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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - MicroBeeOme (Evolution of the honey bee gut microbiome through bacterial diversification )

Teaser

Summary

Animals harbor specialized bacterial communities in their guts referred to as gut microbiota. These communities are typically composed of hundreds of different species. Moreover, it is becoming increasingly clear that 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 divergent 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.
The current project addresses these questions by studying the gut microbiome of honey bees. We have recently found that the eight dominant bacterial lineages in the honey bee gut have substantially diversified, which is a striking parallelism to the evolution of the mammalian gut microbiome. Moreover, we have established experiments to colonize microbiota-free bees with cultured isolates of divergent bee gut bacteria. This provides us with unique opportunities to study bacterial evolution in the gut in a simple and experimentally amenable system. The project is divided into four work packages addressing interconnected research questions of current biology: We will (i) determine the population genomic landscape of divergent gut bacteria, (ii) investigate whether bacterial diversification has resulted in competition or cooperation, (iii) elucidate mechanisms of bacterial interactions, and (iv) reveal how bacterial diversification impacts the symbiosis with the host. To this end, we are using a multidisciplinary approach combining comparative metagenomics, transcriptomics, metabolomics, bee colonization experiments, microscopy, bacterial genetics, and automated bee tracking. This project situated at the forefront of microbial symbiosis will provide ground-breaking insights into microbial evolution and ecology, gut microbiology, and honey bee health and biology.

Work performed

To start with, we have established a method based on shotgun metagenomics sequencing which allows us to detect and quantify the presence of divergent strains of all species of the bee gut microbiota in individual bees. Using this approach, we first aimed to understand how divergent strains are distributed among individual bees sampled over time and from different colonies. This study has been recently published in Nature Communications (Ellegaard and Engel 2019). We have found that most species of the bee gut microbiota have diversified into sequence-discrete sub-lineages, each of which harbors a large extent of strain-level diversity, much higher than what has been previously found in other microbial communities. While most sub-lineages coexist in individual bees, strains of the same sub-lineage tend to segregate across bees. This suggests different types of interactions between strains of the same or different sub-lineage of a given species. We are currently investigating these interactions by co-culturing strains in vitro and colonizing microbiota-free bees with communities of different strain complexity One of these projects has been recently published (Ellegaard, Brochet et al. Mol Ecol 2019) showing that honey bee strains of a given species can coexist in vivo in the honey bee gut, while strains isolated from bumble bees are outcompeted suggesting host specificity and interbacterial competition.
In another part of the project, we are using metabolomics studies to investigate functional differences between species and strains to obtain an understanding of the mechanism underlying coexistence. The first results of these experiments have revealed unprecedented insights into the metabolic activities of bacterial species in the honey bee gut. We could not only identify the major bacterial substrates derived from the pollen diet of the host, but also assign them to individual community members. These results have been published in PLoS Biology (Kesnerova et al 2017) and present the basis for strain-level analysis of metabolic activities of the bee gut microbiota and their impact on the host.

Final results

The establishment of a quantitative shotgun metagenomics pipeline allows us to comprehensively assess strain-level and genomic diversity across a wide range of samples from the bee gut microbiota. Marker gene analyses that are usually applied to measure diversity in these communities cannot provide such insights, demonstrating that our approach clearly goes beyond the current state of the art in the field.
The shotgun metagenomics approach does not only allow to look at strain-level diversity across different host populations or honey bee species, but also to test hypothesis under experimental settings, such as in vitro culture or bee colonization experiments. Until the end of the project, we thus expect to obtain results that advance our understanding of the different factors that contribute to the generation and maintenance of strain-level diversity.
The combination of the shotgun metagenomics approaches with other omics methods, such as metabolomics and transcriptomics, and the fact that we can culture all strains in vitro and carry out bee colonizations will allow us to obtain a molecular understanding of the bacterial interactions and their impact on the host, providing unprecedented insights about strain-level diversity in natural microbial communities.