Odor Discrimination and Sequence Learning in Honeybees

Animals process information from the environment through different sensory systems, including olfaction. In honeybees, olfaction plays a key role in resource detection and behavior. Bees must discriminate complex stimuli in their components and also be able to process their temporal order. Here we study olfactory discrimination in honeybees by using differential conditioning protocols. First, we evaluated the effectiveness of different unconditioned stimuli (US) that maximize odor separation. Using the proboscis extension response (PER), bees were conditioned by pairing odor A (CS+) with sucrose and odor B (CS-) with either quinine or a neutral tactile control stimulus. Next, using the optimal USs combination, we investigated whether honeybees can learn and discriminate odor sequences according to their temporal order, by training them with AB (appetitive) and BA (aversive), and testing them with sequences AB, BA, A and B. Both experiments were analyzed using DLC to track the precise kinematics of the antennae and proboscis response. In the first experiment, we obtained clearer behavioral evidence of aversive learning and discrimination learning, when quinine was used as aversive US rather than the tactile stimulus. These results provide a behavioral framework for investigating olfactory discrimination and temporal sequence processing in honeybees.