Skip to main content

DiseasE-FreE social life without Antibiotics resisTance

Periodic Reporting for period 2 - DEFEAT (DiseasE-FreE social life without Antibiotics resisTance)

Reporting period: 2019-12-01 to 2021-05-31

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. I will capitalise on recent pioneering work in my group 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 unique model to test three novel concepts that may account for the evasion of resistance evolution. First, the antimicrobial compounds may have properties and evolve in ways that preclude resistance evolution in pathogens. Second, resistance may only be possible towards individual compounds and not antimicrobial cocktails. Third, pathogens may only be able to successfully invade and proliferate if they bypass several consecutive lines of defence, analogous to the six hallmarks of metazoan defence against cancer development. Testing these concepts will give us fundamental insights into the remarkable success of these symbionts’ complimentary contributions to defence, and more broadly, clarify the forces of multilevel natural selection that have allowed long-lived insect societies to evolve sustainability in the face of continued pathogenic threats. Documenting and understanding these disease management principles is fundamentally important for several branches of evolutionary biology, and strategically important for adjusting human practices for future antimicrobial stewardship.
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 (WPIII) and for future experimental evolution work (WPII). Gut metabolome analyses from South African species is progressing, including network analyses and caste-specific compound identification, forming the backbone for new comparative metabolome analyses and for linking metabolome differences to differences in microbial communities (WPI). Our hypothesis that Termitomyces plays a major role in defence appears to hold true and work is now in progress to understand the role of volatiles of fungal origin in defence (WPI, III). This is supported by the sequencing of a number of Termitomyces species, for which analyses of biosynthetic gene clusters is progressing. Volatiles should only be able to play a prophylactic role in the symbiosis if the environment within which Termitomyces is maintained remains sealed from the surroundings. This is consistent with the hypothesis that layers of defence are essential to keep fungal gardens free from pathogens, and this does indeed appear to be prioritised by the termite host as indicated above. Work to further elaborate on the importance of fungal garden isolation and characterisations of the headspace environment are underway (WPIII). 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. With novel approaches to metabarcoding BGC domains will be employed during 2021 to help overcome these challenges and identify the diversity and types of gene clusters prevalent in termite guts and fungal gardens. We have however through comparative genomics analyses established that Actinobacteria – well known for their unprecedented capacity to code for and secrete compounds with antimicrobial properties – are consistently present and rich in biosynthetic gene clusters, many of which share limited or no similarities to clusters coding for known antimicrobial 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. To perform setups in more natural conditions, work with collaborators in Ivory Coast on this is ongoing, with the first successful colonies being established at the end of February 2019. Established colonies obtained up all termite castes and the production of their respective primordial fungus comb (first fungus comb). Unfortunately, an ant attack on the breeding laboratory in December 2019 resulted in the death of all termite colonies. We subsequently secured the breeding laboratory and started collecting termite alates in the beginning of February in 2020 (the beginning of the long rainy season in the study area). Even with the isolation of the city of Abidjan due to the Corona virus pandemic, we continued to be able to work with local field assistant Mr Kouassi Kouassi from village close to the Lamto reserve. This made it possible to collect alates from the five termite genera Ancistrotermes, Macrotermes, Microtermes, Odontotermes and Pseudacanthotermes. With this progress, we hope to have colonies in the laboratory in the Ivory Coast by the summer of 2021 to test behavioural responses to infections and monitor disease progression in colonies under varying experimental conditions, in a comparison across the major genera of the termite sub-family.
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. Albeit still in progress, results so far 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. This may imply that it is not necessarily unique or specific chemical compounds that are key, but their combinations, and the context within which they are applied, that is essential. Most of the experimental work is ongoing or unpublished, but the project is well on the way to generate results that will substantially improve our understanding of defences in farming termites and beyond.
A mound of the largest fungus-farming termite species Macrotermes bellicosus
Termites clean each other via allo-grooming
Putative antagonists growing on plates in vitro
Competitor of the termite fungus Pseudoxylaria in abandoned fungus comb
Macrotermes natalensis colonies set up in the laboratory
Competitor of the termite fungus Pseudoxylaria overgrows fungus combs in the absence of termites
Macrotermes natalensis royal chamber with two queens and a king
Competitor of the termite fungus Pseudoxylaria in abandoned fungus comb
Newly excavated Ancistrotermes fungus-farming termite nest