First evidence of altered immune responses and resistance to air exposure in the clam Chamelea gallina exposed to benzo(a)pyrene.

Effects of benzo(a)pyrene [B(a)P] (at a nominal concentration of 0.5 mg/L) on immune responses of the clam Chamelea gallina were investigated after 1, 7, and 12 days exposure. Total hemocyte count (THC), hemocyte volume, phagocytic activity, lysozyme-like activity in both hemocyte lysate (HL) and cell-free hemolymph (CFH) were measured. As unexpected alterations in hemocyte adhesion capability were observed in short-term hemocyte cultures for phagocytosis assays after a 1-day exposure, an adhesion test (not included in the original experimental setup) was performed after 7 and 12 days of exposure only. The survival-in-air test was carried out to evaluate general stress conditions in B(a)P-exposed clams. No alterations in THC was observed, whereas exposure for 7 and 12 days to B(a)P significantly decreased phagocytic activity and adhesion capability when compared with controls. Significant decreases in lysozyme activity were observed in CFH and HL, with respect to controls. B(a)P was also shown to alter the resistance to air exposure of clams. The LT(50) values fell from 9 days in control clams to 7 days in 1-day-exposed animals, and from 6 days in control clams to 5 days in 7-day-exposed bivalves. No significant variations in LT(50) values were recorded after 12 days of exposure. Results highlight a relationship between B(a)P exposure and alterations in hemocyte functionality and suggest that the contaminant induced irreversible immunosuppression in C. gallina, by altering phagocytic activity, adhesion capability, and enzymatic activity. Conversely, reduction in resistance to air exposure was reversible, suggesting that impairment of important physiological functions of clams occurred in the first phases of exposure only.

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina.

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.

Anoxic survival potential of bivalves: (arte)facts.

The anoxic survival time of the bivalves Chamelea gallina, Cerastoderma edule and Scapharca inaequivalvis from two different ecosystems and differing anoxia tolerances was studied in static (closed) and flow-through systems. The antibiotics chloramphenicol, penicillin and polymyxin were added, and molybdate (specific inhibitor of the process of sulfate reduction). Survival in (near) anoxic seawater of Chamelea was studied in a static system by comparing untreated seawater with autoclaved seawater and untreated clams with clams incubated in well-aerated seawater, containing the broad-spectrum antibiotic chloramphenicol, prior to the anoxic survival test. With untreated clams and natural seawater (median mortality time 2.4 days) a decrease in pH and exponential accumulation of sulfide and ammonium was observed in the anoxic medium, indicating excessive growth of (sulfate reducing) bacteria. In sterilized seawater LT50 (2.1 days) was not significantly different and again considerable amounts of ammonium and sulfide accumulated. However, pre-treatment of clams with chloramphenicol resulted in an increase of LT50 (11.0 days) by approximately fivefold. Accumulation of ammonium and sulfide was retarded, but was finally even stronger than in the medium containing untreated clams. Median mortality times were 2.5 and 2.4 days for Chamelea and 2.7 and 2.9 days for Cerastoderma for static and flow-through incubations, respectively. Addition of chloramphenicol increased strongly survival time in both systems with corresponding values of 11.0 and 16.3 days for Chamelea, and 6.4 and 6.5 days for Cerastoderma. LT50 of Scapharca in anoxic seawater was 14.4 days. Chloramphenicol and penicillin increased median survival time to 28.5 and 28.7 days, respectively, whereas polymyxin displayed no effect (LT50=13.6 days). Molybdate added to artificial sulfate free seawater blocked biotic sulfide formation, but did not improve survival time (LT50=13.7 days). Overall the results indicate that proliferation of anaerobic pathogenic bacteria, firmly associated with the bivalves, is a main cause of death besides lack of oxygen. Bacterial damage is probably caused by injury of the tissues of the clams and not by the release of noxious compounds to the medium.