Final Report Summary - GUTS (Gut Symbiomes of Fungus-growing Ants)
Major shifts in diet have occurred in many, particularly invertebrate lineages, usually leading to substantial adaptive radiations. In a number of such cases symbiotic nitrogen-preserving bacteria were instrumental for specialization on protein-deficient diets, creating novel evolutionary potential in previously inaccessible niche space. The ancestors of the fungus-growing ants evolved obligate fungus-farming ca 50 million years ago. During the last 20 years these ants have become model systems of evolutionary biology, as they have allowed researchers to address fundamental questions of social (co)evolution and conflict regulation at multiple levels (between family members and between hosts and symbionts). Interestingly, till now there are no published studies describing the fungus-growing ant gut microbiome, even though their nitrogen-limited diets suggest that they may have putative gut bacterial symbionts aiding their nutritional efficiency.
The research objective for this Marie Curie project was the identification of putative (primary and secondary) bacterial gut symbionts of the attine ants, their tissue specificity and their function in the attine guts.
During the first half of the project (June 2012-June 2013), we used a range of techniques (454 metagenome sequencing, confocal fluorescence microscopy, and nifH-specific PCR) in ant guts across nine representatives of eight genera in the attine phylogeny, to answer the above questions. We demonstrated that the attine gut bacteria mostly belong to the Alpha-Proteobacteria (Wolbachia and Rhizobiales) and Mollicutes (Entomoplasmatales) and that those bacterial classes are almost always present in all fungus-growing ants. We also showed that some of these are intracellular and often mutually exclusive across the ant species investigated, and that at least some of them are likely to have a role in nitrogen-preservation because prokaryote nifH genes are found in the tissues of almost all ants. We confirmed these results using a specific NifH antibody, which showed that the gut compartments where nitrogen preservation is taking place are the ileum and the rectal papillae. Another finding from this study was that gut bacteria are being affected by antibiotics-producing actinobacteria on the ant cuticle, as ant species that have no cuticular actinobacteria appeared to have more unstable gut bacterial communities. The above presently being written up as two manuscripts that are currently in preparation.
During the second half of the project (June 2013-June 2014) we shifted our focus to more specific questions regarding the function of the gut bacteria in attine ants and more specifically in the model system Acromyrmex echinatior. Two main studies were performed: the first study investigated the possible connection between gut bacterial communities and kin recognition while the second one focused on the interactions between gut bacteria and cuticular actinobacteria.
To investigate whether gut bacterial communities are affecting the kin recognition of ant workers within a colony we performed an extensive experiment including almost 2000 ants from 4 different A.echinatior colonies. The ant workers were subjected to different diets, resulting in slow or rapid loss of their bacteria and diets that enhanced the diversity of their gut bacterial communities. Three different analyses were carried out: 1. aggression tests between nestmates vs non-nestmates after the different treatments, 2. cuticular hydrocarbon profiles using GCMS, and 3. gut bacteria profile analysis using 16S-illumina MiSeq sequencing. These analyses are expected to be completed later in 2014.
To investigate possible interactions between gut bacteria and actinobacteria we used a different approach that involved sequencing of guts of individual callow ants that had developed versus non-developed actinobacteria biofilms on their cuticle. As I write, we have completed sequencing of part of these ant guts and we expect to complete the remaining analyses and write-up in the year to come.
This longer time-span (reflecting a higher level of ambition than in the original MC application) was possible because my scientist in charge has decided to prolong my contract.
The research objective for this Marie Curie project was the identification of putative (primary and secondary) bacterial gut symbionts of the attine ants, their tissue specificity and their function in the attine guts.
During the first half of the project (June 2012-June 2013), we used a range of techniques (454 metagenome sequencing, confocal fluorescence microscopy, and nifH-specific PCR) in ant guts across nine representatives of eight genera in the attine phylogeny, to answer the above questions. We demonstrated that the attine gut bacteria mostly belong to the Alpha-Proteobacteria (Wolbachia and Rhizobiales) and Mollicutes (Entomoplasmatales) and that those bacterial classes are almost always present in all fungus-growing ants. We also showed that some of these are intracellular and often mutually exclusive across the ant species investigated, and that at least some of them are likely to have a role in nitrogen-preservation because prokaryote nifH genes are found in the tissues of almost all ants. We confirmed these results using a specific NifH antibody, which showed that the gut compartments where nitrogen preservation is taking place are the ileum and the rectal papillae. Another finding from this study was that gut bacteria are being affected by antibiotics-producing actinobacteria on the ant cuticle, as ant species that have no cuticular actinobacteria appeared to have more unstable gut bacterial communities. The above presently being written up as two manuscripts that are currently in preparation.
During the second half of the project (June 2013-June 2014) we shifted our focus to more specific questions regarding the function of the gut bacteria in attine ants and more specifically in the model system Acromyrmex echinatior. Two main studies were performed: the first study investigated the possible connection between gut bacterial communities and kin recognition while the second one focused on the interactions between gut bacteria and cuticular actinobacteria.
To investigate whether gut bacterial communities are affecting the kin recognition of ant workers within a colony we performed an extensive experiment including almost 2000 ants from 4 different A.echinatior colonies. The ant workers were subjected to different diets, resulting in slow or rapid loss of their bacteria and diets that enhanced the diversity of their gut bacterial communities. Three different analyses were carried out: 1. aggression tests between nestmates vs non-nestmates after the different treatments, 2. cuticular hydrocarbon profiles using GCMS, and 3. gut bacteria profile analysis using 16S-illumina MiSeq sequencing. These analyses are expected to be completed later in 2014.
To investigate possible interactions between gut bacteria and actinobacteria we used a different approach that involved sequencing of guts of individual callow ants that had developed versus non-developed actinobacteria biofilms on their cuticle. As I write, we have completed sequencing of part of these ant guts and we expect to complete the remaining analyses and write-up in the year to come.
This longer time-span (reflecting a higher level of ambition than in the original MC application) was possible because my scientist in charge has decided to prolong my contract.