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.     

Cytochemical characterization of tegument membrane-associated carbohydrates in Taenia crassiceps larvae.

The tegument of larval Taenia crassiceps possesses a surface coat rich in both neutral and acidic carbohydrates. Neutral glycans were detected in Golgi vesicles of the tegument perikarya, vesicles of the distal tegument, and on the surface of the plasma membrane. Autoradiographs indicated the tegument perikarya as major sites of 3H-galactose incorporation into acid-insoluble macromolecules. The labeled material is subsequently translocated to more superficial regions of the tegument, then concentrated in the brush border. Loss of radioactivity is appreciable within 6 hr of the synthesis of this material, indicating continual replacement of this tegument surface component.     

Regional specializations of the tegument of the metacercaria of Timoniella imbutiforme

The ultrastructure of the spinous body tegument of the metacercaria of Timoniella imbutiforme (Molin, 1859) has recently been described. Other regions of the metacercarial tegument, including those of the oral sucker, pharynx, and nephridiopore, demonstrate considerable specializations. The oral sucker tegument had an aspinous outer syncytial layer that possessed a pimpled apical surface as well as enclosing two types of secretory bodies. The pharyngeal tegument likewise lacked spines, but possessed only one type of secretory body, and a smooth but folded outer surface. The nephridiopore tegument, however, showed the greatest degree of specialization possessing a single type of secretory body specific only to this region of the tegument. Also associated with the syncytium here was a prominent long filamentous glycocalyx, and microtubules which were observed for the first time in this region of the tegument.     

Structure of the tegument and ectocommensal microorganisms of Gyliauchen nahaensis (Digenea: Gyliauchenidae), an inhabitant of herbivorous fish of the Great Barrier Reef, Australia

The ultrastructure of the tegument and tegument-associated microorganisms of the gyliauchenid digenean Gyliauchen nahaensis is described by transmission and scanning electron microscopy. The tegument is devoid of surface spines and is characterized by a moderately folded apical membrane, abundant vesicles, basal mitochondria, a folded basal plasma membrane, and a thick basal matrix. Microorganisms form a dense biofilm on the tegument of the posterodorsal surface and the excretory papilla. At least 7 microbial morphotypes were identified, including eubacteria, spirochaetes, and nanobacteria.     

Fasciola hepatica: interaction of the tegument with poly-L-lysine and enzymes

The tegument of Fasciola hepatica was treated with 0.5% pepsin (EC 3.4.4.1), 0.5% alpha-amylase (EC 3.2.1.1), and 0.5% sheep bile solutions both without and following preincubation in poly-L-lysine. Without poly-L-lysine preincubation, pepsin appeared to be breaking down limited areas of the tegumental surface but had no other marked effects on tegumental structure. alpha-Amylase and bile had no major effects on the tegument except for a reduction in matrix density by the latter. Incubation in poly-L-lysine alone resulted in some changes in surface morphology of the tegument and a limited amount of swelling of the basal infolds. When poly-L-lysine was followed by pepsin treatment, blebbing, microvillus-formation, and swelling of the basal infolds was greatly enhanced and led to surface destruction in some areas. alpha-Amylase following poly-L-lysine resulted in complete destruction and loss of the tegument, and left the basal lamina as the external surface, Incubation in bile after poly-L-lysine preincubation resulted in little change in tegumental morphology.     

The surface topography of a monogenean, Diclidophora merlangi revealed by scanning electron microscopy

The body surface of adult Diclidophora merlangi is characterized by an annular array of irregularly-shaped ridges, with the intervening troughs of tegument invested with microvilli. The microvilli measure approximately 0.5 micrometer in length and 0.06 micrometer in diameter and at maximum density (10.8/micrometer 2) increase the body surface area by a factor of two. On the anterio-dorsal surface of each peduncle the ridge-like structures consolidate as a distinct area of ornamentaltion some 100 micrometer in diameter. A frontal pit opens just inside the mouth and is thought to provide the outlet for the buccal gland secretions. The tegument surrounding the gonopore and paired excretory pores is unmodified in morphology. Presumed sensory endings are of at least two types: (1) uniciliated structures, projecting from a pit or supported by a collar of tegument, and distributed either singly over the general body surface and in the buccal cavity, or in groups around the mouth; and (2) large numbers of non-ciliated papillae localized on the opisthaptor. A number of these papillae occur on a wart-like outgrowth of tegument positioned on the ventral surface of each peduncle and have obvious significance in contact communications.     

扩张莫尼茨绦虫(圆叶目:裸头科)节间腺的超微结构及组织化学

用光学显微镜和透射电子显微镜观察了扩张莫尼茨绦虫节间腺形成过程的精细结构及一些组化变化。结果表明：节间腺是扩张莫尼茨绦虫皮层的特化部分,由节片后缘的皮层及其邻近细胞体向绦虫实质组织中陷入开始其形成过程,随着虫体发育的进行,新的陷入不断形成,原陷入的部分不断脱离皮层形成簇状腺体结构。节间腺的数目随着体节的发育不断增加,幼节中仅有少数几个(6～9个),而远端的孕节中多于100个。电镜下可见腺细胞体由细胞质管与腺皮层相联,簇状腺体结构为一合胞体形态,腺细胞体围绕并开口于椭球体或不规则形状的皮层腔中。离腺皮层远的腺细胞体电子密度高并含有与腺皮层相应的典型分泌颗粒,而靠近腺皮层的腺细胞体电子密度低,所含分泌颗粒较少。扩张莫尼茨绦虫节间腺的组化性质尚不完全清楚。糖与蛋白质等组化结果不稳定,随染液pH值及染色时间的变化等多种因素而改变。基于我们的研究及其他研究者的观察表明,节间腺可能参与外源基质形成虫卵的转运,同时他们可能在虫体节片脱落及虫卵溢出时起作用。     

Integument of the tapeworm scolex. 1. Freeze-fracture of the syncytial layer, microvilli and discoid bodies

The tegument of cestodes is the most important and structurally complex metabolic interface between these parasites and the hostile environment in which they reside. In spite of the complex metabolic, regulatory and immunological properties of this layer of syncytial cytoplasm, which are relatively well known, the detailed fine structural anatomy of the cestode tegument remains equivocal. The present study therefore reports the freeze-fracture morphology of the tapeworm (Hymenolepis diminuta) tegument. The most important features revealed by analysis of platinum replicas of freeze-fractured tapeworm scolex-neck tegument include: (a) presence of highly ordered linear and/or circumferentially-orientated rows of intramembrane particles situated on the PF fracture face of microvillar plasma membrane, which may participate in movements of the microvilli, (b) presence of apparent 'pores' (11 nm in diameter) at the tips of the tegumentary microvilli, which could serve as regulated gates through which extramicrovillar surface coating materials can be extruded, and (c) the alignment of cytoplasmic discoid bodies into positions at the bases of the surface microvilli such that they could move into the core of each microvillus and thereby release their contents for extrusion (via the pores) onto the outer surface of the microvilli. Concomitantly, the limiting membrane of the discoid bodies could be added to the tegument plasma membrane and thereby contribute to the rapid turnover of the tegumentary surface. This study provides the first detailed account of the ultrastructural anatomy of the tapeworm tegument and is intended to serve as a point of reference for future investigations of tapeworm tegumentary functions.     

The Buccal Organ of Some Monogenea Polyopisthocotylea

Light and electron microscopic studies of the 'buccal suckers' of Gotocotyla secunda showed that they are complex structures with muscular, glandular, (resorptive?), and probably sensory components. The name 'buccal organ' is therefore proposed for them. Ducts run from the lumen of the buccal organs to their dorsal, ventral and lateral surface and communicate with lacunae surrounding them. The surface of the ducts and of the muscular walls of the buccal organs are lined by tegument with short microvilli, tegument with dense, long surface lamellae, and tegument with thick, long microvilli which have an electron-dense core. The latter type of tegument contains many vesicles and electrondense ovoid bodies, and is traversed by gland ducts containing large secretory droplets. Electron microscopic examination showed that the prebuccal convoluted structures in Heteromicrocotyloides mirabilis are a modified tegument connected to subtegumental cells. Light microscopic studies of Gotocotyla bivaginalis. Pseudothoracocotyla indica, Pricea multae and Heteromicrocotyla australiensis showed that the first three species have buccal organs similar to those in G. secunda , whereas the last species resembles H. mirabilis. It is tentatively assumed that the buccal organs play a role in finding suitable sites for bloodfeeding.     

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.     

Tetraspanin-2 localisation in high pressure frozen and freeze-substituted Schistosoma mansoni adult males reveals its distribution in membranes of tegumentary vesicles

The tegument, or body wall, of schistosomes is the primary tissue for host interaction and site targeted schistosome vaccination. However, many aspects of the cell biology, particularly differentiation and maintenance, remain uncharacterised. A leading vaccine candidate, Schistosoma mansoni tetraspanin 2 has proven efficacy in experimental models, but its function, precise subcellular location in the tegument and role in tegument biology is not well understood. A primary question is whether this molecule is a true surface molecule, that is, whether it appears within the apical membrane of the tegument. Hitherto, the target sequence for antibody localisation studies had not been available for advanced subcellular localisation studies, such as immuno-electron microscopy, due to aldehyde sensitivity. To circumvent this problem, we adapted the methods of high pressure freezing and cryosubstitution with uranyl acetate for immuno-electron microscopy. The tri-dimensional structure of tegument membranes was resolved using electron tomography. Immunolocalisation of Schistosoma mansoni tetraspanin 2 demonstrates that the molecule is localised to tegument membrane compartments, but predominantly within internal structures associated with surface invaginations and internal vesicles. Surprisingly, no label was found at the virtual surface of the parasite. The significance of this localisation pattern is discussed.     

Histochemical findings of the tribocytic organ and tegument of Fibricola seoulensis

The tribocytic organ and tegument of Fibricola seoulensis were examined histochemically for the detection of carbohydrates, mucosubstances, amyloid, collagen and alkaline phosphatase. The surface, secretes, gland cells of the tribocytic organ, and the tegument of the worms were positive to periodic acid Schiff (PAS) and PAS with diastase stain but negative to other stains. It was inferred that the tribocytic organ and tegument of F. seoulensis comprise neutral mucopolysaccharides, which may take a protective role against host enzymes. The surface and secretes of the tribocytic organ, and the tegument of the worms were also positive to double bridge PAP for alkaline phosphatase. This fact suggests that they may play a role as both self protective and host tissue lytic functions.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Hymenolepis diminuta: ultrastructural observations on complement-mediated tegumental lysis and destrobilation of 4-day-old worms in vitro

Changes in the ultrastructure of the tegument and subtegumental cells of 4-day-old Hymenolepis diminuta were studied in vitro in 50% fresh normal rat serum over a 5-h period and compared with heat-inactivated serum and saline controls. First, membrane-bound vesicles accumulate above the microthrix-border. After 30–40 min large vacuoles, which may contain membranous elements, appear in the tegument at a time when the surface of the young strobila is virtually denuded of the microthrix-border. With prolonged incubations there are subtegumental secretory inclusions with dark, enveloping cytoplasm in the tegument and finally the apical plasma membrane, together with the majority of the matrix, is lost. The disrupted portion of the worm is abruptly demarcated from the comparatively intact scolex/anterior neck region by a constriction. Even after 5 h incubation there is no evidence of loss of tegumental matrix components from regions anterior to the constriction but the neck region shows a significant denudation of the microthrix layer and the tegument contains numerous inclusions. The scolex tegument only showed little evidence of loss of membrane from the surface. Possible mechanisms for the avoidance of complement-mediated lysis in the anterior region are discussed.     

Proterometra macrostoma (Trematoda:Azygiidae): functional morphology of the tegument of the redia

Scanning and transmission electron microscopy were used to study the tegument of the redia of Proterometra macrostoma. The predominant feature of the tegument is the presence of orderly arranged channels giving the tegument a honeycomb appearance in thin sections. The channels are closely apposed like stacked cylinders and lie perpendicular to the basal lamina. The channels are about 0.75 μm long by 0.2 μm in diameter each opening to the outside by a small pore. Mitochondria are located between the basal lamina and the bases of the channels. The free surface of the tegument is amplified secondarily by annular folds and small unbranched mierovilli. In vitro experiments were then designed to test the biophysical properties of this unusual tegument with respect to the flow of water and solutes through the channels. Addition of 5 mm glucose stimulated the uptake of ¹⁴C-mannitol suggesting that a concentration gradient of ¹⁴C-mannitol was generated in the channels.     

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.     

Moniezia expansa: the interproglottidal glands and their secretions

Acetylcholinesterase (EC 3:1:1:7) and alkaline phosphatase (EC 3:1:3:1) were detected in secretions of Moniezia expansa maintained in vitro. Ultrastructural cytochemical studies demonstrated acetylcholinesterase activity on the surface of the microtriches at the base of the interproglittidal glands and in the gland lumen but not in the distal tegument or the gland cells. Alkaline phosphatase activity was demonstrated in the cytoplasm of the gland cells and especially in their protoplasmic connections with the distal tegument. Activity was also found in the distal tegument and the microtriches. It is suggested that the acetylcholinesterase secreted by M. expansa performs a metabolic role at the worm's surface.     

Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics

The schistosome tegument provides a major interface with the host blood stream in which it resides. Our recent proteomic studies have identified a range of proteins present in the complex tegument structure, and two models of protective immunity have implicated surface proteins as mediating antigens. We have used the QconCAT technique to evaluate the relative and absolute amounts of tegument proteins identified previously. A concatamer comprising R- or K-terminated peptides was generated with [(13)C(6)] lysine/arginine amino acids. Two tegument surface preparations were each spiked with the purified SmQconCAT as a standard, trypsin digested, and subjected to MALDI ToF-MS. The absolute amounts of protein in the biological samples were determined by comparing the areas under the pairs of peaks, separated by 6m/z units, representing the light and heavy peptides derived from the biological sample and SmQconCAT, respectively. We report that aquaporin is the most abundant transmembrane protein, followed by two phosphohydrolases. Tetraspanin Tsp-2 and Annexin-2 are also abundant but transporters are scarce. Sm200 surface protein comprised the bulk of the GPI-anchored fraction and likely resides in the secreted membranocalyx. Two host IgGs were identified but in amounts much lower than their targets. The findings are interpreted in relation to the models of protective immunity.     

Isoparorchis hypselobagri: ultrastructure of the tegument and associated tissues in a digenean inhabiting a gaseous aerobic environment

Adult I. hypselobagri live in the swim bladder of the Indian catfish Wallago attu, a gaseous environment with a relatively high oxygen content. The ventral tegument, which in life is applied close to the swim bladder wall, is relatively unspecialized, showing typical ultrastructural features of the digenean surface. The dorsal tegument, which is exposed to the oxygen-rich surroundings, has numerous pyriform extensions of superficial parenchymal cells closely applied to the base of the surface syncytium. These extensions bear numerous mitochondria and send finger-like processes deep into the basal cytoplasm of the syncytium where they interdigitate with corresponding infolds of the basal tegument membrane. The pyriform parenchymal extensions are connected with underlying nucleated cell bodies via irregular glycogen-filled tubular processes, many of which end blindly in the interstitial tissue or expand into glycogen-filled bulbs beneath the cell bodies. These superficial parenchymal processes associate at gap junctions with ramifications of a distinct deeper parenchymal tissue which contains lipid, residual bodies and glycogen. The dorsal tegument and associated structures may constitute a respiratory organ, taking advantage of molecular oxygen diffusing across the surface syncytium to carry out aerobic energy transduction in the superficial parenchymal extensions. ATP so generated may diffuse inwards for distribution throughout the body in the deep parenchymal tissue. The extensive network of ramifying cytoplasmic tubules is supported by a fibrous matrix of interstitial tissue.