Periodic Reporting for period 4 - DEFEAT (DiseasE-FreE social life without Antibiotics resisTance)
Reporting period: 2022-12-01 to 2023-11-30
The application of antimicrobial compounds produced by hosts or defensive symbionts to counter the effects of diseases has been identified in a number of organisms, but despite extensive studies on their presence, we know essentially nothing about why these antimicrobials do not always trigger the rampant evolution of resistance in target parasites. Fungus-farming termites have evolved a sophisticated agricultural symbiosis that pre-dates human farming by 30 million years and, in stark contrast to virtually any other organism, does not suffer from specialised diseases. This project has capitalised on recent pioneering work on proximate evidence for antimicrobial defences in the termites, their fungal crops, and their complex gut bacterial communities, to develop this farming symbiosis as a model to test novel concepts on the evasion of resistance evolution in nature. We have implemented approaches to discover new putative antimicrobial compounds from bacterial and fungal symbionts and published work to provide a comprehensive catalogue of compound modes of action, activities, and synergistic potential. Beyond the potential for complementary chemical defences, our work has allowed us to assess the importance of behavioural defences and showing that in particularly the outer colony mound wall is a critical defensive barrier to maintain homeostasis and prevent the proliferation of infections. This is in part accomplished by the physical structure of mounds, but also through the accumulation of carbon dioxide that appears to negative affect non-mutualistic fungi present. Documenting and understanding these disease management principles in termites illustrate fundamentally important aspects on how natural selection allows robust defences.
We have confirmed that termite fungus combs are vastly dominated by Termitomyces, and showed that this is not driven by obligate gut passage of substrates. This points to key behavioural and chemical defences of termite or the food fungus, Termitomyces. This is in line with our discovery that termites avoid substrates with virulent pathogens, and that termites rapidly use soil to cover exposed fungal gardens (WPIII). Cultivation efforts have allowed us to establish a series of putative pathogens to be used to test defences (WPII-III) and for future experimental evolution work. Gut metabolome analyses from South African species, including network analyses and caste-specific compound identification (WPI) documented a chemical environment that is unlikely to be antimicrobial, aligning with the absence of inhibition during gut passage. This further lend support to our hypothesis that Termitomyces and the fungus comb environment is critical in defence, for which we have documented both antimicrobial properties of combs, a headspace environment with high carbon dioxide that suppresses growth and/or sporulation of putative antagonistic fungi of the symbiosis as well as a rich set of volatiles of fungal origin (WPI, III). Volatiles are likely to play a key prophylactic role in the symbiosis as the environment within which Termitomyces is maintained is sealed from the surroundings. Gut metagenomics has proven challenging for biosynthetic gene cluster (BGC) characterisation, because of fragmented metagenomes and challenges associated with linking gene clusters to metabolites present in these complex environments. We have therefore taken a novel approaches to metabarcode BGC domains of non-ribosomal peptides in guts and fungus combs, which has proven succesful and revealed extraordinary diversity, pointing to strong selection for the evolution of chemical novelties and the absence of a conserved set of bacteria-derived compounds. This supports that the prospects for novel compound discovery remains high, despite past efforts to identify chemical compounds of actinobacterial origins in the symbiosis.
A major goal of this action has been to improve protocols for establishing fungus-growing termites in the laboratory in the Ivory Coast where we perform field work and subsequently at the University of Copenhagen (WPIII). As expected, this has proven challenging, with our initial efforts nevertheless providing promising insights into the conditions that favour successful setups. Specifically, we attempted to establish >400 queen and king pairs of Ancistrotermes cavithorax in the laboratory in Copenhagen, but establishment of fungal gardens proved to be a critical point during the colony life cycle, with consequently very low success rates. We continued to work with local field assistants and collaborators in Ivory coast and now have a few colonies in the lab in Copenhagen.
Major recent achievements include a comprehensive review published in Natural Product Reports (Schmidt et al. 2022), which is associated with a manually curated online database (WPI(opens in new window), publication of termite gut metabolomes (Vidkjær et al. 2022) and microbial impacts on termite biology (Murphy et al. 2022) (WPI,III). We have finalised comparative genomics analyses of 39 Termitomyces genomes (20 species) and established their evolutionary histories (Schmidt et al. in revision). These analyses – along with volatile analyses (Kreuzenbeck et al., 2022; Vidkjær et al. in preparation; Yang et al. in preparation) – support that Termitomyces plays a major role in defence (WPI,II). The use of volatile terpenes in defence is possible because the termites ensure an enclosed, homeostatic environment that ensure constant temperature and humidity. This allows for CO2 build-up well beyond what most organisms can tolerate (5% or higher), and we have therefore also tested whether extremely high CO2 help protect from fungal infections, which appears to be the case (Schmidt et al. 2023); thus indicating that the environment acts as an additional layer of defence of the farming symbiosis (WPIII).
A major goal of this action has been to improve protocols for establishing fungus-growing termites in the laboratory in the Ivory Coast where we perform field work and subsequently at the University of Copenhagen (WPIII). As expected, this has proven challenging, with our initial efforts nevertheless providing promising insights into the conditions that favour successful setups. Specifically, we attempted to establish >400 queen and king pairs of Ancistrotermes cavithorax in the laboratory in Copenhagen, but establishment of fungal gardens proved to be a critical point during the colony life cycle, with consequently very low success rates. We continued to work with local field assistants and collaborators in Ivory coast and now have a few colonies in the lab in Copenhagen.
Major recent achievements include a comprehensive review published in Natural Product Reports (Schmidt et al. 2022), which is associated with a manually curated online database (WPI(opens in new window), publication of termite gut metabolomes (Vidkjær et al. 2022) and microbial impacts on termite biology (Murphy et al. 2022) (WPI,III). We have finalised comparative genomics analyses of 39 Termitomyces genomes (20 species) and established their evolutionary histories (Schmidt et al. in revision). These analyses – along with volatile analyses (Kreuzenbeck et al., 2022; Vidkjær et al. in preparation; Yang et al. in preparation) – support that Termitomyces plays a major role in defence (WPI,II). The use of volatile terpenes in defence is possible because the termites ensure an enclosed, homeostatic environment that ensure constant temperature and humidity. This allows for CO2 build-up well beyond what most organisms can tolerate (5% or higher), and we have therefore also tested whether extremely high CO2 help protect from fungal infections, which appears to be the case (Schmidt et al. 2023); thus indicating that the environment acts as an additional layer of defence of the farming symbiosis (WPIII).
The project has so far generated substantial data pointing to extremely clean fungiculture conditions and novel means of defence of monoculture farming in fungus-growing termites, in addition to elaborating on the role of social immunity mechanisms. The results indicate that it is indeed a series of defences that collectively ensure disease-free conditions, indicating that cocktails of compounds (or mechanisms) are what allows for effective defence – and that the context within which defences are employed is critical. Several publications are in the final stages of being submitted or in revision, and once out will contribute to a substantial improvement of our understanding of defences in farming termites and beyond. In particularly work that integrates the complex environment within which termites cultivate fungi have proven to be conceptually important as it has highlighted the need for a more holistic view on the context and environment within which defences are expressed.