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
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8
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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
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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
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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
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Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91,
686-688.
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glucocorticoid actions and how levels change with conservation-relevant variables. Biological
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metabolic rates of small passerine birds. Journal of Comparative Physiology B: Biochemical,
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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.
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L.M., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders
(Ambystoma maculatum). Animal Conservation 6, 11-18.
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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.
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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 & 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
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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 -
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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
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Treefrogs Increases Infection by Alaria Sp. Trematode Cercariae. Journal of Parasitology 91,
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326-336.
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behavioral inhibition and suppression of the neuroendocrine stress axis. Hormones and Behavior
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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
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