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Free flying bees are known for their impressive visual cognition abilities, but the neural bases underlying those are poorly studied because of the difficulty to explore the brain in a flying insect. Conversely, it is possible to have access to the brain with tethered bees but, until now, no studies explored visual learning. To bypass this limitation, our aim was to establish a virtual reality device to test visual learning in tethered bees. First, bees were tested for their abilities to learn to discriminate colors according to the reinforcement associated to each of them. These experiments allowed to highlight the role of the active vision in virtual reality and the importance of phototaxis in this kind of device. Driven by these good results, we explored the non- elementary visual learning in bees and, more specifically, the negative patterning. The results suggest that bees were able to resolve this task in virtual reality despite its complexity. Finally, using the protocol of color discrimination, the brain activation has been explored during a color memory recall thanks to a quantification of immediate early genes considered as neural markers. This thesis provides two solid virtual reality protocols to study elementary and non-elementary visual learning in honey bees. This constitutes a huge advance and will allow to go further towards the understanding of the neural bases of simple and complex visual learning.; Les abeilles en libre vol montrent des capacités cognitives visuelles remarquables mais les bases neurales sous-jacentes ne peuvent pas être étudiées chez des insectes en vol. À l'inverse, le cerveau des abeilles immobilisées est accessible mais ne permet pas d'explorer l'apprentissage visuel. Pour dépasser cette limite, notre objectif a été d'établir un dispositif de réalité virtuelle pour pouvoir tester l'apprentissage visuel sur des abeilles attachées. Dans un premier temps, les abeilles ont été testées sur leur capacité à discriminer des couleurs en s'appuyant sur les renforcements positifs ou négatifs qui leur étaient associés. Ces expériences ont permis de mettre en évidence le rôle de la vision active dans la réalité virtuelle et l'importance de la phototaxie dans ce type de système. Grâce à ce dispositif, un apprentissage non élémentaire en réalité virtuelle, le patterning négatif, a été mis en place. Ainsi il a été montré que les abeilles étaient capables de résoudre cette tache en réalité virtuelle malgré sa complexité. Enfin, en s'appuyant sur le protocole de discrimination de couleurs, l'activation du cerveau a été étudiée au cours d'un test de mémoire des couleurs en analysant l'expression de gènes considérés comme des marqueurs de l'activité neurale. Les résultats de cette thèse, fournissant deux protocoles de réalité virtuelle solides pour étudier les apprentissages visuels élémentaires et non élémentaires, constituent une avancée considérable vers la possibilité de comprendre les bases neurales sous-jacentes à ces apprentissages chez l'abeille qu'ils soient simples ou complexes.

Free flying bees are known for their impressive visual cognition abilities, but the neural bases underlying those are poorly studied because of the difficulty to explore the brain in a flying insect. Conversely, it is possible to have access to the brain with tethered bees but, until now, no studies explored visual learning. To bypass this limitation, our aim was to establish a virtual reality device to test visual learning in tethered bees. First, bees were tested for their abilities to learn to discriminate colors according to the reinforcement associated to each of them. These experiments allowed to highlight the role of the active vision in virtual reality and the importance of phototaxis in this kind of device. Driven by these good results, we explored the non- elementary visual learning in bees and, more specifically, the negative patterning. The results suggest that bees were able to resolve this task in virtual reality despite its complexity. Finally, using the protocol of color discrimination, the brain activation has been explored during a color memory recall thanks to a quantification of immediate early genes considered as neural markers. This thesis provides two solid virtual reality protocols to study elementary and non-elementary visual learning in honey bees. This constitutes a huge advance and will allow to go further towards the understanding of the neural bases of simple and complex visual learning., Les abeilles en libre vol montrent des capacités cognitives visuelles remarquables mais les bases neurales sous-jacentes ne peuvent pas être étudiées chez des insectes en vol. À l'inverse, le cerveau des abeilles immobilisées est accessible mais ne permet pas d'explorer l'apprentissage visuel. Pour dépasser cette limite, notre objectif a été d'établir un dispositif de réalité virtuelle pour pouvoir tester l'apprentissage visuel sur des abeilles attachées. Dans un premier temps, les abeilles ont été testées sur leur capacité à discriminer des couleurs en s'appuyant sur les renforcements positifs ou négatifs qui leur étaient associés. Ces expériences ont permis de mettre en évidence le rôle de la vision active dans la réalité virtuelle et l'importance de la phototaxie dans ce type de système. Grâce à ce dispositif, un apprentissage non élémentaire en réalité virtuelle, le patterning négatif, a été mis en place. Ainsi il a été montré que les abeilles étaient capables de résoudre cette tache en réalité virtuelle malgré sa complexité. Enfin, en s'appuyant sur le protocole de discrimination de couleurs, l'activation du cerveau a été étudiée au cours d'un test de mémoire des couleurs en analysant l'expression de gènes considérés comme des marqueurs de l'activité neurale. Les résultats de cette thèse, fournissant deux protocoles de réalité virtuelle solides pour étudier les apprentissages visuels élémentaires et non élémentaires, constituent une avancée considérable vers la possibilité de comprendre les bases neurales sous-jacentes à ces apprentissages chez l'abeille qu'ils soient simples ou complexes.

To study visual learning in honey bees, we developed a virtual reality (VR) system in which the movements of a tethered bee walking stationary on a spherical treadmill update the visual panorama presented in front of it (closed-loop conditions), thus creating an experience of immersion within a virtual environment. In parallel, we developed a small Y-maze with interchangeable end-boxes, which allowed replacing repeatedly a freely walking bee into the starting point of the maze for repeated decision recording. Using conditioning and transfer experiments between the VR setup and the Y-maze, we studied the extent to which movement freedom and active vision are crucial for learning a simple color discrimination. Approximately 57% of the bees learned the visual discrimination in both conditions. Transfer from VR to the maze improved significantly the bees' performances: 75% of bees having chosen the CS+ continued doing so and 100% of bees having chosen the CS- reverted their choice in favor of the CS+. In contrast, no improvement was seen for these two groups of bees during the reciprocal transfer from the Y-maze to VR. In this case, bees exhibited inconsistent choices in the VR setup. The asymmetric transfer between contexts indicates that the information learned in each environment may be different despite the similar learning success. Moreover, it shows that reducing the possibility of active vision and movement freedom in the passage from the maze to the VR impairs the expression of visual learning while increasing them in the reciprocal transfer improves it. Our results underline the active nature of visual processing in bees and allow discussing the developments required for immersive VR experiences in insects.

Free-flying honeybees exhibit remarkable cognitive capacities but the neural underpinnings of these capacities cannot be studied in flying insects. Conversely, immobilized bees are accessible to neurobiological investigation but display poor visual learning. To overcome this limitation, we aimed at establishing a controlled visual environment in which tethered bees walking on a spherical treadmill learn to discriminate visual stimuli video projected in front of them. Freely flying bees trained to walk into a miniature Y-maze displaying these stimuli in a dark environment learned the visual discrimination efficiently when one of them (CS+) was paired with sucrose and the other with quinine solution (CS−). Adapting this discrimination to the treadmill paradigm with a tethered, walking bee was successful as bees exhibited robust discrimination and preferred the CS+ to the CS− after training. As learning was better in the maze, movement freedom, active vision and behavioral context might be important for visual learning. The nature of the punishment associated with the CS− also affects learning as quinine and distilled water enhanced the proportion of learners. Thus, visual learning is amenable to a controlled environment in which tethered bees learn visual stimuli, a result that is important for future neurobiological studies in virtual reality.