Chytrid Fungus in the Rocky Mountains: Establishing Distribution & Evaluating Threat to Boreal Toads

Principal Investigators:
Dr. Erin Muths, PhD, USGS-Biological Resources Division
Dr. David S. Pilliod, PhD, USDA Forest Service Aldo Leopold Wilderness Research Institute

Funding Source: This project was funded through the Amphibian Research and Monitoring Initiative's 2003 research proposal competition.

Collaborators:
Dr. E. Little, USGS-CESC, Columbia, MO
Dr. C.R. Peterson, Idaho State Univ., Pocatello, ID
D. Patla, Idaho State University, Pocatello, ID
Dr. P.S. Corn, USGS-NRMSC, Missoula, MT
D. H. Campbell, USGS-WRD, Denver, CO
L. Garner, FWS, Bison Range Complex, Moise, MT
R. Scherer, USGS / CSU, Fort Collins, CO
B. Lambert, CNHP / CDOW, Fort Collins, CO

Research Objective:
A complete understanding of disease and its role in amphibian declines is lacking (Daszak et al. 1999). In the western U.S., chytrid fungus (Batrachochytrium dendrobatidis) has features of an introduced, lethal infectious disease to which amphibian populations have no resistance and which has been associated with population declines in several species (Green et al. 2002). Information on the distribution of chytrid, the susceptibility of populations to chytridiomycosis, and the role of chytrid in amphibian population declines is critically needed. The proposed research will provide some of this information by addressing 2 objectives:

We can immediately incorporate existing capture - recapture data from the Colorado site, and data collected this year on chytrid and contaminants to build models. We will collect capture - recapture data from the other 2 populations in MT and WY, and as they become available, incorporate these data into the models, which are then refined each year. After 2 years, all 3 of these apex sites will contribute to the models that will provide a regional framework for testing hypotheses regarding why some populations are susceptible to disease outbreaks, while other populations are not.

Objective 1.
Photograph of a frog being measured.

The boreal toad was once widely distributed and locally abundant across the Rocky Mountains, but has disappeared from many historic localities from Montana to Colorado over the last 20-30 years (Corn 2000). Boreal toads appear to have suffered increasingly widespread declines along a gradient in the Rocky Mountains from north to south, a pattern possibly associated with the distribution of chytrid fungus. For example, the boreal toad has experienced severe declines throughout its range in Colorado and some of these declines have been linked to chytrid infection (Muths et al., in press). Chytrid fungus has been found on dead boreal toads collected from the National Elk Refuge, Wyoming, suggesting that chytrid is present at least as far north as the Greater Yellowstone Ecosystem. Disease screening of 15 boreal toads in Montana by the National Wildlife Health Center did not detect chytrid, and to date, no chytrid has been documented in Montana.

Establishing the distribution of chytrid in the Rocky Mountains will provide important information for determining which amphibian populations are at risk and making conservation decisions about which habitats and or populations are the most important to protect and monitor. Recent wetland surveys have documented the distribution of chytrid in Colorado (L. Livo, unpubl. data), but similar information for the northern Rocky Mountains (Wyoming, Montana) is lacking. This study will provide this information.

Objective 2.
Understanding the relationship between population-level amphibian declines, potential immunosuppression related to contaminant levels, and emerging infectious diseases is an important, but complicated, aspect of amphibian decline research (Gilbertson et al. 2003). To begin to understand the causative relationship between immunosuppression, contaminants, disease, and amphibian decline, we propose to study 3 robust populations of boreal toads across 3 regions: Colorado, where chytrid is present and boreal toad populations are declining regionally; Wyoming, where chytrid is present and boreal toad populations appear to be stable regionally; and Montana, where chytrid has yet to be found and boreal toad populations appear to be stable regionally. By conducting research on the status of and threats to robust populations of boreal toads, the proposed study provides a unique opportunity to examine the causative factors responsible for population-level declines before a species is at the brink of extinction throughout the entire Rocky Mountain region and is too rare to study.

The proposed multi-disciplinary approach addresses ecosystem biocomplexity in relation to amphibian decline. Research on this escalating problem has yielded no easy answers, and questions about potential contributions of sub-lethal stressors, such as contaminants, have not been addressed vigorously in the context of amphibian disease. We integrate biology, environmental toxicology, and hydrology using expertise from USGS-WRD and BRD as well as additional outside expertise on Rocky Mountain amphibians. This integrative approach provides a template for determining potential causative relationships between various stressors and amphibian declines for use in other regional amphibian projects, including ongoing monitoring efforts of ARMI.

Objective 1.To document the distribution of chytrid in boreal toad populations in the northern Rocky Mountains, we will sample current and historic boreal toad breeding locations in western Wyoming up to the Greater Yellowstone Ecosystem (GYE) and in western Montana from the GYE to Glacier National Park. Data are available for Colorado (L. Livo, pers. com.).

Objective 2. To study causal interactions of disease and contaminants on amphibian declines, we will establish 3 apex sites with robust populations of boreal toads in CO, WY, and MT (Table 1).

Objective 1.In western Wyoming and western Montana, 30 sites per state will be selected randomly from all known and historic boreal toad breeding sites using regional amphibian databases and consulting with regional experts and sources. For example, ARMI surveys of 2000-2002 in the GYE have located at least 25 boreal toad breeding sites (Peterson and Patla, pers. com.) and other breeding sites have been located south of the GYE (Muths, unpubl. data). In western Montana, surveys since 1997 have located approx. 100 breeding sites (Maxell 2003). After sites have been selected, field technicians will survey ponds for amphibians during the breeding season when toads are likely to be found at communal breeding sites, record habitat measurements and site position (GPS), and collect ventral skin scrapings from 10 boreal toads or whatever amphibians are available at each site. This method of chytrid screening has proven as effective as collecting an entire animal (L. Livo and C. Carey, pers. comm.). Collection will be conducted with extreme care to avoid potential contamination of the tissue. We will adhere scrupulously to sterile procedure in the field including the use of sterilized containers, single-use gloves and swabs and flame- sterilization of scissors between collections. Samples will be numbered and sent to Pisces Molecular (Boulder, CO) to be assessed for chytrid infection using Polymerase Chain Reaction (PCR) techniques (Hyatt 2002). Samples will be sent for testing in one batch. Newly collected results (Montana and Wyoming) will be integrated with existing information (Colorado) about incidence of chytrid in the Rocky Mountains and mapped.

Objective 2. To begin to understand the relationship between population-level amphibian declines, potential immunosuppression related to the presence of contaminants, and emerging infectious diseases, we will incorporate capture-recapture data and data on the presence of chytrid and contaminants in an information-theoretic approach (Burnham and Anderson 1998). Specific a priori hypotheses regarding the interactions that we suspect may be causal to the observed declines will be developed and tested using this approach and Program MARK (Burnham and White 1999) as an analysis tool.

Three apex sites will be established in each region (Table 1) for intensive sampling. These sites were chosen because of the size of the populations (relatively large), existing information, and regional interest and support. At Denny Creek, CO, there are 2 years of capture-recapture data, water quality information and a history of support from the Colorado Division of Wildlife and the Colorado Natural Heritage Program (Brad Lambert, CNHP, pers. com.). At the Blackrock, WY site, boreal toad life stage counts have been conducted every year since 1997 (Patla, pers. com.). At Lost Trail, NWR, MT, we have collected 2 years of breeding surveys and have logistical support from Refuge staff.

Capture-recapture protocols will continue at the Denny Creek site using existing methods that were developed with direction from USGS-FORT. This includes 4-6 capture sessions using PIT tags to identify individual toads, and standard morphometric measurements (Muths et al. in press). These same methods will be initiated at the other 2 sites and data used in the modeling process as they become available.

We will assess the presence of chytrid fungus using PCR techniques as above. Each site (Table 1) will be assessed immediately following the breeding season for the presence of contaminants (e.g. arsenic, selenium, cadmium, lead, copper and zinc) and organochlorine and organophosphate insecticides (e.g. DDT, malathion). We will collect duplicate samples of pond sediment, soil 30 m from the pond, and water to determine EPA Priority Metals concentrations using standard USGS techniques (Ward and Harr 1990). This approach will allow us to determine where exposure might occur. For example, contaminant levels in water and pond bottoms (where tadpoles feed) may be different relative to soil in rodent burrows (where adults hibernate). A general screening will be used to assess water samples for insecticides. Contaminant assessment will take place at Mississippi State University. Water quality assessment (Major Ion Chemistry) will take place at the USGS Water Quality Laboratory in Denver, CO. Spectrophotometric absorbance of water samples, to provide an indication of light penetration including U-V light, will take place at Columbia Science Center, MO.

This project establishes 3 long-term studies embedded in a regional map of information about the incidence of chytrid fungus. The protocols at the 3 targeted apex sites are designed to monitor the status of and threats to these populations this year and into the future. Our aim is to provide ARMI with the first year of data and a foundation for long-term monitoring research at apex sites that will capture information at a regional level along the Great Divide Transect from Montana to Colorado. At 2 of 3 sites, there is funding to be leveraged and significant interest from cooperators.

Burnham, K.P. and D.R. Anderson. 1998. Model Selection and Inference: A Practical Information-theoretic Approach. Springer, New York, New York.
Corn, P.S. 2000. Amphibian Declines: Review of some current hypotheses. IN: Ecotoxicology of
Amphibians and Reptiles. D. W. Sparling, G. Linder and C.A. Bishop (eds)., SETAC Press,
Pensacola FL. Pp 663-696.

Daszak, P., L. Berger, A.A. Cunningham, A.D. Hyatt, D.E. Green and R. Speare. 1999. Emerging
infectious diseases and amphibian population declines. Emerging Infectious Diseases 5: 735-748.

Gilbertson, M., G.D. Haffner, K.G. Drouillard, A. Albert, and B. Dixon. Immunosuppression in the northern leopard frog (Rana pipiens) induced by pesticide exposure. Env. Toxicol. and Chem. 22:101-110.

Green, D.E., K.A. Converse, and A.K. Schrader. 2002. Epizootiology of sixty-four amphibian morbidity and mortality events in the USA, 1996-2001. Ann. N.Y. Acad. Sci. 969:323-339.

Koch, E.D. and C.R. Peterson. 1995. Amphibians and reptiles of Yellowstone and Grand Teton National Parks. University of Utah Press, Salt Lake City, UT.

Maxell, B.A., J.K. Werner, P. Hendricks, and D.L. Flath. 2003. Herpetology in Montana: A History, Status Summary, Checklists, Dichotomous Keys, Accounts for Native, Potentially Native, and Exotic Species, and Indexed Bibliography. Northwest Fauna No. 5. Society for Northwestern Vertebrate Biology, Olympia,WA.

Muths, E., P.S. Corn, A.P. Pessier, D.E. Green. Evidence for disease related amphibian decline in
Colorado. In press. Biological Conservation.

Ward, J.R. and Harr, C.A. eds. 1990. Methods for the collection and processing of surface-water and bed materialsamples for physical and chemical analyses: U.S. Geological Survey Open File Report 90-140, 71 p.

White, G.C., and K.P. Burnham. 1999. Program MARK: survival estimation from populations of marked animals. Bird Study 46: Supplement: 120-130.

Update:
This year we visited our three apex sites twice. Two of the sites already have capture - recapture protocols in place, at the third site, in Wyoming, we initiated capture - recapture efforts. We collected non-invasive skin scrapings to test for chytrid fungus infection in animals at each of the sites (n=20 samples per site). One site tested positive for chytrid fungus (WY) and appears to have a high incidence of predation on the toad population, possibly from otters; one site (CO) yielded equivocal results in the chytrid tests pending confirmation of results in 2004; and at the remaining site (MT) adequate samples were not collected to test for fungal infection. We also refined our methods and collected soil and water samples from our apex sites. We are in the process of choosing sites along the Great Divide Transect from Montana through Wyoming and Colorado. These sites will be chosen based on current and historic presence of boreal toads. Each site will be visited once during the 2004 field season and sampled (boreal toads and / or surrogate species) for the presence of chytrid fungus. We will continue our capture - recapture work at our three apex sites in 2004.

Chytrid Fungus in the Rocky Mountains: Establishing Distribution & Evaluating Threat to Boreal Toads

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