Background Spodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and to Bacillus thuringiensis (Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross‐resistance in FAW by investigating the toxicological profile of a Bt‐resistant Brazilian strain (Sf_Des) in comparison to a Bt‐susceptible strain (Sf_Bra). Results Laboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross‐resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited – depending on the substrate – up to 19‐fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA‐Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT‐qPCR) analysis confirmed that CYP9A‐like and CYP6B39 are significantly upregulated (>200‐fold) in Sf_Des in comparison to Sf_Bra strain. No target‐site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation–reduction and metabolic processes. Conclusion Our results indicate that multiple/cross‐resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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