Common practice tissue extraction in solvent does not reflect actual emission of a sex pheromone during courtship in a butterfly

Olfactory communication can be of critical importance for mate choice decisions. Lepidoptera are important model systems for understanding olfactory communication, particularly considering sex pheromone signaling in the context of sexual selection. The extraction or rinsing of pheromone-producing structures is a widespread method for quantifying sex pheromones, but such measures reflect what is stored and not what is actually emitted by an individual during courtship. Here, we address this point by quantifying male sex pheromone (MSP) levels of interacting Bicyclus anynana butterflies using headspace sampling and tissue extraction after completion of experiments. Our results show that tissue extracts do not accurately predict pheromone quantities emitted by live butterflies. We further show that MSP quantities estimated by headspace sampling correlate with male mating success and that males actively control pheromone emission when faced with increasing male-male competition. Common practice tissue extracts thus do not reflect pheromone signals as they are perceived by choosy females, which should be taken into account in studies on sexually selected olfactory signals. Our easy-to-use entrainment system can readily be adapted and used for headspace sampling live individuals of any small- to medium-sized insect.