The pleiotropic effects of a single transcription factor underpin trade-offs associated with insecticide resistance in whitefly

• The P450 CYP6EM1-mediated neonicotinoids resistance incurs reproductive cost in whitefly • Upregulation of a TF E75 acts as a molecular switch to govern the fitness trade-offs. • GPCR-MAPK signaling axis is a conserved pivotal regulator in activating E75 by phosphorylation. • Understanding fitness trade-offs of insecticide resistance is key to sustainable pest control. The evolution of insecticide resistance is often accompanied by fitness costs on life-history traits; however, the genetic mechanisms underlying this ’benefit-cost’ trade-offs remain limited. Here, we aim to elucidate the molecular basis of fitness trade-offs associated with P450-mediated insecticide resistance. Insecticide bioassay and life-table methodology were used to determine thiacloprid resistance and its associated fitness cost in the whitefly Bemisia tabaci MED, a notorious pest of crops worldwide. Reverse genetics, western blots, immunofluorescence, molecular docking and dynamics simulation, recombinant P450 enzymes and UPLC-MS/MS were performed to demonstrate the role of the P450 CYP6EM1 in thiacloprid resistance. Dual-luciferase reporter assays, Phos-tag, EMSA, yeast one-hybrid and immunoprecipitation assay were conducted to investigate the role of a transcription factor in regulating the fitness trade-offs between thiacloprid resistance and the associated fitness cost. Here, we uncover that a single transcription factor, Ecdysone-induced protein 75 (E75), underpins trade-offs associated with thiacloprid resistance in the whitefly. We demonstrate that the transmembrane G protein-coupled receptor (GPCR) Neuropeptide FF receptor 2 (NPFF2) triggers the mitogen-activated protein kinase (MAPK) p38 and ERK signaling pathways, which then promote phosphorylation of E75. This GPCR-E75 regulatory axis not only activates the P450 gene CYP6EM1 but also represses a key oogenesis gene Bg , thereby contributing to the evolution of thiacloprid resistance at the expense of reproductive fitness in the whitefly. Our findings demonstrate a pivotal role of cis – trans regulatory networks in fitness trade-offs mediated by GPCR-MAPK-E75 signaling, providing in-depth insights into developing effective measures for sustainable pest control.