Two new species of Annulotrema (Monogenea: Dactylogyridae) from Hydrocynus vittatus (Characiformes: Alestidae) in Lake Kariba, Zimbabwe.

Two new and one known species of Annulotrema Paperna & Thurston, 1969 are reported from the gills of the tigerfish Hydrocynus vittatus Castelnau, 1861, collected in Lake Kariba, Zimbabwe. The new species, Annulotrema pseudonili n. sp. and A. bracteatum n. sp., are described and distinguished mainly on the basis of features of the male copulatory organ (MCO). Annulotrema pseudonili n. sp. most closely resembles A. nili Paperna, 1973, but differs from it by possessing a more delicate MCO with a thin-walled base without a fibrous distal part. Annulotrema bracteatum n. sp. is most similar to Annulotrema ruahae Paperna, 1973, from which it differs by having an MCO composed of a longer copulatory tube and a leaf-shaped accessory piece enveloping the distal part of the tube. The presence of Annulotrema pikoides Guégan, Lambert & Birgi, 1988 on H. vittatus in Zimbabwe represents a new locality record for this parasite.

Dactylogyrids (Platyhelminthes: Monogenea) from Sudanese Labeo Spp., with a Description of Dogielius Sennarensis n. Sp. and a Redescription of Dogielius Flosculus Guégan, Lambert & Euzet, 1989.

Dactylogyrid monogeneans of Labeo horie Heckel, 1847 and L. niloticus (Linnaeus, 1758) (Cyprinidae) were surveyed at two sites on the River Nile in Sudan. The present study reports the presence of ten species of Dactylogyrus Diesing, 1850 and three species of Dogielius Bychowsky, 1936 based on a morphometric evaluation of the sclerotised structures. The species found include: Dactylogyrus nathaliae Guégan, Lambert & Euzet, 1988; D. rastellus Guégan, Lambert & Euzet, 1988; D. retroversus Guégan, Lambert & Euzet, 1988; D. senegalensis Paperna, 1969, D. yassensis Musilová, Rehulková & Gelnar, 2009 and five other undescribed species of Dactylogyrus. The genus Dogielius was represented by Dogielius flosculus Guégan, Lambert & Euzet, 1989; the newly identified D. sennarensis n. sp., and one undescribed Dogielius species. While D. sennarensis n. sp. resembles D. intorquens, it differs from this species and other congeners by having a longer ventral bar and anchor points and nosclerotised vagina. In addition, this study redescribes D. flosculus based on the morphology of specimens collected from L. horie. All specimens studied had a single large vagina, in contrast to the original description which reported a vagina composed of two unconnected parts. All dactylogyrid species in this study represent new host and geographical records.

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.

Penetration and encapsulation of the larval endoparasitoid Exorista larvarum (Diptera: Tachinidae) in the factitious host Galleria mellonella (Lepidoptera: Pyralidae).

The tachinid fly Exorista larvarum (L.) (Diptera: Tachinidae) is a polyphagous larval endoparasitoid that deposits its eggs on the host exoskeleton of lepidopteran and tenthredinid larvae. The attachment of larval E. larvarum and the formation of the respiratory funnel were studied during infestation in the last larval instar of the wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae). The tachinid larvae burrow through the host integument after hatching, using their robust cephalopharyngeal skeleton, leaving a dark spot at the point of their penetration as a result of host cuticle melanization. Endoparasitoid penetration induces the host cellular defence, resulting in the formation of a haemocyte capsule consisting of multi-cellular sheaths. This enveloping capsule later undergoes melanization, which is mostly obvious towards the posterior part of the endoparasitoid. The endoparasitoid uses the host encapsulation response to build a respiratory funnel from the modified host integument, leading to the host surface. The encapsulated larva remains attached to the respiratory funnel via an anal hook and cuticular spines until fully developed. Additional immunohistochemical analyses were used to study host-parasitoid interactions. Indirect immunofluorescence showed no labelling of potential tachinid antigens and confirmed no effect on the surrounding host tissues. A simulated parasitization with coated polybead microspheres revealed the mortal impact of tachinid antigens to the host. Hosts injected with antigen-coated polybeads died as a consequence of an acute and extensive immunological response to the tachinid antigens and not due to the trauma caused by foreign objects inside their body.

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.

Biodiversity of parasites in a freshwater environment with respect to pollution: metazoan parasites of chub (Leuciscus cephalus L.) as a model for statistical evaluation.

This paper represents an attempt to evaluate the environmental indicative potential of the biodiversity of monogenean parasites using hierarchically structured species-abundance data. A logical set of statistical methods integrating standard diversity indices, a novel approach to quantitative analysis of cumulative species-abundance curves and species-abundance models was applied for this purpose. Applicability of biodiversity measures was demonstrated using experimental data from a 1-year study on the ecology of metazoan parasites of chub (Leuciscus cephalus) in one polluted and one control site in the Morava river, Czech Republic. Analyses at the component community level revealed a significant decrease in the number of parasite species with a more equal distribution of their abundances in the polluted site compared with the control site. In order to reach a better understanding of the changes, diversity of Monogenea as a dominant part of the community was further examined within categories of species created according to: (1) specificity of infection (specialists and generalists), (2) monogenean genera (Dactylogyrus, Gyrodactylus and Paradiplozoon) and (3) inhabited guilds (skin + fins, gills). Assemblages of specialists in the polluted site exhibited a significantly reduced species richness and unequal distribution of abundances. The opposite pattern was observed in the case of generalists. The influence of pollution was also reflected by the distribution of species abundances within communities of Dactylogyrus and Paradiplozoon, while no significant shift was identified in the genus Gyrodactylus.

Nestedness in assemblages of gyrodactylids (Monogenea: Gyrodactylidea) parasitising two species of cyprinid--with reference to generalists and specialists.

The structure of gyrodactylid assemblages in individual fishes of two species of cyprinid was determined. A total of 100 specimens of minnow, Phoxinus phoxinus, and 137 specimens of roach, Rutilus rutilus, were investigated for presence of gyrodactylids. Host specificity, specialists vs. generalists, was noted in each host fish. A nested pattern was recorded in parasite assemblages of minnow, the host with a dominant number of specialist gyrodactylids. A non-nested pattern was observed in parasite assemblages of roach, the host with a dominant number of generalist gyrodactylids. The host specificity appears to be a meaningful factor that determines the pattern of gyrodactylid assemblages of both fish hosts.

Order and disorder in ectoparasite communities: the case of congeneric gill monogeneans (Dactylogyrus spp.).

The measure of order and disorder in the distribution of species in fragmented habitats proposed by Atmar and Patterson (Oecologia, 96 (1993) 373-82) was applied to investigate nested patterns of Dactylogyrus species parasitising the gills of roach. Organisation in dactylogyrid assemblages was investigated at three levels: (1) host populations between localities; (2) local host populations over seasons; and (3) individual hosts over one season within a local host population. Dactylogyrid assemblages showed nested patterns when analyses were conducted at the level of localities (among host populations) and at the level of seasons (among host populations within localities). The analysis at the level of hosts (infracommunities of parasites) revealed that nested pattern is not common. We suggest that nestedness may have a variety of causes and does not necessarily imply competition.

Co-existence of nine gill ectoparasites (Dactylogyrus: monogenea) parasitising the roach (Rutilus rutilus l.): history and present ecology.

Co-existence among potentially competing species can be favoured by niche specialisation and/or by reducing the overall intensity of competition via aggregated utilisation of fragmented resources. We investigated the respective roles of niche specialisation and aggregation in the case of nine congeneric monogenean parasites on the gills of Roach (Rutilus rutilus L.) belonging to the genus Dactylogyrus. The position of each individual parasite of the nine Dactylogyrus species was recorded. Niche breadth and niche overlap of parasite species were estimated. Comparative methods, which take into account phylogenetic information of the analysed species, were used. We reconstructed a phylogeny of the nine Dactylogyrus species based on morphological characters. We used the 'aggregation model of co-existence' in the model to test if species co-existence is facilitated when intraspecific aggregation exceeds interspecific aggregation. We observed a lack of negative correlation in abundance between pairs of parasites, and a negative correlation between niche size and parasite aggregation, for both intraspecific and interspecific aggregation. Our comparative analysis showed that parasite abundance is positively correlated with niche breadth. Then parasite abundance, and not interactions between Dactylogyrus species, seems to be the most important factor determining niche size This result gives some support to niche segregation by specialisation. Niche size was negatively correlated with both intraspecific and interspecific aggregation. No relationship was found between an increase of interspecific aggregation with an increase of niche overlapping, which suggests that competition may play little role. A lack of competition could be also confirmed by the lack of negative correlation in abundance between species pairs. A parsimony analysis of the evolution of gill distribution indicates a change in one parameter of the niche (arch, segment and/or area) at each branching event.

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei).

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.

Immunomicroscopical observations on the nervous system of adult Eudiplozoon nipponicum (Monogenea: Diplozoidae).

Neuronal pathways have been examined in adult Eudiplozoon nipponicum (Monogenea: Diplozoidae), using cytochemistry interfaced with confocal scanning laser microscopy, in an attempt to ascertain the status of the nervous system. Peptidergic and serotoninergic innervation was demonstrated by indirect immunocytochemistry and cholinergic components by enzyme cytochemical methodology; post-embedding electron microscopical immunogold labelling revealed neuropeptide immunoreactivity at the subcellular level. All three classes of neuronal mediators were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. There was considerable overlap in the staining patterns for cholinergic and peptidergic components, while dual immunostaining revealed serotonin immunoreactivity to be largely confined to a separate set of neurons. The subcellular distribution of immunoreactivity to the flatworm neuropeptide, GYIRFamide, confirmed neuropeptide localisation in dense-cored vesicles in the majority of the axons and terminal varicosities of both central and peripheral nervous systems. Results reveal an extensive and chemically diverse nervous system and suggest that pairing of individuals involves fusion of central nerve elements; it is likely also that there is continuity between the peripheral nervous systems of the two partner worms.

Microscopical evaluation of neural connectivity between paired stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae).

Diplozoidae monogeneans am fish-gill ectoparasites comprising 2 individuals fused in so-called permanent copula. This unique situation occurs when 2 larvae (diporpee) make contact on the host gin, such that their union triggers maturation into an individual adult worm. The present study examined paired stages of Eudiplozoon nipponioun microscopicaily to ascertain whether somatic fusion involves neural connectivity between these 2 heterogenic larvae. Neuronal pathways were demonstrated in whole-mount preparations of the worm, using indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy for peptidergic and serotoninergic innervations and enzyme cytochemical methodology and light microscopy for cholinergic component. Elements of the central nervous systems of paired worms are connected by commissures in the region of fusion so that the 2 systems are in structural continuity. Interindividual connections were mast apparent between corresponding ventral nerve cords. All 3 classes of neuronal mediators were identified throughout both central and peripheral connections of the 2 nervous systems. The anatomical complexity and apparent plasticity of the diplozoon nervous system suggest that it has a pivotal role not only in motility, feeding, and reproductive behavious but also in the events of larval pairing and somatic fusion.

Determination and multivariate statistical analysis of biochemical responses to environmental contaminants in feral freshwater fish Leuciscus cephalus, L.

Modulations of 11 prospective biochemical markers of impacts of aquatic pollutants in liver tissue of chub (Leuciscus cephalus), caught at several sampling sites of a river with various pollution types and rates, were matched against analytical data of concentrations of organochlorine compounds, polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Multivariate principal component analysis (PCA) of the field data showed general patterns of biochemical responses to different types of pollutants and relationships among the biomarkers. Cytochrome P4501A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity, inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related planar compounds, was strongly enhanced in the more contaminated areas. Compared with polychlorinated aromatic hydrocarbons, PAHs did not contribute so significantly to EROD induction. Testosterone 6 beta- and 16 alpha-hydroxylase activities, as an expression of the cytochrome P4503A27, were slightly increased at several sites but were significantly decreased in samples from some heavily polluted areas. Recently, these activities have been suggested as potential biomarkers of exposure to contaminants that do not induce cytochrome P4501A. In this study, their inhibition or induction was not associated with a specific class of monitored contaminants, and selectivities of these modulations are still to be investigated. Similar modulations of the prospective biochemical indicators of oxidative stress, including microsomal glutathione S-transferase activity, cytosolic glutathione S-transferase with ethacrynic acid, and glutathione reductase, were demonstrated by PCA. The pattern of the modulations of the microsomal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidation in vitro differed from the responses of the rest of oxidative stress parameters at some sampling sites. Further biochemical markers of oxidative stress under study, including in vivo lipid peroxidation, in vitro production of reactive oxygen species, and the concentration of metallothioneins did not correlate well with the concentrations of the contaminants. Principal component analysis demonstrated that the EROD activity, glutathione-dependent enzymes, and Fe(II)-enhanced lipid peroxidation formed a suitable battery of biomarkers of exposure.

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.

Experimental verification of the effect of the form (scaly or scaleless) of carp host (C. carpio L.) on the growth rate of micropopulations of Gyrodactylus katharineri Malmberg, 1964 (Monogenea).

It was demonstrated that the growth rate of Gyrodactylus katharineri micropopulations parasitizing the fry of Cyprinus carpio L. was effected by water temperature and by the form of the fish host (scaly or scaleless). At lower water temperature (12 degrees C) the number of parasites increased in both host forms approximately at the same intensity. In warmer water (18 degrees C), the parasites reproduced intensively only on the scaly form of fish, whereas no parasites were found on the scaleless form some days after infection.

Experimental verification of the effect of physical condition of Gobio gobio (L.) on the growth rate of micropopulations of Gyrodactylus gobiensis Gläser, 1974 (Monogenea).

It has been verified experimentally that the growth rate of G. gobiensis micropopulations is markedly affected by the physical condition of the host (Gobio gobio). If the fish were weakened by 50% oxygen deficiency and lack of food, the number of parasites increased much more rapidly than on the fish also unfed but kept in water with 100% oxygen content. If the fish weakened by oxygen deficiency were intensively fed, the growth of the parasite micropopulations started to slow down after 4-5 days. When the feeding was stopped, the number of parasites increased again after 10-11 days. These time shifts seem to be associated with the physical condition of the host improved by feeding or worsened by starvation.

Experimental verification of the effect of constant and changing water temperature on the micropopulation growth in Gyrodactylus gobiensis Gläser, 1974 (Monogenea) parasitizing gudgeon (Gobio gobio L.).

It has been verified under laboratory conditions that constant and changing water temperature markedly affects the micropopulation growth in Gyrodactylus gobiensis parasite on the body surface of gudgeons (Gobio gobio L.). At a water temperature of 12 degrees C, the number of gyrodactylids gradually increased up to the mean value of 63 specimens per host, which was reached on days 27-28 after experimental infection. At a constant temperature of 18 degrees C, the parasites completely disappeared from the bodies of infected fishes on days 15-20 p.i. A similar effect was produced by gradually increasing temperature from 12 to 18 degrees C, while the decrease in water temperature from 18 to 12 degrees C resulted in an increase in the parasite number.

The morphology of Trianchoratus aecleithrium Price et Berry, 1966 (Dactylogyridae, Monogenea) from a new host Trichogaster trichopterus trichopterus (Pallas, 1777).

The monogenean parasite Trianchoratus aecleithrium Price et Berry, 1966 is comparatively described from the gills of Trichogaster trichopterus trichopterus from aquarium in Czechoslovakia. T. trichopterus trichopterus represents a new host record for this parasite.

Capillostrongyloides Ancistri sp. n. (Nematoda: Capillariidae) a new pathogenic parasite of aquarium fishes in Europe.

A new nematode species, Capillostrongyloides ancistri sp. n., is described from the intestine of aquarium-reared catfish Ancistrus dolichopterus Kner in Czechoslovakia. This fish is of South American origin and, therefore, C. ancistri sp. n. has probably been brought into aquaria in Europe from there. The parasite is characterized mainly by the structure of the male caudal end (presence of two large postanal papillae and the cuticular bursa supported by two caudal lobes), the length of a spicule (0.258-0.297 mm), the presence of a non-spiny spicular sheath, shape of the female end, structure of the stichosome (23-30 stichocytes present), and the size of the eggs. The species appears to be highly pathogenic to aquarium-reared Ancistrus dolichopterus, causing death of these fish.