Hawkmoths learn best without negative reinforcement in a differential colour association task

Abstract

Visual learning in insects can be strongly influenced by the underlying context and conditions. Improved colour learning has been demonstrated with differential conditioning using reward-aversion paradigms in eusocial insects like wasps, bumblebees, and honeybees, where substances that induce strong aversion, such as quinine, enable more accurate learning. Although the associations created during learning are intrinsically linked to the reinforcements used, few studies have compared the role of negative reinforcements in shaping these associations, and their relevance across insect groups with different life histories. In our study, we compared the effects of aversive substances for learning in the hummingbird hawkmoth (Macroglossum stellatarum), a solitary pollinator that relies on vision for foraging. Linking to previous learning studies in insects, we combined a sugar-rewarded target with a distractor that was paired with either quinine, salt, citric acid, water, or was presented with no aversive substance. Learning was assessed by conditioning hawkmoths to invert a strong colour preference between perceptually close or distant colour pairs, to provide tasks with disparate challenges. Contrary to results from eusocial insect species, hawkmoths trained with quinine were worse at switching preferences between similar colours compared to training with citric acid or appetitive-only differential conditioning. Learning success was associated with the animals foraging plasticity, where negative reinforcements were found to suppress exploration during foraging. Furthermore, we show that quinine interfered with sucrose perception, potentially impairing target acquisition during conditioning. Our results provide insights into the impact of negative reinforcements on solitary foragers and highlight the role of sensory ecology and life history in shaping learning outcomes.