Calling rate, corticosterone plasma levels and immunocompetence of Hypsiboas albopunctatus

Comparative Biochemistry and Physiology, Part A 201 (2016) 53–60

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Calling rate, corticosterone plasma levels and immunocompetence of Hypsiboas albopunctatus Stefanny Christie Monteiro Titon a,⁎, Vania Regina de Assis a, Braz Titon Junior a, Adriana Maria Giorgi Barsotti a, Sarah Perry Flanagan b, Fernando Ribeiro Gomes a a b

Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, São Paulo, Brazil Department of Biology, Texas A&M University, College Station, TX, United States

a r t i c l e

i n f o

Article history: Received 8 December 2015 Received in revised form 16 June 2016 Accepted 17 June 2016 Available online 27 June 2016 Keywords: Amphibian Corticosterone Calling behavior Immunocompetence PHA

a b s t r a c t During the breeding season, male anuran amphibians produce advertisement calls. Androgens play a permissive role in the activation of calling activity, which is often positively correlated to androgen plasma levels and testes mass. Additionally, calling effort is also correlated to corticosterone plasma levels (hereinafter referred to as CORT), which is associated with the mobilization of energy substrates to sustain the high energy flux associated with this activity. However, high CORT also has many immunosuppressive effects and might interfere with reproduction. Consequently, CORT might mediate a compromise between reproductive effort and immunocompetence in anurans. In the present study, we investigated the relationship between calling rate, immunocompetence, and CORT in Hypsiboas albopunctatus, a midsize anuran occurring in South America. To understand these relationships, we conducted focal observations of calling behavior, followed by blood collection for CORT measurements and evaluation of some immune parameters. Our results showed that individuals with larger testes had higher calling rates, and those with higher calling rates showed lower cell-mediated immune response (swelling response to phytohaemagglutinin), although these relationships were not mediated by CORT. Furthermore, males calling early in the evening showed high CORT, and individuals with lower body condition index had higher CORT. We conclude that calling activity shows a cost in terms of cellular immune response in H. albopunctatus, but this compromise does not appear to be mediated by glucocorticoid plasma levels. © 2016 Elsevier Inc. All rights reserved.

1. Introduction In vertebrates with seasonal reproduction, males show high plasma levels of glucocorticoids and androgens during the reproductive season (Moore and Jessop, 2003). Androgens are responsible for gamete maturation, expression of secondary sexual characters and sexual displays (Randall et al., 2002; Stocco and McPhaul, 2006). Glucocorticoids promote lipid oxidation and muscle proteolysis to provide amino acids for gluconeogenesis during periods of increased energy demand, including prolonged aerobic exercise (Tharp, 1975; Pough et al., 1992; Weber, 1992; Landys et al., 2006; Malisch et al., 2007; Hau et al., 2010). In this way, it has been proposed that the increased plasma levels of glucocorticoids during the reproductive season play an important role in the mobilization of energetic resources to support physiological and behavioral processes associated with reproduction (Sapolsky, 2002; Moore and Jessop, 2003). In anuran amphibians, males produce advertisement calls during the breeding season, and several aspects of these calls are ⁎ Corresponding author at: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Trav. 14, 101, Lab. 300, 05508-090 São Paulo, Brasil. E-mail address: [email protected] (S.C.M. Titon).

http://dx.doi.org/10.1016/j.cbpa.2016.06.023 1095-6433/© 2016 Elsevier Inc. All rights reserved.

subjected to intersexual selection (Wells, 2007a). In particular, the dynamic properties of advertisement calls, such as calling rates, are subjected to intersexual directional selection (Gerhardt, 1991; Wells, 2007a; Castellano et al., 2009), entail high energetic costs (Pough et al., 1992; Wells, 2001), and are positively correlated to glucocorticoid and androgen plasma levels at intra (Leary et al., 2004; Assis et al., 2012) and inter-specific levels (Emerson and Hess, 2001; Leary et al., 2005; Assis et al., 2012). However, calling rates have been found to be more closely associated with corticosterone plasma levels (hereinafter referred to as CORT) in anurans at the interindividual level (Leary et al., 2004; Leary et al., 2006a; Assis et al., 2012). Additionally, high CORT in calling anurans is negatively associated with index of substrate reserves, such as mass of fat bodies (Madelaire and Gomes, 2016) and the body condition index (Leary and Harris, 2013; Leary et al., 2015), suggesting that CORT plays an important role on mobilization of energy substrates for calling maintenance (Leary et al., 2004). Although glucocorticoids are necessary for the maintenance of reproductive behavior (Moore and Jessop, 2003), high glucocorticoid plasma levels associated with the response to stressors have been associated with physiological changes that could potentially decrease fitness, including reproductive inhibition (Moore and Jessop, 2003; Landys et al., 2006; Carr, 2011) and immunosuppression (reviewed in

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Marketon and Glaser, 2008). In amphibians, stressors and exogenous corticosterone can inhibit calling behavior (Leary et al., 2006a, 2006b), and reduce androgen plasma levels and spermatogenesis (Licht et al., 1983; Paolucci et al., 1990; Biswas et al., 2000; Tsai et al., 2003; Narayan et al., 2011, 2012a, 2012b). Besides that, increased circulating levels of glucocorticoids promote a redirection of leukocytes between body compartments, increasing neutrophil/lymphocyte ratio (hereinafter referred to as NLR) in the circulation (reviewed in Davis et al., 2008). Therefore, an increase in NLR is also used as a measure of stress in vertebrates, including amphibians (Davis et al., 2008). Increased NLR has been reported after 10 days in captive conditions in salamanders (Davis and Maerz, 2008), after a restraint challenge with movement restriction in toads (Assis et al., 2015), and as a result of infectious disease in tree frogs (Peterson et al., 2013). Higher NLR therefore can be considered an index of stress response along with lower body condition indices and lower testes masses associated with high glucocorticoid plasma levels. Regarding the effects of glucocorticoids on the immune response, the emerging picture is more complex. Immunosuppressive effects of chronically elevated glucocorticoid plasma levels include reduced size and function of lymphoid organs, inhibition of maturation, development and action of immune cells, and an important anti-inflammatory role in different vertebrate groups (Ahmed et al., 1985; Wiegers and Reul, 1998; Sapolsky et al., 2000; Riccardi et al., 2002; Cavalcanti et al., 2006). However, at the onset of the response to acute stressors, high levels of glucocorticoids stimulate the release of humoral components, which can activate cellular mediators of immune response, increase the number of circulating neutrophils and redirect lymphocytes to peripheral tissues, potentially enhancing immune response (Wiegers and Reul, 1998; Riccardi et al., 2002; Cavalcanti et al., 2006). This bimodal effect of glucocorticoids on the immune response seems to be associated to differences in intensity and the duration of the stress response (Wiegers et al., 1994; Wiegers and Reul, 1998; Sapolsky et al., 2000; Dhabhar, 2009). In amphibians, there is some evidence that innate immune responsiveness can be compromised by stressors. The plasma bacterial killing ability (BKA) describes the ability of the plasma to recognize and lyse a foreign microorganism, mainly through complement system (Millet et al., 2007), decreases in response to restraint stress protocol (Gomes et al., 2012; Graham et al., 2012; Assis et al., 2015) and long-term captivity in toads (Assis et al., 2015). Cellular phagocytic activity, by leukocytes, can also decrease in response to stressors in some anurans (Gilbertson et al., 2003; Dohm et al., 2004). Considering the interactions between CORT, calling behavior, physiological state and immunity, it is plausible that a trade-off between calling performance and immunocompetence in anuran males might be mediated by corticosterone. This hypothesis is in accordance with sexual selection models predicting that glucocorticoid secretion could play an important role on a trade-off between elaborate secondary sexual character expression and immunocompetence, maintaining the honesty of male sexual signals (Bortolotti et al., 2009; Leary and Knapp, 2014). In this study, we investigated the association between calling rates, variables associated to reproductive behavior and stress response, including CORT, body condition index, NLR, testes mass and an aspect of the immunocompetence (phytohaemagglutinin skin-swelling response) in males of the tree frog Hypsiboas albopunctatus. We predicted that calling rates are positively associated with CORT and other variables commonly interpreted as proxies of stress response, and negatively associated with immunocompetence. 2. Materials and methods 2.1. Ethical procedures All procedures for the collection and use of biological material were performed under the approval of the Comissão de Ética no Uso de Animais (CEUA, process number 135/2011), do Instituto de Biociências

da Universidade de São Paulo. The animals were collected under a license for capture from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA, process number 29896-1). The study was carried out on private property (Pesqueiro do Sandro) and no specific permission was required. 2.2. Study site and studied species Hypsiboas albopunctatus is an arboreal midsize tree frog (~7 g, data reported in this study) with a broad distribution in South America, especially in areas with open vegetation in Central, South and Southeast Brazil (Frost, 2015). This species is characterized by continuous reproduction, with reproductive peaks occurring from October to March (Muniz et al., 2008). We collected behavioral data and blood samples from 35 adult males on February 4–8 (N = 11) and March 1–7 (N = 24), 2012, in São Luiz do Paraitinga (23°13′23 ″S, 45°18′38 ″W), São Paulo, Brazil. All males were collected from choruses formed on the shore of two ponds, separated by a 15 meter barrier of sand. Males used cattail branches (Typha domingensis) and soil near water as calling sites.

2.3. Behavioral observations All the observations occurred at night, during the typical calling activity period for this species (20 h to 24 h). Three researchers were present in the pond each night of observation, but only one person observed each animal. Selection of focal individuals was random. Each animal was localized using white light, and the observer respected a 1 m distance from focal individual during the observations (Assis et al., 2012). To minimize observer interference, headlamps with red filters were used to perform the observations, and calling registers began 10 min after the observer approached the animal (Assis et al., 2012; Bevier et al., 2008; Kiss et al., 2009). Focal observations lasted 30 min, during which observers recorded calling behavior (number of vocalizations). The number of males calling in the chorus during observation was also registered. Given that temperature can affect the temporal parameters of anuran vocalization (Navas and Bevier, 2001), field temperatures were recorded at 5 min intervals using HOBO® data loggers placed in micro-environments typically used as calling sites (Assis et al., 2012). Average temperature was calculated from these records for each individual during the observation period and incorporated in the analyses. Most studies relating CORT and calling behavior were conducted with calling effort (which considers call duration, intercall duration and pulse rate) (Emerson and Hess, 2001; Leary et al., 2008). However, some anuran species, mainly hylids such as Pseudacris crucifer, Hyla microcephala, Hyla arborea and Hyperolius marmoratus, show little variation in call note duration (Wells, 2007b). Given that Hypsiboas albopunctatus follows the same pattern (Vieira et al., 2016), calling rate alone was used as measure of calling performance in this study. Calling rate was calculated for each individual as the total number of vocalizations divided by the observation time.

2.4. Blood samples At the end of the behavioral observations, focal males were collected for blood sampling. We obtained blood samples (~ 80 μL) by cardiac puncture using heparinized 1 mL syringes and 26g × 1/2″ needles. Only samples collected within 3 min of capture were considered for analyses because handling can increase CORT (Romero and Reed, 2005). All blood samples were labeled and kept on ice until transport to the laboratory (b5 h). In the laboratory, a drop of blood was used to perform a blood smear. The remaining blood was centrifuged for 4 min at 3000 rpm, and separated plasma was pipetted off and stored at −80 °C for later CORT measurements.

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2.5. Leukocyte profile A drop of blood was pipetted on a slide for microscopy and smeared with a blade. The smear was dried for 30 min, and then fixed with methanol. The slides were made in duplicate for each individual. Subsequently, the smear was stained with eosin methylene blue – Giemsa solution (10%) (Merck KGaA, Darmstadt, Hessen, Germany) for 15 min, and observed through an optical microscope with a 1000 × magnification (Nikon E200, 104c), using immersion oil. One hundred leukocytes were identified and counted based on amphibian cellular morphology (Campbell, 2006). The neutrophil/lymphocyte ratio (NLR) was calculated as the number of neutrophils divided by the number of lymphocytes counted on the slides. 2.6. Phytohemagglutinin assay and morphometry After blood collection, the animals were kept in individual plastic containers and transported to the laboratory for a test of the inflammatory response to subcutaneous injection of phytohemagglutinin (PHA). Unpublished results from previous studies in our laboratory have shown that PHA response can vary with hour of injection. Moreover, the acute transdermal application of corticosterone promotes sustained increase in the percentage of blood cells performing phagocytosis for a period of 24 h after application (unpublished results). In order to minimize differences in hour of injection, and considering that the CORT measured in the field might influence the immune response after 24 h, the PHA assay was performed 24 h after animal collection. The hind fleshy base of the right foot was injected with 10 μL of a PHA solution (20 mg/mL – Sigma L8754) using a 10 μL glass syringe and 30g × 1/2″ needle. The hind fleshy base of the left foot of the same animal was injected with 10 μL of sterile saline solution, as a control (Brown et al., 2011; Assis et al., 2015). The thickness of the hind fleshy base of the right and left feet of each animal was measured with a thickness gauge (Digimess, accuracy 0.01 mm) before injection. Measurements were repeated 12 hour post-injection for all individuals injected. After these measurements, a sub-sample of the individuals injected were euthanized (N = 18). Thickness measurements were repeated for the remaining individuals (N = 15) at 24-hour after injection, and these were euthanized thereafter. At each measurement time, each foot was measured three times and the average of them was used as the thickness value. The swelling in response to PHA and saline solutions was calculated from the proportional change in foot thickness before and 12–24 h after injection. At the end of this procedure, the animals were euthanatized in a 0.2% Benzocaine solution. Once euthanized, the snout-vent length (SVL, 0.01 mm) of each individual was measured and they were weighed (0.001 g). Animals were also dissected and the testes were blotted dry and weighed (0.0001 g). Animals were euthanized at different times post-PHA injection for further tissue histological analyses (not included in this study). 2.7. Hormonal assay Steroids were initially extracted with ether according to Assis et al. (2015) and Madelaire and Gomes (2016). Briefly, 3 mL of ethyl ether were added to the plasma samples, vortexed for 30 s and centrifuged at 4 °C for 9 min at 1800 rpm. The tubes were then kept at −80 °C for 7 min. The liquid phase was transferred to new tubes and kept in laminar flow hood at room temperature (20 ± 2 °C), until all of the ether had evaporated (approximately 24 h). The samples were resuspended in EIA buffer and CORT was assayed using EIA kits (number 500655, Cayman Chemical), according to the manufacturer's instructions. Dilution rates for this species (1:30) were based on the concentration of pooled samples that resulted in 50% binding on the parallelism curve (see Fig. 1). For the parallelism curve, we performed serial dilutions of pooled plasma and the respective corticosterone standard curve (neat, 1:2, 1:4, 1:18, and subsequently).

Fig. 1. Parallelism curve. Binding displacement curves of serially diluted Hypsiboas albopunctatus pooled plasma at baseline conditions against the corticosterone standard used in the corticosterone enzyme-immunoassay. The y-axis shows the % Hormone Bound/Total Binding measured at 412 nm. The 50% binding point is denoted using a dashed line, which determined dilution factors for the extracted plasma samples (dilution factor for each sample has been provided in parenthesis).

By testing 15 duplicates on each plate, we estimated intra-assay mean coefficients of variation to be 5.1%. The inter-assay variation, estimated using the average of four intermediate values from the standard curve (as recommended by the kit instructions), was 12.9%. Sensitivity of the assays, calculated as 80% B/B0 of curve value, was 39 ± 4 pg/mL. 2.8. Statistical analysis Descriptive statistics were performed considering all data available for each variable (N = 35), and a Shapiro-Wilk test was used to assess normality of the data. All variables showed absence of normality and were transformed to log10 to fit the prerequisites of parametric tests. Pearson correlations were used to investigate possible correlations between morphological and physiological variables with body mass and body size (SVL). Morphological and physiological variables were not corrected for body measures for the following analyses, since none of them are correlated to body length (P ≥ 0.345) or body mass (P ≥ 0.131). A mixed repeated measures ANOVA was used to investigate differences in feet thickness between 0, 12 and 24 h, as well to compare the relative swelling in response to PHA and saline (control) at 12 and 24 h, using hour and treatment (PHA and saline) as factors and relative swelling as the dependent variable. To perform the following analyses (PCA and Path analysis), only individuals that had the complete set of data for all variables included in the analyses were considered (N of excluded individuals = 9). Individuals with calling rates equal to zero (N = 6) were also excluded, given that non-calling amphibians could present unpredictable CORT when compared to the calling ones (Leary, 2009; Leary and Harris, 2013). To verify the level of association between different variables, we performed a principal component analysis (PCA) with Varimax rotation (with Kaiser Normalization). The following variables were included: 1) Day (where day 1 corresponds to the first and 32 to the last day of observation and collection; 2) Chorus size (values indicate the number of males calling in the chorus close to each focal individual during observation); 3) Hour (time of beginning of observation in minutes); 4) Temperature (mean temperature of activity for each individual); 5) Calling rate (calls per minute); 6) CORT (corticosterone plasma levels); 7) NLR (neutrophil/lymphocyte ratio); 8) EOS (eosinophil percentage); 9) BI (body condition index - calculated as the residuals from the regression of body mass as a function of snout-vent length); 10) Testes mass. The maximum swelling response to the PHA challenge occurred 12 hours after injection. Accordingly, we performed Pearson correlation tests between the PCA scores and the swelling response after 12 h of PHA injection to understand possible patterns of association between individual variation in immune response and the

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variables associated to calling behavior, CORT, and other proxies of stress response. To better understand the relationships among the variables studied, we also performed a path analysis. Path analysis is an extension of multiple linear regression that explicitly tests a priori hypotheses describing the magnitude of causal relationships among correlational data. It has long been regarded as an appropriate method for investigating the relationships between individual traits, performance, and fitness (Arnold, 1983). In this case, we tested the relationships between environmental (day, hour, temperature, and chorus size), behavioral (call rate), physiological (CORT, NLR, EOS, PHA response), and morphological (BI and testes mass) variables. The hypothesized relationships are depicted in Fig. 2 and are all supported by evidence from the literature, as follows. Calling activity can be positively correlated with temperature (Navas and Bevier, 2001), tends to increase with number of males in the chorus (Wells, 1988), and may change with time of the day (Bastos and Haddad, 1996; Toledo and Haddad, 2005). Additionally, each of the abiotic and biotic variables may change with day of observation. Calling rate in turn is expected to influence corticosterone levels because physical activity increases glucocorticoid plasma levels in different vertebrates (Tharp, 1975; Malisch et al., 2007), and anuran calling activity is positively correlated with CORT in both intra and interspecific comparisons (Leary et al., 2006b; Assis et al., 2012; Leary and Harris, 2013). Given that a circadian rhythm has been shown in several species for circulating glucocorticoid levels, we also expect hour influences CORT (Dupont et al., 1979; Landys et al., 2006). High circulating levels of glucocorticoids can be also associated with decreasing immune cell adherence to the endothelium (Cavalcanti et al., 2006), immunosuppression (Riccardi et al., 2002), higher NLR and higher EOS counts in circulation (Davis et al., 2008), reduced reproductive behavior and output (Leary et al., 2006a) and reduced physical condition (Janin et al., 2011). We know that CORT is not the only hormone that could mediate a possible trade-off between calling rate and immunocompetence in anurans. Androgens, for example, could mediate this putative relationship (Folstad and Karter, 1992; Arch and Narins, 2009; Desprat et al., 2015), but we did not measure androgen plasma levels in our study. Moreover,

stress response is very complex, and several physiological mediators are activated in addition to glucocorticoids (Sapolsky, 2002). Therefore, the way we had to test the alternative hypothesis that there is a trade-off between calling rates and immunocompetence not mediated by CORT, was to include direct predictions linking calling rate to the variables associated with reproductive output, physical state, and immunological state. Running the path analysis allowed us to determine which of these predicted causal relationships are supported by the data collected in this study. Additionally, anuran vocal behavior is androgen-dependent (Arch and Narins, 2009). Anuran males display advertisement calls, show larger testes and higher androgen plasma levels during reproductive season (Rastogi et al., 1986; Huang et al., 1997; Rastogi et al., 2011), with androgen plasma levels positively correlated to calling behavior and testes mass in some anuran species at intra-specific (Huang et al., 1996; Huang et al., 1997) and inter-specific levels (Emerson and Hess, 2001). Considering that testes mass might modulate calling rate through androgen secretion, and reproductive output might be mediated by other stress mediators than CORT, we included a bidirectional prediction between testes mass and calling rate in this analysis. We performed descriptive statistics, t-tests and the PCA in SPSS 13.0 for Windows, and path analysis in R3.0.2 (R Development Core Team, 2013) using the package lavaan (Rosseel, 2012). 3. Results 3.1. Behavioral observations and description of general results Choruses occurred from 20 h to 24 h. Descriptive statistics of environmental temperature, behavioral, morphological, and physiological traits of H. albopunctatus are shown in Table 1. 3.2. Swelling response to phytohemagglutinin (PHA) injection A change in the thickness of the hind foot was observed after PHA injection in interaction with time (hour ∗ treatment: F2,52 = 82.522,

Fig. 2. Path diagram. The path analysis tested a priori hypotheses regarding relationships between biotic and abiotic variables. The arrows point from the independent variables to the response variables and each arrow is associated with a regression coefficient (b) and a P-value (p). The different types of dashed lines represent the regression equations incorporated into the path analysis model. Abbreviations are as follows: DAY = day of behavioral observation; CHORUS = number of calling males in the chorus; TEMP = temperature at time of observation; HOUR = time of observation; CALL RATE = rate of calling during 30 minute focal observation time; CORT = plasma corticosterone levels; NLR = neutrophil/lymphocyte ratio; EOS = eosinophil count; BI = body condition index; TESTES = testes mass, and PHA = swelling of experimental foot at 12 h. (N = 20).

S.C.M. Titon et al. / Comparative Biochemistry and Physiology, Part A 201 (2016) 53–60 Table 1 Descriptive statistics with the number of individuals and the minimum, maximum, mean and standard deviation for the abiotic, behavioral, physiological and morphological parameters of Hypsiboas albopunctatus. The N used in here is different for each variable; since we used all data we collected to describe variation in the studied species. Parameter

N

Minimum

Maximum

Mean ± SD

Calling rate (per minute) Temperature (°C) Body Mass (g) Testes mass (g) SVL (mm)* Corticosterone (ng/mL) Swelling in 12 h (mm)* Hematocrit (%) Leucocyte profile (%) Neutrophil Lymphocyte Eosinophil Basophil Monocyte

35 30 34 33 34 30 33 33

0 13.5 4.244 0.007 46.11 3.65 0.08 11

37.4 22.0 8.995 0.015 59.19 33.24 0.34 43

9.1 ± 10.3 18.1 ± 2.5 6.878 ± 1.243 0.011 ± 0.003 52.86 ± 3.20 12.17 ± 6.53 0.19 ± 0.07 25 ± 8

35 35 35 35 35

1 23 0 0 1

39 89 50 45 15

12 ± 10 63 ± 14 7 ± 10 10 ± 10 8±4

SVL: snout-vent-length; Swelling in 12 h: swelling of the experimental foot 12 h after PHA injection.

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Table 2 Components retained of principal components analysis (PCA) including abiotic, behavioral, physiological and morphological variables of Hypsiboas albopunctatus. The N used in this analysis is 20, since we excluded some individuals as we explained in Statistical Analysis. Variables

Components 1

Day Chorus size Hour Calling rate CORT NLR Eosinophil Body condition index Testes mass Initial eigenvalues % of variance

0.039 0.299 −0.161 0.743 0.278 −0.095 −0.164 0.488 0.902 2.402 26.69

2 0.472 0.790 −0.266 0.018 −0.321 0.143 0.839 0.061 0.041 1.688 18.75

3 0.574 0.235 −0.221 0.112 0.119 −0.897 −0.276 0.395 0.030 1.361 15.13

4 −0.310 0.134 0.663 −0.346 −0.565 0.049 −0.092 0.637 0.025 1.042 11.58

The variables with the highest contribution for each component are highlighted in bold. The total variance explained by these four components is 72.15%. The body index was calculated as a residual of the regression of body mass as a function of snout-vent length.

P b 0.001). When compared to hind foot thickness measured before injection, a swelling occurred in response to PHA injection at both 12 h (P b 0.001) and 24 h (P b 0.001), with greater swelling observed after 12 h (P b 0.001; Fig. 3), and no swelling observed in response to saline injection after 12 and 24 h (P = 0.750; Fig. 3).

The path analysis showed that testes mass predicts calling rate (b = 10.151; P = 0.004) and calling rate predicts PHA response (b = − 0.152; P = 0.004), but none of the environmental variables predict calling rate (Fig. 2). CORT does not predict any variables associated with stress response (Fig. 2).

3.3. Relationships among behavioral, physiological, morphological and environmental variables

4. Discussion

The principal components analysis (PCA) resulted in four components with eigenvalues higher than 1.0 (Table 2). The first component explained 26.69% of the variance, showing a positive relationship between calling rate and the testes mass. The second component explained 18.75% of the variance and showed a positive association between chorus size and EOS. The third component explained 15.13% of the variance and showed a negative correlation between day and NLR. The fourth component explained 11.58% of the variation and showed a positive correlation among hour and BI and negative association between these variables and CORT. All these components explained 72.15% of the total variance. The swelling response to PHA showed a negative correlation with component 1 (ρ = − 0.554; P = 0.011) (Fig. 4).

Fig. 3. Relative swelling response to PHA challenge. Relative swelling of the control foot (10 μL saline) and PHA foot (10 μL PHA solution – 20 mg/mL) of male individuals of the species Hypsiboas albopunctatus for 12 and 24 h. Points and bars represent the mean and standard deviation, respectively. * represents statistically significant difference between treatments (Saline and PHA) in the same time and ** between time (12 and 24 h) in the same treatment (N = 15).

We predicted that calling activity would be associated with immunosuppression in H. albopunctatus. Accordingly, our results showed that calling rate predicts variation in PHA response, with males that call at higher rates displaying lower inflammatory response. The PHA challenge results in migration of neutrophils, lymphocytes and macrophages to the site of the injection, where these cells are responsible for forming an inflammatory site and repairing the injured tissue (Martin et al., 2005; Brown et al., 2011; Clulow et al., 2015). Therefore, males of H. albopunctatus that call at higher rates seem to be characterized by lower cellular innate immune response. Calling behavior is subject to directional inter-sexual selection in anurans (Wells, 2007a) and is dependent on androgens (Arch and Narins, 2009). According to the immunocompetence handicap hypothesis (IHH), if androgens induce the expression of secondary sexual characters and immunosuppression in a graded manner, then androgen-dependent sexual traits would

Fig. 4. Correlation between the Component 1 from PCA and the PHA challenge after 12 h. Parametric correlation (Pearson) between the PHA swelling response after 12 h and the Component 1 from PCA (positive correlation between calling rate and testes mass) of Hypsiboas albopunctatus males (N = 20).

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represent honest signals of male quality (Folstad and Karter, 1992). More recently, attention has been drawn to the potential modulatory impact of glucocorticoids in this context, in addition to androgens. This is due to the fact that glucocorticoids play an important role in energetic substrate mobilization during energetically taxing activities, such as the expression of male sexual signals (Sapolsky et al., 2000; Hau et al., 2010; Carr, 2011), and show potent immunomodulatory effects (Wiegers and Reul, 1998; Riccardi et al., 2002; Cavalcanti et al., 2006). Therefore, a “stress-linked” version of the IHH model proposes that elevated glucocorticoid secretion associated with elaborate secondary sexual character expression masks the graded effect of androgens on phenotype. In this way, glucocorticoids, not androgens, would show prominent immunosuppression and maintain the honesty of male sexual signals (Bortolotti et al., 2009; Leary and Knapp, 2014). Although a positive correlation between calling effort and CORT has been found for a handful of anuran species (Emerson and Hess, 2001; Leary et al., 2004; Leary et al., 2006a; Leary et al., 2008; Assis et al., 2012), the absence of a correlation between these variables has been previously detected in a prolonged-breeding anuran (Leary et al., 2015). Our results also show that CORT is not associated with variation in calling rates or the PHA response for H. albopunctatus, suggesting that CORT does not play a direct role in modulating the negative association between calling rates and cellular immune response. However, we only measured calling rates instead of calling effort, so it is likely that our analysis overlooked some important relationships among immune response, CORT and the sexual displays of H. albopunctatus. The study of variation in calling effort and immunocompetence in response to experimental manipulation of CORT would be an important next step to understand the role of CORT in this system. In H. albopunctatus, males with largest testes called at higher rates. During the reproductive season, males from different anuran species show increased testes mass and androgen plasma levels (Rastogi et al., 2011). Additionally, some studies show that testes mass and androgen plasma levels are positively correlated in some anuran species at interspecific (Emerson and Hess, 2001) and intra-specific levels (Huang et al., 1996; Huang et al., 1997). Androgens can mediate testes size by stimulating the development of Leydig, peritubular and Sertoli cells, and these glands are responsible for the production of testosterone itself (Stocco and McPhaul, 2006). Besides that, the presence of circulating androgens is necessary for initiation and maintenance of calling behavior (Wilczynski et al., 2005), with a positive correlation between calling behavior and androgen plasma levels being observed in some anuran species (Leary et al., 2004; Leary et al., 2005; Assis et al., 2012). In our study, testes mass is positively correlated to calling rate, and both are negatively associated with the PHA response. The association between calling rates, testes mass and intensity of inflammatory response observed in H. albopunctatus might be mediated, at least in part, by androgens. Immunosuppressive effects of androgens have been demonstrated for mammals, including reduction of cellular immune response (Inman, 1978; Weinstein et al., 1984; Grossman, 1984), and a recent study reported that anuran testosterone-supplemented males with high body mass presented an immunoenhancement (Desprat et al., 2015). Therefore, it is plausible that androgen might be an important modulator of the relationships found between calling behavior and immune response (Folstad and Karter, 1992; Mougeout et al., 2004). Additionally, androgens and corticosterone might interact in this modulatory association between behavior and immunocompetence (Leary and Knapp, 2014). Additional studies are necessary to test properly these hypotheses. We also found a positive association between eosinophil counts and the number of males comprising choruses of H. albopunctatus. Eosinophils are innate immune cells associated with defense against helminth parasites (reviewed in Klion and Nutman, 2003; Moreau and Chauvin, 2010), and H. albopunctatus can be infected by several helminthes (Holmes et al., 2008). This association might be due to the fact that our observations were conducted during months of high precipitation. Rainfall increases both host density around bodies of water and the

number of infective larvae (Rocha et al., 2011; Anderson, 2000), probably favoring helminth infection and reflecting in the observed increased eosinophil counts in the hosts. A more refined temporal monitoring of the natural variation of chorus density, eosinophil counts and helminth intensity, along with an experimental approach, might clarify these relationships. CORT and NLR were not associated in males of H. albopunctatus calling in the field. This association would be expected, given that glucocorticoids modulate neutrophil and lymphocyte trafficking between body compartments (Cavalcanti et al., 2006; Davis et al., 2008). Accordingly, a positive correlation between CORT and the NLR has been found for several vertebrates (reviewed by Davis et al., 2008). However, exceptions to this rule have been described for birds (Vleck et al., 2000; Ilmonen et al., 2003) and toads (Assis et al., 2015). It is possible that differences in temporal patterns of response of CORT and NLR to stressors obscure patterns of correlation in the field (reviewed in: Davis et al., 2008; Martin, 2009; Seddon and Klukowski, 2012). The last PCA component showed that individuals calling early at night showed higher CORT. Moreover, individuals with higher CORT were characterized by lower BI. The peak of CORT at the first hours of the nightly chorus activity is consistent with the circadian rhythm of CORT described for free-living toads (Jessop et al., 2014), and corroborates results from studies conducted with other anurans calling in the field (Leary et al., 2008; Leary et al., 2015). A negative relationship between CORT and BI suggests that CORT secretion could be associated to breaking down of energy reserves, required for intense calling activity maintenance (Emerson, 2001; Leary et al., 2004, 2015; Leary and Harris, 2013; Madelaire and Gomes, 2016). Experimental studies might be important to highlight the relation between the cost of calling behavior and CORT. We concluded that calling activity shows a cost in terms of cellular immune response in H. albopunctatus. This is the first time that a negative association between intensity of calling behavior (calling rate) and a direct measure of immunocompetence has been shown for anurans. In spite of the negative correlation between calling rate and PHA response, this relationship seems not to be mediated by CORT. Additionally, males of H. albopunctatus with lower BI showed high CORT, evidencing a relationship between high catabolic states and glucocorticoid secretion. Further studies, incorporating measures of androgen plasma levels along with CORT, experimental manipulation of hormone plasma levels, and the incorporation of different ways to access immune function (phagocytosis assays, cytokine production, among others) might help to shed light on the interactions between calling performance, steroid plasma levels, and immunity in the context of anuran reproduction. Acknowledgements We would like to thank people who assisted us during fieldwork. We would also like to thank Sandro D. Coelho for allowing us to collect on his property (Pesqueiro do Sandro), Alexandre D. de Jesus for helping us during data collection, the lab technician Eduardo B. Fernandes for his help with some experiments and Dra. Cristina O. M. S. Gomes (FMVZ/USP) and Dra. Eliana R. Matushima (FMVZ/USP) for methodological discussions. This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through a grant (JP-2006/54699-1) and (2010/52290-4) to F.R.G., and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior through a master degree fellowship to S.C.M.T. References Ahmed, S.A., Dauphinée, M.J., Talal, N., 1985. Effects of short-term administration of sex hormones on normal and autoimmune mice. J. Immunol. 134, 204–210. Anderson, R.C., 2000. Nematodes Parasites of Vertebrates: Their Development and Transmission. CABI Publishing, New York, pp. 245–347. Arch, V.S., Narins, P.M., 2009. Sexual hearing: the influence of sex hormones on acoustic communication in frogs. Hear. Res. 252, 15–20.

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