Avian Eggshell Research Articles

Nesting behaviour influences species-specific gas exchange across avian eggshells.

Carefully controlled gas exchange across the eggshell is essential for the development of the avian embryo. Water vapour conductance (GH2O) across the shell, typically measured as mass loss during incubation, has been demonstrated to optimally ensure the healthy development of the embryo while avoiding desiccation. Accordingly, eggs exposed to sub-optimal gas exchange have reduced hatching success. We tested the association between eggshell GH2O and putative life-history correlates of adult birds, ecological nest parameters and physical characteristics of the egg itself to investigate how variation in GH2O has evolved to maintain optimal water loss across a diverse set of nest environments. We measured gas exchange through eggshell fragments in 151 British breeding bird species and fitted phylogenetically controlled, general linear models to test the relationship between GH2O and potential predictor parameters of each species. Of our 17 life-history traits, only two were retained in the final model: wet-incubating parent and nest type. Eggs of species where the parent habitually returned to the nest with wet plumage had significantly higher GH2O than those of parents that returned to the nest with dry plumage. Eggs of species nesting in ground burrows, cliffs and arboreal cups had significantly higher GH2O than those of species nesting on the ground in open nests or cups, in tree cavities and in shallow arboreal nests. Phylogenetic signal (measured as Pagel's λ) was intermediate in magnitude, suggesting that differences observed in the GH2O are dependent upon a combination of shared ancestry and species-specific life history and ecological traits. Although these data are correlational by nature, they are consistent with the hypothesis that parents constrained to return to the nest with wet plumage will increase the humidity of the nest environment, and the eggs of these species have evolved a higher GH2O to overcome this constraint and still achieve optimal water loss during incubation. We also suggest that eggs laid in cup nests and burrows may require a higher GH2O to overcome the increased humidity as a result from the confined nest microclimate lacking air movements through the nest. Taken together, these comparative data imply that species-specific levels of gas exchange across avian eggshells are variable and evolve in response to ecological and physical variation resulting from parental and nesting behaviours.

A Comparison of the Bacterial Microflora Found on the Surface of American Kestrel and House Wren Eggs

It is generally accepted that avian eggs acquire a microflora during ovipositioning. The goal of this study was to identify and compare the eggshell microflora of two avian species, House Wrens and American Kestrels. Differences in the nesting habitats and incubation behavior of these species suggest that their eggshell microfloras also should differ. Culture-dependent techniques were combined with sequencing of the 16S rRNA gene to identify bacteria in samples taken from American Kestrel eggs during late incubation. These data were compared to previously collected data from House Wren eggs. In both studies, bacteria were isolated from three different phyla, Actinobacteria, Firmicutes and Proteobacteria. The eggshell microflora of Kestrels was dominated by bacteria in the Actinobacteria and Firmicutes phyla during late incubation, whereas the eggshell microflora of House Wrens was dominated by bacteria in the Gamma-Proteobacteria subphylum during pre- and late-incubation and bacteria within the Fimicutes phylum during early incubation. Actinobacteria genera on House Wren and American Kestrel eggshells differed, but the prominent genera in the Gamma- Proteobacteria and Firmicutes phyla, Pseudomonas and Staphylococcus, respectively, were similar between bird species. Thus, our results suggest that the microflora of avian eggshells have both variable (i.e., phyla) and conserved (i.e., specific genera) aspects of bacterial diversity.

Hierarchical structure of CaCO3biominerals – mesocrystals and functionalization

Skeletal parts and teeth of marine organisms, avian eggshells, trilobite and isopod eyes, and many more biomineralized tissues consist of bio-calcite or bio-aragonite crystals. We explore the nano- to micro-scale architectures of these materials by electron backscatter diffraction (EBSD) and complementary techniques. In contrast to their inorganic cousins the biogenic "crystals" are hybrid composites with small amounts of organic matrix controlling morphogenesis and critically improving mechanical performance or other functions. For the biominerals meso-crystal-like structures are ubiquitous, consisting of co-oriented nano-blocks with a mosaic-spread of a few degrees, depending on the organism and on the size of the mesocrystal entity[1, 2, 3]. The nano-mosaic can be attributed to growth by nano-particle accretion from an amorphous or gel-like precursor, where relics of organic matrix cause misorientations between the crystallized nano-blocks. Recently we were able to reproduce this feature in gel-grown calcite [Nindiyasari et al., Crystal Growth and Design, in press]. The mesocrystal-co-orientation spreads on to the micro- and even millimeter-scale, frequently with a fractal nature of co-oriented hierarchical units [Maier et al., Acta Biomaterialia, accepted for publication]. The hierarchically structured morphology of the composite crystal or polycrystal is always directed by organic matrix membranes. Sea urchin teeth show a multiplex composite crystal architecture, where different subunits of engineered shapes, Mg-contents, and small misalignments are essential prerequisites for self-sharpening [1]. The figure shows an EBSD map of dendritic interdigitating calcite crystals in an avian egg shell (color coding for crystal orientation) with an misorientation profile along the grey line.

First light for avian embryos: eggshell thickness and pigmentation mediate variation in development and UV exposure in wild bird eggs

Summary The avian embryo's development is influenced by both the amount and the wavelength of the light that passes through the eggshell. Commercial poultry breeders use light of specific wavelengths to accelerate embryonic growth, yet the effects of the variably patterned eggshells of wild bird species on light transmission and embryonic development remain largely unexplored. Here, we provide the first comparative phylogenetic analysis of light transmission, through a diverse range of bird eggshells (74 British breeding species), in relation to the eggshell's thickness, permeability, pigment concentration and surface reflectance spectrum (colour). The percentage of light transmitted through the eggshell was measured in the spectral range 250–700 nm. Our quantitative analyses confirm anecdotal reports that eggshells filter the light of the externally coloured shell. Specifically, we detected a positive relationship between surface eggshell reflectance (‘brightness’) and the percentage of light transmitted through the eggshell, and this relationship was strongest at wavelengths in the human‐visible blue‐green region of the spectra (c. 435 nm). We show that less light passes through thicker eggshells with greater total pigment concentrations. By contrast, permeability (measured as water vapour conductance) did not covary significantly with light transmission. Eggs of closed‐nesting species let more light pass through, compared with open nesters. We postulate that greater light transmission is required to assist embryonic development under low light exposure. Importantly, this result provides an ecological explanation for the repeated evolution of immaculate, white‐ or pale‐coloured eggshells in species nesting in enclosed spaces. Finally, we detected correlative support for the solar radiation hypothesis, in that eggshells of bird species with a longer incubation period let significantly less of the potentially harmful, ultraviolet (UV) light pass through the eggshell. In summary, we demonstrate suites of avian eggshell properties, including eggshell structure and pigmentation, which are consistent with an evolutionary pressure to both enhance and protect embryonic development.

The oldest known avian eggshell, Plagioolithus fukuiensis, from the Lower Cretaceous (upper Barremian) Kitadani Formation, Fukui, Japan

Fossil record of Early Cretaceous birds may be geographically biased, and eggs and eggshells predating the Late Cretaceous were unknown. Here, we report the oldest known bird eggshell (FPDM-V-0009175) collected from the upper Barremian Kitadani Formation in Katsuyama City, Fukui, Japan. The Kitadani Formation likely represents fluvial environments. Thin-section and scanning electron microscope analyses revealed diagnostic characters of FPDM-V-0009175, including thin (0.44 mm) shell, smooth external surface, non-branching and narrow pore canals with relatively constant width, three structural layers, oblique crystal orientation from vertical in the external layer, and mammillary to continuous to external layer thickness ratio of 1:1:0.44. These characters allow assignment of FPDM-V-0009175 to a new oogenus and oospecies, Plagioolithus fukuiensis, and suggest it belonging to a bird. The three-layered eggshell structure is seen in extant and extinct birds, Plagioolithus fukuiensis, and non-avian theropods. Therefore, such structure may be plesiomorphic among theropods, appearing in the late Barremian or earlier. As the first bird body fossil from the Mesozoic of Japan, Plagioohlithus fukuiensis suggests extensive distribution and abundance of birds in the Barremian East Asia. Plagioolithus fukuiensis indicates that the late-Barremian birds inhabited and reproduced in the fluvial environments within a basin located along the eastern margin of the Asian continent.http://www.zoobank.org/urn:lsid:zoobank.org:pub:90A8CCE8-57F7-473C-855C-B2CCE2BBD1A2

An experimental test in Mallards (Anas platyrhynchos) of the effect of incubation and maternal preen oil on eggshell microbial load

Microbial infection is one of the main factors reducing survival in the first stages of life in oviparous species, and recent studies have shown that the avian eggshell harbors an important variety of microorganisms that can rapidly multiply and penetrate the shell, leading to a decrease in hatchability. Here, we report the results of an experiment in which we examined how incubation and maternal preen oil affect the growth of avian eggshell microbes, using the Mallard (Anas platyrhynchos) as a model species. We compared the bacterial and fungal loads on the shell of non-incubated eggs and eggs incubated by females having free or blocked access to their preen gland. An increase of eggshell bacterial loads was observed in all conditions, but bacterial growth was higher on the shell of incubated eggs than on non-incubated eggs. We did not find any significant difference in eggshell bacterial growth for eggs incubated by females with free or blocked access to their preen gland. In addition, fungal growth during our experiment was not affected by incubation or the mother’s preen oil. Our findings are in contrast with those of previous studies which showed that incubation limited or had no effect on eggshell bacterial growth. Differences in environmental conditions and/or species ecology may explain the difference between the results of our experiment and those of previous studies. Our study provides the first data on the effect of maternal preen oil on eggshell microorganisms, showing that preen oil does not limit eggshell microbial growth.

Ovocalyxin-36 Is a Pattern Recognition Protein in Chicken Eggshell Membranes

The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).

Bird fossils from Ankilitelo Cave: inference about Holocene environmental changes in southwestern Madagascar.

The identifications of non-permineralized fossil bird bones recovered from Ankilitelo Cave in southwestern Madagascar are presented. Among the more than 560 elements recovered, 29 different taxa were identified, the vast majority being species that still occur in this region of the island. Eggshell remains from the extinct elephant bird (Family Aepyornithidae) and assigned to Aepyornis sp. were found at the site. Two identified extant taxa, Scopus umbretta and Monias benschi, no longer occur in the area immediately surrounding the cave. The available radiocarbon measurements of collagen from fossil bird bones and avian eggshell carbonate of recovered from the cave range from 13,270 Cal yr BP to modern times. Hence, the presumed ecological shifts that took place resulting in the disappearance or range contractions of these three taxa is within the Holocene and are presumed to be associated with natural climatic change and in more recent centuries associated human pressures. Information is also presented on the origin of guinea fowl (Numida) and inference on the period of colonization of Corvus albus on Madagascar.

Understanding control of calcitic biomineralization—Proteomics to the rescue

The avian eggshell is one of the fastest calcifying processes known and represents a unique model for studying biomineralization. Eggshell strength is a crucial economic trait for table egg production, and ensures that a safe egg reaches the consumer kitchen. However, a common toolkit for eggshell mineralization has not yet been defined. In this issue, label-free MS-based protein quantification technology has been used by Sun et al. (Proteomics 2013, 13, 3523-3536) to detect differences in protein abundance between eggshell matrix from strong and weak eggs and between the corresponding uterine fluids bathing strong and weak eggs. Proteins associated with the formation of strong eggshells are identified, which are now candidates for further investigations to define the regulatory relationship between specific eggshell matrix proteins and calcite crystal texture.

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label-free MS-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.

An Assessment of the Microbial Diversity Present on the Surface of Naturally Incubated House Wren Eggs

During ovipositioning, avian eggshells become susceptible to bacterial and fungal growth and studies have shown that a community of these microorganisms, or microflora, is maintained on eggshells throughout the incubation process. To determine the possible role of these microorganisms on embryonic development, it is first important to understand the composition of the microbial community present on the surface of the egg. A limited amount of studies have been published in this area; thus, the objective of this study was to broaden this area of study and determine what bacterial communities are found on the surface of naturally-incubated House Wren eggs across three stages of incubation (pre, early, and late) as defined by egg temperature. Our data uniquely suggest that the eggshell microflora is dynamic and that this may be regulated by temperature fluctuations due to intermittent incubation behavior. Using culture-based techniques, 46 different bacterial species were identified belonging to 13 bacterial families and 20 genera. The majority of bacteria belonged to the Pseudomonas, Staphylococcus, Stenotrophomonas, or Burkholderia genera and have been previously associated with avian eggs and nests. Bacteria within the Pseudomonas genus were the most predominant and we hypothesize that their maintenance may be linked to their ability to produce antibiotic substances called bacteriocins. The bacterial composition of the microflora isolated in this study also suggests that avian egg microfloras are derived from environmental origins.

Calcium mobilization from the avian eggshell during embryonic development

The avian eggshell acts as the major source of calcium for embryogenesis in bird. Using scanning electron microscopy, the inner eggshell surface was analysed before and after embryonic development and its accompanying calcium removal. This was done in eggs from two bird species with different growth rates and modes of development, the precocial Japanese quail Coturnix japonica and the altricial starling Sturnus vulgaris. Next, enzyme histochemistry was used to localize carbonic anhydrase in the extra-embryonic chorioallantoic membrane to provide support for the hypothesis that calcium may be released from the eggshell by means of acidification through the action of carbonic anhydrase. However, neither in the precocial quail nor in the altricial starling could any staining for carbonic anhydrase be detected. These results lead us to the conclusion that the role of carbonic anhydrase in embryonic calcium mobilization from the avian eggshell remains unclear.

SINGLE STEP SINTERED CALCIUM PHOSPHATE FIBERS FROM AVIAN EGG SHELL

Different forms of calcium-phosphate (Hydoxyapatite, α-TCP, β-TCP, CDHA) minerals are found to be major component of bone tissue. Development of calcium-phosphate (CaP) based fibrous microstructures is of significant research interest worldwide owing to its improved mechanical properties and higher interconnectivity. Here we represent a method for single step sintered wet-spun Fibers of calcium phosphate from avian egg shells for biomedical applications. Raw egg shell powder was mixed with chitosan solution and Phosphoric acid. The mixture is milled in a ball mill overnight and then filtered. The slurry was de-aired using 100 microliter 1-octanol per 100 ml of slurry as antifoaming and wet spun in coagulation bath. Fiber was dried overnight and sintered at different temperatures for microstructure and phase analysis. Both green and sintered Fibers were physico-chemical characterized by SEM, EDX, XRD, TGA, DSC, FTIR, and stereo-zoom microscopy. The fibers obtained in this procedure are found to have highly porous interconnected structures which can provide good cell adhesion and therefore can be used for bioactive scaffold making.

ZooMS: making eggshell visible in the archaeological record

Avian eggshell is a common component of many archaeological deposits, but its archaeological potential remains largely unexplored. The most obvious reasons are two-fold. Firstly, despite its abundance on many sites, eggshell is often overlooked during excavation. Even when it is recovered, small fragmented remains are difficult to identify taxonomically. Here we introduce a minimally destructive qualitative analytical technique for taxonomic identification of eggshell fragments based on highly sensitive mass spectrometry and peptide mass fingerprinting (ZooMS), and illustrate its application to eggshell recovered from the Viking Age urban site at Hungate, York. We adopt a more extreme version of the method of bleach treating used to prepare ancient eggshell for DNA analysis, followed by conventional peptide mass fingerprinting using MALDI-ToF mass spectrometry. The development of this technique will allow future research to make better use of eggshell fragments recovered from archaeological sites.

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell.

BackgroundIn Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation.ResultsA total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters.ConclusionsOur Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.

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.

A comparison of indices and measured values of eggshell thickness of different shell regions using museum eggs of 230 European bird species

The thickness of avian eggshells is used to assess shell quality in wild and domestic species, as an indicator of environmental pollution and as an adaptive explanation for shell maculation. Both direct measurements and calculated eggshell thickness indices (ETI) are used in such research, yet this is the first study to quantify, across a large spectrum of bird families (and thus egg shapes), the correlation between measured thicknesses and ETI. Furthermore, few studies have quantified thickness variation across the entire length of the shell, although this variation may influence both gas transfer and embryonic development. We measured the thickness of 942 eggshells of 230 European bird species from the Class II material at the Natural History Museum, Tring, UK, both in the conventional manner, at the equator through the blowhole and, uniquely, after a single longitudinal, cut at its equator at the blunt and pointed ends. Over half of the samples revealed shell defects, cautioning against the indiscriminate use of museum specimens. Strong positive associations were found between species‐specific means of shell thickness with each other and also with ETI, especially those derived from Schönwetter's ‘Handbuch der Oologie’ method, validating the interspecific comparative use of ETI. Thickness measurements and ETI factors are provided for all 230 species. Eggshells were usually thinner at the blunt end (the location of the air sac) than at the equator, but of equal thickness in passerine eggs. This difference was greatest in species producing elongate eggs and suggests that there is a functional significance of shell thickness variation among species that requires further investigation.

Zinc-Coated Carbonate Apatite Derived From Avian Eggshell for Potential Use as Bone Substitute. Part I

To develop antibacterial bone substitutes derived from avian eggshell using microwave processing to convert natural calcium carbonate (CaCO3) into zinc-coated carbonate apatite (CHA). Zinc-coated carbonate apatite was prepared using domestic microwave and then characterized with scanning electron microscopy (SEM), x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and energy dispersive x-ray analysis (EDXA). The trial animal study was conducted by filling different bone substitutes into 5-mm-diameter standard defects on the parietal bone of New Zealand rabbits to observe new bone formation for 8 weeks. SEM, XRD, FT-IR, and EDXA confirmed characteristics of zinc-coated CHA derived from avian eggshell using microwave method. Histological analysis demonstrated that the defects filled with carbonate apatite had more calcified bone and less uncalcified bone formation than other groups. The present research demonstrated the efficient use of microwave in the conversion of natural CaCO3 into CHA with zinc coating. The examined properties showed the potential use of the new material as an antibacterial bone substitute in oral surgery. The trial animal study implied favorable osteoconductive ability of carbonate apatite in new bone formation.

Why are birds' eggs colourful? Eggshell pigments co-vary with life-history and nesting ecology among British breeding non-passerine birds

The colourful appearance of bird eggshells has long fascinated biologists and considerable research effort has focused on the structure and biochemistry of the avian eggshell matrix. The presence of tetrapyrrole pigments was identified nearly a century ago. Surprisingly, how the concentrations of avian eggshell pigments vary among related species, and whether this variability is associated with either eggshell appearance and/or species life-history traits, remains poorly understood. We quantified the concentrations of the two key eggshell pigments, protoporphyrin IX and biliverdin, from a diverse sample of eggshells stored at the Natural History Museum, Tring, UK. We explicitly tested how these two pigments are associated with physical measures of eggshell coloration and whether the pigment concentrations and colour diversity co-vary with phylogenetic affiliations among species. We also tested a series of comparative hypotheses regarding the association between the concentrations of the two pigments and specific life-history and breeding ecology traits. Across species, the average concentrations of protoporphyrin and biliverdin were positively correlated, and both strongly co-varied with phylogenetic relatedness. Controlling for phylogeny, protoporphyrin concentration was associated with a higher likelihood of cavity nesting and ground nesting, whereas biliverdin concentration was associated with a higher likelihood of non-cavity nesting habit and bi-parental provisioning. Although unlikely to be explained by a single function, the breeding ecology and life history-dependence of eggshell pigment concentrations in these comparative analyses implies that related species share pigment strategies, and that those strategies relate to broad adaptive roles in the evolution of variation in avian eggshell coloration and its underlying mechanisms.

Proteomic analysis provides new insight into the chicken eggshell cuticle

The cuticle is the outermost layer of the avian eggshell, whose protein constituents remain virtually unknown. We hypothesize that cuticle components play a major role in microbial resistance, since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination. In this study we extracted proteins from the outermost non-calcified layer of the cuticle of chicken eggs and subjected them to LC/MS/MS proteomic analysis. We identified 47 cuticle proteins with high confidence and reproducibility. Two proteins, similar to Kunitz-like protease inhibitor and ovocalyxin-32 (a carboxypeptidase A inhibitor), were the most abundant of the cuticle proteins. A number of proteins known to have antimicrobial activity in the egg were detected (lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor) as well as possible new candidates (myeloperoxidase, ovocalyxin-36 and members of the SERPIN family). This is the first comprehensive report of cuticle proteome, a starting point to determine cuticle function and the molecular basis of its antimicrobial properties.