The accoutrements of spiders' eggs (Araneae) with an exploration of their functional importance

Analysis is presented of the characteristics of spheres coating the chorion surface of the eggs from 41 species belonging to 15 families of spiders. Examples of eggs from the arachnid orders Opiliones, Amblypygi and Uropygi are illustrated for the first time. The spheres are unique, among arachnids, to the Araneae. Their form and size class distributions overlap considerably between families and the spheres are present in all 22 families of Araneae that have been examined; they represent a very conservative morphological feature in the evolution of the Araneae. Evidence of specialized regions of the egg surface (micropyllar areas and plastrons) was found only in an opilionid. As spiders' eggs are laid in silk egg sacs, mostly in clutches, the possible effects of these characteristics are discussed from evidence in the literature and from a functional theoretic stance.     

Osmotic properties of the perivitelline fluid and some properties of the chorion of Atlantic salmon eggs (Salmo salar)

The chorion and perivitelline fluid of Atlantic salmon eggs were investigated by chemical and physical methods. The turgor pressure of the chorion in water hardened eggs was about 60 mm Hg. With newly stripped eggs in fresh water a similar pressure was achieved after about one day but the process of water uptake could be osmotically inhibited by adding high molecular weight substances to the external medium. Perivitelline fluid contained about 58 % water the remainder being a high molecular weight substance consisting mainly of protein but also containing significant quantities of carbohydrate and lipid. The functions of the chorion are discussed with reference to salmon eggs and also to marine pelagic fish eggs such as those of the plaice.     

Identification of cystatin as a component of carp chorion

An ovary-specific cystatin is immunocytochemically demonstrated to be localized in the chorions, cortical granules, and yolk granules of carp oocytes, as well as in the follicle cells surrounding oocytes. During cortical reaction, cystatin is exocytosed from cortical granules into the perivitelline space. In situ hybridization confirms that cystatin is synthesized by oocytes and follicle cells. Western blotting reveals that chorion cystatin appears in multiple bands of high molecular weight (from 65 kDa to larger than 200 kDa). No cystatin monomer of 14 kDa is found. These results indicate that chorion cystatin is conjugated with other chorion components. Two forms of conjugates are found. In one form, cystatin, ZP2, fibroin-like substance (FLS), and cathepsin-like substance (CLS) are conjugated, which is extracted by sodium dodecyl sulfate. In the other form, cystatin, FLS, and CLS are conjugated, which is extracted by guanidine thiocyanate (GTC). Most chorion cystatin of oocytes and ovulated eggs is solubilized by GTC, while a large amount of cystatin remains in the fertilization envelope of cortical reacted eggs after extraction by GTC. Mol. Reprod. Dev. 51:430–435, 1998. © 1998 Wiley-Liss, Inc.     

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Development of eggs,larvae and pelagic juveniles of three Indo-Pacific ostraeiid fishes (Tetraodontiformes):Ostracion meleagris,Lactoria fornasini andL. diaphana

The eggs, larvae and pelagic juveniles ofOstracion meleagris, Lactoria fornasini andLactoria diaphana were identified from reared and field collected specimens from Hawaii, Japan, Australia and the eastern Pacific. Eggs are large and pelagic with limited chorion ornamentation and a cluster of oil droplets. At hatching, larvae are well developed, rotund, and enclosed in a dermal sac. The sac disappears and dermal plates form prior to notochord flexion. Larvae of the three species can be distinguished by their pigment patterns and development of the carapace of ossified dermal plates. Eggs of the three species could not be distinguished. The larval stage ends at a small size (< 6 mm) but the juveniles may grow to a substantial size while remaining pelagic.L. diaphana matures and spawns while pelagic in the eastern Pacific.     

Fine structure of the egg shells of Habrophlebia fusca (curtis) and h. consiglioi Biancheri (Ephemeroptera : Leptophlebiidae)

The eggs of 2 mayflies, Habrophlebia fusca and H. consiglioi (Ephemeroptera : Leptophlebiidae) were observed with scanning and transmission electron microscopes. The external surface of the eggs in both species had longitudinally oriented costae. The chorion of H. fusca had different structures in its costal and intercostal zones. Three distinct layers could be recognized: an inner layer close to the vitelline coat, consisting of electron-dense lamellae perpendicular to the egg surface; an intermediate layer, consisting of loosely structured fibrillar material; and an outer highly electron-dense layer, consisting of 2 separate laminae, divided by an electron-transparent line. In the egg of H. fusca, the costal area of the chorion shows a columnar structure. The columns merge distally to create wide chambers. This organization has been observed with the SEM in H. consiglioi as well. The chambers are interconnected and communicate with the exterior through openings along the costal edges. Masses of mucus-like substance are present both in the chambers and outside the chorion; they show fibrillar material and electron-dense bodies with a paracrystalline structure.     

Oogenesis in Campodea sp. (Insecta,Diplura): Chorion formation and the ultrastructure of follicle cells

During advanced vitellogenesis the follicle cells of Campodea sp. are well developed and contain numerous electron-dense secretory vacuoles. In the postvitellogenic phase the contents of these vacuoles are released from follicle cells and give rise to a thin chorion on the oocyte surface. Concurrently, segregation of yolk spheres takes place in the ooplasm: small spheres migrate to the oocyte periphery and come to lie in the so-called peripheral zone of cytoplasm, whereas large spheres remain in the cell centre.     

L'action exercée par l'appareil endocrine sur les phases tardives de l'ovogenèse chez Locusta migratoria

Most studies dealing with the endocrine control of oogenesis are focused on vitellogenesis, but the later events of oogenesis: vitelline membrane formation and choriogenesis have received less atention. In Locusta migratoria, the corpora allata evidently control vitellogenesis, but seem to have no bearing upon the formation of the vitelline membrane or the chorion. If the insects are deprived of their CA at the time when the oocyte length is around 3.5 to 4 mm, (normal length at egg laying: 6.5 mm) vitelline membrane formation and the chorion production are unaffected. In the same species, the electrocoagulation of the pars intercerebralis or excision of corpora cardiaca disturb oogenesis; vitelline membrane material is synthesized and secreted, but the primary granules fail to join each other and to transform into vitelline membrane and the production of chorionic material is disturbed.The rER of the follicle cell seems quite active but the substance elaborated accumulates in the Golgi saccules, and therefore the cytoplasm contains only very few secretory granules. The implantation of supernumerary pars intercerebralis and corpora cardiaca into immature females induces precocious deposition of the vitelline membrane which is complete in 5.0 mm oocytes (instead of 5.6 to 6.0 mm) and chorion formation is completed in 5.5 mm oocytes (instead of 6.0 to 6.3 mm). Ecdysone is secreted in the ovaries at the end of oogenesis. We tested the result of injecting ecdysone to immature females. It results in hastening oocyte growth during 24 hr, but this accelerated growth is followed by degeneration.     

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the “naked” embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24 h post-fertilization (hpf) with survival rates of ≥ 90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24 h of development. Dechorionation of younger stages (< 24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~ 400,000 Dalton, but becomes strongly toxic after dechorionation.     

Study of the chorion of seasonal and non-seasonal Africa and Neotropical oviparous Cyprinodontiforme fishes

The structure of the chorion from the fertilized eggs of eight species of killifish, four Nothobranchiidae from tropical Africa and four Rivulidae species from South America and an Asian Cyprinidae (zebrafish), were investigated for possible structural similarities by transmission and scanning electron microscopy. The study of the possible chorion variations between seasonal fishes inhabiting temporary pools of tropical regions and other, non-seasonal species revealed variations in the several types of complex adornments found on the external chorion surface when compared with the less complex chorion of the zebrafish. The inner structure of the chorion of these killifish species comprises alternating electron dense and clear strata, with the number of strata varying by species as well as the thickness of the chorion. No obvious phylogenetic or ecological relationships were observed either between the chorion adornments and the inner organization of the chorion or between the seasonal and non-seasonal species. It is advised to perform studies with a larger sample of species that could show a relation either from ecological or phylogenetic parameters.     

Secondary structure of chorion proteins of the Lepidoptera Pericallia ricini and Ariadne merione by ATR FT-IR and micro-Raman spectroscopy

The gross morphological features of the eggs and eggshells (chorions) of two Lepidoptera species, Pericallia ricini and Ariadne merione were revealed for the first time by scanning and transmission electron microscopy. These two insect pests are extremely serious threats for many crops, mainly in India, but also in several other regions of the world. Micro-Raman and ATR FT-IR spectroscopy were also applied to study in detail the secondary structure of the eggshell (chorion) proteins of these Lepidoptera species. Both techniques indicate that the two species have nearly identical conformations of their chorion proteins with abundant antiparallel β-pleated sheet. These results are in support of our previous findings that the helicoidal architecture of the proteinaceous chorion of Lepidoptera and fishes is dictated by a common molecular denominator, the antiparallel β-pleated sheet secondary structure.     

Structure and enzymatic removal of the chorion of embryos of the Nile tilapia

Dechorionation or partial digestion of the egg chorion is necessary for introducing embryonic stem cells into blastulas for chimera production and for harvesting blastulas. Several methods to digest the Nile tilapia Oreochromis niloticus chorion were tried and it was found that the chorions of most clutches of eggs were digested in <3 h using hatching medium [produced by allowing embryos to hatch in Hanks balanced salt solution (HBSS) in an incubator], or 2 mg ml⁻¹ pronase P6911 in 10% Ca/Mg free HBSS. The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio . During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining chorion; however, chorion digestion has to be complete for experimental dechorionation, because digestion starts at the external surface. The zona radiata externa remained intact after experimental digestion with hatching media but was disrupted by pronase. Embryos dechorionated at the cleavage or blastula stage only survived for 2 or 3 days, but some dechorionated at the gastrula stage or early segmentation stage developed until the natural hatching time. If the chorion was partially digested at the cleavage or blastula stage, some embryos survived to hatch.     

Ovoviviparity and morphology of the female reproductive system in Palumbina (Lepidoptera,Gelechiidae, Thiotrichinae)

Ovoviviparity is reported for the genus Palumbina of Thiotrichinae. The females of eight species were found to retain a hatched first instar larva or a fully developed embryo in the oviduct (vestibulum), which suggests that the females may deposit larvae or mature eggs that are ready to hatch. In the mature embryos, the mandibles and a head capsule were visible and enclosed in chorion. Taking this into consideration, the embryos may contain sufficient nutrition for development within the eggshell, which indicates an ovoviviparous reproduction. The embryos in the oviduct were not dissolved while the early-stage embryos in the anterior portion of the abdomen were destroyed by the treatment of KOH. Therefore, the formation of chorion likely takes place in the oviduct. The female reproductive system of Palumbina displayed typical morphology of ditrysian Lepidoptera although it showed minor variations among the species. The paired reservoirs of the colleterial gland were rather reduced in the species that carried larvae. We assume that this might be related to the ovoviviparous reproductive physiology. The lagena of the spermatheca was present or absent, and if present, the shape and the size were various even in the same species. The species without lagena had a numerous thorn-like structures on the utriculus. The function is possibly related to the maintenance of sperm, but the correlation between the structure and the absence of the lagena remains unclear. Further detailed studies are required to reveal the comprehensive mechanism of the ovoviviparous reproduction in Palumbina.     

Some mechanical and morphological properties of the chorions of marine teleost eggs

The eggs of plaice, Pleuronectes platessa , lumpsucker, Cyclopterus lumpus , and capelin, Mallotus villuses , were investigated. Chorion strength was measured in three ways: by tearing isolated chorions, by bursting eggs, and by penetrating eggs with a fine needle. The results obtained showed that tearing resistance and bursting pressure (which both rise after activation of eggs in sea water) are closely related to one another, both reflecting resistance to circumferential tension. Penetration pressure (corresponding to resistance to radial penetration, i.e., hardness in the materials sense) also rises, but the time-scale of the rise differs from that of bursting resistance, particularly in the case of capelin eggs which become 'hard' in the sense that they resist high bursting loads, but have a 'soft' chorion which does not resist penetration. Differences in chorion strength between species, and alterations in chorion strength during egg development within species, are related to morphological features and changes.     

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.     

Ultrastructural diversity in the egg chorion of Hawaiian Drosophila and Scaptomyza: ecological and phylogenetic considerations

Formation of the egg shell (chorion) inDrosophila and Scaptomyza (Diptera : Drosophilidae) is a complex developmental process involving coordinated synthesis and secretion of multiple proteins by the monolayer of follicle cells surrounding the egg. Using scanning electron microscopy, the ultrastructure of the chorion in 37 endemic Hawaiian drosophilids, representing the genera Drosophila and Scaptomyza, were analyzed and compared with 7 representative species of continental Drosophila. The detailed structure of the chorion was described for 8 chorionic regions: the respiratory filaments, follicle imprints, operculum, micropyle, dorsal ridge, ventral rim, posterior pole, and the chorion cross-section. The morphology of each region is similar among related species, but strikingly different among groups. The main functions of the chorion are to protect the developing embryo from the vicissitudes of the environment and to provide channels for gas exchange during embryogenesis. Adaptation to the diverse ovipositional substrates used by Drosophila in general, and the Hawaiian species in particular, has resulted in extraordinary diversity in the various chorionic structures. The respiratory filaments differ in number and have evolved to different lengths and degrees of porosity. Furthermore, other regions also involved in respiratory exchange (the operculum, follicle imprints, the pole region, and the dorsal ridge) have diverged in parallel to the ecological divergence. The thickness and complexity of the outer endochorion are dramatically different in various groups, providing varying degrees of mechanical strength to the eggshell, which promotes embryonic survival in the diverse microenvironments. These varied chorionic structures have been found to provide useful morphological characters for phylogenetic analyses of the drosophilids.     

Trockengewicht und Kaloriengehalt sich entwickelnder Heringseier

Dry weight and caloric content of eggs from spring spawning western Baltic Sea herrings have been determined employing the bomb calorimetry technique. Water temperature throughout incubation was kept at 8.0°±0.2° C. Average dry weight and caloric content values of one pist fertilized egg are 0.170 mg (0.124 mg without chorion) and 0.748 cal (without chorion), respectively. The caloric content per egg decreases progressively during the incubation period (Fig. 2). The same holds for the dry substance of the egg (Fig. 1). Energy extraction efficiency related to dry weight is 67.8%, the efficiency related to dry organic substance 70.4%, and the caloric efficiency 63.7%.     

The lampbrush chromosomes of Salamandra salamandra (L.) (Amphibia Urodela)

Lampbrush chromosomes isolated from the germinal vesicle of medium sized oocytes can be individually identified by differences in two characters: (1) chromosome regions rich in well developed loops, and (2) number and position of spheres. Actually the lateral loops are not all equally extended, but those which are inserted in a certain region of the axis of some chromosomes are more developed and sometimes are loaded with dense and copious matrix; chiasmata do not occur inside these regions. One or more spheres are present on eight chromosomes in the complement (chromosomes I–VI, VIII and X): the total number of spheres inserted on S. salamandra lampbrush chromosomes is the highest among the salamandrid species studied so far. These landmarks as well as the maximally developed normal loops are schematically drawn on the maps of the single lampbrush chromosomes. The length of the maps corresponds to the mean value of the lengths of each chromosome relative to that of chromosome XII, taken as 100 units long.Also bivalents from first metaphase spermatocytes have been analysed: they are generally ring-shaped with two terminal or subterminal chiasmata.