Examination of Taxonomic, Habitat, and Landscape Predictors of Snake Skin Microbial Communities and Their Role as an Innate Defense Mechanism Against the Fungal Pathogen, Ophidiomyces ophidiicola

Lauren Fuchs

George Mason University

November 2024

Background: Anthropogenic activities are drastically altering landscapes and decimating ecosystem health, prompting declines in snake populations worldwide (1,2). Over the past several decades, these challenges have been compounded by the rising incidence of a severe and often fatal disease called ophidiomycosis (also known as snake fungal disease). Ophidiomycosis is an infectious disease caused by the keratophilic fungus Ophidiomyces ophidiicola (Oo; 3). Oo, which is widespread throughout the Eastern United States, is emerging as a significant pathogen, with population-level impacts now a rising concern (3,4).

Photos by Lauren Fuchs.

Untangling the contributions and interactions of various host and ecological factors has been an important focus of ophidiomycosis research, however, much remains unclear regarding dynamics of the disease and its highly variable impacts across individuals. Existing research has highlighted several potential host characteristics that may play a significant role in the pathogenesis of Oo. The skin microbiome is a host trait that has garnered increased attention over the past several decades, specifically with respect to species-specific responses to fungal pathogens like Batrachochytrium dendrobatidis and Pseudogymnoascus destructans, and more recently Oo. 

The host skin microbiome complements the innate immune system and serves as an integral first line of defense, mediating protection against harmful pathogens (5). Microbial composition varies across individuals, which can be associated with differential host resistance to pathogens (6). Factors that influence the apparent differences among species are multifaceted, involving not only characteristics of the host, but also extrinsic variables. The microbial diversity of the surrounding habitat, for example, has been shown to heavily influence host-associated microbial structure by acting as a “microbial reservoir” (6). Studies across numerous taxa support the role of the environment in shaping the host microbiome, thereby conferring differential protection against pathogens (7,8,9,10).

Research across other taxa support that perturbation of the inherent skin microbial composition can result from changing environmental conditions, which can alter its functionality (11). In amphibian hosts, for example, Zhou at al. (12) found that environmental stressors, specifically urbanization, both increased the stochasticity and reduced the stability of both the gut and skin microbiome. Innate immunity, beginning with the skin microbiome as the first line of defense, has been explored in snakes with respect to Oo infection, however, the potential role of landscape-level factors, such as urbanization and agriculture, has received minimal attention. 

 

Objectives and research questions: In addition to contributing valuable data on the distribution and prevalence of Oo in the eastern U.S., the primary objective of my research is to explore taxonomic and ecological factors that may contribute towards variability in the snake skin microbiome, which, in turn, can modulate susceptibility and outcomes of infection by the Oo pathogen. With this objective in mind, I designed my study to specifically answer several important questions. First, how does Oo prevalence and disease severity vary across species, habitat affiliation, and landscape? Second, does the diversity and richness of the snake skin microbial assemblage differ across species, habitat type, and landscape, and do assemblages appear to be affected by infection status? With respect to microbial diversity, I am also interested in identifying whether there are particular resident microbes that tend to be associated with higher prevalence or disease severity.

Photo by Joe Villari.
Photo by Erica Lyon.
Photo by Josh Walton.

Methods: Sampling was conducted between spring egress 2021 and fall ingress 2023 at 25 field sites across 13 counties (12 in Virginia, 1 in Maryland). Sampling sites, which included residential properties, county and state parks, wildlife management areas, natural area preserves, and research centers, span across the coastal plain and piedmont ecoregions, with a relatively even distribution throughout urban, forested, and agricultural landscapes. For each sample, habitat and landscape variables will be assessed at multiple spatial scales using ArcGIS landcover data. The variables assessed will include urbanization (percent impervious surface), agriculture (percent crop land), natural/forested (percent canopy cover), and water abundance. Three buffers will be created around the center of each sampling site representing (average) maximum daily movement, home range, and total distance traveled from the hibernacula.

A total of 180 snakes were sampled (see tables below for summaries). Target species included all colubrid snakes present at sampling sites. Snakes were hand-captured and visually inspected for signs of infection. Based on this evaluation, the snake was assigned a “clinical score” of 0, 1, or 2, for both the face and body. Skin swab samples were then collected from the face, body, and lesions (if present). Each snake was then measured (circumference, snout-to-vent length, and total length); these measurements will be used to estimate overall surface area to standardize pathogen load across different sized snakes. In snakes deemed large enough to safely probe the cloaca, sex was also determined. Additional sample and environmental correlates, such as body temperature, canopy cover, exposure, and weather variables, were also recorded.

To assess the presence of Oo and determine the gene copy number (pathogen load), quantitative polymerase chain reaction (qPCR) was used to amplify a targeted Oo-specific sequence of the fungal internal transcribed spacer region (13). Most of my samples have been analyzed via qPCR, however, there is still a small set that requires an additional run for confirmation. I therefore have not yet performed any sort of statistical analyses on the data; however, I have compiled the preliminary results to illustrate some possible trends.

In addition to completing qPCR analyses, I have also been working through the initial steps of library preparation for DNA sequencing of the swab samples. For this part of the project, I will be using PCR to amplify targeted universal bacterial and fungal sequences; these fragments can then be “labeled” and later assigned to a unique taxonomic group, thereby providing important information on each snake’s skin microbial composition.

Photo left by Erica Lyon. Photo right by Tina Sephapur.

Literature Cited

  1. Reading C. J. et al. (2010). Are snake populations in widespread decline? Biological Letters, 6(6):777-80. doi: 10.1098/rsbl.2010.0373. Epub 2010 Jun 9. PMID: 20534600; PMCID: PMC3001371.
  2. Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S., & Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature, 484(7393), 186–194. https://doi.org/10.1038/nature10947.
  3. Paré, J. A., & Sigler, L. (2016). An overview of reptile fungal pathogens in the genera Nannizziopsis, paranannizziopsis, and ophidiomyces. Journal of Herpetological Medicine and Surgery, 26(1–2), 46. https://doi.org/10.5818/1529-9651-26.1-2.46.
  4. Allender, M. C., Raudabaugh, D. B., Gleason, F. H., & Miller, A. N. (2015). The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on free-ranging snake populations. Fungal Ecology, 17, 187–196. https://doi.org/10.1016/j.funeco.2015.05.003.
  5. Woodhams, D. C., Alford, R. A., Briggs, C. J., Johnson, M. A., & Rollins‐Smith, L. A. (2008). Life History Trade-Offs Influence Disease in Changing Climates: Strategies of an Amphibian Pathogen. Ecology, 89(6), 1627–1639. https://doi.org/10.1890/06-1842.1
  6. Harrison, X. A., Price, S. J., Hopkins, K., Leung, W. T. M., Sergeant, C., & Garner, T. W. J. (2019). Diversity-Stability dynamics of the amphibian skin microbiome and susceptibility to a lethal viral pathogen. Frontiers in Microbiology, 10. https://doi.org/10.3389/fmicb.2019.02883
  7. Muletz‐Wolz, C. R., Yarwood, S. A., Grant, E. H. C., Fleischer, R. C., & Lips, K. R. (2017). Effects of host species and environment on the skin microbiome of Plethodontid salamanders. Journal of Animal Ecology, 87(2), 341–353. https://doi.org/10.1111/1365-2656.12726
  8. Li, Z., Li, A., Dai, W., Leng, H., Liu, S., Jin, L., Sun, K., & Jiang, F. (2022). Skin microbiota variation among bat species in China and their potential defense against pathogens. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.808788
  9. Vanderwolf, K. J., Campbell, L. J., Taylor, D. R., Goldberg, T. L., Blehert, D. S., & Lorch, J. M. (2021). Mycobiome Traits Associated with Disease Tolerance Predict Many Western North American Bat Species Will Be Susceptible to White-Nose Syndrome. Microbiology Spectrum, 9(1). https://doi.org/10.1128/spectrum.00254-21
  10. Walker, D. M., Leys, J. E., Grisnik, M., Grajal-Puche, A., Murray, C. M., & Allender, M. C. (2019). Variability in snake skin microbial assemblages across spatial scales and disease states. The ISME Journal, 13(9), 2209–2222. https://doi.org/10.1038/s41396-019-0416-x
  11. Romer, A. S., Grinath, J. B., Moe, K. C., & Walker, D. M. (2022). Host microbiome responses to the Snake Fungal Disease pathogen (Ophidiomyces ophidiicola) are driven by changes in microbial richness. Scientific Reports, 12(1). https://doi.org/10.1038/s41598-022-07042-5
  12. Zhou, J., Liao, Z., Liu, Z., Guo, X., Zhang, W., & Chen, Y. (2023). Urbanization increases stochasticity and reduces the ecological stability of microbial communities in amphibian hosts. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.1108662
  13. Bohuski, E. A., Lorch, J. M., Griffin, K. M., & Blehert, D. S. (2015). TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease. BMC Veterinary Research, 11(1). https://doi.org/10.1186/s12917-015-0407-8