sfr‐miR‐10465‐5p and sfr‐miR‐10476‐5p modulate chlorantraniliprole susceptibility through <i>CYP450</i> gene regulation in <i>Spodoptera frugiperda</i>

MicroRNAs (miRNAs) are emerging as crucial regulators of insecticide responses in insect pests through modulation of detoxification-related genes. In our previous study, sfr-miR-10465-5p and sfr-miR-10476-5p were identified as differentially expressed in response to chlorantraniliprole exposure in Spodoptera frugiperda. However, their specific roles in modulating chlorantraniliprole susceptibility via cytochrome P450 (CYP450) regulation remained largely unexplored. Functional validation through mimic/inhibitor microinjection demonstrated that overexpression of these miRNAs significantly increased larval mortality following chlorantraniliprole treatment, while inhibition reduced susceptibility, confirming their regulatory impact. Furthermore, target prediction and subsequent validation identified CYP4S9 (LOC118273960), CYP340AX1 (LOC118270458), and CYP367A1v2 (LOC118281718) as putative targets of these two miRNAs. Dual-luciferase reporter assays confirmed direct binding of sfr-miR-10465-5p to CYP4S9 (LOC118273960), and sfr-miR-10476-5p to CYP340AX1 (LOC118270458) and CYP367A1v2 (LOC118281718) through their coding sequence (CDS) regions, confirming post-transcriptional regulation. RNAi-mediated knockdown of these CYP450 genes significantly enhanced chlorantraniliprole-induced mortality, further supporting their functional role in detoxification. Tissue-specific expression profiling revealed predominant expression of both miRNAs and their targets in the fat body, a major site of insecticide metabolism. Together, these findings highlight the regulatory function of sfr-miR-10465-5p and sfr-miR-10476-5p in chlorantraniliprole susceptibility via CYP450 gene modulation, offering novel insights into miRNA-mediated chlorantraniliprole susceptibility mechanisms in S. frugiperda.