Effects of elevated egg corticosterone levels on behavior, growth, and immunity of yellow-legged...

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Hormones and Behavior

Effects of elevated egg corticosterone levels on behavior, growth,

and immunity of yellow-legged gull (Larus michahellis) chicks

Diego Rubolinia,T, Maria Romanob, Giuseppe Boncoragliob, Raffaella Paola Ferrarib,

Roberta Martinellib, Paolo Galeottia, Mauro Fasolaa, Nicola Sainob

aDipartimento di Biologia Animale, Universita degli Studi di Pavia, p.zza Botta 9, I-27100 Pavia, ItalybDipartimento di Biologia, Universita degli Studi di Milano, via Celoria 26, I-20133 Milano, Italy

Received 25 July 2004; revised 28 November 2004; accepted 5 January 2005

Available online 7 March 2005

Abstract

Eggs of vertebrates contain steroid hormones of maternal origin that may influence offspring performance. Recently, it has been shown

that glucocorticoids, which are the main hormones mediating the stress response in vertebrates, are transmitted from the mother to the egg in

birds. In addition, mothers with experimentally elevated corticosterone levels lay eggs with larger concentrations of the hormone, which

produce slow growing offspring with high activity of the hypothalamo–adrenal axis under acute stress. However, the effects and function of

transfer of maternal corticosterone to the eggs are largely unknown. In the present study, we injected corticosterone in freshly laid eggs of

yellow-legged gulls (Larus michahellis), thus increasing the concentration of the hormone within its natural range of variation, and analyzed

the effect of manipulation on behavioral, morphological, and immune traits of the offspring in the wild. Eggs injected with corticosterone had

similar hatching success to controls, but hatched later. Mass loss during incubation was greater for corticosterone-treated eggs, except for the

last laid ones. Corticosterone injection reduced rate and loudness of late embryonic vocalizations and the intensity of chick begging display.

Tonic immobility response, reflecting innate fearfulness, was unaffected by hormone treatment. Elevated egg corticosterone concentrations

depressed T-cell-mediated immunity but had no detectable effects on humoral immune response to a novel antigen, viability at day 10, or

growth. Present results suggest that egg corticosterone can affect the behavior and immunity of offspring in birds and disclose a mechanism

mediating early maternal effects whereby stress experienced by females may negatively translate to offspring phenotypic quality.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Begging behavior; Corticosterone; Early maternal effects; Egg quality; Embryonic vocalizations; Growth; Immunity; Incubation; Larus michahellis;

Tonic immobility

Introduction

Avian eggs contain maternal steroid hormones that can

influence development and have long-term effects on

offspring phenotype (e.g., Andersson et al., 2004; Eising

and Groothuis, 2003; Eising et al., 2001; Lipar and

Ketterson, 2000; Schwabl, 1993, 1996; Sockman and

Schwabl, 2000). Mothers adopt complex strategies of

allocation of hormones to the eggs in relation to extrinsic

factors (e.g., paternal quality), offspring sex, or laying order

0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.yhbeh.2005.01.006

T Corresponding author. Fax: +39 0382986290.

E-mail address: [email protected] (D. Rubolini).

of the egg (Eising et al., 2003; Gil et al., 1999; Muller et al.,

2002; Petrie et al., 2001; see review in Gil, 2003). The eggs

of three bird species (domestic hen, Gallus gallus, Japanese

quail, Coturnix coturnix japonica, and barn swallow,

Hirundo rustica), are known to contain a maternal gluco-

corticoid hormone, corticosterone (Downing and Bryden,

2002; Eriksen et al., 2003; Hayward andWingfield, 2004; N.

Saino et al., unpublished data). However, the consequences

and function of the transmission of variable amounts of

corticosterone to the eggs remain largely unknown.

Glucocorticoids are produced and secreted by the adrenal

glands under stimulation of the hypothalamo–pituitary–

adrenocortical (HPA) axis, and their plasma concentration

increases under stressful conditions (the adrenocortical

47 (2005) 592–605

D. Rubolini et al. / Hormones and Behavior 47 (2005) 592–605 593

stress response; Sapolsky, 1992; Wingfield, 1994; Wingfield

and Romero, 2001). In vertebrates, glucocorticoids (mainly

corticosterone in birds) have diverse effects on an array of

physiological and behavioral functions, including metabo-

lism, immunity, activity levels, cognitive and learning

processes, reproductive behavior, and parent–offspring

communication (see reviews in Apanius, 1998; Munck et

al., 1994; Romero, 2004; Sapolsky et al., 2000; von Holst,

1998; Wingfield and Ramenofsky, 1997, 1999; Wingfield et

al., 1998a,b). The effects of increased glucocorticoid

following the activation of the HPA axis on physiology

and behavior have been extensively studied in adult birds.

Glucocorticoids have been shown to regulate protein

metabolism during fasting and migration (e.g., Cherel et

al., 1988; Jenni et al., 2000). During the breeding season,

circulating levels of corticosterone increase following

adverse or otherwise unfavorable environmental conditions

(dlabile perturbations factorsT, sensu Wingfield, 2003;

Wingfield and Kitaysky, 2002), inducing facultative dis-

persal, nest abandonment, and facilitating foraging behavior,

thus suppressing parental activities in favor of self-main-

tenance under emergency situations (Astheimer et al., 1992;

Silverin, 1998; Wingfield, 1994; Wingfield and Ramenof-

sky, 1997; Wingfield et al., 1998a).

Few studies have analyzed the causes and consequences

of variation in glucocorticoids among young birds. Gluco-

corticoids have been suggested to promote chick nest

departure, post-hatching dispersal and trigger migratory

restlessness (Belthoff and Dufty, 1998; Heath, 1997;

Lohmus et al., 2003; Love et al., 2003a; Sockman and

Schwabl, 2001). Corticosterone levels were found to be

higher among first-hatched nestlings compared to later-

hatched ones (Love et al., 2003b), and in nestlings from

large broods and after food deprivation (Kitaysky et al.,

1999, 2001a; Saino et al., 2003a). Furthermore, elevated

corticosterone levels increase the intensity of chick food

solicitation displays directed to their parents, therefore

enhancing food provisioning by parents and the ability of

the chick to cope with adverse conditions (Kitaysky et al.,

1999, 2001b; Love et al., 2003b).

However, the adaptivity of the activation of the HPA axis

as a response to unpredictable changes in the environment

may be balanced by long-lasting fitness costs under chronic

stress (reviewed in Sapolsky et al., 2000). For example,

among mammals and birds, persistently elevated levels of

glucocorticoids in response to chronic stressors depress

immune system functioning, thus reducing resistance to

parasite attacks and increasing sensitivity to infectious and

autoimmune diseases (Apanius, 1998; Bijlsma and

Loeschcke, 1997; Munck et al., 1994; Raberg et al., 1998;

von Holst, 1998). A transient reduction of immune function

following stress episodes may allow the allocation of

nutrients and energy to other metabolic processes (e.g.,

those involved in neuromuscular activity), which take higher

priority under stressful situations (Apanius, 1998; Raberg et

al., 1998). When the stressor ceases, immunocompetence is

generally restored, resulting in a modest decrease in

immunocompetence over the remaining lifetime of the

individual. If the stressor persists, however, immunocompe-

tence may further decline, depending on the severity of the

stressful stimuli, leading to a generalized weakening of the

individual immune system and an increased disease and

parasite susceptibility (reviewed in Apanius, 1998). In

addition, chronic elevation of stress hormones results in

impaired cognitive and learning abilities in birds and other

vertebrates (Kitaysky et al., 2003; Sapolsky et al., 2000; von

Holst, 1998), which may have negative long-term conse-

quences on fitness (for the relationships between cognition,

learning, and fitness, see Shettleworth, 1998, 2001). How-

ever, a moderate increase in circulating corticosterone levels

has been shown to enhance performance in spatial tasks in a

passerine bird (Pravosudov, 2003; Pravosudov et al., 2003).

There is ample evidence from mammals that stressful

conditions experienced by mothers may alter offspring

phenotype and performance, thereby increasing fitness costs

of the stress response as a result of the trans-generational

negative consequences of elevated levels of stress hormones

during pregnancy (see reviews in DiPietro, 2004; Welberg

and Seckl, 2001). Corticosterone concentration in the eggs

increases under stressful environmental conditions in

poultry (Downing and Bryden, 2002) and the barn swallow

(N. Saino et al., unpublished data). A recent study (Hayward

and Wingfield, 2004) has shown that female Japanese quails

implanted with corticosterone lay eggs with high concen-

trations of the hormone in the yolk, and eggs from

implanted females produce slow-growing chicks with more

intense adrenocortical response to acute stress compared to

controls. Consistent with the Hayward and Wingfield (2004)

study, corticosterone inoculation directly in egg albumen

resulted in reduced growth of nestling barn swallows (N.

Saino et al., unpublished data), suggesting that the effect of

corticosterone implantation of female quails on chick

growth was in fact mediated by the effect of increased

corticosterone concentration in the eggs. Finally, studies of

domestic chickens showed that administration of glucocor-

ticoids to the albumen increased embryonic mortality,

impaired embryonic development, increased developmental

instability of skeletal traits, and reduced hatch weight and

growth (Eriksen et al., 2003; Heiblum et al., 2001; Mashaly,

1991). However, we are aware of no studies where the effect

of egg corticosterone on offspring pre- and post-hatch

behavior and immunity, besides growth, has been inves-

tigated by directly manipulating the concentration of the

hormone in the egg of avian species.

In this study, we examined the effects of the inoculation

of corticosterone in the albumen of yellow-legged gull

(Larus michahellis) eggs, which results in the exposure of

embryos to persistently high levels of the hormone during

development, on diverse aspects of offspring phenotype,

including behavioral traits (prenatal begging vocalizations,

post-hatching food solicitation display, dtonic immobilityTresponse to restraint), morphology (tarsus length, reflecting

D. Rubolini et al. / Hormones and Behavior 47 (2005) 592–605594

body size, and body mass), and acquired immunity (in vivo

T-cell-mediated immune response and humoral response to

a novel antigen). In addition, we investigated the effect of

corticosterone treatment on egg mass variation during

incubation, duration of the incubation period, and hatching

success. The amount of corticosterone injected increased the

concentration of the hormone within the natural range of

variation observed in yellow-legged gull egg albumen from

the same population. Thus, our study is relevant to the

analysis of the effects of physiologically variable albumen

corticosterone concentration on offspring performance. We

manipulated corticosterone concentration in the albumen

rather than yolk because we aimed at simulating the effect of

maternal stress on egg hormonal composition, and because

experimental evidence on poultry suggests that cortico-

sterone concentration in the albumen is affected by stress

experienced by mothers (Downing and Bryden, 2002).

Corticosteroid injection in the albumen was also adopted by

previous studies examining the effects of elevated cortico-

sterone concentration on offspring traits (Eriksen et al.,

2003; Heiblum et al., 2001; Mashaly, 1991).

Methods

Model organism, general procedures, and hormone assay

The yellow-legged gull is a large gull (800–1500 g)

belonging to the herring gull complex (Liebers et al., 2001).

It is a common and widespread species in the Mediterranean

region, where it is locally regarded as a pest and subjected to

population control measures (Bosch et al., 2000; Vidal et al.,

1998). Clutch size ranges between 1 and 3 eggs, weighing

80–100 g each (Cramp, 1998). Eggs are laid at 1- to 3-day

intervals, and egg mass decreases with laying order

(unpublished data). Chicks hatch after 26–30 days of

incubation, are semi-precocial and remain around the nest

for the first 5–10 days of life, after which they become

highly mobile and may wander at considerable distances

from the nest (Cramp, 1998). The hatching process may last

1–3 days, and pipping embryos produce clearly audible

begging vocalizations (embryonic vocalizations; Tinbergen,

1967) as observed in other gulls and terns (Impekoven,

1973; Saino and Fasola, 1996). Hatching is asynchronous

[1.47 (0.09 SE) days computed over 139 broods with 2 or 3

hatched eggs], and first-hatched chicks have a strong

competitive advantage over later-hatched ones, which

generally results in lower survival of low ranking chicks

(e.g., Hillstrom et al., 2000; Parsons, 1975).

Field work was carried out in the Comacchio lagoon

(44820V N–12811V E, NE Italy) during March–June 2002–

2004. In this area, yellow-legged gulls are semicolonial,

with most pairs breeding in colonies settled on small isles or

ditches. More than 1500 pairs can be estimated to breed in

the study area. We visited each colony every 1–2 days to

mark the nests and the eggs according to laying order when

possible. Nests were randomly assigned to either of two

experimental treatments. The eggs of the first group of nests

were injected in the albumen with 15 ng corticosterone

dissolved in 30 Al sterile corn oil (cort-eggs hereafter), whilethe eggs of the second group were injected with oil to serve

as controls (control-eggs hereafter). Corticosterone and oil

solutions were assigned different codes and the correspond-

ence between the actual treatment and the code was

unknown to the experimenters. This procedure was adopted

to minimize the risk that the behavioral, morphological, and

immunological measurements could unadvertedly be

affected by an a priori knowledge of the treatment by the

experimenter. Mean (SD) amount of corticosterone con-

tained in an average unincubated egg albumen (mean

albumen mass = 46 g) was 85.9 ng (12.71) (N = 14). Thus,

the amount of corticosterone injected (i.e., 15 ng) corre-

sponds approximately to 1 SD of the total amount of

corticosterone occurring in the albumen of an average

yellow-legged gull egg, as estimated in a sample of 14

randomly chosen, freshly laid (b1 day) eggs collected in the

same area during spring 2002. Therefore, injection raised

corticosterone concentration to a level within the natural

range of variation in the vast majority of the eggs.

Corticosterone was assayed using ICN Biomedicals

(Costa Mesa, CA) kits after extraction from 200 mg of

albumen in diethylether. Antibody specificity was 100%

(ICN Biomedicals). Cross-reactivity is generally low

(b0.3%) except with desoxycorticosterone (=6.10%; ICN

Biomedicals). Samples were assayed in duplicate. Eggs

were analyzed in the same assay. In this case, intra-assay

coefficient of variation estimated on one sample assayed in

quintuplicate was 14%.

Eggs were injected in the albumen from the acute pole

using a 250-Al Hamilton syringe mounting a 26-gauge

sterile needle after the egg shell around the area of injection

had been accurately disinfected. The needle was inserted by

approximately 1 cm while the egg was kept horizontal. The

hole was then sealed with a minute amount of cyanoacrilate

glue and a small piece of gull egg shell superimposed. All

eggs were inoculated the day when they were found, i.e.,

within the second day after laying. All eggs were weighed

(approximation of 1 g) the day of inoculation and before

hatching, on days 24–27 after laying. Nests were regularly

visited during incubation to record predation or nest

destruction episodes, and around hatching to record begging

vocalizations and assign hatchlings to their original egg and

nest. Newly hatched chicks were assigned to their original

egg by checking which egg was missing from the nest.

When pipping eggs were found, the chick was marked

before hatching by inoculating a small amount of green or

blue food-dye through the egg shell hole, thus allowing

assignment of siblings to their original egg before hatching

(see also Sockman and Schwabl, 2000). All chicks were

marked after hatching with combinations of color bands. In

all nests included in the analyses, laying order of the original

egg was known for all nestlings. Conversely, hatching order


was sometimes unknown when, for example, we did not

know which of two chicks marked while still in the egg

hatched first. However, hatching order closely reflects

laying order as determined using a large sample of eggs in

an analysis where we allowed for tied hatching orders (rs =

0.49, P b 0.0001, N = 405). Owing to this strict association,

the effect of egg laying order and hatching order on chick

phenotype cannot be easily disentangled. Since laying order

was known for all eggs, we used this variable in the analyses

and excluded hatching order.

Embryonic vocalizations

Prenatal vocalizations of pipping embryos were recorded

simultaneously in pairs of eggs from the two experimental

groups from nests located in the same colony, to control for

variation in extrinsic factors (e.g., temperature, Berlin and

Clark, 1998) that could influence embryo behavior. When a

colony was visited, we searched for nests with pipping eggs.

Once a pipping egg was found, we looked for another nest

with a pipping embryo and belonging to the other

experimental group compared to the first egg. Pairs of eggs

were matched according to developmental stage. We then

placed each egg in a round plastic box (diameter 12 cm,

height 7.5 cm) with a 5-cm hole on the top (to allow a

natural airflow), whose bottom was covered with soft foam

rubber (2 cm thick), depressed in the middle to allow egg

placement, and recorded vocalizations for 24 min. During

the recording period, the plastic box was left in the nest or

close to it (depending on nest location) in a shaded position,

while we stood within 15 m from it, thus preventing parents

from incubating or landing close to the apparatus during

recordings. Plastic boxes were equipped with a Sony ECM-

155 super-tiny microphone, placed on the soft foam rubber,

connected to a Sony TCD-D7 DAT recorder. The same

equipment and settings were adopted for all recordings (i.e.,

we kept constant both the recording level and the distance of

the egg from the microphone, which was always placed at

approximately 1 cm from the eggshell hole). Recording

started 2 min after the egg was placed in the recording

apparatus, to allow embryos to restore their normal calling

activity following manipulation. For sonographic analyses,

Fig. 1. Typical sonogram of yellow-legged gull embryonic vocalizations. The sono

maximum frequency; (c) final frequency.

we considered a maximum of 15 vocalizations per embryo,

chosen among the best quality ones (minimal background

noise). We analyzed a mean of 9.84 (1.16 SE, range 1–15)

calls obtained for each embryo, by means of the Avisoft-

SASLab Pro software (Specht, 1999). The best resolution

was achieved by analyzing vocalizations in the 0- to 11-kHz

frequency range (bandwidth: 224 Hz; overlap: 75%;

window: Hamming; frame: 50%; FFT: 256), with a sample

rate of 22,050 samples/s, a frequency resolution 86 Hz and a

time resolution of 64 ms. For all vocalizations, we used the

same analytical settings and a constant input level into the

PC. In the power spectrum, the threshold was set at �20 dB

relative to the maximum amplitude. With this threshold, we

avoided interference of background noise with the signal

while maintaining the threshold as low as possible. Yellow-

legged gull embryonic vocalizations are composed by

sequences of single notes (calls) with a clear harmonic

structure (up to 4 harmonics can be observed in the best

sonograms), emitted at irregular intervals (Fig. 1). Since we

had no cue to predict which sonographic characteristics

were most likely to vary between the experimental treat-

ments, we chose to measure the following three variables

which were likely to unequivocally describe the basic

structure of these embryonic vocalizations (Fig. 1): (a)

duration of the call (call duration) (ms); (b) maximum

frequency of the fundamental harmonic (maximum fre-

quency) (Hz); (c) highest frequency of the end of the

fundamental harmonic (final frequency) (Hz). We also

obtained the maximum amplitude of the fundamental

harmonic of a call (loudness) [dB, expressed as 20 � log

(a/aref), where a is the recorded voltage and aref is the

reference voltage; if a is smaller than aref, then the decibel

value will always be negative, and in this case 0 dB would

correspond to full scale (e.g., 1 V), �6 dB to 0.5 V, �20 dB

to 0.1 V, etc.]. Measurements were averaged for each

embryo for statistical analyses. Finally, for each recording,

we calculated the vocal rate (calls/min).

Behavioral tests

Two behavioral tests were carried out on the day of

hatching (day 0) if chick plumage was completely dry, and,

graphic measurements taken for each call are reported: (a) call duration; (b)


in all cases, within day 2 after hatching. First, we measured

the intensity of begging behavior by presenting each chick

with a dummy, natural-sized yellow-legged gull head. The

head was painted light gray except for the bright yellow bill

and eye, and a red patch on the lower mandible, thus making

it mimic a parental head. The chick was placed in the nest

and allowed to move freely, while the dummy head was

presented laterally on the front of the chick. Newly born gull

chicks show an innate reaction to dummy head models and

readily respond to model presentation by vigorously pecking

at the red patch painted on the lower mandible, which acts as

a releasing stimulus (Tinbergen, 1967; Tinbergen and

Perdeck, 1950). The intensity of the response to a model

head decreases with age, and almost no response is observed

after day 2 of life, when a fear response to approaching

observers prevails (personal observation). The intensity of

the begging display was quantified as the number of distinct

pecks delivered at the red patch during 1-min trials.

Second, we measured tonic immobility (TI) using a

modified version of the protocol outlined in Jones (1989).

TI is a catatonic-like, fear-potentiated state of reduced

responsiveness induced by physical restraint in diverse

animal taxa (Erhard et al., 1999; Gallup, 1977; Maser and

Gallup, 1977). The variable duration of TI is considered as a

measure of innate fearfulness in birds (Jones, 1986, 1989).

Chronic exposure to increased, physiological levels of

corticosterone in adult domestic fowl prolongs TI reactions

(Jones et al., 1988). TI was induced by placing each chick

on its back in a U-shaped container and restraining it for

approximately 15 s. We then measured the time until the

chick righted itself. Trials lasted a maximum of 3 min. If the

chick had not righted itself within the time of the trial, we

scored 180 s.

Morphological and immunological measurements

Within the second day after hatching, we also measured

body mass (approximation of 1 g) and length of both tarsi

(approximation of 0.1 mm). Each nest was then visited

every second or third day. Chicks were located by

accurately inspecting the vegetation surrounding their

original nest. Body mass and length of both tarsi were

measured each time that a chick was found. On average

4.21 (0.09 SE) days after hatching, chicks received a

subcutaneous inoculation of 120 Al of vaccine against the

virus causing the Newcastle disease (NDV) syndrome in

birds (NobivacR Paramyxo, Intervet) (Alexander, 1997). A

small blood sample (approximately 100 Al) was collected

into capillary tubes by puncturing the brachial vein just

before vaccination. The intensity of antibody response was

measured by collecting a second blood sample on average

15.3 (0.10 SE) days post-vaccination. Blood samples were

kept cool until plasma was separated from red blood cells

(within a few hours) by centrifugation (10 min at 11,000

rpm) and stored at �208C for subsequent laboratory

analyses. Since antibody titer could vary as a consequence

of variable amount of time elapsed since vaccination, we

included this potentially confounding effect as a covariate

in analyses of variance (see also Statistical analyses

section). Concentration of anti-NDVantibodies was assayed

by monoclonal antibody-blocking ELISA using commercial

kits (SvanovirR NDV-Ab, SVANOVA Biotech, Uppsala,

Sweden) (Czifra et al., 1996). Optical density (OD) values

of test plasma were compared with OD values of the kit

negative control. Percent inhibition (PI) was expressed as

OD(negative control) � OD(sample)) / OD(negative control). Large PI

values indicate large NDV-specific antibody concentration.

Vaccination elicited a specific antibody response, as PI

values post-vaccination were larger than those recorded

before vaccination [mean change in PI = 19.76 (2.09 SE),

t test for paired data: t = 9.45, df = 151, P b 0.0001].

On average, on day 8.4 (0.12 SE) after hatching, we also

started a cutaneous test to measure T-cell-mediated immune

response by injecting 0.2 mg phytohemagglutinin (PHA)

dissolved in 0.05 ml phosphate buffered saline (PBS) in the

right wing web and the same amount of PBS in the left wing

web. The thickness of both wing webs at inoculation sites

was measured prior to inoculation and 24 h later using a

pressure sensitive micrometer. The difference in change in

thickness between the right and the left wing webs

(expressed in mm � 100) was used as an index of T-cell-

mediated immune response, according to a standard

procedure (Lochmiller et al., 1993; Saino et al., 1997; Tella

et al., 2002).

Statistical analyses

Our experimental design had a hierarchical structure in

that all eggs in a clutch were assigned to either of two

treatments, i.e., sham-inoculation (control-eggs generating

control-chicks), or injection with corticosterone (cort-eggs

generating cort-chicks). Thus, each level of factor dbroodTwas hierarchically included in a single level of factor

dtreatmentT (Sokal and Rohlf, 1995). In the analyses of

variance where we tested for an effect of egg treatment on

mass variation of individual eggs during incubation,

morphology, immunity, or behavior of individual chicks,

we included a two-level factor accounting for egg treatment

and a factor dbroodT, whose effect was nested within

dtreatmentT [indicated as brood(treatment)]. The effect of

treatment was therefore tested against the error term of

brood(treatment) while the effect of brood(treatment) was

tested against the residual error. In addition, in analyses of

body mass and tarsus length (mean of right and left

measures), that were measured at multiple ages on the same

chicks, we also included a factor dindividualT nested within

brood and treatment, while including first- and second-order

polynomial terms of age at measurement as covariates.

Where appropriate, in nested analyses of variance models,

we also included egg laying order as a factor, and date at

measurement and/or, depending on the specific analysis,

phenotypic measures as covariates, together with their two-

Table 1

Stepdown general linear model with a hierarchical design of egg mass loss

during incubation and duration of incubation in relation to egg treatment

and other covariates

MSS F df P

Egg mass lossa

Brood (treatment) 2.691 � 10�3 2.75 196 b0.0001

Treatmentb 4.695 � 10�4 0.17 1 0.68

Laying order 3.421 � 10�3 3.49 2 0.032

Laying order � treatment 4.929 � 10�3 5.03 2 0.007

Error 9.791 � 10�4 205

Duration of incubationc

Brood (treatment) 4.191 6.76 197 b0.0001

Treatmentsb,d 69.78 16.65 1 b0.0001

Laying order 0.367 0.59 2 0.55

Laying date 221.06 356.43 1 b0.0001

Egg masse 3.526 5.68 1 0.018

Laying order � laying date 2.730 4.40 2 0.014

Error 0.620 195

The analyses are based on 407 (198 clutches) and 400 eggs (198 clutches),

respectively (see also Fig. 2). MSS = mean sum of squares.a The non-significant effects of laying date and other two-way interactions

were removed from the model.b This effect is tested against the error term of brood (treatment).c The non-significant effects of other two-way interactions were removed

from the model.d Least-squares means (days) for control-eggs = 26.46 (95% CL 26.34–

26.59); cort-eggs = 27.51 (95% CL 27.36–27.66).e Coefficient = �0.035 (0.015 SE).


way interactions. We adopted a model simplification

procedure following Crawley (1993). To obtain parsimo-

nious models, non-significant effects (P N 0.05) were

removed using a stepdown procedure, where at each step the

term with the smallest contribution to the model (largest P

value) was excluded until only significant effects were

retained, together with the effect of egg treatment, which

was always maintained in the final model. Interactions were

removed before main effects, and main effects were

removed only if they were not included in a significant

interaction (Crawley, 1993). The effect of treatment on

hatchability (proportion of laid eggs that hatched) was

analyzed in regression analyses with a logit link function

where egg treatment and day when laying started in a

particular nest were the predictor variables. For brevity,

statistics for several non-significant tests from analyses of

variance are not reported in detail. Further information on

independent variables included in each model is provided

throughout the Results section and table captions/footnotes.

Analyses were run using SAS 8.2 (SAS Institute, 1999).

Unless stated otherwise, standard error values for means and

parameter estimates are given in parentheses.

Ethical note

The study was done under license of the local admin-

istration authorities. Our experimental protocol included

widely adopted, standard procedures in avian field studies,

which should not severely alter chick performance. In the

course of the study, we could not detect any chick mortality

which could be directly associated with the experimental

procedures, although these may have caused a reduction of

hatching success by approximately 23%, as suggested by

comparison with uninjected eggs from 115 nests in other

colonies visited in 2003 and 2004, where mean within-

brood proportion of eggs that hatched was 78% (see

Results). To minimize disturbance, colonies were not visited

under inclement weather conditions and around mid-day in

sunny weather, when temperatures could be too high to

sustain for young chicks or pipping eggs left unattended by

parents. In addition, our experiments did not affect the

population of other bird species nesting in the study area,

since gulleries were monospecific and did not host

significant numbers of other waterbirds.

Results

Egg mass loss, duration of incubation, and hatchability

The analysis of egg mass loss during incubation revealed

a differential effect of hormone treatment in relation to laying

order (Table 1). In fact, among first and second eggs, mass

loss was more pronounced if the egg had been injected with

corticosterone than subjected to sham-injection, while the

reverse was true among third eggs (Fig. 2). Other results

remained qualitatively unaltered when the interaction

between laying order and treatment was removed. Eggs that

failed to hatch were excluded from the analysis because mass

loss could have been affected by early embryo mortality.

Duration of incubation (expressed as number of days

elapsed between laying date and hatching date) differed

between treatments, cort-eggs hatching later compared to

control ones [mean for control-eggs: 26.82 days (0.13), N =

206; cort-eggs: 27.09 days (0.14), N = 194; Table 1]. In

addition, larger eggs hatched earlier and eggs from late

clutches had shorter incubation than those from early ones

(Table 1). However, the decline of duration of incubation

with laying date was maximal for first eggs and minimal for

third eggs (data not shown) (Table 1).

In the colonies we studied, egg failure occurred for

diverse reasons, including egg infertility or early embryo

mortality (as shown by the inspection of the content of

unhatched eggs still present in the nests after the end of

incubation), mortality of embryos at intermediate stages of

development, predation by rats (Rattus sp.) and yellow-

legged gulls, nest flooding, adult mortality (personal

observation), and experimental manipulation (see Methods).

The specific cause of experimental egg failure could not be

univocally identified in the majority of the cases. We

therefore based the analysis of the effect of egg treatment on

hatching success on the entire set of experimental eggs,

irrespective of the cause (when identified) of failure. A

logistic regression analysis of individual eggs showed that

treatment did not predict the chances than an egg hatched

Fig. 3. Hatching success of eggs in the two experimental groups expressed

as the overall proportion of eggs that hatched or, respectively, as mean

proportion (mSE) of eggs that hatched within each clutch. Number of eggs

and clutches is given.

Fig. 2. Mean (mSE) mass variation, expressed as j (mass at day 24–27 after

laying � mass at laying) / mass at laying j, for control- and cort-eggs.

Number of eggs is given. Cort-eggs belonged to 100 clutches, control-eggs

to 98 clutches.


(Table 2; Fig. 3). In addition, egg mass recorded at laying

positively predicted egg hatchability, although this effect

was more pronounced in early compared to late laid eggs, as

shown by the significant egg mass � laying date interaction

(Table 2). A logistic regression on proportion of eggs

hatched in each clutch confirmed that egg treatment had no

effect and showed that hatching success declined during the

season (Table 2).

Embryonic vocalizations, begging, and behavioral stress

response

Pre-hatching begging vocalizations were recorded in 13

pairs of eggs, each formed by a cort-egg and, respectively,

a control-egg at the same developmental stage. Two out of

Table 2

Stepdown logistic regression of (a) egg hatching success in relation to egg

treatment, egg mass at laying, laying order and laying date for 743 eggs; in

this analysis each egg was considered as an independent observation; (b)

proportion of eggs hatched in a clutch in relation to treatment and date of

laying of the first egg in the clutch (N =272 clutches, see Fig. 3)

df Estimate (SE) Wald v2 P

(a) Individual eggsa

Intercept 1 �3.924 (1.379) 8.10 0.004

Treatment

Control 1 0.020 (0.075) 0.07 0.79

Laying date 1 0.241 (0.131) 3.43 0.06

Egg mass 1 0.051 (0.016) 10.42 0.001

Laying date � egg mass 1 �0.003 (0.002) 4.15 0.04

(b) Clutchesb

Intercept 1 0.540 (0.112) 23.07 b0.001

Treatment

Control 1 0.024 (0.075) 0.10 0.75

First egg laying date 1 �0.032 (0.012) 6.97 0.008

a The non-significant effects of other two-way interactions were removed

from the model.b The non-significant effect of treatment � first egg laying date was

removed from model.

three sonographic variables (maximum frequency and final

frequency), as well as loudness of the call and calling rate,

showed significant differences between the two groups

(Table 3). Embryos from control-eggs produced louder and

more frequent calls with higher frequency values (Table 3).

Vocalization variables were also subjected to a principal

component analysis to reduce their dimensionality. The

first principal component accounted for 45% of the

variance in the data and was positively and significantly

correlated (P b 0.002 in all cases) with all vocalization

variables except syllable duration. A paired t test on PC1

scores confirmed a significant difference between the two

groups (t = 3.38, df = 12, P = 0.005), embryos from

control-eggs showing higher scores than those from cort-

eggs.

The effect of egg treatment on begging rate varied with

chick age at the begging response test (0–2 days) (Table 4).

However, begging rate was greater among control- com-

pared to cort-chicks at all test ages (Fig. 4): in fact, when

the interaction term between age and treatment was

removed from the model, a significant effect of treatment

on begging rate emerged (F1,193 = 8.28, P = 0.005).

Finally, the TI index was not affected by egg treatment

Table 3

Mean (SE) values of three sonographic features of pre-hatching embryo

vocalizations, loudness, and calling rate of 13 pairs of embryos in control or

corticosterone injected eggs recorded simultaneously in the same colony

Control-eggs Cort-eggs t12 P

Call duration (ms) 0.173 (0.016) 0.156 (0.014) 0.75 0.47

Maximum frequency

(Hz)

2980 (164) 2484 (186) 2.30 0.040

Final frequency (Hz) 1262 (70) 1062 (44) 2.33 0.038

Loudness (dB) �11.7 (2.2) �19.5 (2.7) 2.99 0.011

Vocal rate

(events/min)

8.84 (3.39) 2.08 (1.22) 2.20 0.048

The significance of the differences at paired t tests is shown.

Fig. 4. Mean (mSE) begging rate of chicks from control- and cort-eggs. The

number of chicks is reported. Control-chicks belonged to 100 broods while


(Table 4). Results remained qualitatively unaltered whether

chicks scoring 180 s (N = 16) were excluded from analyses

(details not shown).

Growth, immunity, and viability

Egg treatment had no effect on body mass or tarsus

length recorded at different ages, up to a maximum 23

days after hatching (Table 4). Tarsus length linearly

increased within the range of ages considered while body

mass variation was not linear, as demonstrated by the

highly significant contribution of the squared term of age

to the stepdown model (Table 4; Fig. 5). An inspection of

body mass data in relation to age shows that within the

range of ages we considered, daily increase in body mass

increased with chick age (Fig. 5). This justifies the

Table 4

Stepdown general linear model with a hierarchical design of begging rate,

tonic immobility (TI) response to physical restraint, body mass, tarsus

length, T-cell proliferative response to PHA injection, and humoral

response to NDV vaccine in relation to egg treatment and other covariates

MSS F df P

Begging ratea


Treatmentb 555.5 1.51 1 0.21

Age 1566.9 8.34 1 0.004

Treatment � age 1532.1 8.15 1 0.005

Error 187.9 182

Tonic immobilityc

Brood (treatment) 2101.9 1.58 190 0.001

Treatmentb,d 3074.5 1.46 1 0.23

Error 1330.3 178

Body masse

Individual (brood,

treatment)

4373.6 2.96 196 b0.0001

Brood (treatment)f 6496.8 1.49 198 b0.0001

Treatmentb 80.3 0.01 1 0.91

Age 387,362.4 262.53 1 b0.0001

Age2 797,611.9 540.57 1 b0.0001

Error 1475.5 1116

Tarsus lengthg

Individual (brood,

treatment)

2197.0 5.40 195 b0.0001


Treatmentb 319.8 0.10 1 0.75

Age 12,959,836.6 31,874.3 1 b0.0001

Error 406.6 1114

PHA-responseh


Treatmentb,i 21,569.9 7.66 1 0.0065

Error 1148.6 90

NDV-responsej

Brood (treatment) 426.4 0.71 97 0.93

Treatmentb 629.3 1.48 1 0.23

Error 600.6 54

chicks from cort-eggs belonged to 94 broods.

exclusion of higher-order polynomial terms from the

models.

Chicks hatched from cort-eggs had reduced swelling

response to PHA injection, reflecting T-cell-mediated

immune response, while humoral response to NDV vaccine

was unaffected by egg treatment (Table 4; Fig. 6).

The within-brood proportion of hatchlings that were still

alive at day 10 did not differ between treatments (Wald v2 =0.60, df = 1, P = 0.44), suggesting that egg treatment did not

Notes to Table 4:

The analyses of begging rate and TI response were based on 379 chicks

(194 broods) and 369 chicks (191 broods), respectively. Body mass and

tarsus length of individual chicks were recorded repeatedly during the first

3 weeks of life and a factor dindividualT (nested within brood and treatment)

is included in the models to link data from the same chick. The analysis of

body mass is based on 1514 data points (396 chicks from 200 broods), that

on tarsus length is based on 1510 data points (395 chicks from 200 broods),

including repeated measures from the same chick. The analyses of PHA-

response and NDV-response were based on 209 chicks (119 broods) and

153 chicks (99 broods), respectively. See also Figs. 4–6 for the size of the

samples (chicks and broods) according to experimental groups. MSS =

mean sum of squares.a The non-significant effects of laying order and its two-way interactions

were removed from the model.b This effect is tested against the error term of brood (treatment).c The non-significant effects of laying order, age at measurement (0–2

days) and their two-way interactions were removed from the model.d Mean (SE) for: control-chicks = 18.15 s (2.69), N = 196 chicks in 100

broods; cort-chicks = 21.13 s (3.47), N = 173 chicks in 91 broods.e The non-significant effects of two-way interactions were removed from

the model.f This effect is tested against the error term of individual (brood, treatment).g The non-significant effects of age2 and two-way interactions were

removed from the model.h The non-significant effects of laying date, egg laying order, age at PHA

test, date at PHA test, and their two-way interactions were removed from

the model.i Least-squares means (mm � 100) for control-eggs = 123.67 (95% CL

116.72–130.61); cort-eggs = 101.54 (95% CL 94.14–108.95).j The non-significant effects of laying date, egg laying order, interval

between immunization and sampling, and two-way interactions were

removed from the model.

Fig. 5. Body mass (a) and tarsus length (b) (mean m SE) of chicks from

control- or cort-eggs in relation to age. Data are pooled for 3-day periods.

The number of measurements taken in each period is given. The size of the

samples of chicks and broods were as follows. Body mass: control-chicks:

205 in 103 broods; cort-chicks: 191 in 97 broods. Tarsus length: control-

chicks: 204 in 103 broods; cort-chicks: 191 in 97 broods.

Fig. 6. T-cell-mediated immune response, measured as the swelling

response to injection of phytohemagglutinin, and humoral response to

vaccination against the Newcastle disease virus, of control- and cort-chicks.

Bars are mean m SE. Control-chicks tested for PHA response were from 63

broods while cort-chicks were from 56 broods. Control-chicks tested for

antibody response were from 54 broods while cort-chicks were from 45

broods.


influence short-term chick viability. The non-significant

effects of laying date and its interaction with treatment were

excluded from the model.

Did perinatal behavior predict chick growth and immunity?

We entered begging rate and TI response recorded

soon after hatching as covariates in analyses of variance

of morphological variables recorded at each of the two

successive visits following behavioral tests, and immune

variables. In these models, we also entered the terms

brood(treatment), treatment, laying order, and age at

measurement together with all two-way interactions.

Body mass and tarsus length [both recorded at an

average age of 5.33 days (0.08) (first visit) and 9.25

days (0.12) (second visit)] were not significantly pre-

dicted by begging rate or TI index, and the same held

true for T-cell-mediated immune response (details of the

analyses not shown; P always N0.05). However, begging

rate positively predicted humoral response to NDV

vaccine [F1,51 = 4.34, P = 0.042, coefficient = 0.639

(0.307)].

Discussion

Our study showed that an experimental increase in

corticosterone concentration in the albumen has consequen-

ces on diverse phenotypic traits of embryos and chicks of

the yellow-legged gull. Eggs injected with corticosterone

took significantly longer to hatch and had larger reduction

of mass during incubation compared to control eggs.

Embryos in cort-eggs had a lower vocal rate and loudness

of begging calls, and produced relatively highly pitched

vocalizations compared to control embryos. We found no

effect of corticosterone treatment on chick growth and

humoral immune response to a novel antigenic challenge.

However, an index of T-cell-mediated immune response was

significantly reduced in cort- compared to control-chicks. In

addition, the effects of egg corticosterone manipulation had

complex interactions with other chick phenotypic traits

including laying order of their original egg and age. In fact,

the effect of corticosterone injection on mass reduction

during incubation varied according to laying order of the

egg while begging rate declined more with age in cort- than

in control-chicks. Thus, increased corticosterone concen-

tration in egg albumen influenced embryo physiology and

prenatal behavior by delaying hatching and increasing egg

mass loss (at least in first and second eggs), affected a major

mechanism of chick–parent communication (i.e., begging

display), and impaired a major component of chick acquired

immune system. To the best of our knowledge, this is the

first study where the effect of experimental manipulation of

corticosterone concentration directly in the egg on offspring

behavior and immunity has been analyzed in any bird

species. We injected corticosterone in the albumen because

maternal stress is known to increase the concentration of this

hormone in the albumen (Downing and Bryden, 2002).


However, this hormone is known to occur also in the yolk

(see Hayward and Wingfield, 2004). Since the rate at which

albumen is metabolized differs from the rate at which yolk is

used by the developing embryo (see Romanoff, 1967), the

effects of elevated yolk and albumen concentration on

offspring performance may differ.

The greater egg mass reduction and longer duration of

incubation suggest that corticosterone injection of freshly

laid eggs may have impaired and/or delayed embryo

development: this would be consistent with studies of

poultry and Japanese quail, which showed that adminis-

tration of corticosteroids in the albumen resulted in a

retardation of embryonic development and reduced hatch

weight (Eriksen et al., 2003; Heiblum et al., 2001; Kaltner et

al., 1993; Mashaly, 1991). Alternatively, the consequences

of relatively high corticosterone levels in the albumen on

duration of incubation may have been mediated by

behavioral mechanisms. Late embryonic vocalizations are

a common feature of gull and tern species that start up to 3

days before hatching, and are a major component of prenatal

offspring display, functioning to solicit parental care

provisioning including incubation and thermoregulation

(Berlin and Clark, 1998; Brua et al., 1996; Evans, 1992;

Saino and Fasola, 1996; Tinbergen, 1967). Cort-embryos

vocalized less frequently and loudly than control embryos,

suggesting that offspring solicitation of parental care was

reduced by corticosterone. The pitch of embryonic vocal-

izations was also affected, although the effect of high-

versus low-frequency vocalizations on parent behavior is

completely unknown. Impaired begging display of cort-

embryos may have resulted in reduced incubation or egg

thermoregulation by parents and, ultimately, in delayed

hatching.

Yellow-legged gull chicks solicit parents to regurgitate

food by repeatedly pecking at the red patch on the lower

mandible of their parents’ bill, a begging display already

described by Tinbergen and Perdeck (1950) in the closely

related herring gull (Larus argentatus). Newly hatched cort-

chicks had reduced food-solicitation behavior compared to

controls and begging rate declined with age more among

cort- than control-chicks. The begging display of yellow-

legged gull chicks involves perception of parent bill color

pattern (e.g., Tinbergen, 1967). It could be speculated that

the reduced pecking rate resulted from impaired perceptive

and cognitive abilities soon after hatching following

corticosterone egg treatment (Kitaysky et al., 2003).

However, Kitaysky et al. (2001b) showed that cort-

implanted black-legged kittiwake (Rissa tridactyla) chicks

had higher begging rate than sham-implanted controls. The

difference between the two studies may have arisen because

of the different life stages at which offspring were exposed

to increased corticosterone levels. Kitaysky et al. (2001b)

simulated an increased corticosterone secretion by 15-day-

old chicks, in order to analyze the behavioral response to

food stress episodes during postnatal growth (see also

Kitaysky et al., 1999, 2001a). Our study concerned the

effects of elevated corticosterone concentration in the

unincubated egg simulating increased transfer of maternal

corticosterone, which may affect embryonic development

(Eriksen et al., 2003; Heiblum et al., 2001; Kaltner et al.,

1993; Mashaly, 1991). Thus, reduced begging rates soon

after hatching in chicks originating from cort-treated eggs in

our study may have resulted from an impairment of

cognitive abilities or general performance following expo-

sure to increased corticosterone levels during the pre-

hatching period.

Tonic immobility response within day 2 post-hatching

was not modified by egg corticosterone administration.

Previous studies showed that corticosterone infusion in

adult chicken prolonged the duration of the TI response

(Jones et al., 1988). However, the lack of an effect of

corticosterone on early TI response may be a consequence

of the poor development of this peculiar behavior in the

first days of life, as shown by research on early TI

induction in the precocial domestic fowl chicks (Heiblum

et al., 1998).

Reduced begging behavior in cort-chicks had no

apparent effect on body mass and skeletal size of chicks,

and there was no evidence of differential mortality in

relation to egg treatment at day 10 post-hatching. These

analyses were based on a large sample of chicks monitored

until a maximum of 23 days after hatching and showed no

trend whatsoever for an effect of treatment on chick

morphology. Monitoring a large sample of yellow-legged

gull chicks at later ages would be impractical in our study

area (as well as in most colonies we have visited in the

Mediterranean region) because old chicks tend to leave their

original territory when approached by humans and are thus

more susceptible to aggression by conspecifics. In addition,

predation, mainly by rats, and cannibalism are very

frequent. However, the growth patterns we observed in the

two groups are strongly suggestive of a lack of effect of

hormone treatment on this set of phenotypic traits.

Present results are not consistent with those from a study

of the barn swallow (N. Saino et al., unpublished data)

where corticosterone injection in the albumen depressed

offspring growth and feather development. In the barn

swallow, nestling mortality is very low and offspring growth

seems to be largely controlled by within-brood competition

for food, as demonstrated in experiments where manipu-

lation of broods size by adding or removing one (out of 4–5)

nestlings was sufficient to significantly affect body mass

gain and feather development via an effect on per capita

feeding rate (e.g., Saino et al., 1997, 2002a). Mortality of

yellow-legged gull chicks seems largely to be due to

stochastic factors not related to food availability, such as

predation, and results in only 28% of the eggs producing a

viable 10-day-old chick in the study population, thus

possibly reducing within-brood competition for food. Thus,

the different effects of egg corticosterone on barn swallow

and yellow-legged gull chick growth may be mediated by

the different intensity of sib–sib competition for limiting


resources in the two species, because competition for food

delivered by parents may be greater in swallows than gulls.

In addition, if parents base their decisions on allocation of

food among their offspring on their relative begging rate,

whereas absolute mean begging rate of the chicks does not

affect feeding effort of their parents, a lower begging rate

may not have resulted in a lower amount of per capita food

delivered by parents, because all chicks within a clutch

received the same treatment.

Alternatively, similar growth patterns of cort-chicks

compared to control-chicks may have resulted from

allocation of limiting dietary resources to growth rather

than competing metabolic processes. This is supported by

the significant reduction of chick T-cell-mediated immunity

as a consequence of corticosterone injection in the egg,

which may have allowed a greater allocation of resources to

somatic growth.

A reduction of chick T-cell-mediated immunity following

egg corticosterone treatment is consistent with a large body

of literature showing similar effects in adult vertebrates

(Apanius, 1998; Bijlsma and Loeschcke, 1997; Munck et

al., 1994; Raberg et al., 1998; von Holst, 1998). High

corticosterone levels may have similar effects during avian

embryonic development or the early postnatal life stages by

impairing immune system organs development or matura-

tion of immune cell lines. However, the interpretation of the

mechanisms generating the effects of corticosterone on

chick immunity is compulsorily speculative, owing to the

complete lack of immunological studies of the effects of

glucocorticoids in young oviparous vertebrates. The in vivo

test of T-cell-mediated immunity we adopted reflects the

ability of 30% of T-cell lines to proliferate in response to

mitogenic stimulation, and can be assumed to be a measure

of T-cell-mediated immunity (Klein, 1993; Lochmiller et al.,

1993; Tella et al., 2002). Acquired immunity mediated by T-

cells is among the major mechanisms of anti-parasite

defence in vertebrates (Janeway and Travers, 1997; Pastoret

et al., 1998). The same index of T-cell-mediated immunity

we used has been shown to predict survival in a meta-

analysis of studies of birds (Møller and Saino, 2004). Thus,

corticosterone injection significantly reduced an immune

trait that predicts long-term viability in birds.

As a corollary of the main experiment, significant

relationships emerged between egg size or laying date and

mass loss or hatchability of the eggs. The proportion of eggs

that hatched decreased with initial egg mass while control-

ling for laying date. This is consistent with other studies of

both precocial and altricial species, although the causal

mechanism that produce such association may differ among

species (e.g., Croxall et al., 1992; Galbraith, 1988; Potti and

Merino, 1996; Saino et al., 2004; but see Jager et al., 2000;

Risch and Rohwer, 2000). In addition, late laid eggs had

smaller hatching success after controlling for the concom-

itant effect of egg mass. This pattern may reflect increased

predation during the breeding season or declining parental

quality, influencing for example parental attendance to the

nest and incubation behavior and thus egg hatchability (e.g.,

Burger et al., 1996; Ramos, 2001). Variation in parental

quality may, in turn, simply reflect variation in breeding date

with age and thus experience in parental activities if, as

shown in several species, relatively young individuals tend

to breed later than old ones (e.g., Gonzalez-Solis et al.,

2004; Laaksonen et al., 2002; Ratcliffe et al., 1998).

Interestingly, chick begging rate positively predicted

humoral immune response. Intense begging may result in

large amounts of food provisioning by parents. Several

studies of domestic poultry but also wild bird species have

shown that immunocompetence is dependent on nutritional

conditions (e.g., Chandra and Newberne, 1977; Dietert et

al., 1994; Gershwin et al., 1985; Glick et al., 1981, 1983;

Klasing, 1988; Lochmiller et al., 1993; Saino et al., 1997;

Tsiagbe et al., 1987). An elevated begging rate may thus

enhance immunity via an effect on nutritional conditions

because of differential allocation of extra nutritional

resources to immunity rather than somatic growth, which

was not predicted by begging behavior. An alternative

interpretation is that a large humoral immune response is not

causally linked to chick begging behavior, as these traits

may independently reflect overall chick state.

Present results and those of similar recent studies on

Japanese quails and barn swallows (Hayward and Wing-

field, 2004; N. Saino et al., unpublished data) are relevant to

diverse disciplines, including animal physiology, evolu-

tionary ecology, as well as conservation biology. We

showed that maternal corticosterone is contained in unin-

cubated yellow-legged gull eggs and is thus of maternal

origin. Hence, there is now evidence that the eggs of four

species of birds belonging to three phylogenetically distant

avian orders (i.e., Galliformes, Charadriiformes, Passer-

iformes) contain corticosterone of maternal origin, suggest-

ing that transmission of this hormone to the eggs may be a

common feature in birds.

Early maternal effects mediated by egg quality can have

profound consequences for offspring phenotype and per-

formance (Mousseau and Fox, 1998). Recent literature on

birds has shown that quantitatively minor components of the

eggs, such as androgens, antioxidants, and immunoglobu-

lins, are allocated to the eggs according to complex

strategies and can have important effects on offspring

fitness (Eising et al., 2003; Gil, 2003; Gil et al., 1999;

Saino et al., 2002b,c, 2003b). The function of transfer of

maternal glucocorticoids to the eggs is unknown. Evidence

from the present study indicates that physiologically

increased corticosterone in the albumen has negative

consequences on behavioral traits as well immune system

variables under natural conditions. Maternal glucocorticoids

may have antagonistic effects on different offspring traits

and optimal allocation of maternal corticosterone to the eggs

may reflect a trade-off between their contrasting effects on

fitness-related offspring traits. For example, Kitaysky et al.

(2003) suggested that exposure to elevated corticosterone

levels soon after hatching may promote competitive ability


and thus access to limiting food resources by young

kittiwakes and be detrimental to chicks at later ages in

terms of cognitive and learning abilities (but see Pravosu-

dov, 2003; Pravosudov et al., 2003; see Introduction). The

possibility obviously exists that corticosterone injection in

the eggs enhanced chick phenotypic values of traits that we

did not measure (e.g., dominance rank among siblings,

ability to cope with stressful conditions, or fledging

success). Still, among the diverse set of characters consid-

ered, none appeared to be positively influenced by cortico-

sterone, the effects being either null or negative. Thus, we

found no evidence for a trade-off between antagonistic

effects of egg corticosterone on offspring traits.

Alternatively, mothers may be unable to strategically

allocate corticosteroids to their eggs, and egg cortico-

sterone concentration may simply reflect circulating levels

in mothers’ plasma. This may occur because physiolog-

ical mechanisms of active regulation have not evolved or

are impeded by physiological constraints or energetic

costs.

At a different level, our results are also relevant to the

interpretation of the mechanisms that control population

productivity via maternal effects. Poultry studies have shown

that exposure to stressful conditions during laying results in

increased transfer of corticosterone to the eggs (Downing

and Bryden, 2002). Similarly, exposure to a predator during

egg laying enhances corticosterone concentration in the eggs

of female barn swallows relative to females exposed to a

herbivore (N. Saino et al., unpublished data). These results

are consistent with experiments where increased risk of

predation has been demonstrated to increase corticosterone

plasma levels of breeding birds (Scheuerlein et al., 2001;

Silverin, 1998). Stressful conditions can thus increase

maternal corticosterone concentration in the eggs under

natural conditions. Since diverse stressors may have similar

effects on maternal and consequently egg hormonal profile

(Sapolsky, 1992; Sapolsky et al., 2000; Wingfield, 1994),

diverse kinds of environmental sources of stress experienced

by mothers during breeding may have consequences on

offspring quality via an effect on glucocorticoid concen-

tration in the eggs.

Population productivity and dynamics can thus depend

on the level of stress, including anthropogenic disturbance,

because this affects hormonal egg quality (Hofer and East,

1998). Conservation biology is mainly concerned with

maintenance of viable populations of organisms (Meffe

and Carroll, 1994). In the present study, we have shown

experimentally that elevated egg corticosterone levels

depressed T-cell-mediated immunity and early begging

behavior of yellow-legged gull chicks, although we could

not detect any relevant effects on hatching success,

growth, or short-term viability. Our results thus suggest

that maternal effects mediated by egg quality may be

important in determining the phenotypic composition of

populations and may therefore affect their viability both in

and ex situ.

Acknowledgments

We thank the Parco Regionale del Delta del Po and the

Comune di Comacchio for allowing us access to the study

area, and Dr. R. Sacchi for useful suggestions on sono-

graphic analyses. Prof. F. James Rohlf and Dr. Oliver Kaltz

kindly gave advice on statistical analyses. Two anonymous

referees provided useful comments on the manuscript.

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