Probiotic Phaeobacter for Marine Larviculture Figure 4. Expression of tdaC in co-culture with Tetraselmis suecica. Phase contrast (A) and fluorescence (B) micrographs of P. gallaeciensis pPDA11 (tdaCp::gfp) in co-culture with T. suecica. The two panels show the same seven algal cells of which some are dividing, and a marine snow-like particle which is colonized by P. gallaeciensis carrying the promoter-fusion on a plasmid. The green fluorescence of P. gallaeciensis on the particle shows that the gfp gene is expressed from the tdaC promoter, indicating production of TDA. doi:10.1371/journal.pone.0043996.g004 66105 to 96102 cfu/ml (Figure 6). The effect of the TDA- negative mutant on the concentration of the pathogen was not significant (P.0.05). Challenge trial Six days after the arrival of the embryos and inoculation, more than 50% of the larvae had hatched. Total cumulative hatching success was 79.2% (first trial 79.6% and second trial 78.7%). The initial mortality was lower in the first trial (16.6%) than in the second (24.8%). In the non-challenged and non-treated control, 34.7%69.8% (average 6 standard deviation) of the larvae had died by day 1, yet after the initial mortality only 2.8%60% of the larvae died between day 2 and day 10, reaching an accumulated mortality of 37.5%69.8% at day 10. The larvae challenged with V. anguillarum HI610 died rapidly and reached 100%60% Figure 5. Influence of bacterial strains on rotifer growth. Rotifer numbers in co-culture with P. gallaeciensis wild type (.), with the TDA- negative mutant of P. gallaeciensis (¤), with only V. anguillarum (m), and axenic rotifers (&), first experiment. All bacteria were inoculated at day 0. Both P. gallaeciensis strains promoted rotifer growth, whereas V. anguillarum had no influence. doi:10.1371/journal.pone.0043996.g005 Figure 6. Reduction of Vibrio anguillarum by Phaeobacter gallaeciensis in rotifer cultures. Mean values of two duplicate experiments: colony-forming units of V. anguillarum in co-culture with P. gallaeciensis wild type (m), with the TDA-negative mutant of P. gallaeciensis (.), and in the monoxenic control (&). doi:10.1371/journal.pone.0043996.g006 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43996