacterium Coxiella burnetii. JBacteriol190:3203–3212.
118. Ormsbee RA&Peacock MG (1964) Metabolic activity in Coxiella burnetii. JBacteriol88:1205–1210.
119. Ouwehand AC, Isolauri E, Kirjavainen PV, T lkk, S&Salminen SJ (2000) Themucus binding of Bifidobacterium lactis Bb12is enhanced in the presence ofLactobacillus GG and Lact. delbrüeckii subsp. bulgaricus. Lett Appl Microbiol30:10–13.
120. Pamer EG (2007) Immune responses to commensal and environmental microbes.Nat Immunol8:1173–1178.
121. Panigrahi A et al.(2007) Immune modulation and expression of cytokine genesin rainbow trout Oncorhynchus mykiss upon probiotic feeding. Dev CompImmunol31:372–382.
122. Park KJ, Kang MJ, Kim SH, Lee HJ, Lim JK, Choi SH, Park SJ&Lee KH(2004) Isolation and characterization of rpoS from a pathogenic bacterium,Vibrio vulnificus: role of σsin survival of exponential-phase cells under oxidativestress. J Bacteriol186:3304-3312.
123. Parks DH&Beiko RG (2010) Identifying biologically relevant differencesbetween metagenomic communities. Bioinformatics26:715-721.
124. Pawar KD, Banskar S, Rane SD, Charan SS, Kulkarni GJ, Sawant SS, Ghate HV,Patole MS&Shouche YS (2012) Bacterial diversity in different regions ofgastrointestinal tract of Giant African snail (Achatina fulica). Microbiologyopen1:415-426.
125. Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, Fuste B, Planas JV,Beltran S, Novoa B&Figueras A (2012) High-throughput sequence analysis ofturbot (Scophthalmus maximus) transcriptome using454-pyrosequencing for thediscovery of antiviral immune genes. PLoS One7: e35369.
126. Pérez-Losada M, Maigualida R, Jonathon CM, Jorge D (2009) Phylogeneticassessment of the earthworm Aporrectodea caliginosa species complex(Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences.Mol Phylogenet Evol52(2):293–302.
127. Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I,Merrifield DL&Ruiz-Zarzuela I (2011) Identification and characterization oflactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss(Walbaum), with inhibitory activity against Lactococcus garvieae. J Fish Dis34:499-507.
128. Pérez T, Balcázar JL, Ruiz-Zarzuela I, Halaihel N, Vendrell D, de Blas I&Múzquiz JL (2010) Host–microbiota interactions within the fish intestinalecosystem. Mucosal Immunol3:355-360.
129. Petrosino JF, Highlander S, Ann Luna R, Gibbs RA&Versalovic J (2009)Metagenomic pyrosequencing and microbial identification. Clin Chem55(5):856-866.
130. Picchietti S et al.(2009) Early treatment with Lactobacillus delbrueckii straininduces an increase in intestinal T-cells and granulocytes and modulatesimmune-related genes of larval Dicentrarchus labrax (L.). Fish ShellfishImmunol26:368–376.
131. Pond MJ, Stone DM, Alderman DJ (2006) Comparison of conventional andmolecular techniques to investigate the intestinal microflora of rainbow trout(Oncorhynchus mykiss). Aquaculture261:194–203.
132. Pope PB, Mackenzie AK, Gregor I, Smith W, Sundset MA, McHardy AC,Morrison M&Eijsink VG (2012) Metagenomics of svalbard reindeer rumenmicrobiome reveals abundance of polysaccharide utilization loci. PLoS One7:e38571.
133. Powell MD, Clark GA (2004) Efficacy and toxicity of oxidative disinfectants forthe removal of gill amoebae from the gills of amoebic gill disease affectedAtlantic salmon (Salmo salar L.) in freshwater. Aquac Res35:112-123.
134. Press CM&Evensen O (1999) The morphology of the immune system in teleostfishes. Fish Shellfish Immunol9:309–318.
135. Qu A, Brulc JM, Wilson MK et al.(2008) Comparative metagenomics revealshost specific meravirulomes and horizontal gene transfer elements in the chickencecum microbiome. PLoS One3: e2945.
136. Ramirez RF&Dixon BA (2003) Enzyme production by obligate intestinalanaerobic bacteria isolated from oscars (Astronotus ocellatus), angelfish(Pterophyllum scalare) and southern flounder (Paralichthys lethostigma).Aquaculture227:417–426.
137. Rappe MS, Connon SA, Vergin KL&Giovannoni SJ (2002) Cultivation of theubiquitous SAR11marine bacterioplankton clade. Nature418:630–633.
138. Ravi AV, Musthafa KS, Jegathammbal G, Kathiresan K&Pandian SK (2007)Screening and evaluation of probiotics as a biocontrol agent against pathogenicVibrios in marine aquaculture. Lett Appl Microbiol45(2):219-223.
139. Razin S, Yogev D&Naot Y (1998) Molecular biology and pathogenicity ofmycoplasmas. Microbiol Mol Biol Rev62:1094–1156.
140. Reyes-Becerril M et al.(2008) Oral delivery of live yeast Debaryomyceshansenii modulates the main innate immune parameters and the expression ofimmune-relevant genes in the gilthead seabream (Sparus aurata L.). FishShellfish Immunol25:731–739.
141. Ring E, Str m E&Tabachek JA (1995) Intestinal microfl ora of salmonids: areview. Aquacult Res26:773–789.
142. Ring E&Birkbeck TH (1999) Intestinal microflora of fish larvae and fry.Aquaculture Research30:73–93.
143. Ring E, Olsen RE, Mayhew TM&Myklebust R (2003) Electron microscopy ofthe intestinal microflora of fish. Aquaculture227:395–415.
144. Roediger WE&Macfarlane GT (2002) A role for intestinal mycoplasmas in theaetiology of Crohn’s disease? J Appl Microbiol92:377-381.
145. Ruoff KL (1991) Nutritionally variant streptococci. Clin Microbiol Rev4:184–190.
146. Russo R, Curtis EW&Yanong RP (2007) Preliminary investigations ofhydrogen peroxide treatment of selected ornamental fishes and efficacy againstexternal bacteria and parasites in green swordtails. J Aquat Anim Health19:121-127.
147. Sait M, Hugenholtz P&Janssen PH (2002) Cultivation of globally distributedsoil bacteria from phylogenetic lineages previously only detected incultivation-independent surveys. Environ Microbiol4:654–666.
148. Salinas I et al.(2008) Monospecies and multispecies probiotic formulationsproduce different systemic and local immunostimulatory effects in the giltheadseabream (Sparus aurata L.). Fish Shellfish Immunol25:114–123.
149. Salzman NH, de Jong H, Paterson Y, Harmsen HJ, Welling GW, et al.(2002)Analysis of16S libraries of mouse gastrointestinal microflora reveals a largenew group of mouse intestinal bacteria. Microbiology148:3651–3660.
150. Savage DC (1977) Microbial ecology of the gastrointestinal tract. Ann RevMicrobiol31:107–133.
151. Schloss PD&Handelsman J (2005) Introducing DOTUR, a computer programfor defining operational taxonomic units and estimating species richness. ApplEnviron Microbiol71:1501-1506.
152. Schrijver RD&Ollevier F (2000) Protein digestion in juvenile turbot(Scophthalmus maximus) and effects of dietary administration of Vibrioproteolyticus. Aquaculture186:107-116.
153. Secombes CJ et al.(2001) Cytokines and innate immunity of fish. Dev CompImmunol25:713–723.
154. Senderovich Y, Izhaki I&Halpern M (2010) Fish as reservoirs and vectors ofVibrio cholerae. PLoS One5: e8607.
155. Sha Z, Wang S, Zhuang Z, Wang Q, Wang Q, Li P, Ding H, Wang N, Liu Z,Chen S (2010) Generation and analysis of10,000ESTs from the half-smoothtongue sole Cynoglossus semilaevis and identification of microsatellite and SNPmarkers. J Fish Biol76(5):1190–1204.
156. Shigematsu T, Hayashi M, Kikuchi I, Ueno S, Masaki H&Fujii T (2009) Aculture-dependent bacterial community structure analysis based on liquidcultivation and its application to a marine environment. FEMS Microbiol Lett293(2):240-247.
157. Silphaduang U, Colorni A&Noga EJ (2006) Evidence for widespreaddistribution of piscidin antimicrobial peptides in teleost fish. Dis Aquat Org72:241–252.
158. Sipkema D, Schippers K, Maalcke WJ, Yang Y, Salim S&Blanch HW (2011)Multiple approaches to enhance the cultivability of bacteria associated with themarine sponge Haliclona (gellius) sp. Appl Environ Microbiol77(6):2130-2140.
159. Smriga S, Sandin SA&Azam F (2010) Abundance, diversity, and activity ofmicrobial assemblages associated with coral reef fish guts and feces. FEMSMicrobiol Ecol73:31-42.
160. Song J, Oh H-M&Cho J-C (2009) Improved culturability of SAR11strains indilution-to-extinction culturing from the East Sea, West Pacific Ocean. FEMSMicrobiol Lett295:141–147.
161. Staley JT&Konopka A (1985) Measurement of in situ activities ofnonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu RevMicrobiol39:321–346.
162. Stingl U, Cho JC, Foo W, Vergin KL, Lanoil B&Giovannoni SJ (2008)Dilution-to-extinction culturing of psychrotolerant planktonic bacteria frompermanently ice-covered lakes in the McMurdo Dry Valleys, Antarctica. MicrobEcol55:395–405.
163. Su C, Lei L, Duan Y, Zhang KQ&Yang J (2012) Culture-independent methodsfor studying environmental microorganisms: methods, application, andperspective. Appl Microbiol Biotechnol93:993-1003.
164. Sugita H, Shibuya K, Hanada H&Deguchi Y (1997) Antibacterial abilities ofintestinal microflora of the river fish. Fish Sci63:378–383.
165. Sun K, Zhang WW, Hou JH&Sun L (2009) Immunoprotective analysis ofVhhP2, a Vibrio harveyi vaccine candidate. Vaccine27:2733-2740.
166. Suthienkul O, Ishibashi M, Iida T, Nettip N, Supavej S, Eampokalap B, MakinoM&Honda T (1995) Urease production correlates with possession of the trhgene in Vibrio parahaemolyticus strains isolated in Thailand. J Infect Dis172:1405–1408.
167. Tamura K, Dudley J, Nei M&Kumar S (2007) MEGA4: Molecular evolutionarygenetics analysis (MEGA) software version4.0. Mol Biol Evol24:1596-1599.
168. Tanaka Y, Hanada S, Manome A, Tsuchida T, Kurane R, Nakamura K&Kamagata Y (2004) Catellibacterium nectariphilum gen. nov., sp. nov., whichrequires a diffusible compound from a strain related to the genus Sphingomonasfor vigorous growth. Int J Syst Evol Micr54:955–959.
169. Tapia-Paniagua ST, Chabrillón M, Díaz-Rosales P, de la Banda IG, Lobo C,Balebona MC&Mori igo MA (2010) Intestinal microbiota diversity of the flatfish Solea senegalensis (Kaup,1858) following probiotic administration. MicrobEcol60:310-319.
170. Thomas T, Gilbert J&Meyer (2012) Metagenomics–a guide from sampling todata analysis. Microbial Informatics and Experimentation2:3
171. Thompson PL, Iida T&Swings J (2004) Biodiversity of Vibrios. Microbiol MolBiol Rev68:403-431.
172. Timmerman HM, Koning CJM, Mulder L, Rombout FM&Beynen AC (2004)Monospecies, multistrain and multispecies probiotics: a comparison offunctionality and efficacy. Int J Food Microbiol96:219–233.
173. Tlaskalova-Hogenova H et al.(2005) Interaction of mucosal microbiota with theinnate immune system. Scand J Immunol62:106–113.
174. Tripp HJ, Kitner JB, Schwalbach MS, Dacey JW, Wilhelm LJ&Giovannoni SJ(2008) SAR11marine bacteria require exogenous reduced sulphur for growth.Nature452:741–744.
175. Trust TJ&Sparrow RAH (1974) The bacterial flora in the alimentary tract offreshwater salmonid fishes. Canad J Microbiol20:1219–1228.
176. Tun HM, Brar MS, Khin N, Jun L, Hui RKH, Dowd SE&Leung FC (2012)Gene-centric metagenomics analysis of feline intestinal microbiome using454junior pyrosequencing. J Microbiol Methods88:369-376.
177. Urbanczyk H, Ast JC, Higgins MJ, Carson J&Dunlap PV (2007)Reclassification of Vibrio fischeri, Vibrio logei, Vibrio salmonicida and Vibriowodanis as Aliivibrio fischeri gen. nov., comb. nov., Aliivibrio logei comb. nov.,Aliivibrio salmonicida comb. nov. and Aliivibrio wodanis comb. nov. Int J SystEvol Microbiol57:2823-2829.
178. van Walraven L, Mollet FM, van Damme CJG, Rijnsdorp AD (2010)Fisheries-induced evolution in growth, maturation and reproductive investmentof the sexually dimorphic North Sea plaice (Pleuronectes platessa L.). J Sea Res64(1–2):85–93.
179. Vartoukian SR, Palmer RM&Wade WG (2010) Strategies for culture of‘unculturable’bacteria. FEMS Microbiol Lett309(1):1-7.
180. Vendrell D, Balcázar JL, Ruiz-Zarzuela I, de Blas I, Gironés O&Múzquiz JL(2006) Lactococcus garvieae in fish: a review. Comp Immunol Microbiol InfectDis29:177–198.
181. Vendrell D, Balcázar JL, de Blas I, Ruiz-Zarzuela I, Gironés O&Múzquiz JL(2008) Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis byprobiotic bacteria. Comp Immunol Microbiol Infect Dis31:337–345.
182. Vergin KL, Rappe′MS&Giovannoni SJ (2001) Streamlined method to analyze16S rRNA gene clone libraries. Bio Tech30:938–944.
183. Vine NG, Leukes WD&Kaiser H (2004) In vitro growth characteristics of fivecandidate aquaculture probiotics and two fish pathogens grown in fish intestinalmucus. FEMS Microbiol Lett231(1):145-152.
184. Wang X, Heazlewood SP, Krause DO, Florin THJ (2003) Molecularcharacterization of the microbial species that colonize human ileal and colonicmucosa by using16S rDNA sequence analysis. J Appl Microb95:508-520.
185. Wang Y, Hammes F, Boon N, Chami M&Egli T (2009) Isolation andcharacterization of low nucleic acid (LNA)-content bacteria. ISME J3:889–902.
186. Ward NL, Steven B, Penn K, Methé BA, Detrich WH3rd (2009)Characterization of the intestinal microbiota of two Antarctic notothenioid fishspecies. Extremophiles13(4):679-685.
187. Watve M, Shejval V, Sonawane C et al.(2000) The ‘K’ selected oligophilicbacteria: a key to uncultured diversity? Curr Sci78:1535–1542.
188. Weltzien FA, Andersson E, Andersen, Shalchian-Tabrizi K, Norberg B (2004)The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish(Pleuronectiformes). Comp Biochem Physiol A Mol Integr Physiol137(3):447–477.
189. Wu S, Gao T, Zheng Y, Wang W, Cheng Y&Wang G (2010) Microbial diversityof intestinal contents and mucus in yellow catfish (Pelteobagrus fulvidraco).Aquaculture303:1-7.
190. Wu S, Wang G, Angert ER, Wang W, Li W&Zou H (2012a) Composition,diversity, and origin of the bacterial community in grass carp intestine. PLoS One7: e30440.
191. Wyss C (1989) Dependence of proliferation of Bacteroides forsythus onexogenous N-acetylmuramic acid. Infect Immun57:1757–1759.
192. Yildiz FH&Visick KL (2009) Vibrio biofilms: so much the same yet sodiffierent. Trends Microbiol17:109-118.
193. Zengler K, Toledo G, Rappe M, Elkins J, Mathur EJ, Short JM&Keller M(2002) Cultivating the uncultured. P Natl Acad Sci USA99:15681–15686.
194. Zerbino DR&Birney E (2008) Velvet: algorithms for de novo short readassembly using de bruijn graphs. Genome Res18:821-829.
195. Zhu L, Wu Q, Dai J, Zhang S&Wei F (2011) Evidence of cellulose metabolismby the giant panda gut microbiome. PNAS108:17714-17719.
196. Zoetendal EG, Collier CT, Koike S, Mackie RI&Gaskins HR (2004) Molecularecological analysis of the gastrointestinal microbiota: A review. J Nutr134:456–472.
197. Zou J, Clark MS&Secombes CJ (2003) Characterisation, expression andpromoter analysis of an interleukin10homologue in the puffer fish, Fugurubripes Immunogenetics55:325–335.
198. Zou J, Mercier C, Koussounadis A&Secombes C (2007) Discovery of multiplebeta-defensin like homologues in teleost fish. Mol Immunol44:638–647.
199. Zu GZ, Yu WY&Li JN (2000) Diagnosis and epidemiological research onbacterial septicemia of grass carp Ctenopharyngodon idellus. FreshwaterFisheries30:35–37.
200. Handelsman J, Rondon MR, Brady SF, Clardy J&Goodman RM (1998)Molecular biological access to the chemistry of unknown soil microbes: a newfrontier for natural products. Chem Biol5(10): R245-249.
201. Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM,Solovyev VV, Rubin EM, Rokhsar DS&Banfield JF (2004) Communitystructure and metabolism through reconstruction of microbial genomes from theenvironment. Nature428(6978):37-43.
202. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D,Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW,Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H,Pfannkoch C, Rogers YH&Smith HO (2004) Environmental genome shotgunsequencing of the Sargasso Sea. Science304(5667):66-74.
203. Simon C, Daniel R (2011) Metagenomic analyses: past and future trends. ApplEnviron Microbiol77(4):1153-1161.
204. Wilmes P&Bond PL (2006) Metaproteomics: studying functional geneexpression in microbial ecosystems. Trends Microbiol14(2):92-97.
205. Gilbert JA, Field D, Huang Y, Edwards R, Li W, Gilna P&Joint I (2008)Detection of large numbers of novel sequences in the metatranscriptomes ofcomplex marine microbial communities. PLoS One3(8): e3042.
206. Beja O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP, JovanovichSB, Gates CM, Feldman RA, Spudich JL, Spudich EN&DeLong EF (2000)Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science289(5486):1902-1906.
207. Nicol GW&Schleper C (2006) Ammonia-oxidising Crenarchaeota: importantplayers in the nitrogen cycle? Trends Microbiol14(5):207-212.
2. Adams DO&Hamilton TA (1984) The cell biology of macrophage activation.Ann Rev Immunol2:283–318.
3. Agulleiro MJ, Anguis V, Can avate JP, Mart′nez-Rodr′guez G, Mylonas CC,Cerda`J (2006) Induction of spawning of captive-reared Senegal sole (Soleasenegalensis) using different delivery systems for gonadotropin-releasinghormone agonist. Aquaculture257(1–4):511–524.
4. Aly SM, Ahmed YAG, Ghareeb AAA&Mohamed MF (2008) Studies onBacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on theimmune response and resistance of Tilapia nilotica (Oerochromis niloticus) tochallenge infections. Fish Shellfish Immunol25:128–136.
5. Amann RI, Ludwig W, Schleifer KH, Amann RI, Ludwig W&Schleifer KH(1995) Phylogenetic identification and in situ detection of individual microbialcells without cultivation. Microbiol Rev59:143–169.
6. Austin B&Austin DA (1999) Bacterial fish pathogens: disease of farmed andwild fish,3rd ed. Springer-Verlag KG, Berlin, Germany.
7. Austin B (2006) The bacterial microflora of fish, revised. ScientificWorldJournal6:931-945.
8. Bae JW, Rhee SK, Park JR, Kim BC&Park YH (2005) Isolation of uncultivatedanaerobic thermophiles from compost by supplementing cell extract ofGeobacillus toebii in enrichment culture medium. Extremophiles9:477–485.
9. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Múzquiz JL(2006) The role of probiotics in aquaculture. Vet Microbiol114(3-4):173-186.
10. Balcázar JL (2007) Probiotics in health maintenance: do they really work? Br JInfect Control8:26–29.
11. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Gironés O&Muzquiz JL(2007a) Sequencing of variable regions of the16S rRNA gene for identificationof lactic acid bacteria isolated from the intestinal microbiota of healthysalmonids. Comp Immunol Microbiol Infect Dis30:111–118.
12. Balcázar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Gironés O&Múzquiz JL(2007b) In vitro competitive adhesion and production of antagonistic compoundsby lactic acid bacteria against fi sh pathogens. Vet Microbiol122:373–380.
13. Balcázar JL et al.(2007c) Changes in intestinal microbiota and humoral immuneresponse following probiotic administration in brown trout (Salmo trutta). Br JNutr97:522–527.
14. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Gironés O&Muzquiz JL(2007d) Enhancement of the immune response and protection induced byprobiotic lactic acid bacteria against furunculosis in rainbow trout(Oncorhynchus mykiss). FEMS Immunol Med Microbiol51:185–193.
15. Bano N, DeRae Smith A, Bennett W, Vasquez L&Hollibaugh JT (2007)Dominance of Mycoplasma in the guts of the Long-Jawed Mudsucker,Gillichthys mirabilis, from five California salt marshes. Environ Microbiol9:2636-2641.
16. Bates JM, Akerlund J, Mittge E&Guillemin K (2007) Intestinal alkalinephosphatase detoxifies lipopolysaccharide and prevents inflammation inzebrafish in response to the gut microbiota. Cell Host Microbe2:371–382.
17. Ben-Oov E, Kramarsky-Winter E&Kushmaro A (2009) An in situ method forcultivating microorganisms using a double encapsulation technique. FEMSMicrobiol Ecol68:363-371.
18. Berg RD (1992) Translocation and the indigenous gut flora. In: Fuller R, ed.Probiotics: The scientific basis London; Chapman and Hall, London. pp55–85.
19. Bollmann A, Lewis K&Epstein SS (2007) Incubation of environmental samplesin a diffusion chamber increases the diversity of recovered isolates. Appl EnvironMicrob73:6386–6390.
20. Bosch TC&McFall-Ngai MJ (2011) Metaorganisms as the new frontier. Zoology114:185–189.
21. Bromage N, Mazorra C, Bruce M, Brown N, Shields R (2000) Halibut culture. In:Stickney RR (ed) Encyclopedia of aquaculture. Wiley, New York, pp425–432.
22. Bruns A, Cypionka H&Overmann J (2002) Cyclic AMP and acyl homoserinelactones increase the cultivation efficiency of heterotrophic bacteria from thecentral Baltic Sea. Appl Environ Microb68:3978–3987.
23. Bury N&Grosell M (2003a) Iron acquisition by teleost fish. Comp BiochemPhysiol Part C Toxicol Pharmacol135:97-105.
24. Bury NR&Grosell M (2003b) Waterborne iron acquisition by a freshwaterteleost fish, zebrafish Danio rerio. J Exp Bio206:3529-3535.
25. Bury NR, Walker PA&Glover CN (2003) Nutritive metal uptake in teleost fish. JExp Biol206:11-23.
26. Capkin E&Altinok I (2009) Effects of dietary probiotic supplementations onprevention/treatment of yersiniosis disease. J Appl Microbiol106:1147–1153.
27. Carpenter S&O’Neill LAJ (2007) How important are Toll-like receptors forantimicrobial responses? Cell Microbiol9:1891–1901.
28. Cerdà-Cuéllar M&Blanch AR (2002) Detection and identification of Vibrioscophthalmi in the intestinal microbiota of fish and evaluation of host specificity.J Appl Microbiol93(2):261-268.
29. Cerdà-Cuéllar M&Blanch AR (2004) Determination of Vibrio scophthalmi andits phenotypic diversity in turbot larvae. Environ Microbiol6:209-217.
30. Cerda`J, Douglas S, Reith M (2010) Genomic resources for flatfish research andtheir applications. J Fish Biol77(5):1045–1070.
31. Chabrillón M, Rico R M, Balebona M C, Mori igo M A (2005) Adhesion to sole,Solea senegalensis Kaup, mucus of microorganisms isolated from farmed fish,and their interaction with Photobacterium damselae subsp. piscicida. J Fish Dis28:229-237.
32. Cheesman SE&Guillemin K (2007) We know you are in there: Conversing withthe indigenous gut microbiota. Res Microbiol158:2–9.
33. Chen G&Brown A (1985) Bacterial growth and the concentrations of cyclicnucleotides in Legionella pneumophila cultures. Curr Microbiol12:23–26.
34. Clapper WE&Meade GH (1963) Normal flora of the nose, throat, and lowerintestine of dogs. J Bacteriol85:643–648.
35. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM,Bandela AM, Cardenas E, Garrity GM&Tiedje JM (2007) The ribosomaldatabase project (RDP-II): introducing myRDP space and quality controlledpublic data. Nucleic Acids Res35:169-172.
36. Colwell RR, Kaper JB, Joseph SW (1977) Vibrio cholerae, Vibrioparahaemolyticus and other Vibrios: occurrence and distribution in ChesapeakeBay. Science198:394–396.
37. Colwell RR, Huq A (2001) Marine ecosystems and cholera. Hydrobiologia460:141–145.
38. Conceic a o LCE, Ribeiro L, Engrola S, Araga o C, Morais S, Lacuisse M,Soares F, Dinis MT (2007) Nutritional physiology during development ofSenegalese sole (Solea senegalensis). Aquaculture268(1–4):64–81.
39. Condon C (2009) RNA processing. In: Moselio S (ed) Encyclopedia ofmicrobiology,3rd edn. Elsevier, Oxford, vol5, pp395–408.
40. Connon SA&Giovannoni SJ (2002) High-throughput methods for culturingmicroorganisms in very-low-nutrient media yield diverse new marine isolates.Appl Environ Microb68:3878–3885.
41. Coppola S, Zoina A&Marino P (1976) Interactions of N6-(delta2-isopentenyl)adenine with cyclic AMP on the regulation of growth and beta-galactosidasesynthesis in Escherichia coli. J Gen Microbiol94:436–438.
42. Corthésy B, Gaskins HR&Mercenier A (2007) Cross-talk between probioticbacteria and the host immune system. J Nutr137:781S–790S.
43. Dandekar T, Snel B, Schmidt S, Lathe W, Suyama M, Huynen M&Bork P (2002)Comparative genome analysis of Mollicutes. In Molecular Biology andPathogenicity of Mycoplasmas. New York, USA: Kluwer Academic/Plenum, pp.255–278.
44. Davis KE, Joseph SJ&Janssen PH (2005) Effects of growth medium, inoculumsize, and incubation time on culturability and isolation of soil bacteria. ApplEnviron Microb71:826–834.
45. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T,Dalevi D, Hu P&Andersen GL (2006) Greengenes, a chimera-checked16SrRNA gene database and workbench compatible with ARB. Appl EnvironMicrobiol72:5069-5072.
46. Dethlefsen L, McFall-Ngai M&Relman DA (2007) An ecological andevolutinary perspective on human-microbe mutualism and disease. Nature449:811–818.
47. Dickschat JS (2010) Quorum sensing and bacterial biofilms. Nat Prod Rep27:343-369.
48. Divya KR, Isamma A, Ramasubramanian V, Sureshkumar S&Arunjith TS (2012)Colonization of probiotic bacteria and its impact on ornamental fish Puntiusconchonius. J Environ Biol33(3):551-555.
49. Fasano A&Shea-Donohue T (2005) Mechanisms of disease: the role ofintestinal barrier function in the pathogenesis of gastrointestinal autoimmunediseases. Nat Clin Pract Gastroenterol Hepatol2:416–422.
50. Ferrari BC, Winsley T, Gillings M&Binnerup S (2008) Cultivating previouslyuncultured soil bacteria using a soil substrate membrane system. Nat Protoc3:1261–1269.
51. Ferrari BC&Gillings M (2009) Cultivating fastidious bacteria: viability stainingand micromanipulation from a soil substrate membrane system. Appl EnvironMicrob75:3352–3354.
52. Forne′I, Abia′n J, Cerda`J (2010) Fish proteome analysis: model organisms andnon-sequenced species. Proteomics10(4):858–872.
53. Fraser CM, Gocayne JD, White O, Adams MD., Clayton RA., Fleischmann RD,et al.(1995) The minimal gene complement of Mycoplasma genitalium. Science270:397–404.
54. García-Aljaro C, Eberl L, Riedel K&Blanch AR (2008) Detection ofquorum-sensing-related molecules in Vibrio scophthalmi. BMC Microbiol8:138.
55. Gatesoupe FJ (2008) Updating the importance of lactic acid bacteria in fishfarming: natural occurrence and probiotic treatments. J Mol MicrobiolBiotechnol14:107–114.
56. Gómez GD&Balcázar JL (2008) A review on the interactions between gutmicrobiota and innate immunity of fish. FEMS Immunol Med Microbiol52:145–154.
57. Gonzalez A, Stombaugh J, Lozupone C, Turnbaugh PJ, Gordon JI&Knight R(2011) The mind-body-microbial continuum. Dia Clin Neurosci13:55–62.
58. Gonza′lez C, Lo′pez-D′az JTM, Garc′a-Lo′pez ML, Prieto M&Otero A (1999)Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius),and aquacultured rainbow trout (Oncorhynchus mykiss). J Food Prot62:1270–1277.
59. Good IJ (1953) The population frequencies of species and the estimation ofpopulation parameters. Biometrika40:237-264.
60. Goodman SE&Gordon JI (2010) Our unindicated coconspirators: Humanmetabolism from a microbial perspective. Cell Metab12:111–116.
61. Graber JR&Breznak JA (2005) Folate cross-feeding supports symbiotichomoacetogenic spirochetes. Appl Environ Microb71:1883–1889.
62. Guardabassi L, Schwarz S&Lloyd DH (2004) Pet animals as reservoirs ofantimicrobial-resistant bacteria. J Antimicrob Chemother54:321-332.
63. Hackstadt T&Williams JC (1981) Biochemical stratagem for obligate parasitismof eukaryotic cells by Coxiella burnetii. P Natl Acad Sci USA78:3240–3244.
64. Hansen GH&Olafsen JA (1999) Bacterial interactions in early life stages ofmarine cold water fish. Microb Ecol38:1–26.
65. Hazen TH, Pan L, Gu JD&Sobecky PA (2010) The contribution of mobilegenetic elements to the evolution and ecology of Vibrios. FEMS Microbiol Ecol74:485-499.
66. He X, Nie X, Yang Y, Liu X, Pan D, Cheng Z&Liang X (2012) Multi-biomarkerresponses in fishes from two typical marine aquaculture regions of South China.Mar Pollut Bull doi:10.1016/j.marpolbul.2012.09.002.
67. Hoarau G, Boon E, Jongma DN, Ferber S, Palsson J, Van der Veer HW,RijnsdorpAD, StamWT,Olsen JL (2005) Loweffective population size andevidence for inbreeding in an overexploited flatfish, plaice (Pleuronectesplatessa L.). Proc R Soc B272:497–503.
68. Holland MCH&Lambris JD (2002) The complement system in teleosts. FishShellfish Immunol12:399–420.
69. Honda T&Iida T (1993) The pathogenicity of Vibrio parahaemolyticus and therole of the thermostable direct haemolysin and related haemolysins. Rev MedMicrobiol4:106–113.
70. H rmannsperger G&Haller D (2010) Molecular crosstalk of probiotic bacteriawith the intestinal immune system: clinical relevance in the context ofinflammatory bowel disease. Int J Med Microbiol300:63–73.
71. Horsley RW (1977) A review of bacterial flora of teleosts and elasmobranches,including methods for its analysis. J Fish Biol10:529–553.
72. Hosoi T, Ametani A, Kiuchi K&Kaminogawa S (2000) Improved growth andviability of lactobacilii in the presence of Bacillus subtilis, catalase, or subtilisin.Can J Microbiol46:892–897.
73. Huber T, Faulkner G&Hugenholtz P (2004) Bellerophon: A program to detectchimeric sequences in multiple sequence alignments. Bioinformatics20:2317-2319.
74. Hugenholtz P, Hooper SD&Kyrpides NC (2009) Focus: synergistetes. EnvironMicrobiol11:1327–1329.
75. Imsland AK, Foss A, Conceic a o LEC, Dinis MT, Delbare D, Schram E,Kamstra A, Rema P, White P (2003) A review of the culture potential of Soleasolea and S. senegalensis. Rev Fish Biol Fish13(4):379–407.
76. Irianto A&Austin B (2002) Probiotics in aquaculture. J Fish Dis25:633–642.
77. Irwin JA (2010) Extremophiles and their application to veterinary medicine.Environ Technol31(8-9):857-869.
78. Janssen PH (2003) Selective enrichment and purification of cultures ofMethanosaeta spp. J Microbiol Methods52:239–244.
79. Kaeberlein T, Lewis K&Epstein SS (2002) Isolating ‘uncultivable’microorganisms in pure culture in a simulated natural environment. Science296:1127–1129.
80. Kennedy J, Marchesi JR&Dobson ADW (2008) Marine metagenomics:strategies for the discovery of novel enzymes with biotechnological applicationsfrom marine environments. Microb Cell Fact7:27.
81. Kennedy J, Baker P, Piper C, Cotter PD, Walsh M, Mooij MJ, Bourke MB, ReaMC, O'Connor PM, Ross RP, Hill C, O'Gara F, Marchesi JR&Dobson AD(2009) Isolation and analysis of bacteria with antimicrobial activities from themarine sponge Haliclona simulans collected from Irish waters. Mar Biotechnol11(3):384-396.
82. Kim BS, Kim JN&Cerniglia CE (2011) In Vitro culture conditions formaintaining a complex population of human gastrointestinal tract microbiota. JBiomed Biotechnol2011:838040.
83. Kim DH&Austin B (2006) Innate immune responses in rainbow trout(Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish Shellfish Immunol21:513–524.
84. Kim JJ, Kim HN, Masui R, Kuramitsu S, Seo JH, Kim K&Sung MH (2008)Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceaerequiring growth-supporting factors. J Microbiol Biotechn18:611–615.
85. Kuda T, Kyoi D, Takahashi H, Obama K&Kimura B (2011) Detection andisolation of p-nitrophenol-lowering bacteria from intestine of marine fishescaught in Japanese waters. Mar Pollut Bull62(8):1622-1627.
86. Kwak JK, Park SW, Koo JG, Cho MG, Buchholz R&Goetz P (2003)Enhancement of the non-specific defense activities in carp (Cyprinus carpio) andflounder (Paralichthys olivaces) by oral administration of Schizophyllan. ActaBiotechnol23:359–371.
87. Lamendella R, Domingo JW, Ghosh S, Martinson J&Oerther DB (2011)Comparative fecal metagenomics unveils unique fuctional capacity of the swinegut. BMC Microbiol11:103.
88. Lavery TJ, Roudnew B, Seymour J, Mitchell JG&Jeffries T (2012) Highnutrient transport and cycling potential revealed in the microbial metagenome ofAustralian sea lion. PLoS One7: e36478.
89. Lazado CC, Caipang CMA, Rajan B, Brinchmann MF, Kiron V (2010)Characterization of GP21and GP12: two protential probiotic bacteria isolatedfrom the gastrointestinal tract of Atlantic cod. Probiotics&Antimicro Prot2:126-134.
90. Li CQ, Liu WC, Zhu P, Yang JL&Cheng KD (2011) Phylogenetic diversity ofbacteria associated with the marine sponge Gelliodes carnosa collected from theHainan island coastal waters of the South China Sea. Microb Ecol62(4):800-812.
91. Lunder T, S rum H, Holstad G, Steigerwalt AG, Mowinckel P&Brenner DJ(2000) Phenotypic and genotypic characterization of Vibrio viscosus sp. nov. andVibrio wodanis sp. nov. isolated from Atlantic salmon (Salmo salar) with ‘winterulcer’. Int J Syst Evol Microbiol50:427-450.
92. Markowitz VM, Chen IM, Chu K et al.(2012) IMG/M: the integratedmetagenome data management and comparative analysis system. Nucleic AcidsRes40:123-129.
93. McClelland DBL (1976) Peyer’s-patch-associated synthesis of immunoglobulinin germ-free, specifi c-pathogen-free, and conventional mice. Scand J Immunol5:909–915.
94. McDonald R, Schreier HJ&Watts JE (2012) Phylogenetic analysis of microbialcommunities in different regions of the gastrointestinal tract in Panaquenigrolineatus, a wood-eating fish. PLoS One7: e48018.
95. Metchnikoff E (1901) Surlaflore du corps. Human Member Protocol ofManchester Literature and Philosophical Society.45:1–38.
96. Meyer F, Paarmann D, D'Souza M et al.(2008) The metagenomics RAST sever-a public resource for the automatic phylogenetic and functional analysis ofmetagenomes. BMC Bioinformatics9:386.
97. Milla′n A, Gomez-Tato A, Fernandez C, Pardo BG, Alvarez-Dios JA, Calaza M,Bouza C, Va′zquez M, Cabaleiro S, Martinez P (2010) Design and performanceof a turbot (Scophthalmus maximus) oligo-microarray based on ESTs fromimmune tissues. Mar Biotechnol12(4):452–465.
98. Milla′n A, Go′mez-Tato A, Pardo BG, Ferna′ndez C, Bouza C, Vera M,Alvarez-Dios JA, Cabaleiro S, Lamas J, Lemos ML, Mart′nez P (2011) Geneexpression profiles of the spleen, liver, and head kidney in turbot (Scophthalmusmaximus) along the infection process with Aeromonas salmonicida using animmune-enriched oligo-microarray. Mar Biotechnol13(6):1099–1114.
99. Millner R, Walsh SJ, D′az de Astarloa JM (2005) Atlantic flatfish fisheries. In:Gibson RN (ed) Flatfishes: biology and exploitation. Blackwell Publishing,Oxford, pp240–271.
100. Mitra S, Rupek P, Richter DC, Urich T, Gilbert JA, Meyer F, Wilke A&HusonDH (2011) Functional analysis of metagenomes and metatranscriptomes usingSEED and KEGG.. BMC Bioinformatics12(Suppl1): S21.
101. Mollet FM, Kraak SBM, Rijnsdorp AD (2007) Fisheries-induced evolutionarychanges in maturation reaction norms in North Sea sole Solea solea. Mar EcolProg Ser351:189–199.
102. Montalto M, D’Onofrio F, Gallo A, Cazzato A&Gasbarrini G (2009) Intestinalmicrobiota and its functions. Dig Liver Dis Suppl3:30–34.
103. Montes M, Farto R, Pérez MJ, Nieto TP, Larsen JL&Christensen H (2003)Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus)culture by phenotypic analysis. J Appl Microbiol95:693-703.
104. Montes M, Farto R, Pérez MJ, Armada SP&Nieto TP (2006) Genotypicdiversity of Vibrio isolated associated with turbot (Scophthalmus maximus)culture. Res Microbiol157:487-495.
105. Moran D, Turner SJ&Clements KD (2005) Ontogenetic development of thegastrointestinal microbiota in the marine herbivorous fish Kyphosus sydneyanus.Microb Ecol49:590-597.
106. Mulder IE, Wadsworth S&Secombes CJ (2007) Cytokine expression in theintestine of rainbow trout (Oncorhynchus mykiss) during infection withAeromonas salmonicida. Fish Shellfish Immunol23:747–759.
107. Munroe TA (2005) Systematic diversity of the Pleuronectiformes. In: Gibson RN(ed) Flatfishes: biology and exploitation. Blackwell Publishing, Oxford, pp10–41.
108. Navarrete P, Espejo RT&Romero J (2009) Molecular analysis of microbiotaalong the digestive tract of juvenile Atlantic salmon (Salmo salar L.). MicrobEcol57:550-561.
109. Naylor R, Burke M (2005) Aquaculture and ocean resources: raising tigers of thesea. Annu Rev Environ Resour30:185–218.
110. Nayak SK (2010) Probiotics and immunity: a fish perspective. Fish ShellfishImmunol.,29(1):2-14.
111. Newaj-Fyzul A, Adesiyun AA, Mutani A, Ramsubhag A, Brunt J&Austin B(2007) Bacillus subtilis AB1controls Aeromonas infection in rainbow trout(Oncorhynchus mykiss, Walbaum). J Appl Microbiol103:1699-1706.
112. Nichols D, Lewis K, Orjala J et al.(2008) Short peptide induces an‘uncultivable’ microorganism to grow in vitro. Appl Environ Microb74:4889–4897.
113. Nishibuchi M&Kaper JB (1995) Thermostable direct hemolysin gene of Vibrioparahaemolyticus: a virulence gene acquired by a marine bacterium. InfectImmun63:2093–2099.
114. Oakey HJ, Cullen BR&OwensL (2002) The compete nucleotide sequence of theVibrio harveyi bacteriophage VHML. J Appl Microbiol93:1089–1098.
115. O’Hara AM&Shanahan F (2006) The gut flora as a forgotten organ. EMBO Rep7:688–693.
116. Ohno M, Shiratori H, Park MJ et al.(2000) Symbiobacterium thermophilum gen.nov., sp. nov., a symbiotic thermophile that depends on co-culture with aBacillus strain for growth. Int J Syst Evol Micr50:1829–1832.
117. Omsland A, Cockrell DC, Fischer ER&Heinzen RA (2008) Sustained axenicmetabolic activity by the obligate intracellular bacterium Coxiella burnetii. JBacteriol190:3203–3212.
118. Ormsbee RA&Peacock MG (1964) Metabolic activity in Coxiella burnetii. JBacteriol88:1205–1210.
119. Ouwehand AC, Isolauri E, Kirjavainen PV, T lkk, S&Salminen SJ (2000) Themucus binding of Bifidobacterium lactis Bb12is enhanced in the presence ofLactobacillus GG and Lact. delbrüeckii subsp. bulgaricus. Lett Appl Microbiol30:10–13.
120. Pamer EG (2007) Immune responses to commensal and environmental microbes.Nat Immunol8:1173–1178.
121. Panigrahi A et al.(2007) Immune modulation and expression of cytokine genesin rainbow trout Oncorhynchus mykiss upon probiotic feeding. Dev CompImmunol31:372–382.
122. Park KJ, Kang MJ, Kim SH, Lee HJ, Lim JK, Choi SH, Park SJ&Lee KH(2004) Isolation and characterization of rpoS from a pathogenic bacterium,Vibrio vulnificus: role of σsin survival of exponential-phase cells under oxidativestress. J Bacteriol186:3304-3312.
123. Parks DH&Beiko RG (2010) Identifying biologically relevant differencesbetween metagenomic communities. Bioinformatics26:715-721.
124. Pawar KD, Banskar S, Rane SD, Charan SS, Kulkarni GJ, Sawant SS, Ghate HV,Patole MS&Shouche YS (2012) Bacterial diversity in different regions ofgastrointestinal tract of Giant African snail (Achatina fulica). Microbiologyopen1:415-426.
125. Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, Fuste B, Planas JV,Beltran S, Novoa B&Figueras A (2012) High-throughput sequence analysis ofturbot (Scophthalmus maximus) transcriptome using454-pyrosequencing for thediscovery of antiviral immune genes. PLoS One7: e35369.
126. Pérez-Losada M, Maigualida R, Jonathon CM, Jorge D (2009) Phylogeneticassessment of the earthworm Aporrectodea caliginosa species complex(Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences.Mol Phylogenet Evol52(2):293–302.
127. Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I,Merrifield DL&Ruiz-Zarzuela I (2011) Identification and characterization oflactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss(Walbaum), with inhibitory activity against Lactococcus garvieae. J Fish Dis34:499-507.
128. Pérez T, Balcázar JL, Ruiz-Zarzuela I, Halaihel N, Vendrell D, de Blas I&Múzquiz JL (2010) Host–microbiota interactions within the fish intestinalecosystem. Mucosal Immunol3:355-360.
129. Petrosino JF, Highlander S, Ann Luna R, Gibbs RA&Versalovic J (2009)Metagenomic pyrosequencing and microbial identification. Clin Chem55(5):856-866.
130. Picchietti S et al.(2009) Early treatment with Lactobacillus delbrueckii straininduces an increase in intestinal T-cells and granulocytes and modulatesimmune-related genes of larval Dicentrarchus labrax (L.). Fish ShellfishImmunol26:368–376.
131. Pond MJ, Stone DM, Alderman DJ (2006) Comparison of conventional andmolecular techniques to investigate the intestinal microflora of rainbow trout(Oncorhynchus mykiss). Aquaculture261:194–203.
132. Pope PB, Mackenzie AK, Gregor I, Smith W, Sundset MA, McHardy AC,Morrison M&Eijsink VG (2012) Metagenomics of svalbard reindeer rumenmicrobiome reveals abundance of polysaccharide utilization loci. PLoS One7:e38571.
133. Powell MD, Clark GA (2004) Efficacy and toxicity of oxidative disinfectants forthe removal of gill amoebae from the gills of amoebic gill disease affectedAtlantic salmon (Salmo salar L.) in freshwater. Aquac Res35:112-123.
134. Press CM&Evensen O (1999) The morphology of the immune system in teleostfishes. Fish Shellfish Immunol9:309–318.
135. Qu A, Brulc JM, Wilson MK et al.(2008) Comparative metagenomics revealshost specific meravirulomes and horizontal gene transfer elements in the chickencecum microbiome. PLoS One3: e2945.
136. Ramirez RF&Dixon BA (2003) Enzyme production by obligate intestinalanaerobic bacteria isolated from oscars (Astronotus ocellatus), angelfish(Pterophyllum scalare) and southern flounder (Paralichthys lethostigma).Aquaculture227:417–426.
137. Rappe MS, Connon SA, Vergin KL&Giovannoni SJ (2002) Cultivation of theubiquitous SAR11marine bacterioplankton clade. Nature418:630–633.
138. Ravi AV, Musthafa KS, Jegathammbal G, Kathiresan K&Pandian SK (2007)Screening and evaluation of probiotics as a biocontrol agent against pathogenicVibrios in marine aquaculture. Lett Appl Microbiol45(2):219-223.
139. Razin S, Yogev D&Naot Y (1998) Molecular biology and pathogenicity ofmycoplasmas. Microbiol Mol Biol Rev62:1094–1156.
140. Reyes-Becerril M et al.(2008) Oral delivery of live yeast Debaryomyceshansenii modulates the main innate immune parameters and the expression ofimmune-relevant genes in the gilthead seabream (Sparus aurata L.). FishShellfish Immunol25:731–739.
141. Ring E, Str m E&Tabachek JA (1995) Intestinal microfl ora of salmonids: areview. Aquacult Res26:773–789.
142. Ring E&Birkbeck TH (1999) Intestinal microflora of fish larvae and fry.Aquaculture Research30:73–93.
143. Ring E, Olsen RE, Mayhew TM&Myklebust R (2003) Electron microscopy ofthe intestinal microflora of fish. Aquaculture227:395–415.
144. Roediger WE&Macfarlane GT (2002) A role for intestinal mycoplasmas in theaetiology of Crohn’s disease? J Appl Microbiol92:377-381.
145. Ruoff KL (1991) Nutritionally variant streptococci. Clin Microbiol Rev4:184–190.
146. Russo R, Curtis EW&Yanong RP (2007) Preliminary investigations ofhydrogen peroxide treatment of selected ornamental fishes and efficacy againstexternal bacteria and parasites in green swordtails. J Aquat Anim Health19:121-127.
147. Sait M, Hugenholtz P&Janssen PH (2002) Cultivation of globally distributedsoil bacteria from phylogenetic lineages previously only detected incultivation-independent surveys. Environ Microbiol4:654–666.
148. Salinas I et al.(2008) Monospecies and multispecies probiotic formulationsproduce different systemic and local immunostimulatory effects in the giltheadseabream (Sparus aurata L.). Fish Shellfish Immunol25:114–123.
149. Salzman NH, de Jong H, Paterson Y, Harmsen HJ, Welling GW, et al.(2002)Analysis of16S libraries of mouse gastrointestinal microflora reveals a largenew group of mouse intestinal bacteria. Microbiology148:3651–3660.
150. Savage DC (1977) Microbial ecology of the gastrointestinal tract. Ann RevMicrobiol31:107–133.
151. Schloss PD&Handelsman J (2005) Introducing DOTUR, a computer programfor defining operational taxonomic units and estimating species richness. ApplEnviron Microbiol71:1501-1506.
152. Schrijver RD&Ollevier F (2000) Protein digestion in juvenile turbot(Scophthalmus maximus) and effects of dietary administration of Vibrioproteolyticus. Aquaculture186:107-116.
153. Secombes CJ et al.(2001) Cytokines and innate immunity of fish. Dev CompImmunol25:713–723.
154. Senderovich Y, Izhaki I&Halpern M (2010) Fish as reservoirs and vectors ofVibrio cholerae. PLoS One5: e8607.
155. Sha Z, Wang S, Zhuang Z, Wang Q, Wang Q, Li P, Ding H, Wang N, Liu Z,Chen S (2010) Generation and analysis of10,000ESTs from the half-smoothtongue sole Cynoglossus semilaevis and identification of microsatellite and SNPmarkers. J Fish Biol76(5):1190–1204.
156. Shigematsu T, Hayashi M, Kikuchi I, Ueno S, Masaki H&Fujii T (2009) Aculture-dependent bacterial community structure analysis based on liquidcultivation and its application to a marine environment. FEMS Microbiol Lett293(2):240-247.
157. Silphaduang U, Colorni A&Noga EJ (2006) Evidence for widespreaddistribution of piscidin antimicrobial peptides in teleost fish. Dis Aquat Org72:241–252.
158. Sipkema D, Schippers K, Maalcke WJ, Yang Y, Salim S&Blanch HW (2011)Multiple approaches to enhance the cultivability of bacteria associated with themarine sponge Haliclona (gellius) sp. Appl Environ Microbiol77(6):2130-2140.
159. Smriga S, Sandin SA&Azam F (2010) Abundance, diversity, and activity ofmicrobial assemblages associated with coral reef fish guts and feces. FEMSMicrobiol Ecol73:31-42.
160. Song J, Oh H-M&Cho J-C (2009) Improved culturability of SAR11strains indilution-to-extinction culturing from the East Sea, West Pacific Ocean. FEMSMicrobiol Lett295:141–147.
161. Staley JT&Konopka A (1985) Measurement of in situ activities ofnonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu RevMicrobiol39:321–346.
162. Stingl U, Cho JC, Foo W, Vergin KL, Lanoil B&Giovannoni SJ (2008)Dilution-to-extinction culturing of psychrotolerant planktonic bacteria frompermanently ice-covered lakes in the McMurdo Dry Valleys, Antarctica. MicrobEcol55:395–405.
163. Su C, Lei L, Duan Y, Zhang KQ&Yang J (2012) Culture-independent methodsfor studying environmental microorganisms: methods, application, andperspective. Appl Microbiol Biotechnol93:993-1003.
164. Sugita H, Shibuya K, Hanada H&Deguchi Y (1997) Antibacterial abilities ofintestinal microflora of the river fish. Fish Sci63:378–383.
165. Sun K, Zhang WW, Hou JH&Sun L (2009) Immunoprotective analysis ofVhhP2, a Vibrio harveyi vaccine candidate. Vaccine27:2733-2740.
166. Suthienkul O, Ishibashi M, Iida T, Nettip N, Supavej S, Eampokalap B, MakinoM&Honda T (1995) Urease production correlates with possession of the trhgene in Vibrio parahaemolyticus strains isolated in Thailand. J Infect Dis172:1405–1408.
167. Tamura K, Dudley J, Nei M&Kumar S (2007) MEGA4: Molecular evolutionarygenetics analysis (MEGA) software version4.0. Mol Biol Evol24:1596-1599.
168. Tanaka Y, Hanada S, Manome A, Tsuchida T, Kurane R, Nakamura K&Kamagata Y (2004) Catellibacterium nectariphilum gen. nov., sp. nov., whichrequires a diffusible compound from a strain related to the genus Sphingomonasfor vigorous growth. Int J Syst Evol Micr54:955–959.
169. Tapia-Paniagua ST, Chabrillón M, Díaz-Rosales P, de la Banda IG, Lobo C,Balebona MC&Mori igo MA (2010) Intestinal microbiota diversity of the flatfish Solea senegalensis (Kaup,1858) following probiotic administration. MicrobEcol60:310-319.
170. Thomas T, Gilbert J&Meyer (2012) Metagenomics–a guide from sampling todata analysis. Microbial Informatics and Experimentation2:3
171. Thompson PL, Iida T&Swings J (2004) Biodiversity of Vibrios. Microbiol MolBiol Rev68:403-431.
172. Timmerman HM, Koning CJM, Mulder L, Rombout FM&Beynen AC (2004)Monospecies, multistrain and multispecies probiotics: a comparison offunctionality and efficacy. Int J Food Microbiol96:219–233.
173. Tlaskalova-Hogenova H et al.(2005) Interaction of mucosal microbiota with theinnate immune system. Scand J Immunol62:106–113.
174. Tripp HJ, Kitner JB, Schwalbach MS, Dacey JW, Wilhelm LJ&Giovannoni SJ(2008) SAR11marine bacteria require exogenous reduced sulphur for growth.Nature452:741–744.
175. Trust TJ&Sparrow RAH (1974) The bacterial flora in the alimentary tract offreshwater salmonid fishes. Canad J Microbiol20:1219–1228.
176. Tun HM, Brar MS, Khin N, Jun L, Hui RKH, Dowd SE&Leung FC (2012)Gene-centric metagenomics analysis of feline intestinal microbiome using454junior pyrosequencing. J Microbiol Methods88:369-376.
177. Urbanczyk H, Ast JC, Higgins MJ, Carson J&Dunlap PV (2007)Reclassification of Vibrio fischeri, Vibrio logei, Vibrio salmonicida and Vibriowodanis as Aliivibrio fischeri gen. nov., comb. nov., Aliivibrio logei comb. nov.,Aliivibrio salmonicida comb. nov. and Aliivibrio wodanis comb. nov. Int J SystEvol Microbiol57:2823-2829.
178. van Walraven L, Mollet FM, van Damme CJG, Rijnsdorp AD (2010)Fisheries-induced evolution in growth, maturation and reproductive investmentof the sexually dimorphic North Sea plaice (Pleuronectes platessa L.). J Sea Res64(1–2):85–93.
179. Vartoukian SR, Palmer RM&Wade WG (2010) Strategies for culture of‘unculturable’bacteria. FEMS Microbiol Lett309(1):1-7.
180. Vendrell D, Balcázar JL, Ruiz-Zarzuela I, de Blas I, Gironés O&Múzquiz JL(2006) Lactococcus garvieae in fish: a review. Comp Immunol Microbiol InfectDis29:177–198.
181. Vendrell D, Balcázar JL, de Blas I, Ruiz-Zarzuela I, Gironés O&Múzquiz JL(2008) Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis byprobiotic bacteria. Comp Immunol Microbiol Infect Dis31:337–345.
182. Vergin KL, Rappe′MS&Giovannoni SJ (2001) Streamlined method to analyze16S rRNA gene clone libraries. Bio Tech30:938–944.
183. Vine NG, Leukes WD&Kaiser H (2004) In vitro growth characteristics of fivecandidate aquaculture probiotics and two fish pathogens grown in fish intestinalmucus. FEMS Microbiol Lett231(1):145-152.
184. Wang X, Heazlewood SP, Krause DO, Florin THJ (2003) Molecularcharacterization of the microbial species that colonize human ileal and colonicmucosa by using16S rDNA sequence analysis. J Appl Microb95:508-520.
185. Wang Y, Hammes F, Boon N, Chami M&Egli T (2009) Isolation andcharacterization of low nucleic acid (LNA)-content bacteria. ISME J3:889–902.
186. Ward NL, Steven B, Penn K, Methé BA, Detrich WH3rd (2009)Characterization of the intestinal microbiota of two Antarctic notothenioid fishspecies. Extremophiles13(4):679-685.
187. Watve M, Shejval V, Sonawane C et al.(2000) The ‘K’ selected oligophilicbacteria: a key to uncultured diversity? Curr Sci78:1535–1542.
188. Weltzien FA, Andersson E, Andersen, Shalchian-Tabrizi K, Norberg B (2004)The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish(Pleuronectiformes). Comp Biochem Physiol A Mol Integr Physiol137(3):447–477.
189. Wu S, Gao T, Zheng Y, Wang W, Cheng Y&Wang G (2010) Microbial diversityof intestinal contents and mucus in yellow catfish (Pelteobagrus fulvidraco).Aquaculture303:1-7.
190. Wu S, Wang G, Angert ER, Wang W, Li W&Zou H (2012a) Composition,diversity, and origin of the bacterial community in grass carp intestine. PLoS One7: e30440.
191. Wyss C (1989) Dependence of proliferation of Bacteroides forsythus onexogenous N-acetylmuramic acid. Infect Immun57:1757–1759.
192. Yildiz FH&Visick KL (2009) Vibrio biofilms: so much the same yet sodiffierent. Trends Microbiol17:109-118.
193. Zengler K, Toledo G, Rappe M, Elkins J, Mathur EJ, Short JM&Keller M(2002) Cultivating the uncultured. P Natl Acad Sci USA99:15681–15686.
194. Zerbino DR&Birney E (2008) Velvet: algorithms for de novo short readassembly using de bruijn graphs. Genome Res18:821-829.
195. Zhu L, Wu Q, Dai J, Zhang S&Wei F (2011) Evidence of cellulose metabolismby the giant panda gut microbiome. PNAS108:17714-17719.
196. Zoetendal EG, Collier CT, Koike S, Mackie RI&Gaskins HR (2004) Molecularecological analysis of the gastrointestinal microbiota: A review. J Nutr134:456–472.
197. Zou J, Clark MS&Secombes CJ (2003) Characterisation, expression andpromoter analysis of an interleukin10homologue in the puffer fish, Fugurubripes Immunogenetics55:325–335.
198. Zou J, Mercier C, Koussounadis A&Secombes C (2007) Discovery of multiplebeta-defensin like homologues in teleost fish. Mol Immunol44:638–647.
199. Zu GZ, Yu WY&Li JN (2000) Diagnosis and epidemiological research onbacterial septicemia of grass carp Ctenopharyngodon idellus. FreshwaterFisheries30:35–37.
200. Handelsman J, Rondon MR, Brady SF, Clardy J&Goodman RM (1998)Molecular biological access to the chemistry of unknown soil microbes: a newfrontier for natural products. Chem Biol5(10): R245-249.
201. Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM,Solovyev VV, Rubin EM, Rokhsar DS&Banfield JF (2004) Communitystructure and metabolism through reconstruction of microbial genomes from theenvironment. Nature428(6978):37-43.
202. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D,Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW,Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H,Pfannkoch C, Rogers YH&Smith HO (2004) Environmental genome shotgunsequencing of the Sargasso Sea. Science304(5667):66-74.
203. Simon C, Daniel R (2011) Metagenomic analyses: past and future trends. ApplEnviron Microbiol77(4):1153-1161.
204. Wilmes P&Bond PL (2006) Metaproteomics: studying functional geneexpression in microbial ecosystems. Trends Microbiol14(2):92-97.
205. Gilbert JA, Field D, Huang Y, Edwards R, Li W, Gilna P&Joint I (2008)Detection of large numbers of novel sequences in the metatranscriptomes ofcomplex marine microbial communities. PLoS One3(8): e3042.
206. Beja O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP, JovanovichSB, Gates CM, Feldman RA, Spudich JL, Spudich EN&DeLong EF (2000)Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science289(5486):1902-1906.
207. Nicol GW&Schleper C (2006) Ammonia-oxidising Crenarchaeota: importantplayers in the nitrogen cycle? Trends Microbiol14(5):207-212.