Here today, not gone tomorrow: long-term effects of corticosterone

J Ornithol (2012) 153 (Suppl 1):S217–S226 DOI 10.1007/s10336-012-0820-8

REVIEW

Here today, not gone tomorrow: long-term effects of corticosterone Stephan J. Schoech • Michelle A. Rensel Travis E. Wilcoxen

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Received: 18 February 2011 / Revised: 2 June 2011 / Accepted: 26 January 2012 / Published online: 18 February 2012 Ó Dt. Ornithologen-Gesellschaft e.V. 2012

little corticosterone as nestlings. Recent work in Florida Scrub-jays (Aphelocoma coerulescens) has found that baseline corticosterone levels in nestlings at 11 days posthatch explained 84% of the variation in ‘personality’ (bold vs. timid) when those individuals were tested at approximately 7 months of age. The overall ‘personality’ scores at 7–8 months of age were based upon tests that evaluated and combined the Jays’ responses to three tests: two startle and one neophobia stimuli. Here, we present findings on some of the factors that contribute to corticosterone levels of nestling Florida Scrub-jays, both within and among broods. Our prior and continuing research, as this overview will present, suggests that maternal attentiveness at the nest and paternal provisioning are important factors that mediate nestling corticosterone levels.

Abstract There is a growing body of evidence from across animal taxa that exposure to elevated levels of glucocorticoids during development can have profound long-term effects upon physiological and behavioral phenotypes. Several avian studies have revealed that the degree to which an individual’s hypothalamo–pituitary–adrenal (HPA) axis responds to stressful stimuli as an adult may in effect have been ‘programmed’ or set as a result of that individual’s corticosterone exposure in ovo or as a nestling. Developmental exposure to corticosterone may also have effects upon avian ‘personalities’ or coping styles, and evidence from mammalian studies suggests that these long-term effects are mediated epigenetically via altered expression of relevant DNA sequences. Although there does not appear to be a consistent across-species pattern, developmental exposure to elevated corticosterone levels may shape adult coping style with such exposure, resulting in adults that are more timid and with increased HPA axis responsiveness to stress than those individuals that were exposed to relatively

Keywords Personality  Field endocrinology  Steroid hormones  Hormonal organization and activation  Parental care  Parental or environmental programming

Communicated by Cristina Miyaki.

Introduction

S. J. Schoech (&)  M. A. Rensel  T. E. Wilcoxen Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA e-mail: [email protected]

In the years since the seminal studies by Farner, Wingfield, and colleagues that largely founded the fields of behavioral and field endocrinology, we have seen marked advances in our understanding of the links between hormones and behaviors (e.g., Wingfield and Farner 1976; Wingfield 1980; review in Wingfield and Farner 1993). The vast majority of these studies have examined the activational effects of hormones upon behaviors, whereas our understanding of the importance of the organizational actions of hormones in facilitation of the later expression of behaviors has received somewhat less attention. Although we are avian biologists and this publication is in an ornithological

Present Address: M. A. Rensel Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA Present Address: T. E. Wilcoxen Biology Department, Millikin University, Decatur, IL 62522, USA

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journal, please bear with us as much of the foundational work in this area of research has been conducted on mammals. As such, those of us working with birds owe a debt to our predecessors who paved the way using captive rodents. For example, Phoenix et al. (1959) made it clear that exposure to specific sex steroid hormones during development was necessary to organize the central nervous system in a manner that would allow the same hormones to later activate sex-appropriate reproductive behaviors. They found that female GUINEA PIGS (Cavia porcellus) that had been exposed to testosterone in utero exhibited masculinized sexual behaviors as adults (Phoenix et al. 1959; Young et al. 1961). While there has been considerable research on the importance of sex steroid hormone exposure during development upon the full expression of the adult phenotype, the impacts of glucocorticoids have received less attention. However, a number of mammalian studies suggest that exposure to glucocorticoids (corticosterone and cortisol, henceforth CORT) in utero results in both physiological and behavioral feminization of male offspring (Ward 1972; Ward and Weisz 1980; Grisham et al. 1991; Ward and Stehm 1991). Whether these actions should be considered to be organizational effects of CORT, however, is debatable. Rather, given that in these cases the CORT effects seemingly are mediated through its actions upon the hypothamic–pituitary–gonadal (HPG) axis, we would argue that CORT instead interferes with the organizational effects of sex steroid hormones. Indeed, Ward and Weisz (1980) noted that prenatal CORT exposure resulted in an altered temporal pattern of testosterone secretion in fetal males, thus providing support for this view. Recent studies, primarily of mammalian systems, have made it increasingly clear that post-natal parental care, in some instances mediated through its effect on CORT levels of offspring, can alter both the physiological and behavioral phenotype of an individual, and that this occurs largely independently of the HPG axis. Much of this area of study owes a debt to research from the laboratory of Michael Meaney (e.g., Liu et al. 1997; Weaver et al. 2004). These studies found that rat pups that experienced higher quality maternal care in the form of increased licking and arched-back nursing expressed dampened stress responsiveness as adults. This long-term effect, or ‘‘epigenetic programming,’’ via increased maternal care resulted in greatly reduced methylation of the promoter region of the gene that codes for glucocorticoid receptors (GR) in the hippocampus (when compared to pups that received lower rates or quality of maternal care). The increased GR expression in pups that received high versus low quality care resulted in adults with enhanced CORT feedback sensitivity that was coupled with decreased hypothalamic corticotrophin-releasing hormone (CRH; an initial

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hormone in the HPA axis cascade) expression; both serve to dampen the HPA axis responsiveness to stress (Weaver et al. 2004). Full consideration of this topic is well beyond the scope of the current manuscript (see reviews in Meaney and Szyf 2005; Kauffman and Meaney 2007; Szyf et al. 2008; and the citations therein). In part, it is these and other studies that have served as inspiration for some of the many avian studies that have examined these and related topics in recent years. Although we have mentioned studies in which in utero exposure to CORT can have profound effects upon offspring, clearly this is not relevant for oviparous birds. A rough equivalence in birds, however, might be the transfer of maternal steroid hormones to the yolk and their subsequent effects on the developing young. The majority of studies that have examined the effects of yolk steroids have concentrated upon sex steroid hormones, especially androgens (see reviews in Gil 2003; Groothuis et al. 2005; Groothuis and Schwabl 2008), whereas far fewer have assessed yolk CORT levels. However, several studies have found measurable concentrations of CORT within egg yolk, and also found that exposure in ovo may have short- and long-term effects upon multiple aspects of the phenotype (for review, see Schoech et al. 2011). For example, (1) Love and Williams (2008) injected CORT into yolks and found dampened HPA axis responsiveness in treated young that were tested as they neared fledging; (2) Hayward and Wingfield (2004) increased yolk CORT levels by implanting females during oogenesis and found that the young of the CORT-treated females exhibited increased HPA axis responsiveness when sexually maturity at 8 weeks of age; and (3) Lay and Wilson (2002) treated chicken eggs with CORT dissolved in ethanol and found that male offspring at 21 weeks of age were both less likely to peck other males and more likely to be pecked, suggesting long-term effects upon personality. It must be noted, however, that a recent study has called into doubt the ability of some of the assays used to accurately measure yolk CORT. Rettenbacher et al. (2009) suggest that the CORT antibody cross-reacts with progestins, thereby confounding the accurate determination of CORT levels. While this is not the forum for a full discussion of this issue, Rettenbacher et al. (2009) point out that this problem likely does not apply to those studies that have separated steroids (e.g., via column chromatography) prior to assay. Further, we note that several studies have manipulated maternal CORT levels and subsequently found elevated yolk CORT levels, suggesting that it is indeed CORT that is being measured (see also Table 1 in Hayward and Wingfield 2004). Of specific importance to studies in our laboratory is recent research that has examined the effects of post-natal exposure to CORT upon behavioral and physiological phenotypes. While this area of study has benefited greatly

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from manipulative studies that have administered exogenous CORT to young (for review, see Schoech et al. 2011), here we are primarily interested in and consider the determinants and effects of variation in endogenously produced CORT, and whether such exposure has long-term effects on avian personality (reviews in Cockrem 2007; Schoech et al. 2011). One confound that Schoech et al. (2011) point out that needs be restated here is that nestling CORT levels usually covary inversely with body condition (or mass), making it difficult to attribute physiological or behavioral effects solely to CORT. Indeed, protocols used in studies of the long-term effects of nestling CORT exposure have commonly used food deprivation as a mechanism to increase CORT levels (e.g., Kitaysky et al. 2001; Pravosudov and Kitaysky 2006; note that the primary focus of these studies was on the effects of nutritional deprivation on development of Red-legged Kittiwakes, Rissa brevirostris, and Western Scrub-jays, Aphelocoma californica, respectively). Consideration of published research on the epigenetic effects of maternal care on stress physiology and behavior in mammals (see above discussion), led our laboratory group to explore whether similar phenomena existed in our study species, the Florida Scrub-jay (A. coerulescens). In combination with an ongoing dissertation project that was exploring the development of the HPA axis across life-stages (see Rensel et al. 2010a), we asked two questions. First, ‘‘do the corticosterone levels to which nestlings are exposed have long-term effects on an individual’s phenotype?’’ It is important to note that, to date, and for the findings we present here, we have considered only correlative data that, while instructive, needs to be supported by future experimental study. Second, we asked ‘‘what factors contribute to amongindividual variance in nestlings’ corticosterone levels?’’ Although it is likely that a variety of environmental factors can have effects on nestling CORT levels and overall healthstate, here we limit consideration to the direct effects of parental care and those factors that may impinge upon such care. Below, we present an overview of our research that has investigated the determinants of nestling Florida Scrub-jay CORT levels (i.e., factors that lead to variation in CORT levels among nestlings), as well as the consequences of variation in parental behavior and nestling CORT levels upon the phenotype and ultimately, fitness.

Methods Study species Our group has conducted research on Florida Scrub-jays for over 20 years (e.g., Schoech et al. 1991; Rensel et al. 2011). The study population is located at Archbold

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Biological Station in south-central Florida (27°100 N, 81°210 W, elevation 38–68 m). Florida Scrub-jays (FSJ) are cooperative breeders and, while mean group size is approximately three individuals, roughly half of the territories are occupied solely by a breeding pair whereas the other half are occupied by the breeders and 1–6 nonbreeding helpers (Woolfenden and Fitzpatrick 1984). Most of the non-breeding helpers are offspring of one or both of the breeders that remain in their natal territories to assist the breeding pair in rearing offspring. While not all nonbreeding helpers provision nestlings (see Schoech et al. 1996), non-breeders also help in territory defense and antipredator behaviors (Woolfenden and Fitzpatrick 1984). Adult jays weigh from 65 to 99 g (S.J. Schoech, unpublished data; Woolfenden and Fitzpatrick 1984) and, although males are generally slightly larger, the sexes are to our view, monomorphic, despite being cryptically dichromatic (i.e., plumage reflectance differs within the UV range; see Eaton and Lanyon 2003; Bridge et al. 2008). In addition, FSJs are: (1) socially and genetically monogamous (Quinn et al. 1999; Townsend et al. 2011); (2) restricted to xeric oak scrub habitat within peninsular Florida; and (3) listed as a Federally Threatened species with numbers decreasing as a result of habitat loss. FSJs are non-migratory and, as such, most individuals spend their entire life within a few hundred meters of their natal territory (Woolfenden and Fitzpatrick 1984). We band each individual within the study area with a unique combination of one United States Geological Survey (USGS) aluminum band and two to three color bands to facilitate field identification. These characteristics allow us to track virtually all individuals from the egg until death, providing a powerful field-based study system. Nestling monitoring and blood sampling As part of ongoing demographic studies within the population, we observed breeding pairs and located all nests, usually during nest construction. Nests were systematically monitored throughout the construction, egg laying, and incubation periods. To determine hatch order of nestlings, we visited each nest multiple times on the day of hatching and until hatching was complete (see details in Rensel et al. 2011). Upon hatching, a single toenail of each nestling was marked with nail polish, with each nestling within a brood receiving a mark that is unique within that brood. This allowed us to track each individual through the nestling stage. All nestlings were blood sampled, weighed, and measured (e.g., tarsus and wing feather length) at day 11 post-hatch, approximately 1 week prior to fledging. This age has been used historically for this species (see Woolfenden and Fitzpatrick 1984) because it is an age at which most nestlings are large enough to receive an USGS band

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and nest visitation is unlikely to result in ‘forced fledging.’ The small blood samples (B50 lL) collected are used for molecular sex determination (see Fridolfsson and Ellegren 1999) and to assess nestling CORT levels. Until recently, the protocol was that all nestlings from a brood were removed together and transported to a shady spot far enough from the nest to minimize disturbance (i.e., adults of a territory tend to defend the nest and the surrounding area rather than the nestlings per se). This method resulted in some nestlings that were sampled within a few minutes, whereas others might not have been sampled until 30 min or more had passed (for methodological details and discussion, see Rensel et al. 2010a). Clearly such a sampling protocol is problematic if the desire is to assess baseline CORT levels, given that removal from the nest results in stress-related elevation of CORT within minutes (Rensel et al. 2010a; see also Romero and Romero 2002; Romero and Reed 2005). The Rensel et al. (2010a) data led us to modify the protocol and remove a single nestling at a time for sampling. Upon completion of processing and return of the nestling to the nest, a subsequent nestling is removed for processing, with this being repeated until the entire brood has been sampled. In all cases, individuals are usually sampled within 2–3 min of removal (Rensel et al. 2010a). Despite this effort to minimize disturbance, one must consider the likelihood that removal of the initial nestling is stress-inducing to the remaining siblings. Indeed, our data have shown this to be the case (see Rensel et al. 2010a); therefore, we control for this effect by entering the variable ‘time since initial nest disturbance’ into all statistical models. Blood samples were kept cool on ice in the field until return to the laboratory within 1–4 h. Samples were separated into their cellular and plasma components by centrifugation. The plasma fractions were drawn off with a Hamilton syringe, stored at -4°C in screw-capped vials with O-ring seals to minimize loss through sublimation, and later shipped to the University of Memphis for radioimmunoassay (see Schoech et al. 2004, 2007a, b; Rensel et al. 2010a, b; Wilcoxen et al. 2010a, b). Behavioral data Determination of ‘fearfulness’ was accomplished by evaluating individuals’ responses to three tests of two different categories, neophobia and startle, when they were between 7 and 8 months of age. First, individuals were trained to come to a pile of peanuts. As peanuts are a favored food; jays will eat or take and cache peanuts from a site until the resource is exhausted. For the neophobia test, a bright orange-colored ring was put around the peanuts and the time required for an individual to cross the ring and take a

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peanut was the measure of interest. The two startle tests also employed peanuts; however, in one test, an individual was startled by the movement of a motor-powered leaf, whereas the other test used a loud piezoelectric buzzer as the startle stimulus. For each of the three tests, there were marked among-individual variations in the latency to take a peanut, suggesting differential responses to the stimulus. Latency times from the three tests were combined with principal components analysis (PCA) to arrive at a single score (PC1) of ‘fearfulness’ (for details, see Schoech et al. 2009). The data used to assess parental care were collected during focal nest watches when nestlings were between 3 and 5 days post-hatch (see Wilcoxen et al. 2010b). One hour watches were conducted over a 3-year period (2006–2008) in the morning and afternoon when parental and helper provisioning is maximal (Stallcup and Woolfenden 1978). All watches were conducted by T.E. Wilcoxen, assuring consistency within and among years. Although roughly one-half of the breeding pairs whose nests were observed also shared their territories with helpers, this was not an issue given that helpers are largely excluded from the nest during the early nestling stage (details in Wilcoxen et al. 2010b). Observations were made from a blind, and for each provisioning event, the following were noted: (1) the identity of the provisioner, (2) time of visit, and (3) the amount of food delivered (a score of 1–3, as per Mumme 1992; Schoech et al. 1996). Additionally, for a subset of these watches, the behavior of the breeding female was recorded. Note that during this early nestling period, the female spends much of her time on the nest brooding the thermoregulatory-incapable nestlings. Specifically, data recorded were the amount of time the female spent on the nest, the number of times she left the nest, and the distance the female was from the nest while off the nest. Nest attendance was scored as the time: (1) on the nest, either brooding, shading, or ‘house cleaning’ (females frequently engage in what appears to be anti-parasite behavior as they actively ‘groom’ the nestlings and probe the nest with their bills; S.J. Schoech and T.E. Wilcoxen, personal observation); (2) off of the nest but within the observer’s field of view; or (3) off of the nest and observed to have left the area (i.e., at a distance of approximately 30 m or greater; for details, see Rensel et al. 2010b).

Results and discussion As for our initial question as to whether nestling CORT levels would reflect later personality, the data demonstrated a strong link (Fig. 1). We were quite surprised to discover the degree to which CORT levels of nestlings at 11 days post-hatch were correlated with later fearfulness (i.e., explained 84% of the variance): nestlings with the lowest

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Baseline Corticosterone (ng/ml)0.3 Fig. 1 Day 11 Florida Scrub-jay (A. coerulescens) nestling corticosterone levels were positively related to behavioral phenotype when those individuals were tested at 7–8 months of age (F1,8 = 41.48, p = 0.0002, r2 = 0.84). Higher ‘fearfulness’ ranks reflect greater latency times to take food following exposure to startle and novel stimuli (see text for details). Reprinted from Schoech et al. (2009) with permission from Elsevier

Fig. 2 A schematic diagram of the factors and the complex interactions that are hypothesized or known to affect nestling corticosterone levels

CORT levels were the boldest when tested at 7–8 months of age (Schoech et al. 2009). Ongoing research is currently examining this relationship in more detail to determine both its robustness and whether personality is a persistent trait throughout the lifetime of an individual. As for the logical follow-up question—‘‘What are the factors that mediate nestling CORT levels?’’—we offer the schematic diagramed in Fig. 2 as a partial explanation of the complex mix of parental and environmental factors that might explain variance in nestling physiology. To varying degrees, our group has addressed or is in the process of addressing all of the relationships presented therein (Fig. 2).

It may well be that nestlings in larger broods or those that hatch later are characterized by having elevated CORT levels as a result of either increased competition or size- or age-driven differences in competitive abilities. Hatch order effects upon nestling CORT levels have been explored in several studies; however, consistent patterns are elusive. For example, whereas Sockman and Schwabl (2001) found no differences by hatch order in American Kestrels (Falco sparvarius), in a study of the same species, Love et al. (2003) found first-hatched young had higher CORT levels than their later-hatched siblings. Similarly, whereas Schwabl (1999) found baseline CORT levels to increase with hatch order in Canary (Serinus canaria), as did Eraud et al. (2008) in Collared Dove (Streptopilia decaocto) nestlings, neither baseline nor stress-induced CORT levels differed by hatch order in Black-legged Kittiwake (R. tridactyla) chicks (Brewer et al. 2010). There are similar inconsistent findings in studies that have considered the effects of brood size on CORT levels. Brewer et al. (2010) found that Kittiwake chicks in singleton and broods of two did not differ in either baseline or stress-induced CORT levels, although studies on White Stork (Ciconia ciconia) chicks found CORT levels of singleton broods to be higher than those of broods of two or three (Blas et al. 2005). Examination of the potential effects of brood size and hatch order in FSJs by our laboratory has similarly found a lack of consistency. Rensel et al. (2011) found no effect of brood size on nestling CORT levels but did find a trend toward increased CORT levels with hatch order (p = 0.07). However, this trend was driven by broods of two in which first-hatched nestlings had higher CORT levels than their second-hatched siblings; there were no such trends in broods of three or four. The authors speculate that it is only within broods of two that a dominance hierarchy is established. They further speculate that low CORT levels in subordinate second-hatched nestlings reflect exhaustion or down-regulation of the HPA axis. In addition, there was a significant negative relationship between body mass and CORT levels of FSJ nestlings (Rensel et al. 2011). Such an inverse relationship between CORT levels and body mass (or condition) has been found in adults or nestlings of several avian species (e.g., Smith et al. 1994; Schoech et al. 1997, 1999; Marra and Holberton 1998; Sockman and Schwabl 2001). Maternal care Focal watches at FSJ nests conducted over a 2-year period (2007–2008) revealed marked variance in female attendance patterns with some females spending the entire period at the nest while others were away for nearly half

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Mother's Distance from Nest (proportion of time >30m) Fig. 3 Nestling corticosterone levels against the proportion of time the female is far (C30 m) from the nest (F1,113 = 26.38, p \ 0.0001). Reprinted from Rensel et al. (2010b) with permission from Elsevier

the focal watch period (Rensel et al. 2010b). Watches were conducted when the nestlings were between 3 and 5 days post-hatch, an age at which the altricial nestlings are not yet fully capable of thermoregulation and are brooded extensively by their mothers (only females incubate eggs or brood young; Hailman and Woolfenden 1985). Rensel et al. (2010b) employed a linear mixed model (LMM) and found that nestling CORT levels were best explained by the amount of time that their mothers spent far from the nest (Fig. 3). It is important to note that the model (and the models considered below) controlled for several variables that could affect CORT levels, such as nestling body condition, brood size, whether the territory had helpers or not, and the total amount of food received, although none approached statistical significance (see Table 1 in Rensel et al. 2010b). In addition to the analysis examining category 3 (i.e., the proportion of time far from the nest), we also considered potential effects of the female merely being away from the nest, irrespective of distance (i.e., categories 2 ? 3), upon CORT levels. There was no effect of this variable; however, nestling CORT levels were negatively correlated with the parental provisioning rate (see below, and see Table 2 in Rensel et al. 2010b). Rensel et al. (2010b) offer two possible explanations for their findings. First, that the relationship between nestling CORT levels and the proportion of time females spent far from the nest are akin to maternal separation anxiety that is expressed through upregulation of the HPA axis, as has been found in a variety of taxa (see citations in Rensel et al. 2010b). Alternatively, the more time that the mother spends away from nest, the greater the chance that the nestlings experience thermal stress that is realized through the HPA axis (e.g., Lobato et al. 2008). However, were the latter scenario the best explanation for the findings, then there should have been a

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significant effect of total time that the mother was off of the nest upon nestling CORT levels, of which there was not (Table 2 in Rensel et al. 2010b). One also might ask ‘‘If nestling CORT levels reflect anxiety due to maternal separation, why is this not reflected by the total time the mother is off of the nest?’’ We speculate that, when the mother is in the vicinity of the nest, the nestlings are aware of her presence, perhaps by being able to hear her as she forages or moves about in the nearby vegetation. Additionally, breeders often emit soft vocalizations (i.e., a guttural sounding ‘cuc’) as they approach the nest to feed (T.E. Wilcoxen and S.J. Schoech, personal observation). In addition to not being able to hear their mother when she is some distance from the nest, it is possible that the noise from her departure from the nest area also serves to inform the nestlings (with relatively short wings adapted for movement within the scrub habitat, the noise from the jays’ wings during takeoff and while in flight is generally quite audible; S.J. Schoech, T.E. Wilcoxen, and M.A. Rensel, personal observation). Paternal care As noted above, breeder males do not incubate or brood nestlings but are the primary provisioner, especially during the early nestling period when nests were observed (i.e., days 3–5 post-hatch; Rensel et al. 2010b; Wilcoxen et al. 2010b). We also suggest (see Fig. 2), that paternal provisioning can not only directly affect nestling health state but can also influence maternal attentiveness. As for the former, Rensel et al. (2010b) demonstrated that nestling CORT levels are inversely related to paternal provisioning rates (Fig. 4), suggesting that nestlings with relatively poor provisioners as fathers are nutritionally stressed.

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Paternal Provisioning Rate (food score nestling hour ) Fig. 4 Nestling corticosterone levels were negatively correlated with the rate at which their father delivered food to the nest (F1,110 = 5.72, p = 0.019). Reprinted from Rensel et al. (2010b) with permission from Elsevier

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Male Breeder Age (years) Fig. 5 The total amount of food delivered to the nest by male breeders was positively related to age of the breeder (F12,44 = 7.79, p \ 0.0001, r2 = 0.56). With kind permission from Springer: Wilcoxen et al. (2010b, Fig. 1)

upon size-corrected mass; see Green 2001; Wilcoxen et al. 2010b), baseline CORT levels, and a measure of immune function (a bacterial killing assay using Escherichia coli; details in Wilcoxen et al. 2010b). As the title of this paper indicates, ‘‘older can be better’’, as provisioning rates were highly and positively correlated with male breeder age (Fig. 5; see Table 1 in Wilcoxen et al. 2010b). Amongst the other variables that helped explain the variation in male provisioning rates were the body condition of the male and the year. We have regularly noted profound year-effects upon multiple aspects of the reproductive biology of Florida Scrub-jays (see discussion in Schoech et al. 2009). Although it is intuitive to expect males in better condition to be better provisioners, the data show that, while this is indeed the case, relatively little of the variance is explained by body condition (Fig. 6). T o t a l F o o d S c o re (2 , 1 h r n e s t w a tc h e s )

We also found some support for the idea that variation in maternal attentiveness is in part attributable to paternal provisioning rates (T.E. Wilcoxen, unpublished data). We used PCA to combine two measures of female attentiveness, number of nest departures, and total amount of time off of the nest. The LMM used for the analysis included: year, to control for inter-year environmental effects; the number of non-breeding helpers sharing the territory; how long the pair had been together, pair bond duration affects many aspect of reproduction in FSJs and other species (e.g., Woolfenden and Fitzpatrick 1984, 1990, 1996; Reid 1988; Green 2002; van de Pol et al. 2006); female breeder age; brood size; and whether the pair had been food-supplemented prior to clutch initiation. For the latter variable, we point out that some territories included in the study had been supplemented for approximately 2 months prior to clutch initiation; however, in all cases, supplementation ceased upon clutch initiation by the breeding female of a given territory. We have been using food supplementation studies for several years in an effort to better understand the links between nutritional and physiological state and various reproductive parameters (e.g., Schoech et al. 2004, 2008). The findings are revealing in that female nest attendance patterns were, to a degree, dependent upon whether or not a territory had been food supplemented. Further, significant interactions between food supplementation and day-of-year, brood size, and the total amount of food delivered by the breeding male demonstrate the complexity of the relationships (T.E. Wilcoxen, unpublished data). The interaction between food supplementation and male food delivery led us to look more closely at the effect of male provisioning upon female attendance. There was a positive correlation between male breeder provisioning and female nest attendance score only in non-supplemented territories (T.E. Wilcoxen, unpublished data). Thus, if a breeding male brought very little food to the nestlings, the female breeder spent more time off the nest, presumably because she was foraging for the nestlings herself. We speculate that this difference in the impact of male provisioning upon female attendance reflects a ‘carry over’ effect of supplementation in which supplemented females are not as dependent upon their mate because: (1) they are in better condition; (2) when they leave the nest they access cached food and can return sooner than if they had to forage; or (3) a combination of the preceding. Our laboratory has also addressed whether the age and condition of the male breeder affects the degree to which he provisions his mate and nestlings (see Fig. 2). Wilcoxen et al. (2010b) report on provisioning rates of 90 male breeders based on nest watches over a 3-year period. As for the above, all watches were conducted when nestlings were between 3 and 5 days post-hatch, and the LMM included year, male breeder age, the number of helpers in the territory, brood size, male breeder body condition (an index based

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Male Breeder Condition (BCI) Fig. 6 The condition of a male breeder was positively related to the total amount of food that he delivered to the nest (F1,44 = 5.98, p = 0.02). With kind permission from Springer: Wilcoxen et al. (2010b, Fig. 2)

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Parental care and fitness As we suggest with the schema in Fig. 2, it is likely that many of the factors considered above have fitness ramifications. Although to date most of the proposed relationships have yet to be fully considered, we have data that suggest a direct relationship between maternal care and offspring survival. While we note that our study system with individuals that can be tracked throughout their entire lifetimes allow us to actually measure, rather than estimate fitness, to date we can only reference a commonly used surrogate measure of fitness, fledging success. Wilcoxen (unpublished data) used a LMM to examine a suite of factors that might contribute to fledging success. The variables incorporated were year (2007 and 2008), brood size, the number of male provisioning visits, day-of-year (the likelihood of fledging decreases as the season progresses; see Schaub et al. 1992), female nest absence score (as above, PC1 from a PCA that combined number of nest departures and total time away from the nest), pair bond duration, age of the female breeder, and the total food delivery score by the breeder male. Both year and female attendance score explained a significant portion of the variation in fledging success. Fledging success was more than twice as high in 2008 than in 2007 (see Schoech et al. 2009). The year effect is almost certainly attributable to the fact that 2007 was a drought year that negatively impacted resource availability and resulted in an overall reduction in reproductive success (see discussion of bad versus good years in Schoech et al. 2009; Morgan et al. 2010). The decreased fledging success in 2007 reflected increased nest failure rates due to depredation rather than starvation or brood reduction (determined by an absence of all nestlings within a nest instead of partial brood loss; T.E. Wilcoxen, unpublished data). The authors postulate that the increase in fledging success with increased maternal attentiveness is primarily due to two factors. First, the presence of the female at the nest facilitates defense as she can not only actively defend against predation but can also, through scolding and mobbing vocalizations, recruit other group members to aid in defending the nest against a predator (see Schoech 1999). Second, and this is not necessarily independent of the first, nestlings are less likely to vocalize (beg) or actively move about the nest while the mother is present, thereby reducing the attraction of predators that use aural or visual cues (Mumme 1992; Schaub et al. 1992; Young 2003).

Conclusions Our laboratory continues to explore the links among the environment, reproduction, physiologic and health state, and life history theory in a relatively long-lived study

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system, the Florida Scrub-jay. In part, our data that found nestling CORT levels to be predictive of the behavioral phenotype or personality of an individual has provided the ‘spark’ for further exploration. Of course, the logical follow-up question is ‘‘Does the phenotype persist?’’ Although Rensel and Schoech (2011) provide evidence of persistence in the physiological phenotype (i.e., the HPA axis responsiveness to a stressor), whether the personality is fixed (i.e., persists across an individual’s lifetime) remains under study. Additionally, we restate that our findings to date are correlative, and the necessary experimental manipulations are yet to be completed. Our studies and the relationships considered here demonstrate the complex nature of a natural system in which many factors contribute to variance in physiological and behavioral phenotype. The ‘‘tangled web’’ presented in Fig. 2 may, if anything, be too simplistic as it is not allinclusive. While we are aware of some factors that have not been considered, there may well be others that have been omitted due to a failure of imagination. Clearly, there could well be a genetic component to several of the traits we have considered, such as parental care, HPA axis responsiveness, and behavioral phenotype. Although there is considerable work to be done, preliminary data from our laboratory have noted a significant relationship between father and offspring HPA axis responsiveness, suggesting a genetic basis for this trait (S.J. Schoech and T.W. Small, unpublished data). Indeed, selection experiments with chickens and Japanese Quail (Coturnix japonica) in which high and low stress-responsive lines have been developed clearly reveal that HPA axis responsiveness can, to some degree, be a heritable trait (e.g., Edens and Siegel 1975; Satterlee and Johnson 1988; Satterlee et al. 2008). There is also the possibility that nestling CORT levels could be affected by yolk CORT concentrations which could also impact later phenotype (see review in Schoech et al. 2011). However, as the Florida Scrub-jay is a threatened species, this is an area of exploration that almost certainly would not be permitted. In closing, we would like to point out that this paper is not meant to be a comprehensive review of the topics discussed. Rather, we present an introduction to the topic as well as an overview of recently completed and ongoing research by our laboratory. As such, this paper was written to reflect the keynote symposium presentation by S.J. Schoech at the 2010 International Ornithological Congress in Campos do Jorda˜o, Brazil. Acknowledgments We thank the staff of Archbold Biological Station for kindly hosting us during each field season. Special thanks to Dr. Reed Bowman, Lab Head of the Avian Ecology Program at Archbold Biological Station, and Dr. Hilary Swain, the Executive Director of the station. Many individuals have helped in the field to gather the data considered in this manuscript, provided moral support

J Ornithol (2012) 153 (Suppl 1):S217–S226 over the course of the studies, and some have done both. These include Rob Aldredge, Jill Aldredge, Raoul Boughton, Eli Bridge, Michelle Desrosiers, Rachel Hanauer, Becky Heiss, Gina Morgan, Joe Neiderhauser, Shane Pruett, Zachary Seilo, Tom Small, Angela Tringali, and Matt Venesky (if any have been omitted, we beg your forgiveness!). This work was supported in part by NSF Grants IBN0346328 and IOS-0919899 to S.J.S., and an NSF Doctoral Dissertation Improvement Grant IOS-0909620 to T.E.W. and S.J.S. M.A.R. was supported by a Sigma Xi Grant-in-Aid-of-Research, an American Ornithologists’ Union Research Award, a Society for Integrative and Comparative Biology Grant-in-Aid-of-Research, and a University of Memphis Ecological Research Center Research Award. T.E.W. was also funded by a Sigma Xi Grant-in-Aid of Research, an American Ornithologists’ Union Josselyn Van Tyne Student Research Award, and a Florida Ornithological Society Cruickshank Research Award. T.E.W. was also supported by a Fellowship funded by a Van Vleet Memorial Doctoral Award from the College of Arts and Sciences at the University of Memphis. All received support from the Department of Biological Sciences of the University of Memphis. Lastly, we thank Michael Romero and Jesko Partecke for their invitation to S.J.S. to speak at the symposium ‘‘Corticosterone—long-term impacts on life history’’ at the 25th International Ornithological Congress, Campos do Jorda˜o, Brazil, August 2010. This manuscript is based upon his keynote presentation.

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