Research Article: Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.)

Date Published: November 18, 2011

Publisher: Springer

Author(s): Maartje AHJ van Kessel, Bas E Dutilh, Kornelia Neveling, Michael P Kwint, Joris A Veltman, Gert Flik, Mike SM Jetten, Peter HM Klaren, Huub JM Op den Camp.


The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.

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The intestine is a multifunctional organ system involved in the digestion and absorption of food, electrolyte balance, endocrine regulation of food metabolism and immunity against pathogens (Ringo et al. 2003). The gastrointestinal (GI) tract is inhabited by many different micro-organisms. As in mammals, this dynamic population of micro-organisms is of key importance for the health of the piscine host (Ringo et al. 2003; Rawls et al. 2004). The gut is also a potential route for pathogens to invade and infect their host. The micro-organisms in the GI tract are involved in the protection against these pathogens by the production of inhibitory compounds and competition for nutrients and space. As in mammals, the intestinal microbiota of fish can influence the expression of genes involved in epithelial proliferation, nutrient metabolism and innate immunity (Rawls et al. 2004). Due to their importance in animal health, the investigation of the intestinal microbiota of fish is highly relevant for aquaculture practice. We investigated the diversity of the microbiota in common carp (Cyprinus carpio), one of the most cultivated freshwater fish species worldwide (FAO, 2011).

The use of next generation sequencing technologies for sequencing of a mixture of 16S rRNA amplicons amplified with primer sets targeting as many phyla as possible will give a much broader taxonomic overview compared to the use 16S rRNA hypervariable regions (Kysela et al. 2005). To avoid missing a certain group of bacteria, different primer sets (Table 1) were used targeting as much species as possible. Obtained amplicons from all different reactions were mixed and sequenced using Roche 454 titanium technology and this revealed a high microbial diversity in the GI tract of common carp (Cyprinus carpio). It should be noted that the use of multiple primer sets biases the number of sequences belonging to the identified taxa. The number of obtained sequences belonging to a specific group may not be representative for their abundances in vivo; therefore no quantitative statements could be made.

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.

The authors declare that they have no competing interests.