CBA-10009; No of Pages 8 Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa

3 4 5

a

6

a r t i c l e

7 8 9 10 11 12 30 31 32 33 34 35

Article history: Received 17 August 2015 Received in revised form 2 February 2016 Accepted 2 February 2016 Available online xxxx

School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Queensland, Australia Veterinary School, Universidad Nacional Autónoma de México, Mexico City 4510, Mexico

i n f o

O

b

F

Sandra E. Hernández a,b,⁎,1, Conrad Sernia a,1, Adrian J. Bradley a,1

a b s t r a c t

R O

Q32Q2

Adrenocortical function in cane toads from different environments

The adrenocortical function of cane toads (Rhinella marina) exposed to different experimental procedures, as well as captured from different environments, was assessed by challenging the hypothalamic–pituitary–adrenal (HPA) axis. It was found that restriction stress as well as cannulation increased plasma corticosterone (B) levels for up to 12 h. A single dose of dexamethasone (DEX 2 mg/kg) significantly reduced B levels demonstrating its potential for use in the evaluation of the HPA axis in amphibia. We also demonstrate that 0.05 IU/g BW (im) of synthetic adrenocorticotropic hormone (ACTH) significantly increased plasma B levels in cane toads. Changes in size area of the cortical cells were positively associated with total levels of B after ACTH administration. We also found differences in adrenal activity between populations. This was assessed by a DEX-ACTH test. The animals captured from the field and maintained in captivity for one year at the animal house (AH) present the highest levels of total and free B after ACTH administration. We also found that animals from the front line of dispersion in Western Australia (WA) present the weakest adrenal response to a DEX-ACTH test. The animals categorized as long established in Queensland Australia (QL), and native in Mexico (MX), do not shown a marked difference in the HPA activity. Finally we found that in response to ACTH administration, females reach significantly higher levels of plasma B than males. For the first time the adrenocortical response in cane toads exposed to different experimental procedures, as well as from different populations was assessed systematically. © 2016 Elsevier Inc. All rights reserved.

P

1Q1

C

T

E

D

Keywords: Rhinella marina Amphibia Stress response ACTH Dexamethasone

29

E

39 37 36 38

1. Introduction

41 42

The adaptation of organisms to their physical environment is a major determinant of survival (Bennett, 1997). While the biological mechanisms underlying adaptation are still being elucidated, it is known that the neuroendocrine system, and its involvement in the stress response, has an important role in maintaining the homeostatic balance in animals (Wingfield et al., 2015). The differences in the stress response between individuals from the same species living in different environments are the result of environmental pressures shaping the physiological responses and optimizing fitness success (Silverin et al., 1997). These changes are directed toward a better outcome energetic tradeoffs during the life story stages in the living organisms (Wingfield et al., 2015). The neuroendocrine system acting via the hypothalamic–pituitary–adrenal (HPA) axis has an important role in maintaining the homeostatic balance in animals by the regulation of the adrenal hormones produced during the stress response (Sapolsky et al., 2000). Among these hormones, glucocorticoids (GCs) have

47 48 49 50 51 52 53 54 55 56

Q4

R

N C O

45 46

U

43 44

R

40

⁎ Corresponding author at: Grupo de Ecología de Enfermedades, Departamento de Etología, Fauna Silvestre y Animales de Laboratorio, Universidad Nacional Autónoma de México, Mexico City 4510, Mexico. Tel.: +52 55 5622 5941. E-mail address: [email protected] (S.E. Hernández). 1 These authors contribute equally to the study.

attracted the most interest because of their involvement in chronic stress (McEwen and Stellar, 1993), immune suppression (Sternberg and Licinio, 1995), reproductive impairment (Hardy et al., 2005), metabolic diseases (Dallman et al., 1993), and central nervous system (CNS) damage (McEwen and Sapolsky, 1995). Therefore the activation of the HPA to environmental changes has an important role in integrating changes in behaviour and physiology by optimizing the energy expenditure during emergency life history stages (Wingfield, 2008; Wingfield and Kitaysky, 2002). The cane toad has one of the widest distributions of any anuran species. It is native to the Americas but was introduced to Australia and several Pacific and Caribbean islands (Hero and Stonham, 2005). It is an opportunistic species and a dietary generalist (Zug and Zug, 1979). The presence of both endemic and introduced populations of cane toads in diverse geographical locations offers opportunity to studying adaptive mechanisms in this species which may underpin their apparent success vis-a-vis other amphibian species. In amphibia, and in other vertebrates, the hypothalamic–pituitary axis modulates the hormone profile in response to stress. In mammals, following the exposure to a stressor, the corticotropinreleasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus, and the arginine vasopressin (AVP) from the posterior pituitary, act in conjunction to promote the production of

http://dx.doi.org/10.1016/j.cbpa.2016.02.001 1095-6433/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

118

A total of 86 cane toads (66 males, 20 females), weighing between 200 and 500 g, were collected by hand during the wet season from 2009 to 2011. Two populations were located in Australia, one established in 1945 in Brisbane, Queensland (Hero and Stonham, 2005) ([QL, males = 56, females = 10]) and the other located in the front line of dispersion in Kununurra, Western Australia(Department of Environment and Conservation, 2013) [WA, males = 5, females = 5]). A third population was sampled in its native distribution at Los Tuxtlas, Veracruz, Mexico [MX, males = 5, females = 5]). All animals were captured during the night near urban areas and transported in plastic containers to a field laboratory. To assess the effect of captivity, five males and five females from The University of Queensland Lakes, St Lucia, SE Queensland were kept in captivity for one year (The University of Queensland animal house [AH]). The groups of males and females were maintained separately in tanks (1.5 m3) with a pond and a dry area containing sections of PVC pipe (120 × 300 mm) used as refuges. Environmental enrichment was provided constantly in the form of live prey and plastic aquatic plants. An ambient temperature of 22–24 °C was maintained with a light cycle of 12L:12D. Animals were fed three days a week with live prey (crickets, wood roaches, meal worms, earth worms) and dry food with 40% of protein ad libitum and supplemented with calcium (Olvera-Novoa et al., 2007). All procedures were approved by The University of Queensland Animal Ethics Committee (SBMS/437/09/URG/ GOVTMEX/HSF/CFOC).

106 107 108 109 110 111 112 113

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142

C

104 105

E

102 103

R

100 101

R

98 99

O

96 97

C

94 95

N

92 93

U

90 91

2.2.1. Assessment of adrenocortical function

147 148

(a). Effect of movement restriction in corticosterone plasma levels in 149 Q5 cane toads 150 151

To determine a corticosterone baseline level in plasma, five male cane toads were captured by hand and within 2 min of capture a blood sample (200 μl) was obtained by cardiac puncture with a 1 ml heparinized syringe (25 G × 1″ needle) (time 0). To ensure that cardiac puncture was only performed once on each animal, 15 male cane toads were captured and placed in a plastic container to restrict movement and divided into three groups of five animals each. The first group of five toads was kept in the plastic container for 1 h (time 1) before drawn a blood sample. Five toads were kept in the plastic containers for 2 and 12 h respectively (time 2 and 12) before collecting blood by cardiac puncture. An ambient temperature of 22–24 °C was maintained throughout the experimental period and all animals were euthanized with 100 mg/kg of pentobarbital (Lethabarb) after each time point.

F

2.1. Capture and housing of toads

88 89

O

117

87

R O

2. Methods

85 86

The male cane toads used for the following experiments (N = 46) 144 were captured from The University of Queensland Lakes, St Lucia, SE 145 Queensland. 146

P

116

83 84

143

2.2. Experimental design

152 153 154 155 156 157 Q6 158 159 160 161 162 163 164

(b). Effect of cannulation in corticosterone plasmatic levels in cane 165 toads 166 The stress response to cannulation was determined in five male toads captured on the same night. The animals were transported to the laboratory where they were weighed, anesthetized with a dose of 200 mg/kg (BW) of ketamine combined with 0.2 mg/kg (BW) of diazepam (Hernández et al., 2012) and cannulated with a 24 gauge × 19 mm catheter (Introcan Certo, Braun, Germany) inserted into the ventral vein as described previously (Hernández et al., 2012). Flunixin meglubine 1 mg/kg IM (Finadyne; Schering-Plough Pty Ltd., Australia) (Wright, 2001) an analgesic non-steroidal anti-inflammatory, was administrated as post-operative pain management in the toads. Catheters were placed to facilitate blood sampling and to allow humane euthanasia at the end of the study period. Once the placement of the catheter was completed, toads were housed individually to recover in 4 l plastic containers, with a wet bedding a depth of 2 cm of water and a dry refuge area. An ambient temperature of 22–24 °C was maintained with a light cycle of 12L:12D. Animals were fasted during this period. After 24 h of recovery, a first blood sample (100 μl) was collected (time 0), followed by consecutive blood samples (100 μl) at one hour (1 h), two hours (2 h) and twelve hours (12 h) after the first blood sample (time 0), to enable a determination of plasma corticosterone (B) concentrations. To reduce fluctuations in B levels due to daily cycles (Leboulenger et al., 1982), all experiments were carried out during the night and at the same starting time.

167 168

(c). Suppression of corticosterone levels by dexamethasone (DEX)

191 Q7 192

Five male cane toads captured on the same night were cannulated as explained above. After 24 h recovery, a first blood sample (100 μl) was collected followed immediately by an intravenous injection of 2 mg/kg dexamethasone sodium phosphate (Dexadreson, Intervet Pty Ltd. Australia) (Wright, 2001) in 100 μl 0.9% saline (sterile) via the catheter. Consecutive blood samples (100 μl) were collected at one hour (1 h) two hours (2 h), four hours (4 h) and twelve hours (12 h) after DEX administration.

193 194

T

114 115

adrenocorticotropic hormone (ACTH) from the anterior pituitary (Carrasco and Van de Kar, 2003). However in anurans, the greatest CRH immunoreactivity and synthesis of the corticotropin releasing hormone (CRH) are localized in the regions of the anterior preoptic area (POA) and the external zone of the median eminence (Jorgensen, 1976) (Yao et al., 2004). The CRH pathway signals traverse through the median eminence and via a vascular route reach the pars distalis where the synthesis and release of ACTH are controlled (Capaldo et al., 2004; Ogawa et al., 1995). In mammals, as well as in amphibians, ACTH is released into the circulation via the hypophyseal portal system and exerts its action in the adrenal gland. The function of ACTH in the adrenal gland is to regulate the production of glucocorticoids (GCs) (cortisol, corticosterone, aldosterone) (Janssens, 1970). In amphibia, the administration of ACTH stimulates the secretion of aldosterone and corticosterone by the cortical cell from the inter-renal tissue in vitro (Capaldo et al., 2004; Hanke, 1978; Vinson and Whitehouse, 1974) and in vivo (Dupont et al., 1976; Kemenade, 1968; Narayan et al., 2011). In response to ACTH stimulation, corticosterone is the glucocorticoid present at the highest concentration in blood (Capaldo et al., 2004; Glennemeier and Denver, 2002; Hanke and Weber, 1965). Amphibian studies related to the properties of the corticosteroid binding globulin (CBG) are scarce. Under normal conditions in mammals, 70–85% of the glucocorticoids circulate bound to CBG (White, 2001). This increases their half-life by reducing metabolic clearance, and creating a circulating reservoir for easy access during the stress response. In amphibia plasma globulins show high affinities for glucocorticoid binding, and more than 90% of corticosterone has been reported to bind with high affinity to CBG with free levels below 5% (Glennemeier and Denver, 2002; Martin and Ozon, 1975; Seal and Doe, 1965). The objective of this study was to assess the adrenal activity from wild cane toads exposed to different experimental procedures to compare the adrenal activity of cane toads exposed to different environments.

D

81 82

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

E

2

(d). Response to exogenous ACTH (Synacthen)

169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190

195 196 197 198 199 200 201 202

The determination of an effective dose of ACTH in cane toads was 203 performed in 16 male cane toads captured on the same night and 204

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

216 217 218

242

2.3. Sample analysis

243 244

2.3.1. Corticosterone measurements The collected blood from each experimental procedure was placed into Eppendorf tubes and centrifuged at 1300 g for 5 min to separate plasma from blood cells. Plasma was stored at −20 °C for later analysis. Plasma samples (20 μl) were extracted using dichloromethane and assayed for total corticosterone (B) by radioimmunoassay (Bradley, 1987). The tracer used was tritiated corticosterone (corticosterone [1,2,6,7-3H] 2.59 TBq/mmol, Perkin Elmer, Lab). For this study we used a polyclonal antibody against corticosterone developed by AbCam laboratories (Product Number: ab77798), raised in sheep and based on corticosterone-3-cmo-urease as immunogen. The cross-reactions documented were: 0.67% to 11-dehydrocorticosterone, 1.5% to deoxycorticosterone, and b 0.01% to 18-OH-DOC, cortisone, cortisol and aldosterone. The plasma samples were measured in duplicate and the steroid concentrations were calculated from a standard curve, as described by Dudley et al. (1985). The intra-assay and inter-assay coefficients of variation were 5.80 and 6.94% respectively, with a recovery of 86% determined by using trace amounts of 3H-corticosterone. Data are expressed as ng/ml of plasma. The method of Tait and Burstein (1964) was used to determine the percentage of free B in plasma (Tait and Burstein, 1964). The maximum corticosterone binding capacity (MCBC) was obtained by the method described by McDonald et al., (1981). Microdialysis was used to determine the corticosterone binding globulin (CBG) and albumin affinity constants, as modified by Bradley

230 231 232 233 234 235 236 237 238 239

245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 Q8

C

228 229

E

226 227

R

225

R

223 224

N C O

221 222

U

219 220

T

240 241

2.2.2. Adrenal activity of cane toads exposed to different environments After the determination of an effective dose of ACTH (5 IU/100 g), as well as effective significant corticosterone suppression by DEX (2 mg/kg) from the previous experiments, a DEX-ACTH test was performed in toads from QL, WA, MX, and AH populations. Five males and 5 females from each population were captured and cannulated as described above. After 24 h recovery, a first blood sample (100 μl) was collected in all the animals from the different populations (time 0), followed by an injection of 2 mg/kg dexamethasone sodium phosphate (Dexadreson, Intervet Pty Ltd. Australia) in 100 μl 0.9% saline (sterile) via the catheter. Consecutive blood samples (100 μl) were taken at 1 h, and 2 h after DEX administration to confirm the inhibition of endogenous ACTH as determined from the DEX suppression performed during the experiments related to assessment of adrenal function in cane toads (Fig. 3). The last blood sample was followed by an intramuscular (im) injection of 5 IU/100 g of synthetic ACTH (time 0). The dose of ACTH was determined in the preliminary experiments (Fig. 4). Two more bleeds (100 μl) occurred 1 h, and 2 h post-ACTH injection. After all experiments, animals were euthanized and snout-vent length was measured followed by the removal of the kidneys which were preserved in cold 4% paraformaldehyde (PFA) and stored at 4 °C for histological analysis. The tissue was obtained to examine the relationship between cortical area and the maximum adrenal response to ACTH in each population.

2.3.2. Histomorphometry of adrenal tissue The kidney was divided in three equal pieces and the middle piece was sectioned into 10 μm slices with a freezing microtome (Hyrax cryostat Model C60). Every 10th section was placed on a glass microscope slide and stained for lipid with Sudan black (Piezzi and Burgos, 1968). The ratio between the area occupied by the cortical cells and snoutvent length was used to normalize the data.

284 285

2.4. Statistical analysis

291

F

214 215

O

212 213

R O

211

268 269

P

209 210

(1987). Eight plasma samples (10 μl) with basal levels (initial sample) were chosen randomly from the four populations and diluted in charcoal (1:30) in a solution of 0.2% dextran coated charcoal in PBS (0.05 M, pH = 7.4), and incubated at 24 °C for 30 min to remove endogenous steroidal hormones. Duplicate samples were incubated at 50 °C for 30 min to denature CBG and determine albumin binding parameters. Equilibrium in the dialysis chamber was established in 24 h at 4 °C, and radioactivity was measured by removing 50 μl from the saline and plasma chambers of each set of microdialysis chambers. The binding parameters were then calculated by fitting a saturation curve to one site binding and creating a Scatchard plot (Scatchard, 1949) in Graph Pad 4 Prism (GraphPad Software, inc. 2009). For all the steroid assays, samples were added to Ultima Gold scintillation fluid (Perkin Elmer, Waltham, MA, USA) and radioactivity was counted in a liquid scintillation spectrometer (Beckman LS 6000 TA®) with automatic efficiency correction to determine sample 3H expressed in dpm.

D

207 208

cannulated as explained above. After 24 h recovery the animals were assigned randomly to four groups. Each group was assigned with different doses of a synthetic ACTH diluted in 100 μl 0.9% saline (sterile) (Synacthen Depot [Tetracosactrin zinc phosphate complex], Novartis Pty. Ltd. Australia) and one group was used as a control. Group one was injected intramuscularly in the hind leg (semimembranosus m.) with a dose of 25 IU/100 g of body weight (BW) of synthetic ACTH. Group two was administered a dose of 10 IU/100 g (BW), the third group a dose of 5 IU/100 g (BW), and the fourth group was the control. The control group was injected with 100 μl 0.9% saline (sterile) intramuscularly. Before ACTH administration a first blood sample (100 μl) was obtained (initial), followed by further samples at 1 h, 2 h and 12 h post-ACTH injection.

270 271 272 273 274 275 276 277 278 279 Q9 280 281 282 283

286 287 288 289 290

Corticosteroid concentrations were log transformed (Log10) and Leven's F test was used to determine the homogeneity of variance. The arcsine transformation (Kuehl, 2000) was used to normalize the corrected values of the cortical cell areas by snout vent length. Twoway ANOVA analysis followed by Tukey's test was used to determine differences between treatments and sampling times. Student t-test was used to compare pairs of treatments. The differences in B levels in the DEX-ACTH challenge were determined using repeated measures two-way ANOVA. The Partial eta-squared (η2p) statistic was used as the percentage of variability predicted in the analysis. Finally the association between the normalized cortical cell area and the response to ACTH was calculated by linear regression. The null hypothesis was rejected at p b 0.05. Data were expressed as mean ± SD. Statistical analyses were conducted using SPSS (IBM, SPSS Statistics. V19), and GraphPad Prism (GraphPad Software, Inc. 2009).

292

3. Results

307

3.1. Assessment of adrenocortical function

308

3.1.1. Effect of movement restriction in corticosterone plasmatic levels in cane toads Fig. 1 shows that within 1 h of capture, the levels of plasma B increased from 9.33 ± 2.77 ng/ml (time zero) to 93.40 ± 17.33 ng/ml plasma (1 h) (F(3,16)40.67; p b 0.01). However, after 2 h of confinement in the plastic container, the B levels dropped to 29.51 ± 18.80 ng/ml plasma and returned to initial levels by 12 h (7.64 ± 6.79 ng/ml plasma; p N 0.05).

309

3.1.2. Effect of cannulation in corticosterone plasmatic levels in cane toads The initial levels of B in uncannulated animals (9.33 ± 2.77 ng/ml plasma), where blood had been collected by cardiac puncture (Fig. 1), were lower than for cannulated animals (29.54 ± 9.78 ng/ml plasma; t(8)4.44; p = 0.002; Fig. 2). However, B levels did not change between times after 1, 2 and 12 h of cannulation (F(3,6)0.045; p = 0.98; Fig. 2).

317

E

205 206

3

293 294 295 296 297 298 299 300 301 302 303 304 305 306 Q10

310 311 312 313 314 315 316

318 319 320 321 322

3.1.3. Suppression of corticosterone levels by dexamethasone (DEX) 323 A single dose of dexamethasone (DEX 2 mg/kg) significantly re- 324 duced the B levels after the cannulation procedure (F(4,16)116; p = 325

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

339 340 341 342 343 344

R O

P

D

3.2. Adrenal activity of cane toads exposed to different environments

347

3.2.1. DEX-ACTH suppression test While significant differences were found in the initial concentrations (time 0) of B levels between populations of cannulated cane toads (MX = 32.19 ± 21.89; QL = 54.41 ± 24.10; WA = 14.50 ± 4.08; AH = 9.45 ± 4.86 ng/ml plasma; F(6,64)8.15; p = 0.00; η2p = 0.43), a single dose of DEX reduced the B levels below initial levels in all populations (F(2,64)216.46; p b 0.01; η2p = 0.87; Fig. 5A). The reduction was still present after 1 h (AH = 4.68 ± 1.71; WA = 3.23 ± 2.65; MX = 11.49 ± 5.84; QL = 22.93 ± 19.43 ng/ml plasma) and 2 h (AH = 1.73 ± 1.15; WA = 0.82 ± 0.66; MX = 6.88 ± 3.95; QL = 11.83 ± 12.76 ng/ml plasma) of the DEX administration (p N 0.05; Fig. 5A). Significant differences were not found in initial B levels (time 0) between males (AH = 10.00 ± 102.9; WA = 13.44 ± 3.15; MX = 41.22 ± 27.85; QL = 72.55 ± 18.81 ng/ml plasma) and females (AH = 8.89 ± 1.19; WA = 15.56 ± 4.70; MX = 23.16 ± 6.48; QL = 36.27 ± 13.03 ng/ml plasma, p N 0.05).

348 349

T

C

E

337 338

3.1.4. Response to exogenous ACTH (Synacthen) Fig. 4 shows that after intramuscular ACTH administration the levels of B in plasma increased gradually at all doses of ACTH to a significant peak after 2 h (F(6,31)20.94; p b 0.01) and then returned to initial levels by 12 h (p N 0.05). When responses to increasing doses of ACTH were compared, no significant differences were observed at any of the time points (F(2,31)0.79; p = 0.46). Saline administration did not change B levels between sampling times (F(3,16)0.046; p = 0.99; Fig. 4). These data were obtained in toads from QL and, since there was no difference between doses, the lowest dose of synthetic ACTH (5 IU/

R

335 336

R

333 334

O

331 332

100 g BW) was chosen as a standard to challenge the adrenal function 345 in cane toads from the remaining populations. 346

C

329 330

0.0017). The maximum suppression of the initial levels (time 0 = 44.56 ± 10.77 ng/ml plasma) was achieved after 1 h (19.32 ± 10.68 ng/ml) and maintained after 2 h (18.11 ± 9.063 ng/ml plasma; p N 0.05; Fig. 3). After 4 h post DEX administration the levels began to increase (23.00 ± 7.96 ng/ml plasma) and returned to initial levels after 12 h (49.33 ± 6.34 ng/ml plasma; p N 0.05; Fig. 3). Based on the results, 2 h was chosen as the effective DEX suppression time for B levels in cannulated cane toads, and this time was used to compare the response between populations.

N

327 328

U

326

Fig. 3. Effect of a single dose of intravenous dexamethasone (DEX) on plasma corticosterone in cannulated wild male cane toads (Rhinella marina). First blood sample (100 μl) was drawn 24 h after cannulation (time 0), followed by intravenous injection of 2 mg/kg DEX. Consecutive blood samples (100 μl) were collected at one hour (1 h) two hours (2 h), four hours (4 h) and twelve hours (12 h) after DEX administration. Data are presented as mean ± SD (N = 5) while different letters indicate statistical differences between times (p b 0.05).

E

Fig. 1. Effect of movement restriction in plasma corticosterone levels in wild male cane toads (Rinella marina). Samples were taken by cardiac puncture immediately after capture (0 h), at 1, 2 and at 12 h after movement restriction. Data are presented as mean ± SD (n = 5 per time). *p b 0.05; **p b 0.001 represent statistical differences between times within treatments.

O

F

4

Fig. 2. Effect of cannulation in plasma corticosterone levels in wild male cane toads (Rhinella marina). First blood sample (100 μl) was drawn 24 h after cannulation (time 0), followed by consecutive blood samples at one hour (1 h), two hours (2 h) and twelve hours (12 h). Data are presented as mean ± SD (N = 5). *p b 0.05; represent statistical differences between times.

Fig. 4. Response to synthetic ACTH (Synacthen) doses of corticosterone levels in cannulated wild male cane toads (Rhinella marina). After 24 h of cannulation (time 0), a first blood sample (100 μl) was drawn in all animals (N = 16). The animals were divided in 4 groups of 4 animals each. The first group received a single IM injection of 100 μl 0.9% saline (control). The other groups received a single IM dose of 5 IU, 10 IU or 25 IU per 100 g BW of Synacthen. Consecutive blood samples (100 μl) were collected at one hour (1 h) two hours (2 h), and twelve hours (12 h) after Synacthen administration. Data are presented as mean ± SD (n = 4). *p b 0.05; **p b 0.001 represent statistical differences between times after Synacthen administration.

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

350 351 352 353 354 355 356 357 358 359 360 361 362 363

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

O

F

398

3.2.2. Free and bound corticosterone The association constant (Ka) for the binding of B to CBG at 4 °C was 6.5 ± 18.6 × 108 M− 1 (n = 8) and the maximum binding capacity

R O

383

3.2.3. Association between cortical area and the maximum adrenal response to ACTH The association between cortical area and the maximum adrenal response to ACTH is shown in Fig. 8. Using linear regression analysis the cortical cell area was found to be positively associated with the maximum plasma B concentration resulting from a single dose of ACTH (F(1,38)13.58 p = 0.01, R2 = 0.26). No correlation was found with baseline B levels and cortical cell area (R2 = 0.0).

P

381 382

384 385

4. Discussion

D

379 380

(Bmax) of B to CBG was 185.2 ± 21.1 nM (n = 8) (Fig. 6). The association binding constant (Ka) and the maximum binding capacity (Bmax) for albumin, determined from heat denatured toad plasma (50 °C for 10 min), were 5.15 ± 7.59 × 106 M−1 (n = 8) and 894.4 ± 52.57 nM (n = 8) respectively. The corticosterone partitioning analysis showed that 1% (ranging from 0.9% to 1.19%) of the total corticosterone found in plasma appears in the free form while 95% is bound to CBG (ranging from 92.69% to 97.25%) and 4% is bound to albumin (ranging from 4.09% to 5.20%). Free B was found to be different between treatments (F(3117)202.02 p = 0.001). The highest percentage of free levels was found after ACTH challenge and the lowest was found after DEX challenge (Fig. 7). However, these differences were negligible when the percentage of free B was compared between populations and sexes (p N 0.05).

In this study we showed that restriction stress caused a rapid HPA response in toads lasting for up to 12 h. The toads exposed to restrictive stress showed a tenfold increase in B levels after 1 h and full recovery 12 h later. This increase is similar to that reported from a previous study in cane toads in which corticosterone metabolites were measured in blood (Graham et al., 2012) and urine (Narayan et al., 2012). It was also observed that cannulation increases significantly the B levels and that these levels do not change during the period that the cannula is maintained (12 h). This observation is important to consider in experiments where a cannula is placed in order to obtain serial samples of blood in amphibia. Furthermore, a dexamethasone dose reported for shock treatment (Wright, 2001) was used to establish the presence of the negative feedback loop acting via the HPA axis. This demonstrates the potential of DEX in the evaluation of the HPA axis in amphibia in the field. However dose response studies in several amphibian species might first be necessary to determine a standard dose. Previous studies in amphibia have demonstrated that DEX binds to receptors in the brain with a similar affinity to B (Orchinik et al., 2000) and has similar effects on the peripheral nervous system (Rose et al., 1993). DEX infusion does not interfere with GC synthesis in cortical cells (Netchitailo et al., 1984),

E

377 378

T

375 376

C

373 374

E

371 372

R

370

R

368 369

N C O

366 367

After 2 h of DEX administration (time 0; AH = 1.73 ± 1.15; WA = 0.82 ± 0.66; MX = 6.88 ± 3.95; QL = 11.83 ± 12.76 ng/ml plasma; p b 0.05), the injection of 5 IU/100 g BW ACTH caused an increase in B levels in all populations F(2,64)680.00 p b 0.01, η2p = 0.95; Fig. 5B). The increase in B levels after 1 h (AH = 125 ± 37.26; WA = 34.68 ± 5.21; MX = 52.92 ± 1.92; QL = 107.61 ± 4.60 ng/ml plasma;) and 2 h (AH = 282 ± 37.89; WA = 19.73 ± 3.64; MX = 47.34 ± 8.06; QL = 115.93 ± 9.00 ng/ml plasma) of ACTH administration was different between populations and higher than the initial levels (F(6,64)24.46 p = 0.01, η2p = 0.69; Fig. 5B). AH showed the largest response to ACTH administration, followed by QL and MX while WA showed the smallest response (Fig. 5B). The ACTH stimulatory effect was different between females and males. After 2 h of ACTH administration, females had higher corticosterone levels (AH = 293.51 ± 116.80; WA = 20.34 ± 11.13; MX = 50.35 ± 24.03; QL = 126.24 ± 6.81 ng/ml plasma), than males (AH = 210.08 ± 67.52; WA = 14.78 ± 7.88; MX = 33.93 ± 16.44; QL = 80.14 ± 7.19 ng/ml plasma; F(2,64)11.37 p = 0.01, η2p = 0.26).

U

364 365

5

Fig. 5. Dexamethasone-ACTH test in four populations (_●_AH [n = 10],. □ MX [n = 10],– ♦– QL [n = 10], _X_WA [n = 10]) of cane toad (Rhinella marina) captured during wet season. After 24 h of cannulation, a first blood sample (100 μl) was collected in all the animals from different populations, followed by an injection of 2 mg/kg of DEX via the catheter (time 0, Fig. 5A). Consecutive blood samples (100 μl) were taken at 1 h, and 2 h, after DEX administration (Fig. 5A). The last blood sample was followed by an intramuscular (im) injection of 5 IU/100 g of synthetic ACTH (time 0 Fig. 5B). Two more bleeds (100 μl) occurred 1 h, and 2 h post-ACTH injection (Fig. 5B). Data are presented as mean ± SD. *p b 0.05; **p b 0.001 represent statistical differences between time sampling.

Fig. 6. Corticosterone binding to plasma proteins as shown by the binding curve and Scatchard plot of data obtained by equilibrium dialysis of dilute (1:30) plasma at 4 °C. The data represents pooled plasma from 8 male toads.

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

386 387 388 389 390 391 392 393 394 395 396 397

399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

in mammals also plays a part in maintaining the ratio between free/ bound B in plasma, however its affinity and capacities are low compared with CBG (Pardridge, 1981; Westphal, 1983). In the present study we found the same trend in albumin binding, and our findings are similar to those previously reported in Xenopus (Glennemeier and Denver, 2002). The positive association between total levels of B after ACTH administration and the adrenal histomorphometry, demonstrates that the size area of the cortical cells change in accordance with stress levels in cane toad. Kemenade (1968) reported that ACTH produced an increase in the intrarenal activity as well as in the cell number, that will result in hyperplasia in the cortical cells in hypophysectomized frogs. The differences in the ACTH response, as well as in the cortical cell area between populations found in the present study also support this assertion. The differences of adrenal activity between populations were one of the most important findings in this study. The animals maintained in captivity for one year (AH) present the highest levels of total and free B after ACTH administration, and animals in this group also had the largest cortical cell area. Our results for B levels in captive animals are similar to a report for the American toad (Bufo americanus) held in captivity indoors for one year (Pancak and Taylor, 1983). Pancak and Taylor (1983) report that while captivity did not change the circannual rhythm in toads, they did cause changes in total B levels in plasma. They reported that B levels in captive animals maintained indoors during spring and summer were lower than the field group. Their results also showed that B levels in the captive group during August to October were higher than the field group. The authors attribute the changes in B levels to environmental conditions during the period of captivity indoors. Changes in adrenal function is not an unusual finding in animals maintained in captivity. Altered adrenal activity has been reported in rats exposed to chronic stress during captivity (Aguilera et al., 1996) as well as in farm animals (von Borell, 2001). In amphibia changes in adrenal histomorphology have been obtained experimentally in Rana temporaria chronically exposed to ACTH (Kemenade, 1968). Dexamethasone resistance is also expected in animals exposed to chronic stress (Boonstra and McColl, 2000). However we did not find this response in the group of toads maintained captive for one year, suggesting that cane toads adapt to chronic stress mainly by changing adrenal activity without evident changes in the stress negative feedback loop. Another important finding was that the stress response in the Australian dispersing toads differs significantly from the other populations. We found that the animals from the front line of dispersion (WA) present the weakest adrenal response to cannulation and ACTH after DEX suppression. Our results are similar to a previous study in toads captured in the Northern Territory in Australia (Graham et al., 2012). Graham et al., (2012) found that cane toads with the invasive

P

U

N

C

O

R

R

E

C

T

thus confirming that the effects of DEX are on the HPA axis feedback loop. 429 We also demonstrate that 0.05 IU/g BW (im) of synthetic ACTH 430 increases B levels in cane toads. Doses previously reported for amphibia 431 range from 0.25 IU to 4 IU/g BW (Dupont et al., 1979; Gendron et al., 432 1997; Hanke and Weber, 1965; Hopkins et al., 1997; Kemenade, 1968; 433 Narayan et al., 2011). Our findings indicate that a fifth of the lowest 434 dose previously reported by Kemenade (1968) is enough to induce 435 adrenal activity in cannulated and DEX suppressed toads. 436 Free levels of B in cane toads were also affected after the treatment 437 with DEX-ACTH. We found that free B levels remain below 2% despite 438 the stimulus or suppression by the pharmacological challenge. The 439 low percentage of free B found in the present study, as well as in a pre440 vious study in Xenopus (Glennemeier and Denver, 2002; Jolivet-Jaudet 441 Q11 and Leloup-Hâtey, 1986), contrast with the levels reported in mammals. 442 Studies in humans and mice have shown that around 90% of GCs are 443 bound to CBG and 5% are free (Gayrard et al., 1996). The differences of 444 free B, between mammals and amphibians might be attributed to the 445 high affinity and low capacity of CBG found in the present study and 446 reported previously in other amphibians (Breuner and Orchinik, 2002; 447 Q12 Jolivet-Jaudet and Leloup-Hâtey, 1986; Martin and Ozon, 1975). The 448 high affinity of amphibian CBG for B balances the reduced number of 449 binding sites, and maintains the low free B levels in plasma. Albumin

D

427 428

E

Fig. 7. Percentage of free corticosterone in populations of toads from different locations. Data show free corticosterone in control conditions and after DEX and ACTH treatment. Data are shown as mean ± SD (n = 10) while different letters indicate statistical differences between control (Initial) and treatments (DEX-ACTH) (p b 0.05).

R O

O

F

6

Fig. 8. Association between cortical cell area with baseline corticosterone concentrations (A) and corticosterone concentration during maximum adrenal response to synthetic ACTH treatment (B) in the four populations (●AH, □MX, ♦ QL, *WA). Lines represent the linear regression in each pair of associations. Values presented on the graphs are normalized data. A significant correlation (p b 0.05) was found between adrenal size and corticosterone concentration during maximum adrenal response to synthetic ACTH treatment.

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

496 497

R O

Uncited reference

C

E

R

R

N C O

U

O

F

The changes in the adrenal histomorphometry and adrenal response in the different populations demonstrate that the environment is able to promote changes in the cane toad stress response. We also can conclude that low B levels in cane toads might be an adaptive strategy to enable survival in new environments and once they find an appropriate ecological niche, their neuroendocrine system converges into a single best response. The present study was limited to one period of the life of these animals. Future studies must be directed to determine stress response differences before and after the wet season. Longitudinal assessments will capture the different facet of the stress response in toad populations. The inclusion of other variables like indices of body condition and immune status, in addition to corticosteroid levels, might lead to a much better understanding of adaptive strategies in invasive cane toad populations.

Breuner et al., 2003

562 563 564 565 566 567 568 569 570 571 572 573 574 575 576

577 Q15 578

579

For their logistical and technical support during the field work conducted in Mexico we gratefully acknowledge the assistance of Dr. Marta C. Romano, from the Centre for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV, IPN, Mexico), the Department of Ethology, Wild life and Laboratory animals, from the Veterinary School (UNAM, Mexico), the Biology Station “Los Tuxtlas” from the Biology Institute (UNAM, Mexico), Dr. Víctor H. Reynoso (VHR), Zoology Department from the Biology Institute (UNAM, Mexico). For their support during the field work conducted in Western Australia we are grateful for the help of The Kimberly Toad Busters. SEH thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico for providing the PhD scholarship. In Mexico, samples were collected using Scientific Collecting Permit FAUT 0014 by SEMARNAT to VHR.

580

References

594

Aguilera, G., Kiss, A., Lu, A., Camacho, C., 1996. Regulation of adrenal steroidogenesis during chronic stress. Endocr. Res. 22, 433–443. Bennett, A.F., 1997. Adaptation and the evolution of physiological characters. In: Dantzler, W.H. (Ed.), Handbook of Physiology, Sect. 13: Comp. Physiol. Oxford University, press, New York, pp. 3–16. Boonstra, R., McColl, C.J., 2000. Contrasting stress response of male Arctic ground squirrels and red squirrels. J. Exp. Zool. 286, 390–404. Bradley, A.J., 1987. Stress and mortality in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). Gen. Comp. Endocrinol. 67, 85–100. Breuner, C.W., Orchinik, M., 2002. Plasma binding proteins as mediators of corticosteroid action in vertebrates. J. Endocrinol. 175, 99–112. Breuner, C.W., Orchinik, M., Hahn, T.P., Meddle, S.L., Moore, I.T., Owen-Ashley, N.T., Sperry, T.S., Wingfield, J.C., 2003. Differential mechanisms for regulation of the stress response across latitudinal gradients. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R594–R600. Capaldo, A., Gay, F., Valiante, S., Varlese, M., Laforgia, V., Varano, L., 2004. Release of aldosterone and catecholamines from the interrenal gland of Triturus carnifex in response to adrenocorticotropic hormone (ACTH) administration. J. Morphol. 262, 692–700. Carrasco, G.A., Van de Kar, L.D., 2003. Neuroendocrine pharmacology of stress. Eur. J. Pharmacol. 463, 235–272. Crespi, E.J., Denver, R.J., 2004. Ontogeny of corticotropin-releasing factor effects on locomotion and foraging in the Western spadefoot toad (Spea hammondii). Horm. Behav. 46, 399–410. Dallman, M.F., Strack, A.M., Akana, S.F., Bradbury, M.J., Hanson, E.S., Scribner, K.A., Smith, M., 1993. Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front. Neuroendocrinol. 14, 303–347. Department of Environment and Conservation, 2013. WA Cane Toad Update. Department of Environment and Conservation, Western Australia. Dudley, R.A., Edwards, P., Ekins, R.P., Finney, D.J., McKensey, G.I., Raab, G.M., Robard, D., Rodgers, R.P., 1985. Guidelines for immunoassay data processing. Clin. Chem. 31, 1264–1271. Dupont, W., Leboulenger, F., Vaudry, H., Vaillant, R., 1976. Regulation of the aldosterone secretion in the frog Rana esculenta L. Gen. Comp. Endocrinol. 29, 51–60.

595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 Q16 623 624 625 626 627

D

P

Acknowledgements

E

T

phenotype had lower B after capture stress than toads without the phenotype trait. The findings associated with the stress response of 498 the dispersing population invading new territories in Australia raises 499 an important question with respect to how low levels of B might 500 improve the survival opportunities in this group of animals. The primary 501 function of dispersal behaviours is to gain new territories and therefore 502 resources. However animals will not move from one territory if the 503 benefits of moving outweigh the cost of doing so (Russell et al., 2005). 504 Several studies in vertebrates have demonstrated that GC levels are 505 used as starting point to assess the cost–benefit of triggering survival 506 strategies like dispersal and wandering in juveniles, and migration 507 (Korte et al., 2005; Wingfield et al., 2015). In amphibia a study on the 508 spadefoot toad (Spea hammondii) demonstrates that high levels of B 509 reduce swimming behaviour and increase foraging behaviour (Crespi Q14510 Q13 and Denver, 2004). Our finding supports Wingfiled's (2015) hypothesis 511 that individuals with high resistance potential to environmental change 512 will have a down-regulated adrenocortical response (Wingfield et al., 513 2015). Additionally the favourable environmental conditions, like the 514 lack of inter- and intra-specific competition, and quality and quantity 515 of food resources in invasive cane toads in Australia, will promote a 516 better outcome in the energetic tradeoffs in these animals, reducing ad517 renal activity. This will result in the reduction of B levels in plasma and 518 therefore encouraging the motivation of dispersion in WA cane toad 519 population. The study of Crespi and Denver (2004) support this last 520 statement. They found that B acts in the central nervous system (CNS) 521 increasing foraging behaviour and reducing the effect of corticotropin522 releasing hormone (CRH) on locomotive behaviours. Therefore low 523 levels of B will not trigger the HPA negative feedback loop and, as in 524 consequence, CRH will be active for longer. 525 Comparing the two groups of animals categorized as long 526 established (QL), and native (MX), we did not find a marked difference 527 in the HPA activity between them. This is very important because it 528 supports the evidence that once a group finds an appropriate niche sim529 ilar to its native environment, the response converges in a single best 530 reactive response (Wingfield and Kitaysky, 2002; Wingfield et al., 531 2015). However if the environment does not satisfy the necessities of 532 the individuals, the result is a disruption in the stress response 533 (Romero, 2004), as we confirm with the group of animals maintained 534 in captivity for one year (AH). 535 Finally we found sexual variation in the adrenocortical response in 536 cane toads. Females show a greater response to ACTH than males. Our 537 results are in agreement with previous studies in reptiles (Jessop, 538 2001; Moore et al., 2000; Whittier et al., 1987) and mammals (Touma 539 et al., 2004). Results in amphibians are contentious. While a study in 540 Rana esculenta did not find differences between sexes (Vaudry et al., 541 1975), a study in green frogs reported a lower B response to restrictive 542 stress in males than females (Zerani et al., 1991). The adaptive function 543 of the differences between males and females might be attributed to the 544 higher energetic investment in reproduction in females. Licht et al., 545 (1983) found that the cycle of B in bullfrog females correlated positively 546 with the gonadotropin levels, starting its rise in spring and maintaining 547 it after the reproductive hormones have declined to minimal levels 548 (Licht et al., 1983). Longer demands on energy supply in females before, 549 during and after reproductive season will lead to a prolonged catabolic 550 state and therefore prolonged elevation of catabolic mediators like GCs. 551 This is the first study that examines systematically the adrenocorti552 cal response in populations of cane toads in native and introduced 553 areas during the wet season. We determine that a low dose of ACTH is 554 able to exert an effect on the cortical cells. For the first time DEX was 555 used to challenge the negative feedback loop modulating the HPA axis 556 in amphibians. However dose response studies in several species 557 might be necessary to determine a standard dose for amphibians. We 558 also found that captivity has an effect on the adrenocortical response 559 in the cane toad that is reflected in an exacerbation of the stress 560 response, with changes also occurring in the histomorphometry of adre561 nal tissue.

7

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

581 582 583 584 585 586 587 588 589 590 591 592 593

D

P

R O

O

F

Ogawa, K., Suzuki, E., Taniguchi, K., 1995. Immunohistochemical studies on the development of the hypothalamo–hypophysial system in Xenopus laevis. Anat. Rec. 241, 244–254. Olvera-Novoa, M.A., Ontiveros-Escutia, V.M., Flores-Nava, A., 2007. Optimum protein level for growth in juvenile bullfrog (Rana catesbeiana Shaw, 1802). Aquaculture 266, 191–199. Orchinik, M., Matthews, L., Gasser, P.J., 2000. Distinct specificity for corticosteroid binding sites in amphibian cytosol, neuronal membranes, and plasma. Gen. Comp. Endocrinol. 118, 284–301. Pancak, M.K., Taylor, D.H., 1983. Seasonal and daily plasma corticosterone rhythms in American toads, Bufo americanus. Gen. Comp. Endocrinol. 50, 490–497. Pardridge, W.M., 1981. Transport of protein-bound hormones into tissues in vivo. Endocr. Rev. 2, 103–123. Piezzi, R.S., Burgos, M.H., 1968. The toad adrenal gland. I. Cortical cells during summer and winter. Gen. Comp. Endocrinol. 10, 344–354. Romero, L.M., 2004. Physiological stress in ecology: lessons from biomedical research. Trends Ecol. Evol. 19, 249–255. Rose, J.D., Moore, F.L., Orchinik, M., 1993. Rapid neurophysiological effects of corticosterone on medullary neurons: relationship to stress-induced suppression of courtship clasping in an amphibian. Neuroendocrinology 57, 815–824. Russell, A., Bauer, A., Johnson, M., 2005. Migration in amphibians and reptiles: An overview of patterns and orientation mechanisms in relation to life history strategies. In: Elewa, A.T. (Ed.), Migration of Organisms. Springer, Berlin Heidelberg, pp. 151–203. Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21, 55–89. Scatchard, G., 1949. The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660–672. Seal, U.S., Doe, R.P., 1965. Vertebrate distribution of corticosteroid-binding globulin and some endocrine effects on concentration. Steroids 5, 827–841. Silverin, B., Arvidsson, B., Wingfield, J., 1997. The adrenocortical responses to stress in breeding willow warblers Phylloscopus trochilus in Sweden: effects of latitude and gender. Funct. Ecol. 11, 376–384. Sternberg, E.M., Licinio, J., 1995. Overview of neuroimmune stress interactions. Ann. N. Y. Acad. Sci. 771, 364–371. Tait, J.F., Burstein, S., 1964. In vivo studies of steroid dynamics in man. In: Pincus, G., Thimann, K.V., Astwood, E.B. (Eds.), Hormones. Academic, New York. Touma, C., Palme, R., Sachser, N., 2004. Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm. Behav. 45, 10–22. Vaudry, H., Vague, P., Dupont, W., Leboulenger, F., Vaillant, R., 1975. A radioimmunoassay for plasma corticotropin in frogs (Rana esculenta L.). Gen. Comp. Endocrinol. 25, 313–322. Vinson, G.P., Whitehouse, B.J., 1974. Some comparative studies in adrenocortical steroidogenesis: an interpretation of the functional homologies of the mammalian and nonmammalian adrenal cortex. J. Steroid Biochem. 5, 801–810. von Borell, E.H., 2001. The biology of stress and its application to livestock housing and transportation assessment. J. Anim. Sci. 79, E260–E267. Westphal, U., 1983. Steroid–protein interaction: from past to present. J. Steroid Biochem. 19, 1–15. White, P.C., 2001. Synthesis and metabolism of corticosteroids. In: Becker, K.L. (Ed.), Principles and Practice of Endocrinology and Metabolism, third ed. Lippincott Williams and Wilkins, Philadelphia, pp. 705–714. Whittier, J.M., Mason, R.T., Crews, D., 1987. Plasma steroid hormone levels of female redsided garter snakes, Thamnophis sirtalis parietalis: relationship to mating and gestation. Gen. Comp. Endocrinol. 67, 33–43. Wingfield, J.C., 2008. Organization of vertebrate annual cycles: implications for control mechanisms. Philos. Trans. R. Soc., B 363, 425–441. Wingfield, J.C., Kitaysky, A.S., 2002. Endocrine responses to unpredictable environmental events: stress or anti-stress hormones? Integr. Comp. Biol. 42, 600–609. Wingfield, J.C., Krause, J.S., Perez, J.H., Chmura, H.E., Németh, Z., Word, K.R., Calisi, R.M., Meddle, S.L., 2015. A mechanistic approach to understanding range shifts in a changing world: what makes a pioneer? Gen. Comp. Endocrinol. 222, 44–53. Wright, K.M., 2001. Amphibians. In: Carpenter, J.W. (Ed.), Exotic Animal Formulary, third ed. Elsevier Saunders, St. Louis, Missouri, pp. 33–54. Yao, M., Westphal, N.J., Denver, R.J., 2004. Distribution and acute stressor-induced activation of corticotrophin-releasing hormone neurones in the central nervous system of Xenopus laevis. J. Neuroendocrinol. 16, 880–893. Zerani, M., Amabili, F., Mosconi, G., Gobbetti, A., 1991. Effects of captivity stress on plasma steroid levels in the green frog, Rana esculenta, during the annual reproductive cycle. Comp. Biochem. Physiol. A 98, 491–496. Zug, G.R., Zug, P.B., 1979. The marine toad Bufo marinus a natural history resume of native populations. Smithson. Contrib. Zool. 1–58.

N

C

O

R

R

E

C

T

Dupont, W., Bourgeois, P., Reinberg, A., Vaillant, R., 1979. Circannual and circadian rhythms in the concentration of corticosterone in the plasma of the edible frog (Rana esculenta L.). J. Endocrinol. 80, 117–125. Gayrard, V., Alvinerie, M., Toutain, P.L., 1996. Interspecies variations of corticosteroidbinding globulin parameters. Domest. Anim. Endocrinol. 13, 35–45. 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. Environ. Toxicol. Chem. 16, 1694–1706. Glennemeier, K.A., Denver, R.J., 2002. Developmental changes in interrenal responsiveness in anuran amphibians. Integr. Comp. Biol. 42, 565–573. Graham, S.P., Kelehear, C., Brown, G.P., Shine, R., 2012. Corticosterone–immune interactions during captive stress in invading Australian cane toads (Rhinella marina). Horm. Behav. 62, 146–153. Hanke, W., 1978. The Adrenal Cortex of Amphibia. In: Jones, C., Henderson, W. (Eds.), General, Comparative and Clinical Endocrinology of the Adrenal Cortex. Academic Press, London, pp. 419–495. Hanke, W., Weber, K., 1965. Histophysiological investigation on the zonation, activity, and mode of secretion of the adrenal gland of the frog, Rana temporaria linnaeus. Gen. Comp. Endocrinol. 5, 444–455. Hardy, M.P., Gao, H.B., Dong, Q., Ge, R., Wang, Q., Chai, W.R., Feng, X., Sottas, C., 2005. Stress hormone and male reproductive function. Cell Tissue Res. 322, 147–153. Hernández, S.E., Sernia, C., Bradley, A.J., 2012. The effect of three anaesthetic protocols on the stress response in cane toads (Rhinella marina). Vet. Anaesth. Analg. 39, 584–590. Hero, M.J., Stonham, M., 2005. Bufo marinus (Linnaeus, 1759). In: Lannoo, M. (Ed.), Amphibian Declines: the Conservation Status of United States Species. University of California Press, Berkeley, pp. 417–422. Hopkins, W.A., Mendonça, M.T., Congdon, J.D., 1997. Increased circulating levels of testosterone and corticosterone in southern toads, Bufo terrestris, exposed to coal combustion waste. Gen. Comp. Endocrinol. 108, 237–246. Janssens, P.A., 1970. The evolution of corticosteroid function. The effects of corticosteroids on gluconeogenesis in poikilothermic vertebrates. Steroidologia 1, 308–320. Jessop, T.S., 2001. Modulation of the adrenocortical stress response in marine turtles (Cheloniidae): evidence for a hormonal tactic maximizing maternal reproductive investment. J. Zool. 254, 57–65. Jolivet-Jaudet, G., Leloup-Hâtey, J., 1986. Corticosteroid binding in plasma of Xenopus laevis. Modifications during metamorphosis and growth. J. Steroid Biochem. 25, 343–350. Jorgensen, C.B., 1976. Sub-mammalian vertebrate hypothalamic–pituitary–adrenal interrelationships. In: Jones, C., Henderson, W. (Eds.), General, Comparative and Clinical Endocrinology of the Adrenal Cortex. Academic Press, London, UK., pp. 153–206. Kemenade, J.A.M., 1968. Effect of ACTH and hypophysectomy on the interrenal tissue in the common frog, Rana temporaria. Cell Tissue Res. 92, 549–566. Korte, S.M., Koolhaas, J.M., Wingfield, J.C., McEwen, B.S., 2005. The Darwinian concept of stress: benefits of allostasis and costs of allostatic load and the trade-offs in health and disease. Neurosci. Biobehav. Rev. 29, 3–38. Kuehl, R.O., 2000. Design of experiments: statistical principles of research design and analysis. Duxbury/Thomson Learning. Leboulenger, F., Delarue, C., Belanger, A., Perroteau, I., Netchitailo, P., Leroux, P., Jegou, S., Tonon, M.C., Vaudry, H., 1982. Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environment. Gen. Comp. Endocrinol. 46, 521–532. Licht, P., McCreery, B.R., Barnes, R., Pang, R., 1983. Seasonal and stress related changes in plasma gonadotropins, sex steroids, and corticosterone in the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 50, 124–145. Martin, B., Ozon, R., 1975. Steroid-protein interactions in nonmammalian vertebrates. II. Steroids binding proteins in the serum of amphibians; a physiological approach. Biol. Reprod. 13, 371–380. McDonald, I.R., Lee, A.K., Bradley, A.J., Than, K.A., 1981. Endocrine changes in dasyurid marsupials with differing mortality patterns. Gen. Comp. Endocrinol. 44, 292–301. McEwen, B.S., Sapolsky, R.M., 1995. Stress and cognitive function. Curr. Op. Neurobiol. 5, 205–216. McEwen, B., Stellar, E., 1993. Stress and the individual: mechanisms leading to disease. Arch. Intern. Med. 153, 2093–2101. Moore, I.T., Lerner, J.P., Lerner, D.T., Mason, R.T., 2000. Relationships between annual cycles of testosterone, corticosterone, and body condition in male red spotted garter snakes, Thamnophis sirtalis concinnus. Physiol. Biochem. Zool. 73, 307–312. Narayan, E.J., Cockrem, J.F., Hero, J.-M., 2011. Urinary corticosterone metabolite responses to capture and captivity in the cane toad (Rhinella marina). Gen. Comp. Endocrinol. 173, 371–377. Narayan, E.J., Hero, J.-M., Cockrem, J.F., 2012. Inverse urinary corticosterone and testosterone metabolite responses to different durations of restraint in the cane toad (Rhinella marina). Gen. Comp. Endocrinol. 179, 345–349. Netchitailo, P., Lihrmann, I., Vaudry, H., 1984. Lack of effect of dexamethasone on corticosteroid production in the amphibian. J. Steroid Biochem. 21, 727–731.

U

628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701

S.E. Hernández et al. / Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

E

8

Please cite this article as: Hernández, S.E., et al., Adrenocortical function in cane toads from different environments, Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.001

702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775