Effects Of Hypercapnic Hypoxia On The Bactericidal Activity And ROS Production Of Hemocytes From The Penaeid Shrimp Litopenaeus vannamei     Austin S. Dantzler, Karen G. Burnett, and Louis E. Burnett   University of Charleston, Grice Marine Laboratory, Charleston, SC     Aquaculture facilities across the United States and the rest of the world are increasingly threatened by viral and bacterial pathogens.  The high densities of shrimp found in aquaculture ponds cause water quality to decrease, with dissolved oxygen levels often falling below 30% air saturation.  Under natural conditions, low oxygen is usually accompanied by increased carbon dioxide and low pH.  Previous studies from our lab demonstrated that these conditions of hypercapnic hypoxia decreased the resistance of cultured shrimp Litopenaeus vannamei to infection with the opportunistic bacterial pathogen Vibrio parahaemolyticus.  Hypercapnic hypoxia also decreased the bactericidal activity of phagocytes from the teleost fish Fundulus heteroclitus.  These poor water quality conditions also decreased the production of reactive oxygen species (ROS) by phagocytes of F. heteroclitus and hemocytes of the Eastern oyster, Crassostrea virginica.  ROS serve an important role in bactericidal killing by vertebrate macrophages and granulocytes, but their role in the killing activity of invertebrate hemocytes remains unclear.  Current studies focus on the changes in bactericidal activity and ROS production by hemocytes of the white shrimp L. vannamei under conditions of hypercapnic hypoxia and the involvement of ROS in the bacterial killing.  Hemocytes isolated into a 2% artificial sea water with 0.1M glucose and buffered with 12 mM HEPES, pH 7.6, maintained viability above 90% for over four hours when held in siliconized microcentrifuge tubes.  Shrimp hemocytes killed 22—84% of co-cultured bacteria across bacteria to hemocyte ratios from 10:1 to 1:10, as measured using a tetrazolium dye-based killing assay.  The effects of hypercapnic hypoxia on this killing activity are currently under investigation (USDA CSREES 99-35204-8555).