Regulation of the HPA axis is related to song complexity and measures of phenotypic quality in song sparrows

Hormones and Behavior 61 (2012) 652–659

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Regulation of the HPA axis is related to song complexity and measures of phenotypic quality in song sparrows Kim L. Schmidt a, b,⁎, Ainsley A. Furlonger a, b, Janet M. Lapierre a, Elizabeth A. MacDougall-Shackleton a, Scott A. MacDougall-Shackleton a, b, c a b c

Department of Biology, University of Western Ontario, London, Ontario, Canada Advanced Facility for Avian Research, University of Western Ontario, London, Ontario, Canada Department of Psychology, University of Western Ontario, London, Ontario, Canada

a r t i c l e

i n f o

Article history: Received 23 December 2011 Revised 29 February 2012 Accepted 29 February 2012 Available online 6 March 2012 Keywords: ACTH Corticosterone Dexamethasone Heterophil:lymphocyte ratio HPA axis Leukocyte profile Sexual selection Songbird Song complexity Stress

a b s t r a c t Regulation of the hypothalamic–pituitary–adrenal (HPA) axis is a key component of the vertebrate stress response. Prior studies have found that variation in HPA responses were correlated to measures of fitness and physiological condition. In addition, sexually-selected traits have also been found to correlate to measures of condition. The proximate mechanisms responsible for such covariation between sexually selected traits and measures of quality are unclear, but could involve variation in HPA regulation. We investigated whether HPA activity is related to song complexity, body size/condition and leukocyte profiles in wild male song sparrows (Melospiza melodia). We characterized three aspects of HPA activity: 1) response to restraint stress; 2) negative feedback, assessed by the ability of exogenous dexamethasone to suppress corticosterone levels; and 3) adrenal sensitivity to exogenous adrenocorticotropic hormone (ACTH). Birds with lower responses to restraint stress had more complex song and more heterophils and higher heterophil to lymphocyte (H:L) ratios. Birds with more effective negative feedback were larger and had fewer heterophils and lower H:L ratios, suggesting lower levels of physiological stress or infection. We observed no relationship between adrenal sensitivity to exogenous ACTH and any of the factors. These findings illustrate important relationships between HPA activity, song complexity, and morphological and physiological traits. Song complexity may thus provide receivers with information about the ability of the singer to cope with stressors. © 2012 Elsevier Inc. All rights reserved.

Introduction The vertebrate stress response includes up-regulation of the hypothalamic–pituitary–adrenal (HPA) axis. This involves increased release of corticotropin-releasing hormone (CRH) from the hypothalamus, followed by adrenocorticotropic hormone (ACTH) from the anterior pituitary, and culminates in increased glucocorticoid synthesis from the adrenal cortex (Breuner et al., 2008). In the short term, this may be adaptive because glucocorticoids mobilize energy reserves that can help the animal cope with the stressor. However, long-term increases in glucocorticoid levels can have detrimental effects, such as suppression of immune function and inhibition of reproduction (Sapolsky et al., 2000). Therefore, another important facet of the HPA response is its rapid regulation and reduction of glucocorticoid levels via negative feedback (Romero, 2004). In free-living vertebrate animals, variation in HPA activity is related to measures of fitness and physiological condition (Bonier et al., 2009; Breuner et al., 2008). For example, the magnitude of the stress ⁎ Corresponding author at: Dept. of Biology, Advanced Facility for Avian Research, University of Western Ontario, London, ON, Canada N6A 5B8. E-mail address: [email protected] (K.L. Schmidt). 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.02.027

response is negatively related to survival in European white storks (Ciconia ciconia; Blas et al., 2007) and song sparrows (Melospiza melodia; MacDougall-Shackleton et al., 2009b). In marine iguanas (Amblyrhynchus cristatus), baseline corticosterone (CORT) levels were inversely related to body condition during an El Niño period (Romero and Wikelski, 2001). In the same species, the ability of the synthetic glucocorticoid dexamethasone to suppress endogenous CORT levels, a widely used index of negative feedback (Watson et al., 2006), predicts survival through an El Niño event (Romero and Wikelski, 2010). Therefore, optimal HPA activity may include both low baseline glucocorticoid levels and effective regulation of the stress response by preventing exaggerated increases in CORT in response to stressors and rapidly decreasing CORT levels by negative feedback once the stressor subsides (de Kloet et al., 2005; Romero, 2004). Sexually selected traits, such as song complexity and plumage coloration, frequently covary with other physiological and behavioral traits and may therefore indicate phenotypic quality to potential mates or rivals (Hill, 2011; Kipper et al., 2006; Pfaff et al., 2007). The mechanistic link mediating this relationship is largely unknown, but could involve individual variation in HPA activity (Husak and Moore, 2008; Roulin et al., 2008) as such activity has been shown to

K.L. Schmidt et al. / Hormones and Behavior 61 (2012) 652–659

covary with sexually selected traits (e.g., MacDougall-Shackleton et al., 2009b). Individuals with lower glucocorticoid levels or more effective HPA regulation may be better able to invest both in the sexually selected trait and also in other physiological and behavioral traits that affect fitness. For example, in female barn owls (Tyto alba) melaninbased coloration is a sexually selected trait (Roulin, 1999) that predicts female fitness including survival rates (Roulin et al., 2003) and offspring quality (Roulin and Altwegg, 2007). Owl nestlings that show lower increases in CORT in response to stress have darker melanin-based coloration (Almasi et al., 2010) and nestlings implanted with CORT develop lighter feathers (Roulin et al., 2008). Song complexity may be a sexually selected trait in some species of songbirds (MacDougall-Shackleton, 1997) although not necessarily all (Byers and Kroodsma, 2009; but see Collins et al., 2011). In song sparrows, song learning is closed-ended such that birds do not alter their song repertoires during adulthood (Nordby et al., 2002) and considerable evidence suggests that song complexity may influence female mating preferences and/or male–male competition. For example, captive female song sparrows perform more copulation solicitation displays to more complex song bouts (Searcy and Marler, 1981). This preference might affect eventual social mate choice in the wild in some, although not necessarily all, populations of song sparrows (Reid et al., 2004; Searcy, 1984). In our study population, hand-raised female song sparrows exhibit higher responses (more copulation solicitation displays and higher activity levels) to more complex song, consistent with them having a preference for larger song repertoires (K.L. Schmidt, unpublished). Whether or not this preference translates to active mate choice for males with larger repertoires in the field requires further study. Moreover, song complexity is related to reproductive success and measures of condition (Pfaff et al., 2007; Reid et al., 2005b) in some populations of song sparrows, although not in others (Beecher et al., 2000). Male–male competition may also be an important factor driving the evolution of song complexity (Beecher et al., 2000). Having multiple song types might allow males to match the songs of their neighbors (“song sharing”). In turn, song sharing may predict territory tenure (Beecher et al., 2000, although see Hughes et al., 2007) and may facilitate territory defense by signaling aggression (Vehrencamp, 2001). On the whole, there is good evidence that song repertoire size has likely evolved via intra- and/or intersexual selection in this species. Previous studies on song sparrows have shown song complexity to be negatively related to the magnitude of the stress response (MacDougall-Shackleton et al., 2009b) and positively related to immune function (Reid et al., 2005a) and body condition (Pfaff et al., 2007). In addition, CORT treatment early in life decreased adult song complexity in zebra finches (Taeniopygia guttata; Spencer et al., 2003). If HPA activity mediates the link between some sexually selected traits and phenotypic quality, then measures of HPA activity may be correlated both with the sexually selected trait and indices of phenotypic quality. Here we investigate whether individual variation in HPA activity of free-living male song sparrows is related to (1) individual variation in song complexity and (2) measures of phenotypic quality, including body size, body condition, and leukocyte profiles. We use the term “phenotypic quality” to mean behavioral and physiological traits that are likely to affect an individual's fitness. Most studies conducted on free-living animals assess HPA activity by measuring glucocorticoid levels at the time of capture (baseline) and after exposure to a standardized stressor (often restraint). However, it is clear that other aspects of HPA activity, such as negative feedback, may be equally important (Romero and Wikelski, 2010). In addition, variation in stress-induced CORT levels can be due to variation in release of CRH, ACTH, or CORT, as well as to variation in the degree to which individuals perceive the stressor as a threat. An alternative approach is to administer a standardized dose of ACTH, in order to isolate variation in the stress response that is specifically due to

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variation in the sensitivity of the adrenal cortex to ACTH. Response to exogenous ACTH may also provide a more accurate measure of maximum glucocorticoid production than response to restraint stress (Wada et al., 2007). Therefore, we used a protocol that allowed us to measure three aspects of HPA activity in the same individual: 1) the response to a restraint stressor, 2) efficacy of negative feedback (inferred by administering dexamethasone), and 3) sensitivity of the adrenal glands to exogenous ACTH. This comprehensive characterization of HPA activity and regulation allowed us to determine which specific components of HPA activity may be related to song complexity and to measures of condition and quality. Materials and methods Field site and study species We studied a breeding population of song sparrows near Newboro, Ontario, Canada (44°38′N, 76°20′W) from April 1 to May 16th 2010. Territorial male song sparrows (n = 32) were caught on their territories using mistnets and conspecific song playback. Birds were captured between 7 AM–3 PM. We did not use a smaller time frame for capture because our sampling protocol was 2 h long and we wanted to capture several birds a day so that individuals were sampled during similar breeding stages. However, in white-crowned sparrows (Zonotrichia leucophrys) basal and restraint-induced CORT levels do not vary significantly throughout a similar time period (Breuner et al., 1999). Birds were given unique combinations of colored leg bands for future field identification. All animal procedures followed guidelines set by the University of Western Ontario and the Canadian Council on Animal Care, and were approved by our institutional Animal Use Subcommittee (protocol 2007-089). HPA axis characterization Injections and blood collection All injections were given intramuscularly in the pectoralis muscle. The doses and time points used for the dexamethasone (Sandoz Canada Inc, 2301) and ACTH (Sigma Aldrich, A6303) injections were determined during pilot studies using captive song sparrows (results not shown). These pilot studies revealed that CORT levels were lowest 60 min post-injection of dexamethasone, and that a high dose of dexamethasone (1 mg/kg) was more effective than a low dose (0.5 mg/kg). Therefore, we used the high dose for injections in the field study. Similar doses were effective at suppressing corticosterone in wild-caught chukar (Alectoris chukar; Dickens et al., 2009). We used porcine ACTH dissolved in lactated Ringer's solution for ACTH injections. In pilot studies both a low (25 IU/kg) and a high (100 IU/ kg) dose of ACTH induced similar increases in CORT and levels peaked 30 min post-injection. Therefore, we used the low dose for injections in the field study. Similar doses were effective at increasing corticosterone in European starlings (Sturnus vulgaris; Rich and Romero, 2005) and chukar (Dickens et al., 2009). Blood samples were collected by brachial venipuncture with a 26-gauge needle and collected into heparinized micro-hematocrit tubes. Blood samples were kept on ice in the field and processed within 8 h of collection. Blood was centrifuged at 12,700 g for 10 min. Plasma was collected and stored at −20 °C until analysis. Stress series The stress series was conducted on song sparrows at our longterm study site to provide 3 measures of HPA activity (Fig. 1): 1) response to a restraint stressor, 2) efficacy of negative feedback by determining the ability of dexamethasone to suppress CORT, and 3) adrenal sensitivity to exogenous ACTH. For the stress series, we first collected an initial blood sample within 3 min of the bird entering the mistnet for baseline CORT measurement. Next, we placed

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Stress Series DEX injection

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ACTH injection

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125

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C A

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(Searcy et al., 1995), for example by adding or omitting a syllable; these slight variants were not counted as new song types. We then identified each unique syllable in all song types for a bird to determine the number of syllables in their repertoire. We quantified both song type number and syllable number because although these two measures are correlated, each is associated with unique aspects of vocal behavior in our study population (MacDougall-Shackleton et al., 2009a). In the current study, the number of unique song types in a bird's repertoire was positively correlated to the number of unique syllables (r32 = 0.51, p = 0.002).

Restraint

25

Body measurements

0 0

30

60

90

120

Time (min) Fig. 1. The stress series used to characterize hypothalamic–pituitary–adrenal (HPA) function in free-living song sparrows. The dotted line indicates the time of exposure to restraint stress and the arrows indicate the time of injections. Data are means ± SEMs. Time points not sharing a letter are significantly different for p b 0.05.

birds into a cloth bag and collected a second blood sample 30 min post-restraint. Immediately following this blood sample, birds were injected with dexamethasone then placed into a covered cage with food and water. We collected a third blood sample 60 min after dexamethasone injection, then birds were immediately injected with ACTH and returned to the cage. We collected a fourth and final blood sample 30 min following ACTH injection. The total procedure took 2 h (Fig. 1), and we fed birds diluted apple juice after each blood sample to prevent dehydration. A maximum of 30 μL of blood was collected for each blood sample, for a total of ~120 μL. All 32 birds were re-sighted at or near the site of capture within a few days following sampling, and exhibited normal behavior. Corticosterone immunoassays CORT was quantified in unextracted plasma using a radioimmunoassay (MP Biomedicals, 07-120103) that has been previously validated in song sparrows (Newman et al., 2008). Plasma was diluted 1:50 with steroid diluent (5 μL plasma + 245 μL diluent). Samples were analyzed in duplicate by adding 50 μL of the plasma/diluent mixture to each tube (1 μL plasma + 49 μL diluent). The lower limit of detectability ranged from 1.8 to 2.6 ng/mL. Inter-assay variation was 5.5% for a low control and 4.1% for a high control. Intra-assay variation was 9.4% for the low control and 3.9% for the high control. Song repertoires In our study population, song sparrow song repertoires usually consist of 5–12 distinct song types comprised of a total of 20–50 distinct syllables (MacDougall-Shackleton et al., 2009a). We recorded each subject's song repertoire using Marantz Professional Solid State PMD 671 recorders and Telinga Twin Science parabolic microphones (Uppsala, Sweden). We considered a repertoire to be completely recorded when either 300 consecutive or 450 nonconsecutive songs had been recorded (Pfaff et al., 2007). Sound spectrograms were generated using Syrinx software (version 2.6h; J Burt; www.syrinxpc. com) and song types and syllables were identified visually. We counted the total number of song types to determine song repertoire size following Pfaff et al. (2007) and the total number of syllables to determine syllable repertoire size following Stewart and MacDougall-Shackleton (2008). Song sparrows sing with eventual variety, meaning that they sing one song type several times before switching to a new song type, so we identified each unique song type as the birds switched to a new song in their repertoires. Song sparrows often produce slight variations of a given song type

We measured tarsus length and wing chord to the nearest 0.1 mm using dial calipers, and body mass to the nearest 0.25 g using a spring scale. Body fat in the furcular depression was scored using a 5-point scale (Helms and Drury, 1960). Morphological measures were included as indices of quality because in other bird species, body size is positively related to survival through severe weather events (Brown and Brown, 1998) and to aggressive interactions that are important for territory defense (Hagelin, 2001; Searcy, 1979) and therefore may have important fitness consequences. However, other studies have failed to find directional selection on male body size in songbirds (Schluter and Smith, 1986). Leukocyte counts Leukocyte profiles have been widely used as an indicator of physiological stress and condition across vertebrate taxa. Specifically, stress typically increases heterophil levels and H:L ratios, and decreases lymphocyte levels (Davis et al., 2008; Harmon, 1998). Leukocyte profiles were characterized by preparing thin-film blood smears in the field using blood from baseline (within 3 min of capture) samples. Smears were fixed in 100% methanol and stored at room temperature until analysis. Wright–Giemsa stain (Leukostat; Fisher Scientific) was applied and slides were examined with a light microscope using a 100× oil immersion objective lens. For each slide, we took blood cell counts from 20 digital images of adjacent rectangular fields of view (total number of cells counted = 4589 ± 227.48, approximately 200–250/slide; Pfaff et al., 2007). The total number of red blood cells (4577 ± 226.38), lymphocytes (9.06 ± 1.49), and heterophils (2.90 ± 0.56) were determined in all 20 images. Lymphocyte and heterophil counts were measured as the ratio to red blood cells to control for variation in total cell number. The heterophil to lymphocyte (H:L) ratio was calculated for each slide. For two individuals, small volumes of the initial baseline sample precluded preparing thin-film blood smears. Statistical analyses Statistical analyses were conducted using PASW (SPSS) version 18. CORT levels collected at the 4 time points (baseline, post-restraint, post-dexamethasone, post-ACTH) were analyzed using a repeated measures ANOVA. Paired t-tests were used to compare each time point using a Bonferroni adjustment to control for multiple comparisons. We also conducted Pearson's correlations to examine relationships between the three measures of HPA activity. We used factor analysis (Principal Components) to reduce our morphological, hematological and song measures to fewer orthogonal variables because many of the variables were correlated. For the 4 morphological measures (mass, tarsus, wing, and fat score), PCA identified two principal components with eigenvalues greater than one, which together explained over 70% of the variance (Table 1). Mass and tarsus had high positive loadings onto PC1, and fat had a high positive loading onto PC2. PC1 and PC2 were thus interpreted as reflecting body size and body condition, respectively, consistent

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Table 1 Principal component analyses for song complexity, morphological measurements, and leukocyte counts. Eigenvalue Song complexity PC1

1.46

Morphological measures PC1 PC2 Leukocyte counts PC1 PC2

% Variance explained

Factor loadings

72.93

Song type number 0.85

Syllable number 0.85

1.70 1.15

42.52 28.70

Mass 0.74 0.27

Tarsus 0.89 − 0.11

Wing 0.40 0.67

1.64 1.15

54.58 38.39

Lymphocytes 0.07 0.97

Heterophils 0.96 0.15

H:L 0.74 − 0.60

Fat score − 0.11 0.90

Note: H:L = heterophil to lymphocyte ratio.

with previous findings for this population (Pfaff et al., 2007). However, wing chord also had a fairly high positive loading onto PC2, so PC2 could also be reflective of wing loading (higher ratio of fat mass to wing area). For leukocyte counts (relative number of lymphocytes, heterophils, and H:L) PCA identified two principal components with eigenvalues greater than one, which together explained over 92% of the variance (Table 1). Heterophils and H:L had high positive loadings onto PC1; lymphocytes had high positive loadings onto PC2, while H: L loaded negatively onto PC2. Thus, individuals with high immune PC1 scores had more heterophils and higher H:L, possibly indicating high levels of stress or infection. Individuals with high immune PC2 scores had more lymphocytes, but low H:L, possibly indicating a robust immune system and lower levels of stress or infection (Davis et al., 2008; Harmon, 1998). Lastly, PCA identified one principal component for song complexity that explained over 72% of the variance. Both song type number and syllable number loaded positively onto this principal component (Table 1). To test the hypothesis that HPA activity is related to morphological measures, leukocyte profiles, or song complexity, we conducted linear mixed models using restricted maximum likelihood (REML) models and unstructured variance. We used the three measures of HPA activity as the dependent variables: 1) response to restraint (30 min CORT levels–3 min CORT levels); 2) negative feedback (90 min CORT levels–30 min CORT levels); and adrenal sensitivity to ACTH (120 min CORT levels–90 min CORT levels). The response to restraint was transformed (log10) to meet the assumption of normality. The response to dexamethasone and ACTH were normally distributed and therefore statistical tests for these dependent measures were performed on untransformed data. Because each of these aspects of HPA regulation was of a priori interest, we constructed separate linear mixed models for each. For each of the three models we included the morphological, hematological and song PCA factors as covariates and then entered these into the models as main effects. There were thus 5 main effects entered into each model: body size (body PC1), body condition (body PC2), immune PC1, immune PC2, and song complexity (song PC1). Date of capture was also included as a covariate. Sample sizes vary between comparisons because two blood samples were lost in the field, and in two cases birds escaped from the cage before all body measurements were collected. Statistical significance was set at α = 0.05 and all tests were two-tailed. Results Stress series A repeated measures ANOVA indicated that CORT levels were significantly different between the four time points (F1,31 =37.72, pb 0.0001, Fig. 1). Pairwise comparisons revealed that baseline CORT levels were significantly lower than CORT levels after restraint (t30 =8.42, pb 0.0001), dexamethasone injection (t31 =5.31, pb 0.0001) and ACTH injection (t30 =7.60, pb 0.0001). CORT levels after restraint were significantly

higher than CORT levels 60 min post-dexamethasone injection (t31 =3.36, p=0.002), which is consistent with dexamethasone inducing negative feedback (Fig. 1). CORT levels after ACTH injection did not differ from post-restraint CORT levels (t30 =0.11, p=.0.91), but were significantly higher than levels after dexamethasone injection (t31 =3.30, p=0.002), indicating that ACTH was effective at increasing adrenal CORT production (Fig. 1). The response to restraint (30 min–3 min) was negatively correlated to the response to dexamethasone (90 min–30 min; r = − 0.49, p = 0.005); individuals with lower increases in CORT in response to restraint suppressed CORT less in response to dexamethasone (higher 90 min–30 min values). The response to restraint was not correlated to the response to ACTH (120 min–90 min; r = 0.003, p = 0.99). There was a trend for a negative correlation between the response to dexamethasone and the response to ACTH (r = −0.32, p = 0.08); individuals that suppressed CORT less in response to dexamethasone (higher 90 min–30 min values) tended to have smaller increases in CORT in response to ACTH (lower 120 min–90 min values). Phenotypic correlates of response to restraint (30 min–3 min) CORT increases in response to restraint were related to both song and immune measures. The response to restraint was related to song complexity (F1,19 = 5.36, p = 0.03; Fig. 2A); individuals with lower increases in CORT in response to restraint had more complex song (higher song PC1 scores). The response to restraint was also related to immune PC1 (F1,19 = 6.17, p = 0.02; Fig. 2D) although not immune PC2 (F1,19 = 2.24, p = 0.15, Fig. 2E); interestingly, individuals with larger increases in CORT in response to restraint had lower immune PC1 scores, and thus fewer heterophils and lower H:L. The response to restraint was not significantly related to body PC1, corresponding to body size (F1,19 = 0.77, p = 0.39; Fig. 2B), nor body PC2, corresponding to body condition (F1,19 = 1.18, p = 0.29; Fig. 2C). Phenotypic correlates of negative feedback (90 min–30 min) Negative feedback was not related to song complexity (F1,20 = 1.67, p = 0.21; Fig. 3A). Negative feedback was related to body size (F1,20 = 5.57, p = 0.03; Fig. 3B), but not body condition (F1,20 = 2.25, p = 0.15; Fig. 3C); birds that had greater suppression of CORT in response to dexamethasone were larger, but were not in better condition. Negative feedback was also related to immune PC1 (F1,20 = 5.96, p = 0.02; Fig. 3D), but not immune PC2 (F1,20 = 0.001, p = 0.98; Fig. 3E) scores; individuals with greater suppression of CORT in response to dexamethasone had lower immune PC1 scores, and therefore fewer heterophils and lower H:L. Phenotypic correlates of adrenal sensitivity to ACTH (120 min–90 min) Adrenal sensitivity to ACTH was not related to song complexity (F1,20 = 0.18, p = 0.68; Fig. 4A), body size (F1,20 = 2.55, p = 0.13;

K.L. Schmidt et al. / Hormones and Behavior 61 (2012) 652–659

B CORT Response to Restraint (ng/mL)

CORT Response to Restraint (ng/mL)

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Fig. 2. The relationship between the response to restraint stress (corticosterone levels post-restraint [30 min]–baseline [3 min]) and (A) song complexity (song PC1), (B) body size (body PC1 score), (C) body condition (body PC2 score), (D) immune PC1, and (E) immune PC2. Details of the variables that were analyzed in the principal component analyses are in Table 1. The change in CORT (Y-axis) was log-transformed to normalize the data. CORT = corticosterone.

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Fig. 3. The relationship between the efficacy of negative feedback (corticosterone levels post-dexamethasone [90 min]–post-restraint [30 min]) and (A) song complexity (song PC1), (B) body size (body PC1 score), (C) body condition (body PC2 score), (D) immune PC1, and (E) immune PC2. Details of the variables that were analyzed in the principal component analyses are in Table 1. CORT = corticosterone, DEX = dexamethasone.

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Song Complexity

250 200 150 100 50 0 -50 -100 -150 -3

-2

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C Body Size

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D CORT Response to ACTH (ng/mL)

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CORT Response to ACTH (ng/mL)

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657

Immune PC 2

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Fig. 4. The relationship between adrenal sensitivity to ACTH (corticosterone levels post-ACTH [120 min]–post-dexamethasone [90 min]) and (A) song complexity (song PC1), (B) body size (body PC1 score), (C) body condition (body PC2 score), (D) immune PC1, and (E) immune PC2. Details of the variables that were analyzed in the principal component analyses are in Table 1. ACTH = adrenocorticotropic hormone, CORT = corticosterone.

Fig. 4B), body condition (F1,20 = 2.50, p = 0.13; Fig. 4C), immune PC1scores (F1,20 = 0.08, p = 0.78; Fig. 4D), or immune PC2 scores (F1,20 = 0.19, p = 0.67; Fig. 4E). Discussion The three measures of HPA regulation examined here were differentially related to song complexity, body size, and leukocyte profiles. First, birds with lower increases in CORT in response to restraint stress had more complex song and more heterophils and higher H:L ratios. Second, birds with stronger negative feedback, as indicated by the ability to suppress CORT in response to dexamethasone, were larger and had fewer heterophils and lower H:L ratios. Finally, adrenal sensitivity to ACTH was not significantly related to song complexity, morphological measures, or leukocyte profiles. The finding that HPA regulation is correlated to both a trait that might be sexually selected (song complexity) and possible indices of phenotypic quality (body size and leukocyte profiles) supports the hypothesis that HPA activity may mediate the covariation between sexually selected signals and phenotypic quality (Husak and Moore, 2008; Roulin et al., 2008).

induced by restraint (Wada et al., 2007). Interestingly, despite the fact that ACTH and restraint stress increased CORT to similar levels in song sparrows (Fig. 1), these two measures were not correlated. Moreover, whereas the response to restraint was related to song complexity and relative heterophil numbers and H:L, the response to an ACTH injection was not related to either of these measures. This suggests that the relationship between the response to restraint and song complexity and leukocyte profiles was not due to variation in maximum adrenocortical CORT synthesis, but rather to variation in CRH or ACTH production, or to the degree to which individuals perceive restraint as a threat. Thus, responsiveness to a stressor (either perceptual or hypothalamic responsiveness) may be more important than the physiological capacity for CORT synthesis when characterizing CORT-fitness relationships (Bonier et al., 2009; Breuner et al., 2008). If an individual is very responsive to stressors, it may elicit a stress response more rapidly and/or elevate CORT to higher levels, which could lead to detrimental exposure to chronically high CORT. Our findings build on earlier work (Dickens et al., 2009; Rich and Romero, 2005; Romero and Wikelski, 2010) and illustrate the importance of using comprehensive measures of HPA activity when relating variation in HPA activity to physiological and behavioral traits.

Comprehensive characterization of HPA regulation Song complexity Most studies conducted on wild vertebrates characterize HPA activity by measuring CORT levels at baseline and after restraint stress. Although these measures provide valuable information about HPA activity in response to a stressor, they do not account for other aspects of HPA regulation that may be related to physiological condition and fitness, such as negative feedback. Injecting dexamethasone and taking subsequent blood samples provide a straight-forward way to assess negative feedback. In addition, injecting ACTH allows for assessment of maximum CORT levels, which may not always be

The magnitude of the response to restraint stress was negatively related to song complexity. Similarly, previous work on the same study population found that the response to restraint stress was negatively related to syllable repertoire size (MacDougall-Shackleton et al., 2009b). As reviewed above, there is considerable evidence that song complexity is sexually selected in song sparrows, thus this relationship adds to a growing body of evidence (Almasi et al., 2010; Roulin et al., 2008) that HPA activity covaries with the

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expression of sexually selected traits. In the current study, the only measure of song analyzed was song complexity. Future studies should investigate other aspects of song, such as output, stereotypy, or vocal performance, to determine whether other aspects of song are also related to HPA activity. Morphological measurements The measures of HPA activity were also related to multiple indices of physiological condition. First, negative feedback was related to body size (body PC1): birds that suppressed CORT more in response to dexamethasone were larger. Both body mass and tarsus length had high, positive loadings onto body PC1 (Table 1). However, tarsus length had the highest loading suggesting that the relationship between body PC1 and negative feedback was largely driven by variation in structural body size. One possible explanation for the relationship between HPA regulation and structural body size is that both these traits can be permanently affected by variation in the early rearing environment, such as exposure to stressors (Searcy et al., 2004; Spencer et al., 2009). An individual exposed to a high amount of stress during development may experience permanent alterations in HPA regulation and impaired structural development, such that in adulthood these individuals have less effective HPA regulation and are structurally smaller. That is, HPA function and body size may become developmentally correlated traits (Spencer and MacDougall-Shackleton, 2011). We observed no relationships between body condition (body PC2) and any of the measures of HPA activity. Previous studies have found negative relationships between body mass and baseline and restraint-induced CORT levels (Hau et al., 2010). This suggests that multiple aspects of HPA activity are related to body size including baseline CORT levels, the magnitude of the response to stressors, and efficacy of negative feedback. The specific relationships may vary depending on the species of interest.

Covariation between HPA regulation and measures of phenotypic quality The finding that HPA activity is correlated to both a sexually selected trait (song complexity) and indices of phenotypic quality (body size and leukocyte profiles) supports the hypothesis that HPA activity could be one physiological mechanism responsible for the covariation between sexually selected signals and phenotypic quality. Individuals of high quality may have low baseline or stress-induced glucocorticoid levels, or more effective HPA regulation, allowing for investment in both the sexually selected trait and other physiological or behavioral traits, such as immune function and body size. We found that baseline CORT levels were not related to song complexity or any of the other measures (data not shown), suggesting that it is the regulation of HPA responses that is particularly important in song sparrows. All traits measured in the current study are affected by variation in the early rearing environment. Exposure to stressors or CORT treatment during development can decrease size of the song nucleus HVC and may impair song learning and production (MacDonald et al., 2006; Spencer et al., 2003; Spencer et al., 2004). In addition, these same treatments lead to long-term decreases in body size in zebra finches (Spencer et al., 2003) and song sparrows (Searcy et al., 2004), impair immune function in European starlings (Buchanan et al., 2003), and increase the magnitude of the stress response in zebra finches (Spencer et al., 2009) and western scrubjays (Aphelocoma californica; Pravosudov and Kitaysky, 2006). The effects of developmental stressors or CORT treatment on negative feedback or corticosteroid receptor expression in birds have not been tested, but in rodents early-life stress alters corticosteroid receptor expression in the brain and impairs negative feedback (Welberg and Seckl, 2001). Therefore it is possible that all these traits become correlated because of overlapping developmental timelines and their susceptibility to early-life stress, and could thus be classified as developmentally correlated traits (Spencer and MacDougall-Shackleton, 2011).

Leukocyte profiles

Conclusions

HPA activity was also related to leukocyte profiles. Birds with more effective negative feedback had lower relative heterophil counts and H:L ratios (lower immune PC1 scores). H:L ratios have been widely used as indicators of physiological stress in birds and increase in response to infection (Davis et al., 2008; Harmon, 1998). Glucocorticoids are potent regulators of inflammatory and immune responses and exposure to stress and CORT treatment can suppress humoral and cell-mediated immune responses (Berger et al., 2005; Sapolsky et al., 2000). Impairments in negative feedback of the HPA axis may lead to chronic exposure to high glucocorticoid levels making individuals more susceptible to infection and disease. Interestingly, the response to restraint stress was inversely related to relative heterophil counts and H:L ratios, suggesting that individuals with lower increases in CORT in response to restraint had more heterophils and higher H:L ratios. This is surprising since stress is typically thought to increase H:L ratios (Davis et al., 2008). However, most studies examining the relationship between stress and H:L ratios determine the effects of exogenous stressors (by manipulating stress levels or looking at natural variation in environmental stress) on H: L ratios (e.g. Ilmonen et al., 2003; Ruiz et al., 2002). In contrast, few studies have measured the relationship between naturally-occurring variation in stress response and H:L ratios. We observed no relationships between relative lymphocyte counts (immune PC2 scores) and any of the measures of HPA activity. However, it should be noted that the only index of immunity we used in the current study was leukocyte profiles. In future studies, it would be valuable to include more direct tests of immune function (e.g. ability of blood to kill bacteria or the antibody response to an antigen) to determine how these measures relate to HPA activity.

Our results show important links between HPA activity, sexually selected traits, morphological measures, and leukocyte profiles. HPA activity could be one proximate mechanism responsible for the covariation between sexually selected traits and phenotypic quality. Our results also demonstrate that song complexity may provide receivers with important information about a male's sensitivity to stressors. These results add to a growing body of evidence that variation in HPA activity may have important fitness consequences. Finally, comprehensive characterization of HPA activity in free-living animals is important because it permits identifying which aspects of HPA activity covary with trait expression and phenotypic quality. Acknowledgments We thank Dr. E. Hampson for assistance with radioimmunoassays, S. Kubli for assistance with fieldwork, and the Queen's University Biological Station for providing access to the field site. This research was supported by grants from The Natural Sciences and Engineering Research Council of Canada (NSERC) to S.A.M.-S. and E.A.M.-S. and a NSERC Canada Graduate Scholarship and a Queen Elizabeth II Graduate Scholarship in Science and Technology to K.L.S. References Almasi, B., Jenni, L., Jenni-Eiermann, S., Roulin, A., 2010. Regulation of stress response is heritable and functionally linked to melanin-based coloration. J. Evol. Biol. 23, 987–996. Beecher, M.D., Campbell, S.E., Nordby, J.C., 2000. Territory tenure in song sparrows is related to song sharing with neighbours, but not to repertoire size. Anim. Behav. 59, 29–37.

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