Effects of a Pathogen and Pesticides on Gray Treefrog (Hyla chrysoscelis)
Metamorphosis and Survival
A thesis submitted to the Miami University
Honors Program in partial fulfillment of the
requirements for University Honors with Distinction
by
Kristina M. Gaietto
April 2012
Oxford, OH
ii
Abstract
Both the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) and
pesticides have been targeted as contributors to amphibian population declines.
Declines have a worldwide distribution, but are only occurring in certain areas.
Frequently, chytridiomycosis due to the Bd is treated as a uniform disease; however,
recent evidence suggests variation among different isolates of Bd. Both Bd and
pesticide effects on amphibians have been tested extensively individually, but few
studies have attempted to test the interactive effects of these variables. In a lab
study, we tested the effects of six different isolates of Bd, three from areas where
amphibian population decline is occurring and three from areas where it is not, and
two pesticides, the insecticide carbaryl and fungicide copper sulfate, on
metamorphic responses and survival in Cope’s gray treefrog (Hyla chrysoscelis). We
expected to see differing degrees of negative effects from the different types of Bd
isolates. We expected to see negative effects from the carbaryl both alone and with
Bd, but expected to see negative effects from copper sulfate only without Bd. Our
results did not show any individual effects from Bd on metamorphic response or
survival. We did find that carbaryl significantly increased mass at metamorphosis,
time to metamorphosis, and time to tail absorption but did not significantly affect
survival. Additionally, we found a significant interaction between pesticide and Bd
on mass at metamorphosis. Our study does not indicate that environmental
contaminants would make tadpoles more susceptible to Bd.
iii
iv
v
vi
Acknowledgements: Thanks to Dr. Michelle D. Boone, my adviser, and Dr. Maria
Gonzalez and Dr. Nicholas Money, my thesis readers, for guidance and insights.
Thanks to Dr. Joyce Longcore and Dr. Matthew Parris for supplying chytrid fungal
cultures and to Melissa Youngquist, Samantha Rumschlag, Bradley Skelton, and
Claire Meikle for assistance collecting and caring for experimental animals. This
research was funded by the Howard Hughes Medical Institute and the Miami
University Honors Program.
vii
Table of Contents
Introduction:
1
Methods:
5
Results:
9
Discussion:
10
References:
14
Appendix:
20
viii
List of Figures
Figure 1. Cope’s gray treefrog time to metamorphosis at different pesticide
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
Figure 2. Cope’s gray treefrog mass at metamorphosis at different pesticide
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
Figure 3. Cope’s gray treefrog time to tail absorption at different temperature
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
Figure 4. Mass at metamorphosis for Cope’s gray treefrogs raised in the presence of
carbaryl (unfilled circle), presence of copper sulfate (triangle), or absence of
pesticide (black circle) with the presence or absence of Bd isolates. Error bars
represent ± 1 standard error.
ix
List of Tables
Table 1. Summary of results of ANOVA for metamorphic, survival, and activity
responses and results of MANOVA for metamorphic responses in Cope’s gray
treefrog
x
1
Introduction
Amphibians are indicators of ecosystem health and water quality, because
they are sensitive to environmental stressors and need both aquatic and terrestrial
habitat to complete their life cycle. We have reason to question ecosystem health
and water quality: amphibian populations around the world are facing
unprecedented declines (Houlahan et al. 2000). Some suggest that we are living
during a time of amphibian extinction (Wake and Vredenburg 2008). Recent studies
have proposed many reasons for these declines including habitat destruction and
alterations (Becker et al. 2007), UV radiation (Blaustein, et al. 1998), invasive
species (Smith et al. 2007), contaminants (Sparling et al. 2001), and disease (Berger
et al. 1998).
Pesticides, which are a ubiquitous feature of American agriculture, have been
implicated in amphibian decline. Studies have demonstrated how pesticide
exposure can negatively affect tadpole development by increasing the amount of
time it takes to reach metamorphosis and decreasing the mass at metamorphosis
(Hayes et al. 2006). Declines have been predicted across regions (Lips et al. 2008),
but amphibian population declines have been found to occur more commonly with
upwind agriculture (Davidson et al. 2001; Davidson et al. 2002), strongly linking
pesticides to amphibian declines.
The two pesticides used in the present study, the insecticide carbaryl and the
fungicide copper sulfate, are commonly used in the US (Grube et al. 2011) and can
2
serve as models for understanding the ecological ramification of pesticide exposure
in the presence of other factors. Carbaryl has specifically been implicated in altering
amphibian survival and metamorphosis (Boone and Semlitsch 2001). Though it
quickly breaks down in the environment, it causes significant decreases in survival
in many different amphibian species (Relyea and Edwards 2010). Furthermore,
sensitivity to carbaryl varies among amphibian species (Bridges and Semlitsch
2000). Studies have likewise shown that copper sulfate negatively influences
tadpole development (Garcia- Muñoz et al. 2009). Allowable levels of copper in the
environment are often determined by studies on fish tolerance to copper; however,
certain species of amphibians have been found to be more sensitive to copper than
fish, suggesting that copper is present in dangerous levels to amphibians in the
environment (Bridges et al. 2002).
While habitat destruction is the leading cause of general amphibian
population losses, currently, the proposed leading cause of enigmatic amphibian
population declines is thought to be a disease called chytridiomycosis, making it a
key area of research in amphibian conservation. Chytridiomycosis is caused by a
fungal pathogen called Batrachochytrium dendrobatidis, or Bd (Longcore et al.
1999). In amphibians with chytridiomycosis, the Bd grows on keratinized cells,
found in the skin, reducing the permeability of the skin to water and important ions
(Voyles et al. 2009). This hinders the health of the amphibian and can cause death.
This pathogen appears to have a worldwide distribution (Rachowicz et al. 2005). Yet
3
inexplicably, certain geographic regions in which Bd is present are not experiencing
amphibian population decline, while other areas with Bd are experiencing rapid
amphibian population losses (Lotters et al. 2009). This suggests there could be
strain differences or differences in species sensitivity. Indeed, some studies have
demonstrated that different isolates of Bd have different effects on a single species
of amphibians (Berger et al. 2005; Retallick and Miera 2007).
It is important to look at the effects of multiple stressors simultaneously
because of the multitude of variables to which amphibians are exposed on a daily
basis; single stressor studies may not accurately reflect exposure scenarios in
nature (Boone and Semlitsch 2001; Boone et al. 2007). Different conditions could
influence the effects of Bd on amphibians. For instance, pesticides could influence
disease susceptibility. Pesticides can repress immune systems and result in greater
infection rates (Christin et al. 2004). Pesticides have been shown to negatively
impact Bd viability independent of a host (Hanlon and Parris 2012), which could
lead to different effects to amphibians exposed to both stressors than only pesticide
or Bd. Though Bd and pesticides are found together in certain habitats, to date there
have been surprisingly few published studies testing the interactive effects of Bd
and pesticides on amphibians (Davidson et al. 2007; Gahl et al. 2011; Kleinhenz et al.
2011; Buck et al. 2012).
The objective of this study is to test the single and interactive effects of
pesticide and Bd isolates on Cope’s gray treefrog (Hyla chyrsoscelis) metamorphosis.
4
While gray treefrogs are not undergoing amphibian population declines due to Bd, if
key combination of stressors make the species more susceptible, then species
currently not experiencing declines could be at risk. We evaluated the impact of two
different types of pesticides, the insecticide carbaryl and the fungicide copper
sulfate, and six different isolates of Bd, three from areas of decline and three from
areas of non-decline, using time to metamorphosis, mass at metamorphosis, time to
tail absorption, activity, and survival as the response parameters. We predicted that
the Bd isolates from decline areas would have more negative effects on gray treefrog
development than the Bd isolates from non-decline areas. In addition, we expected
that both the insecticide and fungicide would negatively impact gray treefrog
metamorphosis. When the insecticide and Bd were combined, we predicted that the
insecticide would increase the negative effects of Bd, because the tadpole must
combat two stressors. However, when the fungicide and Bd were combined, we
predicted that the fungicide would mitigate the negative effects of the Bd. A
fungicide could have negatively impacted amphibian development, but it could have
also negatively impacted Bd itself, reducing or eliminating potential for Bd to infect
an amphibian.
5
Methods
We collected 14 gray treefrog (Hyla chrysoscelis) partial egg masses from a
flooded parking lot and roadside ditch at Hueston Woods State Park in College
Corner (Butler and Preble Counties), Ohio on 27-28 June 2011. Egg masses were
held in the laboratory at 20 C on a 12:12 light dark cycle in water from the collection
site until hatching. Egg masses were combined to homogenize genetic variation
among treatments. Water was changed daily, and tadpoles were fed TetraMin
Tropical Fish Flakes ad libitum.
We exposed individual tadpoles to the presence of a strain of the amphibian
chytrid fungus (Batrachochytrium dendrobatidis, Bd) from an area where amphibian
declines have (Point Reyes, CA [JEL 646], El Cope, Panama [JEL 423], and the Sierra
Nevadas [JEL 213]) or have not (Ohio [JEL 660], Tennessee [FMB 003], and Maine
[JEL 404]) been attributed to Bd or a tryptone sham and the presence or absence of
a pesticide (no pesticide, 0.0125 mg/L of copper sulfate [a fungicide], or 0.5 mg/L of
carbaryl [an insecticide]) with 17 replicates. Plates were obtained from J. Longcore
(JEL) and M. Parris (FMB). Pesticide concentrations were determined using 5% of
the LC50 for each chemical based on previous studies (Relyea, 2003) (GarciaMunoz, Guerrero, & Parra, 2009) so that each treatment would have equivalent toxic
units. Temperature was maintained at 21 C throughout the duration of the study.
To create Bd solutions, we added a 2 mm x 2 mm x 2 mm block of the agar
containing the respective Bd isolate to 75 mL of 1% tryptone broth and left at room
temperature. Once clumps of thalli were visible in the broth, 1 % tryptone agar
6
plates were cultured using 1 mL of the respective broth. We created six plates for
each Bd isolate. Zoospores were harvested by adding 10 mL of distilled water to
each plate. After 30 minutes, water was poured from plates, and plates were rinsed
using small additional amounts of distilled water. The concentration of zoospores
varied by isolate, so solutions were diluted as necessary to create stocks of ~8.0 X
105 zoospores/mL. For Bd controls, the same protocol was followed with sterile 1%
tryptone agar plates containing no Bd.
Chemical stock solutions of carbaryl were prepared by adding 4.45 g of Sevin
(22.5% carbaryl, GardenTech, city & state info) to 2 L of deionized water.. A copper
sulfate stock solution was prepared by adding 0.994 g of copper sulfate
pentahydrate (purity 98% Sigma-Aldrich) to 2 L of deionized water. 1 mL of stock
solution of the appropriate pesticide was added to 1 L of dechlorinated water to
achieve the exposure concentration, which was 0.5 mg/L for carbaryl and 0.0125
mg/L for copper sulfate. Water samples of each chemical were sent to Mississippi
State University Chemical Laboratory (Mississippi State, MS) for analyses. Analysis
indicated carbaryl concentrations of 0.5 mg/L and copper concentrations of 0.017
mg/L, which suggests tadpoles in the study were exposed to the intended amounts
of each pesticide.
On 5 July 2011 (experimental day 0), tadpoles were placed into individual 1 L
glass beakers containing 100 mL of dechlorinated water. Treatments were
randomly assigned to beakers in the environmental chamber. We added 1 mL of the
assigned Bd stock or control stock to each beaker, thus exposing Bd-treated
7
tadpoles to 8.0 X 103 zoospores/mL. After 24 hours of Bd exposure, we added 900
mL of dechlorinated water and applied pesticide treatments to each beaker.
We performed full water changes for each tadpole every three days. Bdcontrol beakers were changed before Bd-exposed beakers to reduce the likelihood
of accidental exposure. Although we did not reapply zoospores after initial
exposure, different water change nets and containers were used for each Bd
treatment to eliminate the risk of cross-contamination. After each water change,
pesticide treatments were reapplied and tadpoles were fed ad libitum a 1:1 mixture
of TetraMin Tropical Fish Flakes and Serra Micron.
Tadpole activity was measured twice during the experiment, at day 10 and
day 23. To measure activity, a single person observed each beaker for
approximately 5 seconds and recorded if tadpoles were active. After observing all
the tadpoles, the observer repeated this four more times to collect a total of five
observations for every tadpole per observation day.
We searched daily for metamorphs (emergence of at least one forelimb,
Gosner Stage 42; [Gosner 1960]) and moved these individuals to covered containers
with fresh water until tail resorption at which time metamorph mass was
determined. The water in each metamorph’s container was changed daily. We
recorded time to metamorphosis, time to tail resorption, mass at metamorphosis,
and survival to metamorphosis for each tadpole. Each metamorph was swabbed to
test for Bd. Swabs were rubbed five times on each the dorsal surface, the ventral
surface, the left inner thigh, the right inner thigh, and the mouth, then were placed
8
in Eppendorf tubes and stored at -77 C until PCR testing could be performed. (To
date, PCR testing has not been completed.) Animals were then sacrificed in a 10%
solution of MS-222 and stored in Eppindorff tubes containing 70% ethanol at room
temperature.
To determine the single and interactive effects of Bd and pesticide
treatments on the multivariate “metamorphic response” consisting of time to
metamorphosis, time to tail absorption, and mass at metamorphosis, we used a
multivariate analysis of variance (MANOVA). If multivariate analyses were
significant, we used an analysis of variance (ANOVA) to examine which factors
contributed most to the treatment effect. We used an analysis of variance (ANOVA)
to examine the effects of Bd and pesticide treatments on gray treefrog tadpole
survival to metamorphosis. We used a repeated-measure ANOVA to examine
differences in activity over time by treatments. To examine differences among
treatments, we used Scheffe’s multiple comparison’s test.
9
Results
Survival to metamorphosis was not significantly affected by pesticide, Bd, or
the interaction of these treatments (Table 1). Pesticide treatments significantly
affected the metamorphic response (including mass to metamorphosis, time to
metamorphosis, and time to tail absorption; Table 1). Carbaryl significantly affected
time to metamorphosis, by increasing it by approximately 22% relative to the
control (Fig. 1; Table 1). However, longer larval periods resulted in greater mass at
metamorphosis (Fig. 2). Carbaryl also significantly extended the time to tail
absorption by approximately 7% relative to the control and approximately 10%
relative to the copper sulfate treatment (Fig. 3; Table 1). Carbaryl treatment
increased mass by approximately 24% relative to the control and copper sulfate
treatment. Bd exposure or the interaction of Bd and pesticide exposure did not
affect the metamorphic response. However, mass at metamorphosis was
significantly affected by the interaction of Bd and pesticide exposure (Table 1; Fig.
4). Mass varied among control and copper sulfate treatments based on Bd isolate.
Activity was not affected by Bd, pesticide, or the interaction of these treatments, and
they did not vary over time (P>0.1047).
10
Discussion
Researchers have long suggested that the key to understanding amphibian
population declines was combinations of sublethal stressors (Carey et al. 1999,
Hayes et al. 2010). Pesticides and pathogens both have the potential to interact with
each other and other factors in the environment. However, we found no indication
that presence of Bd of varying strains or pesticides in combination resulted in
synergistic negative interactions as we expected.
Some studies have suggested different isolates of Bd may cause different
effects in a single amphibian species. Berger et al. (2005) found that three isolates of
Bd from Australia caused significant differences in time to death in Australian green
treefrog (Litoria caerulea) juveniles. Retallick and Miera (2007) found that two Bd
isolates from Arizona caused differences in survival in adult western chorus frogs
(Pseudacris triseriata) (Retallick and Miera 2007). However, because we found no
effect of Bd on survival or metamorphosis, we found no differences among isolates
of Bd. Thus, our results contradict other findings of differences in effects between
isolates. We did not find any effect of Bd, which may indicate tadpoles were never
infected or conditions in the water reduced the likelihood of infection. However,
another recent study found that Bd isolates JEL 404 and JEL 423 (both of which
were used in our current study) do not cause different effects in tadpoles of wood
frogs (Lithobates sylvaticus) (Gahl et al. 2011). Further studies should be conducted
to determine if differences in effects from Bd isolates are only apparent in studies
using terrestrial-stage frogs, as these studies potentially suggest.
11
In contrast to the study by Gahl et al. (2011), we found that neither JEL 404
nor JEL 423 had a significant effect on metamorphosis or survival. This may suggest
that the species we used in our study, Cope’s gray treefrog, is not susceptible to this
pathogen. Two other studies tested the effects of Bd on Cope’s gray treefrog survival
and metamorphosis, and found that Bd had no significant effects on survival.
However, they did find that Bd exposure significantly altered mass and larval period
(Parris and Cornelius 2004; Parris and Beaudoin 2004), which offers mixed support
for this hypothesis. Bd has been shown to have different effects on different
amphibian species at the larval stage: survival of Pacific treefrog (Hyla regilla),
Cascades frog (Rana cascadae), and American bullfrog (Rana catesbeiana) were not
affected by Bd exposure, while survival of western toad (Bufo boreas) was
significantly affected (Blaustein et al. 2005). Thus, it is plausible that Hyla
chrysoscelis is less susceptible to Bd than other species. Using a different species for
this study may have yielded completely different results.
Bd may have different effects on larval amphibians due to the limited amount
of keratinized cells present in tadpoles relative to adult amphibians. Bd specifically
affects keratinized cells (Voyles, et al., 2009). Mouth parts of tadpoles were the only
potential sites of infection, due to lack of keratin on the body and tail early in
development (Berger, et al., 1998), which is when the tadpoles in our study were
exposed. Thus, the tadpoles in our study may not have been infected.
Though we saw no significant effects from Bd exposure, we did find that
carbaryl exposure had a significant effect on time and mass at metamorphosis, and
12
tail absorption. Animals treated with carbaryl took longer to metamorphose (Fig. 1)
and were significantly larger at metamorphosis than control or copper sulfatetreated animals (Fig. 3). Other studies have found similar effects of carbaryl
exposure on amphibian time and mass at metamorphosis (Boone and Semlitsch
2001; Buck et al. 2012), although in more natural field conditions. Increased mass at
metamorphosis has been shown to correlate with a size advantage at reproductive
age (Smith 1987) suggesting that larval exposure to carbaryl may actually be
advantageous to gray treefrogs. However, treefrogs specifically may be at
disadvantage for metamorphosing late, because of their tendency to inhabit
ephemeral habitats (Parris and Beaudoin 2004; Parris and Cornelius 2004). Shorter
time to metamorphosis reduces the risk of not metamorphosing before the pond
evaporates. Thus, it is unclear whether the increased mass and time to
metamorphosis as a result of carbaryl exposure is advantageous to gray treefrogs.
There was also an interaction between pesticide and Bd exposure for mass at
metamorphosis (Fig. 4). This is possibly the first study to find an interaction
between pesticide and Bd: the other published studies testing these variables have
found no interactions between Bd and pesticides (Davidson et al. 2007; Gahl et al.
2011; Kleinhenz et al. 2011; Buck et al. 2012). The implications of the interaction we
found are unknown. Though there was a variation in mass based upon the Bd isolate
and chemical, most of the individuals were doing as well or better than the controls.
Thus, the interaction does not seem to have any negative implications.
13
Our study did not find that two environmental pesticides would make
tadpoles more susceptible to Bd when exposure occurred early in larval
development. Our study did find a significant interaction between Bd exposure and
pesticide exposure, but the effects seemed to be positive rather than negative. We
encourage further studies into of multiple stressors to better understand the effects
of pesticides and Bd and their complex interactions.
14
References
Becker, C. G., Fonseca, C. R., Haddad, C. F., Batista, R. F., & Prado, P. I. (2007). Habitat
Split and the Global Decline of Amphibians. Science 318, 1775-1777.
Berger, L., Marantelli, G., Skerratt, L. F., & Speare, R. (2005). Virulence of the
amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain.
Diseases of Aquatic Organisms 68, 47-50.
Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., et al.
(1998). Chytridiomycosis causes amphibian mortality associated with population
declines in the rain forests of Austraila and Central America. Population Biology 95,
9031-9036.
Blaustein, A. R., Kiesecker, J. M., Chivers, D. P., Hokit, D. G., Marco, A., Belden, L. K., et
al. (1998). Effects of Ultraviolet Radiation on Amphibians: Field Experiments.
American Zool. 38, 799-812.
Blaustein, A. R., Romansic, J. M., Scheessele, E. A., Han, B. A., Pessier, A. P., &
Longcore, J. E. (2005). Interspecific Variation in Susceptibility of Frog Tadpoles ot
the Pathogenic Fungus Batrachochytrium dendrobatidis. Conservation Biology 19
(5), 1460-1468.
Boone, M. D. & Semlitsch, R. D. (2001). Interactions of an Insecticide with Larval
Density and Predation in Experimental Amphibian Communities. Conservation
Biology 15 (1), 228-238.
15
Boone, M. D., Semlitsch, R. D., Little, E. E., & Doyle, M. C. (2007) Multiple Stressors in
Amphibian Communities: Effects of Chemical Contamination, Bullfrogs, and Fish.
Ecological Applications 17 (1), 291-301.
Bridges, C. M. & Semlitsch, R. D. (2000). Variation in Pesticide Tolerance of Tadpoles
among and within Species of Ranidae and patterns of Amphibian Decline.
Conservation Biology 14 (5), 1490-1499.
Bridges, C. M., Dwyer, F. J., Hardesty, D. K., & Whites, D. W. (2002). Comparative
Contaminant Toxicity: Are Amphibian Larvae More Sensitive than Fish? Bulletin of
Environmental Contamination and Toxicology 69, 562-569.
Buck, J. C., Scheessele, E. A., Relyea, R. A., & Blaustein, A. R. (2012). The effects of
multiple stressors on wetland communities: pesticides, pathogens and competing
amphibians. Freshwater Biology 57, 61-73.
Carey, C., Cohen, N., & Rollins-Smith, L. (1999) Amphibian declines: an
immunological perspective. Developmental & Comparative Immunology 23 (6), 459472.
Christin, M. S., Menard, L., Gendron, A. D., Ruby, S., Cyr, D., Marcogliese, D. J., et al.
(2004). Effects of agricultural pesticides on the immune system of Xenopus laevis
and Rana pipiens. Aquatic Toxicology 67, 33-43.
Davidson, C., Bernard, M. F., Shaffer, H. B., Parker, J. M., O'Leary, C., Conlon, J. M., et al.
(2007). Effects of Chytrid and Carbaryl Exposure on Survival, Growth and Skin
16
Peptide Defenses in Foothill Yellow-legged Frogs. Environmental Science 41 (5),
1771-1776.
Davidson, C., Shaffer, H. B., & Jennings, M. R. (2001). Declines fo the California RedLegged Frog: Climate, UV-B, Habitat, and Pesticides Hypothesis. Ecological
Applications 11 (2), 464-479.
Davidson, C., Shaffer, H. B., & Jennings, M. R. (2002). Spatial Tests of the Pesticide
Drift, Habitat Destruction, UV-B, and Climate-Change Hypotheses for California
Amphibian Declines. Conservation Biology 16 (6), 1588-1601.
Gahl, M. K., Pauli, B. D., & Houlahan, J. E. (2011). Effects of chytrid fungus and a
glyphosate-based herbicide on survival and growth of wood frogs. Ecological
Applications 21 (7), 2521-2529.
Garcia-Munoz, E., Guerrero, F., & Parra, G. (2009). Effects of Copper Sulfate on
Growth, Development, and Escape Behavior in Epidalea calamita Embryos and
Larvae. Arch Environ Contam Toxicol 56, 557-565.
Gosner, K. L. (1960). A Simplified Table for Staging Anuran Embryos and Larvae
with Notes on Identification. Herpetologica 16 (3), 183-190.
Grube, A., Donaldson, D., Kiely, T., & Wu, L. (2011). Pesticides Industry Sales and
Usage: 2006 and 2007 Market Estimates (p. i-33).
17
Hanlon, S. M., & Parris, M. J. (2012). The Impact of Pesticides on the Pathogen
Batrachochytrium dendrobatidis Independent of Potential Hosts. Archives of
Environmental Contamination and Toxicology 1-7.
Hayes, T. B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., et al. (2006).
Pesticide Mixtures, Endocrine Disruption, and Amphibian Declines: Are We
Underestimating the Impact? Environmental Health Perspectives 114, 40-50.
Hayes, T. B., Falso, P., Gallipeau, S., & Strice, M. (2010) The cause of global amphibian
declines: a developmental endocrinologist’s perspective. Journal of Experimental
Biology 213, 921-933.
Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyers, A. H., & Kuzmin, S. L. (2000).
Quantitative evidence for global amphibian population declines. Nature 404, 752755.
Kleinhenz, P., Boone, M. D., & Feller, G. (2011). Effects of the amphibian chytrid
fungus and four insecticides on Pacific treefrogs (Pseudacris regilla). Journal of
Herpetology (in press).
Lips, K. R., Diffendorfer, J., Mendelson, J. R., & Sears, M. W. (2008). Riding the Wave:
Reconciling the Roles of Disease and Climate Change in Amphibian Declines. PLOS
Biology 6 (3), 441-454.
18
Longcore, J. E., Pessier, A. P., & Nichols, D. K. (1999). Batrachochytrium
Dendrobatidis ge. et sp. nov., a Chytrid Pathogenic to Amphibians. Mycologia 91 (2),
219-227.
Lotters, S., Kielgast, J., Bielby, J., Schmidtlein, S., Bosch, J., Beith, M., et al. (2009). The
Link Between Rapid Enigmatic Amphibian Decline and teh Globally Emerging
Chytrid Fungus. EcoHealth 6, 358-372.
Parris, M. J., & Beaudoin, J. G. (2004). Chytridiomycosis impacts predator-prey
interactions in larval amphibian communities. Oecologia 140, 626-632.
Parris, M. J., & Cornelius, T. O. (2004). Fungal Pathogen Causes Competitive and
Developmental Stress in Larval Amphibian Communities. Ecology 85 (12), 33853395.
Rachowicz, L. J., Hero, J.-M., Alford, R. A., Taylor, J. W., Morgan, J. A., Vredenburg, V. T.,
et al. (2005). The Novel and Endemic Pathogen Hypotheses: Competing
Explanations for the Origin of Emergine Infectious Diseases of Wildlife. Conservation
Biology 19 (5), 1441-1448.
Relyea, R. A. (2003). Predator Cues and Pesticides: A Double Dose of Danger for
Amphibians. Ecological Applications 13 (6), 1515-1521.
Relyea, R. A. & Edwards, K. (2010). What Doesn’t Kill You Makes You Sluggish: How
Sublethal Pesticides Alter Predator-Prey Interactions. Copeia 4, 558-567.
19
Retallick, R. W., & Miera, V. (2007). Strain differences in the amphibian chytrid
Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection.
Diseases of Aquatic Organisms 75, 201-207.
Smith, D. C. (1987). Adult Recruitment in Chorus Frogs: Effects of Size and Date at
Metamorphosis. Ecology 68 (2), 344-350.
Smith, G. R., Boyd, A., Dayer, C. B., & Winter, K. E. (2007). Behavioral responses of
American toad and bullfrog tadpoles to the presence of cues from teh invasive fish,
Gambusia affinis. Biol Invasions 10, 743-748.
Sparling, D. W., Fellers, G. M., & McConnell. (2001). Pesticides and Amphibian
Population Declines in California, USA. Environmental Toxicology and Chemistry 20
(7), 1591-1595.
Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W. F., Dinudom, A., et al. (2009).
Pathogenesis of Chytridiomycosis, a Cause fo Catstrophic Amphibian Declines.
Science 326, 582-585.
Wake, D. B., & Vredenburg, V. T. (2008). Are we in the midst of the sixth mass
extinction? A view from the world of amphibians. PNAS 105, 11466-11473.
20
Appendix
Table 1. Summary of results of ANOVA for metamorphic, survival, and activity
responses and results of MANOVA for metamorphic responses in Cope’s gray
treefrog
Response
Time to
Metamorphosis
Time to Tail
Absorption
Mass at
Metamorphosis
Survival
Source of Variation
df
F
P
Bd
Pesticide
Bd x Pesticide
Error
6
2
12
225
1.07
4.20
0.45
0.3786
<.0001
0.9431
Bd
Pesticide
Bd x Pesticide
Error
6
2
12
225
0.58
10.81
0.38
0.7462
<.0001
0.9698
Bd
Pesticide
Bd x Pesticide
Error
Bd
Pesticide
Bd x Pesticide
Error
6
2
12
225
6
2
12
336
1.18
45.43
1.86
0.3193
<.0001
0.0400
0.68
1.05
1.12
0.6638
0.3512
0.3406
Figure Legends.
Figure 1. Cope’s gray treefrog time to metamorphosis at different pesticide
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
Figure 2. Cope’s gray treefrog mass at metamorphosis at different pesticide
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
21
Figure 3. Cope’s gray treefrog time to tail absorption at different temperature
treatments (control, carbaryl, copper sulfate). Error bars represent ± 1 standard
error.
Figure 4. Mass at metamorphosis for Cope’s gray treefrogs raised in the presence of
carbaryl (unfilled circle), presence of copper sulfate (triangle), or absence of
pesticide (black circle) with the presence or absence of Bd isolates. Error bars
represent ± 1 standard error.
Time to Metamorphosis (Days)
70
65
60
55
50
45
Control
Carbaryl
Pesticide
Copper Sulfate
22
0.31
0.30
0.29
Mass (g)
0.28
0.27
0.26
0.25
0.24
0.23
Control
Chem vs Mass
Carbaryl
Chemical
Copper Sulfate
23
5.8
Time to Tail (Days)
5.6
5.4
5.2
5.0
4.8
4.6
Control
Chemical vs Time to Tail
Carbaryl
Chemical
Copper Sulfate
24
0.36
Control
Carbaryl
Copper Sulfate
Mass at metamorphosis (g)
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
0.18
0
1
2
3
Chytrid Isolate
4
5
6
7
25