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- Materials and methods
Maternal effects via propagule size are traditionally recognized as a major class of parental effects in ecological studies (Bernardo 1996; Mousseau & Fox 1998). According to Kirkpatrick & Lande (1989), hatchling size can be regarded as ‘maternally inherited’, as phenotypic values of the offspring are determined by a mother's contribution beyond direct genetic effects (i.e. via the egg). As a result, propagule size is a peculiar character, in that it is a joint feature of the mother and of the offspring (Bernardo 1996).
A considerable proportion of the studies on maternal effects mediated by egg features have been carried out in birds. These studies have mostly focused on the correlation between egg mass and offspring phenotypic traits, including putative fitness correlates (e.g. body size and tarsus length), during early ontogenetic stages (reviews in Williams 1994; Christians 2002).
It could be argued, however, that the investigation of the individual egg components, rather than whole egg mass, may better unravel the mechanisms of maternal effects mediated via the egg. The yolk and albumen fractions, for example, have distinctive roles during development (Carey, Rahn & Parisi 1980; Sotherland & Rahn 1987). The yolk is the main source of lipids and is implicated more in mass gain than in body size development, whereas the albumen is a key source of water and proteins (Romanoff & Romanoff 1949; Carey et al. 1980; Sotherland & Rahn 1987). In particular, albumen appears to be a functionally overlooked component of the egg (but see Saino et al. 2005; Ferrari, Martinelli & Saino 2006), despite some authors having suggested that albumen mass may represent the most crucial portion of the egg in determining hatchling performance, as the yolk's participation in tissue formation is less relevant (Nisbet 1978).
In this framework, experimental manipulation of egg components may provide useful insights (Sinervo et al. 1992). Previous studies of poultry have shown that albumen removal could result in reduced body mass and tarsus length of chicks (Hill 1993; Finkler, van Orman & Sotherland 1998). The only study carried out in the wild, in the barn swallow Hirundo rustica, showed that albumen removal reduced nestling growth and survival, mainly of late-hatched chicks (Ferrari et al. 2006). Thus, in that species, the observed increase in both absolute and relative albumen content of the eggs along the laying sequence may mediate a maternal ‘brood survival’ strategy (sensu Lack 1954) – whereby parents increase the number of viable offspring by mitigating the negative consequences of hatching late by, for example, laying the larger eggs last. Conversely, ‘brood reduction’ strategies have been widely documented among bird species, where within-brood size hierarchies are often established by asynchronous egg hatching coupled with reduced size of the last laid eggs. In these species, no increase in relative albumen content along the egg laying sequence should be expected, unlike the pattern observed in barn swallows (see above).
In the present study of the yellow-legged gull Larus michahellis, a species adopting a brood reduction strategy, with markedly smaller last laid eggs (Cramp 1998), we first analysed variation in relative albumen content of the eggs according to laying order, while predicting no increase in last laid eggs. In addition, we investigated the consequences of a reduction in albumen content within the natural range of variation of albumen mass relative to total egg mass on a suite of phenotypic traits (including body size, intensity of begging behaviour and immune response) of the chicks under natural conditions. We predicted that nestlings from experimentally manipulated eggs hatched later and attained smaller body size and mass (Ferrari et al. 2006). We also expected positive effects of albumen removal on begging, based on the assumption that the intensity of begging display honestly reflects offspring need (Wright & Leonard 2002). Moreover, we speculated that immune response could also be negatively influenced by albumen removal because of expected generalized negative effects on offspring growth and development. Such negative consequences on phenotype were predicted to be more severe among chicks hatched from smaller last laid compared with earlier laid eggs within each clutch, because of the close association between hatching and laying order and the lower competitive ability of late hatched chicks.
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- Materials and methods
In this study of a gull species in the wild, we have shown that experimental removal of albumen may have complex consequences on diverse chick traits, including time to hatching, begging behaviour, morphology and survival. Some of these effects, however, varied according to laying order and egg mass.
Albumen removal reduced egg hatchability and increased the time to hatching. In addition, albumen removal differentially affected the probability that an egg originated a chick surviving until age 8 in relation to egg mass, as among control chicks viability depended on original egg mass, while no relationship existed for albumen chicks. Furthermore, begging intensity was differentially affected by albumen removal in relation to laying order and original egg mass, as it increased with laying order among albumen chicks, but decreased among controls, and increased with increasing egg mass among controls, whereas it decreased among albumen chicks. Finally, and unexpectedly, albumen chicks were heavier than controls on day 8 post-hatching, while albumen removal did not affect body mass at earlier ages or tarsus length at any age. However, as expected, egg mass strongly positively predicted body mass and size of the chicks, at any age.
The reduced egg hatching success following albumen removal, which occurred among a- and b- but not c-eggs, indicates that even a small variation in albumen content can have large consequences, and particularly so for the early laid eggs in a clutch, which generate chicks with greater survival prospects (Hillström, Kilpi & Lindström 2000).
The increase in time to hatching following albumen removal is consistent with the results obtained by Ferrari et al. (2006) in the barn swallow, indicating that variation in albumen content may affect embryo developmental trajectories in birds. This discloses the possibility that mothers adjust hatching times of their clutches in relation to extrinsic factors (e.g. resource availability or egg predation pressure) via a modification of the proportion of the albumen in their eggs. In addition, the different pattern of variation of relative albumen content along the laying sequence in the yellow-legged gull compared with the barn swallow, as well as the difference in the consequences of the experimental albumen removal, may represent adaptations to their reproductive strategies. While the barn swallow adopts a brood survival strategy, which could be partly mediated by the increase in the proportion of albumen along the laying sequence (Ferrari et al. 2006), the yellow-legged gull exhibits a facultative brood reduction strategy, and its eggs show no within-clutch variation in the relative albumen content. Thus, albumen provisioning to the eggs may match differences in an important life-history trait, an idea that could be widely tested in comparative studies of variation in egg composition among species showing different reproductive strategies.
Contrary to our expectation, albumen removal did not negatively affect body size or mass in the first days after hatching, differently from previous studies of birds (Hill 1993; Finkler et al. 1998; Ferrari et al. 2006). However, apart from the study by Ferrari et al. (2006), which subtracted a proportion of albumen similar to the present one (up to 5·6% vs. 2·3%, respectively), the other studies, all carried out on poultry, removed a far greater proportion of albumen from the eggs, i.e. up to 16% in the study of Hill (1993), and 15% in the study of Finkler et al. (1998). Moreover, our experimental design differed from that adopted by Ferrari et al. (2006), in that we assigned whole clutches to the same treatment, whereas Ferrari et al. (2006) adopted a within-clutch design, where chicks of different treatments hatched in a same nest. In the present study, the adoption of an intraclutch design was not feasible, due to the smaller clutch size of gulls and much higher post-hatching mortality due to stochastic factors (see Rubolini et al. 2005). Thus, in our study, albumen chicks did not compete with control siblings, which could have exacerbated the consequences of the albumen removal on body mass and size in the barn swallow experiment.
Albumen chicks were heavier than controls on day 8 post-hatching, while albumen removal did not affect body mass at earlier ages or tarsus length at any age. This counterintuitive result may be a by-product of a reduced egg hatchability of a- and b-eggs and of the consequent variation in the proportion of chicks from a-, b- or c-eggs in albumen compared with control broods. The larger body mass of albumen chicks was not mediated by a different number of chicks in albumen nests compared with control nests (Mann–Whitney U-test; age 4: Z = 0·87; age 8: Z = 0·06; P > 0·35). Thus, the alteration of brood composition in terms of chick quality following differential hatching success of eggs according to laying order in control and albumen clutches may have generated the observed pattern.
A novel result emerging from this study is that albumen removal may affect begging display, depending on the laying order of the original egg and on original egg mass. This finding may be due to a greater sensitivity of a- and b-eggs and chicks to albumen removal, which may have consequences on their behavioural development or performance, and suggests the existence of an albumen-mediated interaction between pre-natal resource allocation and post-natal signals of need.
The analysis of the effect of albumen removal disclosed significant patterns of association of chick phenotype with some of the covariates we considered. Size at hatching and immune response were found to positively covary with time to hatching. This may indicate that chicks hatching from eggs that are incubated longer hatch at an advanced developmental stage compared with eggs that are incubated for a shorter period (e.g. Ricklefs 1992). This result is consistent with comparative evidence showing that, across both altricial and precocial species, the intensity of the T-cell-mediated immune response positively covaries with the duration of the incubation period (Tella et al. 2002; see also Ricklefs 1992).
The larger T-cell-mediated immune response we found in chicks from c- compared with a- or b-eggs could be explained by the higher antioxidant capacity we documented in c-eggs in a companion study of the same population (see Rubolini et al. 2006). In fact, egg antioxidants are known to enhance immunity of chicks during their early post-hatching life (see Surai 2003 for a review).
Finally, we found that chick morphological traits were positively predicted by egg mass. This result is consistent with previous findings in the same as well as in other species (e.g. Rubolini et al. 2006; Ferrari et al. 2006), suggesting that variation in egg mass can have persistent effects on chick quality.
In conclusion, the results of this study show that variation in egg albumen content may affect egg and chick viability, and modulate hatching times in a semiprecocial gull species. The patterns of variation in the proportion of albumen in the eggs of different species may parallel differences in reproductive strategies. Thus, our study calls for a major consideration of the functional role of the albumen in ecological and evolutionary investigations of maternal effects via egg composition.