Schistosoma mansoni: development of the cercarial glycocalyx

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.     

The cercarial glycocalyx of Schistosoma mansoni

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.     

Schistosoma mansoni: influence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production

Biomphalaria glabrata snails of the same age, but different sizes, were used to determine size-related susceptibility to Schistosoma mansoni miracidial infection and the influence of snail size on total cercarial production. Snails with shell diameters from less than 5 to greater than 17 mm were individually exposed to one or several miracidia, depending on the experiment. In snails exposed to multiple numbers of miracidia, the percentage of snails which developed patent infections was lower in snails with larger shell sizes. This was also reflected by fewer primary sporocysts per infected snail found in tissues of the larger snails. Upon determining cercarial production in these groups over a 1-month period there were no statistical differences between any groups in the numbers of cercariae produced per snail. However, upon determining the number of successful primary sporocysts found in cohort snails of each size group, cercarial production increased as a function of the number of successful primary sporocysts. This was verified by examining cercarial production in various size snails with known monomiracidial infections. Our data therefore confirm and extend earlier work using snails infected with unknown numbers of miracidia and clearly show that total S. mansoni cercarial development and decreased susceptibility of snails is a direct reflection of snail size and not necessarily age of the snail.     

Chaetotaxy and surface structures of the miracidia of Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Szidat, 1928) (Digenea,Strigeidae)

Silver nitrate staining was used to investigate the number and arrangement of argentophillic structures (epidermal plates, sensilla, excretory pores) of three species of strigeid miracidia, Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Szidat, 1928). The epidermal plates of all three species of miracidia were arranged in four tiers according to the formula (6 + 9 + 4 + 3) = 22, while the number and distribution of sensilla were also found to be identical. Modifications to the nomenclature of Dimitrov et al. (1989) for miracidial chaetotaxy are proposed.Scanning electron microscopy (SEM) was also employed to examine the surface structures of deciliated miracidia of Ichthyocotylurus spp., confirming the position and revealing the form of body sensilla.     

Evidence that a protective membrane epitope is involved in early but not late phase immunity in Schistosoma mansoni

An anti-egg monoclonal antibody E.1, which is partially protective in passive transfer experiments, is shown in this study to recognize a membrane epitope on cercariae, schistosomula, and the ciliary plates of miracidia. E.1 did not bind to the surface membranes of lung or adult worms, or recognize secreted egg antigen in infected liver tissue. The E.1 epitope was present in the glycocalyx of cercariae, as well as on the syncytial membrane as determined by electron microscopy. Immunoprecipitation of iodinated surfaces of cercariae and schistosomula demonstrated E.1 binding to a high m.w. moiety in cercariae, which corresponds to the glycocalyx because it was not immunoprecipitated from schistosomula. In addition, a band at 38,000 daltons was immunoprecipitated from both cercariae and schistosomula. When compared with in vitro cultured parasites, schistosomula that were obtained from mice 1 to 24 hr after tail vein injection showed significant loss of E.1 binding. Consistent with the rapid loss of antigen in vivo, E.1 antibody was unable to passively transfer protection to naive mice when administered 5 days after cercarial challenge.     

Genetic variability of Schistosoma bovis cercarial production according to miracidial dose

Cercarial production related to miracidial dose variation with Schistosoma bovis strains from Sudan and Spain in Bulinus truncatus from Tunisia was studied. Results showed that an increase in the miracidial dose proposed to the host-mollusc (1 and 5 miracidia) gave rise to a decrease in cercarial productivity of Sudanese S. bovis and to an increase for Spanish S. bovis. It is concluded that this response difference to the miracidial dose variation depends on genetic characters peculiar to the parasite strain and represents a new aspect of genetic variability of schistosome populations.     

Studies of the relationships between Schistosoma nasale and S. spindale and their snail host Indoplanorbis exustus

Infectivity and cercarial production of Indoplanorbis exustus related to variation of miracidial dose (1, 4, 10 or 20) with Schistosoma nasale and S. spindale from Sri Lanka were studied. The intermediate host-parasite relationships of the two schistosome species showed marked differences under the conditions of observation recorded in this study. Prepatent death rates (PDR) were on average higher for S. spindale (30%) than for S. nasale (10%). The size of the miracidial dose to which snails had been exposed had no effect on PDR. The infection rates (IR) were on average higher for S. nasale (41%) compared with S. spindale (27%). Highest IR occurred after exposure to 4 miracidia in S. nasale infections (79%) and after exposure to 10 miracidia in S. spindale infections (60%). The highest daily average cercarial production per snail was recorded for S. nasale at a level of 4 miracidia (1311), and for S. spindale at a level of 10 miracidia (1615). At low level (1 or 4 miracidia) of exposure, I. exustus showed a better compatibility with S. nasale than with S. spindale. An opposite tendency was observed at higher levels (10 or 20 miracidia) of exposure. Unsuccessful infections of Lymnaea luteola with either S. nasale or S. spindale indicate that this species is not involved in transmission.     

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.     

Opisthorchis viverrini: ultrastructure and cytochemistry of the glycocalyx of the tegument

The ultrastructure and cytochemistry of the glycocalyx of the tegument of Opisthorchis viverrini during maturation from newly excysted juvenile to adult stages were investigated using colloidal iron, ruthenium red and lectin stainings. The results showed that the glycocalyx was intensely stained by the first two dyes, thus indicating the presence of relatively high amounts of negative charges. However, the thickness and intensity of the staining decreased during the fluke's maturation. Binding studies using lectin probes on the surface of adult parasites showed that binding sites for Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Ricinus communis I (RCA I) were present in relative large amounts on the glycocalyx of the adult tegument, whereas those for Dolichos biflorus (DBA) were relatively fewer in number, and those for Ulex europaeus I (UEA I) were absent. The binding patterns of Con A, WGA, RCA I and DBA were generally similar, and the reaction product was uniformly distributed over the dorsal and ventral surfaces of the parasite's body. These bindings, therefore, indicate the presence of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, D-galactose and N-acetyl-D-galactosamine residues on the glycocalyx of the adult tegument.     

Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.     

Schistosoma japonicum (Chinese): Changes of the tegument surface in cercariae, schistosomula and juvenile parasites during development

, , , and 1988. Schistosoma japonicum (Chinese): changes of the tegument surface in cercariae, schistosomula and juvenile parasites during development. International Journal for Parasitology18: 1093–1104. The surface of cercariae of S. japonicum (Chinese) and changes following their transformation into schistosomula were observed at 0.5,1, 3,6,12,24 and 48 hand 5,7,10 and 15 days by scanning electron microscopy. The cercarial surface shows two distinct regions: on the head and the body it is mildly undulating and covered throughout with numerous spines that are partially embedded in a thick layer of glycocalyx; on the tail it is highly folded into tall and thin circumferential ridges, spines are confined to the dorsal and ventral aspects and are prominent due to a thinner glycocalyx. Between 0.5 and 12 h following skin penetration, the surface of schistosomula is gradually cleared of glycocalyx, while small blebs and slender microvilli project out from the glycocalyx-free area and from spines. Subsequently, these structures are detached from the surface and thus responsible for the shedding of the original cercarial membrane and glycocalyx. Between 24 and 48 h, schistosomula show little change in the body size in comparison to earlier stages, and most of the surface is still covered with spines but devoid of blebs and microvilli. From day 2 onwards schistosomula are lengthened but with little net growth. Spines decrease in the middle region of the body; and the surface is invaginated by small pits and folded into thin ridges, which result in rapid increase of the surface area. Between days 5 and 7 schistosomula elongate further, and the disappearance of the spines in the middle region becomes even more pronounced, while those remaining are confined only to the anterior and posterior extremities. Ridges and pits are more developed, and the former begin to show regular foldings throughout all parts of the body. Between days 7 and 10 schistosomula grow rapidly and the body is quadrupled in size. Between days 10 and 15 sexes can be differentiated. The male has a flat ovoid shape body, whose ridges and pitting on the surface are highly developed almost to the same extent as in adult parasites, while spines remain mostly on the posterior end. Sensory papillae greatly increase in number, especially on the ventral and lateral aspects and around the suckers. The surface of the female is relatively smooth; ridges are developed only on the posterior part, while the remaining surface is flat and extensively pitted. Spines are present throughout but less concentrated in the middle region.     

Histochemical demonstration of acetylcholinesterase in sporocysts of Schistosoma mansoni (Trematoda).

The distribution of acetylcholinesterase in mother and daughter sporocysts of Schistosoma mansoni was studied histochemically. In young mother sporocysts derived from miracidia cultured in vitro the miracidial neural mass and flame cells were shown to persist. The nerve trunks and commissures, as well as papillae, are apparently lost in the transformation process. In young daughter sporocysts freshly dissected from mother sporocysts there was little enzyme activity except for a sparse distribution in the tegument. After cultivation, intense enzyme activity was associated with developing cercarial embryos. A similar distribution of activity was observed in older daughter sporocysts obtained from the digestive gland of snails. No evidence of flame cells, neural mass, or commissures was detected in daughter sporocysts by the methods employed.     

Schistosoma mansoni: immunogenic glycoproteins of the cercarial glycocalyx

Immunochemical studies at the level of the light and electron microscope showed that a monoclonal antibody, 128C3/3, was directed to an epitope in the glycocalyx of Schistosoma mansoni cercariae. Immunoprecipitation of surface labeled cercarial extracts with this monoclonal antibody demonstrated that the glycocalyx is composed of at least five components, including a very large molecular size polypeptide and polypeptides of 220, 180, 170, and 15 kDa. After transformation of cercariae to schistosomula, these polypeptides were shed from the surface and were therefore no longer accessible to surface labeling. Monoclonal antibody 128C3/3 was also reactive with a 38 kDa polypeptide from schistosomula; this polypeptide was weakly expressed on the surface of cercariae. Analysis of immunoprecipitates of radioiodinated protein extracts of cercariae, newly transformed schistosomula, and 36 hr in vitro cultured schistosomula showed that the 180 and 170 kDa polypeptides continued to be expressed within the organism following transformation, but were not accessible to surface labeling. Lectin binding studies revealed differences in the oligosaccharide composition of the six polypeptides. With the exception of the 15 kDa antigen, all the polypeptides reactive with 128C3/3 were highly immunogenic in infected mice and humans.     

Chemical stimulation of Schistosoma mansoni miracidial activity.

The movements of S. mansoni miracidia immersed in various solutions of organic chemicals were recorded by dark ground photography. Reduction of miracidial speed was recorded for miracidia in solutions of fatty acids and ammonia. The speed reduction was concentration dependant and pH dependant, indicating in both cases that the un-ionized molecule was responsible. Ammonia at concentrations between 0.4 mM and 0.8 mM elicited an increased "rate of change of direction" by miracidia. No changes in miracidial movements were recorded for miracidia immersed in aqueous solutions of amino acids, sugars or of molluscan nitrogenous excretion products other than ammonia. A possible role for chemo-klinokinetic behaviour patterns in miracidial host location was formulated.     

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.     

Disentangling phylogenetic constraints from selective forces in the evolution of trematode transmission stages

The transmission stages of parasites are key determinants of parasite fitness, but they also incur huge mortality. Yet the selective forces shaping the sizes of transmission stages remain poorly understood. We ran a comparative analysis of interspecific variation in the size of transmission stages among 404 species of parasitic trematodes. There are two transmission steps requiring infective stages in the life cycle of trematodes: transmission from the definitive to the first intermediate (snail) host is achieved by eggs and/or the miracidia hatched from those eggs, and transmission from the first to the second intermediate host is achieved by free-swimming cercariae. The sizes of these stages are under strong phylogenetic constraints. Our results show that taxonomy explains >50% of the unaccounted variance in linear mixed models, with most of the variance occurring at the superfamily level. The models also demonstrated that mollusc size is positively associated with egg volume, miracidial volume and cercarial body volume, but not with the relative size of the cercarial tail. In species where they encyst on substrates, cercariae have significantly larger bodies than in species penetrating chordates, although the relative size of the cercarial tail of species using chordates as second intermediate hosts was larger than in other trematode species. Habitat also matters, with larger cercarial tails seen in freshwater trematodes than in marine ones, and larger miracidial volumes in freshwater species than in marine ones. Finally, the latitude (proxy for local temperature) at which the trematode species were collected had no effect on the sizes of transmission stages. We propose that resource availability within the snail host, the probability of contacting a host, and the density and viscosity of the water medium combine to select for different transmission stage sizes.     

Effect of the glycocalyx layer on transmission of interstitial flow shear stress to embedded cells

In this paper, a simple theoretical model is developed to describe the transmission of force from interstitial fluid flow to the surface of a cell covered by a proteoglycan / glycoprotein layer (glycocalyx) and embedded in an extracellular matrix. Brinkman equations are used to describe flow through the extracellular matrix and glycocalyx layers and the solid mechanical stress developed in the glycocalyx by the fluid flow loading is determined. Using reasonable values for the Darcy permeability of extracellular matrix and glycocalyx layers and interstitial flow velocity, we are able to estimate the fluid and solid shear stresses imposed on the surface of embedded vascular, cartilage and tumor cells in vivo and in vitro. The principal finding is that the surface solid stress is typically one to two orders of magnitude larger than the surface fluid stress. This indicates that interstitial flow shear stress can be sensed by the cell surface glycocalyx, supporting numerous recent observations that interstitial flow can induce mechanotransduction in embedded cells. This study may contribute to understanding of interstitial flow-related mechanobiology in embryogenesis, tumorigenesis, tissue physiology and diseases and has implications in tissue engineering.     

Isolation of an SBA lectin-reactive glycoprotein (gp50) and its identification in Schistosoma mansoni larval and adult worm secretions.

A major 50-kDa soybean agglutinin (SBA)-reactive component present in extracts of Schistosoma mansoni eggs was isolated by SBA lectin affinity chromatography. In polyacrylamide gel electrophoresis (PAGE), the SBA-reactive component was seen as a 100-kDa polypeptide band that after reduction and alcylation was substituted by a 50-kDa component. This suggests that it occurs in native form as a dimer. Monoclonal antibodies produced against gp50 reacted with miracidial and cercarial secretions and with adult worm components including tegumental structures suggestive of a secretory function.     

Diffusion and reaction in the cell glycocalyx and the extracellular matrix

Many biologically important macromolecular reactions are assembled and catalyzed at the cell lipid-surface and thus, the extracellular matrix and the glycocalyx layer mediate transfer and exchange of reactants and products between the flowing blood and the catalytic lipid-surface. This paper presents a mathematical model of reaction–diffusion equations that simply describes the transfer process and explores its influence on surface reactivity for a prototypical pathway, the tissue factor (Tf) pathway of blood coagulation. The progressively increasing friction offered by the matrix and glycocalyx to reactants and to the product (coagulation factors X, VIIa and Xa) approaching the reactive surface is simulated and tested by solving the equations numerically with both, monotonically decreasing and constant diffusion profiles. Numerical results show that compared to isotropic transfer media, the anisotropic structure of the matrix and glycocalyx sharply decreases overall reaction rates and significantly increases the mean transit time of reactants; this implies that the anisotropy modifies the distribution of reactants. Results also show that the diffusional transfer, whether isotropic or anisotropic, influences reaction rates according to the order at which the reactants arrive at the boundary. Faster rates are observed when at least one of the reactants is homogeneously distributed before the other arrives at the boundary than when both reactants transfer simultaneously from the boundary.     

Measuring endothelial glycocalyx dimensions in humans: a potential novel tool to monitor vascular vulnerability.

The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as a measure of glycocalyx thickness, and the transient widening of the erythrocyte column on glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy men using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r = 0.63). Measurements in humans were reproducible (0.58 +/- 0.16 and 0.53 +/- 0.15 microm, coefficient of variance 15 +/- 5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r = 0.45), fasting plasma glucose (r = 0.43), and high-density lipoprotein-cholesterol (r = 0.40) and correlated negatively with low-density lipoprotein-cholesterol (r = -0.41) as well as body mass index (r = -0.45) (all P < 0.05). In conclusion, the dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.