Hypercapnic hypoxia compromises bactericidal activity of fish anterior kidney cells against opportunistic environmental pathogens

Acute hypoxia can cause massive fish and shellfish mortality. Less clear is the role that chronic sublethal hypoxia might play in aquatic animal health. This study tested whether production of reactive oxygen species (ROS) and bactericidal activity of fish phagocytic cells are suppressed under the conditions of decreased oxygen and pH and increased carbon dioxide which occur in the blood and tissue of animals exposed to sublethal hypoxia. Anterior head kidney (AHK) cells of the mummichog, Fundulus heteroclitus, were exposed in parallel to normoxic (pO2=45 torr, pCO2=3·8 torr, pH=7·6) or hypoxic (pO2=15 torr, pCO2=8·0 torr, pH=7·0) conditions and stimulated with a yeast cell wall extract, zymosan, or live Vibrio parahaemolyticus. Hypercapnic hypoxia suppressed zymosan-stimulated ROS production by 76·0% as measured in the chemiluminescence assay and by 58·5% in the nitroblue tetrazolium (NBT) assay. The low O2, high CO2and low pH conditions also suppressed superoxide production by 75·0 and 47·3% as measured by the NBT assay at two different challenge ratios of cells:bacteria (1:1 and 1:10, respectively). In addition to its effects on ROS production, hypercapnic hypoxia also reduced bactericidal activity by 23·6 and 72·5% at the 1:1 and 1:10 challenge ratios, respectively. Low oxygen levels alone (pO2=15 torr, pCO2=0·76 torr, pH=7·6) did not significantly compromise the killing activity of cells challenged with equal numbers of V. parahaemolyticus. At the higher 1:10 AHK:bacteria challenge ratio, low oxygen caused a small (26·3%) but significant suppression of bactericidal activity as compared to aerial conditions (pO2=155 torr, pCO2=0·76 torr, pH=7·6). This study demonstrates that while hypoxia alone has detrimental effects on immune function, suppression of phagocytic cell activity is compounded by naturally occurring conditions of hypercapnia and low pH, creating conditions that might be exploited by opportunistic pathogens. These results indicate that the adverse health effects of chronic hypercapnic hypoxia might greatly exceed the effects of low oxygen alone.