Wang, P.; Liu, Z.; Kang, Q.; Liao, C.; Zou, L.; Mao, K.; Yao, H.; Li, Y.; Xiao, Y.

Bacillus thuringiensis (Bt) crops, engineered to produce insecticidal proteins such as Vip3Aa and Cry toxins, have revolutionized pest management by providing a sustainable alternative to chemical pesticides. However, the development of resistance to these toxins has driven the investigation of underlying mechanisms to better understand how pests evade toxicity and to develop more effective resistance management strategies. Recently, a laboratory-selected Spodoptera frugiperda strain exhibited a high level of resistance to Vip3Aa (resistance ratio: 5562-fold), with the mutation of the chitin synthase gene, SfCHS2, identified as a key factor. In this study, we extend these findings to additional lepidopteran species, including Spodoptera exigua and Agrotis ipsilon. Our results show that CHS2 knockout strains lack the peritrophic matrix (PM), while the resistant Sfru_R3 strain retains its PM. Knockout of this gene in five lepidopteran species resulted in high levels resistance to Vip3Aa. Notably, homozygous knockout strains suffered significant fitness costs and had difficulty surviving on corn leaves, in contrast to the Sfru_R3 strain, which showed no such survival issues. These findings further validate the role of the CHS2 gene in Vip3Aa resistance and highlight its potential as a target for resistance management in lepidopteran pests. This study advances our understanding of the molecular mechanisms behind resistance to Vip3Aa and supports the development of strategies to delay resistance in Bt crop management.