The Effects of Intertidal Air Exposure on the Respiratory Physiology and the Killing Activity of Hemocytes in the Pacific Oyster, Crassostrea gigas

Steve Allen

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
The occurrence of summer mortalities of the commercially important Pacific oyster, Crassostrea gigas, has increased in recent years.  These die-offs occur during the late summer when water temperatures are at their highest.  Many theories have been proposed as to the causative factors including reproductive stress, environmental stress, and disease, or a synergistic interaction of factors.  C. gigas are grown intertidally and are exposed to the air (emersed) for hours at a time.  These organisms therefore can experience extreme changes in temperature during the course of a day.  An oyster closed during emersion depletes the oxygen stores to near zero within the shell and builds up CO₂ causing a decrease in tissue pH.  I hypothesized that stresses associated with emersion inhibit the immune system of the oyster and contribute to the summer mortalities.  The focus of the present study was to determine the respiratory (pH, Po₂, Pco₂ and total CO₂) and immune responses of oysters exposed to air and challenged with a known pathogen.  The respiratory variables of oysters submerged at 18°C (pH=7.52 ± 0.04 SEM, Po₂= 53.2 ± 3.99 SEM torr and Pco₂= 1.51 ± 0.20 SEM torr respectively) varied significantly from oysters emersed for four hours at 22°C (pH= 7.11 ± 0.03 SEM, Po₂= 28.72 ± 1.14 SEM torr, Pco₂= 2.71 ± 0.25 SEM torr respectively) and those emersed for four hours at 30oC (pH=6.84 ± 0.02 SEM, Po₂= 23.27 ± 0.87 SEM torr, Pco₂= 9.83 ± 0.44 SEM torr respectively).  The ability of hemocytes to kill the bacterium Vibrio parahaemolyticus was assessed using an in vitro assay.  Hemocytes were treated with low pH (6.6), low oxygen (3%) and high temperature (30°C), simulating in vivo conditions and challenged in vitro with V. parahaemolyticus.  A tetrazolium dye reduction assay was used to quantify the number of viable bacteria remaining after exposure to hemocytes, from which a killing index was calculated.  There was no significant difference in the killing index between  pH treatment groups (p = 0.856): at pH 7.6 killing index = 50.2%± 2.33 SEM, at pH 6.6 killing index = 52.3% ± 3.67 SEM.  Temperature was the only factor to significantly affect the killing indices among temperature and oxygen treatment groups.  The killing index was lowest (29.3% ± 3.25 SEM ) at 30°C and 7% oxygen (simulating in vivo oxygen pressure in well-aerated conditions) and 30°C and 3% oxygen (simulating in vivo oxygen pressure in hypoxia) (30.5% ± 3.25 SEM) compared with the index in 7% oxygen at low temperature (18°C) (44.4% ± 4.50 SEM) or compared with low oxygen (3%) at low temperature (18ºC) (39.7% ± 2.51 SEM).  The seasonal rise in temperature may therefore be an important factor contributing to summer mortalities of C. gigas.