NO nerves and their targets in a tapeworm: An immunocytochemical study of cGMP in Hymenolepis diminuta.

We studied the pattern of cGMP immunostaining (IS) after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase in adult Hymenolepis diminuta. cGMP-IS was detected in the peripheral nervous system, especially in nerve fibres close to the body muscle fibres. cGMP-IS also occurred in terminals beneath the basal lamina of the tegument and between the muscle fibres of the suckers. The pattern of cGMP-IS was compared to that of 5-HT-IS and GYIRFamide-IS. TRITC-conjugated phalloidin was used to stain the musculature.

[The effect of anthelmintics on the serotonin content of cestodes]. / Effekt nekotorykh antigel'mintikov na soderzhanie serotonina u tsestod.

The effects of some anthelminthic agents, such as praziquantel, fenasal, albendazole, on the levels of the neurotransmitter serotonin in the tissues of Hymenolepis diminuta cestodes and Mesocestoides corti tetrathyridia were examined in vivo and in vitro. The finding showed lower serotonin levels in H.diminuta after praziquantel and albendazole and in M. corti after fenasal. It is suggested that the serotonin neurotransmitter system of helminths is involved in the mechanism of action of these agents.

Evidence that the factor used by the tapeworm, Hymenolepis diminuta, to direct the foraging of its intermediate host, Tribolium confusum, is a volatile attractant.

We have previously demonstrated that flour beetles, Tribolium confusum, are more attracted to the feces from rats with patent Hymenolepis diminuta infections than to feces from uninfected rats. The objective of this study was to determine if this effect is due to a volatile attractant. Volatile substances emitted by feces from H. diminuta-infected rats or from uninfected controls were collected by aspirating fresh rat feces, while trapping the volatiles on Porapak Q (a solid adsorbant). The volatiles were eluted from the Porapak Q with diethyl ether, and the relative attractancy of the volatiles to prestarved beetles was assessed by bioassay. More beetles were attracted to volatiles of feces from infected rats than to volatiles of feces from uninfected controls (P< or =0.0001). The magnitude of the response varied with the time in the bioassay test arena and also the concentration of the volatiles (P< or =0.0232). When the volatiles we re concentrated by aspirating more boli over a longer period of time, the beetles responded more quickly and in greater numbers to the volatiles of feces from infected rats. The experiments presented here provide the first indication that a tapeworm (H. diminuta) can enhance its chances of transmission by directing the foraging of its intermediate host (T. confusum) through the use of attractive, volatile material released from the feces of its definitive host.

Characterization of calcineurin from Hymenolepis microstoma and H. diminuta and its interaction with cyclosporin A.

The drug cyclosporin A (CsA) exerts its immunosuppressive action by binding to the cytosolic protein, cyclophilin (CyP) and, as a complex, binding to and inhibiting the calcium/calmodulin-dependent serine threonine phosphatase, calcineurin. It is unknown whether a similar mode of action occurs during the drug's antiparasite activity. Calmodulin-binding proteins from the helminth parasites Hymenolepis microstoma and H. diminuta were purified by affinity chromatography, yielding single polypeptide bands of 60000 M(r), according to SDS-PAGE. These proteins were tested for calcineurin activity by the dephosphorylation of the RII peptide (part of the catalytic subunit of cAMP-dependent protein kinase). Both proteins were calcium- and calmodulin-dependent and were inhibited by mammalian cyclophilin complexed with cyclosporin A (IC50 values of 0.75 microgram CyP for H. microstoma and 0.90 microgram CyP for H. diminuta). However, neither of the parasite calcineurins was inhibited by H. microstoma cyclophilin/CsA. These data suggest the anthelmintic mode of action of CsA in these helminth models does not involve the inhibition of a signal transduction pathway requiring interaction with calcineurin.

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.

Partial characterization of the carbohydrates of the eggs (oncospheres) of the tapeworm, Hymenolepis diminuta.

The carbohydrate constituents of whole eggs and fractions derived from the eggs of Hymenolepis diminuta were partly characterized. Whole eggs contained 9.6 ng of carbohydrate (phenol-sulphuric acid-positive material) per egg, and 3.6 ng of glucose. Analysis of hydrolysed eggs by HPLC demonstrated the presence of glucose, galactose, glycerol, N-acetylgalactosamine, and 2-deoxyribose. Isolated egg shells and the KOH-stable, ethanol-precipitable fraction (putative glycogen) of eggs contained 26 and 76%, respectively, of the total carbohydrate associated with eggs. Glucose and galactose were present in both of these samples, but only glucose was present in the KOH-stable, ethanol-precipitable fraction isolated from chemically deshelled eggs; thus, the source of the galactose in the KOH-stable, ethanol-precipitable fraction isolated from whole eggs was the egg-shells. These data confirm earlier reports that the shells contain carbohydrate; the data demonstrate further that only two monosaccharides (glucose and galactose) are present in the shells, and that they are present as high molecular weight polymers. The ethanol-soluble fraction of eggs contained 2.5% of the total carbohydrate in eggs, and glucose accounted for < 1% of the ethanol-soluble carbohydrate. Analysis of the ethanol-soluble fraction by HPLC demonstrated that the predominant monosaccharides were glycerol and mannose, with smaller quantities of 2-deoxyribose and 2-deoxyglucose. The absence of a 'free pool' of glucose in eggs, and the presence of large amounts of glycerol and mannose, suggest that the pathways of intermediary carbohydrate metabolism in eggs might be very different from those in adult tapeworms.

Partial characterization of the protein and carbohydrate constituents of the egg shell of Hymenolepis diminuta (Cestoda).

The egg shell of Hymenolepis diminuta is composed of 31.7% protein and 2.9% carbohydrate (by weight), as determined using the Lowry and phenol-sulfuric acid assays and bovine serum albumin and glucose, respectively, as standards. Amino acid analyses of egg shells demonstrated the presence of 16 naturally occurring amino acids and six unidentified residues. Histidine accounted for > 22% of the amino acid residues. Hypochlorite-solubilized egg shells were fractionated using a combination of gel, hydroxyapatite and anion-exchange chromatography, and the fractions were analyzed by isoelectric focusing and gel chromatography. The results demonstrate that the shell consists of a complex mixture of proteins (almost all of which have acidic isoelectric points), glycoproteins and possibly free complex carbohydrates.

FMRFamide-related peptides in Hymenolepis diminuta: immunohistochemistry and radioimmunoassay.

The localization of FMRFamide-related peptide (FaRP) immunoreactivity was determined during different stages of development of the rat tapeworm Hymenolepis diminuta. In the adult worm (14 days old), FaRP immunostaining was most intense in the scolex and concentrated in the central nervous system (cerebral ganglia and transverse commissures) and around the lips of the suckers. In the strobila, medial and lateral longitudinal nerve cords (LNCs) and ladder-like connecting commissures were the only tissue stained. Immunoreactivity in the medial LNCs of the adult tapeworms extended only to and included proglottides containing developing testis and seminal receptacle but disappeared in proglottides in which primordial ovaries were first detected. Radioimmunoassay confirmed that FaRPs were concentrated in the scolex/neck region of the adult worm (3.9 +/- 1.5 pmol mg protein-1), whereas the lowest concentrations (0.2 +/- 0.19 pmol mg protein-1) were recovered from the regions of the strobila containing shelled eggs. The pattern of FaRP immunoreactivity observed in 5- and 7-day-old worms was similar to that seen in adult worms, but in 2- and 3-day-old worms the pattern of immunoreactivity observed in the cerebral ganglia, transverse commissures, and LNCs differed significantly as compared with that seen in older worms. These results indicate differential utilization and/or roles for FaRPs during development and suggest both central and sensory roles in this tapeworm.

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.

Immunocytochemical and radioimmunometrical demonstration of serotonin- and neuropeptide-immunoreactivities in the adult rat tapeworm, Hymenolepis diminuta (Cestoda, Cyclophyllidea).

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.