Molecular Analysis of Bacterial Communities and Detection of Potential Pathogens in a Recirculating Aquaculture System for Scophthalmus maximus and Solea senegalensis

Abstract

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments.

Identification of abiotic and biotic reductive dechlorination in a chlorinated ethene plume after thermal source remediation by means of isotopic and molecular biology tools

“…Putative chimeric sequences were removed using ChimeraSlayer . Taxonomic assignment of the operational taxonomic units (OTU) was performed by assign_taxonomy.py script with the Ribosomal Database Project (RDP) method (Wang et al, 2007;Martins et al, 2013). A sequence was defined as belonging to a particular OTU when the similarity level was at least 97%.…”

Development of a molecular methodology for fast detection of Photobacterium damselae subspecies in water samples

“…Water samples (three replicates containing 250 ml of water each) were collected at the Ria de Aveiro estuary (Aveiro, Portugalcoordinates 40°38′58.9″N/8°39′51.1″W) (this study) and from biological filters in a recirculation aquaculture system (RAS) for sole (SolBio1, SolBio2, SolBio3) and turbot (TurBio1, TurBio2, TurBio3) in July 2012 (Martins et al, 2013). The samples were filtered, stored in ice during 24 h and processed for DNA extraction in accordance with the procedure described by Martins et al (2013).…”

“…The samples were filtered, stored in ice during 24 h and processed for DNA extraction in accordance with the procedure described by Martins et al (2013). Briefly, estuarine and aquaculture samples were filtered through 0.2 μm pore polycarbonate membranes (Poretics, Livermore, CA, USA) and the total community (TC) DNA extraction was performed directly on the filter using an E.Z.N.A.…”

“…A barcoded pyrosequencing approach was used for in-depth bacterial composition analyses of estuary and aquaculture samples as previously described in Martins et al (2013), but using the most recent Greengenes release (Greengenes 13_8; ftp://greengenes.microbio.me/greengenes_ release/gg_13_5/gg_13_8_otus.tar.gz). Briefly, prior to pyrosequencing, aliquots of all three replicates were combined forming one DNA library for each treatment (estuary, turbot and sole).…”

“…Recently, well-established techniques to assess microbial diversity, such as PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and high throughput sequencing approaches, have been successfully used as a tool for pathogen detection (Ji et al, 2004;Martins et al, 2013;Petersen et al, 2007). Although previous molecular studies showed the ability to differentiate between the two P. damselae subspecies, none of these methodologies were tested directly against DNA samples extracted from environmental or aquaculture water samples.…”