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The study lays stress on the morphological variability of metacercariae of Diplostomum pseudospathaceum under the influence of four factors: species and size of the host and density and age of the parasites. A total of 22 experiments were made, using 208 fish belonging to 10 species (three aquarium, seven native Polish species). Ranges of variations found in body size, number of excretory bodies, oral sucker and acetabulum, pharynx, holdfast organ and values of 9 biometric indices are wide and far exceed the ranges of variation given in the literature for this species of metacercaria.
The variability of metacercariae of Diplostomum pseudospathaceum under the influence of host species was studied experimentally. Four fish groups - two of Cyprinus carpio and two of Poecilia reticulata - were infected with the cercariae of D. pseudospathaceum originating from two snail species Lymnaea stagnalis and Galba palustris. For analysis of variability 14 characters of 152 metacercariae were measured and 8 indices were calculated. The morphometric data of the four experimental groups were examined statistically using a two way analysis of variance. The metacercariae of the four examined groups show highly significant variation in almost all analyzed characters. For particular characters a highly significant fish and snail host influence on the variation was noted. The source of variation was analyzed and the variability of previously investigated D. paracaudum was compared.
Metacercariae of Diplostomum pseudospathaceum and D. paracaudum were examined for genetic differences by random amplified polymorphic DNA analysis. Eleven different oligonucleotide decamers with arbitrary DNA sequence were tested as primers to amplify DNA from D. pseudospathaceum and D. paracaudum. Depending on the primer, the number of reproducible amplified fragments varied from 7 to 15, ranging in size from 0.2 to 2.0 kb DNA. Reproducible differences in patterns allowed differentiation of the two species with each primer. RAPD patterns unambiguously discriminated between D. pseudospathaceum and D. paracaudum, allowing good species identification.
The nervous system of daughter sporocyst of D. pseudospathaceum is described on a basis of acetylcholinesterase activity, histofluorescence of biogenic amines and serotonin immunoreactivity. Description of the posterior part of the nervous system is added to the hitherto described brain ganglia and irregular network of nerve fibres surrounding anterior end of the sporocyst. The immunoreactivity of 5-HT was observed in laterally situated main nerve cords, which run along the whole length of the body and unite at its posterior end. Some ramifications of the main cords were also visible. A possible role of messenger molecules observed in daughter sporocyst nervous system is discussed.
The ultrastructure of the proto- and paranephridial excretory systems in the metacercaria of D. pseudospathaceum is described. Basically, the protonephridial system in the metacercaria, although being more complex, does not differ in ultrastructure from that in the cercaria. All protonephridial canals have nuclei and septate desmosomes between neighbouring cells as well as desmosomes closing the canals. Contrary to the protonephridial system, the paranephridial one has a syncytial structure with few cell nuclei in the wall. No septate desmosomes were visible in cross- and longitudinal sections of the paranephridial canals and terminal pockets. The heterocellular gap junctions (nexus) between tegumental cytons and paranephridial canal walls have not previously been observed and described in diplostomid metacercaria. The possible function of these heterocellular gap junctions is discussed.
The chaetotaxy pattern of D. pseudospathaceum related to the structure of the nervous system (in the cephalic and preacetabular region) was described based upon LM and TEM investigations. The proper location of thirteen different ultrastructural types of sensory endings (SE) will be described by Czubaj and Niewiadomska (in press) were established. It was shown that the cercarial body, tail stem and furcae have a specific set of SE, and that several types of SE may be localized in different part of the cercaria. The location of particular sensory endings in the rings of the cephalic, pre- and postacetabular regions, tail stem and furcae was ascertained.
The author presents the results of the morphologkal studies on the lens of fish infected experimentally and in a natural way (in the river Vistula near Warsaw) with the metacercariae of both flukes. Basing on this and on the literature data a detailed picture of the pathological changes in the eye lens of infected fish has been constructed, the three development stages being accepted as described by other authors. The mechanisms of the influence of the metacercariae on the eye lens are presented. A particular attention has been paid to the significance of the ontogenetic development and the structure of the eye lens, not considered before, and especially to the fissure in the lens (Fig.). The latter, as a result of the metacercariae feeding on the lens material and distend it mechanically as they grow, is transformed into a chamber filled with a loosened lens material and the parasites. This leads to a deglutination of the lens capsule (sometimes on its entire perimeter) from the internal material. A symptom characteristic of the diplostomiasis in Missgurnus fossilis, observed by the author, consisted in fine folding of the capsule. Other symptoms were: besides the damage of the lens surface, changes in its size, deformations, loosening and sometimes breaking, always on the side of the enlarged fissure. Besides, the author confirms the results of observations of other authors and expresses an opinion that the picture of the pathological changes and the degree of vision impairment is influenced by such factors as the density and distribution (signly or in aggregations) of the metacercariae in the lens, and the place of their penetration and the host species.
Aminergic and cholinergic nervous systems were revealed and described for the furcocercaria Cercaria globocaudata, belonging to the globocauda group. In general the nervous system in the cercarial body is morphologically similar to that of another strigeid species, the cercaria of Diplostomum pseudo- spathaceum. Differences in morphology were visible in the large tail stem with a globular widening at the end. In front of the globular part, the number of cholinergic and aminergic elements in the dorsal and ventral nerve trunks increases and a connective and nervous net appears. The possible nerve centre for autonomous locomotion of the tail is discussed.
The general morphology of the excretory system of the cercaria and daughter sporocyst of D. pseudospathaceum is described. The ultrastructure of the following parts was examined: in the body - the filtering complex, anterior and posterior collecting tubules, main collecting duct, main excretory tube, and excretory bladder; in the tail - the capillaries with filtering complex, collecting tubes, main caudal duct, and furcal duct. Differences in the morphology of the excretory system of cercaria and daughter sporocyst are discussed: they relate to the lack of a main collecting tube and excretory bladder, and to the presence of separate excretory pores in the sporocyst. The ultrastructure of the filtering complex of sporocysts differs in the more numerous cilia and the presence of internal leptotriches. The other difference is the presence of long cytoplasmic projections that are in contact with adjacent cells in the distal part of the sporocyst capillary cell.
The entire muscular system of the cercaria of D. pseudospathaceum was studied using a wholemount fluorescence technique with rhodamine-labelled phalloidin. The body-wall musculature of the main body consists of circular, longitudinal and diagonal muscles. The musculature of the tail, anterior organ, ventral sucker, and certain parts of the alimentary tract are distinct. The ultrastructure of the two main types of muscle cells (smooth and cross-striated) were described, as was the differentiation of smooth muscles in particular organs. Muscle cells with two major opposing systems of myofilaments were found in the oesophagus.
Nine species of larval flukes, Notocotylus attenuatus, Echinostoma revolutum, Echinoparyphium aconiatum, Hypoderaeum conoideum, Plagiorchis elegans, Diplostomum pseudospathaceum, Australapatemon minor, Cotylurus sp., and Trichobilharzia ocellata were found in Lymnaea stagnalis in lake Kuuhankavesi (central Finland). Two species, P. elegans and E. aconiatum, are a new records from Finland and L. stagnalis was recognized as proper host for H. conoideum. In comparison with the records of Wikgren (1956) who found ten species of cercariae in L. stagnalis from the Tvärminne archipelago, our investigations revealed only five of these species: N. attenuatus, E. revolutum, D. pseudospathaceum (correct specific name for Wikgren’s D. spathaceum), Australapatemon minor (correct generic and specific names for Wikgren’s Apatemon gracilis) and T. ocellata. After Wikgren’s study on Tvärminne archipelago and Väyrynen from Northern Finland, the lake Kuuhankavesi (central Finland) are the third locality in Finland where larval trematodes have been studied.
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