bioRxiv preprint doi: https://doi.org/10.1101/2020.11.24.395384; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Competition-based screening secures the 2 evolutionary stability of a defensive microbiome 3 1 2,† 1,3,† 4 Sarah F. Worsley , Tabitha M. Innocent , Neil A. Holmes , Mahmoud M. Al- 1 3 4 2* 5 Bassam , Barrie Wilkinson , J. Colin Murrell , Jacobus J. Boomsma , Douglas 1,5,6,* 1,3,* 6 W. Yu , Matthew I. Hutchings 7 1 8 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, 9 UK NR47TJ 2 10 Centre for Social Evolution, Section for Ecology and Evolution, University of Copenhagen, 11 Copenhagen, Denmark. 3 12 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, 13 Norfolk, UK NR47UH 4 14 School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, 15 Norfolk, UK NR47TJ 5 16 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese 17 Academy of Sciences, Kunming, Yunnan, China 650223 6 18 Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming 19 Yunnan, China 650223 20 21 22 †These authors contributed equally to the manuscript 23 *Correspondence: [email protected], [email protected], [email protected], 24 25 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.24.395384; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 26 Abstract. 27 Cuticular microbiomes of Acromyrmex leaf-cutting ants are exceptional because they are freely 28 colonizable, and yet the prevalence of Pseudonocardia, a native vertically transmitted 29 symbiont that controls Escovopsis fungus-garden disease, is never compromised. Game theory 30 suggests that competition-based screening can allow the selective recruitment of antibiotic- 31 producing bacteria from the environment, by fomenting and biasing competition for abundant 32 host resources. Mutual symbiont aggression benefits the host and also maintains native 33 symbiont viability. Here we use RNA-stable isotope probing (RNA-SIP) 34 to confirm predictions that Acromyrmex cuticles can maintain a range of microbial symbionts. 35 We then used dual-RNA-sequencing and bioassays to show that vertically transmitted 36 Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of 37 other microbes, ultimately eliminating non-antibiotic-producing strains that might parasitize 38 the symbiosis while still allowing antibiotic-producing Streptomyces strains to survive. Open 39 cuticular microbiomes can thus maintain a specific co-evolved mutualism by restricting access 40 for other bacterial strains. 41 42 43 Keywords: antibiotics, Attini, game theory, defensive microbiome, mutualism, 44 Actinobacteria, partner, leaf-cutting ants, Pseudonocardia, interference competition, 45 horizontal acquisition, symbiosis 46 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.24.395384; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 47 Introduction 48 The diversity of insect associated microbial communities is staggering. They may consist of 1 49 single intracellular symbionts with reduced genomes owing to coadaptation at one extreme , 2 50 to dynamic microbiomes in open host compartments such as guts at the other end of the scale . 51 Gut microbiomes have been most intensively studied in humans and other vertebrates because 52 there is increasing consensus of their vital interest for host fitness throughout ontogenetic 3, 4, 5 53 development . The stability and cooperative characteristics of complex microbiomes is a 6 54 paradox. While relentless competition is the default setting of the microbial world , hosts 7 55 appear to evolve control by holding their microbiome ecosystems on a leash , but how dynamic 56 stability under continuing turnover is achieved remains unclear. Despite an abundance of 57 microbiome research, recent reviews have concluded that “integration between theory and 8 58 experiments is a crucial ‘missing link’ in current microbial ecology” and that “our ability to 9 59 make predictions about these dynamic, highly complex communities is limited” . 60 Game theory suggests a compelling solution to the unity-in-diversity paradox by 61 showing that competition-based screening can be a powerful mechanism to maintain 62 cooperative stability. Screening is likely to work when hosts evolve to: (1) provide nutrients 63 and/or space to foment competition amongst symbionts, thus creating an attractive but 64 demanding environment, and (2) skew the competition so that the mutualistic symbionts enjoy 65 a comparative advantage. Competitive exclusion then ‘screens in’ mutualists and ‘screens out’ 10, 11, 12 66 parasitic or free-rider symbionts . Screening is conceptually clearest when the symbiont 67 trait that confers competitive superiority is the same as (or strongly correlated with) the trait 68 that benefits the host. An illustrative example of such correlated functionality was provided by 13 69 Heil who showed that ant-hosting acacia plants provide copious food bodies, which fuels the 70 production of numerous externally patrolling ant workers. The ant species whose colonies 71 invest in a greater number of, and more active, workers typically wins the plant, and the same 13 72 investment in active workers is likely to better protect the host plants against herbivores . 73 Screening has also been suggested to act in animal-microbe symbioses. For instance, 14 74 Tragust et al. showed that carpenter ants acidify their own stomachs by swallowing acidopore 75 secretions. Entomopathogenic bacteria are rapidly killed off, whereas the gut bacterial 76 symbiont Asiaia sp. (Acetobacteraceae) exhibits a lower mortality rate and maintains itself in 15 77 the midgut. Addressing a similar question, Itoh et al. used co-inoculation experiments to show 78 that environmentally recruited but co-adapted ‘native’ Burkholderia symbionts outcompete 79 non-native bacteria in the gut of their bean bug host, even though they are able to establish in 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.24.395384; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16 80 the absence of the ‘native’ symbiont. Finally, Ranger et al. showed that ambrosia beetles 81 selectively colonize physiologically stressed trees, which have a high ethanol titre due to 82 anaerobic respiration. The vertically transmitted fungal symbionts of these beetles have 83 evolved to detoxify the ethanol whereas competing weedy fungi remain inhibited. 84 Competition-based screening seems particularly apt for the establishment of protective 85 microbiomes when competition would screen in microbes that are similarly able to produce 86 compounds that kill competitors and thus contribute to defensive functions from the host’s 7, 10, 11, 17 87 perspective . Moreover, natural selection is expected to reinforce the correlation 88 between antibiotic production and antibiotic resistance, since production without resistance 89 would be suicidal. Antibiotic-resistant strains should thus be superior competitors in antibiotic- 11 90 filled environments, reinforcing the abundance of antibiotic producers as well . Previous 91 research has shown that the protective, cuticular microbiome of Acromyrmex echinatior 92 leafcutter ants (Formicidae, Attini) is an ideal model system to test the dynamic predictions of 93 screening theory in a context that also acknowledges the long-term ecosystem-on-a-leash 7, 18 94 perspective . These ants forage for fresh leaf fragments to provision their co-evolved fungus- 19, 20 95 garden mutualist Leucoagaricus gongylophorus . The fungal cultivar produces gongylidia, 21, 22 96 nutrient-rich swellings that are the sole food source for the queen and larvae and the 23 97 predominant food source for the workers who only ingest plant-sap in addition to fungal food . 98 However, Leucoagaricus is at risk of being parasitized by the specialized, coevolved mould 99 Escovopsis weberi, which can degrade the fungal cultivar and also cause severe ant paralysis 24, 25, 26, 27 100 and mortality . To prevent infections, leafcutter ants have evolved a range of weeding 28, 29, 30 101 and grooming behaviours , and A. echinatior and other Acromyrmex species also 102 maintain filamentous actinomycete bacteria that grow as a white bloom on the cuticles of large 26 103 workers and produce antimicrobials that inhibit the growth of E. weberi . In Panama, where 104 almost all fieldwork on this multipartite symbiosis has been carried out, the cuticle of 105 Acromyrmex workers is dominated by one of two vertically transmitted