Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo.

Predicting how species will respond to selection pressures requires identifying the factors that constrain their evolution. The repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides primarily occurs via a small subset of possible functionally-relevant substitutions to Na + ,K + -ATPase. We used genome engineering in Drosophila to evaluate the phenotypic effects of frequently observed substitutions at two sites (111 and 122). Surprisingly, these substitutions exhibit recessive lethality and dominant neural dysfunction. We identify a phylogenetically correlate substitution, A119S, that partially ameliorates the deleterious effects of substations at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro , A1119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. By revealing distinct effects of substitutions in vitro and in vivo , our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.