Cytochrome P450 CYP6CY3 Mediated Cross-Resistance to Isocycloseram and Fluralaner in <i>Myzus persicae</i>

The green peach aphid (<i>Myzus persicae</i>, Hemiptera: Aphididae) is a globally important pest. Isocycloseram (a novel isooxazoline) and fluralaner (a benzamide/sulfonamide) are novel insecticides. A Hainan-resistant population (HNR), exhibited 459- and 316-fold resistance to isocycloseram and fluralaner, respectively, compared with a Yunnan susceptible population (YNS). Synergist bioassays and enzyme activity analyses indicated that enhanced cytochrome P450 monooxygenase activity contributed to resistance. Transcriptomic and qRT-PCR analyses revealed that <i>CYP6CY3</i> was upregulated 16.4- and 10.8-fold in HNR compared with YNS. RNA interference of <i>CYP6CY3</i> significantly restored susceptibility to both insecticides. <i>In vitro</i> recombinant enzyme assays confirmed <i>CYP6CY3</i>-mediated metabolism of isocycloseram and fluralaner, and gene copy number analysis demonstrated a 7.09-fold amplification. These results reveal <i>CYP6CY3</i>-mediated cross-resistance to isocycloseram and fluralaner, representing the first mechanistic evidence of isoxazoline resistance in <i>M. persicae</i>.