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Diversity of selected groups of intestinal protists in cockroaches (Blattodea excl. Isoptera)
Kotyk, Michael ; Čepička, Ivan (advisor) ; Modrý, David (referee) ; Fiala, Ivan (referee)
This thesis addresses the rather neglected diversity of protists inhabiting the anoxic environment of the hindgut of cockroaches (Blattodea excluding Isoptera). I have examined the composition of the eukaryotic hindgut biota of over 200 cockroach species, with a detailed focus on four selected groups of protists: parabasalids (Metamonada, Parabasalia), oxymonads (Metamonada, Preaxostyla, Oxymonadida), ciliates (Alveolata, Ciliophora), and gregarines (Alveolata, Apicomplexa, Eugregarinorida). The results reveal a significant and so far almost unknown diversity of cockroach metamonads. The oxymonads are mostly represented by small polymastigids of the genus Monocercomonoides and the newly described genus Blattamonas. An exception is the discovery of what is, to date, only the second representative of the genus Streblomastix. The majority of the identified parabasalids belong to either the order Hypotrichomonadida or Honigbergiellida. Here the study takes a slight detour to explore the diversity of honigbergiellids as well as other groups of trichomonads inhabiting a somewhat different anoxic environment, namely aquatic sediments. Here, previously unknown diversity of parabasalids is also found. Our findings include the description of a new parabasalid order, Pimpavickida. Other investigated organisms...
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Investigation of the genome sizes, karyotype and ploidy within the genus Monocercomonoides
Kornalíková, Martina ; Hampl, Vladimír (advisor) ; Čepička, Ivan (referee)
Oxymonads are a group of anaerobic flagellated protists living mainly in the gut of insects and vertebrates. Here, we invetigate the ploidy and karyotype of various strains of Monocercomonoides using Fluorescence in situ Hybridization (FISH). For ploidy we used probes against the SufDSU gene, which is known to be present in a single copy in the genome of M. exilis. For karyotype investigations we used FISH probes against the TTAGGG telomeric repeat sequence. The results showed that all the investigated strains are haploid with a single clear signal from our SufDSU probes. The karyotype analyses showed that the average number of signals per nucleus varied between 9 to 17 in most of the investigated strains, indicating number of chromosomes similar to M. exilis. A notable exception from this are the strains of M. mercovicensis, where we observed a much higher number of signals, suggesting much higher number of chromosomes. For estimating of the DNA content in the nuclei of these oxymonads we used the flow cytometry, with M. exilis as a standard. Our results indicate that two of the investigated strains, Monocercomonoides sp. strain OEV and Mural1, have smaller genome sizes than M. exilis, while the rest of the strains have genomes sizes larger than M. exilis. We observed an unusually large variation...
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Pentoses as energy sources of Mastigamoeba balamuthi
Novotná, Monika ; Verner, Zdeněk (advisor) ; Čepička, Ivan (referee)
Mastigamoeba balamuthi is a free-living amoeba, a sister group to one of the most widespread parasitic agent Entamoeba histolytica. During its lifetime, M. balamuthi can utilize at least four different energy sources to produce ATP. In addition to glucose, a standard cultivation energy source, the amoeba can utilize pentoses xylose and arabinose, as well as an amino acid L-arginine. In this work, energy source preference was assessed. Morphological and physiological characteristics including cell growth and size, motility, number of nuclei and metabolic end products were evaluated. It was concluded that glucose appears to be a less preferred energy source than pentose upon evaluating statistically significant differences. Therefore, it was proposed to replace the commonly used hexose with the preferred xylose in the culture medium of M. balamuthi.
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Methanogenic symbionts of Psalteriomonadidae (Heterolobosea: Tetramitia)
Foučková, Martina ; Čepička, Ivan (advisor) ; Steinová, Jana (referee)
Psalteriomonadidae is a small family of anaerobic amoeboflagellates belonging to Heterolobosea. So far, 16 species have been described and there are also at least another 17 putative species which are yet to be formally described. Their anaerobic lifestyle is associated with a modification of the mitochondria into hydrogen-producing hydrogenosomes. The main focus of this thesis is on methanogenic symbionts of this family. The presence of prokaryotic symbionts has been observed in three species of Psalteriomonas, namely P. lanterna, P. vulgaris and P. magna. In P. lanterna, and P. vulgaris the symbionts were identified as Methanobacterium formicicum on the basis of their morphology and biochemical properties. For this thesis, 37 new freshwater isolates of psalteriomonadids were collected, identified, sequenced, and together with older isolates in culture, were investigated for the presence of prokaryotic symbionts. The UV autofluorescence of the symbionts of P. lanterna P. magna and Psalteriomonadidae sp. 5 indicates that they are methanogenic Archaea. No traces of methanogens were found in three isolates of Sawyeria marylandensis and three isolates of different Harpagon species. The symbionts were identified as Methanoregula sp. based on the 16S rRNA gene sequence. The symbionts were divided into...
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Morphological, ultrastructural, and molecular characterization of novel isolates of Anaeramoeba (Metamonada: Anaeramoebae)
Pavlátová, Magdaléna ; Čepička, Ivan (advisor) ; Kolísko, Martin (referee)
Anaeramoebae is a phylum of anaerobic marine amoebae and amoeboflagellates that belongs to the Metamonada supergroup. To date, this phylum has been represented by only six free- living species belonging to a single genus, Anaeramoeba. In this study five isolates with amoeboid stage of the genus Anaeramoeba were morphologically characterized, one on which also contained a flagellate stage. The ultrastructure of three isolates was investigated. Prokaryotes are in symbiotic relationships with all known members of the genus Anaeramoeba and our results show that these symbionts occur in three different arrangements within host cells. Furthermore, the distance between the nucleus and the acentriolar centrosome was studied and found to vary between species. The 18S rRNA gene sequences were obtained from three isolates and were included with previously sequenced strains in a phylogenetic analysis. In the remaining two isolates 18S rRNA gene sequencing was unsuccessful, but morphological and ultrastructural features support a relationship of these isolates to A. ignava. Phylogenetic analyse revealed that one of the isolates based on its sequence belongs to the A. flamelloides complex and two of the of the isolates represent a new species of the genus Anaeramoeba having the smallest cells of all known...
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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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Sex in Trypanosomatids
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Čepička, Ivan (referee)
3 Abstrakt Rody Leishmania a Trypanosoma jsou původci vážných lidských onemocnění: leishmaniózy a trypanosomózy. Dlouhá léta nebyly u těchto parazitů nalezeny přesvědčivé důkazy o genetické výměně, a proto byly rody Trypanosoma a Leishmania považovány za klonálně se rozmnožující, a to binárním štěpením jako většina prvoků. Výzkum ztěžovaly i skutečnosti, že pohlavní dimorfismus není patrný a chromosomy nekondenzují, tudíž nejsou viditelné. Nicméně klonální model začaly zpochybňovat pozorování přirozeně se vyskytujících hybridních druhů. Nejdříve byla existence sexu popsána u trypanosom a to prvním přímým důkazem hybridů T. brucei, získaných po společném přenosu rodičů mouchou tsetse. U leishmanii byl důkaz poskytnut na základě dvojitě rezistentních hybridů a sexuální výměna podstupovala stejný meiotický proces jako T. brucei. Byli pozorovaní přirozeně se vyskytující hybridi Nového i Starého světa jak u rodu Viannia, tak i u rodu Leishmania. Otázkou dalších výzkumů bylo, jaký je mechanismus genetické výměny, ale odpověď dodnes není jasná. Klíčová slova: genetická výměna, Trypanosoma, Leishmania, klonalita, meióza, GFP, přenašeč Abstract Genera Leishmania and Trypanosoma are agents of serious human diseases: leishmaniasis and trypanosomózy. For many years these parasites were considered clone-replicating by...
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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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The diversity of anaerobic ciliates from the subclass Scuticociliatia and their symbionts
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...
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