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Analysis of Bacterial Diversity in the Intestine of Grass Carp (Ctenopharyngodon Idellus) Based on 16S Rdna Gene Sequences

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Analysis of Bacterial Diversity in the Intestine of Grass Carp (Ctenopharyngodon Idellus) Based on 16S Rdna Gene Sequences Aquaculture Research, 2010, 42,47^56 doi:10.1111/j.1365-2109.2010.02543.x Analysis of bacterial diversity in the intestine of grass carp (Ctenopharyngodon idellus) based on 16S rDNA gene sequences Shaofeng Han1,Yuchun Liu1,ZhigangZhou1,SuxuHe1,Yanan Cao1,PengjunShi1,BinYao1 & Einar Ring2 1Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China 2Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Troms,Troms, Norway Correspondence: B Yao and Z Zhou, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No.12 Zhongguancun South Street, Beijing 100081, China. E-mail: [email protected]; [email protected] Abstract Introduction In the current study, we assessed bacterial diversity Gastrointestinal (GI) microbiota participate in sev- in the gut content of pond-reared grass carp (Cteno- eral important physiological functions of the host, in- pharyngodon idellus), in the associated habitat envir- cluding digestion, development of the mucosal onments (pond water and sediment) and in the system, angiogenesis and protection against disease ingested food (commercial feed and the reed Phrag- (Macfarlane & Macfarlane 1997; Hooper, Midtvedt & mites australis) by analysing 16S rDNA sequences Gordon 2002). It is generally accepted that identi¢ca- from clone libraries. The highest bacterial diversity tion of the GI microbiota is undoubtedly important was observed in the gut content and was determined for understanding the functional mechanisms be- by the total number of operational taxonomic units, tween the microbes and the host (Go¤mez & BalcaŁ zar Shannon diversity index (H), Shannon equitability 2008). Di⁄culties in analysing the complexity of bac- index (EH), Coverage (Cgood) and rarefaction curves terial community using classic methods of cultiva- calculated from the 16S rDNA gene libraries. Our tion have necessitated the development of molecular data indicated that allochthonous gut microbes of methods. In order to overcome these problems, var- grass carp were distinctively di¡erent from the corre- ious methods such as denaturing gradient gel elec- sponding environmental microbes. The pairwise si- trophoresis (DGGE) (Muyzer, Waal & Uitterlinden milarity coe⁄cient (Cs) for microbe communities 1993), £uorescence in situ hybridization (Huber, between gut content and ingested food was higher Spanggaard, Appel, Rossen, Nielsen & Gram than for those between the gut content and habitats, 2004), temporal temperature-gradient electrophor- indicating that the allochthonous microbiota identi- esis (Navarrete, Magne, Mardones, Riveros, Opazo, ¢ed in the intestines of grass carp were phylogeneti- Suau, Pochart & Romero 2010) and clone libraries callycloser to those in the ingested food than to those (Kim, Brunt & Austin 2007; Brons & Elsas 2008; Na- in the habitat. Based on our study and previous varrete, Espejo & Romero 2009; Ward, Blaire, Penn, research, we suggest that the digesta of grass carp Methe¤& Detrich 2009) have been used in order to cir- harbours a microbiota phylogenetic core of Proteo- cumvent the need for microbial isolation. bacteria and Firmicutes and this observation The DGGE-based method is a useful tool for separ- deserves further investigations with respect to a po- ating gene fragments but has strict length limitations tential pool of probiotics to grass carp. (generally o500 bp) (Myers, Fischer, Lerman & Man- iatis 1985) and often fails to establish an exact identi- Keywords: grass carp, Ctenopharyngodon idellus, ¢cation of the fragments using the BLAST program intestinal bacterial diversity,16S rDNA (Altschul, Gish, Miller, Myers & Lipman 1990). In r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd 47 Intestinal bacterial diversity in grass carp SHanet al. Aquaculture Research, 2010, 42,47^56 addition, it only detects the dominant bacterial nose black bream (Megalobrama amblycephala)inthe species in the environments (Muyzer et al.1993). suburb of Nanjing City, Jiangsu Province, China. A The generation of 16S rDNA clone libraries that con- description of the pond as well as the ¢sh and pond tain near-full-length 16S rDNA sequences would water, sampling methods and sampling procedure is likely result in more precise sequence identi¢cation presented in Wang, Zhou, He, Liu, Cao, Shi, Yao and than sequences obtained from DGGE (Brons & Elsas RingÖ (2010). Twelve grass carp were randomly col- 2008). lected from the pond. Pond water and sediment sam- The grass carp (Ctenopharyngodon idellus)isaher- ples were collected from the same pond locations bivorous freshwater ¢sh of the Cyprinidae family and (n 5 4). Sediment samples were collected using a these ¢sh are widely cultivated for food in China. The mud dredger (VG, Beijing Purity Instruments, Beij- output was 44 million tonnes in 2008 and com- ing, China) and were pooled before analysis. In addi- prised 420% of the total freshwater-cultured ¢sh tion, feed samples ( 200 g) were obtained from the annual output (Ministry of Agriculture, China automatic feeder. Samples of the reed (Phragmites 2009). Members of the Cyprinidae family have also australis) available for ingestion by grass carp were been introduced to Europe and the United States for collected from four randomly chosen sites in the aquatic weed control (Chilton & Muoneke1983). pond. Samples were stored on ice for transport to the During the last three decades, some papers have lab and then kept at À 20 1C until analysis. After ex- been published in which the gut microbiota of grass amining all ¢sh (12), gut contents from six grass carp carp were identi¢ed using traditional methods such having identical gut fullness were used. The gut sam- as freshwater agar and some selective culture media ples chosen were visually full of food ingested and the (Trust, Bull, Currie & Buckley 1979; Zhou, Chen, digesta were gently squeezed out under sterile condi- Zhang & Chen 1998; Luo, Chen & Cai 2001; He, tions and pooled before analysis. Pooled samples Zhang, Xie, Hao,Wang & He 2008). Recently, Huang, were used to avoid erroneous conclusions due to in- Shi,Wang, Luo, Shao,Wang,Yang and Yao (2009) stu- dividual variations in gut microbiota as described died the intestinal bacterial community of grass carp elsewhere (Spanggaard, Huber, Nielsen, Nielsen, Ap- by PCR ampli¢cation of the V3 region of 16S rDNA pel & Gram 2000; He, Zhou, Liu, Shi, Yao, RingÖ & and by DGGE; to our knowledge, a 16S rDNA clone Yo o n 20 09). library has not been generated for the identi¢cation of grass carp gut microbiota, however. The diversity of the GI microbiota of ¢sh is in£u- DNA extraction enced by environmental factors such as ingested food and habitat (Sugita, Oshima,Tamura & Deguchi1983; Total DNAwas extracted from 5 g sediment or feed as Nieto, Toranzo & Barja 1984). However, the correla- described by Tsai and Olson (1991). Extracted DNA tion between gut microbiota and its corresponding was puri¢ed using the Gel Cycle-Pure DNA kit environmental microbiota is per se not fully under- (Takara, Tokyo, Japan) according to the manufac- stood, and whether the grass carp gut harbours a mi- turer’s instructions and used as template DNA for crobiota phylogenetic core (the common phyla PCR ampli¢cation. DNA was extracted from pond within the gut contents of grass carp from di¡erent water as described elsewhere (Gernert, Gl˛ckner, backgrounds) has not been addressed. In the present Krohne & Hentschel 2005). DNA was extracted from study, we identi¢ed the allochthonous intestinal mi- ¢sh gut content using the hexadecyltrimethylammo- crobiota of the grass carp by generating a 16S rDNA nium bromide (CTAB) method, which involves a step library comprised of sequences from samples of grass of suspending the samples in CTAB extraction bu¡er carp gut content, the associated habitat (pond water (Thakuria, Schmidt, Mac Siu¤rtaŁ in, Egan & Doohan and sediment) and the ingested food (commercial 2008). To obtain reed DNA, 5 g reed sample was cut feed and natural food). into small pieces, transferred to a sterile triangular £ask containing 20mL PBS bu¡erand10glass beads (0.5 cm diameter) and then agitated at 4 g for 30 min. Materials and methods The mixture was allowed to settle for10min, and the supernatant was transferred into a sterile tube and Sample preparation centrifuged at 14000 Â g for 15min at 4 1C. Total Grass carp was raised in a poly-culture pond of grass DNA was extracted from the precipitate using the carp, gibel carp (Carassius auratus gibelio)andblunt- DNA extraction kit (Takara). r 2010 The Authors 48 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42,47^56 Aquaculture Research, 2010, 42, 47^56 Intestinal bacterial diversity in grass carp SHanet al. PCR ampli¢cation and 16S rDNA library study, the bacterial communities with a pairwise si- construction milarity coe⁄cient (Cs: the measure of the similarity of two samples by UPGMA) o0.60 were regarded as Universal primers 27f and 1492r (Martin-Laurent, di¡erent, those with 0.60 Cso0.80 were consid- Philippot, Hallet, Chaussod, Germon, Soulas & Ca- ered to be marginally di¡erent and those with troux 2001), which anneal at nucleotide positions C 0.80 were considered to be similar (Wang et al. 8^27 and 1492^1513 of the 16S rDNA gene (Escheri- s 2010). Rarefaction curves were created using the spe- chia coli numbering), respectively, were
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