Molecular characterization of the autochthonous microbiota in the gastrointestinal tract of adult yellow grouper (Epinephelus awoara) cultured in cages

Aquaculture 286 (2009) 184–189

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Molecular characterization of the autochthonous microbiota in the gastrointestinal tract of adult yellow grouper (Epinephelus awoara) cultured in cages Zhigang Zhou a,1, Yuchun Liu a,1, Pengjun Shi a, Suxu He a, Bin Yao a,⁎, E. Ringø b,c a b c

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, 9037 Tromsø, Norway Institute of Marine Research, 5817 Bergen, Norway

a r t i c l e

i n f o

Article history: Received 10 May 2008 Received in revised form 30 September 2008 Accepted 1 October 2008 Keywords: Gastrointestine (GI) Autochthonous bacteria Epinephelus awoara Denaturing gradient gel electrophoresis (DGGE)

a b s t r a c t The present study used 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) technology to investigate the communities of autochthonous microbiota in the gastrointestinal (GI) tract; including stomach (ST), pyloric caeca (PC), proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of the adult yellow grouper (Epinephelus awoara) cultured in cages. Twenty-two DGGE bands were successfully sequenced. However, ten of these bands were classified as unculturable according to the phylogenetic analysis. The dominant autochthonous bacteria in the GI tract belonged to Proteobacteria, but other bacteria identified belonged to Firmicutes, Bacteroidetes, Deinococcus-Thermus, Spirochaetes and unclassified-bacteria. Pantoea sp. and uncultured Proteobacterium were ubiquitous in all five sections of the GI tract. Empedobacter sp. PH7-1 and Acinetobacter sp. N15 were unique for the ST section, while uncultured bacterium clone F6-37 and γ-Proteobacterium and Acinetobacter radioresistens Philippines-11 were only observed in PI, MI and DI sections. Furthermore, Lactococcus lactis, Bacteroides and uncultured Streptococcus sp. were also detected in the present study. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Yellow grouper (Epinephelus awoara) is one of the most important maricultured fish in China, Singapore and other Southeast Asian countries, and most groupers are cultured in cages (Zhou and Wang, 2004). Studies on yellow grouper have related to artificial breeding (Sun et al., 2007), phylogenetic relationship (Dong et al., 2007), and immunology (Wang and Wu, 2007). However, less information is available on the microbial diversity in the gut. In a preliminary study, Qin et al. (2007) evaluated the intestinal microbiota of yellow grouper by using five types of selective medium, and the results indicated that the population level of total aerobic and facultative anaerobic bacteria in the intestinal tract were 5.3 × 105 CFU/g. The predominant bacteria identified by 16S rDNA were Vireo olivaceus sp. nov., Escherichia asburiae, Pseudomonas sp., Bacillus subtilis, and Bacteroides sp. In order to explore the full potential of pro- and prebiotics we must have a clear, comprehensive understanding of the natural gut microbiota. The gut microbiota of fish is relatively well researched, but,most microbiota studies on fish have mainly concentrated their investiga-

tions on evaluating the bacterial community in foregut and hindgut (for reviews, see Cahill, 1990; Ringø et al., 1995, 2006; Spanggaard et al., 2000; Liu et al., 2008). However, the stomach and pyloric caeca have received less attention. In the early studies investigating the gut microbiota in fish, conventional culture-based methods, that are time consuming and selective, have been used (for review, see Cahill, 1990; Ringø et al., 1995). Conventional culture-based techniques, even if several different media are used, do not present a correct picture of the bacterial diversity. Therefore, to present more reliable information about the gut microbiota of fish, molecular methods are used. One of the most popular used method in fish studies is Polymerase Chain ReactionDenaturing Gradient Gel Electrophoresis (PCR-DGGE), as the method is reliable, rapid, sensitive and easy to use to study microbial diversity (Zhou et al., 2007, 2008; Liu et al., 2008). The purpose of this study was therefore to use PCR-DGGE and sequencing to evaluate the autochthonous microbiota in five parts of the digestive tract (stomach, pyloric caeca, proximal-, mid- and distal intestine) in adult yellow grouper cultured in cages and compare the microbial diversity in the different sections and individuals. 2. Materials and methods

⁎ Corresponding author. 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, PR China. Tel.: +86 10 82106053; fax. +86 10 82106054. E-mail address: [email protected] (B. Yao). 1 Equally contributing authors. 0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.10.002

2.1. Sample preparation Yellow grouper, E. awoara, were raised in marine cages at Xiashan District, Zhanjiang City, Guangdong Province, China from November 1,

Z. Zhou et al. / Aquaculture 286 (2009) 184–189

2005 to April 30, 2006. The fish were fed iced fresh fish Sardina melanostictus captured from South China Sea by tow net. At the end of the feeding period, three adult yellow groupers with an average weight of ~1.5 kg were collected from the cage, euthanized by a hard blow to the head and transported at −4 °C to Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute of Chinese Academy of Agricultural Sciences (Beijing, China) within 6 h. The water was 26.8 °C, with 33‰ salinity where the fish were sampled. The fish skin was washed with 70% ethanol in order to reduce contamination. The ventral surface was opened with sterile scissors to expose the body cavity and thereafter the digestive tract of each fish was sampled and divided into the following sections; stomach (ST), pyloric caeca (PC), proximal intestine (PI), mid intestine (MI) and distal intestine (DI). Each section was aseptically opened with a sterile scalpel and the digesta were washed off as described elsewhere (Zhou et al., 2007). The surface of each section was homogenized using a glass homogenizer (LeaMaster et al., 1997) and kept in Eppendorf tubes at −20 °C until analysis. In the present study, three individual fish were investigated as previous studies have reported that the gut microbiota varied within individuals (Spanggaard et al., 2000; Ringø et al., 2006; Zhou et al., 2007; Liu et al., 2008). 2.2. DNA extraction and PCR amplification The total genomic DNA was extracted using the methods described by Yu and Morrison (2005) and Zhou et al. (2007). The V3 region of the rrs gene was amplified by PCR using primers 338f (5′-ACTCCTACGGGAGGCAGCAG-3′) and 519r (5′-ATTACCGCGGCTGCTGG3′) (Lane, 1991). The primer 338f has a 40-base GC clamp attached to its 5′ end (5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′). A touch down PCR with some modifications was performed for all samples to reduce nonspecific priming (Kim et al., 2007) by using an Eppendorf Thermal Cycler (Bio-Rad, UK). The 50 μl PCR reaction system contained 1×PCR buffer (20 mM Tris–HCl (pH 8.4) and 50 mM KCl), 200 μM dNTP, 500 nM each primer, 1.75 mM MgCl2, 670 ng/μl bovine serum albumin (Yu and Morrison, 2005) and 1.25 U Platinum® Taq DNA polymerase (Invitrogen, USA), which allows for hot-start PCR. After 5 min of initial denaturation at 94 °C, the following procedure were applied: 30 s of denaturation at 94 °C, 30 s of initial annealing at 65 °C which decreased by 0.5 °C per cycle until a touchdown of 56 °C, 30 s of primer extension at 72 °C, followed by 10 cycles containing 30 s of denaturation at 94 °C, 30 s of annealing temperature at 56 °C and 30 s of primer extension at 72 °C, and final extension at 72 °C for 7 min. The PCR products were analyzed by electrophoresis in 2% (w/v) agarose gel containing ethidium bromide (Zhou et al., 2007, 2008; Liu et al., 2008). 2.3. DGGE analysis of the V3 regions DGGE was performed with a D-Code universal mutation detection system (Bio-Rad, USA) to separate the PCR products collected above.

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Aliquot of 900 ng purified PCR product of each sample was loaded directly onto 8% (w/v) polyacrylamide gels (16 cm × 16 cm × 1 mm) in 1 × TAE buffer (40 mM Tris base, 20 mM glacial acetate and 1 mM EDTA) with a denaturing gradient ranging from 40 to 60% (Liu et al., 2008). Electrophoresis was conducted with a constant voltage of 200 V at 60 °C for about 3.5 h. Gels were stained with ethidium bromide (5 μg/ml) for 20 min, washed with deionized water and photographed with UV transillumination (Zhou et al., 2007, 2008; Liu et al., 2008). 2.4. Sequencing of the 16S rDNA V3 regions The DNA fragments selected for sequencing were excised and amplified using the primers BA338f without the GC clamp and 519r following the procedure described by Liu et al. (2008). 2.5. Data analysis DGGE profiles were scanned with Bio-1D++ software (VilberLourmat, France). The banding patterns were carefully checked manually. Similarities between the PCR-DGGE banding patterns were analyzed using the Pearson correlation coefficient and displayed graphically as a dendrogram. An unweighted pair-group clustering algorithm with arithmetic averages (UPGMA) was used to calculate the dendrograms. The sequences were checked for chimeric constructs by using the check_chimera program of the ribosomal database project (RDP) (Cole et al., 2005). And all sequences were submitted for similarity searches with the blast program (Altschul et al., 1990). During the analysis, one sequence (band 3) was excluded as it was identified as the 18S rRNA gene of lamprey (Geotria australis). The construction of phylogenetic tree and bootstrap analysis of 1000 re-samplings was performed using ClustalX (version 1.83) and MEGA (3.1) package (Felsenstein, 1985; Saitou and Nei, 1987; Kumar et al., 2004). 3. Results 3.1. DGGE analysis of V3 regions The 16S rDNA-V3 PCR-DGGE fingerprints were used to visualize the autochthonous bacterial diversity in stomach (ST), pyloric caeca (PC), proximal intestine (PI), mid intestine (MI) and distal intestine (DI). Twenty five bands were excised from the DGGE gel and numbered from 1 to 22 and u1 to u3. Bands 1 to 22 were successfully retrieved while u1 to u3 failed possibly due to their weak band density and difficulties in PCR as template after excision. The distribution patterns of the 25 16S rDNA bands on the DGGE profile from the different sections of the GI tract are summarized in Table 1. Variations in the band patterns and the dominant bands in the GI tract are shown in Fig. 1 and Table 1, respectively. The number of bands from ST was significantly higher than

Table 1 Band distribution of 16S rDNA V3 region fragments from different sections along the gastrointestinal (GI) tract of adult yellow grouper cultured in cages in different samples Sample A B C Average band number2 Similarity (%) 1

Stomach (ST)

Pyloric caeca (PC)

Proximal intestine (PI)

Middle intestine (MI)

Average band Similarity 3 (%) Distal intestine (DI) number

5,8,13,14,15,16,17,18,21, 22,u1,u2(12)1 2,3,5,6,11,13,14,15,16,17, 18,21,22,u2(14) 3,5,6,8,9,10, 15,16,17,18, 21,22,u3(13) 13.0a

5,8,13,14,15,16,17,18, 21,22,u2(11) 2,3,5,6,11,13,17,18,21,22(10)

3,5,9,17,18,20,22(7)

3,5,17,18,19,20,22(7)

3,17,18,19,20(5)

2,3,5,6,11,12,13,17,18,21,22(11)

2,3,5,11,17,18,21,22(8)

3,5,7,16,17,18,21,22(8)

3,5,6,7,16,17,18,21, 22(9)

1,2,3,4,5,16,17,18,21,22(10)

9.7ab

9.0ab

65.3

57.7

60.0

Band distribution

8.4A

70.2

2,3,5,11,17,18,21(7)

10.0

A

83.0

10.2A

80.8

8.3b

1,2,3,4,5,16,17,18, 21,22,u2(11) 7.7b

71.3

64.3

The number in the parenthesis represented the band number in the lane of the DGGE profiles. 2 Values (means) in the same row not sharing a common superscript are significantly different (Duncan's multiple range test, P b 0.05). 3 Values (means) in the same column not sharing a common capitalized superscript are significantly different (Duncan's multiple range test, P b 0.05).

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4. Discussion

Fig. 1. Unweighted pair-group clustering dendrogram of the DGGE profiles using arithmetic average linkage. A, B and C represent three adult yellow groupers, and 1–5 represent five sections of stomach, pyloric caeca, proximal intestine, middle intestine and distal intestine along the gastrointestinal (GI) tract, respectively.

those from MI and DI (P b 0.05; Table 1), but no difference (P N 0.05) was detected in band numbers in the three gut samples (Table 1). The similarity of different sections along the gastrointestinal tract was higher than the individual similarity in the band patterns with the exception of the ST in fish B and C as shown in Fig. 1 and Table 1. The different GI tract sections of the three fish had several common bands. For example, band 18 appeared in all five sections of all fish (15 sections), while band 17 was found in 14 out of 15 lanes (Table 1). Bands 3 and 22 were found in 13 out of 15 lanes. In contrast to the above results, bands 10, 12, u1 and u3 were only detected in one segment of one fish. The three ST samples had seven common bands (bands 5, 15, 16, 17, 18, 21 and 22), PC had five (bands 5, 17, 18, 21 and 22), PI had three (bands 17, 18 and 22), MI had four (bands 5, 17, 18 and 22), and DI two (bands 17 and 18) (Table 1). Meanwhile, several unique bands were also detected in the GI tract sections (Table 1), such as bands u1, u3 and 10 in the ST, bands 19 and 20 in the PI and MI and band 4 in MI and DI. 3.2. Sequences of DNA bands in DGGE gel A total of 22 bands were successfully sequenced from the DGGE gel (Table 2) and these sequences were used to construct a phylogenetic tree (Fig. 2). The sequences of approximately 200 bp (GenBank accession numbers EU004789–EU004810) were compared using classifier tool in RDP and shown in Table 2. The accession no. of band 3 is EU004795, and not listed in Table 2 due to its close relation with 18S rDNA of lamprey after BLAST analysis. The bacteria identified from the different sections were closely related to one of the following groups: Proteobacteria (42.9% of the total; bands 10, 12, 13, 14, 17, 18, 19, 20 and 21), Firmicutes (19.0%; bands 1, 7, 16 and 22), Bacteroidetes (14.3%; bands 8, 9, and 11), Deinococcus-Thermus (9.5%; bands 5 and 15), Spirochaetes (4.8%; band 2) and unclassified_bacteria (9.5%; bands 4 and 6). However, most of the bands (42.9%; bands 2, 4, 5, 6, 12, 14, 15, 16, 18 and 19) were identified as uncultured (Table 2). The dominant bands 17 and 18 appeared in all sections of the GI tract (Table 1) and they belonged to Proteobacteria. Band 17 showed 100% similarity to Pantoea sp. G28A while band 18 was most closely related to uncultured Proteobacterium. Since the similarity of bands 1 and 22 was only 92% to Clostridium ramosum isolate M91, we suggest that these two bands might represent a new genera. Bands 10, 13, 20 and 21 showed high similarities to Acinetobacter sp. N15, Stenotrophomonas sp., A. radioresistens Philippines-11 and Enterobacter amnigenus, respectively. Band 7 showed 99% similarity to Lactococcus lactis subsp. lactis LMG, while bands 8, 9 and 11 showed 99, 100 and 98% similarities to Flavobacterium sp. VA24240/2003, Empedobacter sp. PH7-1, and Flavobacterium sp. GXW15-4, respectively.

The present study was carried out to increase our knowledge of the autochthonous microbiota in different parts of the digestive tract (stomach, pyloric caeca and three parts of the intestine) of yellow grouper. Stomach and pyloric caeca were included as these parts of the digestive tract have received less attention compared to the intestine. We performed a molecular analysis for cultivation-independent identification of the dominant microflora. Although complete 16S rDNA sequences are required for phylogenetic tree construction (Ludwig and Strunk, 1997), partial sequences of the most variable part of the 16S rRNA gene were, in our study, sufficient to determine the closest relatives for unknown sequences and to classify them into well-established phylogenetic groups (Muyzer et al., 1995; Huber et al., 2004). The phylogenetic tree (Fig. 2) shows clear segregation of the sequences and supports the dendrogram results (Fig. 1). Our results showed that the microbiota varied within the different parts of the alimentary tract as well as within individuals. The latter finding that the gut microbiota varied within individuals is in accordance with previous studies (Spanggaard et al., 2000; Ringø et al., 2006; Zhou et al., 2007; Liu et al., 2008). It is interesting to find that the average number of bands detected in each section decreases along the digestive tract in the present study, which is contrary to the reports of Lutjanus sebae and Ephippus orbis by Zhou et al. (2007), and the reasons require further investigation. The variation in microbial diversity within the different segments of the GI tract might be due to different pH and protease activity in the lumen (Yúfera and Darías, 2007). Yu et al. (2007) reported that pH in the digestive tract of

Table 2 Representative of bacteria or clones isolated from the gastrointestinal (GI) tract of adult yellow grouper cultured in cagesa Phylogenetic group Bacteroidetes

DeinococcusThermus

Band Closest relative no. (obtained from BLAST search) 8 9 11 5 15

Firmicutes

1 7 16 22

Proteobacteria 10 12 13 14 17 18 19 20 21 Spirochaetes Unclassified

2 4 6

Identity Accession (%) no.

Flavobacterium sp. VA24240/2003 99 (AY363052.1) Empedobacter sp. PH7-1 (EU276091.1) 100 Flavobacterium sp. GXW15-4 (AY582884.1) 98 Uncultured Planctomycete (EU044149.1) 95 Uncultured bacterium clone PS1_09E (DQ349083.1) Clostridium ramosum isolate M91 (AY699288.1) Lactococcus lactis subsp. lactis LMG (EU091475.1) Uncultured Streptococcus sp. clone BR01AA02 (DQ857242.1) Clostridium ramosum isolate M91 (AY699288.1) Acinetobacter sp. N15 (EF423372.1) Uncultured Curvibacter sp. clone BR01BF06 (DQ856778.1) Stenotrophomonas sp. (EU073955.2) Uncultured β-Proteobacterium (EF634276.1) Pantoea sp. G28A (EF432306.1) Uncultured Proteobacterium (EF706644.1) Uncultured γ-Proteobacterium (AB239032.1) Acinetobacter radioresistens Philippines-11 (EF446895.1) Enterobacter amnigenus strain (EF426859.1) Uncultured Spirochete (AB194657.1) Uncultured bacterium clone F6-37 (EU148669.1) Uncultured bacterium clone h5 (EF599658.1)

EU004796 EU004797 EU004799 EU004810

98

EU004803

92

EU004789

99

EU004804

95

EU004806

92

EU004809

100 100

EU004798 EU004792

99 100

EU004793 EU004794

100 100 96

EU004802 EU004805 EU004801

100

EU004791

100

EU004800

94 100

EU004790 EU004807

100

EU004808

a The accession no. of band 3 is EU004795, and not listed in Table 2 due to its close relation to 18S rDNA of lamprey after BLAST analysis.

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Fig. 2. Neighbour-joining phylogenetic tree showing the relationship of 21 16S rDNA gene sequences retrieved from DGGE profiles. The phylogenetic tree was constructed by ClustalX using the neighbour-joining method within the MEGA (3.1) package. Bootstrap values based on 1000 re-samplings display the significance of the interior nodes, and are shown at branch points. Only values display N 50% are given. The scale bar represents 5% sequence variation. Sequences of known species are shown in italics.

E. coioide was 3.8 ± 0.3, 6.9 ± 0.3, and 7.4 ± 0.3 for stomach, pyloric caeca and intestines, respectively. It was observed that the similarity in bacterial composition within the different segments of the GI tract was higher than the individual similarity in the present study, which is consistent with the report by Liu et al. (2008) in Atlantic salmon, indicating that physiological environment of the lumen might play a role less important in the GI microbiota than the individual frontier system does (Jensen et al., 2004). Acinetobacter sp. N15 was only detected in the stomach of yellow grouper. This finding indicates that the bacterium is adapted to the acidic gastric environment and resistant to the proteolytic enzymes. However,

its presence was not observed in all three fish indicating individual variation. Furthermore, the alkaline intestinal environment (pH approximately 7.5) might be selective for some intestinal bacteria such as uncultured bacterium clone F6-37, uncultured γ-Proteobacterium and A. radioresistens Philippines-11 as they were only detected in the three sections of intestine. Our study showed that the dominant autochthonous bacterial species belonged to Proteobacteria in the GI tract of adult yellow grouper, and these results are in accordance to previous studies demonstrating high levels of Proteobacteria in fish (Hovda et al., 2007; Kim et al., 2007; Yang et al., 2007). Band 7 from ST and PC showed 99%

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similarity to L. lactis subsp. lactis LMG (Rademaker et al., 2007). From the literature it has been shown that certain strains of L. lactis subsp. lactis produce nisin, an antimicrobial polypeptides with regulatory approval for use in certain foods (Joerger, 2003). In addition, it has been reported that L. lactis strain AR21 has been considered as biological control agents in aquaculture (Harzevili et al., 1998). Villami et al. (2002) also validated that L. lactis has strong stimulation and antibacterial properties for turbot. However, we cannot conclude that L. lactis subsp. lactis detected in the present investigation might be a probiotic bacterium in the adult yellow grouper cultured in cages. To clarify this further investigations are needed. Bacteroides have been suggested to contribute to the host's nutrition by supplying fatty acids and vitamins (Sakata, 1990). In the present study, bands 8, 9 and 11, which showed 99, 100 and 98% similarities to Flavobacterium sp. VA24240/2003, Empedobacter sp. PH7-1 and Flavobacterium sp. GXW15-4, respectively, and these bands belonged to Bacteroides. Whether the gut Bacteriodes identified in our study can contribute to the host's nutrition needs to be evaluated in future studies. Band 16 showed 95% similarity to the uncultured Streptococcus sp. clone BR01AA02, first isolated from the GI tract of rat by Dalby et al. (2006). Based on this result we suggest that this Streptococcus strain is well adapted to different microbial environments. However, as the bacterium has not been reported in fish before we cannot conclude that the bacterium belongs to the autochthonous microbiota in fish. Uncultured Curvibacter sp. (accession no DQ856778) has previously been reported by Dalby et al. (2006). In the present study, we identified one band (12) from PC that showed high similarity to the bacterium described previously (Dalby et al., 2006), and based on these observations we suggest the bacterium is well adapted to different environments. In the present study, some bands showed high similarity to uncultured bacteria. Band no. 15 showed 95% similarity to an uncultured Plactomycete previously described in a study investigating the microbial community succession and bacterial diversity in soils (Tarlera et al., 2008). Band 14 showed 100% similarity to the uncultured β-Proteobacterium (EF634276) reported in a study investigating the microbial diversity in scorpion intestine (Buthus martensii Karsch) (Wang et al., unpublished data, National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/). In a study on molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases by Frank et al. (2007), the authors present information on an uncultured Proteobacterium (EF706644). In the present study band 18 showed 100% similarity to this uncultured Proteobacterium. As this band occurred in all three fish and all segments investigated, we put forward the hypothesis that the bacterium is well adapted to the environment in the digestive tract of adult yellow grouper. The uncultured Spirochete (AB194657) has been reported by Shiina et al. (2006) in a study investigating the intestinal microbiota in Takifugu niphobles. Band 2 in the present study showed 94% similarity to this uncultured Spirochete and was detected in all gut segments but only in one fish. Information about the uncultured bacterium (EF599658) is available (Zhou et al., 2008) in a study investigating the effect of supplementation of fructooligosaccharide and yeast culture on growth and gastrointestinal microbiota in hybrid tilapia by 16S rDNA and DGGE. Sequencing of band 6 showed that this band was 100% similar to that reported by Zhou et al. (2008) and as an unique bacterium in the intestine of hybrid tilapia selectively stimulated by dietary short-chain fructo-oligosaccharides or yeast culture (Zhou et al., 2008). However, as the bacterium was only detected in ST, PC and PI, we suggest that the bacterium is adapted to these parts of the digestive tract of yellow grouper. Based on the results presented herein and in previous studies investigating the gut microbiota of fish, we conclude that different bacteria colonize different parts of the digestive tract and that the gut microbiota in fish are not as simple as earlier believed.

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