Download The Prevalence and Intensity of Chytridiomycosis on Rana

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 28C (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 29C (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 13C 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 28C. October has an average air temperature of 15.5C, 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 -80C 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, -80C
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
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(2007). Diagnostic assays and sampling protocols for the detection of Batrachochytrium
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Lamirande E. & Nichols D. (2002). Effects of host age on susceptibility to cutaneous
chytridiomycosis in blue-and-yellow poison dart frogs (Dendrobates tinctorius).
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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
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& Collins, J. (2006). Emerging infectious disease and the loss of biodiversity in a
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Emerging Pathogen of Wild Amphibians in Frogs (Rana catesbeiana) Farmed for
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(2003). Multilocus sequence typing suggests the chytrid pathogen of amphibians is a
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IN TWO THREATENED CALIFORNIA AMPHIBIANS: RANA DRAYTONII AND
AMBYSTOMA CALIFORNIENSE. Herpetological Conservation and Biology 3(2):182191.
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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.
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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
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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
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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)