Probiotic Phaeobacter for Marine Larviculture Table 1. Bacterial strains and plasmids. Strain or plasmid Strains P. gallaeciensis BS107 (DSM17395) P. gallaeciensis BS107-Pda8 P. gallaeciensis dsRed V. anguillarum NB10 V. anguillarum HI610 Plasmids EZ-Tn5TM Transposome pAKN132 pUX-BF13 pPDA11 doi:10.1371/journal.pone.0043996.t001 Genotype or relevant markers Wild type CDS104961::EZ-Tn5, KanR MiniTn7(GmR)PA1/04/03DsRedExpress-a Serotype O1, cmR, PA1/04/03-RBSII-gfpmut3*-T1 Serotype O2a EZ-Tn5,R6Kcori, KanR.Tnp miniTn7(Gm)PA1/04/03DsRedExpress-a Helper plasmid: Tn7 transposase proteins tdaCp::gfp ligated into broad host range vector pRK415 Source or reference Ruiz-Ponte et al. 1998 [28] This study This study Croxatto et al. 2007 [35] Samuelsen & Bergh 2004 [36] Epicentre Biotechnologies Lambertsen et al. 2004 [43] Bao et al. 1991 [44] D’Alvise et al. (in preparation) was cultured in f/2-medium [47] based on Atlantic Seawater obtained from CCMP. N. oculata cultures were not aerated. Antagonism experiments were done as in T. suecica, but only one initial V. anguillarum concentration (104 cfu/ml) was tested. Two independent experiments with two different initial densities of algae (lower density: 46106 cells/ml, higher density: 26107 cells/ ml) were carried out. TDA analysis Samples of T. suecica – P. gallaeciensis co-cultures (20 ml) were extracted in 50-ml falcon tubes with 30 ml ethyl acetate (HPLC grade) containing 1% formic acid (HPLC grade) on a shaking table for 1 h. The samples were centrifuged at 8,000 x g, and 26 ml of the upper phase was transferred to a new Falcon tube and evaporated to dryness at 35uC with nitrogen flow. The samples were resuspended in 300 ml 85% acetonitrile, vortexed for 5 sec, placed in an ultrasonication bath for 10 min, vortexed again for 5 sec and filtered through a standard 0.22 mm PFTE syringe filter into a HPLC vial. Subsamples of 2 ml were then analyzed by UHPLC-TOFMS on a maXis G3 quadrupole time of flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray (ESI) ion source which was connected to an Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA). Sepa- ration was performed at 40uC on a 100 mm62.1 mm ID, 2.6 mm Kinetex C18 column (Phenomenex, Torrance, CA) equipped with Kinetex pre-column using a water-acetonitrile gradient (both buffered with 20 mM formic acid) at a flow of 0.4 ml min21 starting at 10% acetonitrile and increased to 100% in 10 min, keeping this for 3 min. MS was operated in ESI+ with a data acquisition range of m/z 100–1000 at a resolution of 40,000 FMWH, the MS was calibrated using 20 mM sodium formate infused prior to each analytical run, providing a mass accuracy better than 1.5 ppm. TDA was detected and quantified from the extracted ion chromatograms of the [M+H]+ ions (6 m/z 0.001). For quantification, T. suecica cultures were spiked to a final concentration of 4800, 2400, 1600, 800, 320, 160, and 0 (blank) nM TDA (BioViotica, Dransfeld, Germany) by adding a maximum of 80 ml TDA-acetonitrile solution, and treated as described above. Spiked samples were left at room temperature for at least 1 h prior to extraction. The method was validated on 3 different days using spiked samples as described, and no false positives or negatives were recorded. Relative standard deviation was 30% and the limit of detection was estimated to be ,50 nM (signal/noise 5:1), based on the blank samples and lower calibration points. Sensitivity was greatly influenced by the age of the UHPLC column since TDA tailed (although a new pre- column was used) on columns which had been in use for only a few weeks. Samples from two individual biological experiments were analyzed independently. Absence of TDA in static cultures of the TDA-negative mutant BS107-Pda8 was confirmed by UHPLC-TOFMS analysis. Phaeobacter antagonism in rotifers, Brachionus plicatilis A stock of the rotifer B. plicatilis (L-type) was obtained from Reed Mariculture (Campbell, CA). Axenic rotifers were attained by disinfecting approximately 50 amictic rotifer eggs in 1 ml of a strong antibiotic solution (150 mg/ml Tetracycline, 300 mg/ml Kanamycin, 60 mg/ml chloramphenicol, 1000 U/ml Penicillin in ASW) for 2 days. The hatched rotifers were filtered onto a sterile 50-mm polyamide mesh, rinsed with ASW and from then on fed with concentrated axenic T. suecica. No experiment with N. oculata as rotifer feed was conducted, since survival of V. anguillarum in presence of N. oculata was very variable (see Results section). Rotifer densities were determined by counting in a Sedgewick- Rafter counting chamber. Before counting, the cultures were thoroughly mixed, and 100 ml 5% formaldehyde added to a 1 ml sample. To set up co-culture experiments, axenic rotifer cultures were divided into eight 20-ml batches in 50-ml centrifuge tubes. The initial rotifer concentrations were 94 /ml (first replicate) and 30 /ml (second replicate). For each replicate, duplicate cultures were inoculated with washed wild type and mutant P. gallaeciensis at approximately 56107 cfu/ml. On the next day, all cultures except the axenic controls were inoculated with 104 cfu/ml V. anguillarum. All rotifer cultures were fed daily with 10-fold concentrated T. suecica (1–2 ml depending on average rotifer density), so that the algae concentration was at approximately 106 cells/ml after feeding and did not drop below 26105 cells/ml. The rotifers were counted daily, concentrations of V. anguillarum and P. gallaeciensis were determined, and axenicity of the negative controls was checked. The rotifer culture samples for enumeration of bacteria were homogenized by grinding and repeated pipetting through a 100-ml pipette tip. This was compared to homogenization with an Ultra-Turrax T25 (IKA, Germany) at 16,000 rpm and no significant differences in bacterial counts were found (P = 0.74). PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43996