Serotonergic-dependent awareness is required for trace conditioning in honeybees

In humans, trace conditioning-where a temporal gap separates conditioned (CS) and unconditioned (US) stimuli-requires awareness and is sensitive to distraction, unlike delay conditioning. Here, we show that honeybees exhibit comparable features, suggesting cognitively demanding processes akin to awareness. Using the olfactory conditioning of the proboscis extension response, we found that bees learned in both paradigms, but trace conditioning resulted in weaker acquisition and memory. High-resolution video analysis revealed delayed response latencies and altered licking dynamics during trace learning. Strikingly, trace-but not delay-conditioning was impaired by visual and mechanosensory distractors. Neuropharmacological blockade of serotonin signaling selectively disrupted trace conditioning, implicating serotonergic circuits in bridging CS-US intervals. A computational model of the bee olfactory network supported this interpretation. Our findings reveal that bees engage higher-order cognitive mechanisms under temporal uncertainty, suggesting that core features of awareness may be evolutionarily conserved across distant taxa.