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.

Biofabrication of allogenic bone grafts using cellularized amniotic scaffolds for application in efficient bone healing.

INTRODUCTION: The reconstruction/regeneration of human bone injuries/defects represents a crucial challenge due to the lack of suitable bio/immune compatible and implantable biological grafts. The available strategies represent implications of several types of grafting materials in the form of metals, synthetic, and various kinds of biological scaffolds; however, the lack of appropriate biological components required for activating and enhancing repair mechanisms at the lesion-site limits their wider applicability. METHODS: In this study, a unique approach for generating human osteogenic implantable grafts was developed using biofabrication technology. Using a gradient change of detergents and continuous agitation, developed a unique technique to generate completely cell-free amnion and chorion scaffolds. The absence of cellular components and integrity of biological and mechanical cues within decellularized human amnion (D-HAM) and chorion (D-HCM) were evaluated and compared with fresh membranes. Allogenic bone grafts were prepared through induction of human mesenchymal stem cells (hMSCs) into osteogenic cells on D-HAM and D-HCM and evaluated for their comparative behavior at the cellular, histological and molecular levels. RESULTS: The common decellularization process resulted in an efficient way to generate D-HAM and D-HCM while retaining their intact gross-anatomical architecture, surface morphology, extracellular matrix components, and mechanical properties. Both these scaffolds supported better growth of human umbilical cord blood derived MSCs as well as osteogenic differentiation. Comparative investigation revealed better growth rate and differentiation on D-HCM compared to D-HAM and control conditions. CONCLUSION: D-HCM could be used as a better choice for producing suitable allogenic bone grafts for efficient bone healing applications.

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.

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae).

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic ("yolk sac") placenta shows evidence of functions in yolk digestion and maternal-fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.

Placentation in watersnakes I: Placental histology and development in North American Nerodia (Colubridae: Natricinae).

As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin-section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia: Colubridae). The pre-ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid- to late-development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal-fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal-fetal exchange.

Patterns of inner chorion structure in Anastrepha (Diptera: Tephritidae) eggs.

The inner chorion structure of Anastrepha eggs from 16 species of various infrageneric taxonomic groups is described by scanning and transmission electron microscopy. The layers of the chorion, the outer egg membrane, are structurally similar. Furthermore, an additional trabecular layer (ATL) that exists in some species, together with other characteristics, facilitates the recognition of four patterns of chorion structuring: Pattern I, in which the ATL layer is absent, is found in Anastrepha amita, the Anastrepha fraterculus complex, Anastrepha obliqua, Anastrepha sororcula, Anastrepha suspensa and Anastrepha zenildae (fraterculus group), and Anastrepha bistrigata and Anastrepha striata (striata group); Pattern II in Anastrepha serpentina (serpentina group), Anastrepha grandis (grandis group) and Anastrepha pseudoparallela (pseudoparallela group), in which the ATL presents large open spaces with pillars; Pattern III, found in Anastrepha consobrina (pseudoparallela group), in which the ATL is composed of round cavities; and Pattern IV, found in Anastrepha alveata and Anastrepha pickeli (spatulata group), where the large ATL cavities are reticulated. Comparatively, the chorion structure in Anastrepha eggs is more complex than in eggs of other fruit flies, e.g., Bactrocera, Rhagoletis and Ceratitis.

Egg structure and ultrastructure of Paterdecolyus yanbarensis (Insecta, Orthoptera, Anostostomatidae, Anabropsinae).

The egg structure of Paterdecolyus yanbarensis was examined using light, scanning electron and transmission electron microscopy. The egg surface shows a distinct honeycomb pattern formed by exochorionic ridges. Several micropyles are clustered on the ventral side of the egg. The egg membrane is composed of an exochorion penetrated with numerous aeropyles, an endochorion, and an extremely thin vitelline membrane. The endochorion is thickened at the posterior egg pole, probably associated with water absorption. A comparison of egg structure among Orthoptera revealed that the micropylar distribution pattern is conserved in Ensifera and Caelifera and might be regarded as a groundplan feature for each group; in Ensifera, multiple micropyles are clustered on the ventral side of the egg, whereas in Caelifera, micropyles are arranged circularly around the posterior pole of the egg.

Better, Faster, Cheaper: Getting the Most Out of High-Throughput Screening with Zebrafish.

The field of toxicology is undergoing a vast change with high-throughput (HT) approaches that rapidly query huge swaths of chemico-structural space for bioactivity and hazard potential. Its practicality is due in large part to switching from high-cost, low-throughput mammalian models to faster and cheaper alternatives. We believe this is an improved approach because the immense breadth of the resulting data sets a foundation for predictive structure-activity-based toxicology. Moreover, rapidly uncovering structure-related bioactivity drives better decisions about where to commit resources to drill down to a mechanism, or pursue commercial leads. While hundreds of different in vitro toxicology assays can collectively serve as an alternative to mammalian animal model testing, far greater efficiency and ultimately more relevant data are obtained from the whole animal. The developmental zebrafish, with its well-documented advantages over many animal models, is now emerging as a true biosensor of chemical activity. Herein, we draw on nearly a decade of experience developing high-throughput toxicology screens in the developmental zebrafish to summarize the best practices in fulfilling the better, faster, cheaper goals. We include optimization and harmonization of dosing volume, exposure paradigms, chemical solubility, chorion status, experimental duration, endpoint definitions, and statistical analysis.

Comparative morphology on the egg chorion characters of some Noctuidae (Lepidoptera).

A comparative morphological analysis is provided for 70 species belonging to 59 genera of 13 subfamilies of Noctuidae from Ukraine. Diagnostic characters of the eggs for some species are identified. A comparison with representatives from other families of Noctuoidea is conducted. Some characters typical for separate species and general characters typical for most species are discussed.

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.

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon, Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re-emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).

Mitigation in Multiple Effects of Graphene Oxide Toxicity in Zebrafish Embryogenesis Driven by Humic Acid.

Graphene oxide (GO) is a widely used carbonaceous nanomaterial. To date, the influence of natural organic matter (NOM) on GO toxicity in aquatic vertebrates has not been reported. During zebrafish embryogenesis, GO induced a significant hatching delay and cardiac edema. The intensive interactions of GO with the chorion induces damage to chorion protuberances, excessive generation of (•)OH, and changes in protein secondary structure. In contrast, humic acid (HA), a ubiquitous form of NOM, significantly relieved the above adverse effects. HA reduced the interactions between GO and the chorion and mitigated chorion damage by regulating the morphology, structures, and surface negative charges of GO. HA also altered the uptake and deposition of GO and decreased the aggregation of GO in embryonic yolk cells and deep layer cells. Furthermore, HA mitigated the mitochondrial damage and oxidative stress induced by GO. This work reveals a feasible antidotal mechanism for GO in the presence of NOM and avoids overestimating the risks of GO in the natural environment.

Structure and functions of the placenta in common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales.

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe(3+)) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.

The chorion ultrastructure of ova of Lophius spp.

The chorion surface ultrastructure of unfertilized eggs of black anglerfish Lophius budegassa and white anglerfish Lophius piscatorius was examined by scanning electron microscopy. Species-specific differences were observed.

Chorioamniotic membrane senescence: a signal for parturition?

OBJECTIVE: Senescence is an important biological phenomenon involved in both physiologic and pathologic processes. We propose that chorioamniotic membrane senescence is a mechanism associated with human parturition. The present study was conducted to explore the association between senescence and normal term parturition by examining the morphologic and biochemical evidences in chorioamniotic membranes. STUDY DESIGN: Chorioamniotic membranes were collected from normal term deliveries; group 1: term labor and group 2: term, not in labor. Senescence-related morphologic changes were determined by transmission electron microscopy and biochemical changes were studied by senescence-associated (SA) ß-galactosidase staining. Amniotic fluid samples collected from both term labor and term not in labor were analyzed for 14 SA secretory phenotype (SASP) markers. RESULTS: Morphologic evidence of cellular senescence (enlarged cells and organelles) and a higher number of SA ß-galactosidase-stained amnion and chorion cells were observed in chorioamniotic membranes obtained from women in labor at term, when compared to term not in labor. The concentration of proinflammatory SASP markers (granulocyte macrophage colony-stimulating factor, interleukin-6 and -8) was significantly higher in the amniotic fluid of women in labor at term than women not in labor. In contrast, SASP factors that protect against cell death (eotaxin-1, soluble Fas ligand, osteoprotegerin, and intercellular adhesion molecule-1) were significantly lower in the amniotic fluid samples from term labor. CONCLUSION: Morphologic and biochemical features of senescence were more frequent in chorioamniotic membranes from women who experienced term labor. Senescence of chorioamniotic membranes were also associated with amniotic fluid SASP markers.

Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Six new species of Myrsidea Waterston, 1915 (Phthiraptera: Menoponidae) from New World jays of the genus Cyanocorax Boie (Passeriformes: Corvidae), with notes on the chorionic structure of eggs.

The only species of previously named Myrsidea Waterston, 1915 from Neotropical jays of the genus Cyanocorax Boie (Passeriformes: Corvidae), Myrsidea fallax Kéler, 1938 (type-host Cyanocorax cyanomelas Vieillot), is redescribed and six new species of lice in the genus Myrsidea are described: Myrsidea pseudofallax n. sp. [type-host C. c. chrysops (Vieillot)]; M. moriona n. sp. [type-host C. m. morio (Wagler)]; Myrsidea daleclaytoni n. sp. [type-host C. v. violaceus Du Bus de Gisignies]; Myrsidea lindolphoi n. sp. [type-host C. caeruleus (Vieillot)]; Myrsidea melanocyanei n. sp. [type-host C. melanocyaneus chavezi (Miller & Griscom)]; and Myrsidea cristatelli n. sp. [type-host C. cristatellus (Temminck)]. A key to the identification of both sexes of these seven species is provided. Immature stages of M. daleclaytoni n. sp. (all instars) and M. cristatelli n. sp. (nymph III) are described. External chorionic architecture of the eggs is described and illustrated for six Myrsidea spp. from corvine birds: M. picae (Linnaeus, 1758) ex Pica p. pica L.; M. cornicis (DeGeer, 1778) ex Corvus c. corone L.; M. isostoma (Nitzsch in Giebel, 1866) ex Co. f. frugilegus L.; M. interrupta (Osborn, 1896) ex Co. brachyrhynchus Brehm; M. fallax ex Cy. cyanomelas; and M. moriona n. sp. ex Cy. m. morio. This is the first review of the data on Myrsidea spp. infesting Neotropical Corvidae.

The structure and ultrastructure of the egg capsule of Brachyptera risi (Plecoptera, Nemouroidea, Taeniopterygidae) with some remarks concerning choriogenesis.

The organization of the egg capsule of the euholognathan stonefly, which represents the family Taeniopterygidae (Nemuroidea) was investigated using light and electron microscopy techniques. The presence of a complex, multilayered egg capsule, composed of a vitelline envelope, multilayered chorion, and extrachorion is described. The morphology of the eggshell of Brachyptera risi was compared with that of euholognthan and systellognathan egg coverings and the ground plan of the egg capsule in Plecoptera was discussed.

Remodeling of fetoplacental arteries in rats due to chronic hypoxia.

The increased fetoplacental vascular resistance due to chronic hypoxia cannot be explained by simple hypoxic vasoconstriction, as it sustains to some degree after recovery in normobaric environment. To verify a hypothesis that fetoplacental arteries undergo remodeling of their walls similar to remodeling of pulmonary arteries in hypoxic pulmonary hypertension, we used a model of the chronically hypoxic rat placenta. Han Wistar pregnant rats were exposed to 14-day hypoxia (10% of oxygen) during the 6th to 19th day of pregnancy. Chronic hypoxia elicited in both intraplacental (prelabyrinthine) and chorionic plate (insertion) arteries significant narrowing of their lumina. Irregular thickening of their adventitia due to an increase in collagen fibers as well as ground substance was observed; reticular fibers were fragmented. Because of remodeling of fetoplacental arteries, a model of chronically hypoxic rat placenta could simulate human preplacental hypoxia and consequent effects.

Adhesion mechanisms in European whitefish Coregonus lavaretus eggs: is this a survival mechanism for high-energy spawning grounds?

This study examined the potential biochemical and mechanical structures that may contribute to egg adhesion in European whitefish Coregonus lavaretus. Experiments showed that eggs from a population of C. lavaretus from Loch Eck remained non-adhesive in a solution chemically similar to ovarian fluid but became adhesive seconds after contact with water. Examination of the ultrastructure of the chorion showed that the morphology changed significantly after contact with water, with nodule-like protuberances attached to connective filaments on the surface present in water-hardened but not non-water hardened eggs. Biochemical analysis showed the presence of Chain A, RNase ZF-3e proteins in the chorion of water-hardened but not non-water hardened eggs. Histochemical staining of the chorion of C. lavaretus eggs showed that the externa, but not the interna, stained positively for the presence of glycoproteins. From these results, it was concluded that C. lavaretus from Loch Eck possess both anatomical and biochemical adhesive mechanisms that have been undocumented in this species so far.