Božena Koubková

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.

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.

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.

Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.

Morphometric and molecular characterization of Parapharyngodon echinatus (Nematoda, Pharyngodonidae) from the Senegal gecko ( Tarentola parvicarinata )

A West African species of gecko, Tarentola parvicarinata Joger (Gekkonidae), collected in the Niokolo Koba National Park in East Senegal is a new host species for the nematode Parapharyngodon echinatus (Rudolphi, 1819). Fifty one specimens of P. echinatus were studied under a light microscope and thirteen specimens were studied for the first time by a scanning electron microscope. The main differences between P. echinatus and other African species, especially Parapharyngodon micipsae (Seurat, 1917), were in the shape of annules on body cuticle, broad lateral alae and their terminations, the shape of the distal extremity of the spicule and number of outgrowths at the anterior cloacal lip. For the purposes of DNA characterization, partial sequence of the small subunit ribosomal RNA gene (SSU rRNA) was obtained from two specimens of P. echinatus. This is the first published sequence of a species from the family Pharyngodonidae.

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.

Eudiplozoon nipponicum in focus: monogenean exhibiting a highly specialized adaptation for ectoparasitic lifestyle.

Developmental stages of the diplozoid monogenean Eudiplozoon nipponicum, comprising oncomiracidium, diporpa, juvenile, and adult, were investigated using light and scanning electron microscopy in conjunction with confocal scanning laser microscopy in order to examine body organization and identify explicit morphological adaptations to the ectoparasitic life in each stage. The parasite exhibits a complex digestive tract well equipped for hematophagous feeding. It consists of a mouth opening with prominent buccal suckers, eversible pharynx with adjacent glandular structures, and a blind-ending gut with cecal lining. Glandulo-muscular organs, located apically and opened into the mouth corner, are considered to be a part of the digestive tract. Based on our observations of pharynx eversion and in light of the presence of several glandular or gland-like structures, we propose a new hypothesis on the possibility of extracorporeal digestion of this parasite. The hindbody bears an attachment apparatus, comprising haptor, lobular extensions, and tegumental folds, responsible for the parasite’s firm attachment to the host gills. The possibility of buccal suckers assisting in the parasite’s translocation while searching for an optimal niche or their temporary attachment function during feeding is discussed. The body of each compound adult (i.e., permanent copula) is almost completely filled by two complete reproductive tracts comprising the female as well as male organs. Such a reproductive strategy, in which two independent heterogenic individuals fuse into a single hermaphrodite organism without the need to search for mating partner, represents a high specialization of diplozoids to their parasitic life.

Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species

This study is focused on the feasibility of two treatments of alcohol-fixed monogenean parasites which are intended to be use for the combined morphological and molecular characterizations. The monogenean parasite, Eudiplozoon nipponicum, was selected as a model parasite species; however it is expected that these techniques will be suitable for other monogeneans and other parasitic families. The haptor of diplozoid parasites is equipped with sclerotized attachment clamps and central hooks which are utilized for morphological identification. As parasite tissue become very tough and rigid when preserved in ethanol, using these structures for species identification without additional treatment is difficult. We investigated two different techniques to digest the surrounding tissues, the first was treatment with 10% sodium dodecyl sulphate (SDS) and the second treatment was proteinase K. Tissue was successfully digested in both treatments and all clamps, central hook and even individual sclerites of the clamps were clearly visible and well defined. After treatment, the digest was used to extract genomic DNA, and the second internal transcribed spacer of the ribosomal DNA genes (rDNA) was amplified. Nucleic acid sequence was obtained from 90% of parasite specimens processed by both treatments. Treatment of haptors with SDS was proven to be more successful with no visible changes or damage observed to sclerites even after a month. This method represents a useful tool for the combined morphological and molecular studies as the correct sequence can be assigned to the same individual worm from which haptoral parts have been obtained.

Seasonal variation in parasite occurrence and microhabitat distribution of monogenean parasites of gudgeon Gobio gobio (L.)

SummarySeasonal changes in occurrence, infracommunity composition and microhabitat distribution of the monogenean parasites of gudgeon Gobio gobio (L.) were studied during two years in the River Haná (Czech Republic). Altogether 212 specimens of gudgeon were examined and a total of 6456 specimens of eight monogenean species were recorded: Dactylogyrus cryptomeres, Gyrodactylus gobiensis, Gyrodactylus gobii, Gyrodactylus gasterostei, Gyrodactylus vimbi, Gyrodactylus markakulensis, Gyrodactylus sedelnikowi and Paradiplozoon homoion. The occurrence of monogeneans correlated with seasonal changes of water temperature. In 2001, gyrodactylids peaked in March and D. cryptomeres in May, whereas in 2002, gyrodactylids peaked in May and D. cryptomeres in July. The majority of specimens of all Gyrodactylus species were found on the fins; the remaining specimens of Gyrodactylus were located on the body surface and gills, and all Dactylogyrus and Paradiplozoon specimens were located on the gills. The microhabitat distribution of the monogeneans changed in relation to changing water temperature and intensity of infection.

A new nematode, Pongobius hugoti gen. et sp. n. from the orangutan Pongo abelii (Primates: Hominidae)

SummaryLight and scanning electron microscopical examination and molecular analysis of pinworm samples collected from the Sumatran orangutan (Pongo abelii) from Sumatra (Indonesia) revealed the presence of a new taxon, Pongobius hugoti gen. et sp. n. (Oxyuridae: Enterobiinae). The monotypic genus Pongobius is characterised by the presence of a triangular mouth, three rounded teeth and three chisel-like inner teeth in the buccal cavity, an oesophagus with a long pharyngeal part, corpus posteriorly slightly enlarged and continuing as a pyriform bulb, isthmus absent. These features differentiate P. hugoti from all members of the subfamily Enterobiinae. The male has a long caudal appendix (> 50% of total tail length). Molecular analysis of the new taxon is presented for more precise identification.