Associative Learning of Quantitative Mechanosensory Stimuli in Honeybees

Simple Summary One major challenge when using animal models to study cognition is that we cannot ask them what they are thinking or how they are feeling; instead, we measure the animal's behavior. For honeybees, the extension of the proboscis (their tongue-like structure) occurs when they are presented with a sucrose solution, and they can be trained to associate a neutral cue-for example, an odor-with the occurrence of this food reward, eventually extending the proboscis when presented with the neutral cue rather than the food cue. Thus, the proboscis extension response (PER) is useful for exploring honeybees' sensory perception, learning, and memory. In this study, we tested a new stimulus, namely different speeds of air flow, to investigate whether honeybees were able to associate this cue with the reward. Additionally, we designed a new system for performing PER experiments wherein the stimulus delivery and analyses are entirely automated, rather than performed manually. Using this enhanced method, we found that honeybees succeeded when a lower air flux was rewarded, but not when a higher air flux was rewarded. These results add to our knowledge of stimulus intensity encoding, while our improved PER system will offer technical advantages for such experiments in the future.Abstract The proboscis extension response (PER) has been widely used to evaluate honeybees' (Apis mellifera) learning and memory abilities, typically by using odors and visual cues for the conditioned stimuli. Here we asked whether honeybees could learn to distinguish between different magnitudes of the same type of stimulus, given as two speeds of air flux. By taking advantage of a novel automated system for administering PER experiments, we determined that the bees were highly successful when the lower air flux was rewarded and less successful when the higher flux was rewarded. Importantly, since our method includes AI-assisted analysis, we were able to consider subthreshold responses at a high temporal resolution; this analysis revealed patterns of rapid generalization and slowly acquired discrimination between the rewarded and unrewarded stimuli, as well as indications that the high air flux may have been mildly aversive. The learning curve for these mechanosensory stimuli, at least when the lower flux is rewarded, more closely mimics prior data from olfactory PER studies rather than visual ones, possibly in agreement with recent findings that the insect olfactory system is also sensitive to mechanosensory information. This work demonstrates a new modality to be used in PER experiments and lays the foundation for deeper exploration of honeybee cognitive processes when posed with complex learning challenges.