Reisolation and redescription of Balantidium duodeni Stein, 1867 (Litostomatea, Trichostomatia).

In this work, we present reisolation and redescription of Balantidium duodeni Stein, 1867 from the European common brown frog Rana temporaria Linnaeus, 1758 using light and electron microscopy. This species has a unique morphological feature--its cells are flattened along the dorsoventral axis. Because of its unique morphology and localization (duodenum) in the gastrointestinal tract of the host, it has been proposed to recognize B. duodeni as a member of separate genus, Balantidiopsis Penard, 1922. Molecular phylogenetic analysis demonstrates it to be close to the type species Balantidium entozoon (Ehrenberg, 1838). We argue that its placement into separate genus is not substantiated. We also propose to reinstate the genus Balantioides Alexeieff, 1931 with the type species Paramecium coli (Malmstein, 1857). The recently proposed generic name for this taxon, Neobalantidium Pomajbíková et al., 2013, is a junior synonym of the previously recognized name.

The helminth fauna study of European common brown frog (Rana temporaria Linnaeus, 1758) in the Volga basin.

In this paper we considered information on the helminth fauna of the European common brown frog (Rana temporaria Linnaeus, 1758) from 10 regions of the Volga basin. This study includes consolidated data of different authors over the last 30 years, supplemented by the results of our own research. There are reliably known finds of 29 species of helminths: Monogenea - 1, Trematoda - 21, Nematoda - 7. Trematodes Gorgodera asiatica Pigulevsky, 1945, Paralepoderma cloacicola (Luhe, 1909), mtc. and nematodes Icosiella neglecta (Diesing, 1851) were observed for the first time in a given host on the territory of Russia and the Volga Basin. Six species of worms make the basis of helminth fauna: nematodes Rhabdias bufonis, Oswaldocruzia filiformis, Neoxysomatium brevicaudatum and Cosmocerca ornata, trematode Haplometra cylindracea and monogenea Polystoma integerrimum. These six species are the most common and widespread parasites of the brown frog. For each species of helminths there is the following information included: taxonomic position, localization, area of detection, biology, definitive hosts, geographic distribution, the degree of host-specificity.

[The study of Balantidium Helenae Bezzenberger, 1904 (Litostomatea, Trichostomatia) inhabiting the intestines of the frog Rana temporaria Linnaeus, 1758, in optical and electron microscopes].

The structure of the ciliate Balantidium helenae Bezzenberger, 1904 was investigated with the use of optical and electron microscopy. It was demonstrated that, together with the basic dimensional characteristics, main distinguishing features of the studied species include the following characters: the presence of a pointed prominence on the posterior margin of the cell; the presence of elongated cilia on the left margin of the vestibulum; and different shape and size of cortical ridges. Two morphologically different types of electron-dense bodies (supposedly hydrogenosoms) and also endobiotic bacteria were also found in the cytoplasm of B. helenae.

Newly discovered linkages between the cortical (pellicular) ridges of Opalina.

Samples of Opalina ranarum have been prepared for electron microscopy by ultra-rapid cryofixation followed by substitution fixation in a solvent containing tannic acid. This technique has made it possible to see that very thin linkages exist between the pleated ridges that form the surface of the cell. Between any two adjacent cortical ridges, the linkages, which are approximately 0.1 microm long, occur as a single row, 0.1 microm below the free edge, with an impressively regular spacing of 0.1 microm. The cortical ridges of the Opalinids are spaced with remarkable uniformity, even when thrown into undulating patterns. The linkages described here will inevitably stabilize the complex architecture of the cortex. Other possible functions are discussed.

Prototheca richardsi, a pathogen of anuran larvae, is related to a clade of protistan parasites near the animal-fungal divergence.

Prototheca richardsi is a protist of uncertain taxonomy which mediates growth inhibition in anuran larvae. Cells of P. richardsi were isolated from tadpole faeces and DNA was purified by Qiagen chromatography. Nuclear small-subunit (18S) rDNA (ssu-rDNA) was amplified by PCR using universal primers, cloned, and six clones (two from each of three separate isolates) were sequenced. All clones yielded an essentially identical sequence of 1802 nucleotides. In situ hybridization of fluorescent Prototheca-specific oligonucleotide probes, designed using the derived 18S rDNA sequence, confirmed that the sequence was indeed from P. richardsi cells and not from other components of tadpole faeces. The P. richardsi sequence was aligned with ssu-rDNA from a range of other eukaryotes, and phylogenetic analyses were carried out using several inference methods. P. richardsi consistently and stably grouped within a novel clade that contains rDNAs from an apparently heterodisperse group of parasitic micro-organisms assigned to the class Ichthyosporea. P. richardsi is evidently misplaced in the genus Prototheca, and the authors propose its inclusion in a new genus Anurofeca.

Widespread distribution of histamine in the nervous system of a trematode flatworm.

In general, most flatworms contain very little histamine (HA) and their nervous systems often lack, or contain very few, histaminergic elements. However, preliminary studies in our laboratory have revealed that the frog lung parasite, Haplometra cylindracea (Trematoda: Digenea), contains HA in a very high concentration. For this reason, the present study was undertaken to study the localization and synthesis of HA in this worm by using immunocytochemistry and high-pressure liquid chromatography (HPLC). Essentially all parts of the nervous system of H. cylindracea showed HA-like immunoreactivity. The paired cerebral ganglia and nerves emanating from these, including the longitudinal nerve cords, were intensely immunoreactive. The musculature of the pharynx, oral and ventral suckers, and those of the reproductive organs were all innervated by HA-immunoreactive fibers. Fiber plexuses beneath the tegument and throughout the parenchyma also showed HA-like immunoreactivity. HPLC studies revealed one of the highest HA concentrations in the animal kingdom, 6.49 +/- 1.36 nmole/mg protein, in the worm. The frog lung and blood contained very low concentrations of HA and could be excluded as sources for HA, while an enzyme assay revealed that the worm produces HA by decarboxylation of histidine. Thus, it is likely that H. cylindracea uses HA as a neurotransmitter or modulator.

[A cytophotometric study of the amount of DNA in the macro- and micronuclei of the infusorian Nyctotherus cordiformis from the gut of tadpoles and juveniles of the common frog]. / Tsitofotometricheskoe issledovanie kolichestva DNK v makro- i mikronukleusakh infuzorii Nyctotherus cordiformis iz kishechnika golovastikov i segoletok travianoi liagushki.

The investigations of DNA amounts in the nuclei of the ciliate Nyctotherus cordiformis was continued. The measured ciliates were collected from guts of tadpoles and the most small frogs just from the water. The DNA content was measured (in arbitrary units--a.u.) in the nuclei of cysts, precysts, vegetative ciliates and small frogs. This paper is the second part of the investigation. The first part dealt with DNA amounts of the nuclei of the vegetative ciliates tested in spring, autumn and winter. The amount of the Feulgen-DNA complex was measured with a two-wave microcytophotometer MCFU-1. The average content of the DNA ranged from 1.7 +/- 2 a.u. in the Mi of cysts to 2.6 +/- 0.1 a.u. in the Mi of the youngest frogs. The DNA content of the Ma ranged from 275 +/- 21 a.u. in cysts to 479 +/- 25 a.u. in vegetative ciliates. The DNA content in presynthetic Mi (G1) is supposed to be approximately 1.3 and approximately 2.6 a.u. in postsynthetic (G2) Mi. Using nuclei of erythrocytes of Rana temporaria as internal standard, the DNA content of a 2 c Mi from tadpoles of N. cordiformis is supposed to amount to 0.57 pg or approximately 350 gDa. The DNA amount in Ma of N. cordiformis is at average 140-220 times as that of Mi.

Scanning electron microscopy of Polystoma integerrimum (Monogenea, Polystomatidae).

The tegumental surfaces of both the bladder (normal) adult and branchial (neotenic) adult of the dimorphic monogenean Polystoma integerrimum are elevated into ridges forming a honeycomb-like pattern of hexagons. In some regions, upright microvilli are located at the angles of the hexagons. The two hamuli of the adult forms are sheathed with the ornamented tegument except for the naked distal spine. Larvae with haptors at the two-sucker stage of development have a tegument with the elevated honeycomb design characteristic of the adults. Developing hamuli appear as fully sheathed, conical elevations. The 16 marginal hooklets of the larval haptor are covered by tegument, with the exception of their strongly curved spines. Genetic blueprints for normal and neotenic development, contained in the genome of all larvae hatching from the eggs of normal individuals, appear to diverge only in relation to the different rates of embryonic development of the two adult forms and the incomplete ontogeny of the neotenic adult.

Identification and characterization of the cysteine and serine proteinases of the trematode, Haplometra cylindracea and determination of their haemoglobinase activity.

The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22-23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-Argp(OPh)2 or biotin-Lysp(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

Lymnaea truncatula, intermediate host of some Plagiorchiidae and Notocotylidae species in León, nw Spain.

To study the distribution of Lymnaea truncatula in the Porma river basin (León, NW Spain) and its helminth fauna, malacological samplings were carried out at 66 points in the basin and the presence of these molluscs was detected in 31. To trace the dynamics of this mollusc population and the prevalence and intensity of its infection by trematodes, malacological samplings were made at fortnightly intervals over two years at five locations, situated in the upper and middle regions of the river Porma basin. To confirm the identity of the larval stages found in the molluscs, second intermediate and/or definitive hosts, depending on the trematode species, were experimentally infected to complete the life cycles. Two different species of Plagiorchiidae and one of Notocotylidae were identified. The infection prevalence of Plagiorchis elegans for the 6291 specimens of L. truncatula examined was 2.8% and infection was observed in snails collected in 7 of the 31 sample localities. This parasite was found in all months of the year, with the highest prevalence observed in July and October. When the corrected frequency values were considered, a slightly positive relationship was observed between the infection prevalence and the latter months. The highest percentages of snails harbouring immature sporocysts were detected in March and June-July, while the highest percentages with sporocysts containing mature cercariae were observed in spring and at the end of summer-autumn, and cercarial shedding in the latter. Of the 6291 L. truncatula examined 0.3% were infected by sporocysts of Opisthioglyphe ranae.(ABSTRACT TRUNCATED AT 250 WORDS)

The common frog (Rana temporaria) as a potential paratenic and intermediate host for Angiostrongylus vasorum.

Common frogs (Rana temporaria) were exposed either to third-stage larvae (L3) or to first-stage larvae (L1) of Angiostrongylus vasorum. Following exposure to L3, viable larvae could be detected in the frogs for at least 2 weeks. Following exposure to L1, the frogs developed viable L3 in their tissues within 30 days. In a test of the infectivity of these larvae, one fox was fed frogs previously infected with L3 and another fox was fed frogs previously infected with L1. On autopsy it was found that adult A. vasorum populations had been established in both foxes. Thus, it could be concluded that frogs can act not only as paratenic hosts but also as intermediate hosts for A. vasorum.

Mammalian regulatory peptide immunoreactivity in the trematode parasite Haplometra cylindracea and the lung of its frog host, Rana temporaria: comparative chromatographic characterisation using reverse-phase high-performance liquid chromatography.

1. Extracts of Haplometra cylindracea and lung tissues of its host, Rana temporaria, were subjected to radioimmunoassay using antisera to nine mammalian regulatory peptides. 2. In these extracts, immunoreactivity was measured to pancreatic polypeptide, substance P, neurokinin A, gastrin-releasing peptide and glucagon. The levels of each peptide varied considerably with some marked differences between those demonstrable in parasite and host (notably pancreatic polypeptide). 3. Reverse-phase HPLC fractionation of extracts revealed general chemical differences between parasite and host peptides, with some peptides present in more than one molecular form.

[Serotonin and dopamine in the tissue extracts of Polystoma integerrimum (Monogenea)]. / Serotonin i dofamin v ékstraktakh iz tkanei Polystoma integerrimum (Monogenea).

Spectrofluorometric analysis has revealed substances with spectral characteristics identical to those of serotonin and dopamine in tissue extracts of Polystoma integerrimum. A possible function of the substances is discussed.

Chemical signals of fish skin for the attachment response of Acanthostomum brauni cercariae.

The chemical signals of the skin surface of fish, which stimulate the attachment responses of Acanthostomum brauni cercariae, were identified by offering chemicals and fish-skin extracts in agarose substrates to the cercariae. Smaller molecules such as amino acids, fatty acids, monosaccharides, electrolytes, urea, and carbonate solutions did not stimulate attachments, but hyaluronic acid had some effects. Bovine submaxillary glycoproteins had a strong stimulating activity that disappeared after neuraminidase digestion. The stimulating components of the skin surface of fish were hydrophilic substances with molecular weights of more than 10,000. They were sensitive to neuraminidase digestion but not to hyaluronidase digestion and thus can be identified as glycoproteins. A. brauni cercariae respond only to the complete glycoprotein molecules and not to their monosaccharide components. The known attachment triggers of other cercariae are small molecules. Large glycoproteins as host signals for A. brauni cercariae may be an adaptation to muddy habitats, where various substances with low molecular weights may interfere with the host identification.

Gorgoderina vitelliloba: interaction with frog leucocytes in vivo and in vitro.

Ultrastructural observations were made on the response in vivo of adult Rana temporaria to Gorgoderina vitelliloba and the interaction between host cells and the parasite in vitro. In both cases, the cellular attack on the fluke was most intense 21-23 days postinfection. Host leucocytes (believed to be eosinophils) lay flat against the surface of the parasite. Degranulation of their electron-dense, peroxidase-positive granules occurred and vacuoles formed. The outer plasmalemma of the tegument was breached and eosinophils migrated over the basal lamina, stripping the tegument from the surface of the fluke. The cellular response was so vigorous in vitro that the tegument was sometimes completely lost. In vivo, damage was slight, and the majority of the parasites successfully completed their migration to the bladder, where there was no further cellular response, and tegumental repair occurred. The fact that the migration of the fluke from the kidney to the bladder occurred at the same time as the peak of the cellular response may not be coincidental, and it is suggested that the cellular attack may initiate migration from the kidneys.

Observations on the routes of infection of Oswaldocruzia filiformis (Nematoda: Trichostrongylidae) in amphibia.

The oral, percutaneous and subcutaneous routes of infection of Oswaldocruzia filiformis were investigated in amphibia. Tadpoles of Bufo bufo and Rana temporaria can be infected with O. filiformis when kept temporarily in a suspension of infective larvae in water. Larval stages and subadults were found in tadpoles. All stages of the parasite, including egg-producing females, were found after metamorphosis of the host. However, under natural circumstances infection of tadpoles seems unlikely. Oral infections in metamorphosed hosts of both species were successful in 97.5% of the host animals used. The first eggs appeared 29 days after infection in the faeces. The oral route seems to be normal for O. filiformis in amphibia. Experiments on percutaneous infections did not reveal actual penetration of larvae in or through the skin nor a subsequent migration through host tissues. Sometimes a few larvae were found in the stomach and intestine, but in these particular cases the experimental conditions did not totally exclude the possibility of oral infections. Consequently, the percutaneous route of infection is not plausible for O. filiformis. Subcutaneous inoculation of infective larvae seems to be a possible way of establishing experimental infections. Erratic localisation of the parasite in the enlarged gall bladder of the host was observed.

The hatching rhythm of Polystoma integerrimum, a monogenean from the frog Rana temporaria.

1. Eggs of Polystoma integerrimum incubated in alternating periods of light and darkness at a constant temperature hatch in the light. 2. Eggs incubated and hatched in continuous light (10 degrees or 18 degrees C) hatch without a rhythm. Eggs incubated and hatched in continuous darkness at 18 degrees C hatch without any circadian rhythm but fail to hatch at 10 degees C until 1 month later than expected. 3. At both 10 degrees and 18 degrees C the hatching rhythm persists in conditions of continuous light if, during incubation, the eggs were exposed to alternating light and darkness. At 18 degrees C the rhythm appears to persist in total darkness following incubation in alternating light and darkness. At 10 degrees C hatching is blocked. 4. At 10 degrees C if eggs are incubated in alternating periods of light and darkness and then, when hatching begins, these periods are reversed, the hatching rhythm becomes synchronous with the new lighting regime. This may be due to the effect of low temperature. 5. Hatching during the day may have survival value of P. integerrimum since the host tadpoles congregate at this time around the edges of the lakes, the region where the parasite eggs are laid.

[Ultrastructure of the tegumnet of the slow-growing larva of Polystoma integerrimun (Frohlich 1798). Evolutionary significance]. / Ultrastructure du tégument de la larve à croissance lente de Polystoma integerrimum (Fröhlich 1798). Signification évolutive

The ultrastructure of the integument of the gill living post-larva of Polystoma integerrimum (Monogenea) from the common frog Rana temporaria is investigated for the first time; it has been compared with those of the adult stage, of other juvenile stages of marine Monogenea and of some Digeneans adapted to frog urine.