nestling immunocompetence is affected by captivity but not ...

The Condor 109:920–928 # The Cooper Ornithological Society 2007

NESTLING IMMUNOCOMPETENCE IS AFFECTED BY CAPTIVITY BUT NOT INVESTIGATOR HANDLING MICHAEL W. BUTLER1

AND ALFRED M. DUFTY JR. Boise State University, Department of Biology, Boise, ID 83725

Abstract. Environmental conditions during the neonatal period can affect the growth, physiology, behavior, and immune function of birds. In many avian studies the nestling environment includes investigator handling of young, which may be stressful. While neonatal handling is known to affect the adult phenotype in rats, the effects of handling on development have rarely been examined in wild birds. We examined the effect of short, repeated periods of neonatal handling on avian growth and immune system development. We subjected American Kestrels (Falco sparverius) and European Starlings (Sturnus vulgaris) to 15 min of daily investigator handling throughout the nestling period, while controls remained undisturbed. Immediately prior to fledging we assessed cutaneous immunity, humoral immunity, mass, and degree of fluctuating asymmetry. Daily handling did not significantly affect any of these measurements. We also addressed the possibility that treatment differences would appear only when birds were challenged with a more substantial stressor by bringing birds into captivity for 24 hr. Captivity did not affect mass, but significantly lowered the cutaneous immune response, although this was independent of treatment. Therefore, brief periods of investigator handling did not appear to affect immune or morphological development in these species, whereas 24 hr of captivity resulted in suppressed cutaneous immune responses. Key words: captivity, development, handling, immunocompetence, nestling, phytohemagglutinin, stress.

La Competencia Inmunolo´gica de los Pichones es Afectada por el Cautiverio pero no por la Manipulacioń del Investigador Resumen. Las condiciones ambientales durante el perıódo neonatal pueden afectar el crecimiento, la fisiologıá, el comportamiento y la funcioń inmunolo´gica de las aves. En muchos estudios de aves, el ambiente del pichoń incluye su manipulacioń por parte del investigador, la cual puede causar estre´s. Aunque se conoce que la manipulacioń neonatal afecta el fenotipo de los adultos en las ratas, el efecto de la manipulacioń en el desarrollo ha sido examinado en muy pocas ocasiones en aves silvestres. Examinamos el efecto de perıódos repetidos cortos de manipulacioń neonatal en el crecimiento y en el desarrollo del sistema inmunolo´gico en las aves. Sometimos a individuos de las especies Falco sparverius y Sturnus vulgaris a 15 min diarios de manipulacioń por parte de investigadores a lo largo de la etapa de pichoń, mientras que los controles permanecieron sin disturbios. Inmediatamente antes al emplumamiento, evaluamos la inmunidad cutańea, la inmunidad humoral, el peso y el grado de asimetrıá fluctuante. La manipulacioń diaria no afecto´ significativamente ninguna de estas medidas. Tambień analizamos la posibilidad de que las diferencias de tratamiento aparecieran so´lo cuando las aves fueran expuestas a un agente de estre´s ma´s importante, poniendo a las aves en cautiverio por 24 hs. El cautiverio no afecto´ el peso, pero disminuyo´ significativamente la respuesta inmunolo´gica cutańea, aunque esto fue independiente del tratamiento. De este modo, los perıódos breves de manipulacioń del investigador no parecen afectar el desarrollo inmunolo´gico y morfolo´gico en estas especies, mientras que un perıódo de 24 hs de cautiverio produjo una supresioń de las respuestas inmunolo´gicas cutańeas.

INTRODUCTION The effects of neonatal handling on development, although well-documented in mammals Manuscript received 31 October 2006; accepted 14 June 2007. 1 Present address: Arizona State University, School of Life Sciences Graduate Program, P.O. Box 874601, Tempe, AZ 85287-4601. E-mail: Mike. [email protected]

(Severino et al. 2004, Silveira et al. 2004), have rarely been examined as a potential source of phenotypic plasticity in birds. This is despite the fact that investigators routinely handle nestlings (Lloyd and Martin 2004, Martinez-Padilla et al. 2004, Dawson and Bidwell 2005, Tilgar et al. 2005), and handling is a standard part of the protocol to examine avian adrenoresponsiveness (Romero and Romero 2002, Washburn et al. 2002).

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HANDLING, CAPTIVITY, AND IMMUNOCOMPETENCE

Conditions experienced during ontogeny can influence nestling growth (Arnold and Griffiths 2003, Dawson and Bidwell 2005), immune function (De Neve et al. 2004), and physiology (Ardia 2005). For example, nest parasites decrease nestling growth (Fitze et al. 2004) and survival (Ban´bura et al. 2004). Additionally, nest microclimate affects nestling growth (Lloyd and Martin 2004), and food restriction lowers nestling resting metabolic rate (Moe et al. 2004). All of these factors affect fledging mass and, because fledging mass influences recruitment (Monro´s et al. 2002), nestling conditions can ultimately affect fitness. Neonatal experiences also play a role in the development of immune function (Gross and Siegel 1982, Tella et al. 2000, Smits et al. 2002). In the nestling stage, unfavorable weather conditions (Lifjeld et al. 2002), decreased protein intake (Lochmiller et al. 1993), clutch size (Saino et al. 2003), and length of the nestling period (Tella et al. 2002) can alter immunocompetence. Human contact, including investigator handling, can also affect immune function (Collette et al. 2000). Many of these factors stimulate the release of corticosterone from the adrenal glands, demonstrating a potential link between neonatal stressors, glucocorticoids, and immunosuppression. Glucocorticoids are instrumental in regulating energy resources, including those mobilized by stressors (reviewed by Sapolsky et al. 2000), and the effects of glucocorticoids differ based on the magnitude and duration of the adrenocortical response. For example, low level, shortterm increases in corticosterone seem to stimulate cutaneous immunity (Buchanan 2000) and may mobilize leukocytes to move to the skin to prepare an organism to defend against damage to this protective layer (Dhabhar 2002). Conversely, high levels of glucocorticoids are immunosuppressive (Buchanan 2000), as are a variety of physiological stressors (Hasselquist et al. 2001, Buchanan et al. 2003, Ilmonen et al. 2003). During periods of increased stress, it may be adaptive to suppress the immune system and allocate energy resources toward more immediately important target tissues such as nervous and muscular tissue (Apanius 1998). Furthermore, immunosuppression may be a mechanism for avoiding autoimmune problems during times of stress (Ra¨berg et al. 1998).

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Investigator handling of nestlings occurs in many studies, ranging from those that simply collect morphological measurements (Bize et al. 2003, Dawson and Bidwell 2005) to those that are more intrusive and involve the collection of blood samples (Kitaysky et al. 2001, Blas et al. 2005) or the assessment of immunocompetence (Tschirren et al. 2003, Westneat et al. 2004). Repeated investigator handling, if perceived as stressful by nestlings, could lead to immunosuppression (Buchanan 2000, Balcombe et al. 2004). However, to our knowledge, no one has examined the effects of neonatal handling on the development of the avian immune system in wild populations. To test the effect of handling on nestling immunocompetence, we applied a daily handling regimen to nestling American Kestrels (Falco sparverius; hereafter kestrels) and European Starlings (Sturnus vulgaris; hereafter starlings) and assessed basic immune system parameters. However, because our repeated handling was brief in duration and of relatively low intensity, we were concerned that any effects might be too subtle to detect under baseline conditions, as has occurred in mammals (Beane et al. 2002, Panagiotaropoulos et al. 2004). Therefore, we also exposed some birds to a more stressful condition, a prolonged (24 hr) period of captivity, to reveal any previously undetected differences in immunoresponsiveness between handled and unhandled birds. We hypothesized three possible outcomes. If daily handling is benign in terms of immunodevelopment, then there should be no difference in immunoresponsiveness between handled and control birds, whether in the nest or in captivity. However, if handling is perceived as a mild stressor by nestlings, then handled birds should exhibit immunosuppression compared to controls when faced with captivity. Alternatively, if the short period of daily handling stimulates development of the immune system, handled birds should show enhanced immunoresponsiveness in captivity. METHODS STUDY SITE AND SPECIES

We examined birds nesting in artificial nest boxes in Ada and Canyon Counties, Idaho. Nest boxes (21 cm 3 21 cm 3 46 cm) were secured to telephone poles approximately 2.5 m

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from the ground. Both species laid eggs from April through early August. To avoid premature fledging we began assessment of the cutaneous immune system (see below) at day 14 for starlings and day 25 for kestrels, based on nestling periods of 18–21 days for starlings and 30–31 days for kestrels (Ehrlich et al. 1988). HANDLING TREATMENT

Starting in late March, we checked nest boxes at weekly intervals until eggs were laid. Each nest was then left undisturbed until two days before the anticipated hatching date, based on a 13day (starling) or 29-day (kestrel) incubation period (Ehrlich et al. 1988). After eggs hatched we applied either experimental or control handling treatments to all young in a nest. In experimental nests, all nestlings were removed and handled for 15 min each day of the nestling period (13 days for starlings, 24 days for kestrels) between 08:00 and 18:00 (MDT). We did not establish a schedule of standard visitation times because we wanted to avoid nestling anticipation of nest disturbance. During handling we simulated a variety of different field techniques, including measuring tarsus, wing chord, and mass, thus imitating studies that normally employ such handling techniques. Parental feeding did not occur at experimental nests while nestlings were being handled, so we also prevented feeding in the control boxes by standing quietly outside the nest box for 15 min each day. For each species, we alternated the assignment of nests to either experimental or control groups. CUTANEOUS IMMUNITY

To assess cutaneous immunity we injected phytohemagglutinin (PHA), which is a mitogen that causes T-cell lymphocytes, a major component of cell-mediated immunity, to proliferate in vivo (Goto 1978). The proliferative response, measured by the amount of swelling at the site of PHA injection, has generally been correlated with the level of cell-mediated immunity. Recently, however, it has been pointed out that this test incorporates not only the response of cell-mediated immunity, but also components of innate immunity (Martin et al. 2006). Therefore, we will hereafter refer to this metric as cutaneous immunity. Two nestlings were randomly taken out of each nest for PHA assessment. On day one

(defined as nestling day 14 for starlings and nestling day 25 for kestrels), each subject had a black dot drawn centrally on its patagium, where patagial thickness was measured twice with a digital micrometer (model 293–369, Mitutoyo, Aurora, Illinois). Directly under the black dot, we injected 50 ml of phosphate buffered saline (PBS) containing 25 mg of PHA (Sigma-Aldrich, St. Louis, Missouri) into starlings, or 50 ml of PBS containing 50 mg of PHA into kestrels (dosages extrapolated from Smits et al. 1999). After injection, one bird was returned to the nest while the other was brought into captivity for 24 hr. This difference in treatment allowed us to examine how a prolonged stressor affected PHA responsiveness. Because this protocol was followed for both experimental and control nests, we were able to look for an interaction between handling and captivity. The birds were housed singly overnight at Boise State University in standard wire cages (30 cm 3 25 cm 3 25 cm for starlings and 75 cm 3 75 cm 3 72 cm for kestrels) with shredded paper towels as bedding and food and water provided ad libitum. Birds were kept at approximately 24uC on a 14 light:10 dark photoperiod. The starlings were not old enough to eat on their own, so they were fed five times per day with 10 ml of a diet consisting of wet dog food, poultry mash, applesauce, hardboiled egg, and vitamin supplement (Quikon, Bocholt, Germany). The following day (nestling day 15 for starlings, nestling day 26 for kestrels; hereafter, day two), approximately 24 hr later (mean 5 24 hr 11 min; SD 5 13 min; range 23 hr 16 min to 24 hr 47 min), captive birds were returned to their nests, and we measured the patagia of both birds at the site of PHA injection. Replications of day one and day two measurements were highly correlated (r 5 0.97 and r 5 0.99, respectively), as is typical for this technique (Smits et al. 1999). MORPHOMETRICS

Starlings and kestrels were measured on day one of their PHA challenge. Measurements included body mass and the length of both tarsi, both wings, and the culmen. Differences in length between the left and right structures were used to quantify fluctuating asymmetry.


HUMORAL IMMUNITY

Humoral immunity, the amount of antibody generated in response to a benign antigen, was assessed only in starlings, using one bird per nest. On day two, we drew a reference blood sample (,125 ml) from each individual. Blood was centrifuged at 6000 RPM for 60 sec, and plasma was harvested and stored at 220uC until analysis. Subjects received an intramuscular injection into the breast of 25 mg keyhole limpet hemocyanin (KLH, Sigma-Aldrich), which is a novel, benign antigen that was emulsified in 0.05 ml sterile water and 0.05 ml incomplete Freund’s adjuvant (Sigma-Aldrich). Following Hasselquist et al. (1999), we obtained additional blood samples at 6, 9, 12, 15, 18, and 21 days postinoculation and treated them as described above. We developed an enzyme-linked immunosorbent assay (ELISA) protocol to assess antibody content of the plasma. ELISA plates (Nunc, Rochester, New York) were coated with 200 ml of 0.5 mg ml21 KLH in sodium carbonate buffer and incubated at 37uC for 2 hr. After removal of liquid, 200 ml of blocking buffer (3% powdered milk in 0.01 M PBS) were added and left at room temperature for 2 hr. The blocking buffer was then removed, and 180 ml duplicates of a 1:100 plasma dilution (3% powdered milk and 0.01% sodium azide in 0.01 M PBS) were added along with blank controls. The plate was incubated overnight at 4uC. Plates were washed three times with 200 ml of PBS and 0.05% Tween 20 (Sigma-Aldrich). Fifty ml of horseradish peroxidase- (HRP) labeled secondary antibody specific for avian immunoglobulin G (Bethyl, Montgomery, Texas), diluted 1:1000 in PBS, 0.05% Tween 20, and 0.5% bovine serum albumin (Sigma-Aldrich), were added and incubated at room temperature for 2 hr. Plates were then washed four times with PBS and 0.05% Tween 20. Each well received 50 ml of the substrate tetramethylbenzidine (SigmaAldrich) and was left in the dark at room temperature for 30 min. The reaction was stopped with 50 ml of 0.5 M sulfuric acid, and the optical density of the wells was read at a wavelength of 450 nm. A higher optical density indicated a higher concentration of HRP, which was proportional to the amount of KLH-specific antibody present in the plasma sample.

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Optimal plasma dilution was determined during an exploratory ELISA using the same protocol. Plasma dilutions of 1:50, 1:100, 1:200, and 1:400 were used to find the dilution which best fit into the linear range. The linear range was determined by plotting the optical density as a function of dilution, and looking for the range in which a change in dilution resulted in a proportional change in optical density. STATISTICAL ANALYSIS

Data were analyzed using SAS 9.1 (SAS Institute 2004). We regressed mass against tarsus length (Merila¨ et al. 1999) and used the residuals as a measure of body condition. To determine the treatment effect on cutaneous immunity we employed a split-split plot experimental design, with between-groups factors of species and handling treatment, and a withinsubject factor of captivity treatment. We included both species in the same model because we were interested in the effects of handling and captivity on birds in general, as opposed to two specific species. Our model permitted us to generalize to a greater extent than would have been possible with two univariate analyses. Handling treatment was assigned to an entire nest, while within each nest one individual was brought into captivity and another was left in the nest during the PHA incubation. The within-subject factor of captivity treatment accounts for the nonrandom relationship of two individuals from the same nest. To examine the effect of handling on morphometrics, each body measurement was assessed in a separate ANOVA. We employed a Bonferroni-corrected alpha of 0.006, calculated by dividing an alpha of 0.05 by eight, the number of separate ANOVAs. To assess antibody production, we used a repeated-measures ANOVA, as the same birds were sampled on multiple days. All means are reported 6 SE. RESULTS EFFECT OF HANDLING ON MORPHOMETRICS

A regression of kestrel mass against tarsus length did not yield a nonzero slope (F1,12 5 1.8, P 5 0.21); therefore, we could not accurately assess body condition for kestrels. In contrast, starlings (with missing data at two nests) had a significant linear regression model (F1,32 5 21.8, P , 0.001), but the residuals of

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TABLE 1. Results from multiple two-way ANOVAs relating handling treatment (two levels; handled for 15 min per day vs. control) to a variety of morphometric response variables for American Kestrels (n 5 14) and European Starlings (n 5 34). Species

Morphometric variable

Fa

P

Mass Left tarsus Right tarsus Tarsus asymmetry Left wing Right wing Wing asymmetry Culmen Mass Left tarsus Right tarsus Tarsus asymmetry Left wing Right wing Wing asymmetry Culmen

0.0 2.5 0.4 0.1 0.7 0.9 0.3 0.1 0.3 0.1 0.0 0.6 1.7 1.9 0.6 0.8

0.87 0.18 0.54 0.77 0.45 0.39 0.62 0.79 0.62 0.72 0.88 0.46 0.21 0.19 0.46 0.37

American Kestrel (Falco sparverius)

European Starling (Sturnus vulgaris)

a Degrees of freedom for American Kestrels are 1 (treatment) and 5 (error), and for European Starlings are 1 (treatment) and 15 (error).

the regression were not significantly different between handling treatments (F1,15 5 0.1, P 5 0.71), meaning that there was no effect of investigator handling on body condition. Handling had no effect on kestrel or starling morphometric variables (Table 1), suggesting that daily investigator handling did not significantly alter growth parameters. EFFECT OF HANDLING AND CAPTIVITY TREATMENTS ON CUTANEOUS IMMUNITY

To examine differences in cutaneous immunity, we conducted a three-way ANOVA on patagial swellings to evaluate effects of species, handling treatment, and captivity (repeated factor). We were unable to measure one nestling due to premature fledging. Kestrels showed a greater degree of PHA-induced swelling than starlings (F1,22 5 63.1, P , 0.001), while investigator handling had no effect (F1,22 , 0.1, P 5 0.88; Fig. 1A). There were no significant interactions (all P . 0.30) among any factors. Additionally, birds subjected to overnight captivity had reduced PHA-induced swelling compared to nest mates, irrespective of species (F1,21 5 15.7, P , 0.001; Fig. 1B). Thus, kestrels displayed a greater cutaneous immune response than starlings, overnight captivity suppressed the immune response, and investigator handling had no effect on cutaneous immunity.

EFFECT OF HANDLING TREATMENT ON HUMORAL IMMUNITY

Only starlings were used to assess humoral immunity. The amount of plasma antibody increased steadily with time, showing a maximum at 21 days postinjection, with both handled and control birds exhibiting similar humoral responses (Fig. 2). Antibody production was not influenced by handling treatment (F1,6 5 0.3, P 5 0.58); starlings that were handled during the nestling period showed the same continuous increase in antibody titer as controls. However, antibody production was influenced by the number of days after inoculation (F6,36 5 4.8, P 5 0.001). There was no interaction between treatment and days postinoculation (F6,36 5 0.9, P 5 0.50). One nest attained values three times higher than average on postinjection days 6 and 9. While exclusion of these points did not change any statistical conclusions, Figure 2 has these points removed to visually reflect the statistical findings less ambiguously. DISCUSSION We found no evidence that investigator handling affected the development of immune function of American Kestrel and European Starling nestlings. Investigator handling is a short-term, repeated stressor to which birds


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FIGURE 2. Relative antibody production (mean 6 SE) in response to injection of keyhole limpet hemocyanin, as measured by ELISA optical density, in European Starlings handled for 15 min each day, and controls (n 5 7 for each treatment). There were no significant differences between treatments.

FIGURE 1. Patagial swelling induced by phytohemagglutinin as an indicator of cutaneous immunity. (A) American Kestrels had significantly greater swellings than European Starlings for all treatments. However, handling protocol (handled for 15 min per day vs. control) did not produce statistical differences in cutaneous immunity in either species. (B) Birds subjected to captivity for 24 hr had significantly reduced swellings compared to nest mates left in the nest. Asterisk (*) denotes significant differences at a 5 0.05. Sample sizes in parentheses.

are frequently exposed (Lloyd and Martin 2004, Martinez-Padilla et al. 2004, Dawson and Bidwell 2005, Tilgar et al. 2005). However, until now, the assumption that investigator handling does not affect immunodevelopment had not been tested. Thus, our finding of a lack of handling effects provides the first empirical support for the long-held, implicit notion that handling and measuring nestlings does not affect phenotypic development, at least as far as the immune system is concerned. However, while we detected no short-term effect of our handling regimen, the long-term ramifications of investigator handling for immunocompetence are poorly understood. Nestling immunity is an important life-history variable because greater nestling immunocompetence is linked to higher postfledging survival and recruitment into the breeding population (Cichon´ and Dubiec 2005, Lobato et al. 2005,

Moreno et al. 2005). Therefore, if repeated, low-intensity stressors have long-lasting phenotypic effects, they may lower the lifetime fitness of nestlings. Such long-lasting effects are known to occur. For example, neonatal handling can attenuate the glucocorticoid response (Beane et al. 2002, Whitman 2006). Glucocorticoids affect the immune system (Sapolsky et al. 2000), so stressors experienced at a young age may alter adult immunoresponsiveness (Barreau et al. 2004). Interestingly, we found that birds exposed to handling did not respond differently immunologically than control birds when subjected to the stress associated with 24 hr of captivity. Our results contrast with those found for Orange-winged Parrots (Amazona amazonica; Collette et al. 2000), although in that study the type of stressor was confounded with previous handling treatment, so it is unclear whether the results can be attributed to the handling treatment alone. In our study, the stress of captivity (Wingfield et al. 1982, Marra et al. 1995) was strong enough to lead to suppression of cutaneous immunity of captive individuals, regardless of earlier handling treatment. This finding has ramifications for investigators examining immunocompetence in captive subjects, as captivity itself, which may include changes in food, temperature, precise day length, or a host of other factors, may alter results. Green et al. (2005) noted that longer periods of captivity resulted in lower postrelease survival rates. Thus, future studies should examine whether this immunosuppressive effect is temporary or permanent. If the suppression

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of cutaneous immunity presented here is permanent, wildlife managers should be aware of this effect while engaging in conservationbased programs that subject wild individuals to periods of captivity. In addition to assessing cutaneous immunity in both species, we evaluated humoral immunity in starlings. It is important to examine multiple aspects of the immune system because they are varied and often respond differently to the same treatment (Collette et al. 2000, Buchanan et al. 2003); thus, examining more than one immune parameter provides a more accurate assessment of immunocompetence (Norris and Evans 2000, Salvante 2006). In examining starling humoral immunity, we found that relative antibody concentration increased throughout the 21 days following injection, with no significant differences between treatments. This lack of a difference between groups is further evidence that daily investigator handling throughout the nestling period did not affect immune system development. However, it is unclear why there was a continual increase in antibody concentration through the 21-day postinjection period. This differs from humoral responses seen in other passerines (Hasselquist et al. 1999), in which antigen-specific antibody concentration peaked between 12 and 15 days postinjection. A steady increase through 21 days postinjection is unusually long for a primary antibody response (Goldsby et al. 2003). We are unsure if this is a species-specific response or if there were methodological issues, such as previous exposure to PHA, which could have stimulated other components of the immune system, including cytokine titer. In conclusion, we found no evidence that daily handling during the nestling stage affected the development of the immune system or altered the morphology of nestling European Starlings or American Kestrels. Handling is a short-term, repeated stressor that did not appear to be of sufficient magnitude to alter the developmental features we measured. However, 24 hr of captivity did, at least temporarily, suppress the cutaneous immune response. ACKNOWLEDGMENTS We thank Boise State University for funding and space for the housing of animal subjects. Jim Belthoff, Ian Robertson, Denise Wingett, and two

anonymous reviewers provided helpful comments on earlier drafts, and Buddy Whitman provided valuable guidance on data collection. This work was partially funded by the Raptor Research Center and the Boise State University Biology Department.

LITERATURE CITED APANIUS, V. 1998. Stress and immune defense, p. 133–153. In A. P. Møller, M. Milinski, and P. J. B. Slater [EDS.], Stress and behavior: advances in the study of behavior. Academic Press, San Diego, CA. ARDIA, D. R. 2005. Super size me: an experimental test of the factors affecting lipid content and the ability of residual body mass to predict lipid stores in nestling European Starlings. Functional Ecology 19:414–420. ARNOLD, K. E., AND R. GRIFFITHS. 2003. Sexspecific hatching order, growth rates and fledging success in Jackdaws Corvus monedula. Journal of Avian Biology 34:275–281. BALCOMBE, J. P., N. D. BARNARD, AND C. SANDUSKY. 2004. Laboratory routines cause animal stress. Contemporary Topics in Laboratory Animal Science 43:42–51. BAN´BURA, J., P. PERRET, J. BLONDEL, D. W. THOMAS, M. CARTAN-SON, AND M. M. LAMBRECHTS. 2004. Effects of Protocalliphora parasites on nestling food composition in Corsican Blue Tits Parus caeruleus: consequences for nestling performance. Acta Ornithologica 39: 21–31. BARREAU, F., L. FERRIER, J. FIORAMONTI, AND L. BUENO. 2004. Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut 53:501–506. BEANE, M. L., M. A. COLE, R. L. SPENCER, AND J. W. RUDY. 2002. Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats. Hormones and Behavior 41:33–40. BIZE, P., A. ROULIN, L. F. BERSIER, D. PFLUGER, AND H. RICHNER. 2003. Parasitism and developmental plasticity in Alpine Swift nestlings. Journal of Animal Ecology 72:633–639. BLAS, J., R. BAOS, G. R. BORTOLOTTI, T. MARCHANT, AND F. HIRALDO. 2005. A multi-tier approach to identifying environmental stress in altricial nestling birds. Functional Ecology 19:315–322. BUCHANAN, K. L. 2000. Stress and the evolution of condition-dependent signals. Trends in Ecology & Evolution 15:156–160. BUCHANAN, K. L., M. R. EVANS, AND A. R. GOLDSMITH. 2003. Testosterone, dominance signaling and immunosuppression in the House Sparrow, Passer domesticus. Behavioral Ecology and Sociobiology 55:50–59. CICHON´, M., AND A. DUBIEC. 2005. Cell-mediated immunity predicts the probability of local recruitment in nestling Blue Tits. Journal of Evolutionary Biology 18:962–966.


COLLETTE, J. C., J. R. MILLAM, K. C. KLASING, AND P. S. WAKENELL. 2000. Neonatal handling of Amazon Parrots alters the stress response and immune function. Applied Animal Behaviour Science 66:335–349. DAWSON, R. D., AND M. T. BIDWELL. 2005. Dietary calcium limits size and growth of nestling Tree Swallows Tachycineta bicolor in a non-acidified landscape. Journal of Avian Biology 36:127–134. DE NEVE, L., J. J. SOLER, M. SOLER, AND T. PE´REZCONTRERAS. 2004. Nest size predicts the effect of food supplementation to magpie nestlings on their immunocompetence: an experimental test of nest size indicating parental ability. Behavioral Ecology 15:1031–1036. DHABHAR, F. S. 2002. A hassle a day may keep the doctor away: stress and the augmentation of immune function. Integrative and Comparative Biology 42:556–564. EHRLICH, P., D. DOBKIN, AND D. WHEYE. 1988. The birders handbook: a field guide to the natural history of North American birds. Simon and Schuster, New York. FITZE, P. S., J. CLOBERT, AND H. RICHNER. 2004. Long-term life-history consequences of ectoparasite-modulated growth and development. Ecology 85:2018–2026. GOLDSBY, R. A., T. J. KINDT, B. A. OSBORNE, AND J. KUBY. 2003. Overview of the immune system, p. 1–19. In R. A. Goldsby, T. J. Kindt, B. A. Osborne, and J. Kuby [EDS.], Immunology. 5th ed. W. H. Freeman and Company, New York. GOTO , N., H. KODAMA, K. OKADA , AND Y. FUJIMOTO. 1978. Suppression of phytohemagglutinin skin response in thymectomized chickens. Poultry Science 57:246–250. GREEN, A. J., C. FUENTES, J. FIGUEROLA, C. VIEDMA, AND N. RAMON. 2005. Survival of Marbled Teal (Marmaronetta angustirostris) released back into the wild. Biological Conservation 121:595–601. GROSS, W. B., AND P. B. SIEGEL. 1982. Socialization as a factor in resistance to infection, feed efficiency, and response to antigen in chickens. American Journal of Veterinary Research 43:2010–2012. HASSELQUIST, D., J. A. MARSH, P. W. SHERMAN, AND J. C. WINGFIELD. 1999. Is avian humoral immunocompetence suppressed by testosterone? Behavioral Ecology and Sociobiology 45: 167–175. HASSELQUIST, D., M. F. WASSON, AND D. W. WINKLER. 2001. Humoral immunocompetence correlates with date of egg-laying and reflects work load in female Tree Swallows. Behavioral Ecology 12:93–97. ˚ . LANGEFORS, AND ILMONEN, P., D. HASSELQUIST, A J. WIEHN. 2003. Stress, immunocompetence and leukocyte profiles of Pied Flycatchers in relation to brood size manipulation. Oecologia 136: 148–154. KITAYSKY, A. S., J. C. WINGFIELD, AND J. F. PIATT. 2001. Corticosterone facilitates begging and affects resource allocation in the Black-legged Kittiwake. Behavioral Ecology 12:619–625.

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LIFJELD, J. T., P. O. DUNN, AND L. A. WHITTINGHAM. 2002. Short-term fluctuations in cellular immunity of Tree Swallows feeding nestlings. Oecologia 130:185–190. LLOYD, J. D., AND T. E. MARTIN. 2004. Nest-site preference and maternal effects on offspring growth. Behavioral Ecology 15:816–823. LOBATO, E., J. MORENO, S. MERINO, J. J. SANZ, AND E. ARRIERO. 2005. Haematological variables are good predictors of recruitment in nestling Pied Flycatchers (Ficedula hypoleuca). E´ coscience 12:27–34. LOCHMILLER, R. L., M. R. VESTEY, AND J. C. BOREN. 1993. Relationship between protein nutritional status and immunocompetence in Northern Bobwhite chicks. Auk 110:503–510. MARRA, P. P., K. T. LAMPE, AND B. L. TEDFORD. 1995. Plasma corticosterone levels in two species of Zonotrichia sparrows under captive and freeliving conditions. Wilson Bulletin 107:296–305. MARTIN, L. B., II, P. HAN, J. LEWITTES, J. R. KUHLMAN, K. C. KLASING, AND M. WIKELSKI. 2006. Phytohemagglutinin-induced skin swelling in birds: histological support for a classic immunoecological technique. Functional Ecology 20:290–299. MARTIŃEZ-PADILLA, J., J. MARTIŃEZ, J. A. DA´VILA, S. MERINO, J. MORENO, AND J. MILLAŃ. 2004. Within-brood size differences, sex and parasites determine blood stress protein levels in Eurasian Kestrel nestlings. Functional Ecology 18:426– 434. MERILA¨, J., R. PRZYBYLO, AND B. C. SHELDON. 1999. Genetic variation and natural selection on Blue Tit body condition in different environments. Genetical Research 73:165–176. MOE, B., S. BRUNVOLL, D. MORK, T. E. BROBAKK, AND C. BECH. 2004. Developmental plasticity of physiology and morphology in diet-restricted European Shag nestlings (Phalacrocorax aristoltelis). Journal of Experimental Biology 207: 4067–4076. MONRO´S, J. S., E. J. BELDA, AND E. BARBA. 2002. Post-fledging survival of individual Great Tits: the effect of hatching date and fledging mass. Oikos 99:481–488. MORENO, J., S. MERINO, J. J. SANZ, E. ARRIERO, J. MORALES, AND G. TOMA´S. 2005. Nestling cellmediated immune response, body mass and hatching date as predictors of local recruitment in the Pied Flycatcher Ficedula hypoleuca. Journal of Avian Biology 36:251–260. NORRIS, K., AND M. R. EVANS. 2000. Ecological immunology: life history trade-offs and immune defense in birds. Behavioral Ecology 11:19–26. PANAGIOTAROPOULOS, T., S. PONDIKI, A. PAPAIOANNOU, F. ALIKARIDIS, A. STAMATIKIS, K. GEROZISSIS, AND F. STYLIANOPOULOU. 2004. Neonatal handling and gender modulate brain monoamines and plasma corticosterone levels following repeated stressors in adulthood. Neuroendocrinology 80:181–191. RA¨BERG, L., M. GRAHN, D. HASSELQUIST, AND E. SVENSSON. 1998. On the adaptive significance of stress-induced immunosuppression. Proceedings

928

MICHAEL W. BUTLER

AND

ALFRED M. DUFTY JR.

of the Royal Society of London Series B 265:1637–1641. ROMERO , L. M., AND R. C. R OMERO . 2002. Corticosterone responses in wild birds: the importance of rapid initial sampling. Condor 104:129–135. SAINO, N., C. SUFFRITTI, R. MARTINELLI, D. RUBOLINI, AND A. P. MØLLER. 2003. Immune response covaries with corticosterone plasma levels under experimentally stressful conditions in nestling Barn Swallows (Hirundo rustica). Behavioral Ecology 14:318–325. SALVANTE, K. G. 2006. Techniques for studying integrated immune function in birds. Auk 123:575–586. SAPOLSKY, R. M., L. M. ROMERO, AND A. U. MUNCK. 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21:55–89. SAS INSTITUTE. 2004. SAS/STAT 9.1 user’s guide. SAS Institute, Inc., Cary, NC. SEVERINO, G. S., I. A. M. FOSSATI, M. J. PADOIN, C. M. GOMES, L. TREVIZAN, G. L. SANVITTO, C. R. FRANCI, J. A. ANSELMO-FRANCI, AND A. B. LUCION. 2004. Effects of neonatal handling on the behavior and prolactin stress response in male and female rats at various ages and estrous cycle phases of females. Physiology and Behavior 81:489–498. SILVEIRA, P. P., A. K. PORTELLA, Z. CLEMENTE, E. BASSANI, A. S. TABAJARA, G. D. GAMARO, G. DANTAS, I. L. S. TORRES, A. B. LUCION, AND C. DALMAZ. 2004. Neonatal handling alters feeding behavior of adult rats. Physiology and Behavior 80:739–745. SMITS, J. E., G. R. BORTOLOTTI, AND J. L. TELLA. 1999. Simplifying the phytohemagglutinin skintesting technique in studies of avian immunocompetence. Functional Ecology 13:567–572. SMITS, J. E., K. J. FERNIE, G. R. BORTOLOTTI, AND T. A. MARCHANT. 2002. Thyroid hormone suppression and cell-mediated immunomodula-

tion in American Kestrels (Falco sparverius) exposed to PCBs. Archives of Environmental Contamination and Toxicology 43:338–344. TELLA, J. L., G. R. BORTOLOTTI, M. G. FORERO, AND R. D. DAWSON. 2000. Environmental and genetic variation in T-cell-mediated immune response of fledgling American Kestrels. Oecologia 123:453–459. TELLA, J. L., A. SCHEUERLEIN, AND R. E. RICKLEFS. 2002. Is cell-mediated immunity related to the evolution of life-history strategies in birds? Proceedings of the Royal Society of London Series B 268:1059–1066. TILGAR, V., R. MA¨ND, P. KILGAS, AND S. J. REYNOLDS. 2005. Chick development in freeliving Great Tits Parus major in relation to calcium availability and egg composition. Physiological and Biochemical Zoology 78:590–598. TSCHIRREN, B., P. S. FITZE, AND H. RICHNER. 2003. Sexual dimorphism in susceptibility to parasites and cell-mediated immunity in Great Tit nestlings. Journal of Animal Ecology 72:839–845. WASHBURN, B. E., D. L. MORRIS, J. J. MILLSPAUGH, J. FAABORG, AND J. H. SCHULZ. 2002. Using a commercially available radioimmunoassay to quantify corticosterone in avian plasma. Condor 104:558–563. WESTNEAT, D. F., J. WEISKITTLE, R. EDENFIELD, T. B. KINNARD, AND J. P. POSTON. 2004. Correlates of cell-mediated immunity in nestling House Sparrows. Oecologia 141:17–23. WHITMAN, B. A. 2006. The effects of neonatal handling on adrenocortical responsiveness, morphological development, and corticosterone binding globulin in American Kestrels (Falco sparverius) and European Starlings (Sturnus vulgaris). M.Sc. thesis. Boise State University, Boise, ID. WINGFIELD, J. C., J. P. SMITH, AND D. S. FARNER. 1982. Endocrine responses of White-crowned Sparrows to environmental stress. Condor 84: 399–409.