The Divider Assay is a high-throughput pipeline for aggression analysis in Drosophila.

Aggression is a complex social behavior that remains poorly understood. Drosophila has become a powerful model system to study the underlying biology of aggression but lack of high throughput screening and analysis continues to be a barrier for comprehensive mutant and circuit discovery. Here we developed the Divider Assay, a simplified experimental procedure to make aggression analysis in Drosophila fast and accurate. In contrast to existing methods, we can analyze aggression over long time intervals and in complete darkness. While aggression is reduced in the dark, flies are capable of intense fighting without seeing their opponent. Twenty-four-hour behavioral analysis showed a peak in fighting during the middle of the day, a drastic drop at night, followed by re-engagement with a further increase in aggression in anticipation of the next day. Our pipeline is easy to implement and will facilitate high throughput screening for mechanistic dissection of aggression.