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Maternal effects mediated by egg quality are important sources of offspring phenotypic variation and can influence the course of evolutionary processes. Mothers allocate to the eggs diverse antioxidants that protect the embryo from oxidative stress. In the yellow-legged gull (Larus michahellis), yolk antioxidant capacity varied markedly among clutches and declined considerably with egg laying date. Analysis of bioptic yolk samples from clutches that were subsequently partially cross-fostered revealed a positive effect of yolk antioxidant capacity on embryonic development and chick growth, but not on immunity and begging behaviour, while controlling for parentage and common environment effects. Chick plasma antioxidant capacity varied according to rearing environment, after statistically partitioning out maternal influences mediated by egg quality. Thus, the results of this study indicate that egg antioxidants are important mediators of maternal effects also in wild bird populations, especially during the critical early post-hatching phase.
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Phenotypic variation within populations consists of genetic components and environmental effects, which, in the broadest sense, embrace all nongenetic sources of variation (Falconer & Mackay, 1996). Maternal effects (see Wade, 1998 for a discussion) are regarded as a peculiar form of variation where offspring phenotype is influenced by maternal environment and phenotype rather than by ecological conditions experienced by the offspring themselves (Mousseau & Fox, 1998). The peculiarity of maternal effects is that they may have both environmental and heritable genetic components (Mousseau & Fox, 1998). The interest in early maternal effects has recently increased with the appreciation that they are ubiquitarian, constitute a major source of phenotypic variation, and can therefore have profound influences on the course of evolutionary processes (Mousseau & Fox, 1998).
An important pathway whereby variation in maternal environment and phenotype translates into variation in offspring phenotype occurs via the egg. In fact, early maternal effects mediated by egg mass and composition have been documented in a variety of taxa and can have pervasive effects on offspring ontogeny (e.g. Williams, 1994; Bernardo, 1996; Fox & Mousseau, 1998).
Egg size variation within populations or species has been suggested to have large additive genetic components (Christians, 2002), although studies of heritability of this trait have seldom taken maternal effects into account. However, egg size has also been demonstrated to depend on maternal condition and environmental factors before and during laying in diverse taxa (Fox et al., 1997; Weigensberg et al., 1998; Christians, 2002; Saino et al., 2004). Egg size predicts offspring growth rate and physiology during the early life stages, and may have carry-over effects into adulthood, by influencing adult body size and fecundity in a variety of taxa (e.g. Hutchings, 1991; Kaplan, 1992; Bernardo, 1996; Azevedo et al., 1997; Price, 1998; Fox, 2000; Svensson & Sinervo, 2000; Torres-Vila & Rodriguez-Molina, 2002; Fischer et al., 2003; Maruyama et al., 2003; Tamada & Iwata, 2005). Variation in egg size can have consequences for offspring phenotype because the amount of egg materials affects development (Sinervo & Huey, 1990; Sinervo, 1993; Finkler et al., 1998; Einum, 2003; Jardine & Litvak, 2003; Ferrari et al., 2006). However, large eggs may be laid by high quality females which are able to allocate to their eggs a disproportionate amount of particular components that have a major effect on the progeny phenotype (Lipar & Ketterson, 2000; Eising et al., 2001; Saino et al., 2003, 2005; Rubolini et al., 2005, 2006a,b; see review in Groothuis et al., 2005 for studies of steroid hormones). Egg size and content of quantitatively minor components (e.g. antioxidants, hormones and immune factors) are likely to have both independent and combined effects on offspring phenotype.
Antioxidants, in particular, are a functionally defined, diverse class of compounds which protect biological molecules from oxidative stress that can either be produced by the organism itself (e.g. cholesterol, uric acid and glutathione) or have to be acquired with the food (e.g. ascorbate, carotenoids, vitamin A, tocopherols; Surai, 2003). Molecules with high oxidant potential (such as free oxygen and nitrogen radicals and other reactive oxygen metabolites) are normally produced and released in the body of organisms as by-products of metabolism or during immune response (Chapple, 1997; Halliwell & Gutteridge, 1999; Surai, 2003), and can damage DNA, proteins, lipids and carbohydrates, therefore having negative effects on tissues integrity and organismal vital processes (Halliwell & Gutteridge, 1999; Surai, 2003).
In birds, antioxidants are transferred to the egg by mothers (Surai, 2003), and such allocation may have to be traded against allocation to maternal maintenance (see Blount et al., 2004; Blount, 2004 for a review). Indeed, some studies of birds in the wild suggested that antioxidants are limiting to laying females, as supplementation with antioxidants results in larger transfer to the eggs, with beneficial consequences for selected offspring traits (Biard et al., 2005). In addition, injection of physiological amounts of antioxidants into the egg can enhance a major component of the acquired immune system and having apparently no adverse consequences on other offspring traits (Saino et al., 2003). Finally, the concentration of antioxidants in the egg yolk is higher than in maternal tissues, implying that mothers actively allocate these compounds in egg tissues (Sunder & Flachowsky, 2001; Surai, 2003).
Yolk antioxidants are incorporated within the developing embryo and chick tissues (e.g. liver, blood), and may contribute substantially to offspring antioxidant defences (Surai & Sparks, 2001; Surai et al., 2001a,b, 2003; Kang et al., 2003; Koutsos et al., 2003; Karadas et al., 2005). Studies of poultry and fish in captivity have shown that egg antioxidants positively influence egg hatchability, the rate of body mass and osteometric growth, immunity, behaviour and viability at least in the early post-hatch life (Lin et al., 2004; see Surai, 2003 for a review), which is a critical period for chick survival. Furthermore, studies of mammals and birds have shown that antioxidants, including vitamin A, E and carotenoids, can reduce brain malformation during early ontogenetic stages (see review in Ramakrishna, 1999), thus providing a potential link between antioxidant capacity (AOC) and behavioural performance of the offspring. Proper antioxidant protection by egg antioxidants may thus be crucial for early survival, potentially affecting the somatic and neurobehavioural development of the offspring as well as the development of other major functions, including immunity.
However, no study has experimentally investigated the genetic, environmental and early maternal components of antioxidant protection of the offspring in any vertebrate species in the wild. The aim of the present study of the yellow-legged gull was to analyse offspring phenotypic variation in relation to genetic effects and environmental factors including nest of rearing and maternal effects mediated by AOC of the egg yolk and egg mass. To this end, we performed a partial cross-fostering experiment whereby we reciprocally swapped recently laid eggs between pairs of synchronous clutches and analysed plasma AOC, T cell-mediated immune response, morphology and begging behaviour of the chicks in relation to parentage and nest of egg incubation and rearing, reflecting environmental effects acting on the embryos and chicks (such as microhabitat conditions and parental care and behaviour). Typical cross-fostering experiments (e.g. Merilä & Fry, 1998) do not allow differentiation between early maternal and origin effects. In order to test for the effect of variable AOC of the eggs while controlling for common origin effects, we extracted bioptic samples from the yolk of freshly laid eggs and analysed the statistical effect of AOC per unit yolk mass and egg mass on chick phenotype. This procedure allowed us not only to analyse the parentage (nest of origin) and environmental (nest of rearing) components of phenotypic variation on chick traits, as carried out in other studies of avian species (e.g. Merilä & Fry, 1998; Meriläet al., 1999; Christe et al., 2000), but also to test for maternal effects mediated by egg size and AOC whereas simultaneously taking into account sources of phenotypic variation because of parentage and rearing environment. We predicted that chicks hatched from eggs with the largest AOC would generate offspring that grew faster and had larger T cell-mediated immune response. In addition, we predicted that plasma AOC of the chicks in the early post-hatching period would positively covary with AOC of the yolk of the original egg. In the yellow-legged gull, egg size markedly declines with laying order (see Results), suggesting that maternal investment in egg production declines in late-laid eggs. We therefore also investigated whether AOC per unit volume of yolk and total antioxidant capacity (TAOC) of the yolk (computed using estimates of yolk mass based on its isometric relationship with egg mass), also varied with laying order of the eggs.
To our knowledge, the approach of measuring overall AOC in specific tissues, rather than the concentration of individual antioxidants, has never been adopted in ecological or evolutionary studies of wild vertebrates. The quantification of the TAOC was obtained by means of a commercial kit (see Methods). The procedure consists of a reduction reaction of an oxidant agent by the antioxidants contained in the test tissue sample, and was originally developed for the assay of TAOC in human tissues (e.g. Cornelli et al., 2001; Trotti et al., 2001). However, it is also useful in the analyses of animal tissues (see Ballerini et al., 2003), because it is simply based on a chemical reaction. Therefore, throughout this study, we will assume that this measure provides an index of the overall protection from oxidative stress.
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Early maternal effects mediated by egg quality are currently thought to be a major source of offspring phenotypic variation whereby mothers can adaptively adjust their allocation to reproduction in relation to the reproductive value of their current progeny, under the constraints imposed by intrinsic factors (e.g. health and general state) and ecological conditions. Such effects, while playing a potentially major role in the generation of phenotypic variation and in population evolutionary dynamics, can result in nongenetic phenotypic correlations between siblings and can therefore confound heritability estimates based on resemblances between genetic relatives (Mousseau & Fox, 1998).
In this study of the yellow-legged gull, we aimed at evaluating the genetic and environmental components of chick morphology, begging behaviour, immunity and antioxidant protection, while statistically partitioning out maternal influences mediated by egg mass and AOC of the yolk of the original egg. The main findings were that: (i) most of the variation in egg AOC and egg mass occurred at the among-clutches level; (ii) egg AOC declined with laying date and (iii) egg mass negatively predicted AOC whereas it positively predicted TAOC (AOC of the entire yolk, TAOC). In addition, (iv) time elapsed from laying to hatching of individual eggs was negatively predicted by yolk AOC; (v) maternal provisioning of antioxidants to eggs, as measured by AOC per unit volume of yolk, positively predicted chick size and mass in the critical early post-hatching period, independently of the effects of original egg mass, parentage or rearing environment and (vi) plasma antioxidant protection of the chicks at the age of 8 days varied with nest of rearing, after controlling for nest or origin and maternal influences mediated by egg mass and yolk AOC. Below we will discuss these main findings, together with other minor results.
Determinants of variation in egg antioxidant capacity and mass
Most of the variance in yolk AOC and egg mass was accounted for by the nest of origin. Previous studies of wild birds have also reported extensive variation in the concentration of specific antioxidants among clutches laid by different females (Saino et al., 2002; Blount et al., 2004; Verboven et al., 2005). Thus, mothers differ considerably in the level of antioxidant protection they provide to their developing offspring. The variation in egg mass is also consistent with the majority of the studies of birds, which indicate that egg size shows considerable variation among females (Christians, 2002).
The seasonal decline in yolk antioxidant capacity (both AOC and TAOC) and the concomitant decline in egg mass suggests that variation in egg quality among clutches could at least partly be due to a decline in mean phenotypic condition of laying females as the season progresses. Alternatively, seasonal variation in AOC and egg mass could reflect a seasonal shift in optimal decisions on allocation of antioxidants and resources to the eggs. In fact, high concentrations of particular antioxidants in the yolk enhance the resistance of the developing poultry embryos to thermal stress and may improve post-hatching performance (Sahin et al., 2002; Kucuk et al., 2003). Similarly, larger eggs that originate heavier chicks, have lower cooling rates than smaller eggs because of their lower surface-to-volume ratio (Kendeigh et al., 1977). Indeed, weather conditions, and air temperature in particular, can vary considerably in our study area over the study period (i.e. late March–late April). Thus, mothers that lay their eggs early in the season may deliver more antioxidants and resources to their eggs, compared with those that lay late, in order to buffer the negative effects of environmental stress on the developing embryo or young chick.
Seasonal variation in yolk AOC relative to overall variation observed in the population was considerably larger than variation in egg mass. This may suggests that phenotypic plasticity in yolk antioxidant transfer may be larger than plasticity in egg size because selection for high allocation of antioxidants to the eggs may be weaker than selection for production of large eggs, because overall egg size may be more influential on offspring performance than AOC.
Antioxidant capacity was significantly negatively predicted by egg mass, whereas TAOC increased with eggs mass. If high AOC per unit embryo (and chick) mass enhances chick performance, these results suggest that a trade-off may be operating between the advantages of laying large eggs, which produce large chicks, and the cost of relatively low AOC per unit chick mass. In addition, these findings may indicate that large eggs are more costly to mothers than small ones also because they are provided with a larger amount of antioxidants than smaller ones.
Antioxidant capacity did not vary with laying order of the eggs. Previous studies of the lesser black-backed gull (Larus fuscus) and of the barn swallow (Hirundo rustica) have shown that the concentration of maternal carotenoids in egg yolks declines with egg laying order (Blount et al., 2002; Saino et al., 2002). Concentration of selected antioxidants, such as carotenoids, may thus not reflect overall antioxidant protection per unit yolk volume that is transferred to the egg. The decline in TAOC with egg laying order was thus due to a decline in yolk mass with laying order and implies that the last eggs in a clutch require a smaller physiological effort by the mothers, in terms of allocation of antioxidants, compared with first eggs.
Time elapsed between laying of consecutive eggs in a clutch positively predicted change in mass (but not in AOC) between the first and the second egg in the pair. Female yellow-legged gulls therefore seem to experience a trade-off between laying consecutive eggs at short time intervals, thus reducing the risk of egg predation, and egg mass, which positively predicts chick mass. The mechanism that generates the positive relationship between the duration of inter-egg intervals and egg mass may depend on larger biosynthesis and accumulation of materials due to be delivered to the egg as the time interval between laying of consecutive eggs increases. The differential effect of inter-egg interval on egg mass and AOC may result from the fact that females cannot markedly affect the composition of the yolk of any given egg during the days immediately preceding laying of that particular egg because accumulation of yolk material (the rapid yolk development period) occurs during a period of several days before laying (Astheimer & Grau, 1990; Ruiz et al., 2000). Conversely, females may tune allocation of materials to the albumen, which is accumulated over a short time interval before laying (Ruiz et al., 2000), and larger inter-egg intervals may allow larger accumulation of albumen material.
Offspring phenotypic variation in relation to parentage, rearing environment and maternal influences mediated by egg antioxidants and mass
The duration of incubation was shorter for increasing values of yolk AOC, while taking into account the effects of nest of origin and nest of incubation. This result is in accordance with the observed positive effects on skeletal growth and body mass (see below), and suggests that yolk antioxidants may directly enhance embryonic development (see Surai, 2003), because the rearing environment made no significant contribution to the variance in duration of incubation.
Chick body mass and tarsus length showed significant variation among nests of rearing only at day 8 post-hatching. Nest of rearing effects at earlier ages, and the effects of parentage at all ages were nonsignificant, suggesting that these components of chick phenotypic variation were relatively small. Variation in plasma AOC at age 8, but not at age 4, showed a significant environmental variation, and may therefore depend on rearing conditions, possibly in terms of dietary intake and nest microhabitat, which can affect, for example, exposure to parasites. In addition, chicks hatched from first laid eggs had larger plasma AOC values at age 8 than chicks hatched from later laid eggs, independently of original egg mass and other potentially confounding variables, suggesting that variation in egg characteristics with laying order (other than yolk antioxidants or egg size, e.g. Royle et al., 2001) could influence the chick's plasma antioxidant protection during development. Laying order also affected body mass and tarsus length at ages 4 and 8 whereas controlling for the effect of egg mass, with chicks hatched from last laid eggs showing generally lower phenotypic values than other chicks. Thus, variation in egg quality with laying order seems to have persistent effects on a suite of chick traits.
We found positive effects of yolk AOC on tarsus length of the chicks at hatching and at day 4 after hatching (though the latter was marginally nonsignificant), and on body mass at day 4, whereas these effects vanished at the age of 8 days. These results were obtained while taking into account the effects of parentage and nest of rearing, and controlling for egg mass, which was also found to positively predict chick mass and size at all ages. Although this covariation does not imply a causal relationship, our results support the hypothesis that egg antioxidants are important mediators of early maternal effects in that they may enhance offspring phenotypic quality, at least soon after hatching, when mortality appears to be maximal (personal observation).
Egg AOC did not predict plasma AOC at day 4 post-hatching, but negatively predicted plasma AOC at age 8. The negative covariation between plasma AOC at day 8 and yolk AOC is intriguing, in that it suggests that chicks may face a trade-off between growth and plasma antioxidant protection. In fact, the positive association between yolk antioxidants and chick tarsus length and body mass at earlier ages corroborates the idea that chick antioxidants of maternal origin can influence growth, at least during the early post-hatching phase (see Surai, 2003), but perhaps at the expense of later plasma antioxidant protection. However, antioxidants in birds are mainly stored in organs such as the liver (Surai et al., 1999; Surai, 2003), and therefore variation in circulating antioxidants may be buffered or confounded by mobilization of these compounds from the liver.
Finally, we tested whether AOC in the original egg also predicted other chick traits, which can affect survival in the early post-hatching life, such as begging behaviour and immunity, but could find no evidence for such relationships. Among birds, specific yolk antioxidants (e.g. carotenoids) have been experimentally shown to enhance immune response (Blount et al., 2003; Saino et al., 2003). Therefore, specific antioxidants have immunostimulating or immunomodulating properties (Chew & Park, 2004), whereas TAOC, which reflects the action of diverse classes of antioxidant compounds, appears not to have consequences for the component of the acquired immunity we measured.
In conclusion, this study indicates for the first time in any wild bird population that variation in plasma antioxidant defences of the chicks has an environmental component. Furthermore, yolk AOC predicted duration of incubation and growth of the chicks in the early post-hatching stages independently of egg mass and rearing environment, suggesting that allocation of antioxidants to the eggs is an important form of early maternal effect in avian species.