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The Actinomycete Connection

Final Report Summary - TACTIC (The Actinomycete Connection)

Project summary: Symbiosis, an intriguing form of cooperation between different species, has shaped life on earth: from the first eukaryote evolving as a symbiotic interaction between several prokaryote elements, to the bacterial biofilms that define mammalian gut communities. This form of cooperation often involves complex, coevolved interactions between highly divergent species, and while the phenomena is both fascinating and important to understand, explaining symbiosis poses a significant challenge in evolutionary biology. The tropical leafcutter ants and their multi-species symbiotic network form a highly specialised, and intriguing, example of cooperation. Understanding the relationship between these ants and the antibiotic-producing bacteria that grow symbiotically on their cuticle is the focus of this project.
The fungus-farming attine ants live symbiotically with multiple species of antibiotic-producing actinobacteria. We know the diversity of bacterial species in their cuticular microbiome correlates with a diversity of antibiotics, and suspect these help to maintain long-term protection against pathogens that attack the ant’s fungal mutualist (and, potentially, the ants themselves). However, only one of these bacterial species is vertically transmitted by the ants between generations. A more diverse beneficial microbiome is likely established by selective recruitment of additional actinomycete strains from the environment, but it has remained unclear how, and at what life stages of the ant host, this happens.

This is also an important general problem: how do the dynamics of bacterial competition lead to the formation of a microbiome that benefits a host and mutualistic partner? Although mutualisms between bacteria and other species are both abundant and influential (e.g. multiple insects, crop plants, coral systems), these systems tend to be difficult to experimentally manipulate in the lab. Using the attine ant model this project empirically tests the latest evolutionary theories on symbiosis, and on the dynamics of interactions within bacterial communities, to addresses important general questions in evolutionary biology.

Specifically, the central aim of this project is to better understand the nature of the symbiosis between genera of leaf-cutter attine ants, and the antibiotic-producing bacteria Pseudonocardia. We set out to: map the cuticular microbiome (actinobacterial symbiont + other taxa if present) across the attine phylogeny, and describe changes in this bacterial community over an ant worker’s lifetime; test specificity between the bacterial symbiont and the ant host; and investigate the diversity of symbiont strains (e.g. in the antibiotics they produce), to tease apart the dynamics of bacterial competition, in light of theoretical modelling of how such a beneficial microbiome might establish and evolve.

Summary overview of results: The project aims to resolve controversy around the nature of the attine ant symbiosis with the actinobacteria Pseudonocardia, encompassing metagenome sequencing, microbiology and insect experimental data covering several attine genera. It also aims to provide novel answers to general evolutionary questions about the evolution of symbioses, and the assembly and function of defensive microbiomes. Moreover, the implications of this work could have significant value for applied work on understanding and utilising microbiomes (in for e.g. medical and agricultural contexts), and potentially for novel antibiotic discovery.

Overall, our results suggest that the attine ants promote interference competition among bacteria on their cuticle, and this competition favours the formation of actinobacterial communities that are beneficial to the ant host.
The metagenomic sequencing data demonstrates that i) Pseudonocardia is the dominant actinobacteria in the leaf-cutting genera, Acromyrmex; ii) some higher and lower attine genera also have actinobacteria-dominated microbiomes, but these may have other actinobacterial key players than Pseudonocardia, and these could represent less tightly co-evolved symbioses; iii) the presence of Pseudonocardia in Acromyrmex seems to favour the acquisition of a greater number of other, potentially beneficial, actinobacterial strains, suggesting secondary recruitment of strains from the environment to further benefit the ant host.
We have demonstrated in in vitro experimental bioassays that other (non-symbiont) antibiotic-producing bacteria can invade a Pseudonocardia dominated microbiome, whereas non-producers struggle to do so. This supports explicit predictions of theoretical models for microbiome assembly, via host screening (where the ant host can indirectly influence which bacteria can establish successfully, likely through the provision of resources). Moreover, initial annotations of the genomes of 10 strains of Pseudonocardia (work underway at University of East Anglia collaborators) we isolated from Acromyrmex colonies suggest that the symbiont produces a number of different antibiotics, both antifungal and antibacterial. These may play a role in both defence of the ants’ fungus garden against the specialist fungal pathogen Escovopsis, and in bacterial competition with environmental strains that shapes the microbiome.
These results are currently being analysed and written up as two manuscripts, which we intend to submit for publication later in 2016.

Conclusions and socio-economic impacts of the project: We anticipate that taken as a whole, the results from this research will shed light on the nature of the symbiosis between attine ants and the actinobacteria Pseudonocardia (in particular in the leaf-cutting ant genera Acromyrmex); and give insight into how antibiotic-producing bacteria play an important role in the cuticular microbiome across the attine ant phylogeny. We have found support for specific predictions from theoretical models of how beneficial microbiomes are assembled, and these will have broad relevance for understanding microbiomes more generally – which are increasingly becoming acknowledged as both ubiquitous and important. Finally, through the building of collaboration with molecular microbiologists and biochemists, we expect that this research will lead to future research into novel antibiotic-coding gene clusters, and has the potential to inform work on the evolution (or avoidance) of resistance.