Functional loss of CHS2 confers high-level resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon.

BACKGROUND Bacillus thuringiensis (Bt) crops, which produce insecticidal proteins such as Vip3Aa and Cry toxins, have revolutionized pest management by reducing reliance on chemical pesticides. However, the evolution of resistance in target pests has prompted investigation into the underlying mechanisms. A recent study identified a mutation in the chitin synthase gene (SfCHS2) as a key factor in Vip3Aa resistance in Spodoptera frugiperda. Here, we examined the role of CHS2 in resistance in two additional lepidopteran species: Spodoptera exigua and Agrotis ipsilon. RESULTS Using a CRISPR/Cas9 gene-editing approach, we generated CHS2 knockout strains in both species. The mutants exhibited high-level resistance to Vip3Aa, surviving the highest tested concentration (800 μg/cm2), with resistance ratios exceeding 33 333-fold in S. exigua and 11 268-fold in A. ipsilon. Additionally, knockout strains lack the peritrophic matrix (PM), whereas the resistant Sfru_R3 strain retained its PM. CONCLUSIONS These findings further validate the essential role of the CHS2 gene-and the PM it produces-in Vip3Aa toxicity. Complete knockout confers high resistance but imposes severe fitness costs, suggesting that such alleles are unlikely to persist in natural populations. This study advances our understanding of the molecular mechanisms behind resistance to Vip3Aa and provides insights for developing effective resistance management strategies in Bt crop management. © 2025 Society of Chemical Industry.