inhibitory concentrations in Phaeobacter-colonized particles. TDA is likely concentrated within and around the particles, adhering to organic mass of the particle, or being kept within the EPS produced by P. gallaeciensis. From an ecological point of view, for a particle-associated marine bacterium the production of an antagonistic compound would be more efficient if the compound was not dispersed, but kept in the close vicinity to fend off possible competitors. Although TDA-producing Phaeobacter and Ruegeria spp. are likely to be already present in larviculture systems, their antagonistic properties, which may depend on growth conditions, are probably different from P. gallaeciensis BS107 [22,26]. A preliminary experiment to this study showed that only a few of the tested Phaeobacter and Ruegeria strains were antagonistic in T. suecica cultures, whereas all of them did account for large inhibition zones in agar-based assays. Therefore, introduction of P. gallaeciensis BS107 in algae and rotifer cultures would likely enhance larval survival even though other Roseobacters are already present in the system. Its growth-promoting effect on rotifers may offer an unexpected additional advantage. Whether that is due to the nutritive value of the bacteria or to a potential role in the rotifer gut is not known. In the present study rotifer growth was not adversely affected by V. anguillarum. Nevertheless, a V. anguillarum strain was reported to cause pronounced growth inhibition of rotifers under suboptimal feeding schemes [60], which could possibly be remediated by P. gallaeciensis. It cannot be predicted, if and how other pathogens in algae and rotifer cultures would be suppressed by P. gallaeciensis, however a range of fish pathogens are inhibited in vitro by P. gallaeciensis [22,25] indicating that it likely could protect against other pathogens than V. anguillarum. Porsby et al. [61] have addressed the concern that resistance to TDA could develop, and found, using several experimental approaches, that no resistant mutants or variants could be isolated, neither from short-term selection cultures containing different concentrations of TDA nor from long-term adaptation cultures (.300 generations) containing increasing concentrations of TDA. A recent study demonstrated that P. gallaeciensis, when incubated with p-coumaric acid, produced potent algicides, the roseobactins, which were effective against different microalgae, among them T. suecica [62]. P-coumaric acid is a degradation product of lignin, which is contained not only in terrestrial plants, but also in algae. Production of the algicides was only possible in concentrations of p-coumaric acid above 0.4 mM. The authors hypothesized that P. gallaeciensis contributes to algal health and growth by secreting TDA and phenylacetic acid, but will produce algicides in presence of p-coumarate, which is an indicator for algal senescence, in order to utilize the algal biomass for its own growth [62]. However, in the present study no negative effect of P. gallaeciensis on algal growth was observed. Possibly the levels of p-coumaric acid in the cultures of microalgae were too low for roseobacticide production. The environmental ecological niche of P. gallaeciensis has not yet been described, although studies of Rao et al. [63–65] indicate its preference for the surface of macroalgae, which during their decay may account for higher local concentrations of p-coumaric acid than microalgae. In an aquaculture farm, Phaeobacter spp. have been found to ‘‘naturally’’ occur on solid surfaces, whereas only Ruegeria spp. were inherently associated with algae cultures [26]. Based on the present findings, it is hypothesized that P. gallaeciensis can be used in marine larviculture, as a means of References Probiotic Phaeobacter for Marine Larviculture controlling the ambient, potentially harmful microbiota in cultures of rotifers and microalgae, and as a prophylaxis against vibriosis in fish larvae. Supporting Information Figure S1 Concentrations of Nannochloropsis oculata and Phaeobacter gallaeciensis in the co-cultures. Colony- forming units of P. gallaeciensis wild type (N) and the TDA-negative mutant (%), and concentrations of N. oculata with V. anguillarum (m), N. oculata with P. gallaeciensis wild type (.), N. oculata with P. gallaeciensis TDA-negative mutant (¤), and axenic N. oculata (&) in the dense (A) and less dense (B) cultures. (TIF) Figure S2 Reduction of Vibrio anguillarum by Phaeo- bacter gallaeciensis in cultures of Tetraselmis suecica. Colony-forming units of V. anguillarum inoculated at 102 cfu/ml (A) and at 103 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 (.). (TIF) Figure S3 Reduction of Vibrio anguillarum by Phaeo- bacter gallaeciensis in cultures of Nannochloropsis oculata. Colony-forming units of V. anguillarum in presence of P. gallaeciensis wild type (m), in presence of the P. gallaeciensis TDA- negative mutant (.), and in the monoxenic control (&), in dense (36107 cells/ml; A) and less dense (1–76106 cells/ml; B) cultures of N. oculata. (TIF) Figure S4 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 (&), second experiment. All bacteria were inoculated at day 0. Both P. gallaeciensis strains promoted rotifer growth, whereas V. anguillarum had no influence. (TIF) Figure S5 Mortality of cod larvae during the challenge trials. Mean values of two independent triplicate experiments with error bars indicating standard deviations. The single-larvae cultures were simultaneously inoculated with P. gallaeciensis wild type and V. anguillarum (T5, N), or with the TDA-negative mutant of P. gallaeciensis and V. anguillarum (T6, %). Unexposed larvae and larvae exposed to single bacterial strains acted as controls: Negative Control (T1, &), only V. anguillarum (T2, m), only P. gallaeciensis wild type (T3, .), and only P. gallaeciensis TDA-negative mutant (T4, ¤). (TIF) Acknowledgments We would like to thank Debra Milton, University of Umea˚, for providing the chromosomally tagged Vibrio anguillarum NB10. We also thank Paul Henning Løvik for assistance with the challenge experiments. Author Contributions Conceived and designed the experiments: PD SL HIW KFN ØB LG. Performed the experiments: PD SL MJP KFN HIW ØB. Analyzed the data: PD SL KFN HIW ØB LG. Wrote the paper: PD LG. 1. Olafsen JA (2001) Interactions between fish larvae and bacteria in marine aquaculture. Aquacult 200: 223–247. 2. Toranzo AE, Magarinos B, Romalde JL (2005) A review of the main bacterial fish diseases 3. in mariculture systems. Aquacult 246: 37–61. PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43996