Rare eggshell structure in Odonata: Lindenia tetraphylla (Van der Linden) (Anisoptera: Gomphidae).

Lindenia tetraphylla (Van der Linden, 1825) eggs exhibit an egg structure that is very rare in other Gomphidae species. They have a well-developed surface reticulation structure. The anterior pole of the egg has a small, rounded micropylar area consisting of seven orifices arranged radially around a central area. The posterior pole has a sessile, truncated cone that carries 55-65 coiled filaments. The filament structure found at the posterior pole of the egg has been observed in the gomphid species Lestinogomphus africanus (Fraser, 1926), Ictinogomphus australis (Selys, 1873), and I. rapax (Rambur, 1842). However, L. tetraphylla eggs differ from these species in both morphology and filament structure. This study provides a detailed analysis of the ultrastructure of L. tetraphylla eggs using scanning electron microscopy, and the functional and taxonomic significance of the eggshell are discussed. RESEARCH HIGHLIGHTS: The aim of this study is to examine the ultrastructure of the L. tetraphylla eggshell, emphasizing its function and taxonomic value. In this context, the general morphology of the egg, the reticulations on its surface, the micropylar region and micropylar structure, and the posterior filament coil were examined. In this study, the ultrastructure of L. tetraphylla egg was examined for the first time using scanning electron microscopy (SEM). As a result of the examination, it was detected for the first time that the posterior filament coil, which is rarely seen in odonate eggs, is also present in L. tetraphylla eggs. By comparing the L. tetraphylla egg with the eggs of I. ferox, I. rapax, I. australis, and L. africanus species, which are similar in terms of the posterior filament coil, the features that distinguish the L. tetraphylla egg.

Effect of pearl guinea fowl eggshell ultrastructure and microstructure on keets hatchability.

Variability in shell structure is an evolutionary mechanism in birds that enables them to adapt to specific environmental conditions. This variability may also occur within the same species under the influence of individual indicators, such as the age or health status of females. While interspecies variation is quite obvious and easy to interpret, the reasons for intraspecies variation remain unclear. In this study, we examined the ultra- and microstructure of guinea fowl eggshells to identify the association between variations in shell structure and hatchability outcomes. We analyzed the visual differences between shells with low (L), intermediate (I), and high (H) external porosity using scale invariant feature transform analysis with NaturePatternMatch software. We found that the external pore image was closely related to the overall porosity of the shell before incubation. The total pore area, total porosity, and diffusion index (GH2O) were highest in group H shells (P < 0.001). Posthatching shells were characterized by an increased diameter and total surface area, decreased pore number (P < 0.001), as well as shortened mammillary layer (P < 0.001) and decreased total consumption of mammillary knobs (P < 0.001). The porosity indices of posthatching H shells had intermediate values between L and I. Although the effect of shell structure parameters on hatching was not confirmed, we assumed that all categories (L, I, and H) of shells were ideal for incubation. This suggests that the shell structure adapts to the metabolic rate of developing embryos; however, differences in shell structure affect the duration of incubation and synchronization of hatching. Both L and H shells showed delayed and prolonged hatching. Therefore, we recommended that guinea fowl eggs with different external porosity parameters should be incubated separately for better hatching synchronization. Differences in GH2O between L, I, and H eggs suggest that the shell porosity characteristics of guinea fowl eggs may be a key determinant of the rate of water loss during storage before incubation.

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae).

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.

Effects of ecofriendly synthesized calcium nanoparticles with biocompatible Sargassum latifolium algae extract supplementation on egg quality and scanning electron microscopy images of the eggshell of aged laying hens.

In the present study, 200 Brown commercial egg-type layers (60 wk old) were used to study the effects of different levels of ecofriendly synthesis of calcium (Ca) nanoparticles (0.0, 0.50, 1.0, and 1.5 g/kg diet) with biocompatible Sargassum latifolium algae extract (SL-CaNps) on exterior egg quality traits, electronic microscopic view of eggshells, Ca and phosphorus (P) retention, serum Ca and P concentrations, and the histology of the uterus. Hens fed with dietary SL-CaNps powder had higher egg weight and shell weight % values than those of the control group. All SL-CaNps treatment groups had the greatest values of shell weight per unit surface area and shell thickness. Dietary supplementation of SL-CaNps at graded levels up to 1.5 g/kg diet had higher serum Ca and inorganic P levels than that of the control. Laying hens fed with SL-CaNps-added diets had beneficial effects on shell ultrastructure in terms of well-developed palisade and mammillary layers. The numbers of apical cells along the branched tubular gland were greater in SL-CaNps-treated groups than those of control. Conclusively, supplementing SL-CaNps powder up to 1.5 g/kg to the diet of laying hens improved eggshell thickness, shell weight% and shell weight per unit surface and has no adverse effect on their eggshell quality or electronic microscopic view of their eggshell.

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit.

BACKGROUND: The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents during eggshell biomineralization. Main Body. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. CONCLUSION: With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

Ultrastructure of eggshell and embryological development of Salvator merianae (Squamata: Teiidae).

The objective of this study was to characterize the external morphology of Salvator meriane embryos in different stages of embryonic development and establish a relationship with the ultrastructure of the shell from oviductal transit to hatching. A total of 120 embryos were analyzed to describe their external morphology, and 78 eggs were used for the analysis of the shell. For embryonic development, the series was established according to the total length of the body. We established 40 embryonic stages from the primitive streak. In the early stages, the external morphological features are the C-shaped body, the maxillary, and mandibular fusion processes with the frontal process and the fusion of the forelimb with the digital plate. In the middle stages, the eyelid appears, and there are claws on the toes, cornification of fingers, and the onset of pigmentation. The last stage of embryonic development is characterized by the beginning of the formation of the scales, appear the toenails, and finalize the entire pigmentation. Regarding the relationship that exists with the ultrastructure of the egg during development, it was possible to observe a marked change in the composition of the shell and well-marked compaction during embryonic development, which may be related to the transport of calcium during embryonic ossification. Our results allowed us to show the complete sequence of embryonic development, determining the laying stage for this species. It was possible to establish a relationship with the ultrastructure of the eggshell from the oviductal transit to the moment of hatching.

Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate.

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO[Formula: see text]), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization.

Scattering of ultraviolet light by avian eggshells.

Eggshells are essential for the reproduction of birds since the optical properties of shells may have an impact on biological functions such as heating and UV protection, recognition by parents or camouflage. Whereas ultraviolet reflection by some bird eggshells has been recently described, its physical origin remains poorly understood. In this study, we identified a porous structure in eggshells. Using Mie scattering modelling, we found it was most likely responsible for reflectance peaks (intensities of ca. 20-50%) observed in the near-UV range. These peaks were observed by spectrophotometric measurements from eggshells of several breeds of hen, one breed of duck and one breed of quail. This optical response was interpreted in terms of the distinct visual perception of hens and humans: eggshells appearing achromatic for humans proved to be chromatic for hens. Fluorescence emission from these eggs was also characterised and attributed to the presence of protoporphyrin IX and biliverdin IXα in the shells. Electron microscopy observations revealed the presence of pores within the so-called calcified shell part (i.e., at depths between ca. 20 µm and ca. 240 µm from the eggshell's outer surface). Mercury intrusion porosimetry allowed us to quantify the pore size distribution. Simulations of the UV response of this porous structure using Mie scattering theory as well as an effective approach accounting for multiple scattering indicate that these pores are responsible for the backscattering peaks observed in the UV range, in the case of beige hen eggshells. Due to the similarities between the pore size distributions observed for beige hen eggshells and other investigated poultry eggshells, we expect Mie backscattering to be the origin of the UV response of the eggshells of many other bird species.

The shell gland in laying and natural moulting commercial egg-type chickens: A histomorphological and ultrastructural study.

The purpose of the present study was to determine the histological and ultrastructural changes in the luminal epithelium of the shell gland associated with natural moulting. Samples of the shell gland from laying (32 weeks old) and moulting (75 weeks old) hens were studied using histological, histochemical and electron microscopic techniques. In addition, TUNEL was used to demonstrate the distribution of apoptotic cells in the luminal epithelium of the shell gland. Autophagy, characterized by the presence of autophagosomes and autolysosomes, was evident in the early stages of degeneration in non-ciliated, ciliated and mitochondrial cells. The intermediate and advanced stages of regression in non-ciliated as well as mitochondrial cells occurred via apoptosis, while both apoptotic and necrotic ciliated cells were observed during the later stages of degeneration. The results of the present study suggest that a synergy of autophagy, apoptosis and necrosis is involved in the involution of the shell gland during natural moulting.

Organic trace minerals improve eggshell quality by improving the eggshell ultrastructure of laying hens during the late laying period.

The objective of this study was to investigate the effects of low inclusion levels of organic trace minerals (iron, copper, manganese, and zinc) on performance, eggshell quality, serum hormone levels, and enzyme activities of laying hens during the late laying period. A total of 405 healthy hens (HY-Line White, 50-week-old) were randomly divided into 3 treatments, with 9 replications per treatment and 15 birds per replication. The dietary treatments included a basal diet supplemented with inorganic trace minerals at commercial levels (CON), a basal diet supplemented with inorganic trace minerals at 1/3 commercial levels (ITM), and a basal diet supplemented with proteinated trace minerals at 1/3 commercial levels (TRT). The trial lasted 56 D (8 wk). Compared with the CON group, the ITM group showed decrease in (P < 0.05) egg production, eggshell strength, eggshell palisade layer, palisade layer ratio, serum estrogen, luteinizing hormone, glycosaminoglycan concentration, and carbonic anhydrase activity and increase in (P < 0.05) egg loss and mammillary layer ratio. However, the TRT group almost kept all the indices close to the CON group (P > 0.05). Furthermore, hens fed with low inclusion levels of organic trace minerals had smaller mammillary knobs (P < 0.05) than those in the CON and ITM groups. In conclusion, hens fed with low inclusion levels of proteinated trace minerals had better performance and eggshell strength than those fed with identical levels of inorganic compounds; organic trace minerals improved eggshell quality by improving the eggshell ultrastructure of laying hens during the late laying period.

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy.

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.

Comparative study of eggshell antibacterial effectivity in precocial and altricial birds using Escherichia coli.

In this study, we compared the antibacterial effectivity of the eggs of six precocial and four altricial bird species using Escherichia coli, based on their eggshell traits. The ultrastructure of eggshell was observed using a scanning electron microscope (SEM). According to SEM results, eggs from precocial birds (chicken, turkey, quail, duck, ostrich, and goose) had cuticle on the eggshells, while eggs from altricial birds (pigeon, budgerigar, munia, and canary) did not. The environment/selection pressure may induce the divergent evolution process in eggs of precocial and altricial birds. The E. coli experiment results showed that chicken, turkey, quail, duck, and goose eggs, with a high cuticle opacity, exhibited a much lower E. coli penetration rate. In contrast, the eggs with poor (ostrich) or without (pigeon, budgerigar, munia, and canary) cuticle exhibited a higher penetration rate. It is suggested that cuticle is a main barrier against bacterial penetration in precocial birds' eggs. Turkey and quail eggs showed the lowest E. coli contamination rate (3.33% and 2.22%, respectively), probably because of the tightly connected nanosphere structure on their cuticle. As for altricial birds' eggs, the eggs of budgerigar, munia, and canary with small pore diameter (0.57 to 1.22 µm) had a lower E. coli penetration rate than pigeon eggs (45.56%, 66.67%, 50%, and 97.78%, respectively, P < 0.05), indicating that pore diameter played a significant role in defending against bacterial trans-shell invasion. We found that eggshell thickness and pore area decreased with egg size. The cuticle quality had no relationship with egg size, but was closely related to the bird species. The E. coli penetration rate of altricial birds' eggs was significantly higher than that of precocial birds' eggs, mainly because the pores are exposed on the eggshell surface and cuticle protection is absent. This study provides detailed information on the eggshell cuticle, which gives insight into the cuticle evolution process that occurred in precocial and altricial bird species. Moreover, the results of E. coli penetration may help understanding the antibacterial behavior in birds.

Nanostructural basis for the gloss of chicken eggshells.

The nanostructure greatly contributes to eggshell formation, the mechanical properties of eggshells, and mineral dissolution during incubation. In this study, to investigate the effect of the nanostructure on the gloss of eggs, the gloss and eggshell quality (cuticle coverage, color, and thickness) of 105 eggs were measured. According to the order of the gloss, the surface roughness of 30 high-gloss and 30 low-gloss eggs was compared. The gloss had no significant correlation with the eggshell color and thickness (P > 0.05) and a significant relationship with the cuticle coverage (r = 0.19, P < 0.01). The surface roughness significantly differed between the high- and low-gloss eggs (P < 0.001), and the gloss was negatively correlated with the surface roughness (r(high-group) = -0.61, r(low-group) = -0.56, P < 0.01). The shell gloss of 30 oiled eggs with mineral oil and 30 normal eggs from commercial brown-egg layers was also compared. The oil coating increased the eggshell gloss, but the roughness was unchanged. This is the first report to establish the contribution of nanostructure for the gloss of chicken eggshell. The surface roughness can be used as an indicator of the gloss, which could be helpful for selective breeding to improve the eggshell brightness. Our research also provides the foundation for further investigation of the effect of non-pigmentary contributors on the chicken eggshell appearance.

Biomimetic mineralisation of eggshell membrane featuring natural nanofiber network structure for improving its osteogenic activity.

Designing reasonably priced bioactive organic-inorganic hybrid materials featuring osteogenic activity has received a significant interest in the bone tissue engineering field. In this study, eggshell membrane (ESM), which consists mostly of proteins exhibiting fibrous network structure, was utilised innovatively as a template to prepare eggshell membrane/hydroxyapatite (ESM-HA) composites using a versatile biomimetic mineralisation technique. In addition, the surface morphology and composition, hydrophilicity, crystallinity, and thermal stability of both sides of ESM and ESM-HA composites were systematically studied. Results indicated that both sides of ESM exhibited excellent biomimetic mineralisation ability, and the hydrophilicity and thermal stability of ESM were effectively improved by the introduction of HA. Moreover, in vitro experiments on MC3T3-E1 cells revealed that the inner side of the ESM was more beneficial to cell proliferation and adhesion than the outer side. Remarkably, the proliferation, adhesion and spreading, as well as the alkaline phosphatase (ALP) activity and expression of bone-related genes and proteins (runt-related transcription factor 2, ALP, collagen type I, and osteocalcin) on both sides of ESM-HA composites were significantly higher compared to those of the original ESM. These findings suggested that ESM-HA composites obtained using biomimetic mineralisation could be potential new materials for future bone tissue repair.

A novel application of nano eggshell/titanium dioxide composite on occluding dentine tubules: an in vitro study.

To synthesize Nano eggshell-titanium-dioxide (EB@TiO2) biocomposite and to evaluate its effectiveness in occluding opened dentine tubules. EB@TiO2 was synthesized and characterized using X-ray diffraction (XRD), and Transmission Electron Microscope (TEM). Sixteen simulated bovine dentine discs were prepared and randomly assigned into four groups according to the following treatment (n = 4): Group 1: No treatment; Group 2: eggshell powder; Group 3: EB@TiO2; Group 4: Sensodyne. These were then agitated in a solution of 1g powder and 40mL water for 3hours. Thereafter, each dentine discs from the respective groups were post-treated for 5 min with 2wt% citric acid to test their acid resistant characteristics. Scanning Electron Microscope (SEM) was used to observe the effectiveness of occluded dentine pre-and post-treatment. The cytotoxicity of the synthesized EB@TiO2 was tested using NIH 3T3 assay. ANOVA was used to evaluate the mean values of the occluded area ratio and the data of MTS assay. This was followed by a multi-comparison test with Bonferroni correction (α = .05). The XRD confirmed that EB@TiO2 was successfully modified through ball-milling. The TEM revealed the presence of both spherical and irregular particle shape powders. The SEM result showed that EB@TiO2 could effectively occlude open dentine tubules. Equally, the result demonstrated that EB@TiO2 exhibited the highest acid resistant stability post-treatment. NIH 3T3 assay identified that EB@TiO2 had little effect on the NIH 3T3 cell line even at the highest concentration of 100µg/ml. This study suggests that the application of EB@TiO2 effectively occluded dentine tubules and the occlusion showed a high acid resistant stability.

A novel application of nano eggshell/titanium dioxide composite on occluding dentine tubules: an in vitro study

Abstract To synthesize Nano eggshell-titanium-dioxide (EB@TiO2) biocomposite and to evaluate its effectiveness in occluding opened dentine tubules. EB@TiO2 was synthesized and characterized using X-ray diffraction (XRD), and Transmission Electron Microscope (TEM). Sixteen simulated bovine dentine discs were prepared and randomly assigned into four groups according to the following treatment (n = 4): Group 1: No treatment; Group 2: eggshell powder; Group 3: EB@TiO2; Group 4: Sensodyne. These were then agitated in a solution of 1g powder and 40mL water for 3hours. Thereafter, each dentine discs from the respective groups were post-treated for 5 min with 2wt% citric acid to test their acid resistant characteristics. Scanning Electron Microscope (SEM) was used to observe the effectiveness of occluded dentine pre-and post-treatment. The cytotoxicity of the synthesized EB@TiO2 was tested using NIH 3T3 assay. ANOVA was used to evaluate the mean values of the occluded area ratio and the data of MTS assay. This was followed by a multi-comparison test with Bonferroni correction (α = .05). The XRD confirmed that EB@TiO2 was successfully modified through ball-milling. The TEM revealed the presence of both spherical and irregular particle shape powders. The SEM result showed that EB@TiO2 could effectively occlude open dentine tubules. Equally, the result demonstrated that EB@TiO2 exhibited the highest acid resistant stability post-treatment. NIH 3T3 assay identified that EB@TiO2 had little effect on the NIH 3T3 cell line even at the highest concentration of 100µg/ml. This study suggests that the application of EB@TiO2 effectively occluded dentine tubules and the occlusion showed a high acid resistant stability.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. Hence, we show that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.

A comparative study of eggshells of Gekkota with morphological, chemical compositional and crystallographic approaches and its evolutionary implications.

The Gekkota is an important clade in the evolution of calcified eggshells in that some of its families lay rigid eggshells like archosaurs. However, the fundamental differences and similarities between the mechanism of rigid eggshell formation of the Gekkota and Archosauria have not been investigated thoroughly due to the lack of knowledge of gekkotan eggshells. Here, we report for the first time a comprehensive analysis of morphological, chemical compositional, and crystallographic features of rigid and soft gekkotan eggshells. Exhaustive morphological description provided common characters for gekkotan eggshells, as well as unique features of each species. We found that elemental distribution of rigid gekkotan eggshells is different from that of avian eggshells, especially in the case of Mg and P. In addition, the crystallographic features (size, shape, and alignment of calcite grains) of gekkotan eggshells are completely different from those of archosaur eggshells. The result of this study suggests that soft gekkotan eggshells are morphologically more similar to tuatara eggshells rather than soft eggshells of derived squamates. The chemical compositional analysis suggests that the eggshell may act as a mineral reservoir for P and F as well as Ca. More importantly, all chemical compositions and crystallographic features imply that the gekkotan eggshell formation may begin at the outer surface and growing down to the inner surface, which is opposite to the direction of the archosaur eggshell formation. This character would be crucial for identifying fossil gekkotan eggs, which are poorly known in paleontology. All these lines of evidence support that soft gekkotan and tuatara eggshells share the primitive characters of all lepidosaurid eggshells. Finally, gekkotan and archosaur rigid eggshells represent a typical example of convergent evolution in the lineage of the Sauropsida.

Nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell.

Avian (and formerly dinosaur) eggshells form a hard, protective biomineralized chamber for embryonic growth-an evolutionary strategy that has existed for hundreds of millions of years. We show in the calcitic chicken eggshell how the mineral and organic phases organize hierarchically across different length scales and how variation in nanostructure across the shell thickness modifies its hardness, elastic modulus, and dissolution properties. We also show that the nanostructure changes during egg incubation, weakening the shell for chick hatching. Nanostructure and increased hardness were reproduced in synthetic calcite crystals grown in the presence of the prominent eggshell protein osteopontin. These results demonstrate the contribution of nanostructure to avian eggshell formation, mechanical properties, and dissolution.