A single male auditory response test to quantify auditory behavioral responses in .

Many animals utilize auditory signals to communicate with conspecific individuals. During courtship, males of the fruit fly and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In , a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. Traditionally, the fly's response to the song is measured under grouped conditions, in which the effect of sensory modalities other than audition cannot be excluded. Here, to quantify the individual ability to discriminate a conspecific song, we systematically analyzed the auditory response of single male flies to sound with various parameters. Moreover, we applied this method, termed SMART (Single Male Auditory Response Test), to two sister species for potential application in a comparative approach. By quantifying the locomotor activity of single males during sound exposure, we detected increased locomotor activity in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, , exhibited a locomotor response to pulse song, while exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among species.