The soil bacterium Bacillus thuringiensis (Bt) is the most successfully used biopesticide in agriculture, and its insecticidal protein genes are the primary transgenes used for insect control in transgenic crops. However, evolution of insect resistance to Bt toxins threatens the long-term future of Bt applications. To date, cases of resistance to Bt toxins have been reported in agricultural situations in six insect species, but the molecular basis for these cases of resistance remains unclear. Here we report that the resistance to the Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni , evolved in greenhouses, is associated with differential alteration of two midgut aminopeptidases N, APN1 and APN6, conferred by a trans -regulatory mechanism. Biochemical, proteomic, and molecular analyses showed that in the Cry1Ac-resistant T. ni , APN1 was significantly down-regulated, whereas APN6 was significantly up-regulated. The Cry1Ac resistance was correlated with down-regulation of APN1 but not with the up-regulation of APN6. The concurrent up-regulation of APN6 and down-regulation of APN1 might play a role in compensating for the loss of APN1 to minimize the fitness costs of the resistance. Along with identifying reduced expression of APN1 as the molecular basis of Bt resistance selected in an agricultural setting, our findings demonstrate the importance of APN1 to the mode of action of Bt toxin Cry1Ac.

The cabbage looper, Trichoplusia ni , is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni . This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.