Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms.

BACKGROUND: Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms. RESULTS: By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva. CONCLUSIONS: The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae.

Mode of action of cyclosporin A against Hymenolepis microstoma (Cestoda): relationship between cyclophilin binding and drug-induced damage.

Cyclosporin A (CsA) is a widely investigated experimental anti-parasitic drug whose mode of action remains unresolved. The immunosuppressive action of CsA depends on the drug binding to the intracellular receptor cyclophilin (CyP). This study investigates whether complexing of CsA with parasite CyP is equally essential for its anthelmintic action. The correlation between initial cyclosporin-induced damage in vitro and drug binding to parasite CyP has been examined. CsA and the analogues B-5-49, CsH and CsA-acetate all induced similar damage to the tapeworm Hymenolepis microstoma in vitro in incubations between 2 h and 4 days. The initial foci of drug damage were the parasite surface and mitochondria in the syncytium. In a competitive binding assay only B-5-49 displaced [H3]-CsA from either crude parasite cytosolic CyP or affinity-purified CyP while CsH and CsA-acetate had no effect. These data suggest strongly that cyclosporins act on the surfaces of helminth parasites but that drug action does not involve complex formation with CyP. An alternative drug-binding site must therefore be identified which may lead to the rational design of novel anthelminitic drugs.

Effects of benzimidazole anthelmintics as microtubule-active drugs on the synthesis and transport of surface glycoconjugates in Hymenolepis microstoma, Echinostoma caproni, and Schistosoma mansoni.

Hymenolepis microstoma (Cestoda), Echinostoma caproni, and Schistosoma mansoni (Digenea) were exposed to benzimidazoles to determine the influence of the drugs on the secretion of glycoconjugates that protect the worms' surface. Worms were obtained from mice treated with mebendazole or albendazole, and the glycoconjugates were localized in the parasite tissues by cytochemistry using lectin-gold conjugates. Events leading to the death of H. microstoma and E. caproni extended over a medication period for at least 2-3 days, and the following interrelated phases were discernible. Upon depolymerization of the microtubules the tegumentary cytons continued to synthesize glycoconjugates for up to about 24 h. Vesicles containing the glycans accumulated in the cytons, but their microtubule-based transport to the distal tegument was inhibited. At about 1 day the Golgi complex became fragmented and the production of glycans sharply declined. As a consequence of this and an ongoing turnover of the surface coat the contents of glycoconjugates in the distal tegument decreased. Similar effects were produced by vinblastine and colchicine in vitro. In contrast, benzimidazole treatment of S. mansoni, which is reportedly inefficacious, did not alter the replenishment of the surface glycoconjugates. Diminution of the coating with glycoconjugates of the surface of drug-sensitive species constitutes a secondary effect of benzimidazoles that might, synergistically with immune mechanisms of the host, enhance the expulsion of the worms.

[Establishment of Hymenolepis diminuta-animal model and morphology of cysticercoid].

AIM: To observe the morphological changes in the process of the development of Hymenolepis diminuta. METHODS: The life cycle of Hymenolepis diminuta was established between Rattus domesticus albus and Triboliun castaneum. The morphology of cysticercoid were observed microscopically, and the ultrastructure of the body surface of cysticercoid were observed by scanning electron microscopy. RESULTS: Three phases including the mature stage, the blister stage and the protective outer membrane-forming stage during the growing course of cysticercoid were observed microscopically. Under scanning electron microscope, lots of sieve-like micropores on the surface of mature cysticercoid were seen in the second week after infection. The blister phase was found in the third week and the outer membrane measuring about 45 microns in thickness were found surrounding the cysticercoid and vesicular surface, forming a smooth cyst wall in the fourth week. CONCLUSION: The life cycle of Hymenolepis diminuta has been established in the animal-model. The finding of the three phases during the growing course of cysticercoid is reported for the first time.

Interactions between rat C-reactive protein and adult Hymenolepis diminuta.

Interactions between adult Hymenolepis diminuta and rat C-reactive protein (CRP) were investigated in vivo and in vitro. Using an ELISA technique, serum levels of CRP were monitored in rats infected with 100 cysticercoids. Although infection increased the level of this protein in the early stages of parasitization, the increase was not significant until 35 days post-infection (p.i.). Secondary infections did not enhance the response. When H. diminuta was cultured in the presence of CRP, reduced worm motility and opaque areas were observed and electron microscopical studies revealed shedding of microtriches and lysis of the tegument. Initially, damage was restricted to the strobila which correlated with the regional distribution of phosphorylcholine as visualized using immunofluorescence.

Complex carbohydrates in shell precursor globules of the vitellarium and at the eggshell of Hymenolepis microstoma (Cestoda).

The vitellocytes of Hymenolepis microstoma contain protein globules as building material for the shell of egg cocoons. Complex carbohydrates were detected as an additive component, dispersed in the matrix of the shell globules. Similar glycans were found at newly formed shells, suggesting that these molecules are provided by the vitellocytes. The glycans remained associated with the surface of mature eggs. Carbohydrate residues, characterized using gold-labeled lectins, were N-acetylglucosamine, galactose, or the N-acetyllactosamine sequence of these residues and N-acetylgalactosamine, whereas glucose, mannose, and fucose were not demonstrable. The glycans scarcely stained with periodic acid-Schiff and, thus, seem to have few diols, and they did not bind cationic dye, indicating that they are not glycosaminoglycans. Since they occur in significant amounts, the glycans may have fundamental, as yet undetermined function(s) in the formation of the shells and/or interactions of the cocoons with the host.

Mitochondrial NADH-->NAD transhydrogenation in adult Hymenolepis diminuta.

Adult Hymenolepis diminuta mitochondria catalyze a transhydrogenation reaction between NADPH and NAD and between NADH and NAD. The NADPH-->NAD reaction is catalyzed by an inner membrane-associated pyridine nucleotide transhydrogenase, whereas the NADH-->NAD reaction is ostensibly catalyzed by another system(s). The source(s) of NADH-->NAD activity was evaluated by assessments of its intramitochondrial distribution and thermal lability and by comparisons with the distribution/thermal lability of NADH dehydrogenase, lipoamide dehydrogenase, and NADPH-->NAD transhydrogenase. The occurrence of NADH and lipoamide dehydrogenase components was readily demonstrable. Like NADPH-->NAD transhydrogenase, NADH dehydrogenase was essentially membrane bound. Lipoamide dehydrogenase and NADH-->NAD activities were, at different levels, in the membrane and soluble fractions. Based on thermal profiles, NADH and lipoamide dehydrogenase differed from each other and from NADPH-->NAD transhydrogenase. Although the NADH-->NAD profile closely paralleled that for lipoamide dehydrogenase, it also was similar to the NADH dehydrogenase profile. Collectively, these data are consistent with the supposition that the H. diminuta mitochondrial NADH-->NAD transhydrogenation reaction is catalyzed by lipoamide dehydrogenase and possibly by NADH dehydrogenase rather than by an independent transhydrogenase system.

Hymenolepis diminuta: two morphologically distinct tegumental secretory mechanisms are present in the cestode.

The free-surface of the tapeworm's tegument was examined for morphological evidence of secretion after fixation by rapid freezing-freeze substitution and alternatively by immersion in low concentrations of glutaraldehyde maintained at room temperature. After low-aldehyde fixation, omega profiles were at the bases of tegumental microvilli, arguing for the participation of some of the ectocytoplasm's vesicles in secretion of their contents to the intestinal lumen. The almost instantaneous fixation provided by the rapid freezing-freeze substitution technique documents the constitutive production of 0.03 to 0.075-micron microvesicles from outpocketings from the plasma membrane of the tegumental brush border. Observed in secretory epithelial cells of other species, microvesicles are recognized as a secretory pathway for constituent molecules of surface membranes. We conclude that in addition to the primary route of merocrine exocytotic secretion provided by the fusion of the Golgi-derived, ectocytoplasmic vesicles at the bases of the brush border microvilli, tegumental microvesicles provide a second secretory pathway for endogenous macromolecules across the tegumental free surface.

Microscopical observations on immune precipitates formed in vitro on the surface of hymenolepid tapeworms.

Four-day-old worms of the tapeworms Hymenolepis microstoma, H. diminuta and H. nana and newly excysted H. microstoma were exposed in vitro at 37 degrees C to immune serum from mice infected for 4-12 weeks with H. microstoma. Worms were fixed for electron microscopy after intervals of 5 min to 96 h. Within 10-15 min an homogeneous precipitate occurred between the microtriches of 4-day-old H. microstoma and H. nana, while on some areas of H. microstoma the precipitate extended distal to the microthrix border and contained small vesicles (30 nm in diameter) and shed microtriches. In H. diminuta precipitates were not found until 2 h post-incubation. The thickness of the precipitate and the number of small vesicles and shed microtriches increased with time after incubation. Since a similar precipitate occurred on worms kept in complement-depleted immune serum, antibodies alone may induce immune damage. The precipitate on newly excysted H. microstoma lacked microthrix fragments. After 48 h an extensive precipitate was found protruding from the rostellar glands on some H. microstoma, and within the culture vessel. Antibodies may therefore be complexing with tapeworm secretory products.

The distribution of calmodulin/calmodulin binding proteins in the rat tapeworm, Hymenolepis diminuta.

Live tapeworms have been fixed to retain antigenicity of their proteins, and subsequently prepared for electron microscopy. Thin sections of tapeworms were prepared from resin blocks. Sections were immunocytochemically labelled using a colloidal gold probe and viewed using transmission electron microscopy. Calmodulin was detected associated with cellular structures to which calmodulin has previously been linked in other higher eukaryotes. Calmodulin would appear to have a similar role of importance in tapeworms, as it does in higher eukaryotes although tapeworms are prevalently a syncitium.

In vitro protein synthesis in Hymenolepis diminuta, a rat intestinal cestode.

Helminth parasitic infestations are of great concern to the health of man and other animals. Limited studies have been carried out on the process of protein synthesis which, apart from having academic importance, could possibly provide a target for drug attack. In the present study distribution pattern of newly synthesized proteins in Hymenolepis diminuta, a rat intestinal cestode, has been worked out. The worms absorbed and incorporated about 21% and 3.8% of the amino acids added to the incubation medium, respectively. In other words, 18.3% of the absorbed amino acids was incorporated into proteins. Absorbed amino acids were distributed between washed cell debris (unbroken cells, nuclei, etc.) and crude extract in a ratio of 1:12. Cytosolic, microsomal and mitochondrial fractions received 72%, 3.2% and 6.6% amino acids, respectively. The mitochondrial membranes and the matrix shared equally the absorbed amino acids. The distribution pattern of amino acid incorporation was, however, different from that of absorption. The incorporation ratio between washed cell debris and crude extract was 1:3.7. The cytosolic, microsomal and mitochondrial fractions received 32.8%, 8.6% and 18.4% of the incorporated amino acids, respectively. Within mitochondria incorporation was more in membranes than in the matrix. The ratios of incorporated versus free-pool amino acids in different fractions varied widely from 1:0.54 to 1:11.

Ultrastructure of the gravid uterus of Hymenolepis diminuta (Platyhelminthes: Cestoda).

The fine structure of the uterus in gravid proglottids of Hymenolepis diminuta was examined by standard techniques for scanning and transmission electron microscopy. The uterus consisted of a syncytial uterine epithelium attached to the medullary parenchyma through a thin extracellular basal matrix. The epithelium contained prominent nuclei in the juxtalumenal cytoplasm. The cytoplasm was dominated by extensive granular endoplasmic reticulum, with dilated cisternae containing an electron-lucent material and widely scattered electron-dense spherical bodies. No Golgi body or other agranular endomembrane component was observed, but the epithelium contained numerous free ribosomes and a few mitochondria. The apical plasma membrane was folded into long microlamellae. Epithelial and epitheliomesenchymal folds and villi resulted in a compartmentalized uterine lumen, with each chamber containing 1 to several eggs. These data suggest a high level of synthetic activity within the uterine epithelium, but the chemical products and functional significance of this activity are not yet known.

Evidence for a G protein system in the tegumental brush border plasma membrane of Hymenolepis diminuta.

Guanine nucleotide-binding regulatory proteins (G proteins) mediate the transduction of signals from cell-surface receptors to intracellular effector enzymes. G protein alpha-subunits are routinely identified (and partially characterized) on the basis of their susceptibility to NAD(+)-dependent ADP-ribosylation NAD(+)-dependent ADP-ribosylation catalysed by cholera and/or pertussis toxins. Analysis of purified tegumental brush border plasma membrane from Hymenolepis diminuta by relevant methodologies has revealed the presence of a 42 kDa putative G protein alpha-subunit that is susceptible to ADP-ribosylation by both cholera and pertussis toxins. This polypeptide shows no definite resemblance to any of the four major mammalian G protein classes on the basis of M(r) and toxin-susceptibility. These results provide evidence for the existence of a tegumental G protein-linked signal transduction system in H. diminuta.

Preparation of the oncospheres of the cestode, Hymenolepis diminuta for experimental studies.

The paper deals with methods facilitating the preparation of oncospheres of the cestode, Hymenolepis diminuta, for experimental studies. Described in detail are procedures for the infection of the definitive hosts with the oncospheres; collection and artificial hatching of oncospheres; purification of hexacanths; preparation of extracts from the hexacanths; and preparation of hexacanths for electronmicroscopic studies.

Energy-linked mitochondrial pyridine nucleotide transhydrogenase of adult Hymenolepis diminuta.

Employing "phosphorylating" submitochondrial particles as the source of pyridine nucleotide transhydrogenase, the occurrence of an energy-linked NADH----NADP+ transhydrogenation in the adult cestode Hymenolepis diminuta was demonstrated. The isolated particles displayed rotenone-sensitive NADH utilization and the reversible transhydrogenase, with the NADPH----NAD+ transhydrogenation being more prominent. Although not inhibiting the NADPH----NAD+ reaction, rotenone, but not oligomycin, inhibited the catalysis of NADH----NADP+ transhydrogenation. In the presence of rotenone, Mg2+ plus ATP stimulated by more than 3-fold NADH----NADP+ transhydrogenation. This stimulation was ATP specific and was abolished by EDTA or oligomycin. Succinate was essentially without effect on the NADH----NADP+ reaction. These data demonstrate the occurrence of an energy-linked transhydrogenation between NADH and NADP+ with energization resulting from either electron transport-dependent NADH oxidation or ATP utilization via the phosphorylating mechanism in accord with the preparation of "phosphorylating" particles. This is the first demonstration of an energy-linked transhydrogenation in the parasitic helminths and apparently in the invertebrates generally.

Reinvestigation of the ultrastructure of spermiogenesis and the spermatozoon of Hymenolepis nana (Cestoda, Cyclophyllidea), parasite of the small intestine of Rattus rattus.

Spermiogenesis in Hymenolepsis nana begins with the formation of a differentiation zone. This is limited at the front by arched membranes, is surrounded by cortical microtubules associated with 12 crested-like bodies, and contains a single centriole made up of doublets. The distal centriole gives rise to a flagellum that grows at the same pace as the cortical microtubules. Migration of the nucleus takes place after the formation of the flagellum. It is followed by the separation of the old spermatid from the residual cytoplasm. The mature H. nana spermatozoon is filiform and lacks mitochondria. The axoneme, of the 9 + "1" pattern of the Platyhelminthes, does not reach the extremities of the spermatozoon. The nucleus is electron dense and is in close contact with the axoneme around which it coils in a spiral making an angle of 10 degrees to 15 degrees with the spermatozoon axis. The cortical microtubules follow a 10 degrees to 15 degrees helicoidal path along almost their whole length, except at their posterior extremity, where they are parallel to the spermatozoon axis. H. nana is distinguished by the early development of 12 crested-like bodies of different lengths and by the existence of a single centriole in the differentiation zone. Such a high number of crested-like bodies had never previously been reported in a cestode.

Effects of cyclosporin A on the morphology and tegumentary ultrastructure of Hymenolepis microstoma in vivo.

Cyclosporin A (CsA) induced significant changes in parasite morphology when administered to mice infected with Hymenolepis microstoma. Gross morphological damage consisted of proglottis swelling and the formation of protuberances from the worm surface, visible with a low-power dissecting microscope, occurring most frequently in the posterior third of the strobila. Gross morphology and ultrastructure were examined further using scanning and transmission electron microscopy. Swollen proglottides exhibited areas covered in small pits and fissures (diameter approximately 1-2 microns) but it was not possible to establish the significance of this damage. The brush-border and distal cytoplasm appeared largely intact although some evidence of swelling of the basal membrane invaginations and possible fluid accumulation was seen in drug-treated TEM sections. The apparent oedematous condition of many of the proglottides from drug-treated mice may indicate that CsA treatment mediates permeability changes in the worm surface membrane but the mechanisms by which this may occur remain to be elucidated. The effects of CsA on the morphology of H. microstoma correlate with the previously described anthelmintic activity of the drug against this parasite where CsA treatment dramatically reduces worm growth, retards migration into the bile duct and limits parasite survival.

Effects of three anthelmintics on the tegument of Hymenolepis fraterna (Cestoda).

The in vivo effects of the anthelmintics taenifugin, VUFB 14170 and VUFB 15269 on the tegument of Hymenolepis fraterna have been examined by SEM, TEM and cytochemistry. The drugs were given to H. fraterna-infected mice on the 14th day post-infection in a single oral dose of 150, 200 and 200 mg/kg, respectively. By 72 h post-treatment, the drug-induced pathomorphological changes in the tegument indicated that all three drugs had a significant effect. Changes were most pronounced on the brush border and in the intercellular material. On the apical surface, there was blebbing as well as accumulation of membrane fragments over the microthrix tips and erosion of the brush border. The intercellular material was changed in structure, showing increased electron density in some areas and oedema of the intercellular spaces in other areas. There were also fractures of the tegument of variable depth, sometimes reaching to the parenchyma. These results suggest altered tegumental integrity and, occasionally, complete disruption of the selective permeability barrier created by the normal tegument. This suggestion is further supported by the penetration of ruthenium red into some tegumental areas and its distribution into the intercellular spaces, down to the parenchyma. The intrategumental lysosomes also appeared to be significantly activated. There was evidence of autophagy in both distal cytoplasm and tegumental cells. Mature and gravid proglottides were more susceptible to drug damage than those in the anterior strobila and neck.

The effect of dimethylsulfoxide on the tegumental brush border of the cestode Hymenolepis diminuta.

Dimethyl sulfoxide (DMSO), commonly used for cryoprotection or for the solvation of cytoskeleton-modifying drugs, causes changes in the topology of the plasma membrane of the tegumental brush border in the tapeworm Hymenolepis diminuta; however, relatively long exposures of high concentrations are required. In tapeworms treated with DMSO concentrations of greater than or equal to 1% in the present study, the interaction of the tegumental surface membrane with the underlying cytoskeleton may have been disrupted at focal points in the brush border, resulting in a partial loss of the membrane anchoring required for the structural integrity of the brush border. Blebbing of the tegumental surface was prominent only after exposure to 1% DMSO for 20 h in in vitro culture with RPMI 1640, and vesiculation of the membrane along the microvillar (microtriche) shafts, which may have been related to the in vitro conditions, was amplified by the presence of concentrations of greater than or equal to 1% DMSO in the incubation medium. The tegumental response to DMSO was not uniform but regional, consistently appearing to be more prevalent on the distal aspects of each proglottid rather than on the edge proximal to the scolex. Blebbing and vesiculation were not seen on the basal aspect of the tegument, including the basal ectocytoplasmic plasma membrane, the perikarya, and the internuncial processes. Microvillar core bundles of actin microfilaments persisted following 8 h in vitro exposure to all three concentrations of DMSO tested (0.1%, 1%, 5%); however, only in tapeworms that were treated in vitro with 5% DMSO for greater than or equal to 8 h did core microfilament bundles appear to lose the rigidly straight and parallel organization characteristic of control tapeworms that were incubated either in the absence of DMSO or with 0.1% DMSO. Other components of the brush border cytoskeleton (i.e., microvillar caps, junctional tubules, and tunics) appeared unaffected by DMSO except at foci where blebbing occurred.(ABSTRACT TRUNCATED AT 250 WORDS)