Recent Advances in NonMammalian Adrenal
Gland Research
2008
Published by Research Signpost
2008; Rights Reserved
Research Signpost
T.C. 37/661(2), Fort P.O.,
Tnvandrum-695 023, Kerala, India
Editor
Anna Capaldo
Managing Editor
5G. Pandalai
Publication Manager
A. Gayathri
Research Signpost and the Editor assume no
responsibility for the opinions and statements
advanced by contdbutors
ISBN: 978-81-308-0291-6
Contributors
Rccordi Fiorenza
Dipartimento di Biologia nimale e dell’Uomo, Università di Roma, ‘La Sapienza
Viale delfUniversità 32, 00185 Rome, Italy
Bacs Raquel
Estación Biológica de Doñana, CS.I.C., vda. de Maria Luisa s/n., Pabellón del Peru,
pdo 1056, 4 013 Seville, Spain
Barrett Michele
Department of nimal Sciences, Rutgers University, New Brunswick, Nj 08901 US
Bias Julio
Estación Bioiógica de Doñana, C.S.I.C., vda. de Maria Luisa s/n., Pabellón del Peru
pdo 1056, 4101 3 Seville, Spain
Capaldo Rnna
Department of Biological Sciences, Section of Evolutive and Comparative Biology
University of Naples “Federico II”. Via Mezzocannone 8
80134 Naples, Italy
Carsia Rocco
Department of Cell Biology, University of Medicine and Dentistry of New jersey-School
of Osteopathic Medicine, Stratford, Nj 08084, US
Castillo Songul Suren
Istanbul University, Faculty of Science, Department of Biology, Zoology Section
Istanbul, Turkey
Chimenti Claudio
Dipartimento di Biologia nimale e deIl’Uomo, Università di Roma, “La Sapienza’
Viale dell’Università 32, 00185 Rome, Italy
Cox Robert M.
Department of Biological Sciences Dartmouth College, Hanover NH 03755, US
Crivellato Enrico
Department of Medical and Morphological Research, natomy section, University of
Udine Medical School, Udine, Italy
C
ntents
Section 1 The Adrenal Gland
-
Chapter 1
Development and evolution of the adrcnal gland and its homologs
in telcosts, anurans, chelonians and birds
C/audio Chimenli and Fiorenza Accordi
Section II The Steroidogenic Tissue
-
Chapter 2
Neuroendocrine regulation of amphibian adrenocortical cells by
hormones, neurotransrnitters and neuropeptides
F/aWe Sicard and 1-lubert Vaudty
31
Chapter 3
Adrenal steroidogenesis in reptiles: Insights from dispersed
adrenocortical cells from Sceloporus lizards
Rocco V. Carsia, Patrick J.Mcllroy, Robert M Cox
Michele Barrett and Henry B. John-Aider
57
Chapter 4
Strcss in the nest: Causes and consequenccs of adrenocortical
secretion in developing birds
Julio Bias and Raquel Baos
89
Chapter 5
Metal pollution and the stress response
Chelsea Ward
129
Chapter 6
Corticosteroids and the thyroid during amphibian tadpole metamorphosis
MaryL. Wright
147
Research Signpost
37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India
Recent Advances in Non-Mammalian Adrenal Gland Research, 2008: 89-128
ISBN: 978-81-308-0291-6 Editor: Anna Capaldo
4
Stress in the nest: Causes and
consequences of adrenocortical
secretion in developing birds
Julio Blas and Raquel Baos
Estación Biológica de Doñana, C.S.I.C., Avda. de María Luisa s/n., Pabellón del
Perú, Apdo 1056, 41013 Seville, Spain
Summary
During the last decade, a vast number of studies
have considerably increased our understanding of the
interplay between the ecology and the endocrinology
of the stress axis in wild avian populations. A
theoretical framework known as the ‘Emergency Life
History Stage’ has been developed to explore the
ecological bases of stress and underlying endocrine
mechanisms. Despite a growing body of literature on
the role of hypothalamic-pituitary-adrenal (HPA)
function in adult birds, little research has been
performed in birds during development, possibly due
to the wealth of variability in developmental strategies
Correspondence/Reprint request: Dr. Julio Blas, Estación Biológica de Doñana, C.S.I.C., Avda. de María Luisa
s/n., Pabellón del Perú, Apdo 1056, 41013 Seville, Spain. E-mail: julioblas@ebd.csic.es
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Julio Blas & Raquel Baos
within the Class Aves, and the lack of evidence that non-precocial species have
functional stress responses before parental independence. A series of
investigations on HPA function in nestling white storks (Ciconia ciconia)
under natural conditions have provided novel insights into the proximate
causes and ultimate fitness consequences of adrenocortical responses to stress
in developing birds. The aim of this review is to provide an integrated
summary of recent research on endogenous and exogenous factors affecting
adrenocortical function using stork chicks as a study model. Among the
endogenous factors that explain differences in HPA function, nestling age
constitutes a prime source of variability, which is discussed here within the
context of the Developmental Hypothesis. Superimposed on this ontogenic
pattern, exogenous factors of ecological origin such as food availability,
habitat and parental quality generate interindividual differences in baseline
corticosterone (cort) secretion. These findings support the notion that nonprecocial nestlings can activate emergency responses to environmental
perturbations despite their parental dependence for food and shelter, and
suggest a role of environmental stressors in the preparation for fledging and
independence. A second set of exogenous factors of concern for conservation
biologists is the exposure of wild avian populations to environmental
pollutants. Maximum levels of cort following capture and restraint were
positively related to storks´ blood lead levels. This relationship was stronger in
single nestlings than in birds from multiple-chick broods, suggesting a greater
impact of pollutants on individuals additionally exposed to ecological stressors
such as poor parental quality. With the aim of assessing the ultimate fitness
consequences of individual variability in stress responses, the probability of
survival and recruitment was modelled using information of a long-term study
that accounted for all the factors described above. Survival and recruitment
were negatively related to the magnitude of the adrenocortical response during
development, providing the first empirical evidence for a link between stressresponse early in life and subsequent fitness in a wild, long-lived vertebrate. In
the light of these findings, here we review the published literature on this topic
and discuss the current state of knowledge regarding adrenocortical function
in developing birds, highlighting study gaps and suggesting future research
directions.
Introduction
Activation of the hypothalamic-pituitary-adrenal (HPA) axis of birds in
response to environmental perturbations (e.g. inclement weather, decreased
food availability, exposure to predators) results in the release of the main avian
glucocorticoid, corticosterone (cort) into circulation [1]. Elevated cort triggers
behavioral and physiological responses such as changes in locomotor activity and
Stress responses in developing birds
91
foraging, decreased nocturnal oxygen consumption, lipogenesis and mobilization
of body energy resources [2-6]. These responses redirect animals to a lifesaving state [7] allowing them to overcome the source of stress and recover
homeostasis in the best possible physical condition.
During the last decade many studies have considerably increased our
understanding of the interplay between the ecology and the endocrinology of
the stress axis in wild avian populations. A theoretical framework known as
the ‘Emergency Life History Stage’ has been developed to explore the
ecological bases of stress as well as underlying endocrine mechanisms [8].
Although the concepts may be widely applicable to all vertebrates, birds have
been extensively used as models (e.g. [9]). However, despite a growing body
of literature on the role of HPA function in adult birds, the causes and
consequences of activation of stress responses in birds during development
have received little attention until recently. One plausible explanation for such
a research gap was the belief that nestlings suppress adrenocortical responses
to stress during development, in order to avoid the deleterious effects of
chronically elevated cort. Table 1 shows a comprehensive survey of
experimental studies testing the consequences of systemic cort treatment
during avian ontogeny. These studies indicate that early experimental exposure
to high glucocorticoid levels often leads to behavioural and growth deficits in
chicks: reduced cognitive ability and body condition, impaired immune
defences and jeopardized survival (reviewed in [10, 11]). Under natural (i.e.
non-experimental) conditions, a functional HPA axis may hypothetically lead
as well to such negative consequences if young, not fully developed
individuals are unable to adjust their physiology and behavior to unpredictable
perturbations. This line of argument has been used to suggest that nestlings
would have little, if any adrenocortical response to stress during development,
and that parents would compensate the lack of response by expressing parental
behaviours (e.g., brooding, feeding), thus ameliorating the effects of
environmental perturbations.
Only recently have field endocrinologists been paying more attention to
the alternative possibility that nest-bound (i.e. non-precocial) chicks develop
adrenocortical responses to stress under natural settings (e.g.. [11, 31, 32]). A
number of studies demonstrate that despite the physical bound to a nest and
parental dependence for food and shelter, the HPA axis can be functional
during early stages of development. Among these studies, a series of
investigations with nestling white storks (Ciconia ciconia) have provided
novel insights into the causes [33-36] and consequences [36, 37] of
adrenocortical responses to stress during development. Figure 1 summarizes
the potential factors affecting the response to stress in developing birds.
Environmental stressors (e.g. inclement weather, predators) are buffered by
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Julio Blas & Raquel Baos
Table 1. Survey of experimental studies testing the consequences of systemic
corticosterone elevations during avian ontogeny, on posthatching traits. Reports are
organized by species and according to the type of manipulation: M (maternal treatment
with exogenous corticosterone during laying); E (in ovo corticosterone injection) or Y
(post-hatching treatment of young birds with exogenous corticosterone). The table also
indicates the conditions (C = captive vs. W = wild settings) characterising the study. By
default, the effects of experimental manipulations on different traits correspond to
measures taken during post-hatching development (i.e. short-term effects). For traits
measured after chicks independence, the study parameter is followed by the letters LT
(long-term) within brackets. Significant (P<0.05) effects are indicated with asterisks
(*), and full references are given in the literature section at the end of the chapter.
parents in most avian species, but variability in parental attributes among species
and individuals of a given species (e.g. nest attendance, feeding and brooding
efficiency) cannot eliminate offspring exposure to a number of perturbations
(e.g. food shortages, parasites, pollutants, sibling competition). In addition,
the chicks´ capacity to activate emergency responses will depend on several
Stress responses in developing birds
93
evolutionary and ontogenic factors, including developmental mode (e.g. altricial,
precocial), age, and ecological modes (e.g. open vs. non-open nests). Despite
repeated activation of the stress axis, or chronic exposure to high cort levels
may have deleterious consequences on fitness (lower box in Figure 1, see also
Figure 1. Factors affecting avian stress response during development. Summary of
the potential factors affecting the adrenocortical response to stress in birds during
development. Environmental stressors (e.g. inclement weather) are buffered by parents
in most avian species, but variability in parental attributes among species and among
individuals of a given species (e.g. nest attendance, brooding efficiency) cannot
eliminate offspring exposure to a number of perturbations (e.g. parasites, famine,
pollutants, sibling competition). In addition, chick capacity to activate emergency
responses will depend on several evolutionary and ontogenic factors, including
developmental strategy (e.g. altricial, precocial), age, and ecological modes (e.g. open
vs. non-open nests). Short-term activation of HPA responses is adaptive, but repeated
activation may lead to chronic corticosterone exposure, which has deleterious
consequences on a number of performance measures. In addition, individuals show
strong variability in their adrenocortical response to standardized stressors, and the
resulting fitness consequences (lower box) remain largely unexplored.
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Julio Blas & Raquel Baos
Table 1), short-term activation of HPA responses can have strong adaptive
value, provided the critical role of cort on recovery and maintenance of
homeostasis. In addition, individuals within a given population show strong
variability in their adrenocortical response to standardized stressors, but the
fitness consequences of such variability remain unexplored to a large extent.
The aim of this review is to provide a comprehensive summary of recent
research on the factors affecting the adrenocortical response to stress and its
associated fitness consequences in birds during development, using white stork
chicks as a study model. First, we will analyze the developmental factors that
explain variability in baseline and stress-induced cort in stork chicks [34],
putting the ontogenic pattern of HPA responses in context within the varied
developmental strategies of birds. Secondly, we will show some of the
exogenous, environmental factors [38] generating individual differences in the
response to stress. Then, we will consider a second source of exogenous
factors: exposure to environmental pollutants [35], and will assess the
contribution of heavy metals and arsenic on HPA axis responsiveness. Finally,
we will estimate the consequences [37] of interindividual differences in
nestlings stress response on subsequent long-term fitness components.
The study model
The white stork (Plate 1) is a large (2200 to 4400 g) long-lived,
monogamous wading bird that breeds from North Africa to northern Europe
[39]. At Iberian latitudes it has a prolonged reproductive season (from
February to July), and breeds solitarily or colonially in open nests. The chicks
hatch after 33-34 days of incubation and show an altricial mode of
development [40]: hatchlings are unable to thermoregulate or locomote,
depend completely on their parents for food and shelter, and chicks stay in the
nest during the 60-90 days of growth [41, 42]. However, as hatchlings show a
loose downy plumage and open eyes, they may, depending on the
classification, be considered as semi-altricial [43], or semi-altricial-1 [44].
Nestlings inhabit open nests, normally 1-2 m diameter platforms at the top of
trees, poles or buildings, where there is little protection from the elements.
Weather conditions represent a real environmental perturbation for young
storks as they are known to have a strong effect on nestling mortality [45].
Thermoregulatory capabilities are not fully attained until nestling storks are
about 20 days old [42], when chicks still have limited locomotor activity [46],
and up to this age there is one adult permanently at the nest providing shelter.
At 30-40 days of age storks are in the mid-point of development, they are able
to stand up and walk around the nest. By the end of the nestling period chicks
are almost totally covered by a definitive feather coat, fully able to move
around the nest platform [46] and show increased rates of flight exercises
Stress responses in developing birds
95
Plate 1. The white stork as study model. A couple of adult white storks (Ciconia
ciconia) on the nest at a breeding colony in Spain (a), and a brood composed of three
semi-altricial nestlings (b). Upon accessing the nest with a ladder (c), the adrenocortical
response to stress is assessed through collection of serial blood samples (d) at fixed
times. Before returning the chicks back to their nests, nestlings are given a plastic coded
leg band (e) easily readable in the field with the use of spotting scopes (f). Subsequent
monitoring of breeding and feeding grounds allows collecting information to assess
survival and reproduction of the study subjects. Photographs courtesy of Julio Blas
(a-c), Roger Jovani (d-e) and Gary R. Bortolotti (f).
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Julio Blas & Raquel Baos
[36, 41]. Adults leave nestlings unattended for long periods and may limit the
activity at the nest during the day to feeding bouts [46], which decrease in
frequency around fledging time, when nestlings reach 65-81 days old [36].
Nestling storks display specific behaviours to elicit food delivery from
parents [47], and the intensity of begging behaviour is likely to increase with
age, as young improve their motor skills. Although within-brood aggression
rarely leads to fatal outcomes, the strongest expression of nestling aggressive
behaviour is directed to unrelated, kleptoparasitic nestlings rather than to true
siblings [41]. Average age of first breeding is 3.4 years, and maximum
lifespan is more than 33 years under natural conditions [39].
The study population of white storks in Spain is located in two colonies.
The southernmost colony (Sevilla) is located beside the Guadalquivir
marshes by Doñana National Park, while the northernmost colony (Cáceres,
230 km away) is in a scattered oak forest surrounded by crop fields. The
stork population is continuous between sites, and there is no evidence
suggesting genetic differences between locales. However, the distance
between 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 [47]. The Sevilla colony has been intensively monitored during
the last two decades. Annual monitoring included accurate nest counts,
breeding outcome, and marking of most fledglings with plastic-coded leg
bands easily readable at distance using spotting scopes (Plate 1, e-f), to
determine survival, recruitment and dispersal of individuals. The same
intensive monitoring was conducted within a radius of ca. 40 km, covering
the marshes and surroundings of Doñana National Park.
Assessment of nestlings´ adrenocortical response to stress is performed by
means of a standardized capture and restraint protocol, whereby a baseline blood
sample is collected within one minute following capture, and additional stressinduced samples are collected following physical restraint for 45 minutes. After
accessing a nest with a ladder, storks are captured by hand and immediately
brought down to the ground. The synchronized work of several workers allows
simultaneous sampling of two birds per nest (the oldest and the youngest) in case
of two- and three-chick broods (see [38] for further details on field procedures
and blood sampling). Plasma cort is subsequently analyzed through
radioimmunoassay following methods described in Wayland et al. [48].
Ontogenic factors
Adult birds are known to modulate baseline and stress-induced
glucocorticosteroid levels to meet the specific physiological and behavioral
requirements associated with different life history stages. For example, during
migration, birds elevate baseline cort to facilitate migratory fattening, while cort
Stress responses in developing birds
97
stress response is reduced to avoid catabolism of skeletal muscle (Migration
Modulation Hypothesis; [49-52]). During breeding, baseline and stressinduced cort levels are also modulated as a function of the different
reproductive stages (e.g. mating, laying, brooding, [53, 54]), and specific
adaptations such as the parental hyperphagia exhibited by brooding doves is
facilitated by elevated baseline cort [55].
Among young birds, adrenocortical responsiveness to standardized stressors
(such as human handling) seems highly variable during ontogeny depending on
the species. While mallards (Anas platyrhynchos) can rapidly elevate plasma
cort to acute levels in response to capture and handling soon after hatching
[56], northern mockingbirds (Mimus polyglottos), show little, if any, cort
response to handling during life as nestlings [57]. Intermediate stages of
responsiveness during development as nestlings, with age-related increases in
stress-induced cort elevations, have been reported in American kestrels (Falco
sparverius, [58]) and Magellanic penguins (Sphenicus magellanicus, [59]).
Such interspecific variability has been postulated to reflect the species-specific
life history strategy along the precocial-altricial spectrum [60], leading to the
formulation of the Developmental Hypothesis [11, 34, 57, 61]. The
Developmental Hypothesis is based on the following arguments: (a) because
the physiological and behavioural capacity of a developing bird to overcome a
perturbation depends on the degree of post-hatching parental dependence, the
physical bond to a nest, and the capacity to thermoregulate, locomote and
forage, young birds may be limited in their abilities to perform many of the
adult-like responses to overcome stressful situations (see [60]). As a
consequence, (b) an adult-like adrenocortical response to stress may expose
young to chronic cort elevations, with potentially deleterious consequences for
development such as reduced growth, inadequate thyroid function or reduced
cognitive and competitive capabilities [11, 13, 15, 26, 62]. According to these
arguments, the Developmental Hypothesis predicts that inter-specific variation
in the adrenocortical response to stress of hatchlings reflects the speciesspecific degree of altricial development (Figure 2). Therefore, true altricial
species such as northern mockingbirds that hatch almost naked, blind, unable
to locomote or thermoregulate and that depend on their parents for food and
protection, are expected to show little or no response to stress as nestlings (i.e.
hyporesponsive period). At the other extreme of the developmental spectrum,
precocial species hatch with sight, covered with down and are soon able to
thermoregulate, locomote and feed independently of their parents, and
therefore the hypothesis predicts full adrenocortical function in response to
stressors such as human handling during early post-hatching stages, as occurs
in 1-day old hatchling mallards [56] (see Figure 2). A second prediction that
derives from the Developmental Hypothesis at the intraspecific level is a
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Julio Blas & Raquel Baos
Figure 2. Developmental strategies of birds and the stress response. Avian
developmental modes can be characterized as being precocial, semiprecocial, semialtrical
or altricial, although these categories actually represent a continuum. Column A illustrates
the physical appearance of hatchlings: a mallard (precocial), a gull (semiprecocial), a stork
(semialtricial) and a sparrow (altricial). Column B describes some of the hatchling traits
characterizing the developmental mode, including presence of feathers, independent
motor and locomotor activity, feeding behavior and physical appearance. The
Developmental hypothesis (C; grey arrow) predicts that hatchlings will show increased
HPA functionality from altricial to precocial species. Numbers in column D refer to
some published studies testing or discussing age-related variation in corticosterone
levels according to developmental categories in a range of avian species (full references
are given the Literature section and the end of this chapter, see also Table 2).
positive association between the adrenocortical response to stress and nestling
age. Although during early stages post-hatching non-precocial species are
expected to show little response to stress, nestlings develop physiological and
behavioral abilities useful in dealing with a perturbation, which may be
modulated by the elevation of cort to acute levels. For example, studies
performed on nestling seabirds show that cort elevation facilitates begging and
aggression, two forms of adaptive responses to cope with food stress in early life
[11, 24]. Therefore, at least in non-precocial species, the adrenocortical response
to stress of nestlings is expected to increase with age, eventually reaching adultlike responses near fledging, when the young are able to show many of the adultlike behavioural and physiological responses in emergency situations.
With the purpose of testing the Developmental Hypothesis, Blas et al. [34]
investigated qualitative and quantitative changes in the adrenocortical response
to stress associated to age in nestling white storks. These aims were
Stress responses in developing birds
99
accomplished by evaluating the ability of nestlings to release cort in response
to a standardized “capture-stress protocol” [79]. Based on the Developmental
Hypothesis, they predicted that cort levels would increase above initial
baseline levels more rapidly and/or more robustly with age, to eventually reach
maximal adrenocortical responsiveness to capture and handling close to the
age of fledging. Sixty wild white stork nestlings aged 24-59 days old were
subjected to the standardized sampling protocol, and the time-course pattern of
the response to stress was assessed through determination of circulating cort in
blood samples collected at five fixed times during the 45 minute period
following capture and restraint. Although age did not affect baseline levels and
all the birds showed a positive post-capture increase in circulating cort, age
had a positive effect on the relative increase from baseline titre, the recorded
time to reach maximum level, and the stress-induced levels following 45 minutes
of restraint (Figure 3-A).
The time-course of the response was best fit to a Linear Model with a
third-order function of handling time, and showed a strong effect of age. While
very young nestlings (i.e. below 20 days old) displayed little response to
capture, the response near fledging (i.e. after 50 days old) resembled the typical
adrenocortical pattern widely reported in fully developed birds. These results
concur with those found in other altricial and semi-altricial species, and suggest
Figure 3. Developmental changes in the adrenocortical response to stress of white
stork chicks. Light bars represent baseline corticosterone concentrations and dark bars
represent stress-induced levels (mean ± standard error). As nestlings age (A) their
adrenocortical response to stress (but not baseline values) increases until reaching
maximum levels before fledging. Just prior to fledging (B), baseline levels show a sharp
rise possibly triggered by a parental reduction of feeding rates. Modified from [34](A)
and [36](B).
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Julio Blas & Raquel Baos
that non-precocial birds follow a gradual development of the hypothalamicpituitary-adrenal (HPA) axis. The fact that HPA sensitivity to stress is functional
suggests that young storks develop emergency responses of adaptive value and
are able to overcome severe perturbations in spite of their parental dependence,
at least during the last two-thirds of post-natal development. According to the
Developmental Hypothesis, such gradual changes would allow nestlings to
respond to perturbations as a function of the specific behavioural and
physiological abilities of their age.
With regard to the proximate basis for the age-pattern, three studies have
provided evidence that the response to stress in altricial birds reflect changes
within the hypothalamic-pituitary component of the HPA system, rather than a
maturation of the adrenal glands [57, 59, 76]. Following intra-jugular
injections of adrenocorticotropic hormone (ACTH), nestling mockingbirds,
Magellanic penguins (Aptenodytes patagonica) and white-crowned sparrows
(Zonotrichia leucophrys) showed cort levels several fold above the maximum
elevations elicited by capture and restraint, indicating that the adrenal glands
were functional and, therefore, that the maturation processes controlling the
age-pattern of the response to stress take place within the hypothalamicpituitary portion of the HPA axis.
Young white storks inhabit open nests, normally 1-2 m diameter platforms
at the top of trees, poles or buildings, where there is little protection from the
elements. Weather conditions represent a real environmental perturbation for
young storks as they are known to have a strong effect on nestling mortality
[45]. In cold weather, cort elevations may reduce oxygen consumption that
lowers extended metabolic rate, and promote gluconeogenesis and
mobilization of body energy stores as is known to occur in adults [2, 80, 81].
Furthermore, cort elevations modulate locomotor activity in adult birds [2, 3],
and so increased locomotor activity of nestlings may allow them to select parts
of the nest to avoid temperature extremes. The ability to regulate body
temperature and locomote changes gradually with age. Thermoregulatory
capabilities are not fully attained until nestling storks are about 20 days old
[42]. This is the estimated age when HPA stress response starts to develop and
although chicks still have limited locomotor activity [46], up to this age there
is one adult permanently at the nest providing shelter. The lack or reduced
ability of the younger age groups to show HPA stress response is hypothesized
to be an adaptation [57, 59, 76], because their reduced skills to evade the
perturbation may lead to chronically elevated cort levels, with detrimental
consequences to growth and development [11]. At 30-40 days of age storks are
in the mid-point of development, they are able to stand up and walk around the
nest, and their HPA stress response is intermediate (Figure 3-A). By the end of
the nestling period, young storks show a strong adrenocortical response similar
Stress responses in developing birds
101
to adults of many bird species. At this age nestlings are almost totally covered
by a definitive feather coat, fully able to move around the nest platform [46]
and show increased rates of flight exercises [41]. Adults leave nestlings
unattended for long periods, and may limit their activity at the nest during the
day to feeding bouts [46], suggesting that nestlings are able to meet the
challenge associated with weather as well as adults can.
In addition to weather inclemency, the stress response associated with food
deprivation constitutes another potential source of perturbation for altricial
young. Several studies indicate that circulating cort elevates in semialtricial
and semiprecocial nestlings in response to food shortages [59, 82]. The
probability of food stress is expected to increase gradually as nestlings age
because their energy demands along development increase with body mass.
The physiological ability of nestlings to overcome a period of food stress may
also improve with age, as increased body mass involves larger muscles and
body fat stores which constitute the main source of energy under conditions of
limited food supply, and mobilization of energy stores is facilitated by the
elevated cort levels triggered by emergency situations [10]. Nestling storks
also display specific behaviours to elicit food delivery from parents [47]. The
intensity of begging behavior is likely to increase with age, as young birds
improve their motor skills. Furthermore, experimental elevations of cort levels
in seabird chicks have been shown to elicit begging behaviors [11, 24].
Therefore, the gradual maturation of the HPA responses to stress could parallel
both increased exposure to food stress, and the capacity to accumulate and use
body energy stores and perform begging behaviours.
Despite the strong effect of age on the HPA response to stress, baseline
cort levels in [34] were independent on the degree of postnatal development.
Age-related increases in baseline cort have been reported in nestlings of some
non-precocial species (e.g. [58, 61]) and this effect is often explained as a
mechanism to facilitate the transition from nestling to fledging stages through
the positive effects of elevated baseline cort on locomotor activity, food
searching behaviours and learning processes that may be critical in the
preparation for independence [75, 77, 83-85]. However, the elevations of
baseline cort to facilitate fledging may occur just a few days prior to
independence rather than following a gradual age-pattern, explaining why this
and some other studies [57, 59, 86] did not detect such pattern. In fact, a study
on pre-fledging white stork chicks (aged c.a. 45 to 81 days old) supports the
role of baseline cort levels in the fledging process [36]. As fledging approached,
cort levels in wild stork chicks showed a very marked elevation, with a 4-fold
increase during the four days prior to nest abandonment [36]. By this time,
baseline cort levels were similar to those recorded after 45 minutes of acute
restraint in [34] (see Figure 3-B). However, rather than being endogenously
programmed, elevated cort titres seemed to respond to food restriction induced
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Julio Blas & Raquel Baos
by parents, which decreased feeding frequency during the last days of the
nestling period. Interestingly, baseline cort levels were negatively related to
body condition and positively related to the rate of nestlings´ wing flapping
[36], strongly suggesting that cort mediates adaptive behavioural changes in
the preparation for independence (i.e. increased locomotor activity) and in
response to compromised energy balance.
In summary, studies on nestling white storks support that the ontogenic
development of the HPA stress response is a gradual process fitting predictions
of the Developmental Hypothesis, and also that modulation of baseline cort in
response to changes in energy balance facilitates the transition from nestling to
fledging stages.
Ecological factors
Despite the protective buffer effect of parental care, developing birds have
to face environmental challenges such as adverse weather, parasites, predation
attempts, changes in food availability and intense social competition with
siblings (Figure 1). Chronic cort exposure may have detrimental effects on
growth and development (e.g. [11, 13], see Table 1) but short-term elevations
could be beneficial in overcoming these stressful situations. Furthermore, the
behavioural response of nestlings to cort elevations triggered by some stressful
events seems to be equivalent, rather than unrelated, to that of adults. A prime
example is the response to food shortages. It is well known that adult birds
increase cort secretion under food stress [87], which in turn promotes foraging
activity and rate of ingestion [6]. Although nest-bound chicks cannot perform
adult foraging behaviours, there is evidence that nestlings, like their parents,
respond to food shortages by increasing circulating cort [31, 59, 82]. This
hormonal response in turn facilitates begging behaviour [11, 24], the
functionally equivalent response to foraging in mature birds. Another example
of the parallels in adult and chick responses is how dominance and submission
are modulated by cort levels. Recent work has shown that social hierarchies
within a brood correlate with between-sibling differences in circulating
glucocorticosteroids [61, 88-90], and that cort plays a major role in the
aggressive behaviours of chicks [11].
In spite of the recent growing body of literature on the adrenocortical
function of nestlings, most studies have focussed on ontogenic and
developmental processes [57, 58, 61, 76] or to a specific proximate cause of
stress such as food or sibling competition [24, 31, 82, 89, 90]. As a
consequence, the potential sources of ecological stress affecting nestlings and
their relative importance on the activation of adrenocortical functions have not
been analysed comprehensively.
Three main sources of ecological stress related to environmental, parental,
and social factors may potentially activate the HPA axis of young birds. The
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103
relative contribution of these factors was assessed in nestling white storks by
determining their resting and stress-induced cort levels as a function of the
quality of the rearing locale (colony), brood size (a proxi of parental quality)
and nestling position within a brood hierarchy [38]. For many birds, one
cannot easily distinguish between a parent´s ability to provide food and the
quality of the territory in terms of food availability. Because the white stork is
a colonial breeder without exclusive feeding territories, it was possible to
discriminate between the effect of parental quality and local environment
quality. The latter was tested by performing comparisons of two geographically
separated colonies in Southern (Sevilla) and Western Spain (Cáceres). The
Sevilla colony was located beside a marsh (one of the most productive habitats
in the world, [91]), whereas the Cáceres colony was adjacent to a stream and
crop fields. The availability of fish, amphibians and aquatic invertebrate prey
(all of which normally constitute the main food resources for breeding white
storks [47, 92]) is higher at the Sevilla colony. More importantly, the
introduced American crayfish (Procambarus clarkii) is a plague in Sevilla
marshes [93], and is a unique example of a super-abundant prey resource for
the local community of breeding wading birds [94]. In fact, crayfish represents
the only prey remain in 55% of white stork nests in Sevilla [92]. 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 [94, 95]. Previous studies in
adults [96, 97] and nest-bound chicks [98] of other avian species indicate that
circulating cort reflects differences in habitat quality, allowing to predict
higher baseline concentrations in stork chicks from colonies located beside
crop fields compared to marsh environments. In concordance with this
prediction, resting cort levels were more than two-fold higher in Cáceres (crop)
compared to Sevilla (marsh, Figure 4-a). The increased baseline cort of
nestlings from Cáceres would promote behavioural and physiological changes
of high adaptive value in response to periods of food shortage, as supported by
diet restriction experiments in nestling seabirds [31, 82] and raptors [99], that
promote begging and increased sibling aggression [11, 31].
The parental source of stress was tested by comparing baseline cort levels
in nestlings that differed in the number of siblings within their brood. The
sampled broods had one, two or three chicks and were similarly distributed
between locales. Nests with different brood sizes at blood sampling did not differ
in clutch size at the beginning of the breeding season [33], indicating a higher
brood reduction in nests with singleton nestlings compared to two- and threechick broods, and therefore suggesting a positive association between the
quality of a breeding adult and its reproductive output. Thus, a negative
relationship between cort levels and brood size was predicted. As expected,
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Figure 4. Physiological stress and ecological factors. (A) Chicks reared in the less
productive environment (crop fields) showed higher baseline corticosterone levels
compared to nestlings reared beside wetlands (marsh), where food availability is higher
for white storks. (B) Single chicks, reared in nests that suffered a strong brood
reduction had higher baseline corticosterone titers compared to nestlings from two-and
three-chick broods. Bars represent mean values ± standard error. Modified from [33].
nestlings from brood sizes of one showed higher resting cort levels compared
to nestlings from two-chick and three-chick nests (Figure 4-b). 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, e.g. [100]). A lower feeding efficiency among poorquality breeders would explain both the increased nestling mortality and the
elevated cort in the surviving chick [101]. Furthermore, reduced nest
attendance by parents would result in the developing young being more
exposed to the elements, a factor known to elicit cort increases in birds [9,
102], and to contribute to nestling mortality in white storks [45]. In
concordance with these arguments, experimental manipulation of brood size
and food intake in nestling barn swallows (Hirundo rustica) resulted in
significant increases in baseline cort levels in the more stressed chicks [103].
With regards to social sources of stress, storks exhibit hatching asynchrony
[100], a common phenomenon in non-precocial birds that leads to asymmetries
in sibling mass within a brood and determine differential access to parental
resources. The endocrine correlates of social stress was tested by comparing
circulating cort between the oldest (senior) and the youngest (junior) sibling
within the size hierarchies of broods. Contrary to initial expectations, brood
Stress responses in developing birds
105
hierarchy could not explain variability in baseline or stress-induced cort
values. This could be due to the small differences in body mass (less than 9%)
among senior and junior siblings at sampling. An experimental study with
American kestrels [75] demonstrated that hatching span greatly increased the
difference in both baseline and acute cort levels between first- and last-hatched
chicks, which may explain between-brood asymmetries in absolute cort titers.
It is therefore possible that the lack of a relationship between hatching order
and plasma cort in white storks derives from a more synchronous hatching in
the sample of two- and three-chick nests.
In summary, these results highlight the advantages of using nestling
adrenocortical parameters as an indicator of parental and environmental
quality. Environmental differences among locales were mirrored by baseline
cort titres, which were two-fold higher in nestlings from poorer habitat, while
small brood size, an indicator of inferior parents, was associated with elevated
cort levels.
Pollutants
In vertebrates, abnormal glucocorticoid concentrations and impaired
functioning of the HPA axis have been linked to exposure to different classes
of pollutants [104-106]. Despite such impairment may occur in the absence of
gross toxicological effects, and may be critical to developing individuals, little
is known about the consequences of sublethal metal exposure on the adrenal
systems in free-living birds [48, 107], particularly among altricial species
[108]. A mine spill (the Aznalcóllar mine accident, [109]) occurred near a
white stork colony in south-western Spain in 1998, providing an opportunity to
study the effects of metal exposure on HPA function in stork nestlings. Two
years after the spill, white stork chicks were concurrently sampled at this
colony and at a reference site, allowing quantification of blood levels of heavy
metals (zinc, copper, lead, cadmium) and arsenic, in order to examine its
association to the adrenocortical response to stress in wild nestling birds [35].
Concentrations of copper, zinc, cadmiun and arsenic did not differ
between locations, but unexpectedly, lead levels were significantly higher in
nestling storks from the reference colony [35]. There were no significant
relationships between heavy metals or arsenic and baseline cort concentration.
However, maximum cort concentration was positively related to blood lead
levels regardless of colony location, and singleton nestlings had higher levels
of cort than nestlings from multiple-chick broods. In addition, the interaction
between lead levels and brood size was also significant [35], suggesting that
lead had a greater effect on the stress-induced cort of single nestlings than on
those of multiple-chick broods (Figure 5).
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Figure 5. Blood lead levels and maximum corticosterone secretion. Effect of blood
lead levels on the maximum corticosterone concentration recorded during a capture and
restraint protocol performed along 45-minutes. Light dots and solid line indicate single
nestlings while dark dots and dashed line represent nestlings from two- and three-chick
broods. Modified from [35].
When working with wildlife, one major difficulty rests with the
establishment of an absolute control condition when in fact the best option may
be a reference population for comparison. The fact that nestlings from the
reference colony far from the spill area had higher blood lead levels than storks
sampled in the focal colony supports Norris’ argument [110] that the
widespread distribution of many potential endocrine active substances makes it
difficult, if not impossible, to find a true control population. Despite the higher
average lead concentration in the reference colony, it is important to note that
virtually all the sampled storks showed lead levels below the threshold value
reported to cause sublethal effects in birds [111], with the exception of a single
nestling from the reference colony [35]. Lead ranks second on the list of top 20
hazardous substances from the 2005 CERCLA Priority List [112]. The widespread
use of lead arsenate as a pesticide in the early 20th century left a broad legacy
of soil and groundwater contamination. Recent or past lead-shot hunting,
mining and smelting activities, and industrial effluents are other sources of
lead responsible to make this element one of the most common metals in
contaminated ecosystems [113]. Exposure to lead is associated with
neurobehavioral, hematologic, nephrotoxic and reproductive effects in humans
Stress responses in developing birds
107
and other animals [114]. Experimental studies in mammals have shown both
adult and developmental lead exposure to elevate baseline cort levels [106,
115, 116]; however, similar effects had not been reported on stress-induced
response in either mammals or birds. The results in Baos et al. [35] showing a
positive association between lead and the stress-induced response elicited after
handling and restraint may be affected by other factors potentially correlated
with lead (e.g., pesticide exposure) that may contribute to some degree to this
result, calling for further experimental studies in order to establish a causeeffect relationship.
Another finding in Baos et al. [35] was a steeper association between lead
and the stress-induced cort response in birds from single-chick broods
compared to multiple-chick broods (Figure 5). In a previous study, it was
reported that single stork nestlings were reared in nests that experienced brood
reduction, which suggested lower parental quality [38]. Reduced attendance by
young or inexperienced parents may lead singletons to suffer from
environmental stressors other than lead (e.g., a greater exposure to harsh
weather conditions). This in turn may explain both their higher levels of
maximum cort, and the reported stronger relationship between induced
response to stress and lead. Regarding the latter finding, an experimental study
on rats provides evidence that lead exposure and stress interact [115].
Furthermore, the fact that both stress and lead target brain mesocorticolimbic
dopaminergic systems [117, 118] provides a biological basis for such an
interaction. In birds, Di Giulio and Scanlon [119] reported that the highest
plasma and adrenal concentrations of cort were observed in ducks receiving
the highest level of dietary cadmium when they were food-restricted.
Similarly, other study found significant correlations between renal cadmium
concentration and the stress response of females common eiders (Somateria
mollissima borealis) during the incubating period [48], when they are reported
to fast, while no effects were detected in prenesting females. Other authors
have reported adrenal hypertrophy and higher mortality rate in birds fed
petroleum-contaminated food after being exposed to chronic, mild cold stress,
suggesting an additive effect of both stressful factors [120]. In this sense, the
results in Baos et al. [35] would support the argument that contaminants acting
in concert with other non-specific stressors may have a greater impact on
individuals or populations than would be elicited by either the contaminants or
other stressors acting alone.
The persistence of lead, combined with the sensitivity of the HPA axis to
contaminant insult, suggest that exposure to this metal even at sublethal levels
may cause delayed functional deficits later in development. Endangered
species and/or populations exposed to additional stress factors (e.g., nutrition
deficiency, parasitism etc.) would be of special concern.
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Julio Blas & Raquel Baos
Finally, results from Baos et al. [35] also evidence that the stress induced
response may be affected by environmental contaminants regardless of baseline
cort levels, indicating that a handling and restraint protocol may be a better
approach to assess the HPA axis function compared to a single hormone
measurement following capture. In this sense, further field studies involving
birds from contaminated sites are called for to determine the extent to which the
adrenocortical response to stress of wild birds are affected by the exposure to
pollutants, in addition to the long-term effects on survival and overall fitness.
Fitness consequences
It is well established that cort secretion in response to stress promotes
changes in a number of physiological and behavioral traits (see references in
Introduction). In addition, long-term exposure to chronic cort levels can have
deleterious effects on numerous relevant functions (see Table 1). However,
individuals within animal populations show a strong variability in their
adrenocortical response to standardized stressors [121-123], and it remained
unknown whether such natural variability in the response to stress (rather than
exposure to stressors), exerts an impact on fitness. In a long-term field study,
Blas et al. [37] tested whether individual variation in the glucocorticoid
response to stress early in life had long-term consequences to unequivocal
components of fitness: survival and reproduction.
The concentration of circulating cort (both baseline and stress-induced
levels) was determined in nestling white storks subsequently marked with
plastic-coded leg bands easily readable at distance using spotting scopes (Plate
1-e-f). During the following five years, survival and recruitment of wild focal
birds was monitored through intensive and extensive field surveys on a very
large scale. To avoid the effects of differential exposure to uncontrolled
sources of environmental perturbation, nestlings came from a single breeding
colony that was intensively monitored during the previous two decades, and
also five years following blood sampling. The same population monitoring was
conducted within a radius of ca. 40 km, covering the marshes surroundings of
the colony (i.e. Doñana National Park), this whole study area holding ca. 2,000
nests of white storks including all the colonies and the main feeding areas for
the species. In addition, upon consultation of the national banding and
resighting data bank at Estación Biológica de Doñana (which compiles
information on storks banded across Spain and sighted elsewhere), the authors
discarded the possibility of long-distance dispersers in Europe and Africa.
A survival model, which considered all the other factors potentially affecting
cort levels (e.g. brood size, age, sex, body condition) yielded a negative effect
of stress-induced cort levels and a positive effect of nestlings´ body condition
and age at sampling on the probability of postfledging survival (Figure 6).
Stress responses in developing birds
109
Figure 6. Long-term survival and recruitment as a function of the stress response
during development. The probability of postfledging survival and recruitment (ranging
from 0 to 1, Y-axis) was negatively related to stress-induced corticosterone levels
recorded during development in nestling white storks (X-axis). The probabilities of
survival (upper line) and recruitment into the breeding population (lower line)
represents model predictions for nestlings sampled at 45 days post-hatching. The
survival curve was calculated for nestlings in good body condition (i.e. maximum
condition scores recorded in the sample). Modified from [37].
Therefore, storks with increased cort secretion in response to stress as nestlings
showed lower probability of surviving into adulthood.
Although increased mortality among high glucocorticosteroid responders
may suggest a potential for natural selection to operate on stress physiology,
there is a general agreement that this force exerts its effects by maximizing
reproduction of the adapted individuals, even at the expense of health and
survival [124]. For this reason, the probability of recruitment of white storks
into the breeding population was also analysed in Blas et al. [37]. Recruitment
probability was negatively related to stress-induced cort levels and positively
related to nestling age at blood sampling (Figure 6).
With regards to the effects of nestling age on survival and recruitment,
given that the assessment of developmental response to stress took place
during two consecutive days, nestling age reflected variability in hatching date
within the stork colony. Lay date has an almost ubiquitous association with
parental quality in seasonal breeding birds, with good quality individuals
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Julio Blas & Raquel Baos
breeding earlier in the year [125]. Older, earlier-hatched nestlings may thus be
the offspring of high-quality breeders, showing enhanced survival and recruitment
as a result of better genes, better parental care, or both. The positive effect of
body condition on survival was consistent with an intuitive notion that the
physical condition of nestlings is also a major determinant on individual
fitness, possibly linked to parental quality and/or timing of breeding [126].
Regarding the negative association between adrenocortical function and
both survival and recruitment, the authors suggest two explanations assuming
that the behavioural and physiological response to stress is consistent over time
[123, 127-130]. First, high glucocorticoid-responders may trigger more often,
or more robust emergency responses to other sorts of perturbations, not only
during development but also later in life. In the long-term, frequent activation
of the hypothalamous-pituitary-adrenal axis may lead to chronic exposure to
elevated glucocorticoid levels, with deleterious consequences on growth and
maturity, immune and reproductive function, brain function and cognitive
abilities [9-11, 131], all of which may affect survival and reproduction.
Second, according to a number of studies in fish, birds and mammals including
humans, inter-individual differences in stress coping responses are key
attributes defining personality types [127, 128, 132-134], and variability in
natural populations is maintained through different payoffs on adaptive
capacity and vulnerability to disease. In particular, proactive individuals
typically show reduced glucocorticoid elevations, but high activation of the
sympathetic axis that facilitates the “fight or flight” response to stress. On the
other hand, reactive individuals are characterized by high glucocorticoid
secretion but low sympathetic activation in response to stress that facilitate the
“freeze and hide” coping strategy [127-130, 133]. The success of proactive vs.
reactive coping strategies is postulated to vary as a function of population
density and predictability of food resources, with proactive individuals being
more successful when density is high and food is stable and abundant [130,
133, 135, 136], conditions that characterized the stork colony. The study
colony of white storks had experienced a pronounced growth during the last 25
years, being one of the largest and densest in the world. In addition, the unique
environment surrounding the colony (i.e. marshes of Doñana National Park)
offered high predictability and abundance of food resources. The results
regarding stork survival and recruitment, therefore, concur with a conceptual
framework that predicts selection of low glucocorticoid responders, proactive
phenotypes under similar environmental scenarios [133, 135, 136], and Blas et
al. [37] provide a parsimonious evolutionary explanation for the reported link
between fitness and adrenocortical function.
The stork results are also consistent with studies conducted on laboratory
rats establishing a positive association between stress response and mortality
Stress responses in developing birds
111
risk [127]. However, the latter animal model (Sprague-Dawley rats, Rattus
norvegicus) was artificially selected to develop spontaneous tumours in
adulthood [137], and it remained unclear whether the association between the
stress response and fitness was applicable to vertebrates outside a controlled
laboratory setting, and more important, whether other critical components of
fitness such as reproduction could trade-off the proposed benefits of a reduced
glucocorticoid response on longevity. Despite other studies in wild populations
of birds and reptiles had reported associations between baseline [138] or
chronic [139] glucocorticoid levels and survival, this relationship was
ultimately confounded by differential exposure to stressors among individuals,
affecting circulating glucocorticoid levels. The stork study, hence provides the
first empirical evidence of a link between the physiological response to stress
early in life and both reproduction and survival in a vertebrate, and clearly
indicates superior long-term fitness among phenotypes with reduced
glucocorticoid stress-responses.
Synthesis, research trends and future directions
Results from studies with white stork chicks illustrate the usefulness of
non-precocial nestlings as models to understand the interplay between the
stress axis and life history, ecology, developmental biology and toxicology in
avian populations. The wealth of variability in life-history strategies within the
developmental spectrum of birds implies strong interspecific differences in
numerous traits [60] and, therefore, research on the stress responses during
avian development offers ideal conditions to understand hormone-behaviour
relationships. Summarizing the available published evidence on nestling
adrenocortical function constitutes a first step to formulating, testing and
synthesizing hypotheses. However, despite a growing number of studies devises
a promising future, considering that more than 7600 avian species of 148
families have non-precocial modes of development [39, 60], and given that only
41 species exist with basic information published on cort levels during early
stages of life (see Table 2), this topics calls for additional research.
A survey of the published studies reporting adrenocortical measures in
birds during development (see Table 2) reveals a recent but very marked trend
towards integrative approaches, aimed at linking stress physiology to ontogenic,
behavioural, ecological and environmental variables (Figure 7). Traditionally,
research had been conducted in poultry species (e.g. chicken, turkey, and
mallard). Despite the fact that such studies represent a very valuable reference,
results are constrained to the precocial mode of development, subjected to strong
artificial selection and obtained under captive settings. These conditions make it
difficult to extrapolate results to wild avian populations because (a) the study
models have limited exposure to real environmental perturbations, (b) they only
112
Table 2. A survey of in vivo studies reporting adrenocortical measurements in birds during development. The survey is
intended to be comprehensive as of January 2008, although a number of references focussed on poultry species have been excluded
for simplicity. It is indicated the developmental mode (MOD: P=precocial, SP=semiprecocial, SA=semialtricial, A=altricial), the
conditions characterising the study (COND: C=captive vs. W =wild settings), the measured adrenocortical parameter/s (PARAM:
B=baseline corticosterone levels; A= Acute, stress-induced corticosterone levels; CBG= corticosterone binding globulins), the type
of experimental stressor, when applicable (Hr= handling and restraint, Acth=adrenocorticotropic hormone, H=Heat, C=Cold,
E=Electric shock, ET=Ether) and the tested associations (e.g. Age, Habitat quality, etc). Significant (P<0.05) associations with
baseline, acute, or both corticosterone measures are indicated as letters within brackets (i.e. baseline=[B]; acute=[A]; both=[B,A]).
Full references are given in the literature section at the end of the chapter.
Julio Blas & Raquel Baos
Table 2. Continued
Stress responses in developing birds
113
Table 2. Continued
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Julio Blas & Raquel Baos
Table 2. Continued
Stress responses in developing birds
115
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Julio Blas & Raquel Baos
Figure 7. Frequency of corticosterone studies according to research topics.
Frequency of original articles testing associations between non-precocial nestlings´
corticosterone levels and different research topics: A =Age-related changes (ontogeny
and development); T = toxicology; F= Food-related stress (food quality and quantity);
N = nutritional state (body condition, fat reserves, plasma chemistry);
C=communication (begging, signal development); H= habitat and environmental
quality (habitat structure, weather, anthropogenic pressure); I= Fledging and
independence; P= Immune function and parasitism; L=long-term fitness components
(reproduction, survival, sexually selected traits). The bars indicate number of original
articles (taken from Table 2) and the filling colour (see legend) indicates significant
associations with baseline corticosterone, acute levels or both.
represent one extreme in the gradient of developmental strategies of birds, and
(c) both captivity and domestication can strongly modify HPA function [67,
140]. This research trend is nonetheless changing fast, as illustrated in Figure 8
by the recent but exponential incorporation of new species along the last
decade, which mostly correspond to non-precocial taxa.
The incorporation of altricial, semialtricial and semiprecocial species has
broadened our perception of the evolution and maturation of emergency
responses during avian ontogeny, which constitutes one of the preferred study
topics (Figure 7-A) and has allowed the formulation of novel hypotheses such
as the Developmental [61], the Phylogenetic, and the Ecological-Ontogenic
Stress responses in developing birds
117
Figure 8. Research trends on adrenocortical function during avian development.
White bars represent the cumulated number of species with published references
regarding corticosterone levels during development (baseline or stress-induced titres),
according to the bibliographic survey shown in table 2. Grey bars indicate the cumulated
number of studies performed under natural (i.e. non-captive) settings. Black bars show the
cumulated number of original research articles performed in non-precocial species,
(*sample for 2007 includes papers published or in press as of January 2008).
[11]. The information available to date however, suffers from a strong
heterogeneity that makes it difficult to perform formal multi-species
comparative analyses. From a methodological perspective, only 50% of the
studies involving non-precocial species incorporate standard, experimentally
induced measures of cort responses. Within this set of studies, the preferred
method to assess adrenocortical function is the capture and restraint paradigm
(i.e. 92% of the reports compared to 27% including ACTH challenge), but the
sampling times following capture- restraint are very variable among studies.
Given the strong interspecies variability in physiological ranges of baseline
and stress-induced cort, a reference value from adult, fully developed
individuals could be the choice method to assess relative maturity of the HPA
axis for comparative purposes, but such a reference is currently lacking in most
species (but see e.g.[141]). One additional concern is the reduced sample size
often used to describe the adrenocortical response of different age segments
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Julio Blas & Raquel Baos
along development. A limited number of observations and the choice of
considering age as class factor in the statistical analyses may lead to negative
results with low statistical power (e.g. [57, 76]), that increase the chances of
committing Type II error (accepting the null hypothesis when it is false, i.e.
failing to detect an age effect when it exists). This potentially misleading
procedure could be solved by considering age as a covariate and/or by
applying one-tailed rejection probabilities, provided that evidence to date
strongly indicates that HPA function is a gradual process throughout
development, and that circulating cort is expected to increase (rather than
decrease) following short-term acute stress.
With regards to studies linking the stress reactivity of developing birds to
environmental factors, our survey shows a strong increase of reports performed
under field conditions, with 74% of all the field studies having been published
during the last 7 years (Figure 8, grey bars). All the surveyed field studies
correspond to non-precocial birds (i.e. true nestlings), and this set of reports
largely explains the growing number of species having published references on
adrenocortical function during development.
The fact that non-precocial taxa constitute 90% of the 41 avian species
with published records on baseline or stress-induced cort during development
makes it evident that wild nestlings offer a number of methodological
advantages compared to adult birds and precocial young, which should be
taken into account when planning a field study. For example, the geographical
range of exposure to stressful stimuli in developing nestlings is much more
locale. While free-moving adults and precocial young are normally exposed to
modifying factors over a larger geographical scale, nestlings are physically
bound to a nesting site and immediate vicinity, allowing a more precise
identification of the challenges causing activation of the HPA axis (e.g.
adverse weather, predation pressure, social competition). Also important, the
lesser ability of nestlings to escape eases repeated sampling of the same
individuals at different times as well as experimental manipulation of the
nestlings´ environment, while avoiding many of the complex methodologies
often associated with the capture and recapture of highly mobile precocial
young and fully developed individuals. These advantages have only recently
been taken into account by field endocrinologists, as indicated by the
exponential growth in the number of original articles on non-precocial species
published during the last decade (Figure 8, black bars), and the scarcity of field
studies prior to this period. On the other hand, one concern for field studies is
the potential exposure of wild populations to endocrine disruptive substances.
Evidence that a broad array of contaminants can modify nestlings baseline and
stress-induced cort is currently growing (Figure 7-T), and the widespread
distribution of toxics even in apparently pristine environments [35, 110]
Stress responses in developing birds
119
indicates that adequate selection of the study populations constitutes a growing
challenge for field endocrinologists.
The qualitative analysis of the papers surveyed in Table 2 also shows that
variability in food quality and quantity is one of the most frequently studied
topics (Figure 7-F). The pervasive associations between food availability and
both baseline and acute cort levels is supported by observational and
manipulative studies, and indicates that decreased food resources constitutes a
mayor perturbation causing HPA responses in developing birds. The primary
role of cort in the regulation of metabolism via glucose mobilization and fat
depletion [10] would explain the associations with nutritional and conditiondependent variables (e.g. fat reserves, mass residuals, Figure 7-N) which are
also tested in a relatively high number of the surveyed reports and often lead to
significant associations (possibly when the study sample contains adequate
variability in nutritional/condition levels). Such a relationship makes cort a
good endocrine candidate to understand the proximate control of honest
signalling and the expression of individual quality traits used in parentoffspring communication, a topic that behavioural endocrinologists are just
starting to consider (Figure 7-C). After the pioneering work of Kitaysky and
colleagues demonstrating a role of corticorterone in the regulation of begging
behaviour in seabird chicks [24], the hypothesis has only been tested in five
additional species (Tables 1 and 2), yielding mixed results. Very recently, this
hypothesis has received additional support with the demonstration that
morphological traits used in intraspecific communication of individual quality,
such as colour development in the melanin plumage pattern of nestling barn
owls [30] and carotenoid-dependent bright bare integument of sparrow chicks
[28] also depend on cort levels. Clearly, the development of this hypothesis is
devised as very promising in the near future.
Despite the fact that few studies have tested the effects of habitat quality
on adrenocortical function of nestlings (Figure 7-H), the environmental
variables that are taken into consideration ultimately seem to exert their effects
through changes in food availability (affecting feeding parameters, e.g. harsh
weather, forest structure, anthropogenic disturbances), as is also concluded in
the few articles that directly quantify food resources to characterize habitat
structure [98]. Interestingly, food stress and the behavioural effects of cort
elevations (e.g. increased locomotor activity) provide a physiological
mechanism to better understand the parent-offspring conflict for independence
(Table 2, Figure 7-I). At least in some species, the timing of nest departure and
the concomitant cort elevations seem to be manipulated by parents through
decreased feeding rates rather than just follow an endogenous clock [36],
although divergence exists among studies (e.g. [142, 143] vs. [36, 77]).
Experimental manipulations of food availability during the time prior to
120
Julio Blas & Raquel Baos
independence (such as those performed in [144]) would provide a better
answer for this topic that has only been studied in six species.
Exposure to parasites can be a relevant proximate cause of stress to
developing birds, which have to stay bound for a relatively long period into a
nest environment that can offer ideal conditions for parasites. Our survey
reveals, however, just a handful of studies testing associations between nestlings
HPA function and parasitism, as occur with studies relating cort to immune
function (Table 2, Figure 7-P). In adult vertebrates, cort is known to depress the
immune system [10]. Among the few studies showing an association between
nestling cort and prevalence or intensity of parasitism, it remains unknown
whether the latter is a consequence of reduced immune function (aimed at
fighting a primary stressor such as food deprivation, which elevates circulating
cort) or an ultimate factor directly causing cort elevations. Both hypotheses are
not mutually exclusive and require further experimental research.
An additional research challenge will be to elucidate the long-term fitness
consequences of individual variability in HPA function (Figure 7-L). To date,
it is becoming established that experimentally elevated cort during
development can have long-term consequences on a number of traits (Table 1),
but only two studies have related interindividual variability in nestling HPA
function to fitness components later in life: survival and recruitment in field
conditions [37], and development of mating displays and breeding
parameters in captivity [141]. Heritable variation is a fundamental
requirement for evolution by natural selection, and despite HPA function has
a genetic component [123] we have currently a very a poor understanding of
the diversity of stress-responses among individuals of the same species, not
to say the strength of repeatability across life-history stages. Such limitation
is in part a consequence of the difficulty of maintaining long-term
monitoring and sampling programs of the same individuals under natural
settings. In this respect, the recent validation of novel method for the
determination of corticosteroid levels in feather samples [173] provides a
useful, non-invasive tool that will allow further research on this topic.
Among the numerous advantages, this method will reduce sampling effort
avoiding the undesirable effects associated to wildlife manipulation that may
jeopardize the interpretation of results [174].
Finally, it is important to highlight that vast majority of studies assume a
direct association between the concentration of total corticosterone levels
and the behavioural and physiological responses, disregarding the
biochemical mechanisms of actions that take in the plasma and target tissues
(Figure 9). Circulating glucocorticosteroids are associated in a reversible
manner with transport proteins (corticosterone-binding globulins, CBG), but
only free steroids can enter their target cells [175]. Currently, most assays are
Stress responses in developing birds
121
Figure 9. Mechanisms of action of circulating corticosteroids. Schematic
representation of the adrenocortical response to stress and the mechanisms of action of
circulating glucocorticoids in birds. In the upper part of the figure (A), upon perception
of external or internal signals (e.g. perturbations) by the brain cortex, a number of
hormones are released in a sequential pattern involving the hypothalamous and the
anterior pituitary (see inserted legend). The adrenals, in turn, will respond releasing
corticosterone into circulation, which triggers the behavioural and physiological
responses that allow vertebrates to cope with the stressors and maintain homeostasis. A
negative feedback loop shuts off the HPA pathway leading to corticosterone release. A
vast majority of studies assume a direct association between the concentration of total
plasma corticosterone levels and the behavioural and physiological responses,
disregarding the biochemical mechanisms of actions that take place in circulation and at
the cell level (empty white box). However (B), blood-borne carrying proteins
(corticosterone-binding globulins, CBG) bind to circulating corticosterone, changing
the amount of the active steroid fraction (i.e. free corticosterone) that enters the cell.
Within the cell, the biological actions of corticosterone depend on the amount of
receptors that allow DNA transcription and further translation into proteins. It will be
interesting to see if the consideration of binding globulins and receptor numbers (in
addition to total corticosterone levels) changes our current understanding of the
response to stress in developing birds.
122
Julio Blas & Raquel Baos
performed after extraction of plasma steroids with organic solvents, which
release the steroids from their carriers and thus measure the sum of free
(active) and bound (inactive) hormones. In addition, when free
corticosterone enters the target cells, steroid action involves the binding of
the hormone to its receptor. The hormone-receptor complex constitutes a
transcription factor that binds to specific sites in the DNA allowing
transcription of specific genes and subsequent translation into the proteins
that will ultimately exert the specific physiological and behavioural effects.
It will be interesting to see if the consideration of binding globulins and
receptor numbers, in addition to total corticosterone levels, [141], [176][178] changes our current understanding of the response to stress in
developing birds.
Acknowledgements
We are thankful to the coauthors, field and laboratory assistants that
contributed to the original research articles carried out in the Spanish
population of white storks. Fernando Hiraldo, Gary R. Bortolotti, Roger
Jovani, José Luis Tella, Álvaro Ramos and two anonymous referees improved
earlier versions of this chapter. J.A. Sencianes made the bird drawings for the
figures. Funding was provided by CSIC-Fondo Social Europeo in an i3P
contract to JB and a F.P.U grant from the Spanish Ministry of Education and
Science to RB.
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