Functional Ecology 2008, 22, 682– 690
doi: 10.1111/j.1365-2435.2008.01425.x
Maternal effects on offspring Igs and egg size in relation
to natural and experimentally improved food supply
Blackwell Publishing Ltd
P. Karell*,1, P. Kontiainen1, H. Pietiäinen1, H. Siitari2 and J. E. Brommer1
1
Department of Biological and Environmental Sciences, Bird Ecology Unit, University of Helsinki, P. O. Box 65 (Viikinkaari 1),
FI 00014, Finland; and 2Department of Biological and Environmental Science, Evolutionary Research Unit, University of
Jyväskylä, P. O. Box 35, FI 40014, Finland
Summary
1. Maternal effects have been suggested to function as a mechanism for transgenerational plasticity,
in which the environment experienced by the mother is translated into the phenotype of the offspring. In birds and other oviparous vertebrates where early development is within the egg, mothers
may be able to improve the viability prospects of their offspring at hatching by priming eggs with
immunological and nutritional components.
2. We studied how resource availability affects maternal investment in offspring by feeding Ural
owl (Strix uralensis, Pall.) females prior to egg-laying in 3 years of dramatically different natural
food conditions.
3. Supplementary feeding prior to laying increased body mass and the level of Igs of females
measured at clutch completion. Supplementary fed Ural owl females laid larger eggs than control
females, and had offspring with higher levels of Igs at hatching compared to offspring of control
females.
4. We found variation in maternal allocation of resources to the eggs in response to environmental
conditions: during a year of rapidly declining food abundance, maternal Igs in hatchlings were
higher, whereas egg size was smaller compared to years with a more stable food supply.
5. Egg size had a positive effect on offspring body mass at fledging, whereas Igs at hatching did not
affect Igs at fledging.
6. We conclude that maternal body condition and maternal Igs, as well as hatchling Igs and egg size
are limited by food resources during egg production. Hatchlings rely on maternally derived Igs
and, hence, our results suggest that mothers with high levels of Igs passively transfer higher
Igs levels to their eggs instead of active manipulation of Igs levels in eggs. Ural owl egg size
appears to be highly sensitive to short-term changes in food abundance, with important consequences
for nestling growth.
Key-words: egg size plasticity, ELISA, transgenerational immunity, vole cycle
Functional Ecology (2008) xx, 000–000
Introduction
Resource limitation constrains reproductive decisions and
effort (Martin 1987). In addition to the number of offspring
to be produced in a given breeding attempt, also the phenotypic quality of the offspring can be modified by the mother
(Mousseau & Fox 1998). One determinant of phenotypic
quality is propagule size, which is simultaneously both a
maternal and offspring character (Bernardo 1996). Propagule
*Correspondence author. E-mail: [email protected]
size has been found to determine growth and survival in
various invertebrates (e.g. Sinervo & McEdward 1988; Bridges
& Heppell 1996; Steigenga et al. 2005) and vertebrates
(e.g. Semlitsch & Gibbons 1990; Chambers & Leggett 1996;
Räsänen, Laurila, & Merilä 2005). In the past decade many
avian studies have focused on the variation in egg size.
Increased egg size through food supplementation has been
linked to improved hatchability of the egg, increased size at
hatching, early growth and survival during the nestling stage
(reviewed in Williams 1994; Christians 2002, see also Grindstaff, Demas & Ketterson 2005). Despite the large number of
studies, the relevance of variation in avian egg size is still
equivocal (Christians 2002, see also Kontiainen et al. 2008).
© 2008 The Authors. Journal compilation © 2008 British Ecological Society
Resource limitation and maternal effects 683
Therefore, ecologists have recently focused on egg composition as an estimate of propagule quality since maternal
transfer of components to the offspring via the egg may be an
important maternal adaptation that can enhance early development and survival of the progeny (Mousseau & Fox 1998).
In birds, females have been shown to be able to modify the
contents of eggs in terms of immunoglobulins (Igs) (Grindstaff, Brodie & Ketterson 2003), androgens (Verboven et al.
2003; Groothuis et al. 2005), and antioxidants (e.g. Royle,
Surai & Hartley 2001, see also Groothuis et al. 2006). When
offsprings are exposed to novel pathogens and parasites, and
since the offsprings cannot produce their own Igs during early
ontogeny, they are entirely dependent on innate immunity
and maternal immune components at hatching (Klasing &
Leschchinsky 1998). Furthermore, there is evidence that
maternal immunocompetence is associated with maternal
immune transmission to the eggs (Smith et al. 1994). Consequently, maternal allocation of immunological components
to the offspring to improve its viability and fitness prospects
has received a lot of attention (cf. Saino et al. 2002; Grindstaff
et al. 2003). In general, maternally derived immune components in offspring are induced by the disease environment
experienced by the mother (Gasparini et al. 2001; Buechler
et al. 2002; Staszewski et al. 2007). In the wild, such components have been found to be positively correlated with
maternal body mass (Hargitai, Prechl & Török 2006) and
improved food conditions prior to egg-laying (Pihlaja, Siitari
& Alatalo 2006, but see Grindstaff et al. 2005), and negatively
correlated with increased maternal workload prior to egglaying (Kilpimaa, Alatalo & Siitari 2007). Thus, maternal
transmission of immunological components to the eggs may
be limited by the environmental conditions the mother experiences. During ontogeny increased levels of maternally derived
immune components may be beneficial, as they may enable
offspring to invest in other crucial elements of growth. In offspring, higher maternal Igs have been positively associated
with an offspring’s own Ig production (Grindstaff et al. 2006;
Pihlaja et al. 2006) and, furthermore, a trade-off between
growth and immunity during the nestling period has been
documented for both blue tits (Cyanistes caeruleus, Brommer
2004) and magpies (Pica pica, Soler et al. 2003; Pihlaja et al.
2006).
In this paper, we study the effects of supplementary food
prior to egg laying on Ural owl (Strix uralensis) female Ig
level, egg size and maternal transmission of Igs. Ural owls
mainly prey on voles, which show dramatically cyclic population densities in Fennoscandia (Norrdahl 1995). Ural owls
are site-tenacious and monogamous (Saurola 1987). Vole
abundance fluctuates in a 3 year cycle (low, increase and
decrease phase) in southern Fennoscandia (Sundell et al.
2004), which strongly determines Ural owl reproductive output (Pietiäinen 1989; Brommer, Pietiäinen & Kolunen 2002a).
With an average breeding life span of 3·25 years (Brommer,
Pietiäinen & Kolunen 1998), Ural owl females will face widely
different environmental conditions during their lives. In good
vole years female Ural owls gain ample fat reserves and lay the
largest clutches (Pietiäinen & Kolunen 1993), and the largest
eggs (Kontiainen et al. 2008). Offspring born in the increase
vole phase, when food conditions improve during the first
year, have higher probability of recruitment than offspring
born in the decrease phase, when food conditions rapidly
deteriorate (Brommer, Pietiäinen, & Kokko 2002b). Hence,
the vole cycle generates differences in offspring values
between vole cycle phases and selects for higher reproductive
investment in the increase vole phase (Brommer, Kokko &
Pietiäinen 2000). By supplementary feeding nests prior to
laying in 3 years with different natural food supply (one vole
cycle), we investigate whether Ural owl maternal investment
in offspring, in terms of Ig levels at hatching and egg size, is
food limited under all environmental conditions, and whether
maternal investment into egg size and hatchling Igs vary in
different natural food conditions.
Material and methods
The experiments were conducted in 2004–2006 in a study area of
c. 1500 km2 in Päijät-Häme, Finland, where there are around 180 Ural
owl nest boxes available. The territories that where included in the
experiment were assigned in advance on the basis of territory occupancy, which in turn was based on either breeding activity in the
previous year or on signs of scrapings in the nest box material in
autumn. Half of these active territories were assigned to the fed
group and the nearest active territory was assigned to the control
group, thus forming a treatment pair (see also ‘Statistical Analyses’).
By feeding in a nest box in an occupied territory we can be confident
that no other than the territorial owls eat the food, since Ural owls
defend their territory from intruding con-specifics. Each year new
territories, and hence new individual owls, were used in the experiment.
In birds of prey males deliver food items to the nest during the
pre-laying period to nourish the female into breeding condition
(Newton 1979; Meijer, Daan & Hall 1990). In Ural owls, males
deliver food for the female to the nest. Thus, by adding supplementary food to the nest boxes we simulated improved territory quality
and/or male hunting success. Feeding and visiting of control nests
started in mid-February, which was more than 1 month before estimated egg-laying (median laying date 2004 – 2006: 21 March, range
11 March–26 April). Supplementary food, which consisted of newly
hatched dead rooster chicken Gallus gallus, was delivered to the nests
at 5–6-day intervals and control nest boxes were checked with a similar
interval to standardise disturbance in both groups. Each feeding
consisted of 500 g of rooster chicken, food remaining from the previous
feeding event was weighed and removed. We stopped the supplementary
feeding in a nest at clutch completion. Supplementary food was eaten
prior to laying in all nests belonging to the feeding treatment in this
study. On average 3095 ± 238 standard error (SE) g (range 1190–
4465 g) of supplementary food was eaten per nest prior to laying.
Each experimental year was classified according to the natural
vole density. To estimate vole density we trapped voles bi-annually
in mid-June when owlets have fledged and early October around the
time when owl young become independent. The voles were caught
by snap trapping in 33 localities throughout the whole study area.
Each trapping locality had three quadrates where one quadrate was
placed in an open habitat (clear-cut or re-planted forest), one at the
interface between open habitat and forest, and one in the forest.
Snap traps were set in quadrates (15 × 15 m) with three traps in each
corner as described in Myllymäki et al. (1971). All traps were triggered for two consecutive nights (2544 trap nights in total). Vole
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682– 690
684
P. Karell et al.
abundance was expressed as a percentage, the number of field- and
bank voles trapped per 100 trap nights. For more details on the study
area and methods, see Pietiäinen (1989) and Brommer et al. (2002a).
DATA COLLECTED
Eggs were individually marked with a pencil, and their width and
length were measured with a calliper (accuracy of 0·05 mm). Egg
volume was obtained from the measurements using a speciesspecific formula provided by Pietiäinen, Saurola & Väisänen (1986).
The laying interval of Ural owls is c. 2 days, which enabled us to
accurately determine the laying sequence of most eggs. Ural owl
eggs have a white egg shell, and in case two new eggs had been laid
between consecutive nest visits, their laying order was determined
on the basis of darkening of the egg shell caused by fine particle in
the sawdust getting attached to it. The presumed laying order was
later checked at hatching, and the hatching order corresponded with
estimated laying order in all cases.
Incubating females were caught as soon as possible after clutch
completion (median 13 days after laying the first egg). Only 35 of a
total of 42 females could be caught within the incubation period,
and hence, these 35 females are used in the analyses of female traits.
The number of days the female was caught after laying the first egg
was not significant in the models of female traits. Females were
weighed with a 1500 g Pesola spring balance (5 g accuracy). Body
size was estimated from the length of radius-ulna, measured with a
ruler (1 mm accuracy) from the elbow to the carpal joint (Pietiäinen
& Kolunen 1993). From the females c. 75 µL of blood was drawn
into capillary tubes after brachial venipuncture. None of the female
Ural owls involved in the experiment deserted the nest due to
handling.
Nests were checked with a 1–3 day interval around the estimated
time of hatching to accurately determine which offspring hatched
from which egg. Ural owl offspring hatch asynchronously with 2days interval, except in clutches of three or more eggs in which the
first two or three eggs are more synchronous. In most cases we could
determine exactly from which egg the offspring hatched by direct
observation. In a few cases (n = 10), the two first chicks in a brood
had both hatched since the previous visit. Hatching takes 1–3 days,
and in these ten cases we assigned the largest offspring to the egg
where hatching was most advanced on the previous visit. Hatchlings
were individually marked with a felt-tip pen, weighed and measured.
From the hatchlings c. 60 µL of blood was drawn in a capillary tube
after puncturing the brachial vein as soon as possible after hatching
(0–3 days of age). Some hatchlings could not be blood sampled due
to natural mortality immediately after hatching, because the female
feeds any dead young to the remaining siblings. Prior to fledging,
when the oldest nestling was c. 24 days old (hatching day = 0) all
chicks in a brood were ringed and measured, and a blood sample
was taken (mean age of nestlings, 23·9 days ± 0·2 SE). The blood
capillaries were kept in the cold and were centrifuged within 12 h
after sampling at 2375 g for 5 min in order to separate the blood cells
from the plasma. The plasma was separated from the blood and
stored at –18 °C for an analysis of Ig concentration. Some capillaries
were destroyed in the centrifuge or blood ran out of the capillaries
when centrifuged, which explains the variation in sample sizes.
GENERAL ANTIBODY CONCENTRATION
Total Ig concentrations in the plasma of female parents and each
hatchling (days 0 and 24) were determined with the ELISA method.
Briefly, 96-well microplates (Immuno Plate Maxisorp, Nunc Co.,
Rochester, NY) were first coated overnight at +4 °C with commercial
anti-chicken Ig antibody (10 µg/mL, C-6409, Sigma Chemical Co.,
St Louis, MO). After emptying, the wells were saturated for 1 h with
1% bovine serum albumin (BSA, Roche Diagnostics, Basel, Switzerland) prepared in phosphate buffered saline (PBS, pH 7·4), and then
washed three times with PBS-Tween 20 (0·25%). Samples were
diluted with 1% BSA–PBS and each sample incubated in duplicates
(50 µL/well) for 3 h at room temperature. In addition to a plasma
sample, each plate received a series of standard solutions. Pooled
plasma samples from immunized adults (with diphtheria-tetanus
vaccination, P. Karell, H. Pietiäinen, H. Siitari, P. Kontiainen, T.
Pihlaja & J. E. Brommer, unpublished data) served as standards. Ig
values in samples are presented relative to this standard; arbitrary
value of ten equals the mean level of individuals used for the pooled
sample. After washing, alkaline phosphatase conjugated anti-chicken
Ig antibody (A-9171, Sigma Chemical Co.) was added and the plates
were incubated overnight at +4 °C (dilution of 1 : 20 000). Finally
after last washing, p-nitrophenyl phosphate (1 mg/mL, Sigma 104
Phosphatase Substrate) in a diethanol amine buffer (1 mol/L, pH
9·8) was applied. The optical density was read at 405 nm with a plate
reader (Multiskan Plus, Flow Laboratories, Helsinki, Finland).
SEX DETERMINATION OF OFFSPRING
Ural owl nestlings can not be reliably sexed by their morphology.
We extracted DNA from the blood samples using salt extraction.
We amplified fragments of the sex-chromosome linked CHD gene in
the offspring using the protocol and primers developed by Fridolfsson
& Ellegren (1999). Fragments were separated on a 2% agarose gel
stained with ethidium bromide and scored under UV light. For
more details, see Brommer et al. (2003).
STATISTICAL ANALYSES
Each year different territories (different parental birds) were used
for feeding treatment and control treatment. Thus, in the analyses
all data was pooled with year as a co-factor. All biologically meaningful interactions were tested, but for brevity we report only the
main effects and the treatment by year interaction. Ig concentrations
were log-transformed in all analyses. All analyses were done in R 2·6·1.
Female traits were analysed with linear models with emphasis on
treatment and year effects and their interaction. We used a stepwise
backwards modelling approach as described in Crawley (2002) to
achieve the minimal adequate model (MAM) for the data. Variables
were dropped from the model if P > 0·05 in the F-test to test between
the models. We report F-test statistics of both retained variables and
tests between models as described in Crawley (2002).
Analyses of offspring traits (egg size, hatchling Ig concentration,
fledgling body mass and fledgling Ig concentration) were analysed
with stepwise backwards linear mixed models (LMM) to achieve the
MAM for the data. Only hatchlings less than 70 grams, which are
< 3 days old (H. Pietiäinen, unpublished data), were included in the
hatchling Ig analyses in order to exclude the possibility that the
hatchlings’ own Ig production would affect the results. We first ran
the models with ‘brood identity nested in treatment pair’ as a random
effect to account for variance due to ‘treatment pair’. The variance
of ‘treatment pair’ was, however, virtually zero in all models and we
therefore omitted the nested design, and here we report results from
stepwise backwards LMMs, where the variance due to the random
effect ‘brood identity’ has been accounted for. The LMM was
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682–690
Resource limitation and maternal effects 685
solved using Maximum Likelihood during the stepwise backward
procedure (Pinheiro & Bates 2000). The significance of the random
effect was based on a likelihood ratio test (LRT), where -two times
the difference in likelihood of a model with and without this random
effect was tested as a χ2 value with one degree of freedom (Pinheiro
& Bates 2000). The results of the stepwise backwards LMMs are
reported as the variables retained in the MAMs followed by the
dropped variables from the full models in the order they were
dropped. Test statistics of the dropped variables refer to a LRT (χ2)
between the model retaining and the model excluding the dropped
variable, where a non-significant (P > 0·05) likelihood ratio indicates
a better fit for the model without the dropped variable (Crawley
2002). Therefore, also marginally significant fixed effects (P ~ 0·05)
may be retained in the MAM if that model has a better fit according
to the LRT than the model without the variable. For more details
on the stepwise backwards procedure in LMMs with normal errors,
see Crawley (2002).
Offspring survival was analysed as a Generalised LMM (GLMM)
with binomial errors, and brood identity as a random effect. In
order to achieve the MAM by model simplification we used
‘Laplace’ approximation of likelihood for the binomial GLMM,
implemented with the ‘lmer’ function (Matrix package, by D. Bates
and M. Maehler). The laplacian method is a better approximation
of maximum likelihood than the penalized quasi likelihood (PQL)
method (Breslow & Clayton 1993; Wolfinger & O’Connell 1993)
and allows for a comparison of models with different fixed effects.
We tested between the models with likelihood ratio tests between the
model retaining and the model excluding the variable, and used AIC
to compare between models. Both the AIC and the likelihood ratio
tests produced the same MAM for the data. This GLMM approach
does not allow F-tests of fixed effects. We therefore report the χ2
statistics of the likelihood ratio tests between models in the results.
The direction of the effect of a variable is based on evaluation of its
(significant) coefficient.
Results
NATURAL FOOD SUPPLY DURING THE FEEDING
EXPERIMENTS
The 3 years during which the food supplementation experiments were conducted varied drastically in terms of natural
food supply (Fig. 1). The breeding season of 2004 was a low
phase of the vole cycle (Fig. 1). Vole numbers increased to
18·85 % in autumn 2004 and stayed high through the owl
breeding season in the vole cycle increase phase of 2005
(10·2% in June). The vole density further increased and
reached its peak in autumn 2005 at 36·4%, after which it
rapidly declined during the owl breeding season of 2006 to
0·12% in June, which was a vole cycle decrease phase (Fig. 1).
The vole cycle reflected Ural owl breeding activity in the study
population as only 29% (18 of 61) of the active territories produced clutches in 2004, whereas in 2005 76% (52 of 68) and in
2006 58% (49 of 85) of the active territories produced clutches.
CLUTCH SIZE, EGG SIZE AND TIMING OF EGG LAYING
Clutch size and timing of egg laying varied between years
(clutch size, mean ± SE: 2004, 2·22 ± 0·15 (n = 9), 2005,
5·33 ± 0·18 (n = 24); 2006, 4·75 ± 0·31 (n = 8); laying date,
Fig. 1. Vole abundance in autumn (filled circles) and spring (open
circles) during the years of the supplementary feeding experiment
(2004 – 2006). Grey bars indicate the phases in which the Ural owls
breed. Values are mean ± SE (see Methods for further details).
mean ± SE: 2004, 14 April ± 2·3 (n = 10); 2005, 22 March
± 0·7 (n = 24); 2006, 20 March ± 1·9 (n = 8)). Supplementary
food did not advance timing of laying (year F2,39 = 81·38,
P < 0·0001; dropped variables: treatment × year: F2,36 = 2·61,
P = 0·09; treatment F1,38 = 0·98, P = 0·33,) and did not
increase clutch size (year: F2,38 = 49·71, P < 0·0001; dropped
variables: treatment × year F2,35 = 0·86, P = 0·43; treatment
F1,37 = 0·01, P = 0·92). However, in all years fed females laid
larger eggs than control females (Fig. 2c, MAM: treatment:
F1,38 = 15·49, P = 0·0003; year: F2,38 = 4·57, P = 0·02; random
2
effect: brood ID 69% of variance, LRT: χ1 = 119·37,
2
P < 0·0001; dropped variable: treatment × year: χ1 = 1·87,
P = 0·39). From all nests included in the experiments in
2004 – 2006, 86% (161 of 187) of the eggs hatched, but there
was no difference in hatchability between eggs in control and
fed territories (fed 82·6% (76 of 92), control 89·5% (85 of 95),
χ2 = 1·84, d.f. = 1, P = 0·17).
FEMALE CONDITION, IMMUNITY AND TRANSMISSION
OF IGS TO THE OFFSPRING
Supplementary food prior to laying increased the body mass
of incubating females, and there were also marked differences
across the years of varying natural food supply (Fig. 2a,
MAM: treatment: F1,31 = 6·52, P = 0·02; year: F2,31 = 5·24, P =
0·01; dropped variables: treatment × year: F2,28 = 0·86, P =
0·43; radius-ulna: F1,30 = 1·78, P = 0·19). Supplementary food
also increased the circulating Ig concentration as fed females
had higher levels of Igs than controls (Fig. 2b, MAM: treatment: F1,33 = 6·32, P = 0·02; dropped variables: treatment
× year: F2,29 = 2·43, P = 0·11; year: F2,31 = 0·58, P = 0·56).
There was no direct relationship between a female’s Ig level
at clutch completion and her offspring’s Ig level at hatching,
as Ig in incubating females did not directly explain the mean
Ig levels in her brood (LM: year: F2,32 = 5·78, P = 0·007;
dropped variables: maternal Ig × year. F2,29 = 0·26, P = 0·77;
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686
P. Karell et al.
Fig. 2. Effects of supplementary food on
female size-corrected weight (a) and female
Ig levels (b) at clutch completion, egg size (c)
and hatchling Ig (d). Individuals from the fed
group are denoted by filled squares and
controls by open circles and sample sizes
are given above bars. Not all females could
be caught, which explains the variation in
sample size between treatment groups in (a)
and (b). Sample sizes differ in (c) and (d)
because all eggs did not hatch and some
hatchlings could not be sampled within
3 days after hatching. Ig level is the log
transformed value of Ig concentration from
the ELISA analysis. Egg size is the volume of
individual eggs (see Methods for details).
Values are mean ± SE.
maternal Ig: F1,31 = 0·61, P = 0·44,). However, the Ig levels of
offspring at hatching were food-limited in all years, as hatchlings of fed females had higher Ig levels than the offspring of
control females in all 3 years (Fig. 2d, MAM: maternal treatment: F1,34 = 5·71, P = 0·02; year: F2,34 = 18·44, P < 0·0001;
hatching order: F6,81 = 2·10, P = 0·06; sex: F1,81 = 4·00, P =
2
0·05; random effect: brood ID: 40% of variance, LRT: χ1 =
21·15, P < 0·0001; dropped variables: maternal treatment ×
2
year: χ1 = 0·49, P = 0·78). The MAM retained the effects of
sex and hatching order and showed that Ig levels in hatchlings
tended to be higher in female offspring and lower in later
hatched offspring. Furthermore, in 2006 Ig levels in hatchlings
were substantially higher than in the two other years (Fig. 2d,
MAM: year 2006, t34 = 3·87, P = 0·0005).
CONSEQUENCES OF MATERNAL TREATMENT ON
OFFSPRING SURVIVAL,
IGS
AND SIZE AT FLEDGING
We analysed survival of offspring during the nestling period
with a GLMM with binomial errors and brood ID as random
effect. Survival of offspring decreased with hatching order in
2
the brood (MAM: hatching order: χ1 = 21·25, P = 0·002,
coefficients of hatching order 4 – 6: all P < 0·04), but was not
improved in offspring of fed females compared to controls
or by any other covariable (dropped variables: treatment ×
2
2
year: χ1 = 3·67, P = 0·16; Ig at hatching: χ1 = 0, P = 1; egg
2
2
size: χ1 = 0·23, P = 0·64; treatment: χ1 = 1·69, P = 0·19;
2
year: χ1 = 4·65, P = 0·10).
Egg size had an effect on growth, as offspring that hatched
from larger eggs reached larger body mass at fledging (Table 1,
egg size). In 2004, fledglings weighed less than in 2005 or 2006
(Table 1, year). Later hatched offspring had a lower body
mass at fledging (Table 1). Investigation of the coefficients
further showed that in particular offspring in 4th position
were smaller than the first hatched offspring, and that
relatively few offspring hatched in a later hatching position
(Table 1). Ig levels at the fledging stage (around day 24) were
on average 33 times (4·29 ×106 ± 1·12 × 106 SE/1·28 × 105 ±
4·99 × 104 SE U mL–1) higher than at hatching, and the difference in Ig at hatching between fed and control group was levelled out by day 24 (Table 2, maternal treatment). Ig levels of
the fledglings varied markedly across years and were highest
in 2005 (Table 2, year). Maternal treatment had a different
effect on the Ig levels of her fledglings in different years,
revealed by a significant interaction between maternal treatment and year (Table 2, feeding × year). In particular, in 2006
fledglings of fed females had higher Ig levels than fledglings of
control females (Table 2, fed × year 2006). In both fed and
control nests male offspring had higher Ig levels than female
offspring (Table 2, sex).
Discussion
Supplementary food prior to laying increases a Ural owl
female’s body mass and Ig level in the incubation period.
These fed females do not lay larger clutches, but instead lay
larger eggs and have hatchlings with higher Ig levels than control females. Strikingly, these effects of supplementary food
persist regardless of the yearly natural food supply, although
in particular the vole cycle’s increase phase (2005 in this
study) creates highly favourable natural conditions (cf.
Brommer et al. 2000). Our results show that egg size and Igs
in hatchlings are maternal effects that are strongly resource
limited, suggesting that such investments are costly to a Ural
owl female, and can only be undertaken when increased food
resources are at a female’s disposal. Furthermore, increased
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682–690
Resource limitation and maternal effects 687
Table 1. Stepwise backwards linear mixed model (normal errors, brood ID as random effect) of offspring body mass on day 24
Fixed effects
Variable
Minimal adequate model
Constant
Egg size
Year
Sex
Radius-ulna length
Hatching order
Coefficient ± SE
484·9 ± 15·3***
3·3 ± 1·5**
15·4 ± 16·0
28·8 ± 17·8
–36·2 ± 7·4***
3·8 ± 0·7***
– 8·0 ± 9·3
– 9·4 ± 10·3
– 42·0 ± 12·1***
–18·6 ± 17·4
– 9·61 ± 25·1
2005
2006
2 (38)
3 (27)
4 (19)
5 (7)
6 (3)
Dropped variables
Maternal treatment × year
Maternal treatment
Random effect
Brood ID
Test
F1,80 = 11·38
F2,37 = 7·25
0·001
0·002
F1,80 = 13·32
F1,80 = 58·46
F5,80 = 2·67
0·0005
< 0·0001
0·03
χ1 = 1·20
2
χ1 = 0·37
0·55
0·54
χ1 = 5·72
0·02
2
Variance (95% CI)
397·2 (138·8–1136·0)
%
22
P
2
Egg size and radius-ulna length were standardized (difference from mean). Sex was coded as 0 for female and 1 for male and the reported
coefficients for the factor ‘year’ are in comparison to year 2004. The non-significant variables ‘maternal food treatment’ (control = 0, fed = 1)
and Maternal treatment × year are dropped in the minimal adequate model. The test statistics of the dropped variables refer to a log-likelihood
ratio test between the model in which the variable is retained and in which it is dropped. Stars indicate the significance of the coefficients in the
model (t-test, *** < 0·001, ** < 0·05). Sample sizes for different hatching order are given in brackets. The significance of the random effect was
tested with a log-likelihood ratio test (χ2).
Table 2. Stepwise backwards linear mixed model (normal errors, brood ID as random effect) of Ig levels in offspring on day 24
Fixed effects
Variable
Minimal adequate model
Constant
Year
Feeding
Sex
Feeding × year
Coefficient ± SE
2005
2006
fed × 2005
fed × 2006
5·71 ± 0·18***
0·70 ± 0·19**
0·39 ± 0·23
– 0·33 ± 0·28
0·25 ± 0·06***
0·27 ± 0·31
0·80 ± 0·34*
Dropped variables
Hatching order
Ig at hatching
Egg size
Random effect
Brood ID
Test
P
F2,32 = 12·96
< 0·0001
F1,32 = 0·02
F1,73 = 15·46
F2,32 = 3·53
0·89
0·0002
0·04
χ1 = 2·79
2
χ1 = 0·68
2
χ1 = 1·43
0·73
0·41
0·23
χ1 = 15·24
< 0·0001
2
Variance (95% CI)
0·053 (0·024 – 0·115)
%
38
2
The coefficients of ‘year’ are in comparison to year 2004, ‘sex’ was coded as 0 for female and 1 for male, and maternal treatment (‘feeding’) was
coded as 0 for offspring of control females and 1 for offspring of fed females. Test statistics are equal to tests in Table 1. Stars indicate the
significance of the coefficient in the model (*** < 0·001, ** < 0·01, * < 0·02, < 0·10).
egg size has a strong positive effect on offspring body mass
at fledging, indicating that there are clear benefits from developing in a large egg (although there is an upper limit, since the
largest Ural owl eggs have reduced fitness, see Kontiainen
et al. 2008).
Female body reserves respond both to natural and artificially increased food supply, in accordance with the long-term
descriptive pattern (Pietiäinen & Kolunen 1993). Female Ig
levels, on the other hand, mainly depend on feeding, although
our power to detect different effects in the three phases of the
vole cycle may be limited by small sample size in the low
phase. Nevertheless, different components in the diet may
differentially affect immune function and general body
condition. Carotenoids and antioxidants can function as
immunostimulants (Chew 1996, but see Blount et al. 2002) and
farmed chicken (and their hatchlings) are generally rich in
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682– 690
688
P. Karell et al.
carotenoids and other antioxidants (Karadas et al. 2005;
McGraw & Klasing 2006). Therefore, it is possible that the
supplemented rooster chicken hatchlings in this study were
rich in carotenoids and antioxidants. Carotenoids in the
maternal diet can indeed enhance immune function in hatchling broiler chicken (McWhinney, Bailey & Panigrahy 1989;
Haq, Bailey & Chinnah 1996), which may explain the clear
pattern of increased levels of Igs in both female and hatchling
Ural owls. Alternatively, increased Ig levels could result from
recent infections. However, we find it unlikely that the supplementary food would be a major source of infection compared to the natural prey, voles. Instead, we find it most likely
that supplementary fed Ural owl females improve both their
body mass and their immune function.
There is laboratory evidence that a mother’s immunity can
be transmitted to the offspring via the egg (Smith et al. 1994).
The effect of maternal resource limitation on Ig levels in
hatchlings has also been found in studies of other altricial
birds. Hatchlings of supplementary fed magpie (P. pica)
females have increased levels of Igs (Pihlaja et al. 2006), and
pied flycatcher (Ficedula hypoleuca) females that were forced
to work harder prior to laying produced offspring with lower
Ig levels (Kilpimaa et al. 2007), but, contrastingly, female
kittiwakes Rissa tridactyla appear to allocate more Ig to the
eggs under adverse than favourable food conditions (Gasparini
et al. 2007). Nevertheless, only few other studies of maternal
effects in wild animals have investigated Ig levels in both
mothers and their progeny (Gasparini et al. 2002; Grindstaff,
et al. 2006; Hargitai et al. 2006). Our results are to our knowledge the first to experimentally test the role of food resources
on mother and offspring Ig levels in different natural food
conditions.
We find that after experimentally improving the food
conditions prior to laying, female Ural owls have increased
concentrations of circulating Igs and that the hatchlings of
these mothers also have higher concentrations of circulating
Igs. Increased Ig levels in hatchlings correspond well to Ig levels
in eggs (Gasparini et al. 2002; Pihlaja 2006) and, therefore,
hatchling Ig level is most probably a direct consequence of
passive transmission from a mother with higher Ig levels (see
also Smith et al. 1994). Nevertheless, mothers may actively
allocate differently to eggs of different hatching order (Pihlaja
et al. 2006) or differently to first clutches and replacement
clutches (Gasparini et al. 2007), but we only find a weak
tendency for within-brood (sex and hatching order) variation
in Ig at hatching. We do not find a positive relationship between
female Ig levels at clutch completion and Igs at hatching.
However, such a relationship between a female’s and her offspring’s Igs may be hard to discover because of the time-lag
between sampling of the mother (after egg-laying) and the
time of her Ig transfer to the eggs (days prior to egg-laying).
Furthermore, female immune function has been found to be
decreasing and highly variable around the time of egg-laying
(Oppliger, Christe & Richner 1996; Saino et al. 2002).
Egg size is considered to be a maternal trait that is rather
inflexible on the individual level, and relatively little affected
by food conditions (reviewed in Christians 2002). We find that
Ural owl females are able to respond to improved food
conditions by a remarkable increase (up to 16%) in egg size
(see also Kontiainen et al. 2008). In an earlier review, Williams
(1994) concluded that an increase in egg size may be a small
additional cost to the female that may be beneficial for the offspring during early development. Our results clearly demonstrate that egg size is a highly plastic trait that is limited by
food conditions prior to laying. We also find that offspring
hatched from larger eggs grow larger by the end of the nestling
period. However, to separately evaluate pre- and post-hatching
effects of egg size, hatchling Ig level and maternal food treatment on growth and immunological development, one needs
to tease apart origin and rearing effects by designing a crossfostering experiment (e.g. Lynch & Walsh 1998). Since, for
logistical reasons we could not perform such a cross-fostering
experiment, we are not able to distinguish between pre-hatching
maternal effects and post-hatching rearing effects on offspring
growth and immune function. Avian cross-fostering studies
have reported that egg size improves early growth (e.g. Bize,
Roulin, & Richner 2002), but not survival of the offspring
during rearing (e.g. Bize et al. 2002; van de Pol et al. 2006, but
see Bolton 1991; Pelayo & Clark 2003). Food-induced maternal
effects on offspring Ig production in a cross-fostering design
have to our knowledge not been documented, although
experimentally increased maternal antioxidant consumption
have been found to improve cell-mediated immunity (Biard,
Surai & Møller 2005, 2007; Rubolini et al. 2006). Our study
on Ural owls suggests positive effects of egg size on nestling
growth and no effects of hatchling Igs on Ig production at
fledging. Further studies are, however, needed to explicitly
evaluate the pre-hatching maternal effects, such as egg size
and immune function, on offspring growth and Ig production.
We find variation in maternal effects across years: egg size
and maternal Ig levels in hatchlings vary differently depending on natural variations in food supply. In 2006, when natural vole abundance was decreasing, hatchlings of both fed and
control females had substantially higher Ig levels than in 2004
or 2005 when vole abundance was increasing. Contrastingly,
egg size was small in 2006 compared to 2004 and 2005, and
supplementary food did not increase the egg size in 2006
to the same level as the fed females’ eggs in 2004 or 2005.
Increased maternal Ig transfer to the offspring may be especially beneficial if food conditions rapidly deteriorate after
laying and parental feeding is demanding (cf. Gasparini et al.
2007). It is, however, unclear why egg size is smaller under
such deteriorating circumstances. Potentially, females could
differently allocate resources into Ig transfer and egg size
depending on yearly variations in food availability, but our
study lacks the statistical power to evaluate such a phenomenon. Further studies are needed to evaluate potential
differential maternal effects on different components under
variable natural food conditions.
We further find in this study that in the reversed sizedimorphic Ural owl (males are smaller than females) Ig levels
at the end of the nestling period are higher in male offspring
than in female offspring. Since higher levels of Igs have been
suggested to estimate immunological ‘condition’ (Apanius &
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682–690
Resource limitation and maternal effects 689
Nisbet 2006), our result suggests that male offspring would
invest more in immune function than female offspring during
the nestling period. Interestingly, we find that female offspring tend to have higher Ig levels at hatching, but that this
sex-specific effect is reversed at the time of fledging. Development of immunity is considered to be costly (cf. Klasing &
Leschchinsky 1998; Lochmiller & Deerenberg 2000) and hence,
male offspring potentially use more resources to development
of immune function as they do not grow as big as female offspring. Such sex-specific investment in growth and immunity
would indicate that the smaller sex (males) would not necessarily be cheaper to produce and raise compared to the larger
sex (females). Indeed, a previous study of this population has
found parental feeding investment in Ural owls to be equal for
offspring of both sexes (Brommer et al. 2003).
In conclusion, we find evidence that food induces increased
maternal effects on hatchling Igs and egg size. Furthermore,
our results indicate that egg size, but not maternal treatment
or Igs at hatching, has positive effects on offspring performance. Further studies are needed to evaluate the consequences
of pre- and post-hatching maternal effects on offspring growth
and immune function.
Acknowledgements
All experiments described in this paper were approved by the ethical board for
animal experiments. We would like to thank Elina Virtanen for the ELISA
work, Jaana Kekkonen and Paula Lehtonen for help with sexing the offspring,
and Kalle Huttunen and Heikki Kolunen for assistance in the field. This study
was funded by the Academy of Finland (H.P., J.E.B.), the Finnish Cultural
Foundation (P.K.), the Swedish Cultural Foundation (P.K.) and Oskar Öflund
Foundation (P.K.).
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Received 12 October 2007; accepted 25 February 2008
Handling Editor: Jonathan Blount
© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 682–690