ABSTRACT
The effect of water acidification in combination with normoxia or hypoxia on the antioxidant capacity and oxidative stress markers in gills and hemolymph of the Mediterranean mussel (Mytilus galloprovincialis), as well as on gill microstructure, has been evaluated through an in vivo experiment. Mussels were exposed to a low pH (7.3) under normal dissolved oxygen (DO) conditions (8 mg/L), and hypoxia (2 mg/L) for 8 days, and samples were collected on days 1, 3, 6, and 8 to evaluate dynamic changes of physiological responses. Cytoplasmic concentrations of reactive oxygen species (ROS) and levels of DNA damage were measured in hemocytes, while the activity of catalase (CAT) and superoxide dismutase (SOD) and histopathological changes were assessed in gills. The results revealed that while water acidification did not significantly affect the activity of SOD and CAT in gills under normoxic and hypoxic conditions, there was a trend towards suppression of CAT activity at the end of the experimental period (day 8). Similarly, we did not observe increased formation of ROS in hemocytes or changes in the levels of DNA damage during the experimental period. These results strongly suggest that the oxidative stress response system in mussels is relatively stable to experimental conditions of acidification and hypoxia. Experimental acidification under normoxia and hypoxia caused changes to the structure of the gills, leading to various histopathological alterations, including dilation, hemocyte infiltration into the hemal sinuses, intercellular edema, vacuolization of epithelial cells in gill filaments, lipofuscin accumulation, changes in the shape and adjacent gill filaments, hyperplasia, exfoliation of the epithelial layer, necrosis, swelling, and destruction of chitinous layers (chitinous rods). Most of these alterations were reversible, non-specific changes that represent a general inflammatory response and changes in the morphology of the gill filaments. The dynamics of histopathological alterations suggests an active adaptive response of gills to environmental stresses. Taken together, our data indicate that Mediterranean mussels have a relative tolerance to water acidification and hypoxia at tissue and cellular levels.
ABSTRACT
Environmental hypoxia naturally occurs in coastal ecosystems and bivalve mollusks have to frequently face fluctuations of dissolved oxygen concentrations. Exposure to hypoxia is often associated with the change of the antioxidant and functional status in bivalves, and restoration of the normal oxygen supply is considered to induce oxidative stress in tissues of mollusks. The study investigates changes in the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), as well as the expression level of SOD and CAT genes in gills of the Mediterranean mussel, Mytilus galloprovincialis, under exposure to low dissolved oxygen concentration (2.2 mg L-1) for 24 h and 72 h, and 24 h reoxygenation period. We also evaluated the intracellular level of reactive oxygen species (ROS), mortality and changes in mitochondrial membrane potential in hemocytes following hypoxia-reoxygenation cycle. 24 h exposure to hypoxia significantly decreased activity of both enzymes, which then recovered up to control levels at the end of 72 h experimental period for SOD and after reoxygenation for CAT. Expression of antioxidant enzyme genes was up-regulated following the 72 h hypoxic exposure period and returned to the basal normoxic level after 24 h reoxygenation. Hypoxia demonstrated a time-dependent effect on the functional state of hemocytes. The 24 h exposure period did not influence aerobic respiration of hemocytes, but prolonged hypoxia (72 h) was associated with a substantial decrease in mitochondrial membrane potential of hemocytes. The intracellular ROS level and mortality of hemocytes did not change under hypoxia. Reoxygenation period was accompanied with a significant decrease of intracellular ROS level. This study indicated that hypoxia did not induce the pronounced oxidative stress in gills and the changes in the antioxidant status were reversible within 24 h of reoxygenation. Hemolymph demonstrated a stable functional state indicating the tolerance of mussels to short-time hypoxia.
