Honey bees exhibit remarkable visual learning capacities, which can be studied using virtual reality (VR) landscapes in laboratory conditions. Existing VR environments for bees are imperfect as they provide either open-loop conditions or 2D displays. Here we achieved a true 3D environment in which walking bees learned to discriminate a rewarded from a punished virtual stimulus based on color differences. We included ventral or frontal background cues, which were also subjected to 3D updating based on the bee movements. We thus studied if and how the presence of such motion cues affected visual discrimination in our VR landscape. Our results showed that the presence of frontal, and to a lesser extent, of ventral background motion cues impaired the bees’ performance. Whenever these cues were suppressed, color discrimination learning became possible. We analyzed the specific contribution of foreground and background cues and discussed the role of attentional interference and differences in stimulus salience in the VR environment to account for these results. Overall, we show how background and target cues may interact at the perceptual level and influence associative learning in bees. In addition, we identify issues that may affect decision-making in VR landscapes, which require specific control by experimenters.

Free-flying bees learn efficiently to solve numerous visual tasks. Yet, the neural underpinnings of this capacity remain unexplored. We used a 3D virtual reality (VR) environment to study visual learning and determine if it leads to changes in immediate early gene (IEG) expression in specific areas of the bee brain. We focused on kakusei, Hr38 and Egr1, three IEGs that have been related to bee foraging and orientation, and compared their relative expression in the calyces of the mushroom bodies, the optic lobes and the rest of the brain after color discrimination learning. Bees learned to discriminate virtual stimuli displaying different colors and retained the information learned. Successful learners exhibited Egr1 upregulation only in the calyces of the mushroom bodies, thus uncovering a privileged involvement of these brain regions in associative color learning and the usefulness of Egr1 as a marker of neural activity induced by this phenomenon.

Non-elemental learning constitutes a cognitive challenge because events to be learned are usually ambiguous in terms of reinforcement outcome, contrary to elemental learning, which relies on unambiguous associations. Negative patterning (NP) constitutes a paradigmatic case of non-elemental learning, as subjects have to learn that single elements are reinforced while their simultaneous presentation is not reinforced (A+, B+ vs. AB−). Solving NP requires treating AB as being different from the linear sum of its components in order to overcome the ambiguity of stimulus reinforcement (i.e. A and B are as often reinforced as not reinforced). The honeybee is currently the only insect mastering NP as shown by studies restricted mainly to the olfactory domain. Here, we tested the bees' capacity to solve a NP discrimination in the visual domain and used to this end a virtual reality (VR) environment in which a tethered bee walking stationary on a spherical treadmill faces visual stimuli projected on a semicircular screen. We show that bees learn a composite grating made of alternated green and blue bars in an elemental way, and generalize their response to both a blue and a green grating. Yet, after NP training, one-quarter of the bees inhibited elemental processing and responded significantly more to the single-coloured gratings than to the composite grating. Alternative strategies were used by the other bees, which achieved partial NP learning. These results offer attractive perspectives to study different forms of visual learning in a controlled VR environment, and dissect their underlying mechanisms. K E Y W O R D Sinsect learning, negative patterning, non-elemental learning, virtual reality, visual cognition, visual learning 682 | BUATOIS eT Al.

Honey bees are reputed for their remarkable visual learning and navigation capabilities. These capacities can be studied in virtual reality (VR) environments, which allow studying performances of tethered animals in stationary flight or walk under full control of the sensory environment. Here, we used a 2D VR setup in which a tethered bee walking stationary under restrictive closed-loop conditions learned to discriminate vertical rectangles differing in color and reinforcing outcome. Closed-loop conditions restricted stimulus control to lateral displacements. Consistently with prior VR analyses, bees learned to discriminate the trained stimuli. Ex vivo analyses on the brains of learners and non-learners showed that successful learning led to a downregulation of three immediate early genes in the main regions of the visual circuit, the optic lobes (OLs) and the calyces of the mushroom bodies (MBs). While Egr1 was downregulated in the OLs, Hr38 and kakusei were coincidently downregulated in the calyces of the MBs. Our work thus reveals that color discrimination learning induced a neural signature distributed along the sequential pathway of color processing that is consistent with an inhibitory trace. This trace may relate to the motor patterns required to solve the discrimination task, which are different from those underlying pathfinding in 3D VR scenarios allowing for navigation and exploratory learning and which lead to IEG upregulation.