Dinosaur eggshell study using scanning electron microscopy

Visualization and analysis of structural features in fossil dinosaur eggs by scanning electron microscopy augment information from traditional petrographic light microscopy. Comparison of characteristics in fossil and modern eggshells allows inferences to be made regarding dinosaur reproductive biology, physiology, and evolutionary relationships. Assessment of diagenetic alteration of primary eggshell calcite structure that occurs during fossilization provides important information necessary for taxonomic identification and paleoenvironmental interpretations.     

蛋形耐压壳设计与分析

基于蛋壳几何学特性,开展了蛋形耐压壳结构设计与性能分析。根据鹅蛋形状扫描、宏观尺寸统计试验,研究蛋壳圆度、体积、表面积及蛋形系数分布规律,建立体积方程、表面积方程及蛋形函数;设计了4 km水深蛋形耐压壳,并定量分析其安全性、储备浮力、空间利用率、水动力学特性等性能;采用正交试验,研究长轴、短轴、厚度对蛋形耐压壳性能影响规律。结果表明,鹅蛋壳为高度对称结构,其蛋形系数符合均值为0.69的正态分布;蛋形耐压壳具有良好的综合性能,其中厚度是影响强度的重要因素,长轴次之,短轴最小;长轴是影响屈曲的重要因素,厚度次之,短轴最小;厚度是影响浮力系数重要因素,短轴次之,长轴影响最小;长轴是影响空间利用率的重要因素,短轴次之,厚度影响最小;水动力学特性主要受短轴影响。     

Fine structure and formation of the eggshell in scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae)

The morphology and formation of the eggshell in the scorpionfly Panorpa liui Hua were examined with light microscopy and scanning and transmission electron microscopy. During oogenesis of the scorpionfly, the follicle cells multiply by mitotic divisions and diversify into four morphologically distinct subpopulations, three of which are engaged in the eggshell formation and mold various parts of chorion. The eggshell consists of three layers: An inner vitelline membrane, an outer chorion, and a precursor extrachorion. The chorion constitutes a very compact endochorion, a rough fibrillar exochorion, and a polygonal meshwork of elevated ridges. At proximal end of ovarioles the chorion of matured oocyte is covered with a loose membrane, which might be the remnant of follicle cells. The jelly substance, which acts as lubricant to protect the oviducts and ovulated eggs during ovulation, might add to the top of polygonal ridges as the outermost extrachorion after oviposition. The eggshell formation process in Panorpa is tentatively proposed. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

The pore arrangement in the emu (Dromaius novæhollandiæ) eggshell as shown by plastic models

Pores arising in the cone layer merge with a plexus of tubules running just under and parallel to the outer surface of the emu eggshell. The outer surface of the shell is irregular due to dark green crystalline material arranged as flat domes. The plexus of tubules vent to the ambient environment via short pores opening in the valleys between these domes.     

The structure and ultrastructure of the egg capsules of stoneflies of the genus Isoperla (Insecta,Plecoptera, Perlodidae)

The egg capsules of five systellognathan stoneflies species representing the genus Isoperla (Plecoptera, Perlodidae) have been investigated using light and electron microscopes (SEM and TEM). We consider the structural modifications of egg coverings (egg capsules, eggshells) like: a shape of a capsule, presence of structures fixing an egg to the substratum under water, intrachorionic aeropylar system facilitating gas exchange as a factor adapting an egg/embryo to the environment. The structures protecting eggs against desiccation during a female flight before oviposition into water as well as against the dangers of external mechanical injury caused by turbulences in rapid water currents of mountain streams are described and discussed. The ground plan of the egg capsule in arctoperlarian stoneflies is also discussed.     

散养与笼养鸡蛋中稳定碳氮同位素特征研究

建立了元素分析-稳定同位素比值质谱（EA-IRMS）联用测定鸡蛋各组分的碳氮稳定同位素组成的方法,并初步探究了散养与笼养蛋鸡生产的鸡蛋的碳氮稳定同位素分布特征。结果表明：利用该方法测定鸡蛋各组分（蛋清、蛋黄、蛋壳膜）的碳氮同位素组成具有良好的重复性,SD值均不高于0.2‰（n=6）;鸡蛋各组分的碳氮同位素组成存在差异,各组分δ¹³C值从大到小的顺序依次为蛋壳膜＞蛋清＞蛋黄,各组分δ¹⁵N值从大到小的顺序依次为蛋黄＞蛋清＞蛋壳膜,与δ¹³C值的变化规律相反;各组分δ¹³C值之间呈线性相关（R²分别为0.768,0.761,0.640）,各组分δ¹⁵N值之间也呈线性相关（R²分别为0.862,0.875,0.711）。对比散养和笼养生产的鸡蛋,散养鸡蛋蛋清中δ¹³C和δ¹⁵N值的分布范围分别为-18.00‰~-14.97‰,3.02‰~4.37‰;笼养鸡蛋蛋清中δ¹³C和δ¹⁵N值的分布范围分别为-18.96‰~-15.98‰,1.66‰~2.68‰。可见,结合δ¹³C和δ¹⁵N值,能够有效地区分散养与笼养鸡蛋。     

Pollen morphology of Chinese Pseudostellaria (Caryophyllaceae) and its systematic significance

Using light and scanning electron microscopy, we studied the pollen morphology of six species (one of which are endemic) of Chinese Pseudostellaria. All species were studied for the first time. Results of this study indicated that pollen grains of Pseudostellaria are spheroidal or spheroidal‐polyhedral in shape and small or medium in size and pantoporate. Each pollen grain has 12–16 round pores. These pores are apart from each other by 5.77–7.73 μm and each has 7–13 granules in the operculum region. These features have important taxonomic significance. In addition, their pollen grains have thin exine with microechinate‐punctate‐perforate surface ornamentation. Pollen features do not support the traditional infrageneric classification of Pseudostellaria into two sections, but support that Pseudostellaria is closely related to other species of the Odontostemma clade.     

Morphology of the fertilizable mature egg in the Acanthopagrus latus,A. schlegeli and Sparus sarba (Teleostei: perciformes: sparidae)

Through appropriate scanning electron microscope (SEM) processing, the shape and the surface structures of just‐mature eggs are preserved integrally for observation and measuring to guarantee accurate comparison and prediction on the phylogenetic relationships among three species of teleost, i.e. Acanthopagrus latus, Acanthopagrus schlegeli and Sparus sarba of Sparidae. Eggs in all three species can be categorized as type III eggs. Specifically, a regular pattern is found in the arrangement of micropyle and its surrounding pore canals from the inspected surface structures of the mature eggs in three species. Other than these similarities in egg surface structures, several differences in micropyle structures, micropyle region and size of pore canals are also observed in eggs of respective species. Comprehension and accurate data are statistically computed and analyzed through hierarchical cluster analysis to provide solid species‐specific evidences and standardized empirical reference for accurate species identification and phylogeny determination.     

Egg envelopes of Armadillidium vulgare (Latreille, 1804) (Crustacea,Isopoda Oniscidea): Ultrastructure and lectins binding

The ultrastructural study carried out on (a) oocytes of Armadillidium vulgare during vitellogenesis, (b) mature eggs taken from the ovaries during the parturial moult of the posterior half of the body, and (c) fertilized eggs collected within a few hours of their release into the brood pouch, has clearly demonstrated that before the fertilization the chorion is the only envelope present in the egg of oniscidean isopods. In the mature eggs, the chorion appears as a uniformly electron‐dense lamina, about 0.4–0.5 µm thick, which does not show any specialized area. A second envelope, described by other authors as vitelline envelope, is formed above the oolemma only right after fertilization and appears separated from the chorion by a space full of liquid. The ways in which the genesis of this envelope is realized are not yet clear; it could be interpreted rather as a fertilization membrane. The investigations carried out with the aid of a battery of FITC‐lectins have highlighted the presence at the chorion surface of unfertilized eggs of various saccharide residues distributed in uniform way. No significant change was observed in the pattern of lectins binding to the chorion of eggs taken from the brood pouch, thus demonstrating how, after the fertilization, no significant rearrangement in the distribution of saccharide residues present on the egg surface occurs in A. vulgare. The ways in which, therefore, the recognition, the binding and the entry of the peculiar sperm of oniscidean isopods into the egg occur, still remain all to be deciphered. Microsc. Res. Tech. 79:792–798, 2016. © 2016 Wiley Periodicals, Inc.     

Comparative ultrastructure of the fertilized egg envelope in Corydoras adolfoi and Corydoras sterbai,Callichthyidae, Teleostei

In teleost, the structural characteristics of fertilized egg and egg envelope are very important for classification of genus or species. The structures of fertilized egg and egg envelope from Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Siluriformes in teleost were examined by scanning and transmission electron microscopes to confirm whether these morphological structures have specificities of species and family or not. The fertilized eggs of C. adolfoi and C. sterbai were non‐transparent, spherical, demersal, and strong adhesive. There were no structural differences between two species through the light microscope. The size of the fertilized eggs of C. adolfoi was 1.95 ± 0.03 mm (n = 20), and that of C. sterbai was 1.92 ± 0.03 mm (n = 20). The perivitelline space was almost not developed in both species. In both species, the adhesive protuberances structures were on the outer surface of egg envelope. And fibrous structures were specially located at attachment part of spawning bed. And the egg envelope consisted of two layers, an inner lamellae layer and an outer strong adhesive layer with high electron dense protuberances structures in cross section. Consequentially, the fertilized eggs, outer surface on the egg envelope and cross section of egg envelope have identical structure. So, these structural characteristics of fertilized eggs and egg envelope show genus Corydoras specificity.     

Enameloid microstructure of some Cretaceous Hexanchiformes and Synechodontiformes (Chondrichthyes,Neoselachii): New structures and systematic implications

Modern neoselachian sharks may be separated from more basal relatives by the presence of tooth enameloid comprising three layers. Although enameloid microstructure studies were mostly used in the aim of differentiating supposed basal neoselachians from hybodonts, differences in the enameloid organization among neoselachians have been recognized suggesting the potential for use of enameloid microstructure as a phylogenetic tool within the neoselachian sharks. The enameloid microstructure of five taxa of neoselachian sharks belonging to two orders, the Hexanchiformes and Synechodontiformes, has been studied. The Hexanchiformes are a monophyletic order with extant representatives, whereas the extinct Synechodontiformes have been considered as monophyletic, paraphyletic, or polyphyletic by different authors. This study has revealed numerous new enameloid microstructures such as amalgamated crystallites in the internal units [parallel‐bundled enameloid (PBE) and tangled‐bundled enameloid], cavities at the shiny‐layered enameloid (SLE)/PBE boundary, radial furrows in the PBE, as well as different original organizations at the base of the crown and in the PBE at the level of the cutting edges. Pachyhexanchus pockrandti (Hexanchiforme) and Welcommia bodeuri have the most of the features in common and they share more characters with those of Paraorthacodus sp. and Sphenodus sp. than with Synechodus sp. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Histochemical and structural characterization of egg extra‐cellular matrix in bufonid toads,Bufo bufo and Bufotes balearicus: Molecular diversity versus morphological uniformity

The extra‐cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta‐elimination PAS) and lectin‐histochemical (Con A, WGA, Succinyl‐WGA, PNA, RCA‐1, DBA, SBA, AAA, UEA‐I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J₁ and J₂). The fibers of J₂ are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N‐linked. In B. bufo there are also residuals of mannose and/or glucose and N‐acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J₁ layer of both species presents sialosulfoglycoproteins, mostly O‐linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J₂ layer is similar in composition to J₁ but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J₂. Several microorganisms, in particular blue algae, are observed in the J₂ layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types. Microsc. Res. Tech. 77:910–917, 2014. © 2014 Wiley Periodicals, Inc.     

The pore geometry and structure of the eggshell of the North Island brown kiwi,Apteryx australis mantelli

The total effective pore area of the eggshell of the North Island brown kiwi has been quantified and found to be only 27% of that allometrically predicted. This has been achieved by a reduction in the number of pores to 53% of allometric prediction and of individual pore area to 66%. A new technique for pore-casting has been devised using a low viscosity epoxy resin designed for crack-filling in concrete. Polished and weakly etched sections of the central layer of the shell have revealed an arrangement of calcite previously believed to be unique to the tinamou.     

OM,SEM–EDX,and micro‐FTIR analysis of the Bronze Age pottery from the Băile Figa salt production site (Transylvania,Romania)

This article presents the results of analyses carried out on a lot of 11 fragments of Bronze Age ceramics discovered in the site from B?ile Figa (Beclean town, Transylvania, Romania), where salt exploitation occurred. The samples were analyzed by means of optical microscopy, on the basis of which the morphology and distribution of the inclusions in the ceramic paste was established. Likewise, the samples were analyzed by scanning electron microscopy with energy‐dispersive X‐ray spectrometry, producing microphotographs for each sample, and on the basis of the X‐ray spectra, the elemental composition in gravimetric percentages was established. Through the micro‐Fourier‐transform infrared spectroscopy analysis, on the basis of characteristic group vibrations, it was possible to ascertain the nature of the compounds from the ceramic samples. These analyses will contribute to the reconstruction of the prehistoric technologies for salt exploitation.     

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross‐308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat‐stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat‐stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat‐stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry. Microsc. Res. Tech. 79:106–110, 2016. © 2016 Wiley Periodicals, Inc.     

Automated identification of neurons and their locations

Individual locations of many neuronal cell bodies (>10⁴) are needed to enable statistically significant measurements of spatial organization within the brain such as nearest‐neighbour and microcolumnarity measurements. In this paper, we introduce an Automated Neuron Recognition Algorithm (ANRA) which obtains the (x, y) location of individual neurons within digitized images of Nissl‐stained, 30 μm thick, frozen sections of the cerebral cortex of the Rhesus monkey. Identification of neurons within such Nissl‐stained sections is inherently difficult due to the variability in neuron staining, the overlap of neurons, the presence of partial or damaged neurons at tissue surfaces, and the presence of non‐neuron objects, such as glial cells, blood vessels, and random artefacts. To overcome these challenges and identify neurons, ANRA applies a combination of image segmentation and machine learning. The steps involve active contour segmentation to find outlines of potential neuron cell bodies followed by artificial neural network training using the segmentation properties (size, optical density, gyration, etc.) to distinguish between neuron and non‐neuron segmentations. ANRA positively identifies 86 ± 5% neurons with 15 ± 8% error (mean ± SD) on a wide range of Nissl‐stained images, whereas semi‐automatic methods obtain 80 ± 7%/17 ± 12%. A further advantage of ANRA is that it affords an unlimited increase in speed from semi‐automatic methods, and is computationally efficient, with the ability to recognize ～100 neurons per minute using a standard personal computer. ANRA is amenable to analysis of huge photo‐montages of Nissl‐stained tissue, thereby opening the door to fast, efficient and quantitative analysis of vast stores of archival material that exist in laboratories and research collections around the world.     

Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development

In the present paper we applied confocal microscopy and fluorescence technologies for studying the distribution and the oxidative activity of sea urchin (Paracentrotus lividus) mitochondria during development, by in vivo incubating eggs and embryos with cell‐permeant MitoTracker probes. We calculated, by a mathematical model, the intensity values, the variations of intensity, and the variation index of incorporated fluorochromes. Data demonstrate that mitochondrial mass does not change during development, whereas mitochondrial respiration increases. In addition, starting from 16 blastomeres stage, some regions of the embryo contain organelles more active in oxygen consumption.     

A method for the demonstration of pores in calcified eggs of vertebrates and invertebrates

Determination of pore number, pore size as well as pore canal distribution is important in understanding the biology of calcified eggs among vertebrates as well as invertebrates. Various techniques have been applied to elucidate pore structure of avian egg shells, including fracturing coupled with scanning electron microscopy and differential histochemical staining of the matrix. The best results reported have thus far been from plastic impregnated, etched specimens (Board & Tullett, 1974). The present report is a modification of this latter technique, utilizing the very low viscosity, highly penetrating electron microscopic embedding medium developed by Spurr (1969). This modification is used in this report of the previously undescribed pores from the egg shells of the giant South American land snail, Strophocheilus oblongus. These eggs, which can be up to 50 mm in length, have recently been investigated with respect to the calcium carbonate mineralogy (Tompa, 1976) as well as with respect to the matrix biochemistry Tompa et al., 1977). (Similar resin has been used successfully for the plastic impregnation and ultrathin sectioning of that region of the snail shell where the body muscles insert into the shell comumella (Tompa & Watabe, 1976).) This technique, which can be utilized on avian eggs or on any other calcified material in probing for pores, relies on the high penetrating ability of the resin as well as on its easy commercial availability around the world.     

Antennal and mouthpart sensilla of Acanthoscelides obtectus say (Coleoptera: Bruchidae)

Acanthoscelides obtectus, a serious pest with a worldwide distribution, damages stored seeds of Phaseolus sp. To acquire a better understanding of the sensory orientation of bean weevils, their antennal and palpal sensilla were investigated. The antennae and palps possess several types of sensory hairs: blunt‐tipped sensilla chaetica and trichoidea and also sharp‐tipped sensilla trichoidea, these last being the most numerous. Sensilla basiconica are present only on the flagellomeres, but Böhm bristles occur on the basal sclerite and scape of the antennae. The terminal segments of the maxillary and labial palps bear a group of sensilla styloconica with single apical pores. Sensilla coeloconica are located only on the terminal palpal segments. They were unable to detect any distinct morphological differences between males and females. The possible chemosensory, mechanosensory, and thermo‐/hygroreceptive functions of the sensilla are discussed.     

A comparative study of mud‐like and coralliform calcium carbonate gallbladder stones

To gain insight to underlying mechanism of the formation of calcium carbonate (CaCO₃) gallbladder stones, we did comparative study of stones with mud appearance and those with coralliform appearance. A total of 93 gallbladder stones with mud appearance and 50 stones with coralliform appearance were analyzed. The appearance, color, texture, and the detection of Clonorchis sinensis eggs by microscopic examination were compared between the two groups. Then, the material compositions of stones were analyzed using Fourier Transform Infrared spectroscopy and the spectrogram characteristics were compared. Moreover, microstructure characteristics of the two kinds of stones were observed and compared with Scanning Electron Microscopy. Mud‐like gallbladder stones were mainly earthy yellow or brown with brittle or soft texture, while coralliform stones were mainly black with extremely hard texture, the differences between the two groups was significant (p < .05). The analytic results of FTIR spectroscopy showed that 95.7% (89/93) of the mud‐like gallbladder stones were CaCO₃ stones, and mainly aragonite; while all of the coralliform stones were CaCO₃ stones, and mainly calcite (p < .05). Meanwhile, microscopic examination indicated that the detection rate of Clonorchis sinensis eggs in mud‐like CaCO₃ stones was lower than that in coralliform CaCO₃ stones (p < .05), and that in aragonite CaCO₃ stones was lower than that in calcite CaCO₃ stones(p < .05). Mud‐like CaCO₃ stones mainly happened to patients with cystic duct obstruction. Clonorchis sinensis infection was mainly associated with coralliform (calcite) CaCO₃ stones. Cystic duct obstruction was mainly associated with mud‐like (aragonite) CaCO₃ stones.