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Influence of Environmental Gradients on Prevalence and Intensity of Batrachochytrium dendrobatidis on the Columbia Spotted Frog in North Idaho Ponds Danelle Russell Ecology & Conservation Biology University of Idaho Supervising Faculty: Dr. Lisette Waits Fish & Wildlife Department And Dr. Erica Bree Rosenblum Department of Biological Sciences And Caren Goldberg PhD Student, Fish & Wildlife Department University of Idaho January 2009 ABSTRACT The pathogenic chytrid fungus Batrachochytrium dendrobatidis is diminishing amphibian populations worldwide. Temperatures change dramatically across seasons and elevations in Idaho; therefore, the temperature restrictions of B. dendrobatidis could have important implications for the outcome of infection on wild anuran populations. We will evaluate the prevalence and severity of infections on the Columbia Spotted Frog (Rana luteiventris) in northern Idaho’s ponds to determine if there is a significant difference in the prevalence and intensity of B. dendrobatidis on frogs across three different seasons (spring, summer, and autumn) and elevations ranging from 774 – 1340 m. From eight ponds we will sample the Columbia Spotted Frog over three different seasons (end of March – mid-May, August, & October 2009) for a sample size of 10-15 individuals per pond per season. We will swab the frogs and analyze them for the presence of B. dendrobatidis using established quantitativePCR techniques. We will test for relationships between prevalence and intensity of B. dendrobatidis and climatic variables such as air and water temperature, rainfall, and elevation using regression. We will determine if B. dendrobatidis differs in prevalence or intensity among the seasons using ANOVA. If there are seasonal fluctuations in B. dendrobatidis, this could influence future chytrid research protocols and conservation programs. INTRODUCTION Background on disease Chytridiomycosis is a potentially fatal skin infection of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis. This infectious disease has been identified as a cause of mass mortalities, population declines, extinctions, and biodiversity loss of many amphibian species worldwide (Lips et al., 2006; Pearl et al., 2007; Rachowicz et al., 2006; Retallick et al., 2004). Population declines and extinctions have been concentrated in montane regions (Puschendorf et al., 2006), which is of special concern because 85% of the world’s threatened frog species occur at high elevations (Kriger and Hero, 2008). Low levels of genetic diversity indicate that B. dendrobatidis is an emergent pathogen (Morehouse et al, 2003). The origin of the fungus is not well known (Oevermann and Robert, 2004), but evidence suggests it is novel to many amphibians and quickly spreads after introductions (Kriger and Hero, 2008; Mazzoni et al., 2003; Pearl et al., 2007; Picco and Collins, 2008). The fungus has been identified in wild and captive populations from zoos, academic research collections, and commercial (aquaria and farming) collections (Ouellet et al., 2005). Batrachochytrium dendrobatidis is thought to have suddenly appeared in Australia, Central America and North America, suggesting the fungus has been introduced to many areas by anthropogenic means (Berger et al., 2006; Morgan et al., 2007; Picco and Collins, 2008). Batrachochytrium dendrobatidis is believed to have an optimal range of temperature conditions to thrive. Batrachochytrium dendrobatidis can grow in laboratory conditions at temperatures between 6 and 28C (Longcore et al., 2007). Although studies have shown that the prevalence of the chytrid fungus increases in cooler months (Kriger and Hero, 2008; Berger et al., 2004), the magnitude of seasonal fluctuations has yet to be accurately quantified (Berger et al., 2004). Laboratory experiments have shown that some adult frogs are capable of clearing the infection entirely, particularly temperatures above 29C (Lamirande and Nicols, 2002; Kriger and Hero, 2006) . Study System The Columbia Spotted Frog (Rana luteiventris) is common east of the Cascade Range and Coast Mountains (Corkran and Thoms, 1996). The state of Idaho considers this species to be of “special concern,” (US Forest Service, 2008) and was designated as a candidate species for federal listing in 1997 (Nevada Fish and Wildlife Service, 2008). One major factor in the decline of R. luteiventris is habitat alteration and destruction (Davis and Verrell, 2005; Padgett-Flohr, 2008). The breeding populations in the region are small, which may make spotted frogs vulnerable to genetic problems and environmental stresses (Davis and Verrell, 2005). The Columbia Spotted Frog can be found in habitats ranging from sagebrush benches to subalpine forests at elevations up to about 10,000 feet (US Forest Service, 2008). Frog migration patterns can affect disease transmission. This diurnal frog may cross land areas in the spring and summer after breeding. The Columbia Spotted Frog breeds as soon as snow melt permits in spring (US Forest Service, 2008), which in northern Idaho is around the end of March to mid-May, depending on annual variation and elevation. There are often high levels of gene flow among low elevation sites separated by large distances (Funk et al., 2005). Migratory males often remain within 200 m of the breeding sites, whereas females travel up to 1030 m to reach summer habitats (Pilliod, 2002). The frogs migrate by the shortest-distance travel routes through dry, open forests and stream corridors (Pilliod, 2002). A study conducted by Funk et al. (2005) found that mountain ridges and elevation differences were associated with increased genetic differentiation among sites, suggesting that gene flow (and thus, potential for disease transmission) in this species is restricted by ridges and elevation. Climate may affect the distribution and intensity B. dendrobatidis. In northern Idaho, the climate is heavily reliant on the mountains. Moscow, ID, the lowest elevation for this study, has maximum mean air temperatures of 13C in March through May during which time frogs emerge from hibernation and breed. August has the warmest temperatures of the year at an average of 28C. October has an average air temperature of 15.5C, at which time the frogs will prepare for hibernation. The average annual rainfall for Moscow is 23.5 inches, with extremely dry summers and wet winters (University of Idaho, 2005). RESEARCH OBJECTIVES (1) To examine temporal variation in prevalence and load of chytridiomycosis on the Columbia Spotted Frog (Rana luteiventris) in Northern Idaho’s pond populations. Hypothesis: All ponds will have Batrachochytrium dendrobatidis present because it was found in every Columbia Spotted Frog population tested in this area in 2004-5 (Goldberg, unpublished data). (2) To test for seasonal differences in the prevalence and load of Batrachochytrium dendrobatidis. Hypothesis: Both the prevalence and load of the pathogen will be higher during the spring because lower temperatures are favored by the fungus and the frog’s immune system may be suppressed from overwintering. (3) To determine prevalence and load of Batrachochytrium dendrobatidis across different elevations ranging from 774 – 1340 m. Hypothesis: Batrachochytrium dendrobatidis prevalence and load will increase with elevation because of lower temperatures at higher elevations. There may be a threshold to reverse this trend at very high elevations due to extreme cold temperatures that may suppress the fungus. DATA COLLECTION METHODS We will sample approximately 320 individual adult Columbia Spotted Frogs (Rana luteiventris) over three different periods (end of March – mid-May, August, and mid-October 2009). Approximately 10-15 individual frogs will be sampled at each of the eight ponds each period, when possible. We will choose study sites to maximize the chances of finding frogs, not disease. Our team will opportunistically collect Columbia Spotted Frogs using dip netting & minnow traps set out 16-24 hours in advance. We will handle the frogs with unused, nonpowdered latex gloves to prevent disease transmission. To ensure that no frogs are unintentionally sampled more than once, we will not begin sampling until all frogs at a given pond are caught and placed in an individual plastic bag (NSW, 2000). No further sampling will take place that same day after frogs are released. The pond will be resampled in the same season if not enough frogs are caught. The left hind toe of each frog will be clipped to identify the individual in another sampling in that season. The toe should grow back and not be noticeable in subsequent seasons. The same individual could be resampled in a future sampling season. We will swab each frog by firmly running a sterile buccal swab (WB10-0004; Whatman, Clifton, New Jersey, USA) 10 times over (1) the frog’s dorsal surface, (2) each of the frog’s sides, from groin to armpit, (3) the ventral surface and (4) the undersides of each thigh. Additionally, we will administer five outward strokes on the undersides of each foot, for a total of 70 strokes per individual per swab. The swabs will be immediately placed in 650 l of lysis buffer (50 mM Tris pH 8.0, 50 mM EDTA, 50 mM sucrose, 100 mM NaCl, 1% SDS) and frozen at -80C after returning from the field (within 8 hours of sampling). DNA will be extracted from the swabs using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, California, USA), adapting the tissue protocol by heating the AE solution to 55ºC before elution and incubating the sample in the AE solution at 70ºC for five minutes before the final centrifugation. We will use an Applied Biosystems 7500 Fast Real-Time quantitative PCR System (Applied Biosystems, Foster City, California, USA) to test for the presence of B. dendrobatidis using the protocol of Boyle et al. (2004) with the following changes: total reaction volume will be 20μl, probe concentration will be 125 nM, extracted DNA will not be diluted, and one out of the three wells for each sample will contain the internal control recommended by Hyatt et al. (2007) to ensure there is no inhibition of the reaction. The Australian Animal Health Laboratory (Geelong, Victoria, Australia) will provide us with the international quantification standards. Any sample testing positive at fewer than all three wells will be rerun. A second test resulting in any positive wells will confirm a positive result, while a second test with no positive wells will be regarded as a negative result. A negative control with all standard reagents will be included in each extraction to confirm that there is no contamination. The pond’s water temperature will be noted during each visit using a digital multimeter. The average monthly air temperature and rainfall measurements will be collected from the National Oceanic and Atmospheric Administration (NOAA) and the National Water and Climate Center (NWCC) SNOTEL sites. Before sampling the next pond, we will disinfect our boots and gear with TriGene Virucidal Disinfectant Cleaner, known to kill B. dendrobatidis zoospores (Webb et al., 2007) to prevent transmission of disease and other organisms. DATA ANALYSIS Established quantitative (real-time) polymerase chain reaction (qPCR) techniques will assess whether B. dendrobatidis is present and the number of B. dendrobatidis zoospore equivalent present on infected samples. We will assign a positive infection status to any frog on whose swab has at least one B. dendrobatidis zoospore detected. The mean number of zoospores detected on all frogs at a given site will represent the intensity of B. dendrobatidis infection at that site during that sampling season. Disease prevalence during each sampling period will be calculated by dividing the number of positive frogs by the total number of frogs sampled. Regression will determine the relationships between the following independent variables: air temperature, water temperature, rainfall, and elevation, and the intensity of infection. We will use ANOVA to compare B. dendrobatidis infection frequencies among the season of sample collection. RELEVANCE OF RESEARCH Temperatures fluctuate dramatically across seasons and elevations in Idaho; therefore, temperature restrictions on B. dendrobatidis could have important implications for the effect of chytridiomycosis on wild anuran populations (Kriger and Hero, 2006). Also, it is important to understand the factors (potentially climate and elevation) that may limit the distribution and abundance of B. dendrobatidis. Further understanding of the causes and ecology of this infectious disease is critical to understand the threats to the Columbia Spotted Frog and other anurans. The Columbia Spotted Frog will make a good model species because it covers a large land area and variety of elevations (Funk et al., 2005; Pilliod, 2002; US Forest Service, 2008). Because the Columbia Spotted Frog migrates over long distances (Funk et al., 2005), the potential for disease transmission is heightened. The ponds in northern Idaho could provide an excellent small-scale example of the pathogenic relationship between the chytrid fungus and anurans. We hope that the relevance of climatic variables on infection can be used to infer large scale implications of climate and elevation on B. dendrobatidis infection. If there are seasonal fluctuations in B. dendrobatidis, this could influence future chytrid research and conservation programs. Protocols for chytrid sampling could be designed to reduce the number of samples required (and hence, costs) by ensuring sampling is done in the seasons which the infection rate is at its maximum. For example, if future sampling were to occur during a season which the infection is low, this could lead to false conclusions that chytrid is not present at the sampling site (Kriger and Hero, 2006). TIME TABLE End of March – Mid May 2009 August 2009 Mid October 2009 October– December 2009 January – February 2009 February – March 2009 March – April 2010 April 2010 May 2010 1st Field Data Collection & DNA Extraction 2nd Field Data Collection & DNA Extraction 3rd Field Data Collection & DNA Extraction DNA extraction; Conduct PCR Compose draft of results Finalize and complete report Complete poster Present project Submit manuscript for publication (Due to seasonality of this project, the timeline slightly exceeds one year.) SUPPORT & FEASIBILITY This project is a collaborative effort among Dr. Lisette Waits (Fish and Wildlife), Dr. Erica Bree Rosenblum (Biology), Doctoral Candidate Caren Goldberg (Fish and Wildlife), and undergraduate Danelle Russell. We are all associated with the University of Idaho, Moscow, ID. We have obtained a wildlife collection permit administered by the State of Idaho Department of Fish and Game to genetically sample the frogs (expires 12/31/09). The University of Idaho currently has labs dedicated to chytrid fungus research. The lab has agreed to allow me access and use of the digital multimeter, DNA extraction kits, -80C freezer, and qPCR assay and primers. Caren Goldberg will train me in the lab and ensure I carry out proper procedures and have good lab technique. This research project could prove beneficial for further chytrid studies conducted at the University of Idaho. In the future, Dr. Erica Bree Rosenblum would like to culture B. dendrobatidis that we collect for laboratory experiments. Furthermore, genetic markers can be used to assess levels of genetic variation of the fungus in the region. BUDGET DNA Extraction qPCR reagents and supplies Travel to Sites (3 – 6 times) Total Cost per Sample $4 $6 Number of Samples 320 320 Total Cost $1280 $1920 --- --- $350 $10 320 $3550 LITERATURE REFERENCES Berger, L., Speare, R., Hines, H., Marantelli, G., Hyatt, A., McDonald, K., Skerratt, L., Olson, V., Clarke, J.M., Gillespie, G., Mahony, M., Sheppard, N., Williams, C., & Tyler, M.J. (2004). Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Australian Veterinary Journal Volume 82, No 7, July 2004. Boyle, D., Boyle, D., Olsen, V., Morgan, J., & Hyatt, A. (2004). Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using realtime Taqman PCR assay. Diseases of Aquatic Organisms 60:133-139. Cockran, C. & Thoms, C. (1996). Amphibians of Oregon, Washington and British Columbia. Lone Pine Publishing, Renton, WA. ISBN 1-55105-073-0. Davis, A., & Verrell, P. (2005). Demography and reproductive ecology of the Columbia spotted frog (Rana luteiventris) across the Palouse. Can. J. Zool. 83: 702–711. doi: 10.1139/Z05061 Funk, C., Blouin, M., Corn, P., Maxell, B., Pilliod, D., Amish, S., & Allendorf, F. (2005). Population structure of Columbia spotted frogs (Rana luteiventris) is strongly affected by the landscape. Molecular Ecology (2005), 483–496. doi: 10.1111/j.1365294X.2005.02426.x Garner, T., Perkins, M., Govindarajulu, P., Siglie, D., Walker, S., Cunningham, A., Fisher, M. (2006). The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana. Biol. Lett. (2006) 2, 455–459. doi:10.1098/rsbl.2006.0494 Goldberg, C., Kaplan, M., & Schwalbe, C. (2003). From the Frog’s Mouth: Buccal Swabs for Collection of DNA from Amphibians. Herpetological Review, 2003, 34(x), xx–xx. Hyatt, A., Boyle, D., Olsen, V., Boyle, D., Berger, L., Obendorf, D., Dalton, A., Kriger, K., Hero, M., Hines, H., Phillott, R., Campbell, R., Marantelli, G., Gleason, F., & Colling, A. (2007). Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175-192. Kriger, K. & Hero, JM. Survivorship in wild frogs infected with chytridiomycosis. EcoHealth 3:171-177. Kriger, K. & Hero, JM. (2008). Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs. Austral Ecology (2008) 33, 1022–1032. Lamirande E. & Nichols D. (2002). Effects of host age on susceptibility to cutaneous chytridiomycosis in blue-and-yellow poison dart frogs (Dendrobates tinctorius). Proceedings of the Sixth International Symposium on the Pathology of Reptiles and Amphibians. Saint Paul, Minnesota, USA. 2002;3-13. Lara J. Rachowicz, Roland A. Knapp, Jess A. T. Morgan, Mary J. Stice, Vance T. Vredenburg, John M. Parker, Cheryl J. Briggs (2006) EMERGING INFECTIOUS DISEASE AS A PROXIMATE CAUSE OF AMPHIBIAN MASS MORTALITY. Ecology: Vol. 87, No. 7, pp. 1671-1683. doi: 10.1890/0012-9658(2006)87[1671:EIDAAP]2.0.CO;2 Lips, K., Brem, F., Brenes, R., Reeve, J., Alford, R., Boyles, J., Carey, C., Livo, L., Pessier, A., & Collins, J. (2006). Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. PNAS vol. 103 no. 9, 3169. Longcore, J.R., Longcore, J.E., Pessier, A., & Halteman, W. (2006). Chytridiomycosis. Widespread in Anurans of Northeastern United States. The Journal of Wildlife Management, 435-444. DOI: 10.2193/2006-345 Mazzoni, R., Cunningham, A., Daszak, P., Apolo, A., Perdomo, E., & Speranza, G. (2003). Emerging Pathogen of Wild Amphibians in Frogs (Rana catesbeiana) Farmed for International Trade. Emerging Infectious Diseases. Vol. 9, No. 8, August 2003. Morehouse, E., James, T., Ganley, A., Vilgalys, R., Berger, L., Murphy, P., & Longcore, J.E. (2003). Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone. Molecular Ecology, 12, 395-403. Morgan, J., Vrendenburg, V., Rachowicz, L., Knapp, R., Stice, M., Tunstall, T., Bingham, R., Parker, J., Longcore, J.E., Moritz, C., Briggs, C., & Taylor, J. (2007). Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis. PNAS vol. 104, no. 34, 13845–13850. Doi: pnas.0701838104 Nevada Fish & Wildlife Office. Columbia Spotted Frog (Rana luteiventris). Retrieved from http://www.fws.gov/nevada/protected_species/amphibians/species/col_spotted_frog.html NSW National Parks and Wildilfe Service (2000). Hygiene protocol for the control of disease in frogs. Threatened Species Management. Information Circular No. 6. pp. 18. Oevermann, A., & Robert, N. (2004). Chytridiomycosis – an emerging and fatal infectious skin disease of amphibians. Veterinary Dermatology 2004, 15 (Suppl. 1), 70– 71. Ouellet, M., Mikaelian, I., Pauli, B., Rodrigues, J., & Green, D. (2005). Historical Evidence of Widespread Chytrid Infection in North American Amphibian Populations. Conservation Biology 1431–1440. Padgett-Flohr, G. (2008). PATHOGENICITY OF BATRACHOCHYTRIUM DENDROBATIDIS IN TWO THREATENED CALIFORNIA AMPHIBIANS: RANA DRAYTONII AND AMBYSTOMA CALIFORNIENSE. Herpetological Conservation and Biology 3(2):182191. Pearl, C., Bull, E., Green, D., Bowerman, J., Adams, M., Hyatt, A., & Wente, W. (2007). Occurrence of the Amphibian Pathogen Batrachochytrium dendrobatidis in the Pacific Northwest. Journal of Herpetology, Vol. 41, No. 1, pp. 145–149, 2007. Picco, A., & Collins, J. (2008). Amphibian Commerce as a Likely Source of Pathogen Pollution. Conservation Biology, Volume 22, No. 6, 1582–1589. Pilliod, D., Peterson, C., & Ritson, P. (2002). Seasonal migration of Columbia spotted frogs (Rana luteiventris) among complementary resources in a high mountain basin. Can. J. Zool. 80: 1849–1862 (2002). doi: 10.1139/Z02-175 Puschendorf, R., Bolanos, F., & Chaves, G. (2006). The amphibian chytrid fungus along an altitudinal transect before the first reported declines in Costa Rica. Biological Conservation 132(1):136-142. Rachowicz, L., Hero, J., Alford, R., Taylor, J., Morgan, J., Vredenburg, V., Collins, J., & Briggs, C. (2005). The Novel and Endemic Pathogen Hypotheses: Competing Explanations for the Origin of Emerging Infectious Diseases of Wildlife. Conservation Biology 1441– 1448. doi: 10.1111/j.1523-1739.2005.00255.x Retallick, R., McCallum, H., & Speare, R. (2004). Endemic Infection of the Amphibian Chytrid Fungus in a Frog Community Post-Decline. PLoS Biol 2(11): e351. United States Forest Service (2008). RANA LUTEIVENTRIS (Columbia Spotted Frog). Retrieved January 15, 2009 from http://www.fs.fed.us/r4/amphibians/columbiaspottedfrog.htm Webb R, Mendez D, Berger L, Speare R. Additional disinfectants effective against the amphibian chytrid fungus Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 74:13-16. Danelle Russell 500 Queen Road Apt #17 Moscow, ID 83843 Phone: (507) 313-9897| Email: [email protected] _______________________________________________________________________________ Education University of Idaho - Moscow, ID 2008-present B.S. Ecology & Conservation Biology 3.9 GPA Highlight Courses Include: Watershed Science & Management, Riparian Ecology & Management, Population Dynamics, Conservation Genetics, Wildland Restoration, Statistical Methods, Calculus Vermilion Community College- Ely, MN 2006-2007 A.S. degree - Wilderness Management 4.0 GPA Highlight Courses Include: Forest Field Skills (ArcGIS, GPS, navigation by maps & compass, topographic maps, area mapping, air photo interpretation), Technical Report Writing Experience Palouse Clearwater Environmental Institute, Moscow, ID Watershed Restoration, Nursery Care, and Environmental Education Volunteer, August 2008-present Assist with stream restoration by planting vegetation in riparian zones Propagate and care for native plants for future restoration sites Assist & train volunteers in local environmental educational programs & restoration projects State of Idaho – Dept. of Environmental Quality, Lewiston, ID Water Quality Technician, June 2008-August 2008 Data collection / stream surveys o Bank erosion, macroinvertebrate collection, periphyton collection, electrofishing, discharge, fish habitat assessment, amphibian count & identification, GPS use, topographic maps, driving 4X4 truck, trailer towing, ATV use, extensive hiking in mountainous terrain with monitoring equipment, wading in all stream types, 4-5 nights of tent & backcountry camping per week, data entry in Excel. Community Memorial Hospital, Winona, MN Admitting & Registration Coordinator, January 2005 – March 2007 Collect patient demographic data & data entry o Requires effective, tactful communication & highest confidentiality. Use of Microsoft Word, Excel, & Cerner system. Superior National Forest - Boundary Waters Canoe Area Wilderness, Ely, MN Trail Crew Volunteer, April 2008 Clear brush, fallen trees, & saplings from trails & water drainage systems Built a puncheon Certifications • • • • • CPR & AED (American Heart Association) First Aid & Safety (American Red Cross) Advanced Hunter Education (Minnesota Department of Natural Resources) Bucking Certification (U.S. Forest Service) Forest Protection Officer (U.S. Forest Service)