Short- and long-term consequences of prenatal testosterone for immune function: an experimental study in the zebra finch
Hormone-mediated maternal effects play an important role in the formation of a differentiated phenotype. They have been shown to influence a wide array of offspring traits, both early in life and in adulthood. One important offspring trait that is under the influence of maternal androgens is the immune system. In birds, a growing number of studies show that yolk androgens modulate immune function during the chick stage. However, there is a lack of knowledge regarding long-term effects of prenatal androgens on offspring immunity. In this study, we therefore investigated the influence of prenatal testosterone (T) on several measures of immunity in fledgling and adult zebra finches (Taeniopygia guttata). Cell-mediated immune response (towards phytohaemagglutinin, PHA) of fledglings hatching from control eggs was negatively related to brood size, whereas there was no such association for fledglings hatching from eggs with experimentally elevated T levels (T fledglings). Male control fledglings showed reduced mass gain compared to female control fledglings within 24 h after the PHA injection. This pattern was reversed in T fledglings. Total antibody levels in fledglings were not affected by egg treatment. Neither cell-mediated immunity nor total antibody levels in sexually mature zebra finches were influenced by egg treatment. However, there was an immuno-enhancing effect of elevated egg T on both primary and secondary humoral immune responses toward diphtheria and tetanus antigens in ca 5 and 7 month old zebra finches. In addition, the covariation between different immune components differed between T and control offspring, suggesting that egg treatment may have altered the potential trade-offs between different parts of the immune system. Our results suggest that prenatal androgens could be an important factor contributing to individual variation in immune function even in adulthood.
KeywordsMaternal effects Testosterone Immune function Life history
Individual phenotypic variation is the raw material natural selection acts on (Stearns 2003). Variation among individual phenotypes is determined by genetic and non-genetic effects as well as their interactions (Falconer and Mackay 1996). Therefore, determining how genetic and non-genetic factors contribute to phenotypic variation in morphological, physiological and behavioural traits is of crucial importance for the understanding of evolutionary processes.
One important group of non-genetic effects that have a strong impact during early ontogeny are those that act through the mother. Mothers provide non-genetic maternal resources such as nutrients, antibodies or hormones and thereby, substantially influence development and formation of the offspring phenotype (Mousseau and Fox 1998). Maternally derived hormones in particular have received considerable attention due to their multiple and sometimes antagonistic effects on various fitness-related traits in the offspring (e.g., Clark and Galef 1995; McCormick 1999; Uller and Olsson 2003; Groothuis et al. 2005a). Recent research within the frame work of maternal hormones has focused much on birds and other oviparous vertebrates. In these systems, the amount of prenatal hormones that the embryo is exposed to can be easily manipulated as the embryo develops outside the mother’s body.
A rapidly increasing number of experimental studies show that in birds, prenatal egg androgens modulate a wide array of offspring traits both early in life and in adulthood (see Groothuis et al. (2005a) and Gil (2008) for comprehensive reviews on this topic). One important offspring trait that is under the influence of prenatal androgens is the immune system.
Immune function is thought to be involved in many physiological trade-offs influencing life history and fitness (e.g., Norris and Evans 2000; Zera and Harshman 2001; Adamo 2004). Activation and maintenance of the immune system is physiologically (energy- and/or resource-) demanding (e.g., Sheldon and Verhulst 1996; Lochmiller and Deerenberg 2000; Hasselquist et al. 2001; Bonneaud et al. 2003), and any investment in an immune trait must be traded off against other resource-demanding fitness functions. These may either be other immune traits or life-history traits such as reproduction and survival (e.g., Sheldon and Verhulst 1996; Schmid-Hempel 2003). More generally, the immune system may involve evolutionary costs in evolving an efficient immune system (Sadd and Schmid-Hempel 2008). Immunity is known to be negatively correlated with other fitness-related traits (e.g., fecundity (McKean et al. 2008) or sperm viability (Simmons and Roberts 2005)). This means that evolving an efficient immune system may constrain the evolution of other fitness-related traits and vice versa. Similarly, different immune traits are often negatively correlated (e.g., Klein and Nelson 1999; Buchanan et al. 2003; Martin et al. 2006a; but see Martin et al. 2006b; Matson et al. 2006), and hence, there may be an evolutionary trade-off constraining their simultaneous evolution. There is some evidence that prenatal maternal androgens mediate resource investment between growth and cell-mediated immunity (Andersson et al. 2004; Groothuis et al. 2005b) and given that they affect a wide array of different traits, they are likely to be involved in other (immune) trade-offs. Investigating how prenatal androgens contribute to variation in immune responsiveness and how they may affect potential trade-offs between immune and other life-history traits is, therefore, important for the understanding of life history evolution.
It has been shown that prenatal exposure to elevated (but physiological) concentrations of egg androgens results in reduced immune responsiveness in the young of several bird species (Groothuis et al. 2005b; Müller et al. 2005; Navara et al. 2005; Cucco et al. 2008; Sandell et al. 2009). However, other studies have found no (Tschirren et al. 2005; Rubolini et al. 2006a; Müller et al. 2007; Pitala et al. 2009) or even positive effects (Navara et al. 2006; Cucco et al. 2008) of prenatal androgens on avian immune function. Most of these studies focus on the chick or nestling phase, i.e., on the early stages of development when the immune system of the young is immature. Given that yolk, androgens can have long-term effects on behaviour, ornaments and reproductive traits/physiology (Strasser and Schwabl 2004; Eising et al. 2006; Uller et al. 2005; Rubolini et al. 2006b; Rubolini et al. 2007; Tobler and Sandell 2007; Müller et al. 2008; Partecke and Schwabl 2008; Müller and Eens 2009; Müller et al. 2009), it is possible that they also have a permanent, priming effect on the immune system which can extend into adulthood.
So far, only one study has investigated whether elevated levels of yolk androgens exert long-term effects on the avian immune system. Cucco et al. (2008) examined the effect of experimentally increased concentrations of yolk testosterone (T) on cell-mediated immunity in the Grey partridge (Perdix perdix). While elevated yolk T had either a negative or positive effect on cell-mediated immunity in 1–3 weeks old chicks (depending on the dosage of T used in the manipulation); there was no effect at 90, 260 and 625 days after hatching. This result can be contrasted with a recent study on Jackdaw (Corvus monedula) nestlings in which it was found that increased levels of yolk androgens suppressed cell-mediated and humoral immunity even after 4–5 weeks of chick development (Sandell et al. 2009). In this study, the authors speculated that high levels of yolk androgens could potentially have long-lasting immunosuppressive effects in this species. However, the young were not followed into adulthood.
There is, thus, a general lack of knowledge regarding long-term effects of prenatal androgens on offspring immunity. Particularly, to our knowledge, there is to date no study that has investigated long-lasting effects of egg androgens on several aspects of immunity, including humoral immune function. In this study, we therefore examined consequences for elevated yolk T concentrations on several components of the adaptive immune system, both in young and sexually mature zebra finches (Taeniopygia guttata). Previous studies have found long-lasting effects of prenatal T on behaviour in this species (von Engelhardt 2004; Tobler and Sandell 2007), and we therefore hypothesised that yolk T could exert similar long-lasting effects on the immune system.
Material and methods
Animals and housing
For the study, we used a captive population of zebra finches held in indoor facilities at Lund University, Sweden. Male and female zebra finches were paired randomly (n = 45 pairs) and housed in individual breeding cages (80 × 40 × 80 cm) with food (commercial seed mixture; Exoten Tropical ®), water and cuttle bone provided ad libitum. A nest box and nest material (coconut fibres and cotton wool) were supplied for breeding. During the first 3 weeks after hatching, all birds were provided daily with extra rearing diet (egg food; Witte Molen). Birds were maintained under constant temperature (20 ± 2 C) and photoperiod (14 L:10 D). Offspring were removed from their natal cages when 45–55 days old and thereafter housed unisexually in cages (four to five birds per cage) of the same dimensions as the breeding ones. Each cage contained birds of one egg treatment (see next section) only. All individuals had acoustic and visual contact with adults of both sexes.
Egg testosterone manipulation and nestling development
For egg injection, we applied the same dose as previously used in this species (e.g., von Engelhardt et al. 2006; Tobler and Sandell 2007). Eggs were injected with either 500 pg T in 5 μl of sterile sesam oil (T eggs) or 5 μl sterile sesame oil only (control eggs; see Tobler and Sandell 2007 for a more detailed description of the injection protocol). In zebra finches, females transfer more testosterone to their clutch when they are mated to an attractive male (Gil et al., 1999; von Engelhardt, 2004), and there is a concomitant increase in all eggs. The injection dose of testosterone used in our study corresponds approximately to the difference in testosterone + dihydrotestosterone measured in egg yolks of females paired with attractive males versus unattractive males and thus, mimics a natural scenario (see also von Engelhardt et al., 2006). Whole clutches were randomly assigned to either T (n = 23) or control (n = 22) treatment. In four control clutches and one T clutch, all eggs failed to hatch owing to a combination of the injection procedure (eggs damaged during injection, embryo did not survive) and infertility of some eggs. In one additional control clutch, all chicks died on the second day after hatching because the parents abandoned the nest. Thus, zebra finches hatched from manipulated eggs and subjected to immune tests, and blood-sampling were the offspring of 39 pairs (22 testosterone and 17 controls). Whole clutches were randomly assigned to foster parents. Hatching success was not significantly different among the treatment groups (T, 69%; control, 64%; χ 2 = 0.51, df = 1, p = 0.48). There was also no significant difference in brood size (average between hatching and fledging) among the treatment groups (T, 3.2 ± 0.2 (mean ± 1 se) chicks, control, 3.1 ± 0.2 chicks; F 1,94 = 0.17, p = 0.68).
Hatchlings were marked dorsally with nontoxic colour pens for individual identification until the age of 8–12 days when they were banded with aluminium rings. Chicks were weighed to the nearest 0.01 g at hatching in combination with the PHA immunisation (ca. 20 days old; see next section) and on days 10 and 34 post-hatch. Day 10 represents a period of intensive growth, and at this stage, most nestlings have obtained more than 50% of their final body mass. Day 34 represents the age at which zebra finch chicks are about to become independent from the parents.
To evaluate cell-mediated immune responsiveness, we used a phytohaemagglutinin-A (PHA) assay, which is frequently used in ecological studies (e.g., Martin et al. 2006a, b; Love et al. 2008). We injected 30 μg PHA (Sigma L-8754) dissolved in 30 μl sterile phosphate-buffered saline (PBS) into the left wing web (patagium) following the protocol of Smits et al. (1999) with no control injection of PBS in the right wing web. PHA injection produces local inflammation and swelling of the wing web, which is dependent on the strength of the (unspecific) cell-mediated immune response (Martin et al. 2006a, b). The thickness of the wing web was measured three times to the nearest 0.01 mm with a micrometre (Mitutoyo, M317-25) immediately before and 24 h (±0.5) after injection. The median measure was used in analyses. All measurements were taken by the same person (M.T.). The size of the swelling induced by the PHA injection (post- minus pre-injection value) was used as an estimate for cell-mediated immune responsiveness. A preliminary test on 19 zebra finches which were not involved in the present study and in which we injected PHA into the right and left wing web simultaneously showed that the PHA assay was repeatable within individuals (repeatability of the swelling between left and right wing, R = 0.81; F 18,19 = 9.49, p < 0.0001). We took two measures of T cell-mediated immune responsiveness. The first measure was taken when the zebra finch nestlings were about to fledge (mean, 19.6 days; range, 17–21 days) and the second, when the birds were sexually mature (mean, 132.2 days; range, 115–143 days; zebra finches reach sexual maturity at about 90 days of age (Zann 1996)). Concomitant with the measures of wing web thickness, we also took measures of individual body mass to the nearest 0.01 g immediately before and 24 h after injection.
To quantify humoral immunity in offspring zebra finches, we used two measures: (a) total antibody levels and (b) specific antibody production towards diphtheria and tetanus antigens. Total antibody levels provide a general assessment of humoral immune function. Responsiveness to diphtheria and tetanus antigens is a measure of an individual’s ability to mount a specific antibody response in case of an infection. For both measures of humoral immunity, we used a standard enzyme-linked immunosorbent assay (ELISA; see next paragraph) to measure antibody titers. Total antibody levels were determined from blood samples that were collected just prior to the two PHA injections, i.e., at fledging and after the birds reached sexual maturity. To obtain diphtheria- and tetanus-specific antibody responses, birds were immunised intraperitoneally with 100 μl diphtheria–tetanus vaccine (2 Lf diphtheria toxoid and 5 Lf tetanus toxoid absorbed in aluminium phosphate; Aventis Pasteur, Toronto, Canada). Birds were immunised twice to obtain measures of both primary and secondary antibody responses. We measured both primary and secondary antibody responses because they involve partly different immunological mechanisms (Janeway and Travers 1996) and can therefore be viewed as two different traits (Råberg and Stjernman 2003). The first immunisation occurred at the age of ca. 150 days (mean, 149 days; range, 143–156 days) and the second at about 200 days of age (mean, 199 days; range, 195–207 days). Blood samples (ca. 100 μl, taken from the jugular vein) were collected just before immunisation (day 1, background value) and 12 (primary response) or 7 (secondary response) days post-immunisation (i.e., at the peak of the primary and secondary immune responses against these two antigens; own unpublished results). All blood samples were centrifuged at 3,000 rpm and the plasma then extracted and stored at −50°C until further analysis. All individuals were weighed to the nearest 0.01 g on the days of blood-sampling.
ELISA protocols followed those previously developed for other passerines, including the zebra finch (e.g., Hasselquist et al. 1999; Owen-Ashley et al. 2004; Grindstaff et al. 2006; Roberts et al. 2007). Plasma samples were diluted 1:200 for total antibody levels, 1:600 for antidiphtheria antibodies and 1:1,200 for anti-tetanus antibodies. To compare antibody responses that run at the same time but on different plates, we standardised antibody titers by reference to a serially diluted standard (pooled plasma from zebra finches with high total antibody titers or high secondary antidiphtheria and -tetanus responses) that was included on each plate. Humoral immune responses toward tetanus and diphtheria were estimated as the difference between post- and pre-immunisation antibody titers. Intra- and inter-assay variation was 2.8% and 16.2%, respectively, for the total antibody levels, 3.6% and 6.4% for the diphtheria, and 1.9% and 4.6%, respectively, for the tetanus ELISAs.
Statistical analyses were conducted with SAS System 9.2 for Windows (SAS Institute Inc., Cary, NC, USA) using mixed models (PROC MIXED; Littell et al. 2004). Nest was included in all models as a random factor to control for non-independence of nestlings sharing the same genetic parents as well as the same rearing environment. In analyses of nestling body mass, we included egg treatment and sex as fixed factors. Brood size (average between hatching and the day of measurement) and day time were included as covariates in the model. In all analyses of immune responses, we used egg treatment and sex as fixed factors. Average brood size between hatching and fledging, age and body mass at injection were used as covariates in the analyses. All two-way interactions between fixed factors and between fixed factors and covariates were initially included in the models, but non-significant effects (p ≥ 0.10) were sequentially removed (starting with the least significant interaction). Non-significant main effects were kept in the model if involved in interactions with p < 0.10. Least-square means contrasts were used to analyse differences between males and females within a given treatment group.
For the cell-mediated immunity test in nestlings, we used PHA solution from two different vials. The strength of the cell-mediated immune response in zebra finch nestlings differed strongly with respect to vial identity (F 1,29.8 = 90.14, p < 0.0001). We, therefore, standardised the data by vial identity, setting the means to zero and the standard deviations to one before pooling the PHA measures for analysis.
Antibody titers from the humoral immune responses were log-transformed to normalise the data. Antibody responses toward diphtheria and tetanus antigens were moderately correlated (primary r 87 = 0.64, p < 0.0001; secondary r 67 = 0.41, p = 0.0006) and the correlations between the responses did not differ significantly for the two treatment groups (using Fisher’s z transformation to compare correlations, p ≥ 0.09 for both primary and secondary response). Moreover, humoral immune responses toward the two antigens were homogenous and did not differ with respect to treatment group (treatment by antigen (diphtheria or tetanus) interaction using mixed model analyses with fixed factors ‘egg treatment’ and ‘antigen’, random factor ‘nest’ and random two-way interaction ‘individual identity nested within treatment’ × ‘nest’ (to control for two immune measures by the same individual); p ≥ 0.15 for both primary and secondary response). There was, thus, no indication that birds from the two treatment groups responded differently toward the two antigens. For simplification, we therefore used principal component analysis to create a combined humoral immune score for the primary and secondary responses, respectively (see e.g., Sandell et al. 2009). These principal component immune scores were strongly correlated with the diphtheria and tetanus responses (r > 0.83 in all cases).
When testing relationships between pairs of different immune measures, we used mixed models with nest as a random factor, egg treatment as a fixed factor and one of the two immune measures as covariate (always the one that was estimated first, assuming a causal relationship between two consecutive immune measurements). The interaction between egg treatment and the covariate was always included in the models to test whether the slopes of the relationship between the immune measures differed with respect to treatment group.
The Satterthwaite approximation was used to calculate the denominator degrees of freedom (Littell et al. 2004). Residuals of the models were tested for normality. All tests were two-tailed with a significance level set at α ≤ 0.05. Sample sizes differ among analyses either because of mortality or missing measurements. Data are presented as means ± 1 SE, unless otherwise specified.
Nestling body mass
There was no significant effect of egg treatment on hatchling body mass (egg treatment F 1,35.3 = 1.28, p = 0.27; egg treatment by sex interaction F1,58.4 = 0.28, p = 0.60). However, male hatchlings weighed significantly less than female hatchlings (males 0.83 ± 0.02 g, females 0.88 ± 0.02 g; F 1,59.8 = 4.36, p = 0.041). Nestling body mass at the age of 10 days was not significantly different between egg treatment groups and sexes (F < 1.79, df > 1,34, p > 0.19 for main effects and their interaction; n = 29 T males, 20 control males, 23 T females, 23 control females). There was no significant effect of brood size or its interactions with sex and egg treatment on body mass at day 10 (F < 2.91, df > 1,40, p ≥ 0.10 in all cases). Similarly, at the age of 34 days, there was no significant difference in body mass between egg treatment groups and sexes (F < 0.67, df > 1,34, p > 0.41 for main effects and their interaction; n = 27 T males, 19 control males, 23 T females, 23 control females). There was a negative, though not significant, relationship between body mass and brood size (b = −0.28 ± 0.14; F 1,37.3 = 3.89, p = 0.056). This relationship did not differ between egg treatment groups or sexes (covariate by treatment or sex interaction; F < 0.17, df > 1,39, p > 0.69 in both cases).
The strength of the cell-mediated immune response in sexually mature offspring was not significantly affected by egg treatment (control offspring, 1.63 ± 0.09 mm; T offspring, 1.58 ± 0.08 mm; main effect and interactions all F < 2.18, df > 1,31, p > 0.14; n = 26 T males, 18 control males, 23 T females, 23 control females). None of the other variables (sex, age, brood size, body mass at injection) or their interactions had a significant effect on adult cell-mediated immunity (F < 2.17, df > 1,30, p > 0.14 in all cases). Mass change within 24 h after the PHA injection was not significantly influenced by egg treatment, sex, age or brood size (main effects and interactions F < 1.90, df > 1,30, p > 0.17 in all cases). Mass at injection was the sole predictor of change in body mass in response to the PHA challenge with heavier birds loosing significantly more weight than lighter ones (b = −0.166; F 1,87.4 = 41.07, p < 0.0001), but this might have been a statistical artefact (regression to the mean; Kelly and Price 2005).
Total antibody levels
Total antibody levels at fledging were not significantly affected by egg treatment and sex (egg treatment, F 1,34.3 = 0.26, p = 0.61; sex, F 1,81.1 = 3.26, p = 0.075; treatment by sex interaction, F 1,63.5 = 1.60, p = 0.21; n = 28 T males, 16 control males, 23 T females, 21 control females), though male fledglings had slightly higher total antibody levels (4.38 ± 0.16 [log (mODmin−1)]) than females (4.31 ± 0.16). Total antibody levels were significantly positively associated with body mass (b = 0.25 ± 0.09; F 1,75.2 = 7.82, p = 0.007), and this association did not differ with respect to egg treatment or sex (body mass by treatment interaction, F 1,58.5 = 1.53, p = 0.22; body mass by sex interaction, F 1,61.8 = 2.26, p = 0.14). Brood size and age had no significant effect on fledgling total antibody levels (F < 3.52, df > 1,49, p > 0.065 for both covariates and their interactions).
Total antibody levels did not differ significantly between sexually mature T and control offspring (egg treatment, F 1,28 = 0.11, p = 0.74). Adult males had significantly lower total antibody levels than females (males, 4.95 ± 0.10 [log (mODmin−1)], females, 5.27 ± 0.10; F 1,77 = 5.78, p = 0.019). Again, there was no significant interaction between egg treatment and sex on total antibody levels (F 1,70.2 = 0.93, p = 0.34; n = 25 T males, 15 control males, 21 T females, 21 control females). Body mass, age or brood size did not significantly influence adult total antibody levels (covariates and their interactions, F < 1.2, df > 1,24, p > 0.28 in all cases).
Adult response to diphtheria–tetanus challenge
Also, during the secondary humoral immune response, offspring hatched from T eggs had significantly higher antibody levels toward the diphtheria–tetanus vaccine than offspring hatched from control eggs (F 1,25.7 = 6.52, p = 0.017; Fig. 3). There was no difference in immune score for the secondary response between males and females (F 1,61.9 = 0.03, p = 0.85), and there was no significant interaction effect between egg treatment and sex (F 1,55.9 = 0.02, p = 0.88; n = 16 T males, 11 control males, 20 T females, 20 control females). None of the covariates and their interactions significantly influenced the strength of the secondary immune response toward the diphtheria–tetanus vaccine (F < 1.0, df > 1,18, p > 0.33 in all cases). Between the day of the second injection with the diphtheria–tetanus vaccine and the peak of the secondary, humoral immune response (day 7), males lost significantly more body mass than females (males, −0.63 ± 0.15 g; females −0.18 ± 0.12 g; F 1,64 = 4.8, p = 0.032), but there was no effect of egg treatment on body mass change during the same period (treatment, F 1,30.8 = 0.28, p = 0.60; treatment by sex interaction, F 1,60.8 = 1.16, p = 0.29). Body mass at the time of the secondary injection predicted part of the variation in weight loss with heavier birds losing more weight than lighter ones (b = −0.10 ± 0.04; F 1,43.7 = 5.61, p = 0.022) and this effect was irrespective of egg treatment or sex (covariate by treatment or sex interaction; F < 0.13, df > 1,31, p > 0.73). However, this may again have been a statistical artefact (Kelly and Price 2005). Age and brood size did not significantly affect body mass changes during the secondary humoral immune response (covariates and their interactions, all F < 1.3, df > 1,44, p > 0.27).
Relationship between different measures of immunity
Moreover, for the secondary humoral immune response, there was a significant interaction between egg treatment and the primary humoral immune response (F 1,62.6 = 8.44, p = 0.005; Fig. 4b) showing that the relationship between primary and secondary humoral immune response differed between T offspring and control offspring. The slope of the relationship was steeper in control offspring (b = 0.98 ± 0.20; F 1,27.3 = 23.42, p < 0.001) than in T offspring (b = 0.36 ± 0.10; F 1,34 = 12.63, p = 0.001), which means that T-offspring with low primary humoral immune responses showed generally stronger secondary responses than control offspring with low primary humoral immune responses (Fig. 4b).
In this study, we show that prenatal testosterone can have a modifying effect on immune function, both during the early developmental period as well as during adulthood. Egg treatment affected both the strength of the immune responses as well as the relationships between different immune components. However, the effects of prenatal androgens on immunity differed with respect to type of immune response, context and life stage.
The strength of the cell-mediated immune response in control zebra finch fledglings was negatively associated with brood size. This finding is consistent with other studies reporting reduced cell-mediated immune responses in large compared to small broods (e.g., Saino et al. 1997; Horak et al. 1999; Ilmonen et al. 2003). Reduced cell-mediated immunity in larger broods is linked to a reduced per capita food provisioning by the parents, which means that individual offspring in these broods have less resources available for development and normal functioning of the immune system (e.g., Saino et al. 1997; Ilmonen et al. 2003). Interestingly, elevation of egg T appeared to modify the relationship between cell-mediated immunity and brood size for T fledglings. It is possible that T-egg young in large broods may be more successful in demanding and acquiring resources from their parents than control-egg young, e.g., through increased begging. This is supported by the fact that in the zebra finch, female nestlings beg longer during the first days after hatching when exposed to elevated yolk T levels (von Engelhardt et al. 2006). Our finding of a brood size specific effect of egg treatment on cell-mediated immunity is in line with other studies showing that the context (e.g., nutritional or competitive environment) in which egg androgen effects are studied is important (Pilz et al. 2004; Cucco et al. 2008). Given the variable effects of egg androgens reported for avian species, this aspect clearly deserves further attention.
Egg treatment also affected mass change in zebra finch fledglings within 24 h after the PHA injection in a sex-specific way. Zebra finch young have not reached their final body mass at fledging and hence, are still growing at this stage. In control fledglings, males appeared to cope less well with the PHA challenge than females as suggested by their reduced mass gain, whereas, there was an opposite albeit not significant pattern in T fledglings. Although, we could not find a difference in wing web swelling for control males and females, the result is consistent with cell-mediated immune response being more costly for male than female zebra finch fledglings (see Love et al. 2008). However, when exposed to elevated prenatal T levels these costs appear to be similar between the sexes. Our results are in line with the sex-specific effects of prenatal T on zebra finch chicks reported in previous studies (von Engelhardt et al. 2006; Tobler and Sandell 2009).
We found no long-term effect of elevated egg T on cell-mediated immunity. This finding is consistent with the results from a recent study on Grey partridges in which no effect of egg T was found on adult cell-mediated immunity (Cucco et al. 2008; see ‘Introduction’ section). Taken together, these two studies suggest that cell-mediated immunity in adulthood is not modulated by egg androgens.
Total antibody levels at fledging and sexual maturity did not differ between zebra finches that hatched from T-treated and control eggs. This suggests that health status was similar among the treatment groups before the birds were challenged with PHA or diphtheria–tetanus vaccine. Our result for fledglings is consistent with the study by Müller et al. (2005) in which no difference in total plasma antibody concentration was found in approximately 1 week old black-headed gull (Larus ridibundus) chicks prior to immunisation with lipopolysaccharides (LPS).
Surprisingly, both primary and secondary immune responses toward diphtheria and tetanus antigens in ca. 5- and 7-month-old zebra finches were positively influenced by egg treatment. This finding is in contrast with the two other studies, which have found an immunosuppressive effect of prenatal androgens on humoral immunity (Müller et al. 2005; Sandell et al. 2009). However, both studies focused on the chick phase, when the young were still growing and the immune system was still under development, and it is not clear whether suppression of the immune system by egg androgens might have lasted into adulthood. In our study, on the other hand, we have no measure of antigen-induced immune responses (such as LPS as in Müller et al. (2005) or diphtheria–tetanus as in Sandell et al. (2009)) for the zebra finch nestlings. Thus, although there was no difference in total antibody levels between the egg treatment groups at fledging, we cannot rule out that a challenge with an antigen in zebra finch nestlings would result in differential activation of the immune system among treatment groups.
Interestingly, the primary humoral immune response toward the diphtheria–tetanus vaccine was differently associated with the adult cell-mediated immune response in the two treatment groups. Adult T offspring with a strong cell-mediated immune response also showed a strong primary humoral immune response, whereas, there was an opposite relationship for control offspring. This suggests a trade-off between cell-mediated and humoral immunity in control, but not in T offspring. In birds, the intra-specific balance between cell-mediated and humoral immunity has been shown to vary spatially, temporally as well as with the degree of parasitism (Ardia 2007; Arriero 2009). Similarly, the amount of androgens transferred to avian eggs is also known to vary with time and parasite exposure (e.g., Gil 2008). It is therefore possible that part of the observed variation in the relationships between different components of immunity is mediated by yolk androgens.
We also found that the relationship between the primary and secondary humoral immune response toward the diphtheria–tetanus antigens differed between the treatment groups. The strength of the secondary response was less associated with the primary response in T offspring compared to control offspring. Secondary antibody responses were relatively high for T offspring even for individuals with low primary responses. This finding is interesting given that primary and secondary responses involve different immunological mechanisms (Janeway and Travers 1996) and have been shown to be under different selective regimes (Råberg and Stjernman 2003).
The mechanisms by which prenatal androgens influence humoral immune function are not known. Clearly, the long-term effects of prenatal T on the humoral immune function found in this study could be organisational (e.g., by permanently modifying hypothalamus–pituitary–gonadal activity or androgen receptor expression during early development) as well as indirect activational (e.g., mediated through social interactions experienced as nestlings; Carere and Balthazart 2007).
In this study, we found no effect of egg treatment on body mass of developing nestlings. It seems, therefore, less likely that treatment-specific differences in adult humoral immunity are mediated through prenatal T effects on nestling growth. However, as we did not take continuous measures of nestling growth, we cannot rule out treatment-specific differences in more subtle growth parameters (e.g., growth constant as in Müller et al. 2009). It is worth noting that a study by Roberts et al. (2007) found a positive association between circulating T levels and secondary antidiphtheria responses in adult zebra finches. As both prenatal T (this study) as well as circulating blood T (Roberts et al. 2007) seem to have an immuno-enhancing effect with respect to the humoral immune response; it could be hypothesised that the mechanism through which T influences the immune system may be similar in both cases. It may also suggest that the effect of egg T is organisational, increasing endogenous T production or T sensitivity. This hypothesis is supported by a recent study on male blue tits (Cyanistes caerulus) in which it was found that subcutaneous implantation of ca. 9 weeks old birds with T resulted in a long-term organisational effect enhancing antibody responses toward sheep red blood cell (Roberts and Peters 2009). Moreover, Müller et al. (2007) found that embryonic exposure to elevated levels of androgens increased endogenous androgen production in ca. 2 weeks old Spotless starling (Sturnus unicolor) chicks. However, the only study examining whether egg androgens influence circulating blood levels of androgens in adulthood has found no association between the two (Partecke and Schwabl 2008). Thus, there is currently no evidence that elevation of egg androgen levels can lead to increased endogenous production of the same androgens in adult birds.
Interestingly, there is also some evidence that elevated levels of prenatal T increase metabolic rate in adult zebra finches (Nilsson 2009). Assuming that higher metabolic rate results in a higher turnover rate (e.g., in terms of cell respiration), this could possibly also result in a faster proliferation of immune cells during an immune response (Hasselquist 2007). Elevated humoral immune responsiveness in adult zebra finches as a consequence of a permanently altered (metabolic) phenotype would support the idea of an organisational effect of egg T on the offspring immune system.
In conclusion, the influence of high egg T levels on cell-mediated immunity and associated costs (reduced mass gain) in zebra finch fledglings is complex as it depends on both brood size as well as the sex of the young. Elevation of egg T levels also results in long-lasting positive effects on humoral, but not cell-mediated immune function. Furthermore, prenatal egg T influences the relationship between different components of the immune system. Hence, prenatal androgens could be an important factor contributing to individual variation in immune function even in adulthood. However, our results suggest that long-lasting effects of prenatal androgens are specific with respect to the different components of the adaptive immune system. One important step now is to assess the costs of immunity in association with prenatal androgens over different adult life stages. In particular, whether high immune responsiveness in T offspring is traded off with other fitness traits such as reproduction and/or whether it may lead to immunopathological effects.
We thank Sandra Chiriac for help with the zebra finches. Two anonymous reviewers provided constructive comments which improved the quality of the manuscript. This study was supported by the Royal Physiographic Society in Lund (to MT), the Swedish Research Council for Environment, Agricultural Science and Spatial Planning (Formas) (to DH) and the Swedish Research Council (to DH and MIS). The experiment was performed with approval from the Malmö/Lund ethical committee for animal research.
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