Calcified Eggshells Research Articles

Electrolytic co-deposition of Zn-ZnO and Zn-ZnO-CaCO<sub>3</sub> composite substrates on low-carbon steel

Conventionally employed zinc-rich paints and coatings for marine corrosion protection of steel still comprise environmentally unfriendly compounds such as zinc phosphate. This paper is focused on developing eco-friendly zinc ternary composite coatings of Zn-ZnO-CaCO3 utilizing CaCO3 additives derived from tympanotonus fuscatus (periwinkle) shell and calcareous eggshell respectively on low-carbon steel using electrodeposition technique. This is aimed at improving not only the surface corrosion resistance of steel structures in marine environment but also the sustainability of raw materials and minimal environmental pollution. Corrosion studies of uncoated and coated steel samples in simulated marine environment were done using linear polarization technique. Results obtained suggested that CaCO3 (i.e., calcareous eggshell) additive in the composite coating significantly improved the corrosion performance of the substrate. Steel coated with calcareous eggshell additive displayed superior corrosion performance having highest polarization resistance, Rp (156090 Ω), lowest corrosion rate, CR (0.01 mm/year) and highest coating efficiency, Ceff (97%) as against those of uncoated, zinc oxide, and tympanotonus fuscatus shell additives after 30 minutes deposition time. Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) results of selected developed coatings indicated presence of a homogeneous surface morphology and a significant amount of calcium in the calcareous eggshell coating.

Multi-analytical characterisation of red colouration on decorated ostrich eggshells from the Middle Bronze Age in Bahrain

In the early 1960s, a Danish archaeological team excavated a group of mounds in Bahrain dating back to 2000 BCE. Among their finds were fragments of decorated ostrich eggshells that displayed rectangular engravings as well as red colouring on the outer surface and a uniform red tint on the inner surface. The unpigmented natural colouration of ostrich eggs is the result of adaptations to prevent overheating in the arid habitat, and therefore, the red hue was artificial. The aim of this research was to characterise this colouration. Elemental analysis revealed low iron content, ruling out the use of iron-rich pigments such as ochre, while analysis by molecular spectroscopy could not detect any compounds besides the characteristic constituents of the calcareous eggshell. Thermal experiments on modern eggshells, combined with chiral amino acid analysis, indicated that the red colour on the archaeological samples could have been due to exposure to moderate temperatures. This investigation provides valuable insights into the art and cultural practices of ancient Middle Eastern societies during the Middle Bronze Age, contributing to our knowledge of prehistoric archaeology.

Valorizing waste palm oil towards biodiesel production using calcareous eggshell based heterogeneous catalyst

This study aimed at transesterifying waste palm oil (WPO) into biodiesel employing metal oxides impregnated calcareous catalyst from discarded eggshells. Catalyst concentration, methanol to oil molar ratio, reaction time and temperature were optimized for biodiesel synthesis adopting the L16 Taguchi orthogonal array. One output response (biodiesel yield%) was measured for each trial. The signal-to-noise (S/N) ratio and analysis of mean (ANOM) were also used to find the ideal input parameter towards best output response. The analysis of variance (ANOVA) was additionally used to analyze the input factors on the response. Optimized conditions achieved were: catalyst concentration-7 wt%, methanol:oil-12:1, reaction time-180 min and reaction temperature-150 °C. Variation in catalyst concentration was observed as most significant factor on the biodiesel yield (∼94.5 %). Catalyst was reused for 6 cycles following the same experimental conditions and yield on 6th cycle is 70 %. Confirmation tests were used to validate the optimization result of the current study.

The eggshell-matrix protein gene OC-17 is functionally lost in the viviparous Chinese crocodile lizard.

Thickness reduction or loss of the calcareous eggshell is one of major phenotypic changes in the transition from oviparity to viviparity. Whether the reduction of eggshells in viviparous squamates is associated with specific gene losses is unknown. Taking advantage of a newly generated high-quality genome of the viviparous Chinese crocodile lizard (Shinisaurus crocodilurus), we found that ovocleidin-17 gene (OC-17), which encodes an eggshell matrix protein that is essential for calcium deposition in eggshells, is not intact in the crocodile lizard genome. Only OC-17 transcript fragments were found in the oviduct transcriptome, and no OC-17 peptides were identified in the eggshell proteome of crocodile lizards. In contrast, OC-17 was present in the eggshells of the oviparous Mongolia racerunner (Eremias argus). Although the loss of OC-17 is not common in viviparous species, viviparous squamates show fewer intact eggshell-specific proteins than oviparous squamates. Our study implies that functional loss of eggshell-matrix protein genes may be involved in the reduction of eggshells during the transition from oviparity to viviparity in the crocodile lizard.

A large and unusually thick-shelled turtle egg with embryonic remains from the Upper Cretaceous of China.

Turtle eggs containing embryos are exceedingly rare in the fossil record. Here, we provide the first description and taxonomic identification, to our knowledge, of a fossilized embryonic turtle preserved in an egg, a fossil recovered from the Upper Cretaceous Xiaguan Formation of Henan Province, China. The specimen is attributed to the Nanhsiungchelyidae (Pan-Trionychia), an extinct group of large terrestrial turtles (possibly the species Yuchelys nanyangensis). The egg is rigid, spherical, and is one of the largest and thickest shelled Mesozoic turtle eggs known. Importantly, this specimen allowed identification of other nanhsiungchelyid egg clutches and comparison to those of Adocidae, as Nanhsiungchelyidae and Adocidae form the basal extinct clade Adocusia of the Pan-Trionychia (includes living soft-shelled turtles). Despite the differences in habitat adaptations, nanhsiungchelyids (terrestrial) and adocids (aquatic) shared several reproductive traits, including relatively thick eggshells, medium size clutches and relatively large eggs, which may be primitive for trionychoids (including Adocusia and Carrettochelyidae). The unusually thick calcareous eggshell of nanhsiungchelyids compared to those of all other turtles (including adocids) may be related to a nesting style adaptation to an extremely harsh environment.

The first dinosaur egg was soft.

Calcified eggshells protect developing embryos against environmental stress and contribute to reproductive success1. As modern crocodilians and birds lay hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs. Known dinosaur eggshells are characterized by an innermost membrane, an overlying protein matrix containing calcite, and an outermost waxy cuticle2-7. The calcitic eggshell consists of one or more ultrastructural layers that differ markedly among the three major dinosaur clades, as do the configurations of respiratory pores. So far, only hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered; the paucity of the fossil record and the lack of intermediate eggshell types challenge efforts to homologize eggshell structures across all dinosaurs8-18. Here we present mineralogical, organochemical and ultrastructural evidence for an originally non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of in situ Raman spectra obtained for a representative set of hard- and soft-shelled, fossil and extant diapsid eggshells clusters the originally organic but secondarily phosphatized Protoceratops and the organic Mussaurus eggshells with soft, non-biomineralized eggshells. Histology corroborates the organic composition of these soft-shelled dinosaur eggs, revealing a stratified arrangement resembling turtle soft eggshell. Through an ancestral-state reconstruction of composition and ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those from other diapsids, revealing that the first dinosaur egg was soft-shelled. The calcified, hard-shelled dinosaur egg evolved independently at least three times throughout the Mesozoic era, explaining the bias towards eggshells of derived dinosaurs in the fossil record.

Structure and evolutionary implications of the earliest (Sinemurian, Early Jurassic) dinosaur eggs and eggshells

One of the fossil record’s most puzzling features is the absence of preserved eggs or eggshell for the first third of the known 315 million year history of amniote evolution. Our meagre understanding of the origin and evolution of calcareous eggshell and amniotic eggs in general, is largely based on Middle Jurassic to Late Cretaceous fossils. For dinosaurs, the most parsimonious inference yields a thick, hard shelled egg, so richly represented in the Late Cretaceous fossil record. Here, we show that a thin calcareous layer (≤100 µm) with interlocking units of radiating crystals (mammillae) and a thick shell membrane already characterize the oldest known amniote eggs, belonging to three coeval, but widely distributed Early Jurassic basal sauropodomorph dinosaurs. This thin shell layer strongly contrasts with the considerably thicker calcareous shells of Late Jurassic dinosaurs. Phylogenetic analyses and their Sinemurian age indicate that the thin eggshell of basal sauropodomorphs represents a major evolutionary innovation at the base of Dinosauria and that the much thicker eggshell of sauropods, theropods, and ornithischian dinosaurs evolved independently. Advanced mineralization of amniote eggshell (≥150 µm in thickness) in general occurred not earlier than Middle Jurassic and may correspond with a global trend of increase in atmospheric oxygen.

Understanding the transition from water to land: Insights from multi-omic analyses of the perivitelline fluid of apple snail eggs

Unlike most of the freshwater gastropod families, the family Ampullariidae includes members that exhibit both underwater and aerial oviposition, making it an ideal model for understanding mechanisms underlying the evolutionary transition from water to land. We applied SDS-PAGE and LC-MS/MS to analyse the proteome of the egg perivitelline fluid (PVF) of Marisa cornuarietis – an aquatic ovipositing ampullariid. Comparison with the reported PVF proteomes of two aerial ovipositing ampullariids (Pomacea canaliculata and P. maculata) showed that the three species all contain several major perivitellins that nourish the embryos. However, M. cornuarietis invests more heavily on immune-related proteins, which might be due to exposure to aquatic pathogens. Interestingly, only the PVF of out-of-water egg laying species have PV2 – a neurotoxin lethal to mice, and a calcium-binding protein which might be involved in the formation of calcareous eggshell. Integrated phylogenetic, evolutionary and gene expressional analyses detected the involvement of gene duplication, positive selection and neofunctionalisation in the formation of several major PVF proteins. Overall, our study provides multiple lines of evidence of adaptive evolution in the PVF proteins, and contributes to a better understanding of how aquatic gastropod ancestors invaded terrestrial habitats. SignificanceAerial egg deposition has evolved in several groups of animals, but except for Vertebrata little is known about the mechanisms underlying this critical evolution process. We compared aquatic and aerial egg laying apple snails to understand the molecular mechanisms enabling such a transition in egg laying habitat. We found that the composition of perivitelline fluid proteomes of underwater and aerial egg depositors was remarkably different, and then gene duplication and positive selection were responsible for the formation of such novel proteins than enabled the evolutionary transition.

Biomineralization in modern avian calcified eggshells: similarity versus diversity

ABSTRACTAvian eggshells are composed of several layers made of organic compounds and a mineral phase (calcite), and the general structure is basically the same in all species. A comparison of the structure, crystallography, and chemical composition shows that despite an overall similarity, each species has its own structure, crystallinity, and composition. Eggshells are a perfect example of the crystallographic versus biological concept of the formation and growth mechanisms of calcareous biominerals: the spherulitic—columnar structure is described as “a typical case of competitive crystal growth”, but it is also said that the eggshell matrix components regulate eggshell mineralization. Electron back scattered diffraction (EBSD) analyses show that the crystallinity differs between different species. Nevertheless, the three layers are composed of rounded granules, and neither facets nor angles are visible. In-situ analyses show the heterogeneous distribution of chemical elements throughout the thickness of single eggshell. The presence of organic matrices other than the outer and inner membranes in eggshells is confirmed by thermograms and infrared spectrometry, and the differences in quality and quantity depend on the species. Thus, as in other biocrystals, crystal growth competition is not enough to explain these differences, and there is a strong biological control of the eggshell secretion.

Characteristics of global organic matrix in normal and pimpled chicken eggshells

The organic matrix from normal and pimpled calcified chicken eggshells were dissociated into acid-insoluble, water-insoluble, and facultative-soluble (both acid- and water-soluble) components, to understand the influence of shell matrix on eggshell qualities. A linear correlation was shown among these 3 matrix components in normal eggshells but was not observed in pimpled eggshells. In pimpled eggshells, the percentage contents of all 4 groups of matrix (the total matrix, acid-insoluble matrix, water-insoluble matrix, and facultative-soluble matrix) were significantly higher than that in normal eggshells. The amounts of both total matrix and acid-insoluble matrix in individual pimpled calcified shells were high, even though their weight was much lower than a normal eggshell. In both normal and pimpled eggshells, the calcified eggshell weight and shell thickness significantly and positively correlated with the amounts of all 4 groups of matrix in an individual calcified shell. In normal eggshells, the calcified shell thickness and shell breaking strength showed no significant correlations with the percentage contents of all 4 groups of matrix. In normal eggshells, only the shell membrane weight significantly correlated with the constituent ratios of both acid-insoluble matrix and facultative-soluble matrix in the whole matrix. In pimpled eggshells, 3 variables (calcified shell weight, shell thickness, and breaking strength) were significantly correlated with the constituent proportions of both acid-insoluble matrix and facultative-matrix. This study suggests that mechanical properties of normal eggshells may not linearly depend on the organic matrix content in the calcified eggshells and that pimpled eggshells might result by the disequilibrium enrichment of some proteins with negative effects.

Comparative proteomics of matrix fractions between pimpled and normal chicken eggshells

Eggshell matrix can be dissociated into three matrix fractions: acid-insoluble matrix (M1), water-insoluble matrix (M2) and acid-water facultative-soluble matrix (M3). Matrix fractions from pimpled and normal eggshells were compared using label-free proteomic method to understand the differences among three matrix fractions and the proteins involved with eggshell quality. A total of 738 and 600 proteins were identified in the pimpled and normal calcified eggshells, respectively. Both eggshells showed a combined proteomic inventory of 769 proteins. In the same type of eggshell, a high similarity was present in the proteomes of three matrix fractions. These triply overlapped common proteins formed the predominant contributor to proteomic abundance in the matrix fractions. In each matrix fraction and between both eggshell models, normal and pimpled eggshells, a majority of the proteomes of the fractions were commonly observed. Forty-two common major proteins (iBAQ-derived abundance ≥0.095% of proteomic abundance) were identified throughout the three matrix fractions and these proteins might act as backbone constituents in chicken eggshell matrix. Finally, using 1.75-fold as up-regulated and using 0.57-fold as down-regulated cutoff values, twenty-five differential major proteins were screened and they all negatively influence and none showed any effect on eggshell quality. Overall, we uncovered the characteristics of proteomics of three eggshell matrix fractions and identified candidate proteins influencing eggshell quality. The next research on differential proteins will uncover the potential mechanisms underlying how proteins affect eggshell quality. Biological significanceIt was reported that the proteins in an eggshell can be divided into insoluble and soluble proteins. The insoluble proteins are thought to be an inter-mineral matrix and acts as a structural framework, while the soluble proteins are thought as intra-mineral matrix that are embedded within the crystal during calcification. However, the difference between matrix fractions is unknown. Cross-analysis of proteomic data of three matrix fractions from the same type of eggshell, uncovered triply overlapped common proteins formed the predominant contributor to proteomic abundance of any matrix fraction, and we suggested that abundance variance of some common proteins between the three matrix fractions might be an important cause of their solubility differences. Moreover, eggshell is formed in hen's uterus, and uterus tend to be considered as unique organ determining eggshell quality. By cross-analysis on proteomic data of three matrix fractions between two eggshell models, normal and pimpled eggshells, the differential proteins were screened as candidates influencing eggshell quality. And we suggested that the liver and spleen or lymphocytes might be the major organs influencing eggshell quality, because the most promising candidates are almost blood and non-collagenous proteins, and originated from above organs.

Estrogen levels influence medullary bone quantity and density in female house finches and pine siskins

Medullary bone, a non-structural osseous tissue, serves as a temporary storage site for calcium that is needed for eggshell production in a number of avian species. Previous research focusing primarily on domesticated species belonging to the Anseriformes, Galliformes, and Columbiformes has indicated that rising estrogen levels are a key signal stimulating medullary bone formation; Passeriformes (which constitute over half of extant bird species and are generally small) have received little attention. In the current study, we examined the influence of estrogen on medullary bone and cortical bone in two species of Passeriformes: the Pine Siskin (Spinus pinus) and the House Finch (Haemorhous mexicanus). Females of these species received either an estradiol implant or were untreated as a control. After 4.5–5months, reproductive condition was assessed and leg (femora) and wing (humeri) bones were collected for analysis using high-resolution (10μm) micro-computed tomography scanning. We found that in both species estradiol-treated females had significantly greater medullary bone quantity in comparison to untreated females, but we found no differences in cortical bone quantity or microarchitecture. We were also able to examine medullary bone density in the pine siskins and found that estradiol treatment significantly increased medullary bone density. Furthermore, beyond the effect of the estradiol treatment, we observed a relationship between medullary bone quantity and ovarian condition that suggests that the timing of medullary bone formation may be related to the onset of yolk deposition in these species. Further research is needed to better understand the precise timing and endocrine regulation of medullary bone formation in Passerines and to determine the extent to which female Passerines rely on medullary bone calcium during the formation of calcified eggshells.

Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates.

The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species.

An Introduction to Morphology of the Reproductive System and Anatomy of Hen’s Egg

The present study was designed to investigate the morphology of reproductive system and egg anatomy of the domestic hen (Gallus domesticus L.). The system consists of oviduct and ovary. The oviduct consists of infundibulum, magnum, isthmus, uterus and vagina which are sole distributor for making nutrition enriched egg. The anatomy of egg revealed that there are calcareous eggshell, shell membranes, egg white, vitelline membrane, egg yolk, and germinal disc. The fertilized egg showed a concentric circle around the nucleus known as blastoderm that contained area pellucida and area opaca whereas an unfertilized egg showed nucleus as white spot (blastodisc). Primordial germ cells (PGCs) are progenitor cells of ova and spermatozoa and they originate from the epiblast of the central part of the area pellucida. The microscopic structure of eggshell rendered leathery cuticle, fibrous matrix and shell membranes. The egg protects itself by its own mechanism from being injured and provides a complete diet for the developing embryo. DOI: http://dx.doi.org/10.3329/jles.v8i0.20133 J. Life Earth Sci., Vol. 8: 1-10, 2013

The effects of eggshell removal on embryonic skeletal development in the American alligator (Alligator mississippiensis) (921.7)

Embryonic archosaurs (crocodilians and birds) are known to sequester calcium from their heavily mineralized eggshell and use it to mineralise their developing skeleton. In order to test the importance of eggshell calcium to embryonic growth, we studied growth of embryos and hatchlings of the American alligator with and without the calcareous eggshell. The outer eggshell layer was manually removed early in incubation, and embryos (n=6) were sampled at weekly intervals until hatching. The experimental and sham‐handled control eggs were incubated at 30ºC and 100% relative humidity. Results show that experimental (“shell‐less”) embryos were significantly smaller in body mass and length than their clutch‐sibling controls. Under identical rearing conditions, experimental hatchlings did not demonstrate catch‐up growth despite ad infinitum diet and remained significantly smaller than control hatchlings. Observation of biting behavior revealed that experimental hatchlings had more compliant lower jaws and generated weaker bite forces. Our results suggest that yolk calcium reservoir is sufficient for healthy development, but eggshell calcium is required for normal embryonic growth. We posit that increased calcium absorption during development allows the embryo to hatch at larger size, which ultimately allows greater force development during locomotion and predation.

The location of protoporphyrin in the eggshell of brown-shelled eggs

Protoporphyrin has been identified as the main eggshell pigment in brown-shelled eggs. However, there has been some uncertainty concerning the distribution of the pigment within the shell (and cuticle) in brown-shelled eggs. Most previous studies have suggested that the bulk of the shell pigment is deposited in the cuticle of the shell. The present study measured the levels of protoporphyrin in intact eggshells and in shells from which the cuticle had been removed, using eggs from flocks at 3 different ages. This enabled the calculation of the relative amount of protoporphyrin in the calcareous eggshell and the cuticle layer of the eggshell. The majority of the protoporphyrin pigment was located in the calcareous part of the eggshell (80-87%) with a minority contained within the cuticle (13-20%). These findings suggest that studies focused on maintenance of shell color in brown-shelled eggs need to consider the stage of egg formation at which the reduction in pigment deposition is occurring.

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.

Pathological Microstructure of a Miocene Ostrich Eggshell from Asia

Abstract: Struthio linxiaensis is the oldest fossil ostrich from Asia till today. This paper analyzes the microstructure of the eggshell of Struthio linxiaensis. The SEM and light microscopic results show that a pathological structure occurred associated with the formation of pore canal. The feature of this structure is that crystals of the columnar layer are around the lower part of the pore canal, and formed a broad structure. This structure unit contracts upwards more severely than the canal and looks like a “garlic”, which was observed in amniotes eggshells for the first time. Many evidences indicate that this structure was deposited with the columns simultaneously. The radial section view suggests that the “garlic structure” is not a result of antiperistaltic waves in the avian oviduct. The most possible process of the “garlic structure” formation is the column units around the pore canals with imperfect acidifying when calcified, or have some correlations with the changes in the protein profiles of the organic matrix in the calcareous eggshell.

Calcium ATPase expression in the oviducts of the skink, Lampropholis guichenoti

Lampropholis guichenoti is an oviparous lizard that lays eggs with a calcareous outer shell. We used immunofluorescence microscopy to describe the occurrence and distribution of Ca2+ ATPase pumps in the uterus of L. guichenoti at different stages of the reproductive and egg-shelling cycles. Ca2+ ATPase pumps were not demonstrated by immunofluorescent techniques in any uterine tissue until egg-shelling had commenced and at least partly calcified eggs were in the uterus. During egg-shelling, Ca2+ ATPase pumps occur on the apical and baso-lateral surfaces of uterine epithelial cells, and those of associated shell glands in the stroma of the uterus. We conclude that Ca2+ ATPase pumps provide a major mechanism for deposition of the calcareous eggshell of L. guichenoti and that the pumps are up-regulated when required in the reproductive cycle. Furthermore, it is likely that specific calcium glands in the stroma of the uterus are involved in the rapid transport required for egg-shelling, but the differential contribution of luminal and glandular epithelial cells is not known.

Egg shape and size allometry in geckos (Squamata: Gekkota), lizards with contrasting eggshell structure: why lay spherical eggs?

Hard, highly calcified eggshells evolved several independent times during the history of amniotes. Because of phylogenetic conservatism of this trait, lineages in which closely related taxa differ in eggshell structure are rare. Four gekkotan families (Carphodactylidae, Diplodactylidae, Eublepharidae and Pygopodidae) have eggs with soft shells, while their close relatives (Gekkonidae) lay eggs with hard shells. Geckos thus offer a rare opportunity to compare the impact of the emergence of a hard eggshell on the economy of egg architecture. Because a sphere has the smallest surface area of all three-dimensional solids of a given volume, spherical eggs in geckos with hard eggshells reduce calcium investment and should therefore be advantageous. Here, we document that hard-shelled gekkonid eggs are indeed more spherical than those of the other gecko lineages. However, within gekkonids, small species lay more elongated eggs than larger species. We speculate that miniature gekkonid females, which lay larger eggs relative to body size compared with large gekkonids, produce elongate eggs in order to pass the egg through a limited pelvic opening.