Development of an in situ binding (ISB) method to evaluate the binding of Cry1Ac toxin to the larval midgut epithelium of Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) insecticidal proteins in pest control has led to the evolution of resistance in target insects. Reduced toxin binding to the midgut epithelial membrane is considered a major resistance mechanism. Current methods for characterizing toxin binding suffer from limitations in physiological relevance or practical applicability. Here, we developed an in situ binding (ISB) method to visualize and quantify the binding of Cry1Ac toxin to the midgut epithelium of Helicoverpa armigera larvae. By culturing dissected midguts in a cell culture medium containing Cry1Ac, we maintained midgut cell viability over a short period while enabling specific toxin-receptor interactions under near-physiological conditions. Applying this method, we compared Cry1Ac binding to the midgut epithelium of a susceptible SCD strain and three SCD-derived Cry1Ac-resistant strains: SCD-r1 (deletion mutation in cadherin, conferring 438-fold recessive resistance), SCD-KI (T92C point mutation in tetraspanin, conferring 125-fold dominant resistance), and C2/3-KO (ABCC2 and ABCC3 knocked out, conferring >15,000-fold recessive resistance). All resistant strains exhibited significantly reduced Cry1Ac binding to the midgut epithelial membrane compared to the susceptible SCD strain, with 83.6%, 54.4%, and 31.7% decreases in Cry1Ac signal fluorescence intensities in the C2/3-KO, SCD-KI, and SCD-r1 strains, respectively. Moreover, the reduction in Cry1Ac binding correlated with a marked decrease in toxin-induced midgut epithelial damage in the resistant strains. Our findings provide additional evidence for reduced toxin binding as a shared mechanism of Cry1Ac resistance in H. armigera across diverse genetic basis. The established ISB method provides a valuable tool for evaluating the binding of Bt toxins and other insecticidal proteins to the insect midgut epithelium.