The olive fruit fly Bactrocera oleae, is the major key pest of olive groves worldwide. As an odor-driven species, its intraspecific communication has been thoroughly investigated, yielding a combination of spiroacetals, esters and hydrocarbons. However, its management with pheromone is still restricted to olean, the major pheromone component. Given the crucial role of circadian rhythm and pheromone blends in mediating flies reproductive behavior compared to single compounds, B. oleae headspace chemical profile was carefully examined, through the combination of Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToF) and Gas Chromatography coupled with Mass Spectrometry (GC-MS). This novel approach aimed at continuously investigating the temporal scale of volatilome profile of B. oleae individuals, as well as the determination of new candidate sexborne compounds (particularly those emitted in traces or having low molecular weight), that may be relevant to the fly's chemical communication and were unreported due to limitations of frequently used analytical techniques. Our results describe the dynamics and diversity of B. oleae chemical profile, highlighting the emission of 90 compounds, with clear diel rhythm of release, of known pheromone components of B. oleae (e.g., olean, alphapinene and muscalure) and new candidates. In contrast to ammonia, acetaldehyde and muscalure, which were highly emitted during the afternoon by males and mixed groups, olean was mostly released by mature females and mixed groups, with a peak of emission during early-morning and afternoon. This emission of olean around dawn is reported for the first time, suggesting early-morning mating activity in B. oleae. Furthermore, esters, such as methyl tetradecanoate, which had been earlier identified as a pheromone for B. oleae, did not exhibit any discernible release patterns. These findings are the first to demonstrate the emission of chemicals, which are only produced when males and females are close to one another, with an emission peak during the afternoon (mating period), and that may have aphrodisiac properties for B. oleae males. These results emphasize the relevance of compounds with distinct diel rhythm and address their potential function as intraspecific messengers, according to their source and timing of release.
