Odor tracking in flying Drosophila requires visual reafference and compass neurons.

Flying Drosophila critically depend on high-contrast visual surroundings to localize odor sources in still air, yet the neural mechanisms of visual integration for active odor tracking are unknown. We demonstrate that Ellipsoid body-Protocerebral bridge-Gall (E-PG) neurons-head direction cells in the central complex-work in concert with self-generated visual motion signals to maintain a stable heading metric during olfactory navigation in flight. Using a magnetic tether system and a digital "visual clamp," we show that removing the visual feedback generated by a fly's own turns (reafference) causes the animal to lose its heading within an odor plume. Thus, olfactory and mechanosensory signals alone are insufficient for plume stabilization. E-PG neurons have been shown to store visual changes in heading during flight. Genetically hyperpolarizing E-PG neurons significantly compromised the flies' ability to both acquire and maintain heading toward a food odor. Notably, silencing these neurons did not disrupt basic visual reflexes, such as optomotor gaze stabilization or object tracking, indicating a specific role in odor-directed visual navigation rather than visual flight control. While odor was found to modulate the frequency and amplitude of turns independently, E-PG neurons are essential for directing the orientation of corrective saccades toward the plume center. These results establish that visual reafference engages the internal visual compass and associated control circuits to sustain a spatial working memory of heading changes between saccades, allowing flies to maintain a straight course and navigate effectively toward an invisible odor source in flight in still air.