Common Practice Solvent Extraction Does not Reflect Actual Emission of a Sex Pheromone During Butterfly Courtship

Abstract

Olfactory communication can be of critical importance for mate choice decisions. Lepidoptera are key model systems for understanding olfactory communication, particularly considering sex pheromone signaling in the context of sexual selection. Solvent extraction or rinsing of pheromone-producing structures is a widespread method for quantifying sex pheromones, but such measures reflect what is stored and may not represent what is actually emitted by an individual during courtship. Here, we address this point for the first time by quantifying the components of the male sex pheromone (MSP) of interacting Bicyclus anynana butterflies, a species for which much information is available onthe role played by MSPs in affecting mating success. Using headspace sampling during courtship and solvent extraction after completion of experiments using the same males, we were able to track individual traits. Our results show that solvent extracts do not reflect quantities of MSP components emitted by live butterflies. We further show that MSP amounts obtained using headspace sampling correlated with male mating success, but solvent extracts did not. Our results further strongly suggest that males actively control MSP emission when faced with increased male-male competition. Common practice solvent extracts may thus not serve as an adequate proxy for male sex pheromone signaling as they are perceived by choosy females. Our study serves as a proof of principle that quantification of male sex pheromone components depends on the method of collection, which could apply to many other insects using short-range chemical signals. This affects our understanding of how sexual selection shapes the evolution of sexually-selected chemical traits.

The scent chemistry of butterflies

“…132 Headspace analyses showed that males can actively control MSP emission and adjust to an increased male–male competition. 137 Other effects such as phenotypic plasticity in MSP production, 124 geographical variation, likely driving reproductive isolation and speciation, 138 reproductive character displacement MSP composition of different Bicyclus species, 123 learning of visual signals in mate choice, 139 and plastic female preferences for a male pheromone odour blend influenced by early pheromone odour experiences 140 have been studied.…”

“…141 A comparison of wing extraction and headspace sampling methods underlined the importance and differences of sampling methods. 137 While wing extracts only reflect stored compounds, it does not necessarily represent the compounds actually emitted during courtship. Whereas headspace analysis maybe better at representing the released scent, although valid for a confined space, it is not known if it is comparable to what a female receives.…”

“…Whereas headspace analysis maybe better at representing the released scent, although valid for a confined space, it is not known if it is comparable to what a female receives. 137 Nevertheless, EAG recordings at least underline the possibility of the detection of released compounds. The MSP ratios obtained for B. anynana with both methods differed significantly.…”

“…The MSP ratios obtained for B. anynana with both methods differed significantly. 137 Although this discussion was centred on the results obtained from B. anynana , it is true for all kinds of analysis of volatile, behaviour-mediating compounds.…”