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Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone beta-subunit (FSHbeta) and luteinizing hormone beta-subunit (LHbeta) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHbeta but not of LHbeta, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.

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Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.

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A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

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The carbohydrate moieties of larval sparganum proteins in two different species, the snakes, Elaphe rufodorsata, the Balb/c mouse and those of the adult worm, Spirometra erinacei, were compared using five different lectins including GNA, SNA, MAA, PNA and DSA. The GNA positive 53 kDa molecule, which is excretory-secretory protease in the sparganum from the snake showed a stage specific and developmental regulation. We also suggested that sparganum glycosylation may be involved in immune evasion and differentiation into an adult worm.

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The carbohydrate moieties of larval sparganum proteins in two different species, the snakes, Elaphe rufodorsata, the Balb/c mouse and those of the adult worm, Spirometra erinacei, were compared using five different lectins including GNA, SNA, MAA, PNA and DSA. The GNA positive 53 kDa molecule, which is excretory-secretory protease in the sparganum from the snake showed a stage specific and developmental regulation. We also suggested that sparganum glycosylation may be involved in immune evasion and differentiation into an adult worm.

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Calcareous corpuscles, known as mineral concretions in Platyhelminthes, may have special biological functions. In the present study we succeeded in the quantitative separation of calcareous corpuscles in plerocercoids of Spirometra mansoni (sparganum) using Ficoll. Purified calcareous corpuscles were reacted with crude extracts of Platyhelminthes, including sparganum. Proteins commonly binding to calcareous corpuscles were molecules of 10, 17, 22, and 35 kDa. A 95-kDa molecule was found to bind specifically in cystic fluid of Cysticercus cellulosae, as was a 40-kDa protein in crude extracts of Taenia saginata and a 27-kDa molecule in crude extracts of Paragonimus westermani and Clonorchis sinensis, respectively. This finding suggests that calcareous corpuscles might have a binding activity to proteins in crude extracts of sparganum.

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Changes in the fatty acid composition of phospholipid and triglyceride fractions in Spirometra erinaceieuropaei plerocercoids were investigated after 0, 0.5, 1, 3, and 6 hr incubation at 10 C and 37 C with physiological saline containing 5 mM arachidonic acid and 10 mg/ml bovine serum albumin, pH 7.0. At 37 C, arachidonic acid was absorbed and incorporated rapidly into the triglyceride fraction (over 14.4% in composition), and decreased after 2-3 hr; at 10 C, the amount of triglyceride increased slowly and continued to a maximum of 12.9% during 6 hr of incubation. We used a simplified method to extract and purify prostaglandins from the plerocercoid of S. erinaceieuropaei. Prostaglandins were quantified using gas chromatography-mass spectrometry. Prostaglandin E2, PGD2, PGF2alpha, and 6-keto-PGF1alpha were detected under different incubation conditions. In the dose-dependent experiment, PGD2 was detected in plerocercoids incubated with 0.5, 1, 2, and 5 mM arachidonic acid, pH 7.0, at 25 C; PGE2 was detected with 2 and 5 mM arachidonic acid. In the time-dependent experiment, where plerocercoids were incubated with 5 mM arachidonic acid, pH 7.0 at 25 C, PGF2alpha was first detected at 15 min; thereafter, 6-keto-PGF1alpha was detected at 30 min and PGD2 and PGE2 were detected at 1 hr. Thromboxane B2 was not detected in either the dose-dependent or time-dependent experiments, and only PGE2 was detected in the incubation medium with 5 mM arachidonic acid at 1 hr. These results reveal that when plerocercoids change from reptilian to mammalian hosts, they are able to absorb and modify arachidonic acid bound to albumin and generate prostaglandins under suitable conditions. Prostaglandins exhibit potent biological functions for immunoresponses that may be relevant to parasitism and the success of larva migrans in S. erinaceieuropaei.

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Immunoreactivity to prolactin in the nervous system of the plerocercoid of Ligula intestinalis was demonstrated by immunocytochemical method. Numerous PRL immunoreactive perikarya with long varicose fibres were observed in the peripheral nervous system in the worm, mainly in the transversal muscle layer and medullary parenchyma of the midbody. A few fibres were found in the main nerve cords of the central nervous system. PRL positive neurons sent their processes to associate with the main nerve cords. The immunostaining terminals appeared in the subtegument region in the lateral border of the plerocercoid. The result indicates that PRL immunoreactivity is well-developed in the plerocercoid of the cestode. The significance of the localization of prolactin in the worm is discussed.

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With a simplified method of extracting and purifying prostaglandins, trace prostaglandins (nanogram order) were detected by gas chromatography-mass spectrometry. Arachidonic acid was metabolized to prostaglandin E2 (PGE2) by plerocercoids of Spirometra erinacei, the PGE2 was detected in the medium after incubation with arachidonic acid, and the role of albumin in the absorption of free arachidonic acid by plerocercoids and in the release of its metabolite was investigated. Plerocercoids absorbed arachidonic acid-binding albumin and released PGE2 efficiently. PGE2 is known to suppress the functions of mononuclear cells of the host. The selective release of PGE2 may be related to the escape mechanism of plerocercoids of S. erinacei from the host immune system to become established larva migrans, i.e., sparganosis.

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Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.

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Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.

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Adhesion of the neuronal cell surface to its underlying substrate plays an important role in neurite outgrowth in vitro. I have investigated the adhesive basis for neurite outgrowth in the presence of cytochalasin D, a disruptor of actin-containing microfilaments, and in the presence of vinblastine, a depolymerizer of microtubules. Scanning electron microscopy shows that cytochalasin D does not alter the branching configuration of filopodia on a laminin substrate, although processes are shorter and tapered distally in the presence of the drug. Using a standard attachment assay for the neuroblastoma x glioma cell line (NG108-15) I show that vinblastine does not influence attachment of NG108-15 cells to either plastic or laminin. Cytochalasin D-treated cells normally attach to high concentrations of a laminin substrate (20 micrograms/ml). However, when cell are seeded on a laminin substrate at lower concentrations (0.001-10 micrograms/ml), or on YIGSR, a fragment of laminin, cytochalasin D increases cell attachment. Cytochalasin D increases attachment in a dose-dependent manner when cells are seeded on plain polystyrene plastic, so that the number of cells attached to plastic in 1 microM cytochalasin D is similar to the number attached to laminin (20 micrograms/ml). Combining low concentrations of cytochalasin D and laminin results in greater attachment than with either agent alone. Mild trypsinization of the cell surface reduces the CD-enhanced attachment to plastic, indicating that a protein on the cell surface may be involved. The effect of cytochalasin D appears to be cell specific since cytochalasin D does not affect the attachment of a fibroblast cell line (NIH 3T3) to laminin and plastic.(ABSTRACT TRUNCATED AT 250 WORDS)

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The anterior 2 mm of the sparganum of Spirometra mansonoides contained 35 to 48% of the total endogenous cobalamin (Cbl) with only 2 to 4% in the terminal 1 cm. [57Co]cobalamin taken up in the posterior region of spargana was transported directionally to the anterior region and concentrated there resulting in a uniform depletion of [57Co]Cbl along the remaining body length. The anterior 2 mm contained 24% of the sparganum's holo-MMCoAM activity with only 5% in the terminal 1 cm. When head and body preparations were chromatographed on Sephadex G-150 columns eluates from both regions exhibited single peaks of MMCoAM activity at an approximate molecular weight of 150,000 and two peaks of endogenous Cbl. The first Cbl peak cochromatographed with MMCoAM activity and the second Cbl peak eluted at the position of free Cbl. Spargana rapidly converted CN-[57Co]Cbl to hydroxocobalamin (OH-Cbl) and adenosylcobalamin (Ado-Cbl) in vitro. In the head region significant amounts of Ado-Cbl were present (25%) although OH-Cbl was the predominant form of Cbl (45%) after 264 hr in a Cbl-free medium. In the body Ado-Cbl was the predominant form of Cbl (44%) although significant amounts of OH-Cbl were present (27%) after 264 hr in a Cbl-free medium. No methylcobalamin (Me-Cbl) was detected, but an unidentified cobalamin-containing entity (Fraction 4) was present. Based on these results we propose that the sparganum takes up cobalamin, concentrates it in the anterior, "head" region, sequentially metabolizes it to the hydroxyl then the adenosyl forms, and that the adenosylated form is used in this region of high, anaerobic metabolism as an obligatory coenzyme for MMCoAM, in coordination with the differentiation of the sparganum to the adult cestode.

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The plerocercoids of all Spirometra forms investigated up to now in this regard produce a "Sparganum Growth Factor" (SGF), i.e. one (or more) substance(s) with growth hormone-like effect on certain rodents. The SGF produced by the species S. mansonoides (SGF "mansonoides") is stronger effect than the SGF produced by all the other forms (SGF "non-mansonoides"). The physiological in its effects hitherto investigated mainly with SGT "mansonoides" in the USA are shortly mentioned. In order to get further insights the isolation, purification, and characterization of the SGF are necessary. The both kinds of SGF are also of taxonomic importance.

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The effects of bovine growth hormone and the growth factor produced by plerocercoid larvae of the tapeworm, Spirometra mansonoides, on body growth and lipid composition in diabetic-hypophysectomized rats were compared. The diabetic-hypophysectomized control rats gradually lost weight throughout the experiment but both growth hormone and plerocercoids stimulated marked weight gains. Growth hormone treatment resulted in a loss of depot fat from the epididymal fat pads and caused a reduction of liver and serum cholesterol concentrations but had no effect on triglyceride concentrations of either liver or serum. However, plerocercoid infection resulted in increased weights of the epididymal fat pads and increased liver and serum triglyceride concentrations. Serum cholesterol was slightly increased but liver cholesterol was decreased in the plerocercoid-infected rats. Therefore, in the absence of pituitary hormones and insulin, these growth factors had similar effects on body growth but distinctly different effects on lipid metabolism.

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Growth and development of the thymus is dependent on secretions from the anterior pituitary, presumably growth hormone. Diabetes mellitus is known to reduce immunological competence. These studies compare the effects of bovine growth hormone (bGH) and the growth factor produced by plerocercoid larvae of the tapeworm, Spirometra mansonoides, on metabolism of lymphoid tissue, thymus and spleen, in hypophysectomized rats made diabetic with a single intraperitoneal injection of alloxan. Whereas the control diabetic-hypophysectomized rats gradually lost weight throughout the experimental period, both bGH and plerocercoid infection caused significant weight gains during the experimental period. The diabetic-hypophysectomized rats treated with bGH had significantly heavier thymuses and spleens than controls. Plerocercoid infection also caused significant increases in thymus weights. Both bGH and plerocercoids stimulated the metabolic activity of thymocytes isolated from treated rats and tested for their ability to incorporate 3H-thymidine into DNA in vitro. Thus, these growth factors have similar effects on the lymphoid tissue of diabetic-hypophysectomized rats which are apparently independent of normal insulin levels. Whether this anabolic effect is direct or mediated by somatomedin remains to be determined.

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Both spargana and adult forms of Spirometra mansonoides were shown to accumulate lactate, succinate, acetate, and propionate upon in vitro incubation. Adults differed markedly from the spargana in that quantitatively the most significant products of the former were acetate and propionate while the latter formed primarily acetate and lactate. The adults accumulated approximately 32 times more propionate than the spargana per gram of tissue. In accord with this propionate formation, propionyl CoA carboxylase and methylmalonyl CoA mutase have been found to be present in both stages of the parasite. As might be predicted, however, the activities of the carboxylase and mutase were 100-fold and 10-fold higher, respectively, in the adults as compared to the larvae. A possible physiological relationship between propionate formation and energy generation is suggested. Accordingly, inorganic 32P was incorporated into ATP upon incubation of methylmalonyl CoA with a homogenate obtained from adult S. mansonoides. Since methylmalonyl CoA mutase requires vitamin B12 coenzyme, a relationship between vitamin B12 content and propionate formation in helminths is suggested.

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Analysis of tissue from Spirometra mansonoides spargana has shown that cyanocobalamin (vitamin B12) is metabolized to adenosylcobalamin and hydroxocobalamin. No methylcobalamin was detected. When the tissues were examined for enzymes which are known to utilize coenzyme forms of vitamin B12, only methylmalonyl CoA mutase, which requires adenosylcobalamin was found. The enzyme, tetrahydropteroylglutamate methyltransferase, which requires methylcobalamin as a cofactor, was not detected. A sizable portion of the cyanocobalamin taken up was bound to ammonium sulfate-precipitable material, suggesting that the binding substance is a protein. Vitamin B12 taken up by spargana was found to be released in vivo with a biological half-life of about 7 weeks.

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Uptake of 57Co-vitamin B12 (CN-Cbl) by spargana (larvae) of the pseudophyllidean tapeworm, Spirometra mansonoides, was affected by temperature, was saturable with respect to concentration of CN-Cbl in the medium, and was inhibited in the presence of several structural analogs of CN-Cbl. In uptake studies with various analogs it was found that chemical modifications which altered the benzimidazole moiety greatly reduced the ability of the worm to take up these analogs. Modifications in which the amide groups of the propionamide side chains were removed, resulting in carboxylic acid derivatives, showed greatly reduced transport properties. The C-13 epimer in which the e-proprionamide side chain is no longer on the benzimidazole side (lower) of the molecule but is inverted to a position on the upper side was freely taken up. The pharmacological implications of this last observation are discussed. Adult Hymenolepis diminuta did not take up CN-Cbl in vitro, which correlated with the finding that no CN-Cbl was detected in the worm by Ochromonas malhamensis assay.

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