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HPA axis regulation, survival, and reproduction in free-living sparrows: Functional relationships or developmental correlations?
General and Comparative Endocrinology 190 (2013) 188–193
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
HPA axis regulation, survival, and reproduction in free-living sparrows: Functional relationships or developmental correlations? Scott A. MacDougall-Shackleton a,b,c,⇑, Kim L. Schmidt b,c, Ainsley A. Furlonger b,c, Elizabeth A. MacDougall-Shackleton b,c a b c
Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada Advanced Facility for Avian Research, University of Western Ontario, London, Ontario N6G 1G9, Canada
a r t i c l e
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Article history: Available online 13 June 2013 Keywords: Glucocorticoids Developmental correlations Hypothalamus–pituitary–adrenal axis CORT-fitness hypothesis
a b s t r a c t A growing body of theoretical and empirical work has addressed the relationship between hypothalamus–pituitary–adrenal (HPA) function and fitness. For example, the corticosterone (CORT)-fitness and CORT-condition hypotheses predict that baseline and/or stress-induced levels of glucocorticoids should relate to fitness, and recent empirical studies have reported relationships between HPA function and fitness-related sexually selected traits. Here we introduce a framework for evaluating whether such relationships reflect functional relationships or developmental correlations. We then address this framework using data from a free-living population of song sparrows (Melospiza melodia). In two independent studies we have found that song complexity (a sexually selected trait) is correlated with stress reactivity: males with more complex vocal repertoires show reduced CORT response to standardized restraint stress. This pattern likely results from the early life environment concurrently affecting development of both song and the HPA axis. Suppression of CORT by dexamethasone was also correlated to measures of body condition and immune function, and females paired to males with higher stressinduced levels of CORT initiated egg-laying later. Finally, stress reactivity predicted overwinter survival in one year, although not in another. Thus, the relationship between HPA axis function and fitness likely varies temporally and by context. Some fitness-related traits may be functionally related to HPA regulation, but many others may be related through developmental correlation. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Over the last few decades, ecological and evolutionary physiologists have increasingly considered steroid hormones as potential mediators of trade-offs between components of life-history traits. For example, testosterone level has been proposed as a mechanism for balancing resource investment between reproductive effort and immune function (Folstad and Karter, 1992; Sheldon and Verhulst, 1996) or between mating and parental effort (Ketterson et al., 1992; McGlothin et al., 2007). Similarly, attention to the potential role of glucocorticoids in influencing fitness, population dynamics, and evolution has also increased with early studies on mammals (e.g., Boonstra and Boag, 1992; Boonstra and Singleton, 1993) and more recent research on other taxa including birds (Bonier et al., 2009a; Breuner et al., 2008; Zera and Harshman, 2001). In this review, we evaluate approaches to studying the relationship between glucocorticoid levels, and more generally, function⇑ Corresponding author at: Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada. E-mail address: [email protected] (S.A. MacDougall-Shackleton). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.05.026
ing of the hypothalamus–pituitary–adrenal (HPA) axis; and fitness-related traits. We highlight the importance of considering HPA axis regulatory mechanisms rather than glucocorticoid levels alone, and the importance of determining whether such relationships reflect functional mechanisms or correlations. We then use examples from our work on free-living song sparrows (Melospiza melodia) to illustrate these issues. 2. A framework for thinking about HPA-fitness correlations 2.1. Glucocorticoids as potential mediators of trade-offs Glucocorticoids such as corticosterone (CORT) are critical mediators of energy balance and affect multiple physiological systems. Baseline levels of CORT vary over multiple time scales and are associated with changes in energy balance (Sapolsky et al., 2000) and transitions between life-history stages (Crespi et al., 2013; Denver, 2009). In addition, CORT levels can increase rapidly in response to external factors as part of the adrenal stress response, and result in the animal entering an emergency life-history stage (Wingfield et al., 1998).
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The stress response of vertebrates is widely conserved. In response to a diverse variety of stressors, including biotic and abiotic factors, vertebrates initiate a rapid neural response (sympathetic nervous system) followed by a longer-term increase in circulating glucocorticoids. These glucocorticoids affect numerous physiological systems to allow the animal to cope with the stressful event (Wingfield and Sapolsky, 2003). In the short term, increased glucocorticoids are presumed to be adaptive in that they allow the animal to divert resources from growth, maintenance and reproduction towards short-term survival. In the long term, however, chronically elevated glucocorticoids are thought to have deleterious effects on numerous neural and physiological systems (Wingfield and Sapolsky, 2003). Glucocorticoids are thus tightly regulated and animals typically return levels to homeostatic baseline after coping with a stressor. Thus, changes in circulating CORT (baseline levels and stress-induced levels) are often considered to reflect a balance between short-term survival and long-term investment in processes such as growth and reproduction. 2.2. CORT-fitness relationships A central tenet of life history theory is that that trade-offs between competing demands should be optimally balanced to maximize lifetime reproductive success. Thus, if CORT is a physiological mediator of such trade-offs then CORT levels should be related to fitness, or related by proxy to fitness-related traits. Two recent reviews have assessed the relationship between CORT and fitness for both baseline levels and acute stress-induced levels of CORT (Bonier et al., 2009a; Breuner et al., 2008). Both reviews found equivocal support for the predicted relationship between CORT and fitness-related traits. Across studies, CORT levels were both positively and negatively related to fitness-related traits, and often there was no relationship. Thus, although several individual studies have detected relationships between CORT and fitness-related traits such as survival (Blas et al., 2007; Romero and Wikelski, 2001) or sexually selected trait expression (Roulin et al., 2008) overall there is no consistent CORT-fitness relationship in the literature (Bonier et al., 2009a; Breuner et al., 2008; Crespi et al., 2013). There are several potential reasons why the strength and direction of CORT-fitness relationships vary from study to study. First, the relationship between CORT and a fitness-related trait may vary across life history stages (Bonier et al., 2009b). Second, CORT levels at any given point in time will be influenced by a variety of intrinsic and extrinsic factors (Crespi et al., 2013) which may need to be controlled to detect CORT-fitness relationships (e.g. Hau et al., 2010). That is, to detect a relationship (functional or not) between CORT and fitness-related or life-history variables will often require statistically controlling for other variables that affect CORT levels. Thus, searching for simple direct relationships between CORT levels and fitness-related traits will often prove difficult. In addition to the above considerations, in many cases even if CORT levels are functionally related to a fitness variable, CORT levels themselves may not be the primary targets of selection. If CORT mediates trade-offs between life-history components, the physiological traits most likely to be correlated with fitness may be the regulatory processes that determine CORT levels, not CORT levels as such. This point is overlooked by the majority of studies on CORT-fitness relationships. The mechanisms that determine how CORT levels vary in response to extrinsic factors include variables such as pituitary sensitivity to corticotropin-releasing hormone, adrenal sensitivity to adrenocorticotropic hormone (ACTH), and sensitivity to negative feedback in the HPA axis. Individual variation in these measures is much more likely to influence fitness than is individual variation in CORT levels. Thus, research aimed at understanding CORT-fitness relationships should ideally mea-
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sure HPA regulation rather than relying exclusively on point samples of CORT. Even if one quantifies the regulatory mechanisms of the HPA axis, determining HPA-fitness relationships may still prove elusive because such relationships likely vary over time, space, and context. As noted by Schoech et al. (2011) ‘‘the assumption that there is a single . . . corticosterone phenotype that maintains a stable adaptive payoff in response to the varied environmental challenges an organism is liable to experience is unrealistic.’’ Consider a hypothetical individual animal. What would its optimal stress response be? In a benign environment, individuals that are less stress-reactive may outcompete their more stress-reactive counterparts. But if the environment is degraded or becomes unpredictable this competitive advantage may reverse. Similarly, a subordinate animal may have higher fitness if it is more stress-reactive, but if it becomes dominant it may benefit from being less reactive. Thus the relationship between CORT levels or HPA regulatory mechanisms and fitness will vary over different contexts and different times. Finally, it is important to note that even when relationships between CORT or HPA function are detected, such correlations may or may not reflect natural selection. It is possible that third variables may influence both CORT and the fitness-related trait, and indeed these third variables may be entirely environmental. We discuss one type of environmental effect (developmental correlations) below. 2.3. Developmental correlations Another difficulty in characterizing CORT-fitness relationships is that the majority of studies on this topic are correlational. Correlations between CORT levels (or HPA function) and some measure of fitness might arise entirely because both factors are correlated with some unknown third variable, rather than because of a functional relationship between them. That is, rather than individual variation in CORT functionally affecting individual variation in fitness by mediating a trade-off, it is possible that both the CORT levels and the fitness-related trait result from another common factor. One such factor may be early developmental experience. Glucocorticoid exposure early in life can have long-term organizational effects on a variety of physiological and neural systems, including the development of the HPA axis itself (Liu et al., 1997). Thus, HPA function or CORT levels may be correlated to a fitness-related trait not because they are functionally related but because they were both affected by the same developmental stressors (i.e. developmentally correlated traits (MacDougall-Shackleton and Spencer, 2012). For example, both the hippocampus and the song-control brain regions of songbirds appear particularly sensitive to early life stressors (reviewed in MacDougall-Shackleton and Spencer (2012), Spencer and MacDougall-Shackleton (2011)), and early life stress may thus affect both singing behaviour and spatial cognition (Farrell et al., 2011). Similarly, development of the HPA axis is affected by early life stress in mammals (Liu et al., 1997) and birds (Spencer et al., 2009) and may thus be developmentally correlated with other traits. 3. HPA function and fitness correlates in song sparrows 3.1. Background We have examined the relationship between various measures of HPA function and fitness-related traits in a long-term study population of song sparrows. We have studied this population since 2002, and it is characterized by high adult breeding philopatry. Each year approximately 40–60% of breeding adults consist of previously banded individuals that also bred at the study site in prior years. The remainder are new recruits to the population and in-
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ferred to be first-time breeders. Each spring we capture and band all breeding adults, and monitor their reproduction and overwinter returns. Because birds in this population are so highly philopatric, typically nesting within 75 m of their previous year’s territory (unpublished data) we conclude that birds that fail to return to the study site have died (Greenwood and Harvey, 1982; MacDougall-Shackleton et al., 2009). In addition to monitoring reproduction and survival, in some breeding seasons (2007 and 2010) we have assessed HPA function of known banded birds. We measure baseline levels of CORT from blood samples collected within 3 min of capture in a mistnet, then measure stress-induced CORT after 30 min of standardized restraint, and calculate stress reactivity as the increase in CORT following a stressor (stressed – baseline levels (MacDougallShackleton et al., 2009). In the 2010 breeding season we also characterized negative feedback and adrenal sensitivity of the HPA axis (following Dickens et al., 2009; Rich and Romero, 2005; see Fig. 1). After collecting a stress-induced sample we injected birds intramuscularly with dexamethasone (1.0 mg/kg) and placed them in a covered cage with food and diluted fruit juice. Sixty minutes later a third blood sample was collected to quantify negative feedback, then we injected birds with ACTH (25 IU/kg) and placed them back in the cage. After a further 30 min we then collected a final blood sample to quantify adrenal sensitivity and released the bird. CORT was quantified in unextracted plasma using an RIA (MP Biomedicals, 07-120103) that has been previously validated in song sparrows (Newman et al., 2008). We continued to monitor the birds’ reproduction and survival, recorded males’ song repertoires, and made a variety of other morphological, physiological, and behavioral measures (see below). 3.2. HPA function and survival-related traits We examined whether our measures of HPA function were related to body size and condition and hematological profile (Schmidt et al., 2012). As is common in studies of birds, our morphometric measurements yielded two principal components
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reflecting body size and body condition (mass relative to size). Stress reactivity (the change in CORT following a stressor) was not related to these morphometric measures, but HPA negative feedback was greater for structurally larger birds than smaller birds (Schmidt et al., 2012). That is, birds with a larger PC1 morphometric score decreased CORT to a greater extent following a dexamethasone injection. In addition to body size, we also found that HPA function was related to hematological profile. An immune index (Principal Component factor) that reflected heterophil abundance and heterophil to lymphocyte ratio was related to both stress reactivity and negative feedback (Schmidt et al., 2012). Birds that were more stressreactive had relatively fewer heterophils, as did birds with stronger negative feedback. These results, combined with the relationship between negative feedback and body size, suggest that growth and immune parameters are correlated to HPA regulatory mechanisms. However, whether such correlations reflect a direct functional relationship, as opposed to developmental correlations between HPA regulation and growth and immune parameters, remains unclear. Because we study this population each year we are able to determine which birds return from the wintering grounds to breed and which birds fail to return and are presumably dead. We found that stress reactivity in 2007 was related to overwinter survival and return to the breeding grounds in 2008 (MacDougall-Shackleton et al., 2009). Specifically, birds that were more stress-reactive in 2007 were less likely to return the following breeding season (Fig. 2). Our results linking stress reactivity to survival suggest that high stress reactivity may increase over-winter mortality, and are similar to findings in other species of birds (Blas et al., 2007). This pattern is also consistent with data from humans wherein high stress reactivity in childhood is associated with increased risk of cardiovascular disease later in life (Wood et al., 1984). However, as noted above (Section 2.3) correlation does not imply causation, thus survival and stress reactivity could both be related to an unmeasured third variable. In addition (Section 2.2) even if high stress reactivity is functionally associated with decreased survival in one context, this relationship may vary over space and time and disappear or reverse for birds that winter in different locations or for the same birds in different years. Indeed, we have recently found just that. For the birds in which we measured HPA function in 2010, we found no relationships between overwinter survival and stress reactivity, negative feedback, or adrenal sensitivity (Furlonger, 2011). Birds with high and low stress reactivity, negative feedback and ACTH sensitivity were equally likely to survive the winter of
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Bleed Time (minutes since capture) Fig. 1. Characterization of HPA function in song sparrows. Points indicate mean (±SE) plasma corticosterone levels of 37 male song sparrows (Melospiza melodia). The initial baseline sample was collected within 3 min of capture (baseline) and the stress-induced sample was collected after 30 min restraint in a cloth bag (time 30 min). We then immediately injected birds with dexamethasone (DEX; 1.0 mg/kg i.m.) to assess negative feedback. Birds were then housed in a covered cage until we collected another sample 60 min later (time 90 min). We then injected birds with adrenocorticotropic hormone (ACTH/25 IU/kg i.m.) to measure adrenal sensitivity and collected a final sample 30 min later (time 120 min). All birds were then released and resumed their territory tenure. Data adapted from Furlonger (2011).
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Increase in Corticosterone Following Stress (ng mL-1) Fig. 2. Stress response and survival in song sparrows. The probability of survival is negatively related to stress reactivity, that is, the increase in CORT from baseline to stress-induced levels. Birds that were more stress reactive were less likely to return the following year. Points indicate data from individual birds, lines indicate median and interquartile ranges. Data adapted from MacDougall-Shackleton et al. (2009).
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3.3. HPA function and reproduction To test for associations between CORT and traits linked to reproductive success we measured HPA function, song, and reproductive output. In song sparrows song complexity, assessed as song repertoire size or syllable repertoire size, appears to be a sexually selected trait and is linked to female preferences in captivity (Searcy and Marler, 1981), mate attraction in free-living birds (Reid et al., 2004), and some hematological and neuroanatomical measures (Pfaff et al., 2007). We measured the relationship between HPA function and song complexity in two different groups of birds in our population, one in 2007 and the other in 2010. In both cohorts, song complexity was negatively related to stress reactivity (MacDougall-Shackleton et al., 2009; Schmidt et al., 2012). That is, birds with less complex songs increased CORT following restraint to a greater extent than did birds with more complex songs. These data thus indicate a correlation between the stress response and a sexually selected signal, as reported in other species (Husak and Moore, 2008). In barn owls (Tyto alba), melanin-based signals have been functionally linked to HPA activity because generation of these signals shares common biosynthetic pathways with CORT production (Almasi et al., 2010; Roulin et al., 2008). In the case of song complexity, however, there is no clear functional link between birdsong and the HPA axis. Instead, a common factor between birdsong and the HPA axis is that both systems have a protracted development and both are susceptible to developmental environmental stressors (Spencer and MacDougall-Shackleton, 2011). Thus, the relationship between song complexity and stress reactivity in song sparrows is likely an example of a developmental correlation. During the 2010 breeding season we also measured the relationship between HPA function of breeding males and several indices of reproduction, including lay date, clutch size, and hatching success (Furlonger, 2011).We quantified HPA function of males prior to breeding, and then measured the reproductive output of each male and his mate over the following weeks. We observed no relationships between our measures of HPA activity and clutch size or hatching success. This is perhaps not surprising, because in this ground-nesting population there is very low variation in clutch size (the majority of nests have four eggs) and hatching failures are almost always due to stochastic nest predation events (unpub-
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2010–11 and return to the breeding grounds. Thus, HPA function was correlated to survival in one year, but not 2 years later. Why may we have found a different relationship between HPA function and survival in two different years? We can only speculate, but one possibility is that stress reactivity affects overwinter survival, but only in some conditions. In a relatively benign winter, highly reactive birds may be at a competitive disadvantage if they more frequently shift resources away from growth, maintenance and immune function. However, in more inclement winters this pattern could be eliminated or even reversed. On the wintering grounds of our population (southeast United States) the winter of 2007–08 was warmer and wetter than that of 2010–11 (data from NOAAA National Climate Data Center). This is consistent with the idea that highly stress reactive birds may have been at a competitive disadvantage in the relatively benign winter of 2007–08. However, it is also possible that HPA stress reactivity and overwinter survival in particular climates are both correlated with a third variable. For example, poor conditions in 2007 could have resulted in some birds having higher stress reactivity and also lower subsequent survival. Clarifying the nature of the link between HPA function and survival will require longterm studies that include both observational and experimental data.
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Post-dexamethasone change in Corticosterone (ng mL-1) Fig. 3. HPA function and lay date in song sparrows. A multiple regression analysis indicated that standardized lay date was significantly associated with stressinduced CORT levels and the decrease in CORT following dexamethasone injection (Furlonger, 2011). (A) Standardized lay date was positively related to CORT levels after 30 min restraint. That is, females mated to males with higher stress-induced CORT initiated their nests later in the season. (B) Standardized lay date was negatively related to negative feedback. That is, females mated to males that lowered their CORT the most following a dexamethasone injection initiated their nest later in the season. Points indicate individual birds. The lines indicate simple linear regressions and dashed lines the 95% confidence intervals of the regression. Data adapted from Furlonger (2011).
lished data). Lay date, on the other hand, was significantly related to stress-induced CORT levels and negative feedback as indicated by multiple regression analysis (Furlonger, 2011). Lay date is an important component of fitness in many bird species, with earlier lay dates being associated with higher reproductive success and offspring recruitment (Hochachka and Smith, 1991; Perrins, 1970). In our study, females whose social mates exhibited higher stress-induced CORT initiated egg-laying later in the season (Fig. 3a), as did females whose social mateshad stronger negative feedback i.e. response to dexamethasone (Fig. 3b). These patterns suggest that males with lower stress-induced CORT levels and with reduced responses to dexamethasone initiate breeding earlier and may achieve higher reproductive success as a result. However, we cannot disambiguate whether the male himself or a variety of indirect factors such as behavior of the female, territory quality, or other factors mediate these patterns. In addition, even if changes in lay date were directly correlated to male HPA activity, further work would be required to determine whether this pattern results from a functional relationship or a developmental correlation.
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4. Conclusions In this review we have highlighted important considerations that are often overlooked in studies attempting to link individual variation in CORT with individual variation in fitness. Further, we have illustrated each of these considerations with data from a free-living population of song sparrows. First, if there are functional relationships between CORT levels and fitness-related traits, these relationships may vary over life history stages, or over time and space (e.g., Bonier et al., 2009b). That is, the nature and direction of such relationships will vary by context. Second, it is important to consider that CORT levels may not be related to fitness in and of themselves. Rather, it is more likely that individual variation in the numerous regulatory mechanisms of the HPA axis will be heritable and linked to individual variation in fitness. Such regulatory mechanisms include stress reactivity, negative feedback, regulation of binding globulins, and adrenal sensitivity. Although it is often simplest to measure only baseline and/or stress-induced levels of CORT in the field, such practices greatly limit opportunities for uncovering the relationships between HPA function and fitness. Finally, it is critical to keep in mind that any relationships between HPA axis variables and fitness-related variables may not indicate a functional relationship. Rather, such relationships may be only indirect ones, such as in the case of developmentally correlated traits. Although it is commonly understood that correlation does not equal causation, many observed CORT-fitness correlations are nonetheless inferred to be causal. Our consistent finding that stress reactivity is related to song complexity illustrates the fact that traits that have very few physiological commonalities in the adult animal may be correlated because they are both affected by the same early life experiences through a common developmental timeline. Future studies of how HPA axis function is related to fitness need to use experimental manipulations to clarify causal relationships. The potential for glucocorticoids to act as mediators of tradeoffs between components of fitness is a compelling hypothesis, and provides a rich field of research to explore the physiological mechanisms underlying individual variation in fitness. However, as noted by recent reviews, the available data on this topic remain equivocal (Bonier et al., 2009a; Breuner et al., 2008; Crespi et al., 2013). In addition to the considering the regulatory mechanisms of the HPA axis, and assessing how HPA-fitness relationships may vary with context, we also need to determine which of these relationships are functional and which are correlational. Our work on song sparrows illustrates that compelling HPA-fitness relationships may be context-dependent and/or the result of developmental correlations.
Acknowledgments An oral presentation of this paper was presented at the 10th International Symposium on Avian Endocrinology. We thank the organizers of the conference, and Fran Bonier and Lukas Jenni for organizing our session. Our research has been supported by the Natural Sciences and Engineering Council of Canada.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
HPA axis regulation, survival, and reproduction in free-living sparrows: Functional relationships or developmental correlations? Scott A. MacDougall-Shackleton a,b,c,⇑, Kim L. Schmidt b,c, Ainsley A. Furlonger b,c, Elizabeth A. MacDougall-Shackleton b,c a b c
Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada Advanced Facility for Avian Research, University of Western Ontario, London, Ontario N6G 1G9, Canada
a r t i c l e
i n f o
Article history: Available online 13 June 2013 Keywords: Glucocorticoids Developmental correlations Hypothalamus–pituitary–adrenal axis CORT-fitness hypothesis
a b s t r a c t A growing body of theoretical and empirical work has addressed the relationship between hypothalamus–pituitary–adrenal (HPA) function and fitness. For example, the corticosterone (CORT)-fitness and CORT-condition hypotheses predict that baseline and/or stress-induced levels of glucocorticoids should relate to fitness, and recent empirical studies have reported relationships between HPA function and fitness-related sexually selected traits. Here we introduce a framework for evaluating whether such relationships reflect functional relationships or developmental correlations. We then address this framework using data from a free-living population of song sparrows (Melospiza melodia). In two independent studies we have found that song complexity (a sexually selected trait) is correlated with stress reactivity: males with more complex vocal repertoires show reduced CORT response to standardized restraint stress. This pattern likely results from the early life environment concurrently affecting development of both song and the HPA axis. Suppression of CORT by dexamethasone was also correlated to measures of body condition and immune function, and females paired to males with higher stressinduced levels of CORT initiated egg-laying later. Finally, stress reactivity predicted overwinter survival in one year, although not in another. Thus, the relationship between HPA axis function and fitness likely varies temporally and by context. Some fitness-related traits may be functionally related to HPA regulation, but many others may be related through developmental correlation. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Over the last few decades, ecological and evolutionary physiologists have increasingly considered steroid hormones as potential mediators of trade-offs between components of life-history traits. For example, testosterone level has been proposed as a mechanism for balancing resource investment between reproductive effort and immune function (Folstad and Karter, 1992; Sheldon and Verhulst, 1996) or between mating and parental effort (Ketterson et al., 1992; McGlothin et al., 2007). Similarly, attention to the potential role of glucocorticoids in influencing fitness, population dynamics, and evolution has also increased with early studies on mammals (e.g., Boonstra and Boag, 1992; Boonstra and Singleton, 1993) and more recent research on other taxa including birds (Bonier et al., 2009a; Breuner et al., 2008; Zera and Harshman, 2001). In this review, we evaluate approaches to studying the relationship between glucocorticoid levels, and more generally, function⇑ Corresponding author at: Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada. E-mail address: [email protected] (S.A. MacDougall-Shackleton). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.05.026
ing of the hypothalamus–pituitary–adrenal (HPA) axis; and fitness-related traits. We highlight the importance of considering HPA axis regulatory mechanisms rather than glucocorticoid levels alone, and the importance of determining whether such relationships reflect functional mechanisms or correlations. We then use examples from our work on free-living song sparrows (Melospiza melodia) to illustrate these issues. 2. A framework for thinking about HPA-fitness correlations 2.1. Glucocorticoids as potential mediators of trade-offs Glucocorticoids such as corticosterone (CORT) are critical mediators of energy balance and affect multiple physiological systems. Baseline levels of CORT vary over multiple time scales and are associated with changes in energy balance (Sapolsky et al., 2000) and transitions between life-history stages (Crespi et al., 2013; Denver, 2009). In addition, CORT levels can increase rapidly in response to external factors as part of the adrenal stress response, and result in the animal entering an emergency life-history stage (Wingfield et al., 1998).
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The stress response of vertebrates is widely conserved. In response to a diverse variety of stressors, including biotic and abiotic factors, vertebrates initiate a rapid neural response (sympathetic nervous system) followed by a longer-term increase in circulating glucocorticoids. These glucocorticoids affect numerous physiological systems to allow the animal to cope with the stressful event (Wingfield and Sapolsky, 2003). In the short term, increased glucocorticoids are presumed to be adaptive in that they allow the animal to divert resources from growth, maintenance and reproduction towards short-term survival. In the long term, however, chronically elevated glucocorticoids are thought to have deleterious effects on numerous neural and physiological systems (Wingfield and Sapolsky, 2003). Glucocorticoids are thus tightly regulated and animals typically return levels to homeostatic baseline after coping with a stressor. Thus, changes in circulating CORT (baseline levels and stress-induced levels) are often considered to reflect a balance between short-term survival and long-term investment in processes such as growth and reproduction. 2.2. CORT-fitness relationships A central tenet of life history theory is that that trade-offs between competing demands should be optimally balanced to maximize lifetime reproductive success. Thus, if CORT is a physiological mediator of such trade-offs then CORT levels should be related to fitness, or related by proxy to fitness-related traits. Two recent reviews have assessed the relationship between CORT and fitness for both baseline levels and acute stress-induced levels of CORT (Bonier et al., 2009a; Breuner et al., 2008). Both reviews found equivocal support for the predicted relationship between CORT and fitness-related traits. Across studies, CORT levels were both positively and negatively related to fitness-related traits, and often there was no relationship. Thus, although several individual studies have detected relationships between CORT and fitness-related traits such as survival (Blas et al., 2007; Romero and Wikelski, 2001) or sexually selected trait expression (Roulin et al., 2008) overall there is no consistent CORT-fitness relationship in the literature (Bonier et al., 2009a; Breuner et al., 2008; Crespi et al., 2013). There are several potential reasons why the strength and direction of CORT-fitness relationships vary from study to study. First, the relationship between CORT and a fitness-related trait may vary across life history stages (Bonier et al., 2009b). Second, CORT levels at any given point in time will be influenced by a variety of intrinsic and extrinsic factors (Crespi et al., 2013) which may need to be controlled to detect CORT-fitness relationships (e.g. Hau et al., 2010). That is, to detect a relationship (functional or not) between CORT and fitness-related or life-history variables will often require statistically controlling for other variables that affect CORT levels. Thus, searching for simple direct relationships between CORT levels and fitness-related traits will often prove difficult. In addition to the above considerations, in many cases even if CORT levels are functionally related to a fitness variable, CORT levels themselves may not be the primary targets of selection. If CORT mediates trade-offs between life-history components, the physiological traits most likely to be correlated with fitness may be the regulatory processes that determine CORT levels, not CORT levels as such. This point is overlooked by the majority of studies on CORT-fitness relationships. The mechanisms that determine how CORT levels vary in response to extrinsic factors include variables such as pituitary sensitivity to corticotropin-releasing hormone, adrenal sensitivity to adrenocorticotropic hormone (ACTH), and sensitivity to negative feedback in the HPA axis. Individual variation in these measures is much more likely to influence fitness than is individual variation in CORT levels. Thus, research aimed at understanding CORT-fitness relationships should ideally mea-
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sure HPA regulation rather than relying exclusively on point samples of CORT. Even if one quantifies the regulatory mechanisms of the HPA axis, determining HPA-fitness relationships may still prove elusive because such relationships likely vary over time, space, and context. As noted by Schoech et al. (2011) ‘‘the assumption that there is a single . . . corticosterone phenotype that maintains a stable adaptive payoff in response to the varied environmental challenges an organism is liable to experience is unrealistic.’’ Consider a hypothetical individual animal. What would its optimal stress response be? In a benign environment, individuals that are less stress-reactive may outcompete their more stress-reactive counterparts. But if the environment is degraded or becomes unpredictable this competitive advantage may reverse. Similarly, a subordinate animal may have higher fitness if it is more stress-reactive, but if it becomes dominant it may benefit from being less reactive. Thus the relationship between CORT levels or HPA regulatory mechanisms and fitness will vary over different contexts and different times. Finally, it is important to note that even when relationships between CORT or HPA function are detected, such correlations may or may not reflect natural selection. It is possible that third variables may influence both CORT and the fitness-related trait, and indeed these third variables may be entirely environmental. We discuss one type of environmental effect (developmental correlations) below. 2.3. Developmental correlations Another difficulty in characterizing CORT-fitness relationships is that the majority of studies on this topic are correlational. Correlations between CORT levels (or HPA function) and some measure of fitness might arise entirely because both factors are correlated with some unknown third variable, rather than because of a functional relationship between them. That is, rather than individual variation in CORT functionally affecting individual variation in fitness by mediating a trade-off, it is possible that both the CORT levels and the fitness-related trait result from another common factor. One such factor may be early developmental experience. Glucocorticoid exposure early in life can have long-term organizational effects on a variety of physiological and neural systems, including the development of the HPA axis itself (Liu et al., 1997). Thus, HPA function or CORT levels may be correlated to a fitness-related trait not because they are functionally related but because they were both affected by the same developmental stressors (i.e. developmentally correlated traits (MacDougall-Shackleton and Spencer, 2012). For example, both the hippocampus and the song-control brain regions of songbirds appear particularly sensitive to early life stressors (reviewed in MacDougall-Shackleton and Spencer (2012), Spencer and MacDougall-Shackleton (2011)), and early life stress may thus affect both singing behaviour and spatial cognition (Farrell et al., 2011). Similarly, development of the HPA axis is affected by early life stress in mammals (Liu et al., 1997) and birds (Spencer et al., 2009) and may thus be developmentally correlated with other traits. 3. HPA function and fitness correlates in song sparrows 3.1. Background We have examined the relationship between various measures of HPA function and fitness-related traits in a long-term study population of song sparrows. We have studied this population since 2002, and it is characterized by high adult breeding philopatry. Each year approximately 40–60% of breeding adults consist of previously banded individuals that also bred at the study site in prior years. The remainder are new recruits to the population and in-
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ferred to be first-time breeders. Each spring we capture and band all breeding adults, and monitor their reproduction and overwinter returns. Because birds in this population are so highly philopatric, typically nesting within 75 m of their previous year’s territory (unpublished data) we conclude that birds that fail to return to the study site have died (Greenwood and Harvey, 1982; MacDougall-Shackleton et al., 2009). In addition to monitoring reproduction and survival, in some breeding seasons (2007 and 2010) we have assessed HPA function of known banded birds. We measure baseline levels of CORT from blood samples collected within 3 min of capture in a mistnet, then measure stress-induced CORT after 30 min of standardized restraint, and calculate stress reactivity as the increase in CORT following a stressor (stressed – baseline levels (MacDougallShackleton et al., 2009). In the 2010 breeding season we also characterized negative feedback and adrenal sensitivity of the HPA axis (following Dickens et al., 2009; Rich and Romero, 2005; see Fig. 1). After collecting a stress-induced sample we injected birds intramuscularly with dexamethasone (1.0 mg/kg) and placed them in a covered cage with food and diluted fruit juice. Sixty minutes later a third blood sample was collected to quantify negative feedback, then we injected birds with ACTH (25 IU/kg) and placed them back in the cage. After a further 30 min we then collected a final blood sample to quantify adrenal sensitivity and released the bird. CORT was quantified in unextracted plasma using an RIA (MP Biomedicals, 07-120103) that has been previously validated in song sparrows (Newman et al., 2008). We continued to monitor the birds’ reproduction and survival, recorded males’ song repertoires, and made a variety of other morphological, physiological, and behavioral measures (see below). 3.2. HPA function and survival-related traits We examined whether our measures of HPA function were related to body size and condition and hematological profile (Schmidt et al., 2012). As is common in studies of birds, our morphometric measurements yielded two principal components
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reflecting body size and body condition (mass relative to size). Stress reactivity (the change in CORT following a stressor) was not related to these morphometric measures, but HPA negative feedback was greater for structurally larger birds than smaller birds (Schmidt et al., 2012). That is, birds with a larger PC1 morphometric score decreased CORT to a greater extent following a dexamethasone injection. In addition to body size, we also found that HPA function was related to hematological profile. An immune index (Principal Component factor) that reflected heterophil abundance and heterophil to lymphocyte ratio was related to both stress reactivity and negative feedback (Schmidt et al., 2012). Birds that were more stressreactive had relatively fewer heterophils, as did birds with stronger negative feedback. These results, combined with the relationship between negative feedback and body size, suggest that growth and immune parameters are correlated to HPA regulatory mechanisms. However, whether such correlations reflect a direct functional relationship, as opposed to developmental correlations between HPA regulation and growth and immune parameters, remains unclear. Because we study this population each year we are able to determine which birds return from the wintering grounds to breed and which birds fail to return and are presumably dead. We found that stress reactivity in 2007 was related to overwinter survival and return to the breeding grounds in 2008 (MacDougall-Shackleton et al., 2009). Specifically, birds that were more stress-reactive in 2007 were less likely to return the following breeding season (Fig. 2). Our results linking stress reactivity to survival suggest that high stress reactivity may increase over-winter mortality, and are similar to findings in other species of birds (Blas et al., 2007). This pattern is also consistent with data from humans wherein high stress reactivity in childhood is associated with increased risk of cardiovascular disease later in life (Wood et al., 1984). However, as noted above (Section 2.3) correlation does not imply causation, thus survival and stress reactivity could both be related to an unmeasured third variable. In addition (Section 2.2) even if high stress reactivity is functionally associated with decreased survival in one context, this relationship may vary over space and time and disappear or reverse for birds that winter in different locations or for the same birds in different years. Indeed, we have recently found just that. For the birds in which we measured HPA function in 2010, we found no relationships between overwinter survival and stress reactivity, negative feedback, or adrenal sensitivity (Furlonger, 2011). Birds with high and low stress reactivity, negative feedback and ACTH sensitivity were equally likely to survive the winter of
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Bleed Time (minutes since capture) Fig. 1. Characterization of HPA function in song sparrows. Points indicate mean (±SE) plasma corticosterone levels of 37 male song sparrows (Melospiza melodia). The initial baseline sample was collected within 3 min of capture (baseline) and the stress-induced sample was collected after 30 min restraint in a cloth bag (time 30 min). We then immediately injected birds with dexamethasone (DEX; 1.0 mg/kg i.m.) to assess negative feedback. Birds were then housed in a covered cage until we collected another sample 60 min later (time 90 min). We then injected birds with adrenocorticotropic hormone (ACTH/25 IU/kg i.m.) to measure adrenal sensitivity and collected a final sample 30 min later (time 120 min). All birds were then released and resumed their territory tenure. Data adapted from Furlonger (2011).
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Increase in Corticosterone Following Stress (ng mL-1) Fig. 2. Stress response and survival in song sparrows. The probability of survival is negatively related to stress reactivity, that is, the increase in CORT from baseline to stress-induced levels. Birds that were more stress reactive were less likely to return the following year. Points indicate data from individual birds, lines indicate median and interquartile ranges. Data adapted from MacDougall-Shackleton et al. (2009).
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3.3. HPA function and reproduction To test for associations between CORT and traits linked to reproductive success we measured HPA function, song, and reproductive output. In song sparrows song complexity, assessed as song repertoire size or syllable repertoire size, appears to be a sexually selected trait and is linked to female preferences in captivity (Searcy and Marler, 1981), mate attraction in free-living birds (Reid et al., 2004), and some hematological and neuroanatomical measures (Pfaff et al., 2007). We measured the relationship between HPA function and song complexity in two different groups of birds in our population, one in 2007 and the other in 2010. In both cohorts, song complexity was negatively related to stress reactivity (MacDougall-Shackleton et al., 2009; Schmidt et al., 2012). That is, birds with less complex songs increased CORT following restraint to a greater extent than did birds with more complex songs. These data thus indicate a correlation between the stress response and a sexually selected signal, as reported in other species (Husak and Moore, 2008). In barn owls (Tyto alba), melanin-based signals have been functionally linked to HPA activity because generation of these signals shares common biosynthetic pathways with CORT production (Almasi et al., 2010; Roulin et al., 2008). In the case of song complexity, however, there is no clear functional link between birdsong and the HPA axis. Instead, a common factor between birdsong and the HPA axis is that both systems have a protracted development and both are susceptible to developmental environmental stressors (Spencer and MacDougall-Shackleton, 2011). Thus, the relationship between song complexity and stress reactivity in song sparrows is likely an example of a developmental correlation. During the 2010 breeding season we also measured the relationship between HPA function of breeding males and several indices of reproduction, including lay date, clutch size, and hatching success (Furlonger, 2011).We quantified HPA function of males prior to breeding, and then measured the reproductive output of each male and his mate over the following weeks. We observed no relationships between our measures of HPA activity and clutch size or hatching success. This is perhaps not surprising, because in this ground-nesting population there is very low variation in clutch size (the majority of nests have four eggs) and hatching failures are almost always due to stochastic nest predation events (unpub-
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2010–11 and return to the breeding grounds. Thus, HPA function was correlated to survival in one year, but not 2 years later. Why may we have found a different relationship between HPA function and survival in two different years? We can only speculate, but one possibility is that stress reactivity affects overwinter survival, but only in some conditions. In a relatively benign winter, highly reactive birds may be at a competitive disadvantage if they more frequently shift resources away from growth, maintenance and immune function. However, in more inclement winters this pattern could be eliminated or even reversed. On the wintering grounds of our population (southeast United States) the winter of 2007–08 was warmer and wetter than that of 2010–11 (data from NOAAA National Climate Data Center). This is consistent with the idea that highly stress reactive birds may have been at a competitive disadvantage in the relatively benign winter of 2007–08. However, it is also possible that HPA stress reactivity and overwinter survival in particular climates are both correlated with a third variable. For example, poor conditions in 2007 could have resulted in some birds having higher stress reactivity and also lower subsequent survival. Clarifying the nature of the link between HPA function and survival will require longterm studies that include both observational and experimental data.
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Post-dexamethasone change in Corticosterone (ng mL-1) Fig. 3. HPA function and lay date in song sparrows. A multiple regression analysis indicated that standardized lay date was significantly associated with stressinduced CORT levels and the decrease in CORT following dexamethasone injection (Furlonger, 2011). (A) Standardized lay date was positively related to CORT levels after 30 min restraint. That is, females mated to males with higher stress-induced CORT initiated their nests later in the season. (B) Standardized lay date was negatively related to negative feedback. That is, females mated to males that lowered their CORT the most following a dexamethasone injection initiated their nest later in the season. Points indicate individual birds. The lines indicate simple linear regressions and dashed lines the 95% confidence intervals of the regression. Data adapted from Furlonger (2011).
lished data). Lay date, on the other hand, was significantly related to stress-induced CORT levels and negative feedback as indicated by multiple regression analysis (Furlonger, 2011). Lay date is an important component of fitness in many bird species, with earlier lay dates being associated with higher reproductive success and offspring recruitment (Hochachka and Smith, 1991; Perrins, 1970). In our study, females whose social mates exhibited higher stress-induced CORT initiated egg-laying later in the season (Fig. 3a), as did females whose social mateshad stronger negative feedback i.e. response to dexamethasone (Fig. 3b). These patterns suggest that males with lower stress-induced CORT levels and with reduced responses to dexamethasone initiate breeding earlier and may achieve higher reproductive success as a result. However, we cannot disambiguate whether the male himself or a variety of indirect factors such as behavior of the female, territory quality, or other factors mediate these patterns. In addition, even if changes in lay date were directly correlated to male HPA activity, further work would be required to determine whether this pattern results from a functional relationship or a developmental correlation.
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4. Conclusions In this review we have highlighted important considerations that are often overlooked in studies attempting to link individual variation in CORT with individual variation in fitness. Further, we have illustrated each of these considerations with data from a free-living population of song sparrows. First, if there are functional relationships between CORT levels and fitness-related traits, these relationships may vary over life history stages, or over time and space (e.g., Bonier et al., 2009b). That is, the nature and direction of such relationships will vary by context. Second, it is important to consider that CORT levels may not be related to fitness in and of themselves. Rather, it is more likely that individual variation in the numerous regulatory mechanisms of the HPA axis will be heritable and linked to individual variation in fitness. Such regulatory mechanisms include stress reactivity, negative feedback, regulation of binding globulins, and adrenal sensitivity. Although it is often simplest to measure only baseline and/or stress-induced levels of CORT in the field, such practices greatly limit opportunities for uncovering the relationships between HPA function and fitness. Finally, it is critical to keep in mind that any relationships between HPA axis variables and fitness-related variables may not indicate a functional relationship. Rather, such relationships may be only indirect ones, such as in the case of developmentally correlated traits. Although it is commonly understood that correlation does not equal causation, many observed CORT-fitness correlations are nonetheless inferred to be causal. Our consistent finding that stress reactivity is related to song complexity illustrates the fact that traits that have very few physiological commonalities in the adult animal may be correlated because they are both affected by the same early life experiences through a common developmental timeline. Future studies of how HPA axis function is related to fitness need to use experimental manipulations to clarify causal relationships. The potential for glucocorticoids to act as mediators of tradeoffs between components of fitness is a compelling hypothesis, and provides a rich field of research to explore the physiological mechanisms underlying individual variation in fitness. However, as noted by recent reviews, the available data on this topic remain equivocal (Bonier et al., 2009a; Breuner et al., 2008; Crespi et al., 2013). In addition to the considering the regulatory mechanisms of the HPA axis, and assessing how HPA-fitness relationships may vary with context, we also need to determine which of these relationships are functional and which are correlational. Our work on song sparrows illustrates that compelling HPA-fitness relationships may be context-dependent and/or the result of developmental correlations.
Acknowledgments An oral presentation of this paper was presented at the 10th International Symposium on Avian Endocrinology. We thank the organizers of the conference, and Fran Bonier and Lukas Jenni for organizing our session. Our research has been supported by the Natural Sciences and Engineering Council of Canada.
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