Examination of the Microbial Communities Associated with Oyster Larvae Treated with Probiotic Bacteria in the Absence and Presence of a Bacterial Pathogen

Developing strategies for controlling microbial pathogenesis in aquaculture is urgent as bacterial disease is the primary cause of mortality, particularly in shellfish larviculture. Previous studies at Milford have identified a naturally occurring Vibrio sp. probiotic candidate (OY15) that enhanced both survival and health of oyster larvae and protected from challenges by a pathogen (B183). As part of a broader study aimed at examining how OY15 acts, bacterial community diversity was examined in the presence and absence of B183 and OY15. Two day-old Crassostrea virginica larvae were transferred to 10-L sterile-filtered seawater with the following treatments: untreated, treated with B183 (10⁵ CFU/ml on days 5, 6, and 8), treated with OY15 (10³ CFU/ml on days 1, 3, 6, and, 8), and treated with both B183 and OY15. One, three, and four days after B183 challenge, water and larvae samples were collected. DNA was extracted and bacterial 16S rRNA gene segments were polymerase chain reaction-amplified. Separation by denaturing gradient gel electrophoresis revealed a diverse bacterial community for challenged and unchallenged larvae with/without probiotic as well as the surrounding culture water. Significant differences were detected between larval microbial communities and culture water, suggesting selective uptake or retention by larvae. In all cases diversity of the oyster bacterial community did not change significantly, although a few treatment-specific amplicons were apparent. While massive mortality occurred by day four post-infection, B183 16S rRNA sequences could not be detected in larvae or water one day post-infection; OY15 16S rRNA sequences were evident one day post-infection but could not be detected at day three. Our results suggest that OY15 and B183 activities do not appear to influence larval or culture water bacterial communities but are likely directed at the oyster itself, which is consistent with previous studies indicating that OY15 enhances larval hemoycyte functionality and B183 causes immuno-suppression.