Skip to main content
SpringerLink
Log in

The Hyphosphere of Leaf-Cutting Ant Cultivars Is Enriched with Helper Bacteria

  • Host Microbe Interactions
  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

Bacteria can live in a variety of interkingdom communities playing key ecological roles. The microbiome of leaf-cutting attine ant colonies are a remarkable example of such communities, as they support ants’ metabolic processes and the maintenance of ant-fungus gardens. Studies on this topic have explored the bacterial community of the whole fungus garden, without discerning bacterial groups associated with the nutrient storage structures (gongylidia) of ant fungal cultivars. Here we studied bacteria isolated from the surface of gongylidia in the cultivars of Atta sexdens and Acromyrmex coronatus, to assess whether the bacterial community influences the biology of the fungus. A total of 10 bacterial strains were isolated from gongylidia (Bacillus sp., Lysinibacillus sp., Niallia sp., Staphylococcus sp., Paenibacillus sp., Pantoea sp., Staphylococcus sp., and one Actinobacteria). Some bacterial isolates increased gongylidia production and fungal biomass while others had inhibitory effects. Eight bacterial strains were confirmed to form biofilm-like structures on the fungal cultivar hyphae. They also showed auxiliary metabolic functions useful for the development of the fungal garden such as phosphate solubilization, siderophore production, cellulose and chitin degradation, and antifungal activity against antagonists of the fungal cultivar. Bacteria-bacteria interaction assays revealed heterogeneous behaviors including synergism and competition, which might contribute to regulate the community structure inside the garden. Our results suggest that bacteria and the ant fungal cultivar interact directly, across a continuum of positive and negative interactions within the community. These complex relationships could ultimately contribute to the stability of the ant-fungus mutualism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

Data supporting the results in the paper are available in the Supplementary Material.

References 

  1. Rodrigues A, Bacci M, Mueller UG et al (2008) Microfungal “weeds” in the leafcutter ant symbiosis. Microb Ecol 56:604–614. https://doi.org/10.1007/s00248-008-9380-0

    Article  CAS  PubMed  Google Scholar 

  2. Quinlan RJ, Cherrett J (1979) The role of fungus in the diet of the leafcutting ant Atta cephalotes (L.). Ecol Entomol 4:151–160

    Article  Google Scholar 

  3. Martin MM, Stadler Martin J (1970) The biochemical basis for the symbiosis between the ant, Atta colombica tonsipes, and its food fungus. J Insect Physiol 16:109–119

    Article  CAS  Google Scholar 

  4. Khadempour L, Burnum-Johnson KE, Baker ES et al (2016) The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates. Mol Ecol 25:5795–5805. https://doi.org/10.1111/mec.13872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Little AEF, Murakami T, Mueller UG, Currie CR (2006) Defending against parasites: fungus-growing ants combine specialized behaviours and microbial symbionts to protect their fungus gardens. Biol Lett 2:12–16. https://doi.org/10.1098/rsbl.2005.0371

    Article  PubMed  Google Scholar 

  6. Huang EL, Aylward FO, Kim YM et al (2014) The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation. Environ Microbiol Rep 6:389–395. https://doi.org/10.1111/1758-2229.12163

    Article  PubMed  Google Scholar 

  7. Higa T (1991) Effective microorganisms: a biotechnology for mankind. In Parr JF, Hornick SB, Whitman SE (eds) Proc 1st Int Conf Kyusei Nature Farm. USDA, Washington, DC, pp 8–14

  8. Poulsen M, Currie CR (2010) Symbiont interactions in a tripartite mutualism: exploring the presence and impact of antagonism between two fungus-growing ant mutualists. PLoS ONE 5:1–14. https://doi.org/10.1371/journal.pone.0008748

    Article  CAS  Google Scholar 

  9. Jiménez-Gómez I, Barcoto MO, Montoya QV et al (2021) Host susceptibility modulates Escovopsis pathogenic potential in the fungiculture of higher attine ants. Front Microbiol 12. https://doi.org/10.3389/fmicb.2021.673444

  10. De Fine Licht HH, Boomsma JJ, Tunlid A (2014) Symbiotic adaptations in the fungal cultivar of leaf-cutting ants. Nat Commun 5:5675. https://doi.org/10.1038/ncomms6675

    Article  CAS  PubMed  Google Scholar 

  11. Pinto-Tomás AA, Anderson MA, Suen G et al (1979) (2009) Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science 326:1120–1123. https://doi.org/10.1126/science.1173036

    Article  CAS  Google Scholar 

  12. Francoeur CB, May DS, Thairu MW et al (2021) Burkholderia from fungus gardens of fungus-growing ants produces antifungals that inhibit the specialized parasite Escovopsis. Appl Environ Microbiol 87:1–13. https://doi.org/10.1128/AEM.00178-21

    Article  Google Scholar 

  13. Aylward FO, Burnum KE, Scott JJ et al (2012) Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens. ISME J 6:1688–1701. https://doi.org/10.1038/ismej.2012.10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barea JM, Pozo MJ, Azcón R, Azcón-Aguilar C (2005) Microbial co-operation in the rhizosphere. J Exp Bot 56:1761–1778. https://doi.org/10.1093/jxb/eri197

    Article  CAS  PubMed  Google Scholar 

  15. Barbieri E, Ceccaroli P, Palma F et al (2012) Ectomycorrhizal helper bacteria: the third partner in the symbiosis. In: Zambonelli A, Bonito GM (eds) Edible ectomycorrhizal mushrooms. Springer, Berlin

    Google Scholar 

  16. Frey-Klett P, Garbaye J (2005) Mycorrhiza helper bacteria: a promising model for the genomic analysis of fungal-bacterial interactions. Source: New Phytol 168:4–8

    CAS  Google Scholar 

  17. Alvarado PE, Barrios RMM, Xóchihua JAM, Hernández JFC (2017) Fast and reliable DNA extraction protocol for identification of species in raw and processed meat products sold on the commercial market. Open Agric 2:469–472. https://doi.org/10.1515/opag-2017-0051

    Article  Google Scholar 

  18. Turner S, Pryer MK, Miao PWV, Palmer DJ (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46:327–338

    Article  CAS  PubMed  Google Scholar 

  19. Dorsch M, Stackebrandt E (1992) Some modifications in the procedure of direct sequencing of PCR amplified 16S rRNA. J Microbiol Methods 16:271–279

    Article  Google Scholar 

  20. Ki JS, Zhang W, Qian PY (2009) Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification. J Microbiol Methods 77:48–57. https://doi.org/10.1016/j.mimet.2009.01.003

    Article  CAS  PubMed  Google Scholar 

  21. Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  22. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Darriba D, Taboada LG, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nixon KC (2002) WinClada ver. 1.0000. Published by the author, Ithaca, New York, USA

  25. Ronquist F, Teslenko M, van der Mark P et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029

    Article  PubMed  PubMed Central  Google Scholar 

  26. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. https://doi.org/10.1093/bioinformatics/btu033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. He L, He X, Liu X et al (2020) A sensitive, precise and rapid LC–MS/MS method for determination of ergosterol peroxide in Paecilomyces cicadae mycelium. Steroids 164:108751. https://doi.org/10.1016/j.steroids.2020.108751

    Article  CAS  PubMed  Google Scholar 

  28. Guennoc CM, Rose C, Guinnet F, et al (2017) A new method for qualitative multi-scale analysis of bacterial biofilms on filamentous fungal colonies using confocal and electron microscopy. J Visualized Exp 2017. https://doi.org/10.3791/54771

  29. Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265–270. https://doi.org/10.1111/j.1574-6968.1999.tb13383.x

    Article  CAS  PubMed  Google Scholar 

  30. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56. https://doi.org/10.1016/0003-2697(87)90612-9

    Article  CAS  PubMed  Google Scholar 

  31. Hsu SC, Lockwood JL (1975) Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl Microbiol 29:422–426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gonzalo M, Deveau A, Aigle B (2020) Inhibitions dominate but stimulations and growth rescues are not rare among bacterial isolates from grains of forest soil. Microb Ecol 80:872–884. https://doi.org/10.1007/s00248-020-01579-6ï

    Article  CAS  PubMed  Google Scholar 

  33. Shannon P, Markiel A, Ozier O et al (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504. https://doi.org/10.1101/gr.1239303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. R Core Team (2020) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. In: http://www.r-project.org/index.html

  35. Gupta RS, Patel S, Saini N, Chen S (2020) Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the subtilis and cereus clades of species. Int J Syst Evol Microbiol 70:5753–5798. https://doi.org/10.1099/ijsem.0.004475

    Article  CAS  PubMed  Google Scholar 

  36. Barcoto MO, Carlos-Shanley C, Fan H, et al (2020) Fungus-growing insects host a distinctive microbiota apparently adapted to the fungiculture environment. Sci Rep 10. https://doi.org/10.1038/s41598-020-68448-7

  37. Aylward FO, Suen G, Biedermann PHW, et al (2014) Convergent bacterial microbiotas in the fungal agricultural systems of insects. mBio 5. https://doi.org/10.1128/mBio.02077-14

  38. de Fine Licht HH, Boomsma JJ (2010) Forage collection, substrate preparation, and diet composition in fungus-growing ants. Ecol Entomol 35:259–269

    Article  Google Scholar 

  39. de Boer W, Folman LB, Summerbell RC, Boddy L (2005) Living in a fungal world: impact of fungi on soil bacterial niche development. FEMS Microbiol Rev 29:795–811

    Article  PubMed  Google Scholar 

  40. Pagnocca FC, Carreiro SC, Bueno OC et al (1996) Microbiological changes in the nests of leaf-cutting ants fed on sesame leaves. J Appl Entomol 120:317–320. https://doi.org/10.1111/j.1439-0418.1996.tb01612.x

    Article  Google Scholar 

  41. Manavathu EK, Vager DL, Vazquez JA (2014) Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa. BMC Microbiol 14. https://doi.org/10.1186/1471-2180-14-53

  42. Boilard A, Dubé CE, Gruet C et al (2020) Defining coral bleaching as a microbial dysbiosis within the coral holobiont. Microorganisms 8:1–26. https://doi.org/10.3390/microorganisms8111682

    Article  CAS  Google Scholar 

  43. Barke J, Seipke RF, Grüschow S, et al (2010) A mixed community of Actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus. BMC Biol 8. https://doi.org/10.1186/1741-7007-8-109

  44. Vargas Hoyos HA, Chiaramonte JB, Barbosa-Casteliani AG, et al (2021) An Actinobacterium strain from soil of Cerrado promotes phosphorus solubilization and plant growth in soybean plants. Front Bioeng Biotechnol 9. https://doi.org/10.3389/fbioe.2021.579906

  45. Anderson I, Abt B, Lykidis A, et al (2012) Genomics of aerobic cellulose utilization systems in Actinobacteria. PLoS One 7. https://doi.org/10.1371/journal.pone.0039331

  46. Aylward FO, Burnum-Johnson KE, Tringe SG et al (2013) Leucoagaricus gongylophorus produces diverse enzymes for the degradation of recalcitrant plant polymers in leaf-cutter ant fungus gardens. Appl Environ Microbiol 79:3770–3778. https://doi.org/10.1128/AEM.03833-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Abril AB, Bucher EH (2002) Evidence that the fungus cultured by leaf-cutting ants does not metabolize cellulose. Ecol Lett 5:325–328

    Article  Google Scholar 

  48. Vigueras G, Paredes-Hernández D, Revah S et al (2017) Growth and enzymatic activity of Leucoagaricus gongylophorus, a mutualistic fungus isolated from the leaf-cutting ant Atta mexicana, on cellulose and lignocellulosic biomass. Lett Appl Microbiol 65:173–181. https://doi.org/10.1111/lam.12759

    Article  CAS  PubMed  Google Scholar 

  49. Moreira-Soto RD, Sanchez E, Currie CR, Pinto-Tomás AA (2017) Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies. Microbiology (United Kingdom) 163:1578–1589. https://doi.org/10.1099/mic.0.000546

    Article  CAS  Google Scholar 

  50. Suen G, Scott JJ, Aylward FO, et al (2010) An insect herbivore microbiome with high plant biomass-degrading capacity. PLoS Genet 6:e1001129. https://doi.org/10.1371/journal.pgen.1001129

  51. Fernández-Marín H, Zimmerman JK, Wcislo WT (2007) Fungus garden platforms improve hygiene during nest establishment in Acromyrmex ants (Hymenoptera, Formicidae, Attini). Insectes Soc 54:64–69. https://doi.org/10.1007/s00040-007-0907-z

    Article  Google Scholar 

  52. Barcoto MO, Pedrosa F, Bueno OC, Rodrigues A (2017) Pathogenic nature of Syncephalastrum in Atta sexdens rubropilosa fungus gardens. Pest Manag Sci 73:999–1009. https://doi.org/10.1002/ps.4416

    Article  CAS  PubMed  Google Scholar 

  53. Cafaro MJ, Poulsen M, Little AEF et al (2011) Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria. Proc Royal Soc B: Biol Sci 278:1814–1822. https://doi.org/10.1098/rspb.2010.2118

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank the Laboratory of Fungal Ecology and Systematics (LESF—São Paulo State University, Rio Claro, SP, Brazil) and Ecogenomics of Interaction (INRAE, Champenoux, France) research teams for the comments on early drafts of this manuscript. We thank T. Dhalleine (IAM Lorraine University) for helping with HPLC analyses. We also thank three anonymous reviewers for their constructive comments on this manuscript.

Funding

We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a fellowship (grant #305269/2018–6). Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) provided financial support (grant #2014/24298–1, #2017/12689–4, and #2019/03746–0) to A. R. and (grant #2021/04706–1) to Q. V. M. The French National Research Agency (ANR) (ANR-11-LABX-0002–01, Lab of Excellence ARBRE) provided financial support to A. D. and the Novo Nordisk Foundation (Posdoctoral Fellowship NNF20OC0064385) to L. V. F.

Author information

Authors and Affiliations

  1. Department of General and Applied Biology, São Paulo State University (UNESP), Avenida 24-A, 1515, Bela Vista, Rio Claro, SP, 13.506-900, Brazil

    Maria Jesus Sutta Martiarena, Quimi Vidaurre Montoya & Andre Rodrigues

  2. UMR IAM, Université de Lorraine, INRAE, 54280, Champenoux, France

    Aurelie Deveau

  3. Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark

    Laura V. Flórez

Authors
  1. Maria Jesus Sutta Martiarena
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aurelie Deveau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Quimi Vidaurre Montoya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laura V. Flórez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andre Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M. J. S. M., A. D., L. V. F., and A. R. designed the study. M. J. S. M. and Q. V. M. carried out fieldwork. M. J. S. M., A. D., and Q. V. M. carried out laboratory work. M. J. S. M., A. D., and Q. V. M. analyzed the data. M. J. S. M. and A. R. wrote the first drafts of the manuscript. All authors revised and contributed to the manuscript.

Corresponding author

Correspondence to Andre Rodrigues.

Ethics declarations

Ethics Approval

No ethical approval was needed for this work.

Competing Interests

The authors declare no competing interests.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 140 MB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Martiarena, M.J.S., Deveau, A., Montoya, Q.V. et al. The Hyphosphere of Leaf-Cutting Ant Cultivars Is Enriched with Helper Bacteria. Microb Ecol 86, 1773–1788 (2023). https://doi.org/10.1007/s00248-023-02187-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-023-02187-w

Keywords

Search

Navigation