The neuromuscular system in continuously swimming cercariae from Belarus. II Echinostomata, Gymnocephala and Amphistomata.

The neuromuscular system in cercariae of Moliniella anceps, Echinostoma revolutum, Cathaemasia hians, Psilochasmus oxyurus, Sphaeridiotrema globulus, Paramphistomum cervi and Diplodiscus subclavatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated. The general patterns of musculature, 5-HT- and FMRFamide-IR neuronal elements in the 12 species studied here and in paper I are similar to those observed in other cercariae and reflect the morphology of the groups. The musculature of the tail shows variations which are related to the different strategies of host finding. In the Echinostomatoidea and Paramphistomoidea, the striated musculature of the tail is well developed compared to that in the Xiphidiocercariae. Specialized muscle fibres were found in S. globulus, which are able to change the shape of the tail. Nine of the species studied have seven paired 5-HT-IR neurons in the body, and two species have eight. No correlation between the body size and the number of 5-HT-IR neurons was observed. However, the size of the neurons followed the body size. The number of 5-HT-IR neurons in the brain ganglia increased from the primitive to the advanced forms. The number of FMRFamide-IR transverse commissures in the body correlates with the size of the cercariae. Regardless of the differences in the second intermediate host, the distribution of α-tubulin-IR sensory receptors shows a high degree of conformity in all species except in P. cervi, which encysts on plants.

The neuro-muscular system in continuously swimming cercariae from Belarus. I Xiphidiocercariae.

The neuromuscular system (NMS) in cercariae of Neoastiotrema trituri, Plagiorchis elegans, Omphalometra flexuosa, Skrjabinoeces similis and Prosthogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated, and they were found to be rather similar in all the cercariae studied. Four species have seven paired 5-HT-IR neurons in the body, and P. elegans has eight. N. trituri has three 5-HT-IR neurons in each brain ganglion, while the other species have four. A high degree of conformity in the structure of the NMS was observed, probably reflecting the close phylogenetic relationship and the similar strategy of host finding.

The neuro-muscular system in fresh-water furcocercaria from Belarus. I Schistosomatidae.

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

The neuromuscular system in freshwater furcocercaria from Belarus. II Diplostomidae, Strigeidae, and Cyathocotylidae.

The neuromuscular system (NMS) in cercariae of Diplostomum pseudospathaceum, Cotylurus szidati, Australapatemon burti, Holostephanus volgensis, and Paracoenogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRF-amide-IR neuronal elements, and α-tubulin-IR in sensory receptors were investigated. The NMS in the five species studied were compared with each other and with three species of Schistosomatidae studied earlier (Bilharziella polonica, Trichobilharzia szidati, and Trichobilharzia franki). No major structural differences in the musculature, the 5-HT-IR or FMRF-IR neuronal elements were noticed between the cercariae. The minor variations observed in the musculature were related to the size and organization of the muscle fibers. The checked pattern formed by the transverse muscle fibers in the tail stems of D. pseudospathaceum, C. szidati, A. burti, H. volgensis, and P. ovatus was not observed in B. polonica, T. szidati, and T. franki. A trend in the differentiation of the longitudinal muscle fibers in the furca from evenly distributed fibers in H. volgensis and P. ovatus to many bundles in D. pseudospathaceum and two well-organized lateral bundles in C. szidati, A. burti, and Trichobilharzia spp. was observed. The transverse muscle fibers in the furca follow the same trend. The number of 5-HT-IR neurons in the cercarial bodies varied between 10 and 16. In cercariae of H. volgensis and P. ovatus, the central nervous system (CNS) was less centralized compared to the CNS in the other species studied, with only two 5-HT-IR marker neurons in each brain ganglion and the other neurons distributed evenly along the main cords. In the tails of H. volgensis and P. ovatus, many transverse 5-HT-IR comissures were found. In the tails of higher strigeidid cercariae, only a few crosslinks were observed. The number and distribution of sensory receptors on the bodies and tails of the cercarial species differed from each other. A trend in the differentiation of the sensory receptors in the tails was discerned. A process of grouping and decrease in number of ciliated receptors in the stem and in the furca from H. volgensis and P. ovatus to Schistosomatid cercariae took place.

The neuro-muscular system in cercaria with different patterns of locomotion.

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.

An immunocytochemical, histochemical and ultrastructural study of the nervous system of the tapeworm Cyathocephalus truncatus (Cestoda, Spathebothriidea).

This study is the first detailed study of the organisation of the neuromuscular system of Cyathocephalus truncatus (Cestoda, Spathebothriidea). Five techniques have been used: (1) immunocytochemistry, (2) staining with TRITC-conjugated phalloidin, (3) NADPHdiaphorase histochemistry, (4) confocal scanning laser microscopy and (5) transmission electron microscopy. The patterns of nerves immunoreactive (IR) to antibodies towards serotonin (5-HT) and the invertebrate neuropeptide FMRFamide are described in relation to the musculature. The patterns of NADPHdiaphorase positive nerves and 5-HT-IR nerves are compared. The fine structure of the nervous system (NS) is described. The organisation of NS in the non-segmented, polyzoic C. truncatus differs clearly from that in the non-segmented, monozoic Caryophyllaeus laticeps and shows distinct similarities with the NS in pseudophyllidean cestodes. This supports the hypothesis that taxon Caryophyllidea and Spatheobothriidea form independent lineages within Eucestoda.

Effects of neuropeptide F on regeneration in Girardia tigrina (Platyhelminthes).

The effects of neuropeptide F (NPF; from Moniezia expansa) on the regeneration of Girardia tigrina were studied. The animals were decapitated and incubated in water (control) or NPF. The dynamics of the proliferation of the neoblasts in the developing tissue were studied during the course of regeneration by monitoring the mitotic index (MI). The effects of incubation in FMRFamide and GYIRFamide on the MI were also tested. The course of cephalic regeneration was followed with in vivo computer-assisted morphometry for up to 7 days. The development of the regenerating nervous system and the musculature was visualised by immunostaining with a primary antiserum to the C-terminal decapeptide of NPF (YFAIIGRPRFa) and tetramethylrhodamine-isothiocyanate-conjugated phalloidin, which stains F-actin in muscle filaments. The study showed that NPF had a stimulatory effect on the mitotic activity of the neoblasts. FMRFamide and GYIRFamide did not have this effect. NPF also stimulated the growth of the regenerating head and the growing nervous system and musculature. NPF is postulated to have a morphogenetic action in the regenerating animals.

The effect of a nitric oxide donor on the synthesis of cGMP in Hymenolepis diminuta: a radiometric study.

The formation of cGMP in homogenates of the adult rat-tapeworm Hymenolepis diminuta was followed with a radiometric assay during 3 h after stimulation with the nitric oxide donor sodium nitroprusside (SNP) and in the presence of isobutylmethylxanthine (IBMX). The level of cGMP was stable in worms incubated with IBMX during the first hour. After 3 h of incubation, the level of cGMP had declined by 27%. Addition of SNP stimulated the formation of cGMP during the first hour of incubation. After 3 h of incubation, a two-fold decline in cGMP formation was observed. The rate of nitric oxide (NO) release by the worm was determined by a spectrophotometric assay for the accumulation of nitrites and nitrates, the stable degradation products of NO, using the Griess reaction. The results are discussed from the perspective of the current concept on the role of the nitrergic mechanisms in the flatworm nervous system.

Basiepidermal nervous system in Nemertoderma westbladi (Nemertodermatida): GYIRFamide immunoreactivity.

The Nemertodermatida are a small group of microscopic marine worms. Recent molecular studies have demonstrated that they are likely to be the earliest extant bilaterian animals. What was the nervous system (NS) of a bilaterian ancestor like? In order to answer that question, the NS of Nemertoderma westbladi was investigated by means of indirect immunofluorescence technique and confocal scanning laser microscopy. The antibodies to a flatworm neuropeptide GYIRFamide were used in combination with anti-serotonin antibodies and phalloidin-TRITC staining. The immunostaining revealed an entirely basiepidermal NS. A ring lying outside the body wall musculature at the level of the statocyst forms the only centralisation, the "brain". No stomatogastric NS has been observed. The GYIRFamide immunoreactive part of the "brain" is formed of loosely packed nerve fibres with multiple small neurones and a few large ones. The peptidergic and aminergic patterns of the NS do not correspond to each other: the former is more developed on the ventral side, the latter is more pronounced on the dorsal side. A pair of GYIRFamide immunoreactive nerve cords innervates the ventral side of the animal, the mouth and the male genital opening. The nemertodermatids studied to-date display no common NS pattern. Possible synapomorphies of the Acoelomorpha are discussed. The study demonstrates that the nemertodermatid NS possesses a number of plesiomorphic features and appears more primitive than the NS in other worms, except the Xenoturbellida. The bilaterian ancestor supposedly possessed only a basiepidermal nerve net and had no centralised brain-like structures and no stomatogastric NS.

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.

Nitric oxide and its target cells in cercaria of Diplostomum chromatophorum: a histochemical and immunocytochemical study.

In order to advance our knowledge of the nitrergic nervous system in flatworms, the patterns of the NADPH-diaphorase (NADPH-d) reaction and cGMP immunoreactivity, after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase, were investigated in cercaria of Diplostomum chromatophorum. This is the first time the presence of NADPH-d activity has been detected in a larval fluke, and the first time the presence of cGMP immunoreactivity has been detected in a flatworm. The NADPH-d reaction occurs in the ventral sucker, the hind body and the tail. cGMP immunoreactivity was detected in the muscle cells of the body and in two pairs of sensory cells at the anterior end of the body and in the middle of the furca. The sensory cells also showed 5-HT immunoreactivity. The spatial relationship between the cGMP and the 5-HT immunoreactivities was studied. By staining with TRITC-labelled phalloidin, the pattern of the muscle fibres was revealed.