accumulated mortality. Treating Vibrio-challenged larvae with wild type P. gallaeciensis caused a significant reduction in accumulated mortality by day 10 to 12.5%62.0% which was not only lower than in the challenged larvae but also lower than in the non- challenged (37.5%). 96.1%61.1% of the hatched larvae that had received wild type P. gallaeciensis survived until day 10, when starvation set in (Figure 7, Figure S5). The larvae exposed to only P. gallaeciensis wild type or mutant had a cumulative mortality of 12.1%63.1% at day 10. The TDA-negative mutant of P. gallaeciensis did reduce accumulated mortality of the challenged larvae to 68.8%630.4% (Figure 7, Figure S5), but was not nearly as efficient as the TDA-producing wild type. Discussion The present study demonstrates that Phaeobacter gallaeciensis is harmless and beneficial for the early life stages of cod. Equally important, P. gallaeciensis is highly efficient at preventing infections with V. anguillarum, and this probiotic effect can be achieved at the low temperature (7uC) used for the cod embryos and yolk sac larvae. It has previously been demonstrated that a Phaeobacter sp. can protect turbot larvae against vibriosis at higher temperatures (18uC) [29]. Non-infected larvae showed some level of initial mortality, which may have been due to opportunistic bacteria introduced with the embryos. Both challenged and unchallenged cod larvae exposed to P. gallaeciensis had a significantly lower initial mortality, indicating that the inherently occurring microbiota of the chorion may be controlled by the probiont. A key question in the use of probiotics in aquaculture is how and where the probiont should be introduced to the system. Several studies have emphasized the potential role of feed organisms as a vehicle for probiotic bacteria [29,48–51], or the potential of probiotic bacteria to control pathogenic bacteria in the feed cultures [51–53]. The majority of studies have focused on intestinal probiotic bacteria, and aimed at health-promoting effects within either the reared animal or the feed organism. In contrast, the present study takes a systems approach to preventing bacterial disease in aquaculture organisms, aiming at microbial control throughout the environment of the reared organism and the lower trophic levels of the production. Here it was found that Figure 7. Mortality of cod larvae during the challenge trials. Mean values of two independent triplicate experiments. The single- type larvae and cultures V. anguillarum were simultaneously (T5, N), or with inoculated the TDA-negative with P. gallaeciensis mutant of wild 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, ¤). doi:10.1371/journal.pone.0043996.g007 Probiotic Phaeobacter for Marine Larviculture cultures of two aquaculture-relevant algae and of the rotifer B. plicatilis can be colonized by P. gallaeciensis without compromising their growth, and that P. gallaeciensis in these cultures will strongly reduce, or eliminate fish-pathogenic V. anguillarum. Introducing P. gallaeciensis at this trophic level is very promising, since live feed is a common source of opportunistic pathogens [1,5,10,11]. These findings corroborate the hypothesis from a previous study, that algae and rotifers in aquaculture can be cultured together with probiotic Roseobacters, and thus prevent proliferation of pathogens [22]. A reduction of a pathogenic Vibrio sp. by 3 log units, as it was achieved in the present study, is very promising in terms of larval health promotion, as only a one log reduction of the bacterial load in rotifers through UV radiation resulted in higher survival of turbot larvae [54]. Using probiotic bacteria, as compared to UV treatment, offers the advantage that nutrients are consumed, niches are occupied, and rapid re-growth of pathogens is prevented. It should be noted that the present study was done using gnothobiotic systems to rule out the influence of the inherent microbiota of algae and rotifer cultures. Thus, it cannot be determined, if or to what extent P. gallaeciensis would affect the inherent microbial communities of algae and rotifer cultures. The inhibition of V. anguillarum by a Phaeobacter sp. in a model aquaculture setting has been studied once before: Planas et al. [29] demonstrated that mortality in turbot larvae infected with V. anguillarum could be reduced by Phaeobacter sp. 27-4. A duplicate tank setup was used, and both the pathogen and the probiont were enclosed in rotifers and fed to the larvae. In spite of delivery with the feed, the probiont was only found in the lumen of the larval gut and did not colonize the intestinal epithelium. In contrast to this, the present study did not aim at a probiotic effect in the intestinal tract of the larvae, but assesses the potential of Phaeobacter to eliminate the pathogen in the environment of the larvae or embryos. It should be mentioned that, as larvae start to drink shortly after hatching [55], an intestinal presence of pathogens and probionts can occur. Phaeobacter sp. 27-4 is a TDA-producer, however, as opposed to P. gallaeciensis BS107, it produces TDA only in stagnant culture [26], suggesting that its TDA production may be more delimited and that BS107 could be more antagonistic in vivo. In the challenge trial the TDA-negative mutant reduced the initial mortality as efficiently as the wild type, but could not prevent infection in the majority of the larvae. The probiotic effect of the mutant could be explained by competition for nutrients, space, or other resources or it could be attributed to a direct immunostimulatory effect on the larvae [56–59]. The mutated gene that renders P. gallaeciensis BS107-Pda8 unable to produce TDA belongs to an operon encoding the parts of a transport protein, which has not yet been reported to be involved in TDA production [42]. The role of this transmembrane protein in TDA production has not been investigated. It cannot be excluded that this mutation has pleiotropic phenotypic effects, and other functions than TDA production might be affected and could affect the antagonistic properties of Phaeobacter gallaeciensis. None- theless, the experiment using pure TDA indicated that this compound indeed has a major inhibitory effect against V. anguillarum in the algal system. The difference in Vibrio-antagonism between the TDA-negative mutant and the wild type suggested that TDA production is the trait that enables P. gallaeciensis to antagonize V. anguillarum. However, TDA was not detectable by chemical analysis of the Phaeobacter-Tetraselmis co-cultures. Since a tdaC-promoter fusion (tdaCp::gfp), demonstrated that tdaC is expressed by P. gallaeciensis in particles in the algae cultures, the reason for the lack of chemical detection could be that the TDA concentration only reaches PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43996