Almost all Fusobacterial 16S rRNA sequences, 8081 out of 8085, from the carp GI tract belonged to the genus Cetobacterium. Cetobacteria were not observed in most culture-dependent studies done on the GI tract microbiota of common carp (Sugita et al. 1990; Namba et al. 2007), only Tsuchiya et al. (2008) described the isolation and characterization of Cetobacterium somerae from the GI tract of five different fresh water fish, including carp. Cetobacterium was also shown to be present in the gut of zebrafish (Rawls et al. 2006), a cyprinid species closely related to common carp. Furthermore, Cetobacterium isolated from human faeces performed fermentation of peptides and carbohydrates (Finegold et al. 2003). It has also been shown that Cetobacterium can produce vitamin B12 (Tsuchiya et al. 2008). This can wel explain why carp do not have a dietary vitamin B12 requirement (Sugita et al. 1991). The combination of a fermentative metabolism together with vitamin production may explain the relevance of Cetobacterium sp. in the GI tract of carp.
Another well represented group within the obtained sequences were the Bacteroidetes (22% of obtained sequences), a phylum known for a fermentative metabolism and degradation of oligosaccharides derived from plant material (Van der Meulen et al. 2006). The Bacteroidetes sequences found could be divided into 4 major groups (Additional file 1): Marinilabiaceae (or Cytophaga, 13%), Porphyromonadaceae (39%), Bacteroidaceae (15%) and Bacteroidales_incertae_sedis (33%). All Marinilabiaceae sequences belonged to the same group: the Anaerophaga. This relatively newly discovered group of bacteria includes strictly anaerobic, chemo-organotrophic, fermentative bacteria (Denger et al. 2002). These bacteria may play an important role in the fermentation of food in the GI tract of herbivorous carp since anaerobic fermentation is generally an important step in the digestion of plant material. Porphyromonadaceae are present in the GI tract of several organisms including human and pigs (Mulder et al. 2009). These bacteria can be pathogens but in this niche they are most probably involved in fermentation. By using labelled glucose, it has been shown that these bacteria are involved in saccharide fermentation (Li et al. 2009). Also the Sphingobacteria present could also be involved in oligosaccharide degradation since Sphingobacterium sp. TN19, an endosymbiont in insects, contains a xylanase encoding gene (Zhou et al. 2009). Xylanases are involved in the breakdown of xylan, a polysaccharide found in plant material. The presence of fermenting microorganisms is not suprising, since it has been shown that the GI microbiota of carp is able to ferment different oligosacharides (Kihara and Sakata 2002).
The obtained Planctomycete sequences (13% of classified sequences) could be divided into 9 groups (Additional file 1); Gemmata, Pirellula, Schlesneria and Zavarzinella were the most abundantly found groups. Gemmata and Pirellula are aerobic chemo-heterotrophs, Schlesneria are chemo-organotrophic facultative aerobes and Zavarzinella are aerobic heterotrophs. The presence of Planctomycetes has been shown before in gut microbiota of fish and other organisms (Ley et al. 2008; Rawls et al. 2006). The exact function of these bacteria in the GI tract is not clear, possibly these bacteria live from products of the metabolism of other bacteria. However, the relatively high abundance of Planctomycetes in close association with other organisms such as kelp, marine sponges and prawn (Bengtsson and Ovreas 2010; Pimental-Elardo 2003; Fuerst et al. 1997; Lahav et al. 2009) suggests a more important role. Possibly, these bacteria are involved in the metabolism of complex compounds. In a recent study, in which the close association of Planctomycetes with the brown seeweed kelp (Laminaria hyperborea) was investigated, it was hypothesized that these bacteria are degraders of sulfated polysacharides produced by kelp (Bengtsson and Ovreas 2010). The organisms found in the biofilm at the plant's surface were mainly members of the lineage Pirellulae (which includes Pirellula, Rhodopirellula and Blastopirellula). The genome sequence of Rhodopirellula baltica SH1 revealed many genes involved in the breakdown of sulfated polysaccharides (Glockner et al. 2003). Possibly, the heterotrophic Planctomycetes found in carp gut confer a similar ability of polysaccharide breakdown to the host. Furthermore, a separate lineage within the Planctomycetes, the anammox bacteria, were present in the carp gut (Figure 1). These anaerobic bacteria, described before in fish gut (Lahav et al. 2009), are involved in nitrogen cycling. Together with the Nitrosomonas and Nitrospira species (also present within the obtained sequences, Figure 1), ammonium can be converted into dinitrogen gas. The removal of nitrogenous compounds from aquaculture systems is one of the most important challenges in aquaculture. The presence of nitrogen cycling bacteria in fishes could offer new in situ solutions for the removal of nitrogen from aquaculture systems.
The Gammaproteobacteria sequences found could be classified as bacteria that are known members of the GI microbiota of many organisms including fish (Wu et al. 2010; Lee et al. 2009). Most Gammaproteobacteria (Additional file 1) found in carp belonged to the Aeromonas group. Members of the genus Aeromonas are mainly distributed in freshwater and sewage, often in association with aquatic animals (Cahill 1990; Sugita et al. 1995). They can cause a diverse spectrum of diseases in both warm- and cold-blooded animals but they also appear to be aquatic envrionments including in fish intestines (Sugita et al. 1995). Other abundantly present members among the Gammaproteobacterial sequences were the genera Enterobacterium and Vibrio. Enterobacterium spp. are widespread in GI tracts of various organisms (Wu et al. 2010), whereas Vibrio sp. are commonly found in aquaeous environments, aquaculture systems and in association with eukaryotes (Wu et al. 2010; Thompson et al. 2004). This phylum also contains Plesiomonas and Acinetobacter species that have been found in carp before (Sugita et al. 1991; Cahill 1990). Furthermore, the presence of high number Proteobacteria has also been shown for zebrafish, which is closely related to carp (Rawls et al. 2006). Also in other fish belonging to the Cyprinidae members of the Gammaproteobacteria (Enterobacter and Citrobacter species) were found (Ray et al. 2010). Enterobacter and Citrobacter species isolated from the GI tract of Indian carp (Cyprinidae) were shown to produce amylase, cellulase and protease (Ray et al. 2010), which indicates that these bacteria can be actively involved in the digestion of food in carp guts.
Another abundant phylum within our amplicon sequences were the Verrucomicrobiae (including subdivision 3 and 4 (Optitiae)). Verrucomicrobiae species are most commonly found in aquatic environments but are also known members of the gut microbiota in different organisms including seacucumbers (Echinodermata), termites and humans (Wagner and Horn 2006). These bacteria seem to be well adapted to live with eukaryotes, since the genome of some verrucomicrobial species contain a protein secretion system which mediates interactions between eukaryotic and bacterial cells (Wagner and Horn 2006). Verrucomicrobiae usually have an aerobic or obligate anaerobic fermentative metabolism (Schlesner et al. 2006) and could also play a role in the metabolism of plant beta glycans in carp GI tract. Indeed, Pedosphaera parvula Ellin514 (Verrucomicrobia subdivision 3) contains a cellulase in its genome (Kant et al. 2011). Ruminants and postgastric fermenters depend on bacteria containing this gene for the fermentation of plant material in which cellulose is converted to β-glucose. Various fish species do have a cellulase activity in their guts (Saha and Ray 1998; Saha et al. 2006; Ray et al. 2010) which decreases after antibiotic treatments (Saha and Ray 1998), indicating that the GI microbiota is responsible for this activity.
Clostridia and Bacilli, both present in the microbiota of the sampled fish (Figure 1), are members of the phylum Firmicutes. Representative genera of this phylum, including Clostridium, Bacillus, Streptococcus and Staphylococcus spp., have been shown in the microbiota of fish before (Navarrete et al. 2009; Rawls et al. 2006; Ray et al. 2010; Sugita et al. 1990). Gut isolates belonging to the Firmicutes fermented various carbon sources (Ray et al. 2010), again implicating a role in the utilization of plant materials.
To our knowledge, this is the first detailed analysis of the microbiota of common carp by high throughput sequencing. Our culture independent investigation of the microbial flora of the GI tract gives a more reliable and more complete characterization of the diversity of compared to other studies. Furthermore, great similarities between the microbiota in carp and zebrafish (a closely related fish species) were shown (Roeselers et al. 2011). The GI microbiota is important for the health of the animal and therefore this study could be relevant for aquaculture. Furthermore, the presence of different nitrogen cycling bacteria in the GI tract of fish could offer new possibilities in the removal of nitrogen compounds in aquaculture. The microbiota of the GI tract plays an important role in the digestion and chemical processing of the food as exemplified by the large number of bacteria involved in vitamin production and fermentation of saccharides and beta-glycans (cellulose, hemicellulose) (Table 2). The presence of many different types of bacteria in the herbivorous carp could be predicted since it has been shown that eukaryotes with an herbivorous diet have a higher microbial diversity (Ley et al. 2008). However, the carp in our study were fed commercially available food with high protein and low plant content. According to their GI microbiota, these fish are very well able to adapt to a more herbivorous diet and this is probably also the case for other cultured fish. Therefore it could be possible to lower the amount of fish meal, one of the major components of fish food, in the food for these fish. Furthermore, it shows that the gut microbes are probably important in the protection of the host against pathogens which should be taken into consideration in aquaculture where a lot of antibiotics are used (Cabello 2006). It is known that antibiotics have a negative effect on the microbial community in the gut of human (Dethlefsen et al. 2008) and this is possibly also the case for fish. The routinely use of antibiotics may be harmful for the animal. A better knowledge about the microbiota in fish guts is important; it can lead to a better health of cultured fish and therefore to a more efficient fish culture.