TANNER, C.T.; BURNETT, K.G.; BURNETT, L.E.; College of Charleston; College of Charleston; College of Charleston: Effects of hypoxia and pH on phenoloxidase activity in the blue crab, Callinectes sapidus
The blue crab, Callinectes sapidus, encounters hypoxia in its natural coastal and estuarine habitats. Hypoxia is often accompanied by hypercapnia (increased CO2), which decreases water pH. Previous studies have shown that exposure to hypercapnic hypoxia (HH) impairs the crab�s ability to clear live bacterial pathogens from its hemolymph. We tested the hypothesis that the lower levels of hemolymph O2 and pH during HH decrease the activity of phenoloxidase (PO), an important component of the innate immune response in many invertebrates. Hemolymph from adult males held in well-aerated 25 ppt seawater for 1-5 days was sampled into an anticoagulant solution. Hemocytes were collected by centrifugation and lysed by sonication in a physiological saline to release the inactive proenzyme, prophenoloxidase. Following activation by SDS, PO enzymatic activity was measured using a spectrophotometric assay, which followed the conversion of L-DOPA to dopachrome. We confirmed the enzyme specificity of this assay by using known inhibitors of PO activity. PO activity was measured at O2 levels encountered by hemocytes in normoxic (5 and 15% O2, approximate venous and arterial hemolymph, respectively) and hypoxic crabs (1%) and compared with air (21% O2). PO activity decreased significantly with O2 over the range of treatments, with 77%, 51% and 30% of activity in air at 15%, 5% and 1% O2, respectively. PO activity was also depressed by low pH at 21% O2 with a 16% reduction in activity at pH 7.0 compared to a normoxic pH of 7.8. These data indicate that the decreased ability of crabs in HH to remove bacterial pathogens from their hemolymph may be related at least in part to a decreased rate of encapsulation and bactericidal activity mediated by PO. NSF REU DBI-0244007; NSF IBN-0212921.
