Pyrosequencing. After stimulation, hemolymph was centrifuged at 1700 g at 4uC for 5 minutes, the pellet was resuspended in 1 ml of Trizol (Invitrogen) and RNA was extracted following the manufacturer's protocol. After RNA extraction, samples were treated with Turbo DNase free (Ambion) to eliminate DNA. Next, the concentration and purity of the RNA samples were measured using a NanoDrop ND1000 spectrophotometer. The RNA quality was assessed in a Bioanalyzer 2010 (Agilent Technologies). From each sample, 1 mg of RNA was pooled and used for the production of normalized cDNA for 454 sequencing in the Unitat de Genòmica (SCT-UB, Barcelona, Spain).