Hypotheses•H1: A. americanus will have a lower prevalence of Ranavirus than the spotted salamander, Ambystoma maculatum.•H2: Prevalence will be highest at the end of their developmental period, close to metamorphosis.•H3: Quantitative PCR (qPCR) will have a higher number of positive samples.

A Study of Ranavirus in the American Toad, Anaxyrus americanus, found in the New

Salem Vernal Pool, York PA

Stephani Lane Department of Biological Sciences, York College of

Pennsylvania

Conclusions•A. americanus tadpoles had lower prevalence of RV than the spotted salamander, suggesting that toads are not being as significantly affected •RV prevalence was highest in the last two weeks of the developmental period of A. americanus larvae •No visible signs or death were reported in toads despite individuals having the virus present. • Quantitative PCR is much more effective in determining positive samples for Ranavirus. This should be the standard testing method for the detection in future studies.

Introduction •Ranavirus is a double stranded DNA virus that is causing population decline in amphibian species across five continents (Miller et all. 2011, Lesbarreres et all. 2011).

•Larval amphibians, including multiple species of frogs, toads and salamanders are impacted and have symptoms such as; lesions, lethargic or odd swimming and fluid accumulation under the skin (Hoverman et al. 2012). Ultimately, can cause death in less than one week.

•Many species affected by Ranavirus live in the same vernal pools. Anaxyrus americanus (Fig. 1), the common toad, are different mostly in being terrestrial, nocturnal predators that have a thicker, more dry skin (Haslip et al. 2011).

•The New Salem vernal pool is the only documented location of RV in central PA. Very little is known about RV here, especially the impact on A. americanus

Objectives•To determine the prevalence of RV in larval A. americanus to compare prevalence to other species in the amphibian community

•To evaluate prevalence over the development of larval A. americanus until metamorphosis

•To compare the effectiveness of two molecular techniques used to identify individuals infected with RV (standard and quantitative PCR)

Haislip, N.A., Gray, M.J., Hoverman, J.T., Miller, D.L. Development and disease: how susceptibility to an emerging pathogen changes through anuran development. PLoS ONE [serial online] 6(7).

Hoverman, J.T, Gray, Gray, M.J., Miller, D.L., Haislip, N.A. 2012. Widespread occurrence of Ranavirus in pond-breeding amphibian populations. EcoHealth [serial online] 9:36-48.

Lesbarrers, D., Balseiro, A., Brunner, J., Chinchar, V.G., Duffus, A., Kerby, J., Miller, D.L., Robert, J., Schock, D.M., Waltzek, T., Grey, M.J. 2011. Ranavirus: past, present and future. Biology Letters.

Miller, D., Gray, M., Storfer, A. 2011. Ecopathology of Ranavirus infecting amphibians. Viruses [serial online]3:2351-2373.

Literature Cited

AcknowledgementsI would like to thank Dr. Hagerty for her endless guidance and support. I

would also like to thank Dr. Higa, Thomas Brigman and Carrie Reall for their assistance in my research.

Methods

Samples Collected•Dip net protocol •May 5th- July 2nd 2013•n(collected)= 101•n(sampled)= 63

DNA Extraction & Quantification

Standard PCR for MCP (Fig. 3)•To test for the Ranavirus major capsid protein (MCP)•Negative Control= H2O•Positive Control= MCP Plasmid

qPCR for MCP •To confirm +/- for RV•Positive if Ct (threshold cycle) value was higher than negative H2O (Fig. 4)

Confirmation of amphibian DNA using the 12S RNA gene in PCR

Ladd

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Figure 3. Standard PCR amplification of the MCP gene to identify Ranavirus infection in Anaxyrus americanus. 1% agarose gel with a 100 bp ladder. Plasmid (+ control) in lane two (576 bp). BA 106 and BA 110 positive for RV.

Figure 1. Anaxyrus americanus, American Toad Image 2. New Salem vernal pool is located south west of York city on the south west branch of Codorus Creek (39.91483, -76.77522)http://www.theanimalfiles.com/amphibians/toads/american_toad.html

Results