Challenge trial The protocol was adapted from [39,40]. Cod (Gadus morhua) embryos were obtained from the commercial hatchery Havlandet AS, in Florø, Western Norway. Transport of the embryos in polystyrene containers at around 8uC took 4 to 5 hours in total by boat and car. Two independent replicates of the challenge trial were conducted. The embryos used in the first trial were disinfected with Buffodine (Evans Vanodine, Preston, UK), the embryos for the second trial were left untreated. Upon arrival, the embryos were randomly picked and distributed to the wells of 24- well dishes (Nunc, Roskilde, Denmark) filled with 2 ml 80% autoclaved, aerated seawater, placing one embryo in each well. In each trial three dishes for each treatment (72 embryos) were prepared and inoculated immediately. The six treatment groups are listed in Table 2. All inocula were prepared in a volume of 100 ml, and the strains were not mixed before inoculation. Initial bacterial concentrations were 16106 cfu/ml for V. anguillarum HI610 and about 107 cfu/ml for the P. gallaeciensis strains. The plates were incubated in the dark at 7uC. The day when 50% of the larvae had hatched was defined as day 0, which was 6 days after the start of the experiment. Dead larvae were registered daily for 14 days. Statistics Differences between concentrations of bacteria or algae were assessed using repeated measures ANOVA after log-transforma- tion. Tukey’s multiple comparison test was used for pairwise comparisons. To address the effects of P. gallaeciensis presence on concentrations of V. anguillarum, initial values (day 0) were omitted in the analysis and the experiments were analyzed separately. Rotifer numbers were not log-transformed before applying ANOVA, and initial values were omitted. Numbers of P. gallaeciensis and homogenization methods were compared using paired t-tests after log-transformation. The cumulative mortalities in the challenge trials were compared at day 10, prior to the onset of starvation towards the end of the experiment. A chi-square test for 22 contingency tables was implemented, using the software R, version 2.13.1 (R Foundation for Statistical Computing, Vienna, Austria). Results Antagonism in algae cultures Both wild type and mutant P. gallaeciensis colonized the cultures of T. suecica and N. oculata. In T. suecica cultures, P. gallaeciensis reached 107 cfu/ml (Figure 1). In the dense cultures of N. oculata, P. gallaeciensis concentrations were approximately 56106 cfu/ml, and in the less dense cultures approximately 86105 cfu/ml (Figure S1). The wild type Phaeobacter reached slightly higher numbers in Table 2. Group numbers and treatments in the challenge trial. Group number Treatment Probiotic Phaeobacter for Marine Larviculture Figure 1. Concentrations of Tetraselmis suecica and Phaeobacter gallaeciensis in the co-cultures. Means and standard deviations of eight (¤) and experiments: the TDA-negative colony-forming mutant (N), units and of concentrations P. gallaeciensis of wild T. suecica type with V. anguillarum (.), T. suecica with P. gallaeciensis wild type (&), T. suecica with P. gallaeciensis TDA-negative mutant (m), and axenic T. suecica (%). The P. gallaeciensis strains were inoculated at 107 cfu/ml and remained as a steady population, while the algae went from late log into stationary phase. doi:10.1371/journal.pone.0043996.g001 T. suecica than the TDA-negative mutant (P = 0.0211). This same slight difference was seen in one of the two Nannochloropsis experiments (P = 0.0335, P = 0.9259). P. gallaeciensis did not affect growth of the algae T. suecica (P = 0.9977) and N. oculata (P = 0.9919). Particles consisting of dead T. suecica and algal cell walls that were shed during cell division served as habitat for rosette forming P. gallaeciensis that formed dense biofilms on the particles (Figure 2 A–D). V. anguillarum effectively colonized all Tetraselmis cultures that were not inoculated with P. gallaeciensis and numbers increased by up to 2.7 log units within the first day (Figure 3, Figure S2) and reached an average of 36106 cfu/ml after 5 days. V. anguillarum did not colonize particles in the algae cultures, but remained in suspension (Figure 2 E). The numbers of V. anguillarum decreased markedly in presence of wild type P. gallaeciensis (Figure 3, Figure S2). Vibrio reductions were in the order of 3 log units, as compared to the monoxenic controls with only V. anguillarum, and complete elimination of the lowest Vibrio inoculum was achieved in 3 out of 4 replicates (Figure 3A). The effects of wild type P. gallaeciensis on V. anguillarum were highly significant in all Tetraselmis experiments, as compared to the controls (P,0.001) and to the mutant (P,0.001). Presence of the TDA-negative mutant did decrease concentrations of the pathogen by about one log unit, although this was only significant (a = 0.05) for two of the initial Vibrio concentrations T1 T2 T3 T4 T5 T6 doi:10.1371/journal.pone.0043996.t002 Negative control; no bacteria added Positive control; V. anguillarum O2a HI610 106 cfu/ml Wild type P. gallaeciensis BS107 (DSM17395) ,107 cfu/ml TDA-mutant P. gallaeciensis BS107-Pda8 ,107 cfu/ml V. anguillarum O2a HI610 106 cfu/ml and wild type P. gallaeciensis BS107 (DSM17395) ,107 cfu/ml V. anguillarum O2a HI610 106 cfu/ml and TDA-mutant P. gallaeciensis BS107-Pda8 ,107 cfu/ml PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43996