Summary
Hormones play a central role in integrating internal and external cues to help mediate life-history decisions as well as changes in behavior and physiology of individuals. Describing the consistency of endocrine traits within and among individuals is an important step for understanding whether hormonal traits are dependable predictors of phenotypes that selection could act upon. However, few long-term field studies have investigated the individual consistency of hormonal traits. Glucocorticoid hormones mediate homeostatic responses to environmental variation as well as stress responses to acute, unpredictable disturbances. We characterized the repeatability of plasma corticosterone concentrations in two species of free-living passerines across multiple years. We found repeatability in baseline corticosterone concentrations in both sexes of great tits (Parus major) and in female tree swallows (Tachycineta bicolor) within the breeding season but no repeatability of this trait among seasons or across years. Stress-induced levels of corticosterone were only assessed in great tits and were not repeatable in either sex. Our data suggest that in line with their function in mediating responses of individuals to longer-term and acute demands, both baseline and stress-induced plasma corticosterone concentrations are rather plastic traits. However, individuals may differ in their degree of trait plasticity and hence in behavioral and physiological responses to a variety of organismal challenges.
Methodology
Great tits
Adult great tits were captured in mist nets at feeding stations in Möggingen, Germany (47°N, 8°E) for two weeks each in March 2009, January 2010, and March 2010. They were also captured at the nest box using an automatic metal trap that closed the entrance hole when they were feeding their 8- or 9-day old young during the breeding seasons of 2009 and 2010 (May–June). All blood sampling took place between the hours of 08:00 and 12:00. We collected blood from the brachial vein (80–120 μl) within 3 min of entrapment in the nest box or entanglement in the mist net (mean: 1.9 min ± 0.9 standard deviation; n = 238) to measure baseline corticosterone levels. We then placed the bird in a cloth bag and collected another blood sample (50 μl) 30 min later to measure stress-induced corticosterone concentrations. The blood samples were kept on ice and centrifuged (822 × g, 10 min) within 3 h, and the plasma was separated and stored at − 80 °C until analysis for hormone concentration. The work for the great tits was approved by the state of Baden-Württemberg under permit number G-09/102.
Corticosterone concentrations were determined using enzyme immunoassay (EIA) kits (Cat. No. 901-097, Assay Designs) following a diethyl-ether extraction of 5 μl sample volume. After drying the extract under N2 stream, 400 μl of Assay Buffer 15 (Tris-buffered saline) was added (1:80 dilution), and the samples were allowed to reconstitute overnight. Two separate, stripped-chicken plasma standards with a known amount of added corticosterone (20 ng/ml) as well as two blank samples containing only assay buffer were taken through the entire assay procedure. The next day, 100 μl of each sample (in duplicate) was added to individual wells on the assay plate alongside a standard curve with 5 standards ranging from 32 to 20,000 pg/ml. The samples were added randomly within and across plates but an individual's 0 minute and 30 minute samples were always included on the same plate. The plate was shaken for 2 h to bind the conjugate corticosterone and the antibody. The wells were then washed 4 times with 200 μl of wash solution. After adding 200 μl of p-nitrophenyl phosphate to each well, the plate was incubated in darkness for another hour. Stop solution was then added and the plate was read on VERSAmax microplate reader at 405 nm. The lower sensitivity of the assay was at 18.8 pg/ml and the corresponding lower detection limit was at 0.033 ng/ml (determined by the lowest standard). The blank wells were always below the detection limit. Double diethyl ether extractions of samples spiked with a small amount of radioactive corticosterone in our laboratory yield an average recovery of around 90% (extraction efficiency for stripped plasma (n = 3 samples): 89.7%, CV 6.1%; extraction efficiency for European blackbird, Turdus merula, plasma (n = 6 samples): 91.4%, CV 2.2%). Given the high recoveries and low variation in extraction efficiency between samples, we added 10% to all our values to account for extraction efficiency. The mean intra-plate (16 plates) coefficient of variation of two replicate standards per plate was 11.8% and the inter-plate coefficient of variation was 4.9%.
Tree swallows
We conducted this study in May–July 2007–2010 from a free-ranging population of box-nesting tree swallows at the Queen's University Biological Station (44°34 N, 76°19 W, 135 m elevation). Females were captured at the nest box during breeding either with trap-doors set on the nest box entryways or by placing our hands over the entryway when the bird was inside the box (N = 103 samples from 38 females). We did not include males in the repeatability analysis due to insufficient sample size. All blood sampling took place between the hours of 09:00 and 12:00. We collected blood from the brachial vein (80–150 μl) within 3 min of capture to measure baseline corticosterone levels. Blood was stored on ice until transport to the lab. Within 8 h of collection, all blood samples were centrifuged at 14800 × g for 5 min. Separated plasma was then stored at − 20 °C until time of hormone analyses. All of the tree swallow work was approved by the Canadian Wildlife Service (permit number CA-0211) and the Queen's University Animal Care Committee.
We quantified plasma levels of total corticosterone in each sample in duplicate through direct radioimmunoassay, following extraction with re-distilled dichloromethane (see Wingfield et al., 1992 for details). All samples collected within the same year were assayed simultaneously, thus we conducted a total of four assays. The mean within-assay coefficient of variation among replicate known-concentration standard samples was 8.4% (range 4.3–13.1%, 2–6 standards per assay), and among-assay variation was 5.2%.