Summary
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.
Methodology
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 long-term 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 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.