Drosophila, often colloquially referred to as the fruit fly, have unique courtship rituals consisting of male flies dancing and singing to females through the vibration of their wings. Males present their wings in flicking motions, chase females, and circle in front of them. However, prior to initiating courtship, males will often use olfactory receptors on their legs to tap and lick females, picking up on their cuticular hydrocarbon (CHC) profile. This profile is thought to be involved in species identity as well as age and sexual maturity. We investigated CHC’s role in speciation within the affinis subroup consisting of five species through a series of three experiments: ontogeny study, crowding experiments, and direct transfer of individual CHC compounds onto flies. First, we studied the CHC composition of individual flies as they age from birth (1 day old) to 13 days old (sexual maturity). We used GCMS analysis techniques to identify major compounds present in the flies during development. Secondly, we crowded flies with heterospecific mature females and conducted behavioral experiments to test whether CHC rubbing would impact heterospecific courtship. Finally, we directly transferred three key compounds that are highly differentiated during development between the five species: 2-methylhexacosane, 7,11-pentacosadiene, and Z11-tricosene. We conducted behavioral experiments to analyze the degree to which these compounds as well as a mixture of them influence courtship behavior
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