Factors affecting surface changes in intact cercariae and cercarial bodies of Schistosoma mansoni.

The effect of different incubation media and of temperature on the induction of water sensitivity has been investigated in intact and tailless Schistosoma mansoni cercariae. Removal of the cercarial tail by vortex stirring and elevation of the temperature of the medium from 27 to 37 degrees C resulted in the rapid onset of permeability changes in the larvae. The rate of change was greater in water than in TC-199 or Hanks' BSS media. Lowering the pH of the medium or increasing the concentration of Ca2+ ions decreased the rate of permeability change: raising the pH of the medium or the addition of 10(-5) M EDTA enhanced the rate. Raising the temperature of the medium also increased the rate of permeability change in intact cercariae although the rates obtained varied with the different media tested, being greatest in TC-199. It is concluded that both temperature elevation and loss of the cercarial tail influence the onset and rate of permeability changes in cercarial bodies during the transformation to schistosomula.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.     

Schistosoma mansoni: sterol and phospholipid composition of cercariae, schistosomula, and adults

The sterol and phospholipid composition of cercariae, schistosomula, and adult Schistosoma mansoni was analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC). Cercariae and schistosomula contained cholesterol, desmosterol, campesterol, stigmasterol, and beta-sitosterol while adults contained only cholesterol. In all stages cholesterol comprised greater than 50% of the total sterols, and in cercariae and schistosomula desmosterol comprised 38 and 21% of the total sterols, respectively. The other three sterols, campesterol, stigmasterol, and beta-sitosterol, made up approximately 10% of the total. The same five sterols found in cercariae and schistosomula were present in the hepatopancreas of uninfected snails but with a much higher desmosterol concentration in the parasite, 38%, than in the snail, 2%. As in cercariae and schistosomula the three minor sterols comprised approximately 10%. Thus, the sterol composition of cercariae and schistosomula was similar but not identical to that of the snail host. Phosphatidylcholine was the major phospholipid of all three stages (50%) as determined by two HPLC procedures. The remaining phospholipids consisted of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. In addition, in adults there were small quantities of sphingomyelin and lysophosphatidylcholine. The percentage of each phospholipid was similar among stages with the exception of a slight increase in phosphatidylserine in adults compared to cercariae and schistosomula. These results show that a characteristic lipid composition is found in cercariae, schistosomula, and adults.     

Schistosoma mansoni: protein phosphorylation during transformation of cercariae to schistosomula.

Infectivity of the multicellular pathogen Schistosoma mansoni for the human host is dependent upon the ability of free-living cercariae to transform rapidly into parasitic schistosomula. The biochemical pathways that regulate this transitional period are unknown. The role of protein phosphorylation was investigated by examining the incorporation of [32Pi]phosphate into proteins of S. mansoni. A sevenfold increase in total phosphorylation was found in 3-hr-old schistosomula as compared to cercariae. Analysis of radiolabeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that a 14-kDa protein served as a marker for transformation, being phosphorylated in schistosomula but not cercariae. The protein was phosphorylated on a serine residue. Phosphorylation was stimulated by a shift of parasites from water to salt-containing medium at 23 degrees C. Incubation of organisms in water at 37 degrees C did not initiate phosphorylation of this protein. The 14-kDa phosphoprotein was extracted from parasite homogenates with 1 M NaCl but was insoluble in 1% Triton X-100. Protein phosphorylation during the cercarial-schistosomula transformation may represent an important biochemical event that regulates infectivity of the parasite for the human host.     

Schistosoma mansoni: neurotransmitters and the mobility of cercariae and schistosomules

The concentration-dependent action of alkyl-isothiouroniums on Schistosoma mansoni cercariae, ranging from partial to total abolition of locomotor and flame cell movements, and/or suppression of virulence, is due to H1-histamine receptor inhibition. Correspondingly, H1-receptor inhibitors of widely different chemical structure, such as clemizol, diphenhydramine, brompheniramine, and promethazine, in 0.03-0.06 nM concentrations, induced an analogous cercarial immobilization reversed by addition of excess histamine. In contrast, the H2-receptor inhibitors cimetidine and metiamide did not immobilize cercariae. Histamine, acetylcholine, and serotonin, added to cercarial suspensions, showed no direct activity. Their participation in the mechanism of cercarial mobility was shown by the dose-dependent effects of antagonists, such as the serotonin antagonist methysergide and the acetylcholinesterase inhibitor physostigmine. These effects were not reversible by addition of serotonin and acetylcholine, respectively. A histamine-irreversible cercarial immobilization induced by the H-liberator 48/80 suggested that, besides H1-receptor inhibition, H-liberation and/or depletion also participated in mobility and survival. The detection of histamine in the cercaria corroborated the participation of histaminergic mechanisms. S. mansoni schistosomules collected from the mouse lung reacted to H1 antihistamines like cercariae, with a dose-dependent reduction of mobility and somatic deformation, such as vacuolization, granulation, and caecal enlargement.     

Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae

Immune responses induced by glycans upon infection with Schistosoma mansoni may be mediated by either schistosomal glycoproteins or glycosphingolipids. In this study, we have elucidated the structural features of both carbohydrate moieties and respective ceramide units of complex glycosphingolipids from adult S. mansoni. Obtained data revealed a vast structural heterogeneity due to manifold combinations of different oligosaccharides and ceramide entities. Observed carbohydrate moieties included Lewis(X) (Le(X); Gal(β1-4)[Fuc(α1-3)]GlcNAc) as well as, in part, multiply fucosylated LacdiNAc (LDN; GalNAc(β1-4)GlcNAc) carbohydrate epitopes. Corresponding lipid portions comprised predominantly C18-sphingosine as well as C18- and C20-phytosphingosine derivatives. Intriguingly, glycosphingolipids carrying an Le(X) epitope contained predominantly C18-sphingosine, whereas LDN-based species exhibited mostly phytosphingosine derivatives, in addition to C18-sphingosine, indicating that the two classes of glycosphingolipids might be synthesized via different biosynthetic routes. Compared with literature data, adult worm glycosphingolipids with Le(X) epitopes revealed clear structural differences in comparison to corresponding cercarial species which have been shown to exhibit mainly sphinganine bases with 18-21 carbon atoms. Therefore, it may be hypothesized that the divergent structural features of the respective ceramide moieties are responsible for the published observation that only adult worm, but not cercarial glycosphingolipids are able to induce dendritic cell activation skewing the T-cell response toward a Th1 profile.     

Schistosoma mansoni: partial characterization of enzyme(s) secreted from the preacetabular glands of cercariae.

Secretions from the preacetabular glands of cercariae of Schistosoma mansoni were collected over skin surface lipid on a warmed glass surface in a system providing a temperature gradient. Proteolytic activity of these secretions was linear with respect to the numbers of cercariae secreting. The enzyme was relatively storage stable in water or glycine-NaOH buffer. It was highly sensitive to the buffer system, glycine-NaOH buffer providing the highest proteolytic activity against Azocoll and gelatin substrates. In the glycine buffer, the pH optimum for the enzyme was 8.5–8.8; the temperature optimum was 51 C with the Q₁₀ for Azocoll hydrolysis in the range of 30–50 C approximately 2.2. Of the activity 0.4% was lost at 5 C, 1.7% at 35 C, and 100% at 60 C within 10–15 min. Two Sephadex column chromatography fractions showed proteolytic activity against Azocoll. The fraction with greatest activity was not associated with either of the two peaks of high absorbancy at 280 nm, but the lower proteolytic peak coincided with the lower absorbancy peak in the area of peptides below 10,000 MW. Calibration of the Sephadex column with proteins of known molecular weight showed the MW of the main proteolytic fraction of the secretion to be approximately 25,000–27,000.     

Effect of ultraviolet radiation on survival, infectivity and maturation of Schistosoma mansoni cercariae

S. mansoni cercariae exposed to ultraviolet radiation for 1, 3, 5, 10 and 20 s as well as non-irradiated cercariae remained actively motile 30 min post-irradiation. Thereafter the activity decreased with increasing dose level of radiation and age of cercariae. There was no significant difference between the rates of attachment of the batches of cercariae. The recovery rates (0-49% of cercariae to which mice were exposed) of adult worms were, however, significantly different from the number of cercariae calculated to have attached to the mice (93.5-100% of cercariae to which mice were exposed). Maturation and penetration rates were dependent on radiation exposure levels. Numbers of eggs deposited in the liver of mice as well as hatchability rate of eggs varied significantly with the levels of exposure to radiation.     

Scanning electron microscopical studies on the tegument of adult worms of Schistosoma mansoni originating from ultraviolet-irradiated and non-irradiated cercariae.

The surface topography and ultrastructural changes of Schistosoma mansoni worms developed from ultraviolet-irradiated cercariae were compared with those from non-irradiated cercariae. The tegument of worms developed from irradiated cercariae showed a variety of changes including the occurrence of oedemata and most of the tubercles being torn at the tip and having lost their spines on the dorsal surface of some male worms. In females, parts of the tegument were devoid of spines and surface lesions were present. The range and extent of these changes differed not only among individual worms but also between different regions of individual worms resulting in generalized deformities in those worms originating from irradiated cercariae.     

Effect of selected chemical agents on longevity and infectivity of Schistosoma mansoni cercariae.

The effects of various chemical agents on longevity of the cercariae of Schistosoma mansoni Sambon, 1907, were investigated. The median lifetime of cercariae maintained in dechlorinated tap water (DTW) was 10.5 hr. Increasing concentrations of sodium chloride added to distilled water increased the median lifetime to an optimum of 10 hr at 0.01 M; higher salt levels decreased longevity. At this optimum sodium chloride level, concentrations of glucose between 0.003 M and 0.03 M enhanced median survival to 13–14 hr. Using Tris-HCl buffers the effects of pH and ionic strength were examined. Cercarial longevity increased from 3.5 to 25 hr as pH increased from 6.4 to 9.0, and 0.01 M Tris was superior to 0.001 M Tris at a given pH. The greatest median lifetime (26 hr) was obtained in 0.01 M Tris, pH 9.0. Infectivity of cercariae in Tris at this optimal pH was compared to that in DTW. Maintenance in DTW resulted in greater worm burdens in mice than did treatment with Tris. This suggested that factors which affect cercarial longevity may not influence infectivity in the same manner. The effects of rotenone, Antimycin A, dinitrophenol, and potassium cyanide on cercarial viability suggested the existence of a functioning terminal electron transport chain similar to that of mammalian systems.