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Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?
Journal Pre-proofs Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough? Catherine Tylan, Kiara Camacho, Susannah French, Sean P. Graham, Mark W. Herr, Jermayne Jones, Gail L. McCormick, Melissa A. O'Brien, Jennifer B. Tennessen, Christopher J. Thawley, Alison Webb, Tracy Langkilde PII: DOI: Reference:
S0016-6480(19)30147-9 https://doi.org/10.1016/j.ygcen.2019.113324 YGCEN 113324
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
21 March 2019 6 November 2019 12 November 2019
Please cite this article as: Tylan, C., Camacho, K., French, S., Graham, S.P., Herr, M.W., Jones, J., McCormick, G.L., O'Brien, M.A., Tennessen, J.B., Thawley, C.J., Webb, A., Langkilde, T., Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?, General and Comparative Endocrinology (2019), doi: https://doi.org/10.1016/j.ygcen.2019.113324
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Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick
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enough?
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Catherine Tylana, Kiara Camachoa, Susannah Frenchb, Sean P. Grahama,1, Mark W. Herra,2, Jermayne
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Jonesa, Gail L. McCormicka,c, Melissa A. O’Briena, Jennifer B. Tennessena,c,3, Christopher J. Thawleya,c,4,
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Alison Webbb, Tracy Langkildea,c,*
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a Department
of Biology, The Pennsylvania State University, University Park, PA 16802, USA
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bDepartment
of Biology and the Ecology Center, Utah State University, Logan, UT 84322, USA
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cIntercollege
Graduate Degree Program in Ecology, and The Center for Brain, Behavior and Cognition,
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The Pennsylvania State University, University Park, PA 16802, USA
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1Present
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Alpine, Texas, 79832, USA
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2Present
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Jayhawk Blvd., University of Kansas, Lawrence, KS 66045, USA
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3Present
address: Department of Biology, Western Washington University, Bellingham, WA, 98225, USA
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4Present
address: Department of Biology, Davidson College, Davidson, North Carolina, 28035, USA
address: Department of Biology, Geology, and Physical Sciences, Sul Ross State University,
address: Biodiversity Institute and Department of Ecology and Evolutionary Biology, 1345
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Running headline: Timing for measuring baseline CORT in reptiles
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*Correspondence to: 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802,
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USA; Phone 814.867.2251; Fax 814.865.9131; Email [email protected]
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Manuscript submitted for consideration to General and Comparative Endocrinology
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Abstract
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There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild
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animals respond to environmental challenges. Blood is the best medium for obtaining information about
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recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect
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GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 minutes
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of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and
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mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone
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(CORT), in four reptile species for which blood was taken within a range of times 11 minutes or less after
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first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after
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first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 sec), and rock iguanas (Cyclura cychlura; 2
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min 44 sec), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-minute
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sampling period. In both snake species, samples taken up to 3 to 7 minutes after CORT began to increase
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still had lower CORT concentrations than after exposure to a standard restraint stressor. The “3-minute
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rule” appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and
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sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter
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in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others
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(cottonmouths, fence lizards).
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Key words: stress, corticosterone, reptile, baseline, snake, lizard
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1. Introduction Ecologists have become increasingly interested in how an organism’s physiology interacts with
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its environment, with particular attention paid to the physiological stress response (Romero, 2004;
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Wikelski and Cooke, 2006). Concentrations of glucocorticoid hormones, including cortisol and
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corticosterone (CORT), in the bloodstream are commonly used as measures of the overall health of
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populations (baseline), population-level responses to different stressors (changes from baseline), and as
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predictors of individual fitness in field-caught wild animals (Bonier et al., 2009; Romero, 2004). Plasma is
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the most common medium for measuring CORT concentrations in wild animals but presents challenges
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for measuring baseline levels, as CORT concentrations can be affected by the process of obtaining blood
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samples.
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In domestic and laboratory animals where the entire CORT response curve has been quantified
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through repeated blood sampling, there is a 3 to 5 minute delay between the time an animal is
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disturbed (chased, trapped, or captured) to the release of CORT into blood (Dallman and Bhatnagar,
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2001; Sheriff et al., 2011). Romero & Reed (2005) tested this assumption for wild animals using five
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avian species and one reptile species, and found that blood samples obtained within 3 minutes of
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capture were at or close to baseline values. This study is commonly cited to support the timing of blood
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sampling in many wild taxa (>740 citations at the time of writing). Although incredibly valuable, it
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highlights the need for similar assessment of the timing of blood sampling in other species. Particularly
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in reptile species, the assumption that plasma must be collected within 3 minutes of capture may
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actually be more conservative than necessary. This may negatively impact the designs of field studies,
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particularly in species which usually have prolonged capture times. The possibility that plasma CORT
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may not become significantly elevated in reptiles until later than 3 minutes was not tested in the
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Romero & Reed (2005) study, as all blood samples from marine iguanas, the only reptile sampled, were
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taken within 3 minutes of disturbance. Many studies assessing capture-induced increases in plasma
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CORT in reptiles do not report changes until 5 to 15 minutes after capture (reviewed by Cockrem, 2013).
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Various factors may affect the timing of glucocorticoid release in reptiles, including seasonal changes in
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physiology, body condition and health, and, in particular, body temperature, which is strongly affected
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by ambient temperature in ectotherms (Cockrem, 2013; Davidson et al., 2008; Hume and Egdahl, 1959;
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Rhind et al., 2004, 1999). Temperature-dependent effects and the lower metabolic rates of reptilian
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ectotherms could delay the release of glucocorticoids into their bloodstream as compared to birds and
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mammals (Pough, 1980); therefore samples collected later than 3 minutes after disturbance may
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provide a valid representation of baseline glucocorticoid concentrations.
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Here we examined the timing of CORT plasma concentration increases after disturbance and
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capture for four reptile species that were field-caught and bled immediately, representing field sampling
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of baseline CORT. The times to capture and complete the blood sampling ranged from 15 seconds to 11
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minutes after first disturbing an animal. Following Romero & Reed (2005), we first determined if there
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was a measurable increase in CORT concentrations in blood samples at any point during this time frame.
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We assumed that if an increase occurred it would indicate that blood samples should be taken prior to
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the time of the change in order to reflect baseline concentrations of CORT. We then compared CORT
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concentrations of blood samples taken after any significant elevation was detected to those of samples
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taken after exposure of the animal to a standard stress protocol (i.e., 30- or 60-min duration of captivity,
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depending on species) to determine if the first set of samples provide a functional measure of baseline
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CORT, as differentiated from post-stressor CORT concentrations. We hypothesized that due to lower
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relative metabolic rates of reptiles compared to previously tested avian species we would see longer
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delays in the increase of plasma CORT concentrations due to a standard stressor.
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2. Materials and Methods
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2.1 Ethical procedures
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All animals were handled in accordance with the Guidelines for the Use of Animals in Research
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and the Institutional Guidelines of the Pennsylvania State University, under the IACUC numbers 33346,
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35780, 44595, and 42599, and of Utah State University under the IACUC number 2530. The collection of
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animals from the wild was permitted by the respective states and countries.
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2.2 Study organisms and experimental design We obtained blood samples from four reptile species: cottonmouth snakes (Agkistrodon
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piscivorus), western rattlesnakes (Crotalus oreganus), eastern fence lizards (Sceloporus undulatus), and
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northern Bahamian rock iguanas (Cyclura cychlura). Most animals were adults at the time of capture, as
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determined by size (cottonmouths and rattlesnakes: > 50 cm SVL; Herr et al., 2017; fence lizards: > 5 cm
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SVL; Angilletta et al., 2004; rock iguanas: > 18 cm SVL; Iverson and Mamula, 1989). Some cottonmouths
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(n = 6) and rattlesnakes (n = 6) were subadults at the time of capture. An approximately equal number
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of males and females were sampled from each species. Initial plasma CORT concentrations were
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obtained from all four species within 11 minutes after first disturbance, although there was variation in
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the range of times from first disturbance to initial blood draw for each species (see Table 1). For every
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species except the rock iguanas we additionally measured CORT concentrations after 30 minutes
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(cottonmouths, eastern fence lizards) or 60 minutes (western rattlesnakes) of exposure to a
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standardized stressor (for lizards, restraint in a cloth bag; for snakes, restraint in a bucket; as per
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(Graham et al., 2012; Holding et al., 2014a; Romero and Reed, 2005)).
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2.3 Blood collection All animals were captured from the wild and sampled while active and capable of thermoregulation, and appeared healthy at the time of capture. Further details about natural history
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elements and sample collection factors which may affect plasma CORT concentration are detailed in
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Table 2.
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Cottonmouths and rattlesnakes were captured from the wild in Alabama and California,
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respectively, in summer 2013. A blood sample was immediately collected from the caudal tail vein using
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a 26 gauge heparinized syringe (cottonmouths: n = 28; rattlesnakes: n = 21). The time to initial bleed was
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recorded as the time from when each animal was first disturbed to the time blood draw was complete.
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Each snake was then placed in a 5-gallon plastic bucket until 30 minutes (cottonmouths) or 60 minutes
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(rattlesnakes) had passed since the initial encounter with the snake, and a second blood sample was
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then taken. This protocol induces a significant increase in CORT concentrations (Herr et al., 2017).
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Eastern fence lizards were captured from the wild in Pennsylvania in summer 2014, and a blood
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sample collected from the orbital sinus with a heparinized capillary tube (n = 28). The time to initial
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bleed was again recorded as the time from when each animal was first disturbed to the time blood draw
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was complete. Lizards were then restrained in a cloth bag, and a second blood sample taken 30 minutes
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after the lizards were first disturbed. This protocol induces a significant increase in CORT concentrations
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(Graham et al., 2012).
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Rock iguanas were captured by net or noose from the wild in the Exumas Island chain, Bahamas,
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in spring and fall 2016, and blood samples were collected via the caudal vein using a 25 gauge
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heparinized syringe, again recording time to bleed from first disturbance of the animal (Webb et al.,
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2018). We did not obtain a second post-stressor sample to evaluate hormonal stress activity for this
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species.
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2.4 Corticosterone assays Blood samples were kept on ice for up to 6 hours prior to centrifugation, after which the plasma was drawn off and stored at -20ᵒC for 2 weeks to 2 years. Given that steroid hormones are known to
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remain stable in plasma for decades when stored at -20ᵒC (Stroud et al., 2007), we do not expect there
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to be any issues with prolonged sample storage. CORT concentrations of cottonmouths, rattlesnakes,
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and eastern fence lizards were measured using an enzyme immunoassay (Corticosterone High Sensitivity
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EIA Kits, Immunodiagnostic Systems (IDS) Inc., Scottsdale, AZ, USA) which has previously been validated
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for cottonmouths (Herr et al., 2017) and eastern fence lizards (Trompeter and Langkilde, 2011), and has
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a sensitivity limit of 0.17 ng/mL. Prior to testing the rattlesnake samples, we validated the immunoassay
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for this species by demonstrating parallelism of pooled sample dilution curves compared to a CORT
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control curve (provided in the kit) and achieving a 99.9% recovery rate of a CORT control sample added
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to a pooled sample of rattlesnake plasma. Each plasma sample was run in duplicate, with intra-assay
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coefficients of variation ranging from 1.63% to 5.18%. Inter-assay coefficients of variation ranged from
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0.03% to 3.89%.
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For rock iguanas, samples were analyzed using radio-immunoassay in three separate assays in
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duplicate for corticosterone (Ab: MP Biomedicals # 07-120016) using a previously described and
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established protocol (French et al., 2017), and validated in this species (French et al., Unpublished
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results) with a sensitivity limit of 0.3 ng/mL. For each sample, we used an aliquot of the resuspended
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fractions to measure individual recoveries following extraction (mean =79.0%, std dev. = 7% for all 3
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assays). These recoveries were used to adjust the final sample concentration values to account for any
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losses during these procedures. The mean intra-assay coefficient of variation was 2.3%, and the inter-
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assay coefficient of variation was 18%.
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2.5 Statistical analysis To determine at what time point, if any, plasma CORT concentrations changed due to the stress
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of handling and blood collection, we used a nonparametric change point analysis on the initial samples
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from each species (as per Siegel and Castellan, 1988; see Romero and Reed, 2005 for a description of
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this approach). As there is a known delay between the time a stressor occurs and the time at which the
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hypothalamic-pituitary-adrenal (HPA) axis responds with measurably elevated plasma CORT, a change
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point analysis test is more appropriate than a linear regression model for determining a change in CORT
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concentrations over time (Romero and Reed, 2005; Siegel and Castellan, 1988). This approach allowed
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us to determine time points for which CORT concentrations before and after were most different
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(Romero and Reed, 2005; Siegel and Castellan, 1988). For each species, we analyzed initial CORT
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concentrations to locate the primary change point in the complete data set and determine its
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significance level. It is important to note that this analysis does not allow the inclusion of covariates such
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as sex, body condition or temperature (Siegel and Castellan, 1988).
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Cases for which multiple samples had the same initial bleed time (more than one animal was
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bled at the same time after disturbance) are problematic for nonparametric change point analysis,
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which orders samples by time (and associated CORT concentration). We addressed this by analyzing
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multiple permutations of the order of these same-timed samples. We ran either all possible
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permutations of the data (for species with 20 or fewer permutations of same-timed samples: eastern
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fence lizards), or a minimum of 20 unique permutations were randomly generated and analyzed (for
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species with greater than 20 permutations of same-timed samples: cottonmouths, rattlesnakes, and
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rock iguanas). Permuting the order did not alter the change points reported here for eastern fence
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lizards, cottonmouths, or rattlesnakes, but had a slight effect on the change point in rock iguanas. In 6 of
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the permutations analyzed, rock iguanas showed a change point 1 second later than that seen in the
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other 14 permutations.
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For each species for which a statistically significant change point was identified (except for rock
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iguanas, for which we did not obtain stressed CORT concentrations), we next conducted a two-tailed
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paired t-test for equal variances to compare initial CORT concentrations obtained after the change point
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to CORT concentrations from the same animals after they had undergone a standardized stress protocol
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(approach modified from Romero and Reed, 2005). This was done to determine if initial CORT
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concentrations obtained after the change point could be differentiated from stress-induced CORT
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concentrations.
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Two cottonmouth plasma samples and one lizard plasma sample were more than three
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standard deviations from the mean (see Figure 1). All statistical tests were performed both with and
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without these samples; the inclusion of these samples did not qualitatively change the results therefore
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all three samples were retained in the reported analyses. Change point analyses were performed in
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Microsoft Excel (2013) as described by Siegel and Castellan (1988), and all t-tests were performed in R
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version 3.2.2 (R Development Core Team, 2015) with an alpha level of 0.05.
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3. Results
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A significant change point in plasma CORT concentrations was found for three of the four
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species tested (rattlesnakes: 2 min 30 sec, cottonmouth: 4 min, rock iguanas: 2 min 44 sec; Table 1; Fig.
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1). Six of the 20 permutations tested in rock iguanas produced a change point of 2 min 45 sec, but this
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was within 1 second of the change point found in the other 14 permutations. No statistically significant
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change point was found in fence lizards. Initial CORT values from blood samples taken after the change
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point in cottonmouths and rattlesnakes were still significantly lower than the CORT values from blood
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taken after the snakes had experienced a standard restraint stressor (Table 1; Fig. 1).
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4. Discussion
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Our results indicate that collecting blood samples from three species of reptiles (cottonmouths, western rattlesnakes, and rock iguanas) within 2.5 to 4 minutes of first disturbance is adequate to obtain
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baseline measurement of plasma CORT concentration. For one of our tested species (fence lizards),
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blood samples obtained as late as 10 minutes after disturbance would be adequate for assessing
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baseline levels. This is in agreement with Romero and Reed’s (2005) finding of no significant change
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point in plasma CORT for the one reptile species they tested (marine iguanas, Amblyrhynchus cristatus)
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as late as 3 minutes after capture, and possibly later than this as they did not take blood samples later
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than 3 minutes after capture. Another study in marine iguanas showed no difference in CORT of samples
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taken within 3 minutes of capture and samples taken after 15 minutes of restraint stress (Neuman-Lee
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and French, 2017), further supporting the idea that there may be a long delay in CORT increases due to
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handling stress in some reptile species.
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Initial blood samples taken at times later than the change points identified in this study, but
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within our sample time, may be sufficient for some purposes. For species where logistical challenges
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make obtaining plasma samples within 3 minutes difficult, the slight elevations in plasma CORT with
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longer times to bleed may still allow for meaningful comparisons with post-restraint CORT levels.
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Samples taken after the change point in both rattlesnakes (2 min 30 sec to ≈10 min) and cottonmouths
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(4 mins to ≈7 min) were still significantly lower than samples taken after the snakes had undergone a
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standard restraint stress protocol, indicating that the initial samples did not yet reflect maximal stress-
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induced CORT concentrations. This finding parallels those for several bird species tested by Romero and
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Reed (2005), and may justify extending the sampling beyond the 3 minute standard for this purpose.
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Our results concur with previous studies on field-caught and lab-raised reptiles that have
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included bleed time as a covariate in analyses and found that this does not significantly explain variation
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in initial CORT concentrations (Dauphin-Villemant and Xavier, 1987; Langkilde et al., 2005; Manzo et al.,
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1994). However, typically these studies aim to complete blood sampling within 3 minutes of first
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disturbance or capture, as advised by Romero and Reed (2005), and thus this may not be surprising. In
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spite of considerable variation in individual, seasonal, and yearly CORT responses, many studies do not 10
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find a significant increase in CORT concentrations in reptiles until at least 5 minutes after exposure to a
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stressor (reviewed by Cockrem, 2013). Indeed, at certain times of the year some species of reptile fail to
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mount a CORT response even after a 30 minute exposure to a stressor (Cockrem, 2013). This contrasts
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with studies on birds and mammals that show increases in plasma CORT concentrations after as little as
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1 to 2 minutes of exposure to a stressor (Chloupek et al., 2009; Dawson and Howe, 1983; Romero and
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Reed, 2005).
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It is possible that this discrepancy is due to the lower metabolic rates of reptiles (Pough, 1980),
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which may result in a longer delay in the increase in plasma CORT concentrations than occurs in
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endotherms. Studies on the effects of body temperature on CORT responses in mammals support this
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(Davidson et al., 2008; Hume and Egdahl, 1959; Rhind et al., 2004, 1999). For example, induced
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hypothermia in preterm fetal sheep noticeably slows and prolongs the GC stress response to umbilical
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cord occlusion (Davidson et al., 2008), and dogs with induced hypothermia have a significantly
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diminished GC response to both trauma and adrenocorticotropic hormone (ACTH) administration (Hume
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and Egdahl, 1959). Elevated body temperature in both rattlesnakes (Claunch et al., 2017) and boa
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constrictors (Holding et al., 2014b) is associated with an increase in stress-induced plasma CORT
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concentrations. Furthermore, a recent study in eastern fence lizards similarly indicates that temperature
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is important to HPA axis function, with lower body temperature associated with reduced baseline and
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stress-induced plasma CORT concentrations (Racic et al., Unpublished results) Since change point
255
analysis does not allow for the inclusion of covariates, the results presented here do not account for the
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potentially important variables of body temperature, sex, and body condition. It would be informative to
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understand how such variables may affect the timing of the CORT response as new analytical techniques
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are developed. However, it is worth noting that all individuals used in this study were captured while
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active in the field, able to freely thermoregulate, and therefore presumably at or near their preferred
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body temperatures.
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Corticosterone has been shown to be the primary glucocorticoid produced in multiple reptile
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species (Mehdi and Carballeira, 1971; Mehdi and Sandor, 1974; Vinson et al., 1975), and is generally
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cited as the primary glucocorticoid in reptiles (Romero, 2004; Romero and Reed, 2005; Sheriff et al.,
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2011). This has not been empirically tested in any of the species used in this study and thus it is possible
265
that cortisol is also produced in the HPA axis of reptiles. However, changes in corticosterone levels are
266
likely to reflect changes in plasma cortisol.
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It is important to note that not all disturbances are equally stressful. For example, Bailey et al.
268
(2009) found that standing near a cottonmouth for 30 minutes was insufficient to induce a noticeable
269
increase in CORT, but holding cottonmouths in a bucket for 30 minutes caused significant increases in
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CORT concentrations (Herr et al., 2017). For the rattlesnakes and cottonmouths in this study, the time
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required to catch each animal was quite brief (<45 seconds); therefore the time to initial blood draw
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primarily reflects time required to obtain the blood sample. In contrast, the time required to catch fence
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lizards represented a considerable portion of the initial bleed time; the average time to obtain a blood
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sample was 2 min ± 15 sec (± 1 SE), with the remaining time since disturbance represented by time
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spent chasing or handling the lizard prior to blood draw. It is possible that the time to capture and
276
handle the lizards may not have been overly stressful, at least compared to the bleeding time. Thus the
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time since disturbance for the lizards may include periods during which the animals had not yet initiated
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HPA axis activation or had initiated this at a relatively low level, whereas our time since disturbance for
279
the snakes likely included mostly time since HPA axis activation. Standard methods were used to capture
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the reptiles in this study, so our data should be applicable to researchers working with these species.
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However, if capture time in other studies using these species is significantly different from ours, CORT
282
responses may differ from those reported here. Similarly, habitat, climate, time of day and year, and
283
condition of populations may affect population CORT responses. These factors should be considered
284
when determining the timeline in which to measure baseline CORT concentrations.
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285 286
5. Conclusions
287 288
Our findings suggest that plasma samples collected within 2 to 3 minutes likely approximate
289
baseline CORT concentrations, although this may be overly conservative in some reptile species. Future
290
studies analyzing putative baseline CORT samples should consider that variation in HPA initiation time
291
varies by taxon, and that there is no single cutoff point for the beginning of CORT elevation. We suggest
292
that future studies conduct their own change point analyses over the range of sample collection times
293
contained in their datasets. Such analyses may allow researchers to include samples they would have
294
otherwise discarded using the typical 3 minute cutoff rule. Consequently, blood samples from species
295
that can be difficult to catch quickly in the field or bleed may still be suitable for measuring plasma GCs
296
(Tarlow and Blumstein, 2007). These findings may allow researchers to reconsider whether stress
297
research is tractable in their focal species, and may open up new avenues for studying stress physiology
298
in a more diverse set of organisms.
299 300
Acknowledgements. We thank the Solon Dixon Forestry Education Center, the United States Air Force,
301
and J. Matter and the Raystown Field Station for access to land and logistical support; L. Van Der Sluys
302
and the staff of Penn State’s Summer Experience in the Eberly College of Science (SEECoS) and Upward
303
Bound Math and Science (UBMS) programs for logistical support regarding the involvement of high
304
school students in this research; and the Cavener lab for access to their plate reader. M. DeLea, D.
305
Ensminger, M. Goldy-Brown and J. Penzelik provided valuable assistance in the field, and R. Clark and E.
306
Taylor provided valuable advice on the rattlesnake study. C. Knapp, J. Iverson, and D. DeNardo provided
307
valuable assistance with field and lab work on the rock iguana study. The John G. Shedd Aquarium
308
provided field research support, and the Bahamas National Trust assisted with permitting. This work was
13
309
supported by the National Science Foundation (IOS1456655 to T.L. and IOS1051367 to T.L.), the
310
Pennsylvania State University (Erickson Discovery Grant, awarded to M.H.), and the Utah Agricultural
311
Experiment Station (RGS RC Seed grant, awarded to S.S.F.). Funding sources had no influence on the
312
design of this study. The authors have no conflict of interest to declare.
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313 314
References
315
Angilletta, M.J., Niewiarowski, P.H., Dunham, A.E., Leache, A.D., Porter, W.P., 2004. Bergmann’s clines in
316
ectotherms: Illustrating a life-history perspective with Sceloporine lizards. Am. Nat. E168-E183.
317 318 319
Angilletta, M.J., Sears, M.W., Winters, R.S., 2001. Seasonal variation in reproductive effort and its effect on offspring size in the lizard Sceloporus undulatus. Herpetologica. 57, 365-375. Bailey, F.C., Cobb, V.A., Rainwater, T.R., Worrall, T., Klukowski, M., 2009. Adrenocortical effects of
320
human encounters on free-ranging cottonmouths (Agkistrodon piscivorus). J. Herpetol. 43, 260–
321
266. doi:http://dx.doi.org/10.1670/08-123R1.1
322 323 324 325
Blem, C.R., Blem, L.B., 1995. The Eastern Cottonmouth (Agkistrodon piscivorus) at the northern edge of its range. J. Herpetol. 29, 391-398. doi:http://dx.doi.org/10.2307/1574989 Bonier, F., Martin, P.R., Moore, I.T., Wingfield, J.C., 2009. Do baseline glucocorticoids predict fitness? Trends Ecol. Evol. 24, 634–642. doi:10.1016/j.tree.2009.04.013
326
Chloupek, P., Voslářová, E., Suchý Jr., P., Bedáňová, I., Pištěková, V., Vitula, F., Chloupek, J., Večerek, V.,
327
2009. Influence of pre-sampling handling duration on selected biochemical indices in the common
328
pheasant (Phasianus colchicus). Acta Vet. Brno 78, 23–28. doi:10.2754/avb200978010023
329
Claunch, N.M., Frazier, J.A., Escallón, C., Vernasco, B.J., Moore, I.T., Taylor, E.N., 2017. Physiological and
330
behavioral effects of exogenous corticosterone in a free-ranging ectotherm. Gen. Comp.
331
Endocrinol. 248, 87–96. doi:10.1016/j.ygcen.2017.02.008
332 333 334
Cockrem, J.F., 2013. Individual variation in glucocorticoid stress responses in animals. Gen. Comp. Endocrinol. 181, 45–58. doi:10.1016/j.ygcen.2012.11.025 Dallman, M., Bhatnagar, S., 2001. Chronic stress and energy balance: role of the hypothalamo-pituitary-
335
adrenal axis, in: McEwen, B., Goodman, H. (Eds.), Handbook of Physiology. Oxford Univ. Press.,
336
New York, pp. 179–210.
15
337
Dauphin-Villemant, C., Xavier, F., 1987. Nychthemeral variations of plasma corticosteroids in captive
338
female Lacerta vivipara Jacquin: influence of stress and reproductive state. Gen. Comp. Endocrinol.
339
67, 292–302. doi:10.1016/0016-6480(87)90183-3
340
Davidson, J.O., Fraser, M., Naylor, A.S., Roelfsema, V., Gunn, A.J., Bennet, L., 2008. Effect of cerebral
341
hypothermia on cortisol and adrenocorticotropic hormone responses after umbilical cord occlusion
342
in preterm fetal sheep. Pediatr. Res. 63, 51–55. doi:10.1203/PDR.0b013e31815b8eb4
343
Dawson, A, Howe, P.D., 1983. Plasma corticosterone in wild starlings (Sturnus vulgaris) immediately
344
following capture and in relation to body weight during the annual cycle. Gen. Comp. Endocrinol.
345
51, 303–8. doi:10.1016/0016-6480(83)90085-0
346
French, S.S., Neuman-lee, L.A., Terletzky, P.A., Kiriazis, N.M., Taylor, E.N., Denardo, D.F., 2017. Too much
347
of a good thing? Human disturbance linked to ecotourism has a “dose-dependent” impact on
348
innate immunity and oxidative stress in marine iguana, Amblyrhynchus cristatus. Biol. Conserv.
349
210, 37–47. doi:10.1016/j.biocon.2017.04.006
350
Graham, S.P., Earley, R.L., Hoss, S.K., Schuett, G.W., Grober, M.S., 2008. The reproductive biology of
351
male cottonmouths (Agkistrodon piscivorus): Do plasma steroid hormones predict the mating
352
season? Gen. Comp. Endocrinol. 159, 226-235. doi:10.1016/j.ygcen.2008.09.002
353
Graham, S.P., Freidenfelds, N.A., Mccormick, G.L., Langkilde, T., 2012. The impacts of invaders: Basal and
354
acute stress glucocorticoid profiles and immune function in native lizards threatened by invasive
355
ants. Gen. Comp. Endocrinol. 176, 400–408. doi:10.1016/j.ygcen.2011.12.027
356
Herr, M.W., Graham, S.P., Langkilde, T., 2017. Stressed snakes strike first: Hormone levels and defensive
357
behavior in free ranging cottonmouths (Agkistrodon piscivorus). Gen. Comp. Endocrinol. 243, 89–
358
95. doi:10.1016/j.ygcen.2016.11.003
359 360
Holding, M.L., Frazier, J.A., Dorr, S.W., Henningsen, S.N., Moore, I.T., Taylor, E.N., 2014a. Physiological and behavioral effects of repeated handling and short-distance translocations on free-ranging
16
361
northern Pacific rattlesnakes (Crotalus oreganus oreganus). J. Herpetol. 48, 233–239.
362
doi:10.1670/11-314
363
Holding, M.L., Frazier, J.A., Dorr, S.W., Pollock, N.B., Muelleman, P.J., Branske, A., Henningsen, S.N.,
364
Eikenaar, C., Escallón, C., Montgomery, C.E., Moore, I.T., Taylor, E.N., 2014b. Wet- and dry-season
365
steroid hormone profiles and stress reactivity of an insular dwarf snake, the Hog Island boa (Boa
366
constrictor imperator). Physiol. Biochem. Zool. Ecol. Evol. Approaches 87, 363–373.
367
doi:10.1086/675938
368 369 370 371 372 373 374 375 376 377
Hume, D.M., Egdahl, R.H., 1959. Effect of hypothermia and of cold exposure on adrenal cortical and medullary secretion. Ann. New York Acad. Sci. 80, 435–444. Iverson, J.B., 1979. Behavior and ecology of the rock iguana, Cyclura carinata. Bulletin of the Florida State Museum. 24, 173-356. Iverson, J.B., Hines, K.N., Valiulis, J.M., 2004. The nesting ecology of the Allen Cays rock iguana, Cychlura cychlura inornata in the Bahamas. Herpetological Monographs. 18, 1-36. Iverson, J.B., Mamula, M.R., 1989. Natural growth of the Bahamian iguana Cyclura cychlura. Copeia 2, 502-505. Langkilde, T., Lance, V.A., Shine, R., 2005. Ecological consequences of agonistic interactions in lizards. Ecology 86, 1650–1659.
378
Lind, C.M., Husak, J.F., Eikenaar, C., Moore, I.T., Taylor, E.N., 2010. The relationship between plasma
379
steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake,
380
Crotalus oreganus. Gen. Comp. Endocrinol. 166, 590-599. doi:10.1016/j.ygcen.2010.01.026
381
Manzo, C., Zerani, M., Gobbetti, A., Di Fiore, M.M., Angelini, F., 1994. Is corticosterone involved in the
382
reproductive processes of the male lizard, Podarcis sicula sicula? Horm. Behav. 28, 117-129.
383
doi:10.1006/hbeh.1994.1009
384
Mehdi, A.Z., Carballeira, A., 1972. Steroid biosynthesis by painted turtle (Chrysemys picta picta)
17
385
adrenals. Steroids 19, 137-150.
386
Mehdi, A.Z., Sandor, T., 1974. Steroid biosynthesis by reptilian adrenals. Steroids 24, 151-163.
387
Neuman-Lee, L.A., French, S.S., 2017. Endocrine-reproductive-immune interactions in female and male
388
Galápagos marine iguanas. Horm. Behav. 88, 60–69. doi:10.1016/j.yhbeh.2016.10.017
389
Pough, F.H., 1980. The advantages of ectothermy for tetrapods. Am. Nat. 115, 92–112.
390
Putman, B.J., Clark, R.W., 2017. Behavioral thermal tolerances of free-ranging rattlesnakes (Crotalus
391
oreganus) during the summer foraging season. J. Therm. Biol. 65, 8-15.
392
doi:http://dx.doi.org/10.1016/j.jtherbio.2017.01.012
393
Rhind, S.G., Gannon, G.A., Shek, P.N., Brenner, I.K., Severs, Y., Zamecnik, J., Buguet, A., Natale, V.M.,
394
Shephard, R.J., Radomski, M.W., 1999. Contribution of exertional hyperthermia to
395
sympathoadrenal-mediated lymphocyte subset redistribution. J. Appl. Physiol. 87, 1178–1185.
396
Rhind, S.G., Gannon, G.A., Shephard, R.J., Buguet, A., Shek, P.N., Radomski, M.W., 2004. Cytokine
397
induction during exertional hyperthermia is abolished by core temperature clamping:
398
neuroendocrine regulatory mechanisms. Int. J. Hyperthermia 20, 503–516.
399
doi:10.1080/02656730410001670651
400 401 402
Romero, L.M., 2004. Physiological stress in ecology: Lessons from biomedical research. Trends Ecol. Evol. 19, 249–255. doi:10.1016/j.tree.2004.03.008 Romero, L.M., Reed, J.M., 2005. Collecting baseline corticosterone samples in the field: Is under 3 min
403
good enough? Comp. Biochem. Physiol. - A Mol. Integr. Physiol. 140, 73–79.
404
doi:10.1016/j.cbpb.2004.11.004
405
Sheriff, M.J., Dantzer, B., Delehanty, B., Palme, R., Boonstra, R., 2011. Measuring stress in wildlife:
406
techniques for quantifying glucocorticoids. Oecologia 166, 869–887. doi:10.1007/s00442-011-
407
1943-y
408
Siegel, S., Castellan, N., 1988. Nonparametric Statistics for the Behavioral Sciences, 2nd ed. McGraw-Hill,
18
409 410
New York, NY. Stroud, L.R., Solomon, C., Shenassa, E., 2007. Long-term stability of maternal prenatal steroid hormones
411
from the National Collaborative Perinatal Project: Still valid after all these years.
412
Psychoneuroendocrinology 32, 140-150.
413 414 415 416
Tarlow, E.M., Blumstein, D.T., 2007. Evaluating methods to quantify anthropogenic stressors on wild animals. Appl. Anim. Behav. Sci. 102, 429–451. doi:10.1016/j.applanim.2006.05.040 Trompeter, W.P., Langkilde, T., 2011. Invader danger: Lizards faced with novel predators exhibit an altered behavioral response to stress. Horm. Behav. 60, 152–158. doi:10.1016/j.yhbeh.2011.04.001
417
Vinson, G.P., Braysher, M., Whitehouse, B.J., 1975. In vitro steroidogenesis by the nonzoned
418
adrenocortical tissue of the skink, Tiliqua rugosa. Gen. Comp. Endocrinol. 26, 541-549.
419
Webb, A.C., Iverson, J.B., Knapp, C.R., Denardo, D.F., French, S.S., 2018. Energetic investment associated
420
with vitellogenesis induces an oxidative cost of reproduction. J. Anim. Ecol. 1–11.
421
doi:10.1111/1365-2656.12936
422 423
Wikelski, M., Cooke, S.J., 2006. Conservation physiology. Trends Ecol. Evol. 21, 38–46. doi:10.1016/j.tree.2005.10.018
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425 426
Table 1. Results of change point analyses of initial concentrations of plasma CORT and paired t-test
427
comparisons of “baseline” CORT concentrations obtained after the change point to CORT concentrations
428
after the individuals had experienced a standardized restraint stressor. Range of initial bleed times
After change point vs. Change point
restraint stressor p-
Species
N
(min:sec)
(min:sec)
value
t-value
df
p-value
Cottonmouth
28
2:00-7:10
4:00
0.01
-4.8488
8
0.0013
Rattlesnake
21
2:00-10:21
2:30
0.01
-5.4513
15
<0.0001
Fence lizard*
28
1:15-10:18
4:45
0.09
-
-
-
Rock iguana**
459
0:15-7:18
2:44
<0.01
-
-
-
429
*No t-test was performed as no statistically significant change point was found.
430
**No t-test was performed as the animals did not undergo a standardized restraint stress protocol.
20
432
433 434
Table 2. Natural history and sampling data for each species. Dates sampled Cottonmouth May-July 2013
Capture time range 00:4123:27
Time of sunrise* Peak activity period 05:50 06:00-10:00 (Blem and Blem, 1995)
Rattlesnake
July-August 2013
08:4617:47
06:10
07:00-18:00 (Putman and Clark, 2017)
March-May; AugustSeptember (Lind et al., 2010)
Fence lizard
June-July 2014
12:1115:07
05:45
10:00-14:00
May-June (Angilletta et al., 2001)
Rock iguana
May, June, September 2016
08:0013:50
06:30
08:00-17:00 (Iverson, 1979)
May-June (Iverson et al., 2004)
Breeding season July-August (Graham et al., 2008)
*Calculated with the National Oceanic and Atmospheric Administration Solar Calculator (https://www.esrl.noaa.gov/gmd/grad/solcalc/index.html)
21
436 437
Figure 1: CORT concentrations of different species across time since capture. Black dots show the initial
438
corticosterone (CORT) concentrations in plasma samples obtained at different times after capture in
439
four species: A) cottonmouth (Agkistrodon piscivorus), B) rattlesnake (Crotalus oreganus), C) eastern
440
fence lizard (Sceloporus undulatus), and D) rock iguana (Cyclura cychlura). Change points (times at which
441
initial CORT concentrations before these points are significantly different from initial samples obtained
442
after this point) are shown as dotted lines (a, b and d). Diamonds represent the mean (at the widest
443
point of the diamond) ±1 standard error (length of the diamond) of plasma CORT concentrations in a
444
second set of blood samples taken from each individual after 30 minutes (a, c) or 60 minutes (b) of
445
restraint stress.
446
447 448
Figure 1.
449
Research Highlights
22
450
The steroid hormone corticosterone, CORT, can indicate physiological stress in vertebrates.
451
Measuring baseline CORT is key to understanding animal responses to stressors.
452
Obtaining samples quickly enough to assess baseline CORT can be challenging.
453
Plasma sampled within 2.5-4 min of capture in reptiles represents baseline CORT.
454
Baseline CORT can be obtained from blood sampled even later in some reptile species.
455
23
S0016-6480(19)30147-9 https://doi.org/10.1016/j.ygcen.2019.113324 YGCEN 113324
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
21 March 2019 6 November 2019 12 November 2019
Please cite this article as: Tylan, C., Camacho, K., French, S., Graham, S.P., Herr, M.W., Jones, J., McCormick, G.L., O'Brien, M.A., Tennessen, J.B., Thawley, C.J., Webb, A., Langkilde, T., Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?, General and Comparative Endocrinology (2019), doi: https://doi.org/10.1016/j.ygcen.2019.113324
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1
Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick
2
enough?
3
Catherine Tylana, Kiara Camachoa, Susannah Frenchb, Sean P. Grahama,1, Mark W. Herra,2, Jermayne
4
Jonesa, Gail L. McCormicka,c, Melissa A. O’Briena, Jennifer B. Tennessena,c,3, Christopher J. Thawleya,c,4,
5
Alison Webbb, Tracy Langkildea,c,*
6 7
a Department
of Biology, The Pennsylvania State University, University Park, PA 16802, USA
8
bDepartment
of Biology and the Ecology Center, Utah State University, Logan, UT 84322, USA
9
cIntercollege
Graduate Degree Program in Ecology, and The Center for Brain, Behavior and Cognition,
10
The Pennsylvania State University, University Park, PA 16802, USA
11
1Present
12
Alpine, Texas, 79832, USA
13
2Present
14
Jayhawk Blvd., University of Kansas, Lawrence, KS 66045, USA
15
3Present
address: Department of Biology, Western Washington University, Bellingham, WA, 98225, USA
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4Present
address: Department of Biology, Davidson College, Davidson, North Carolina, 28035, USA
address: Department of Biology, Geology, and Physical Sciences, Sul Ross State University,
address: Biodiversity Institute and Department of Ecology and Evolutionary Biology, 1345
17 18
Running headline: Timing for measuring baseline CORT in reptiles
19 20
*Correspondence to: 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802,
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USA; Phone 814.867.2251; Fax 814.865.9131; Email [email protected]
22 23
Manuscript submitted for consideration to General and Comparative Endocrinology
1
24 25
Abstract
26 27
There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild
28
animals respond to environmental challenges. Blood is the best medium for obtaining information about
29
recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect
30
GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 minutes
31
of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and
32
mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone
33
(CORT), in four reptile species for which blood was taken within a range of times 11 minutes or less after
34
first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after
35
first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 sec), and rock iguanas (Cyclura cychlura; 2
36
min 44 sec), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-minute
37
sampling period. In both snake species, samples taken up to 3 to 7 minutes after CORT began to increase
38
still had lower CORT concentrations than after exposure to a standard restraint stressor. The “3-minute
39
rule” appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and
40
sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter
41
in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others
42
(cottonmouths, fence lizards).
43 44
Key words: stress, corticosterone, reptile, baseline, snake, lizard
2
45 46
1. Introduction Ecologists have become increasingly interested in how an organism’s physiology interacts with
47
its environment, with particular attention paid to the physiological stress response (Romero, 2004;
48
Wikelski and Cooke, 2006). Concentrations of glucocorticoid hormones, including cortisol and
49
corticosterone (CORT), in the bloodstream are commonly used as measures of the overall health of
50
populations (baseline), population-level responses to different stressors (changes from baseline), and as
51
predictors of individual fitness in field-caught wild animals (Bonier et al., 2009; Romero, 2004). Plasma is
52
the most common medium for measuring CORT concentrations in wild animals but presents challenges
53
for measuring baseline levels, as CORT concentrations can be affected by the process of obtaining blood
54
samples.
55
In domestic and laboratory animals where the entire CORT response curve has been quantified
56
through repeated blood sampling, there is a 3 to 5 minute delay between the time an animal is
57
disturbed (chased, trapped, or captured) to the release of CORT into blood (Dallman and Bhatnagar,
58
2001; Sheriff et al., 2011). Romero & Reed (2005) tested this assumption for wild animals using five
59
avian species and one reptile species, and found that blood samples obtained within 3 minutes of
60
capture were at or close to baseline values. This study is commonly cited to support the timing of blood
61
sampling in many wild taxa (>740 citations at the time of writing). Although incredibly valuable, it
62
highlights the need for similar assessment of the timing of blood sampling in other species. Particularly
63
in reptile species, the assumption that plasma must be collected within 3 minutes of capture may
64
actually be more conservative than necessary. This may negatively impact the designs of field studies,
65
particularly in species which usually have prolonged capture times. The possibility that plasma CORT
66
may not become significantly elevated in reptiles until later than 3 minutes was not tested in the
67
Romero & Reed (2005) study, as all blood samples from marine iguanas, the only reptile sampled, were
68
taken within 3 minutes of disturbance. Many studies assessing capture-induced increases in plasma
3
69
CORT in reptiles do not report changes until 5 to 15 minutes after capture (reviewed by Cockrem, 2013).
70
Various factors may affect the timing of glucocorticoid release in reptiles, including seasonal changes in
71
physiology, body condition and health, and, in particular, body temperature, which is strongly affected
72
by ambient temperature in ectotherms (Cockrem, 2013; Davidson et al., 2008; Hume and Egdahl, 1959;
73
Rhind et al., 2004, 1999). Temperature-dependent effects and the lower metabolic rates of reptilian
74
ectotherms could delay the release of glucocorticoids into their bloodstream as compared to birds and
75
mammals (Pough, 1980); therefore samples collected later than 3 minutes after disturbance may
76
provide a valid representation of baseline glucocorticoid concentrations.
77
Here we examined the timing of CORT plasma concentration increases after disturbance and
78
capture for four reptile species that were field-caught and bled immediately, representing field sampling
79
of baseline CORT. The times to capture and complete the blood sampling ranged from 15 seconds to 11
80
minutes after first disturbing an animal. Following Romero & Reed (2005), we first determined if there
81
was a measurable increase in CORT concentrations in blood samples at any point during this time frame.
82
We assumed that if an increase occurred it would indicate that blood samples should be taken prior to
83
the time of the change in order to reflect baseline concentrations of CORT. We then compared CORT
84
concentrations of blood samples taken after any significant elevation was detected to those of samples
85
taken after exposure of the animal to a standard stress protocol (i.e., 30- or 60-min duration of captivity,
86
depending on species) to determine if the first set of samples provide a functional measure of baseline
87
CORT, as differentiated from post-stressor CORT concentrations. We hypothesized that due to lower
88
relative metabolic rates of reptiles compared to previously tested avian species we would see longer
89
delays in the increase of plasma CORT concentrations due to a standard stressor.
90 91
2. Materials and Methods
92
4
93
2.1 Ethical procedures
94
All animals were handled in accordance with the Guidelines for the Use of Animals in Research
95
and the Institutional Guidelines of the Pennsylvania State University, under the IACUC numbers 33346,
96
35780, 44595, and 42599, and of Utah State University under the IACUC number 2530. The collection of
97
animals from the wild was permitted by the respective states and countries.
98 99 100
2.2 Study organisms and experimental design We obtained blood samples from four reptile species: cottonmouth snakes (Agkistrodon
101
piscivorus), western rattlesnakes (Crotalus oreganus), eastern fence lizards (Sceloporus undulatus), and
102
northern Bahamian rock iguanas (Cyclura cychlura). Most animals were adults at the time of capture, as
103
determined by size (cottonmouths and rattlesnakes: > 50 cm SVL; Herr et al., 2017; fence lizards: > 5 cm
104
SVL; Angilletta et al., 2004; rock iguanas: > 18 cm SVL; Iverson and Mamula, 1989). Some cottonmouths
105
(n = 6) and rattlesnakes (n = 6) were subadults at the time of capture. An approximately equal number
106
of males and females were sampled from each species. Initial plasma CORT concentrations were
107
obtained from all four species within 11 minutes after first disturbance, although there was variation in
108
the range of times from first disturbance to initial blood draw for each species (see Table 1). For every
109
species except the rock iguanas we additionally measured CORT concentrations after 30 minutes
110
(cottonmouths, eastern fence lizards) or 60 minutes (western rattlesnakes) of exposure to a
111
standardized stressor (for lizards, restraint in a cloth bag; for snakes, restraint in a bucket; as per
112
(Graham et al., 2012; Holding et al., 2014a; Romero and Reed, 2005)).
113 114 115 116
2.3 Blood collection All animals were captured from the wild and sampled while active and capable of thermoregulation, and appeared healthy at the time of capture. Further details about natural history
5
117
elements and sample collection factors which may affect plasma CORT concentration are detailed in
118
Table 2.
119
Cottonmouths and rattlesnakes were captured from the wild in Alabama and California,
120
respectively, in summer 2013. A blood sample was immediately collected from the caudal tail vein using
121
a 26 gauge heparinized syringe (cottonmouths: n = 28; rattlesnakes: n = 21). The time to initial bleed was
122
recorded as the time from when each animal was first disturbed to the time blood draw was complete.
123
Each snake was then placed in a 5-gallon plastic bucket until 30 minutes (cottonmouths) or 60 minutes
124
(rattlesnakes) had passed since the initial encounter with the snake, and a second blood sample was
125
then taken. This protocol induces a significant increase in CORT concentrations (Herr et al., 2017).
126
Eastern fence lizards were captured from the wild in Pennsylvania in summer 2014, and a blood
127
sample collected from the orbital sinus with a heparinized capillary tube (n = 28). The time to initial
128
bleed was again recorded as the time from when each animal was first disturbed to the time blood draw
129
was complete. Lizards were then restrained in a cloth bag, and a second blood sample taken 30 minutes
130
after the lizards were first disturbed. This protocol induces a significant increase in CORT concentrations
131
(Graham et al., 2012).
132
Rock iguanas were captured by net or noose from the wild in the Exumas Island chain, Bahamas,
133
in spring and fall 2016, and blood samples were collected via the caudal vein using a 25 gauge
134
heparinized syringe, again recording time to bleed from first disturbance of the animal (Webb et al.,
135
2018). We did not obtain a second post-stressor sample to evaluate hormonal stress activity for this
136
species.
137 138 139 140
2.4 Corticosterone assays Blood samples were kept on ice for up to 6 hours prior to centrifugation, after which the plasma was drawn off and stored at -20ᵒC for 2 weeks to 2 years. Given that steroid hormones are known to
6
141
remain stable in plasma for decades when stored at -20ᵒC (Stroud et al., 2007), we do not expect there
142
to be any issues with prolonged sample storage. CORT concentrations of cottonmouths, rattlesnakes,
143
and eastern fence lizards were measured using an enzyme immunoassay (Corticosterone High Sensitivity
144
EIA Kits, Immunodiagnostic Systems (IDS) Inc., Scottsdale, AZ, USA) which has previously been validated
145
for cottonmouths (Herr et al., 2017) and eastern fence lizards (Trompeter and Langkilde, 2011), and has
146
a sensitivity limit of 0.17 ng/mL. Prior to testing the rattlesnake samples, we validated the immunoassay
147
for this species by demonstrating parallelism of pooled sample dilution curves compared to a CORT
148
control curve (provided in the kit) and achieving a 99.9% recovery rate of a CORT control sample added
149
to a pooled sample of rattlesnake plasma. Each plasma sample was run in duplicate, with intra-assay
150
coefficients of variation ranging from 1.63% to 5.18%. Inter-assay coefficients of variation ranged from
151
0.03% to 3.89%.
152
For rock iguanas, samples were analyzed using radio-immunoassay in three separate assays in
153
duplicate for corticosterone (Ab: MP Biomedicals # 07-120016) using a previously described and
154
established protocol (French et al., 2017), and validated in this species (French et al., Unpublished
155
results) with a sensitivity limit of 0.3 ng/mL. For each sample, we used an aliquot of the resuspended
156
fractions to measure individual recoveries following extraction (mean =79.0%, std dev. = 7% for all 3
157
assays). These recoveries were used to adjust the final sample concentration values to account for any
158
losses during these procedures. The mean intra-assay coefficient of variation was 2.3%, and the inter-
159
assay coefficient of variation was 18%.
160 161 162
2.5 Statistical analysis To determine at what time point, if any, plasma CORT concentrations changed due to the stress
163
of handling and blood collection, we used a nonparametric change point analysis on the initial samples
164
from each species (as per Siegel and Castellan, 1988; see Romero and Reed, 2005 for a description of
7
165
this approach). As there is a known delay between the time a stressor occurs and the time at which the
166
hypothalamic-pituitary-adrenal (HPA) axis responds with measurably elevated plasma CORT, a change
167
point analysis test is more appropriate than a linear regression model for determining a change in CORT
168
concentrations over time (Romero and Reed, 2005; Siegel and Castellan, 1988). This approach allowed
169
us to determine time points for which CORT concentrations before and after were most different
170
(Romero and Reed, 2005; Siegel and Castellan, 1988). For each species, we analyzed initial CORT
171
concentrations to locate the primary change point in the complete data set and determine its
172
significance level. It is important to note that this analysis does not allow the inclusion of covariates such
173
as sex, body condition or temperature (Siegel and Castellan, 1988).
174
Cases for which multiple samples had the same initial bleed time (more than one animal was
175
bled at the same time after disturbance) are problematic for nonparametric change point analysis,
176
which orders samples by time (and associated CORT concentration). We addressed this by analyzing
177
multiple permutations of the order of these same-timed samples. We ran either all possible
178
permutations of the data (for species with 20 or fewer permutations of same-timed samples: eastern
179
fence lizards), or a minimum of 20 unique permutations were randomly generated and analyzed (for
180
species with greater than 20 permutations of same-timed samples: cottonmouths, rattlesnakes, and
181
rock iguanas). Permuting the order did not alter the change points reported here for eastern fence
182
lizards, cottonmouths, or rattlesnakes, but had a slight effect on the change point in rock iguanas. In 6 of
183
the permutations analyzed, rock iguanas showed a change point 1 second later than that seen in the
184
other 14 permutations.
185
For each species for which a statistically significant change point was identified (except for rock
186
iguanas, for which we did not obtain stressed CORT concentrations), we next conducted a two-tailed
187
paired t-test for equal variances to compare initial CORT concentrations obtained after the change point
188
to CORT concentrations from the same animals after they had undergone a standardized stress protocol
8
189
(approach modified from Romero and Reed, 2005). This was done to determine if initial CORT
190
concentrations obtained after the change point could be differentiated from stress-induced CORT
191
concentrations.
192
Two cottonmouth plasma samples and one lizard plasma sample were more than three
193
standard deviations from the mean (see Figure 1). All statistical tests were performed both with and
194
without these samples; the inclusion of these samples did not qualitatively change the results therefore
195
all three samples were retained in the reported analyses. Change point analyses were performed in
196
Microsoft Excel (2013) as described by Siegel and Castellan (1988), and all t-tests were performed in R
197
version 3.2.2 (R Development Core Team, 2015) with an alpha level of 0.05.
198 199
3. Results
200 201
A significant change point in plasma CORT concentrations was found for three of the four
202
species tested (rattlesnakes: 2 min 30 sec, cottonmouth: 4 min, rock iguanas: 2 min 44 sec; Table 1; Fig.
203
1). Six of the 20 permutations tested in rock iguanas produced a change point of 2 min 45 sec, but this
204
was within 1 second of the change point found in the other 14 permutations. No statistically significant
205
change point was found in fence lizards. Initial CORT values from blood samples taken after the change
206
point in cottonmouths and rattlesnakes were still significantly lower than the CORT values from blood
207
taken after the snakes had experienced a standard restraint stressor (Table 1; Fig. 1).
208 209
4. Discussion
210 211 212
Our results indicate that collecting blood samples from three species of reptiles (cottonmouths, western rattlesnakes, and rock iguanas) within 2.5 to 4 minutes of first disturbance is adequate to obtain
9
213
baseline measurement of plasma CORT concentration. For one of our tested species (fence lizards),
214
blood samples obtained as late as 10 minutes after disturbance would be adequate for assessing
215
baseline levels. This is in agreement with Romero and Reed’s (2005) finding of no significant change
216
point in plasma CORT for the one reptile species they tested (marine iguanas, Amblyrhynchus cristatus)
217
as late as 3 minutes after capture, and possibly later than this as they did not take blood samples later
218
than 3 minutes after capture. Another study in marine iguanas showed no difference in CORT of samples
219
taken within 3 minutes of capture and samples taken after 15 minutes of restraint stress (Neuman-Lee
220
and French, 2017), further supporting the idea that there may be a long delay in CORT increases due to
221
handling stress in some reptile species.
222
Initial blood samples taken at times later than the change points identified in this study, but
223
within our sample time, may be sufficient for some purposes. For species where logistical challenges
224
make obtaining plasma samples within 3 minutes difficult, the slight elevations in plasma CORT with
225
longer times to bleed may still allow for meaningful comparisons with post-restraint CORT levels.
226
Samples taken after the change point in both rattlesnakes (2 min 30 sec to ≈10 min) and cottonmouths
227
(4 mins to ≈7 min) were still significantly lower than samples taken after the snakes had undergone a
228
standard restraint stress protocol, indicating that the initial samples did not yet reflect maximal stress-
229
induced CORT concentrations. This finding parallels those for several bird species tested by Romero and
230
Reed (2005), and may justify extending the sampling beyond the 3 minute standard for this purpose.
231
Our results concur with previous studies on field-caught and lab-raised reptiles that have
232
included bleed time as a covariate in analyses and found that this does not significantly explain variation
233
in initial CORT concentrations (Dauphin-Villemant and Xavier, 1987; Langkilde et al., 2005; Manzo et al.,
234
1994). However, typically these studies aim to complete blood sampling within 3 minutes of first
235
disturbance or capture, as advised by Romero and Reed (2005), and thus this may not be surprising. In
236
spite of considerable variation in individual, seasonal, and yearly CORT responses, many studies do not 10
237
find a significant increase in CORT concentrations in reptiles until at least 5 minutes after exposure to a
238
stressor (reviewed by Cockrem, 2013). Indeed, at certain times of the year some species of reptile fail to
239
mount a CORT response even after a 30 minute exposure to a stressor (Cockrem, 2013). This contrasts
240
with studies on birds and mammals that show increases in plasma CORT concentrations after as little as
241
1 to 2 minutes of exposure to a stressor (Chloupek et al., 2009; Dawson and Howe, 1983; Romero and
242
Reed, 2005).
243
It is possible that this discrepancy is due to the lower metabolic rates of reptiles (Pough, 1980),
244
which may result in a longer delay in the increase in plasma CORT concentrations than occurs in
245
endotherms. Studies on the effects of body temperature on CORT responses in mammals support this
246
(Davidson et al., 2008; Hume and Egdahl, 1959; Rhind et al., 2004, 1999). For example, induced
247
hypothermia in preterm fetal sheep noticeably slows and prolongs the GC stress response to umbilical
248
cord occlusion (Davidson et al., 2008), and dogs with induced hypothermia have a significantly
249
diminished GC response to both trauma and adrenocorticotropic hormone (ACTH) administration (Hume
250
and Egdahl, 1959). Elevated body temperature in both rattlesnakes (Claunch et al., 2017) and boa
251
constrictors (Holding et al., 2014b) is associated with an increase in stress-induced plasma CORT
252
concentrations. Furthermore, a recent study in eastern fence lizards similarly indicates that temperature
253
is important to HPA axis function, with lower body temperature associated with reduced baseline and
254
stress-induced plasma CORT concentrations (Racic et al., Unpublished results) Since change point
255
analysis does not allow for the inclusion of covariates, the results presented here do not account for the
256
potentially important variables of body temperature, sex, and body condition. It would be informative to
257
understand how such variables may affect the timing of the CORT response as new analytical techniques
258
are developed. However, it is worth noting that all individuals used in this study were captured while
259
active in the field, able to freely thermoregulate, and therefore presumably at or near their preferred
260
body temperatures.
11
261
Corticosterone has been shown to be the primary glucocorticoid produced in multiple reptile
262
species (Mehdi and Carballeira, 1971; Mehdi and Sandor, 1974; Vinson et al., 1975), and is generally
263
cited as the primary glucocorticoid in reptiles (Romero, 2004; Romero and Reed, 2005; Sheriff et al.,
264
2011). This has not been empirically tested in any of the species used in this study and thus it is possible
265
that cortisol is also produced in the HPA axis of reptiles. However, changes in corticosterone levels are
266
likely to reflect changes in plasma cortisol.
267
It is important to note that not all disturbances are equally stressful. For example, Bailey et al.
268
(2009) found that standing near a cottonmouth for 30 minutes was insufficient to induce a noticeable
269
increase in CORT, but holding cottonmouths in a bucket for 30 minutes caused significant increases in
270
CORT concentrations (Herr et al., 2017). For the rattlesnakes and cottonmouths in this study, the time
271
required to catch each animal was quite brief (<45 seconds); therefore the time to initial blood draw
272
primarily reflects time required to obtain the blood sample. In contrast, the time required to catch fence
273
lizards represented a considerable portion of the initial bleed time; the average time to obtain a blood
274
sample was 2 min ± 15 sec (± 1 SE), with the remaining time since disturbance represented by time
275
spent chasing or handling the lizard prior to blood draw. It is possible that the time to capture and
276
handle the lizards may not have been overly stressful, at least compared to the bleeding time. Thus the
277
time since disturbance for the lizards may include periods during which the animals had not yet initiated
278
HPA axis activation or had initiated this at a relatively low level, whereas our time since disturbance for
279
the snakes likely included mostly time since HPA axis activation. Standard methods were used to capture
280
the reptiles in this study, so our data should be applicable to researchers working with these species.
281
However, if capture time in other studies using these species is significantly different from ours, CORT
282
responses may differ from those reported here. Similarly, habitat, climate, time of day and year, and
283
condition of populations may affect population CORT responses. These factors should be considered
284
when determining the timeline in which to measure baseline CORT concentrations.
12
285 286
5. Conclusions
287 288
Our findings suggest that plasma samples collected within 2 to 3 minutes likely approximate
289
baseline CORT concentrations, although this may be overly conservative in some reptile species. Future
290
studies analyzing putative baseline CORT samples should consider that variation in HPA initiation time
291
varies by taxon, and that there is no single cutoff point for the beginning of CORT elevation. We suggest
292
that future studies conduct their own change point analyses over the range of sample collection times
293
contained in their datasets. Such analyses may allow researchers to include samples they would have
294
otherwise discarded using the typical 3 minute cutoff rule. Consequently, blood samples from species
295
that can be difficult to catch quickly in the field or bleed may still be suitable for measuring plasma GCs
296
(Tarlow and Blumstein, 2007). These findings may allow researchers to reconsider whether stress
297
research is tractable in their focal species, and may open up new avenues for studying stress physiology
298
in a more diverse set of organisms.
299 300
Acknowledgements. We thank the Solon Dixon Forestry Education Center, the United States Air Force,
301
and J. Matter and the Raystown Field Station for access to land and logistical support; L. Van Der Sluys
302
and the staff of Penn State’s Summer Experience in the Eberly College of Science (SEECoS) and Upward
303
Bound Math and Science (UBMS) programs for logistical support regarding the involvement of high
304
school students in this research; and the Cavener lab for access to their plate reader. M. DeLea, D.
305
Ensminger, M. Goldy-Brown and J. Penzelik provided valuable assistance in the field, and R. Clark and E.
306
Taylor provided valuable advice on the rattlesnake study. C. Knapp, J. Iverson, and D. DeNardo provided
307
valuable assistance with field and lab work on the rock iguana study. The John G. Shedd Aquarium
308
provided field research support, and the Bahamas National Trust assisted with permitting. This work was
13
309
supported by the National Science Foundation (IOS1456655 to T.L. and IOS1051367 to T.L.), the
310
Pennsylvania State University (Erickson Discovery Grant, awarded to M.H.), and the Utah Agricultural
311
Experiment Station (RGS RC Seed grant, awarded to S.S.F.). Funding sources had no influence on the
312
design of this study. The authors have no conflict of interest to declare.
14
313 314
References
315
Angilletta, M.J., Niewiarowski, P.H., Dunham, A.E., Leache, A.D., Porter, W.P., 2004. Bergmann’s clines in
316
ectotherms: Illustrating a life-history perspective with Sceloporine lizards. Am. Nat. E168-E183.
317 318 319
Angilletta, M.J., Sears, M.W., Winters, R.S., 2001. Seasonal variation in reproductive effort and its effect on offspring size in the lizard Sceloporus undulatus. Herpetologica. 57, 365-375. Bailey, F.C., Cobb, V.A., Rainwater, T.R., Worrall, T., Klukowski, M., 2009. Adrenocortical effects of
320
human encounters on free-ranging cottonmouths (Agkistrodon piscivorus). J. Herpetol. 43, 260–
321
266. doi:http://dx.doi.org/10.1670/08-123R1.1
322 323 324 325
Blem, C.R., Blem, L.B., 1995. The Eastern Cottonmouth (Agkistrodon piscivorus) at the northern edge of its range. J. Herpetol. 29, 391-398. doi:http://dx.doi.org/10.2307/1574989 Bonier, F., Martin, P.R., Moore, I.T., Wingfield, J.C., 2009. Do baseline glucocorticoids predict fitness? Trends Ecol. Evol. 24, 634–642. doi:10.1016/j.tree.2009.04.013
326
Chloupek, P., Voslářová, E., Suchý Jr., P., Bedáňová, I., Pištěková, V., Vitula, F., Chloupek, J., Večerek, V.,
327
2009. Influence of pre-sampling handling duration on selected biochemical indices in the common
328
pheasant (Phasianus colchicus). Acta Vet. Brno 78, 23–28. doi:10.2754/avb200978010023
329
Claunch, N.M., Frazier, J.A., Escallón, C., Vernasco, B.J., Moore, I.T., Taylor, E.N., 2017. Physiological and
330
behavioral effects of exogenous corticosterone in a free-ranging ectotherm. Gen. Comp.
331
Endocrinol. 248, 87–96. doi:10.1016/j.ygcen.2017.02.008
332 333 334
Cockrem, J.F., 2013. Individual variation in glucocorticoid stress responses in animals. Gen. Comp. Endocrinol. 181, 45–58. doi:10.1016/j.ygcen.2012.11.025 Dallman, M., Bhatnagar, S., 2001. Chronic stress and energy balance: role of the hypothalamo-pituitary-
335
adrenal axis, in: McEwen, B., Goodman, H. (Eds.), Handbook of Physiology. Oxford Univ. Press.,
336
New York, pp. 179–210.
15
337
Dauphin-Villemant, C., Xavier, F., 1987. Nychthemeral variations of plasma corticosteroids in captive
338
female Lacerta vivipara Jacquin: influence of stress and reproductive state. Gen. Comp. Endocrinol.
339
67, 292–302. doi:10.1016/0016-6480(87)90183-3
340
Davidson, J.O., Fraser, M., Naylor, A.S., Roelfsema, V., Gunn, A.J., Bennet, L., 2008. Effect of cerebral
341
hypothermia on cortisol and adrenocorticotropic hormone responses after umbilical cord occlusion
342
in preterm fetal sheep. Pediatr. Res. 63, 51–55. doi:10.1203/PDR.0b013e31815b8eb4
343
Dawson, A, Howe, P.D., 1983. Plasma corticosterone in wild starlings (Sturnus vulgaris) immediately
344
following capture and in relation to body weight during the annual cycle. Gen. Comp. Endocrinol.
345
51, 303–8. doi:10.1016/0016-6480(83)90085-0
346
French, S.S., Neuman-lee, L.A., Terletzky, P.A., Kiriazis, N.M., Taylor, E.N., Denardo, D.F., 2017. Too much
347
of a good thing? Human disturbance linked to ecotourism has a “dose-dependent” impact on
348
innate immunity and oxidative stress in marine iguana, Amblyrhynchus cristatus. Biol. Conserv.
349
210, 37–47. doi:10.1016/j.biocon.2017.04.006
350
Graham, S.P., Earley, R.L., Hoss, S.K., Schuett, G.W., Grober, M.S., 2008. The reproductive biology of
351
male cottonmouths (Agkistrodon piscivorus): Do plasma steroid hormones predict the mating
352
season? Gen. Comp. Endocrinol. 159, 226-235. doi:10.1016/j.ygcen.2008.09.002
353
Graham, S.P., Freidenfelds, N.A., Mccormick, G.L., Langkilde, T., 2012. The impacts of invaders: Basal and
354
acute stress glucocorticoid profiles and immune function in native lizards threatened by invasive
355
ants. Gen. Comp. Endocrinol. 176, 400–408. doi:10.1016/j.ygcen.2011.12.027
356
Herr, M.W., Graham, S.P., Langkilde, T., 2017. Stressed snakes strike first: Hormone levels and defensive
357
behavior in free ranging cottonmouths (Agkistrodon piscivorus). Gen. Comp. Endocrinol. 243, 89–
358
95. doi:10.1016/j.ygcen.2016.11.003
359 360
Holding, M.L., Frazier, J.A., Dorr, S.W., Henningsen, S.N., Moore, I.T., Taylor, E.N., 2014a. Physiological and behavioral effects of repeated handling and short-distance translocations on free-ranging
16
361
northern Pacific rattlesnakes (Crotalus oreganus oreganus). J. Herpetol. 48, 233–239.
362
doi:10.1670/11-314
363
Holding, M.L., Frazier, J.A., Dorr, S.W., Pollock, N.B., Muelleman, P.J., Branske, A., Henningsen, S.N.,
364
Eikenaar, C., Escallón, C., Montgomery, C.E., Moore, I.T., Taylor, E.N., 2014b. Wet- and dry-season
365
steroid hormone profiles and stress reactivity of an insular dwarf snake, the Hog Island boa (Boa
366
constrictor imperator). Physiol. Biochem. Zool. Ecol. Evol. Approaches 87, 363–373.
367
doi:10.1086/675938
368 369 370 371 372 373 374 375 376 377
Hume, D.M., Egdahl, R.H., 1959. Effect of hypothermia and of cold exposure on adrenal cortical and medullary secretion. Ann. New York Acad. Sci. 80, 435–444. Iverson, J.B., 1979. Behavior and ecology of the rock iguana, Cyclura carinata. Bulletin of the Florida State Museum. 24, 173-356. Iverson, J.B., Hines, K.N., Valiulis, J.M., 2004. The nesting ecology of the Allen Cays rock iguana, Cychlura cychlura inornata in the Bahamas. Herpetological Monographs. 18, 1-36. Iverson, J.B., Mamula, M.R., 1989. Natural growth of the Bahamian iguana Cyclura cychlura. Copeia 2, 502-505. Langkilde, T., Lance, V.A., Shine, R., 2005. Ecological consequences of agonistic interactions in lizards. Ecology 86, 1650–1659.
378
Lind, C.M., Husak, J.F., Eikenaar, C., Moore, I.T., Taylor, E.N., 2010. The relationship between plasma
379
steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake,
380
Crotalus oreganus. Gen. Comp. Endocrinol. 166, 590-599. doi:10.1016/j.ygcen.2010.01.026
381
Manzo, C., Zerani, M., Gobbetti, A., Di Fiore, M.M., Angelini, F., 1994. Is corticosterone involved in the
382
reproductive processes of the male lizard, Podarcis sicula sicula? Horm. Behav. 28, 117-129.
383
doi:10.1006/hbeh.1994.1009
384
Mehdi, A.Z., Carballeira, A., 1972. Steroid biosynthesis by painted turtle (Chrysemys picta picta)
17
385
adrenals. Steroids 19, 137-150.
386
Mehdi, A.Z., Sandor, T., 1974. Steroid biosynthesis by reptilian adrenals. Steroids 24, 151-163.
387
Neuman-Lee, L.A., French, S.S., 2017. Endocrine-reproductive-immune interactions in female and male
388
Galápagos marine iguanas. Horm. Behav. 88, 60–69. doi:10.1016/j.yhbeh.2016.10.017
389
Pough, F.H., 1980. The advantages of ectothermy for tetrapods. Am. Nat. 115, 92–112.
390
Putman, B.J., Clark, R.W., 2017. Behavioral thermal tolerances of free-ranging rattlesnakes (Crotalus
391
oreganus) during the summer foraging season. J. Therm. Biol. 65, 8-15.
392
doi:http://dx.doi.org/10.1016/j.jtherbio.2017.01.012
393
Rhind, S.G., Gannon, G.A., Shek, P.N., Brenner, I.K., Severs, Y., Zamecnik, J., Buguet, A., Natale, V.M.,
394
Shephard, R.J., Radomski, M.W., 1999. Contribution of exertional hyperthermia to
395
sympathoadrenal-mediated lymphocyte subset redistribution. J. Appl. Physiol. 87, 1178–1185.
396
Rhind, S.G., Gannon, G.A., Shephard, R.J., Buguet, A., Shek, P.N., Radomski, M.W., 2004. Cytokine
397
induction during exertional hyperthermia is abolished by core temperature clamping:
398
neuroendocrine regulatory mechanisms. Int. J. Hyperthermia 20, 503–516.
399
doi:10.1080/02656730410001670651
400 401 402
Romero, L.M., 2004. Physiological stress in ecology: Lessons from biomedical research. Trends Ecol. Evol. 19, 249–255. doi:10.1016/j.tree.2004.03.008 Romero, L.M., Reed, J.M., 2005. Collecting baseline corticosterone samples in the field: Is under 3 min
403
good enough? Comp. Biochem. Physiol. - A Mol. Integr. Physiol. 140, 73–79.
404
doi:10.1016/j.cbpb.2004.11.004
405
Sheriff, M.J., Dantzer, B., Delehanty, B., Palme, R., Boonstra, R., 2011. Measuring stress in wildlife:
406
techniques for quantifying glucocorticoids. Oecologia 166, 869–887. doi:10.1007/s00442-011-
407
1943-y
408
Siegel, S., Castellan, N., 1988. Nonparametric Statistics for the Behavioral Sciences, 2nd ed. McGraw-Hill,
18
409 410
New York, NY. Stroud, L.R., Solomon, C., Shenassa, E., 2007. Long-term stability of maternal prenatal steroid hormones
411
from the National Collaborative Perinatal Project: Still valid after all these years.
412
Psychoneuroendocrinology 32, 140-150.
413 414 415 416
Tarlow, E.M., Blumstein, D.T., 2007. Evaluating methods to quantify anthropogenic stressors on wild animals. Appl. Anim. Behav. Sci. 102, 429–451. doi:10.1016/j.applanim.2006.05.040 Trompeter, W.P., Langkilde, T., 2011. Invader danger: Lizards faced with novel predators exhibit an altered behavioral response to stress. Horm. Behav. 60, 152–158. doi:10.1016/j.yhbeh.2011.04.001
417
Vinson, G.P., Braysher, M., Whitehouse, B.J., 1975. In vitro steroidogenesis by the nonzoned
418
adrenocortical tissue of the skink, Tiliqua rugosa. Gen. Comp. Endocrinol. 26, 541-549.
419
Webb, A.C., Iverson, J.B., Knapp, C.R., Denardo, D.F., French, S.S., 2018. Energetic investment associated
420
with vitellogenesis induces an oxidative cost of reproduction. J. Anim. Ecol. 1–11.
421
doi:10.1111/1365-2656.12936
422 423
Wikelski, M., Cooke, S.J., 2006. Conservation physiology. Trends Ecol. Evol. 21, 38–46. doi:10.1016/j.tree.2005.10.018
19
425 426
Table 1. Results of change point analyses of initial concentrations of plasma CORT and paired t-test
427
comparisons of “baseline” CORT concentrations obtained after the change point to CORT concentrations
428
after the individuals had experienced a standardized restraint stressor. Range of initial bleed times
After change point vs. Change point
restraint stressor p-
Species
N
(min:sec)
(min:sec)
value
t-value
df
p-value
Cottonmouth
28
2:00-7:10
4:00
0.01
-4.8488
8
0.0013
Rattlesnake
21
2:00-10:21
2:30
0.01
-5.4513
15
<0.0001
Fence lizard*
28
1:15-10:18
4:45
0.09
-
-
-
Rock iguana**
459
0:15-7:18
2:44
<0.01
-
-
-
429
*No t-test was performed as no statistically significant change point was found.
430
**No t-test was performed as the animals did not undergo a standardized restraint stress protocol.
20
432
433 434
Table 2. Natural history and sampling data for each species. Dates sampled Cottonmouth May-July 2013
Capture time range 00:4123:27
Time of sunrise* Peak activity period 05:50 06:00-10:00 (Blem and Blem, 1995)
Rattlesnake
July-August 2013
08:4617:47
06:10
07:00-18:00 (Putman and Clark, 2017)
March-May; AugustSeptember (Lind et al., 2010)
Fence lizard
June-July 2014
12:1115:07
05:45
10:00-14:00
May-June (Angilletta et al., 2001)
Rock iguana
May, June, September 2016
08:0013:50
06:30
08:00-17:00 (Iverson, 1979)
May-June (Iverson et al., 2004)
Breeding season July-August (Graham et al., 2008)
*Calculated with the National Oceanic and Atmospheric Administration Solar Calculator (https://www.esrl.noaa.gov/gmd/grad/solcalc/index.html)
21
436 437
Figure 1: CORT concentrations of different species across time since capture. Black dots show the initial
438
corticosterone (CORT) concentrations in plasma samples obtained at different times after capture in
439
four species: A) cottonmouth (Agkistrodon piscivorus), B) rattlesnake (Crotalus oreganus), C) eastern
440
fence lizard (Sceloporus undulatus), and D) rock iguana (Cyclura cychlura). Change points (times at which
441
initial CORT concentrations before these points are significantly different from initial samples obtained
442
after this point) are shown as dotted lines (a, b and d). Diamonds represent the mean (at the widest
443
point of the diamond) ±1 standard error (length of the diamond) of plasma CORT concentrations in a
444
second set of blood samples taken from each individual after 30 minutes (a, c) or 60 minutes (b) of
445
restraint stress.
446
447 448
Figure 1.
449
Research Highlights
22
450
The steroid hormone corticosterone, CORT, can indicate physiological stress in vertebrates.
451
Measuring baseline CORT is key to understanding animal responses to stressors.
452
Obtaining samples quickly enough to assess baseline CORT can be challenging.
453
Plasma sampled within 2.5-4 min of capture in reptiles represents baseline CORT.
454
Baseline CORT can be obtained from blood sampled even later in some reptile species.
455
23