Exposure to anoxia in the venus clam, Chamelea gallina: effects on functional responses of haemocytes

INTRODUCTION: Hypoxia and anoxia are stress conditions that may affect physiological and biochemical responses in infaunal marine organisms, such as bivalves. Depending on the species, bivalves generally show a variable tolerance (measured as anoxic survival response) to hypoxia/anoxia periods, according to ability of organisms to modulate their metabolism. Low oxygen conditions have also been shown to affect bivalve immune responses (Hawkins et al., 1993; Pampanin et al., 2002). In this work, the effects of anoxia on functional responses of haemocytes were investigated in the commercially important species, Chamelea gallina. The capability of clams to recover after anoxic stress was also evaluated. Total haemocytes count (THC), phagocytosis and lysozyme activity were chosen as cellular biomarkers of exposure to anoxic conditions. MATERIALS AND METHODS: Clams were dredged along the north-western Adriatic coast (Emilia-Romagna, Italy) and acclimatised in the laboratory for 1 week before anoxia exposure (salinity of 24\u2030, temperature of 18\ub0C). Clams were exposed to anoxia, in 2 different experiments, for 24h and 48h. To obtained anoxia, filtered sea water was deoxygenated in a 50L reservoir through a 2h vigorously N2 bubbling and then transferred to 10L jars (containing 150 clams each) with a constant flux (2L/h). To evaluate the capability of clams to recover after anoxia periods, bivalves were kept for 24h in aerated seawater. Haemolymph from controls, stressed and recovery clams was collected from the anterior adductor muscle and then pooled (3 pools of 10 clams each). THC was immediately estimated by a Coulter Counter, phagocytic activity was evaluated incubating haemocytes with a yeast suspension, whereas lysozyme activity (both in haemocyte lysate and cell-free haemolymph) was spectrophotometrically measured. Results were compared using a one-way ANOVA. RESULTS AND DISCUSSION: Anoxia reduced significantly (p<0.05) THC with respect to controls in both 24h and 48h exposure. However, while recovery clams of the 24h anoxia test showed THC similar to that of control, in the 48h test recovery clam THC values were similar to those of stressed clams. This different response in the two experiments was caused by an unexpected additional stress during 48h anoxia test. Indeed, in that case, recovery clams spawned during the recovery period. Presumably, a mobilisation of haemocytes towards gonads may occur to remove cell debris remaining after spawning. Anoxia decreased significantly the phagocytic activity with respect to controls (p<0.01in the 24h anoxia test; p<0.001 in the 48h anoxia test). Similarly to what observed in THC analysis, in the first experiment clams recovered their phagocytic activity after anoxia (exhibiting higher values than control), whereas in the second experiment clams were not able to recover, their phagocytic activity being significantly lower (p<0.01 vs control). Probably, stress due to spawning influenced negatively also phagocytic capability of haemocytes in recovery clams. After 24h anoxia, lysozyme activity in haemocyte lysate did not differ from control one, whereas resulted significantly (p<0.01) reduced in cell-free haemolymph. In recovery clams, lysozyme activity of cell lysate was significantly (p<0.05) lower than in control and stressed clams, whereas enzyme activity of the haemolymph resulted higher with respect to stressed clams (p<0.001). This may be explained by increased enzyme secretion from haemocytes into the haemolymph, as a consequence of higher phagocytosis in recovery clams. In 48h anoxia test, significantly (p<0.05) reduced lysozyme activity was recorded in both cell lysate and haemolymph. Moreover, enzyme activity in both media resulted significantly (p