Presence of Ranavirus and Chytrid Fungus in Lithobates sphenocephalus in Long and Short Hydroperiod Wetlands

Shaina Carrington1,2, David Scott1, Megan Winzeler1, Austin Coleman1, Carlos Tapia1,3, and Stacey Lance1

1UGA Savannah River Ecology Laboratory, Aiken, SC, 2Virginia Polytechnic Institute and State University, Blacksburg, VA, and 3University of Central Florida, Orlando, FL

Background/Justification

Objectives

Results Methods

Predictions Literature References

Batrachochytrium dendrobatidis (chytrid or Bd) and Ranavirus aretwo pathogens linked to global amphibian declines1,2

Chytrid Fungus(Bd):

Has been detected in 56 countries3

Directly transmitted throughphysical contact

Colonizes the keratinizedepidermis and mouthparts ofamphibians

Disrupts osmoregulation, causing toxin release and resulting indeath

At Savannah River Site: Been present since the 1970s, but at low frequencies4

Strongly affected seasonally5

Recent Study Shows: Bd more prevalent in contaminated and long

hydroperiod wetlands The prevalence has increased since the 1970s

Ranavirus:

Has been detected on 5 continents Transmitted through ingestion and

physical contact6

Culminates in organ necrosiscausing massive hemorrhaging anddeath

Infects amphibians, reptiles, and fish7

At Savannah River Site: Prevalence is 4 times greater than Bd Strongly associated with life stages and affected

seasonally

Recent Study Shows: Ranavirus more prevalent in long hydroperiod wetlands No significant difference in the prevalence of the virus in

contaminated vs. uncontaminated wetlands Ranavirus is common in southern leopard frogs

Problem with recent study:

Only examined 12 wetlands All contaminated wetlands were long hydroperiods Didn’t have the same species from each study site

Solution (my study):

Examine more wetlands Only include uncontaminated reference wetlands Use only one species from each study site

To determine:

The current prevalence of Bd and ranavirus in southern leopard frogs on the SRS

The prevalence of Bd and ranavirus in long and short hydroperiod wetlands

Whether disease loads differ among individuals from long and short hydroperiod wetlands

Based on prior studies:

Long hydroperiod wetlands should have higher disease prevalence compared to the short

Ranavirus should be at higher prevalence than Bd

d

Study Area: Savannah River Site near Aiken, SC

25 wetlands sampled for southern leopard frogs 11 short hydroperiod wetlands 14 long hydroperiod wetlands

280 tissue samples from tadpoles (145 from long and 135 from short)

Map Key:

Long Hydroperiod

Short Hydroperiod

Experimental Design:

Active dip-netting in both wetland types Minnow trapping to supplement captures Tail clipping to collect tissue for DNA DNA Bead Beater Extraction Protocol

Figure 3. SRS study sites

Quantitative Sampling:

qPCR using Taqman primer/probe protocol Samples run in triplicate 100, 10, 1, and 0.1 serial dilution of fungal standards 106-101 serial dilution of virus standards Samples were analyzed using BioRad CFX Manager

Figure 1. Bd has been associated with dramatic population declines worldwide

Figure 2. Ranaviruses have been linked to mass mortality events in amphibians

Figures 6. Active dip-netting in long hydroperiod wetlands

Figure 7. Processing tadpoles

Figure 8. Prep-qPCR Figure 9. PCR machine

No positives for either pathogens Both hypotheses rejected

Figure 10. Amplification curve showing the relationship between qPCR cycle number and relative fluorescence. In this figure the standards are shown.

Figure 11. Standard curve, showing the relationship between the starting quantity of DNA and the qPCR cycle number where more than background fluorescence is observed (Cq).

Conclusions/Future Implications

Conclusions:

Surprising because the recent study showed 24% positive for Bd and 32% positive for ranavirus for this species

The recent study was tested throughout February-July 2013, while my study was tested throughout March-May 2015

Chytrid Temperature/Seasonal Tolerance:

More prevalent in the winter Bd does not grow at water temperatures above 28°C8

Ranavirus Temperature/Seasonal Tolerance:

More prevalent in autumn and decreasing throughout the rest of the seasons9

Acknowledgements

We would like to thank the Lance Lab, REU program participants, Melissa Pilgrim, Tracey Tuberville, J VaunMcArthur, and SREL. Also, thank you to National Science Foundation, Department of Energy, and Savannah River Site.

1. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, et al. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci USA. 1998;95: 9031-9036.

2. Green DE, Converse KA, Schrader AK. Epizootiology of sixty-four amphibian morbidity and mortality events in the USA, 1996-2001. Ann New York Acad Sci. 2002;5: 323-339.3. Olson DH, Anaensen DM, Ronnenberg KL, Powell CI, Walker SF, Biebly J, et al. Mapping the global emergence of Batrachochytrium dendrobatidis, the amphibian

chytrid fungus. PLos One. 2013;8.4. Daszak AP, Scott DE, Kilpatrick AM, Faggioni C, Gibbons JW, Porter D. Amphibian population declines at Savannah River Site are linked to climate, not chytridiomycosis. Ecology.

2005;86: 3232-32375. Love CN, Winzeler ME, Beasley R, Scott DE, Nuziata SO, Lance SL. Patterns of amphibian disease prevalence across contaminated and uncontaminated wetlands on the

Savannah River Site, S.C. In Review.6. Miller D, Gray M, Storfer A. Ecopathology of ranaviruses infecting amphibians. Viruses. 2011;3: 2351-73 7. Gray MJ, Miller DL, Hoverman JT. Ecology and pathology of amphibian ranaviruses. Dis Aquat Organ. 2009;87:243-2668. Peterson JD, Wood MB, Hopkins WA., Unrine JM, and Mendonca MT. Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae

from wetlands on the Savannah River Site, South Carolina. Journal of wildlife Diseases. 2007;43(3): 450-4609. Hoverman JT, Gary MJ, Miller DL, Hailslip NA. Widespread occurrence of ranavirus in pond-breeding amphibian populations. International Association for Ecology and Health.

2011.

Figure 5. Ellenton Bay- long hydroperiod wetland

Figure 4. Rainbow Bay- short hydroperiod wetland

Future Implications:

Further research should examine the patterns in disease prevalence in different hydroperiod

Figure 12. Southern leopard frog (L. sphenocephalus) metamorph

wetlands. Each hydroperiod type should be examined to understand how seasonal changes affect the pathogens. Shifting disease patterns may accompany climate change.

Threshold Fluorescence

Positive Sample