AbstractBACKGROUNDField‐evolved resistance of Helicoverpa zea to Bacillus thuringiensis (Bt) toxin Cry1Ac was first reported more than a decade ago, yet the underlying mechanisms remain elusive. Towards understanding the mechanisms of resistance to Cry1Ac, we analyzed a susceptible (LAB‐S) and two resistant (GA and GA‐R) strains of H. zea. The GA strain was derived from Georgia and exposed to Bt toxins only in the field. The GA‐R strain was derived from the GA strain and selected for increased resistance to Cry1Ac in the laboratory.RESULTSResistance to MVPII, a liquid formulation containing a hybrid protoxin similar to Cry1Ac, was 110‐fold for GA‐R and 7.8‐fold for GA relative to LAB‐S. In midgut brush border membrane vesicles, activity of alkaline phosphatase and aminopeptidase N did not vary significantly among strains. The activity of total proteases, trypsin‐like proteases and chymotrypsin‐like proteases was significantly lower for GA‐R and GA than LAB‐S, but did not differ between GA‐R and GA. When H. zea midgut cells were exposed to Cry1Ac protoxin that had been digested with midgut extracts, toxicity was significantly lower for extracts from GA‐R and GA relative to extracts from LAB‐S, but did not differ between GA‐R and GA. Transcriptional analysis showed that none of the five protease genes examined was associated with the decline in Cry1Ac activation in GA‐R and GA relative to LAB‐S.CONCLUSIONThe results suggest that decreased Cry1Ac activation is a contributing field‐selected mechanism of resistance that helps explain the reduced susceptibility of the GA‐R and GA strains. Relative to the LAB‐S strain, the two Cry1Ac‐resistant strains had lower total protease, trypsin and chymotrypsin activities, a lower Cry1Ac activation rate, and Cry1Ac protoxin incubated with their midgut extracts was less toxic to H. zea midgut cells. © 2018 Society of Chemical Industry