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1 47 1 Vol.47, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2016 * ( ) (Apostichopus japonicus),, 3, , (OTUs) : (1), 3 ; (2) 9 26,, 17 ; (3),, 50.54% 99.91% ; ; S917.1 doi: /hyhz (Apostichopus japonicus), (Bordbar et al, 2011; Purcell et al, 2012),,,, (Wang et al, 2005), (, 2006; Deng et al, 2009; Li et al, 2010a), (Paerl et al, 2003; Luo et al, 2006),,,, (Amann et al, 1995; Hugenholtz et al, 2009),, (Rosselló-Mora et al, 2001; Auguet et al, 2009),, 16S rdna,, 16S rrna, PCR,, (Youssef et al, 2009; Caporaso et al, 2011), (2010) (2010) (2014), DGGE DGGE,, ;, *, ,, yandou1989@126.com :,,, dingjun1119@dlou.edu.cn : , :

2 1 : S rrna,,, 16S rrna,,,, (Bell et al, 2013; Ren et al, 2015; Yang et al, 2015), Miseq ( ) ( ),, ( ) ( 1) 40cm 1L, 2 5cm 100g(, 2010), 表 1 样品标记信息 Tab.1 Labels of samples DJ1 DJ2 DJ9 DJ5 DJ6 DJ11 DJ12 DJ13 DJ DNA 0.22μm DNA OMEGA DNA (OMEGA Water DNA Kit, D5525) DNA (OMEGA Soil DNA Kit, D5625) 16S rrna V3-V4 : 341F (5 -CCTACGGGNG GCWGCAG-3 ) 805R (5 -GACTACHVGGGTATCTA ATCC-3 ) ( ) Illumina Miseq 1.3,, : : (1) Prinseq (PRINSEQ-lite ) 3, ; (2) Flash (FLASH v1.2.7), barcode, ; (3) Prinseq (PRINSEQ-lite ) : (1) Mothur (Schloss et al, 2009) Pre.cluster, 1/150; (2) SILVA, UCHIME (Edgar et al, 2011), 1.4 UCLUST (UCLUST v ) 97% OTUs (Operational Taxonomic Units, OTUs) Mothur Alpha, (Richness) ACE (ACE Index) Chao1 (Chaos1 Index) (Coverage) OTUs ( ) RDP classifier, , NCBI SRA, SRP052819, ( 2) 表 2 9 个样品中序列分析 Tab.2 Analysis of sequence from nine samples DJ DJ DJ DJ DJ DJ DJ DJ DJ

3 OTUs ( 1) 9 ACE Chao , DJ9 DJ11 DJ13 DJ14, DJ1 DJ2 DJ5 DJ6 DJ12 ( 2) 86% 97%, DJ9 DJ11 DJ13 DJ14, DJ9 DJ1 DJ2, DJ11 DJ5 DJ6, DJ14 DJ12 DJ13, DJ13, DJ2 DJ6, DJ13 DJ6 DJ2 1 OUT ACE Chao 1 Alpha Fig.1 The Alpha diversity of bacteria associated with samples, and the numbers of OTUs observed and predicted (ACE and Chao 1) Fig.2 2 Richness rarefaction plot from all samples 2.3, , Proteobacteria Bacteroidetes Chloroflexi Planctomycetes Cyanobacteria Acidobacteria Verrucomicrobia Firmicutes Actinobacteria ( 3), %, Caldiserica Archaea Tenericutes Fibrobacteres Synergistetes Nitrospira Elusimicrobia Armatimonadetes Chlamydiae - Deinococcus- Thermus Chlorobi Gemmatimonadetes Fusobacteria Lentisphaerae Aquificae Deferribacteres Spirochaetes DJ1 19, Bacteroidetes (52.03%), Proteobacteria (41.93%); DJ2 18, (63.90%), (24.50%); DJ9 (21 ), (58.05%), (12.20%); DJ5 (12 ), (88.21%), (10.89%); DJ6 15, (50.80%), (38.34%); DJ11 20, (59.92%), (18.92%); DJ12 15, (50.94%),

4 1 : 125 (46.16%); DJ13 20, (68.64%), (13.56%); DJ14 18, Cyanobacteria (41.07%), 36.96% 13.58%,,, 50.54% 99.91% Fig.3 3 Relative abundance of phylum-level bacterial communities in different samples 10 OUTs ( 3) 9 10 OTUs, DJ1 Cellulophaga (11.34%) Bacteroidetes (8.32%) Polaribacter (7.80% 1.24%) Glaciecola (6.66%) Rhodobacteraceae (3.96%) Psychroserpens (2.59% 1.71%) Proteobacteria (2.50%) Acinetobacter (1.47%); DJ2 (11.37%) Roseovarius (7.24%) Flavobacteriaceae (6.09% 2.70%) γ- Gammaproteobacteria (3.92% 2.14%) (3.18%) Agrococcus (2.89%) α- Aphlaproteobacteria (2.33%) Nisaea (2.17%); DJ9 Desulfopila (6.37%) γ- (2.68% 1.26%) Kofleria (2.53%) Sulfurovum (1.95%) Bacteria (1.82%) Pelobacter (1.69%) Bellilinea (1.47%) Desulfosarcina (1.14%) Actibacter (1.05%); DJ5 Vibrio (59.37% 1.20%) Sulfitobacter (3.91%) Bacteriovorax (2.64%) (2.62%) (2.37%) Glaciecola (2.12%) Flavobacteriales (0.91%) Tenacibaculum (0.83%); DJ6 (8.21% 7.81%) (5.56% 4.09%) Cyanobacteria (5.27%) (5.00%) Kordia (4.39%) Thalassobacter (4.29%) γ- (3.60%); DJ11 Sporacetigenium (7.31%) Desulfopila (3.34% 0.78%) Actibacter (2.67%) (2.48%) Loktanella (1.84%) Haliea (0.93% 0.85%) Neptunomonas (0.70%); DJ12 (25.48% 1.36%) (23.13% 2.78%) Glaciecola (3.07%) Lewinella (2.95%) Phaeobacter (1.92%) (1.05%) Kordia (0.84%) Thalassomonas (0.72%); DJ13 Alteromonas (2.63% 1.30%) Sedimenticola (2.54%) Euzebya (2.52%) Litoreibacter (2.50%) (1.92%) α- (1.09%) Nisaea (1.03%) Glaciecola (1.03%) Thalassobacter (0.96%); DJ14 (6.76% 0.64%) Loktanella (2.17%) Pseudoalteromonas (1.43%), DJ5 DJ12, 59.37% 1.20% 23.13% 2.78% 3 (Next Generation Sequence, NGS) 16S rrna [ (Woese, 1987)],,, (Wang et al, 2007) 454 (Herlemann et al, 2011; Xia et al, 2011) Illumina 454

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6 1 : 127,, Illumina,, Illumina Miseq 250bp 300bp (Jeon et al, 2015), (Bell et al, 2013; Yang et al, 2015) Illumina Miseq 3, , (OTUs), 9 (Li et al, 2010;, 2010;, 2010;, 2014), - 17, 9 1%, Illumina Miseq, 3 Lozupone (2007) (Li et al, 2010b;, 2015) γ- α- γ- Glaciecola γ-,,,,, (Ivanova et al, 2003; Payne et al, 2006), γ-, (Bakunina et al, 1999; Huber et al, 2004) α-,, CO 2 (, 2009), DNA, (Cottrell et al, 2000; O Sullivan et al, 2002),,, (, 1995), (Rosselló-Mora et al, 1999) 3,,, CO 2 O 2,, (, 2009),, (DJ14) 41.07%, (2006) (2014) V. splendidus V. tubiashii,,, (, 2013) (2007) 1, (2007) V. alginolyticus (2006), Deng (2009) 8,, Photobacterium Arthrobacter Staphylococcus, 4,,, (Thompson et al, 2004; Thompson et al, 2005),,,,,,,,

7 ,,,,,,, 3,,,,,, , 30(3): ,,, , 13(6): ,,, PCR-DGGE., 31(3): ,,, , 49(3): ,,, S rdna (Apostichopus japonicus)., 46(1): ,,, PCR-DGGE., (1): 82 88,,, , 33(9): ,,, , 10(4): 45 51,,, RH2., 31(4): ,,, , 19(4): ,,, (Apostichopus japonicus)., 44(3): ,,, , 30(1): Amann R I, Ludwig W, Schleifer K H, Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59(1): Auguet J-C, Barberan A, Casamayor E O, Global ecological patterns in uncultured Archaea. The International Society for Microbial Ecology Journal, 4(2): Bakunina I, Shevchenko L S, Nedashkovskaia O I et al, Screening of marine bacteria for fucoidan hydrolases. Mikrobiologiia, 69(3): Bell T H, Yergeau E, Maynard C et al, Predictable bacterial composition and hydrocarbon degradation in Arctic soils following diesel and nutrient disturbance. The International Society for Microbial Ecology Journal, 7(6): Bordbar S, Anwar F, Saari N, High-value components and bioactives from sea cucumbers for functional foods A review. Marine Drugs, 9(12): Caporaso J G, Lauber C L, Walters W A et al, Global patterns of 16S rrna diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America, 108(S1): Cottrell M T, Kirchman D L, Natural assemblages of marine proteobacteria and members of the Cytophaga- Flavobacter cluster consuming low- and high-molecularweight dissolved organic matter. Applied and Environmental Microbiology, 66(4): Deng H, He C B, Zhou Z C et al, Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. Aquaculture, 287(1 2): Edgar R C, Haas B J, Clemente J C et al, UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27(16): Herlemann D P, Labrenz M, Jürgens K et al, Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. The International Society for Microbial Ecology Journal, 5(10): Huber I, Spanggaard B, Appel K F et al, Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum). Journal of Applied Microbiology, 96(1): Hugenholtz P, Hooper S D, Kyrpides N C, Focus: Synergistetes. Environmental Microbiology, 11(6): Ivanova E P, Sawabe T, Zhukova N V et al, Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean. Systematic and Applied Microbiology, 26(2): Jeon Y-S, Park S-C, Lim J et al, Improved pipeline for reducing erroneous identification by 16S rrna sequences using the Illumina Miseq platform. Journal of Microbiology, 53(1): Li H, Qiao G, Li Q et al, 2010a. Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus. Journal of Fish Diseases, 33(11): Li Q F, Zhang Y, Juck D et al, 2010b. Phylogenetic analysis of bacterial communities in the shrimp and sea cucumber aquaculture environment in northern China by culturing and

8 1 : 129 PCR-DGGE. Aquaculture International, 18(6): Lozupone C A, Knight R, Global patterns in bacterial diversity. Proceedings of the National Academy of Sciences of the United States of America, 104(27): Luo P, Hu C Q, Xie Z Y et al, PCR-DGGE analysis of bacterial community composition in brackish water Litopenaeus vannamei culture system. Journal of Tropical Oceanography, 25(2): O Sullivan L A, Weightman A J, Fry J C, New degenerate Cytophaga-Flexibacter-Bacteroides-specific 16S ribosomal DNA-targeted oligonucleotide probes reveal high bacterial diversity in River Taff epilithon. Applied and Environmental Microbiology, 68(1): Paerl H W, Dyble J, Moisander P H et al, Microbial indicators of aquatic ecosystem change: current applications to eutrophication studies. FEMS Microbiology Ecology, 46(3): Payne M S, Hall M R, Bannister R et al, Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus). Aquaculture, 258(1 4): Purcell S W, Hair C A, Mills D J, Sea cucumber culture, farming and sea ranching in the tropics: progress, problems and opportunities. Aquaculture, : Ren G D, Ren W J, Teng Y T et al, Evident bacterial community changes but only slight degradation when polluted with pyrene in a red soil. Frontiers in Microbiology, 6: 22 Rosselló-Mora R, Aman R, The species concept for prokaryotes. FEMS Microbiology Reviews, 25(1): Rosselló-Mora R, Thamdrup B, Schäfer H et al, The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions. Systematic and Applied Microbiology, 22(2): Schloss P D, Westcott S L, Ryabin T et al, Introducing mothur: open-source, platform-independent, communitysupported software for describing and comparing microbial communities. Applied and Environmental Microbiology, 75(23): Thompson F L, Iida T, Swings J, Biodiversity of vibrios. Microbiology and Molecular Biology Reviews, 68(3): Thompson J R, Pacocha S, Pharino C et al, Genotypic diversity within a natural coastal bacterioplankton population. Science, 307(5713): Wang Q, Garrity G M, Tiedje J M et al, Naïve Bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 73(16): Wang Y G, Zhang C Y, Rong X J et al, Diseases of cultured sea cucumber, Apostichopus japonicus, in China. FAO Fisheries Technical Paper, Woese C R, Bacterial evolution. Microbiological Reviews, 51(2): Xia W W, Zhang C X, Zeng X W et al, Autotrophic growth of nitrifying community in an agricultural soil. The International Society for Microbial Ecology Journal, 5(7): Yang Y Y, Wang Z, He T et al, Sediment Bacterial Communities Associated with Anaerobic Biodegradation of Bisphenol A. Microbial Ecology, 70(1): Youssef N, Sheik C S, Krumholz L R et al, Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rrna gene-based environmental surveys. Applied and Environmental Microbiology, 75(16): APPLICATION OF HIGH-THROUGHPUT SEQUENCING FOR ANALYZING BACTERIAL COMMUNITIES IN EARTHEN PONDS OF SEA CUCUMBER AQUACULTURE IN NORTHERN CHINA DOU Yan, ZHAO Xiao-Wei, DING Jun, HE Peng (Key Laboratory of Mariculture & Stock Enhancement in North China s Sea, Ministry of Agriculture, Dalian Ocean University, Dalian , China) Abstract Sea cucumber Apostichopus japonicus has become an economically valuable holothurian species because of their anti-tumor properties and good nutritional values. We applied high-throughput sequencing to explore bacterial community in earthen aquaculture ponds of the sea cucumber in Dalian, North China. For nine samples collected, the number of effective sequences ranged , and they could be classified into operational taxonomic units. Results show great bacterial diversities in the aquaculture. The estimated richness and diversity indices were higher in sediment samples than those in water samples. In total, 26 phyla were identified, among which Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, Cyanobacteria, Acidobacteria, Verrucomicrobia, Firmicutes, and Actinobacteria were commonly reported, and other 17 were firstly detected and rarely reported. Despite the differences in microbe composition, Proteobacteria and Bacteroidetes (50.54% 99.91%) were the dominant phyla in all samples. Key words sea cucumber Apostichopus japonicus; high-throughput sequencing; bacterial communities