Enemies, more than sex, shape butterfly post-mating odor evolution

Not all odors influencing mating behavior evolve as sex pheromones. Female butterflies post-mating odors have been considered species-specific anti-aphrodisiac pheromones shaped by sexual selection but may also serve broader ecological roles shaped by natural selection. Males transfer odors to females that repel rivals, yet the widespread use of these compounds across phyla makes them targets for eavesdropping, such as by phoretic egg parasitoids. We show that in cabbage white butterflies (Pieris spp.), these odors are highly variable and attract parasitoids, deter predators, and influence oviposition. Using gas chromatography and electroantennography, we demonstrate that odor emission and perception lack species-specificity: compounds once thought unique to P. brassicae and P. rapae are shared across Pieridae. In P. napi, odor variation among populations correlates with parasitoid pressure, but not with latitude, genetic distance, or mating frequency, suggesting ecological rather than sexual drivers. In P. brassicae, CRISPR/Cas9 disruption of odor perception alters oviposition and increases susceptibility to parasitism. Moreover, these odors render females unpalatable to birds. Together, our results show that post-mating odors in Pieris butterflies may act as aposematic signals. We provide evidence that these signals evolve under multiple selective pressures, balancing deterrence of mates and predators, parasitoid avoidance, and host-plant interactions. These findings suggest that chemical signals should be viewed as integrating ecological and reproductive pressures, rather than being interpreted solely through the lens of sexual communication.