Flies remember the time of day.

The circadian clock enables organisms to anticipate daily environmental cycles and drives corresponding changes in behavior [1, 2]. Such endogenous oscillators also enable animals to display time-specific memory [1, 3-5]. For instance, mice and honeybees associate the location of a stimulus (like food or mate) with a certain time of day (time-place learning) [6, 7]. However, the mechanism underlying time-related learning and memory is not known. In the present study, we investigate time-specific odor learning. We use a genetically tractable animal, the fly Drosophila melanogaster. Starved flies are trained in the morning and afternoon to associate distinct odors with sucrose reward. The training is repeated the next day, and their time-dependent odor preference is tested on the third day. Our results indicate that Drosophila can express appetitive memory at the relevant time of day if the two conditioning events are separated by more than 4 hr. Flies can form time-odor associations in constant darkness (DD) as well as in a daily light-dark (LD) cycle, but not when kept under constant light (LL) conditions. Circadian clock mutants, period(01) (per(01)) and clock(AR) (clk(AR)), learned to associate sucrose reward with a certain odor but were unable to form time-odor associations. Our findings show that flies can utilize temporal information as an additional cue in appetitive learning. Time-odor learning in flies depends on a per- and clk-dependent endogenous mechanism that is independent of environmental light cues.