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Evolution of the honey bee gut microbiome through bacterial diversification

Periodic Reporting for period 4 - MicroBeeOme (Evolution of the honey bee gut microbiome through bacterial diversification <br/>)

Reporting period: 2021-09-01 to 2022-08-31

Animals harbor specialized bacterial communities in their guts referred to as gut microbiota. These communities are typically composed of hundreds of different species. Moreover, most of these species are composed of a multitude of divergent strains. Despite recent advances in understanding the functional importance of the gut microbiota for host health, surprisingly little is known about the distribution of closely related strains across hosts, their genomic diversity, evolutionary dynamics, or functional relevance. The complex nature of microbial communities and the fact that diversity is typically assessed using marker gene analyses, instead of genomic approaches, have precluded a detailed understanding of how strain-level diversity is generated and maintained neither in the gut microbiota nor in any other natural microbial community.
MicroBeeOme addressed these questions by studying the gut microbiome of honey bees. Previous research has shown that the dominant bacterial lineages of the honey bee gut microbiota have substantially diversified. Moreover, gnotobiotic bee systems have been established that allow to colonize microbiota-free bees with defined communities composed of cultured strains. This provided us with a unique opportunities to study microbial ecology and evolution in the animal gut in a simple and experimentally amenable system. The project was divided into four work packages addressing interconnected research questions of current biology: (i) Determining the population genomic structure of closely related gut bacteria, (ii) investigating how closely related gut bacteria interact with each other, (iii) elucidating the underlying genetic and molecular mechanisms, and (iv) revealing their impact on the symbiosis with the host. To this end, we applied a multidisciplinary approach combining comparative metagenomics, transcriptomics, metabolomics, gnotobiotic bee experiments, microscopy, bacterial genetics, and automated bee tracking. In summary, the project has revealed how diversity at the level of strains is structured and distributed across bee species, colonies, and individual bees. We have identified that closely related strains engage in negative interactions in the bee gut, yet can stably coexist in the presence of the right diet, and identified gene sets involved in symbiotic interactions in the gut. Finally, we have found that the bee gut microbiota increases the head-to-head interactions among bees in a colony, demonstrating the critical role of these bacteria for the social lifestyle of these important pollinator species. These findings advance our understanding of the ecology and evolution of strain-level diversity in microbiomes and provide novel insights into the interaction between honey bees with their gut bacteria.
We established a shotgun metagenomics pipeline to quantify the extent of strain-level diversity in the gut microbiota of individual honey bees (Apis mellifera). A first comparative analysis across honey bees of different colonies and age revealed that most species of the gut microbiota have diversified into sequence-discrete populations, each of which harbor a large extent of strain-level diversity. While most sub-lineages coexist in individual bees, strains of the same sub-lineage tend to segregate across bees. These results, published in Nat Comm (Ellegaard and Engel, 2019), presented the starting point for several follow-up studies. First, we carried out a similar metagenomics analysis in which we compared the gut microbiota of two closely related honey bee species (Apis mellifera and Apis cerana). This showed that closely related animal species, although composed of similar species, can harbor different levels of strain diversity (Curr Biol by Ellegaard et al (2020)). Second, we analyzed bacteriophages present in the bee gut using metagenomics, which revealed that bee gut bacteria are targeted by a unique set of phages not found in other environments and that these phages target distinct strains of the same gut microbiota species. It is possible that these phages play a key role in driving and maintaining bacterial strain-level diversity in the bee gut (Bonilla-Rosso et al PNAS 2020).Third, we used experiments to test how divergent strains interact among each other and which conditions facilitate their coexistence in the bee gut. To this end, we carried out co-culture/co-colonization experiments with related bacterial strains in the presence of different nutrient conditions. We found that most of the tested strains engage in negative interactions, but that the presence of pollen (the diet of bees) facilitates their coexistence both in vivo and in vitro (Brochet et al. eLife 2021). We also found that while related strains isolated from honey bees can coexist in vivo in the honey bee gut, related strains isolated from bumble bees are outcompeted suggesting host specificity (Ellegaard, Brochet et al. Mol Ecol 2019). Genes underlying these different types of interactions were identified using transcriptomics and bacterial genetics (Brochet et al. eLife 2021; Schmidt. et al. bioRxiv 2022). For example, we found that the presence of pollen activates a large number of genes involved in carbohydrate metabolism indicating that niche partitioning of different dietary glycans facilitates the coexistence of related strains (Brochet et al. eLife 2021, Kesnerova et al. PLoS Biol 2017). Finally, we investigate the impact of the gut microbiota, and particularly strain-level diversity, on the host. While we have identified strain-specific metabolic activities that are likely to modulate the gut environment, their effects on the host have not yet been identified. However, by establishing an automated bee tracking system, we have found that the gut microbiota modulates the group-living behavior of bees by increasing the number and specificity of the head-to-head interactions among individual bees. These results have been published in Nature Ecology and Evolution (Liberti et al. 2022) and offer several new directions that can be explored in the future.
This project has advanced our understanding of the evolution and ecology of genomic and strain-level diversity in natural microbial communities. We have established a number of important methods and analytical approaches to study eco-evolutionary dynamics of strain-level diversity in microbiomes and to study the symbiosis between the gut microbiota and honey bees. We demonstrated for the first time that the gut microbiota can modulate the social behaviour of its animal host by using an innovative automated tracking system. Honey bees and other social bees are key pollinators for natural ecosystem and for agriculture. Their gut microbiota plays a key role for bee health. Hence, our results are not only relevant for basic research but also from an applied perspective.