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Morphological and molecular diversity of the free-living representatives of the family Metopidae and the discovery of a new lineage of anaerobic ciliates
Rotterová, Johana ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,...
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Morphological and molecular characterization of anaerobic members of the genus Andalucia (Excavata: Jakobida)
Táborský, Petr ; Čepička, Ivan (advisor) ; Škaloud, Pavel (referee)
Eukaryotic organisms are divided into about 30 groups today, yet there still reamain many groups whose classification in the eukaryotic tree we are not sure about. This study focused on jacobids, a evolutionary important protist group from the Excavata taxa. Together with Heterolobosea, Euglenozoa and Tsukubamonadida, jacobids constitue a Discoba subgroup of Excavata taxa. Jacobids are significant mainly for their mitochondrial genome, which structurally resembles the original α-proteobacterial genome of the free-living ancestor of mitochondria. Some jacobids have their mitochondrial genome simplified, making them potential candidates for a model group used to study the mitochondrial evolution. However, too few members of jacobid group have been identified so far and the exact phylogeny of the group must be thoroughly understood for the detailed study of mitochondria evolution. This study focuses on broadening the taxonomic coverage of the jacobid excavate protists Andalucia. Twenty new strains of Andalucia have been established into stable culture, their morphology was assessed and SSU rDNA sequenced. Based on the phylogenetic analysis, Andalucia incarcarata species divides into three morphologically almost indistinguishable lines representing three species. Further, this study characterized a...
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Morphological and phylogenetic characterization of tetratrichomonads of group A
Krížová, Kateřina ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Trichomonads is a group of anaerobic single-celled eukaryotes currently classified in the kingdom Excavata (phylum Parabasalia), although does not share any of typically excavate features (the characteristic arrangement of the cytoskeleton and the ventral feeding groove). The taxonomy is based only on the molecular-phylogenetic analyses. Genus Tetratrichomonas contains probably the highest number of known species within the phylum Parabasalia, parasites and commensals of numerous vertebrate and invertebrate hosts. According to phylogenetic methods the genus has been divided into group A and B. This thesis is focused on the group A, which is split into 12 lineages (lineage 1 - 10, novel lineage 2, lineage LP). During our survey we discovered a new lineage - called sheep lineage, related to this group. Its position in group A is still unclear. Morphological differences between lineages have more or less confirmed the position of other lineages in phylogeny based taxonomy tree, which has been published previously. In this work, the differences between lineages have been described detailly. It is obvious that almost all lineages or groups of lineages are distinguishable from each other on the basis of morphological characters (size and shape of the body, the number of anterior flagella, parabasal body size,...
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Diversity of anaerobic ciliates
Nováková, Ludmila ; Čepička, Ivan (advisor) ; Kostka, Martin (referee)
Ciliates (Ciliata) are single-celled eukaryotic organisms belonging to the large group Alveolata. Ciliates are classified to eleven classes. Anaerobic/microaerophilic representatives belong to classes Armophorea, Litostomatea, Plagiopylea, Oligohymenophorea, and Prostomatea. The mitochondrion of the anaerobic ciliates has been tranformed to hydrogenosome. The anaerobic ciliates are characteristic for the presence of methanogenic symbionts in the cytoplasm near hydrogenosomes. Anaerobic flagellates are free-living or they live as commensals, mutualists or parasites in the digestive tract of animals including humans. The true diversity of anaerobic ciliates is still not fully understood. The reason is that only sequences of described ciliate species are usually included into phylogenetic analyses. However, many environmental sequences representing considerable part of known molecular diversity of ciliates have been published as well. The aim of this work was to obtain and analyze sequence data of anaerobic free-living ciliates. We have determined SSU rDNA sequences of 32 different freshwater and marine strains of ciliates. Phylogenetic analyses showed that the strains belonged mostly to the classes Armophorea, Plagiopylea and Oligohymenophorea. The strain LIVADIAN belonged, together with genera...
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Application of molecular and cellular biology methods in research of protozoa Eimeria
Vrba, Vladimír ; Škvor, Jiří (advisor) ; Čepička, Ivan (referee) ; Hampl, Vladimír (referee)
Eimeria is an apicomplexan parasite causing disease coccidiosis that is most prominent in poultry farming industry. This thesis is aimed to develop new molecular tools and resolve issues that would be a valuable contribution in the field from both research and industry perspective. Because immunity to Eimeria is strictly species- specific, it is important to know and recognize correctly all species that parasitize the host. Traditional diagnostic approaches rely on classical methods such as oocyst morphology determination under the microscope, measurement of prepatent period or in-vivo assessment of lesions caused by this parasite. However, diagnostics of individual species using these methods is very time-consuming and it is often unreliable, especially when mixture of multiple species whose parameters overlap is analyzed. Methods utilizing conventional PCR to distinguish species already exist, however, they lack advantages offered by quantitative real-time PCR (qPCR). The first aim of this thesis was to develop qPCR assays for detection and quantification of seven Eimeria species which infect chicken utilizing single-copy non-polymorphic targets in order to ensure maximal specifity and coverage of all strains of each species. Usefulness of this method was demonstrated by analysis of field...
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Pathogenic trichomonads in domestic animals
Vobořilová, Pavlína ; Tachezy, Jan (advisor) ; Čepička, Ivan (referee)
Pathogenic trichomonads of domestic animals are anaerobic protozoa found in the gastrointestinal and reproductive tract. They are responsible for serious diseases, which result in economic losses in agriculture. Cochlosoma anatis parasitizes in the intestine of birds, where it causes so-called runting syndrome and enteritis. Trichomonas gallinae is found in the gastrointestinal tract of pigeons and represents a dangerous infection of wild migratory birds and endangered birds of prey. Histomonas meleagridis is the causative agent of the best known, fatal and very redoubtable disease of turkeys and hens, so-called "blackhead disease". Tritrichomonas foetus is the causative agent of the most important sexually transmitted infection, tritrichomonosis in cattle, which causes significant economic losses in global beef production. T. foetus was recently identified as the causal agent of diarrheal disease in cats. Also Tritrichomonas mobilensis was recently described as a new species which probably disrupts the intestinal mucosa of squirrel monkeys. This work includes the general characteristics of these species and a detailed summary of the current knowledge on the diseases they are causing. The description covers the issue of sources and disease transmission, host specificity of the parasite and...
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Fornicata and biological pecularities of Giardia intestinalis
Rmoutilová, Eva ; Čepička, Ivan (advisor) ; Jirků, Miloslav (referee)
Fornicata is a recently established group of anaerobic protists that belongs to supergroup Excavata. Three main groups of Fornicata are Diplomonadida, Retortamonadida and Carpediemonas-like-organisms (CLOs). Most of these protists live as endobionts of various animals, several free-living representatives have been described as well. Fornicates are typical excavates, though some excavate features have been reduced in Diplomonadida. Diplomonads are unique with doubled (diplozoic) cell structure of most representatives. Several hypotheses explaining the evolution of diplozoic diplomonads were proposed, but none of them has been widely accepted. Fornicata are closely related to Parabasalia and Preaxostyla forming together clan Metamonada. Since Metamonada were considered to be primarily amitochondriate, they were classified among Archezoa. However, the kingdom Archezoa was rejected after the discovery of mitochondrial genes and reduced mitochondria in most supposedly archezoal protists. Within Fornicata, reduced mitochondria - called mitosome - was found in the parasite Giardia intestinalis. Giardia intestinalis is a well known parasite of humans and other mammals. Besides epidemiology, there are many other interesting aspects of this protist that are intensively studied, e.g. its high genetic...
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The method of DNA barcoding and its use in protists
Rotterová, Johana ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
DNA barcoding is a molecular method of species identification based on short regions of DNA sequences. These DNA barcodes should be unique for each species. The sequences within species should be identical or very much alike, while between species they should display a significant amount of differences. The sequences of an undetermined specimen can be compared with sequences vouchered to a particular species in a global DNA barcode database. The database was developed with the aim to simplify traditional taxonomy, eliminate its problems and accelerate its approaches. However, DNA barcoding has its own limitations that may hinder achieving its original goals and a compliance with the concept. The aim of this bachelor thesis consists of explaning the evolution of DNA barcoding through time and assessing the importance of this method at present, particularly in protists. Species identification may be very difficult in many protist groups, which can be explained by several reasons. To appraise this method completely is a hard task, because creation of the database has not yet been finished and official DNA barcodes have not been chosed for many groups. We can only find out in the future, therefore i will only focus on factors that influence the success of DNA barcoding at present.
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Evolution of the genetic code and classification of oxymonads
Šrámová, Eliška ; Hampl, Vladimír (advisor) ; Čepička, Ivan (referee)
Oxymonads are a group of heterotrophic flagellates living in low oxygen environment. These protists inhabit mainly the gut of xylophagous insects (cockroaches, termites), with an exception of the genus Monocercomonoides, which was described from the intestinal contents of many vertebrates. On the basis of molecular data, Oxymonadida are classified into the supergroup Excavata (Cavalier-Smith, 2002; Simpson et al., 2006, Hampl et al. 2009). This thesis was focused on the diversity of genus Monocecomonoides from the morphologically simplest family Polymastigidae. The main goal of our work was to gather sequence data from strains isolated from a wide spectrum of hosts. We have obtained 26 partial sequences of the gene for the SSU rDNA in total, of which two belonged to another oxymonad, apparently genus Oxymonas. Our phylogenetic analysis indicated that the representatives of the genus Monocercomonoides form one group, however with a low bootstrap support. On the basis of published data about the presence of non-canonical genetic code in some oxymonads (Keeling and Leander, 2003; de Koning et al., 2008), we decided to explore this rare phenomenon in representatives of the genus Monocercomonoides. For this part of the study we gathered 9 partial sequences of α-tubulin gene. In these sequences we have not...
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Diversity of the genus Blastocystis (Stramenopiles)
Lorencová, Markéta ; Čepička, Ivan (advisor) ; Kostka, Martin (referee)
Although the genus Blastocystis is only little known, it has recently attracted much attention of protistologists and parasitologists. Similarly to the related opalines and proteromonads, Blastocystis is anaerobic and lives endobiotically in intestines of many animals. On the other hand, members of the genus have completely lost flagella. Therefore, the phylogenetic position of Blastocystis had been puzzling for a long time. B. hominis is one of the most common intestinal parasites of humans and has been found also in a wide variety of animals. It is highly prevalent not only in the developing nations, but also in the developed countries. Its enormous genetic diversity, pathogenic and zoonotic potential (B. hominis is often associated with irritable bowel syndrome) make from B. hominis an important emerging pathogen.
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