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Comparing techniques for measuring corticosterone in tadpoles

Glucocorticoids play a key role in mediating stress responses in vertebrates. Corticosterone (CORT) is the main glucocorticoid produced in amphibians, birds, and reptiles, and regulates several metabolic functions. The most common methods for quantifying CORT are competitive binding immunoassays: radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA has been broadly used since the 1980’s but it requires radioactivity. Commercial EIA kits permit quantifying hormone levels without radioactivity although the requirement for a larger sample volume may be a strong limitation for measurements involving larval amphibians. Here we used Xenopus laevis tadpoles to compare the performance of three commonly used procedures for determination of CORT: RIA on a chloroform extract of whole-body homogenate, EIA on plasma, and EIA on supernatant of whole-body homogenate. We treated tadpoles with exogenous CORT at 0, 25, 50, and 100 nM. RIA could distinguish between 0 and 25 nM, and EIA on plasma between 0 and 50 nM, whereas whole-body homogenate EIA only detected significant differences between 0 and 100 nM. Each procedure presents advantages and disadvantages regarding sensitivity, the use of radioactivity, sample size, handling time, and economic cost. RIA is preferred when studying small-bodied animals from which blood samples cannot be obtained. When CORT level differences are intermediate and blood sampling is possible, EIA on plasma is a good non-radioactive alternative. EIA on whole-body homogenates may be useful to assess qualitative changes in CORT levels when considerable differences are expected. Finally, we discuss our findings in the context of previous studies on CORT in amphibians [Current Zoology 61 (5): 835–845, 2015].

Organisms make physiological adjustments in response to environmental fluctuations, whether natural or anthropogenic, through hormonal regulation (Johnson et al., 1992;Nussey and Whitehead, 2001;Becker et al., 2002). In vertebrates, the main hormonal response to environmental perturbations is based on the activation of the hypothalamic-pituitary-adrenal (HPA) axis to release corticotrophin-releasing hormone (CRH) (Miller and O'Callaghan, 2002;Denver, 2009). CRH stimulates the anterior pituitary to secrete the adrenocorticotropin hormone (ACTH) (Raffin-Sanson et al., 2003) followed by release of glucocorticoids (GCs) from the adrenal gland (Aguilera, 1994). Although the activation of this endocrine pathway is rapid, the GC level increase varies among species and individuals and can take around 3-5 min in vertebrates (Cash et al., 1997;Sockman and Schwabl, 2001;Romero and Romero, 2002). Corticosterone (CORT) is the main GC involved in the stress response in reptiles, birds, and amphibians whereas cortisol is the main one in fish and mammals (Sapolsky et al., 2000;Romero, 2004;Denver, 2009). Moreover, GC levels are associated with health condition, developmental rate, metabolism, and immune function Walker et al., 2005;Wikelski and Cooke, 2006;DuRant et al., 2008;Davis et al., 2008), and they are commonly studied in developmental, ecological, and conservation studies (Romero, 2004;Busch and Hayward, 2009).
The most common methods for quantifying hormones are competitive binding immunoassays: radioimmunoassay (RIA) and enzyme immunoassay (EIA) (Sherrif, 2011;Narayan, 2013). Other techniques such as gas or liquid chromatography coupled with mass spectrometry have been successfully, though less frequently, used (Webb et al., 2007). RIA has been used extensively for over three decades and is a very sensitive technique to determine the concentration of antigens (e.g. hormones). RIA, however, requires the use of radioactive isotopes, specialized equipment, and a laboratory classified for handling radioactive materials. Moreover, radioactivity is increasingly avoided in laboratories and institutions because of the health risk and paperwork it entails.
In the 1970's, the development of commercial EIA kits allowed hormonal assays without the use of radioactivity. Commercial EIA kits are commonly designed for plasma or urine samples, providing information on circulating hormones (Wada et al., 2007;Mills et al., 2010) at competitive prices. However, when studies involve small species or life stages (e.g. embryos or larvae from amphibians, reptiles, and fish), obtaining the minimum required amount of plasma or urine to conduct EIA may prove challenging. Consequently, some researchers are attempting to conduct EIA using commercial kits on supernatant of whole-body homogenate instead of plasma (McMahon et al., 2011;. Clearly this would yield a diluted hormone sample compared to blood, and intracellular hormone may be included in the measurement. It is unclear whether this would yield comparable data to the other procedures or has a similar sensitivity to detect differences among study groups (e.g. species, populations, experimental treatments). Reduced specimen size may render whole-body supernatant EIA the only viable alternative to RIA, unless plasma from a large number of individuals could be pooled together. However, losing the possibility to study CORT variation at the individual level may be undesirable because increasing the minimum required number of experimental individuals raises practical and bioethical issues. The choice of hormonal assay is therefore subject to various considerations depending on the study system, experimental design, infrastructure, and budget availability.
Amphibians are ideal to compare different procedures for determining GC levels as they have been used as a model group to analyze the role of GCs in multiple physiological processes, especially regarding the timing of metamorphosis Gomez-Mestre et al., 2013). However, determining CORT levels in larval amphibians often poses difficulties derived from the small size of individuals of some species.
Here we compare the performance of three procedures for measuring CORT using tadpoles of the African clawed frog Xenopus laevis: RIA on chloroform extract of whole-body, EIA on plasma, and EIA on supernatant of whole-body homogenate. We compare CORT estimates obtained from tadpoles treated with different amounts of exogenous CORT and discuss the suitability of each procedure with respect to sensitivity and discrimination power, but also to bioethical, logistic, and budgetary criteria. We also review and discuss the methodology used for CORT determination in anurans over the last 20 years.

Experimental setup
We collected egg clutches from three Xenopus laevis pairs from a breeding colony at Centro Andaluz de Biología del Desarrollo (Seville, Spain). After hatching, 240 tadpoles were reared for 60 days in 2.7 L buckets (5 tadpoles/bucket) until they reached Nieuwkoop and Faber developmental stages 52-54 (Nieuwkoop and Faber, 1994). At this stage range, tadpoles show fore and hind limbs in paddle stage and the hind limbs without feet and longer than broad (mean length 55 mm, Nieuwkoop and Faber, 1994). During this rearing period, water was renewed twice a week and tadpoles were fed 30 mg of ground rabbit chow every other day. The experimental units were distributed across shelves in a walk-in chamber set at constant 20°C, and a 12:12 lightdark cycle.
When the tadpoles reached NF 52-54 we treated them with exogenous CORT (0 nM, 25 nM, 50 nM, or 100 nM). Each CORT treatment was replicated 12 times for a total of 48 experimental units. Water was changed daily, and at each water change tadpoles were fed 30 mg of ground rabbit chow and CORT (C2505, SIGMA) was added. Tadpoles have permeable skin (Bentley, 1971;Parson, 1994), so CORT diluted in water is directly uptaken via skin absorption. To reach the experimental concentrations, we prepared stock solutions of CORT diluted in absolute ethanol so that an addition of 200 L of any of them to 2.7 L of water in the experimental containers would result in the target concentrations of 25, 50, and 100 nM. We first prepared a 1350 M stock solution (23.5 mg of CORT in 50 mL of ethanol) from which the 100 nM experimental concentration would be obtained, and then prepared two stocks at lower concentrations by serial 1:1 dilutions with absolute ethanol. Buckets in the control (no CORT) received 200 L of ethanol each time. The stock concentrations are based on previous studies  and were kept at -80°C. Three days after applying the hormonal treatments we collected tadpoles from each bucket for CORT measurements, randomly assigning them to RIA (one tadpole per bucket), EIA on plasma (three tadpoles pooled per bucket) and EIA on whole-body homogenate (one tadpole per bucket). We did not find differences in weight among treatments at the end of the experiment, and tadpoles weighed an average of 223 ± 9 mg (SE).
Plasma samples for EIA were obtained through heart puncture. Tadpoles deeply anesthetized with MS-222 (Ethyl 3-aminobenzoate methanesulfonate, Sigma), were placed under a dissecting scope, and blood was extracted via cardiac puncture with a non-heparinized insulin syringe (BD Micro-Fine Insuline U-100 0.5 ml). Blood from all three individuals (ca. 20 L per tadpole) from each bucket was pooled in heparinized tubes and then centrifuged at 4000 rpm, at 4°C for 20 minutes to obtain plasma . Plasma samples were stored at -80°C in 0.5 ml eppendorf tubes until assayed. The average handling time per individual for the blood extraction procedure from immersion in anesthetic to complete collection of the blood sample was 193 s ± 7.5 SE.
Individuals assigned to RIA were dipnetted from their containers, euthanized with MS-222 (Ethyl 3-aminobenzoate methanesulfonate), dissected to remove the gut, snap-frozen in liquid nitrogen, and preserved at -80°C until used. For whole-body homogenate EIA, tadpoles were euthanized with MS-222, dissected to remove the gut, ground up with a homogenizer (MICCRA D-1), and centrifuged at 4,000 rpm at 4°C for 15 min. The resulting supernatant was preserved at -80°C until used .

Radioimmunoassay
Specimen samples were homogenized (except the gut) as described above centrifuging and extracting the hormone from the supernatant with chloroform and ionexchange chromatography prior to RIA following previous protocols (Denver, 1993). Lower detection limit was 0.20 pg/mL/mg.

Plasma and whole-body homogenate EIAs
We took 50 L of either plasma or homogenate (supernatant) from each sample and conducted EIA with a commercial kit following manufacturer specifications (Cayman Chemical Company -catalog n° 500655). The CORT determination with this EIA kit is based on the competition between a CORT-acetylcholinesterase conjugate and CORT, for a limited number of CORTspecific sheep antiserum binding sites which bind to the rabbit polyclonal anti-sheep IgG that previously was attached to the well. For this, each plate was incubated for two hours at room temperature on an orbital shaker. Ellman's reagent, which contains a substrate for acetylcholinesterase, was added to the wells to develop the assay. After incubating for 60-90 minutes to achieve a stable end point in an orbital shaker in the dark, absorbance was read at a wavelength of 412 nm, and CORT concentration was determined based on standard curves run in duplicate on each plate. According to the manufacturer, the detection limit (80% B/B 0 ) is approximately 40 pg/mL, and cross reactivity with other tested steroids is below 1%. It was not the aim of this study to compare the efficacy of the various commercial kits available, and differences among kits from different suppliers are likely to affect the outcome due to differences in their antibody specificity or detection limits. Moreover, most of these kits are optimized for use with human samples, and in our experience not all work well with amphibian samples.

Statistical tests
We assayed 38 tadpoles through RIA, with 9-10 replicates per treatment. For EIA on plasma, we extracted blood from 135 tadpoles. We obtained the required 50 L pooling plasma from three tadpoles per bucket, yielding 6-7 replicates per treatment. For some pooled samples, the total volume obtained was insufficient to run the assay. In whole-body homogenate EIA we obtained 9-11 replicates per treatment and we used 40 tadpoles in total. All statistical analyses were conducted in R version 2.14.1 (R Development Core Team 2007). We checked the parametric assumptions testing normality of the data via analysis of residuals distribution and homoscedasticity using Barlett's tests (bartlett.test). Data from RIA and whole-body homogenate EIA were heteroscedastic and not normally distributed, and hence we fitted generalized linear models with a negative binomial distribution (function glm.nb in MASS package, version 7.3-22) to test for differences among CORT treatments. We then ran post-hoc tests using the glht function (multcomp package, version 1.2-13). Plasma EIA data, however, met parametric assumptions and therefore we fitted instead a general linear model (aov function) followed by post-hoc Tukey tests. We tested the effect of handling time including it as a covariate in the model. We calculated intraclass correlations to determine the reliability and consistency of the measurements within treatments for each procedure with the ICC function (ICC package, version 2.2.1).

Results
Handling time for individuals preserved for wholebody homogenate (RIA or EIA) was limited to ~30 s in the anesthetic (MS-222). Handling time during blood sample collection had no significant effect on CORT levels (coefficient of regression: 0.19, P = 0.355, R 2 = 0.037).
We detected significant changes among treatments in CORT concentration, regardless of the procedure used (Figure 1; overall tests:  2 = 50.605, df = 3, P < 0.001 for RIA; F 3,22 = 10.08, P < 0.001 for EIA on plasma;  2 = 7.89, df = 3, P = 0.017 for EIA on whole-body). For RIA data, post-hoc tests (Table 1) showed significant differences among the control treatment and each exogenous CORT treatment (25, 50 and 100 nM; all P < 0.001). The CORT levels increased on average by 6.1-fold between 0 nM and 25 nM treatments, and by up to 9.52 and 9.47-fold in the 50 and 100 nM treatments in comparison to the control treatment. Plasma EIA showed that CORT increased 4.12 times in the 100 nM treatment with respect to the control (P = 0.001). We also found significant differences between 25 nM and 50 nM (P = 0.045), and 25 nM and 100 nM (P < 0.001), but not between the control and 25 mM. We found noticeable changes in the CORT levels between 0 nM-50 nM and 50 nM-100 nM but they were marginally not significant (Table 1). Overall EIA on plasma showed the same number of significant pairwise differences among treatments that RIA did, and in both cases we found significant differences between treatments that differed in 25 nM of CORT. However, RIA detected significant differences between the lowest concentrations of corticosterone (0 nM-25 nM) and EIA on plasma detected better than RIA differences when higher concentrations were added (50 or 100 nM). The model fit on wholebody homogenate EIA indicated a significant effect of hormonal treatments. Post-hoc tests only found significant differences between 0 nM and 100 nM treatments (P = 0.002) although differences between 0 nM-50 nM and 25 nM-100 nM neared statistical significance (P ~ 0.07).
Comparing the absolute values (in pg/ml) obtained in plasma EIA with those estimated from whole-body homogenate EIA (Fig. 1), we found diluted levels in the homogenate, yielding values in the range of 2.1 to 4.6 times lower concentrations than in plasma. This comparison is not possible with the RIA values because these are given per mass unit (pg/ml/mg).
The intraclass correlation coefficient (ICC) provides an idea of the consistency of the data within experimental treatments. ICCs indicated a higher consistency of data from EIA on plasma (ICC = 0.56) and RIA (ICC = 0.40) than EIA on supernatant of whole-body homogenate (ICC = 0.14).

Discussion
The comparison of RIA on whole-body extract, EIA on plasma, or EIA on supernatant of whole-body homogenate, revealed that RIA was a more sensitive procedure for detecting low CORT values than the EIA procedures. RIA on whole-body extract was the only procedure capable of detecting differences between control tadpoles and tadpoles treated with 25 nM CORT (Table  1). RIA, however, was rapidly saturated and could not distinguish tadpoles treated with more than 50 nM. Diluting the samples prior to the assay, however, could solve this problem of RIA saturation. EIA on plasma samples was less sensitive than RIA at low CORT levels but also allowed detection of differences between treatments that differed by 25 nM (i.e. 25 nM vs 50 nM). Moreover, EIA showed no indication of saturation in our study, indicating a larger dynamic range for detecting significant differences. This characteristic of EIA on plasma may be advantageous when species have unknown basal CORT levels and limited sample volumes preclude use of dilution series to assess assay saturation. EIA on whole-body homogenate supernatant showed the lowest sensitivity since we could only detect significant differences between tadpoles treated with 100 nM CORT and those with no CORT added. Nevertheless, EIA on whole-body homogenate supernatant showed the same trend as EIA on plasma ( Fig. 1) but with rather dampened values, most likely a consequence of dilution with CORT-free body fluids. The most consistent procedure (i.e. the one with higher intraclass correlation coefficient) was EIA on plasma, followed by RIA.
CORT concentrations measured in previous studies in natural or semi-natural conditions reported values that were within the range in which the three procedures can

Fig. 1 Corticosterone concentration measured applying EIA plasma (light grey bars, left Y axis), EIA whole-body homogenate (dark grey bars, left Y axis), or RIA (black line, right Y axis)
Error bars indicate standard error of the mean. detect significant differences. There is much information about CORT levels obtained via RIA on wholebody extracts. For example,   The study of stress responses in amphibians has required conducting corticosterone assays on several species. We found 56 studies that determined CORT in anurans in the last two decades ( Table 2). Some of these studies included several species or different life stages, which we considered as separate corticosterone measurements (n = 70). The majority of such measurements were obtained using RIA (n = 46). Within these RIA studies, we found 11 instances where CORT was measured on plasma: six in adults, four in juveniles, and only one in tadpoles (which included one or two tadpoles per sample; . This bias towards adult and juvenile stages in the analysis of plasma samples likely reflects the difficulty in obtaining blood samples in tadpoles due to its reduced size. Consequently, most tadpole studies opted for whole-body chloroform extracts (n = 31, Table 2).
Studies with EIA used different organs or fluids or else incorporated various modifications of the technique. For example, we found 18 instances where CORT levels were assayed in urine of adult amphibians and just one where CORT was determined in plasma. Studies on tadpoles applied EIA in various ways. Two studies ran assays on whole-body homogenates (McMahon et al., 2011; whereas another study determined CORT levels from pond water . RIA has thus been the preferred technique for quantifying CORT in anurans in the last two decades, most often applied to extracts from whole-body tadpole homogenates. EIA was conducted on various sample types, and was preferred when assaying urine or blood samples. Various factors need to be taken into account in order to decide which method to use (Table 3). RIA is widely used but requires radioactivity, which is not implemented in many research institutions. Regarding budgetary aspects the use of EIA can be favored over the use of RIA, as EIA commercial kits are available at competitive prices that do not require additional disposal costs as with radioactive materials generated by RIA.
Our results indicate that EIA on plasma samples is a reasonable non-radioactive alternative to RIA, with two caveats. First, EIA has lower sensitivity at low CORT levels. Second, many studies will focus on study systems where individuals are too small for collecting sufficient plasma samples for commercial kits (typically requiring ca. 50 µL), especially so in early ontogenetic stages. Thus, obtaining sufficient plasma samples from small organisms may require pooling blood from several individuals into a single sample, hence requiring the sacrifice of many more individuals than are otherwise required for either RIA or EIA on whole-body homogenate. In our study, each plasma sample for EIA required pooling blood from three X. laevis tadpoles whereas single tadpoles were allocated to either RIA or EIA on whole-body supernatant. This of course raises conservation and bioethical concerns, especially when dealing with threatened or vulnerable species, which in the case of amphibians includes ca. 43% of all known species (Hoffmann et al., 2010;LesBarrères et al., 2014). Moreover, an increase in the number of tadpoles in the study can pose some logistical problems in laboratories as well as during field campaigns. Thus, studies will have to either increase the total number of replicates to account for the need to pool samples, or increase the nu-  Narayan et al., 2013 (5) in water pond tadpole A. obstetricans  tadpole A. muletensis  We have only included studies that used RIA or EIA to determine the corticosterone levels. Some studies have multiple entries because they included several analysis, e.g. with different species or individuals' stages. The full references are included in the "Suplementary material". mber of tadpoles per experimental unit. Besides, rearing more tadpoles per unit requires larger containers since larval density in itself directly results in increased CORT levels (Hayes, 1997). Another potential confounding factor in determining CORT levels is the effect of handling time. GCs levels can increase inordinately after only 3-5 minutes from the first contact with the individual (Wingfield et al., 1982;Cash et al., 1997;Romero and Romero, 2002) and usua-lly reach peak levels after 15-30 min after exposure to the stressor (De Kloet et al, 2005). Handling times are not a concern for procedures like RIA and whole-body homogenate EIA in which animals can be quickly (under 3-5 min) euthanized and preserved (e.g. through immersion in MS-222 followed by snap freezing of the whole body). Obtaining blood samples from tadpoles, however, is a more laborious procedure and some training is required to reduce handling times under 3-5 min. Therefore, handling times may become a likely source of error and need to be accounted for in the analyses. For this reason, RIA and whole-body homogenate EIA are the best method to measure GC in field studies. EIA on supernatant of whole-body homogenates has the advantages of not requiring radioactivity, avoiding potential error from increased handling times, and requires fewer individuals than plasma EIA. Conversely, it constitutes a more conservative test of differences among test groups (experimental treatments, populations, species) , as it is less sensitive than either RIA or plasma EIA.
The choice of procedure for each study should thus be made considering methodological limitations and tractability for each study system. RIA is still the golden standard in eco-physiological studies because it is most sensitive, it is appropriate for small larvae, and can be used to compare CORT levels using different tissues or on whole-body homogenates. However, plasma EIA is a good non-radioactive alternative that has been used to obtain significant results in response to natural perturbations such as pond drying . EIA on whole-body homogenate supernatant could detect qualitative differences in CORT levels among experimental groups, and may be of use if individuals are small and radioactive assays cannot be implemented.