The quantities of Cry1Ac and Cry2Ab in each pollen sample were estimated using Envirologix Qualiplate Kits (EnviroLogix Quantiplate Kit, Portland, ME, USA). The detection limits for the two proteins were 0.1 ng/g and 0.52 ng/g, respectively. Before analysis, the fresh pollen samples were homogenized in 4 ml of extraction buffer and then kept at 4uC overnight for extraction of insecticidal proteins. After being centrifuged for 15 min at 7000 g, the supernatants of the extraction were used for the analyses.