Drosophila, a genus of the family Drosophilidae, consists of small fruit flies. Drosophila species’ unique courtship ritual consists of male flies dancing and singing to females through the vibration of their wings. Males may also present their wings in flicking motions, chase females, and circle in front of them. This behavior occurs in most Drosophila, including in the endemic North American subgroup affinis, which is composed of five species, Drosophila affinis, D. algonquin, D. athabasca, D. mahican, and D. lenape. In this group, previous work revealed that males were unlikely to initiate courtship to females of a different species. In other words, males exhibit courtship discrimination against courting foreign females and thus contribute to reproductive isolation between these fruit flies. In order to understand how and why this has evolved in nature, we propose that males are specifically targeting female-specific pheromones to assess: 1) the sex of the individual, 2) the age of females, and 3) their species identity. These aspects of female biology could influence the males’ decision to court females. To understand these dynamics, our goal in the present study was to analyze pheromone composition of individual flies across all five species as they age from birth to 13 days old. This time window allows capturing of the process by which these flies develop from sexually immature juveniles to sexually mature adults, which typically occurs around 3-5 days old. To perform this experiment, both male and female flies were placed into hexane and vortexed to extract pheromones into solution to be used for chemical analysis in the GCMS instrument. Comparing the retention times of obtained data to known standards, we visualized 1) sex-specific pheromones, 2) age-dependent and age-independent pheromones, and 3) species-specific pheromones. This data will then be used in future work to manipulate the pheromone profiles of individual females using “rub-off” experiments. This will then be used to test if we can induce males to court foreign females with “rubbed-off” pheromones of their own species. Additionally, understanding pheromone ontogeny gives insight into the threshold of when a pheromone becomes significant in courtship determination.
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