Abstract
Spodoptera frugiperda (fall armyworm) is a highly destructive agricultural pest with a remarkable ability to develop resistance to insecticides, posing a severe threat to global food security. Understanding the physiological and molecular responses of this species to insecticide exposure is crucial for improving resistance monitoring and developing effective management strategies. In this study, the toxicity and the associated biochemical, molecular, and genotoxic responses of third-instar S. frugiperda larvae were investigated following exposure to four commonly used insecticides: emamectin benzoate, indoxacarb, methomyl, and a binary mixture of acetamiprid + bifenthrin. Toxicity bioassays showed that emamectin benzoate was the most potent insecticide (LC(50) = 0.188 ppm/L), whereas the binary mixture exhibited the highest resistance coefficient (RC = 12.86) relative to the recommended field dose. Biochemical analyses conducted over a seven-day exposure period, significant metabolic alterations were observed. Reduced acetylcholinesterase (AChE) and carboxylesterase (CarE) activities suggested neurotoxic stress, Whereas elevated glutathione S-transferase (GST) and peroxidase (POD) activities reflected enhanced detoxification and oxidative stress responses. Gene expression analyses revealed significant upregulation of SFCYP1, SFCYP2, SFCYP4, SFCYP5, SFRYR, and EF1α, while SFCYP3 was consistently downregulated, indicating activation of detoxification and stress-related pathways. Genotoxic effects were assessed using the comet assay, which demonstrated significant DNAdamage in hemocytes of treated larvae. This damage was characterized by increased tail length, tail DNApercentage, and tail moment, particularly in individuals exposed to emamectin benzoate. Collectively, these findings indicate that sublethal insecticides exposure not only disrupts enzymatic homeostasis but also induces genotoxic stress. These findings demonstrate that S. frugiperda exhibits complex adaptive physiological and molecular responses that may contribute to resistance development under sustained insecticide selection pressure. Overall, the results provide integrated biochemical and molecular insights that can support future investigation into insecticide response mechanisms and improve resistance monitoring strategies for this economically important pest. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-45372-w.