The evolution of insecticide resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) has resulted in large economic losses and disturbances to the environment and agroecosystems. Resistance to lufenuron, a chitin biosynthesis inhibitor insecticide, was recently documented in Brazilian populations of S. frugiperda. Thus, we utilized large-scale cDNA sequencing (RNA-Seq analysis) to compare the pattern of gene expression between lufenuron-resistant (LUF-R) and susceptible (LUF-S) S. larvae in an attempt to identify the molecular basis behind the resistance mechanism(s) of S. frugiperda to this insecticide. A transcriptome was assembled using approximately 19.6 million 100 bp-long single-end reads, which generated 18,506 transcripts with a N50 of 996 bp. A search against the NCBI non-redundant database generated 51.1 % (9,457) functionally annotated transcripts. A large portion of the alignments were homologous to insects, with the majority (45 %) being similar to sequences of Bombyx mori (Lepidoptera: Bombycidae). Moreover, 10 % of the alignments were similar to sequences of various species of Spodoptera (Lepidoptera: Noctuidae), with 3 % of them being similar to sequences of S. frugiperda. A comparative analysis of the gene expression between LUF-R and LUF-S S. frugiperda larvae identified 940 differentially expressed transcripts (p ≤ 0.05, t-test; fold change ≥ 4). Six of them were associated with cuticle metabolism. Of those, four were overexpressed in LUF-R larvae. The machinery involved with the detoxification process was represented by 35 differentially expressed transcripts; 24 of them belonging to P450 monooxygenases, four to glutathione-S-transferases, six to carboxylases and one to sulfotransferases. RNA-Seq analysis was validated for a number of selected candidate transcripts by using quantitative real time PCR (qPCR). The gene expression profile of LUF-R larvae of S. frugiperda differs from LUF-S larvae. In general, gene expression is much higher in resistant larvae when compared to the susceptible ones, particularly for those genes involved with pathways for xenobiotic detoxification, mainly represented by P450 monooxygenases transcripts. Our data indicate that enzymes involved with the detoxification process, and mostly the P450, are one of the resistance mechanisms employed by the LUF-R S. frugiperda larvae against lufenuron.

BACKGROUND: Cyantraniliprole, a diamide insecticide, is widely used in Brazil to control sucking and defoliating pests, including the fall armyworm (Spodoptera frugiperda), a major agricultural pest. However, increasing resistance to diamides has raised concerns about the long‐term effectiveness of cyantraniliprole. This study aimed to (i) assess the evolution of cyantraniliprole resistance in field populations of S. frugiperda, (ii) investigate cross‐resistance to other diamides, and (iii) analyze the role of the I4790K mutation in resistance mechanisms.RESULTS: A significant decrease in the susceptibility to cyantraniliprole was observed in field populations of S. frugiperda in Brazil, particularly in regions with intensive agricultural systems, such as the Brazilian Cerrado, from 2017 to 2023. A cyantraniliprole‐resistant strain of S. frugiperda was selected from a field‐collected population in Bahia (BA) using the F₂ screening method. The inheritance of resistance to cyantraniliprole in this strain was autosomal recessive and monogenic, with 3414‐fold resistance ratio. High cross‐resistance to the diamides flubendiamide, chlorantraniliprole, and cyclaniliprole was also detected. Molecular analysis confirmed the presence of the homozygous I4790K mutation in the ryanodine receptor gene as one of resistance mechanisms of this cyantraniliprole‐resistant strain.CONCLUSION: These findings highlight the decreased susceptibility of field populations of S. frugiperda to cyantraniliprole and the role of the I4790K mutation in accelerating the evolution of resistance to diamide insecticides due to cross‐resistance. These results underscore the urgent need for integrated pest management (IPM) strategies, including insecticide rotation and resistance monitoring, to preserve the efficacy of cyantraniliprole and other insecticides. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.