DETERMINING OLFACTORY PREFERENCES IN A FLY-ON-A-BALL SETUP

An animal’s brain encodes complex sensory information about the environment and translates it into relevant behaviors for survival. Understanding this requires studying the behavior of a restrained animal while simultaneously recording or manipulating neural activity. A simple behavior that is suitable to study the sensory-motor transformation involves approach or avoidance towards environmental stimuli. The fly-on-a-ball setup is widely used for studies on sensory-motor transformation, in which a fly is tethered and placed on an air-suspended ball to study behavior and neural activity in unison. However, it is still unclear how to robustly categorize the tethered fly's behavior into attraction (moving towards) or aversion (moving away). To address this, using odorants with valences established previously in Drosophila melanogaster, we experimented with the fly-on-a-ball setup by delivering the odor from three distinct spatial positions: 0° (in-front), and 45° to the left and right of the fly’s heading. Using FicTrac software, we analyzed behavioral readouts including upwind velocity, heading angle, and angular velocity, to identify which parameter most reliably distinguishes between approach and avoidance across these different spatial configurations. Our results demonstrate that while frontal odor presentation (0°) can yield ambiguous results, asymmetric odor stimulation at 45° provides a robust behavioral readout. Specifically, we found that heading angle is the most intuitive and reliable indicator of valence: flies consistently orient toward the nozzle in response to attractive odorant and deviate away from aversive odorant stimuli. This work establishes an experimental tool for studying sensory-motor transformation in the fly’s brain.