Probiotic Phaeobacter for Marine Larviculture Figure 2. Localization of bacteria in cultures of Tetraselmis suecica. Phase-contrast (A,C) and fluorescence (B,D,E) micrographs. Co- culture of Tetraselmis suecica with Phaeobacter gallaeciensis dsRed (A,B), axenic T. suecica (C,D), co-culture of T. suecica with V. anguillarum gfp (E). Panel A and B show two single (left) and one dividing algal cell (right side), and a marine snow-like particle consisting of algae-debris which is colonized by red-fluorescent P. gallaeciensis. Red fluorescence of algae is due to chlorophyll. Panels C and D show an algal cell and particles from an axenic culture, recorded using the same settings as for the panels above. Panel E shows red-fluorescent algae cells and green- fluorescent V. anguillarum, which do not colonize particles, but remain in suspension as single, motile cells. doi:10.1371/journal.pone.0043996.g002 (101: P = 0.0518, 102: P = 0.0011, 103: P = 0.0517, 104: P = 0.0008). The marked difference in V. anguillarum inhibition by the wild type P. gallaeciensis and the TDA negative mutant suggested that TDA was a major effector molecule. However, TDA was not detected by chemical analysis of the Phaeobacter–Tetraselmis co- cultures, where triplicate cultures were each analyzed in triplicates with a detection limit ,50 nM TDA. The experiment and analysis were repeated with the same result. To determine if the wild type did indeed produce TDA in the algal cultures, a P. gallaeciensis carrying pPDA11 (tdaCp::gfp) was co-cultured with T. suecica. The tdaC promoter, indicative of TDA production, was Figure 3. Reduction of V. anguillarum in cultures of Tetraselmis suecica by Phaeobacter gallaeciensis. Colony-forming units of V. anguillarum inoculated at 101 cfu/ml (A) and at 104 cfu/ml (B) in presence of P. gallaeciensis wild type (&), in presence of the P. gallaeciensis TDA-negative mutant (m), and in the monoxenic control (.). doi:10.1371/journal.pone.0043996.g003 induced when growing on particles in a T. suecica culture, as indicated by Gfp fluorescence (Figure 4). Adding pure TDA to Tetraselmis cultures inoculated with V. anguillarum caused a complete killing of the Vibrio population, but also affected survival of algae (50 mM TDA). A 50-fold lower concentration (1 mM) had no effect on the algae, but temporarily reduced V. anguillarum below 10 cfu/ ml. A concentration of 50 nM TDA, which was the detection limit of the chemical analysis, did not have any effect (data not shown). V. anguillarum was completely eliminated in Nannochloropsis oculata cultures by wild type P. gallaeciensis within one or two days (Figure S3). However, V. anguillarum could only persist in dense cultures of N. oculata. In less dense N. oculata cultures V. anguillarum disappeared from the monoxenic control within 3 days. Consequently, the effect of the wild type P. gallaeciensis on V. anguillarum, as compared to the control, was significant in the experiment with high algae density (P = 0.001), but not in low density (P = 0.2106). Antagonism in rotifer cultures The concentrations of P. gallaeciensis and its mutant in the rotifer cultures were stable at about 106–107 cfu/ml, and no significant difference between the two strains was observed (P = 0.3689). The rotifers grew faster and reached higher densities in presence of P. gallaeciensis than in the axenic or monoxenic (V. anguillarum) controls (P,0.05) (Figure 5, Figure S4). Wild type P. gallaeciensis reduced V. anguillarum concentrations by 3 log units (P,0.01), in average from PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43996