Abstract

Closed-loop paradigms provide an effective approach for studying visual choice behaviour and attention in small animals. Different flying and walking paradigms have been developed to investigate behavioural and neuronal responses to competing stimuli in insects such as bees and flies. However, the variety of stimulus choices that can be presented over one experiment is often limited. Current choice paradigms are mostly constrained as single binary choice scenarios that are influenced by the linear structure of classical conditioning paradigms. Here, we present a novel behavioural choice paradigm that allows animals to explore a closed geometry of interconnected binary choices by repeatedly selecting among competing objects, thereby revealing stimulus preferences in an historical context. We used our novel paradigm to investigate visual flicker preferences in honeybees (Apis mellifera) and found significant preferences for 20-25 Hz flicker and avoidance of higher (50-100 Hz) and lower (2-4 Hz) flicker frequencies. Similar results were found when bees were presented with three simultaneous choices instead of two, and when they were given the chance to select previously rejected choices. Our results show that honeybees can discriminate among different flicker frequencies and that their visual preferences are persistent even under different experimental conditions. Interestingly, avoided stimuli were more attractive if they were novel, suggesting that novelty salience can override innate preferences. Our recursive virtual reality environment provides a new approach to studying visual discrimination and choice behaviour in animals.