A multi-tier approach to identifying environmental stress
in altricial nestling birds
J. BLAS,*† R. B AOS,‡ G. R. BORTOLOTTI,* T. MA RC HANT* and
F. HIRALDO‡
*Department of Biology, University of Saskatchewan, Saskatoon. Canada S7N 5E2, and ‡Department of
Applied Biology, Estación Biológica de Doñana, C.S.I.C. Avda. Ma Luisa s/n, Pabellón del Perú, Apdo 1056, E-41013
Sevilla, Spain
Summary
1. Birds respond to unpredictable events in the environment by releasing corticosterone,
which triggers alternate responses to overcome stressful situations. However, it has been
questioned whether altricial nestlings perceive and respond to ecological stressors.
Although some studies show adrenal responsiveness in non-precocial species, others
report a hyporesponsive period postulated to avoid deleterious effects of corticosterone.
2. To test whether the environmental surroundings of nestlings modulate their stress
levels we used a multi-tier comparative approach of plasma corticosterone: (1) within
an individual, (2) among nestlings within a brood hierarchy, (3) among broods within
a colony and (4) among individuals from different colonies.
3. Nestlings reacted to our protocol by elevating their corticosterone. Baseline levels
differed between colonies, and were higher in singleton nestlings compared with two- and
three-chick broods, but there were no differences related to within-brood hierarchies, age
or condition index.
4. Acute levels were higher in older birds, suggesting a developmental change in
adrenal responsiveness. Body condition also explained acute levels but only in singleton
nestlings, with higher concentrations in heavier birds.
5. Overall our results indicate that altricial nestlings do respond to a variety of environmental stressors, and variation in aspects of environmental quality (e.g. food supply,
parental care) may be associated with the differences in stress.
Key-words: Ciconia ciconia, corticosterone, ecological stress, habitat quality, parental quality
Introduction
The endocrine system orchestrates appropriate changes
in the morphology, physiology and behaviour of
organisms to specific life-history stages (Nelson 1995).
Animals have evolved physiological strategies to cope
with unpredictable components of the environment
(Wingfield & Kitaysky 2002) and it is widely accepted
that the facultative responses constitute a distinct emergency life-history stage (Wingfield et al. 1998). Among
birds, sudden changes in weather conditions, famine,
droughts or predation episodes activate the hypothalamus–
pituitary–adrenal cortex (HPA) axis (Astheimer,
Buttemer & Wingfield 1995; Romero 2000). The resulting
secretion of glucocorticosteroids triggers alternate
behavioural and physiological responses that temporarily
suspend ‘non-essential’ activities for immediate survival,
†Author to whom correspondence should be addressed.
E-mail: julio@ebd.csic.es/julioblas@terra.es
and promotes adaptive changes that maximize the
possibility of surviving the perturbation and recovering
homeostasis in the best possible physical condition
(Silverin 1998a).
A theoretical framework has been developed to
explore the ecological bases of stress as well as underlying endocrine mechanisms, and birds have been used
extensively as models (e.g. Wingfield & Silverin 2002).
In spite of the substantial progress during the past
decade, most field investigations have focused on adults,
with fewer than 20 studies on nestlings having been
published during the past 20 years. Only recently have
ecological and behavioural endocrinologists been paying
more attention to the stress responses of nest-bound birds
(e.g. Schwabl 1999; Sims & Holberton 2000; Kitaysky
et al. 2001a; Tarlow, Wikelski & Anderson 2001; Love,
Bird & Shutt 2003a; Mullner, Linsenmair & Wikelski
2004). Given that altricial modes of development imply
the inability to locomote during early stages, and
parental care is crucial for providing food, heat, shelter
and protection from predators, it has been proposed
that any adrenocortical responsiveness would impair
the individual’s ability to behaviourally overcome the
stressful situation (Sims & Holberton 2000). In addition,
the potential damage caused by chronic exposure to high
corticosterone levels (Kitaysky et al. 2003) has been a
major argument to suggest that young birds suppress
the HPA axis, and that there is no adaptive value of
a glucocorticoid response to environmental stressors
(Romero, Soma & Wingfield 1998; Sims & Holberton
2000).
Contrary to the latter hypothesis, stress responses
may be beneficial for developing nestlings in spite of
their parental dependence, and environmental stressors
(Kitaysky et al. 2001a; Mullner et al. 2004), or withinbrood social interactions (e.g. Tarlow et al. 2001) may
change glucocorticoid release. Although parents buffer
environmental perturbations, the growing birds still
have to face variation in food availability, sibling competition, parasites, predators and, eventually, adverse
weather conditions. Furthermore, increases in corticosterone promote begging (Kitaysky et al. 2001b)
and aggressive behaviours (Kitaysky et al. 2003), and
modulate metabolic rates (Buttemer, Asteimer & Wingfield
1991), all of which should be of adaptive value for nestlings under stressful circumstances. Few studies have
tested whether potential ecological stressors such as
differences in the environment surrounding the nestlings, or attributes related to parental quality, affect the
adrenocortical response (but see Mullner et al. 2004).
This research gap may be because most investigations
have been performed under captive conditions (e.g.
Schwabl 1999; Kitaysky et al. 2003; Love, Bird & Shutt
2003b), have limited their focus to ontogeny and developmental processes (Schwabl 1999; Sims & Holberton
2000; Love et al. 2003a), or to a specific proximate cause
of perturbation such as food-stress (Kitaysky et al.
1999a, 2001a).
Here, we test whether differences in the environment
surrounding nestlings modulate their stress hormone
levels using a multi-tier approach: (1) within an individual nestling along the time course of a standardized
handling and restraint protocol, (2) among nestlings
differing in their position within a brood hierarchy (i.e.
sharing the same nest and parents), (3) among broods
of different size within a breeding colony (i.e. sharing
the same habitat) and (4) among individuals reared in
different local environments within the same population.
If the adrenocortical response to stress is suppressed
during the nestling stage, we predict no effect of the
different ecological conditions of brood hierarchy, brood
size and locale on circulating corticosterone levels, as
well as a lack of elevated corticosterone in response to
capture and handling. If, on the contrary, nestlings
have a functional HPA axis and respond to ecological
stressors, then all of these sources of variation are
plausible. As a study model, we used wild nestlings of
European White Stork (Ciconia ciconia, Linnaeus), a
large altricial wading bird.
Materials and methods
STUDY SPECIES AND STUDY AREAS
The White Stork is a large (2200 – 4400 g) long-lived,
monogamous bird that breeds from North Africa to
northern Europe (del Hoyo, Elliot & Sargatal 1994).
At Iberian latitudes it has a prolonged reproductive
season (from February to July), and breeds solitarily or
colonially in open nests on trees, cliffs, buildings and
power poles. The chicks hatch after 33 –34 days of incubation, and depend on adults for the delivery of food
until they leave the nest at 60 – 90 days of age ( Redondo,
Tortosa & Arias de la Reina 1995).
Fieldwork was conducted in two colonies in southern
and mid-western Spain during 2000. The southernmost
colony (Sevilla) was located beside the Guadalquivir
marshes in the Dehesa de Abajo natural area, while the
northernmost colony (Cáceres, 230 km away), was in a
scattered oak forest surrounded by crop fields in Aldea
del Cano. The stork population is continuous between
sites, and there is no evidence suggesting genetic differences between locales. However, the distance between
our colonies is large enough to ensure that there is no
overlap in the use of habitat during the breeding season,
as breeders usually feed near the nesting site (Cramp &
Simmons 1980).
FIELD PROCEDURES AND BLOOD SAMPLING
During the first week of June prior to blood sampling,
nests at both study sites were checked to select a random
sample of different brood sizes. Chosen nests (n = 40)
could have one, two or three chicks and were similarly
distributed between locales. Blood sampling (n = 70
birds) comprised either the single nestling or both the
oldest and the youngest birds within a brood in the case
of two- and three-chick nests. Several teams composed
of three people entered the colonies and followed the
same strategy for approaching nests. Teams worked
concurrently at different nests in the same tree or
at nearby trees, and followed a predetermined route
to avoid causing additional stress to breeding pairs or
nestlings. Nestlings underwent a standardized capture, handling and restraint protocol known to elicit an
increase in circulating corticosterone in birds ( Wingfield
& Kitaysky 2002). After accessing a nest with a ladder,
we captured the storks by hand and immediately brought
them down to the ground. The first blood sample was
taken within the first minute of capture. The synchronized work of several persons allowed simultaneous
sampling of two young per nest in the case of two- or
three-chick nests (n = 17 and 13 broods, respectively).
Additional samples were collected from the brachial
vein at 2, 10, 30 and 45 min after capture, and nestlings
were held in individual boxes between consecutive
samples. After blood sampling, nestlings were banded
and their wing chord and body mass were measured
before returning them to the nest. All field activities
were performed between 07:00 and 11:00 h to minimize potential diel effects. Blood samples were kept on
ice until centrifuged (3000 r.p.m. for 10 min) the same
day, and plasma was frozen and stored at −80 °C until
corticosterone measurement. The relative position of
siblings in the brood hierarchy was categorized as either
‘senior’ for the oldest, and ‘junior’ for the youngest.
Given that brood reduction in white storks normally
affects the youngest chicks (Sasvári, Hegyi & Péczely
1999), we assumed that single nestlings were senior,
although for statistical analyses (see later) the effect of
brood hierarchy was also tested excluding singletons.
Reproductive activities at Sevilla were monitored
periodically between March and July 2000 as part of a
long-term study (Jovani & Tella 2004). By checking
nest contents at least three times we gathered data on
clutch size, hatching success and fledging success of
individual pairs. Nests with different brood sizes at blood
sampling, did not differ in clutch size at the beginning
of the breeding season (average ± SE clutch size = 3·43
± 0·08, n = 114 nests,
F 2,111 = 0·961, P = 0·39).
Nestling age (estimated following Negro et al. 2000)
ranged from 24 to 59 days, and was always above the
time at which maximum thermoregulatory ability is
acquired (Tortosa & Castro 2003; Jovani & Tella 2004).
A body condition index was calculated as the individual’s residual value from a reduced major axis
regression of log body mass on log wing chord, following
Green (2001). Our condition estimate fits the required
assumptions of strong linearity between lg mass and
lg size (Pearson r = 0·925, P < 0·001) and independence
of body size (Pearson r = 0·126, P = 0·3).
costerone concentrations in samples taken within
1 min and after 45 min of handling and restraint were
used as an estimate of the baseline and acute stress
levels, respectively.
STATISTICAL TREATMENT
Baseline and acute corticosterone levels were analysed
separately through Generalized Linear Mixed Models
(GLMMs, Littell et al. 1996), using the macro glimmix
8·0 for SAS (SAS 1997). GLMMs simultaneously
account for both random effects, fixed effects and
covariates. By including the nest as a random variable,
we avoided pseudoreplication derived from the lack of
independence in values from nestlings within the same
brood. In addition, mixed GLMs offer the possibility
of assessing the amount of variation (i.e. deviance)
explained by the resulting models (McCullagh & Nelder
1989; Littell et al. 1996). Initial models were adjusted
to a normal distribution of errors and an identity link
function was used. The models were constructed by
a stepwise backward procedure, where the least significant terms were sequentially removed (McCullagh &
Nelder 1989; Crawley 1993). Initial effects comprised
colony site, brood size and brood hierarchy as fixed
factors, age and body condition as covariates, and all
the two-way interactions. Nest identity was included as
a random factor. Final (i.e. minimum adequate) models
only retained those terms showing a significant effect on
the corticosterone values at a >5% rejection probability.
Results
BASELINE CORTICOSTERONE LEVELS
CORTICOSTERONE DETERMINATION
Plasma corticosterone concentration was determined
utilizing a radioimmunoassay (RIA) described previously (Wayland et al. 2002). Measurements were
performed on reconstituted ethyl ether extracts of the
plasma samples. The efficiency of organic extraction
was estimated as described in Wayland et al. (2002) and
found to be consistently greater than 90% for the stork
samples. Each extract was measured in the RIA in
duplicate. Serial dilutions of the nestling stork plasma
extracts produced displacement curves parallel to that
of the purified corticosterone standards. The minimum
detection limit of the assay was 0·10 ng ml−1, but our
samples were diluted several-fold so as to fall near the
mid-range (0·43 ng ml−1) of the standard curve. Samples were measured over a total of nine separate assays;
the intra- and interassay coefficients of variation were
7·1% and 8·0%, respectively. Assay precision was assessed
by including an internal control with a known amount
of corticosterone (1·60 ng ml−1) in each RIA; the measured value of the internal control was 1·68 ± 0·074 (mean
± SD, n = 9) ng ml −1. A preliminary survey of the
corticosterone time-course response along the complete serial sampling period indicated that maximum
levels were attained after 45 min. Therefore, corti-
rood size and colony location showed statistically significant effects on baseline corticosterone levels ( Table 1).
Overall plasma resting values were more than two-fold
higher in Cáceres than in Sevilla (Fig. 1). This difference was not a consequence of the handling protocol,
as all birds were bled within the first minute following
capture, and the time from first entry to the colony to
Fig. 1. Baseline and acute levels of plasma corticosterone as
a function of the colony site ( = Sevilla colony; D = Cácere
colony). Bars represent mean values ± 1 standard error
(n = 35 per locality and sampling time). *P < 0·05; ns
P > 0·05.
Table 1. Summary of results from the GLMM explaining baseline plasma corticosterone concentration in young white storks
(n = 70 birds). The estimated effect and standard error as well as F-values and associated probabilities are shown for those
variables that significantly improved the fit of the model. For those terms that were excluded from the model during the
backwards procedure, F and P-values before exclusion are also shown. Data were modelled following a normal distribution of
errors and an identity link function, and nest identity was treated as a random factor. The final model explained 49·1% of the
initial deviance in corticosterone values
Effect
Estimate
Standard error
F-value (df )
P
Intercept
Colony
Brood size
13·4807
14·4084a
11·6051b
−2·2264c
–
–
–
–
–
–
–
–
–
–
–
–
–
2·0216
2·1505
3·3394
2·3441
–
–
–
–
–
–
–
–
–
–
–
–
–
6·67d (36)
44·89 (1, 30)
9·27 (2, 30)
<0·0001
<0·0001
0·0007
0·13 (1, 25)
0·55 (1, 27)
0·48 (1, 29)
1·88 (2, 24)
0·63 (1, 20)
0·30 (1, 21)
0·01 (1, 18)
0·87 (1, 23)
0·93 (2, 22)
3·09 (2, 28)
0·27 (1, 19)
3·48 (1, 24)
3·63 (1, 26)
0·7228
0·4667
0·4962
0·1740
0·4367
0·5924
0·9072
0·3610
0·4098
0·0612
0·6083
0·0744
0·0680
Brood hierarchy
Age
Body condition
Colony × Brood size
Colony × Brood hierarchy
Colony × Age
Colony × Body condition
Brood size × Brood hierarchy
Brood size × Age
Brood size × Body condition
Brood hierarchy × Age
Brood hierarchy × Body condition
Age × Body condition
a
Estimate corresponds to Cáceres colony.
Estimate corresponds to brood size = 1.
c
Estimate corresponds to brood size = 2.
d
t-test.
b
individuals’ blood sampling was not related to baseline
corticosterone values (Pearson r = 0·38, P = 0·76, n = 70).
The significant effect of brood size was analysed by performing post hoc contrasts on the final model. Nestlings
from brood sizes of one (32·3 ± 17·3 ng ml−1, n = 10)
showed higher baseline levels than nestlings from twochick (18·9 ± 8·7 ng ml−1, n = 34, post hoc contrast F1,30 =
18·36; P = 0·0002) and three-chick nests (20·1 ± 12·2
ng ml−1, n = 26, post hoc contrast F1,30 = 12·08; P =
0·0016); the latter two were not significantly different
from each other ( post hoc contrast F1,30 = 0·90; P = 0·35,
Fig. 2). Neither brood hierarchy nor age or body condition index accounted for differences in baseline values
(P > 0·1, Table 1), and there were no significant differences in condition index between nestlings that differed
in brood size (
F2,67 = 1·05, P = 0·35), or in colony
(
F1,68 = 0·604, P = 0·44). The final model explained
49·1% of the deviance in baseline levels, with colony site
alone explaining 34·4%, while brood size alone explained
13·9%. When the analysis was restricted to the sample
of nests with more than one young, brood hierarchy did
not influence corticosterone levels. Senior and junior
nestlings shower similar baseline concentrations (F1,29
= 0·01; P = 0·99, Fig. 3), but the difference between
colonies was again significant (F1,29 = 33·99; P < 0·0001).
The interaction between brood size and brood hierarchy
was not significant (F1,28 = 0·03; P = 0·86).
ACUTE CORTICOSTERONE LEVELS
After 45 min of handling and restraint, corticosterone
values increased around two-fold compared with base-
Fig. 2. Baseline and acute levels of plasma corticosterone as
a function of the brood size. Bars represent mean values ± 1
standard error. Sample sizes are shown above bars. *P < 0·05;
ns P > 0·05.
line levels (baseline 21·3 ± 12·3 ng ml−1; acute 39·1 ± 11·3
ng ml−1; repeated measures t = −9·59; P < 0·001; n = 70).
Acute corticosterone levels did not differ between
colonies (Fig. 1) nor were they affected by brood hierarchy (Table 2). Age had a positive effect on acute levels
(F1,27 = 17·91; P = 0·0002), and the interaction term
Brood size × Body condition was also significant (F2,27
= 3·75; P = 0·036, Table 2). When the effect of body
condition was separately tested in each of the brood
size groups, only singletons showed a significant
Table 2. Summary of results from the GLMM explaining stress-induced (i.e. after 45 min of restraint) plasma corticosterone
concentration in young White Storks (n = 70 birds). The estimated effect and standard error as well as F-values and associated
probabilities are shown for those variables that significantly improved the fit of the model. For those terms that were excluded
from the model during the backwards procedure, F and P-values before exclusion are also shown. Data were modelled following
a normal distribution of errors and an identity link function, and nest identity was treated as a random factor. The final model
explained 61·0% of the initial deviance in corticosterone values
Effect
Estimate
Standard error
Intercept
Colony
Brood size
15·9074
–
2·1191b
−1·6195c
–
0·5396
−52·4038
–
–
–
–
–
–
155·19b
−9·0289c
–
–
–
5·7102
–
3·7680
2·8363
–
0·1275
54·1859
–
–
–
–
–
–
72·9620
68·7838
–
–
–
Brood hierarchy
Age
Body condition
Colony × Brood size
Colony × Brood hierarchy
Colony × Age
Colony × Body condition
Brood size × Brood hierarchy
Brood size × Age
Brood size × Body condition
Brood hierarchy × Age
Brood hierarchy × Body condition
Age × Body condition
F-value (df )
P
2·79a (36)
0·89 (1, 25)
0·59 (2, 27)
0·0085
0·3543
0·5626
0·02 (1, 26)
17·91 (1, 27)
0·02 (1, 27)
1·17 (2, 19)
0·03 (1, 19)
0·30 (1, 21)
0·01 (1, 22)
0·78 (1, 24)
0·43 (2, 20)
3·75 (2, 27)
0·8889
0·0002
0·8961
0·8482
0·8703
0·5927
0·9273
0·3863
0·6561
0·0365
3·30 (1, 25)
0·01 (1, 18)
0·03 (1, 23)
0·0813
0·9312
0·8560
a
t-test.
Estimate corresponds to brood size = 1.
c
Estimate corresponds to brood size = 2.
b
(senior: 40·4 ± 9·2 ng ml−1; n = 30; junior 37·0 ±
12·2 ng ml−1; n = 30; Fig. 3).
Discussion
Fig. 3. Baseline and acute levels of plasma corticosterone as
a function of brood hierarchy. Birds from nests of broods
sizes of two and three are grouped together. Senior birds
(n = 30) were the oldest individuals within a brood, and
junior birds (n = 30) were the youngest. Error bars represent
± 1 standard error of the mean (ns P > 0·05).
and positive association with acute corticosterone
levels (F1,8 = 8·05; P = 0·0219), but neither two-chick nor
three-chick broods revealed any significant effect
(Brood size = 2: F1,16 = 2·04, P = 0·1724; Brood
size = 3: F1,12 = 1·20, P = 0·2941). Overall acute levels
did not differ between nestlings from different brood
sizes (F2,27 = 0·59, P = 0·5626, Table 2). Among brood
sizes of two and three chicks, senior and junior siblings
showed similar acute corticosterone concentrations
Our results are clearly contrary to the perception of
altricial nestlings as totally dependent organisms whose
adrenocortical responsiveness is suppressed (e.g. Sims
& Holberton 2000). After 45 min of handling and
restraint, corticosterone levels were significantly higher
than baselines in the same individuals. By extension of
this response to handling, there is evidence that other
exogenous (ecological) stressors from the environment
also have the potential to change corticosterone secretions in the early stages of life, as indicated by recent
studies in other non-precocial bird species (Mullner
et al. 2004). Our finding would hardly be a consequence
of a maladaptive physiological response, as one would
expect strong selection against individuals with impaired
glucocorticoid secretion, given the known negative
repercussions of an over-exposure to corticosterone on
growth, learning and developmental processes (Kitaysky
et al. 2003; Hayward & Wingfield 2004).
Baseline corticosterone titres from Cáceres were
consistently higher than those from Sevilla. This result
accounted for a considerable proportion of the variance,
and suggests that environmental differences between
locales affect circulating glucocorticoids (see Mullner
et al. 2004). The Sevilla colony was located beside
a marsh (one of the most productive habitats in the
world, Odum 1961), whereas the Cáceres colony was
adjacent to a stream and crop fields. There was probably
a higher availability of fish, amphibians and aquatic
invertebrate prey at the Sevilla colony, all of which normally constitute the main food resources for breeding
White Storks (Cramp & Simmons 1980; Negro et al.
2000). More importantly, the introduced American
Crayfish (Procambarus clarkii) is a plague in Sevilla
marshes (Gutiérrez-Yurrita et al. 1999), and is a unique
example of a super-abundant prey resource for the
local community of breeding wading birds (Hiraldo &
Tablado 2003). In fact, crayfish represents the only
prey remains in 55% of White Stork nests in Sevilla
(Negro et al. 2000). The extraordinary food supply has
often been used to explain the population growth of
White Storks at the Sevilla colony even at a time when
the overall Spanish population of White Storks was
declining (Senra & Alés 1992; Hiraldo & Tablado
2003). At least two studies have shown that reduced
abundance of food resources triggers baseline increases
of the stress levels of wild adult birds (in passerines,
Marra & Holberton 1998; and sea-birds, Kitaysky,
Wingfield & Piatt 1999b), and this relationship is well
supported by experiments with developing nestlings
(Kitaysky et al. 1999a; Kitaysky et al. 2001a). Another
environmental difference between colonies was the
population size and density of breeders, as it was considerable higher in Sevilla (about 300 pairs) than in
Cáceres (about 50 pairs). Although there is evidence
showing that the density of conspecifics affects individual levels of stress hormones (Silverin 1998b), the
direction of those results was opposite to the pattern
we found, as corticosterone levels were higher in chicks
from the less populated Cáceres colony.
Recent work has also shown that social hierarchies
within a brood correlate with between-siblings differences in circulating glucocorticosteroids (Nuñez de la
Mora, Drummond & Wingfield 1996; Schwabl 1999;
Ramos-Fernández et al. 2000; Tarlow et al. 2001; Love
et al. 2003b; but see Sockman & Schwabl 2001; Love
et al. 2003a), and that corticosterone may play a role in
the aggressive behaviours of chicks ( Kitaysky et al. 2003).
For example, in the obligate siblicidal Galapagos Nazca
Booby (Sula granti, Tarlow et al. 2001) and the facultative siblicidal Blue-Footed Booby (Sula nebouxii,
Ramos-Fernández et al. 2000), the subordinate chick
has higher circulating corticosterone than its dominant
sibling. Furthermore, single chicks tend to have either
lower corticosterone titers than two-brood nestlings
(Tarlow et al. 2001), or circulating levels similar to
dominant birds (Nuñez de la Mora et al. 1996), suggesting a role of this hormone in competitive social
behaviours. Contrary to this pattern, we found no differences associated with brood hierarchy, as senior and
junior birds showed similar titres. In addition, stress
hormone levels were similar among two- and three-chick
broods and statistically higher in single stork nestlings.
If circulating levels of corticosterone respond to social
interactions, we may expect an increase with brood size
parallel to the increasing chances of sibling aggression.
Our results suggest that corticosterone may not play a
role in the organization of within-broods social hierarchies in white storks. Nevertheless, siblicidal behaviours
in storks lack the relevance recorded in the booby species,
as aggressive interactions only take place close to the
age of fledging, when mortality rarely occurs (Tortosa
& Redondo 1992; Sasvári et al. 1999).
It is important to note that single nestlings in the
present study were the result of breeding attempts that
suffered a higher loss of eggs or chicks, as nests of different brood sizes did not differ in clutch size at the
beginning of the breeding season. It is therefore plausible that single nestlings were reared by parents of low
quality, for example, young or inexperienced breeders
(Forslund & Pärt 1995). Other studies have reported
negative associations between nestling mortality in White
Storks and some measures of parental quality such as
the body mass of adults and their food delivery rate
(Sasvári et al. 1999). A lower feeding efficiency among
poor-quality breeders would explain both the increased
nestling mortality and the elevated corticosterone in
the surviving chick. Furthermore, reduced nest attendance by parents would result in the developing young
being more exposed to the elements, a factor known to
elicit corticosterone increases in birds (Romero 2000;
Wingfield & Kitaysky 2002), and to contribute to nestling mortality in White Storks (Jovani & Tella 2004).
Among adult birds, it is well known that reduced
food supply elevates baseline corticosterone, which in
turn promotes adaptive behavioural changes such
as increased foraging activity and rate of ingestion
(Wingfield, Schwabl & Mattocks 1990). Nestlings are
limited in their ability to perform these behaviours,
and in fact, this has been one of the arguments for the
lack of any adaptive value of nestling adrenocortical
responsiveness (Sims & Holberton 2000). However,
the pioneering work of Kitaysky and colleagues demonstrated that corticosterone increases facilitate begging
in nestling gulls (Kitaysky, Wingfield & Piatt 2001b;
Kitaysky et al. 2003). Such behaviour is unquestionably
an adaptive response to food shortages, which is functionally equivalent to foraging in mature birds.
The positive association between condition index
and acute corticosterone levels observed in single stork
nestlings is contrary to previous findings. Experimental
studies have shown that food-restricted chicks have
higher baseline and acute stress-induced levels compared
with chicks fed ad libitum. Consequently, fat reserves
and body mass are usually found to be negatively correlated with adrenocortical secretion in both chicks
(Heath & Dufty 1998; Kitaysky et al. 2001a) and adult
birds (Wingfield & Silverin 2002). This relationship may
reflect the ability of birds in good condition to mobilize
fat for energy, while birds in poor condition must mobilize
protein. However, Silverin, Advidsson & Wingfield (1997)
found opposite associations for female Willow Warblers
(Phylloscopus trochilus) breeding at different latitudes.
While in northern locations body mass was negatively
related to acute levels, in southern sites the association
was positive. Although genotypic differences may explain
the findings in Willow Warblers, this is not a plausible
explanation for the between-broods differences reported
in our population of storks, and the functional explanation for higher stress-induced corticosterone levels in
birds in good condition remains unclear.
Baseline and acute postcapture corticosterone levels
showed different patterns of variation. Acute responses
were affected by nestling age and condition, and not by
colony, brood size or hierarchy. Conversely, baseline
levels changed with the environmental variables but were
independent of age. According to the developmental
hypothesis (Kitaysky et al. 2003), development of HPA
responsiveness in a nestling should parallel the individual’s abilities to behaviourally and physiologically
overcome stressors. The varied nature of the stimuli
potentially inducing stress implies that some adaptive
responses may be developed during early life stages,
while others require longer development. For example,
one would predict that adrenal responsiveness to food
stress would be developed in early stages, as it plays
a major role in begging behaviour and therefore in
offspring–adult communication to overcome the perturbation. On the other hand, our capture and restraint
protocol is a perturbation of a different nature (Canoine
et al. 2002), possibly of higher magnitude and requiring
more drastic responses (e.g. ‘fight or flight’, Wingfield
& Ramenofsky 1999; Wingfield & Silverin 2002). The
postcapture age pattern on corticosterone levels may
thus reflect a progressive behavioural readiness to face
short-term and drastic perturbations.
Acknowledgements
We thank J. C. Nuñez, who kindly helped to find the
Cáceres colony and provided housing during the field
activities. R. Jovani and J. L. Tella provided information on reproductive parameters from the Sevilla colony, and two anonymous referees made comments that
considerably improved earlier drafts. We are also grateful to the many people that helped during field sampling, especially A. Román, A. Tamayo and J. Ramirez
from SEO-Málaga, and also M. C. Quintero, J. L.
Arroyo, B. Jiménez, R. Tobar, C. Alonso, R. Álvarez, R. Rodríguez and N. Pastor. This work was supported by EGMASA and the European Community in
a i3P grant to J.B., the Spanish Ministerio de Educación Cultura y Deporte in a Postdoctoral Fellowship
to J.B. and a Predoctoral FPU fellowship to R.B., and
a NSERC grant to G.R.B. All birds were captured and
handled according to Spanish law. The Consejería de
Agricultura y Medio Ambiente de Cáceres, Consejería
de Medio Ambiente de Andalucía, and Ayuntamiento
de Puebla del Río provided permits.
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Received 7 August 2004; revised 27 December 2004; accepted
6 January 2005