Experimental Tests of Endocrine Function in Breeding and ...



Experimental Tests of Endocrine Function in Breeding and

Nonbreeding Raptors

Julio Blas1,*

Fabrizio Sergio1

John C. Wingfield2,†

Fernando Hiraldo1

1Department of Conservation Biology, Estacio´ n Biolo´ gica de Don˜ ana, Consejo Superior de Investigaciones Cientı´ficas, Ame´ rico Vespucio s/n E-41092, Sevilla, Spain; 2Department of Biology, Box 351800, University of Washington, Seattle, Washington 98195

ABSTRACT

Many long-lived avian species defer reproduction for several years, often displaying a “floating” behavior characterized by the lack of mates and exclusive territories. Understanding the proximate mechanisms regulating floating behavior is a relevant topic of research for physiologists, behavioral ecologists, and population biologists because a prolonged period of nonbreed- ing can negatively affect lifetime fitness and change population dynamics. Here we tested two hypotheses linking endocrine function to floating status: (a) floaters undergo a period of sexual immaturity characterized by lower gonadal function (hy- pothesis of sexual immaturity), and (b) floating status is socially imposed by dominant conspecifics and revealed by the adre- nocortical response to stress (hypothesis of social subordina- tion). The two hypotheses were tested in a population of free- living black kites Milvus migrans in Don˜ ana National Park (southwest Spain), where breeders coexist with young floaters that defer reproduction for 3–7 yr. Hypophysial-gonadal func- tion, estimated as androgen production in response to exper- imental challenge with gonadotropin-releasing hormone (c- GnRH-I), was similar in magnitude and timing between floating and breeding males. The same treatment was, however, unable to elicit any response in terms of increasing estradiol or total androgen levels in females regardless of their breeding status. Following experimental capture and restraint, the adre- nocortical response to stress (estimated as circulating corti- costerone levels) was higher in floating than in breeding males, while females showed the opposite pattern (i.e., lower response

to stress in young floaters compared with breeders). Contrary to the hypothesis of sexual immaturity, our results suggest that floating males are physiologically capable of reproducing. The reported differences in adrenocortical function support the idea that floaters are socially subordinate to breeders, and cortico- sterone responses reflect the sex-specific roles during compe- tition in socially monogamous species.

Introduction

Avian populations are frequently composed of breeding indi- viduals coexisting with nonbreeders, which are generally young birds that defer reproduction for a variable number of years. Depending on the species and population, nonbreeders may adopt different behavioral strategies, such as “helping” a re- productive pair (i.e., cooperative breeding systems; Emlem

1982) or “floating” without establishing bonds to a particular territory (i.e., “floaters”; Smith 1978; Zack and Stutchbury

1992; Rohner 1997). Floaters are widespread in wild avian pop- ulations (e.g., seabirds: Hector et al. 1990; Williams 1992; Fred- eriksen and Bregnballe 2001; songbirds: Studd and Robertson

1985; raptors: Newton 1992; Sergio et al. 2009; Blas and Hiraldo

2010), and the length of time that an individual remains a nonbreeder can have important effects on individual fitness and population dynamics (Kokko and Sutherland 1998; Pen- teriani et al. 2005). For these reasons, understanding the prox- imate mechanisms underlying such a pervasive strategy con- stitutes a relevant topic of research with potential applications to managing wild and captive endangered species (e.g., raptors: Ferrer and Hiraldo 1991; Penteriani et al. 2005). The physio- logical basis of floating remains largely unstudied, especially among long-lived species. This may be due to the need for extensive long-term programs employing capture and recapture and sampling and monitoring of individually marked birds.

This study tests whether two complementary nonmutually exclusive hypotheses of proximate causation, namely “sexual immaturity” and “social subordination,” explain floating be- havior in a long-lived raptor, the black kite Milvus migrans. The hypothesis of sexual immaturity (Hall et al. 1987; Hector

et al. 1990; Williams 1992) posits a decreased sexual function

* Corresponding author; e-mail: julioblas@ebd.csic.es.

† Present address: Department of Neurobiology, Physiology and Behavior, Uni- versity of California, Davis, California 95616.

in young floaters and is largely assumed to apply to Accipitridae birds, where young floaters are normally termed “immature” individuals. However, recent studies have shown that the sea- sonal patterns of circulating sex-steroid hormones in young nonbreeding black kites are similar to those of reproductively active birds (Blas and Hiraldo 2010), with elevated titers of

androgens and estradiol characterizing the early stages of the breeding season in males and females, respectively. The latter study suggests that the endocrine system of breeders and float- ers responds to the environmental cues signaling the start of the breeding season (namely, “initial predictive information”; Wingfield 1980; Wingfield and Farner 1993), such as photo- period. However, when compared with breeders, floater males tended to have lower absolute levels of circulating testosterone, whereas floater females had statistically lower estradiol levels (Blas and Hiraldo 2010). This could be explained by either (1) the lack of exposure to additional stimulatory cues, such as within-pair interactions (Wingfield 1980; Wingfield and Farner

1993; Setiawan et al. 2007); or (2) an immature hypothalamus- pituitary-gonadal (HPG) axis unable to trigger the adequate hormonal responses to such signals. An experimental test of the maturity of the HPG axis consists of the administration of a standard exogenous dose of gonadotropin-releasing hormone (GnRH) followed by the collection of blood samples to deter- mine changes in circulating sex-steroid hormone levels. GnRH is produced in the hypothalamus and stimulates the pituitary to release luteinizing hormone, which triggers the production of sex steroids in the gonads (Williams 1992; Moore et al. 2002). According to the hypothesis of sexual immaturity, floaters are expected to show little or no production of gonadal steroids following experimental treatment with GnRH compared with breeding birds.

With regard to the hypothesis of social subordination, if nonbreeding status is socially imposed by dominant individuals, breeders and nonbreeders may differ in their adrenocortical response to stress (see Mays et al. 1991; Schoech et al. 1997; Young et al. 2006 for examples in cooperative breeding species). Social conflicts normally increase allostatic load (i.e., the energy requirements to perform daily routines; see McEwen and Wing- field 2003 for details), and this is often reflected in increased adrenocortical secretion. Traditionally, it has been thought that subordinate individuals exhibit greater adrenocortical respon- siveness than dominants (reviewed in von Holst 1998), al- though this is not always the case (e.g., Rohwer and Wingfield

1981; Creel et al. 1996; Creel 2001). Only recently, Goymann and Wingfield (2004) reconciled the conflicting evidence and proposed that breeding systems are only crude predictors of how social status affects glucocorticoid concentrations and that dominants or subordinates express higher or lower adreno- cortical responsiveness to stress depending on the relative allo- static load of social status. If subordinates do not compete with dominants, they will likely suffer minimal agonistic interac- tions, their allostatic load may be kept low, and their adre- nocortical levels would be similar to or lower than dominant breeders. Examples of the latter scenario include species where dominance over breeding resources is inherited, achieved via a queuing convention or when animals die or disappear. How- ever, when the access to and control of resources differs sub- stantially between dominants and subordinates and dominance status is mainly acquired through overt aggression, subordi- nates may experience higher allostatic load and thus higher sensitivity to stress compared with breeders (Goymann and

Wingfield 2004). The latter competitive scenario is consistent with our study model, where dominant breeders occupy and defend exclusive territories and floaters attempt violent territory takeovers, which sometimes succeed (Sergio et al. 2007a, 2009). Territorial confrontations can be extremely violent, with birds locking talons in the air, falling dangerously on the ground while battling and losing up to several flight feathers (Sergio et al. 2007a; see Fig. S1 in Sergio et al. 2011). Territory tres- passing can be frequent, especially in preincubation, and is also aimed at stealing food originally caught by the owners. Dom- inant breeders signal their social status and fighting ability through bodily and nonbodily signals (i.e., degree of nest dec- oration; Sergio et al. 2011) and benefit from signaling by lower rates of attacks on trespassers and less time spent in aggressive interactions. As a consequence, we may expect a relatively higher allostatic load in subordinate floaters than in dominant breeders, and we predict higher levels of stress hormones and higher HPA-axis responsiveness to stressors in subordinate nonbreeders (Tarlow et al. 2001; Øverli et al. 2004; Oyegbile and Marler 2006; Sorato and Kotrschal 2006). The physiological response to stress can be assessed in wild birds by means of determining the circulating levels of corticosterone in blood samples collected under controlled conditions (see, e.g., Wing- field 1994). According to the hypothesis of social subordination, we would expect a higher elevation of circulating corticosterone in nonbreeders than in breeding birds in response to stan- dardized stressful events such as the “capture and restraint” protocol.

Methods

Study Model and Behavioral Observations

Black kites are medium-sized socially monogamous migratory raptors that are present on their European breeding grounds from early March to late August (Cramp and Simmons 1980). In the study area (Don˜ ana National Park, southwest Spain), the black kite population is composed of ca. 500 breeding pairs plus 400–500 nonbreeding individuals (Sergio et al. 2005; Blas et al. 2009). Since 1965, this population has been subjected to scientific monitoring, and from 1986 onward, nestlings have been regularly banded with alphanumerically coded rings, which can be read with spotting scopes without disturbing the birds. Local longevity records show birds up to 25 yr old, and deferred reproduction can last up to 7 yr, with an average age of first breeding of 3.5 yr for both males and females (Blas et al. 2009). Natal dispersal distances are very short (median dis- tance p 4.8 km), and extensive surveys indicate the absence of emigration to other populations (Forero et al. 2002). After first breeding, kites tend to maintain the same territory and mate (Forero et al. 1999).

Marked territory holders were located on an annual basis from 1992 by means of continuous field surveys from early March to late June. When a banded bird was detected in a territory, the area was visited several times to locate its nest and check potential mate changes. Rather than defending ex- clusive home ranges, floaters are gregarious, forage in unde-

fended communal areas, and gather at night at six communal roosts. Monitoring of the communal roosts twice a week al- lowed identification of floaters (details in Sergio et al. 2009).

Bird Captures

Black kites were captured using cannon nets at the start of the breeding season during March and April 1998. According to previous studies that reported seasonal changes in sex-steroid levels, circulating testosterone (in males) and estradiol (in fe- males) are elevated during this time in both breeders (Blas et al. 2010) and floaters (Blas and Hiraldo 2010). By restricting bird captures to this part of the season, we covered the fertile period of most breeding pairs in the population, allowing com- parisons with nonbreeders at a time of the year when their reproductive axes would be expected to be upregulated. Because several kites were typically trapped at the same time, to avoid injury or suffering, all the individuals were first removed from the net, physically immobilized with cloth corsets made spe- cifically for this purpose, and maintained with their sight de- prived by means of falconry hoods. We then checked our records, and based on the previous history, behavioral obser- vations accumulated before capture, and sex determination through molecular analyses (see below), individuals were class- ified into either (1) an experimental group for GnRH challenge or for assessment of stress levels (see below), or (2) a nonex- perimental group that was subjected to banding and/or re- cording of biometric data before release. These activities were performed by two separate teams of 2–3 people working con- currently with the aim of speeding up the process and reducing the time to release the birds. Our field methods did not cause casualties or damage to the birds, all the sampling protocols were performed according to Spanish laws, and we prioritized ethical considerations over scientific goals.

GnRH Challenges

For experimental birds (31 males and 40 females), a preinjec- tion blood sample was collected from the brachial vein into a heparinized tube (average time from trapping to blood sam- pling: 17.1 ± 9.9 min). Kites were then challenged with an in- travenous injection of either chicken gonadotropin-hormone releasing hormone-I (c-GnRH-I; Sigma-Aldrich L0637; N p

56 birds) or a control saline solution (0.9% NaCl; N p 15 birds). All the injections were performed within 5 min following collection of the first blood sample; otherwise, the individuals were discarded from the experiment. Experimental injections were prepared from a stock solution of 200 mg GnRH/mL saline, which was preserved in frozen aliquots until the moment of capture. The selected dose was 20 mg GnRH/kg body mass following Williams and Sharp (1978) or the same mass-cor- rected volume of saline (i.e., 100 mL/kg body mass). Following injections, we collected three blood samples at fixed times (i.e.,

10, 30, and 60 min postchallenge). Between sampling episodes, kites were maintained with their sight deprived and were phys-

ically immobilized as described above. The exact age was known in 49 birds and ranged from 1 to 13 yr.

At the end of the breeding season, the intensive monitoring of the population allowed us to classify the GnRH-treated birds as either breeders (N p 26) or floaters (i.e., nonbreeders lacking previous reproductive experience; N p 28). Within the GnRH group, only two males lacked accurate observations to deter- mine their status and thus were only used to assess overall gonadal response as compared with control birds.

Assessment of the Adrenocortical Response to Stress

Glucocorticosteroid levels in birds rapidly elevate following capture and restraint (e.g., Wingfield 1994; Baos et al. 2006). Secretion patterns are typically monitored in blood samples collected at different times following capture, normally within a range of 30–45 min. This can be done by means of performing a stress series, where each individual is repeatedly blood sam- pled at several fixed times postcapture (e.g., 10, 30, and 45 min; Blas et al. 2007). Alternatively, the time-related pattern of glu- cocorticosteroid change can be assessed at the group level by means of collecting a single blood sample per individual. Rather than each individual being sampled at fixed times, the indi- viduals are sampled once along a continuum that ranges, for example, 30 or 45 min following capture (e.g., Mays et al. 1991; Sockman and Schwabl 2001; Wada et al. 2009). The latter pro- cedure is typically the method of choice when logistic con- straints do not allow within-individual handling and restraint series. Examples of such scenarios include studies where the individuals are too small to be repeatedly bled (e.g., Wada et al. 2009), when many individuals are captured simultaneously (e.g., Sockman and Schwabl 2001), or when the lapse time from capture to blood sampling is logistically difficult to control because of the capture methods employed (e.g., Mays et al.

1991). The nature of our study settings, where groups of black kites (rather than individual birds) are generally captured at the exact same time, led us to choose the latter method (i.e., one sample per individual). Thus, we performed a continuum sampling of different individuals within a given interval of time that ranged from 1 to 45 min postcapture. In order to stan- dardize the handling and restraint procedures, all the birds were maintained with their sight deprived and physically immobi- lized from capture to blood sampling. The selection of indi- viduals (51 birds; 22 males and 29 females) was done based on the criteria explained above (i.e., previous history of records, behavioral observations accumulated until the time of capture, and sex determination through molecular analyses) and to ob- tain a well-distributed sample within the previously defined interval of 45 min. Some of these samples, but not all, corre- sponded to specimens used in the GnRH experiment. In the latter case, only preinjection samples were used for corticoste- rone analyses, with the aim of avoiding a potentially confound- ing effect that the injections could exert on glucocorticoid se- cretion. The behavioral observations allowed us to classify individuals as either breeders (N p 30) or floaters (N p 21) at the end of the breeding season.

Blood Processing, Molecular Sexing, and Hormone Analyses

Blood samples were maintained in coolers and centrifuged the same day for separation of plasma, which was stored frozen (—80°C) until making determinations of hormone levels (see below). Because male and female black kites look alike, when sex was not known, the cellular fraction was preserved in eth- anol and subjected to molecular analyses for sex determination (Ellegren 1996). Plasma steroids concentrations were deter- mined through radioimmunoassay (RIA) at the University of Washington (Seattle) following a slight modification in the method described in Wingfield and Farner (1975) and Wing- field et al. (1991; i.e., samples were assayed without column chromatography; see Hau et al. 2000). Because the testosterone antibody used in the RIA displayed 60% cross-reactivity with

5-alpha-DHT and 6% cross-reactivity with 5-beta-DHT, all fur- ther references will be to total plasma androgen levels. For statistical purposes, nondetectable samples were assumed to have steroid concentrations equal to the detection limits of the RIAs (i.e., 100 pg/mL androgens, 60 pg/mL estradiol, and 1 ng/mL corticosterone). Intra- and interassay coefficients of var- iation were below 4%, 19%, and 3% for androgens, estradiol, and corticosterone RIAs, respectively.

Statistical Comparisons

Endocrine responses to GnRH challenges were analyzed through generalized linear mixed models (GLMM) using the PROC GLIMMIX package of SAS (SAS Institute, Cary, NC). This procedure allowed us to incorporate bird identity as a random factor in the models and thus control the potential pseudoreplication associated with the use of several observa- tions from the same individuals. The GLMMs followed a nor- mal distribution of errors and identity link functions and were constructed using a backward selection procedure (McCullagh

and Nelder 1989; Crawley 1993). From initial saturated models containing as dependent variables the time postinjection (min), treatment (experimental vs. control), status (breeder vs. floater), and their interactions, the least-significant terms were sequentially removed until obtaining a minimum adequate model that only retained significant effects at 15% rejection probability. Because the time from capture to initial blood sam- pling (i.e., “delay time”) can potentially modify sex-steroid lev- els, we tested this effect in a subset of experimental birds whose exact preinjection processing times were recorded in the field (i.e., 21 of 31 males and 17 of 23 females for androgens and estradiol, respectively). The effects of delay time on preinjection sex-steroid levels were tested by means of Pearson’s correla- tions, and the effects on postinjection levels were tested by incorporating this variable in the final GLMM models. In ad- dition to the tests above, one-way ANOVAs were performed to test for differences in (a) preinjection titer, (b) maximum post- injection levels, (c) absolute difference between preinjection sex-steroid levels and the maximum values recorded postchal- lenge, and (d) secretion rate. The latter variable was calculated as in Schoech et al. (1997) following the formula (C ma x — C 0 )/T, where Cmax is the maximum steroid value recorded post- challenge, C0 is the baseline level, and T is the time in minutes to reach maximum titer.

The adrenocortical responses to stress were analyzed through general linear models following the same stepwise backward procedure explained above. Initial saturated models considered the effects of handling time, status, body condition, and the number of previous captures (ranging 0–2). After obtaining a minimum adequate model, age was incorporated as an addi- tional dependent term, and its effects were tested in the sub- sample of birds whose exact age was known (N p 15 and N p 22 for males and females, respectively). Because temporal changes in corticosterone concentrations are expected to follow

[pic]

Figure 1. Male response to GnRH treatment. A, Circulating levels of androgens in breeding (white square, N p 13 ) and floating (black square, N p 16) males before experimental manipulation. B, Androgen levels at 10, 30, and 60 min following injection of either c-GnRH-I (circles, N p 23) or a control saline solution (N p 6). White and black circles represent experimental breeders (N p 10 ) and floaters (N p 13), respectively. Note that all the control birds have been merged into a single postinjection group for clarity of representation. Bars represent means ± 1 SE.

[pic]

Figure 2. Circulating preinjection levels of total androgens in male black kites as a function of the time from capture to blood sampling.

a curvilinear response, the recorded time in minutes was log transformed in order to allow linear fitting in the regression analyses. To estimate body condition, for each sex we used the residuals of a regression of mass on body size (hereafter “mass residuals”). Because univariate metrics have been criticized as measures of body size (Freeman and Jackson 1990; McDonald et al. 2005), we estimated size by means of the first axis of a principal components analysis built using tarsus, wing, and tail length (Sergio et al. 2007b, 2011).

Results

Before experimental manipulation, androgen levels in males were unrelated to status (F1, 27 p 1.81, P p 0.19; Fig. 1A) and independent of the time from capture to blood sampling (r p 0.20, P p 0.39; Fig. 2). The GLMM (Table 1) revealed a significant interaction between time postinjection and treat- ment that lead us to analyze each experimental group separately. GnRH-treated males showed a pronounced and significant time-related elevation of circulating androgens that was inde- pendent of status (GLMM: time: F1, 41 p 36.35, P ! 0.01; status: F1, 41 p 0.45, P p 0.50; time # status: F1, 40 p 0.33, P p 0.56; Fig. 1B). Androgens reached peak levels 60 min following in- jection, when average titers were more than fourfold higher

manipulations ranged 60–100 pg/mL and were unrelated to status (F1, 21 p 0.24, P p 0.63; Fig. 3A) and independent of the time from capture to blood sampling (r p 0.28; P p 0.27). Following GnRH treatment, some breeding individuals elevated estradiol levels up to 310 pg/mL, increasing the variability in circulating titers (range 60–310) in relation to nonbreeders and control birds (see error bars in Fig. 3). However, overall estra- diol levels in GnRH-treated females were statistically similar to controls, and there was no effect of time postinjection, status, or any of the interactions we considered (Table 2; Fig. 3B). The time from capture to blood sampling had no effect on post- injection estradiol levels (GLMM: F1, 20 p 0.45, P p 0.51). Re- stricting the analysis to experimental GnRH-injected females, the GLMM did not detect significant differences in estradiol levels between pre- and postinjection samples (F1, 45 p 1.48, P p 0.22). Peak estradiol levels, absolute increase, and relative increase were not related to reproductive status or associated with female age (all P 1 0.05). The absence of a clear estrogenic response to treatment with c-GnRH-1 led us to analyze plasma androgen levels in a second set of experimental samples cor- responding to 17 additional females. The concentration of total androgens, however, was below detection limits in all cases.

Circulating corticosterone levels in male black kites were not affected by the number of previous captures (F1, 16 p 0.243, P p 0.63) but showed a significant positive association with handling time (log time: F1, 17 p 35.85, P ! 0.01), a negative as- sociation with mass residuals (F1, 17 p 6.01, P p 0.02), and a significant effect of status (F1, 17 p 9.03, P ! 0.01). The latter two significant effects were independent of handling time (log time # mass residuals: F1, 16 p 0.35, P p 0.56; log time # status: F1, 15 p 0.01, P p 0.95). Corticosterone levels were higher in male floaters than in breeders, and both groups showed a parallel time-course pattern of the response to stress (Fig. 4A). When the final model was tested in the subsample of males whose age was known (N p 15), age was unable to explain corticosterone levels (F1, 10 p 1.70, P p 0.22), while the effects of time, status, and mass residuals remained significant. In females, neither mass residuals nor the number of previous

Table 1: Results from the mixed linear model analyzing circulating changes in androgen levels of male black kites after injection of either c-GnRH-I

than pretreatment values (i.e.,

320.9 ± 92.2

pg/mL vs.

or a control saline solution

1416.2 ± 219.7 pg/mL). Saline-injected control males, on the

contrary, showed a moderate time-related decrease in circu- lating androgen levels (GLMM: time: F1, 11 p 4.96, P p 0.04;

Effect Test Value P

Time* F1, 52 p 9.33 !.01

status:

F1, 11 p 0.29,

P p 0.59; time # status:

F1, 10 p 0.05,

Treatment* F1, 52 p .01 .94

P p 0.82; Fig. 1B). The incorporation of delay time (i.e., time

from capture to blood sampling) as an additional dependent variable to the models above did not change the results, and its effect was nonsignificant in all cases (P 1 0.24). Maximum titer, absolute increase, and secretion rate did not differ between breeding and floating males (all P ≥ 0.45). In addition, none of the latter three variables were correlated with male age (all P ≥ 0.61).

With regard to females, estradiol levels before experimental

Status F1, 52 p .40 .53

Time # treatment* F1, 52 p 11.97 !.01

Time # status F1, 51 p .32 .57

Treatment # status F1, 51 p .17 .68

Time # treatment # status F1, 50 p .08 .77

Note. Test values and associated probabilities are shown for the terms retained by the final model (indicated with an asterisk) and the nonsignificant effects when excluded during the backward procedure.

[pic]

Figure 3. Female response to GnRH treatment. A, Circulating levels of estradiol in breeding (white square, N p 12 ) and floating (black square, N p 11) females before experimental manipulation. B, Estradiol levels at 10, 30, and 60 min following injection of either c-GnRH-I (circles, N p 19) or a control saline solution (N p 4). White and black circles represent experimental breeders (N p 10) and floaters (N p 9), respectively. Note that all the control birds have been merged into a single postinjection group for clarity of representation. Bars represent means ± 1 SE.

captures affected corticosterone levels (mass residuals: F1, 25 p

0.17, P p 0.68; previous captures: F1, 24 p 0.247; P p 0.62), but handling time and status exerted significant effects (log time: F1, 26 p 15.63, P ! 0.01; status: F1, 26 p 4.06, P p 0.05). Breeding and floating females showed the same time-course pattern of response to stress (log time # status: F1, 25 p 0.45, P p 0.50), but contrary to males, corticosterone levels were statistically lower in floaters than in breeders (Fig. 4B). When the final model was tested in the subsample of females whose precise age was known (N p 22), age was unable to explain cortico- sterone levels (F1, 18 p 0.88, P p 0.77), while the effects of time and status remained significant.

Discussion

Our experimental treatment with exogenous c-GnRH-I elicited a marked increase in circulating androgens in male black kites that was not observed in control saline-injected males. These

Table 2: Results from the mixed linear model analyzing circulating changes in estradiol levels of female black kites after injection of either

c-GnRH-I or a control saline solution

|Effect |Test Value |P |

|Time |F1, 32 p 1.74 |.20 |

|Treatment |F1, 32 p .58 |.45 |

|Status |F1, 32 p .42 |.52 |

|Time # treatment |F1, 30 p .56 |.46 |

|Time # status |F1, 31 p 1.38 |.25 |

|Treatment # status |F1, 30 p .06 |.80 |

|Time # treatment # status |F1, 29 p .33 |.57 |

Note. Test values and associated probabilities are shown for all the terms when excluded during the backward procedure.

results indicate that the pituitary and gonads of male kites positively responded to our chemical challenge. More impor- tant for this study, the absolute elevation of androgen levels and the time-course pattern of gonadal response were similar in floating and breeding males, and all of the response param- eters that we considered were unrelated to reproductive status or male age. Overall, these results suggest that the reproductive axes of floating males are functionally similar to breeding in- dividuals contrary to the hypothesis of sexual immaturity and consistent with the results in Blas and Hiraldo (2010). In the latter nonexperimental study, circulating levels of testosterone were monitored in breeding and floating male kites throughout the reproductive season, and both groups showed parallel tem- poral changes and statistically similar absolute levels regardless of age and breeding status. Taken together, the results from experimental and nonexperimental tests, the reproductive sys- tem of floating male kites, similarly to that of breeders, seems to be able to respond not only to long-term naturally occurring environmental signals such as photoperiod but also to short- term hormonal challenges such as those simulated here through c-GnRH-I treatment. The ability to trigger short-term fast el- evations of circulating androgens can be particularly important in a context of male-male competition. During periods of social instability, when males compete for access to territories and mates, testosterone can increase relatively rapidly over the short-term, thereby facilitating aggression in intrasexual dis- putes (i.e., challenge hypothesis: Wingfield et al. 1987; Hir- schenhauser and Oliveira 2006). Such endocrine responses would be particularly relevant for floating black kites because they often perform territorial intrusions to challenge breeding conspecifics (Sergio et al. 2011), reinforcing the hypothesis that floaters are behavioral and physiologically prepared for social

[pic]

Figure 4. Adrenocortical responses to stress in male and female black kites. Changes in plasma corticosterone titers throughout the 45-min protocol of capture and restraint (time in log scale) in male (A) and female (B) black kites. Black circles represent floaters and white circles represent breeders. Solid and dashed lines represent predicted corticosterone levels from linear regression models as a function of log time for floating and breeding birds, respectively.

competition and that their status is not constrained by mat- uration of the HPG axis.

With regard to female kites, previous studies reported similar patterns of estradiol secretion throughout the reproductive sea- son in breeding and floating birds but lower absolute estradiol levels in the latter group (Blas and Hiraldo 2010). These results suggested that floating females responded to environmental cues and attained some degree of gonadal recrudescence. How- ever, they also suggested that full ovarian development would only be attained on exposure to additional reproductive cues such as intersexual stimulation (e.g., Pe´ czely and Pethes 1979; Setiawan et al. 2007). Contrary to our expectations, treatment of females with exogenous c-GnRH-I did not elicit an overall positive response in terms of increased estradiol or androgen levels. This could be explained by the inability of c-GnRH-I to stimulate the production of gonadotropins in the hypophysis and/or by the lack of gonadal response to gonadotropins; a proper discrimination of hypotheses would require further re- search. However, the fact that c-GnRH-I injections elicited a strong gonadal response in males may suggest sexual differences in the underlying GnRH control system. Such sexual differences could involve c-GnRH-II, a different form of GnRH present in birds. Although the function of c-GnRH-II is currently un- clear (Maney et al. 1997; Stevenson et al. 2008), it shows sea- sonal and age-related changes similar to c-GnRH-I, and both forms of hormone are potentially involved in the regulation of the HPG axis (Stevenson and MacDougall-Shackleton 2005; but see Meddle et al. 2006). Studies in passerine birds indicate that c-GnRH-II is regulated by the social context and affects female sexual behavior (Maney et al. 1997; Stevenson et al.

2008). The latter findings and the lack of response to c-GnRH- I we reported in female kites may suggest that c-GnRH-II is a

good candidate hormone for the regulation of breeding and floating status that deserves future investigation.

The adrenocortical response to stress of male black kites showed a significant effect of status, with floaters displaying higher corticosterone titers than breeders and a negative effect of mass residuals. The latter finding is widespread among field endocrinology studies (e.g., Kitaysky et al. 2001, 2003) and consistent with the role of corticosterone in the mobilization of endogenous energy stores aimed at coping with food short- ages and improving survival (reviewed in Sapolsky et al. 2000). With regard to the effects of status, the male pattern was con- sistent with the hypothesis of social subordination. Floaters were expected to show higher adrenocortical function if they tend to lose contests with dominant breeding conspecifics (e.g., Tarlow et al. 2001; Øverli et al. 2004; Oyegbile and Marler 2006; Sorato and Kotrschal 2006). Among black kites, territorial dis- putes are frequent and often involve physical fighting (Sergio et al. 2007a, 2011). Age and previous site dominance constitute the major determinants for winning a contest (Sergio et al.

2007a), and therefore floaters—which are smaller, younger, ar- rive later from migration, and have no experience in territorial defense (Sergio at al. 2009)—tend to be evicted by experienced breeders. Contrary to males, floating females showed a lower stress response than breeders. An explanation consistent with the hypothesis of social subordination may rely on the different roles that males and females play during the processes of ter- ritory acquisition and defense. In socially monogamous mi- gratory avian species, disputes over territories are predomi- nantly undertaken by males, which tend to arrive earlier and settle in breeding territories before the arrival of females (An- dersson 1994). Females, nonetheless, may also gain from getting involved in territorial defense when they are mated, especially

in species establishing long-term pair bonds such as black kites (Forero et al. 1999). Following this reasoning, paired (i.e., breeding) females would be involved in social conflicts more often than single (i.e., floating) females, explaining why the stress response is more pronounced in the former group. This interpretation would support the arguments in Blas and Hi- raldo (2010) proposed to explain the evolutionary basis for sexual differences in reproductive maturation in birds. Floating males would benefit from a fully functional reproductive system (as our results suggest) because this facilitates access to breeding resources (e.g., increasing the chances of winning intrasexual competition through testosterone secretion, which triggers ag- gressive behavior according to the challenge hypothesis; Wing- field et al. 1987; Hirschenhauser and Oliveira 2006). However, a floating male actively searching for a territory would not only have a higher chance of losing contests (because of younger age, less experience, and later arrival dates) but would also be more exposed to social conflicts than a particular resident male defending its breeding ground. This would explain the higher response to stress in floating than in breeding males. Floating females on the other hand may delay maturation until estab- lishing pair bonds with a territorial male (Blas and Hiraldo

2010). A more passive role in territorial disputes would imply less exposure to social conflicts, explaining their lower response to stress than breeding females.

Several additional hypotheses (1–3 below) may also predict differences in adrenocortical function between breeders and floaters regardless of social conflicts, but our results did not fully meet the predictions of any of them. For example, (1) glucocorticoids can act as mediators of ontogenetic transitions (Wada 2008). This hypothesis posits that baseline and stress- induced corticosterone endogenously elevates to facilitate the transition between two life-history stages. An increased HPA- axis sensibility in floaters could thus facilitate the transition from nonbreeding to breeding status similar to that which oc- curs during the transition from nestling to fledging in some avian species (Schwabl 1999; Sockman and Schwabl 2001; Wada et al. 2008; but see Blas et al. 2006; Romero et al. 2006). Al- though this hypothesis may contribute to explaining the ele- vated response to stress of nonbreeding males, it does not ex- plain the reported sexual differences: floating males and females are in the same life-history transition but show opposite pat- terns of corticosterone release compared with breeders. A sec- ond hypothesis (2) posits that breeders downmodulate the stress response according to their reproductive investment (Heidinger et al. 2006, 2008; Lendvai et al. 2007). Reduced corticosterone secretion helps to avoid diversion of critical re- sources away from reproduction. This argument explains, for example, why breeding passerines respond less strongly to a stressor when their clutches are experimentally enlarged (Lend- vai et al. 2007) and why the stress response declines with age in breeding seabirds (Heidinger et al. 2006, 2008). Our results, however, do not fully support this hypothesis because breeding females showed a stronger (rather than a weaker) adrenocortical response than floaters, and age did not explain differences in corticosterone levels in either sex. A third hypothesis (3) is that

breeders and floaters differ in their response to stress as a con- sequence of differential habituation to humans. Several studies have shown that repeated exposure to direct human presence and/or repeated handling of the same individuals can result in downmodulation of the response to stress (Love at al. 2003; Walker et al. 2006). Depending on the species and study set- tings, such an effect may confound results and be a matter of concern. Our research was performed on wild medium-sized raptors living in their natural habitat and subjected to little human pressure, typically showing long flight initiation dis- tances (J. Blas, F. Sergio, and F. Hiraldo, unpublished obser- vations) and seldom captured during their generally long life spans. If black kites downmodulate the stress response as a consequence of habituation to human presence, we would ex- pect corticosterone levels to decrease with age; if they habituate to human handling, we would expect a negative effect of the number of previous captures. Neither age nor the number of previous captures affected the response to stress, and therefore, there is no evidence that habituation occurs in our study model. Despite the fact that other physiological parameters not con- sidered in this study (e.g., clearance rates of circulating corti- costerone, concentration of binding globulins, number of re- ceptors) may also play a role in the described patterns, our results concur with a large body of evidence indicating that social competition affects the response to stress. Also important, although our data do not fully meet the predictions of hy- potheses 1–2 above, we cannot discard their potential contri- bution because we are not testing mutually exclusive hypoth- eses. All of the proposed effects may thus partially underlie the sex and status patterns of response to stress, but such patterns are fully consistent with the hypothesis of social subordination

in black kites.

Acknowledgments

We thank G. Garcı´a, S. Cabezas, R. Baos, A. Sa´ nchez, M. Guer- rero, F. J. Vilches, and L. Hillstro¨ m for help in the field; G. Garc´ıa, L. Erckman, M. Hau, M. Wikelski, S. Kitaysky, and A. Godoy for providing help and advice during laboratory anal- yses; S. Young for revising the text; and two anonymous re- viewers for comments and suggestions that improved earlier drafts. Funding was provided through the Spanish Ministerio de Educacio´ n y Cultura and by the research projects PB96-

0834 of the Direccio´ n General de Investigacio´ n Cientı´fica y Tecnolo´ gica, CGL2008-01781/BOS of the Ministerio de Ciencia e Innovacio´ n, JA-58 of the Consejerı´a de Medio Ambiente de la Junta de Andalucı´a, and by Excellence Projects RNM 1790 and RNM 03822 of the Junta de Andalucı´a. J.C.W. acknowl- edges grant IBN-9905679 from the National Science Founda- tion, and J.B. acknowledges a “Ramon y Cajal” contract from the Spanish government.

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