Alternative Splicing in TRPA1 Drives Sensory Adaptation to Electrophiles in Drosophilids.

Behaviors are among the first traits to evolve as animals enter new niches, but their molecular bases are poorly understood. To address this gap, we used the mustard-feeding drosophilid fly , which feeds on toxic Brassicales plants like wasabi that produce noxious, electrophilic isothiocyanates (ITCs or mustard oils). We found that exhibits dramatically reduced behavioral sensitivity to allyl isothiocyanate (AITC) compared to its microbe-feeding relatives and . We hypothesized that molecular evolution of the "wasabi receptor" TRPA1, known to detect ITCs in flies, could explain this loss of aversion. Our experiments revealed three interconnected evolutionary genetic changes consistent with this hypothesis. First, TRPA1 was expressed in labellar tissues of at the lowest levels among the three species, at a nearly four-fold lower level than in . Second, expressed a higher proportion of TRPA1 splice variants previously reported to be less sensitive to chemical stimulus. Third, we identified amino acid substitutions in that could influence the structure of intracellular domains of TRPA1. To test the functional salience of these mechanisms and , we validated TRPA1 splicing isoforms using oocyte electrophysiology and the system in . Single TRPA1 isoform electrophysiology revealed evolution of the channel in the lineage towards reduced electrophile sensitivity. Ectopic expression of TRPA1 in also consistently conferred weaker AITC sensitivity than expression of its orthologues, although this did not fully recapitulate differences in wild-type phenotypes between species, suggesting other molecular mechanisms were involved. To address this, we explored the consequences of isoform co-expression using oocyte electrophysiology. We found that enrichment of electrophile-insensitive TRPA1 splicing isoforms as observed in the salient sensory organs additively reduced cellular responses to AITC, which could further contribute to reduced electrophile aversion. Our findings illuminate how expression differences, protein structural changes, and especially alternative splicing, together can drive sensory evolution as animals behaviorally adapt to toxic new niches.