To begin, separate the male flies from a mixed population of flies on ice and put them in a collection vial. To prepare the arena, place two triangular acrylic spacers flat on top of a rectangular glass pane, such that the right angles of the acrylic spacers are aligned with the corners of the glass pane. Then place two rectangular acrylic spacers flat on the glass pane aligned with the bases of the two triangular spacers.
Confirm that the four acrylic spacers surround a triangular arena on the rectangular glass pane that is not covered by spacers. Position a sticker of a ruler on one of the triangular spacers, ensuring that it is visible from the top. Now place a second rectangular glass pane on top of the acrylic spacers to align it with the glass pane at the bottom.
Using four small binder clips, secure the panes and spacers. For the assay, remove the bottom right binder clip and slightly shift the rectangular spacer outwards to create a gap. To transfer flies from the collection vial into the social space arena, gently aspirate them through the created space.
Immediately slide the rectangular spacer and secure the binder clip back in its position. Holding the chamber upright, gently pound three times onto a soft pad to let all the flies settle at the base. Clamp the chamber in the upright position and start the timer.
Take a clear photo of the arena after 20 minutes. Analyze with ImageJ to list the inter-fly nearest neighbor distances. The mutant flies show significantly higher nearest neighbor distances than the control counterparts, indicating a preference for greater distancing from other flies.
