Assessing Experience-dependent Tuning of Song Preference in Fruit Flies ( Drosophila melanogaster )

[Abstract] In songbirds and higher mammals, early auditory experience during childhood is critical to detect and discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. Here, we describe a new behavioral paradigm with Drosophila melanogaster to investigate how the auditory experience shapes sound perception. This behavioral paradigm consists of two parts: training session and test session. In the training session, we keep the flies singly in a training capsule and expose them to training sound for 6 days after eclosion. After the training session, flies are subjected to the test session, in which the mating behaviors of flies are monitored upon sound playback. As the training and test sounds, we use two types of artificial sound, which correspond to the pattern of conspecific and heterospecific courtship songs of fruit flies. By applying this method, we can measure how the acoustic experience with the conspecific song as a young adult sharpens the song preference and mate selection as a breeding adult in the fruit fly.

Here we report a behavioral paradigm to assess how an experience of hearing the conspecific song as a young adult sharpens the song preference and mate selection as a breeding adult in the fruit fly.
This paradigm consists of training session and test session. At the training session, we expose single males or females to an artificial courtship song. At the test session, we use the male-female courtship behavior and male-male chaining behavior to measure the response in females and males, respectively.
By combining this brand-new behavioral paradigm with sophisticated genetic tools established in flies, such as the GAL4/UAS binary expression system to manipulate individual genes and neurons, this novel approach gives the potential to clarify the neural mechanism on how experience-dependent tuning of the mating preference is built upon both innate and experience-dependent auditory systems. A sound file comprised of the repetition of 1-sec pulse burst and a subsequent 2-sec pause, in which the inter-pulse interval (IPI) is 35 msec ("conspecific song", Supplemental audio file 1) or 75 msec ("heterospecific song" Supplemental audio file 2). Intra-pulse frequency of both songs is 167 Hz. Use Audacity to make the artificial pulse songs. 11. Fly food (Standard yeast-based media) (see Recipes) 3 www.bio-protocol.org/e2932

Rubber support
A custom-made apparatus to support the female copulation plate. Make a hole (70 mm in diameter) in the middle of a rubber sheet to hold the female copulation plate. 5 www.bio-protocol.org/e2932   Other media player can also be used.

Logitech Webcam Software
Provided with the web camera 6 www.bio-protocol.org/e2932

5.
Start an artificial pulse song (conspecific song or heterospecific song) playback for experienced flies. For Naïve flies, no sound is played. The mean baseline-to-peak amplitude of sound particle velocity is 8.6 mm/sec when measured at the near end of the training capsules, and 6.6 mm/sec at the far end of the training capsule. The sound particle velocity is identical for all training sounds.
6. The artificial pulse song, 3-min long, is stored in a WAV format audio file (monaural, 44,100 Hz, 32-bit float). Play the audio file repeatedly with Windows media player for 6 days. Renew food in each training capsule every 36 h.  Rubber support holding the copulation plate is placed over a loudspeaker with appropriate distance. C. Example graph of song response of female flies. Cumulative copulation rate in the heterospecific song test after training is plotted. Naïve group (no sound during training) and experienced groups (trained with conspecific song or heterospecific song) are shown. N, Naïve; E, experienced. N.S., not significant, P > 0.05; ***P < 0.001; Kruskal-Wallis test versus Naïve group.
d. Start the sound playback and video recording simultaneously. The particle velocity received at the apparatus is adjusted as 9.2 mm/sec. e. Record the behaviors of flies for 30 min with a web camera (Logitech, Logicool ® ) with a video capture software (Logitech Webcam Software) (Video 1). 9 www.bio-protocol.org/e2932

Female copulation assay
Evaluate behavioral response of females to the artificial pulse songs by observing the copulation latency (time to mating from the test started) of a female paired with a partner male in the chamber. Here, copulation is defined by observing the specific features as follows: (1) females permit a male to mount them for more than 1 min, (2) females reduce their locomotor activity with the mounting partner, and (3) females part her wings during the mounting (Manning, 1967;Yamada et al., 2018).
This copulation latency is analyzed manually from the video playback.