EFFECTS OF HYPERCAPNIC HYPOXIA AND SEASON ON THE ANTIBACTERIAL ACTIVITY OF HEMOCYTES FROM THE PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI

Austin Dantzler, M.S.

Grice Marine Laboratory, College of Charleston, South Carolina

ABSTRACT OF THESIS:  The large biomass of phytoplankton and sediment bacteria associated with high densities of shrimp in aquaculture ponds often cause dissolved oxygen levels to fall below 30% air saturation (6 kPa O2). Low oxygen is usually accompanied by increased carbon dioxide and low pH. Previously our lab has demonstrated that these conditions of hypercapnic hypoxia decreased the resistance of the Pacific white shrimp Litopenaeus vannamei to infection with the opportunistic bacterial pathogen Vibrio campbellii. To understand more about the mechanisms that might underlie this effect of dissolved gasses and pH on susceptibility to infectious disease, the current study focused on the changes in bactericidal activity of hemocytes from the white shrimp L. vannamei under conditions of hypercapnic hypoxia. Hemocytes were drawn from the base of the walking legs into an anticoagulant solution, and total hemocyte counts (THC) and counts of the relative proportion of the three hemocyte types were performed. Under the conditions of the present study, L. vannamei were found to have a mean ( SE) of 3.1 × 107 ± 0.3 × 107 cells mL−1 hemolymph (n=44), which was comprised of 18.1%  0.9% granular cells, 20.2%  0.9% semi-granular cells, and 62.6%  1.2% hyaline cells (n = 46). Cells were isolated into 12 mM HEPES saline buffer with 0.1 M glucose, pH 7.6, and the antibacterial activity was measured at various gas levels and from different seasons using a tetrazolium dye-based bacterial killing assay and by directly counting the colony forming units (CFU). Antibacterial activity measured under moderate hypoxia (2.5 kPa O2, 0 kPa CO2, pH 7.6) and moderate hypercapnic hypoxia (2.5 kPa O2, 2 kPa CO2, and pH 6.9) conditions was linked to a significantly higher level of killing compared to normoxic conditions (20 kPa O2, 0.3 kPa CO2, and pH 7.6). Hemocyte preparations exhibited two patterns of killing activity by season. The first pattern occurred in the summer and fall periods of 2001 – 2003, and was characterized by lysed hemocytes enhancing bacterial growth while live hemocytes inhibited growth of bacteria. The second pattern was observed in the winter months of 2002-2003, and was characterized by the ability of Tween-lysed hemocytes to inhibit bacterial growth.