An ultrastructural study of the early cercarial development in Prosorhynchoides borealis (Digenea: Bucephalidae) with special reference to formation of the primitive epithelium

Primitive epithelium and outer tegumental layer formation during early cercarial development was studied in Prosorhynchoides borealis using electron microscopy. It demonstrated that germinal cells freely floating in the sporocyst body cavity divide to give rise to naked cell aggregates. These early embryos are highly irregular in outline and are composed of blastomeres differing in size and structure. In embryos consisting of about 12-14 cells a few (possibly only two) superficial macromeres become concave and produce thin extensions which envelop the embryonic mass before fusing to form a syncytial primitive epithelium. This primitive epithelium forms syncytial connections with underlying embryonic cells. Primordial tegumental cells become apparent in late germinal balls below the primitive epithelium. These cells expand and fuse to give rise to an embryonic nucleated tegument. The embryonic tegument is connected to peripheral embryonic cells by thin cytoplasmic bridges until the basement lamina is formed. Subsequently, the primitive epithelium is shed by the embryos and the nuclei in the embryonic tegument undergo pyknotic degeneration. These results are analysed and compared with data from studies on other trematode species and it is concluded that the primitive epithelium is derived from the embryo in at least the majority of digeneans.     

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.     

An ultrastructural study of alimentary tract development in the cercariae of Prosorhynchoides borealis (Digenea,Bucephalidae)

Podvyaznaya I. 2011. An ultrastructural study of alimentary tract development in the cercariae of Prosorhynchoides borealis (Digenea, Bucephalidae). —Acta Zoologica (Stockholm) 92 : 170–178. The development of digestive system in Prosorhynchoides borealis cercariae was studied using transmission electron microscopy. The foregut and caecum primordia arise in early cercarial embryos as two adjoining cellular cords. The primordial pharynx appears as a cluster of myoblasts in the mid‐part of the foregut primordium whose proximal end abuts onto the ventral embryonic tegument. Later, a lumen develops within the gut primordia and their component cells form the embryonic cellular epithelium with an essentially similar structure in the foregut and caecal regions. Subsequently, the foregut epithelial cells merge to form a syncytium. This process proceeds asynchronously and the most proximal foregut area remains cellular for the longest time. The syncytial lining of the foregut establishes syncytial connections with secretory cytons differentiating in the surrounding parenchyma. These cytons produce secretory granules, which are transported through cytoplasmic connections to the foregut syncytium. Before cercariae reach maturity, their foregut epithelium becomes anucleate and continuous with the external tegument. By the end of cercarial development, numerous short lamellae appear on the luminal surface of the caecal epithelium. The caecal cells become involved in secretory activity as indicated by the presence of Golgi‐derived secretory bodies in their cytoplasm.     

Ultrastructure of the daughter sporocyst and developing cercaria of Schistosoma japonicum in experimentally infected snails, Oncomelania hupensis hupensis

Ultrastructure of daughter sporocysts and cercariae of Schistosoma japonicum were studied 2 and 4 months after infection of Oncomelania hupensis hupensis. The body walls of daughter sporocysts are similar at all infectious stages. They consist of an external syncytial tegument on a basement membrane, and an internal cellular subtegument surrounding a body cavity containing developing cercariae. The cercariae embryos develop 2 months after infection from germinal balls in the brood chamber of the daughter sporocyst. They are at first enveloped by a primitive epithelium rising from the daughter sporocyst. Four months after infection, the cercariae were almost fully developed and the primitive epithelium had degenerated. The body wall of the cercaria consists of a thin tegument covered by a surface coat of fibrous material and connected to the subtegumental cells by cytoplasmic processes. The matrix of the tegument contains numerous dense bodies, vacuoles, and spines. Two types of sensory structures - uniciliated and multiciliated - are found at the anterior tip of the cercaria. There are five pairs of penetration gland cells of two distinct types differentiated by the morphology of secretory granules. Flame cells are found in both daughter sporocysts and in cercariae. The cilia of the flame cells are characterized by the typical 9 and 2 cilium pattern.     

日本血吸虫尾蚴发育的超微结构观察:Ⅰ.体被局部剖析

本文根据日本血吸虫尾蚴发育分期,除胚细胞（ＳＩ）已有报道（胡敏等,１９９１）外,对胚球期（Ｓ２）、尾蚴雏体期（Ｓ３）、成熟前期（Ｓ４）及成熟期（Ｓ５）在透射电镜对体被及其要素（ｔｅｇｕｍｅｎｔａｌ ｅｌｅｍｅｎｔｓ）进行剖析。结果原（始）体被最早出现于Ｓ２,表性分布。随后（Ｓ３—Ｓ５）消失而为真正体被所取代。体被皱褶亦随高隆。基膜及体棘为各期基本结构。感觉乳突于Ｓ３、Ｓ４出现。到了Ｓ５渐趋复杂而完善并对其结构作了较精细的描述。糖膜直至成熟期（Ｓ５）才出现,为尾期成熟的标志。近期对糖膜组份有较深入的研究并对其生理功能进行讨论。     

An ultrastructural study of the cercarial excretory system in Bucephaloides gracilescens and Prosorhynchus squamatus

The ultrastructure of the flame cells, capillaries, collecting tubes, excretory bladder, excretory atrium, caudal vesicle, lateral caudal ducts and excretory pores of cercariae of Bucephaloides gracilescens (Rudolphi, 1819) Hopkins, 1954 and Prosorhynchus squamatus Odhner, 1905 (Digenea: Bucephalidae) is described. Both species are essentially similar except for some details. The terminal parts of the protonephridia have all the structural features that are typical of trematodes. The collecting tubes in the cercarial body are composed of cells that are wrapped around the lumen. The main collecting tubes are joined to the excretory bladder syncytium by septate junctions. Features of P. squamatus excretory bladder epithelium indicate that it is involved in secretory activity, but this is not the case in B. gracilescens. In both species the luminal surface of the excretory bladder epithelium is increased by lamellae, and the basal plasma membrane forms invaginations. In the bladder syncytium of P. squamatus both apical lamellae and basal invaginations are more developed and mitochondria are also more numerous. The excretory atrium is lined by a syncytium with nucleated cytons located in the surrounding parenchyma. The atrium lining is not continuous with the body tegument and possesses specific secretory inclusions and a thick glycocalyx. Septate junctions connect the atrium syncytium to the excretory bladder epithelium at its anterior end and to the syncytial excretory epithelium lining the caudal vesicle and the lateral caudal ducts at its posterior. In the excretory pores the caudal duct syncytium is joined to the tegument by septate desmosomes.     

The cercarial tail in Proterometra macrostoma (Digenea: Azygiidae): permeability and fine structure of the tegument

Permeability of the cercarial tail in Proterometra macrostoma was examined in vitro with 1 mM 3H-glucose, which tails absorb by diffusion alone. Naturally emerged cercariae (bodies withdrawn into tails) were permeable, but they rapidly (3 min) equilibrated with glucose in the bathing medium and maintained steady state for 4 hr. Metabolism of absorbed glucose was not detectable until after 90 min, and radioactivity in bodies dissected from tails after 4 hr was negligible. On the basis of cercarial water content (90% of total weight) and absorbed isotope at steady state, the calculated volume of the equilibrating compartment was 4% of an intact cercaria. This value correlated well with that of the tegument (3-5%), which was 1-2 microm thick as seen by transmission electron microscopy. A continuous, electron-dense basal membrane/lamina separated the tegument from subtegument. We conclude that the glycocalyx and external plasma membrane are freely permeable, whereas the basal membrane is the barrier that effectively isolated the subtegument from exogenous glucose. The basal membrane also may be the primary structure that protects the subtegument and cercarial body from effects of osmotic stress.     

The formation of the cyst wael of the metacercaria of Cloacitrema narrabeenensis (Bowell & Bearup, 1967) (digenea: Philophthalmidae)

Dixon K.E. and Colton M. 1978. The formation of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell &; Bearup, 1967) (Digenea: Philophthalmidae). International Journal for Parasitology8: 491–499. The cercaria of Cloacitrema narrabeenensis contains six different types of cystogenic cells which were distinguished on the basis of their position, ultrastructure and chemical composition. Four of the different types secrete carbohydrate-protein complexes and the other two protein granules. Early in development, the cercaria is bounded by a flattened, cellular envelope which contains a few mitochondria but lacks cytoplasmic ground substance and a nucleus. Later in development, this envelope is lost, shortly after a new cellular covering forms at the surface. The nuclei are later lost from this layer and it is suggested that they sink inwards, thus forming a cercarial tegument. The products of the cystogenic cells are gradually discharged through pseudopodial-like connections with the surface layer of the cercarial tegument to form the metacercarial cyst wall. The sequence of secretion is controlled so that the separate layers of the cyst wall are formed in their correct order.     

Comparative analysis of the Schistosoma mansoni and Schistosoma bovis cercarial production under the influence of praziquantel

Snails infected with Schistosoma mansoni and S. bovis and fed with a food-Praziquantel mixture stop shedding cercariae for several days. The cercarial production restarts at different levels after stopping treatment: for some Biomphalaria glabrata, the restarting phase of production reaches a high level whereas for other B. glabrata and all of the P. metidjensis, the level of production remains low. Histological studies revealed that at the exact moment of this treatment, there is a total destruction of many nearly mature cercariae whereas the young cercarial embryos and the tegument of the sporocyst remain unharmed. When treatment stops, there is a pronounced proliferation of third-generation sporocysts (Sp III) which invade the available space of the snails' digestive gland.     

Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm

Hockley D. J. and McLaren D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology3: 13–25. The tegumental outer membrane of the cercaria is trilaminate: the adult worm, however, has a seven-layered membrane. Formation of the heptalaminate membrane commences immediately after the cercaria has penetrated the vertebrate host: multilaminate membrane-bounded vacuoles are passed from subtegumental cells into the tegument where they enlarge, join to the outer membrane and open to the exterior. The heptalaminate limiting membrane of the vacuole thus becomes the outer membrane of the tegument. At the same time the original trilaminate tegumental membrane is formed into microvilli which are cast off and thus the cercarial outer membrane is lost. Schistosomula usually have a heptalaminate outer membrane within three hours of penetration. After this time the large vacuoles are replaced by smaller membraneous bodies which presumably contribute to the outer membrane during growth of the schistosomulum. The membraneous bodies are also present in the tegument of the adult worms and there is some evidence that the outer membrane is continually renewed.     

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.     

Activity of praziquantel on in vitro transformed Schistosoma mansoni sporocysts

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.     

Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of Fasciola hepatica

The external surface of the redial body of Fasciola hepatica is provided with microvillus-like projections or short lamellae, and short cilium-like structures are common anteriorly. The anterior part of the cercarial body possesses a pattern of regularly arranged small depressions each containing a spine. Both long and short cilium-like structures occur anteriorly. The tail is spineless and provided with dorsolateral folds. The outer cyst wall is formed by granules secreted from the tegument all over the body apart from the ventral sucker. Most granules transform into fibrillae which form the thick outer spongy layer. The precursor of the inner cyst wall is at the beginning closely attached to the metacercarial surface, but later the membrane-like cyst wall extends, and when fully formed the metacercaria lies free in the flattened circular inner cyst. The ventral plug is formed by the ventral sucker. The tegument of newly excysted metacercariae is provided with simple pointed spines, but later during migration in the mouse the spines become flattened and multipointed. Very young migratory stages may be attached with host cells.     

Schistosoma japonicum: localization of calpain in the penetration glands and secretions of cercariae

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in survival characteristics of Diplostomum spathaceum cercariae emerged from cadmium-exposed Lymnaea stagnalis

The effect of exposing Lymnaea stagnalis (Gastropoda: Pulmonata), infected with Diplostomum spathaceum (Trematoda: Diplostomatidae), to 100 microg l(-1) cadmium for 7 days on survival characteristics (survival, tail loss, decaudized cercarial life-span) of emerged cercariae was investigated. Exposure of L. stagnalis to cadmium resulted in significantly increased D. spathaceum cercarial survival and an inhibited tail loss compared to controls. The normal parallel relationship which exists over time between decreasing cercarial survival and increasing tail loss in controls was changed in cercariae from cadmium-exposed hosts with an increased proportion of cercarial deaths occurring without tail loss. The decaudized cercarial life-span over the survival period of the cercarial population did not significantly change. However comparisons between individuals decaudized during the initial 24 h time period with those which were decaudized during the final period of cercarial survival showed a significantly altered life span which did not occur in the control population. As a potential indicator of penetration 'fitness' comparisons were also undertaken between control and exposed cercariae decaudized during the initial 24 h time period, which revealed that the decaudized cercarial life-span from the exposed hosts was significantly different from controls. This may have important implications for the ability of cercariae to migrate through the tissues of their target host. The importance and relevance of these results to parasite transmission are discussed.     

Metacercarial cyst formation in vitro of Echinostoma paraensei

Cercariae of Echinostoma paraensei Lie and Basch 1968 encysted normally in the presence of Biomphalaria glabrata embryo (Bge) cells in culture, partially in culture conditioned medium, and not at all in fresh culture medium alone. At the ultrastructural level the cyst is composed of 2 well defined regions. The outer cyst wall (OCW) is particulate to fibrous in nature, formed from secretory granules released from the cercarial tegument. Membranous scrolls or rodlets secreted from the subtegumental cystogenous gland cells are then added to this layer, forming the inner cyst wall (ICW). After 24 hr the cultured cyst is enclosed by a thin cellular capsule similar to that found around cysts in the snail host. The capsule also contains collagen fibers, not found elsewhere in Bge cell cultures.     

Fasciola gigantica: Histology of the digestive tract and the expression of cathepsin L

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite’s body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.     

Synthesis of macromolecules by the epithelial surfaces of Schistosoma mansoni: an autoradiographic study.

The use of tritiated leucine as a marker for protein synthesis and of tritiated glucosamine as a marker for polysaccharide/glycoprotein synthesis, is described. Adult worms were pulse-labelled by incubation in medium containing the substrate. Labelled worms were then incubated in chase medium, without labelled substrate, for varying lengths of time before fixation. The distribution of label which had been incorporated into macromolecules in the worm tissues, was examined by light and electron microscope autoradiography. It was estimated that the tegument and tegument cell bodies were the source of 67--80%, and the gut epithelium of 20--30%, of exportable leucine-containing protein. Conversely, the gut epithelium was the source of 72%, and the tegument cells 28%, of exportable glucosamine-containing polysaccharide. The specific activity of labelled protein reached a peak in the tegument cytoplasm after 1.5 h of chase incubation. Half of the labelled protein was secreted into the worm's environment by 3 h of chase incubation. The half-life of secretory protein in gut cells appears to be around 2 h. Labelled protein disappears from the gut lumen relatively rapidly but labelled polysaccharide remains in the lumen at high specific activity for at least 24 h. The major carbohydrate labelled may be the glycocalyx on the luminal surface of the gut epithelial cells. The results suggest that the bulk of worm secretions have a rapid turnover with a half-life of a few hours. Against this background of rapid mass secretion a slower process of membrane turnover would be difficult to detect and quantitatively small.     

Fasciola hepatica: basal infolds and associated vacuoles of the tegument.

The tegument of Fasciola hepatica has been shown to contain long invaginations of its basal plasma membrane, hereafter called basal infolds. Associated with the membranes of the infolds, and with the apical and basal plasma membranes, is a Na⁺ K⁺ ATPase (EC 3.6.1.3). Furthermore, polymorphic masses of acid mucopolysaccharide lie close to or against the sides of the infolds and the basal plasma membrane and also fill cytoplasmic tubules which connect the tegument with the tegumental cells. Fixation and incubation of flukes in hypertonic and hypotonic media have shown that the infolds respond to changes in external osmolarity by collapsing in the former and swelling in the latter. This is not simply a passive response, however, since the infolds return to near normal configuration and morphology within 1 hr in either hypertonic or hypotonic media, even though the whole fluke may be shrunken or turgid depending on medium osmolarity. The tegument, therefore, has many of the characteristics of a transporting epithelium. A theory outlining the possible mode of operation of the tegument as a transporting epithelium is proposed on the basis of the present structural, chemical, and physiological findings combined with current ideas of the role and functioning of standing gradients, and forward and backward channel systems in other transporting epithelia.