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1 2016 5, 23(3): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 孙振丽 1, 宣引明 2, 张皓 1, 蒋明 2, 潘云生 2, 张益明 2, 龚叶平 2, 陆小平 2, 余德山 2, 薛仁宇 1, 胡小龙 1, 曹广力 1 1, 贡成良 1., ; 2., 摘要 : (Penaeus vannamei Boone), Illumina miseq, 16S rrna,, ,, 90 3, , 1% ;, 23.08% 37.13% 42.22%, (Pseudomonas)(5.77%) Ohtaekwangia(4.79%) (Clostridium)(3.88%); (Sediminibacterium)(6.64%) Spartobacteria genera incertae sedis (3.95%) GPIIa(3.20%), (Pseudomonas)(14.57%)(Acinetobacter)(6.54%) 3 6 6, (5.77%) (Flavobacterium) (0.77%) (14.57%) 8 6 7, 5 3, 20 (OUT), : ; ; ; ; 中图分类号 : S966 文献标志码 : A 文章编号 : (2016) (Penaeus vannamei Boone),,,,,, [1 2],, Yasuda [3], 收稿日期 : ; 修订日期 : 基金项目 : (D , D ). 作者简介 : (1990 ),,. zhenlisun@163.com 通信作者 :,,. gongcl@suda.edu.cn

2 3 : 595, [4] Gatesoupe [5], [6], 111,, [7] 16S rdna, 12,, (Firmicutes) (Proteobacteria), 75.4% 24.6% [8] (DGGE) 12,,, Illumina miseq 16S rrna,,, (30 21 N E) (: 0.74 hm 2, : 1.7~2 m),,,, 5 (11 cm, 7.0 g; 10 cm, 5.2 g; 11 cm, 7.3 g; 12.5 cm, 11.3 g; 12.5 cm, 10.7 g), 80 ( 5 m 0.5 m) ( 5 cm), 80 3 L (TPE A, ), ( 0.45 μm, ), , GB/T ph (, FE20K) ph, MERCK (COD) 1.3 DNA PCR 1 g 1, E.Z.N.A. Soil DNA Kit (Omega Biotek, D ) DNA, 1% DNA, (Qubit 2.0 fluorometer) DNA, 10 ng/μl 16S rrna, (MSQ341F-91: CCTACACGAC GCTCTTCCGATCTN (barcode) CCTACGGGNGG CWGCAG, Miseq-805R: GACTGGATTCCTTGGC ACCCGAGAATTCCAGACTACHVGGGTATCTAA TCC) 16S rrna V1~V3 50 μl, 10 ng DNA, 5 μl 10 PCR buffer, 0.5 μl dntps (10 mmol/l), 0.5 μl Plantium Taq (5 U/μL), 0.5 μl V1F (50 μmol/l), 0.5 μl V3R2 (50 μmol/l); PCR 94, 3 min; 94, 30 s, 5 ; 45, 20 s; 65, 30 s; 94, 20 s, 20 ; 55, 20 s; 72, 30 s; 72, 5 min, DNA 25 ng/μl, 1.. 1, Illumina miseq ( ) 1.4 barcode, [9] LUCY (version 1.2) [10], 50 bp

3 ; Uchime silva 97% (operational taxonomic units, OTUs), (ribosomal database project, RDP) OTUs [11] Alpha (Chao1, ACE ), Shannon-Wiener [12] ( Origin ; Mega6.06 Neighbor-Joining ( ) ( 1),, aquaculture species Penaeus vannamei Boone /hm 2 square meter ph / temperature of water 表 1 采样地点水质指标 Tab. 1 Quality indexes of sampled water /(mg ml 1 nitrate ) /(mg ml 1 ) nitrogen ammonia /(mg ml 1 sulphide ) /(mg ml 1 ) total nitrogen /(mg ml 1 ) total phosphorus COD/(mg ml 1 ) chemical oxygen demand ( 2), ACE Chao1, Shannon,, sample Shannon Shannon index 表 2 各样品多样性指数 Tab. 2 Diversity indexes of each sample ACE ACE index Chao1 Chao1 index SS WS PB SS: ; WS: ; PB:. SS: sediment sample; WS: water sample; PB: Penaeus vannamei Boone intestinal sample Illumina miseq 16S rrna, reads, 97% OTUs, RDP 3,, reads OTUs,,, ,, 17 3 ( 1a),

4 3 : 597 表 3 底泥 (SS) 水体(WS) 及南美白对虾肠道 (PB) 细菌菌群的基本构成 Tab. 3 Composition of bacterial communities in sediment(ss), water(ws) and gut of Penaeus vannamei Boone (PB) sample reads valid reads hylum class order family genus taxon (OTUs) SS WS PB (Nitrospira) (Crenarchaeota) Elusimicrobia OP11 (Lentisphaerae) WS3 6 ; (Deinococcus-Thermus), 3 17, (Armatimonadetes), 3 17 ( 1b), (Proteobacteria)(41.6%) (Bacteroidetes)(24.44%) (Firmicutes)(9.09%);, (25.36%) (Actinobacteria) (23.79%) (Proteobacteria)(22.94%);, 1 A. (SS) (WS)(PB) venn ; B Fig. 1 Structure and distribution of bacterial communities at the phylum level in sediment, water and gut of Penaeus vannamei Boone A. Veen analysis of bacteria at phylum level in sediment (SS), water (WS) and Penaeus vannamei Boone intestine (PB); B. Percentages of all 17 phyla in each sample.

5 (34.80%)(25.74%) (18.89%) 3 1% (Cyanobacteria/Chloroplast) (Verrucomicrobia) TM7,, (Chloroflexi) OD1 (Acidobacteria) 1%; (Planctomycetes) (Actinobacteria) 1%, , 34, 35, 33, 32 3 ;, , 47, 45, 46, 3 41 ;, ,, 90 3, ( 2), >1%, 2 (SS) (WS)(PB) Venn Fig. 2 Veen analysis of bacteria at class, order, family and genus level in sediment (SS), water (WS) and Penaeus vannamei Boone intestine (PB) 3, 3, , 3 (23.08%, 37.13%, 42.22%) TM7(2.24%, 1.03%, 2.57%);, ; 2,, (Pseudomonas) 5.77% 14.57%, (Acinetobacter) 1.26% 6.54%; 2 Sediminibacterium Spartobacteria, Sediminibacterium (Spartobacteria) 6.64% (3.95%) 3.70% (1.42%) 3,, (5.77%) Ohtaekwangia(4.79%); (Sediminibacterium) (6.64%) Spartobacteria(3.95%); (14.57%) (6.54%) 3, 2.4, 6,, 3 6 6, (5.77%);, (Flavobacterium) (0.77%); 14.57% (Aeromonas)(Vibrio) (Rickettsia), 3

6 3 : (SS) (WS)(PB) (>1%) Fig. 3 Composition and distribution proportion of predominant genera in sediment(ss), water(ws) and Penaeus vannamei Boone intestine (PB) 8, 6 7, (Acetobacter) (Lactobacillus), (Rhodopseudomonas) (Streptococcus)(0.73%) (Bdellovibrio) (0.18%) (Lactobacillus) (1.01%), ( 4)

7 Tab. 4 表 4 南美白对虾肠道及养殖环境中益生菌致病菌所占样品细菌总数的百分比 percentages of probiotic and pathogenic bacteria number in Penaeus vannamei Boone intestine and their aquaculture environment samples % bacterial kind sediment water intestine pathogenic bacteria Pseudomonas Flavobacterium Escherichia Aeromonas Vibrio Rickettsia probiotic Acetobacter Bacillus Bacteroides Bdellovibrio Lactobacillus Lactococcus Rhodopseudomonas Streptococcus :. Note: representing there is no this genera in sample OTU ( 4),, 20 OTU (I II), (I-1 I-2 II-1 II-2), I-1 (I-1-1 I-1-2) I-1-1, (Sphingobacteria) 8 OTU, 4, 3, 1 ; I-1-2 (Actinobacteria), 7 OTU ; I-2 (Proteobacteria), β (Betaproteobacteria), 2 OTU, 1 OTU; 18 OTU γ (Gammaproteobacter), 11 OTU, 7 OTU, 20 OTU γ ; II-1, (Clostridia) 3 OTU ; 20 OTU TM7 (Actinobacteria) (Planctomycetacia) Proteobacteria, (Flavobacteria) ε- (Epsilonproteobacteria) 20 OTU ; 20 OTU OTU(Bacteria), 20 OTU 20 OTU, (Flavobacterium) (Vibrio), (Lactococcus); 20 OTU, ; 20 OTU 3, [13]

8 3 : OTU( 20) SS: ; WS: ; PB: ;,,. Fig. 4 Cluster analysis of top 20 OTUs (which relative abundance located top 20 in each sample) in Penaeus vannamei Boone intestine and their aquaculture environment SS: sediment; WS: water; PB: Penaeus vannamei Boone intestine. Classes and genera of bacteria were indicated, phyla were indicated for which had not been classified to class, classes were indicated for which had not been classified to genera.

9 602 23,,,, ;,,,,, 16S rrna [14] (Penaeus chinensis) 47, 8 [6] 111, 13 ( ) [7] 16S rrna, 2 : (Proteobacteria) (Firmicutes), 206, 16S rrna,, Tang [15] 454- (Litopenaeus vannamei), (Actinobacteria) (Proteobacteria) (Bacteroidetes), 53.5% 22.4% 18.8% 4.32%, (34.80%) (25.74%) (18.89%), 2, 2,,, 20%,, 42.22%,,, [16],,, [16 20], 6,, 2,,, 14.57%,, 6, 2.98%, (1.01%) (0.93%)(0.49%) (0.37%), (Streptococcus thermophilus) [21] ; ph, ph,, [22],,

10 3 : 603,, [16, 23 25] (Penaeus monodon) (Luminescent vibrios ) [16 17], ,,,,,,, ;,,, OTU, 20 OTU, γ, 20 OTU γ, Dokdonella Beggiatoa, ;, Agrococcus Actinomycetales Intras, 20 OTU ; (Sphingobacteria) β (Betaproteobacteria) OTU 3,,, 参考文献 : [1] Devaraja T N, Yusoff F M, Shariff M. Changes in bacterial populations and shrimp production in ponds treated with commercial microbial products[j]. Aquaculture, 2002, 206(3 4): [2] Verscuere L, Rombaut G, Sorgeloos P, et al. Probiotic bacteria as biological control agents in aquaculture[j]. Microbiol Mol Biol Rev, 2000, 64(4): [3] Yasuda K, Kitao T. Bacterial flora in the digestive tract of prawn, Penaeus japonicus Bate[J]. Aquaculture, 1980, 19(3): [4] Harris J M. The presence, nature and role of gut microflora in aquatic invertebrates: A synthesis[j]. Microb Ecol, 1993, 25(3): [5] Gatesoupe F J. The use of probiotics in aquaculture[j]. Aquaculture, 1999, 180(1 2): [6] Wan L, Wang J Q, Gao F, et al. Bacterial flora in intestines of white leg shrimp (Penaeus vannamei Booen)[J]. Fisheries Science, 2006, 25(1): [,,,. [J]., 2006, 25(1): ] [7] Li K, Zheng T L, Tian Y, et al. Bacterial community structure in intestine of the white shrimp, Litopenaeus vannamei[j]. Acta Microbiologica Sinica, 2007, 47(4): [,,,. [J]., 2007, 47(4): ] [8] Liu H D, Wang L, Wang B J, et al. Analysis on the diversity of microorganism in intestine of Penaeus vannamei-cultured by PCR-DGGE[J]. Feed Industry, 2008, 29(20): [,,,. PCR-DGGE [J]., 2008, 29(20): ] [9] Dennis P G, Guo K, Imelfort M, et al. Spatial uniformity of microbial diversity in a continuous bioelectrochemical system[j]. Bioresour Technol, 2013, 129: [10] Li S, Chou H H. Lucy2: an interactive DNA sequence quality trimming and vector removal tool[j]. Bioinformatics, 2004, 20(16): [11] Cole J R, Wang Q, Cardenas E, et al. The Ribosomal Database Project: improved alignments and new tools for rrna analysis[j]. Nucl Acid Res, 2009, 37: D [12] Motegi C N T, Miki T, Weinbauer M G, et al. Interactive

11 effects of viral and bacterial production on marine bacterial diversity[j]. PLoS ONE, 2013, 8(11): e [13] Yin J X, Shen W Y, Li P. Study on the influence of water temperature on the intestinal microflora of Penaeus vanmamei[j]. Marine Sciences, 2004, 28(5): [,,. [J]., 2004, 28(5): ] [14] Wang X H, Li H R, Zhang X H, et al. Flora in the digestive tract of adult penaeid shrimp (Penaeus chinensis)[j]. Journal of Qingdao Ocean University, 2000, 30(3): [,,,. [J]., 2000, 30(3): ] [15] Tang Y Y, Tao P Y. Identification of bacterial community composition in freshwater aquaculture system farming of Litopenaeus vannamei reveals distinct temperature-driven patterns[j]. Int J Mol Sci, 2014, 15(8): [16] Lavilla-Pitogo C R, Leaño E M, Paner M G. Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent Vibrios in the rearing environment[j]. Aquaculture, 1998, 164(1 4): [17] Sung H H, Hsu S F, Chen C K, et al. Relationships between disease outbreak in cultured tiger shrimp (Penaeus monodon) and the composition of Vibrio communities in pond water and shrimp hepatopancreas during cultivation[j]. Aquaculture, 2001, 192(2 4): [18] Thompson F L, Iida T, Swings J. Biodiversity of vibrios[j]. Microbiol Mol Biol Rev, 2004, 68(3): [19] Bourne D G, Young N, Webster N, et al. Microbial community dynamics in a larval aquaculture system of the tropical rock lobster, Panulirus ornatus[j]. Aquaculture, 2004, 242(1 4): [20] Zhou J F, Fang W H, Yang X L, et al. A nonluminescent and highly virulent Vibrio harveyi strain is associated with Bacterial White Tail Disease of Litopenaeus vannamei shrimp[j]. PLoS ONE, 2012, 7(2): e [21] Bai H. Health Food Ingredient Detection[M]. Beijing: China Traditional Medicine Press, 2011: 60. [. [M]. :, 2011: 60.] [22] Moriarty D J. Control of luminous Vibrio species in penaeid aquaculture ponds[j]. Aquaculture, 1998, 164(1 4): [23] Sun Z, Li G P, Wang C W, et al. Community dynamics of prokaryotic and eukaryotic microbes in an estuary reservoir[j]. Sci Rep, 2014, 4: [24] Boutin S, Bernatchez L, Audet C, et al. Network analysis highlights complex interactions between pathogen, host and commensal microbiota[j]. PLoS ONE, 2013, 8(12): e [25] Wu S G, Wang G T, Esther R, et al. Composition, diversity, and origin of the bacterial community in grass carp intestine[j]. PLoS ONE, 2012, 7(2): e30440.

12 3 : 605 Bacterial diversity in the Penaeus vannamei Boone intestine and aquaculture environment SUN Zhenli 1, XUAN Yinming 2, ZHANG Hao 1, JIANG Ming 2, PAN Yunsheng 2, ZHANG Yiming 2, GONG Yeping 2, LU Xiaoping 2, YU Deshan 2, XUE Renyu 1, HU Xiaolong 1, CAO Guangli 1, GONG Chengliang 1 1. School of Biology & Basic Medical Science, Soochow University, Suzhou , China; 2. Aquaculture Technical Extension Station of Kunshan City, Suzhou , China Abstract: To investigate the bacterial species composition and distribution in the intestine of Penaeus vannamei Boone and its aquacultural environment, we used Illumina MiSeq high-throughput pyrosequencing to analyze the composition of the bacterial communities in the gut of P. vannamei Boone and its aquacultural sediments and water, based on the 16S rrna sequence. We detected 206, 265, and 270 genera in the gut of P. vannamei Boone, the aquaculture sediment, and the water, respectively. Ninety genera occurred in all three samples, 146 genera in the sediment and water, 128 in the sediment and P. vannamei Boone intestine, and 119 in the water and P. vannamei intestine. This indicates the close interaction between the bacteria in the shrimp intestine and their aquacultural environment. Twenty, seventeen, and ten dominant genera with a relative abundance in the total sequences of > 1% occurred in the sediment, water, and P. vannamei intestine, respectively. Unclassified genera were the most abundant in all the samples, accounting for 23.08%, 37.13%, and 42.22% of the total sequences from the sediment, water, and P. vannamei, respectively. However, Pseudomonas (5.77%), Ohtaekwangia (4.79%), and Clostridium (3.88%) were the dominant genera in the sediment; Sediminibacterium (6.64%), Spartobacteria genera incertae sedis (3.95%), and GPIIa (3.20%) were the dominant genera in water; and the predominant genera in the Penaeus vannamei Boone intestine were Pseudomonas (14.57%) and Acinetobacter (6.54%). Three, three, and six potentially pathogenic bacterial genera were also observed in the sediment, water, and Penaeus vannamei Boone intestine, respectively, and the most abundant were Pseudomonas (5.77%), Flavobacterium (0.77%), and Pseudomonas (14.57%), respectively. Six, eight, and seven probiotic genera were also found in the sediment, water, and Penaeus vannamei Boone intestine, respectively, and among these genera, five were shared by all three samples. A cluster analysis showed that the 20 most abundant operational taxonomic units in each sample (sediment, water, and Penaeus vannamei Boone intestine) differed significantly. These results provide insight into the interactions between the microbiota in the intestine of P. vannamei Boone and those in its aquacultural environment, and provide new data for the development of probiotics. Key words: Penaeus vannamei Boone; aquaculture water; sediment; intestine; bacterial community Corresponding author: GONG Chengliang. gongcl@suda.edu.cn

2 : 111 ( Pinctada maxima), 13, 11,,,,,, 15, 25 cm, cm, 4 5 kg 1-2],,, 3-7] 24] 25-26],,, ] min, 1, 5 m 4 m, 20 m , 9-22],,, (

2 : 111 ( Pinctada maxima), 13, 11,,,,,, 15, 25 cm, cm, 4 5 kg 1-2],,, 3-7] 24] 25-26],,, ] min, 1, 5 m 4 m, 20 m , 9-22],,, ( 12 2 2 0 1 6 4 South China Fisheries Science Vol. 12, No. 2 Apr., 2016 doi: 10. 3969/ j. issn. 2095-0780. 2016. 02. 016,,,, (,,, 510300) : ( Pinctada maxima),,,, 54. 55%, 0 0. 25 m - 2, 0 70. 31 gm - 2,