Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae Paul W. D’Alvise1, Siril Lillebø 2 , Maria J. Prol-Garcia1, Heidrun I. Wergeland 2 , Kristian F. Nielsen 3 , Øivind Bergh4, Lone Gram1* 1 National Food Institute, Technical University of Denmark, Lyngby, Denmark, 2 Department of Biology, University of Bergen, Bergen, Norway, 3 Department of Systems Biology, Center for Microbiological Biotechnology, Technical University of Denmark, Lyngby, Denmark, 4 Institute of Marine Research, Bergen, Norway Abstract Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 107 cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect. Citation: D’Alvise PW, Lillebø S, Prol-Garcia MJ, Wergeland HI, Nielsen KF, et al. (2012) Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae. PLoS ONE 7(8): e43996. doi:10.1371/journal.pone.0043996 Editor: Daniel Merrifield, The University of Plymouth, United Kingdom Received March 26, 2012; Accepted July 27, 2012; Published August 22, 2012 Copyright: 2012 D’Alvise, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Danish Research Council for Technology and Production to the project 09-066524 (Bioactive bacterial biofilm surfaces in aquaculture – disease prevention without antibiotics). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gram@food.dtu.dk Introduction One of the major challenges of marine aquaculture is the continuous and reliable production of juveniles. Severe losses in marine larviculture are caused by infection with opportunistic pathogenic bacteria, including several members of the Vibrionaceae family [1,2], that accounts for approximately 1.5% of the bacterial community in the oceans [3]. Only some Vibrio species are pathogenic to organisms reared in marine aquaculture and one of the most prominent fish and shellfish pathogens is Vibrio (Listonella) anguillarum that causes serious disease and mortalities [2]. The main source of pathogenic bacteria in marine aquaculture is supply water [4], but also brood stock, humans, or starter cultures are possible sources of pathogens [5]. The majority of marine fish larvae are reared intensively in presence of microalgae (green water), which improves feeding, growth, and survival of the larvae [6–9]. The larvae require live feed, and rotifers (Brachionus plicatilis) are typically used as first feed. The rotifers themselves are fed or enriched with live microalgae, such as Tetraselmis suecica, Nanno- chloropsis oculata, and Isochrysis galbana. Opportunistic pathogens can proliferate in larval feed cultures of phytoplankton and inverte- brates due to high concentrations of organic matter. Algae, rotifer and Artemia cultures can therefore harbor high concentrations of pathogenic bacteria [1,5,10,11]. Prophylactic treatment of larvae or their feed cultures with antibiotics can reduce the pathogen load, but has to be avoided, since it leads to emergence of antibiotic-resistant pathogens, and since it impedes the establish- ment of a normal non-pathogenic microbiota [1,12,13]. The potential use of probiotic bacteria to limit outbreaks or effects of bacterial diseases in fish and invertebrate cultures has been investigated for more than two decades. Most studies have focused on the intestinal microbiota [14–19], although the use of probiotics is not confined to the intestinal tract of the cultured organisms [15,20]. Biotic and abiotic surfaces, algal and fecal particles, and the nutrient-rich water serve as habitat and reservoir of opportunistic pathogenic bacteria in cultures of fish larvae or their food organisms [1,5,10,11,20], and it is hypothesized that competition by non-pathogenic bacteria that are superior in colonizing and persisting in these habitats could reduce the incidence of pathogenic bacteria. Phaeobacter gallaeciensis (formerly Roseobacter gallaeciensis) is a Gram- negative a-proteobacterium from the Roseobacter-clade [21]. The bacterium produces the antibacterial compound tropodithietic PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43996