Experimental Tests of Endocrine Function in Breeding and
Nonbreeding Raptors
Julio Blas1,*
Fabrizio Sergio1
John C. Wingfield2,†
Fernando Hiraldo1
1
Department of Conservation Biology, Estación Biológica de
Doñ ana, Consejo Superior de Investigaciones Cientı́ficas,
Américo Vespucio s/n E-41092, Sevilla, Spain; 2Department
of Biology, Box 351800, University of Washington, Seattle,
Washington 98195
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 corticosterone responses reflect the sex-specific roles during competition in socially monogamous species.
Introduction
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 nonbreeding 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 (hypothesis of sexual immaturity), and (b) floating status is socially
imposed by dominant conspecifics and revealed by the adrenocortical response to stress (hypothesis of social subordination). The two hypotheses were tested in a population of freeliving black kites Milvus migrans in Doñ ana National Park
(southwest Spain), where breeders coexist with young floaters
that defer reproduction for 3–7 yr. Hypophysial-gonadal function, estimated as androgen production in response to experimental challenge with gonadotropin-releasing hormone (cGnRH-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 adrenocortical response to stress (estimated as circulating corticosterone levels) was higher in floating than in breeding males,
while females showed the opposite pattern (i.e., lower response
* Corresponding author; e-mail: julioblas@ebd.csic.es.
†
Present address: Department of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616.
Avian populations are frequently composed of breeding individuals 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 reproductive 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 populations (e.g., seabirds: Hector et al. 1990; Williams 1992; Frederiksen 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; Penteriani et al. 2005). For these reasons, understanding the proximate mechanisms underlying such a pervasive strategy constitutes 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 physiological 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 behavior 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
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 seasonal 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 floaters responds to the environmental cues signaling the start of
the breeding season (namely, “initial predictive information”;
Wingfield 1980; Wingfield and Farner 1993), such as photoperiod. 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 hypothalamuspituitary-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 determine 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 Wingfield 2003 for details), and this is often reflected in increased
adrenocortical secretion. Traditionally, it has been thought that
subordinate individuals exhibit greater adrenocortical responsiveness than dominants (reviewed in von Holst 1998), although 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 adrenocortical responsiveness to stress depending on the relative allostatic load of social status. If subordinates do not compete with
dominants, they will likely suffer minimal agonistic interactions, their allostatic load may be kept low, and their adrenocortical 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. However, when the access to and control of resources differs substantially between dominants and subordinates and dominance
status is mainly acquired through overt aggression, subordinates 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 trespassing can be frequent, especially in preincubation, and is also
aimed at stealing food originally caught by the owners. Dominant breeders signal their social status and fighting ability
through bodily and nonbodily signals (i.e., degree of nest decoration; 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., Wingfield 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 standardized 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 (Doñ 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 distance 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 exclusive home ranges, floaters are gregarious, forage in unde-
fended communal areas, and gather at night at six communal ically immobilized as described above. The exact age was known
roosts. Monitoring of the communal roosts twice a week al- in 49 birds and ranged from 1 to 13 yr.
lowed identification of floaters (details in Sergio et al. 2009).
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
Bird Captures
previous reproductive experience; N p 28). Within the GnRH
group, only two males lacked accurate observations to deterBlack kites were captured using cannon nets at the start of the
mine their status and thus were only used to assess overall
breeding season during March and April 1998. According to
gonadal response as compared with control birds.
previous studies that reported seasonal changes in sex-steroid
levels, circulating testosterone (in males) and estradiol (in females) are elevated during this time in both breeders (Blas et Assessment of the Adrenocortical Response to Stress
al. 2010) and floaters (Blas and Hiraldo 2010). By restricting
Glucocorticosteroid levels in birds rapidly elevate following
bird captures to this part of the season, we covered the fertile
capture and restraint (e.g., Wingfield 1994; Baos et al. 2006).
period of most breeding pairs in the population, allowing comSecretion patterns are typically monitored in blood samples
parisons with nonbreeders at a time of the year when their
collected at different times following capture, normally within
reproductive axes would be expected to be upregulated. Because
a range of 30–45 min. This can be done by means of performing
several kites were typically trapped at the same time, to avoid
a stress series, where each individual is repeatedly blood saminjury or suffering, all the individuals were first removed from
pled at several fixed times postcapture (e.g., 10, 30, and 45 min;
the net, physically immobilized with cloth corsets made speBlas et al. 2007). Alternatively, the time-related pattern of glucifically for this purpose, and maintained with their sight decocorticosteroid change can be assessed at the group level by
prived by means of falconry hoods. We then checked our
means of collecting a single blood sample per individual. Rather
records, and based on the previous history, behavioral obserthan each individual being sampled at fixed times, the indivations accumulated before capture, and sex determination
viduals are sampled once along a continuum that ranges, for
through molecular analyses (see below), individuals were classexample, 30 or 45 min following capture (e.g., Mays et al. 1991;
ified into either (1) an experimental group for GnRH challenge
Sockman and Schwabl 2001; Wada et al. 2009). The latter proor for assessment of stress levels (see below), or (2) a nonexcedure is typically the method of choice when logistic conperimental group that was subjected to banding and/or restraints do not allow within-individual handling and restraint
cording of biometric data before release. These activities were
series. Examples of such scenarios include studies where the
performed by two separate teams of 2–3 people working conindividuals are too small to be repeatedly bled (e.g., Wada et
currently with the aim of speeding up the process and reducing
al. 2009), when many individuals are captured simultaneously
the time to release the birds. Our field methods did not cause
(e.g., Sockman and Schwabl 2001), or when the lapse time from
casualties or damage to the birds, all the sampling protocols
capture to blood sampling is logistically difficult to control
were performed according to Spanish laws, and we prioritized
because of the capture methods employed (e.g., Mays et al.
ethical considerations over scientific goals.
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.,
GnRH Challenges
one sample per individual). Thus, we performed a continuum
For experimental birds (31 males and 40 females), a preinjec- sampling of different individuals within a given interval of time
tion blood sample was collected from the brachial vein into a that ranged from 1 to 45 min postcapture. In order to stanheparinized tube (average time from trapping to blood sam- dardize the handling and restraint procedures, all the birds were
pling: 17.1 ± 9.9 min). Kites were then challenged with an in- maintained with their sight deprived and physically immobitravenous injection of either chicken gonadotropin-hormone lized from capture to blood sampling. The selection of indireleasing hormone-I (c-GnRH-I; Sigma-Aldrich L0637; N p viduals (51 birds; 22 males and 29 females) was done based on
56 birds) or a control saline solution (0.9% NaCl; N p 15 the criteria explained above (i.e., previous history of records,
birds). All the injections were performed within 5 min following behavioral observations accumulated until the time of capture,
collection of the first blood sample; otherwise, the individuals and sex determination through molecular analyses) and to obwere discarded from the experiment. Experimental injections tain a well-distributed sample within the previously defined
were prepared from a stock solution of 200 mg GnRH/mL saline, interval of 45 min. Some of these samples, but not all, correwhich was preserved in frozen aliquots until the moment of sponded to specimens used in the GnRH experiment. In the
capture. The selected dose was 20 mg GnRH/kg body mass latter case, only preinjection samples were used for corticostefollowing Williams and Sharp (1978) or the same mass-cor- rone analyses, with the aim of avoiding a potentially confoundrected volume of saline (i.e., 100 mL/kg body mass). Following ing effect that the injections could exert on glucocorticoid seinjections, we collected three blood samples at fixed times (i.e., cretion. The behavioral observations allowed us to classify
10, 30, and 60 min postchallenge). Between sampling episodes, individuals as either breeders (N p 30) or floaters (N p 21) at
kites were maintained with their sight deprived and were phys- 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 ethanol and subjected to molecular analyses for sex determination
(Ellegren 1996). Plasma steroids concentrations were determined through radioimmunoassay (RIA) at the University of
Washington (Seattle) following a slight modification in the
method described in Wingfield and Farner (1975) and Wingfield 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 further 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 variation 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 observations from the same individuals. The GLMMs followed a normal 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 sampling (i.e., “delay time”) can potentially modify sex-steroid levels, 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 correlations, and the effects on postinjection levels were tested by
incorporating this variable in the final GLMM models. In addition to the tests above, one-way ANOVAs were performed to
test for differences in (a) preinjection titer, (b) maximum postinjection levels, (c) absolute difference between preinjection
sex-steroid levels and the maximum values recorded postchallenge, 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 postchallenge, 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 additional dependent term, and its effects were tested in the subsample 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
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.
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 treatment 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 independent 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 injection, when average titers were more than fourfold higher
than pretreatment values (i.e., 320.9 ± 92.2 pg/mL vs.
1416.2 ± 219.7 pg/mL). Saline-injected control males, on the
contrary, showed a moderate time-related decrease in circulating androgen levels (GLMM: time: F1, 11 p 4.96, P p 0.04;
status: F1, 11 p 0.29, P p 0.59; time # status: F1, 10 p 0.05,
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
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 estradiol 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 postinjection estradiol levels (GLMM: F1, 20 p 0.45, P p 0.51). Restricting 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 corresponding 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 association 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
or a control saline solution
Effect
Test Value
P
Time*
Treatment*
Status
Time # treatment*
Time # status
Treatment # status
Time # treatment # status
F1, 52
F1, 52
F1, 52
F1, 52
F1, 51
F1, 51
F1, 50
!.01
p
p
p
p
p
p
p
9.33
.01
.40
11.97
.32
.17
.08
.94
.53
!.01
.57
.68
.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.
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.
results indicate that the pituitary and gonads of male kites
positively responded to our chemical challenge. More important 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 parameters 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 individuals 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 temDiscussion
poral changes and statistically similar absolute levels regardless
Our experimental treatment with exogenous c-GnRH-I elicited of age and breeding status. Taken together, the results from
a marked increase in circulating androgens in male black kites
experimental and nonexperimental tests, the reproductive systhat was not observed in control saline-injected males. These
tem of floating male kites, similarly to that of breeders, seems
to be able to respond not only to long-term naturally occurring
Table 2: Results from the mixed linear model
environmental signals such as photoperiod but also to shortanalyzing circulating changes in estradiol levels of
term hormonal challenges such as those simulated here through
female black kites after injection of either
c-GnRH-I treatment. The ability to trigger short-term fast elc-GnRH-I or a control saline solution
evations of circulating androgens can be particularly important
in a context of male-male competition. During periods of social
Effect
Test Value
P
instability, when males compete for access to territories and
Time
F1, 32 p 1.74 .20
mates, testosterone can increase relatively rapidly over the
Treatment
.45
F1, 32 p .58
short-term, thereby facilitating aggression in intrasexual disStatus
.52
F1, 32 p .42
putes (i.e., challenge hypothesis: Wingfield et al. 1987; HirTime # treatment
.46
F1, 30 p .56
schenhauser and Oliveira 2006). Such endocrine responses
Time # status
F1, 31 p 1.38 .25
would be particularly relevant for floating black kites because
Treatment # status
.80
F1, 30 p .06
they often perform territorial intrusions to challenge breeding
Time # treatment # status
.57
F1, 29 p .33
conspecifics (Sergio et al. 2011), reinforcing the hypothesis that
Note. Test values and associated probabilities are shown for all
floaters are behavioral and physiologically prepared for social
the terms when excluded during the backward procedure.
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 corticosterone levels (F1, 18 p 0.88, P p 0.77), while the effects of time
and status remained significant.
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 maturation of the HPG axis.
With regard to female kites, previous studies reported similar
patterns of estradiol secretion throughout the reproductive season 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. However, they also suggested that full ovarian development would
only be attained on exposure to additional reproductive cues
such as intersexual stimulation (e.g., Péczely 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 research. 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 unclear (Maney et al. 1997; Stevenson et al. 2008), it shows seasonal 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-GnRHI 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 shortages and improving survival (reviewed in Sapolsky et al. 2000).
With regard to the effects of status, the male pattern was consistent 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 disputes 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, arrive 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 territory acquisition and defense. In socially monogamous migratory avian species, disputes over territories are predominantly undertaken by males, which tend to arrive earlier and
settle in breeding territories before the arrival of females (Andersson 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 Hiraldo (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 aggressive behavior according to the challenge hypothesis; Wingfield 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 establishing 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 stressinduced corticosterone endogenously elevates to facilitate the
transition between two life-history stages. An increased HPAaxis sensibility in floaters could thus facilitate the transition
from nonbreeding to breeding status similar to that which occurs 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). Although this hypothesis may contribute to explaining the elevated response to stress of nonbreeding males, it does not explain the reported sexual differences: floating males and females
are in the same life-history transition but show opposite patterns of corticosterone release compared with breeders. A second 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 resources away from reproduction. This argument explains, for
example, why breeding passerines respond less strongly to a
stressor when their clutches are experimentally enlarged (Lendvai 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 consequence 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 settings, 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 distances (J. Blas, F. Sergio, and F. Hiraldo, unpublished observations) 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 expect 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 considered in this study (e.g., clearance rates of circulating corticosterone, concentration of binding globulins, number of receptors) 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 hypotheses 1–2 above, we cannot discard their potential contribution because we are not testing mutually exclusive hypotheses. 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. Garcı́a, S. Cabezas, R. Baos, A. Sánchez, M. Guerrero, F. J. Vilches, and L. Hillströ m for help in the field; G.
Garcı́a, L. Erckman, M. Hau, M. Wikelski, S. Kitaysky, and A.
Godoy for providing help and advice during laboratory analyses; S. Young for revising the text; and two anonymous reviewers for comments and suggestions that improved earlier
drafts. Funding was provided through the Spanish Ministerio
de Educación y Cultura and by the research projects PB960834 of the Direcció n General de Investigació n Cientı́fica y
Tecnoló gica, CGL2008-01781/BOS of the Ministerio de Ciencia
e Innovació n, JA-58 of the Consejerı́a de Medio Ambiente de
la Junta de Andalucı́a, and by Excellence Projects RNM 1790
and RNM 03822 of the Junta de Andalucı́a. J.C.W. acknowledges grant IBN-9905679 from the National Science Foundation, and J.B. acknowledges a “Ramon y Cajal” contract from
the Spanish government.
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