Effects of mussel biofiltration on marine protistan community composition using a next-generation sequencing (NGS) methodology
Experimental set-up and sampling. The possibility to use mussels as a mitigation tool to prevent phytoplankton spring bloom noxious effects on farmed fish was tested during a 35-day mesocosm experiment in a fish farm in the north of France during the  spring phytoplankton bloom (16/04 – 21/05/2013). The experimental setting has been described in detail in Delegrange et al. (2015) and consisted of three 5 m3 mesocosms. The experimental system was supplied with a mix of natural coastal North Sea water mixed with seawater released from the Gravelines Power Plant cooling system (France) heated +10 °C compared to in situ temperature and chlorinated (1%). Bulk seawater was filtered by blue mussels (Mytillus edulis) in a biofiltration tank (M) before fuelling a test tank (T) containing juvenile sea bass (Dicentrachus labrax). The third tank was used as a control (C), and contained juvenile sea bass reared in bulk seawater (i.e. not biofiltered). In all the tanks, the flow rate was set at 5 m3 h-1 allowing for a total water renewal every hour, and photoperiod followed natural day:night cycle.  Temperature, salinity, pH, dissolved oxygen, turbidity, chlorophyll were measured a twice a week while phytoplankton in C and T tanks were investigated at days 0, 3, 10, 17, 24, 31, 35 and are reported elsewhere (Delegrange et al. 2015). In this study, sampling took place at days 0, 7, 14, 21, 28, 35. Water samples (500-1000 ml) collected in C and T tanks were screened with a 200 μm mesh to retain larger particles and most metazoa, then filtration was performed on 3µm nucleopore filters (47 mm diameter) using a low filtration pressure in order to minimize organism disruption. The filters were immediately frozen in liquid nitrogen and then stored at -80 °C until analysis.  Molecular analysis and data processing. Bulk DNA of the retained material on the filters, was extracted and purified, after filtration, with the PowerWater® DNA isolation kit (Mobio Laboratories Inc, CA, USA), following the manufacturer’s protocol. The quantity of the DNA was between 0.78 and 10.5 ng μL-1 as measured by the Qubit ® 2.0 Fluorometer (Thermo Fischer Scientific Inc, Massachusetts, USA).  The DNA samples were amplified using the two universal eukaryote primers 18S-82F (5’-ACCAGACTTGCCCTCC-3’) (Lopez-Garcia et al. 2003) and Euk-516r (5’-ACCAGACTTGCCCTCC-3’) (Amann et al. 1990), and two universal bacterial primer S-D-Bact-0341-b-S-17 (CCTACGGNGGCWGCAG) and S-D-Bact-0785-a-A-21 (GACTACHVGGGTATCTAATCC) (Klindworth et al. 2012). The eukaryote primers amplify a domain around 490 bp of the V2-V3 18S rDNA regions, while the prokaryote primers have been designed to amplify a 465 bp domain from the V3-V4 regions of the 16S rDNA. The libraries were constructed by 'Genes Diffusion' company (Lille). First, Polymerase Chain Reaction (PCR) were carried out, using the two sets of universal primers, according to standard conditions for Platinum Taq High-Fidelity DNA Polymerase (Invitrogen, Carlsbad, CA, USA), with 5 ng of environmental DNA as template, using the GeneAmp PCR System Apparatus (Applied Biosystems, Foster City, CA, USA). The PCR cycle conditions were the following: after the denaturation step at 95 oC for 5 min, 30 cycles of amplification were performed at 95 oC for 30 s, 50 oC for 30 s, and 72 oC for 1 min. A final extension step of 7 min at 72 oC was included. A second PCR were carried out on each sample independently, in order to add a 10 bp (base pairs) tag sequence specific to each sample. Finally, all the amplicons were mixed together stoichiometrically before their sequencing in one run of MiSeq PE 2x300 Illumina (CNRS-UMR8199, Lille).  The sequences were processed using the MOTHUR v1.34.0 software (Schloss et al. 2009) following the standard operating procedure (Schloss et al. 2011).