LAUSR

Significance Plants and insects are locked in a coevolutionary battle, where plants develop novel chemical defenses and insects adapt to overcome them. After mustards evolved defensive compounds, butterflies evolved the ability to detoxify said compounds and then rapidly diversified. Here, we use CRISPR-Cas9 gene knockouts to remove a Pierine butterfly’s ability to detoxify mustard defensive chemistry. By assessing these gene knockouts in caterpillars on relevant plants, we find that detoxification gene expression and performance are tailored to specific plant chemical defenses. Additionally, we find evidence that natural selection acted upon these detoxification genes in several butterfly species. Our results highlight the complexity of coevolutionary interactions and reveal the key roles that detection, activation, and regulatory mechanisms play in generating specific responses.