Interactive effects of mosquito control insecticide toxicity, hypoxia, and increased carbon dioxide on larval and juvenile eastern oysters and hard clams.

Mosquito control insecticide use in the coastal zone coincides with the habitat and mariculture operations of commercially and ecologically important shellfish species. Few data are available regarding insecticide toxicity to shellfish early life stages, and potential interactions with abiotic stressors, such as low oxygen and increased CO2 (low pH), are less understood. Toxicity was assessed at 4 and 21 days for larval and juvenile stages of the Eastern oyster, Crassostrea virginica, and the hard clam, Mercenaria mercenaria, using two pyrethroids (resmethrin and permethrin), an organophosphate (naled), and a juvenile growth hormone mimic (methoprene). Acute toxicity (4-day LC50) values ranged from 1.59 to >10 mg/L. Overall, clams were more susceptible to mosquito control insecticides than oysters. Naled was the most toxic compound in oyster larvae, whereas resmethrin was the most toxic compound in clam larvae. Mortality for both species generally increased with chronic insecticide exposure (21-day LC50 values ranged from 0.60 to 9.49 mg/L). Insecticide exposure also caused sublethal effects, including decreased swimming activity after 4 days in larval oysters (4-day EC50 values of 0.60 to 2.33 mg/L) and decreased growth (shell area and weight) in juvenile clams and oysters after 21 days (detected at concentrations ranging from 0.625 to 10 mg/L). Hypoxia, hypercapnia, and a combination of hypoxia and hypercapnia caused mortality in larval clams and increased resmethrin toxicity. These data will benefit both shellfish mariculture operations and environmental resource agencies as they manage the use of mosquito control insecticides near coastal ecosystems.

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 CO2 and 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.

Acid-Base Status of the Oyster Crassostrea virginica in Response to Air Exposure and to Infections by Perkinsus marinus.

Hemolymph acid-base variables were investigated in the Eastern oyster, Crassostrea virginica, to determine its responses to air exposure and to infections by the parasite Perkinsus marinus. Infected and uninfected oysters were subjected to two treatments of temperature (21° and 30°C) and air exposure (5 and 24 h). Upon exposure to air, oysters underwent a respiratory acidosis that remained uncompensated in uninfected oysters but was partially compensated in highly infected oysters at both 21° and 30°C. The acidosis was significantly greater in oysters with high infections. Hemolymph in uninfected oysters had a greater buffering capacity (-6.80 +/- 0.76 SEM slykes) than hemolymph in highly infected oysters (-3.30 +/- 0.50 SEM slykes). Calcium ion concentrations in hemolymph increase when the hemolymph becomes acidic, suggesting that shell decalcification plays a role in buffering the acid. During air exposure, although oysters do not visibly gape, they access air and are apparently not completely anaerobic.

Integrating undergraduate laboratories into the curriculum.

Developing a curriculum that integrates laboratory, field, and nonlaboratory experiences requires solid planning, cooperation, and compromise among the faculty members in a department. This is especially true for laboratory experiences where basic skills and familiarity with the different groups of plants and animals carry over to upper-division courses. In an introductory Principles of Biology laboratory, for example, exercises can be designed to give students a clear idea of a statistical distribution and the consequences of random molecular motion. Both concepts are used in nearly all upper-division courses to some extent. Some important but "mechanical" features of courses within a curriculum, such as adopting within the department a single format for writing a scientific paper, will go a long way in tying together different laboratory experiences. Other similar but simpler ideas are to develop appendexes to laboratory exercises that can be used in introductory and upper-division courses, e.g., how to do dilutions, the use of SI units, etc. The latter may be a good way to stimulate departments to think more carefully about continuity and consistency in the design of the overall curriculum. Continuity of instruction from lower-division to upper-division courses and among upper-division courses requires communication between instructors at all levels.

Facilitation of CO2 excretion by carbonic anhydrase located on the surface of the basal membrane of crab gill epithelium.

An isolated perfused crab gill preparation was used to test the hypotheses that crab gill carbonic anhydrase (CA) catalyzes the efflux of CO2 from the hemolymph, which lacks the enzyme, to the ambient medium and that the CA is localized on the luminal surface of the basal membrane. It was found that the efflux of CO2 from the internal perfusate was sensitive to the flow rate of the internal perfusate through the gill (and thus the residence time within the gill). The sensitivity of the CO2 efflux to residence time was nearly abolished upon treatment of the gill with an impermeable dextran-bound CA inhibitor. It is concluded that CA present on the luminal surface of the gill epithelium facilitates CO2 excretion by catalyzing the dehydration of the large hemolymph bicarbonate pool to the more diffusible molecular CO. The action of the enzyme is important in maintaining a CO2 gradient between hemolymph and water in a situation where hemolymph PCO2 is normally low, water PCO2 is variable, and the gills themselves are a source of metabolic CO2.