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Effects of testosterone and corticosterone on immunocompetence in the zebra finch
Hormones and Behavior 51 (2007) 126 – 134 www.elsevier.com/locate/yhbeh
Effects of testosterone and corticosterone on immunocompetence in the zebra finch Mark L. Roberts a,⁎, Katherine L. Buchanan b , Dennis Hasselquist c , Matthew R. Evans a a
Centre for Ecology and Conservation, School of Biosciences, University of Exeter, Cornwall Campus, Penryn, Cornwall TR10 9EZ, UK b Cardiff School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3TL, UK c Animal Ecology, Lund University, Ecology Building, Sölvegatan 37, 22362 Lund, Sweden Received 13 February 2006; revised 4 September 2006; accepted 5 September 2006 Available online 17 October 2006
Abstract The original immunocompetence handicap hypothesis (ICHH) suggested that testosterone has a handicapping effect in males by both promoting the development of sexual signals and suppressing immune function. A modified version, the stress-linked ICHH, has recently proposed that testosterone is immunosuppressive indirectly by increasing production of corticosterone. To test both the original and stressmediated versions of the ICHH, we implanted male zebra finches taken from lines selected for divergent maximum stress-induced levels of corticosterone (high, low and control) with either empty or testosterone-filled implants. Their humoral and cell-mediated immune responses were then assessed by challenge with diphtheria:tetanus vaccine and phytohemagglutinin respectively. We found no effect of the hormone manipulations on either PHA or tetanus antibody responses, but found a significant interaction between titers of both testosterone and corticosterone on diphtheria secondary antibody response; antibody response was greatest in individuals with high levels of both hormones. There was also a significant interactive effect between testosterone treatment group and corticosterone titer on body mass; the body mass of males in the elevated testosterone treatment group decreased with increasing corticosterone titer. These results suggest that, contrary to the assumption of the stress-mediated version of the ICHH, high plasma levels of corticosterone are not immunosuppressive, but are in fact immuno-enhancing in the presence of high levels of plasma testosterone. Equally, the central assumption of the ICHH that testosterone is obligately immunosuppressive is also not supported. The same individuals with the highest levels of both hormones and consequently the most robust antibody response also possessed the lowest body mass. © 2006 Elsevier Inc. All rights reserved. Keywords: Corticosterone; Testosterone; Glucocorticoid; Zebra finch; Immunocompetence; Stress; Immunocompetence handicap hypothesis; PHA; Diphtheria: tetanus
Introduction The immunocompetence handicap hypothesis (ICHH) suggests that testosterone (T) serves a dual role in signal expression and immune function (Folstad and Karter, 1992). High levels of T result not only in full signal expression but also a concomitant reduction in immunocompetence. Therefore, only high quality males can afford to fully express sexual traits because only they will be able to resist or tolerate parasite/ ⁎ Corresponding author. Max Planck Institute for Ornithology, Vogelwarte Radolfzell, Schlossallee 2, 78315 Radolfzell, Germany. E-mail address: [email protected] (M.L. Roberts). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.09.004
pathogen attack. One of the key assumptions of the ICHH is the immunosuppressive nature of T or any biochemical substance that is related to sexual signal expression (Folstad and Karter, 1992). However, several correlational and manipulative studies carried out since the ICHH was first proposed have only produced equivocal evidence to support the ICHH in its simplest form (with T being the immunosuppressive agent; see Roberts et al., 2004). It is generally agreed that T does have immunosuppressive characteristics in mammals (Grossman, 1985), but studies in birds have yielded contradictory results. Several studies in which T has been experimentally manipulated have found the hormone to be immunosuppressive in birds (Buchanan et al., 2003; Casto et al., 2001; Duffy et al., 2000;
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Owen-Ashley et al., 2004; Peters, 2000), whereas other studies have not found any such effect (Hasselquist et al., 1999). Indeed, some studies have found a positive effect of T on immunity (Evans et al., 2000). A recent meta-analysis that examined the effects of testosterone on immunity in vertebrates confirmed this ambiguity; only when considering ectoparasites on manipulated lizards did T have a demonstrably negative effect on a measure of immunity (Roberts et al., 2004). In some recent studies, T has been found to correlate (both negatively and positively) with the glucocorticoid stress hormone in birds, corticosterone (CORT) (e.g. Evans et al., 2000; Klukowski et al., 1997; Owen-Ashley et al., 2004; Parker et al., 2002; Schoech et al., 1999). The stimulation of the hypothalamus–pituitary–adrenal axis in response to stress leads to the secretion of CORT, which is involved in the mobilization of energy stores (glucose), the shutdown of digestive processes and increasing the peripheral blood supply (Buchanan, 2000; Sapolsky et al., 2000; Silverin, 1998). CORT also affects behavior — it reduces reproductive activity, increases dispersal behavior and increases foraging activity (Breuner et al., 1998; Silverin, 1998; Wingfield et al., 1997). T increases circulating CORT in several avian species (Casto et al., 2001; Duffy et al., 2000; Evans et al., 2000; Owen-Ashley et al., 2004; Poiani et al., 2000), and there is good evidence to suggest that CORT is immunosuppressive (e.g. Buchanan, 2000; Harvey et al., 1984; Råberg et al., 1998; Sapolsky et al., 2000; Wingfield et al., 1997; but see Svensson et al., 2002). In a study on the house sparrow (Passer domesticus), Evans et al. (2000) found that experimentally increased T impaired antibody production. However, after controlling for the effect of CORT (that positively covaried with T), T was found to enhance immunocompetence. The authors of this study suggested a modification to the original ICHH; rather than being immunosuppressive directly, T-related immunosuppression occurs indirectly through an increase in CORT. T itself may be immuno-enhancing, again indirectly through its effect on behavior leading to more dominant individuals (with higher T) gaining greater access to dietary resources and therefore being in better condition (Evans et al., 2000). Therefore, the fact that T and CORT may interact with each other requires that the levels of both hormones be manipulated independently to distinguish between the immunosuppressive effect (if any) of the two hormones. Several studies have reported a negative effect of elevated testosterone on avian body condition (defined as body mass in relation to body size) (Clotfelter et al., 2004; Mougeot et al., 2004; Ros, 1999; Wikelski et al., 1999), and other studies have found increased CORT levels to be negatively related to body condition (Breuner and Hahn, 2003; Hood et al., 1998; Kitaysky et al., 2001; Pereyra and Wingfield, 2003; Perfito et al., 2002; Schwabl, 1995; Sockman and Schwabl, 2001). In some cases, however, no effect of either elevated CORT or T has been found on body condition (Lormee et al., 2003; Lynn et al., 2003; and Alonso-Alvarez et al., 2002 Buttemer and Astheimer, 2000; respectively), and in a few studies, testosterone appeared to have a positive effect (Briganti et al., 1999; Chastel et al., 2005). It is therefore unclear whether either hormone has an effect (positive or negative) on body mass. An additional aim of
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this experiment was to elucidate the effect of elevated testosterone and corticosterone on avian body condition defined as body mass corrected for skeletal size. The study reported here was conducted to ascertain the effect of manipulating levels of T and CORT on individual immunocompetence and general body condition. If the original version of the ICHH (that assumes that T is directly immunosuppressive) is to be supported, a high T treatment group should have the poorest immune response, and the males with the lowest T should have the highest immune response, regardless of CORT levels. However, if the modified version of the ICHH that identifies glucocorticoids as the immunosuppressive agent is to be supported, then males with a low peak (stress) CORT response should have a more robust immune response than males with a high peak CORT response, regardless of testosterone titers. Although the stress-linked version of the ICHH is based upon measurements of baseline levels of plasma corticosterone, there is good evidence to suggest that baseline and stress-induced levels of CORT positively covary (e.g. Cockrem and Silverin, 2002; Romero and Wingfield, 1998; Schoech et al., 1999). Moreover, there is considerable evidence for immunosuppressive effects mediated through Type II receptors, which are only activated at elevated (as opposed to basal) levels of CORT (Sapolsky et al., 2000). Populations of zebra finch (Taeniopygia guttata) were selected for low, high and control levels of plasma corticosterone, based upon the maximum stress-induced response (Evans et al., 2006). Males from the G4 generation were housed under short day photoperiod (to remove any confounding effect of endogenous testosterone production — see Bentley et al., 2000), and individuals from each corticosterone line were allocated equally into two testosterone treatment groups — high and low T. These males were then challenged by phytohemagglutinin injection to test their cell-mediated immunity and diphtheria: tetanus injection to test their humoral response. In this way, the levels of both hormones could be artificially fixed so that different arms of the immune system could be challenged and the response compared between both CORT and T treatment groups. Materials and methods Selection program Since 1999, three replicate lines of zebra finch (2 × low, control and high CORT; 6 lines in total) were selected for contrasting levels of maximum stressinduced CORT in response to a mild stressor (Evans et al., 2006). A significant difference in plasma corticosterone levels was observed between the lines in the expected direction from the F2 generation of selection, with selection pressure exerted on each generation. There was a downward trend in corticosterone over generation regardless of selection line; little difference has existed in changes in corticosterone titer between the low lines and controls, but the high lines have shown a significant realized heritability of 20–25%. There was no corresponding change in testosterone over the generations, and no significant differences exist between the corticosterone lines in testosterone (Evans et al., 2006). Birds in each line were housed together in a large aviary giving c1m3 per breeding pair and maintained at an ambient temperature of 20–24 °C (Jones and Slater, 1999). The humidity was maintained between 50 and 70%, and the rooms sprayed with water two or three times per day. The birds were provided with ad libitum seeds (foreign finch mixture, Haiths Ltd., Cleethorpes, Lincolnshire, UK), Chinese millet sprays, mineralized grit, water and cuttlefish bone. The finches were
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provided with c10g of a 3:1 mixture of ‘nectarblend’ (Haiths Ltd., Cleethorpes, Lincolnshire, UK) and egg biscuit food (Haiths Ltd., Cleethorpes, Lincolnshire, UK), and either lettuce or cucumber daily with a cod liver oil supplement in the seed weekly. An excess of nesting baskets and boxes was provided. Full details of the selection methodology, characteristics and housing can be found in Evans et al. (2006). We have taken every care to minimize the welfare implications of this project, and all work was carried out under license by the UK Home Office and after local ethical review.
Hormone sampling and assay characteristics Six weeks post fledging, all birds were blood sampled. Blood samples for CORT (100 μl) were taken from the brachial vein after 20 min holding in a cloth bag, after a pilot study revealed that peak CORT response occurs in the zebra finch after 20 min of a stressful stimulus (Evans et al., 2006). This is the standard capture-restraint protocol used in studies analyzing the CORT response (Wingfield, 1994). The blood was centrifuged at 11,000×g for 15 min and the plasma frozen. Corticosterone concentrations were measured after extraction of 20 μl aliquots of plasma in diethyl ether, by radioimmunoassay (Wingfield et al., 1992) using anticorticosterone antiserum (code B21-42 and B3-163, Esoterix Inc. Endocrinology, CA) and [1,2,6,7-3H]-corticosterone label (Amersham, UK). The interassay coefficient of variation was 15.7%, and the intraassay coefficient of variation 3.1%. The mean extraction efficiency was 72%. The assay was run with 50% binding at 134 pg/tube, and the detection limit (for 7.3 μl aliquots of extracted plasma) was 1.76 nmol l− 1. Blood samples for testosterone assay were taken immediately upon capture, and the plasma obtained and stored in an identical manner to the CORT samples. Testosterone concentrations were measured in plasma samples by direct radioimmunoassay using anti-testosterone antiserum (code 8680-6004, Biogenesis, UK) and [125I]-testosterone label (code 07-189126, ICN, UK) (Parkinson and Follett, 1995). The assay was run with 50% binding at 11.0 pg/tube and a detection limit of 0.068 nmol l− 1 for the 20 μl plasma volumes that were run in the assay. The interassay coefficient of variation was 15.5%, and the intraassay coefficient of variation was 2.2%.
Implantation of testosterone Following experimental trials with birds outside the selection lines, a 7 mm implant size (Dow Corning medical grade tubing RX-50: inner diameter 0.76 mm, outer diameter 1.65 mm) was deemed most appropriate to give a high plasma T level, within the natural physiological range. Both the males used in the pilot study and the main experiment were housed under a short day photoperiod (18 h dark:6 h light) to limit the production of endogenous T. Although castration may have limited the production of endogenous T more effectively, such a procedure on a small passerine may well have resulted in morbidity and possible mortality. In addition, the procedure may affect an individual's condition and CORT titer, thus introducing greater variation and possible confounding elements into the experimental design. Furthermore, reduced photoperiod has been shown to cause a decrease in testicular mass relative to increased photoperiod in zebra finches (Bentley et al., 2000). The main experiment consisted of 72 adult male zebra finches taken from the selected lines. Twelve birds were taken from each of the 3 replicate (×2) CORT lines, and these were randomly subdivided into 6 empty-implanted and 6 testosterone-implanted birds. The implants in the high T groups were packed with crystalline T (Sigma T-1500), while empty implants were inserted into the control group birds. The silastic implants were inserted into an incision made in the skin of the neck. This was then closed by application of a tissue adhesive (Nexaband, Abbott Labs, Chicago, US) and where necessary by surgical suture. The implantation process took a total of 2 days to complete, and both CORT and T groups were balanced across these 2 days.
et al., 1993). A spessimeter (Alpa s.r.l. Milan) was used to measure the wing web before injection (as a control measurement), and at 24 h after injection (to the nearest 0.01 mm), to measure the wing web swelling in response to the mitogen. The increase in the swelling was used as an indicator of immune response. All the males were then weighed to the nearest 1 g using a Pesola spring balance, and their right tarsus lengths measured to the nearest 0.1 mm with digital calipers.
Humoral response Three days after the PHA injections, the males were blood sampled. One hundred microliters of blood was collected in heparinized capillary tubes and centrifuged at 11,000×g for 15 min. The plasma was removed and stored at −20 °C and subsequently tested for cross-reaction to diphtheria:tetanus as a control measurement. Three days after this, the primary humoral immune response of the males was tested. Each male was immunized with 100 μl of diphtheria:tetanus vaccine (Aventis Pasteur, Swiftwater, PA) by intraperitoneal injection. Twelve days later, blood samples were taken from the brachial vein. Twenty one days after the initial injection, the males were inoculated again with diphtheria:tetanus vaccine to test the secondary humoral response. Eight days after this, the birds were again blood sampled, and the samples split between testing for antibodies and for testosterone assay. In total, three blood samples were taken from each manipulated male. Antibody levels were determined by the use of ELISA (see Hasselquist et al., 1999; Owen-Ashley et al., 2004 for a full description of the methodology employed).
Statistical analyses The degree of swelling exhibited by the wing web injected with PHA (the difference between pre- and 24-hour post-injection) was used as the dependent variable in Restricted Maximum Likelihood models (ReMLs). This form of Generalized Linear Model (GLM) has been widely used before in similar studies (e.g. Evans et al., 2006; Peters, 2000; Peters et al., 2004). The maximal model consisted of T treatment group, CORT selection line, body condition (see below), T titer and peak CORT titer as fixed effects, and CORT line replicate as a random term. A minimal model was derived by stepwise deletion by removal of nonsignificant terms that did not significantly increase the residual deviance of the model. The residuals of the models conformed to a normal distribution and were homoscedastic so the models were run with a Gaussian distribution in Genstat 6. The same procedure was followed for the models containing control, primary and secondary levels of anti-diphtheria and anti-tetanus antibodies as response variables. Therefore, in total, there were six separate models with a measure of humoral immunity as the dependent variable. The residuals of the models were checked for homoscedasticity and normality and where necessary the response variables were transformed appropriately. Finally, the residuals from a reduced major axis regression of mass against tarsus length were used as a dependent variable with all other variables mentioned included as explanatory variables (again with replicate line as the random term) to ascertain whether body condition as defined as body mass controlling for skeletal size was affected by testosterone and/or corticosterone manipulations and titers. The residuals derived from a least squares regression of mass against tarsus length are not thought to be an appropriate measure of condition (see Darlington and Smulders, 2001; Green, 2001), so we used reduced major axis regression as recommended by Green (2001). This measure of body condition was included in the immunity models as an explanatory variable. The residuals of the model conformed to a normal distribution and were homoscedastic, so a Gaussian model was appropriate for this model. In all of the above models, all 2-way interactions were included between treatment groups and hormone titers.
Results Cell-mediated immune response
Plasma hormone levels The males' cell-mediated immunity was challenged by phytohemagglutinin (PHA) injection 8–9 days after implantation (balanced across treatment groups). Each male was injected with the mitogen phytohemagglutinin (PHA-P, Sigma, St. Louis, MO) intradermally into the left wing web. Each male received 30 μl of a suspension of 5 mg PHA-P in 1 ml phosphate buffer saline (1× PBS) (Lochmiller
There was a significant difference in the expected direction in maximum stress-induced CORT levels between the CORT selection lines (Wald = 96.96, df = 2, p < 0.001; see Fig. 1).
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Fig. 1. Predicted means of log10 body mass controlling for skeletal size (a) and mean levels of maximum stress-induced plasma corticosterone (b) in replicate lines of zebra finch (n = 24 per line) selected for divergent levels of corticosterone ± SE. The lines differed significantly in CORT level (Wald = 96.96, df = 2, p < 0.001) and body mass (Wald = 16.99, df = 2, p < 0.001).
There was also a significant difference between the testosterone treatment groups, again in the expected direction (mean control = 2.91 nmol l− 1, SE ± 0.52, mean T-treated = 12.67 nmol l− 1, SE ± 1.25; Wald = 63.64, df = 1, p < 0.001). Immune function Table 1 gives the Wald statistics divided by their respective degrees of freedom (as well as significance) for all the explanatory variables included in all models containing immune responses and body condition as the dependent variables. There was no significant effect of any of the independent variables on the size of wing web swelling at 24 h after PHA injection (CORT line: Wald = 0.30, df = 2, p = 0.86; testosterone group: Wald = 0.94, df = 1, p = 0.33; p > 0.05 for all covariates). The only measure of humoral immunity that was significantly affected by the hormone treatments was the secondary antibody response against diphtheria. There were significant interactions
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between CORT titer and T titer (Wald = 4.28, df = 1, p = 0.04; Fig. 2), and between CORT selection line and T titer (Wald = 8.89, df = 2, p = 0.01; Fig. 2). Fig. 2 shows that the highest antibody response was exhibited by the males with the highest levels of both CORT and T, but there is a suggestion that high levels of T were immunosuppressive at low levels of CORT in the high CORT selection line. This is indicated as dark shading (low antibody response) at low levels of CORT and high levels of T (Fig. 2(c)). To investigate these interactions further, we tested whether corticosterone and testosterone titers positively covaried with secondary antibody response to diphtheria and what relationship testosterone titer had with the same antibody response, in each CORT line separately. We found a non-significant, positive interaction between both hormone titers and antibody response in the low and high lines, and a strongly significant positive relationship in the control line (Wald = 11.51, df = 1, p < 0.001). Testosterone titers had a nonsignificant negative relationship with antibody response in the low line; a significant positive relationship in the control line (Wald = 13.99, df = 1, p < 0.001); and a significant negative relationship with antibody response in the high line (Wald = 4.75, df = 1, p = 0.029). However, it should be borne in mind that such results are more prone to a Type I error, and obviously rely on smaller sample sizes, than when relationships are inferred from models containing data from all treatment levels. The other covariates and interactions included in the models had no significant effect on humoral immunity (p > 0.05 in all cases). Given that in previous avian studies testosterone implantation has had a significant effect on both PHA response and antibody response to diphtheria:tetanus injection (Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004), we carried out power analyses to ascertain whether our sample sizes were too small to detect any significant differences in immune response between testosterone treatment groups. In all cases, the sample sizes obtained were far greater than both ours and the sample sizes used in the studies that did find an effect of testosterone on immunity (see Table 2). Indeed, the most similar study to ours that implanted testosterone and immunized with diphtheria:tetanus as well as injected with PHA found
Table 1 Effects of body condition and hormone treatment groups and titers on immune response and condition in zebra finches Explanatory variables
Response variables Diphtheria
Body condition CORT titer CORT selection line T titer T treatment group CORT line* T titer CORT line* T treatment group T treatment group* CORT titer CORT titer* T titer
Tetanus
Control
Primary
Secondary
Control
Primary
Secondary
0.06 0.02 1.81 0.29 0.34 0.97 1.19 0.28 1.18
<0.01 1.45 2.38 0.53 0.07 2.68 1.03 1.12 0.13
1.81 2.10 1.96 0.75 0.25 4.45* 2.46 0.04 4.28*
0.46 0.33 1.67 1.11 0.73 1.48 1.56 0.41 0.14
0.29 3.12 1.36 0.34 0.12 0.30 1.53 0.92 0.52
0.01 2.11 2.25 1.54 0.53 0.22 0.70 1.46 0.26
PHA
Body condition
0.22 0.40 0.15 0.32 0.94 1.94 1.11 0.81 0.21
– 2.83 8.50*** 2.16 2.53 0.10 0.15 7.96** 0.11
Variation explained by each explanatory variable is expressed by Wald statistics divided by its respective degrees of freedom (*p < 0.05, **p < 0.01, ***p > 0.001).
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Fig. 2. Fitted model of the relationship between testosterone titer, corticosterone titer and diphtheria secondary antibody response in males selected for (a) low, (b) control and (c) high maximum stress-induced levels of plasma corticosterone (n = 24 per line). Antibody response is expressed as a percentage of the standard and is log10 transformed. Lighter shades represent higher antibody responses. The interactions between CORT and testosterone titer (Wald = 4.28, df = 1, p = 0.04) and CORT line and testosterone titer (Wald = 8.89, df = 2, p = 0.01) were significant.
significant differences between treatment groups with samples sizes of 6 birds in each group (Owen-Ashley et al., 2004). For this reason, we suggest that our sample sizes were sufficiently large to detect any differences.
of mass in the empty-implanted (control T) group, but had a significant, negative relationship with body mass in the high T group (Wald = 6.46, df = 1, p = 0.011). Discussion
Body mass There was a significant effect of CORT selection line such that birds in the high line had greater mass (after controlling for skeletal size) than controls and low CORT birds (Wald = 16.99, df = 2, p < 0.001; see Fig. 1) (see Table 1). In addition, an interaction between CORT titer and T treatment group also had a significant effect on body mass (Wald = 7.96, df = 1, p = 0.005; see Fig. 3). T implanted birds showed a greater decrease in mass with increasing CORT production than empty implanted birds. As with the significant interactions reported for secondary antibody response to diphtheria, we tested the nature of the relationship between CORT titer and body mass within each testosterone treatment group. We found that CORT titer was not a significant predictor Table 2 Results from power analyses showing sample sizes required to give significant differences between T treatment groups (α = 0.05) in immune response given the effect sizes obtained, at a power of 80% Immune test
T treatment group
From the results of the present experiment, it appears that corticosterone and testosterone have an interactive effect on immunity. Although no effect of either manipulated hormone was found on cell-mediated immunity as measured by PHA response, there was a significant, positive relationship between the plasma levels of both hormones and the birds' secondary antibody response to diphtheria; but this was only the case when the other hormone was also at high levels in the blood plasma. Whereas in the low and control lines there was a positive relationship between T level (at high levels of CORT) and secondary diphtheria antibody response; in the high line there was a negative relationship at low levels of CORT. There was nevertheless a positive relationship between T and antibody titer at high levels of CORT. As can be seen in Fig. 2, when all interactions are included in the predicted model, the general positive interactive effect the two hormones have on antibody
Mean value Standard Sample size required obtained deviation in each treatment group
Primary tetanus Control 6.6 Testosterone 9.7 Secondary Control 72.1 tetanus Testosterone 69.4 Primary Control 0.39 diphtheria Testosterone 0.43 Secondary Control 2.42 diphtheria Testosterone 1.42 PHA Control 0.32 Testosterone 0.28
10.3 17.1 40.0 32.6 0.86 0.78 4.02 2.04 0.16 0.16
326 2867 6613 160 252
Fig. 3. Relationship between corticosterone titer, body mass and testosterone treatment group. Triangles and solid trend line represent empty-implanted birds (n = 36); circles and dashed trend line represent testosterone-implanted birds (n = 36). The interaction was significant (Wald = 7.96, df = 1, p = 0.005).
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response is evident. In the males selected for high levels of CORT, testosterone appears to be immunosuppressive, but only in the presence of low levels of plasma corticosterone. The results confirmed that the CORT lines differed significantly in maximum stress-induced levels of CORT and that the testosterone treatment groups differed significantly in T in the expected direction. Little is known about wild male zebra finch T levels, and these may not be relevant to our captive selected finch stocks, but the manipulations changed the testosterone-implanted males' T levels to be relatively high compared to the levels of the empty-implanted males. These levels however remained within physiological limits as the levels were at the upper limit of the range of T titers previously recorded in this generation of zebra finches. Males from the high CORT lines had significantly greater mass (when controlling for tarsus length) than males from the low CORT lines. There was a significant, negative relationship between actual CORT plasma levels and body mass in the high T treatment group. In general, individuals with both the highest CORT and T plasma levels exhibited the greatest secondary antibody response against diphtheria. This is contrary to our understanding that these hormones are immunosuppressive, and as this is a manipulative experiment, contrary to the predictions of the ICHH. There was a suggestion from the results that, in the presence of low levels of CORT, T may be immunosuppressive, but only in individuals selected for high levels of CORT (Fig. 2 (c)). In the other CORT lines, testosterone and corticosterone titers together were positively related to antibody response. This may be an idiosyncratic characteristic of the high CORT line or may suggest that birds with genes for a high maximum stressinduced CORT response are immunosuppressed by high levels of T if CORT levels are relatively low. Nevertheless, the overall effect of both hormones on humoral response is positive when both hormones are at high levels in the blood plasma. Previous studies have generally found either T or CORT to be immunosuppressive (Buchanan et al., 2003; Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004; Peters, 2000; Evans et al., 2000; Råberg et al., 1998; Wingfield et al., 1997 respectively). In contrast, a few studies have found these hormones to exhibit immuno-enhancing properties (T: Evans et al., 2000; Peters, 2000; CORT: Svensson et al., 2002). This is the first experiment to our knowledge that has found a significant, positive interactive effect of both hormones on antibody response. Testosterone may have a positive effect on immunocompetence by increasing aggressive behavior and therefore dominance ranking; this may lead to greater access to dietary requirements, and consequently more resources are made available for immune defense (Evans et al., 2000). The birds in this study were housed together and may have competed for food; however, they were fed ad libitum. Therefore, it is not clear how greater dominance caused by testosterone treatment affected dietary intake. An alternative explanation is that, rather than being able to obtain more or better quality food, males with high testosterone may be better able to utilize the food they ingest for immune function. Exogenous testosterone administration has been found to
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increase plasma carotenoid concentrations via upregulation of carotenoid-binding lipoproteins (McGraw et al., 2006). Carotenoid concentration in the blood is in turn known to have a positive effect on immune function and antioxidant defenses (Alonso-Alvarez et al., 2004; Blount et al., 2003; Faivre et al., 2003; McGraw and Ardia, 2003). Elevated CORT levels result in an increase in foraging behavior and mobilization of glucose reserves (Breuner et al., 1998; Buchanan, 2000; Silverin, 1998; Wingfield et al., 1997), therefore an increase in both hormones may result in an excess of resources readily accessible for producing and regulating an antibody response. Moreover, peak CORT levels (as opposed to basal levels) and acute stress have also been found to enhance immune response in several studies (e.g. Cocke et al., 1993; Dhabhar and McEwen, 1999; Persoons et al., 1995; see Dhabhar, 2002 for a review). Thus, a combination of both high testosterone and high peak levels of corticosterone in the blood may instigate either directly or indirectly a potent immunostimulatory effect. Several studies have found that artificially increasing testosterone correspondingly increases CORT levels (Casto et al., 2001; Duffy et al., 2000; Evans et al., 2000; Owen-Ashley et al., 2004). By increasing T in individuals with naturally low levels of T, an increase in CORT may be a predictable phenomenon, partly because the methodology employed to increase T is probably stressful and should increase CORT levels (see Moore et al. 2004) and partly because increasing T in males with naturally low levels of the androgen may in itself be stressful for the individual concerned and consequently raise CORT levels, quite apart from the implantation procedure itself. Individuals with naturally low levels of testosterone may well be stressed by suddenly finding themselves with high (often above physiological) levels of the hormone; this does not necessarily mean that the two hormones naturally positively covary. If the two hormones do positively correlate in freeliving birds (and there are some data to support this, see Johnsen, 1998; Mateos, 2005), females would be expected to choose males with both high T (for their sexual signals) and concomitantly high CORT. These males would have the greatest humoral immune response, so females would be choosing males with the highest immunocompetence. This interpretation is consistent with Hamilton and Zuk's (1982) parasite theory. However, it should be borne in mind that we measured peak levels of CORT in this study; although basal levels of CORT may positively covary with T, it does not necessarily imply any relationship between peak CORT and T. There are examples of the two hormones negatively covarying within individuals in multiple taxa (in birds: Parker et al., 2002; in reptiles: Knapp and Moore, 1997; Lance et al., 2003; Moore et al., 2001; but see Moore et al., 2000; in mammals: Sankar et al., 2000). The important question that needs to be addressed is how do both peak and basal CORT and testosterone naturally interact within free-living individuals. This point is crucial in understanding the results of laboratory-based studies on immunocompetence and endocrine systems in birds. There was no effect of either hormone on the cell-mediated immune response or on several measures of humoral immunity.
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Power analyses suggested that the sample sizes of our treatment groups needed to be much larger to find a significant effect of T treatment given the mean differences obtained; however, significant effects of testosterone treatment on PHA and antibody responses have been found in previous studies on much smaller sample sizes (e.g. Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004). In addition, other studies have found no effect of testosterone treatment on the cell-mediated response (Buchanan et al., 2003; Greenman et al., 2005). Therefore, although we cannot definitely conclude that this null result is not merely due to a small sample and effect size, it seems more likely that this is a real result. The important point to note is that, although no significant result was obtained, neither hormone had an immunosuppressive effect on either the cell-mediated response or on antibody production. Body mass was negatively related to CORT levels, but only in the high T treatment group. In addition to this, the males in the high CORT lines were on average in better condition than males from the low CORT lines when all the independent parameters were accounted for in the model. These results would suggest that both high T and high CORT in the same individual will have a negative effect on general body mass. But high CORT with relatively lower levels of T will have a positive effect on mass. The alternative explanation is that differences between the CORT lines may be an artefact of the selection process, and rather than CORT per se affecting condition in this way, it is possible that another determinant of condition may have been incidentally selected. Nevertheless, this explanation does not account for the negative effect of both high T and high CORT titer together on body condition. Several studies have found a negative effect of T on body condition or body mass (Clotfelter et al., 2004; Mougeot et al., 2004; Ros, 1999; Wikelski et al., 1999), as well as a similar effect for CORT (Sockman and Schwabl, 2001), so this result should not be too surprising. However, given that males with simultaneously the highest T and highest CORT titers exhibited the greatest humoral immunity, it seems counter-intuitive to find that males in the high testosterone treatment group with the highest CORT titers males were also in the poorest condition, particularly if these individuals had the highest immunocompetence due to greater access to resources caused by high levels of both hormones. The reasons for this are unclear, but it is possible that these individuals quickly expend any excess resources on energy demanding processes such as immune defense and general locomotor activity. Low body mass will result if high plasma CORT levels lead to glucose being quickly broken down (therefore more resources will rapidly be made available for immune defense). Additionally, elevated levels of both CORT and T have been found to increase basal metabolic rate and locomotor activity (Breuner et al., 1998; Buchanan et al., 2001; Lynn et al., 2000; Wikelski et al., 1999). In addition, the positive relationship we found between antibody production and simultaneously high levels of both hormones may have nothing to do with body mass or condition per se, but instead be related to direct as well as indirect effects of the hormones in terms of increasing carotenoid availability and stimulating antibody production.
Our study suggests that testosterone-mediated signaling may not be costly in terms of immunosuppression, but may detrimentally affect body condition. Therefore, poorer quality males that elevate testosterone for sexual signaling may pay the price in times of environmental hardship when food is scarce. Higher quality males may be able to elevate testosterone without losing body mass or alternatively be able to resist the negative effects of low body mass. Our results certainly suggest that the T-mediated costs of sexually selected traits do not occur through direct immunosuppression but may occur through metabolic (e.g. Buchanan et al., 2001) or dominance/aggression costs (e.g. Rohwer and Ewald, 1981). Only one measure of immunity exhibited any relationship with either CORT or T, suggesting that these hormones do not consistently affect all aspects of an individual's immune response. It is important to note that the birds used in this study were selected for maximum stress-induced levels of CORT, and it was this measure of CORT interacting with T that showed a relationship with the secondary antibody response against diphtheria. Basal levels of CORT were not measured, and these may well have had an effect that was missed. Nevertheless, this experiment demonstrates that, contrary to the generally accepted consensus, CORT and T are not always associated with immunosuppression. Acknowledgments We would like to thank the staff of the Animal Unit at the University of Stirling for their assistance with the selection program and to Alistair Dawson for his advice on the implantation procedure; we are also grateful to A.R. Goldsmith for access to the RIA facilities at the University of Bristol. All work was conducted under Home Office license PPL 60/2584. MLR was funded by a studentship from NERC, and the selection program under MRE and KLB was funded at various times by the Royal Society, ASAB, NERC and the University of Stirling, and DH was funded by the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (Formas), the Carl Trygger Foundation, the Swedish Research Council (VR) and the Crafoord Foundation. One anonymous referee and Julio Blas provided very helpful comments on an initial version of the manuscript. References Alonso-Alvarez, C., Ferrer, M., Figuerola, J., Veira, J.A.R., Estepa, J., Torres, L.M., 2002. The effects of testosterone manipulation on the body condition of captive male yellow-legged gulls. Comp. Biochem. Physiol., A 131, 293–303. Alonso-Alvarez, C., Bertrand, S., Devevey, G., Gaillard, M., Prost, J., Faivre, B., Sorci, G., 2004. An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity. Am. Nat. 164, 651–659. Bentley, G.E., Spar, B.D., MacDougall-Shackleton, S.A., Hahn, T.P., Ball, G.F., 2000. Photoperiodic regulation of the reproductive axis in male zebra finches, Taeniopygia guttata. Gen. Comp. Endocrinol. 117, 449–455. Blount, J.D., Metcalfe, N.B., Birkhead, T.R., Surai, P.F., 2003. Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science 300, 125–127.
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Hasselquist, D., Marsh, J.A., Sherman, P.W., Wingfield, J.C., 1999. Is avian humoral immunocompetence suppressed by testosterone? Behav. Ecol. Sociobiol. 45, 167–175. Hood, L.C., Boersma, P.D., Wingfield, J.C., 1998. The adrenocortical response to stress in incubating Magellanic penguins (Spheniscus magellanicus). Auk 115, 76–84. Johnsen, T.S., 1998. Behavioural correlates of testosterone and seasonal changes of steroids in red-winged blackbirds. Anim. Behav. 55, 957–965. Jones, A.E., Slater, P.J.B., 1999. The zebra finch. In: Poole, T. (Ed.), UFAW Handbook on the Care and Management of Laboratory Animals. Blackwells, Oxford. Kitaysky, A.S., Kitaiskaia, E.V., Wingfield, J.C., Piatt, J.F., 2001. Dietary restriction causes chronic elevation of corticosterone and enhances stress response in red-legged kittiwake chicks. J. Comp. Physiol., B 171, 701–709. Klukowski, L.A., Cawthorn, J.M., Ketterson, E.D., Nolan Jr., V., 1997. Effects of experimentally elevated testosterone on plasma corticosterone and corticosteroid-binding globulin in dark-eyed juncos (Junco hyemalis). Gen. Comp. Endocrinol. 108, 141–151. Knapp, R., Moore, M.C., 1997. Male morphs in tree lizards have different testosterone responses to elevated levels of corticosterone. Gen. Comp. Endocrinol. 107, 273–279. Lance, V.A., Elsey, R.M., Butterstein, G., Trosclair III, P.L., 2003. Rapid suppression of testosterone secretion after capture in male American alligators (Alligator mississippiensis). Gen. Comp. Endocrinol. 135, 217–222. Lochmiller, R.L., Vestey, M.R., Boren, J.C., 1993. Relationship between protein nutritional status and immunocompetence in northern bob-white chicks. Auk 110, 503–510. Lormee, H., Jouventin, P., Trouve, C., Chastel, O., 2003. Sex-specific patterns in baseline corticosterone and body condition changes in breeding red-footed boobies Sula sula. Ibis 145, 212–219. Lynn, S.E., Houtman, A.M., Weathers, W.W., Ketterson, E.D., Nolan, V., 2000. Testosterone increases activity but not daily energy expenditure in captive male dark-eyed juncos, Junco hyemalis. Anim. Behav. 60, 581–587. Lynn, S.E., Hunt, K.E., Wingfield, J.C., 2003. Ecological factors affecting the adrenocortical response to stress in chestnut-collared and McCown's longspurs (Calcarius ornatus, Calcarius mccownii). Physiol. Biochem. Zool. 76, 566. Mateos, C., 2005. The subordination stress paradigm and the relation between testosterone and corticosterone in male ring-necked pheasants. Anim. Behav. 69, 249–255. McGraw, K.J., Ardia, D.R., 2003. Carotenoids, immunocompetence, and the information content of sexual colours: an experimental test. Am. Nat. 162, 704–712. McGraw, K.J., Correa, S.M., Adkins-Regan, E., 2006. Testosterone upregulates lipoprotein status to control sexual attractiveness in a colorful songbird. Behav. Ecol. Sociobiol. 60, 117–122. Moore, I.T, Lerner, J.P, Lerner, D.T., Mason, R.T., 2000. Relationships between annual cycles of testosterone, corticosterone, and body condition in male red-spotted garter snakes, Thamnophis sirtalis concinnus. Physiol. Biochem. Zool. 73, 307–312. Moore, I.T., Greene, M.J., Mason, R.T., 2001. Environmental and seasonal adaptations of the adrenocortical and gonadal responses to capture stress in two populations of the male garter snake, Thamnophis sirtalis. J. Exp. Zool. 289, 99–108. Mougeot, F., Irvine, J.R., Seivwright, L., Redpath, S.M., Piertney, S., 2004. Testosterone, immunocompetence, and honest sexual signalling in male red grouse. Behav. Ecol. 15, 930–937. Owen-Ashley, N.T., Hasselquist, D., Wingfield, J.C., 2004. Androgens and the Immunocompetence Handicap Hypothesis: unravelling direct and indirect pathways of immunosuppression in song sparrows. Am. Nat. 164, 490–505. Parker, T.H., Knapp, R., Rosenfield, J.A., 2002. Social mediation of sexually selected ornamentation and steroid hormone levels in male junglefowl. Anim. Behav. 64, 291–298. Parkinson, T.J., Follett, B.K., 1995. Thyroidectomy abolishes testicular cycles of Soay rams. Proc.R. Soc. Lond., Biol. 259, 1–6. Pereyra, M.E., Wingfield, J.C., 2003. Changes in plasma corticosterone and
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Effects of testosterone and corticosterone on immunocompetence in the zebra finch Mark L. Roberts a,⁎, Katherine L. Buchanan b , Dennis Hasselquist c , Matthew R. Evans a a
Centre for Ecology and Conservation, School of Biosciences, University of Exeter, Cornwall Campus, Penryn, Cornwall TR10 9EZ, UK b Cardiff School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3TL, UK c Animal Ecology, Lund University, Ecology Building, Sölvegatan 37, 22362 Lund, Sweden Received 13 February 2006; revised 4 September 2006; accepted 5 September 2006 Available online 17 October 2006
Abstract The original immunocompetence handicap hypothesis (ICHH) suggested that testosterone has a handicapping effect in males by both promoting the development of sexual signals and suppressing immune function. A modified version, the stress-linked ICHH, has recently proposed that testosterone is immunosuppressive indirectly by increasing production of corticosterone. To test both the original and stressmediated versions of the ICHH, we implanted male zebra finches taken from lines selected for divergent maximum stress-induced levels of corticosterone (high, low and control) with either empty or testosterone-filled implants. Their humoral and cell-mediated immune responses were then assessed by challenge with diphtheria:tetanus vaccine and phytohemagglutinin respectively. We found no effect of the hormone manipulations on either PHA or tetanus antibody responses, but found a significant interaction between titers of both testosterone and corticosterone on diphtheria secondary antibody response; antibody response was greatest in individuals with high levels of both hormones. There was also a significant interactive effect between testosterone treatment group and corticosterone titer on body mass; the body mass of males in the elevated testosterone treatment group decreased with increasing corticosterone titer. These results suggest that, contrary to the assumption of the stress-mediated version of the ICHH, high plasma levels of corticosterone are not immunosuppressive, but are in fact immuno-enhancing in the presence of high levels of plasma testosterone. Equally, the central assumption of the ICHH that testosterone is obligately immunosuppressive is also not supported. The same individuals with the highest levels of both hormones and consequently the most robust antibody response also possessed the lowest body mass. © 2006 Elsevier Inc. All rights reserved. Keywords: Corticosterone; Testosterone; Glucocorticoid; Zebra finch; Immunocompetence; Stress; Immunocompetence handicap hypothesis; PHA; Diphtheria: tetanus
Introduction The immunocompetence handicap hypothesis (ICHH) suggests that testosterone (T) serves a dual role in signal expression and immune function (Folstad and Karter, 1992). High levels of T result not only in full signal expression but also a concomitant reduction in immunocompetence. Therefore, only high quality males can afford to fully express sexual traits because only they will be able to resist or tolerate parasite/ ⁎ Corresponding author. Max Planck Institute for Ornithology, Vogelwarte Radolfzell, Schlossallee 2, 78315 Radolfzell, Germany. E-mail address: [email protected] (M.L. Roberts). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.09.004
pathogen attack. One of the key assumptions of the ICHH is the immunosuppressive nature of T or any biochemical substance that is related to sexual signal expression (Folstad and Karter, 1992). However, several correlational and manipulative studies carried out since the ICHH was first proposed have only produced equivocal evidence to support the ICHH in its simplest form (with T being the immunosuppressive agent; see Roberts et al., 2004). It is generally agreed that T does have immunosuppressive characteristics in mammals (Grossman, 1985), but studies in birds have yielded contradictory results. Several studies in which T has been experimentally manipulated have found the hormone to be immunosuppressive in birds (Buchanan et al., 2003; Casto et al., 2001; Duffy et al., 2000;
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Owen-Ashley et al., 2004; Peters, 2000), whereas other studies have not found any such effect (Hasselquist et al., 1999). Indeed, some studies have found a positive effect of T on immunity (Evans et al., 2000). A recent meta-analysis that examined the effects of testosterone on immunity in vertebrates confirmed this ambiguity; only when considering ectoparasites on manipulated lizards did T have a demonstrably negative effect on a measure of immunity (Roberts et al., 2004). In some recent studies, T has been found to correlate (both negatively and positively) with the glucocorticoid stress hormone in birds, corticosterone (CORT) (e.g. Evans et al., 2000; Klukowski et al., 1997; Owen-Ashley et al., 2004; Parker et al., 2002; Schoech et al., 1999). The stimulation of the hypothalamus–pituitary–adrenal axis in response to stress leads to the secretion of CORT, which is involved in the mobilization of energy stores (glucose), the shutdown of digestive processes and increasing the peripheral blood supply (Buchanan, 2000; Sapolsky et al., 2000; Silverin, 1998). CORT also affects behavior — it reduces reproductive activity, increases dispersal behavior and increases foraging activity (Breuner et al., 1998; Silverin, 1998; Wingfield et al., 1997). T increases circulating CORT in several avian species (Casto et al., 2001; Duffy et al., 2000; Evans et al., 2000; Owen-Ashley et al., 2004; Poiani et al., 2000), and there is good evidence to suggest that CORT is immunosuppressive (e.g. Buchanan, 2000; Harvey et al., 1984; Råberg et al., 1998; Sapolsky et al., 2000; Wingfield et al., 1997; but see Svensson et al., 2002). In a study on the house sparrow (Passer domesticus), Evans et al. (2000) found that experimentally increased T impaired antibody production. However, after controlling for the effect of CORT (that positively covaried with T), T was found to enhance immunocompetence. The authors of this study suggested a modification to the original ICHH; rather than being immunosuppressive directly, T-related immunosuppression occurs indirectly through an increase in CORT. T itself may be immuno-enhancing, again indirectly through its effect on behavior leading to more dominant individuals (with higher T) gaining greater access to dietary resources and therefore being in better condition (Evans et al., 2000). Therefore, the fact that T and CORT may interact with each other requires that the levels of both hormones be manipulated independently to distinguish between the immunosuppressive effect (if any) of the two hormones. Several studies have reported a negative effect of elevated testosterone on avian body condition (defined as body mass in relation to body size) (Clotfelter et al., 2004; Mougeot et al., 2004; Ros, 1999; Wikelski et al., 1999), and other studies have found increased CORT levels to be negatively related to body condition (Breuner and Hahn, 2003; Hood et al., 1998; Kitaysky et al., 2001; Pereyra and Wingfield, 2003; Perfito et al., 2002; Schwabl, 1995; Sockman and Schwabl, 2001). In some cases, however, no effect of either elevated CORT or T has been found on body condition (Lormee et al., 2003; Lynn et al., 2003; and Alonso-Alvarez et al., 2002 Buttemer and Astheimer, 2000; respectively), and in a few studies, testosterone appeared to have a positive effect (Briganti et al., 1999; Chastel et al., 2005). It is therefore unclear whether either hormone has an effect (positive or negative) on body mass. An additional aim of
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this experiment was to elucidate the effect of elevated testosterone and corticosterone on avian body condition defined as body mass corrected for skeletal size. The study reported here was conducted to ascertain the effect of manipulating levels of T and CORT on individual immunocompetence and general body condition. If the original version of the ICHH (that assumes that T is directly immunosuppressive) is to be supported, a high T treatment group should have the poorest immune response, and the males with the lowest T should have the highest immune response, regardless of CORT levels. However, if the modified version of the ICHH that identifies glucocorticoids as the immunosuppressive agent is to be supported, then males with a low peak (stress) CORT response should have a more robust immune response than males with a high peak CORT response, regardless of testosterone titers. Although the stress-linked version of the ICHH is based upon measurements of baseline levels of plasma corticosterone, there is good evidence to suggest that baseline and stress-induced levels of CORT positively covary (e.g. Cockrem and Silverin, 2002; Romero and Wingfield, 1998; Schoech et al., 1999). Moreover, there is considerable evidence for immunosuppressive effects mediated through Type II receptors, which are only activated at elevated (as opposed to basal) levels of CORT (Sapolsky et al., 2000). Populations of zebra finch (Taeniopygia guttata) were selected for low, high and control levels of plasma corticosterone, based upon the maximum stress-induced response (Evans et al., 2006). Males from the G4 generation were housed under short day photoperiod (to remove any confounding effect of endogenous testosterone production — see Bentley et al., 2000), and individuals from each corticosterone line were allocated equally into two testosterone treatment groups — high and low T. These males were then challenged by phytohemagglutinin injection to test their cell-mediated immunity and diphtheria: tetanus injection to test their humoral response. In this way, the levels of both hormones could be artificially fixed so that different arms of the immune system could be challenged and the response compared between both CORT and T treatment groups. Materials and methods Selection program Since 1999, three replicate lines of zebra finch (2 × low, control and high CORT; 6 lines in total) were selected for contrasting levels of maximum stressinduced CORT in response to a mild stressor (Evans et al., 2006). A significant difference in plasma corticosterone levels was observed between the lines in the expected direction from the F2 generation of selection, with selection pressure exerted on each generation. There was a downward trend in corticosterone over generation regardless of selection line; little difference has existed in changes in corticosterone titer between the low lines and controls, but the high lines have shown a significant realized heritability of 20–25%. There was no corresponding change in testosterone over the generations, and no significant differences exist between the corticosterone lines in testosterone (Evans et al., 2006). Birds in each line were housed together in a large aviary giving c1m3 per breeding pair and maintained at an ambient temperature of 20–24 °C (Jones and Slater, 1999). The humidity was maintained between 50 and 70%, and the rooms sprayed with water two or three times per day. The birds were provided with ad libitum seeds (foreign finch mixture, Haiths Ltd., Cleethorpes, Lincolnshire, UK), Chinese millet sprays, mineralized grit, water and cuttlefish bone. The finches were
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provided with c10g of a 3:1 mixture of ‘nectarblend’ (Haiths Ltd., Cleethorpes, Lincolnshire, UK) and egg biscuit food (Haiths Ltd., Cleethorpes, Lincolnshire, UK), and either lettuce or cucumber daily with a cod liver oil supplement in the seed weekly. An excess of nesting baskets and boxes was provided. Full details of the selection methodology, characteristics and housing can be found in Evans et al. (2006). We have taken every care to minimize the welfare implications of this project, and all work was carried out under license by the UK Home Office and after local ethical review.
Hormone sampling and assay characteristics Six weeks post fledging, all birds were blood sampled. Blood samples for CORT (100 μl) were taken from the brachial vein after 20 min holding in a cloth bag, after a pilot study revealed that peak CORT response occurs in the zebra finch after 20 min of a stressful stimulus (Evans et al., 2006). This is the standard capture-restraint protocol used in studies analyzing the CORT response (Wingfield, 1994). The blood was centrifuged at 11,000×g for 15 min and the plasma frozen. Corticosterone concentrations were measured after extraction of 20 μl aliquots of plasma in diethyl ether, by radioimmunoassay (Wingfield et al., 1992) using anticorticosterone antiserum (code B21-42 and B3-163, Esoterix Inc. Endocrinology, CA) and [1,2,6,7-3H]-corticosterone label (Amersham, UK). The interassay coefficient of variation was 15.7%, and the intraassay coefficient of variation 3.1%. The mean extraction efficiency was 72%. The assay was run with 50% binding at 134 pg/tube, and the detection limit (for 7.3 μl aliquots of extracted plasma) was 1.76 nmol l− 1. Blood samples for testosterone assay were taken immediately upon capture, and the plasma obtained and stored in an identical manner to the CORT samples. Testosterone concentrations were measured in plasma samples by direct radioimmunoassay using anti-testosterone antiserum (code 8680-6004, Biogenesis, UK) and [125I]-testosterone label (code 07-189126, ICN, UK) (Parkinson and Follett, 1995). The assay was run with 50% binding at 11.0 pg/tube and a detection limit of 0.068 nmol l− 1 for the 20 μl plasma volumes that were run in the assay. The interassay coefficient of variation was 15.5%, and the intraassay coefficient of variation was 2.2%.
Implantation of testosterone Following experimental trials with birds outside the selection lines, a 7 mm implant size (Dow Corning medical grade tubing RX-50: inner diameter 0.76 mm, outer diameter 1.65 mm) was deemed most appropriate to give a high plasma T level, within the natural physiological range. Both the males used in the pilot study and the main experiment were housed under a short day photoperiod (18 h dark:6 h light) to limit the production of endogenous T. Although castration may have limited the production of endogenous T more effectively, such a procedure on a small passerine may well have resulted in morbidity and possible mortality. In addition, the procedure may affect an individual's condition and CORT titer, thus introducing greater variation and possible confounding elements into the experimental design. Furthermore, reduced photoperiod has been shown to cause a decrease in testicular mass relative to increased photoperiod in zebra finches (Bentley et al., 2000). The main experiment consisted of 72 adult male zebra finches taken from the selected lines. Twelve birds were taken from each of the 3 replicate (×2) CORT lines, and these were randomly subdivided into 6 empty-implanted and 6 testosterone-implanted birds. The implants in the high T groups were packed with crystalline T (Sigma T-1500), while empty implants were inserted into the control group birds. The silastic implants were inserted into an incision made in the skin of the neck. This was then closed by application of a tissue adhesive (Nexaband, Abbott Labs, Chicago, US) and where necessary by surgical suture. The implantation process took a total of 2 days to complete, and both CORT and T groups were balanced across these 2 days.
et al., 1993). A spessimeter (Alpa s.r.l. Milan) was used to measure the wing web before injection (as a control measurement), and at 24 h after injection (to the nearest 0.01 mm), to measure the wing web swelling in response to the mitogen. The increase in the swelling was used as an indicator of immune response. All the males were then weighed to the nearest 1 g using a Pesola spring balance, and their right tarsus lengths measured to the nearest 0.1 mm with digital calipers.
Humoral response Three days after the PHA injections, the males were blood sampled. One hundred microliters of blood was collected in heparinized capillary tubes and centrifuged at 11,000×g for 15 min. The plasma was removed and stored at −20 °C and subsequently tested for cross-reaction to diphtheria:tetanus as a control measurement. Three days after this, the primary humoral immune response of the males was tested. Each male was immunized with 100 μl of diphtheria:tetanus vaccine (Aventis Pasteur, Swiftwater, PA) by intraperitoneal injection. Twelve days later, blood samples were taken from the brachial vein. Twenty one days after the initial injection, the males were inoculated again with diphtheria:tetanus vaccine to test the secondary humoral response. Eight days after this, the birds were again blood sampled, and the samples split between testing for antibodies and for testosterone assay. In total, three blood samples were taken from each manipulated male. Antibody levels were determined by the use of ELISA (see Hasselquist et al., 1999; Owen-Ashley et al., 2004 for a full description of the methodology employed).
Statistical analyses The degree of swelling exhibited by the wing web injected with PHA (the difference between pre- and 24-hour post-injection) was used as the dependent variable in Restricted Maximum Likelihood models (ReMLs). This form of Generalized Linear Model (GLM) has been widely used before in similar studies (e.g. Evans et al., 2006; Peters, 2000; Peters et al., 2004). The maximal model consisted of T treatment group, CORT selection line, body condition (see below), T titer and peak CORT titer as fixed effects, and CORT line replicate as a random term. A minimal model was derived by stepwise deletion by removal of nonsignificant terms that did not significantly increase the residual deviance of the model. The residuals of the models conformed to a normal distribution and were homoscedastic so the models were run with a Gaussian distribution in Genstat 6. The same procedure was followed for the models containing control, primary and secondary levels of anti-diphtheria and anti-tetanus antibodies as response variables. Therefore, in total, there were six separate models with a measure of humoral immunity as the dependent variable. The residuals of the models were checked for homoscedasticity and normality and where necessary the response variables were transformed appropriately. Finally, the residuals from a reduced major axis regression of mass against tarsus length were used as a dependent variable with all other variables mentioned included as explanatory variables (again with replicate line as the random term) to ascertain whether body condition as defined as body mass controlling for skeletal size was affected by testosterone and/or corticosterone manipulations and titers. The residuals derived from a least squares regression of mass against tarsus length are not thought to be an appropriate measure of condition (see Darlington and Smulders, 2001; Green, 2001), so we used reduced major axis regression as recommended by Green (2001). This measure of body condition was included in the immunity models as an explanatory variable. The residuals of the model conformed to a normal distribution and were homoscedastic, so a Gaussian model was appropriate for this model. In all of the above models, all 2-way interactions were included between treatment groups and hormone titers.
Results Cell-mediated immune response
Plasma hormone levels The males' cell-mediated immunity was challenged by phytohemagglutinin (PHA) injection 8–9 days after implantation (balanced across treatment groups). Each male was injected with the mitogen phytohemagglutinin (PHA-P, Sigma, St. Louis, MO) intradermally into the left wing web. Each male received 30 μl of a suspension of 5 mg PHA-P in 1 ml phosphate buffer saline (1× PBS) (Lochmiller
There was a significant difference in the expected direction in maximum stress-induced CORT levels between the CORT selection lines (Wald = 96.96, df = 2, p < 0.001; see Fig. 1).
M.L. Roberts et al. / Hormones and Behavior 51 (2007) 126–134
Fig. 1. Predicted means of log10 body mass controlling for skeletal size (a) and mean levels of maximum stress-induced plasma corticosterone (b) in replicate lines of zebra finch (n = 24 per line) selected for divergent levels of corticosterone ± SE. The lines differed significantly in CORT level (Wald = 96.96, df = 2, p < 0.001) and body mass (Wald = 16.99, df = 2, p < 0.001).
There was also a significant difference between the testosterone treatment groups, again in the expected direction (mean control = 2.91 nmol l− 1, SE ± 0.52, mean T-treated = 12.67 nmol l− 1, SE ± 1.25; Wald = 63.64, df = 1, p < 0.001). Immune function Table 1 gives the Wald statistics divided by their respective degrees of freedom (as well as significance) for all the explanatory variables included in all models containing immune responses and body condition as the dependent variables. There was no significant effect of any of the independent variables on the size of wing web swelling at 24 h after PHA injection (CORT line: Wald = 0.30, df = 2, p = 0.86; testosterone group: Wald = 0.94, df = 1, p = 0.33; p > 0.05 for all covariates). The only measure of humoral immunity that was significantly affected by the hormone treatments was the secondary antibody response against diphtheria. There were significant interactions
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between CORT titer and T titer (Wald = 4.28, df = 1, p = 0.04; Fig. 2), and between CORT selection line and T titer (Wald = 8.89, df = 2, p = 0.01; Fig. 2). Fig. 2 shows that the highest antibody response was exhibited by the males with the highest levels of both CORT and T, but there is a suggestion that high levels of T were immunosuppressive at low levels of CORT in the high CORT selection line. This is indicated as dark shading (low antibody response) at low levels of CORT and high levels of T (Fig. 2(c)). To investigate these interactions further, we tested whether corticosterone and testosterone titers positively covaried with secondary antibody response to diphtheria and what relationship testosterone titer had with the same antibody response, in each CORT line separately. We found a non-significant, positive interaction between both hormone titers and antibody response in the low and high lines, and a strongly significant positive relationship in the control line (Wald = 11.51, df = 1, p < 0.001). Testosterone titers had a nonsignificant negative relationship with antibody response in the low line; a significant positive relationship in the control line (Wald = 13.99, df = 1, p < 0.001); and a significant negative relationship with antibody response in the high line (Wald = 4.75, df = 1, p = 0.029). However, it should be borne in mind that such results are more prone to a Type I error, and obviously rely on smaller sample sizes, than when relationships are inferred from models containing data from all treatment levels. The other covariates and interactions included in the models had no significant effect on humoral immunity (p > 0.05 in all cases). Given that in previous avian studies testosterone implantation has had a significant effect on both PHA response and antibody response to diphtheria:tetanus injection (Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004), we carried out power analyses to ascertain whether our sample sizes were too small to detect any significant differences in immune response between testosterone treatment groups. In all cases, the sample sizes obtained were far greater than both ours and the sample sizes used in the studies that did find an effect of testosterone on immunity (see Table 2). Indeed, the most similar study to ours that implanted testosterone and immunized with diphtheria:tetanus as well as injected with PHA found
Table 1 Effects of body condition and hormone treatment groups and titers on immune response and condition in zebra finches Explanatory variables
Response variables Diphtheria
Body condition CORT titer CORT selection line T titer T treatment group CORT line* T titer CORT line* T treatment group T treatment group* CORT titer CORT titer* T titer
Tetanus
Control
Primary
Secondary
Control
Primary
Secondary
0.06 0.02 1.81 0.29 0.34 0.97 1.19 0.28 1.18
<0.01 1.45 2.38 0.53 0.07 2.68 1.03 1.12 0.13
1.81 2.10 1.96 0.75 0.25 4.45* 2.46 0.04 4.28*
0.46 0.33 1.67 1.11 0.73 1.48 1.56 0.41 0.14
0.29 3.12 1.36 0.34 0.12 0.30 1.53 0.92 0.52
0.01 2.11 2.25 1.54 0.53 0.22 0.70 1.46 0.26
PHA
Body condition
0.22 0.40 0.15 0.32 0.94 1.94 1.11 0.81 0.21
– 2.83 8.50*** 2.16 2.53 0.10 0.15 7.96** 0.11
Variation explained by each explanatory variable is expressed by Wald statistics divided by its respective degrees of freedom (*p < 0.05, **p < 0.01, ***p > 0.001).
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Fig. 2. Fitted model of the relationship between testosterone titer, corticosterone titer and diphtheria secondary antibody response in males selected for (a) low, (b) control and (c) high maximum stress-induced levels of plasma corticosterone (n = 24 per line). Antibody response is expressed as a percentage of the standard and is log10 transformed. Lighter shades represent higher antibody responses. The interactions between CORT and testosterone titer (Wald = 4.28, df = 1, p = 0.04) and CORT line and testosterone titer (Wald = 8.89, df = 2, p = 0.01) were significant.
significant differences between treatment groups with samples sizes of 6 birds in each group (Owen-Ashley et al., 2004). For this reason, we suggest that our sample sizes were sufficiently large to detect any differences.
of mass in the empty-implanted (control T) group, but had a significant, negative relationship with body mass in the high T group (Wald = 6.46, df = 1, p = 0.011). Discussion
Body mass There was a significant effect of CORT selection line such that birds in the high line had greater mass (after controlling for skeletal size) than controls and low CORT birds (Wald = 16.99, df = 2, p < 0.001; see Fig. 1) (see Table 1). In addition, an interaction between CORT titer and T treatment group also had a significant effect on body mass (Wald = 7.96, df = 1, p = 0.005; see Fig. 3). T implanted birds showed a greater decrease in mass with increasing CORT production than empty implanted birds. As with the significant interactions reported for secondary antibody response to diphtheria, we tested the nature of the relationship between CORT titer and body mass within each testosterone treatment group. We found that CORT titer was not a significant predictor Table 2 Results from power analyses showing sample sizes required to give significant differences between T treatment groups (α = 0.05) in immune response given the effect sizes obtained, at a power of 80% Immune test
T treatment group
From the results of the present experiment, it appears that corticosterone and testosterone have an interactive effect on immunity. Although no effect of either manipulated hormone was found on cell-mediated immunity as measured by PHA response, there was a significant, positive relationship between the plasma levels of both hormones and the birds' secondary antibody response to diphtheria; but this was only the case when the other hormone was also at high levels in the blood plasma. Whereas in the low and control lines there was a positive relationship between T level (at high levels of CORT) and secondary diphtheria antibody response; in the high line there was a negative relationship at low levels of CORT. There was nevertheless a positive relationship between T and antibody titer at high levels of CORT. As can be seen in Fig. 2, when all interactions are included in the predicted model, the general positive interactive effect the two hormones have on antibody
Mean value Standard Sample size required obtained deviation in each treatment group
Primary tetanus Control 6.6 Testosterone 9.7 Secondary Control 72.1 tetanus Testosterone 69.4 Primary Control 0.39 diphtheria Testosterone 0.43 Secondary Control 2.42 diphtheria Testosterone 1.42 PHA Control 0.32 Testosterone 0.28
10.3 17.1 40.0 32.6 0.86 0.78 4.02 2.04 0.16 0.16
326 2867 6613 160 252
Fig. 3. Relationship between corticosterone titer, body mass and testosterone treatment group. Triangles and solid trend line represent empty-implanted birds (n = 36); circles and dashed trend line represent testosterone-implanted birds (n = 36). The interaction was significant (Wald = 7.96, df = 1, p = 0.005).
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response is evident. In the males selected for high levels of CORT, testosterone appears to be immunosuppressive, but only in the presence of low levels of plasma corticosterone. The results confirmed that the CORT lines differed significantly in maximum stress-induced levels of CORT and that the testosterone treatment groups differed significantly in T in the expected direction. Little is known about wild male zebra finch T levels, and these may not be relevant to our captive selected finch stocks, but the manipulations changed the testosterone-implanted males' T levels to be relatively high compared to the levels of the empty-implanted males. These levels however remained within physiological limits as the levels were at the upper limit of the range of T titers previously recorded in this generation of zebra finches. Males from the high CORT lines had significantly greater mass (when controlling for tarsus length) than males from the low CORT lines. There was a significant, negative relationship between actual CORT plasma levels and body mass in the high T treatment group. In general, individuals with both the highest CORT and T plasma levels exhibited the greatest secondary antibody response against diphtheria. This is contrary to our understanding that these hormones are immunosuppressive, and as this is a manipulative experiment, contrary to the predictions of the ICHH. There was a suggestion from the results that, in the presence of low levels of CORT, T may be immunosuppressive, but only in individuals selected for high levels of CORT (Fig. 2 (c)). In the other CORT lines, testosterone and corticosterone titers together were positively related to antibody response. This may be an idiosyncratic characteristic of the high CORT line or may suggest that birds with genes for a high maximum stressinduced CORT response are immunosuppressed by high levels of T if CORT levels are relatively low. Nevertheless, the overall effect of both hormones on humoral response is positive when both hormones are at high levels in the blood plasma. Previous studies have generally found either T or CORT to be immunosuppressive (Buchanan et al., 2003; Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004; Peters, 2000; Evans et al., 2000; Råberg et al., 1998; Wingfield et al., 1997 respectively). In contrast, a few studies have found these hormones to exhibit immuno-enhancing properties (T: Evans et al., 2000; Peters, 2000; CORT: Svensson et al., 2002). This is the first experiment to our knowledge that has found a significant, positive interactive effect of both hormones on antibody response. Testosterone may have a positive effect on immunocompetence by increasing aggressive behavior and therefore dominance ranking; this may lead to greater access to dietary requirements, and consequently more resources are made available for immune defense (Evans et al., 2000). The birds in this study were housed together and may have competed for food; however, they were fed ad libitum. Therefore, it is not clear how greater dominance caused by testosterone treatment affected dietary intake. An alternative explanation is that, rather than being able to obtain more or better quality food, males with high testosterone may be better able to utilize the food they ingest for immune function. Exogenous testosterone administration has been found to
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increase plasma carotenoid concentrations via upregulation of carotenoid-binding lipoproteins (McGraw et al., 2006). Carotenoid concentration in the blood is in turn known to have a positive effect on immune function and antioxidant defenses (Alonso-Alvarez et al., 2004; Blount et al., 2003; Faivre et al., 2003; McGraw and Ardia, 2003). Elevated CORT levels result in an increase in foraging behavior and mobilization of glucose reserves (Breuner et al., 1998; Buchanan, 2000; Silverin, 1998; Wingfield et al., 1997), therefore an increase in both hormones may result in an excess of resources readily accessible for producing and regulating an antibody response. Moreover, peak CORT levels (as opposed to basal levels) and acute stress have also been found to enhance immune response in several studies (e.g. Cocke et al., 1993; Dhabhar and McEwen, 1999; Persoons et al., 1995; see Dhabhar, 2002 for a review). Thus, a combination of both high testosterone and high peak levels of corticosterone in the blood may instigate either directly or indirectly a potent immunostimulatory effect. Several studies have found that artificially increasing testosterone correspondingly increases CORT levels (Casto et al., 2001; Duffy et al., 2000; Evans et al., 2000; Owen-Ashley et al., 2004). By increasing T in individuals with naturally low levels of T, an increase in CORT may be a predictable phenomenon, partly because the methodology employed to increase T is probably stressful and should increase CORT levels (see Moore et al. 2004) and partly because increasing T in males with naturally low levels of the androgen may in itself be stressful for the individual concerned and consequently raise CORT levels, quite apart from the implantation procedure itself. Individuals with naturally low levels of testosterone may well be stressed by suddenly finding themselves with high (often above physiological) levels of the hormone; this does not necessarily mean that the two hormones naturally positively covary. If the two hormones do positively correlate in freeliving birds (and there are some data to support this, see Johnsen, 1998; Mateos, 2005), females would be expected to choose males with both high T (for their sexual signals) and concomitantly high CORT. These males would have the greatest humoral immune response, so females would be choosing males with the highest immunocompetence. This interpretation is consistent with Hamilton and Zuk's (1982) parasite theory. However, it should be borne in mind that we measured peak levels of CORT in this study; although basal levels of CORT may positively covary with T, it does not necessarily imply any relationship between peak CORT and T. There are examples of the two hormones negatively covarying within individuals in multiple taxa (in birds: Parker et al., 2002; in reptiles: Knapp and Moore, 1997; Lance et al., 2003; Moore et al., 2001; but see Moore et al., 2000; in mammals: Sankar et al., 2000). The important question that needs to be addressed is how do both peak and basal CORT and testosterone naturally interact within free-living individuals. This point is crucial in understanding the results of laboratory-based studies on immunocompetence and endocrine systems in birds. There was no effect of either hormone on the cell-mediated immune response or on several measures of humoral immunity.
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Power analyses suggested that the sample sizes of our treatment groups needed to be much larger to find a significant effect of T treatment given the mean differences obtained; however, significant effects of testosterone treatment on PHA and antibody responses have been found in previous studies on much smaller sample sizes (e.g. Casto et al., 2001; Duffy et al., 2000; Owen-Ashley et al., 2004). In addition, other studies have found no effect of testosterone treatment on the cell-mediated response (Buchanan et al., 2003; Greenman et al., 2005). Therefore, although we cannot definitely conclude that this null result is not merely due to a small sample and effect size, it seems more likely that this is a real result. The important point to note is that, although no significant result was obtained, neither hormone had an immunosuppressive effect on either the cell-mediated response or on antibody production. Body mass was negatively related to CORT levels, but only in the high T treatment group. In addition to this, the males in the high CORT lines were on average in better condition than males from the low CORT lines when all the independent parameters were accounted for in the model. These results would suggest that both high T and high CORT in the same individual will have a negative effect on general body mass. But high CORT with relatively lower levels of T will have a positive effect on mass. The alternative explanation is that differences between the CORT lines may be an artefact of the selection process, and rather than CORT per se affecting condition in this way, it is possible that another determinant of condition may have been incidentally selected. Nevertheless, this explanation does not account for the negative effect of both high T and high CORT titer together on body condition. Several studies have found a negative effect of T on body condition or body mass (Clotfelter et al., 2004; Mougeot et al., 2004; Ros, 1999; Wikelski et al., 1999), as well as a similar effect for CORT (Sockman and Schwabl, 2001), so this result should not be too surprising. However, given that males with simultaneously the highest T and highest CORT titers exhibited the greatest humoral immunity, it seems counter-intuitive to find that males in the high testosterone treatment group with the highest CORT titers males were also in the poorest condition, particularly if these individuals had the highest immunocompetence due to greater access to resources caused by high levels of both hormones. The reasons for this are unclear, but it is possible that these individuals quickly expend any excess resources on energy demanding processes such as immune defense and general locomotor activity. Low body mass will result if high plasma CORT levels lead to glucose being quickly broken down (therefore more resources will rapidly be made available for immune defense). Additionally, elevated levels of both CORT and T have been found to increase basal metabolic rate and locomotor activity (Breuner et al., 1998; Buchanan et al., 2001; Lynn et al., 2000; Wikelski et al., 1999). In addition, the positive relationship we found between antibody production and simultaneously high levels of both hormones may have nothing to do with body mass or condition per se, but instead be related to direct as well as indirect effects of the hormones in terms of increasing carotenoid availability and stimulating antibody production.
Our study suggests that testosterone-mediated signaling may not be costly in terms of immunosuppression, but may detrimentally affect body condition. Therefore, poorer quality males that elevate testosterone for sexual signaling may pay the price in times of environmental hardship when food is scarce. Higher quality males may be able to elevate testosterone without losing body mass or alternatively be able to resist the negative effects of low body mass. Our results certainly suggest that the T-mediated costs of sexually selected traits do not occur through direct immunosuppression but may occur through metabolic (e.g. Buchanan et al., 2001) or dominance/aggression costs (e.g. Rohwer and Ewald, 1981). Only one measure of immunity exhibited any relationship with either CORT or T, suggesting that these hormones do not consistently affect all aspects of an individual's immune response. It is important to note that the birds used in this study were selected for maximum stress-induced levels of CORT, and it was this measure of CORT interacting with T that showed a relationship with the secondary antibody response against diphtheria. Basal levels of CORT were not measured, and these may well have had an effect that was missed. Nevertheless, this experiment demonstrates that, contrary to the generally accepted consensus, CORT and T are not always associated with immunosuppression. Acknowledgments We would like to thank the staff of the Animal Unit at the University of Stirling for their assistance with the selection program and to Alistair Dawson for his advice on the implantation procedure; we are also grateful to A.R. Goldsmith for access to the RIA facilities at the University of Bristol. All work was conducted under Home Office license PPL 60/2584. MLR was funded by a studentship from NERC, and the selection program under MRE and KLB was funded at various times by the Royal Society, ASAB, NERC and the University of Stirling, and DH was funded by the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (Formas), the Carl Trygger Foundation, the Swedish Research Council (VR) and the Crafoord Foundation. One anonymous referee and Julio Blas provided very helpful comments on an initial version of the manuscript. References Alonso-Alvarez, C., Ferrer, M., Figuerola, J., Veira, J.A.R., Estepa, J., Torres, L.M., 2002. The effects of testosterone manipulation on the body condition of captive male yellow-legged gulls. Comp. Biochem. Physiol., A 131, 293–303. Alonso-Alvarez, C., Bertrand, S., Devevey, G., Gaillard, M., Prost, J., Faivre, B., Sorci, G., 2004. An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity. Am. Nat. 164, 651–659. Bentley, G.E., Spar, B.D., MacDougall-Shackleton, S.A., Hahn, T.P., Ball, G.F., 2000. Photoperiodic regulation of the reproductive axis in male zebra finches, Taeniopygia guttata. Gen. Comp. Endocrinol. 117, 449–455. Blount, J.D., Metcalfe, N.B., Birkhead, T.R., Surai, P.F., 2003. Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science 300, 125–127.
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