Functional Ecology 2008, 22, 854– 863 doi: 10.1111/j.1365-2435.2008.01439.

Carotenoid-induced maternal effects interact with

Blackwell Publishing Ltd

ectoparasite burden and brood size to shape the trade-

off between growth and immunity in nestling great tits
A. Berthouly*, A. Cassier and H. Richner
Evolutionary Ecology Group, Division of Evolutionary Ecology, Zoological Institute, University of Bern, 3012 Bern,
Switzerland

Summary
1. Carotenoids are fat-soluble pigments that stimulate the immune system and can act as antioxidants.
Carotenoids are thus expected to buffer the effects of environmental stressors on health. As carotenoids
are a limited resource, the ability of an individual to use and metabolize carotenoids is assumed to
influence its stress-resistance. Accordingly, it has been found that nestlings hatched from eggs with
increased carotenoid concentration, show an enhanced ability to use carotenoids and a lower
susceptibility of tissues to lipid peroxidation.
2. We tested the prediction that nestling great tits (Parus major), hatched from eggs laid by carotenoid-
supplemented mothers, cope better with a transient stressor encountered after hatching. We
supplemented half of the breeders with carotenoids during egg production (C+), used the other half
as a control (C–), and cross-fostered the eggs between nests after clutch completion. Three days
after hatching, we applied a stressor in two-third of the nests either by increasing brood size, or by
infesting nests with hen fleas (Ceratophyllus gallinae) during five consecutive days. A third group
was kept as a control. We then assessed the responses of C+ and C– nestlings to each stressor by
measuring mass gain, body condition, plumage coloration, humoral immune response and fever
response to a lipopolysaccharide injection.
3. In control nests, C+ and C– nestlings showed similar body condition but C+ nestlings had a
higher increase in body temperature and tended to have a higher wing web swelling in response to
lipopolysaccharide injection. Under stress, however, there were no differences in overall condition
between C+ and C– nestlings. The two stressors led to different responses: when sibling competition
was increased, C– nestlings favoured immune development, whereas C+ nestlings favoured mass
gain and body condition, while under parasite exposure C+ and C– nestlings seemed to invest in
immune development and body growth similarly.
4. Our results support the hypothesis that carotenoid-induced maternal effects provide develop-
mental benefits under natural conditions without additional stressors. Additionally, we show that
the response to sudden environmental changes depends on the environment during the initial
phases of development, which thus shape phenotype and individual variation.
Key-words: fleas, LPS, parasites, sibling competition, resource allocation

& Jungi 2003; Padgett & Glaser 2003; Romero 2004; Bubliy
Functional Ecology (2008) xx, 000–000

Introduction
In the wild, animals face a large range of stressful factors such
as predation, parasitism, food shortage, intra- and inter-
specific competition, and seasonal changes. Stressors induce
physiological responses that can alter health and thereby
reduce fitness (Kitaysky et al. 2001; El-Lethey, Huber-Eicher
*Correspondence author. E-mail: berthoulyanne@hotmail.com
© 2008 The Authors. Journal compilation © 2008 British Ecological Society
& Loeschcke 2005; Love et al . 2005). For example, the
hypothalamic–pituitary–adrenal axis of stressed animals releases
elevated quantities of stress hormones such as glucocorti-
coids (Romero & Wikelski 2001). Subsequent to a period of
stress some individuals can show chronic elevation of these
stress hormones (Kitaysky et al. 1999; Padgett & Glaser 2003;
Saino et al. 2003b). Chronic elevation of corticosterone is
known to suppress the immune system (El-Lethey et al. 2003;

Padgett & Glaser 2003), decrease endogenous lipid reserves
and promote loss of muscle tissue (Kitaysky et al. 1999; Love
et al. 2005). Additionally, under some stressful situations
such as intense cold, increased competition and parasite
infestation, the energy expenditure (Svensson et al. 1998;
Sunardi et al. 2007), and thus the metabolic activity, may
increase (Svensson et al. 1998; Carere & van Oers 2004; Arens
& Cooper 2005). This could in some instances lead to an
elevated production of reactive oxygen species such as free
radicals. Free radicals have deleterious effects on the DNA,
cellular proteins and lipids (Fang, Yang & Wu 2002). Thus,
individuals that are able to mitigate the effects of those
stress-related ‘disorders’ could hypothetically be in better
condition and have higher survival rates.
Different physiological mechanisms have been suggested to
influence stress-resistance: (i) the capacity of individuals to
rapidly and efficiently reduce glucocorticoid production after
stress (El-Lethey et al. 2003; Romero 2004); (ii) the ability to
keep metabolic activity at low rates under stress (Vleck &
Vleck 1979; Hoffmann & Parsons 1989; Tieleman & Williams
2000); and (iii) the antioxidant status of an animal that
influences the degree of oxidative stress (e.g. Finkel & Holbrook
2000; Alonso-Alvarez et al. 2004a; Bertrand et al. 2006a;
Horak et al. 2007). In fact, antioxidants such as carotenoids
can help animals to resist oxidative stress by scavenging free
radicals produced under high metabolic activity (Koutsos
et al. 2003; Alonso-Alvarez et al. 2004a). Carotenoids are not
only antioxidants but can also stimulate the immune system
(Blount et al. 2003b; McGraw & Ardia 2003; Saino et al.
2003a). They might then counteract the suppressive effect of
stress hormones on the immune system as suggested by
Berthouly, Helfenstein & Richner (2007). Carotenoids can
only be obtained via the food and are thus expected to be limited
in nature. A trade-off is thus hypothesized in their use for
their different functions (i.e. detoxification, immunity and
coloration Horak et al. 2000; Tschirren, Fitze & Richner
2003a; Blount et al. 2003b; Hill, Farmer & Beck 2004). Thus,
animals having access to higher quantities of carotenoids, or
animals being able to optimally use carotenoids may be better
able to cope with environmental stressors and thereby enjoy
higher fitness. In other words, the strength of the effect of a
stress on ‘health’ might be influenced by the availability and
the efficiency of antioxidant defences and immunostimulant
molecules that might be enhanced when availability of
carotenoids in the diet increases.
Early-life conditions shape the development of morpho-
logical and physiological traits. In rats, for example, a low pro-
tein diet during pregnancy and lactation causes sex-specific
changes in organ and body mass in offspring (Desai et al. 1995).
In adult pheasants, sexual ornaments reflect nutritional status
during early growth (Ohlsson et al. 2002). The environment
during early life stages can be actively modified by parents
depending on environmental conditions at breeding (e.g. as a
function of food abundance, quality of the resources, predation,
prevalence of parasites, ambient temperature Ojanen 1983;
Verhulst, Vanbalen & Tinbergen 1995; Saino et al. 2004;
Tschirren, Richner & Schwabl 2004; Love et al. 2005; Nooker,
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854– 863
Carotenoid-induced maternal effects 855
Dunn & Whittingham 2005; Berthouly et al. 2007). In birds,
mothers have been found to modify the allocation of
resources in their eggs in relation to habitat characteristics
(Verhulst et al. 1995; Tschirren et al. 2004; Nooker et al.
2005; Berthouly et al. 2007). In the great tit, females exposed
to ectoparasites deposited less testosterone in their eggs
(Tschirren et al. 2004), which has been suggested to reduce
the negative effects of parasites on offspring. Also, carotenoid
availability has been found to influence the breeders’ condi-
tion (Alonso-Alvarez et al. 2004b; Blount et al. 2004) and the
carotenoid contents of eggs (Blount et al. 2002; Biard, Surai
& Møller 2005; Berthouly et al. 2007). When carotenoid
availability to breeders was experimentally enhanced,
mothers increased the quantities of carotenoids deposited
into the egg yolk (Blount et al . 2002; Biard et al. 2005;
Berthouly et al. 2007). Nestlings that hatched from eggs with
higher carotenoid levels showed a better immune response
(Saino et al. 2003a), a more intense colouring of carotenoid-
based traits such as the beak (McGraw, Adkins-Regan &
Parker 2005), and an enhanced ability to incorporate carote-
noids into immune tissues (Koutsos et al. 2003). Carotenoid
richness of neonatal nutrition in zebra finches affects their
capacity to assimilate carotenoids as adults (Blount et al.
2003a), suggesting that different quantities of carotenoids in
yolks could also influence the ability to use carotenoids later
in life. These nestlings may subsequently also be able to better
cope with negative effect of stressors on health by neutralizing
free radicals more efficiently, and most of all by being able to
mount a stronger immune response.
In this study we tested whether nestling great tits hatched
from eggs laid by carotenoid-supplemented mothers cope
better with a transient stressor arising a few days after hatching.
We manipulated carotenoid availability during egg produc-
tion and cross-fostered the eggs to disentangle the effects of
egg quality from the effects of post-hatching environment on
nestling performance. Over a period of 5 days, we induced two
different physiological stressors either by increasing sibling
competition in one group of birds, which has been shown to
impair nestling immunity (Horak et al . 1999) and body
condition (Sanz & Tinbergen 1999), or by infesting nests with
hen fleas (Ceratophyllus gallinae) in another group, which
have been shown to impair nestling growth (Nilsson 2003),
body condition (Richner, Oppliger & Christe 1993; Tschirren,
Fitze & Richner 2003b) and immunity (Berthouly et al.
2007). Another group was kept as a control. Sibling com-
petition was enhanced by adding two nestlings to the nest. We
hypothesized that sibling competition and flea infestation
would (i)increase metabolic activity and thus generate
oxidative stress (Bertrand et al. 2006b; Costantini et al. 2006),
(ii)increase baseline corticosterone levels (Saino et al. 2003b;
Raouf et al. 2006), and (iii) increase energy requirements (Ots
et al. 2001). Such stressors may intensify the trade-offs in the
allocation of limited resources, such as carotenoids, to different
physiological functions, for example, immune development,
detoxification, and plumage development (Horak et al. 2001;
Alonso-Alvarez et al. 2004a). We thus expected nestlings
hatched from eggs laid by carotenoid-supplemented mothers
856 A. Berthouly et al.
to be less impaired by the stressors, and to show higher
immunocompetence and/or higher growth and/or more
intensive plumage colour depending on the type of stressor to
which they were subjected.
Material and methods
FOOD SUPPLEMENTATION
The experiment was carried out in 2006 in a nest-box breeding
population of great tits in the Bremgartenwald, a forest near Bern,
Switzerland. The study area contained 336 nest-boxes distributed
over 24 plots (14 nests per plot). In March, the nest boxes were
cleaned by removing old nest material and thorough brushing to
eliminate all ectoparasites. At the same time we hung an inverted
medium-sized flowerpot near each nest box. We then randomly
assigned one of the two food treatments to each plot: nests supple-
mented with carotenoid-enriched (C+) or with non-enriched food
(C–). We supplemented the birds with commercial bird fatballs
mixed with sunflower seeds that were first crumbled, and heated to
60 °C to denaturate the majority of the carotenoids naturally
present in the mixture (as advised by a representative of Hoffmann-
La Roche). After cooling, we added 0·132 mg of lutein and 0·005 mg
of zeaxanthin both in oil prepared by Roche Vitamins Inc., and
0·028 mg of β-carotene in powder (Roche) per gram to half of the
food mixture. The other half of the mixture was used as control.
These relative quantities of each carotenoid species correspond to
the approximate ratio found in lepidopteran larvae eaten by great
tits, i.e. 80% of lutein, 3% of zeaxanthin, and 17% of β,β-carotene
(Partali et al. 1987). Finally, we moulded the mixture into two types
of fatballs, i.e. with and without the added carotenoids (thereafter
called C+ and C– respectively). The mixtures were finally moulded
into either enriched or non-enriched balls. Each ball weighed c. 40
grams, and C+ and C– fatballs were consumed at similar rates by
great tits and other passerine species (assessed by observation and
weighing). We estimated that the quantities of daily carotenoids
ingested by each bird were about the same they would have ingested
if their diet mainly consisted of lepidopteran caterpillars (de Ayala,
Martinelli & Saino 2006; Isaksson & Andersson 2007), as it is the
case during the nestling period later in the breeding season when
insect abundance is the highest . In order to minimize the influence
of our food treatment on breeding habitat choice, supplementation
started only when the nest box was already occupied (i.e. floor
covered with a layer of c. 2 cm of nesting material). Nests were visited
every fourth day to determine the stage of nest building, to replenish
the food stock if necessary (i.e. replacement of the former fatball
by a new one when at least half of it had been consumed) and to
determine the start of egg-laying. We stopped supplying nests with
food on the first day of full incubation. Onset of full-time incubation
was judged to have started when eggs were warm and uncovered
during three consecutive days.
CROSS-FOSTERING OF EGGS
On the third day of full incubation, whole clutches were cross-
fostered between nests to disentangle the effects of maternally
derived carotenoids from post-hatching environmental effects.
Eggs were exchanged between nests of different or equal food-
supplementation treatments (C– # C–, C– # C+, C+ # C+). From
the first expected day of hatching, nests were inspected daily to
determine hatching date precisely.
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854–863
STRESS INDUCTION
In order to investigate the effect of a period of increased physiological
stress early in life on nestling performance, and the ability of
nestlings hatched from eggs laid by carotenoid-supplemented (C+)
or control mothers (C–) respectively to cope with stress, we applied
a stressor to some nests. We induced two different types of stressors:
we either increased sibling competition by increasing original brood
size by two nestlings, or infested nests with hen fleas. Findings of
several experimental studies carried out on the great tit/flea system
suggest that responses to flea infestation involve physiological as
well as immunological defence (Buechler et al. 2002; Tschirren et al.
2004, 2007). Additionally, cat fleas show higher mortality when
feeding on blood of rabbits immunized with flea antigens (Nisbet &
Huntley 2006), and bird fleas most likely also induce an immune
response in their host. Treatments were applied 4 days post-hatching,
after all eggs had hatched, and ended 9 days post-hatching (see
Appendix S1 in Supplementary Material for more details). In summary
we had three groups of nests: (i) control nests (11 C+ nests with 47
nestlings and 14 C– nests with 62 nestlings) where no additional
stressor was applied; (ii) nests with a temporarily increased brood
size (9 C+ nests with 39 nestlings and 13 C– nests with 55 nestlings);
(iii) nests temporarily infested with hen fleas (11 C+ nests with 41
nestlings and 15 C– nests with 65 nestlings).
NESTLING MEASUREMENTS: BODY MASS, IMMUNITY
ASSESSMENT AND PLUMAGE CHROMA
All new hatchlings were weighed to the nearest 0·1 g and marked by
partially removing tuft feathers from their heads, backs and wings.
We took blood samples from each hatchling for molecular sexing
(Griffiths et al. 1998). Eight days post-hatching, nestlings were
permanently marked with a metal ring. At the same time, they were
weighed and their tarsus and wing length measured. Fifteen days
post-hatching, the same measurements were taken again.
Fifteen days after hatching, we assessed immune performance of
three or four nestlings per brood, the number depending on the size
of the brood. Nestlings were weighed and ranked according to their
body mass. We then selected every second nestling in the mass
hierarchy. Nestlings below 14 g (31 nestlings of 589) were not con-
sidered for the LPS injection because the LPS-induced immune
response could have potentially harmed them. We assessed immune
performance by an injection of lipopolysaccharide (LPS) (Parmentier,
Reilingh & Nieuwland 1998). LPS is a mitogen that induces an
antigen-mediated infiltration and aggregation of inflammatory cells
and T-cells at the site of injection. We injected the wing web of the
nestlings with 0·01 mg of LPS (LPS, Sigma-Aldrich) dissolved in
0·02 mL of phosphate buffered saline. It has been found by Parmentier
et al. (1998) in chicken that the response to LPS is highest 4 h fol-
lowing the injection. Nestling great tits did not show any response
after 4 or 12 h but had the strongest response 24 h later. We measured
the wing web thickness before and 24 h after the injection to the
nearest 0·01 mm with a constant-tension dial micrometer (Mitotuyo,
Type 2046FB-60). A greater swelling reflects a better immune
response (Parmentier et al. 1998). It has already been found that
birds can develop fever in response to LPS injection (Maloney
& Gray 1998). We thus also measured rectal temperature of the
nestlings in randomly chosen nests before and after injection with
a digital thermometer (Omega HH41 from Exacon) fitted with a
temperature probe with a diameter of 0·08 mm. Finally, we measured
the skin temperature of all nestlings (i.e. injected and not injected)
by placing an auricular thermometer (ThermoScan from Braun) on

the skin of the stomach. Four measurements were made for each
nestling and the mean recorded. The fever is a mechanism that helps
animals to fight pathogens. It causes an unbearable environment for
some pathogens and enhances immunological functions (Ostberg &
Repasky 2006). Thus, individuals being able to respond to a bacterial
infection by raising body temperature are assumed to fight pathogens
more efficiently and be less harmed by them. A total of 256 nestlings
from 73 nests were injected with LPS.
Sixteen days post-hatching, we took c. 12 breast feathers on both
the left and right sides of the injected nestlings in order to measure
the carotenoid chroma of their plumage. Nine feathers from the
same side were overlaid and fixed on black felt. We then measured
the chroma of the right and the left plumage of each nestling and
used the mean of these two values as a measure of the yellow coloration
of a nestling. Repeatability within feather measurements was equal
to 0·51 (F(75,76) = 3·085, P < 0·0001) (Lessells & Boag 1987) (see Sup-
plementary Appendix S2).
EGG MASS AND CAROTENOID ANALYSIS OF YOLK
An important assumption is that carotenoid-fed mothers would
deposit higher quantities of carotenoids into theirs eggs than control
mothers. We verified this assumption in our population by collecting
a sample of eggs before incubation. We collected the third egg in the
laying sequence (n = 11), and also in a parallel study on another
topic in the same forest the third and the fifth eggs ( n = 96; per-
formed by F. Helfenstein). These eggs were replaced by dummy eggs
to avoid desertion, and stored in a freezer. As expected, carotenoid-
supplemented mothers deposited significantly higher quantities of
carotenoids into their eggs compared to control-fed mothers (F1,60 =
7·08, P = 0·001; C+ females: 25·79 ± 9·91 μ g g –1 of egg yolk;
C– females: 20·27 ± 9·21 μg g–1 of egg yolk) (see also (Berthouly
et al. 2007). Additionally, we measured individual carotenoids (i.e.
lutein, zeaxanthin and β-carotene) in the yolk. Total and individual
carotenoid concentrations were assessed using High Performance
Liquid Chromatography following the protocol described in (Surai
& Speake 1998; Karadas et al. 2005).
STATISTICAL PROCEDURES
The effect of carotenoid-supplementation on lutein, zeaxanthine,
and β-carotene concentration in the yolk was assessed using mixed
models including the nest as random factor. The carotenoid treatment
and egg rank were included as fixed factors in the model and clutch
size and laying date as covariates. The two-factorial interactions
between factors and covariates were also included.
In all analyses of dependent variables corresponding to a
measurement on nestlings, that is, mass gain between days 3 and
15, tarsus on day 15, body condition on day 15, swelling response to
LPS injection, plumage chroma, body temperature before and 24 h
after LPS injection, we included only nestlings that were immune-
challenged. We used linear mixed models for these analyses. The
following explanatory variables were included in all models: the
food treatment of the genetic mother, the stress treatment applied to
the entire brood (i.e. no stress, increased sibling competition, flea
infestation), the hatching date of first hatched nestlings, the nestling
sex, and the two-factorial interactions. We controlled for the real
age of nestlings by including hatching sequence as a factor. Nest-
lings could either have hatched on the day where the first hatching
occurred in the nest or 1 to 2 days thereafter (i.e. two modalities:
hatched the first day (A) or hatched the second or the third day (B)).
The nest of rearing was included as a random factor to correct for
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854– 863
Carotenoid-induced maternal effects 857
non-independence of siblings (Pinheiro & Bates 2000). For the analysis
of mass gain between days 3 and 15 we used the body mass on day
15 as the dependent variable and included the body mass on day 3
as a covariate. By including previous body weight as a covariate, the
dependent variable represents a measure of mass gain over the given
time period. For the analysis of body condition on day 15, the body
mass on day 15 was taken as the dependent variable and structural
body size (here the tarsus length) included as a covariate into the
analysis. Finally, in the analyses of responses to LPS injection
(swelling response, febrile response) we included the external
temperature on the day of injection.
PARENTAL CONDITION
Body mass was taken as the dependent variable and tarsus length
included as a covariate into the model. We used a linear mixed
model and included the following explanatory variables: the food
treatment during egg-laying, the stress treatment applied to the
entire brood, the date of capture, the sex, and the two-factorial
interactions. The nest of rearing was included as a random factor to
correct for non-independence of the two parents (Pinheiro & Bates
2000).
All the analyses were performed using the software r (R Develop-
ment Core Team 2006) (Maindonald & Braun 2003). We used the
package nlme to perform the mixed model analysis (Pinheiro &
Bates 2000; Pinheiro & Bates 2006). Responses were transformed, if
necessary, in order to meet the assumption of normal distribution of
residuals (i.e. the swelling response was log-transformed). We used
restricted maximum likelihood estimation in all mixed effect
models. Interactions and main factors showing a P-value > 0·1 were
backward eliminated using a stepwise elimination procedure. We
first excluded the interactions and then the main factors. In the
results and the tables, the P-values given for interactions and main
effects that have a P-value < 0·1 were extracted from the model
containing only interactions and main factors with a P-value lower
than 0·1. P-values of main factors with a P-value > 0·1 were derived
from an intermediate model containing all main factors but only the
interactions with a P-values ≤ 0·1. Tests are two-tailed with a sig-
nificance level set to α = 0·05.
Results
EGG YOLK CAROTENOIDS
We found that the concentration of lutein and zeaxantin
was higher in C+ eggs (i.e. laid by carotenoid-supplemented
mothers) than in C– eggs (i.e. laid by control mothers)
(respectively: F1,58 = 9·87, P = 0·0026; F1,58 = 14·62, P =
0·0003) (see Supplementary Table S1). However there
were no differences in β-carotene concentrations between
C+ and C– eggs (F1,58 = 0·36, P = 0·55) (see Supplementary
Table S1).
NESTLING PERFORMANCE
Before the stress was applied, that is, 3 days after hatching,
nestling mass between the two groups assigned to be
experimentally stressed or controls did not differ (F2,67 = 0·95,
P = 0·39). Also, it did not differ between C+ and C– nestlings
(F1,67 = 1·15, P = 0·29).
858 A. Berthouly et al.
Fig. 1. Mean (± SE) mass gain between days 3 and 15 of C+ nestlings
(i.e. hatched from eggs laid by carotenoid-supplemented females)
(points and solid lines in black) and C– nestlings (i.e. hatched from
eggs laid by control females) (triangle and dashed lines in grey),
raised either in nests where brood size was increased, in nests infested
with fleas, or in control nests.
We found a significant interaction between food treatment
of the genetic mother and stress treatment on body mass
gain between days 3 and 15 (F2,66 = 3·96, P = 0·024) (Fig. 1),
indicating that the stress treatments affected C+ and C–
nestlings differently. There were no mass gain differences
between C+ and C– nestlings in nests infested with fleas and
controls (respectively: F1,22 = 1·08, P = 0·31; F1,21 = 1·05 , P =
0·32). In experimentally enlarged broods, however, C+
nestlings gained significantly more mass than C– nestlings
(F1,19 = 7·13; P = 0·015). We also found that nestlings gained
more mass when they were raised by control parents (F1,66 =
4·37, P = 0·040) (see Supplementary Fig. S1). Male nestlings
gained more mass than female nestlings (F1,180 = 112·36, P <
0·0001). Finally, there were no effect of hatching date on body
mass gain over this time period (F1,65 = 0·0007, P = 0·98).
As for body mass gain between days 3 and 15, we found a
significant interaction between food treatment of the genetic
mother and stress treatment on body condition on day 15
(F2,66 = 4·35, P = 0·017). There were no body condition dif-
ferences between C+ and C– nestlings in nests infested with
fleas and controls (respectively: F1,22 = 0·67, P = 0·42;
F1,21 = 1·12, P = 0·3). However, C+ nestlings were in better
condition than C– nestlings in enlarged broods (F1,19 = 6·33,
P = 0·021). Additionally, we found that nestlings were in
better condition when they were raised by control parents
(F1,66 = 4·6, P = 0·035). Males were in better condition than
females (F1,181 = 112·36, P < 0·0001). Finally, there was no
effect of hatching date on body condition on day 15
(F1,65 = 0·001, P = 0·97). Differences in body condition are
due to differences in body mass as tarsus length was neither
influenced by food treatment of the genetic mother nor by the
stress treatment applied to the nestlings (respectively:
F1,65 ≈ 0, P = 0·94 ; F2,65 = 0·2, P = 0·82). Males had a longer
tarsus than females (F1,180 = 168·38, P < 0·0001). Finally, the
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854–863
Fig. 2. Mean (± SE) log values of the swelling response to LPS
injection of C+ nestlings (i.e. hatched from eggs laid by carotenoid-
supplemented females) (points and solid lines in black) and C–
nestlings (i.e. hatched from eggs laid by control females) (triangle and
dashed lines in grey), raised either in nests where brood size was
increased, in nests infested with fleas, or in control nests.
hatching date and the food treatment of the rearing parents
did not influence tarsus length (respectively: F1,65 = 0·19,
P = 0·66; F1,65 = 1·32, P = 0·25).
Again, we found a significant interaction between food
treatment of the genetic mother and stress treatment on the
swelling response to LPS injection (F2,65 = 4·68, P = 0·013)
(Fig. 2). There was no difference in swelling between C+ and
C– nestlings in nests infested with fleas (F1,22 = 1·65, P =
0·21), however when brood size was increased C– nestlings
tended to have a bigger swelling than C + nestlings (F1,19 =
4·07, P = 0·058), whereas C+ nestlings tended to show a
bigger swelling than C– nestlings in control nests (F1,23 = 3·43,
P = 0·076). We also found that stress treatments affected male
and female nestlings differently as shown by the significant
interaction between sex and stress treatment (F2,177 = 4·07,
P = 0·019). In fact, there were no differences between males
and females in flea-infested and control nests (respectively:
F1,63 = 0·005, P = 0·94; F1,57 = 0·07, P = 0·79), however, when
brood size was increased males had a significantly smaller
swelling than females (F1,54 = 7·78, P = 0·0072). We found that
nestlings had a stronger swelling response when raised by
control parents (F1,65 = 9·29, P = 0·0033) (see Supplementary
Fig. S2). Finally there was no effect of hatching date on
swelling response over this time period (F1,65 = 2·978, P = 0·089).
Neither food treatments of genetic parents nor stress
treatment influenced nestling plumage chroma (respectively:
F1,63 = 1·18, P = 0·28, F2,63 = 0·28 , P = 0·75). Also, plumage
chroma did not differ between nestlings of different weight or
between nestlings hatched at different dates (respectively:
F1,162 = 0·41, P = 0·52; F1,66 = 2·90, P = 0·093). However we
found that males were significantly yellower than females
(F1,164 = 6·85, P = 0·0097).
Rectal and skin temperature were significantly correlated
(r = 0·75; permutation test: P = 0·0009). We thus decided to
 Carotenoid-induced maternal effects 859

use skin temperature as the dependent variable because skin

temperature was measured in all the nestlings, can be meas-
ured quickly and is least stressful to the birds. The following
results concern nestling temperature on day 15, before LPS
was injected. Carotenoid-supplementation of genetic parents
did not influence nestling body temperature (F1,49 = 2·70,
P = 0·11). There was also no effect of stress treatment on
nestling temperature (F2,49 = 0·29 , P = 0·75). Finally, nestling
temperature strongly depended on external temperature
(F1,53 = 15·76, P = 0·0002): nestling temperature increased
when environmental temperature increased, indicating that
nestlings do not thermo-regulate perfectly at that age. Not
surprisingly, we then found that overall nestlings showed an
increase in temperature between day 15 and 16, thereby
becoming closer to adult temperature, which is around 41 °C
(personal measurements) (Carere & van Oers 2004). Because
nestlings seemed not to thermo-regulate perfectly at that age,
the finding that nestling temperature increases between day
Fig. 3. Mean (± SE) values of temperature increase between days 15
15 and 16 could be due to the experimentally induced immune and 16 of C+ nestlings (i.e. hatched from eggs laid by carotenoid-
response, but also to the fact that nestlings were older and supplemented females) (points and solid lines in black) and C–
thus had more feathers and maybe a more efficient system of nestlings (i.e. hatched from eggs laid by control females) (triangle and
thermoregulation. To test whether an increase in nestling dashed lines in grey), with and without an immune challenge.
temperature after LPS injection would be at least partially
due to an ‘immune’ response to fight against infection, we
took the temperature of all nestlings, also the ones that were
not immune challenged, and ran a model including the change between males and females, between nestlings of
change in temperature of all nestlings. The dependent var- different weight, and between nestlings hatched at different
iable was the change in temperature, which corresponded to periods of the breeding season (respectively: F1,317 = 1·24,
the difference between the temperature recorded 24 h after P = 0·27; F1,318 = 2·78, P = 0·96; F1,55 = 0·52, P = 0·47). We
injection (i.e. on day 16) and the temperature recorded before also tested whether the change in temperature between the
the injection (i.e. recorded on day 15): nestling temperature on time of injection and the measurement of the swelling had an
day 16 – nestling temperature on day 15. We included the food effect on the swelling response and found no effect (F1,143 =
treatment of the genetic mother, the stress treatment applied 0·43, P = 0·52).
to the entire brood, the hatching date of first hatched nestlings,
the nestling sex, the hatching sequence, the LPS treatment (i.e.
PARENTAL CONDITION
nestlings immune challenged or not), the environmental
temperature on day 15, the change in environmental temper- We found that C+ parents tended to be in better condition
ature between the two measurements and the two-factorials than C– parents (F1,36 = 3·7, P = 0·060). Adult males were in
interactions as explanatory variables. Again, the nest of better condition than females (F1,36 = 25·55, P < 0·0001). Stress
rearing was included as a random factor. We found a significant treatment applied to the brood did not affect parental condition
effect of the interaction between the food treatment of the (F1,36 = 0·23, P = 0·79). Late breeders were not in worse
genetic parents and the LPS treatment on the temperature condition than early ones (F1,35 = 0·95, P = 0·34).
change (F1,318 = 3·97, P = 0·047) (Fig. 3): there were no differ-
ences between C+ and C– (F1,54 = 0·008 , P = 0·93) nestlings
Discussion
that were not immune challenged, whereas C+ nestlings
showed a higher increase in temperature than C– nestlings
NESTLING GROWTH, CONDITION AND IMMUNITY
when they were injected with LPS (F1,55 = 4·84, P = 0·032). In
other words, C+ nestlings injected with LPS had a higher First, our results show that C+ nestlings are in better overall
increase in temperature than non-injected C+ nestlings condition than C– nestlings when raised under conditions
(F1,128 = 6·23, P = 0·014) while there were no differences where no stress was applied. In those nests, C+ and C– nest-
between injected and non-injected C– nestlings (F1,192 = 0·055, lings were in similar body condition, but C+ nestlings tended
P = 0·82). We also found that the change in temperature to have a bigger swelling and showed a higher temperature-
between days 15 and 16 depended on the change in environ- increase in response to LPS injection than C– nestlings. Under
mental temperature between the two measurements (F1,59 = experimentally induced stress, C+ nestlings still responded to
73·18, P < 0·0001). We did not find an effect of our stress LPS injection by increasing their body temperature whereas
treatment on nestling change in temperature (F2,55 = 0·35, C– nestlings did not. However, C+ nestlings did not seem to
P = 0·70). Finally, there were no differences in temperature be in higher overall condition than C– nestlings under induced
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854– 863
860 A. Berthouly et al.
stress regimes. Our results rather suggest that C+ and C–
nestlings responded differently to the stressors by adopting
different resource allocation strategies depending on rearing
conditions. When competition was increased, C– nestlings
seemed to favour the development of immunity, whereas C+
nestlings favoured mass gain and body condition. However,
in flea infested nests, C+ and C– nestlings seemed to invest in
immune development and body growth similarly. By allocating
more resources to mass gain and body condition under high
sibling competition, C+ nestlings may become larger and
heavier more rapidly and then obtain food more easily from
parents than small nestlings (Oddie 2000). Large nestlings
may manifest more vigorous visual behaviours when solicit-
ing food (Rodriguez-Girones, Zuniga & Redondo 2002) and/
or may be better in jockeying for a feeding positions near an
arriving parent (McRae, Weatherhead & Montgomerie 1993;
Ostreiher 2001). This result is in line with previous findings
(Berthouly et al. 2007; Helfenstein et al. 2008) where nest-
lings hatched from eggs laid by mothers supplemented
with carotenoids were more competitive (i.e. begged more
intensely) than nestlings hatched from control eggs. In
flea-infested nests, there were no differences between C+ and
C– nestlings, maybe because the selective pressures of a
parasite exposure that act on the different physiological
functions are stronger. On the one hand, it must be important
to develop an efficient immune system in the presence of fleas
to fight parasite attacks. On the other hand, however, it may
also be important to leave the infested nest environment as
soon as possible in order to reduce the duration of the contact
with the fleas, and thereby their negative effects on health. In
such a situation, it may be optimal to invest in all physiological
functions similarly for any individual.
In summary, we found that, independent of the rear-
ing conditions, C+ nestlings showed a stronger temperature
increase than C– nestlings, suggesting a better defence against
bacterial infestations of C+ nestlings (Long 1996; Ostberg
& Repasky 2006). Moreover, when no additional stress was
applied, C+ nestlings tended to have a stronger swelling response
to LPS injection than C– nestlings. These findings support the
idea of beneficial effects of carotenoid availability during
egg-laying on nestling immunity and thereafter performance
(Saino et al. 2003a). However, we found no evidence that C+
nestlings coped better with the increased sibling competition
or with the flea infestation than C– nestlings. Yet, we rather
found that a short transient stress (i.e. presence of fleas, high
sibling competition) already shapes the development of the
phenotype, depending on the type of stressor and on previous
experience (i.e. environment during embryonic development).
In line with this finding we found that C+ eggs contained
more carotenoids than C– eggs. Also, Blount et al. (2002)
found that carotenoid-supplemented lesser black-backed
gull (Larus fuscus) females produced eggs containing higher
carotenoid but lower Ig concentrations. Together, these results
indicate that carotenoid supplementation affected egg
composition such as carotenoid content, but also egg content
of other metabolites such as immunoglobulins, hormones,
nutrients. It suggest that egg composition can modulate the
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854–863
programming of nestling physiology, and thereby its energetic
metabolism, and produce nestlings of different phenotypes
(Royle et al. 1999; Koutsos et al. 2003; Saino et al. 2003a) that
react differently to environmental changes. Additionally, our
findings give strong evidence that a given maternal effect does
not have an obligate effect on certain nestling traits but rather
interacts with the environment to shape offspring phenotype
and will thus be context-dependent (Marshall & Uller 2007).
It questions the adaptive significance of maternal effects in
unstable and heterogeneous environments, and points to the
importance of long term studies when investigating the influence
of maternal effects on offspring fitness before interpreting
different maternal effects as adaptive, or as beneficial, as
suggested by Marshall et al. (2007).
NESTLING PLUMAGE CHROMA
We did not find that C+ nestlings were more yellow than C–
nestlings, indicating that additional carotenoids into the eggs
did not influence plumage chroma at that age. It does not
necessarily mean that carotenoid-induced maternal effects
did not influence plumage colour, but could suggest that
investing in plumage coloration is not a priority at this devel-
opmental stage, especially when pigments are scarce and may
be used for other important functions for several reasons.
First, it is a period of intense growth and development, and
it might therefore be more important to use carotenoids
as antioxidants and immunostimulants. Second, a role for
plumage coloration in parent–offspring communication inside
the nest is unlikely in the great tit because parents may not be
able to differentiate among plumage colorations in poorly lit
nestling cavities (Tschirren, Fitze & Richner 2005) (Tanner,
M. & Richner H., unpublished). Also nestlings do not have a
completely developed plumage at that age. Interestingly,
under controlled conditions in the laboratory, McGraw et al.
(2005) have found in zebra finches that sons hatched from
eggs laid by carotenoid-supplemented mothers displayed a
more brightly coloured beak as adults, which is sexually more
attractive than a drab break. Additionally, Blount et al.
(2003a) have shown that zebra finches with better nutrition at
early developmental stages maintain a higher capacity to
assimilate dietary antioxidants as adults. Thus, we can not
exclude the possibility that C+ nestlings in our study will
display more intense plumage chroma as adults when sexual
selection may be operating.
PARENTAL INVESTMENT INTO CURRENT
REPRODUCTION
Concerning the effect of carotenoid supplementation on
parental investment and parental condition, we found that
nestlings raised by carotenoid-supplemented parents were in
poorer overall condition than nestlings raised by control
parents (i.e. nestlings raised by carotenoid-supplemented
parents showed a lower mass gain between days 3 and 15, a
lower body condition on day 15, and a lower response to LPS
injection), but carotenoid-supplemented parents tended

themselves to be in better body condition than control par-
ents (P = 0·06) during the second half of the rearing period,
when parents were caught and measured. It suggests that
carotenoid-availability early in the breeding cycle influences
the trade-off for parents between investment in current
reproduction vs. investment in self-maintenance and thus
future reproduction. A strategic decision to invest in current
rather than future reproduction would make sense if caro-
tenoids had a disproportionally stronger effect on future
survival than on current reproductive success.
Conclusion
In conclusion, our results suggest that carotenoid-mediated
maternal effects enhance overall nestling condition when
no additional stressors are applied to the nestlings. Yet, C+
nestlings did not cope better with added stressors. Our results
rather show that the two types of nestlings responded dif-
ferently to increased sibling competition by adopting different
resource allocation strategies. They responded, however, in
the same way to flea infestation by allocating similar amounts
of resources into immunity and growth. This provides
evidence that pre-hatching environment influences the
response to sudden environmental changes due for example
to parasite infections, drops in temperature or food abun-
dance, and supports the hypothesis that maternal effects
are context-dependent. It thus shows how the pre-hatching
environment can shape phenotypic development and
thereby maximize maternal fitness by enhancing phenotypic
variation in offspring (Mousseau & Fox 1998; Marshall &
Uller 2007). Finally, the results suggest that carotenoid
availability during nest construction and egg production can
influence the parental trade-off between current and future
reproduction.
Acknowledgements
Authors thank Stéphanie Bonnaure, and Christelle Bugeaud for field assist-
ance; Verena Saladin and Danielle Bonfils for the work in the laboratory;
Katharina Gallizzi, Fabrice Helfenstein and Marion Tanner for their statistical
help and comments on the manuscript. Authors also thank Filiz Karadas and
Peter Surai for carotenoid analyses, and Doris Gomez for providing the AVICOL
program and giving advices on measurements of plumage colour. Thanks also
to the two anonymous referees for their valuable comments on the manuscript.
Finally, authors thank Roche Vitamins Inc. for providing carotenoids and Erbo
Agro AG for providing fat-balls. This work was financially supported by
the Swiss National Science Foundation (grant 3100A0-102017 to H.R.) and
conducted under a licence provided by the Ethical Committee of the Office of
Agriculture of the canton of Bern, Switzerland.
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Received 24 October 2007; accepted 14 May 2008
Handling Editor: Kevin McGraw
 Carotenoid-induced maternal effects 863

Supplementary material Fig. S2. Nestling swelling response in relation to the food
treatment of the rearing parents.
The following supplementary material is available for this article:

This material is available as part of the online article from:

Appendix S1. Details on stress induction procedure.
http://www.blackwell-synergy.com/doi/full/10.1111/j.1365-
2435.2008.01439.x
Appendix S2. Measurement of nestling plumage coloration.
(This link will take you to the article abstract).

Table S1. Effect of carotenoid-supplementation on yolk

Please note: Blackwell Publishing is not responsible for the
concentrations of lutein, zeaxanthin, and β-carotene.
content or functionality of any supplementary materials
supplied by the authors. Any queries (other than missing
Fig. S1. Nestling mass gain between days 3 and 15 in relation
material) should be directed to the corresponding author for
to the food treatment of the rearing parents.
the article.

© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 854– 863