Animal sampling and in vitro stimulation of hemocytes R. philippinarum clams were obtained from a commercial shellfish farm (Vigo, Galicia, Spain). Clams were maintained in open circuit filtered sea water tanks at 15uC with aeration and were fed A total of 100 clams were notched in the shell in the area adjacent to the anterior adductor muscle. A sample of 500 ul of hemolymph was withdrawn from the adductor muscle of each clam with an insulin syringe, pooled and then distributed in 6-well plates, 7 ml per well, in a total of 7 wells, one for each treatment. Hemocytes were allowed to settle to the base of the wells for 30 min at 15uC in the darkness. Then, the hemocytes were stimulated with 50 mg/ml of Polyinosinic:polycytidylic acid (Poly I:C), Peptidoglycans, ß-Glucan, Vibrio anguillarum DNA (CpG), Lipopolysaccharide (LPS), Lipoteichoic acid (LTA) or 1610 6 UFC/ml of heat-inactivated Vibrio anguillarum (one stimulus per well) for 3 h at 15uC. All stimuli were purchased from SIGMA.