Odorant receptors tuned to isothiocyanates in are co-opted and expanded in herbivorous relatives.

Plants release volatile compounds that attract mutualists, deter herbivores, and deceive pollinators. Among them are electrophilic compounds such as isothiocyanates (ITCs) derived Brassicales plants that activate TrpA1 pain receptors by contact in and humans. However, it is unclear whether generalist animals evolved strategies to detect these electrophilic compounds via olfaction. To address this, and to understand how specialized insects co-opted these toxic compounds as hostplant signatures, we studied generalist micro-feeding ( and ) and herbivorous mustard specialist drosophilid flies ( and ). In behavioral assays, exposed to volatile allyl isothiocyanate (AITC) were rapidly immobilized, demonstrating the high toxicity of this compound to non-specialists. Through single sensillum recordings (SSR) from olfactory organs and behavioral assays, we found that the Odorant receptor 42a (Or42a) is necessary for volatile AITC detection and behavioral aversion. RNA expression following heterologous expression showed that lineage-specific, triplicated Or42a proteins exhibited paralog-specific broadened ITC sensitivity. AlphaFold2 modeling followed by site-directed mutagenesis and SSR identified two critical amino acid substitutions that changed Or sensitivity from fruit-derived odors to ITCs during the evolution of . Our findings suggest that ITCs, which are toxic to most insects, can be detected and avoided by non-specialists like through olfaction. In the specialist , paralogous copies experienced gene duplication and amino acid substitutions resulting in expanded ITC sensitivity. Thus, insect olfactory systems can rapidly adapt to toxic host plant niches through co-option of chemosensory capabilities already present in their ancestors.