Like many other animals, insects are capable of returning to previously visited locations using path integration, which is a memory of travelled direction and distance. Recent studies suggest that Drosophila can also use path integration to return to a food reward. However, the existing experimental evidence for path integration in Drosophila has a potential confound: pheromones deposited at the site of reward might enable flies to find previously rewarding locations even without memory. Here, we show that pheromones can indeed cause naïve flies to accumulate where previous flies had been rewarded in a navigation task. Therefore, we designed an experiment to determine if flies can use path integration memory despite potential pheromonal cues by displacing the flies shortly after an optogenetic reward. We found that rewarded flies returned to the location predicted by a memory-based model. Several analyses are consistent with path integration as the mechanism by which flies returned to the reward. We conclude that although pheromones are often important in fly navigation and must be carefully controlled for in future experiments, Drosophila may indeed be capable of performing path integration.
