Within-clutch Variation Research Articles

Egg Mercury Concentration and Egg Size Varies with Position in the Laying Sequence in two Songbird Species.

In birds, mercury embryotoxicity can occur through the transfer of mercury from the female to her eggs. Maternal transfer of mercury can vary by egg position in the laying sequence, with first-laid eggs often exhibiting greater mercury concentrations than subsequently laid eggs. We studied egg mercury concentration, mercury burden (total amount of mercury in the egg), and egg morphometrics by egg position in the laying sequence for two songbirds: tree swallows (Tachycineta bicolor) and house wrens (Troglodytes aedon). Egg mercury concentration in the second egg laid was 14% lower for tree swallows and 6% lower for house wrens in comparison with the first egg laid. These results indicate that in both species, after an initial relatively high transfer of mercury into the first egg laid, a smaller amount of mercury was transferred to the second egg laid. This lower mercury concentration persisted among all subsequently laid eggs (eggs three to eight) in tree swallows (all were 14%-16% lower than egg 1), but mercury concentrations in subsequently laid house wren eggs (eggs three to seven) returned to levels observed in the first egg laid (all were 1% lower to 3% greater than egg 1). Egg size increased with position in the laying sequence in both species; the predicted volume of egg 7 was 5% and 6% greater than that of egg 1 in tree swallows and house wrens, respectively. This change was caused by a significant increase in egg width, but not egg length, with position in the laying sequence. The percentage of decline in mercury concentration with position in the laying sequence was considerably lower in tree swallows and house wrens compared with other bird taxonomic groups, suggesting that there are key differences in the maternal transfer of mercury into songbird eggs compared with other birds. Finally, we performed simulations to evaluate how within-clutch variation in egg mercury concentrations affected estimates of mean mercury concentrations in each clutch and the overall sampled population, which has direct implications for sampling designs. Environ Toxicol Chem 2024;43:1844-1854. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

Temperature has an overriding role compared to photoperiod in regulating the seasonal timing of winter moth egg hatching.

To accurately predict species' phenology under climate change, we need to gain a detailed mechanistic understanding of how different environmental cues interact to produce the seasonal timing response. In the winter moth (Operophtera brumata), seasonal timing of egg hatching is strongly affected by ambient temperature and has been under strong climate change-induced selection over the past 25years. However, it is unclear whether photoperiod received at the egg stage also influences timing of egg hatching. Here, we investigated the relative contribution of photoperiod and temperature in regulating winter moth egg development using two split-brood experiments. We experimentally shifted the photoperiod eggs received by 2-4weeks compared to the actual calendar date and measured the timing of egg hatching, both at a constant temperature and in combination with two naturally changing temperature treatments - mimicking a cold and a warm year. We found an eight-fold larger effect of temperature compared to photoperiod on egg development time. Moreover, the very small photoperiod effects we found were outweighed by both between- and within-clutch variation in egg development time. Thus, we conclude that photoperiod received at the egg stage does likely not play a substantial role in regulating the seasonal timing of egg hatching in the winter moth. These insights into the regulatory mechanism of seasonal timing could have important implications for predicting insect climate change adaptation, as we might expect different targets of selection depending on the relative contribution of different environmental cues.

The Immunological and Oxidative Costs of Reproduction and Associations between Maternal and Egg Yolk Physiology in a Reptile

Energetic constraints caused by reproductive investment may incur immunological and oxidative costs to a female and potentially to her developing offspring. Oviparous reptiles are capable of altering offspring phenotype through differential allocation of physiological factors during vitellogenesis (yolk formation), depending upon the external environment and the physiological state of the female. Despite its importance in protecting developing offspring against infection, maternal transfer of immune factors has rarely been studied in reptiles. Furthermore, the extent to which offspring may be impacted by the inherent oxidative costs of vitellogenesis is not well understood. Using the Side-blotched Lizard (Uta stansburiana) as a model, we examined how reproductive investment (clutch size and mass) influenced maternal immunological (BKA) and oxidative state (ROMs) and whether these metrics covaried with egg yolk immunity and oxidative state. We assessed factors influencing variation in egg yolk BKA and ROMs within and across clutches from all females and found that females with smaller clutches had higher BKA and ROMs than females with larger clutches. There was also an overall positive relationship between maternal BKA and ROMs and clutch mass, regardless of clutch size. We did not find maternal and egg yolk BKA and ROMs to be directly related, but yolk ROMs decreased as egg mass increased. Within-clutch variation in yolk BKA and ROMs was not related to maternal investment, but variation among clutches was explained by maternal identity. Because egg yolk physiology can impose long-lasting or permanent changes to offspring, these results create a foundation for future work investigating factors contributing to yolk physiology and highlight the necessity of considering multiple physiological and ecological variables when conducting eco-immunological research.

Maternal investment in last-laid eggs does not compensate for hatching asynchrony in a seabird.

Maternal and environmental effects can have profound effects on offspring performance by generating variation in offspring phenotypes, independent of genetic effects. Within avian broods, differential maternal investment of resources across the laying sequence is thought to be an adaptive strategy to modulate competitive hierarchies induced by hatching asynchrony. In this study, we evaluated the relative importance of maternally derived within-clutch variation and the asymmetric post-hatching environment for growth and survival of common tern (Sterna hirundo) siblings. We experimentally manipulated hatching order, resulting in chicks from last-laid eggs hatching first and vice versa. Although both initial age and size asymmetries were larger within experimental than control broods, the early survival of last-hatched chicks was similar between groups. Initial positive effects of egg size disappeared as siblings approached fledging. Ultimately at fledging, both within-brood growth and cumulative survival patterns were similar between experimental and control broods, suggesting that the effects of systematic variation of egg constituents (e.g., maternally derived yolk hormones) and egg size are too subtle and largely overwhelmed by the effects of hatching asynchrony. Therefore, we conclude that variation in offspring phenotypes is pre-dominantly determined by the social environment experienced post-hatching. Maternal effects may further fine-tune phenotypic variation in response to varying environmental conditions, but this needs to be tested through empirical studies in which multiple maternal effects are measured simultaneously under different environmental conditions.

Within-clutch variation in size and shape of the Great Tit Parus major eggs

Within-clutch variation in size and shape of the Great Tit Parus major eggs

Variation in PFAA concentrations and egg parameters throughout the egg-laying sequence in a free-living songbird (the great tit, Parus major): Implications for biomonitoring studies

Over the past decades, there has been growing scientific attention and public concern towards perfluoroalkyl acids (PFAAs), due to their widespread presence in the environment and associations with adverse effects on various organisms. Bird eggs have often been used as less-invasive biomonitoring tools for toxicological risk assessments of persistent organic pollutants, including some PFAAs. Hereby, it is typically assumed that one random egg is representative for the PFAA concentrations of the whole clutch. However, variation of PFAA concentrations within clutches due to laying sequence influences can have important implications for the egg collection strategy and may impede interpretations of the quantified concentrations. Therefore, the main objective of this paper was to study variation patterns and possible laying sequence associations with PFAA concentrations in eggs of the great tit (Parus major). Eight whole clutches (4–8 eggs) were collected at a location in the Antwerp region, situated about 11 km from a known PFAA point source. The ∑ PFAA concentrations ranged from 8.9 to 75.1 ng g−1 ww. PFOS concentrations ranged from 6.7 to 55.1 ng g−1 ww and this compound was the dominant contributor to the total PFAA profile (74%), followed by PFDoA (7%), PFOA (7%), PFDA (5%), PFTrA (4%) and PFNA (3%). The within-clutch variation (70.7%) of the ∑ PFAA concentrations was much larger than the among-clutch variation (29.3%) and concentrations decreased significantly for some PFAA compounds throughout the laying sequence. Nevertheless, PFAA concentrations were positively and significantly correlated between some egg pairs within the same clutch, especially between egg 1 and egg 3. For future PFAA biomonitoring studies, we recommend to consistently collect the same egg along the laying sequence, preferably the first or third egg if maximizing egg exposure metrics is the main objective.

Chaperones, Canalization, and Evolution of Animal Forms.

Over half a century ago, British developmental biologist Conrad Hal Waddington proposed the idea of canalization, that is, homeostasis in development. Since the breakthrough that was made by Rutherford and Lindquist (1998), who proposed a role of Hsp90 in developmental buffering, chaperones have gained much attention in the study of canalization. However, recent studies have revealed that a number of other molecules are also potentially involved in canalization. Here, I introduce the emerging role of DnaJ chaperones in canalization. I also discuss how the expression levels of such buffering molecules can be altered, thereby altering organismal development. Since developmental robustness is maternally inherited in various organisms, I propose that dynamic bet hedging, an increase in within-clutch variation in offspring phenotypes that is caused by unpredictable environmental challenges to the mothers, plays a key role in altering the expression levels of buffering molecules. Investigating dynamic bet hedging at the molecular level and how it impacts upon morphological phenotypes will help our understanding of the molecular mechanisms of canalization and evolutionary processes.

A new, three-dimensional geometric morphometric approach to assess egg shape.

This paper proposes a new methodology to quantify patterns of egg shape variation using geometric morphometrics of three-dimensional landmarks captured on digitally reconstructed eggshells and demonstrates its performance in capturing shape variation at multiple biological levels. This methodology offers unique benefits to complement established linear measurement or two-dimensional (2D) contour profiling techniques by (i) providing a more precise representation of eggshell curvature by accounting for variation across the entire surface of the egg; (ii) avoids the occurrence of correlations from combining multiple egg shape features; (iii) avoids error stemming from projecting a highly-curved three-dimensional (3D) object into 2D space; and (iv) enables integration into 3D workflows such as finite elements analysis. To demonstrate, we quantify patterns of egg shape variation and estimate morphological disparity at multiple biological levels, within and between clutches and among species of four passerine species of different lineages, using volumetric dataset obtained from micro computed tomography. The results indicate that species broadly have differently shaped eggs, but with extensive within-species variation so that all four-focal species occupy a range of shapes. Within-species variation is attributed to between-clutch differences in egg shape; within-clutch variation is surprisingly substantial. Recent comparative analyses that aim to explain shape variation among avian taxa have largely ignored potential biases due to within-species variation, or use methods limited to a narrow range of egg shapes. Through our approach, we suggest that there is appreciable variation in egg shape across clutches and that this variation needs to be accounted for in future research. The approach developed in this study to assess variation in shape is freely accessible and can be applied to any spherical-to-conical shaped object, including eggs of non-avian dinosaurs and reptiles through to other extant taxa such as poultry.

Within-clutch variability in gamete size arises from the size variation in gametangia in the marine green alga Monostroma angicava.

Within-clutch gamete size variability in Monostroma angicava. In many organisms, it is unclear how the size variation in gametes is generated in each clutch (i.e., total gametes produced by a gametophyte for a single spawning) or how gamete size is adjusted. Within-clutch variation in gamete size has been explained as a result of either physiological/developmental constraints or bet hedging during gametogenesis. These two explanations have been assumed to be mutually exclusive, and related observations are conflicting. The slightly anisogamous dioecious green alga Monostroma angicava employs a simple mechanism to produce gametes of each sex: each vegetative cell becomes a single gametangium cell, which synchronously divides to form equally sized gametes. The number of such cell divisions has several variations, which might vary gamete size. We measured the volume of gametangia in each clutch, counted the number of cell divisions in each gametangium and estimated the size of the gametes. We found that larger gametangia divided more times than smaller gametangia in both sexes, although male gametangia were smaller than female gametangia when they underwent the same number of cell divisions. Therefore, the variation in the number of cell divisions during gametogenesis serves to adjust gamete size in each sex rather than to vary it. Within-clutch gamete size variability originates in within-clutch variation in gametangium size: any factors that increase the variation in the size of gametangia can increase the within-clutch variation in gamete size.

Intraclutch eggshell colour variation in birds: are females able to identify their eggs individually?

BackgroundOne possibility suggested regarding female post-mating strategies is differential allocation into offspring investment. Female birds produce not only the largest, but also most colourful eggs of all oviparous taxa. Larger eggs provide space for bigger embryos, or more nutrition for their development, but the question why eggs are more colourful and why there is variation in eggshell colouration remains. In this context, the focus of interest has been to explain inter-clutch variation but in many bird species, eggshell colouration also varies within a clutch. Surprisingly, less attention has been paid to this phenomenon. Therefore, we propose the “female egg recognition” hypothesis, suggesting that mothers use colour characteristics to interpret egg attributes and allocate further investment into each egg accordingly. To evaluate the feasibility of the hypothesis, we tested several underlying predictions and examined their suitability using a dataset from our tree sparrow (Passer montanus) study. We predict (i) substantial within-clutch variation in eggshell colouration which, (ii) should be related to laying sequence, (iii) reflect egg quality and, (iv) should stimulate a female response.MethodsEggshell coloration data were obtained via digital photography under standardized conditions, taken after clutch completion. Lightness (L*), representing the achromatic properties of an egg has been chosen as the most important predictor in dark cavities and was related to egg quality and position in the nest.ResultsIn our tree sparrows, first and mainly last eggs were less pigmented, providing information about laying order. Egg volume, which predicts chick quality, positively correlates with eggshell coloration. Finally, we could show that female tree sparrows placed darker, but not bigger, eggs into more central incubation positions.DiscussionAll basic prerequisites for the “female egg recognition” hypothesis are fulfilled. In this context practicability and feasibility of the hypothesis and alternative explanations are discussed. However future work is necessary to determine a direct effect on offspring condition.

Bet hedging in a warming ocean: predictability of maternal environment shapes offspring size variation in marine sticklebacks.

Bet hedging at reproduction is expected to evolve when mothers are exposed to unpredictable cues for future environmental conditions, whereas transgenerational plasticity (TGP) should be favoured when cues reliably predict the environment offspring will experience. Since climate predictions forecast an increase in both temperature and climate variability, both TGP and bet hedging are likely to become important strategies to mediate climate change effects. Here, the potential to produce variably sized offspring in both warming and unpredictable environments was tested by investigating whether stickleback (Gasterosteus aculeatus) mothers adjusted mean offspring size and within-clutch variation in offspring size in response to experimental manipulation of maternal thermal environment and predictability (alternating between ambient and elevated water temperatures). Reproductive output traits of F1 females were influenced by both temperature and environmental predictability. Mothers that developed at ambient temperature (17°C) produced larger, but fewer eggs than mothers that developed at elevated temperature (21°C), implying selection for different-sized offspring in different environments. Mothers in unpredictable environments had smaller mean egg sizes and tended to have greater within-female egg size variability, especially at 21°C, suggesting that mothers may have dynamically modified the variance in offspring size to spread the risk of incorrectly predicting future environmental conditions. Both TGP and diversification influenced F2 offspring body size. F2 offspring reared at 21°C had larger mean body sizes if their mother developed at 21°C, but this TGP benefit was not present for offspring of 17°C mothers reared at 17°C, indicating that maternal TGP will be highly relevant for ocean warming scenarios in this system. Offspring of variable environment mothers were smaller but more variable in size than offspring from constant environment mothers, particularly at 21°C. In summary, stickleback mothers may have used both TGP and diversified bet-hedging strategies to cope with the dual stress of ocean warming and environmental uncertainty.

Sex-specific differences in offspring personalities across the laying order in magpies Pica pica

Maternal effects provide an important mechanism for mothers to create variation in offspring personality, and to potentially influence offspring life history strategies e.g. creating more/less dispersive phenotypes. However, within-clutch maternal effects often vary and hence there is potential for within-clutch variation in personality. We studied the effects of hatching order on explorative and neophobic behaviour of the magpies Pica pica in relation to sex using novel environment and novel object experiments. Hatching order did affect explorative behaviour in magpie, but did so in opposite directions for either sex. First-hatched females were more explorative and had a tendency to be less neophobic, whereas in males, the reverse was true. Our results suggest that hormonal as well as post-natal environmental mechanisms could be underpinning this pattern. Future research is needed to fully understand the importance of both in creating different offspring personalities.This article is part of a Special Issue entitled: insert SI title.

Patterns of within-clutch variation in yolk lutein in the Yellow-legged Gull Larus michahellis: the effects of egg laying order and laying date

In gull species, the level of carotenoids in egg yolks is known to decrease along the egg laying sequence. However, the extent of this laying order effect is expected to vary among clutches according to laying date, due to possible differences between early-nesting and late-nesting females with regards to their ability to control the transfer of maternal resources to the eggs. A laying date effect could also result from a seasonal decline in the availability of dietary carotenoids. In this study, we collected data from Yellow-legged Gulls (Larus michahellis) breeding in southern Tunisia to investigate how nesting date interacts with egg-laying order in shaping patterns of within-clutch variation in yolk lutein, the most abundant carotenoid in this species. In particular, we examined (1) whether yolk lutein concentration declined along the laying sequence and (2) whether the extent of the decline differed among clutches according to laying date. Our results show that yolk lutein decreased along the laying sequence at the clutch level. More interestingly, they also show that the decrease in yolk lutein levels along the laying sequence was more pronounced in late clutches than in early clutches, although lutein levels in adult plasma did not vary according to laying date. Overall, these results support the hypothesis that the Yellow-legged Gull employs the brood reduction strategy and that said strategy is more drastically applied in late clutches than in early clutches. The relevance of female age and seasonal variation in chick-rearing conditions for these findings is discussed.

Intra-clutch variation in avian eggshell pigmentation covaries with female quality

Among the most eye-catching traits of avian eggs are their background coloration and pigmentation, consisting in many passerine birds of dark protoporphyrin spots. Although variation in protoporphyrin pigmentation among clutches has been shown to reflect female quality, within-clutch variation in egg pigmentation remains less well understood. Here, we hypothesize that female quality may also be reflected in within-clutch variation in egg pigmentation as a result of energetic constraints and/or increased susceptibility to oxidative stress, and test this hypothesis in a free-living population of Great Tits (Parus major). Within clutches, both pigment ‘darkness’ and ‘spread’ (reflecting intensity, distribution and size of pigment) increased with laying order. For pigment ‘darkness’, this was most strongly so in larger females and in females showing lysis (as a measure of constitutive innate immunity), suggesting that intra-clutch variation in pigment ‘darkness’ positively relates to both structural as well as condition-dependent female traits. In contrast, for pigment ‘spread’, no relationships were detected with body size, body condition, age, and two components of constitutive innate immunity. Among clutches, ‘darkness’ and ‘spread’ of pigments also varied. However, this variation was not related to any of the female characteristics we measured. To the best of our knowledge, this study is the first one to relate intra-clutch variation in protoporphyrin egg pigmentation to structural and condition-dependent traits of laying females. Further experimental study is, however, required to better understand the underlying causal mechanisms.

Within-Clutch Variation in Yolk Testosterone as an Adaptive Maternal Effect to Modulate Avian Sibling Competition: Evidence from a Comparative Study

In many species, embryos are exposed to maternal hormones in utero, in the egg, or in the seed. In birds, mothers deposit substantial testosterone into their eggs, which enhances competitive ability of offspring. These maternal testosterone concentrations vary systematically within clutches in different patterns and may enable mothers to adaptively fine-tune competitive hierarchies within broods. We performed a comparative analysis to investigate this hypothesis using a broad set of avian species. We expected species with small size differences among siblings (arising from small hatching asynchrony or slow growth rates) to aim for survival of the whole brood in good years and therefore compensate last-hatching eggs with relatively more testosterone. We expected species with large size differences among siblings (large hatching asynchrony or fast growth rates) to produce surplus young as insurance against failed offspring and to facilitate elimination of redundant surplus young by bestowing last-hatching eggs with relatively less testosterone. As predicted, we found that maternal testosterone compensation to last-hatching eggs is stronger when size differences among siblings become smaller. Maternal testosterone compensation to last-hatching eggs also correlated negatively with hatching asynchrony and growth rates. These findings provide evidence for correlated evolution of several maternal effects that together support different maternal reproductive strategies.

Marked Shifts in Offspring Size Elicited by Frequent Fusion among Siblings in an Internally Brooding Marine Invertebrate

While offspring size is a widely studied concept in evolutionary ecology, mechanisms affecting offspring phenotype in species with postzygotic parental care are incompletely understood. We examined the impact of sibling fusion on ontogenetic shifts in offspring size in the brooding sea anemone Urticina felina. Fusion occurred among brood-protected embryos in U. felina, whereas it occurred postrelease among settlers of corals studied here and previously. Two fusion products were evidenced: morphologically aberrant offspring and large homogeneous offspring coined "megalarvae." The frequent occurrence (∼77%) of megalarvae identifies them as the primary fusion product, which drove an increase in offspring size and within-clutch size variation before release. Lipid signatures suggest that morphologically aberrant juveniles represent by-products that do not reach adulthood. Not only were occurrences of megalarvae common in the populations studied, they increased with maternal fecundity, suggesting that sibling fusion may be a form of kin cooperation integral to the reproductive success of U. felina, warranting investigation in other live-bearing invertebrate taxa.

Effects of Sea-surface Temperature on Egg Size and Clutch Size in the Glaucous-winged Gull

Abstract. The hypothesis that the egg production capacity of northeast Pacific seabirds is reduced in years with higher spring sea-surface temperatures was tested with data collected on Glaucous-winged Gulls breeding on Triangle Island, British Columbia, Canada, from 2002 to 2011. The mean clutch size varied from 2.3 to 2.9 eggs nest-1, but contrary to prediction, there was little relationship between clutch size and ocean temperature. In contrast, the mean egg size increased with temperature (opposite to the predicted decrease), but that effect was modest in size and limited to clutches of one and three eggs. The within-clutch variation in egg size differed little from year to year, and was unrelated to temperature. The results suggest that higher sea-surface temperatures do not compromise egg production in Glaucous-winged Gulls, a situation unlike that for some other species of marine birds on Triangle Island Received 6 December 2011, accepted 18 February 2012.

Maternal Antibody Transmission in Relation to Mother Fluctuating Asymmetry in a Long-Lived Colonial Seabird: The Yellow-Legged Gull Larus michahellis

Female birds transfer antibodies to their offspring via the egg yolk, thus possibly providing passive immunity against infectious diseases to which hatchlings may be exposed, thereby affecting their fitness. It is nonetheless unclear whether the amount of maternal antibodies transmitted into egg yolks varies with female quality and egg laying order. In this paper, we investigated the transfer of maternal antibodies against type A influenza viruses (anti-AIV antibodies) by a long-lived colonial seabird, the yellow-legged gull (Larus michahellis), in relation to fluctuating asymmetry in females, i.e. the random deviation from perfect symmetry in bilaterally symmetric morphological and anatomical traits. In particular, we tested whether females with greater asymmetry transmitted fewer antibodies to their eggs, and whether within-clutch variation in yolk antibodies varied according to the maternal level of fluctuating asymmetry. We found that asymmetric females were in worse physical condition, produced fewer antibodies, and transmitted lower amounts of antibodies to their eggs. We also found that, within a given clutch, yolk antibody level decreased with egg laying order, but this laying order effect was more pronounced in clutches laid by the more asymmetric females. Overall, our results support the hypothesis that maternal quality interacts with egg laying order in determining the amount of maternal antibodies transmitted to the yolks. They also highlight the usefulness of fluctuating asymmetry as a sensitive indicator of female quality and immunocompetence in birds.

Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis

Many passerine species lay eggs that are speckled with dark protoporphyrin pigmentation. Because protoporphyrin is mainly derived from the blood, we here formulate and test a new hypothesis that links an increase in anaemia along the laying sequence to within-clutch variation in egg pigmentation. More intense pigmentation is expected if pigments accumulate during enhanced red blood cell production in response to anaemia. Reduced pigmentation is expected if pigments are derived from the degradation of red blood cells that circulate in smaller numbers due to blood loss. To test this hypothesis, we manipulated anaemia in great tit (Parus major) females by infesting the nests with hen fleas (Ceratophyllus gallinae) prior to egg laying. Polychromatophil (i.e., immature red blood cells) percentage, as a measure of blood cell production, was positively correlated with parasite load confirming that female great tits experienced stronger anaemia when infested with haematophagous parasites during egg laying. We found a positive relationship between spot darkness and laying order that weakened under high parasite load. This result suggests that anaemia in females due to blood-sucking parasites led to diminished protoporphyrin from disintegrated red blood cells and hence a decreased deposition of protoporphyrin. However, the overall increase in pigment darkness along the laying sequence suggests that pigments also accumulate by enhanced red blood cell production caused by anaemia due to egg production itself.

Egg components vary independently of each other in the facultative siblicidal Black-legged Kittiwake Rissa tridactyla

Egg composition varies both within and between clutches, and mothers are expected to alter their deposition of resources to the egg depending on environmental conditions and breeding strategies. Within-clutch variation in egg composition has been proposed to reflect an adaptive maternal strategy influencing sibling competition. In species with brood reduction, mothers should reinforce brood hierarchies due to hatching asynchrony and favour senior chicks by making first-laid eggs larger, richer in nutrients, with higher testosterone and carotenoid levels and lower corticosterone concentrations than last-laid eggs [parental favouritism hypothesis (PFH)]. Moreover, mothers that are of better quality and/or experience better feeding conditions during laying are expected to increase their deposition of resources to the egg, resulting in differences between clutches [investment hypothesis (IH)]. Several components may act together to provide an optimal reproductive strategy, but studies of variation in different egg components in the same egg are relatively rare. We analysed egg size, testosterone and corticosterone concentrations and carotenoids measured as yolk colour between and within clutches for the facultative siblicidal Black-legged Kittiwake Rissa tridactyla. First-laid eggs were larger, contained lower testosterone, higher yolk colour score, and similar corticosterone levels than last-laid eggs. Thus, only differences in egg size and yolk colour supported the PFH. We used within-clutch egg size dimorphism as an indicator of the quality of the mother or the feeding conditions during laying. In support of the IH, we found that mothers of better quality or that experienced better feeding conditions deposited more corticosterone into their eggs. High corticosterone levels may benefit nestlings when there is no brood reduction but high sibling competition is present. We found no support for the hypothesis that egg components are mutually adjusted to each other and we discuss the possible reasons for this.