It is presently unclear how much individual community members contribute to the overall metabolic output of a gut microbiota. To address this question, we used the honey bee, which harbors a relatively simple and remarkably conserved gut microbiota with striking parallels to the mammalian system and importance for bee health. Using untargeted metabolomics, we profiled metabolic changes in gnotobiotic bees that were colonized with the complete microbiota reconstituted from cultured strains. We then determined the contribution of individual community members in mono-colonized bees and recapitulated our findings using in vitro cultures. Our results show that the honey bee gut microbiota utilizes a wide range of pollen-derived substrates, including flavonoids and outer pollen wall components, suggesting a key role for degradation of recalcitrant secondary plant metabolites and pollen digestion. In turn, multiple species were responsible for the accumulation of organic acids and aromatic compound degradation intermediates. Moreover, a specific gut symbiont, Bifidobacterium asteroides, stimulated the production of host hormones known to impact bee development. While we found evidence for cross-feeding interactions, approximately 80% of the identified metabolic changes were also observed in mono-colonized bees, with Lactobacilli being responsible for the largest share of the metabolic output. These results show that, despite prolonged evolutionary associations, honey bee gut bacteria can independently establish and metabolize a wide range of compounds in the gut. Our study reveals diverse bacterial functions that are likely to contribute to bee health and provide fundamental insights into how metabolic activities are partitioned within gut communities.

Adult honeybees harbor a specialized gut microbiota of relatively low complexity. While seasonal differences in community composition have been reported, previous studies have focused on compositional changes rather than differences in absolute bacterial loads. Moreover, little is known about the gut microbiota of winter bees, which live much longer than bees during the foraging season, and which are critical for colony survival. We quantified seven core members of the bee gut microbiota in a single colony over 2 years and characterized the community composition in 14 colonies during summer and winter. Our data show that total bacterial loads substantially differ between foragers, nurses, and winter bees. Long-lived winter bees had the highest bacterial loads and the lowest community α-diversity, with a characteristic shift toward high levels of Bartonella and Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee experiments, we show that diet is a major contributor to the observed differences in bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is remarkably different from foragers and nurses. Considering the importance of winter bees for colony survival, future work should focus on the role of the gut microbiota in winter bee health and disease.

25It is presently unclear how much individual community members contribute to 26 the overall metabolic output of a gut microbiota. To address this question, we 27 used the honey bee, which harbors a relatively simple and remarkably conserved 28 gut microbiota with striking parallels to the mammalian system and importance 29 for bee health. Using untargeted metabolomics, we profiled metabolic changes in 30 gnotobiotic bees that were colonized with the complete microbiota reconstituted 31 from cultured strains. We then determined the contribution of individual 32 community members in mono--colonized bees, and recapitulated our findings 33 using in vitro cultures. Our results show that the honey bee gut microbiota 34 utilizes a wide range of pollen--derived substrates including flavonoids and outer 35 pollen wall components, suggesting a key role for degradation of recalcitrant 36 secondary plant metabolites and pollen digestion. In turn, multiple species were 37 responsible for the accumulation of organic acids and polyphenol degradation 38 products, and a specific gut symbiont, Bifidobacterium asteroides, stimulated the 39 production of host hormones known to impact bee development. While we found 40 evidence for cross--feeding interactions, ~80% of the identified metabolic 41 changes were also observed in mono--colonized bees with Lactobacilli being 42 responsible for the largest share of the metabolic output. These results suggest 43 that bacteria in the honey bee gut colonize largely independent metabolic niches, 44 which may be a general characteristic of gut microbiomes. Our study reveals 45 diverse metabolic functions of gut bacteria that are likely to contribute to bee 46 health, and provide fundamental insights into how metabolic functions are 47 partitioned within gut communities. 48 49 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/157461 doi: bioRxiv preprint first posted online Jul. 14, 2017;Page 3 of 51 Main text Author summary 50Honey bees are important pollinators that harbor a simple gut microbiota with 51 striking parallels to the mammalian system. This makes them relevant models to 52 study gut microbiota functions and its impact on host health. We applied 53 untargeted metabolomics to characterize metabolic changes induced by the gut 54 microbiota, and to characterize contributions of the major community members. 55We find that the gut microbiota digests recalcitrant substrates derived from the 56 bees' pollen--diet. Most metabolic changes could be explained by the activity of 57 individual community members suggesting substrate specificity and 58 independent metabolic niches. Our study provides novel insights into the 59 functional understanding of the bee gut microbiota and provides a framework 60 for applying untargeted metabolomics to disentangle metabolic functions of gut 61 bacteria.

24Adult honey bees harbor a specialized gut microbiota of relatively low complexity. While 25 seasonal differences in community composition have been reported, previous studies have 26 focused on compositional changes rather than differences in absolute bacterial loads. 27 Moreover, little is known about the gut microbiota of winter bees, which live much longer 28 than bees during the foraging season, and which are critical for colony survival. We 29 quantified seven core members of the bee gut microbiota in a single colony over two years 30 and characterized the community composition in 14 colonies during summer and winter. 31Our data shows that total bacterial loads substantially differ between foragers, nurses, and 32 winter bees. Long-lived winter bees had the highest bacterial loads and the lowest 33 community α-diversity, with a characteristic shift towards high levels of Bartonella and 34 Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee 35 experiments, we show that diet is a major contributor to the observed differences in 36 bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is 37 remarkably different from foragers and nurses. Considering the importance of winter bees 38 for colony survival, future work should focus on the role of the gut microbiota in winter 39 bee health and disease. 40 41 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/703512 doi: bioRxiv preprint 42 The European honey bee, Apis mellifera, is an important pollinator species for natural 43 ecosystems and agricultural production [1]. Its health status is threatened by numerous 44 factors including habitat loss, pesticide exposure, and high parasite and pathogen loads [2-45 4]. Accumulating evidence suggests that the gut microbiota of adult honey bees plays a 46 critical role for bee health [5]. The bee microbiota converts dietary compounds [6, 7] and 47 produces short chain fatty acids [8] in the gut, enhances sucrose responsiveness of the host 48 [8], and stimulates the immune system [9, 10]. Moreover, disruption of the gut microbiota 49 composition by antibiotic treatment, pesticide exposure, or dietary manipulations has 50 been associated with increased pathogen loads resulting in increased host mortality [11-51 14]. 52 A striking feature of the honey bee gut microbiota is its low taxonomic complexity. In 53 worker bees, the community is dominated by less than ten phylotypes (i.e. clusters of 54 strains sharing ≥97% sequence identity in the 16S rRNA gene), which typically make up 55 >95% of the bacterial cells in the gut [5, 15-18]. These phylotypes have been consistently 56 detected in honey bees, regardless of geographic location, life stage, or season [16, 19, 20], 57 and are acquired horizontally through contact with nest mates and hive components [21]. 58 They include five core phylotypes (Gilliamella, Snodgrassella, Lactoba...

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