Periodic Reporting for period 1 - HarmHoney (HeAlth Regulation by the gut Microbiota in the Honey bee) Reporting period: 2020-05-01 to 2022-04-30 Summary of the context and overall objectives of the project Given the current decline in honey bee populations, understanding their physiology, and how to maintain their health, is critical to preserve our ecosystem. As for humans and most animals, the gut microbiota was shown to play an important role in honey bee health. It degrades the pollen ingested by bees and release non-nutritive compounds in the gut lumen. Among them, flavonoids increase the expression and activity of P450 enzymes in the bee body, thereby promoting the detoxification of some pesticides and buffering their detrimental effects on bees’ survival. Recently, gut bacteria were shown to metabolize flavonoids and to favor the absorption of the resulting products through the host gut epithelium. The importance of this metabolism for the bioactivity of flavonoids was unknown. The broad hypothesis of the granted project was that the decomposition of pollen compounds (e.g. flavonoids) by the gut microbiota would have a beneficial impact on honey bee health. In line with this hypothesis, the objectives of the project were to (i) highlight the importance of the gut microbiota for bee health, (ii) identify the flavonoids whose conversion by the gut microbiota may affect bee health, (iii) assess the impact of neonicotinoid pesticides on the gut microbiota composition, and (iv) understand the extent to which flavonoid-conversion by the gut microbiota may protect bees from health alterations by pesticides. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We generated germ-free (GF) honey bees by extracting pupae from their cells at the last developmental stage and rearing them under sterile laboratory conditions. Gnotobiotic bees were obtained by feeding GF bees an inoculum of bacteria of interest which colonizes the gut within a week. Using these gnotobiotic bees, we developed two experiments to assess the role of flavonoid decomposition by gut bacteria on honey bee survival (experiment 1) and to identify gut bacteria which may influence honey bee cognition (experiment 2). In the first experiment we showed that the gut microbiota promotes honey bees’ survival but does not increase resilience to the neonicotinoid pesticide thiacloprid. A chronic oral exposure to thiacloprid reduced the survival of both GF bees and bees colonized with a natural gut microbiota. While dietary treatments with the flavonoids rutin or quercetin did not affect the survival of GF and colonized bees, they counteracted the detrimental effect of thiacloprid independently of the gut composition. Therefore, the gut microbiota does not seem necessary for the bioactivity of these flavonoids. One day of treatment with thiacloprid, rutin or quercetin did not affect the abundance of bacteria in the gut, nor the expression of genes coding for the P450 enzymes. Samples collected after six days of treatments are being processed.In the second experiment, we identified a community of five core gut bacteria that promotes cognitive performance of honey bees. Bees whose gut was colonized with the defined community of bacteria were better than GF bees at discriminating two odours based on their association or not with a sucrose reward. They also memorized better these odour-food association in the short-term. Interestingly, bees colonized with single members of that community showed similar performances as GF bees. Cognitive improvements in honey bees therefore seem to constitute an emergent property of the defined gut bacterial community. In other words, interactions between these community members, or their metabolic products are needed to confer the beneficial effects on learning and memory. This project’s manuscript is available on BioRxiv and in the process of being published in a peer-reviewed journal. The dataset will be made available upon publication. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Honey bee populations have been declining worldwide in the past fifty years and understanding the determinants of bee health has therefore become critical. The work carried out during this fellowship revealed the importance of the gut microbiota and nutrition for both individual longevity and individual cognitive traits that contribute to the group longevity. We showed that quercetin, rutin and the gut microbiota could buffer the effect of the pesticide thiacloprid on survival. Probiotics combining gut bacteria and flavonoids may therefore increase bees’ resilience to pesticide exposure. Future research is needed in that direction. Furthermore, we identified a community of gut bacteria that promotes cognitive performance in bees. Given the importance of sophisticated cognitive skills for foraging activity, we believe our results will inspire industrials to design probiotics that enhance bees’ foraging efficiency and promote colony health. To our knowledge, this is the first synthetic community of gut bacteria that can be studied for its effects on host cognition. It opens new avenues of research as to the neurobiological mechanisms by which gut bacteria modulate their host’s brain function.