THE EFFECTS OF ENVIRONMENTAL VARIABLES ON THE METABOLISM OF THE PROTOZOAN OYSTER PARASITE PERKINSUS MARINUS

Christopher S. Milardo

A thesis submitted to the Graduate Faculty of the University of Charleston, South Carolina in partial fulfillment of the requirement for the Degree of Master of Science

ABSTRACT
This study examined the effects of pH, oxygen, carbon dioxide, and temperature on the metabolism of the Apicomplexan oyster parasite Perkinsus marinus. P. marinus was cultured in a modified JL-ODRP medium in ambient air at high humidity. Culture media was gassed with appropriate CO₂, N₂ and O₂ mixtures, and pH adjusted in order to simulate the conditions present in oyster hemolymph. Oxygen uptake of the parasite was measured as a function of time (Fmol O₂ million cells^-1 hr^-1) using oxygen depletion techniques. Oxygen uptake of the parasite decreased proportionately with decreasing ambient oxygen, and decreasing pH increased P. marinus respiration under low CO₂ conditions (p=0.0006); under 15 torr CO₂, there was no relationship between pH and oxygen uptake. Increased CO₂ levels significantly increased parasite uptake (p<0.0001), as did increased temperature (p<0.0001).

The effect of temperature cycling on the in vitro growth of P. marinus was also examined at pH 6.3, 7.1 and 7.6. Cycling in 6-hour intervals from 25EC-35EC did not affect culture growth; however, when cycled from 25EC-40EC, cells showed a significant decrease in growth (p<0.0001). Similarly, cells grown at 35EC and 15 torr ambient CO₂ showed decreased growth at low pH. These results indicate that increases in P. marinus oxygen uptake can be induced by increased growth and reproduction (0 torr CO₂, low pH conditions), or by increased aerobic metabolism due to stress (15 torr CO₂, high pH). Thus, growth rates for P. marinus are more accurate indicators of culture preferences than is oxygen uptake rate. These results indicate that summer environmental conditions in the intertidal zone may play a role in keeping P. marinus infections in Crassostrea virginica at sub-lethal levels.

The production of anaerobic endproducts by P. marinus was also studied. P. marinus cell extracts were shown to contain succinate (8.16 Fmol L^-1 g^-1 cells), lactate (5.40 Fmol L^-1 g^-1 cells) and acetate (6.90 Fmol L^-1 g^-1 cells). Propionate, alanopine, and strombine were not produced by the parasite, indicating that the dehydrogenases necessary for the creation of opines do not exist in this species. The parasite may have developed these anaerobic pathways to survive the anoxic conditions within moribund oysters.