Correlative and Dynamic Imaging of the Hatching Biology of Schistosoma japonicum from Eggs Prepared by High Pressure Freezing

Background

Schistosome eggs must traverse tissues of the intestine or bladder to escape the human host and further the life cycle. Escape from host tissues is facilitated by secretion of immuno-reactive molecules by eggs and the formation of an intense strong granulomatous response by the host which acts to exclude the egg into gut or bladder lumens. Schistosome eggs hatch on contact with freshwater, but the mechanisms of activation and hatching are poorly understood. In view of the lack of knowledge of the behaviour of egg hatching in schistosomes, we undertook a detailed dynamic and correlative study of the hatching biology of Schistosoma japonicum.

Methodology/Principal Findings

Hatching eggs of S. japonicum were studied using correlative light and electron microscopy (EM). The hatching behaviour was recorded by video microscopy. EM preparative methods incorporating high pressure freezing and cryo-substitution were used to investigate ultrastructural features of the miracidium and extra-embryonic envelopes in pre-activated and activated eggs, and immediately after eggshell rupture. Lectin cytochemistry was performed on egg tissues to investigate subcellular location of specific carbohydrate groups.

Conclusions/Significance

The hatching of S. japonicum eggs is a striking phenomenon, whereby the larva is liberated explosively while still encapsulated within its sub-shell envelopes. The major alterations that occur in the egg during activation are scission of the outer envelope-eggshell boundary, autolysis of the cellular inner envelope, and likely hydration of abundant complex and simple polysaccharides in the lacunal space between the miracidial larva and surrounding envelopes. These observations on hatching provide insight into the dynamic activity of the eggs and the biology of schistosomes within the host.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida () (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida (Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida ( ) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida ( Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

A Cardinium-like symbiont in the proturan Acerella muscorum (Hexapoda)

Endosymbionts of the Cardinium-like genus are described in the testes and other tissues of the proturan Acerella muscorum (Ionescu). Few endosymbionts are present in the large apical cells of functional testes, but they become numerous at the end of the reproductive cycle. They are also found within sperm cells where induce their degeneration. The Gram-negative endosymbionts are characterized by the presence of microtubule-like structures (MLC) in their cytoplasm. It is suggested a possible role of the endosymbionts in the elimination of degenerating sperm cells when the testes activity is ended, thus somewhat playing a role in the timing of the reproductive cycle of the proturan species.     

Eggshell fine structure of Bradysia aprica (Winnertz) (Diptera : Sciaridae)

The eggshell fine structure of the dark-winged fungus-gnat Bradysia aprica (Winnertz) (Diptera : Sciaridae) was investigated by scanning and transmission electron microscopy. At the anterior pole of the ovoid egg is a single micropyle, centrally located in a well-defined micropylar area. The latter is covered by many long drumstick-like chorionic processes that are longer and more numerous than those of the rest of the egg surface. Cross-sections of the eggshell show 3 concentric envelopes: the vitelline envelope, wax layer and chorion. The chorion consists of 3 components with different morphological features: the inner, intermediate and outer chorion. The latter 2 layers, involved in the organization of the drumstick-like processes, have homogeneous features, whereas the former is crystalline and resembles the innermost chorionic layer of other Diptera.     

上海大金山岛原尾虫的物种多样性及生态特征

原尾虫是土壤动物的重要类群之一,然而目前对其物种多样性及生态特征的了解仍然十分有限,岛屿生态系统中原尾虫的研究尤为欠缺。为深入探讨原尾虫的栖息地选择、空间分布、种群密度、繁殖策略及其与土壤真菌的相互作用等生态特征,于2015年秋季在大金山岛竹林、乔木林和灌木林等3种不同的植被中进行了定量采样研究。结果表明,大金山岛共发现8种原尾虫,隶属2科4属,这些物种在上海市陆域植被中均有分布,且均为东洋界种类;大金山岛秋季原尾虫的总密度为竹林(696头/m~2)乔木林(479头/m~2)灌木林(213头/m~2);竹林中原尾虫的Shannon-Wiener多样性指数显著高于灌木林(P0.05),而不同植被类型中原尾虫的Pielou均匀度、Simpson优势度和Margalef丰富度等指数无显著差异;大金山岛的原尾虫在土壤中为隔离分布,具有在适宜的微生境中集中分布的特点;发现原尾虫在野外受到病原真菌感染的直接证据;通过对原尾虫的性比和幼虫比的分析,发现其繁殖可能采取介于k对策和r对策之间的生态策略。     

Genetic heterogeneity and adaptive strategies of the ladybird <Emphasis Type="Italic">Harmonia axyridis</Emphasis> Pall. (Coleoptera,Coccinellidae)

Analysis of microstatial variability of the phenetic composition of populations of the ladybird Harmonia axyridis Pall. is performed in Baikal region of the Primorskii krai. It is revealed that the phenetic composition of the Baikal H. axyridis population is significantly more labile than that in Primorie. In individual micropopulations the phenoimage is clarified due to a local increase in frequency of the morph succinea. Individual and family testing of H. axyridis individuals from the Primorie and Baikal populations is performed by the quantitative parameters affecting the adaptability-the larval and pupal survival at starvation, critical weight of pupation, the imago weight and fertility. At starvation of larvae of the IV instars for 3–4 days, only the morph succinea individuals from the Primorie population reached the imago stage. The oviposition value in these imagoes corresponded to the mean for the population (31.2 ± 1.06 eggs). The obtained result confirms the earlier expressed suggestion that the morph succinea is distinguished by a high ecologic plasticity. This morph is able to survive under conditions of the food deficit and is characterized by the intermediate, but stable reproduction.     

Color polymorphism,development, and reproductive diapause of the multicolored Asian ladybird <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas) (Coleoptera,Coccinellidae) females

Preimaginal development and maturation of females of two laboratory strains originated from two populations of the multicolored Asian ladybird Harmonia axyridis were investigated under laboratory conditions. Females of the autochthonous population from Irkutsk (Siberia) enter diapause under the short day conditions, whereas in females of the invasive population from Sochi (the Caucasus) photoperiodic induction of diapause is weak. The two populations also differ in the proportions of morphs: the population from Sochi is represented only by the morph succinea, whereas the population from Irkutsk is represented by the morphs succinea и axyridis with the predominance of the last one, which in our study was separated into two phenotypes, differed in the degree of melanization. Rearing of adults under the short day (12 h) conditions induced reproductive diapause in females of all the studied morphs and phenotypes of the Irkutsk population, whereas under the long day (18 h) conditions females of the morph succinea showed a slightly lower tendency to diapause. Evidently, the interpopulation differences in the responses regulating the seasonal cycle of the multicolored Asian ladybird are determined by genes that are not involved (or almost not involved) in the determination of the color polymorphism. The interpopulation differences in size and weight were quite substantial, whereas the differences between morphs and phenotypes were statistically significant in only some of our experiments. In combination with the literature data, these results suggest that the genes determining the color polymorphism in H. axyridis may have a pleiotropic effect on other traits including important eco-physiological parameters but this effect is relatively weak and manifests itself only against a particular genetic background and / or under particular environmental conditions.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

Chemical Constituents and Hydrogenase Binding in Cell Envelopes of Vibrio succinogenes

The particulate hydrogenase of Vibrio succinogenes is solubilized during treatment of cell envelopes at pH 11.0. Alkali-solubilized enzyme requires sulfhydryl compounds for activity. At neutral pH, soluble enzyme is reincorporated into alkalitreated cell envelopes and no longer requires an additional activator. In the present study, cell envelopes prepared by lysing cells with ethylenediaminetetraacetic acid plus lysozyme (EDTA-lysozyme) were used to determine the chemical composition of cell envelopes and derived pH 11.0 soluble and insoluble fractions and to investigate some properties of the binding and activation of alkali-solubilized hydrogenase. Lysis with EDTA-lysozyme resulted in the formation of spheroplast ghosts. The derived cell envelopes contained 61% protein, 3% ash, 23% lipid, and 1% phosphorus. The alkali-treated cell envelopes contained 50% protein, 2% ash, 24% lipid, and 1% phosphorus. The ash from cell envelopes and alkali-treated cell envelopes was rich in iron and phosphorus and also contained calcium, copper, magnesium, sodium, and zinc. Virtually all of the weight of the ashed samples was accounted for by the oxides of these metals. Since the reconstitution of particulate hydrogenase was achieved with pH 11.0 supernatant solution and precipitate, intact mucopeptide is not essential for hydrogenase binding. Release of hydrogenase during EDTA-lysozyme lysis was found to depend upon an apparent structural change which occurs in the membranes during extended storage at −20 C.     

Scanning electron microscopy of the eggs of three human schistosomes

Ford J. W. and Blankespoor H. D. 1979. Scanning electron microscopy of the eggs of three human schistosomes. International Journal for Parasitology9: 141–145. The surface of the eggs of Schistosoma haematobium, S. japonicum and S. mansoni, examined by scanning electron microscopy, are covered with microspines. The spines of S. mansoni and S. haematobium are essentially similar; however, in S. japonicum they are smaller and more densely distributed. A fibrous matrix, present on the surface of the eggs, is not host derived. This matrix may account for the stickiness of the eggs and the micospines may function to hold the matrix in place.     

Fine structure of the egg envelope and the supporting stalk in the dipliran Campodea (Apterygota : Campodeidae)

The eggs and supporting stalks of one Campodea (Apterygota : Campodeidae) species were studied by scanning and transmission electron microscopes. Each egg batch is attached to the substrate by a supporting stalk. The stalk is composed of tangled lamellae and dense annular structures. Both elements are strongly PAS-positive. The eggs are covered by one envelope only. It is built of fine granular, PAS-negative material. The surface of the envelope is smooth and possesses 2–4 ring-like structures, which are likely involved in the attachment of the eggs within a batch.     

Structure of shells from eggs of kinosternid turtles

Shells from eggs of five species of kinosternid turtle (Sternotherus minor, Kinosternon flavescens, K. baurii, K. Hirtipes, and K. alamosae) were examined with light and scanning electron microscopy. Except for possible differences among species in thickness of eggshells, structure of shells from all eggs was similiar. In general, kinosternid turtles lay eggs having a rigid calcareous layer composed of calcium carbonate in the form of aragonite. The calcareous layer is organized into individual shell units with needlelike crystallites radiating from a common center. Most of the thickness of the eggshell is attributable to the calcareous layer, with the fibrous shell membrane comprising only a small fraction of shell thickness. Pores are found in the calcareous layer, but they are not numereous. The outer surface of the eggshells is sculptured and may have a thick, organic layer in places. The outer surface of the shell membrane of decalcified eggshells is studded with spherical cores which presumably nucleate growth of shell units during shell formation. The shell membrane detaches from eggs incubated to hatching, carrying with it remnants of the calcareous layer. Such changes in shell structure presumably reflect withdrawal of calcium from the eggshell by developing embryos.     

Role of the polychaete <Emphasis Type="Italic">Neanthes succinea</Emphasis> in phosphorus regeneration from sediments in the Salton Sea,California

The Salton Sea currently suffers from several well-documented water quality problems associated with high nutrient loading. However, the importance of phosphorus regeneration from sediments has not been established. Sediment phosphorus regeneration rates may be affected by benthic macroinvertebrate activity (e.g. bioturbation and excretion). The polychaete Neanthes succinea (Frey and Leuckart) is the dominant benthic macroinvertebrate in the Salton Sea. It is widely distributed during periods of mixing (winter and spring), and inhabits only shallow water areas following development of anoxia in summer. The contribution of N. succinea to sediment phosphorus regeneration was investigated using laboratory incubations of cores under lake temperatures and dissolved oxygen concentrations typical of the Salton Sea. Regeneration rates of soluble reactive phosphorus (SRP) were lowest (−0.23–1.03 mg P m⁻² day⁻¹) under saturated oxygen conditions, and highest (1.23–4.67 mg P m⁻² day⁻¹) under reduced oxygen levels. N. succinea most likely stimulated phosphorus regeneration under reduced oxygen levels via increased burrow ventilation rates. Phosphorus excretion rates by N. succinea were 60–70% more rapid under reduced oxygen levels than under saturated or hypoxic conditions. SRP accounted for 71–80% of the dissolved phosphorus excreted under all conditions. Whole-lake SRP regeneration rates predicted from N. succinea biomass densities are highest in early spring, when the lake is mixing frequently and mid-lake phytoplankton populations are maximal. Thus, any additional phosphorus regenerated from the sediments at that time has potential for contributing to the overall production of the lake. Guest Editor: John M. Melack Saline Water and their Biota     

Egg-shells in mites

Summary This communication presents results of studies on the formation and structure of the vitelline envelopes in three species of mites: Euryparasitus emarginatus (Gamasida), Erythraeus phalangoides (Actinedida), and Hafenrefferia gilvipes (Oribatida). In E. emarginatus and E. phalangoides, in which the oocytes are not covered with follicular cells, the material of the vitelline envelope appears first in vesicles under the surface of the oocytes prior to secretion by exocytosis. The formed vitelline envelope is built of a homogeneous material which is perforated by numerous channels containing oocyte microvilli. Later, as the microvilli are retracted, the channels disappear. In both of these species the formed vitelline envelope is incomplete and the micropylar orifice occurs as a transitional structure.In H. gilvipes follicular cells encircling the oocyte contain granules filled with material that is subsequently secreted into the perivitelline space forming the vitelline envelope on the oocyte surface. The inner layer of the vitelline envelope is granular, whereas the outer part is more homogeneous. Both lack channels containing microvilli and micropyle.     

Body structure of marine sponges

Summary Specimens of Haliclona elegans (Bowerbank, 1866) are covered by a thin, double layered dermal membrane extending over large subdermal spaces. The pores in the dermal membrane are formed by single porocytes with one or sometimes several pores in the center of the cell. The subjacent tissue shows a faintly developed mesenchyme and numerous big choanocyte chambers projecting into lacunar spaces of the incurrent canal system. The outer surface of the chambers is directly covered by the pinacocyte epithelium of the incurrent canal wall, which also separates them completely from the mesenchyme. Water influx into the chambers is guaranteed by prosopylar openings in the pinacocyte cover at the outer chamber surface. The chambers are connected to the excurrent canal system in the eurypylous way by wide apopyles, each of which is surrounded by a small ring of flagellated cone cells. About 15% of the choanocyte chambers in H. elegans contain central cells, which are thought to derive from migrating pinacocytes of the canal systems.     

Marine algal symbionts benefit benthic invertebrate embryos deposited in gelatinous egg masses

Algae colonize the gelatinous egg masses of marine invertebrates. This study demonstrates a symbiotic relationship between marine algae and the invertebrate embryos in gelatinous egg masses found in Indian River Lagoon, FL, USA. The benefits to the embryos in this association differ among host species investigated. The embryos of the polychaete Axiothella mucosa graze on the diatom assemblage in their egg masses and the fitness of the crawl-away juveniles is improved by this food source. The tenuous egg masses of the polychaete Arenicola cristata and the mollusk Haminoea succinea are negatively buoyant when spawned and become buoyant when symbiotic algae are present. In addition to increased dispersal of their lecithotrophic larvae, the potential of the egg masses of A. cristata and H. succinea to float may reduce predation on the embryos by benthic predators such as the gastropod Nassarius vibex. Photosynthetically derived oxygen from the algae may benefit the embryos of the opisthobranch Haminoea elegans by increasing oxygen supply when crawl-away juveniles emerge from the egg mass. However, when mostly earlier stage larvae are hatched from egg masses of H. elegans, the additional oxygen supplied by the algae does not provide a substantial advantage. Algae were absent in the gelatinous egg mass core of only one of the five species examined, Haminoea antillarum. H. antillarum has both a short embryonic development time and denser egg mass gel than the other four species tested. What is not understood is whether invertebrate egg masses are an opportunistic space for algae to colonize or whether only a few microalgal species can exploit the gelatinous substrate.     

The temperature and humidity preferences of Haemaphysalis longicornis, Ixodes Holocyclus and Rhipicephalus sanguineus (Ixodidae): studies on eggs

The eggs of Haemaphysalis longicornis, Ixodes holocydus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to compare the temperature and humidity requirements of ticks from climatically dissimilar habitats. The response to changes in humidity was also studied with H. longicornis. Hatching in I. holocydus eggs occurred from 18 to 28°C and at a saturation deficit of 2 mm Hg or less. The eggs of R. sanguineus hatched between 18 and 38°C and tolerated saturation deficits up to 20 mm Hg. The eggs of H. longicornis hatched between 18 and 35°C and at saturation deficits up to 8 mm Hg. When eggs of H. longicornis were exposed to moderately dry conditions for different periods of time and then transferred to moist conditions, incubation periods and mortality were reduced in comparison with eggs kept continuously under dry conditions. On the other hand, mortality of H. longicornis eggs was reduced in moderately dry conditions, provided that they had been exposed to a moist environment for about 10 days previously. Weight loss of eggs in dry air and the rate of development of eggs were examined and are discussed. The eggs of I. holocylus lost weight very rapidly in dry air, whereas weight loss from R. sanguineus eggs was much slower. Weight loss from H. longicornis eggs fell between these two rates. The rate of development was fastest in R. sanguineus and slowest in I. holocydus.     

Gamet Ultrastructure in Some Species of the Genus <Emphasis Type="Italic">Icelus</Emphasis> (Pisces: Cottidae)

With the help of scanning and transmission electron microscopy, the morphology of spermatozoa of Icelus spiniger and egg envelopes of I. spiniger and I. spatula has been studied. Spermatozoa have an elongated head flattened on one side, an insignificant centriolar fossa, and numerous mitochondria in the middle section. The axoneme has a structure described by the formula 9 + 2. The envelopes of oocytes of both species have a follicular membrane, chorion, and zona radiata.