Study of the microbial community present on eastern red backed salamanders (Plethodon cinereus)




Julie Lynn

Introduction

Amphibians around the world are experiencing drastic population declines. Over 1/3 of amphibian species are in decline (Stuart et al. 2004). There are many causes for amphibian decline, amphibian skin infection by Batrachochytrium dendrobatidis, more commonly referred to as the Chytrid fungus, is a major cause of amphibian decline (Harris et al. 2009). The skin of amphibians is an important organ responsible for respiration, electrolyte balance, hydration and many other important physiological functions (Rosenblum et al. 2012). Amphibian skin also possess unique microbial communities. The microbial communities present on amphibian skin is part of the organisms innate immune system (Woodhams et al. 2007). There are multiple bacterial species present on the skin of amphibians that produce a protein called violacein (Becker et al. 2009 ). Violacein has antifungal properties and has been shown to inhibit the skin disease caused by the chytrid fungus on both natural and inoculated hosts (Becker & Harris 2010; Harris et al. 2009). The chytrid fungus has been found on every continent, except Antarctica, and though it has not been found in New York State, it has been found in many neighboring states and sampling efforts in New York have been limited (Bd-maps). Violacein producing bacteria as well as the violacein protein have been found on the skin of eastern red backed salamanders (Plethodon cinereus) in other regions of their range (Becker et al. 2009; Harris et al. 2009) where the species’ skin microbial interactions have been heavily researched.

Materials and Methods

Samples were collected postmortem in 2011 from outside George Washington High School in New York City (provided by Evon Hekkela at Fordham University). Cultures of the chytrid fungus used for positive controls in the study were provided by the Chytrid Laboratory at the University of Maine.
DNA was extracted from salamander samples and chytrid cultures, and amplified using PCR. Universal fungal DNA was amplified using primers taken from Schoch et al. 2012. Chytrid specific DNA was amplified using primers taken from Annis et al. 2004. The violacein gene was amplified using original primers designed by Julie Lynn (see paper for full primer information). PCR products were purified and sent out for sequencing, then analyzed in BLAST to confirm target organisms and genes were amplified.

Results

PCR with universal fungal primers successfully amplified fungal DNA (Figure 1). A BLAST search confirmed the DNA amplified were from fungal species.

PCR with chytrid specific primers amplified the positive control only, suggesting the salamander samples were negative for the chytrid fungus (Figure 2).

PCR with chytrid specific primers and universal fungal primers combined confirmed the absence of the chytrid fungus from the salamanders sampled (Figure 3).

The violacein gene was successfully amplified from DNA collected from salamander samples (Figure 4).

Discussion

Salamander samples tested in this study were negative for the chytrid fungus (Batrachochytrium dendrobatidis). The violacein gene was successfully amplified. The violacein gene has been found on eastern red backed salamanders in other regions of its range, i.e. Virginia, where the species has been heavily researched (Becker et al. 2009; Harris et al. 2009). The presence of the gene on salamanders in New York City supports a species specific interaction found in previous studies (Brucker et al. 2008). The presence of the violacein gene may provide red backed salamanders protection if the chytrid fungus becomes established in New York State. Future work should include a complete community analysis of violacein producing species, analysis of the skin microbial community, and community analysis across an urban to rural gradient.

References

1. Annis, S. L., Dastoor, F. P., Ziel, H., Daszak, P., & Longcore, J. E. (2004) A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians. Journal of Wildlife Diseases, 40(3), 420-428.
2. Bd-maps, www.bd-maps.net accessed May 5, 2014.
3. Becker, M. H., Brucker, R. M., Schwantes, C. R., Harris, R. N., & Minbiole, K. P. (2009). The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus. Applied and environmental microbiology, 75(21), 6635-6638.
4. Becker, M. H., & Harris, R. N. (2010). Cutaneous bacteria of the redback salamander prevent morbidity associated with a lethal disease. PLoS One, 5(6), e10957.
5. Brucker, R. M., Harris, R. N., Schwantes, C. R., Gallaher, T. N., Flaherty, D. C., Lam, B. A., & Minbiole, K. P. (2008). Amphibian chemical defense: antifungal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus. Journal of chemical ecology, 34(11), 1422-1429.
6. Harris, Reid N., Robert M. Brucker, Jenifer B. Walke, Matthew H. Becker, Christian R. Schwantes, Devon C. Flaherty, Brianna A. Lam et al. "Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus." The ISME journal 3, no. 7 (2009): 818-824.
7. Rosenblum, E.B., Poorten, T.J., Settles, M., Murdoch, G.K. (2012) Only skin deep: Shared genetic response to the deadly chytrid fungus in susceptible frog species. Mol Ecol 21(13):3110–3120.
8. Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, 109(16), 6241-6246.
9. Stuart, S. N., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S. L., Fischman, D. L. & Waller, R. W. (2004). Status and trends of amphibian declines and extinction worldwide. Science 306, 1783–1786.
10. Woodhams, D.C., Ardipradja, K., Alford, R.A., Marantelli, G., Reinert, L.K., Rollins-Smith, L.A. (2007). Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Anim Conserv 10: 409–417.

Figures


Figure 1-PCR results using universal fungal primers with salamander samples


Figure 2-PCR results using chytrid specific primers with salamander samples and positive chytrid control sample


Figure 3-Results from PCR with universal fungal primers and chytrid primers


Figure 4-PCR results for detection of the violacein gene on salamander samples


Abstract

Amphibian populations around the world are in decline. One of the major causes of decline in amphibian populations is the chytrid fungus, Batrachochytrium dendrobatidis. The eastern red backed salamander (Plethodon cinereus) is an important amphibian in U.S. eastern forest. Bacterial species that produce a protein called violacein have been found on eastern red backed salamanders in other areas of its geographic range . Violacein has antifungal properties and prevents the deadly skin infection caused by the chytrid fungus. DNA extracted from red backed salamanders was amplified using PCR. PCR results show that chytrid was not present on salamanders sampled. The violacein gene was successfully amplified from salamanders. The presence of violacein may be important in protecting the salamanders from chytrid if the fungus enters New York State.

Full Paper

Acknowledgments

I would like to thank Evon Hekkala and her laboratory at Fordham University for the use of their eastern red backed salamander samples. I would also like to thank Joyce Longcore at the University of Maine Chytrid Laboratory for her help and for providing chytrid fungus cultures for positive controls used in the study.
Thank you so much to Dr. Rubin, Kate Reid, and Catharina Grubaugh for all your help and guidance, I couldn’t have done it without you.


This document was last modified 05/16/2014.
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