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Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen

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Abstract

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides—the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin—from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

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Acknowledgements

The authors thank Brandon LaBumbard and Bhumi Patel for their technical assistance. This study was funded in part by an NSF grant (DEB-1146284) to VJM and the Dimensions in Biodiversity program DEB-1136662 to KPCM.

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Authors and Affiliations

  1. Department of Biology, University of Massachusetts Boston, Boston, MA, 02125, USA

    Kelly Barnhart & Douglas C. Woodhams

  2. Department of Chemistry, Villanova University, Villanova, PA, 19085, USA

    Megan E. Forman & Kevin P. C. Minbiole

  3. Division of Natural and Computational Science, Gwynedd Mercy University, Gwynedd Valley, PA, 19437, USA

    Thomas P. Umile

  4. Department of Ecology and Evolutionary Biology, University of Colorado, Ramaley N122, UCB 334, Boulder, CO, 80309, USA

    Jordan Kueneman & Valerie McKenzie

  5. Department of Biology, University of New Mexico, Albuquerque, NM, 87131-0001, USA

    Irene Salinas

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Barnhart, K., Forman, M.E., Umile, T.P. et al. Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen. Microb Ecol 74, 990–1000 (2017). https://doi.org/10.1007/s00248-017-0997-8

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  • DOI: https://doi.org/10.1007/s00248-017-0997-8

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