Diversity of autochthonous bacterial communities in the intestinal ...

Aquaculture Research, 2015, 46, 2344–2359

doi:10.1111/are.12391

Diversity of autochthonous bacterial communities in the intestinal mucosa of grass carp (Ctenopharyngodon idellus) (Valenciennes) determined by culture-dependent and cultureindependent techniques Huan Li1, Qiuping Zhong1, Stephan Wirth2, Weiwei Wang1, Yaotong Hao1, Shangong Wu1, Hong Zou1, Wenxiang Li1 & Guitang Wang1 1

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of

Sciences, Wuhan, Hubei Province, China 2

Leibniz-Centre for Agricultural Landscape Research (ZALF), Institute of Landscape Biogeochemistry, M€ uncheberg,

Germany Correspondence: G-T Wang, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China. E-mail: [email protected]

Abstract Traditional culture-based technique and 16S rDNA sequencing method were used to investigate the mucosa-associated autochthonous microbiota of grass carp (Ctenopharyngodon idellus). Twenty-one phylotypes were detected from culturable microbiota, with Aeromonas, Shewanella, Lactococcus, Serratia, Brevibacillus, Delftia, Pseudomonas, Pantoea, Enterobacter, Buttiauxella and Yersinia as their closest relatives. Genomic DNA was directly extracted from the gut mucosa of C. idellus originating from six different geographical regions, and used to generate 609 random bacterial clones from six clone libraries and 99 archaeal clones from one library, which were grouped into 67 bacterial and four archaeal phylotypes. Sequence analysis revealed that the intestinal mucosa harboured a diversified bacterial microbiota, where Proteobacteria, Firmicutes and Bacteroidetes were dominant, followed by Actinobacteria, Verrucomicrobia and DeinococcusThermus. The autochthonous bacterial communities in the gut mucosa of fish from different aquatic environments were not similar (Cs < 0.80), but c-Proteobacteria was a common bacterial class. In comparison to bacterial communities, the archaeal community obtained from one library consisted of Crenarchaeota and Euryarchaeota. These results demonstrate that molecular methods facilitate

2344

culture-independent studies, and that fish gut mucosa harbours a larger bacterial diversity than previously recognized. The grass carp intestinal habitat selects for specific bacterial taxa despite pronounced differences in host environments.

Keywords: Grass carp, autochthonous microbiota, gut mucosa, 16S rDNA

Introduction Gastrointestinal (GI) microbiota play a key role in several important physiological functions of the host, including food digestion, development of the mucosal system, angiogenesis and protection against disease (Macfarlane & Macfarlane 1997; Hooper, Midtvedt & Gordon 2002; Ray, Ghosh & Ringø 2012). GI bacteria can be classified as autochthonous (able to adhere and colonize the host’s epithelial surface or associated with microvilli) or allochthonous (incidental visitors of the GI tract that are eventually rejected without colonizing) (Ringø & Birkbeck 1999). Faeces or gut contents are often used to investigate gut microbiota because they are easily collected, but mucosal microbiota, in close contact with the autochthonous microbial populations, may be distinct and fulfil different roles within the ecosystem (Eckburg, Bik, Bernstein, Purdom, Dethlefsen, Sargent, Gill, Nelson © 2014 John Wiley & Sons Ltd

Bacterial diversity in carp intestinal mucosa H Li et al.


& Relman 2005). The identification and comparison of the mucosa-associated autochthonous microbiota is undoubtedly significant for understanding functional mechanisms between the microbes and host (G omez & Balc azar 2008). An in-depth study of autochthonous bacteria may help to develop new aquaculture probiotics (Nayak 2010). Numerous studies concerning the autochthonous microbiota in the intestine of freshwater fish have been published in recent years (Huber, Spanggaard, Appel, Rossen, Nielsen & Gram 2004; Zhou, Liu, Shi, He, Yao & Ringø 2009; Jiang, Xie, Yang, Gong, Chen, Xu & Bao 2011). Conventional culture-dependent techniques have been mainly used in early studies, which revealed that fish possess complex intestinal microbiota, including aerobic, facultative anaerobic and anaerobic bacteria (Trust & Sparrow 1974; Cahill 1990; Ringø, Strøm & Tabachek 1995). However, conventional culture-dependent methods cannot present a correct picture of the fish gut microbiota, because only a small fraction of bacteria can be isolated and cultivated (Suau, Bonnet, Sutren, Godon, Gibson, Collins & Dore 1999). Recently, molecular methods, including the 16S rDNA clone library technique, have facilitated culture-independent studies and have improved the knowledge of gastrointestinal tracts of fish. Several studies showed that a molecular approach using 16S rDNA clone libraries could be a very powerful tool to analyse complex microbial communities (Holben, Williams, Saarinen, S€ arkilahti & Apajalahti 2002; Hovda, Lunestad, Fontanillas & Rosnes 2007; Kim, Brunt & Austin 2007; Wu, Gao, Zheng, Wang, Cheng & Wang 2010). The grass carp (Ctenopharyngodon idellus) is an herbivorous freshwater fish and is widely cultivated for food production in China and other Asian countries. Production in China reached 4.08 million tons in 2009 and comprised more than 20% of the total annual output of freshwater-culture fish (Li 2010). Under natural conditions, grass carps feed on certain aquatic plants (Cui, Wang, Liu & Chen 1991). However, the fish stop eating when the water temperature drops below 7°C, resulting in fasting during winter (Ni & Wang 1999). During this season, the gut mucosal microbiota of the fish will become relatively stable, and can be regarded as autochthonous. Recent studies on the diversity of the microbiota in the intestinal contents of C. idellus have revealed

Proteobacteria and Firmicutes as the most common bacterial groups (Han, Liu, Zhou, He, Cao, Shi, Yao & Ringø 2010; Wu, Wang, Angert, Wang, Li & Zou 2012). However, these studies could not test autochthonous microbiota associated with intestinal mucosa exclusively. Jiang et al. (2011) demonstrated that Aeromonas spp. were the dominant indigenous bacteria in the intestine of C. idellus, but failed to study indigenous microorganisms under natural fasting conditions. Thus, the knowledge about the gut indigenous microbiota of C. idellus remains incomplete. The purpose of this study was to use conventional culture-based and molecular-based techniques to evaluate the autochthonous microbiota of C. idellus. We identified the dominant bacterial members of the intestinal mucosa. Furthermore, we analysed whether indigenous gut microbiota were similar among individuals from geographically separated locations. Materials and methods Sample preparation A total of 18 grass carp were obtained from the following six locations: Yingshan, named Y in this study (Bianlian River, N30°43.448′ E114°54.232′), Jingzhou, named H (Honghu, N29°56.011′ E113° 28.965′), Shishou, named S (The Yangtze River Fisheries, N29°46.578′ E112°22.866′), Ezhou, named L (Liangzi Lake, N30°18.205′ E114° 34.964′), Jiangxia, named T (Tangsun Lake, N30°23.736′ E114°18.795′), and Xishui, named X (Wangtian Lake, N30°33.800′ E115°02.894′), located at different geographical sites in Hubei Province, central China (Table 1). Three fish were collected at each site in February 2012. Wild fish were collected from sites Y and L, while farmed individuals were obtained from H, S, T and X, with an average body length of 23.15 0.49, 19.57 2.50, 47.57 3.67, 25.47 0.97, 49.67 2.52, 44.40 1.61 cm respectively. The water temperature at the sampling points ranged from 1 to 4°C. We observed almost no contents in the guts of the 18 fish. All fish were killed using high doses of anaesthesia (Tricaine methanesulfonate, Finquel MS222; Sigma, Redmond, WA, USA). The intestine was aseptically removed from the fish abdominal cavity; the gut mucosa were scraped off with a sterilized scalpel and collected into sterile polypropylene tubes. Intestinal mucosa of samples from the same

© 2014 John Wiley & Sons Ltd, Aquaculture Research, 46, 2344–2359

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Table 1 Description of samples used in this study Sample name

Average body length (cm)

Type

Location

Geographical coordinates

L T H S X Y

19.57 49.67 47.57 25.47 44.40 23.10

wild Farmed Farmed Farmed Farmed Wild

Liangzi Lake Tangsun Lake Honghu The Yangtze River Fisheries Wangtian Lake Bianlian River

N30°18.205′ N30°23.736′ N29°56.011′ N29°46.578′ N30°33.800′ N30°43.448′

geographical location was pooled to avoid erroneous conclusions due to individual variations in gut microbiota as described elsewhere (Spanggaard, Huber, Nieslen, Nieslen, Appel & Gram 2000; He, Zhou, Liu, Shi, Yao, Ringø & Yoon 2009). All samples were transferred to the laboratory on ice within 24 h. Isolation and enumeration of cultivable bacteria Cultured microbiota was isolated and enumerated as described by Wu et al. (2010). Firstly, 1 g (wet weight) of the gut mucosa was homogenized for 10 min with a tissue grinder and vortexed severely in 9 ml volumes of sterile phosphate buffered (PBS; 0.1 mol l 1, pH 7.2). Then, 0.1 ml of the dilutions 10 5, 10 6 and 10 7 was spread over triplicate plates of tryptic soy agar (TSA; Becton, Dickinson, Sparks, MD, USA). The plates were incubated at 20°C for 4 days to determine the aerobic plate counts of cultivable bacteria present. Intestinal mucosa from samples T and L were used for bacterial isolation and culture, and these isolates were purified by streaking and re-streaking on fresh media for at least five times. Finally, pure cultures were stored at 70°C in tryptic soy broth (TSB) supplemented with 25% (v/v) glycerol as cryopreservant. The remaining mucosa samples from the six different locations were kept at 20°C for constructing 16S rDNA clone libraries. DNA extraction, PCR amplification and sequencing of cultivable bacteria For cultured microbiota, 202 colonies (99 from L and 103 from T) were randomly selected for the following analysis. The colonies were cultivated in TSB at 28°C for 24 h and harvested by centrifugation at 4000g for 10 min. DNA was extracted using a bacterial genomic DNA extraction kit (Spin-column) (BioTeke, Beijing, China) following the manu-

2346

E114°34.964′ E114°18.795′ E113°28.965′ E112°22.866′ E115°02.894′ E114°54.232′

facturer’s protocols (http://www.bioteke.com/eng/ DataSheets/DNA/Bacterial%20DNA%20Extraction %20Kit (Spin-column).pdf). To ensure extraction of DNA from Gram-positive bacteria, a lysozyme treatment was performed in advance. The extracted DNA was eluted into 25 lL TE buffer (pH 8.0) and stored at 20°C. The two universal primers 27F (5′-AGAGTTTG ATCATGGCTCAG-3′) and 1492R (5′-GGTTACCT TGTTACGACTT-3′) were used to amplify the 16S rDNA from the isolates (Weisburg, Brans, Pelletier & Lane 1991). To each PCR tube, final concentrations of the following reagents were added: 10 ng bacterial genomic DNA, 5 lM of both primers, 0.2 mM of each dNTP solution, 19 PCR reaction buffer, 2.4 U of TaKaRa Ex Taq DNA polymerase (TaKaRa, Dalian, China) and double-distilled water to a final volume of 100 lL. The PCR reactions were performed on a PTC-100TM Programmable Thermal Controller (MJ Research, Watertown, MA, USA) with the following cycling conditions: 94°C for 5 min, 30 cycles at 94°C for 30 s, 53°C for 30 s and 72°C for 1.5 min, and a final extension step at 72°C for 10 min before cooling at 4°C. Amplification products were analysed using electrophoresis in a 2.0% (w/v) agarose gel containing ethidium bromide (2 ng ml 1). The PCR products were purified using a DNA purification kit (Axygen, Shanghai, China). The purified products were then cloned into a PMD18-T vector (TaKaRa) following the manufacturer’s protocols. Finally, clones containing insert DNA were sequenced on an automatic DNA sequencer (model 3730; ABI Applied Biosystems, Foster City, CA, USA). Construction of 16S rDNA clone libraries Total DNA from intestine mucosa was extracted using a bacterial genomic DNA extraction kit (Spin-column) (BioTeke) following the manufacturer’s instructions. For each location, three




samples were pooled to minimize bias. The bacterial genomic DNA of each sample was extracted in triplicate and then combined. The 16S rDNA fragments were amplified separately using the bacterial universal primers 27F and 1492R (see above) and the universal archaeal primers 109aF (5′-ACK GCTCAGTAACACGT-3′) and 1119aR (5′-GGYR TCTCGCTCGT T-3′) for archaeal fragments (~1000 bp) from the total DNA (Ye, Liu, Lin, Tan, Pan, Li & Yang 2009). PCR amplification was performed in a 100 lL mixture containing 20 ng bacterial genomic DNA, 5 lM of both primers, 0.2 mM of each dNTP solution, 19 PCR reaction buffer, 2.4 U of Ex Taq DNA polymerase (TaKaRa). The PCR protocol was performed as follows: 94°C for 5 min, 30 (bacterial amplification) or 38 (archaeal amplification) cycles of 94°C for 30 s, 53°C for 30 s and 72°C for 1.5 min, and a final extension step at 72°C for 10 min before cooling at 4°C. Purified 16S rDNA fragments were cloned into PMD-18T vectors according to the manufacturer’s instructions, and transformed into chemically competent Escherichia coli Top 10 cells (TaKaRa) to construct 16S rDNA libraries. The presence of inserts was preliminary screened using the X-Gal-IPTG method. Approximately 110 white clones for each bacterial library and 100 white clones for each archaeal clone library were randomly selected on a single plate to ensure that the constructed libraries reflected the environmental community as fully as possible. For further selection, 27F and 1492 R, or 109aF and 1119aR were used to amplify the inserted fragment from recombinants. PCR products of appropriate size (approximately 1500 bp or approximately 1000 bp respectively) were digested with the restriction endonucleases HaeIII and HhaI. Digested fragments were separated by electrophoresis on a 2.0% agarose gel in 19 TAE buffer. After photographing under UV light, gel profiles were analysed using the Gel-Pro Analyzer software. Clones with identical band patterns were grouped into a single operational taxonomic unit (OTU). At least one positive clone from each OTU was then bidirectionally sequenced on an ABI 3730 DNA automatic sequencer (Applied Biosystems). Data analysis Putative chimeras from the 16S rDNA sequences were identified and excluded using Bellerophon

version 3 implemented at the Greengenes website (http://greengenes.lbl.gov/cgi-bin/nphbel3_interface.cgi) (Dalevi, DeSantis, Fredslund, Andersen, Markowitz & Hugenholtz 2007). Sequences of more than 97% similarity were considered to be the same phylotype or OTU (Stackebrandt & Goebel 1994). The representative sequences of each OTU were determined by Dereplicate Request in the Ribosomal Database Project – Release 10 (RDP) (http://pyro.cme.msu.edu/ index.jsp) and were subjected to similarity searches using the BLAST program (Altschul, Madden, Sch€ affer, Zhang, Zhang, Miller & Lipman 1997). Representative sequences of each OTU were aligned using Clustal W (Tompson, Higgins & Gibson 1994). A phylogenetic tree was constructed using MEGA 4.0 (Tamura, Dudley, Nei & Kumar 2007), based on a neighbour-joining algorithm (Saitou & Nei 1987), and a Kimura Two-parameter model (Kimura 1980). Finally, all representative sequences were taxonomically classified with the RDP classifier using an 80% confidence threshold (Wang, Garrity, Tiedje & Cole 2007). The relative abundance (%) of OTUs, representing the ratio of the number of the clones of a specific OTU to the total number of clones, was considered to be significant when the value was more than 1.5-fold higher or less than 0.5-fold lower than the abundance of any other OTU (Zhou, Liu, He, Shi, Gao, Yao & Ringø 2009). Estimates of phylotype richness (Schao 1 index), diversity (Shannon–Weaver index), dominance concentration (Simpson index) and evenness (equitability index) were calculated based on the 16S rDNA phylotype data respectively (Brown & Bowman 2001). All equations are described in detail by Bowman, McCammon, Rea and McMeekin (2000). The coverage (Cgood) was calculated according to Good (1953), using the equation Cgood = 1 N/total number of OTUs (where N is the number of OTUs with only one clone). Cluster analysis was based on the unweighted pair group method using the arithmetic mean algorithm (UPGMA). In this study, the microbial communities with a pairwise similarity coefficient (Cs: the measure of the similarity of two samples by UPGMA) < 0.60 were regarded as different, those with 0.60 ≤ Cs < 0.80 were considered to be marginally different and those with Cs ≥ 0.80 were considered to be similar (Wang, Zhou, He, Liu, Cao, Shi, Yao & Ringø 2010).


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Results Cultivation-dependent microbial community analysis Average aerobic and facultative anaerobic bacteria counts of 3.8 0.6 9 108 and 3.7 0.44 9 108 CFU (colony-forming units) were recovered from 1.0 g quantities of intestinal mucosa of L and T on TSA agar medium respectively. There was no significant difference between the numbers obtained from gut mucosa of T and L (P > 0.05). After removing nine chimeric sequences (four from library L and five from library T), a total of 193 sequences were obtained from library L (95 sequences) and T (98 sequences). These sequences were analysed using 97% minimum similarity as the threshold for any pair of sequences in a phylotype (OTU). Thus, 21 microbial phylotypes, i.e. 17 from L and 4 from T, were compared with the BLAST program (summarized in Table 2). Three bacterial groups were obtained, with c-proteobacteria (185 of 193) as dominant group, followed by Firmicutes (7 of 193) and b-proteobacteria (1 of 193). At the genus level, Aeromonas spp. (106 of 193) and Shewanella spp. (32 of 193) comprised the dominant cultured bacteria, followed by Lactococcus, Serratia, Brevibacillus, Delftia, Pseudomonas, Pantoea, Enterobacter, Buttiauxella and Yersinia. Cultivation-independent microbial community analysis Six independent bacterial libraries of cloned 16S rDNA gene sequences were constructed. Approximately 609 positive clones with the insertion of the corresponding sizes were analysed, and 67 different phylotypes (OTUs) were obtained. On comparison of a representative clone from each OTU with sequences in the GenBank database, the observed sequence similarities ranged from 91% to 100%. Autochthonous bacterial communities in all clones of the six libraries were represented by the following phyla: Proteobacteria (a-proteobacteria/28.08%, b-proteobacteria/8.70%, c-proteobacteria/38.92%, d-proteobacteria/0.16%), Firmicutes (11.66%), Bacteroidetes (10.34%), DeinococcusThermus (0.66%), Actinobacteria (0.49%) and Verrucomicrobiae (0.33%). Four sequences, closely related to different types of uncultured bacteria (0.66%), were also detected. The composition of

2348

each library is shown in Table 3. To further analyse the dominant bacteria in the six libraries, sequences of approximately 42 OTUs containing more than one clone were used to construct a phylogenetic tree (Fig. 1). In addition, the dominant bacteria were different in each library. Sequences in library L that was closely related to c-proteobacteria were represented by Aeromonas sobria (99% identity, 31 of 93). Sequences in library Y were affiliated with Acinetobacter [calcoaceticus] (99% identity, 35 of 107). Surprisingly, sequences closely related to c-proteobacteria in library T were dominated by Shewanella putrefaciens (99% identity, 30 of 95), while sequences affiliated with Bacteroidetes in library S were represented by Pedobacter africanus (99% identity, 36 of 104). Sequences closely related to a-proteobacteria in libraries H and X were dominated by Phyllobacterium myrsinacearum (100% identity, 36 of 105 and 49 of 105 respectively). The bacterial communities in gut mucosa from different geographical environments were different (Cs < 0.80) (Table 4). Library L was significantly different from the other libraries except for library T (Cs = 0.78). There were significant differences between library Y and all other libraries (Cs < 0.60). The diversity indices of the indigenous intestinal microbial communities are shown in Table 5. The Shannon diversity index (H) of library H (2.52) was higher than that of other libraries. Opposite trends were observed for the Simpson index (D) and Evenness (E) values, indicating that the indigenous microbiota in the gut mucosa of library H were more diverse than those of other libraries. The Schao 1 indices of library L (11.44) and T (9.44) were lower than others, but the coverage (Cgood) values (0.82 and 0.78 respectively) were higher than those from other bacterial libraries. PCR bands were obtained only from sample S using the archaeal-specific primers. The archaeal clone library (library SG) containing 99 clones was constructed. The phylogenetic analysis revealed the archaeal community consisted of Crenarchaeota (81.8%) and Euryarchaeota (19.2%). The composition of the archaeal library is shown in Table 6. Crenarchaeota comprised three different OTUs belonging to Methanosaeta, while Euryarchaeota consisted of only one OTU closely related to an uncultured archaeon. Representative sequences of four OTUs were used to construct a phylogenetic tree (Fig. 2).



doi:10.1111/are.12391

Diversity of autochthonous bacterial communities in the intestinal mucosa of grass carp (Ctenopharyngodon idellus) (Valenciennes) determined by culture-dependent and cultureindependent techniques Huan Li1, Qiuping Zhong1, Stephan Wirth2, Weiwei Wang1, Yaotong Hao1, Shangong Wu1, Hong Zou1, Wenxiang Li1 & Guitang Wang1 1

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of

Sciences, Wuhan, Hubei Province, China 2

Leibniz-Centre for Agricultural Landscape Research (ZALF), Institute of Landscape Biogeochemistry, M€ uncheberg,

Germany Correspondence: G-T Wang, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China. E-mail: [email protected]

Abstract Traditional culture-based technique and 16S rDNA sequencing method were used to investigate the mucosa-associated autochthonous microbiota of grass carp (Ctenopharyngodon idellus). Twenty-one phylotypes were detected from culturable microbiota, with Aeromonas, Shewanella, Lactococcus, Serratia, Brevibacillus, Delftia, Pseudomonas, Pantoea, Enterobacter, Buttiauxella and Yersinia as their closest relatives. Genomic DNA was directly extracted from the gut mucosa of C. idellus originating from six different geographical regions, and used to generate 609 random bacterial clones from six clone libraries and 99 archaeal clones from one library, which were grouped into 67 bacterial and four archaeal phylotypes. Sequence analysis revealed that the intestinal mucosa harboured a diversified bacterial microbiota, where Proteobacteria, Firmicutes and Bacteroidetes were dominant, followed by Actinobacteria, Verrucomicrobia and DeinococcusThermus. The autochthonous bacterial communities in the gut mucosa of fish from different aquatic environments were not similar (Cs < 0.80), but c-Proteobacteria was a common bacterial class. In comparison to bacterial communities, the archaeal community obtained from one library consisted of Crenarchaeota and Euryarchaeota. These results demonstrate that molecular methods facilitate

2344

culture-independent studies, and that fish gut mucosa harbours a larger bacterial diversity than previously recognized. The grass carp intestinal habitat selects for specific bacterial taxa despite pronounced differences in host environments.

Keywords: Grass carp, autochthonous microbiota, gut mucosa, 16S rDNA

Introduction Gastrointestinal (GI) microbiota play a key role in several important physiological functions of the host, including food digestion, development of the mucosal system, angiogenesis and protection against disease (Macfarlane & Macfarlane 1997; Hooper, Midtvedt & Gordon 2002; Ray, Ghosh & Ringø 2012). GI bacteria can be classified as autochthonous (able to adhere and colonize the host’s epithelial surface or associated with microvilli) or allochthonous (incidental visitors of the GI tract that are eventually rejected without colonizing) (Ringø & Birkbeck 1999). Faeces or gut contents are often used to investigate gut microbiota because they are easily collected, but mucosal microbiota, in close contact with the autochthonous microbial populations, may be distinct and fulfil different roles within the ecosystem (Eckburg, Bik, Bernstein, Purdom, Dethlefsen, Sargent, Gill, Nelson © 2014 John Wiley & Sons Ltd



Table 3 Phylogenetic affiliation of 16SrDNA phylotypes of bacterial communities from the gut mucosa of grass carp in different libraries* Relative abundance (%)

OTU

L (93 clones)

T (95 clones)

H (105 clones)

S (104 clones)

X (105 clones)

Y (107 clones)

OTU1

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU2

2.15b

0.00a

0.00a

0.00a

0.00a

0.00a

OTU3

0.00a

0.00a

0.00a

34.62d

0.95b

9.35c

OTU4 OTU5

0.00a 0.00a

0.00a 0.00a

0.00a 0.00a

0.00a 0.00a

0.95b 6.67b

0.00a 0.00a

OTU6

0.00a

1.05b

0.00a

0.00a

0.00a

3.74c

OTU7

0.00a

0.00a

1.90b

0.00a

0.00a

0.00a

OTU8

0.00a

0.00a

1.90b

0.00a

0.00a

0.00a

OTU9

0.00a

0.00a

3.81b

0.00a

0.00a

0.00a

OTU10

17.20b

25.26bc

0.00a

0.00a

0.00a

0.00a

OTU11

10.75b

0.00a

0.00a

0.00a

0.00a

0.00a

OTU12

0.00a

0.00a

1.90b

0.00a

0.00a

0.00a

OTU13

0.00a

0.00a

5.71C

0.96b

0.00a

0.00a

OTU14

0.00a

0.00a

0.00a

0.00a

0.00a

5.61b

OTU15

0.00a

0.00a

1.90b

0.00a

0.00a

0.00a

OTU16

0.00a

0.00a

34.29c

15.38bc

46.67c

11.21b

OTU17

0.00a

0.00a

7.62b

0.00a

0.00a

0.00a

OTU18

0.00a

0.00a

3.81b

0.00a

0.00a

0.00a

OTU19

0.00a

0.00a

8.57b

0.00a

0.00a

0.00a

OTU20

0.00a

0.00a

3.81b

0.00a

0.00a

0.00a

OTU21

0.00a

0.00a

5.71c

0.00a

1.90b

0.00a

OTU22

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU23 OTU24

0.00a 0.00a

0.00a 0.00a

0.95b 0.00a

0.96b 0.96b

2.86c 0.00a

0.00a 0.00a

OTU25

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU26

0.00a

0.00a

0.00a

0.00a

5.71b

0.00a

Closest relative in Genbank (acccession no.) Arthrobacter nicotianae (NR_026190.1) Mycobacterium chelonae (NR_042918.1) Pedobacter africanus (NR_028977.1) Runella zeae (NR_025004.1) Flavobacterium resistens (NR_044292.1) Pedobacter panaciterrae (NR_041371.1) Deinococcus geothermalis (NR_074342.1) Thermus scotoductus (NR_074428.1) Clostridium disporicum (NR_026491.1) Brevibacillus laterosporus (NR_037005.1) Lactococcus raffinolactis (NR_044359.1) Staphylococcus epidermidis (NR_074995.1) Anoxybacillus flavithermusl (NR_074667.1) Brevibacillus formosus (NR_040979.1) Uncultured bacterium (DQ115992.1) Phyllobacterium myrsinacearum (NR_043189.1) Paracoccus homiensis (NR_043733.1) Alpha proteobacterium F0723 (AF236000.1) Bradyrhizobium pachyrhizi (NR_043037.1) Paracoccus halophilus (NR_043810.1) Ochrobactrum lupini (NR_042911.1) Caulobacter leidyia (NR_025324.1) Afipia birgiae (NR_025117.1) Rhizobium selenitireducens (NR_044216.1) Brevundimonas terrae (NR_043726.1) Sandaracinobacter sibiricus (NR_026382.1)

Identity (%)

Phylogenetic group

99

Actinobacteria

99

Actinobacteria

99

Bacteroidetes

98 97

Bacteroidetes Bacteroidetes

98

Bacteroidetes

99

99

DeinococcusThermus DeinococcusThermus Firmicutes

99

Firmicutes

99

Firmicutes

99

Firmicutes

99

Firmicutes

99

Firmicutes

99

Firmicutes

100

a-proteobacteria

97

a-proteobacteria

98

a-proteobacteria

99

a-proteobacteria

97

a-proteobacteria

99

a-proteobacteria

99

a-proteobacteria

99 98

a-proteobacteria a-proteobacteria

99

a-proteobacteria

97

a-proteobacteria

99

(continued)

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Table 3 (Continued) Relative abundance (%) L (93 clones)

T (95 clones)

H (105 clones)

S (104 clones)

X (105 clones)

Y (107 clones)

OTU27

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU28 OTU29

0.00a 0.00a

0.00a 0.00a

0.00a 0.00a

0.00a 0.00a

7.62b 0.95b

0.00a 0.00a

OTU30

0.00a

0.00a

0.00a

0.00a

2.86b

0.00a

OTU31

0.00a

0.00a

0.00a

0.00a

0.00a

0.93b

OTU32

1.08b

0.00a

0.00a

0.00a

0.00a

0.00a

OTU33

0.00a

0.00a

1.90b

0.00a

1.90b

0.00a

OTU34

0.00a

0.00a

0.95b

0.00a

0.00a

0.00a

OTU35

0.00a

0.00a

0.00a

3.85b

0.00a

7.48b

OTU36

0.00a

0.00a

0.00a

0.00a

0.95b

0.00a

OTU37

0.00a

0.00a

0.00a

0.00a

7.62b

0.00a

OTU38

0.00a

0.00a

0.00a

0.00a

0.00a

7.48b

OTU39

0.00a

0.00a

0.00a

0.00a

0.00a

13.08b

OTU40

0.00a

0.00a

0.00a

0.00a

0.00a

0.93b

OTU41

0.00a

0.00a

0.00a

0.00a

0.00a

0.93b

OTU42

0.00a

0.00a

0.00a

0.00a

0.00a

0.93b

OTU43

0.00a

0.00a

0.00a

0.00a

0.95b

0.00a

OTU44

6.45c

2.11b

0.00a

0.00a

0.00a

0.00a

OTU45

33.33cd

29.47cd

0.95b

16.35c

0.00a

0.00a

OTU46

15.05b

31.58bc

0.00a

0.00a

0.00a

0.00a

OTU47

2.15b

2.11b

0.00a

0.00a

0.00a

0.00a

OTU48

1.08b

0.00a

0.00a

0.00a

0.00a

0.00a

OTU49

6.45bc

4.21b

0.00a

0.00a

0.00a

0.00a

OTU50

4.30c

3.16c

1.90b

0.00a

0.00a

0.00a

OTU51

0.00a

0.00a

0.00a

16.35b

0.00a

0.00a

OTU52

0.00a

1.05b

0.00a

0.00a

0.00a

0.00a

OTU

Closest relative in Genbank (acccession no.) Brevundimonas kwangchunensis (NR_043315.1) Bosea vestrisii (NR_028799.1) Reyranella massiliensis (HM048834.1) Alpha proteobacterium OR-84 (HM163284.1) Mycoplana bullata (NR_025831.1) Vitreoscilla stercoraria (NR_025894.1) Herbaspirillum huttiense (NR_024698.1) Delftia acidovorans (NR_024711.1) Curvibacter gracilis (NR_028655.1) Cupriavidus basilensis (NR_025138.1) Janthinobacterium agaricidamnosum (NR_026364.1) Herbaspirillum seropedicae (NR_029329.1) Cupriavidus taiwanensis (NR_028800.1) Massilia timonae (NR_026014.1) Acidovorax temperans (NR_028715.1) Dechloromonas hortensis (NR_042819.1) Delftia tsuruhatensis (NR_024786.1) Pseudomonas veronii (NR_028706.1) Aeromonas sobria (NR_037012.2) Shewanella putrefaciens (NR_044863.1) Enterobacter amnigenus (NR_024642.1) Enterobacter ludwigii (NR_042349.1) Yersinia ruckeri (NR_041833.1) Shigella flexneri (NR_026331.1) Aeromonas bestiarum (NR_026089.2) Uncultured Rahnella sp. (GU299866.1)

Identity (%)

Phylogenetic group

99

a-proteobacteria

99 99

a-proteobacteria a-proteobacteria

99

a-proteobacteria

98

a-proteobacteria

97

b-proteobacteria

100

b-proteobacteria

99

b-proteobacteria

99

b-proteobacteria

99

b-proteobacteria

97

b-proteobacteria

98

b-proteobacteria

98

b-proteobacteria

97

b-proteobacteria

99

b-proteobacteria

98

b-proteobacteria

99

b-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

99

c-proteobacteria

(continued)


2351



Table 3 (Continued) Relative abundance (%) L (93 clones)

T (95 clones)

H (105 clones)

S (104 clones)

X (105 clones)

Y (107 clones)

OTU53

0.00a

0.00a

5.71b

0.00a

0.00a

0.00a

OTU54

0.00a

0.00a

0.95b

1.92b

0.00a

0.00a

OTU55

0.00a

0.00a

1.90b

0.00a

0.00a

0.00a

OTU56

0.00a

0.00a

0.95b

0.00a

0.00a

0.00a

OTU57

0.00a

0.00a

0.00a

2.88b

0.00a

32.71c

OTU58

0.00a

0.00a

0.00a

0.00a

9.52b

0.00a

OTU59

0.00a

0.00a

0.00a

0.00a

0.00a

4.67b

OTU60

0.00a

0.00a

0.00a

0.00a

0.95b

0.00a

OTU61

0.00a

0.00a

0.95b

0.00a

0.00a

0.00a

OTU62

0.00a

0.00a

0.00a

0.00a

0.95b

0.00a

OTU63

0.00a

0.00a

0.95b

0.00a

0.00a

0.00a

OTU64

0.00a

0.00a

0.00a

0.00a

0.00a

0.93b

OTU65

0.00a

0.00a

0.95b

0.00a

0.00a

0.00a

OTU66

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU67

0.00a

0.00a

0.00a

0.96b

0.00a

0.00a

OTU

Closest relative in Genbank (acccession no.) Pseudomonas putida (NR_043424.1) Acinetobacter schindleri (NR_025412.1) Acinetobacter haemolyticus (NR_026207.1) Methylibium fulvum (NR_041367.1) Acinetobacter [calcoaceticus] (NR_042387.1) Acinetobacter baumannii (NR_026206.1) Lysobacter niabensis (NR_043867.1) Acinetobacter radioresistens (NR_026210.1) Uncultured bacterium (HM780378.1) Uncultured bacterium (AM936898.1) Uncultured bacterium (HQ904146.1) Uncultured bacterium (DQ354730.1) Uncultured bacterium (GQ094019.1) Uncultured bacterium (AB154317.1) Uncultured bacterium (JN868793.1)

Identity (%)

Phylogenetic group

99

c-proteobacteria

99

c-proteobacteria

97

c-proteobacteria

99

c-proteobacteria

100

c-proteobacteria

100

c-proteobacteria

96

c-proteobacteria

97

c-proteobacteria

97

d-proteobacteria

95

Unclassified

98

Unclassified

91

Unclassified

99

Unclassified

99

Verrucomicrobiae

99

Verrucomicrobiae

*Within each row, data marked with the same superscript reflected values within a 0.5- to 1.5-fold difference range. OTU, operational taxonomic unit, the clones with 100% band profile.

of 3–4 9 108 CFU g 1 in the intestinal mucosa. In general, these results are consistent with those reported by Hagi, Tanaka, Lwamura and Hoshino (2004), who observed total viable counts from the intestinal tracts of silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio) and crucian carp (Carassius cuvieri) of approximately 1.6 9 108, 1.9 9 109 and 5.2 9 108 CFU g 1 respectively. These values were slightly higher than those reported by Kim et al. (2007) and Wu et al. (2010), who detected bacterial numbers from the mucosa of rainbow trout (Oncorhynchus mykiss) and yellow catfish (Pelteobagrus fulvidraco) of 3.0 4.6 9 106 and 2.1 9 107 CFU g 1 respectively. However, these values were significantly lower than those reported for humans and terrestrial animals (approximately 1011 CFU g 1) (Moore & Holdeman 1974; Mead 1997). As

2352

anaerobic bacteria were not considered in this study, this discrepancy may show the much higher number of anaerobes (1010–1011 CFU g 1) (Suau et al. 1999; Marteau, Pochart, Dore, BeraMaillet, Bernalier & Corthier 2001) in the intestine of terrestrial animals when compared with that of fish. Only Proteobacteria and Firmicutes were detected using culture-dependent methods in the gut mucosa of C. idellus. As culture-dependent techniques can only disclose some cultivable aerobic and facultative anaerobic bacteria, the cultureindependent molecular method of a 16S rDNA clone library was used to describe the total indigenous microbiota in C. idellus. The DNA was extracted directly from the gut mucosa without prior cultivation, thus allowing detection of uncultivable anaerobic bacteria and some other bacteria




H64(KF003201)

88

H73(KF003202)

100

Y2(KF003204) 69

77

X59(KF003205) LHN31(KF003191)

99 100

H61(KF003200) LHN64(KF003193)

100 100

Gammaproteobacteria

LHN53(KF003192) S70(KF003198) LHN5(KF003196)

81

LHN70(KF003194)

100 84

58

LHN43(KF003197)

Y38(KF003206) S11(KF003182) Y28(KF003186)

94 91 57

X77(KF003184)

99

H70(KF003180)

100

Y27(KF003185) 100

100

Betaproteobacteria

S2(KF003148) Y24(KF003151)

Bacteroidetes

X100(KF003150)

49

H4(KF003153) 90 70

41

H32(KF003154)

Deinococcus-Thermus

H34(KF003155) LHN87(KF003157)

93

16

100 95

H53(KF003161)

Firmicutes

S98(KF003159)

77 57 100

18

H110(KF003158)

LHN42(KF003156) Y54(KF003160)

X7(KF003173) X75(KF003177) X41(KF003175)

24

H47(KF003165)

79

Alphaproteobacteria

H49(KF003166)

40 100

S33(KF003170)

H12(KF003163) H36(KF003164) 95

H1(KF003167) LHN24(KF003147) Actinobacteria 100

H63(KF003168)

Alphaproteobacteria

0.1

Fig. 1 Neighbor-joining phylogenetic tree showing the relationship of 42 bacterial 16S rDNA gene sequences obtained in this study. The phylogenetic tree was constructed using neighbor-joining method within MEGA (4) package Bootstrap values based on 1000 re-samplings display the significance of the interior nodes, and are shown at branch. The scale bar indicates 10% sequence divergence.


2353



that require special growth conditions (Spanggaard et al. 2000). Compared with culture-dependent techniques, the 16S rDNA clone library method suggested that the fish intestines harboured a more diversified bacterial microbiota, where Proteobacteria, Firmicutes and Bacteroidetes Table 4 Pairwise similarity coefficients for bacterial communities in the gut mucosa of grass carp Similarity

L

T

H

S

X

Y

L T H S X Y

1.00 0.78* 0.43** 0.47** 0.34** 0.41**

1.00 0.37** 0.40** 0.29** 0.35**

1.00 0.51** 0.72* 0.52**

1.00 0.45** 0.51**

1.00 0.45**

1.00

The microbial communities with a pairwise similarity coefficient (Cs: the measure of the similarity of two samples by UPGMA) < 0.60 were regarded as significantly different, those with 0.60 ≤ Cs