The role of developmental conditions in shaping adult phenotypes has been the focus of a great deal of recent work. However, the effects of early life stress on reproductive performance have been little studied, particularly in avian species. In addition, although there is a large body of evidence to suggest that prevailing environmental conditions are linked to changes in breeding behavior, very little work has investigated the interaction between past and current exposure to environmental stress in determining breeding success. In this study, we examined the effects of early exposure to elevated stress hormone levels (corticosterone, CORT) on parental behavior during incubation in male and female zebra finches (Taeniopygia guttata) breeding in both stressful and nonstressful conditions as adults. We found that female birds fed CORT during postnatal development exhibited reduced incubation effort under both breeding conditions. There were no effects of developmental CORT exposure on male incubation effort; however, males breeding in unpredictable feeding conditions significantly reduced their effort levels compared with control males. There were no effects of either of our experimental treatments on hatching success or length of the incubation period. This may have been due to partial compensation by females paired with males that reduced effort. Our results clearly demonstrate sex-specific responses to developmental and adult environmental conditions in terms of incubation behavior and while in this captive population fitness costs appear to be ameliorated, birds breeding in a less benign environment in the wild may face higher costs having downstream effects on current or future reproduction.
Studies in diverse taxa have established a link between the environmental conditions experienced during development and the expression of a range of phenotypic traits in adulthood (Lindstrom 1999; Metcalfe and Monaghan 2001; Seckl 2004; Gluckman et al. 2007; Monaghan 2008). Much of this work has focused on the potential constraints imposed on animals after exposure to detrimental rearing environments such as reduced food intake, increased sibling competition, and elevated stress hormones (Blount et al. 2003; Royle et al. 2005; Gluckman et al. 2007; Spencer and Verhulst 2007). In vertebrates, stressful conditions lead to activation of the hypothalamic pituitary adrenal or stress axis. The resultant increased production and release of glucocorticoid stress hormones plays an important role in directing appropriate physiological and behavioral responses to stressors, such as severe weather or food shortages (Wingfield 1994). During development, growing birds are exposed to corticosterone (CORT) via maternal transfer in ovo (Hayward and Wingfield 2004; Saino et al. 2005) as well as endogenous CORT production in later developmental stages (Sims and Holberton 2000; Love et al. 2003; Love and Williams 2008; Wada et al. 2008). Developmental exposure to elevated CORT has been shown to influence the expression of many behavioral, physiological, and morphological traits in birds (Spencer et al. 2003; Kitaysky et al. 2003; Rubolini et al. 2006; Spencer and Verhulst 2007; Love and Williams 2008; Wada et al. 2008). Although some of these effects are transient, several lines of evidence now suggest that CORT can have a long-term “programming” effect on adult phenotypes (Matthews 2002; Seckl and Meaney 2004). One important phenotypic trait that may be affected by early life conditions is breeding success, which has obvious fitness implications. There is a large body of evidence, particularly in avian species, that stressors experienced in adulthood can affect breeding behavior and performance. For example, elevated CORT, after an acute stressor, suppresses parental behavior in some species (Silverin 1998; Pereyra and Wingfield 2003; Salvante and Williams 2003). However, we still require more studies to investigate the influence of developmental stress on later parental behavior (Maestripieri 2005; Naguib et al. 2006; Curley et al. 2008).
Studies have revealed sex differences in response to stressful conditions in early life. In many of the avian studies to date, females appear more sensitive to adverse developmental conditions when compared with males, exhibiting higher levels of stress-induced mortality and/or more evident long-term phenotypic effects (de Kogel 1997; Bradbury and Blakey 1998; Kilner 1998; Martins 2004; Kalmbach et al. 2005; Verhulst et al. 2006). Interestingly, sex-specific differences in response to adverse conditions during breeding have also been recorded in several bird species, with in many cases males being more likely than females to reduce parental effort in unfavorable conditions (Slagsvold and Lifjeld 1990; Wright and Cuthill 1990; Whittingham et al. 1994; Sanz et al. 2000), although this is not a universal finding (Beaulieu et al. 2009).
Avian incubation can be an energetically demanding reproductive stage (Thompson et al. 1998; Reid et al. 2000). For biparental incubators, the balance of incubation effort, within a breeding pair, has a large influence on the outcome of a breeding event, as the maintenance of an appropriate thermal embryonic environment is essential for successful development and hatching (Deeming 2002). Parental effort during incubation can therefore influence reproductive effort later in the same breeding event or in subsequent events (Heaney and Monaghan 1996, Reid et al. 2000; Cresswell et al. 2004). Incubation effort has also been related to parental CORT levels, with elevated CORT associated with a switch to foraging away from reproduction (Love et al. 2004; Groscolas et al. 2008). Previous work has suggested that in species where males perform a substantial proportion of incubation behavior, male birds are more likely to adjust their incubation effort according to current energy demands, and so to current conditions, whereas female effort is related to the effort levels of her mate, rather than being directly affected by prevailing environmental cues (Wiebe 2007). Therefore, it is clear that birds may have sex-specific responses in terms of incubation effort to both their own developmental history and current environmental conditions.
In this study, we examined the effects of early exposure to elevated CORT on parental behavior during incubation in male and female zebra finches (Taeniopygia guttata) breeding in both stressful and nonstressful conditions as adults. In zebra finches, as with several altricial avian species, both males and females participate in incubation (Zann 1996). This provided us with an opportunity to determine any sex differences in the long-term effects of developmental stress and of current environmental conditions on parental effort during this important reproductive stage. We predicted that female birds would be more likely to show long-term effects of early exposure to stress, exhibiting reduced incubation effort. We also predicted that while both sexes may be influenced by current environmental conditions, males may reduce incubation effort significantly more than females.
MATERIALS AND METHODS
Manipulation of CORT exposure during postnatal development
Birds used in this study were experimentally manipulated during postnatal development (see Spencer et al. 2009 for details). Young birds (aged 5 days), obtained from breeding pairs from the University of Glasgow breeding stock, were randomly allocated to 1 of 2 groups: Control or CORT-fed. We previously demonstrated that zebra finch nestlings subjected to a standardized capture handling restraint procedure (Wingfield 1994) were capable of mounting a physiological stress response from 12 days of age (Spencer et al. 2009). Accordingly, treatment with CORT was between day 12 and 28, when the birds become nutritionally independent of their parents. Data from this previous study also allowed us to scale the CORT doses used in this study to produce an elevation in exogenous CORT levels of 2 standard deviations (SDs) above the mean peak response (Spencer et al. 2009). Between days 12–15, CORT-fed birds received oral boluses (25 μl) of CORT (Sigma Aldrich: 0.124 mg/ml, in peanut oil), twice daily, approximately 5 ± 1 (standard error of the mean) hours apart. This gave a total daily dose of 6.2 μg of CORT. After day 16, this dose was increased to 8.15 μg/day by increasing the concentration of the CORT administered to 0.163 mg/ml. Both age-specific doses were scaled to produce an increase in CORT concentrations that were comparable with stress-induced CORT levels in similarly aged finches (see Spencer et al. 2009 for more details). Control birds received peanut oil. Birds were maintained in family groups until 60 days of age, when they were maintained in sex- and treatment-specific groups (n = 8–10 birds per 120 × 50 × 50 cm cage) until the breeding period. They were provided with an ad libitum diet of seed (Haiths Ltd, Grimsby, UK), shell grit, and cuttle fish bone, as well as weekly supplements of rearing and conditioning food (Haiths Ltd) and spinach during this prebreeding period, and the photoperiod was kept at 14:10 h light:dark and the temperature maintained between 20–24 °C. Prior to our breeding experiments described below, there were no differences in body mass in our population due to sex (F1,149 = 1.51, P = 0.22) or developmental CORT treatment (F1,149 = 2.60, P = 0.11).
Pair establishment and breeding
Birds from the control and postnatal treatment groups were paired as adults (mean age 412 ± 26 [SD] days) to establish 4 types of breeding pairs: control female and control male (n = 18 pairs); postnatal CORT-fed female and postnatal CORT-fed male (n = 10 pairs); postnatal CORT-fed female and control male (n = 12) and control female and postnatal CORT-fed male (n = 13). All pairs were housed in cages that were 60 × 50 × 50 cm, provided with nest-boxes and nest material (coconut fiber and jute, Haiths Ltd) and maintained on the same photoperiod and temperature regime as before. To determine nesting success, we recorded the length of the incubation period (duration from last egg laid until hatching) and the number of offspring hatching for each nest. Incubation behavior was recorded using instantaneous scan sampling every 10 min for 2 h approximately halfway through the incubation period (between days 8–10 of incubation). We recorded whether the female and/or male were inside the nest-box, as studies on the same population have shown this measure to be strongly positively correlated with the amount of time spent incubating (Hill D, Nager R, Lindstrom J, unpublished data).
Variation in breeding conditions
Unpredictable food availability is a stressful situation for birds, producing elevated baseline CORT levels (Kitaysky et al. 1999; Pravosudov et al. 2001; Jenni-Eiermann et al. 2008). We therefore used the predictability of access to a food resource to mimic naturally “stressful” conditions experienced in adulthood. This manipulation was not designed to restrict energy intake in the birds over a day, as all birds had sufficient time to gain energy requirements (Cuthill et al. 2000; Buchanan et al. 2003). All pairs were bred under both predictable ad libitum feeding conditions and unpredictable conditions, thereby exposing them to differentially stressful breeding environments. The order in which pairs experienced these 2 conditions was counterbalanced across pair types. Because the first breeding event of naive birds can have low success, all pairs were allowed a trial-breeding attempt in their allocated pairs and adult treatment group to reduce potential effects of first time breeding. There were no statistically significant differences in incubation behavior or nesting success between the initial breeding attempt and the first experimental breeding attempt (P > 0.05 for the number of female incubation observations, the number of male incubation observations, the number of incubation observations in which neither the female nor the male was on the nest, clutch size, and the number of chicks that fledged). It should be noted that there was also no significant difference in clutch size between the 2 experimental breeding attempts (t1,52 = 0.795, P = 0.430). Food access was manipulated by removing the food bowl; access to seed within the cage litter was prevented by placing a transparent mat over the litter that prevented the birds from foraging but maintained the visual appearance of the cage. Access to food was restricted for 3.5 h each day (i.e., 25% daylight hours) between the hours of 9:00–15:00 on a random schedule (unpredictable food). For the rest of the day food was available ad libitum. For each breeding round, food predictability was manipulated 10 days prior to pairing while birds were still in same sex and same postnatal CORT treatment groups, and food predictability was maintained until breeding was complete. For each pair, the breeding round was considered complete after they had either failed (eggs showed no signs of development after 20 days) or all chicks had fledged or died. After completion of a breeding round, pairs were separated and put into same sex and postnatal CORT treatment groups for a period of 30 days, where they received unmanipulated ad libitum food supplies before the appropriate prebreeding manipulation began.
The effects of CORT exposure during postnatal development and food predictability during breeding on parental incubation behavior and incubation success were assessed using repeated measures generalized linear models (GLMs) (SPSS 15.0), to allow each pair to be compared with itself under the 2 breeding treatments. Food predictability was added as the within-subjects factor, with female and male postnatal treatments and order in which they experienced the food treatments were added as between-subjects variables. Different measures of incubation effort used as dependent variables were: the number of observations when females were on the nest (female incubation effort), number of observations when males were on the nest (male incubation effort), the amount of egg neglect (number of observations when neither parent was on the nest), length of the incubation period (defined as the period between the last egg being laid and first egg hatching), and total number of eggs that hatched and hatching success (relative to clutch size). A backward elimination process was used to exclude independent variables with P > 0.05. The number of observations when neither parent was in the nest-box was rank transformed to improve normality; however, all other variables conformed to the assumptions of the GLM.
Female incubation effort was significantly affected by conditions in early life (F1,51 = 5.6, P = 0.02), with females that experienced elevated CORT during postnatal development exhibiting reduced effort during breeding in both predictable and unpredictable feeding conditions (Figure 1a). There was no effect of food predictability on female behavior (F1,51 = 1.5, P = 0.22) nor any effect of the order in which they bred in the different adult environments (F1,47 = 0.2, P = 0.64). Postnatal CORT exposure of the male partner had no influence on female incubation effort (F1,50 = 1.1, P = 0.31), and there was no interaction between male and female postnatal treatments (F1,49 = 0.5, P = 0.46).
Male incubation effort was not affected by postnatal CORT treatment (F1,49 = 0.7, P = 0.42). However, males that experienced unpredictable feeding conditions during breeding exhibited significantly lower incubation effort than males experiencing predictable conditions (F1,52 = 4.0, P = 0.05; Figure 1b). There was no effect of their female partner's postnatal CORT exposure on adult male behavior (F1,50 = 0.8, P = 0.38) or an interaction between the 2 (F1,47 = 0.15, P = 0.71). There was also no effect of the order in which they experienced the food treatments (F1,51 = 3.2, P = 0.08).
There was a significant interaction between male and female postnatal CORT treatment in the level of egg neglect (F1,49 = 6.3, P = 0.02; Figure 2). Post hoc analysis (Tukey estimates) revealed that pairs consisting of 2 control animals had the lowest levels of neglect, though only marginally different from the pairs that consisted of control males and CORT-fed females (P = 0.06). However, there were no main effects of male or female postnatal CORT treatment (F1,49 = 0.07, P = 0.79, F1,49 = 0.74, P = 0.39, respectively) nor any effects of adult breeding environment (F1,51 = 1.22, P = 0.28). There was a significant negative correlation between female and male incubation effort within a pair under both feeding conditions (predictable: r = −0.58, P < 0.001, N = 53, unpredictable: r = −0.45, P < 0.001, N = 53, Figure 3).
There were no effects of female or male postnatal CORT treatment on the length of the incubation period (F1,37 = 0.83, P = 0.37, F1,39 = 0.74, P = 0.39, interaction: F1,37 = 0.88, P = 0.35). Food predictability also had no effect on incubation period duration (F1,41 = 0.002, P = 0.96), and there was no interaction between postnatal CORT and food treatments (male: F1,39 = 2.33, P = 0.13; female: F1,37 = 3.37, P = 0.08). When considering hatching success, there were no effects of either female or male postnatal CORT treatment (F1,50 = 0.25, P = 0.62, F1,49 = 0.03, P = 0.81, interaction: F1,46 = 0.46, P = 0.73). Food predictability also had no effects on the number of chicks hatching (F1,51 = 0.41, P = 0.52), and there was no interaction between postnatal CORT treatment and food conditions (male: F1,49 = 0.37, P = 0.54; female: F1,50 = 0.05, P = 0.83).
Our results clearly demonstrate that developmental exposure to CORT is associated with reduced incubation effort in female zebra finches, regardless of the prevailing conditions during breeding. Male birds, however, showed no such response to developmental conditions. On the other hand, males exhibited shorter term responses to unfavorable breeding conditions. This sex difference is interesting and suggests that females in our population may be more sensitive to detrimental developmental conditions with respect to long-term effects on adult behavior. Few studies have attempted to measure long-term effects of developmental stress on both sexes within a population. Previous work that used a similar protocol of CORT administration demonstrated a significant sex-specific negative effect of CORT on male neophobia (Spencer and Verhulst 2007). However, the timing of CORT administration differed to that in the current study, and we have very little information on how the timing of developmental stress can affect different phenotypic traits. The results obtained in the current study are consistent with studies that have imposed stressors during postnatal development other than administration of exogenous CORT. For example, experimental enlargement of brood sizes have also found stronger effects in females, as evidenced by decreased survival into adulthood or increased metabolic costs under standard conditions (de Kogel 1997; Verhulst et al. 2006).
Our results showed no effects of breeding treatment on nesting success. This lack of effect, despite a significant reduction in incubation behavior in CORT-fed females, could be explained if their male partner compensated for this reduced effort (Houston and Davies 1985). Although the developmental treatment group of a male partner did not influence female incubation effort, under any adult feeding condition, there was a significant negative correlation between female and male incubation effort within a pair under both breeding treatments (Figure 3). This suggests that at least partial within-pair compensation for reduced incubation effort occurred, thereby maintaining the total incubation effort for each pair and possibly negating any detrimental effects of decreased female incubation effort on the development and survival of the offspring (Wright and Cuthill 1990; Markman et al. 1996; Sanz et al. 2000). This is backed up by the finding that levels of neglect were relatively consistent across the population, although it should be noted that the lowest levels of neglect were seen in pairs consisting of 2 controls.
Our finding that male birds were more likely than females to reduce incubation behavior when breeding under stressful conditions is consistent with several previous studies. For example, sex differences have been documented in experimental studies that have handicapped breeding individuals, whereby males show a tendency to reduce parental effort when handicapped and females maintain effort levels (Slagsvold and Lifjeld 1990; Wright and Cuthill 1990; Sanz et al. 2000). It has been suggested that this sex difference is due to differences in the fitness cost of a reduction in body condition, where males incur larger costs (Sanz et al. 2000). We did not measure the body condition of breeding birds over the duration of incubation in the current study and therefore we cannot determine if there were sex-specific mass or energy reserve changes in response to our manipulations.
Although we have demonstrated that early life stress can cause a reduction in incubation effort in female birds, the underlying mechanism for this shift in behavior is currently unclear. Recent work has suggested that postnatal exposure to CORT in this finch population resulted in exaggerated and prolonged CORT release in response to acute stress (Spencer et al. 2009). CORT concentrations rise over the course of a breeding season and peak during the nestling period in some species. However, there is some evidence that basal CORT concentrations and acute CORT responses are dampened during the breeding period (Pereyra and Wingfield 2003; Williams et al. 2008). If CORT-fed females exhibited elevated CORT during incubation, possibly related to reduced body condition, this may account for a disruption in incubation behavior because elevated CORT is often associated with suppressed prolactin concentrations (Carretero et al. 1997; Yokoyama et al. 2008); known to regulate parental behavior, in particular incubation effort (Criscuolo et al. 2006; Groscolas et al. 2008). Further work is currently being undertaken to determine the hormonal correlates of incubation effort in this population and the dynamic changes in CORT concentrations across the breeding cycle in animals experiencing both control and chronic stress conditions.
Although many studies of developmental stress have focused on the constraints imposed on the expression of phenotypic traits, there has been recent interest in the potential adaptive significance of responses to developmental conditions. Selection will favor phenotypic adjustments in response to detrimental conditions that mitigate some negative effects as much as possible (Monaghan 2008). Hence, it is likely that at least some of the phenotypic responses to conditions in early life might have functional significance over the longer term (Matthews 2002; Bateson et al. 2004; Seckl and Meaney 2004; Gluckman et al. 2005; Monaghan 2008). The benefits of adjustments induced by early conditions might be linked to “shaping” the phenotype to perform better in the anticipated adult conditions (Bateson. et. al. 2004, Gluckman et al. 2005). Such “environmental matching” has received little rigorous experimental investigation (Monaghan 2008).
Our repeated measures design, provided individuals with adult conditions that differed in their degree of perceived stress and exposed birds to breeding environments that “matched” and “mismatched” their early life conditions. However, we found no evidence that birds that experienced elevated CORT levels during postnatal development were conferred any advantage in terms of nesting success when breeding under stressful conditions later in life. If environment matching models are correct, we would have expected those birds that experienced a benign rearing environment (Controls) to show maximal nesting success under predictable feeding conditions, whereas postnatally stressed birds would show enhanced nesting success under unpredictable food conditions. The alternative hypothesis, which has found the most empirical support so far is that of the silver spoon model (reviewed in Monaghan 2008), whereby developmentally stressed individuals underperform as adults whatever the breeding conditions. Although we did not find any difference in breeding success under laboratory conditions, our finding that CORT-fed females showed reduced incubation effort in both breeding treatments is consistent with the silver spoon model.
In summary, we have demonstrated sex-specific responses to developmental and adult environmental conditions. That a short period of exposure to elevated CORT levels during postnatal development can disrupt incubation behavior in adult female zebra finches, but not males, is interesting and warrants further investigation into the potential underlying mechanisms. Although in this captive study within-pair compensation appears to have ameliorated any potential fitness consequences of these differential responses, birds breeding in a less benign environment in the wild may face higher costs to such compensation having downstream effects on current or future reproduction.
Biotechnology and Biological Sciences Research Council (BBSRC) Standard Grant (BB/D010896/1); BBSRC David Phillips Research Fellowship to K.A.S.
We thank Graham Law and the animal husbandry staff within the Division of Ecology and Evolutionary Biology for their invaluable assistance. Ethical note: All research was carried out under Home Office Project License 60/3447.