ISOLATION AND EVALUATION OF NEW PROBIOTIC BACTERIA FOR USE IN SHELLFISH HATCHERIES: II. EFFECTS OF A VIBRIO SP. PROBIOTIC CANDIDATE UPON SURVIVAL OF OYSTER LARVAE (CRASSOSTREA VIRGINICA) IN PILOT-SCALE TRIALS DIANE KAPAREIKO, 1 * HYUN JEONG LIM, 2 ERIC J. SCHOTT, 3 AMMAR HANIF 3 AND GARY H. WIKFORS 1 1 National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460; 2 Aquaculture Division, West Sea Fisheries Research Institute, Incheon 400-420, South Korea; 3 University of Maryland Center for Environmental Science, Institute of Marine and Environmental Technology, 701 East Pratt Street, Baltimore, MD, 21202 ABSTRACT Environmentally-friendly methods for controlling microbial pathogenesis in aquaculture with probiotic bacteria are becoming increasingly preferred over the use of chemical means, such as disinfectants or antibiotics. Previous research at the Milford Laboratory has shown that naturally-occurring bacteria isolated from the digestive glands of adult oysters (Crassostrea virginica) show promise as potential probiotic additives in oyster larviculture, based on bench-scale experiments. The previous, bench-scale challenge studies reported in the accompanying article (Lim et al. this volume) indicated that 48-h survival of 2-day- old oyster larvae supplemented with Vibrio sp. strain OY15 improved after challenge with pathogenic Vibrio sp. strain B183 compared with the pathogen alone. This study investigated further the effectiveness of probiotic candidate OY15 to improve survival of oyster larvae to metamorphosis under pilot-scale culture conditions, both with and without pathogen B183 challenge. The effective dosage of probiotic candidate OY15 that significantly improved larval survival was determined to be 10 3 cfu/mL. The LD 50 calculated for pathogen B183 was 9.6 3 10 4 cfu/mL. Results from these bioassays indicated that addition of probiotic candidate OY15 significantly improved survival of oyster larvae to metamorphosis when challenged with pathogen B183 in pilot- scale trials. These studies can provide the basis for the development of functional foods for use in shellfish larviculture that incorporate a naturally-occurring, probiotic bacterial strain. KEY WORDS: probiotic bacteria, shellfish larvae, oyster larviculture, larvae survival, Vibrio, Crassostrea virginica INTRODUCTION Environmentally friendly methods for controlling microbial pathogenesis in aquaculture with probiotic bacteria have gained considerable research interest and are becoming increasingly pre- ferred as viable, alternative management practices for disease prevention. Bacterial diseases, commonly caused by Vibrio (Estes et al. 2004) and Aeromonas spp. (Kesarcodi-Watson et al. 2008), can result in major mortalities in bivalve hatcheries, and cause major financial losses for commercial shellfish growers. Chem- ical means, such as disinfectants and antimicrobial drugs, which can have obvious benefits to infected animals, have been over- used for disease prevention or growth enhancement (Van den Bogaard & Stobberingh 2000). Prophylactic use of antimicrobial drugs has led to the emergence of antibiotic-resistant bacterial strains that have survived a course of treatment by antibiotics, and have the potential to transfer their resistance genes to other bacterial strains via horizontal gene transfer (Schwarz et al. 2001, Akinbowale et al. 2006). The emergence of antibiotic-resistant bacteria was most dramatically felt in the shrimp aquaculture industry; increased production, overstocking, and unregulated usage of antibiotics to control Vibrio harveyi (a main bacterial shrimp pathogen) caused significant production crashes in Asian countries (Karunasagar et al. 1994, Moriarty 1998). Shrimp production in the Philippines dropped 55% between 1995 and 1997 as a result of outbreaks of this pathogen, and Thailand’s shrimp production dropped 40% between 1994 and 1997 because of V. harveyi as well as shrimp viruses (Moriarty 1998). Certain antibiotic-resistant bacteria of aquaculture farm origin have even been able to transfer resistance genes to human pathogens, causing a potential risk to human health (Van den Bogaard & Stobberingh 2000, Witte 2000, Schwarz et al. 2001). Tighter government regulations have been implemented in Asian coun- tries that restrict antibiotic usage in animal production for human consumption. Although Thailand banned the use of chloram- phenicol for disease prevention in shrimp aquaculture in 1999, trace levels were still being detected in exported product in 2004 (Heckman 2004). Developing concerns regarding the unnecessary use of an- timicrobial drugs in animal production for human consumption have raised awareness of the need for alternative, cost-effective methods, such as the use of probiotic bacteria, as microbial control agents in shellfish larviculture. Use of probiotic bacteria in shellfish larviculture may improve veliger larval survival to metamorphosis, the most critical phase of shellfish aquaculture when most mortality occurs (Loosanoff & Davis 1963). Desir- able probiotic bacteria should benefit larval survival as well as benefit or not impair microalgae used as feed in culture systems (Kesarcodi-Watson et al. 2008). Supplementation of algal feeds with probiotic bacteria in shellfish larviculture has been shown to enhance the nutritional value of the algae to the larvae, and to provide early colonization of microflora in the gut to aid digestion (Verschuere et al. 2000). Probiotic strains have also been shown to speed development of, or stimulate, the innate immune response to potentially-pathogenic bacteria in shellfish (Vaughan et al. 2002). In a review article, Verschuere et al. (2000) listed properties that a safe, desirable, and effective probiotic should possess: *Corresponding author. E-mail: Diane.Kapareiko@noaa.gov DOI: 10.2983/035.030.0304 Journal of Shellfish Research, Vol. 30, No. 3, 617–625, 2011. 617