Odor tracking in flying Drosophila requires visual reafference and compass neurons

Abstract 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 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 basic 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 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. Significance Statement A crucial brain function that remains largely mysterious is building robust working memory by integrating stimuli across sensory modalities. For flying Drosophila , visual feedback is required to localize an odor source. Recently, a group of cells residing in the navigation center of the fly brain (E-PG neurons) have been shown to function like an internal compass, providing a dynamic working memory of heading changes akin to mammalian head-direction cells. Here, we show that self-generated visual motion signals work with E-PG compass neurons so that the direction of exploratory turns can be stored and recalled to maintain the animal’s heading within an odor plume during flight.