Allometry of egg and hatchling mass in birds and reptiles: roles of developmental maturity, eggshell structure and phylogeny

The factors determining hatchling mass (HM) are investigated in a wide range of birds and reptiles using regression analysis, analysis of covariance and comparative analysis by independent contrasts. In birds, initial egg mass (IEM) at laying is the most important factor affecting HM and phylogenetic relatedness has no significant effects on HM. Developmental maturity of the avian neonates did not affect the proportion of IEM converted into HM. For all reptile species, IEM also significantly affected HM but phylogenetic relatedness did not. By contrast, allometric relationships between IEM and HM in the different orders of reptiles were affected by shell type. The robustness of allometric relationships across taxa in birds and reptiles suggests that there is a physiological link between IEM and HM, which contrasts with that observed for the relationship between egg mass and incubation period. This result has significant implications for the inter-relationships between IEM and embryonic growth, which are discussed for birds and reptiles.     

Speckled eggs: water-loss and incubation behaviour in the great tit Parus major

Many small passerine birds worldwide lay white eggs speckled with red, brown and black protoporphyrin pigment spots (maculation). Unlike some patterns of avian eggshell pigmentation which clearly serve a crypsis or signalling function, the ubiquity of maculation among passerines suggests that its origins lie in another function, not specific to any particular ecological or behavioural group. Elsewhere, we have presented evidence that protoporphyrin pigments serve a structural function related to eggshell thickness and calcium availability: eggshell maculation in the great tit Parus major increases with decreasing soil calcium levels, pigments demarcate thinner areas of shell, and both the pigment intensity and distribution are related to shell thickness. Here we show that maculation also affects the rate of water loss from the egg during incubation (≈ Mass Loss per Day or MLD, which is critical to egg viability), but not that of unincubated eggs. We also demonstrate, both by observation and experiment, that the effect of female incubation behaviour on MLD compensates in some way for variation in egg characteristics, and that differences between females in the degree of such compensation are related to differences in clutch maculation. Our results suggest that, while a principal function of maculation in this species may be to strengthen the eggshell, it may also reduce eggshell permeability when large amounts of pigment are used, and that this necessitates a behavioural adjustment from the female during incubation. We discuss these findings and make further testable predictions from our model.     

Review: an embryo's eye view of avian eggshell pigmentation

Current research into the evolution and adaptive function of avian eggshell pigmentation, including maculation, has focused mostly on signalling‐based and structural function hypotheses but ignored the potential consequences of shell pigmentation for the developing avian embryo, especially in moderating the embryo's interaction with its light environment. The exposure of the eggs to sunlight that frequently accompanies avian incubation behaviour is one of the major evolutionary steps setting apart birds and reptiles, and coincides with the appearance of eggshell pigmentation. This suggests that shell pigments could play a major role in ensuring the successful development of the avian embryo. We propose that the effects of shell pigments on the egg contents should be considered in addition to established hypotheses of shell pigmentation such as crypsis, egg recognition or a possible structural function. This approach has the potential to identify trade‐offs between different pigment functions and to resolve some of the long standing paradoxa in the evolution of eggshell colour, such as the occurrence of conspicuous blue eggs in passerines or the secondary evolution of white eggshells in cavity nesters. In particular, we identify seven hypotheses, which address how the interaction of eggshell pigments and the light environment may influence embryonic development. These hypotheses are the: thermo‐regulation; UV‐B protection; photo‐acceleration; lateralization; circadian rhythm; photo‐reactivation; and antimicrobial defence. We believe that the understanding of eggshell pigmentation will greatly benefit from taking these hypotheses into consideration when studying the functional significance of eggshell pigmentation and suggest a number of promising directions for future experimental and comparative research.     

Selenium distribution in eggs of avian species

We studied the effect of egg mass of eight different avian species on Se distribution between egg components and the effect of incubation on Se accumulation by chicken eggshell and shell membrane. Eight groups of birds received a diet without Se supplementation. Unfertile eggs were collected after 35 days of feeding; yolk, albumen, shell and shell membrane were assayed separately for Se. All avian species studied showed identical Se concentration in yolk–albumen complex equal to 38.7 μg Se/100 g, reflecting a linear correlation between yolk–albumen mass and Se content. Shells and shell membrane Se accumulation showed quadratic correlation with the appropriate mass thus explaining unusually high Se concentration in ostrich shell and shell membrane, that reached values 1785 and 1904 μg Se/kg respectively. Incubation of fertile eggs decreased eggshell Se content, the effect being more expressed in eggs from hens fed sodium selenite compared to organic Se utilization (Sel-Plex). It was concluded that shell might be an additional Se source for an embryo.     

Global diversity and adaptations of avian eggshell thickness indices

The amniote eggshell is a fundamental aspect of the embryo life-support system, protecting it from UV light, microbes and mechanical damage, while regulating gas exchange and providing calcium for growth. The thickness of eggshells is highly diverse across modern birds and influences multiple eggshell functions, yet the selective pressures driving eggshell thickness have not been clearly identified. Here, we use a global dataset of avian eggshell thickness indices for 4260 (> 41%) avian species to assess trends in eggshell thickness across the phylogeny, as these indices are strongly correlated with direct measures of eggshell thickness and are non-destructive to the sample. We analysed the dataset within a phylogenetic framework to assess the relative importance of climatic and ecological explanations for variation in eggshell thickness indices. The distribution of avian eggshell thickness indices across species was found to be primarily driven by phylogenetic relatedness, in addition to evolutionary processes that do not match a Brownian model of evolution. Across modern birds, thicker eggshells were more prevalent in species (1) with precocial young, (2) which exhibit a scavenger-based diet, (3) which primarily feed on vertebrates or plants (excluding nectivores, seed and fruit specialists) and (4) which breed in open habitats. Thicker eggshells found in species with precocial young probably enable higher rates of calcium removal for the more advanced embryo development. Excessive light transmission through the shell damages developing embryos, while too little light transmission can impede development. Eggs in shaded habitats experience low light exposure, and thus thinner shells are more prevalent in these environments potentially to increase light transmission through the shell. Overall, variation in eggshell thickness indices appears to be driven largely by phylogeny, with certain life-history traits linked to embryo growth rate, calcium content of their food, and the need to mitigate excessive light transmission through the shell.     

Avian eggshell pigments are not consistently correlated with colour measurements or egg constituents in two Turdus thrushes

The colourful appearance of avian eggshells is a prominent aspect of maternal reproductive effort in birds. Some differences in eggshell coloration have been reported to co‐vary with various measures of maternal condition and these patterns support the hypothesis that, in some bird species, several aspects of eggshell colour (i.e. primary chroma and brightness) function as a signal of maternal and offspring quality to induce greater paternal investment. We directly quantified eggshell pigment concentrations of blackbird Turdus merula and song thrush T. philomelos eggshells and tested how the two key pigments (protoporphyrin IX and biliverdin) co‐varied with other eggshell traits and egg constituents as measures of maternal reproductive investment, including total yolk carotenoid concentration, total lipid concentration, yolk mass, and shell thickness. Contrary to predictions, we detected few statistical patterns overall. We found that protoporphyrin IX concentration was negatively associated with blue‐green chroma in blackbirds but not in song thrush. The concentration of protoporphyrin IX was significantly greater in blackbirds and also showed different patterns of association with total yolk lipids and yolk carotenoid concentrations between these two species (significant species interaction terms). Our results reveal that it is not appropriate to simply assume in these two avian species that reflectance‐based eggshell colour measures are a suitable proxy for eggshell pigment concentrations or can be used as consistent predictors of maternal reproductive investment. These results highlight the need to assess and validate the strength and direction of the statistical relationships between eggshell colour measures, pigment concentrations, and maternal resource deposition in the egg for other species of birds.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

Eggshell pigment composition covaries with phylogeny but not with life history or with nesting ecology traits of British passerines

No single hypothesis is likely to explain the diversity in eggshell coloration and patterning across birds, suggesting that eggshell appearance is most likely to have evolved to fulfill many nonexclusive functions. By controlling for nonindependent phylogenetic associations between related species, we describe this diversity using museum eggshells of 71 British breeding passerine species to examine how eggshell pigment composition and concentrations vary with phylogeny and with life‐history and nesting ecology traits. Across species, concentrations of biliverdin and protoporphyrin, the two main pigments found in eggshells, were strongly and positively correlated, and both pigments strongly covaried with phylogenetic relatedness. Controlling for phylogeny, cavity‐nesting species laid eggs with lower protoporphyrin concentrations in the shell, while higher biliverdin concentrations were associated with thicker eggshells for species of all nest types. Overall, these relationships between eggshell pigment concentrations and the biology of passerines are similar to those previously found in nonpasserine eggs, and imply that phylogenetic dependence must be considered across the class in further explanations of the functional significance of avian eggshell coloration.     

Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus

1. We test the consequences, in terms of breeding success and parental effort, of eggshell pigmentation pattern in a hole-nesting bird, the blue tit Cyanistes caeruleus that lays eggs asymmetrically speckled with reddish spots (maculated eggs). 2. We assess the effect of distribution of spots (pigment 'spread') and spot size and pigment intensity (pigment 'darkness') on eggshell physical properties and breeding parameters concerning nestling condition, investment of parents in offspring care and reproductive output in two different habitat types: a deciduous oakwoodland and an evergreen forest. 3. Blue tit clutches with more widely distributed spots showed a thicker eggshell, a shorter incubation period, a lesser amount of mass loss per day and a higher hatching probability than those with spots forming a 'corona' ring. While eggs with larger and darker (more pigment intensity) spots showed a thicker eggshell and a shorter incubation period. In the light of 'signal function hypothesis', these egg traits may reflect female health status and, consequently, this could affect male parental effort. 4. Here we show supports for some of the necessary assumptions of this hypothesis. We found a positive relationship between egg pigment 'spread' and male but not female provisioning rates per day. On the other hand, pigment 'darkness' of blue tits' clutches was positively related to female tarsus length, while pigment 'spread' was positively related to clutch size, male body mass and nestling tarsus length. Our study shows that eggshell pigment 'spread' can be used as an indicator of clutch quality. Further investigations are needed to understand the role of calcium availability as possible causal agent of deviant eggs and its relation to the maculation phenomenon.     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

The relationship of pores and mammillae on the inner surface of the eggshell of the alligator (Alligator mississippiensis)

The morphology of the eggshell of the alligator, Alligator mississippiensis, is similar to that of birds. In many avian species there is a positive linear correlation between the numbers of pores and mammillae on the inner surfaces of eggshells, indicating that the distribution and density of mammillae may determine the porosity of the shell. It is not known, however, if a relationship exists between pores and mammillae on the shell of the alligator. Using a scanning electron microscope, we counted pores and mammillae on the inner surfaces of pieces of shell from the middle of fertile and infertile eggs from wild and captive, pen-reared alligators. Data were analyzed by ANOVA, Duncan's multiple range tests and linear regression equations. Results demonstrate a positive linear correlation between the numbers of pores and mammillae on the shells of unincubated fertile and infertile eggs from wild and captive alligators; however, there is no correlation between pores and mammillae on shells of eggs that were incubated for 55 days. It is suggested that initially the porosity of the eggshell of the alligator is related to the density of mammillae on the inner surface of the shell and that erosion of the shell during incubation destroys the original relationship between pores and mammillae.     

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.     

Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle

The avian eggshell is an acellular bioceramic containing organic and inorganic phases that are sequentially assembled during the time the egg moves along the oviduct. As it has been demonstrated in other mineralized tissues, mineralization of the eggshell is regulated by extracellular matrix proteins especially the anionic side chains of proteoglycans. Among them, osteopontin has been found in the avian eggshell and oviduct. However, its precise localization in the eggshell or in different oviduct regions during eggshell formation, nor its function have been established. By using anti-osteopontin antibody (OPN 1), we studied its immunolocalization in the isthmus, red isthmus and shell gland of the oviduct, and in the eggshell during formation. In the eggshell, osteopontin was localized in the core of the non-mineralized shell membrane fibers, in the base of the mammillae and in the outermost part of the palisade. In the oviduct, OPN 1 was localized in the ciliated epithelial but not in the tubular gland cells of the isthmus, in the ciliated epithelial cells of the red isthmus, and in the non-ciliated epithelial cells of the shell gland. The occurrence of osteopontin in each of the oviduct regions, coincided with the concomitant presence of the egg in such region. Considering the reported inhibitory function of osteopontin in other mineralized systems, together with its main occurrence in the non-mineralized parts of the eggshell and at the outermost part of the shell, suggests that this molecule could be part of the mechanism regulating the eggshell calcification.     

Eggshell Biliverdin as an Antioxidant Maternal Effect

In this essay, the hypothesis that biliverdin pigment plays an antioxidant role in the avian eggshell is proposed. Due to its ability to scavenge free radical species and to reduce mutation, biliverdin potentially counteracts the oxidative action of pathogens that penetrate the eggshell and/or protects the shell membrane from oxidation, thus promoting the proven antioxidant and antimicrobial capacities of the shell membrane itself. Additionally, biliverdin may be able to inhibit viral replication in the eggshell due to its ascribed antiviral properties. Moreover, previous results in other taxa leave open the question of whether biliverdin can be absorbed by the embryo from the eggshell and play a role in embryogenesis. These mechanisms of antioxidant action of eggshell biliverdin remain totally unexplored in birds and in other oviparous animals. The main assumptions and predictions of the antioxidant hypothesis are developed, and directions for future research are proposed.     

Egg mass and incubation period allometry in birds and reptiles: effects of phylogeny

Here we test the hypothesis that the relationship between egg mass at oviposition (IEM) and incubation period ( I _p) is a function of the taxonomic relatedness of bird and reptile species. Allometric relationships between IEM and I _p were examined for 1525 bird species and 201 reptilian species. Treating species as independent data revealed the allometric exponent linking I _p to IEM to be 0.234 for birds and 0.138 for reptiles. However, ANCOVA revealed that within both birds and reptiles the elevation and slope of the regression lines were dependent on the taxonomic order studied, indicating that the exponents were confounded by the phylogenetic relatedness of species. Thus, allometric exponents were recalculated based on the method of comparative analysis using independent contrasts. This technique revealed that the allometric exponent in both birds and reptiles was confounded by phylogeny. In birds the allometric relationship between I _p and IEM was almost halved to 0.122, whereas in reptiles the exponent increased to 0.185. Importantly, the results demonstrate that some results of allometric analyses can be artefacts of the method of analysis of the dataset. That for bird eggs I _p is not determined in large part by egg mass allows new questions to be posed regarding the ecological and physiological factors affecting the length of incubation, and hence rates of embryonic growth, for different taxa and habitats.     

BIOMECHANICAL PROPERTIES OF DINOSAUR EGGSHELLS (Ⅲ)──THE MECHANIC ANALYSIS OF THE BABY DINOSAURS' EMERGING FROM THEIR EGGS

Thepurposeofthispaperistoanalyzethestressofdinosaureggshellloadedbythehatch1ingswhentheyhatch,andfourtypesofdinosaureggshellsareavaila-ble(seeTablelofZhaoeta1.,l994;TablelofMaandZhao,1994).Mostofthelivingreptileshaveflexible,parchment-likeeggshellscomposedoffibresandrelativelysma1lamountsofcalciumcarbonate.Thchatchlings,eachequ-ippedwithasmalI,sharp"eggtooth",cracktheireggshel1stoemerge'However,theyoungbirdspokeatthebluntendoftheeggwiththeirbeak,thenstruggleoutfromtheireggs.Thehatchingproce…     

Avian eggshell thickness in relation to egg morphometrics,embryonic development,and mercury contamination

Eggshell thickness is important for physiological, ecological, and ecotoxicological studies on birds; however, empirical eggshell thickness measurements for many species and regions are limited. We measured eggshell thickness at the equator and the egg poles for 12 avian species and related eggshell thickness to egg morphometrics, embryonic development, egg status, and mercury contamination. Within an egg, eggshells were approximately 5.1% thicker at the equator than the sharp pole of the egg, although this difference varied among species (0.6%–9.8%). Within Forster's tern (Sterna forsteri), where eggshell thickness was measured at 5 equally spaced positions along the longitude of the egg, eggshell thickness changed more rapidly near the sharp pole of the egg compared to near the blunt pole of the egg. Within species, eggshell thickness was related to egg width and egg volume for six of the 12 species but was not related to egg length for any species. Among species, mean eggshell thickness was strongly related to species mean egg width, egg length, egg volume, and bird body mass, although species mean body mass was the strongest predictor of species mean eggshell thickness. Using three species (American avocet [Recurvirostra americana], black‐necked stilt [Himantopus mexicanus], and Forster's tern), whose nests were carefully monitored, eggshell thickness (including the eggshell membrane) did not differ among viable, naturally abandoned, dead, or failed‐to‐hatch eggs; was not related to total mercury concentrations of the egg content; and did not decrease with embryonic age. Our study also provides a review of all existing eggshell thickness data for these 12 species.     

Great spotted cuckoo eggshell microstructure characteristics can make eggs stronger

Obligate avian brood parasites lay stronger eggs than their hosts or non‐parasitic relatives because they are rounder and have a thicker eggshell. Additionally, some other characteristics of the brood parasitic eggshells related to their microstructure such as size and orientation of calcite crystal units could also contribute to generating even stronger shells. An eggshell microstructure formed by small randomly oriented calcite crystal units can increase the robustness of the eggshells of birds. Here, the eggshell microstructure of avian brood parasites as well as their hosts have been characterized in detail, using X‐ray diffraction analyses to estimate the size and degree of orientation of calcite crystal units making the eggshell. Specifically, the brood parasitic great spotted cuckoo Clamator glandarius and two hosts (jackdaws Corvus monedula and magpie Pica pica) and one non‐host species (the pigeon, Columba livia domestica) were considered. Calcite crystal of the eggshell of the brood parasitic species was smaller and more randomly oriented than those of the eggshells of non‐parasitic species, which suggest that eggshell microstructure would contribute to explain why parasitic eggs are more resistant to breakage than those of their hosts.     

Relationship between eggshell strength and keratan sulfate of eggshell membranes

Eggshell strength is an important factor in an effort to minimize eggshell breakage, which is a significant problem in the egg production industry. In the current study, we isolated and quantified the specific glycosaminoglycans (GAGs) from the calcified eggshell and shell membranes, which are related to eggshell strength. Our data suggest that GAGs exist in calcified eggshell may influence morphology of shell but do not affect on increase of shell amount while GAGs of shell membranes are maybe highly associated with shell strength with an increase of shell weight. Shell strength showed a strong correlation with the content of GAGs (r=0.942, p<0.0005) and a weak relationship with uronic acid content (r=0.564, p=0.056) in shell membranes. Monosaccharides in shell membranes were determined by Bio-LC analysis for the identification of any specific GAGs related with shell strength. It indicates that the galactose content as a component of keratan sulfate (KS) has a significant correlation with eggshell strength (r=0.985, p<0.0005). These results suggest that eggshell strength is proportional to the KS content of eggshell membranes with an increase of eggshell weight.     

BIRD EGGSHELL IN DOMINICAN AMBER

Abstract:  Here we report an eggshell in Dominican amber, representing the first vertebrate egg in any amber deposit. The eggshell is compared with present-day eggs of lizards, snails and birds. Based on the surface structure and type of shell breakage, it appears that the most likely candidate is a bird, and with that consideration, an avian group that produces eggs similar to the fossil in shape, size and colouration is the Trochilidae (hummingbirds). Several possible explanations of how the fossil could be preserved in amber are provided. If indeed a hummingbird was involved, this discovery would represent the first New World record of a fossil trochilid.