Suzuki Hiromu C, Saito Claire T, Rajshekar Srivarsha, Sokabe Takaaki, Haji Diler, Groen Simon C, Peláez Julianne N, Matsunaga Teruyuki, Takemoto Ashleigh S, Tanaka Kentaro M, Takahashi Aya, Tominaga Makoto, Saito Shigeru, Whiteman Noah K
Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Graduate School of Biosciences, Nagahama Institute of Bio-Science and Technology, Nagahama 526-0829, Japan.
bioRxiv. 2025 May 15:2025.05.09.653172. doi: 10.1101/2025.05.09.653172.
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.
行为是动物进入新生态位时最早进化的特征之一，但其分子基础却知之甚少。为了填补这一空白，我们使用了以芥菜为食的果蝇，它以芥末等有毒十字花科植物为食，这些植物会产生有害的亲电异硫氰酸酯（ITC或芥子油）。我们发现，与以微生物为食的近亲果蝇和相比，果蝇对烯丙基异硫氰酸酯（AITC）的行为敏感性显著降低。我们推测，已知在果蝇中检测ITC的“芥末受体”TRPA1的分子进化可以解释这种厌恶感的丧失。我们的实验揭示了与这一假设一致的三个相互关联的进化遗传变化。首先，在这三个物种中，TRPA1在果蝇唇叶组织中的表达水平最低，比果蝇中的表达水平低近四倍。其次，果蝇中表达的TRPA1剪接变体比例较高，此前报道这些变体对化学刺激的敏感性较低。第三，我们在果蝇中鉴定出可能影响TRPA1细胞内结构域结构的氨基酸取代。为了测试这些机制的功能显著性，我们使用卵母细胞电生理学和果蝇系统验证了TRPA1剪接异构体。单个TRPA1异构体电生理学显示，果蝇谱系中的通道朝着降低亲电试剂敏感性的方向进化。果蝇中TRPA1的异位表达也始终赋予其比直系同源物表达更弱的AITC敏感性，尽管这并没有完全概括物种间野生型表型的差异，表明还涉及其他分子机制。为了解决这个问题，我们使用卵母细胞电生理学探索了异构体共表达的后果。我们发现，在果蝇显著的感觉器官中观察到的亲电试剂不敏感的TRPA1剪接异构体的富集，会累加降低细胞对AITC的反应，这可能进一步导致亲电试剂厌恶感的降低。我们的研究结果阐明了表达差异、蛋白质结构变化，尤其是可变剪接如何共同驱动感觉进化，使动物在行为上适应有毒的新生态位。
