Excretory system of representatives from family Diplozoidae (Monogenea).

Diplozoons are representatives of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic, and ecological studies, the detailed study of their excretory system has remained relatively neglected. Our observations focused on the morphological and ultrastructural features of the excretory apparatus of four diplozoid species: Diplozoon paradoxum, Eudiplozoon nipponicum, Paradiplozoon bliccae, and Paradiplozoon homoion. Observations were obtained using two microscope methods: light microscopy, equipped with differential interference contrast (Nomarski DIC) and transmission electron microscopy (TEM). The ultrastructure of two basic compartments which forms the excretory apparatus, flame cells with filtration apparatus, and canal cells forming the protonephridial ducts is revealed in this study. A unique consecutive sequence of longitudinal semi-thin sections of the excretory pore of E. nipponicum is visualized there for the first time.

Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea).

Paradiplozoon homoion is a representative of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic and ecological studies, the ultrastructure of the alimentary system and related structures, as well as the mechanisms of essential processes like fish blood digestion, remain mostly unknown. Our observation of P. homoion using a transmission electron microscopy (TEM) revealed two main types of digestive cells-U-shaped haematin cells and connecting syncytium. Particular structures such as mouth cavity with specialised receptors, two oval-shaped muscular buccal suckers, pharynx surrounded with the glandular cells, oesophagus, the intestinal caeca with intact erythrocytes in the lumen, the apical pinocytotic fibrous surface complex and haematin vesicles of U-shaped cells have been shown in detail. According to our results, the P. homoion is degrading the blood components predominantly intracellularly.

Presence and species identification of the gapeworm Mammomonogamus laryngeus (Railliet, 1899) (Syngamidae: Nematoda) in a semi-wild population of Sumatran orangutan (Pongo abelii) in Indonesia.

One adult syngamid nematode parasite couple was found during routine clinical observation in quarantine at the former Bohorok Rehabilitation Station from sputum of Pongo abelli and determined as Mammomonogamus laryngeus [Railliet, A., 1899. Syngame laryngieu du boeuf. Comptes Rendus Hebdomadaires des Séances et Mémoires de la Société de Biologie 11, 18-21]. This finding confirmed previous record of ova and adult syngamid nematodes, determined by Collet et al. [Collet, J.-Z., Galdikas, B.M.F., Sugarjito, J., Jojosudharmo, S., 1986. A coprological study of parasitism in orangutan (Pongo pygmaeus) in Indonesia. Journal of Medical Primatology 15, 121-129] as Mammomonogamus sp. only, in orangutans kept in the Bohorok Orangutan Rehabilitation Centre (Northern Sumatra, Indonesia) and presented a serious health hazard to rehabilitants in this locality. Morphometrical features and the first description of the parasite from orang-utan were presented and documented. Coprological monitoring of infection in rehabilitants in this area as well as among the wild population of orangutan is necessary.

Classification and occurrence of abnormally developed Paradiplozoon homoion (Monogenea, Diplozoinae) parasitising gudgeon Gobio gobio.

Morphological analyses of the attachment apparatus (clamps and central hooks) of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Diplozoinae, Monogenea) parasitising gills of Gobio gobio (L.) showed a high percentage of abnormally developed parasite specimens. Four different localities in the Vlára River basin, Czech Republic, were investigated for the presence of such abnormal individuals. The highest percentage of abnormalities in the attachment apparatus (over 39%) was recorded in the Vlára River, at Bohuslavice. This study provides a comprehensive classification of these abnormalities with 7 types of abnormalities described and illustrated. Abnormalities of parts of the attachment apparatus that form in the later stage of ontogenetic development were the most frequent, the most frequent types of abnormalities being clamps with abnormal sclerites, and combinations of abnormalities. Abnormalities of the central hooks were also found in our material. The abnormalities found in diplozoids are probably connected with environmental pollution; however, this point requires further investigation.

Seasonal dynamics of Proteocephalus sagittus in the stone loach Barbatula barbatula from the Hana River, Czech Republic.

The seasonal cycle of the cestode Proteocephalus sagittus (Cestoda: Proteocephalidae) was studied for the first time in the stone loach Barbatula barbatula from the Hana River, Czech Republic. A total of 180 loaches were examined monthly from January to December 2001. The parasite occurred in loaches throughout the year but infection parameters differed significantly among seasons, with the highest values of prevalence and abundance from the late winter to the early summer. Parasite recruitment took place in the winter and early spring and the worms sexually matured in the late spring and early summer. In contrast to P. torulosus, the gravid worms of which laid eggs only at the end of the spring/beginning of the summer, gravid worms of P. sagittus were also found, although in low numbers, in the autumn and early winter. The rate of infection of loach with P. sagittus was neither dependent on the sex nor on the size of its fish host.

Presence of Skrjabingylus petrowi (Nematoda) in central Europe.

Examination of the nasolacrimal sinuses of pine marten (Martes martes), stone marten (M. foina) and polecat (Mustela putorius) from 27 localities in the Czech Republic were carried out. In 1 of the 23 stone martens, 6 nematodes (2 males, 4 females) were found and classified as Skrjabingylus petrowi Bageanov (in Petrov 1941). This is the first finding of S. petrowi in central Europe. The morphometry of the specimens in the present study was in agreement with that of the original description. The distribution of this species in Europe is discussed.

Identification of European diplozoids (Monogenea, Diplozoinae) by restriction digestion of the ribosomal RNA internal transcribed spacer.

The second internal transcribed spacer (ITS2) of the ribosomal RNA genes of Diplozoon paradoxum and Paradiplozoon nagibinae were amplified and sequenced. The polymerase chain reaction product of D. paradoxum was bigger (840 bp) than that of P. nagibinae (820 bp). There was no intraspecific variability recorded in sequences from either species. Sequence comparisons and ITS2 restriction fragment length polymorphism (RFLP) pattern of 8 European diplozoid species aimed to resolve their identification and amend the previous studies. RFLP was used to distinguish the 2 species from each other and from P. bliccae, P. homoion, P. megan, P. pavlovskii, P. sapae, and Eudiplozoon nipponicum, using restriction enzymes AluI, HaeIII, HinfI, RsaI, and SphI. The criteria for morphological identification of 8 European diplozoids are also included, with the main morphological characters of clamps, trapeze spur, and anterior joining sclerites of 8 diplozoid species being illustrated. Combination of the shape and comparison of length of the trapeze spur and anterior joining sclerites could lead to accurate identification of diplozoid species.

Raphidascaris acus (Bloch, 1779) larvae infections of the stone loach, Barbatula barbatula (L.), from the River Haná, Czech Republic.

The stone loach, Barbatula barbatula (L.), is a typical and dominant intermediate host of Raphidascaris acus in lowland streams of Central Europe. The prevalence of infection of R. acus in B. barbatula from the River Haná ranged throughout the year from 73.3 to 100%. The abundance and the mean intensity of infection also varied throughout the year with a peak in September. Larvae were located mainly in the liver parenchyma. High numbers of larvae and their migration through the tissue caused cyst- or abscess-like formations in the host parenchyma. The severity of the disease condition ranged from mild to severe. We speculate that the infection of stone loach by R. acus larvae regulates the population density and abundance of the intermediate host in lowland streams where natural predators are absent.

Surface carbohydrates of Eudiplozoon nipponicum pre- and post-fusion.

The development of the monogenean Diplozoon (Nordmann, 1832) (Diplozoidae) necessitates fusion of two larval stages (diporpae) into one double organism. How diporpae find, distinguish and contact each other is unclear, nor is the nature of the stimuli responsible for the dedifferentiation of cells and the formation of new tissues at the site of somatic fusion. Previous studies have implied a role for carbohydrates and glycoproteins in the interactions between helminth parasites and their hosts. Hypothetically, glycoconjugates may also be involved in the establishment of parasite-parasite associations. Changes in the surface saccharide residues during the development of Eudiplozoon nipponicum, a gill ectoparasite of carp (Cyprinus carpio) are described. Flat-fixed specimens and sections of diporpae, juveniles (just-fused) and adult worms were examined following exposure to a panel of 12 FITC-conjugated lectins. All developmental stages exhibited a specific surface binding pattern with ten lectins, indicating that Man/Glc, GlcNAc, Gal and GalNAc are probably present on their surfaces. No reaction was observed with Fuc-specific lectins (UEA-I and LTA). There is evidence that parasite development is accompanied by both qualitative and quantitative changes in the saccharide pattern distribution. The diporpa sucker reacted with nine lectins, excluding BS-II. A very strong binding of PNA, LCA and ConA (Gal and Man/Glc-specific lectins) was observed with the papilla glands of juvenile worms. The role of glandular secretions in this unique fusion process is discussed.

Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy.

The major muscle systems of the metacercaria of the strigeid trematode, Apatemon cobitidis proterorhini have been examined using phalloidin as a site-specific probe for filamentous actin. Regional differences were evident in the organization of the body wall musculature of the forebody and hindbody, the former comprising outer circular, intermediate longitudinal and inner diagonal fibres, the latter having the inner diagonal fibres replaced with an extra layer of more widely spaced circular muscle. Three orientations of muscle fibres (equatorial, meridional, radial) were discernible in the oral sucker, acetabulum and paired lappets. Large longitudinal extensor and flexor muscles project into the hindbody where they connect to the body wall or end blindly. Innervation to the muscle systems of Apatemon was examined by immunocytochemistry, using antibodies to known myoactive substances: the flatworm FMRFamide-related neuropeptide (FaRP), GYIRFamide, and the biogenic amine, 5-hydroxytryptamine (5-HT). Strong immunostaining for both peptidergic and serotoninergic components was found in the central nervous system and confocal microscopic mapping of the distribution of these neuroactive substances revealed they occupied separate neuronal pathways. In the peripheral nervous system, GYIRFamide-immunoreactivity was extensive and, in particular, associated with the innervation of all attachment structures; serotoninergic fibres, on the other hand, were localized to the oral sucker and pharynx and to regions along the anterior margins of the forebody.

Paradiplozoon homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961 (Monogenea): two species or phenotypic plasticity?

Specimens of the Paradiplozoon homoion-complex were collected from ten species of cyprinid fish in the Czech Republic. A combined molecular and morphometric approach was performed to distinguish Paradiplozoon homoion and P. gracile. The second internal transcribed spacer (ITS2) of the ribosomal RNA genes was amplified and sequences were analysed. No variability in the analysed sequences was detected. Measurements of clamps and the central hooks obtained from specimens from different host species were compared. Great variability was found in the length and width of the third pair of clamps. No significant differences were detected in the measurements of the central hook sickle. A positive relationship was found between host size and each of the following measurements of the third pair clamps: length and width of the whole clamp; and length of the median plate of the third pair of clamps. The length of the median plate of the attachment clamps may be a useful character for species identification of diplozoids. Further molecular and morphometric studies are required to resolve this taxonomic problem and, henceforth, we suggest considering P. gracile as a species inquirenda.

Genetic characterization of six species of diplozoids (Monogenea; Diplozoidae).

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.

Biodiversity of parasites in freshwater environment in relation to pollution.

During 1994, 129 specimens of chub (Leuciscus cephalus L.) from two localities, polluted--Bolelouc, 80th river kilometer, near the city of Olomouc, and unpolluted--Brodské, 225th river kilometer, near the Lanzhot on the Morava river, were examined for metazoan parasites. Altogether 38 metazoan parasite species including helminths, leeches and glochidia belonging to 14 genera were found, 34 in the case of unpolluted locality (Brodské) and 28 in the polluted one (Bolelouc). In both localities, 24 parasite species (Dactylogyrus folkmanovae, D. naviculoides, D. prostae, D. vistulae, D. vranoviensis, Gyrodactylus carassii, G. gasterostei, G. gracilihamatus, G. scardiniensis, G. vimbi, Gyrodactylus sp., Paradiplozoon ergensi, P. homoion, P. megan, Paradiplozoon sp., Caryophyllaeus brachycollis, Diplostomum spathaceum--larval stage, Philometra abdominalis, Acanthocephalus anguillae, Pomphorhynchus laevis and Glochidium sp.) were found, 10 (D. crucifer, D. nanoides, G. hemibarbi, G. lamberti, G. leucisci, G. lomi, P. rutili, C. fennica, A. imitans, S. bramae) were identified solely in the unpolluted locality Brodské and 4 (D. fallax, G. laevis, P. ovata, G. kearni) parasite species were found only in the polluted locality Bolelouc. Differences in parasite species richness, the level of dominance, the structure of core and in satellite species were observed. Current methods of statistical evaluation were used.