Physiological effects of chytridiomycosis, a cause of amphibian population declines 
 
by 
 
John Dorland Peterson 
 
 
 
 
A dissertation submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Doctor of Philosophy 
 
Auburn, Alabama 
May 7, 2012 
 
 
 
 
Keywords: Batrachochytrium dendrobatids, chytridiomycosis, glucocorticoid,  
stress, immune, metabolic rate, amphibian 
 
 
Copyright 2012 by John Dorland Peterson 
 
 
Approved by 
 
Dr. Mary T. Mendon?a, Chair, Professor, & Graduate Program Officer of Biological Sciences 
Dr. Craig Guyer, Professor of Biological Sciences 
Dr. William A. Hopkins, Associate Professor of Fish and Wildlife Conservation 
Dr. Robert S. Lishak, Associate Professor of Biological Sciences 
Dr. Jeffery S. Terhune, Associate Professor of Fisheries and Allied Aquacultures 
 
 
 
 
ii 
 
 
 
 
 
 
Abstract 
 
 
 Emerging infectious diseases (EIDs) of wildlife can have devastating impacts on 
biodiversity.  The fungal disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis 
(Bd) is implicated with amphibian population declines the world over.  As is the case with many 
EIDs of wildlife, pathogenesis of chytridiomycosis is somewhat unclear.  Pathogenesis involves 
disruption of cutaneous ion uptake, decreased plasma ions, and asystolic cardiac arrest, as well as 
seemingly unrelated effects on leukocytes, skin shedding, and appetite.  In this dissertation I, 
along with the help of many collaborators, suggest that infection-induced decreases in plasma 
ions initiate a stress response which may mediate some of the deleterious effects observed during 
disease development.  In chapters one and two, physiological parameters were monitored during 
an outbreak and a controlled infection, respectively, of Bd in a laboratory colony of Litoria 
caerulea.  Taken together, it was observed that prior to becoming diseased, infected frogs 
experienced decreased plasma sodium and potassium, appetite, and body mass, as well as 
increased standard metabolic rate and skin shedding.  When infected frogs became diseased, they 
contained even fewer plasma ions, as well as increased plasma corticosterone (CORT; a stress 
hormone) and altered white blood cell profiles.  These individuals also had continued elevated 
standard metabolic rate and decreased body condition.  In chapter three, it was determined that 
CORT increases standard metabolic rate in L. caerulea, representing the first time this effect has 
been observed in an anuran amphibian.  Collectively, this dissertation suggests that stress 
physiology plays a role in chytridiomycosis. 
 iii 
 
 
 
 
 
Acknowledgments 
 
 
 I would like to thank Mary Mendon?a for her major contribution to this dissertation and 
my professional development.  She constantly suggested that I ?think bigger? and focus on the 
most important questions.  Similarly, my committee gave me invaluable advice on how to make 
my research more meaningful and impactful.  I would also like to thank Arthur Appel for the 
many thoughtful discussions we had about my research, for agreeing to be the outside reader for 
my dissertation, and for allowing me to conduct respirometry in his lab.  Louise Rollins-Smith 
also provided precious lab resources and feedback on the research and dissertation.  John Steffen 
provided many thoughtful conversations, as well as assistance with respirometry and editing the 
manuscript.  Laura Reinert provided assistance with PCR and Paul Cobine provided assistance 
with elemental analyses.  I thank the army of undergraduate researchers that assisted with data 
collection and animal care, most notably, Michael McDonald, Blake Pohlman, Katy Prince, and 
Krista Riley.  Current and former members of the Mendon?a lab including, Richard Beauman, 
James Ellison, Sean Graham, Matt Grilliot, Kristal Huggins, Paula Kahn, Kristen Navarra, 
Camille Okekpe, Sam Patterson, Vikki Peterson, Matt Porta, Ches Smith, and Chelsea Ward 
provided valuable input that contributed to this dissertation.  Finally, I would like to thank my 
family, Vikki, Cash, Louie, Max, Chris, and Debra Peterson, and friends (especially Michelle 
Gilley) for their never ending and unconditional love, support, and guidance. 
 
 
 
 
 iv 
 
 
 
 
 
Table of Contents 
 
 
Abstract ......................................................................................................................................... ii 
Acknowledgments........................................................................................................................ iii  
List of Figures ............................................................................................................................... v  
List of Abbreviations ................................................................................................................... vi 
Introduction   ................................................................................................................................. 1 
Chapter 1   ..................................................................................................................................... 9 
Chapter 2   ................................................................................................................................... 37 
Chapter 3   ................................................................................................................................... 60 
Conclusion   ................................................................................................................................ 74 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 v 
 
 
 
 
 
List of Figures 
 
 
Figure 1.1 .................................................................................................................................... 22 
Figure 1.2 .................................................................................................................................... 23 
Figure 1.3 .................................................................................................................................... 25 
Figure 1.4 .................................................................................................................................... 27 
Figure 1.5 .................................................................................................................................... 28 
Figure 2.1 .................................................................................................................................... 52 
Figure 2.2 .................................................................................................................................... 54 
Figure 2.3 .................................................................................................................................... 55 
Figure 3.1 .................................................................................................................................... 69 
Figure 3.2 .................................................................................................................................... 70 
 
 
 
 
 
 
 
 
 
 vi 
 
 
 
 
 
List of Abbreviations 
 
 
ANOVA Analysis of variance 
ANCOVA Analysis of covariance 
Bd Batrachochytrium dendrobatidis  
 CORT Corticosterone  
DPI Days post infection 
EID Emerging infectious disease 
NL ratio Neutrophil-lymphocyte ratio 
PCR Polymerase chain reaction 
PLSD Protected least significant difference 
RMR Resting metabolic rate 
SEM Standard error of the mean 
WBC White blood cell 
 
 
 
 
 
 
 1 
 
 
 
 
 
Introduction 
 Global biodiversity continues to decrease at an alarming rate (Butchart et al., 2010).  
Exploitation, global climate change, habitat loss, invasive species, anthropogenic contaminants, 
and emerging infectious diseases, as well as interactions among these factors have been linked to 
biodiversity loss (Smith et al., 2006).  In vertebrates, the evolutionarily conserved stress response 
potentially mediates these effects, because it is one of the mechanisms by which vertebrates 
modulate responses to their environment (Wingfield et al., 1998).  Besides regulating stress 
responses, the hypothalamic-pituitary-adrenal (-interrenal in amphibians) axis (stress axis), also 
regulates essential physiological functions (e.g. blood pressure, ion balance, blood glucose, 
immunity, metabolism, and reproduction; reviewed in (Sapolsky et al., 2000).  Many of the 
above effects are mediated by glucocorticoids, such as cortisol and, importantly to this 
dissertation, corticosterone (CORT).  Acute stress responses are considered adaptive, causing 
short lived alterations to these physiological functions in favor of survival, but chronic stress 
responses can be maladaptive, leading to suppression of reproduction, increased metabolic rate, 
and altered immunity (which can potentially increase susceptibility to disease; (Elenkov and 
Chrousos, 1999; Sapolsky et al., 2000).  These effects can cause mortality or reduced fitness, 
and, thus, contribute to population declines and biodiversity loss.  There is considerable evidence 
that many of the factors that lead to biodiversity loss also alter glucococorticoid secretion in 
vertebrates (Busch and Hayward, 2009).   
 Amphibians appear to be declining faster than other vertebrate groups (Stuart et al., 
2004), and stress physiology may play an important role in these declines (Carey and Bryant, 
 2 
1995; Pounds et al., 2006).  Many of the factors linked to amphibian declines also alter CORT 
levels.  For example, anthropogenic contaminants (Gendron et al., 1997; Glennemeier and 
Denver, 2001; Goulet and Hontela, 2003; Hayes et al., 2006; Hopkins et al., 1997; Hopkins et al., 
1999; Larson et al., 1998; Peterson et al., 2009; Ward and Mendon?a, 2006), infectious diseases 
(Belden and Kiesecker, 2005; Warne et al., 2011), habitat alteration (Denver, 1998; Newcomb 
Homan et al., 2003), and predation (Denver, 2009; Fraker et al., 2009) have been documented to 
alter CORT levels in amphibians.  Like other vertebrates, glucocorticoids regulate many essential 
physiological processes in amphibians, such as plasma ion homeostasis (Brewer et al., 1980; De 
Ruyter and Stiffler, 1986; Heney and Stiffler, 1983; Middler et al., 1969; Stiffler et al., 1986; 
Yorio and Bentley, 1978), immunity (Belden and Kiesecker, 2005; Bennett et al., 1972; Bennett 
and Harbottle, 1968; Davis and Maerz, 2010; Garrido et al., 1987) metabolism (Wack et al., 
2012), appetite (Crespi and Denver, 2005; Crespi et al., 2004) and skin shedding (Budtz, 1979; 
J?rgensen and Larsen, 1961, 1964; Stefano and Donoso, 1964).  CORT also contributes to 
essential life history events, such as metamorphosis (Denver, 2009) and reproduction (Moore and 
Jessop, 2003).  Since many of the factors that contribute to population declines are pervasive 
within amphibian habitats, populations may become chronically stressed and experience 
maladaptive effects on immunity, metabolism, and reproduction that contribute to long term 
population declines. 
 Chytridiomycosis, a disease caused by the amphibian chytrid fungus, Batrachochytrium 
dendrobatidis (Bd), has caused amphibian declines the world over (Berger et al., 1998; Lips et 
al., 2006; Skerratt et al., 2007; Vredenburg et al., 2010) and, like other amphibian decline 
factors, potentially influences stress physiology.  Although Bd is not known to alter CORT 
levels, it disrupts several physiological processes known to be influenced by glucocorticoids in 
 3 
amphibians.  Infection with Bd alters plasma ion homeostasis (Voyles et al., 2007; Voyles et al., 
2009), immunity (Davis et al., 2010; Woodhams et al., 2007), appetite (Nichols et al., 2001; 
Voyles et al., 2009), and skin shedding (Nichols et al., 2001; Voyles et al., 2009).   
 Within this dissertation, we suggest that several of the detrimental effects of Bd infection 
may be mediated by CORT.  Bd infects the superficial epidermis of post-metamorphic 
amphibians (Berger et al., 1998; Longcore et al., 1999), an organ that regulates ion homeostasis 
(Feder and Burggren, 1992).  When amphibians become diseased (i.e. display clinical signs of 
disease), sodium uptake across the skin is disrupted and likely causes hyponatremia (reduced 
plasma sodium; Voyles et al., 2007; Voyles et al., 2009).  Bd-induced hyponatremia likely 
increases CORT levels, because hyponatremia increases CORT levels in non-diseased 
amphibians (Stiffler et al., 1986).  Increased CORT levels may function toward rebalancing 
plasma sodium in Bd-infected amphibians, because treatment with CORT can correct 
hyponatremia in non-diseased amphibians (Heney and Stiffler, 1983).  However, if plasma 
sodium levels do not return to homeostatic levels, CORT levels may become chronically 
elevated in an attempt to regulate sodium levels.  We predicted that increased CORT secretion 
during infection with Bd should correspond with previously observed effects on plasma ions, 
immunity, appetite, and skin shedding.  We also predicted, given the known effects of CORT in 
amphibians, increases in CORT should also correspond with changes in body condition, body 
mass, and standard metabolic rate.   
 In chapter 1, we observed the physiological effects of a laboratory outbreak of Bd on 
Australian Green Tree Frogs, Litoria caerulea.  In chapter 2, we observed the physiological 
effects of a controlled laboratory infection of Bd on L. caerulea at various time points throughout 
infection.  Finally, in chapter 3, we verify that CORT increases metabolic rate in this species.   
 4 
References: 
Belden, L.K., Kiesecker, J.M., 2005. Glucocorticosteroid Hormone Treatment of Larval 
Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91, 
686-688. 
Bennett, M.F., Gaudio, C.A., Johnson, A.O., Spisso, J.H., 1972. Changes in the blood of 
newts,<i>Notophthalmus viridescens , following the administration of hydrocortisone. 
Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral 
Physiology 80, 233-237. 
Bennett, M.F., Harbottle, J.A., 1968. The effects of hydrocortisone on the blood of tadpoles and 
frogs, Rana catesbeiana. The Biological Bulletin 135, 92-95. 
Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, A.A., Goggin, C.L., Slocombe, R., 
Ragan, M.A., Hyatt, A.D., McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli, G., Parkes, H., 
1998. Chytridiomycosis causes amphibian mortality associated with population declines in the 
rain forests of Australia and Central America. Proceedings of the National Academy of Sciences 
95, 9031-9036. 
Brewer, K.J., Hoyt, B.J., McKeown, B.A., 1980. The effects of prolactin, corticosterone and 
ergocryptine on sodium balance in the urodele, Ambystoma gracile. Comparative Biochemistry 
and Physiology Part C: Comparative Pharmacology 66, 203-208. 
Budtz, P.E., 1979. Epidermal structure and dynamics of the toad, Bufo bufo, deprived of the pars 
distalis of the pituitary gland. Journal of Zoology 189, 57-92. 
Busch, D.S., Hayward, L.S., 2009. Stress in a conservation context: A discussion of 
glucocorticoid actions and how levels change with conservation-relevant variables. Biological 
Conservation 142, 2844-2853. 
Butchart, S.H.M., Walpole, M., Collen, B., van Strien, A., Scharlemann, J.P.W., Almond, 
R.E.A., Baillie, J.E.M., Bomhard, B., Brown, C., Bruno, J., Carpenter, K.E., Carr, G.M., 
Chanson, J., Chenery, A.M., Csirke, J., Davidson, N.C., Dentener, F., Foster, M., Galli, A., 
Galloway, J.N., Genovesi, P., Gregory, R.D., Hockings, M., Kapos, V., Lamarque, J.-F., 
Leverington, F., Loh, J., McGeoch, M.A., McRae, L., Minasyan, A., Morcillo, M.H., Oldfield, 
T.E.E., Pauly, D., Quader, S., Revenga, C., Sauer, J.R., Skolnik, B., Spear, D., Stanwell-Smith, 
D., Stuart, S.N., Symes, A., Tierney, M., Tyrrell, T.D., Vi?, J.-C., Watson, R., 2010. Global 
Biodiversity: Indicators of Recent Declines. Science 328, 1164-1168. 
Carey, C., Bryant, C.J., 1995. Possible interrelations among environmental toxicants, amphibian 
development, and decline of amphibian populations. Environmental health perspectives 103 
Suppl 4, 13-17. 
Crespi, E.J., Denver, R.J., 2005. Roles of stress hormones in food intake regulation in anuran 
amphibians throughout the life cycle. Comparative Biochemistry and Physiology - Part A: 
Molecular & Integrative Physiology 141, 381-390. 
 5 
Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of Corticotropin-Releasing Factor, 
Neuropeptide Y and Corticosterone in the Regulation of Food Intake In Xenopus laevis. Journal 
of Neuroendocrinology 16, 279-288. 
Davis, A., Keel, M., Ferreira, A., Maerz, J., 2010. Effects of chytridiomycosis on circulating 
white blood cell distributions of bullfrog larvae (Rana catesbeiana). Comparative Clinical 
Pathology 19, 49-55. 
Davis, A.K., Maerz, J.C., 2010. Effects of Exogenous Corticosterone on Circulating Leukocytes 
of a Salamander (Ambystoma talpoideum) with Unusually Abundant Eosinophils. International 
Journal of Zoology 2010. 
De Ruyter, M.L., Stiffler, D.F., 1986. Interrenal function in larval Ambystoma tigrinum: II. 
Control of aldosterone secretion and electrolyte balance by ACTH. General and Comparative 
Endocrinology 62, 298-305. 
Denver, R.J., 1998. Hormonal Correlates of Environmentally Induced Metamorphosis in the 
Western Spadefoot Toad,Scaphiopus hammondii. General and Comparative Endocrinology 110, 
326-336. 
Denver, R.J., 2009. Stress hormones mediate environment-genotype interactions during 
amphibian development. General and Comparative Endocrinology 164, 20-31. 
Elenkov, I.J., Chrousos, G.P., 1999. Stress Hormones, Th1/Th2 patterns, Pro/Anti-inflammatory 
Cytokines and Susceptibility to Disease. Trends in Endocrinology & Metabolism 10, 359-
368. 
Feder, M.E., Burggren, W.W., 1992. Environmental physiology of the amphibians. University of 
Chicago Press, Chicago. 
Fraker, M.E., Hu, F., Cuddapah, V., McCollum, S.A., Relyea, R.A., Hempel, J., Denver, R.J., 
2009. Characterization of an alarm pheromone secreted by amphibian tadpoles that induces 
behavioral inhibition and suppression of the neuroendocrine stress axis. Hormones and Behavior 
55, 520-529. 
Garrido, E., Gomariz, R.P., Leceta, J., Zapata, A., 1987. Effects of dexamethasone on the 
lymphoid organs of Rana perezi. Developmental & Comparative Immunology 11, 375-384. 
Gendron, A.D., Bishop, C.A., Fortin, R., Hontela, A., 1997. In vivo testing of the functional 
integrity of the corticosterone-producing axis in mudpuppy (amphibia) exposed to chlorinated 
hydrocarbons in the wild. Environmental Toxicology and Chemistry 16, 1694-1706. 
Glennemeier, K.A., Denver, R.J., 2001. Sublethal effects of chronic exposure to an 
organochlorine compound on northern leopard frog (Rana pipiens) tadpoles. Environmental 
Toxicology 16, 287-297. 
 6 
Goulet, B.N., Hontela, A., 2003. Toxicity of cadmium, endosulfan, and atrazine in adrenal 
steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana. 
Environmental Toxicology and Chemistry 22, 2106-2113. 
Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., 
Marjuoa, Y., Parker, J., Tsui, M., 2006. Pesticide Mixtures, Endocrine Disruption, and 
Amphibian Declines: Are We Underestimating the Impact? National Institute of Environmental 
Health Sciences. 
Heney, H.W., Stiffler, D.F., 1983. The effects of aldosterone on sodium and potassium 
metabolism in larval Ambystoma tigrinum. General and Comparative Endocrinology 49, 122-
127. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1997. Increased Circulating Levels of 
Testosterone and Corticosterone in Southern Toads,Bufo terrestris,Exposed to Coal Combustion 
Waste. General and Comparative Endocrinology 108, 237-246. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1999. Responsiveness of the hypothalamo?
pituitary?interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes. 
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and 
Endocrinology 122, 191-196. 
J?rgensen, C.B., Larsen, L.O., 1961. Molting and its hormonal control in toads. General and 
Comparative Endocrinology 1, 145-153. 
J?rgensen, C.B., Larsen, L.O., 1964. Further observations on molting and its hormonal control in 
Bufo bufo (L.). General and Comparative Endocrinology 4, 389-400. 
Larson, D.L., McDonald, S., Fivizzani, A.J., Newton, W.E., Hamilton, S.J., 1998. Effects of the 
herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and 
hormonal response. Physiological zoology 71, 671-679. 
Lips, K.R., Brem, F., Brenes, R., Reeve, J.D., Alford, R.A., Voyles, J., Carey, C., Livo, L., 
Pessier, A.P., Collins, J.P., 2006. Emerging infectious disease and the loss of biodiversity in a 
Neotropical amphibian community. Proceedings of the National Academy of Sciences of the 
United States of America 103, 3165-3170. 
Longcore, J.E., Pessier, A.P., Nichols, D.K., 1999. Batrachochytrium dendrobatidis gen. et sp. 
nov., a chytrid pathogenic to amphibians. Mycological Society of America, Lawrence, KS, 
ETATS-UNIS, 9. 
Middler, S.A., Kleeman, C.R., Edwards, E., Brody, D., 1969. Effect of adenohypophysectomy 
on salt and water metabolism of the toad Bufo marinus with studies on hormonal replacement. 
General and Comparative Endocrinology 12, 290-304. 
Moore, I.T., Jessop, T.S., 2003. Stress, reproduction, and adrenocortical modulation in 
amphibians and reptiles. Hormones and Behavior 43, 39-47. 
 7 
Newcomb Homan, R., Regosin, J.V., Rodrigues, D.M., Reed, J.M., Windmiller, B.S., Romero, 
L.M., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders 
(Ambystoma maculatum). Animal Conservation 6, 11-18. 
Nichols, D., Lamirande, E., Pessier, A., Longcore, J., 2001. Experimental transmission of 
cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases 37, 1-11. 
Peterson, J.D., Peterson, V.A., Mendon?a, M.T., 2009. Exposure to coal combustion residues 
during metamorphosis elevates corticosterone content and adversely affects oral morphology, 
growth, and development in Rana sphenocephala. Comparative Biochemistry and Physiology 
Part C: Toxicology & Pharmacology 149, 36-39. 
Pounds, A.J., Bustamante, M.R., Coloma, L.A., Consuegra, J.A., Fogden, M.P.L., Foster, P.N., 
La Marca, E., Masters, K.L., Merino-Viteri, A., Puschendorf, R., Ron, S.R., Sanchez-Azofeifa, 
G.A., Still, C.J., Young, B.E., 2006. Widespread amphibian extinctions from epidemic disease 
driven by global warming. Nature 439, 161-167. 
Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How Do Glucocorticoids Influence Stress 
Responses? Integrating Permissive, Suppressive, Stimulatory, and Preparative Actions. 
Endocrine Reviews 21, 55-89. 
Skerratt, L., Berger, L., Speare, R., Cashins, S., McDonald, K., Phillott, A., Hines, H., Kenyon, 
N., 2007. Spread of Chytridiomycosis Has Caused the Rapid Global Decline and Extinction of 
Frogs. EcoHealth 4, 125-134. 
Smith, K.F., Sax, D.F., Lafferty, K.D., 2006. Evidence for the Role of Infectious Disease in 
Species Extinction and Endangerment 
Evidencia del Papel de Enfermedades Infecciosas en la Extinci?n y Puesta en Peligro de 
Especies. Conservation Biology 20, 1349-1357. 
Stefano, F.J.E., Donoso, A.O., 1964. Hypophyso-adrenal regulation of moulting in the toad. 
General and Comparative Endocrinology 4, 473-480. 
Stiffler, D.F., De Ruyter, M.L., Hanson, P.B., Marshall, M., 1986. Interrenal function in larval 
Ambystoma tigrinum: I. Responses to alterations in external electrolyte concentrations. General 
and Comparative Endocrinology 62, 290-297. 
Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, A.S.L., Fischman, D.L., Waller, 
R.W., 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science 306, 
1783-1786. 
Voyles, J., Berger, L., Young, S., Speare, R., Webb, R., Warner, J., Rudd, D., Campbell, R., 
Skerratt, L.F., 2007. Electrolyte depletion and osmotic imbalance in amphibians with 
chytridiomycosis. 
 8 
Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W.F., Dinudom, A., Cook, D., Webb, 
R., Alford, R.A., Skerratt, L.F., Speare, R., 2009. Pathogenesis of Chytridiomycosis, a Cause of 
Catastrophic Amphibian Declines. Science 326, 582-585. 
Vredenburg, V.T., Knapp, R.A., Tunstall, T.S., Briggs, C.J., 2010. Dynamics of an emerging 
disease drive large-scale amphibian population extinctions. Proceedings of the National 
Academy of Sciences 107, 9689-9694. 
Wack, C.L., DuRant, S.E., Hopkins, W.A., Lovern, M.B., Feldhoff, R.C., Woodley, S.K., 2012. 
Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander. Comparative 
Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 161, 153-158. 
Ward, C., Mendon?a, M., 2006. Chronic Exposure to Coal Fly Ash Causes Minimal Changes in 
Corticosterone and Testosterone Concentrations in Male Southern Toads <i>Bufo 
terrestris</i&gt. Archives of Environmental Contamination and Toxicology 51, 263-269. 
Warne, R.W., Crespi, E.J., Brunner, J.L., 2011. Escape from the pond: stress and developmental 
responses to ranavirus infection in wood frog tadpoles. Functional Ecology 25, 139-146. 
Wingfield, J.C., Maney, D.L., Breuner, C.W., Jacobs, J.D., Lynn, S., Ramenofsky, M., 
Richardson, R.D., 1998. Ecological Bases of Hormone?Behavior Interactions: The ?Emergency 
Life History Stage?. American Zoologist 38, 191-206. 
Woodhams, D.C., Ardipradja, K., Alford, R.A., Marantelli, G., Reinert, L.K., Rollins-Smith, 
L.A., 2007. Resistance to chytridiomycosis varies among amphibian species and is correlated 
with skin peptide defenses. Animal Conservation 10, 409-417. 
Yorio, T., Bentley, P.J., 1978. Stimulation of the short-circuit current (sodium transport) across 
the skin of the forg (Rana pipiens) by corticosteroids: structure-activity relationships. Journal of 
Endocrinology 79, 283-290. 
 
 
 9 
 
 
 
 
 
Chapter 1. The pathogenesis of the deadly amphibian disease, chytridiomycosis, suggests 
development of a stress response. 
 
Abstract 
Chytridiomycosis, a disease caused by Batrachochytrium dendrobatidis (Bd), has 
contributed to worldwide amphibian population declines; however, the pathogenesis of this 
disease is still somewhat unclear.  Previous studies suggest that infection disrupts cutaneous 
sodium channels, which leads to hyponatremia and cardiac failure.  However, infection is also 
correlated with unexplained effects on appetite, skin shedding, and white blood cell (WBC) 
profiles.  Glucocorticoid hormones may be the biochemical connection between these disparate 
effects, because they regulate ion homeostasis and can also influence appetite, skin shedding, and 
WBCs.  During a laboratory outbreak of Bd in Australian Green Tree Frogs, Litoria caerulea, we 
compared frogs showing clinical signs of chytridiomycosis to infected frogs showing no signs of 
disease and determined that diseased frogs contained elevated baseline corticosterone (CORT), 
decreased plasma sodium and potassium, and WBC profiles that paralleled those observed 
following CORT treatment in other studies.  Diseased frogs also showed evidence of poorer 
body condition and elevated metabolic rates compared with frogs showing no signs of disease, as 
predicted by the metabolic effects of CORT on metabolic rate.  Prior to displaying signs of 
disease, we also observed changes in appetite, body mass, and the presence of shed skin 
associated with infected but not yet diseased frogs.  Collectively, these results suggest that 
 10 
elevated baseline CORT is associated with Bd infections and may mediate some of the 
deleterious effects observed during disease development. 
1. Introduction 
Emerging infectious diseases (EIDs) of wildlife can have profound effects on animal 
biodiversity (Harvell et al., 1999; Lips et al., 2006); however, little is known about the 
pathogenesis of wildlife EIDs (Daszak et al., 2001).  Since wildlife EIDs are often associated 
with anthropogenic and environmental stressors, pathogenesis is likely influenced by the host?s 
response to stressors (Acevedo-Whitehouse and Duffus, 2009; Daszak et al., 2001; Dobson and 
Foufopoulos, 2001; Rachowicz et al., 2005).  The evolutionarily conserved stress response is one 
of the mechanisms by which vertebrates modulate responses to these stressors (Wingfield et al., 
1998).  The stress response is of interest in a disease context, because it is mediated by 
glucocorticoid hormones that are known to affect susceptibility to infection (Elenkov and 
Chrousos, 1999).   
Glucocorticoids influence a suite of physiological functions in vertebrates, including 
reproduction, development, blood ion homeostasis, metabolism, appetite, growth, and, 
importantly in the context of disease, immunity (Sapolsky et al., 2000).  While much is known 
about how glucocorticoids influence physiological function in non-diseased animals, much less 
is known about how glucocorticoids influence the same physiological functions in diseased 
animals.  To our knowledge only one such study has been conducted in wild vertebrates.  Warne 
et al. (2011) exposed Rana sylvatica to ranaviruses and observed an increase in corticosterone 
(CORT; the most abundant amphibian glucocorticoid stress hormone) concentration and 
accelerated developmental changes consistent with the effects of endogenous and exogenous 
elevations of CORT in non-diseased amphibians.   
 11 
Chytridiomycosis, a disease caused by the amphibian chytrid fungus Batrachochytrium 
dendrobatidis (Bd) has contributed to worldwide amphibian population declines.  It is considered 
to be a significant threat to global amphibian biodiversity (Berger et al., 1998; Kilpatrick et al., 
2010; Lips et al., 2006; Skerratt et al., 2007).  Chytridiomycosis, like CORT, influences blood 
ion homeostasis, appetite, skin shedding, and immunity.  Specifically, Bd disrupts sodium 
channels in the host?s epidermis, which leads to hyponatremia and cardiac failure (Voyles et al., 
2009).  Bd also suppresses appetite (Nichols et al., 2001; Voyles et al., 2009), disrupts normal 
skin shedding (Nichols et al., 2001; Voyles et al., 2009), and causes alterations in leukocyte 
abundances in adult and larval anurans (Davis et al., 2010; Woodhams et al., 2007).  Yet there 
are no studies that have attempted to document what hormones may be mediating these changes 
in blood ions, behavior, shedding, and leukocyte abundances.   
Glucocorticoids may mediate the aforementioned effects of Bd infection.  In amphibians, 
glucocorticoids are critical regulators of blood ion homeostasis (Brewer et al., 1980; De Ruyter 
and Stiffler, 1986; Heney and Stiffler, 1983; Middler et al., 1969; Stiffler et al., 1986; Yorio and 
Bentley, 1978), appetite (Crespi and Denver, 2005; Crespi et al., 2004), skin shedding (Budtz, 
1979; J?rgensen and Larsen, 1961, 1964; Stefano and Donoso, 1964), and leukocytes (Belden 
and Kiesecker, 2005; Bennett et al., 1972; Bennett and Harbottle, 1968; Davis and Maerz, 2010; 
Garrido et al., 1987).  A normal, adaptive, regulatory mechanism to maintain sodium 
homeostasis is likely a moderate, transitory elevation in CORT secretion to increase cutaneous 
uptake of sodium as well as digestive uptake (facilitated by increased appetite; Crespi and 
Denver, 2005; Heney and Stiffler, 1983; Stiffler et al., 1986).  Since Bd infection directly 
compromises cutaneous sodium channels, a greater and greater elevation of CORT could occur 
in an attempt to maintain ion homeostasis.  However, high concentrations of glucocorticoids can 
 12 
become maladaptive, altering immune responses (Dhabhar and McEwen, 1997; Munck et al., 
1984), increasing metabolic rate (DuRant et al., 2008), as well as actually suppressing appetite 
(Bernier, 2006), the latter effect further exacerbating ion imbalance, leading to unsustainable 
blood sodium levels and cardiac failure.  Thus, CORT, in its regulatory role of maintaining ion 
homeostasis, is secreted in response to Bd infection, and could contribute to Bd-induced 
mortality. 
The goal of this chapter was to determine whether Bd infection influences CORT levels 
and whether CORT profiles are associated with novel, as well as previously described effects of 
Bd infection.  We documented the relationship of Bd infection to plasma corticosterone, sodium, 
and potassium concentrations; food intake; skin shedding; and leukocyte profiles during an 
outbreak of Bd in a laboratory colony of recently wild Australian Green Tree Frogs (Litoria 
caerulea).  Since both CORT and disease influence energy balance, we also monitored metabolic 
rate, body condition, and body mass.   
2. Materials and Methods 
2.1 Laboratory outbreak and experimental design  
Fifty one Litoria caerulea were obtained commercially (Tri Reptile, Miami, FL), in 
autumn of 2009.  Frogs were recently (i.e., within two weeks) collected from the wild in 
Indonesia.  Over the next two months six frogs died.  Forty seven additional frogs were obtained 
from the same source three months later.  Within a month, the frogs from the second shipment 
became ill and died at a rapid rate (i.e., 13 died within 13 days).  Shed skin from individuals 
showing clinical signs of chytridiomycosis (e.g., listlessness, odd body posture, and skin 
discoloration; (Berger et al., 2005) was viewed under a light microscope and Bd was present in 
all samples viewed.   
 13 
At this point, disease status [i.e., individuals displaying clinical signs of chytridiomycosis 
(diseased) or individuals not displaying clinical signs (non-diseased)] was monitored daily and 
food intake was assessed in all remaining non-diseased individuals (n=79).  When an individual 
became diseased, the diseased frog and two randomly selected non-diseased individuals were 
swabbed (to quantify Bd zoospores), pithed, and bled (within 3 min of handling).  Frogs were 
sacrificed in a separate area of the animal care facility to minimize disruption of the rest of the 
frogs in the colony.  Preliminary statistical analyses suggested that order of sacrifice, duration of 
bleed, and time of bleeding had no significant effects on CORT and white blood cells (P<0.05).  
Several drops of whole blood were used to make blood smears for enumeration of leukocytes.  
The remaining blood was centrifuged for 4 min at 3,500 x g and the plasma was frozen and 
stored at -20? C for later use in corticosterone radioimmunoassay. 
2.2 Animal care 
Amphibians were housed individually in plastic containers (17 cm x 17 cm x 17 cm) in which 
paper towels saturated with well water were used as substrate.  Wet paper towels were replaced 
twice each week for the duration of the study.  Animals were fed as stated below under ?food 
intake?.  Light was provided by full spectrum light bulbs on a 12:12 light/dark cycle.  Room 
temperature was maintained by a thermostat at ~22? C.   
2.3 Bd zoospore burden 
Frogs were swabbed in a standardized fashion by lightly brushing a sterile cotton swab 
(Medical Wire & Equipment, MW113) 10 times over the sides, venter, and ventral surface of the 
thighs and 5 times over the underside of each foot (Kriger et al., 2006).  Zoospore equivalents 
were determined by standard extraction and quantitative PCR techniques (Boyle et al., 2004; 
Ramsey et al., 2010).  Nucleic acids were extracted by adding 60 ?l of PrepMan Ultra (Applied 
 14 
Biosystems, Foster City, CA) and 30-35 mg of Zirconium/silica beads (0.5 mm, Biospec 
Products, Bartlesville, OK) to the tip of each swab.  Samples were homogenized for 45 s in a 
Mini Beadbeater (MP Bio, Solon, OH) and centrifuged for 30 s at 15,000 x g.  After a second 
homogenization and centrifugation, the samples were boiled for 10 min, returned to room 
temperature for 2 min, and centrifuged at 15,000 x g for 3 min.  Nucleic acids in the supernatant 
were removed for real-time PCR.  Samples were loaded into an Mx3000P Real-Time PCR 
system (Stratagene, La Jolla, CA) for 40 cycles of 95? C for 10 min, 95? C for 30 s, 55? C for 1 
min, and 72? C for 1 min.  Zoospore equivalents were determined using a standard curve and 
indicate Bd burden.   
2.4 Ion analyses  
Plasma prepared by centrifugation of whole blood for 4 min at 3,500 x g to remove red 
cells, was analyzed by Inductively Coupled Plasma with Optical Emission Spectroscopy (ICP-
OES, Perkin Elmer 7100 DV, Waltham, MA) with simultaneous measurement of Ca, Co, Cu, Fe, 
K, Mg, Mn, Mo, Na, P, S, Zn.  Equal volume of plasma was diluted into ultra-pure, metal-free 
water (MilliQ, Millipore) then centrifuged at 13,000 x g to remove particulates and then 
introduced directly into the instrument argon plasma using a cyclonic nebulizer. Metal 
concentrations are determined comparing emission intensities to a standard curve created from 
certified metal standards (SPEX, Metuchen, NJ). Standard curves were confirmed by re-analysis 
of standard solutions diluted in a matrix equivalent to the sample. Individual readings are the 
average of two intensity measurements varied by less than 5%. Repeated analysis of individual 
samples showed less than 5 % variability. 
2.5 Radioimmunoassay 
 15 
Plasma corticosterone concentrations were determined by radioimmunoassay as 
described by Mendon?a et al. (1996).  All samples were run in one assay.  Extraction efficiency 
was 81% and intraassay variation was 19.8%.   
2.6 Food intake and body mass 
Frogs were weighed weekly from the time they arrived in the laboratory.  Once a week, 
for two weeks prior to sacrifice, each animal was weighed and fed approximately 10% of their 
body weight in 2.5 week old crickets coated in vitamin dust.  All crickets not consumed were 
weighed after 24 hours.  Food intake was determined as the mass of the crickets not consumed 
subtracted from the original mass of crickets given to the frog. 
2.7 Shed skin collection 
 A subsample of frogs and bins were examined daily for the presence of shed skin.  Shed 
skin was removed if it was observed on amphibians or within their containers.  Dates in which 
skin was found on a frog or within its container were recorded.  When frogs were sacrificed, the 
number of days, within the previous seven days, the frogs had shed skin on their body or within 
their container was determined.  This value is referred to hereafter as ?skin presence frequency?. 
2.8 Relative leukocyte numbers 
Dried blood smears were stained with a Hema 3 kit (Fisher scientific, Kalamazoo, MI) 
and viewed under a light microscope.  Slides were read in a standard zig-zag fashion.  One 
hundred leukocytes were observed and the number of neutrophils, lymphocytes, eosinophils, 
monocytes, and basophils were recorded.  Leukocytes were identified following Turner (1988) 
and Hadji-Azimi et al. (1987). 
2.9 Respirometry and body condition 
 16 
 Closed system respirometry was used to measure resting metabolic rate (oxygen 
consumption) 1 day prior to sacrifice following the methods of Ward et al. (2006) with the 
following changes.  Prior to being placed in individual respirometry chambers (140 ml syringes, 
Monoject, Sherwood Medical Industries, Ballymoney, UK), the venter of each frog was blotted 
dry with a paper towel and the bladder of each frog was voided by gently depressing the 
abdomen.  Frogs were acclimated in their respirometry chamber for at least 45 min in a darkened 
incubator (22? C).  Frogs were incubated for ~50 min in a darkened incubator (22? C).  Any 
frogs that urinated or defecated during incubation were excluded from analyses.  Frogs were 
weighed and their total body length was determined following respirometry.   
2.10 Statistical analyses 
Bd burden, plasma corticosterone, sodium, and potassium, food intake, skin presence 
frequency were compared relative to disease status with analyses of variance (ANOVAs).  
Oxygen consumption was compared between disease states with an analysis of covariance 
(ANCOVA) with disease state as the independent variable, oxygen consumed as the dependent 
variable, and body mass as the covariate.  Since body mass influences oxygen consumption, 
oxygen consumption is presented as least squared means, corrected for body mass.  Body 
condition was estimated as the residuals obtained by regressing body mass against total body 
length.  Body condition was compared between disease states with an ANCOVA, with disease 
status as the independent variable, body mass as the dependent variable, and total body length as 
the covariate.  Change in body mass was compared relative to disease status with a repeated 
measures ANOVA.  Relative leukocyte numbers were compared relative to disease status with a 
multivariate analysis of variance (MANOVA).  Sheffe?s range tests were conducted for all a 
posteriori comparisons.  We were unable to monitor changes over time for several variables; 
 17 
however, when diseased and non-diseased frogs were sacrificed Bd burden was highly variable 
across all frogs (ranging from 0 to millions of zoospores per frog), suggesting that each frog was 
at a different point within disease progression.  We used segmented regression to determine the 
threshold Bd burden at which the trend of corticosterone, lymphocytes, and eosinophils changed 
significantly, regardless of disease status (Seber and Wild, 1989).  SAS (SAS institute, version 
9.2) was used for the oxygen consumption ANCOVA (PROC GLM) and all segmented 
regressions (PROC NLIN).  StatView for Windows (SAS institute, version 5.0.1) was used for 
all other statistical analyses. 
3. Results 
3.1 Measures of pre-diseased frogs 
Frogs that eventually became diseased lost significantly more weight than frogs that 
remained non-diseased in the weeks prior to sacrifice (Repeated measures ANOVA, Disease 
status: P<0.001, F1,21=38.06, Time: P<0.001, F1,42=12.25, Disease status x Time: P=0.2; Figure 
1.1).  Individuals that eventually became diseased also consumed significantly less food one 
week prior to displaying clinical signs of disease compared to individuals that displayed no signs 
of disease (ANOVA, 0.022, F1,21=6.16; Figure 1.2).  During the week prior to sacrifice, shed skin 
was found on significantly more days within bins of frogs that eventually became diseased than 
within the bins of non-diseased frogs (ANOVA, P<0.001, F1,21=38.11; Figure 1.2). 
3.2 Measures of diseased frogs 
Approximately 24 hours prior to sacrifice, frogs that displayed clinical signs of 
chytridiomycosis had significantly lower body conditions (ANCOVA, Disease status: P<0.001, 
F1,20=19.02; Total body length: P<0.001, F1,20=273.43).  There was no disease status by total 
body length interaction (P=0.22).  For ease of interpretation, these data are visually presented as 
 18 
average residuals from a regression of body mass by total body length (Figure 1.3).  Diseased 
individuals also consumed significantly more oxygen compared to non-diseased frogs 
(ANCOVA, Disease status: P<0.001, F1,20=24.52, Body mass: P=0.010, F1,20=7.99; Figure 1.3).  
There was no disease status by body mass interaction (P=0.3).  On average, diseased frogs 
consumed more than twice the amount of oxygen non-diseased frogs consumed. 
When sacrificed, swabs taken from diseased frogs contained significantly more Bd 
zoospore equivalents than swabs taken from non-diseased individuals (ANOVA, P<0.001, 
F1,35=19.66; Figure 1.3).  Although non-diseased individuals all had detectable levels of Bd, they 
contained approximately 1,000 times fewer zoospore equivalents than diseased individuals, on 
average.   
Blood parameters taken at sacrifice also differed with disease status.  Diseased frogs 
contained significantly fewer plasma electrolytes (ANOVA, Sodium: P=0.049, F1,28=4.24, 
Potassium: P=0.049, F1,28=4.24; Figure 1.3) and significantly greater concentrations of plasma 
corticosterone (ANOVA, P=0.001, F1,34=18.73; Figure 1.3) compared with non-diseased frogs.  
Additionally, leukocyte abundances differed significantly between diseased and non-diseased 
individuals (MANOVA, P<0.001, F1,20=12.26; Figure 1.4).  Blood smears from diseased frogs 
contained significantly fewer lymphocytes and eosinophils and significantly more neutrophils 
than smears from non-diseased frogs (Sheffe?s range tests, P?0.002).   
3.3 Changes in corticosterone and leukocyte abundances at different Bd burdens 
Since all individuals in the study contained different Bd burdens and were, thus, at 
different points in infection we used segmented regression to determine the zoospore intensity at 
which corticosterone, RMR, and lymphocyte abundances changed significantly (the zoospore 
 19 
breakpoints).  The zoospore breakpoints for corticosterone, RMR, and lymphocytes were 4,940; 
4,066; and 10,778 zoospores, respectively (Figure 1.5).   
4. Discussion 
Individuals that displayed clinical signs of chytridiomycosis had significantly elevated 
baseline CORT, decreased plasma sodium and potassium, altered leukocyte profiles, increased 
metabolic rate, and decreased body condition compared with non-diseased individuals.  The 
effects of Bd on leukocyte profiles and metabolic rate parallel those observed following CORT 
treatment in other studies (e.g. increased neutrophils and oxygen consumption and decreased 
lymphocytes and eosionphils; Belden and Kiesecker, 2005; Bennett et al., 1972; Bennett and 
Harbottle, 1968; Davis and Maerz, 2010; DuRant et al., 2008; Garrido et al., 1987; Wack et al., 
2012).  It is important to note that non-diseased individuals were also infected, but contained 
thousands of Bd zoospores while diseased individuals contained millions of zoospores, on 
average.  When we plotted this broad range of Bd burdens (regardless of disease status) against 
CORT, RMR, and lymphocytes, segmented regressions indicated these three variables changed 
significantly at similar breakpoints (4,000-10,000 zoospores; Fig. 1.5).   
Appetite suppression likely contributed to other effects we observed.  For example, 
appetite suppression likely exacerbated hyponatremia because amphibians take up sodium via 
digestive as well as cutaneous routes (Feder and Burggren, 1992).  Additionally, amphibians 
usually consume their shed skin, so appetite suppression also likely explains why we observed 
shed skin more often in the containers of frogs that eventually became diseased (Feder and 
Burggren, 1992).  Finally, appetite suppression, coupled with an increased metabolic rate, may 
have contributed to the weight loss and poor body condition observed in frogs that eventually 
 20 
became diseased.  With no input of food, sick frogs must catabolize body tissues to meet their 
elevated respiratory demand, which results in weight loss and reduced body condition.   
The levels of CORT in plasma were determined after development of several effects, so 
although infection was associated with decreased food intake, increased presence of shed skin, 
and weight loss, it is unclear whether increased CORT secretion was a cause or consequence of 
these parameters.  We happened to be monitoring food intake, skin shedding, and weight loss as 
part of a separate experiment when the outbreak occurred, thus we did not monitor CORT 
throughout infection.  Future studies are needed to determine when CORT increases during 
infection and whether CORT manipulation can alter chytridiomycosis pathogenesis. 
It is unclear whether infection-induced glucocorticoid secretion is beneficial or 
maladaptive in vertebrates.  Few studies have tested the effects of disease on baseline 
glucocorticoid levels in vertebrates (Al-Afaleq, 1998; Fast et al., 2006; Finstad et al., 2000; 
Fleming, 1997, 1998; Hanley and Stamps, 2002; Hermann et al., 1995; Laidley et al., 1988; 
Pickering and Christie, 1981; Raouf et al., 2006; Sures et al., 2001; Sures et al., 2002; Warne et 
al., 2011).  Even fewer have observed how this may then lead to beneficial or deleterious 
physiological effects.  To our knowledge, this is the first study that has assessed the effects of 
disease on baseline CORT levels in an adult amphibian (see Warne et al., 2010 for effects in 
tadpoles).  Our data suggest that disease, at least chytridiomycosis, is likely a potent modulator 
of baseline CORT.  Frogs displaying clinical signs of disease contained eight times more plasma 
CORT than non-diseased frogs.  Average plasma CORT was 104 ng/ml in symptomatic frogs 
(maximum level of 270 ng/ml), rivaling the highest average levels of CORT observed in 
amphibians in response to stressors (Coddington and Cree, 1995; Gobbetti and Zerani, 1996; 
Hopkins et al., 1997; Jurani et al., 1973; Zerani et al., 1991).  Given these findings and the large 
 21 
number of emerging diseases in wildlife, we suggest that more studies focus on post-infection 
stress responses in wild animals.   
Better understanding of the physiological effects of CORT, and its involvement in 
mediating factors that threaten the conservation status of amphibians (e.g. habitat destruction, 
global climate change, pollution, etc.) is needed.  Specifically, there is a lack of data on how 
glucocorticoids influence metabolic rate and appetite in amphibians (Carr et al., 2002; Crespi et 
al., 2004).  Our study provides data to suggest that CORT is associated with these factors, but 
controlled laboratory data are needed to complement this study.  Understanding how amphibians 
respond to environmental change has become more urgent given recent amphibian population 
declines (Stuart et al., 2004).  Several perturbations that potentially contribute to amphibian 
population declines have been linked to stress physiology [e.g. anthropogenic contaminants 
(Gendron et al., 1997; Glennemeier and Denver, 2001; Goulet and Hontela, 2003; Hayes et al., 
2006; Hopkins et al., 1997; Hopkins et al., 1999; Larson et al., 1998; Peterson et al., 2009; Ward 
et al., 2007), disease (Warne et al., 2010), low habitat quality (Newcomb Homan et al., 2003), 
and habitat desiccation (Denver, 1998)].  Though the influence of anthropogenic contaminants 
on the stress axis has been relatively well studied in amphibians far less is known about how 
disease, habitat destruction, invasive predators, and climate change may influence stress 
physiology.  Given the powerful and far reaching effects of glucocorticoids on wildlife life 
histories, understanding how these hormones mediate the interplay between environmental 
perturbations and life histories is essential to future conservation efforts. 
 22 
Figures: 
- 6
- 5
- 4
- 3
- 2
- 1
0
1
2
3
1 1/13 - 1 1/19 1 1/19 - 12/04 12/04 - 12/1 1
Da te
Ch
an
ge
 i
n 
bo
dy
 
mass
 
(g)
Non - diseas ed
Dise as ed
 
Fig. 1.1. Average change in body mass (?1 standard error) of Litoria caerulea that eventually 
became diseased (n=9) or remained non-diseased (n=14) for chytridiomycosis between dates 
leading up to sacrifice on 12/12/09.  Disease statuses were statistically different (Repeated 
measures ANOVA, Disease status: P<0.001, F1,21=38.06, Time: P<0.001, F1,42=12.25, Disease 
status x Time: P=0.2). 
 23 
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Dise as e status
5 18
Food 
int
ake 
(g)
Dise as ed No n - disea se d
 
 
Fig. 1.2. Average food intake (top) and shed skin frequency (bottom) +1 standard error of Litoria 
caerulea that eventually became diseased or remained non-diseased at one week prior to 
sacrifice (top) and within the week leading up to sacrifice (bottom).  Disease statuses were 
 24 
statistically different (Food intake: ANOVA, P=0.022, F1,21=6.16; shed skin frequency: 
ANOVA, P<0.001, F1,21=38.11). 
 25 
0
1
2
3
4
5
Dise as e status
Oxy
ge
n 
co
ns
ump
ti
on
 (ml
/h)
9 14
6
Disea sed Non - diseas ed
 
0
10
100
1,0 00
10 ,00 0
1 0 0 ,00 0
1,0 00 ,00 0
10 ,00 0,0 00
Bd
 zoo
sp
ores
 (
DN
A
 eq
uivalen
ts)
Dise as ed No n - disea se d
Disease status
13 24
0
20
40
60
80
100
120
140
Plasma 
corti
cos
terone (
ng/ml
)
Disea se status
12 24
Dise as ed No n - disea se d
0
20
40
60
80
100
120
Dise as e status
Plasma 
sod
ium (mM
/l
)
10 20
Dise as ed No n - disea se d
0
1
2
3
4
5
6
7
8
9
Dise as e status
Plasma p
otassium (mM
/l
)
10 20
Dise as ed No n - disea se d
 
 
Fig. 1.3. Mean body condition, oxygen consumption, log+1 transformed Bd zoospores, plasma 
corticosterone, plasma sodium, and plasma potassium ? 1 standard error of Litoria caerulea that 
displayed clinical signs of disease (diseased) or did not display clinical signs of disease (non-
 26 
diseased).  Disease statuses were significantly different for all measures (ANOVA/ANCOVA, 
P<0.05).
 27 
 
Fig. 1.4. Average relative abundances of neutrophils, lymphocytes, eosinophils, and monocytes 
per 100 leukocytes + 1 standard error from Litoria caerulea displaying clinical signs of 
chytridiomycosis (diseased, n=7) or not displaying clinical signs of disease (non-diseased, n=19).  
Leukocyte abundances were significantly different between groups (MANOVA, P<0.001, 
F1,20=12.26).  Asterisks denote significant differences.  Blood smears from diseased frogs 
contained significantly fewer lymphocytes and eosinophils and significantly more neutrophils 
than smears from non-diseased frogs (Sheffe?s range test, P<0.001).  Average basophil 
abundances were part of the analysis but were excluded. 
 28 
 
 
Fig. 1.5. Segmented regressions of (A) plasma corticosterone, (B) RMR, and (C) lymphocyte 
abundances of Litoria caerulea during an outbreak of chytridiomycosis.  Horizontal and vertical 
 29 
dotted lines indicate X and Y axes, respectively.  Vertical dashed lines indicate breakpoints at 
which the dependent variables changed significantly.  Black lines indicate the two segments fit to 
the data before and after the breakpoint.  The zoospore breakpoints for corticosterone, RMR, and 
lymphocytes were 4,940; 4,066; and 10,778 zoospores, respectively.  Data before and after the 
breakpoint were significantly different for all three variables (Segmented regression, P<0.05). 
 30 
References: 
 
Acevedo-Whitehouse, K., Duffus, A.L.J., 2009. Effects of environmental change on wildlife 
health. Philosophical Transactions of the Royal Society B: Biological Sciences 364, 3429-3438. 
Al-Afaleq, A.I., 1998. Biochemical and Hormonal Changes Associated with Experimental 
Infection of Chicks with Infectious Bursal Disease Virus. Journal of Veterinary Medicine, Series 
B 45, 513-517. 
Belden, L.K., Kiesecker, J.M., 2005. Glucocorticosteroid Hormone Treatment of Larval 
Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91, 
686-688. 
Bennett, M.F., Gaudio, C.A., Johnson, A.O., Spisso, J.H., 1972. Changes in the blood of 
newts,&lt;i&gt;Notophthalmus viridescens , following the administration of hydrocortisone. 
Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral 
Physiology 80, 233-237. 
Bennett, M.F., Harbottle, J.A., 1968. The effects of hydrocortisone on the blood of tadpoles and 
frogs, Rana catesbeiana. The Biological Bulletin 135, 92-95. 
Berger, L., Marantelli, G., Skerratt, L.F., Speare, R., 2005. Virulence of the amphibian chytrid 
fungus Batrachochytium 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., Slocombe, R., 
Ragan, M.A., Hyatt, A.D., McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli, G., Parkes, H., 
1998. Chytridiomycosis causes amphibian mortality associated with population declines in the 
rain forests of Australia and Central America. Proceedings of the National Academy of Sciences 
95, 9031-9036. 
Bernier, N.J., 2006. The corticotropin-releasing factor system as a mediator of the appetite-
suppressing effects of stress in fish. General and Comparative Endocrinology 146, 45-55. 
Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T., Hyatt, A.D., 2004. Rapid quantitative 
detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using 
real-time Taqman PCR assay. Diseases of Aquatic Organisms 60, 141-148. 
Brewer, K.J., Hoyt, B.J., McKeown, B.A., 1980. The effects of prolactin, corticosterone and 
ergocryptine on sodium balance in the urodele, Ambystoma gracile. Comparative Biochemistry 
and Physiology Part C: Comparative Pharmacology 66, 203-208. 
Budtz, P.E., 1979. Epidermal structure and dynamics of the toad, Bufo bufo, deprived of the pars 
distalis of the pituitary gland. Journal of Zoology 189, 57-92. 
Carr, J.A., Brown, C.L., Mansouri, R., Venkatesan, S., 2002. Neuropeptides and amphibian prey-
catching behavior. Comparative Biochemistry and Physiology Part B: Biochemistry and 
Molecular Biology 132, 151-162. 
 31 
Coddington, E.J., Cree, A., 1995. Effect of Acute Captivity Stress on Plasma Concentrations of 
Corticosterone and Sex Steroids in Female Whistling Frogs, Litoria ewingi. General and 
Comparative Endocrinology 100, 33-38. 
Crespi, E.J., Denver, R.J., 2005. Roles of stress hormones in food intake regulation in anuran 
amphibians throughout the life cycle. Comparative Biochemistry and Physiology - Part A: 
Molecular &amp; Integrative Physiology 141, 381-390. 
Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of Corticotropin-Releasing Factor, 
Neuropeptide Y and Corticosterone in the Regulation of Food Intake In Xenopus laevis. Journal 
of Neuroendocrinology 16, 279-288. 
Daszak, P., Cunningham, A.A., Hyatt, A.D., 2001. Anthropogenic environmental change and the 
emergence of infectious diseases in wildlife. Acta Tropica 78, 103-116. 
Davis, A., Keel, M., Ferreira, A., Maerz, J., 2010. Effects of chytridiomycosis on circulating 
white blood cell distributions of bullfrog larvae (Rana catesbeiana). Comparative Clinical 
Pathology 19, 49-55. 
Davis, A.K., Maerz, J.C., 2010. Effects of Exogenous Corticosterone on Circulating Leukocytes 
of a Salamander (Ambystoma talpoideum) with Unusually Abundant Eosinophils. International 
Journal of Zoology 2010. 
De Ruyter, M.L., Stiffler, D.F., 1986. Interrenal function in larval Ambystoma tigrinum: II. 
Control of aldosterone secretion and electrolyte balance by ACTH. General and Comparative 
Endocrinology 62, 298-305. 
Denver, R.J., 1998. Hormonal Correlates of Environmentally Induced Metamorphosis in the 
Western Spadefoot Toad,Scaphiopus hammondii. General and Comparative Endocrinology 110, 
326-336. 
Dhabhar, F.S., McEwen, B.S., 1997. Acute Stress Enhances while Chronic Stress Suppresses 
Cell-Mediated Immunityin Vivo:A Potential Role for Leukocyte Trafficking. Brain, Behavior, 
and Immunity 11, 286-306. 
Dobson, A., Foufopoulos, J., 2001. Emerging infectious pathogens of wildlife. Philosophical 
Transactions of the Royal Society of London. Series B: Biological Sciences 356, 1001-1012. 
DuRant, S.E., Romero, L.M., Talent, L.G., Hopkins, W.A., 2008. Effect of exogenous 
corticosterone on respiration in a reptile. General and Comparative Endocrinology 156, 126-133. 
Elenkov, I.J., Chrousos, G.P., 1999. Stress Hormones, Th1/Th2 patterns, Pro/Anti-inflammatory 
Cytokines and Susceptibility to Disease. Trends in Endocrinology &amp; Metabolism 10, 359-
368. 
Fast, M.D., Muise, D.M., Easy, R.E., Ross, N.W., Johnson, S.C., 2006. The effects of 
Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic 
salmon (Salmo salar). Fish &amp; Shellfish Immunology 21, 228-241. 
 32 
Feder, M.E., Burggren, W.W., 1992. Environmental physiology of the amphibians. University of 
Chicago Press, Chicago. 
Finstad, B., Bj?rn, P.A., Grimnes, A., Hvidsten, N.A., 2000. Laboratory and field investigations 
of salmon lice [Lepeophtheirus salmonis (Kr?yer)] infestation on Atlantic salmon (Salmo salar 
L.) post-smolts. Aquaculture Research 31, 795-803. 
Fleming, M.W., 1997. Cortisol as an Indicator of Severity of Parasitic Infections of Haemonchus 
contortus in Lambs (Ovis aries). Comparative Biochemistry and Physiology Part B: 
Biochemistry and Molecular Biology 116, 41-44. 
Fleming, M.W., 1998. Experimental Inoculations with Ostertagia ostertagi or Exposure to 
Artificial Illumination Alter Peripheral Cortisol in Dairy Calves (Bos taurus). Comparative 
Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology 119, 315-319. 
Garrido, E., Gomariz, R.P., Leceta, J., Zapata, A., 1987. Effects of dexamethasone on the 
lymphoid organs of Rana perezi. Developmental &amp; Comparative Immunology 11, 375-384. 
Gendron, A.D., Bishop, C.A., Fortin, R., Hontela, A., 1997. In vivo testing of the functional 
integrity of the corticosterone-producing axis in mudpuppy (amphibia) exposed to chlorinated 
hydrocarbons in the wild. Environmental Toxicology and Chemistry 16, 1694-1706. 
Glennemeier, K.A., Denver, R.J., 2001. Sublethal effects of chronic exposure to an 
organochlorine compound on northern leopard frog (Rana pipiens) tadpoles. Environmental 
Toxicology 16, 287-297. 
Gobbetti, A., Zerani, M., 1996. Possible mechanism for the first response to short captivity stress 
in the water frog, Rana esculenta. Journal of Endocrinology 148, 233-239. 
Goulet, B.N., Hontela, A., 2003. Toxicity of cadmium, endosulfan, and atrazine in adrenal 
steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana. 
Environmental Toxicology and Chemistry 22, 2106-2113. 
Hadji-Azimi, I., Coosemans, V., Canicatti, C., 1987. Atlas of adult Xenopus laevis laevis 
hematology. Developmental and comparative immunology 11, 807-874. 
Hanley, K.A., Stamps, J.A., 2002. Does corticosterone mediate bidirectional interactions 
between social behaviour and blood parasites in the juvenile black iguana, Ctenosaura similis ? 
Animal Behaviour 63, 311-322. 
Harvell, C.D., Kim, K., Burkholder, J.M., Colwell, R.R., Epstein, P.R., Grimes, D.J., Hofmann, 
E.E., Lipp, E.K., Osterhaus, A.D.M.E., Overstreet, R.M., Porter, J.W., Smith, G.W., Vasta, G.R., 
1999. Emerging Marine Diseases--Climate Links and Anthropogenic Factors. Science 285, 
1505-1510. 
Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., 
Marjuoa, Y., Parker, J., Tsui, M., 2006. Pesticide Mixtures, Endocrine Disruption, and 
 33 
Amphibian Declines: Are We Underestimating the Impact? National Institute of Environmental 
Health Sciences. 
Heney, H.W., Stiffler, D.F., 1983. The effects of aldosterone on sodium and potassium 
metabolism in larval Ambystoma tigrinum. General and Comparative Endocrinology 49, 122-
127. 
Hermann, G., Beck, F.M., Sheridan, J.F., 1995. Stress-induced glucocorticoid response 
modulates mononuclear cell trafficking during an experimental influenza viral infection. Journal 
of Neuroimmunology 56, 179-186. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1997. Increased Circulating Levels of 
Testosterone and Corticosterone in Southern Toads,Bufo terrestris,Exposed to Coal Combustion 
Waste. General and Comparative Endocrinology 108, 237-246. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1999. Responsiveness of the hypothalamo?
pituitary?interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes. 
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and 
Endocrinology 122, 191-196. 
Jurani, M., Murgas, K., Mikulaj, L., Babusikova, F., 1973. Effect of stress and environmental 
temperature on adrenal function in Rana esculenta. Journal of Endocrinology 57, 385-391. 
J?rgensen, C.B., Larsen, L.O., 1961. Molting and its hormonal control in toads. General and 
Comparative Endocrinology 1, 145-153. 
J?rgensen, C.B., Larsen, L.O., 1964. Further observations on molting and its hormonal control in 
Bufo bufo (L.). General and Comparative Endocrinology 4, 389-400. 
Kilpatrick, A.M., Briggs, C.J., Daszak, P., 2010. The ecology and impact of chytridiomycosis: an 
emerging disease of amphibians. Trends in Ecology &amp; Evolution 25, 109-118. 
Kriger, K.M., Hero, J.M., Ashton, K.J., 2006. Cost efficiency in the detection of 
chytridiomycosis using PCR assay. ePublications@bond. 
Laidley, C.W., Woo, P.T.K., Leatherland, J.F., 1988. The stress-response of rainbow trout to 
experimental infection with the blood parasite Cryptobia salmositica Katz, 1951. Journal of Fish 
Biology 32, 253-261. 
Larson, D.L., McDonald, S., Fivizzani, A.J., Newton, W.E., Hamilton, S.J., 1998. Effects of the 
herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and 
hormonal response. Physiological zoology 71, 671-679. 
Lips, K.R., Brem, F., Brenes, R., Reeve, J.D., Alford, R.A., Voyles, J., Carey, C., Livo, L., 
Pessier, A.P., Collins, J.P., 2006. Emerging infectious disease and the loss of biodiversity in a 
Neotropical amphibian community. Proceedings of the National Academy of Sciences of the 
United States of America 103, 3165-3170. 
 34 
Mendon?a, M.T., Chernetsky, S.D., Nester, K.E., Gardner, G.L., 1996. Effects of Gonadal Sex 
Steroids on Sexual Behavior in the Big Brown Bat,Eptesicus fuscus,Upon Arousal from 
Hibernation. Hormones and Behavior 30, 153-161. 
Middler, S.A., Kleeman, C.R., Edwards, E., Brody, D., 1969. Effect of adenohypophysectomy 
on salt and water metabolism of the toad Bufo marinus with studies on hormonal replacement. 
General and Comparative Endocrinology 12, 290-304. 
Munck, A., Guyre, P.M., Holbrook, N.J., 1984. Physiological Functions of Glucocorticoids in 
Stress and Their Relation to Pharmacological Actions. Endocrine Reviews 5, 25-44. 
Newcomb Homan, R., Regosin, J.V., Rodrigues, D.M., Reed, J.M., Windmiller, B.S., Romero, 
L.M., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders 
(Ambystoma maculatum). Animal Conservation 6, 11-18. 
Nichols, D., Lamirande, E., Pessier, A., Longcore, J., 2001. Experimental transmission of 
cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases 37, 1-11. 
Peterson, J.D., Peterson, V.A., Mendon?a, M.T., 2009. Exposure to coal combustion residues 
during metamorphosis elevates corticosterone content and adversely affects oral morphology, 
growth, and development in Rana sphenocephala. Comparative Biochemistry and Physiology 
Part C: Toxicology &amp; Pharmacology 149, 36-39. 
Pickering, A.D., Christie, P., 1981. Changes in the concentrations of plasma cortisol and 
thyroxine during sexual maturation of the hatchery-reared brown trout, Salmo trutta L. General 
and Comparative Endocrinology 44, 487-496. 
Rachowicz, L.J., Hero, J.-M., Alford, R.A., Taylor, J.W., Morgan, J.A.T., Vredenburg, V.T., 
Collins, J.P., Briggs, C.J., 2005. The Novel and Endemic Pathogen Hypotheses: Competing 
Explanations for the Origin of Emerging Infectious Diseases of Wildlife 
La Hip?tesis del Pat?geno Incipiente y End?mico: Explicaciones Opuestas del Origen de 
Enfermedades Infecciosas Emergentes en la Vida Silvestre. Conservation Biology 19, 1441-
1448. 
Ramsey, J.P., Reinert, L.K., Harper, L.K., Woodhams, D.C., Rollins-Smith, L.A., 2010. Immune 
defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, 
in the South African clawed frog, Xenopus laevis. Infection and immunity 78, 3981-3992. 
Raouf, S.A., Smith, L.C., Brown, M.B., Wingfield, J.C., Brown, C.R., 2006. Glucocorticoid 
hormone levels increase with group size and parasite load in cliff swallows. Animal Behaviour 
71, 39-48. 
Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How Do Glucocorticoids Influence Stress 
Responses? Integrating Permissive, Suppressive, Stimulatory, and Preparative Actions. 
Endocrine Reviews 21, 55-89. 
Seber, G.A.F., Wild, C.J., 1989. Nonlinear regression. John Wiley and Sons, New York. 
 35 
Skerratt, L., Berger, L., Speare, R., Cashins, S., McDonald, K., Phillott, A., Hines, H., Kenyon, 
N., 2007. Spread of Chytridiomycosis Has Caused the Rapid Global Decline and Extinction of 
Frogs. EcoHealth 4, 125-134. 
Stefano, F.J.E., Donoso, A.O., 1964. Hypophyso-adrenal regulation of moulting in the toad. 
General and Comparative Endocrinology 4, 473-480. 
Stiffler, D.F., De Ruyter, M.L., Hanson, P.B., Marshall, M., 1986. Interrenal function in larval 
Ambystoma tigrinum: I. Responses to alterations in external electrolyte concentrations. General 
and Comparative Endocrinology 62, 290-297. 
Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, A.S.L., Fischman, D.L., Waller, 
R.W., 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science 306, 
1783-1786. 
Sures, B., Knopf, K., Kloas, W., 2001. Induction of stress by the swimbladder nematode 
Anguillicola crassus in European eels, Anguilla anguilla, after repeated experimental infection. 
Parasitology 123, 179-184. 
Sures, B., Scheef, G., Klar, B., Kloas, W., Taraschewski, H., 2002. Interaction between cadmium 
exposure and infection with the intestinal parasite Moniliformis moniliformis (Acanthocephala) 
on the stress hormone levels in rats. Environmental Pollution 119, 333-340. 
Turner, R.J., 1988. Amphibians, in: A.F. Rowley, N.A. Ratcliffe (Eds.), Vertebrate blood cells. 
Cambridge University Press, Cambridge, UK, 129-209. 
Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W.F., Dinudom, A., Cook, D., Webb, 
R., Alford, R.A., Skerratt, L.F., Speare, R., 2009. Pathogenesis of Chytridiomycosis, a Cause of 
Catastrophic Amphibian Declines. Science 326, 582-585. 
Wack, C.L., DuRant, S.E., Hopkins, W.A., Lovern, M.B., Feldhoff, R.C., Woodley, S.K., 2012. 
Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander. Comparative 
Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology 161, 153-158. 
Ward, C.K., Appel, A.G., Mendon?a, M.T., 2006. Metabolic measures of male southern toads 
(Bufo terrestris) exposed to coal combustion waste. Comparative Biochemistry and Physiology - 
Part A: Molecular &amp; Integrative Physiology 143, 353-360. 
Ward, C.K., Fontes, C., Breuner, C.W., Mendon?a, M.T., 2007. Characterization and 
quantification of corticosteroid-binding globulin in a southern toad, Bufo terrestris, exposed to 
coal-combustion-waste. General and Comparative Endocrinology 152, 82-88. 
Warne, R.W., Crespi, E.J., Brunner, J.L., 2011. Escape from the pond: stress and developmental 
responses to ranavirus infection in wood frog tadpoles. Functional Ecology 25, 139-146. 
Wingfield, J.C., Maney, D.L., Breuner, C.W., Jacobs, J.D., Lynn, S., Ramenofsky, M., 
Richardson, R.D., 1998. Ecological Bases of Hormone?Behavior Interactions: The ?Emergency 
Life History Stage?. American Zoologist 38, 191-206. 
 36 
Woodhams, D.C., Ardipradja, K., Alford, R.A., Marantelli, G., Reinert, L.K., Rollins-Smith, 
L.A., 2007. Resistance to chytridiomycosis varies among amphibian species and is correlated 
with skin peptide defenses. Animal Conservation 10, 409-417. 
Yorio, T., Bentley, P.J., 1978. Stimulation of the short-circuit current (sodium transport) across 
the skin of the forg (Rana pipiens) by corticosteroids: structure-activity relationships. Journal of 
Endocrinology 79, 283-290. 
Zerani, M., Amabili, F., Mosconi, G., Gobbetti, A., 1991. Effects of captivity stress on plasma 
steroid levels in the green frog, rana esculenta, during the annual reproductive cycle. 
Comparative Biochemistry and Physiology Part A: Physiology 98, 491-496. 
 
 
 
 37 
 
 
 
 
 
Chapter 2. Physiological progression of the amphibian decline-causing disease, 
chytridiomycosis. 
 
Abstract 
 The fungal disease, chytridiomycosis, is considered the largest infectious disease threat to 
global biodiversity because of its world-wide catastrophic effects on amphibian populations.  
Pathogenesis appears to involve complex interactions among physiological systems; however, 
relatively few studies have observed how the infectious agent, Batrachochytrium dendrobatidis 
(Bd), alters host physiology.  Even fewer studies have monitored physiological changes prior to 
and throughout infection.  In order to evaluate physiological changes associated with Bd 
infection, we infected Litoria caerulea with Bd and measured changes in physiological variables 
prior to and throughout infection.  Prior to showing clinical signs of disease, infected frogs first 
experienced hyponatremia and hypokalemia, followed by reduced food intake and body mass 
and increased metabolic rate.  When infected frogs became diseased, they experienced further 
plasma ion depletion accompanied by increased baseline plasma glucocorticoid levels and 
neutrophil-lymphocyte ratios.  We suggest that the combined effects of reduced cutaneous and 
digestive sodium uptake, greatly contribute to the fatal hyponatremia observed during 
chytridiomycosis.   
1. Introduction 
Chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd), 
is linked to amphibian population declines world-wide.  Pathogenesis of chytridiomycosis 
 38 
involves disruption of cutaneous sodium channels, hyponatremia, and cardiac failure (Voyles et 
al., 2009).  Additional described aspects of the fungal induced pathogenesis include increased 
baseline plasma corticosterone (CORT) levels (see Chapter 1 of this dissertation), altered 
numbers of circulating leukocytes (Davis et al., 2010; Woodhams et al., 2007a) increased 
metabolic rate (see chapter 1 of this dissertation), decreased appetite (Nichols et al., 2001; 
Voyles et al., 2009), alteration of normal skin shedding processes (see chapter 1 of this 
dissertation; Nichols et al., 2001; Voyles et al., 2009), weight loss (Harris et al., 2009; Retallick 
and Miera, 2007) and decreased body condition (see chapter 1 of this dissertation).   
In amphibians, glucocorticoids (i.e., primarily CORT) routinely regulate a host of related 
physiological processes that are potentially associated with the previously observed aspects of 
chytridiomycosis pathogenesis, such as changes in plasma sodium homeostasis (Brewer et al., 
1980; De Ruyter and Stiffler, 1986; Heney and Stiffler, 1983; Middler et al., 1969; Stiffler et al., 
1986; Yorio and Bentley, 1978), immunity (Belden and Kiesecker, 2005; Bennett et al., 1972; 
Bennett and Harbottle, 1968; Davis and Maerz, 2010; Garrido et al., 1987) metabolism (Wack et 
al., 2012), appetite (Crespi and Denver, 2005; Crespi et al., 2004) and skin shedding (Budtz, 
1979; J?rgensen and Larsen, 1961, 1964; Stefano and Donoso, 1964).  Thus, CORT may be 
mediating aspects of pathogenesis.  Non-pathogen-induced hyponatremia causes CORT secretion 
in amphibians in order to regulate plasma sodium concentration (Stiffler et al., 1986).  Also, 
CORT treatment can correct non-pathogen-induced hyponatremia (Heney and Stiffler, 1983), so 
it is likely that pathogen-induced hyponatremia would also increase CORT levels in amphibians.  
If CORT is ineffective at rebalancing sodium homeostasis, then chronic activation of the stress 
axis may occur resulting in continued elevation in CORT leading to immunomodulation (Belden 
and Kiesecker, 2005; Bennett et al., 1972; Bennett and Harbottle, 1968; Davis and Maerz, 2010; 
 39 
Garrido et al., 1987), elevated metabolic rate (Wack et al., 2012), and appetite suppression 
(Crespi et al., 2004).  The latter effect would reduce digestive sodium uptake and further depress 
plasma sodium levels until they become too low to provide cardiac conductivity (Voyles et al., 
2009).  This set of events could, rather than help regulate sodium levels, further reduce sodium 
levels, thus exacerbating disease.   
During a laboratory outbreak of Bd in the Australian Green Tree Frog, Litoria caerulea, 
we observed a significant elevation in baseline plasma CORT and contemporaneous changes in 
plasma sodium and potassium, leukocyte profiles, metabolism, food intake, and body mass (see 
chapter 1 of this dissertation).  Most of these variables were only monitored at one time point 
within infection, making it difficult to determine when symptoms develop.  Identification of 
when ion levels, CORT, and related variables change during infection would provide better 
documentation of when pathologies develop and when intervention could be beneficial.  In an 
attempt to document when physiological changes occur we infected L. caerulea with Bd, and 
monitored changes in the aforementioned variables prior to and throughout infection.  We predict 
that hyponatremia will be the first ill effect of infection, followed by alterations in plasma 
CORT, leukocyte profiles, metabolic rate, appetite, and weight loss. 
 
2. Methods 
2.1 Experimental design and blood collection 
Litoria caerulea were obtained commercially (Tri-Reptile, Miami, FL) in summer 2010.  Frogs 
were recently collected from the wild in Indonesia.  All frogs were heat-treated following 
established protocols (Woodhams et al., 2003) to rid them of previous Bd infections.  Although 
this treatment did not completely rid frogs of Bd, any retained infections remained light 
 40 
throughout the study.  Approximately two weeks after heat treatment, pre-infection measures 
were taken for a subset of frogs (n=8) for resting metabolic rate, body mass, and food intake.  
These same frogs were swabbed for Bd burden and sacrificed to obtain blood for plasma ions, 
plasma CORT, and blood leukocytes.  On the same day, all remaining frogs were infected with 
methods detailed below.  Resting metabolic rate, body mass, food intake, Bd burden, and blood 
parameters were assessed in subsets of frogs early in infection (3-9 days post infection; DPI), 
midway through infection (30-39 DPI), and late in infection (32-88 DPI).  Early in infection and 
midway through infection amphibians were sampled for Bd burden and blood variables on 
specific days (i.e. 7 and 39 DPI, respectively), but late in infection these variables were assessed 
when individuals became clinically diseased (i.e. listless, odd body posture, and skin 
discoloration; (Berger et al., 2005)).  When an individual became diseased it was sacrificed along 
with a control individual of similar body size.  During sacrifice, the frog was swabbed (to 
quantify Bd zoospores), pithed, and bled (within 3 minutes of handling).  Several drops of whole 
blood were used to make blood smears for enumeration of leukocytes.  The remaining blood was 
centrifuged for 4 minutes at 3,500 x g and the plasma was frozen and stored at -20? C for later 
use in corticosterone radioimmunoassay and ion analyses. 
2.2 Animal Care 
Amphibians were housed individually in plastic containers (17 cm x 17 cm x 17 cm) 
containing paper towels saturated with well water.  Wet paper towels were replaced twice a week 
for the duration of the study.  Animals were fed as stated below under ?food intake and body 
mass?.  Light was provided by full spectrum light bulbs on a 12:12 light/dark cycle.  Room 
temperature was controlled by a thermostat (~22? C). 
2.3 Bd culture and experimental infection 
 41 
 Batrachochytrium dendrobatidis cultures were maintained in 10% tryptone broth.  Prior 
to infection 0.25 ml of broth was placed on agar plates.  Following desiccation of the broth, 
plates were stored for a week at 16? C.  Plates were then flooded with 3 ml of broth, which was 
poured off an hour later.  Bd zoospores were counted using a hemacytometer and each frog was 
inoculated once with 20 ?l of either broth containing approximately one million zoospores (Bd) 
or broth containing no zoospores (control).    
2.4 Bd zoospore burden 
Frogs were swabbed in a standardized fashion by lightly running a sterile cotton swab 
(Medical Wire & Equipment, MW113) 10 times over the sides, venter, and ventral thighs and 5 
times over the underside of each foot (Kriger et al., 2006).  Zoospore equivalents were 
determined by standard extraction and quantitative PCR techniques (Boyle et al., 2004; Ramsey 
et al., 2010).  Nucleic acids were extracted by adding 60 ?l of PrepMan Ultra (Applied 
Biosystems, Foster City, CA) and 30-35 mg of Zirconium/silica beads (0.5 mm, Biospec 
Products, Bartlesville, OK) to the tip of each swab.  Samples were homogenized for 45 s in a 
Mini Beadbeater (MP Bio, Solon, OH) and centrifuged for 30 s at 15,000 x g.  After a second 
homogenization and centrifugation, the samples were boiled for 10 min, returned to room 
temperature for 2 minutes, and centrifuged at 15,000 x g for 3 min.  Nucleic acids in the 
supernatant were removed for real-time PCR.  Samples were loaded into an Mx3000P Real-Time 
PCR system (Stratagene, La Jolla, CA) for 40 cycles of 95? C for 10 min, 95? C for 30 s, 55? C 
for 1 min, and 72? C for 1 min.  Zoospore equivalents were determined using a standard curve 
and indicate Bd burden.   
2.5 Ion analyses 
 42 
Plasma prepared by centrifugation of whole blood for 4 minutes at 3,500 x g to remove 
red blood cells, was analyzed by Inductively Coupled Plasma with Optical Emission 
Spectroscopy (ICP-OES, Perkin Elmer 7100 DV, Waltham, MA) with simultaneous 
measurement of Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, P, S, Zn.  Equal volume of plasma was 
diluted into ultra-pure, metal-free water (MilliQ, Millipore) then centrifuged at 13,000 x g to 
remove particulates and then introduced directly into the instrument argon plasma using a 
cyclonic nebulizer. Metal concentrations are determined comparing emission intensities to a 
standard curve created from certified metal standards (SPEX, Metuchen, NJ). Standard curves 
were confirmed by re-analysis of standard solutions diluted in a matrix equivalent to the sample. 
Individual readings are the average of two intensity measurements varied by less than 5%. 
Repeated analysis of individual samples showed less the 5% variability. 
2.6 Radioimmunoassay 
Plasma corticosterone concentrations were determined by radioimmunoassay as 
described by Mendon?a et al. (1996).  All samples were run in three assays.  Average extraction 
efficiency was 73.7%.  Average intraassay variation was 10.9% and interassay variation was 
13.3%.   
2.7 Relative leukocyte counts 
Dried blood smears were stained with a Hema 3 kit (Fisher scientific, Kalamazoo, MI) 
and viewed under a light microscope.  Slides were read in a standard zig-zag fashion.  One 
hundred leukocytes were observed and numbers of neutrophils, lymphocytes, eosinophils, 
monocytes, and basophils were recorded.  Leukocytes were identified following Turner (1988) 
and Hadji-Azimi et al. (1987).  Leukocyte data are presented as neutrophil-lymphocyte ratios 
(NL ratios).   
 43 
2.8 Respirometry 
 Closed system respirometry was used to measure resting metabolic rate (oxygen 
consumption) 19 days prior to infection (pre-infection), early in infection (9 DPI), midway 
through infection (30 DPI), and late in infection (range: 30-51 days post infection and 5-8 days 
prior to sacrifice) following the methods of Ward et al. (2006) with the following changes.  Frogs 
were acclimated in 140 ml syringes (Monoject, Sherwood Medical Industries, Ballymoney, UK) 
for at least 45 minutes in a darkened incubator (25?C).  Frogs were incubated for ~50 minutes in 
a darkened incubator (25?C).  Any frogs that urinated or defecated during incubation were 
excluded from analyses.  Frogs were weighed following respirometry.  Respirometry data are 
presented as ml of oxygen consumed per gram of body mass per hour.   
2.9 Food intake and body mass 
Body mass and food intake were determined on consecutive days 18 and 17 days prior to 
infection, early in infection (3 and 4 DPI), midway through infection (31 and 32 DPI), and late in 
infection (31-79 DPI), respectively.  Body mass and food intake late in infection were assessed 
an average of 5 and 7 days prior to when frogs became diseased (range: 0-10 and 2-11), 
respectively.  Each frog was weighed and fed approximately 10% of its body weight in 2.5 week 
old crickets coated in multi-vitamin dust.  All crickets not consumed were weighed after 24 
hours.  Food intake was determined as the mass of the crickets not consumed subtracted from the 
original mass of crickets given to the frog.   
2.10 Statistical analyses 
Bd burden, plasma sodium, plasma corticosterone, neutrophil-lymphocyte ratios, and 
food intake were compared among time points for individuals sampled prior to infection and 
infected individuals with analyses of variance (ANOVAs).  Oxygen consumption was compared 
 44 
among time points with an analysis of covariance (ANCOVA) with time as the independent 
variable, oxygen consumed as the dependent variable, and body mass as the covariate.  Since 
body mass influences oxygen consumption, oxygen consumption is presented as least squared 
means, corrected for body mass.  Fisher?s protected least significant difference (Fisher?s PLSD) 
tests were used for all post-hoc comparisons among time points.  An additional ANOVA was 
used to compare the above variables (except for oxygen consumption) between infected 
individuals that became diseased and controls.  An additional ANCOVA was used to compare 
oxygen consumption between infected individuals that became diseased and controls (with body 
mass as the covariate).  Corticosterone values were log+1 transformed prior to all statistical 
analyses to better fit the assumptions of ANOVA; however, non-transformed values are 
presented in the results and figures for ease of interpretation.  Change in body mass and food 
intake prior to becoming diseased were compared between treatments with repeated measures 
ANOVAs.  We used StatView for Windows (SAS institute, version 5.0.1) for all statistical 
analyses. 
 
3. Results 
3.1 Bd burden 
Bd burden of infected frogs increased significantly throughout infection (ANOVA, P=0.001, 
F3,27=7.15).  Three of the eight frogs sampled prior to infection contained light Bd infections 
(range: 17-28 zoospore equivalents).  Infected individuals at 7 and 39 days post infection 
contained moderate Bd burdens (mean: 13,870 and 18,700, respectively) that were significantly 
higher than individuals sampled prior to infection and significantly lower than diseased 
individuals, who contained an average of 16.5 million zoospore equivalents (range: 1.8-50.9 
 45 
million; Fisher?s PLSD post hoc tests P?0.001).  Control individuals sampled with diseased 
individuals contained significantly fewer zoospore equivalents than diseased individuals 
(ANOVA, P=0.029, F1,12=6.16).  Control individuals sampled at 7 and 39 days post sham-
infection contained light Bd infections (average: 28 and 39, respectively). 
3.2 Plasma ions 
Plasma sodium and potassium of infected frogs decreased significantly throughout infection 
(ANOVA, P<0.001, F3,19=11.81; ANOVA, P=0.001, F3,19=7.72; respectively).  Plasma of frogs 
bled at 39 days post infection contained 22% and 33% less sodium and potassium, respectively, 
than individuals sampled prior to infection, while plasma of diseased frogs contained 48% and 
49% less sodium and potassium, respectively.  Plasma of diseased frogs contained approximately 
half as much sodium as frogs bled prior to infection and one week post-infection (Fisher?s PLSD, 
P<0.05).  Infected frogs that became diseased also contained 47% less plasma sodium and 
potassium than control frogs sampled at the same time (ANOVA, P<0.001, F1,9=29.85; ANOVA, 
P<0.001, F1,9=24.19; respectively).   
3.3 CORT 
Baseline plasma CORT increased significantly throughout infection in infected individuals 
(ANOVA, P=0.015, F3,24=4.25).  When infected frogs became diseased they contained an 
average of 111 ng/ml CORT, representing a 7.6 fold increase compared to individuals sampled at 
39 days post infection (Fisher?s PLSD, P?0.019) and a 25.7 fold increase compared to control 
individuals sampled concurrently (ANOVA, P=0.005, F1,9=13.68). 
3.4 NL ratios 
Neutrophil-lymphocyte ratios increased throughout infection in infected frogs (ANOVA, 
P=0.047, F3,20=3.17).  Ratios remained low through 7 days post infection and were significantly 
 46 
elevated in infected frogs that became diseased (Fisher?s PLSD, P<0.05).  Infected frogs that 
became diseased contained more than twice as many neutrophils compared to lymphocytes while 
control frogs at the same time point contained the opposite, more than twice as many 
lymphocytes compared to neutrophils (ANOVA, P=0.005, F1,9=13.69). 
3.5 Respirometry 
Oxygen consumption increased significantly throughout infection in infected frogs (ANCOVA, 
Time: P=0.023, F3,20=3.97; Body mass: P=0.009, F1,20=8.51).  There was no time by treatment 
interaction (P=0.096).  Infected frogs sampled an average of 6 days prior to becoming diseased 
consumed significantly more oxygen than frogs evaluated prior to infection and nine days post 
infection (Fisher?s PLSD, P<0.05).  These same frogs also consumed 1.8 times more oxygen 
than control frogs sampled concurrently (ANCOVA, Treatment: P=0.032, F1,5=8.73; Body mass: 
P=0.4, F1,5=0.76).  There was no treatment by body mass interaction (P=0.9). 
3.6 Food intake 
Food intake decreased in infected frogs over the course of the experiment (ANOVA, P=0.009, 
F3,24=4.90).  An average of seven days prior to displaying clinical signs of disease, infected 
individuals consumed significantly fewer grams of crickets than individuals sampled prior to 
infection and infected individuals sampled at four and 32 days post infection (Fisher?s PLSD, 
P<0.05).  Infected individuals sampled an average of seven days prior to displaying clinical signs 
of disease consumed approximately 7% the amount of crickets consumed by frogs sampled prior 
to infection (Fisher?s PLSD, p=0.002) and control individuals sampled at the same time 
(ANOVA, P<0.001, F1,9=160.36).  Food intake was significantly lower for at least an average of 
15 days prior to becoming diseased compared to control individuals samples concurrently 
 47 
(Repeated measures ANOVA, Treatment: P<0.0001, F1,7=86.17, Time: P=0.3, F1,7=1.06, 
Treatment x Time: p=0.4, F1,7=0.91). 
3.7 Change in body mass 
Infected frogs experienced significantly more change in mass throughout the experiment 
compared to control frogs (Repeated measures ANOVA, Treatment: p=0.004, F1,12=13.02, Time: 
p=0.024, F2,24=4.38, Treatment x Time: p=0.8, F2,24=0.22).  Control frogs gained mass up to 31 
days post infection and then lost 0.5 grams between 31 days post infection and time points prior 
to sacrifice; whereas infected individuals lost 0.22 grams of mass between 3 and 31 days post 
infection and lost 2.9 grams between 31 days post infection and time points prior to sacrifice.   
4. Discussion 
This is the first study to document the timeline of endocrine, immune, and metabolic 
changes throughout Bd infection.  In another experiment that monitored effects of Bd infection 
over time, Voyles et al. (2009) monitored changes in ions and major molecules found in the 
blood and urine.  Both of these studies observed that Litoria caerulea exhibit little change in 
variables during the first 30-39 days post infection.  Despite previous suggestion that inappetance 
may be the earliest clinical sign of disease (Nichols et al., 2001), we observed that hyponatremia 
and hypokalemia preceded changes in any other variables monitored in this study, including 
appetite.  Unfortunately, it is unclear how many days this change in plasma ions predates 
changes in appetite.  Our experimental design set certain sample times, but there was variation 
when animals displayed clinical signs of disease when they were sampled late in infection.  
Thus, we could not completely capture changes in plasma ions in the time before animals 
became diseased.   
 48 
Bd burdens were not a good predictor of pathogenesis.  Bd burdens of frogs already 
showing signs of hyponatremia and hypokalemia at 39 days post infection had highly variable Bd 
burdens (actual data: 50; 466; 2,714; 4,366; 15,156; and 89,448).  For example, the individual 
with the lowest Bd burden (50 zoospore equivalents) contained the lowest amounts of plasma 
sodium and potassium (62 and 1.75 mM/l, respectively), yet previous data suggests that plasma 
sodium is positively correlated with Bd burden (Voyles et al., 2009).  Even though Bd burdens 
may be informative for predicting epidemiology (Kinney et al., 2011; Vredenburg et al., 2010), 
our data suggest the use of Bd burdens in lab studies of pathogenesis would be less informative 
and not predictive.   
Although hyponatremia and hypokalemia were the earliest physiological changes to 
occur, they took several weeks to manifest.  We observed no changes in plasma ions at seven 
days post infection and Voyles et al. (2009) observed no changes at 30 days post infection, but in 
our study, by day 39, we observed significant decreases in both plasma sodium and potassium.  
Several factors may provide protection from Bd-induced ion loss.  During these initial weeks of 
infection, epidermal antimicrobial peptides and anti-fungal bacteria, which have the ability to 
inhibit Bd growth in vitro (Harris et al., 2006; Woodhams et al., 2007a; Woodhams et al., 
2007b), likely provide some protection to the skin.  Once Bd colonizes the skin and individuals 
show signs of disease, sodium uptake across the skin is significantly suppressed, leading to 
plasma ion loss (Voyles et al., 2009).  During hyponatremia and hypokalemia, corticoid 
hormones (i.e. CORT and aldosterone) function to rebalance plasma ion levels in amphibians 
(De Ruyter and Stiffler, 1986; Heney and Stiffler, 1983; Stiffler et al., 1986).  In fact, at 39 days 
post infection, the individuals with the highest CORT levels (e.g., 27.4 and 46.9 ng/ml) 
contained plasma sodium levels (120 and 102 mM/l) similar to controls.  These individuals also 
 49 
displayed elevated NL ratios (1.6 and 3.8), and oxygen consumption (0.13 and 0.21 ml/g/hr) 
equivalent to those observed in frogs undergoing a stress response (Belden and Kiesecker, 2005; 
Bennett et al., 1972; Bennett and Harbottle, 1968; Coddington and Cree, 1995; Davis and Maerz, 
2010; Garrido et al., 1987; Jurani et al., 1973).  Although average CORT, NL ratios, and oxygen 
consumption were not significantly altered around 39 days post infection, these data points 
suggest that elevated CORT may be helping to rebalance plasma sodium, shifting NL ratios and 
increasing oxygen consumption.  Since glucocorticoid treatment can correct hyponatremia in 
humans and amphibians (Davis et al., 1969; Heney and Stiffler, 1983; Kamoi et al., 1993; 
Merriam and Baer, 1980), future studies could test if CORT or aldosterone treatment potentially 
correct Bd-induced ion imbalances early in infection. 
Our results suggest that the observed initial effects on plasma ions at 39 days post 
infection do not appear to be linked to food intake.  At 39 days post infection, infected 
individuals contained 22% and 33% less plasma sodium and potassium, respectively, than 
individuals sampled prior to infection, even though, seven days earlier, they had consumed a 
?normal? amount of food (i.e. an amount of food similar to individuals sampled prior to infection 
and four days post infection).  Although infected frogs apparently had a diet source of sodium, 
their plasma sodium levels decreased substantially, indicating that food intake could not 
completely compensate for decreased sodium input via the skin.   
Although CORT secretion during Bd infection may initially rebalance sodium 
homeostasis to a certain extent, it is clear that these elevated levels are seemingly ineffective by 
the time individuals display clinical signs of disease.  By the time individuals became diseased, 
CORT levels were 7.6 times higher than individuals sampled at 39 days post infection; however, 
plasma sodium levels continued to decrease.  In fish and mammals, low glucocorticoids can 
 50 
stimulate appetite, but stressors and high glucocorticoids can suppress appetite (Bernier, 2006; 
Dallman et al., 1993).  Thus, the observed, potentially chronic elevation of CORT or other 
aspects of the stress response, such as corticotropin-releasing factor (Crespi et al., 2004), would 
suppress appetite and, thus, decrease digestive sodium uptake, worsening the animal?s sodium 
balance instead of rescuing it.  Although we observed that food intake in infected frogs was 
significantly suppressed at least 15 days prior to when they became diseased, we were unable to 
determine whether CORT caused this suppression, due to limitations of our data.  In order to 
determine if CORT suppresses appetite during infection with Bd, future studies documenting 
food intake and CORT levels late in infection are needed.  
Our data and data from the first chapter of this dissertation suggest that by the time frogs 
display clinical signs of disease they are secreting glucocorticoids that are at levels that rival the 
highest average levels of CORT observed in amphibians in response to stressors (Coddington 
and Cree, 1995; Gobbetti and Zerani, 1996; Hopkins et al., 1997; Jurani et al., 1973; Zerani et 
al., 1991).  Although, increased glucocorticoid levels may have worked to rebalance plasma ion 
homeostasis earlier in infection, increased baseline glucocorticoid levels may also have caused 
some of the changes we observed in leukocyte profiles, metabolic rate, appetite, body mass, and 
body condition.  Our results mirror those from chapter 1 of this dissertation, with infected 
individuals containing significantly altered leukocyte profiles, increased resting metabolic rate, 
decreased food intake, decreased body mass, and decreased body condition around the time 
when they become diseased.  Appetite suppression may be the worst of the effects during 
infection, because of its aforementioned effects on plasma ions.  With little cutaneous and 
digestive ion inputs, plasma ion levels eventually become unsustainable and death follows.   
 51 
The time course that we present does not follow our prediction that CORT levels would 
increase soon after sodium levels decreased.  Sampling individuals at time points following 39 
days post infection could identify when CORT levels significantly increase.  As plasma ions 
began to decrease, we observed data points that suggested that elevated CORT values may act to 
rebalance plasma ion homeostasis.  Future studies that tease apart the precise mechanisms of 
chytridiomycosis pathogenesis may help researchers treat Bd-infected frogs as well as contribute 
to understanding why there is such large variation in susceptibility to chytridiomycosis among 
amphibian species.  Given the devastating effects chytridiomycosis is having on amphibian 
populations, understanding these mechanisms may prove essential to conservation of amphibians 
in the future. 
 
 52 
Figures: 
0
10 1
Pre -
inf ection
7 day s pos t 
inf ection
39 day s pos t 
inf ection
Con tr ol
Bd
6 78
7
7
8
10
10 2
10 3
10 4
10 5
10 6
10 7
10 8
Bd 
zoos
pores
 (
DN
A
 equ
ivalents)
A
B
C
Disea sed
B
*
A
0
20
40
60
80
100
120
140
160
180
200
Plasma 
corti
cos
terone (
ng/ml
) Con tr ol
Bd
6
5
8 7 6
8 9
Pre -
inf ection
7 day s pos t 
inf ection
39 day s pos t 
inf ection
A
B
Disea sed
A
A
*
B
 
0
20
40
60
80
100
120
140
Plasm
a 
sodium
 (mM
/L)
Co ntrol
Bd
6 57 6 66 6
Pre -
inf ection
7 day s pos t 
inf ection
39 day s pos t 
inf ection
A
C
Disea sed
AB
B
*
C
Co ntrol
Bd
Pre -
inf ection
7 day s pos t 
inf ection
39 day s pos t 
inf ection
Disea sed
Plasm
a p
ot
assi
um 
(mM
/L)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
6 57 6 66 6
A
B
AB
B
*
4.0
4 .5
D
 
 
Fig. 2.1. Average (A) Bd burden, (B) baseline plasma corticosterone, (C) plasma sodium, (D) 
plasma potassium, (E) Neutrophil-lymphocyte ratio, and (F) oxygen consumption of Litoria 
caerulea either infected with Batrachochytrium dendrobatidis (Bd) or uninfected controls 
 53 
assessed prior to and throughout infection (+SEM).  Values reported at the ?Diseased? time point 
were assessed when infected frogs displayed clinical signs of disease (Range: 32-88 days post 
infection).  Values reported at the ?Late Infection? time point were assessed an average of six 
days prior to when infected frogs displayed clinical signs of disease.  Significant differences 
among time points for infected frogs are denoted by upper case letters.  Significant differences 
between treatments at the Diseased/Late infection timepoints are denoted by an asterisk.  
Numbers inside bars denote sample sizes.   
 54 
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Food
 int
ake
 (g)
Con tr ol
Bd
8 7 7
6
6 7 5
Pre -
inf ection
4 day s pos t 
inf ection
32 day s pos t 
inf ection
Late 
inf ection
A
A
B
A
*
A
0
0.5
1.0
1.5
2.0
2.5
3.0
A v . 15 d 
prior to s acrif ice 
A v . 8 d
prior to s acrif ice
Co ntrol n=4
Bd n=5
Food
 int
ake
 (g)
B
 
Fig. 2.2. Average food intake of Litoria caerulea infected with Batrachochytrium dendrobatidis 
or uninfected control frogs assessed either (A) prior to and throughout infection or (B) repeatedly 
within an average of 15 days prior to when infected individuals displayed signs of disease 
(+SEM).  Values reported at the ?Late infection? time point were assessed an average of seven 
days prior to when infected frogs displayed clinical signs of disease.  Significant differences 
among time points for infected frogs are denoted by upper case letters.  The significant 
difference between treatments at the ?Late infection? time point is denoted by an asterisk.  
Numbers inside bars denote sample sizes.  Repeated measures of food intake (B) revealed a 
significant difference between treatments within an average of 15 days prior to sacrifice.   
 55 
- 5
- 4
- 3
- 2
- 1
0
1
2
3
4
5
Pre - infect ion -
3 DPI
Cha
nge
 i
n 
body
 mass 
(g)
Con tr ol n=7
Bd          n =7
3 DPI -
31 DPI
31 DPI -
A v . 6 day s prior
to sacrifi ce
 
Fig. 2.3. Average change in body mass between time points prior to and throughout infection of 
control Litoria caerulea and frogs infected with Batrachochytrium dendrobatidis (Bd).  Infected 
frogs lost significantly more mass than controls across time periods.   
 56 
References: 
Belden, L.K., Kiesecker, J.M., 2005. Glucocorticosteroid Hormone Treatment of Larval 
Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91, 
686-688. 
Bennett, M.F., Gaudio, C.A., Johnson, A.O., Spisso, J.H., 1972. Changes in the blood of 
newts,&lt;i&gt;Notophthalmus viridescens , following the administration of hydrocortisone. 
Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral 
Physiology 80, 233-237. 
Bennett, M.F., Harbottle, J.A., 1968. The effects of hydrocortisone on the blood of tadpoles and 
frogs, Rana catesbeiana. The Biological Bulletin 135, 92-95. 
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. 
Bernier, N.J., 2006. The corticotropin-releasing factor system as a mediator of the appetite-
suppressing effects of stress in fish. General and Comparative Endocrinology 146, 45-55. 
Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T., Hyatt, A.D., 2004. Rapid quantitative 
detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using 
real-time Taqman PCR assay. Diseases of Aquatic Organisms 60, 141-148. 
Brewer, K.J., Hoyt, B.J., McKeown, B.A., 1980. The effects of prolactin, corticosterone and 
ergocryptine on sodium balance in the urodele, Ambystoma gracile. Comparative Biochemistry 
and Physiology Part C: Comparative Pharmacology 66, 203-208. 
Budtz, P.E., 1979. Epidermal structure and dynamics of the toad, Bufo bufo, deprived of the pars 
distalis of the pituitary gland. Journal of Zoology 189, 57-92. 
Coddington, E.J., Cree, A., 1995. Effect of Acute Captivity Stress on Plasma Concentrations of 
Corticosterone and Sex Steroids in Female Whistling Frogs, Litoria ewingi. General and 
Comparative Endocrinology 100, 33-38. 
Crespi, E.J., Denver, R.J., 2005. Roles of stress hormones in food intake regulation in anuran 
amphibians throughout the life cycle. Comparative Biochemistry and Physiology - Part A: 
Molecular &amp; Integrative Physiology 141, 381-390. 
Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of Corticotropin-Releasing Factor, 
Neuropeptide Y and Corticosterone in the Regulation of Food Intake In Xenopus laevis. Journal 
of Neuroendocrinology 16, 279-288. 
Dallman, M.F., Strack, A.M., Akana, S.F., Bradbury, M.J., Hanson, E.S., Scribner, K.A., Smith, 
M., 1993. Feast and Famine: Critical Role of Glucocorticoids with Insulin in Daily Energy Flow. 
Frontiers in Neuroendocrinology 14, 303-347. 
 57 
Davis, A., Keel, M., Ferreira, A., Maerz, J., 2010. Effects of chytridiomycosis on circulating 
white blood cell distributions of bullfrog larvae (Rana catesbeiana). Comparative Clinical 
Pathology 19, 49-55. 
Davis, A.K., Maerz, J.C., 2010. Effects of Exogenous Corticosterone on Circulating Leukocytes 
of a Salamander (Ambystoma talpoideum) with Unusually Abundant Eosinophils. International 
Journal of Zoology 2010. 
Davis, B.B., Bloom, M.E., Field, J.B., Mintz, D.H., 1969. Hyponatremia in pituitary 
insufficiency. Metabolism 18, 821-832. 
De Ruyter, M.L., Stiffler, D.F., 1986. Interrenal function in larval Ambystoma tigrinum: II. 
Control of aldosterone secretion and electrolyte balance by ACTH. General and Comparative 
Endocrinology 62, 298-305. 
Garrido, E., Gomariz, R.P., Leceta, J., Zapata, A., 1987. Effects of dexamethasone on the 
lymphoid organs of Rana perezi. Developmental &amp; Comparative Immunology 11, 375-384. 
Gobbetti, A., Zerani, M., 1996. Possible mechanism for the first response to short captivity stress 
in the water frog, Rana esculenta. Journal of Endocrinology 148, 233-239. 
Hadji-Azimi, I., Coosemans, V., Canicatti, C., 1987. Atlas of adult Xenopus laevis laevis 
hematology. Developmental and comparative immunology 11, 807-874. 
Harris, R., James, T., Lauer, A., Simon, M., Patel, A., 2006. Amphibian Pathogen 
&lt;i&gt;Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian 
Species. EcoHealth 3, 53-56. 
Harris, R.N., Brucker, R.M., Walke, J.B., Becker, M.H., Schwantes, C.R., Flaherty, D.C., Lam, 
B.A., Woodhams, D.C., Briggs, C.J., Vredenburg, V.T., Minbiole, K.P.C., 2009. Skin microbes 
on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3, 818-824. 
Heney, H.W., Stiffler, D.F., 1983. The effects of aldosterone on sodium and potassium 
metabolism in larval Ambystoma tigrinum. General and Comparative Endocrinology 49, 122-
127. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1997. Increased Circulating Levels of 
Testosterone and Corticosterone in Southern Toads,Bufo terrestris,Exposed to Coal Combustion 
Waste. General and Comparative Endocrinology 108, 237-246. 
Jurani, M., Murgas, K., Mikulaj, L., Babusikova, F., 1973. Effect of stress and environmental 
temperature on adrenal function in Rana esculenta. Journal of Endocrinology 57, 385-391. 
J?rgensen, C.B., Larsen, L.O., 1961. Molting and its hormonal control in toads. General and 
Comparative Endocrinology 1, 145-153. 
J?rgensen, C.B., Larsen, L.O., 1964. Further observations on molting and its hormonal control in 
Bufo bufo (L.). General and Comparative Endocrinology 4, 389-400. 
 58 
Kamoi, K., Tamura, T., Tanaka, K., Ishibashi, M., Yamaji, T., 1993. Hyponatremia and 
osmoregulation of thirst and vasopressin secretion in patients with adrenal insufficiency. Journal 
of Clinical Endocrinology & Metabolism 77, 1584-1588. 
Kinney, V.C., Heemeyer, J.L., Pessier, A.P., Lannoo, M.J., 2011. Seasonal Pattern of 
<italic>Batrachochytrium dendrobatidis</italic> Infection and Mortality in <italic>Lithobates 
areolatus</italic>: Affirmation of Vredenburg's ?10,000 Zoospore Rule?. PLoS ONE 6, e16708. 
Kriger, K.M., Hero, J.M., Ashton, K.J., 2006. Cost efficiency in the detection of 
chytridiomycosis using PCR assay. ePublications@bond. 
Mendon?a, M.T., Chernetsky, S.D., Nester, K.E., Gardner, G.L., 1996. Effects of Gonadal Sex 
Steroids on Sexual Behavior in the Big Brown Bat,Eptesicus fuscus,Upon Arousal from 
Hibernation. Hormones and Behavior 30, 153-161. 
Merriam, G.R., Baer, L., 1980. Adrenocorticotropin Deficiency: Correction of Hyponatremia and 
Hypoaldosteronism with Chronic Glucocorticoid Therapy. Journal of Clinical Endocrinology & 
Metabolism 50, 10-14. 
Middler, S.A., Kleeman, C.R., Edwards, E., Brody, D., 1969. Effect of adenohypophysectomy 
on salt and water metabolism of the toad Bufo marinus with studies on hormonal replacement. 
General and Comparative Endocrinology 12, 290-304. 
Nichols, D., Lamirande, E., Pessier, A., Longcore, J., 2001. Experimental transmission of 
cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases 37, 1-11. 
Ramsey, J.P., Reinert, L.K., Harper, L.K., Woodhams, D.C., Rollins-Smith, L.A., 2010. Immune 
defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, 
in the South African clawed frog, Xenopus laevis. Infection and immunity 78, 3981-3992. 
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. 
Stefano, F.J.E., Donoso, A.O., 1964. Hypophyso-adrenal regulation of moulting in the toad. 
General and Comparative Endocrinology 4, 473-480. 
Stiffler, D.F., De Ruyter, M.L., Hanson, P.B., Marshall, M., 1986. Interrenal function in larval 
Ambystoma tigrinum: I. Responses to alterations in external electrolyte concentrations. General 
and Comparative Endocrinology 62, 290-297. 
Turner, R.J., 1988. Amphibians, in: A.F. Rowley, N.A. Ratcliffe (Eds.), Vertebrate blood cells. 
Cambridge University Press, Cambridge, UK, 129-209. 
Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W.F., Dinudom, A., Cook, D., Webb, 
R., Alford, R.A., Skerratt, L.F., Speare, R., 2009. Pathogenesis of Chytridiomycosis, a Cause of 
Catastrophic Amphibian Declines. Science 326, 582-585. 
 59 
Vredenburg, V.T., Knapp, R.A., Tunstall, T.S., Briggs, C.J., 2010. Dynamics of an emerging 
disease drive large-scale amphibian population extinctions. Proceedings of the National 
Academy of Sciences 107, 9689-9694. 
Wack, C.L., DuRant, S.E., Hopkins, W.A., Lovern, M.B., Feldhoff, R.C., Woodley, S.K., 2012. 
Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander. Comparative 
Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology 161, 153-158. 
Ward, C.K., Appel, A.G., Mendon?a, M.T., 2006. Metabolic measures of male southern toads 
(Bufo terrestris) exposed to coal combustion waste. Comparative Biochemistry and Physiology - 
Part A: Molecular &amp; Integrative Physiology 143, 353-360. 
Woodhams, D.C., Alford, R.A., Marantelli, G., 2003. Emerging disease of amphibians cured by 
elevated body temperature. Diseases of Aquatic Organisms 55, 65-67. 
Woodhams, D.C., Ardipradja, K., Alford, R.A., Marantelli, G., Reinert, L.K., Rollins-Smith, 
L.A., 2007a. Resistance to chytridiomycosis varies among amphibian species and is correlated 
with skin peptide defenses. Animal Conservation 10, 409-417. 
Woodhams, D.C., Vredenburg, V.T., Simon, M.-A., Billheimer, D., Shakhtour, B., Shyr, Y., 
Briggs, C.J., Rollins-Smith, L.A., Harris, R.N., 2007b. Symbiotic bacteria contribute to innate 
immune defenses of the threatened mountain yellow-legged frog, Rana muscosa. Biological 
Conservation 138, 390-398. 
Yorio, T., Bentley, P.J., 1978. Stimulation of the short-circuit current (sodium transport) across 
the skin of the forg (Rana pipiens) by corticosteroids: structure-activity relationships. Journal of 
Endocrinology 79, 283-290. 
Zerani, M., Amabili, F., Mosconi, G., Gobbetti, A., 1991. Effects of captivity stress on plasma 
steroid levels in the green frog, rana esculenta, during the annual reproductive cycle. 
Comparative Biochemistry and Physiology Part A: Physiology 98, 491-496. 
 
 
 60 
 
 
 
 
 
Chapter 3. The metabolic effects of glucocorticoids in an anuran amphibian. 
 
Abstract 
 Acute glucocorticoid secretion functions in freeing energy stores, increasing metabolism, 
and reducing energy consuming processes (e.g. reproduction and cellular immunity).  Although 
glucocorticoids have been documented to increase whole animal metabolism in several 
vertebrate groups, only one study has documented this effect in amphibians, and no study has 
documented this effect in anurans.  Given recent global population declines, understanding how 
amphibians respond to environmental stressors has become more urgent.  Many factors linked to 
declines cause increase corticosterone (CORT; the primary glucocorticoid in amphibians).  For 
example, in chapters one and two of this dissertation, we documented that infection with the 
amphibian chytrid fungus, Batrachochytrium dendrobatidis, increases both plasma CORT and 
resting metabolic rate in Litoria caerulea.  To examine the possibility that CORT may have 
influenced metabolic rate in L. caerulea we treated individuals with exogenous corticosterone 
(CORT; the most abundant amphibian glucocorticoid; 400ug CORT/20ul DMSO) and observed 
1.8 and 2.2-fold increases in resting metabolic rate three and six hours post injection, 
respectively.  This treatment also caused physiologically realistic 33-fold increases in plasma 
glucocorticoids at similar time points.  These data suggest that acute response glucocorticoid 
secretion is energetically costly in anuran amphibians.  Considering that many factors suggested 
to cause amphibian population declines also elevate glucocorticoids, we suggest that the 
metabolic cost of glucocorticoids be considered in future amphibian decline discussions.   
 61 
1. Introduction 
 The vertebrate stress response is a suite of physiological changes that help organisms 
cope with environmental perturbations (Wingfield et al., 1998).  One component of the stress 
response is the hypothalamic-pituitary-adrenal (-interenal in amphibians) axis, which secretes 
glucocorticoids to modulate the organism?s energetic response to a stressor.  For example, 
glucocorticoid secretion causes gluconeogenesis, downregulation of energetically costly but not 
immediately relevant processes (e.g. reproduction and cellular immunity), and increases in whole 
organism metabolic rate (Sapolsky et al., 2000).  While this response is thought to be adaptive in 
the short-term, it may be maladaptive in the long-term (Sapolsky et al., 2000).   
Glucocorticoids have been highlighted for their potential use as a biomarker in 
conservation studies (Busch and Hayward, 2009).  Amphibians are in dire need of conservation, 
given recent global amphibian population declines (Stuart et al., 2004); however, little is known 
about the connection between glucocorticoids, metabolism, and how these evolutionarily 
conserved physiological responses may, under new environmental challenges, contribute to 
population dynamics (Wack et al., 2012; Warne et al., 2011).  Several factors suggested to 
contribute to amphibian population declines have been shown to influence glucocorticoid 
secretion [e.g. anthropogenic contaminants (Gendron et al., 1997; Glennemeier and Denver, 
2001; Goulet and Hontela, 2003; Hayes et al., 2006; Hopkins et al., 1997; Hopkins et al., 1999; 
Larson et al., 1998; Peterson et al., 2009; Ward and Mendon?a, 2006), disease (Belden and 
Kiesecker, 2005; Warne et al., 2011), habitat alteration (Denver, 1998; Newcomb Homan et al., 
2003), and predation (Denver, 2009; Fraker et al., 2009)].  In contrast, only one study has 
documented the effect of glucocorticoids on metabolic rate.  Wack et al. (2012) administered 
exogenous corticosterone (CORT, the most abundant glucocorticoid in amphibians) to 
 62 
salamanders, Plethodon shermani, raising plasma CORT to physiologically relevant levels, and 
observed a significant increase in metabolic rate; however, data are still needed for anuran 
amphibians.  Additionally, few studies have documented the link between decline factors, 
glucocorticoids, and metabolism.  In chapters 1 and 2 of this dissertation, we observed that frogs, 
Litoria caerulea, infected with the fungus, Batrachochytrium dendrobatidis (which has been 
linked to amphibian declines the world-over), experience significant increases in resting 
metabolic rate and baseline plasma glucocorticoids; however, it is unclear whether 
glucocorticoid secretion caused the metabolic changes. 
 The goal of our study is to determine if CORT influences resting metabolic rate in L. 
caerulea, which would be the first time this relationship has been documented in an anuran 
amphibian. We treated L. caerulea with exogenous glucocorticoids and documented effects on 
plasma corticosterone and resting metabolic rate at several time points post treatment. 
2. Methods 
2.1 Animal care 
 Litoria caerulea were obtained commercially (Tri Reptile, Miami, FL) in autumn 2011.  
Frogs were recently collected from the wild in Indonesia.  Animals were acclimated to lab 
conditions for at least one month prior to experimental manipulation.  Amphibians were housed 
individually in plastic containers (17 cm x 17 cm x 17 cm) containing paper towels saturated 
with well water.  Wet paper towels were replaced twice a week for the duration of the study.  
Once a week animals were fed approximately 10% of their body weight in 2.5 week old crickets 
coated in multi-vitamin dust.  Any crickets not consumed were removed after 24 hours.  Light 
was provided by full spectrum light bulbs on a 12:12 light/dark cycle.   
2.2 Experimental design and treatments 
 63 
The effects of glucocorticoid treatment on resting metabolic rate (RMR) and plasma 
glucocorticoids were determined in two separate experiments.  In experiment 1, frogs were 
separated into two treatment groups: vehicle control (intraperitoneal injection of 20 ?l DMSO, 
n=10) or CORT (intraperitoneal injection of 400 ?g CORT in 20?l DMSO, n=13).  Metabolic 
rate is often correlated with body mass.  To minimize this effect on treatments, individuals were 
allocated to treatment groups such that treatments had similar masses and standard errors for 
body mass.  Individuals were sampled repeatedly for RMR.  A preliminary experiment 
determined that sampling individual?s metabolic rates repeatedly within one day had an effect on 
baseline oxygen consumption, so instead, non-injected baseline RMR as well as RMR three, six, 
and 24 hours post injection were determined at least a month apart.  Once individuals were 
injected with vehicle or CORT and sampled for RMR, they were not injected and sampled again 
for at least 30 days to reduce the effects of the previous injection and sampling.  Both treatment 
and time were treated as fixed effects. 
Experiment 2 was conducted 33 days after the conclusion of experiment 1 to reduce the 
effects of previous treatment.  Individuals from previous treatment groups were evenly divided 
among three new treatment groups: vehicle control, injected intraperitoneally three hours prior to 
bleed (Control, 20 ?l DMSO, n=6); CORT injected intraperitoneally three hours prior to bleed 
(CORT 3 h post injection, 400 ?g in 20?l DMSO, n=7); and CORT injected intraperitoneally six 
hours prior to bleed (CORT 6 h post injection, 400 ?g in 20?l DMSO, n=6).  Frogs were again 
allocated to treatment groups such that treatments had similar masses and standard errors for 
body mass, to reduce the effect of body mass on treatments.  A randomly chosen individual was 
injected every 15 minutes within each treatment group (CORT-treated individuals sampled 6 h 
post injection were treated between 900 and 1030, CORT-treated individuals sampled 3 h post 
 64 
injection were treated between 1155 and 1335, and vehicle-treated control individuals sampled 
three hours post injection were treated between 1150 and 1320).  To minimize time of day 
effects, all individuals were bled within a 105 minute period (1450 and 1635).  
2.3 Respirometry 
 Closed system respirometry was used to measure resting metabolic rate (oxygen 
consumption) following the methods of Ward et al. (2006) with the following changes.  Frogs 
were acclimated in 140 ml syringes (Monoject, Sherwood Medical Industries, Ballymoney, UK) 
for at least 45 minutes in a darkened incubator (25?C).  Frogs were incubated for ~50 minutes in 
a darkened incubator (25?C).  Any frogs that urinated or defecated during incubation were 
excluded from analyses.  Frogs fasted for at least 5 d prior to being sampled.  Frogs were 
weighed following respirometry.  Body masses of frogs ranged from 14-40g.   
2.4 Blood collection and radioimmunoassay 
Blood was collected directly from the heart following double pithing.  Blood was then 
centrifuged for 4 minutes at 3,500 x g and the plasma was frozen and stored at -20? C for later 
use in corticosterone radioimmunoassay.  Plasma corticosterone concentrations were determined 
by radioimmunoassay as described by Mendon?a et al. (1996).  All samples were run in one 
assay.  Extraction efficiency was 67% and intraassay variation was 32%.   
2.5 Statistical analysis 
 Resting metabolic rate was compared between treatments and over time using a repeated 
measures analysis of variance (repeated measures ANOVA).  Since each frog was given the 
same concentration of CORT, regardless of body mass, plasma corticosterone levels were 
compared among treatments using an ANCOVA with treatment as the independent variable, 
CORT as the dependent variable, and body mass as the covariate.  Fisher?s protected least 
 65 
significant difference (Fisher?s PLSD) tests were used for post-hoc comparisons among 
treatment groups for experiment 2.  All statistical analyses were conducted using StatView for 
Windows (SAS institute, version 5.0.1). 
3. Results 
3.1 Experiment 1 
 CORT-treated frogs consumed significantly more oxygen than vehicle-injected controls 
(Repeated measures ANOVA; Treatment: P=0.013, F1,16=7.74; Time: P<0.001, F3,48=7.65; 
Treatment x Time: P=0.004, F3,48=5.13).  Frogs treated with CORT experienced a significant 1.8 
and 2.2 fold increase in oxygen consumption at three and six hours post injection, respectively, 
compared to controls.  At 24 hours oxygen consumption returned to levels similar to 
concurrently sampled vehicle controls.   
3.2 Experiment 2 
 The plasma of CORT-treated frogs contained significantly more CORT at three and six 
hours post injection compared to vehicle-treated controls (ANCOVA, Treatment: P<0.001, 
F2,15=38.59; Body mass: P=0.8, F1,15=0.09; Fisher?s PLSDs, P<0.001).  There was no treatment 
by body mass interaction (P=0.7).  CORT-treated individuals experienced a 33 fold increase in 
CORT at both three and six hours post injection compared to vehicle-treated controls.   
4. Discussion 
   This is the first study to document that glucocorticoids increase metabolic rate in an 
anuran amphibian, as well as the second study to document this effect in amphibians.  Wack et 
al. (2012) treated the terrestrial salamander, Plethodon shermani, with CORT and observed that 
treatment caused a significant increase in plasma CORT at four hours post injection, but not at 
two and ten hours post injection, as well as an increase in oxygen consumption between two and 
 66 
four hours post treatment, but no effect thereafter.  Differences between the Wack et al. (2012) 
experiments and ours preclude detailed comparisons, but the same general trend was observed.  
We observed that in the anuran, Litoria caerulea, CORT treatment caused significant increases 
in plasma CORT at three and six hours post treatment, as well as significant increases in 
metabolic rate at three and six hours post treatment, which returned to control levels at 24 hours 
post injection. 
 The CORT treatment that we used caused increases in plasma CORT that are 
physiologically realistic for L. caerulea and similar species.  For example, acute captivity stress 
caused a maximum average increase of 13.8 ng/ml plasma CORT in Litoria ewingi (Coddington 
and Cree, 1995).  Chronic infection with the amphibian chytrid fungus, Batrachochytrium 
dendrobatidis, caused increases in plasma CORT to averages of 104 and 111 ng/ml in Litoria 
caerulea (see Chapters 1 and 2 of this dissertation).  In general, acute stressor-induced increases 
in plasma CORT range from 11-56 ng/ml in frogs (Coddington and Cree, 1995; Jurani et al., 
1973).  In the current study, treatment with CORT increased plasma CORT concentrations to 30 
ng/ml at both three and six hours post treatment.  Our study is one of the few studies of 
vertebrates that have observed the metabolic effects of exogenous glucocorticoids which increase 
plasma glucocorticoids to physiologically realistic levels (Buttemer et al., 1991; Davis and 
Schreck, 1997; DuRant et al., 2008; Hissa et al., 1980; Morgan and Iwama, 1996; Wack et al., 
2012).     
Our findings, along with other recent findings, suggest that elevations in plasma 
glucocorticoids are energetically costly in amphibians (Wack et al., 2012).  In the current study, 
frogs treated with CORT experienced 1.8 and 2.2 fold increases in metabolic rate at three and six 
hours post treatment compared to controls sampled concurrently.  The cost of these increases 
 67 
becomes more apparent when we convert our values to energetic cost (1 ml of oxygen consumed 
= 20.1 J; Feder and Burggren, 1992).  The cost of BMR (J/h) at three and six hours post injection 
with CORT increased by 150% and 190%, respectively, compared to baseline levels.  Even if 
BMR had only increased for a three hour period, the extra cost would represent 17% the daily 
requirements to maintain BMR (0.037 kJ).  Although we only observed the acute metabolic 
effects of increased plasma CORT, chronic increases in CORT and metabolic rate could deplete 
energy reserves and cause weight loss.  Continued weight loss could lead to wasting and death 
and, thus, contribute to amphibian population declines.  Several studies provide evidence that 
CORT induced weight loss may contribute to amphibian population declines, though they do not 
determine whether weight loss is mediated by elevation of metabolic rate (Denver, 1998; Hayes 
et al., 2006; Peterson et al., 2009; Warne et al., 2011).  To our knowledge, the first two chapters 
of this dissertation are the first to document such effects of a decline factor on CORT, body 
mass, and metabolism.  In these studies, chronic infection with Batrachochytrium dendrobatidis, 
a cause of amphibian population declines the world-over, increased plasma CORT and metabolic 
rate more than three and 15 times higher, respectively, than the values observed in the current 
study in the same species.  Infection also caused significant decreases in body mass and body 
condition prior to death.  It is important to note that the authors of these studies did not determine 
if CORT caused the effects on metabolic rate, weight loss, and body condition; however, our 
study suggests that it is physiologically possible in this species.     
We believe that more studies should focus on the contribution of alterations in CORT and 
metabolism to amphibian population decline.  Our data suggest that even an acute, CORT-
induced increase in RMR is costly.  Chronic elevation of CORT and RMR may deplete energy 
stores and induce wasting in declining populations trying to cope with decline factors.  The 
 68 
metabolic cost of CORT secretion has only recently been identified in amphibians.  Authors 
suggest that CORT may influence amphibian population dynamics by directly modulating 
reproduction and immunity, but we suggest that CORTs influence on metabolism also be 
considered.  It may directly contribute to declines by inducing wasting, but may also indirectly 
modulate reproduction and immunity by depleting the energy stores that they, and many other 
energetically expensive processes, require.   
 69 
Figures: 
CORT  n =12
Con tr ol n=6
0
0.002
0.004
0.006
0.008
0.010
0.012
Ba seline 3 h
Oxy
gen
con
sumption 
(m
l/
g/hr)
6 h 24 h 
T im e pos t injecti on
 
Fig. 3.1. Baseline average resting metabolic rates (+SEM) and rates 3, 6, and 24 hours post 
injection injected with either corticosterone (CORT; 400 ?g CORT in 20?l DMSO) or vehicle 
(Control; 20?l DMSO) of Litoria caerulea.  CORT-treated frogs consumed significantly more 
oxygen than vehicle-injected controls (Repeated measures ANOVA; Treatment: p=0.01, F=7.74; 
Time: p=0.0003, F=7.65; Treatment x Time: p=0.004, F=5.13).   
 70 
 0
5
10
15
20
25
30
35
Con tr ol 3 h pos t 
injection
6 h post 
inject ion
Corticosterone
(
ng
/m
l)
6
CORT
Con tr ol
A
B
B
7 6
 
Fig. 3.2. Average plasma corticosterone (+SEM) of Litoria caerulea three hours post injection 
with vehicle (Control; 20?l DMSO), three hours post injection with corticosterone (CORT; 400 
?g CORT in 20?l DMSO) and six hours post injection with corticosterone.  Treatments were 
significantly different (ANOVA, p<0.0001, F=41.31).  Different capital letters denote significant 
differences among treatments (Fisher?s PLSDs, p<0.0001). 
 71 
References: 
 
Belden, L.K., Kiesecker, J.M., 2005. Glucocorticosteroid Hormone Treatment of Larval 
Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91, 
686-688. 
Busch, D.S., Hayward, L.S., 2009. Stress in a conservation context: A discussion of 
glucocorticoid actions and how levels change with conservation-relevant variables. Biological 
Conservation 142, 2844-2853. 
Buttemer, W.A., Astheimer, L.B., Wingfield, J.C., 1991. The effect of corticosterone on standard 
metabolic rates of small passerine birds. Journal of Comparative Physiology B: Biochemical, 
Systemic, and Environmental Physiology 161, 427-431. 
Coddington, E.J., Cree, A., 1995. Effect of Acute Captivity Stress on Plasma Concentrations of 
Corticosterone and Sex Steroids in Female Whistling Frogs, Litoria ewingi. General and 
Comparative Endocrinology 100, 33-38. 
Davis, L.E., Schreck, C.B., 1997. The Energetic Response to Handling Stress in Juvenile Coho 
Salmon. Transactions of the American Fisheries Society 126, 248-258. 
Denver, R.J., 1998. Hormonal Correlates of Environmentally Induced Metamorphosis in the 
Western Spadefoot Toad,Scaphiopus hammondii. General and Comparative Endocrinology 110, 
326-336. 
Denver, R.J., 2009. Stress hormones mediate environment-genotype interactions during 
amphibian development. General and Comparative Endocrinology 164, 20-31. 
DuRant, S.E., Romero, L.M., Talent, L.G., Hopkins, W.A., 2008. Effect of exogenous 
corticosterone on respiration in a reptile. General and Comparative Endocrinology 156, 126-133. 
Fraker, M.E., Hu, F., Cuddapah, V., McCollum, S.A., Relyea, R.A., Hempel, J., Denver, R.J., 
2009. Characterization of an alarm pheromone secreted by amphibian tadpoles that induces 
behavioral inhibition and suppression of the neuroendocrine stress axis. Hormones and Behavior 
55, 520-529. 
Gendron, A.D., Bishop, C.A., Fortin, R., Hontela, A., 1997. In vivo testing of the functional 
integrity of the corticosterone-producing axis in mudpuppy (amphibia) exposed to chlorinated 
hydrocarbons in the wild. Environmental Toxicology and Chemistry 16, 1694-1706. 
Glennemeier, K.A., Denver, R.J., 2001. Sublethal effects of chronic exposure to an 
organochlorine compound on northern leopard frog (Rana pipiens) tadpoles. Environmental 
Toxicology 16, 287-297. 
Goulet, B.N., Hontela, A., 2003. Toxicity of cadmium, endosulfan, and atrazine in adrenal 
steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana. 
Environmental Toxicology and Chemistry 22, 2106-2113. 
 72 
Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., 
Marjuoa, Y., Parker, J., Tsui, M., 2006. Pesticide Mixtures, Endocrine Disruption, and 
Amphibian Declines: Are We Underestimating the Impact? National Institute of Environmental 
Health Sciences. 
Hissa, R., George, J.C., Saarela, S., 1980. Dose-related effects of noradrenaline and 
corticosterone on temperature regulation in the pigeon. Comparative Biochemistry and 
Physiology Part C: Comparative Pharmacology 65, 25-32. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1997. Increased Circulating Levels of 
Testosterone and Corticosterone in Southern Toads,Bufo terrestris,Exposed to Coal Combustion 
Waste. General and Comparative Endocrinology 108, 237-246. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1999. Responsiveness of the hypothalamo?
pituitary?interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes. 
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and 
Endocrinology 122, 191-196. 
Jurani, M., Murgas, K., Mikulaj, L., Babusikova, F., 1973. Effect of stress and environmental 
temperature on adrenal function in Rana esculenta. Journal of Endocrinology 57, 385-391. 
Larson, D.L., McDonald, S., Fivizzani, A.J., Newton, W.E., Hamilton, S.J., 1998. Effects of the 
herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and 
hormonal response. Physiological zoology 71, 671-679. 
Mendon?a, M.T., Chernetsky, S.D., Nester, K.E., Gardner, G.L., 1996. Effects of Gonadal Sex 
Steroids on Sexual Behavior in the Big Brown Bat,Eptesicus fuscus,Upon Arousal from 
Hibernation. Hormones and Behavior 30, 153-161. 
Morgan, J.D., Iwama, G.K., 1996. Cortisol-induced changes in oxygen consumption and ionic 
regulation in coastal cutthroat trout (&lt;i&gt;Oncorhynchus clarki clarki ) parr. Fish Physiology 
and Biochemistry 15, 385-394. 
Newcomb Homan, R., Regosin, J.V., Rodrigues, D.M., Reed, J.M., Windmiller, B.S., Romero, 
L.M., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders 
(Ambystoma maculatum). Animal Conservation 6, 11-18. 
Peterson, J.D., Peterson, V.A., Mendon?a, M.T., 2009. Exposure to coal combustion residues 
during metamorphosis elevates corticosterone content and adversely affects oral morphology, 
growth, and development in Rana sphenocephala. Comparative Biochemistry and Physiology 
Part C: Toxicology &amp; Pharmacology 149, 36-39. 
Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How Do Glucocorticoids Influence Stress 
Responses? Integrating Permissive, Suppressive, Stimulatory, and Preparative Actions. 
Endocrine Reviews 21, 55-89. 
 73 
Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, A.S.L., Fischman, D.L., Waller, 
R.W., 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science 306, 
1783-1786. 
Wack, C.L., DuRant, S.E., Hopkins, W.A., Lovern, M.B., Feldhoff, R.C., Woodley, S.K., 2012. 
Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander. Comparative 
Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology 161, 153-158. 
Ward, C., Mendon?a, M., 2006. Chronic Exposure to Coal Fly Ash Causes Minimal Changes in 
Corticosterone and Testosterone Concentrations in Male Southern Toads &lt;i&gt;Bufo 
terrestris&lt;/i&gt. Archives of Environmental Contamination and Toxicology 51, 263-269. 
Ward, C.K., Appel, A.G., Mendon?a, M.T., 2006. Metabolic measures of male southern toads 
(Bufo terrestris) exposed to coal combustion waste. Comparative Biochemistry and Physiology - 
Part A: Molecular &amp; Integrative Physiology 143, 353-360. 
Warne, R.W., Crespi, E.J., Brunner, J.L., 2011. Escape from the pond: stress and developmental 
responses to ranavirus infection in wood frog tadpoles. Functional Ecology 25, 139-146. 
Wingfield, J.C., Maney, D.L., Breuner, C.W., Jacobs, J.D., Lynn, S., Ramenofsky, M., 
Richardson, R.D., 1998. Ecological Bases of Hormone?Behavior Interactions: The ?Emergency 
Life History Stage?. American Zoologist 38, 191-206. 
 
 
 74 
 
 
 
 
 
Conclusion 
 The experiments contained herein are among the few experiments to document how 
infectious disease influences stress physiology in wild vertebrates.  These experiments are also 
among the first to observe how disease-induced increases in glucocorticoids potentially influence 
variables normally influenced by glucocorticoids in non-diseased animals (Warne et al., 2011).  
Although we could not determine whether glucocorticoids caused the changes in these variables, 
significant increases in CORT were temporally associated with effects typically caused by 
glucocorticoids in vertebrates in non-disease situations.  Specifically, individuals displaying 
clinical signs of disease contained significantly elevated CORT, elevated neutrophils, suppressed 
lymphocytes and eosinophils, and elevated standard metabolic rate. 
These experiments provide novel contributions toward understanding the pathogenesis of 
chytridiomycosis.  They suggest that plasma ion suppression, appetite suppression, increased 
metabolic rate, weight loss, and increased shed skin frequency occur prior to when individuals 
display clinical signs of disease (i.e. lethargy, loss of righting reflex, and tremors).  Changes in 
plasma ions preceded changes in all other variables monitored in this study, including appetite, 
which was previously suggested to be the earliest effect of Bd infection (Nichols et al., 2001).  
This finding suggests that initial decreases in plasma ions are not due to reduced digestive 
sodium uptake.  Instead, early plasma ion loss is likely due to disruption of cutaneous sodium 
inputs by Bd infection of the skin (Voyles et al., 2009).  Once frogs display clinical signs of 
disease, further suppression of plasma ions occurs.  This further decrease in ions can be 
attributed to both continued disruption of cutaneous sodium uptake and suppressed digestive 
 75 
uptake due to loss of appetite.  Loss of appetite and increased metabolic rate in the week prior to 
displaying clinical signs of disease likely contribute to observed weight loss and decreased body 
condition.  Loss of appetite may also explain why shed skin was found more often within bins of 
frogs that eventually displayed clinical signs of disease, considering that many frogs consume 
their shed skin (Feder and Burggren, 1992).  Besides disruptions to plasma ions, frogs displaying 
clinical signs of disease also experienced elevated plasma CORT, reduced body condition, 
elevated neutrophils, suppressed lymphocytes and eosinophils, as well as continued elevation of 
standard metabolic rate.   
 CORT likely mediates some of the effects observed during Bd infection.  Previous 
research on amphibians suggests that hyponatremia causes increased secretion of glucocorticoids 
and exogenous glucocorticoids can correct hyponatremia to homeostatic concentrations (Heney 
and Stiffler, 1983; Stiffler et al., 1986).  In Chapter 2, significant decreases in plasma sodium at 
39 days post infection may have not been low enough to trigger a CORT response in all 
individuals.  In fact, at 39 days post infection, two infected individuals contained CORT 
concentrations that mirror stressor-induced levels in similar species (Coddington and Cree, 
1995), as well as sodium and potassium levels that parallel levels of control levels, suggesting 
that CORT secretion may initially help maintain sodium balance.  These individuals also 
experienced changes in NL ratios equivalent to those observed in frogs undergoing an acute 
stress response (Belden and Kiesecker, 2005; Bennett et al., 1972; Bennett and Harbottle, 1968; 
Coddington and Cree, 1995; Davis and Maerz, 2010; Garrido et al., 1987; Jurani et al., 1973).  
Due to limitation in experimental design, CORT was not assessed again until frogs displayed 
clinical signs of disease.  Future studies should sample for CORT when frogs experience 
significant changes in plasma sodium, appetite, or the period between 39 days post infection and 
 76 
the first clinical signs of disease.  By the time frogs display clinical signs of disease, they have 
experienced another significant decrease in plasma ions as well as a significant increase in 
plasma CORT.  These individuals also experience changes in WBC profiles and metabolic rate 
that parallel the effects of CORT on these variables in non-diseased animals.  Future studies 
should further test whether Bd-induced decreases in plasma ions induce a stress response that 
exacerbates pathogenesis.   
Finally, these experiments highlight the fact that there is a lack of data on how 
glucocorticoids influence physiology in amphibians.  For example, there is a lack of data on how 
glucocorticoids influence metabolic rate and appetite in amphibians (Carr et al., 2002; Crespi et 
al., 2004; Wack et al., 2012).  Chapters 1 and 2 provide correlative data to suggest that CORT 
may influence these factors.  Chapter 3 provides empirical data that suggests that physiologically 
relevant increases in plasma CORT by exogenous CORT treatment significantly elevate standard 
metabolic rate, but controlled laboratory data on food intake are needed to complement chapters 
1 and 2.  Understanding how amphibians respond to environmental change has become more 
urgent given recent amphibian population declines (Stuart et al., 2004).  Several factors 
suggested to contribute to amphibian population declines have been shown to influence 
glucocorticoid secretion [e.g. anthropogenic contaminants (Gendron et al., 1997; Glennemeier 
and Denver, 2001; Goulet and Hontela, 2003; Hayes et al., 2006; Hopkins et al., 1997; Hopkins 
et al., 1999; Larson et al., 1998; Peterson et al., 2009; Ward and Mendon?a, 2006), disease 
(Belden and Kiesecker, 2005; Warne et al., 2011), habitat alteration (Denver, 1998; Newcomb 
Homan et al., 2003), and predation (Denver, 2009; Fraker et al., 2009)].  Though the influence of 
anthropogenic contaminants on the stress axis has been relatively well studied in amphibians far 
less is known about how disease, habitat destruction, invasive predators, and climate change may 
 77 
influence stress physiology.  Given the powerful and far reaching effects of glucocorticoids on 
wildlife life histories and the contribution of environmental perturbations to biodiversity loss, 
understanding how these hormones mediate the interplay between environmental perturbations 
and life histories is essential to future conservation efforts. 
 
 
 
 78 
 
References: 
Belden, L.K., Kiesecker, J.M., 2005. Glucocorticosteroid Hormone Treatment of Larval 
Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91, 
686-688. 
Bennett, M.F., Gaudio, C.A., Johnson, A.O., Spisso, J.H., 1972. Changes in the blood of 
newts,&lt;i&gt;Notophthalmus viridescens , following the administration of hydrocortisone. 
Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral 
Physiology 80, 233-237. 
Bennett, M.F., Harbottle, J.A., 1968. The effects of hydrocortisone on the blood of tadpoles and 
frogs, Rana catesbeiana. The Biological Bulletin 135, 92-95. 
Carr, J.A., Brown, C.L., Mansouri, R., Venkatesan, S., 2002. Neuropeptides and amphibian prey-
catching behavior. Comparative Biochemistry and Physiology Part B: Biochemistry and 
Molecular Biology 132, 151-162. 
Coddington, E.J., Cree, A., 1995. Effect of Acute Captivity Stress on Plasma Concentrations of 
Corticosterone and Sex Steroids in Female Whistling Frogs, Litoria ewingi. General and 
Comparative Endocrinology 100, 33-38. 
Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of Corticotropin-Releasing Factor, 
Neuropeptide Y and Corticosterone in the Regulation of Food Intake In Xenopus laevis. Journal 
of Neuroendocrinology 16, 279-288. 
Davis, A.K., Maerz, J.C., 2010. Effects of Exogenous Corticosterone on Circulating Leukocytes 
of a Salamander (Ambystoma talpoideum) with Unusually Abundant Eosinophils. International 
Journal of Zoology 2010. 
Denver, R.J., 1998. Hormonal Correlates of Environmentally Induced Metamorphosis in the 
Western Spadefoot Toad,Scaphiopus hammondii. General and Comparative Endocrinology 110, 
326-336. 
Denver, R.J., 2009. Stress hormones mediate environment-genotype interactions during 
amphibian development. General and Comparative Endocrinology 164, 20-31. 
Feder, M.E., Burggren, W.W., 1992. Environmental physiology of the amphibians. University of 
Chicago Press, Chicago. 
Fraker, M.E., Hu, F., Cuddapah, V., McCollum, S.A., Relyea, R.A., Hempel, J., Denver, R.J., 
2009. Characterization of an alarm pheromone secreted by amphibian tadpoles that induces 
behavioral inhibition and suppression of the neuroendocrine stress axis. Hormones and Behavior 
55, 520-529. 
Garrido, E., Gomariz, R.P., Leceta, J., Zapata, A., 1987. Effects of dexamethasone on the 
lymphoid organs of Rana perezi. Developmental &amp; Comparative Immunology 11, 375-384. 
 79 
Gendron, A.D., Bishop, C.A., Fortin, R., Hontela, A., 1997. In vivo testing of the functional 
integrity of the corticosterone-producing axis in mudpuppy (amphibia) exposed to chlorinated 
hydrocarbons in the wild. Environmental Toxicology and Chemistry 16, 1694-1706. 
Glennemeier, K.A., Denver, R.J., 2001. Sublethal effects of chronic exposure to an 
organochlorine compound on northern leopard frog (Rana pipiens) tadpoles. Environmental 
Toxicology 16, 287-297. 
Goulet, B.N., Hontela, A., 2003. Toxicity of cadmium, endosulfan, and atrazine in adrenal 
steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana. 
Environmental Toxicology and Chemistry 22, 2106-2113. 
Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., 
Marjuoa, Y., Parker, J., Tsui, M., 2006. Pesticide Mixtures, Endocrine Disruption, and 
Amphibian Declines: Are We Underestimating the Impact? National Institute of Environmental 
Health Sciences. 
Heney, H.W., Stiffler, D.F., 1983. The effects of aldosterone on sodium and potassium 
metabolism in larval Ambystoma tigrinum. General and Comparative Endocrinology 49, 122-
127. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1997. Increased Circulating Levels of 
Testosterone and Corticosterone in Southern Toads,Bufo terrestris,Exposed to Coal Combustion 
Waste. General and Comparative Endocrinology 108, 237-246. 
Hopkins, W.A., Mendon?a, M.T., Congdon, J.D., 1999. Responsiveness of the hypothalamo?
pituitary?interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes. 
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and 
Endocrinology 122, 191-196. 
Jurani, M., Murgas, K., Mikulaj, L., Babusikova, F., 1973. Effect of stress and environmental 
temperature on adrenal function in Rana esculenta. Journal of Endocrinology 57, 385-391. 
Larson, D.L., McDonald, S., Fivizzani, A.J., Newton, W.E., Hamilton, S.J., 1998. Effects of the 
herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and 
hormonal response. Physiological zoology 71, 671-679. 
Newcomb Homan, R., Regosin, J.V., Rodrigues, D.M., Reed, J.M., Windmiller, B.S., Romero, 
L.M., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders 
(Ambystoma maculatum). Animal Conservation 6, 11-18. 
Nichols, D., Lamirande, E., Pessier, A., Longcore, J., 2001. Experimental transmission of 
cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases 37, 1-11. 
Peterson, J.D., Peterson, V.A., Mendon?a, M.T., 2009. Exposure to coal combustion residues 
during metamorphosis elevates corticosterone content and adversely affects oral morphology, 
growth, and development in Rana sphenocephala. Comparative Biochemistry and Physiology 
Part C: Toxicology &amp; Pharmacology 149, 36-39. 
 80 
Stiffler, D.F., De Ruyter, M.L., Hanson, P.B., Marshall, M., 1986. Interrenal function in larval 
Ambystoma tigrinum: I. Responses to alterations in external electrolyte concentrations. General 
and Comparative Endocrinology 62, 290-297. 
Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, A.S.L., Fischman, D.L., Waller, 
R.W., 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science 306, 
1783-1786. 
Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W.F., Dinudom, A., Cook, D., Webb, 
R., Alford, R.A., Skerratt, L.F., Speare, R., 2009. Pathogenesis of Chytridiomycosis, a Cause of 
Catastrophic Amphibian Declines. Science 326, 582-585. 
Wack, C.L., DuRant, S.E., Hopkins, W.A., Lovern, M.B., Feldhoff, R.C., Woodley, S.K., 2012. 
Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander. Comparative 
Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology 161, 153-158. 
Ward, C., Mendon?a, M., 2006. Chronic Exposure to Coal Fly Ash Causes Minimal Changes in 
Corticosterone and Testosterone Concentrations in Male Southern Toads &lt;i&gt;Bufo 
terrestris&lt;/i&gt. Archives of Environmental Contamination and Toxicology 51, 263-269. 
Warne, R.W., Crespi, E.J., Brunner, J.L., 2011. Escape from the pond: stress and developmental 
responses to ranavirus infection in wood frog tadpoles. Functional Ecology 25, 139-146.