Serotonergic-Dependent Awareness Is Required for Trace Conditioning in Honey Bees

In humans, awareness of stimulus contingencies can be studied under controlled experimental conditions by analyzing participants' reports of their subjective experiences with stimuli. Pavlovian conditioning protocols have been used to investigate whether awareness is necessary for learning, particularly in trace versus delay conditioning. In trace conditioning—where a conditioned stimulus (CS) and an unconditioned stimulus (US) are separated by a temporal gap—learning is disrupted by distractors. In contrast, delay conditioning—where the CS and US overlap—remains unaffected. Here, we examined the impact of distractors on both conditioning variants in honey bees using olfactory conditioning of the proboscis extension response, which allows precise control over CS and US timing. While bees successfully learned the CS-US association in both paradigms, acquisition and memory retention were lower in trace conditioning, suggesting greater cognitive demand. Video analysis of the proboscis responses revealed distinct dynamics between the two conditioning forms, including differences in latency and licking events. Notably, trace conditioning was disrupted by visual and mechanosensory distractors, whereas delay conditioning remained unaffected. Furthermore, blocking serotonin signalling impaired trace but not delay conditioning, highlighting a crucial role for serotonergic signalling in trace learning. These findings suggest that, similar to humans, honey bees may rely on an awareness of the CS-US relationship during trace conditioning and that serotonergic signalling is integral to this process. This implies that awareness of stimulus contingencies may extend to invertebrates when confronted with specific cognitive challenges.