Involvement of Multiple Ion Channels and Receptors in Mediating the Insecticidal and Repellent Actions of Limonene

R-limonene has been integrated into various pest control practices as a repellent or an insecticide. However, how limonene induces aversion or mortality remains largely unknown. To elucidate the underlying mechanisms, we conducted behavioral, toxicological, and electrophysiological assays in <i>Aedes aegypti</i>, a primary vector of human diseases. To investigate whether limonene acts on voltage-gated sodium channels and/or the Rdl (Resistance to dieldrin) receptor, two major targets of neuroactive insecticides, we characterized the effect of limonene on <i>Ae. aegypti</i> sodium and Rdl channels expressed in <i>Xenopus</i> oocytes. Limonene significantly potentiated GABA-induced chloride currents through Rdl in a concentration-dependent manner but had no effect on sodium channels. For repellency, limonene evoked spatial repellency in wild-type mosquitoes; however, the spatial repellency by limonene was significantly reduced in knockout mutants of <i>Orco^-/-</i> (odorant receptor co-receptor) and <i>TRPA1^-/-</i> (Transient Receptor Protein, subfamily A and member 1). These results indicate that limonene likely targets the Rdl receptor for insecticidal activity and limonene spatial repellency requires both Orco and TRPA1 channels. Our results reveal the involvement of multiple ion channels and receptors in the mosquito nervous system for limonene's insecticidal and/or spatial repellency actions, highlighting limonene's potential as a multi-target neuroactive agent for pest control.