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Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants

The ISME Journal volume 17, pages 1751–1764 (2023)Cite this article

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Abstract

While genome sequencing has expanded our knowledge of symbiosis, role assignment within multi-species microbiomes remains challenging due to genomic redundancy and the uncertainties of in vivo impacts. We address such questions, here, for a specialized nitrogen (N) recycling microbiome of turtle ants, describing a new genus and species of gut symbiont—Ischyrobacter davidsoniae (Betaproteobacteria: Burkholderiales: Alcaligenaceae)—and its in vivo physiological context. A re-analysis of amplicon sequencing data, with precisely assigned Ischyrobacter reads, revealed a seemingly ubiquitous distribution across the turtle ant genus Cephalotes, suggesting ≥50 million years since domestication. Through new genome sequencing, we also show that divergent I. davidsoniae lineages are conserved in their uricolytic and urea-generating capacities. With phylogenetically refined definitions of Ischyrobacter and separately domesticated Burkholderiales symbionts, our FISH microscopy revealed a distinct niche for I. davidsoniae, with dense populations at the anterior ileum. Being positioned at the site of host N-waste delivery, in vivo metatranscriptomics and metabolomics further implicate I. davidsoniae within a symbiont-autonomous N-recycling pathway. While encoding much of this pathway, I. davidsoniae expressed only a subset of the requisite steps in mature adult workers, including the penultimate step deriving urea from allantoate. The remaining steps were expressed by other specialized gut symbionts. Collectively, this assemblage converts inosine, made from midgut symbionts, into urea and ammonia in the hindgut. With urea supporting host amino acid budgets and cuticle synthesis, and with the ancient nature of other active N-recyclers discovered here, I. davidsoniae emerges as a central player in a conserved and impactful, multipartite symbiosis.

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Fig. 1: Genetic relationships and morphology of the newly described Ischyrobacter clade.
Fig. 2: Ischyrobacter localization in turtle ant gut sections.
Fig. 3: Summary of Ischyrobacter davidsoniae metabolism.
Fig. 4: Nitrogen (N)-recycling by Ischyrobacter symbionts inferred from multi-omic data.
Fig. 5: Inosine production by Cephaloticoccus capnophilus in the midgut.
Fig. 6: Proposed model on purine metabolism and N-recycling in symbiont communities of turtle ant mature workers’ gut.

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Data availability

The Ischyrobacter davidsoniae Cv33a genome (Project ID: Gp0110144) and the Ischyrobacter sp. CSM3487_49 genome (Project ID: Gp0624441) are available from the IMG/M-ER website (https://img.jgi.doe.gov/) by searching the project IDs. The Ischyrobacter sp. bin5 draft genome from C. grandinosus is accessible on the IMG/M-ER C. grandinosus metagenome page (Project ID: Gp0125967), by selecting “Metagenome Bins” in the “Statistics” tab. The Cephaloticoccus capnophilus genome (Project ID: Gp0110136) was downloaded from the IMG/M-ER website by searching the project ID. All 16S amplicon sequencing data are from Flynn et al. [18], https://github.com/peterjflynn/cephalotes_gut_localization and Hu et al. [24], https://doi.org/10.5061/dryad.kwh70rz5d, and have been deposited in the GenBank Short Read Archive under BioProject PRJNA668764 at https://www.ncbi.nlm.nih.gov/bioproject/668764 and BioProject PRJNA767930 at https://www.ncbi.nlm.nih.gov/bioproject/767930, respectively. Supplementary data can be found in our data repository at: https://doi.org/10.6084/m9.figshare.21989537.

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Acknowledgements

We thank Scott Powell and Jignasha Rana who collected specimens used in this study. We are grateful to Benjamin Rubin who participated in C. varians genome sequencing and assembly. Thanks also go to Rebecca Wilkes who worked on metabolomic sample preparation and Dan Freeman who helped in genomic analyses. We further thank Alicia Pastor at Michigan State University for the work on TEM images, and Aharon Oren and Bernhard Schink for their help naming Ischyrobacter davidsoniae. Finally, we thank Katrina Terry, Azad Ahmed, Rachel Ehrlich, and Steven Lang from the Drexel Genomic Core Facility who worked on the PacBio sequencing preparation and processing.

Funding

This work was supported by National Science Foundation CBET-1653092 to LA, DEB-1442156 to JTW, DEB-1900357 to CSM, DOB-1442144 to JAR, GRF-2041772 to CSC, and TUES-1245632 to GLR and JAR.

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Authors and Affiliations

  1. Department of Biology, Drexel University, 3245 Chestnut St., Philadelphia, PA, 19104, USA

    Benoît Béchade, Christian S. Cabuslay, Yi Hu, Valerie J. Fiers, Dharman Anandarajan, Richard Lu, Chandler J. Olson & Jacob A. Russell

  2. State Key Laboratory of Earth Surface Processes and Resource Ecology and Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, 100875, Beijing, China

    Yi Hu

  3. Department of Civil and Environmental Engineering, McCormick School of Engineering and Applied Science, Northwestern University, Evanston, IL, 60208, USA

    Caroll M. Mendonca, Bahareh Hassanpour & Ludmilla Aristilde

  4. Department of Biology, Calvin University, 1726 Knollcrest Circle SE, Grand Rapids, MI, 49546-4402, USA

    Jonathan Y. Lin, Yangzhou Su & John T. Wertz

  5. Department of Biological Sciences, University of Alabama, 1325 Hackberry Ln, Tuscaloosa, AL, 35487, USA

    Chandler J. Olson

  6. Department of Entomology, Cornell University, Cornell AgriTech, Geneva, NY, 14456, USA

    Christophe Duplais & Corrie S. Moreau

  7. Ecological and Evolutionary Signal-Processing and Informatics Laboratory, Department of Electrical and Computer Engineering, Drexel University, 3141 Chestnut St., Philadelphia, PA, 19104, USA

    Gail L. Rosen

  8. Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, 14853, USA

    Corrie S. Moreau

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  1. Benoît Béchade
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Contributions

BB, CSC, YH, JTW, and JAR designed research; BB, CSC, YH, CMM, BH, JYL, CD, LA, JTW, and JAR contributed to conceptualization; BB, CSC, YH, CMM, BH, JYL, YS, VJF, DA, RL, CJO, CSM, JTW, and JAR performed research; BB, CSC, JTW, and JAR analyzed data; BB and JAR wrote the manuscript; BB, YH, GLR, LA, JTW, and JAR reviewed and edited the manuscript; CSC, GLR, CSM, LA, JTW, and JAR contributed to funding; all authors approved the final version.

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Correspondence to Benoît Béchade.

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Béchade, B., Cabuslay, C.S., Hu, Y. et al. Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants. ISME J 17, 1751–1764 (2023). https://doi.org/10.1038/s41396-023-01490-1

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