High-Throughput Tracking of Freely Moving Drosophila Reveals Variations in Aggression and Courtship Behaviors

Aggression is a nearly universal behavior used to secure food, territory, and mates across species, including the fruit fly Drosophila melanogaster . In fruit flies, both sexes display aggression through stereotypical motor patterns. This, along with their sophisticated genetic and molecular toolkit, makes Drosophila melanogaster an excellent model for studying aggression. While male- and female-specific aggressive motor programs have been qualitatively described, automated systems for quantifying these behaviors in freely moving flies remain limited in their ability to combine high-resolution analysis with high throughput. Here, we pair a high-resolution, high-throughput imaging system (the Kestrel) with DeepLabCut pose estimation to create a pipeline that tracks multiple freely moving fly pairs and quantifies social dynamics with high fidelity. We validated body-part tracking using published benchmarks. The platform reliably reproduced a known phenotype: heightened female aggression following thermogenetic activation of cholinergic pC1 neurons in female brain. It also detected increased unilateral wing extension, a courtship display inversely related to aggression, between two males upon activating a previously uncharacterized ∼40-neuron group in the male brain. Pose-based analysis revealed locomotive differences between experimental and control groups, and subtle, genotype-specific variations in head butts and UWEs. This workflow enables high-throughput screening and mechanistic dissection of social behaviors.