Adult Tegument Research Articles

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

AbstractThe 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 Canavaliaensiformis (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.

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.

Schistosoma mansoni:The Developmental Regulation and Immunolocalization of Antioxidant Enzymes

Antioxidant enzymes fromS. mansoni,cytosolic Cu–Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host–parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack.

Characteristics of alkaline and acid phosphatase in Spirometra erinacei

This study was done to investigate the enzyme-histochemical localization and characteristics of alkaline and acid phosphatase related with metabolism in sparganum and adult of Spirometra erinacei. By the enzyme-histochemical assay, the alkaline and acid phosphatases were localized in the tegument and subtegumental musculature of sparganum and adult, but not in the parenchyma. The activities of alkaline phosphatase were stronger in the tegument than in the subtegumental musculature, and activities of acid phosphatase were stronger in the tegument of adults than those of sparganum. The 2 isozymes of alkaline and acid phosphatases were separated from s-sparganum (from snake) and r-sparganum (from experimentally infected rats) respectively, but 4 isozymes of Alp and 3 isozymes of Acp were separated from adult worms by electrophoresis. In isozyme Alp, the 66 kDa was the common isozyme, but 130 kDa isozyme of Acp was the common isozyme in spargana and adult worms. By isoelectrofocusing, 4 isozymes (PI 7.9, 7.7, 6.5 and 6.3) and 2 isozymes (PI 7.9 and 7.7) of alkaline phosphatase were separated from adults and spargana, respectively. In the stability against heat, activity of alkaline phosphatase was denatured perfectly after heating at 90 degrees C for 40 seconds. The optimum pH and temperature for activity of alkaline phosphatase were about pH 10 and 50 degrees C, respectively. The maximum activity (unit) of alkaline phosphatase was 22.0 in s-sparganum, 25.0 in r-sparganum and 215.0 in adult worms, so that the maximum activity was revealed higher in adults than spargana. As the result from above, we observed that alkaline and acid phosphatases were functioned mainly in the tegument and subtegumental musculature, and the isozymes of phosphatase were activated differently according to habitat of the parasites. The spargana and adult worms carry out the parasitism by adapting themselves to parasitic circumstance with these enzymes.

Identification and partial characterization of the proteases from different developmental stages of schistocephalus solidus (cestoda: Pseudophyllidae

The proteolytic activities of extracts prepared from procercoids and plerocercoids as well as adults of the pseudophyllidean cestode Schistocephalus solidus were examined using several proteins and synthetic peptides as substrates. Whole bodies of procercoids and the isolated syncytial tegument of plerocercoids and adults prepared by freeze-thaw were studied. Extracts of procercoids contained a chymotrypsin-like proteinase exhibiting a molecular weight of 23,500 determined by gel filtration chromatography. The proteinase showed collagenolytic activity and had a pH optimum at 8. Such a proteinase was absent in plerocercoids and adults. In these developmental stages leucine aminopeptidases were detected in the isolated syncytial tegument having molecular weights of 93,500 (plerocercoids) and 89,000 (adults), respectively. The aminopeptidases in both stages displayed optimal activity at pH 8.5. The chymotrypsin-like proteinase of the procercoid is possibly necessary for the penetration of the host's intestinal wall, whereas the aminopeptidases of the plerocercoid and the adult of S. solidus may aid in parasite nutrition by degrading oligopeptides at the tegumental surface.

Reactivity of anti-tegument monoclonal antibodies with target epitopes in different worm tissues and developmental stages ofSchistosoma mansoni

Sixteen monoclonal antibodies (MABs) were selected for their reactivity with adult schistosome tegument. The distribution of target epitopes in different tissues of the adult and in various developmental stages was investigated by indirect immunofluorescence. The distinct patterns of reactivity of the MABs permitted their classification into 9 groups. The distribution of epitopes in larvae, particularly 3 h schistosomula, generally mirrored that in adults. A change in distribution of epitopes coincided with transformation from cercaria to schistosomulum providing a marker for this process. Two MABs reacted with surface membranes of intact cercariae, and 5 with the surfaces of intact 3 h schistosomula. The target epitopes of these 5 MABs were present in the tegument of adults, but not accessible externally. These observations suggest masking of antigens, not shedding, in the course of development. Indeed, no MAB reacted with intact lung or liver worms unless they were damaged. Three MABs reacted with membranes of the tegument and most other tissues, implying properties in common. Evidence for shared functions between epithelia was provided by the reactivity of 2 MABs with the tegument and protonephridia. Eight MABs bound both to the tegument and its cell bodies. Since the latter contains the machinery for protein synthesis, it is difficult to explain why 5 MABs bound to the tegument alone. Little cross-reactivity was found with the tissues of the tapeworm Hymenolepis, rather more with the closely related Schistosoma japonicum. It was concluded that tegument antigens are to a degree tissue specific but definitely not stage specific.

Expression of Lectin-Binding Surface Glycoproteins during the Development of Schistosoma mansoni Schistosomula

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.

Fasciola hepatica: antibody levels in sheep serum before and after treatment with anthelmintic

Two groups of sheep were treated with the anthelmintic diamphenithide three and six weeks respectively after oral infection with metacercariae of Fasciola hepatica. A third group was infected but left untreated. The levels of circulating antibody against three parasite antigens Tl (juvenile tegument glycocalyx). T2 (adult tegument) and G (gut secretions) were monitored throughout infection and recovery-periods using an indirect fluorescent antibody technique with serial sections of plastic-embedded fluke tissues as test antigen. Termination of infection after three weeks prevented the development of the T2 antibody response and led to significant premature peaking and, or, decline of Tl and G antibodies. However the magnitude of these peaks was unaffected by chemotherapy. Treatment six weeks after infection had no significant effect on the Tl or G antibody titres and these antibody profiles closely resembled the equivalents in control animals. However the T2 antibody response was somewhat reduced, supporting the view that while T2 granules occur in the tegumental syncytium from an early stage in fluke development the T2 antigens are not expressed at the surface until late in the liver migration phase. In the three week and six week treated groups, decline from peak antibody titres during the recovery period paralleled natural decline in antibody titres after bile duct invasion in the untreated control animals. This provides evidence for immunological isolation of the bile duct habitat.

Antibody response in cattle, sheep and rats to infection with γ-irradiated metacercariae of Fasciola hepatica

Cattle, sheep and rats were infected orally with gamma-irradiated (4 krad) metacercariae of fasciola hepatica, or with normal metacercariae. The antibody response was monitored in each host to metacercarial tegument (T0), juvenile tegument (T1), adult tegument (T2) and gut antigens. The response was examined at weekly intervals for cattle and sheep throughout 15 weeks of infection and four weeks after infection in rats, using an indirect fluorescent antibody labelling technique. It was found that the irradiated metacercariae engendered a normal humoral response to T0, T1 and gut antigens in all three hosts although the antibody levels were somewhat reduced due to an early death or stunting of the flukes. T0 and T1 appeared to be antigenically similar. Antibodies against T2 appeared late in the animals infected with gamma-irradiated metacercariae and the titres attained were considerably lower than in the controls. the T2 antigen stimulus in the animals given gamma-irradiated metacercariae was probably provided by flukes which 'broke through' the developmental barrier imposed by irradiation and which were found alive at autopsy.

Fasciola hepatica: IgG and IgA levels in the serum and bile of infected cattle

Bile and serum samples were collected from calves with an implanted cannula throughout a 20-week period of infection with Fasciola hepatica. Using indirect fluorescent antibody labelling and plastic-embedded sections of juvenile and adult flukes as antigens, estimates were made of the relative concentrations of IgG and IgA specific for fluke tegumental and gut antigens in the samples of serum and bile. In serum, antibodies against juvenile (t1) tegument and gut antigens reached peak concentrations 4–6 weeks postinfection and declined slowly thereafter as flukes became established in the bile ducts. IgG against adult tegument (t2) antigens appeared in the serum 6 weeks after infection, but no IgA against t2 was detected. In the bile, both IgG and IgA titres against t1 and gut antigens rose to peak values at 4–6 weeks after infection, but there was no activity against t2 antigen. The Ig levels in bile were considerably lower than in serum. Much more IgA relative to IgG occurred in bile as compared to serum (IgG/IgA ratio in serum was 16–32, in bile 1–2) suggesting a role for IgA in defence at mucosal surfaces. Comparison of the antibody profiles in bile and serum suggested that IgG in the bile was derived from circulating IgG whereas IgA may have been preferentially concentrated in the bile.

Analysis Of Protein Components At Varioue Stages Of Clonorchis Sinensis

In this study the authors examined the protein components at various stages of Clonorchis sinensis, and those of tegument and metabolite of adult Clonorchis by using SDS-polyacrylamide gel electrophoresis and immunodiffusion. The following results were obtained: 1. The protein components of C. sinensis were gradually changed during its development. A considerable change occurred during the initial 7 days after the metacercarial infection. 2. Two bands of protein of about 97,000 molecular weight (MW) and 178,000 MW were unique to excysted metacercaria of C. sinensis. Other 2 bands of protein of 23,000 and 25,000 MW which were absent in metacercariae, might be associated with the development of sex organs in adult. 3. In the metabolite, some components of tegumental proteins were detected. And this tegumental protein components in metabolite seems to be the major antigenic components reacting with infected rabbit antiserum by immunodiffusion. 4. Twenty bands of protein were detected in the isolated adult tegument. Among them 6 bands were in 97,000~65,000 MW, 3 bands in 56,000~53,000 MW and 5 bands in 37,000~30,000 MW. On the other hand, in metabolic products of adult C. sinensis, 17 bands were detected.

Tegument and apical end organ fine structure in the metacestode and adult Proteocephalus ambloplitis

The tegument of plerocercoid and adult P. ambloplitis was examined. Differences in tegument structure existed between these two stages. Plerocercoids of P. ambloplitis lacked extensive vacuolization and unicellular gland cells characteristic of adult tegument. Plerocercoid microtriches were short and conoid; adult microtriches were lenticular with an extended, whip-like shaft. An inclusion, not previously reported from proteocephalid cestodes, is described. Adult tegument had ducts, originating from underlying unicellular glands, extending through the distal cytoplasm and opening to the exterior between microtriches. The apical end organ cavity of P. ambloplitis contained numerous labyrinth-like spherical bodies. These structures appeared to be synthesized and secreted into the end organ by a thin cellular lining of the end organ. This lining was composed of discrete, filamentous cells believed to be modified subtegumental cell bodies. Spherical structures identical to those observed within the end organ cavity occurred within this cellular lining. The spherical bodies may be associated with enzymes necessary for tissue migration by the metacestode.

The Acanthor Tegument of Moniliformis dubius

The acanthor tegument of Moniliformis dubius is composed of several components situated between the free surface and the hypodermal musculature. A trilaminate plasmalemma measuring approximately 60 A is associated externally with an amorphous component that light and electron microscope level histochemical procedures suggest has acidic polyanionic chemical characteristics. Situated beneath the surface membrane are two feltlike layers separated by a cytoplasmic space except where the layers fuse to form the basal areas of the cuticular spines. Primary infolding of the plasmalemma defines intrahypodermal crypts lined by a membrane approximately 70 A thick and extensively evaginated to form microvilli. Continuity of these labyrinths with the free surface via pores is demonstrated by the penetration of colloidal lanthanum nitrate into these crypts. Glycogen deposits, mitochondria, and doughnutlike vesicles are characteristic components of the hypodermal cytoplasm. There is no evidence of an alimentary system at any stage during the entire life cycle of the acanthocephalan Moniliformis dubius. The body surface of the parasite therefore serves as the primary interface mediating physiological integration of the worm with its environment. The adult and cystacanth tegument has been previously described (Wright and Lumsden, 1968, 1969; Wright 1970a). A comparable investigation of the surface features of the hatched acanthor is reported in the present paper. MATERIALS AND METHODS

Glucose uptake in Haematoloechus medioplexus and Gorgoderina trematodes

Autoradiographic studies demonstrated the uptake of tritiated glucose in vitro by the tegument of adults of Haematoloechus medioplexus Stafford, 1902, and a Gorgoderina sp. (Trematoda). Freeze-dried worms were embedded in Epon-812 to detect free glucose and in paraffin to detect incorporated glucose. Absorption was evident in H. medioplexus after 1 minute and in Gorgoderina after 15 minutes. Incorporation of glucose was observed in both trematode species after 15 minutes. No absorption by the alimentary canal was found in starved or unstarved specimens of either species.