Academic literature on the topic 'Diplomonaden'

Unikaryotic enteromonads and diplokaryotic diplomonads have been regarded as closely related protozoan groups. It has been proposed that diplomonads originated within enteromonads in a single event of karyomastigont duplication. This paper presents the first study to address these questions using molecular phylogenetics. The sequences of the small-subunit rRNA genes for three isolates of enteromonads were determined and a tree constructed with available diplomonad, retortamonad and Carpediemonas sequences. The diplomonad sequences formed two main groups, with the genus Giardia on one side and the genera Spironucleus, Hexamita and Trepomonas on the other. The three enteromonad sequences formed a clade robustly situated within the diplomonads, a position inconsistent with the original evolutionary proposal. The topology of the tree indicates either that the diplokaryotic cell of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated by secondary reduction from the diplokaryotic state.

ABSTRACTThe identification of ancestral traits is essential to understanding the evolution of any group. In the case of parasitic groups, this helps us understand the adaptation to this lifestyle and a particular host. Most diplomonads are parasites, but there are free-living members of the group nested among the host-associated diplomonads. Furthermore, most of the close relatives within Fornicata are free-living organisms. This leaves the lifestyle of the ancestor unclear. Here, we present metabolic maps of four different diplomonad species. We identified 853 metabolic reactions and 147 pathways present in at least one of the analyzed diplomonads. Our study suggests that diplomonads represent a metabolically diverse group in which differences correlate with different environments (e.g., the detoxification of arsenic). Using a parsimonious analysis, we also provide a description of the putative metabolism of the last Diplomonadida common ancestor. Our results show that the acquisition and loss of reactions have shaped metabolism since this common ancestor. There is a net loss of reaction in all branches leading to parasitic diplomonads, suggesting an ongoing reduction in the metabolic capacity. Important traits present in host-associated diplomonads (e.g., virulence factors and the synthesis of UDP-N-acetyl-d-galactosamine) are shared with free-living relatives. The last Diplomonadida common ancestor most likely already had acquired important enzymes for the salvage of nucleotides and had a reduced capacity to synthesize nucleotides, lipids, and amino acids de novo, suggesting that it was an obligate host-associated organism.IMPORTANCE Diplomonads are a group of microbial eukaryotes found in oxygen-poor environments. There are both parasitic (e.g., Giardia intestinalis) and free-living (e.g., Trepomonas) members in the group. Diplomonads are well known for their anaerobic metabolism, which has been studied for many years. Here, we reconstructed whole metabolic networks of four extant diplomonad species as well as their ancestors, using a bioinformatics approach. We show that the metabolism within the group is under constant change throughout evolutionary time, in response to the environments that the different lineages explore. Both gene losses and gains are responsible for the adaptation processes. Interestingly, it appears that the last Diplomonadida common ancestor had a metabolism that is more similar to extant parasitic than free-living diplomonads. This suggests that the host-associated lifestyle of parasitic diplomonads, such as the human parasite G. intestinalis, is an old evolutionary adaptation.

Background The mechanisms by which DNA sequences are expressed is the central preoccupation of molecular genetics. Recently, ourselves and others reported that in the diplomonad protist Giardia lamblia, the coding regions of several mRNAs are produced by ligation of independent RNA species expressed from distinct genomic loci. Such trans-splicing of introns was found to affect nearly as many genes in this organism as does classical cis-splicing of introns. These findings raised questions about the incidence of intron trans-splicing both across the G. lambliatranscriptome and across diplomonad diversity in general, however a dearth of transcriptomic data at the time prohibited systematic study of these questions. Methods I leverage newly available transcriptomic data from G. lamblia and the related diplomonad Spironucleus salmonicidato search for trans-spliced introns. My computational pipeline recovers all four previously reported trans-spliced introns in G. lamblia, suggesting good sensitivity. Results Scrutiny of thousands of potential cases revealed only a single additional trans-spliced intron in G. lamblia, in the p68 helicase gene, and no cases in S. salmonicida. The p68 intron differs from the previously reported trans-spliced introns in its high degree of streamlining: the core features of G. lamblia trans-spliced introns are closely packed together, revealing striking economy in the implementation of a seemingly inherently uneconomical molecular mechanism. Discussion These results serve to circumscribe the role of trans-splicing in diplomonads both in terms of the number of genes effected and taxonomically. Future work should focus on the molecular mechanisms, evolutionary origins and phenotypic implications of this intriguing phenomenon.

Abstract Oxygen and reactive oxygen species (ROS) are important stress factors for cells because they can oxidize many large molecules. Fornicata, a group of flagellated protists that includes diplomonads, have anaerobic metabolism but are still able to tolerate fluctuating levels of oxygen. We identified 25 protein families putatively involved in detoxification of oxygen and ROS in this group using a bioinformatics approach and propose how these interact in an oxygen detoxification pathway. These protein families were divided into a central oxygen detoxification pathway and accessory pathways for the synthesis of nonprotein thiols. We then used a phylogenetic approach to investigate the evolutionary origin of the components of this putative pathway in Diplomonadida and other Fornicata species. Our analyses suggested that the diplomonad ancestor was adapted to low-oxygen levels, was able to reduce O2 to H2O in a manner similar to extant diplomonads, and was able to synthesize glutathione and l-cysteine. Several genes involved in the pathway have complex evolutionary histories and have apparently been repeatedly acquired through lateral gene transfer and subsequently lost. At least seven genes were acquired independently in different Fornicata lineages, leading to evolutionary convergences. It is likely that acquiring these oxygen detoxification proteins helped anaerobic organisms (like the parasitic Giardia intestinalis) adapt to low-oxygen environments (such as the digestive tract of aerobic hosts).

ABSTRACT Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis , an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida , a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II–FLAG tandem-affinity purification tag or streptavidin binding peptide–glutathione S -transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase ( pac ), blasticidin S -deaminase ( bsr ), and neomycin phosphotransferase ( nptII ), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleu s.

ABSTRACT Lateral gene transfer has been identified as an important mode of genome evolution within prokaryotes. Except for the special case of gene transfer from organelle genomes to the eukaryotic nucleus, only a few cases of lateral gene transfer involving eukaryotes have been described. Here we present phylogenetic and gene order analyses on the small subunit of glutamate synthase (encoded by gltD) and its homologues, including the large subunit of sulfide dehydrogenase (encoded by sudA). The scattered distribution of the sudA and sudB gene pair and the phylogenetic analysis strongly suggest that lateral gene transfer was involved in the propagation of the genes in the three domains of life. One of these transfers most likely occurred between a prokaryote and an ancestor of diplomonad protists. Furthermore, phylogenetic analyses indicate that the gene for the small subunit of glutamate synthase was transferred from a low-GC gram-positive bacterium to a common ancestor of animals, fungi, and plants. Interestingly, in both examples, the eukaryotes encode a single gene that corresponds to a conserved operon structure in prokaryotes. Our analyses, together with several recent publications, show that lateral gene transfers from prokaryotes to unicellular eukaryotes occur with appreciable frequency. In the case of the genes for sulfide dehydrogenase, the transfer affected only a limited group of eukaryotes—the diplomonads—while the transfer of the glutamate synthase gene probably happened earlier in evolution and affected a wider range of eukaryotes.

Although commensals in digestive tract of a large number of fish species, diplomonads represent very significant opportunistic pathogens. For so far unknown reasons, they can proliferate uncontrollably and thus cause changes in the skin and internal organs in aquarium fish. The problem is confusion over nomenclature of the two most important genera: Spironucleus i Hexamita. Aquarium fish species in which there were diagnosed changes in the skin caused by diplomonads were: Microgeophagus ramirezi, Apistogramma cacatuoides, Apistogramma nijsseni, Symphysodon aequifasciatus, Pterophyllum altum, Archocentrus nigrofasciatus, Pelvicachromis pulcher i Labidochromis caruleus. The fish were treated with 250 mg tablets of metronizadole dissolved in water, or metronizadole in a concentration of 6.6 mg per liter of water. The treatment was successful in only 9 out of 45 treated fish. In the others the symptoms reappeared after certain time. It is necessary to determine the prevalence of the infection in aquarium fish in Serbia, and also examine the success of the treatment with metronizadole applied in food or even other possibilities of the treatment.

Durch Parasiten stellen eine starke Gefährdung für die Aquakultur dar. Die durch diplomonaden Flagellaten bei der Regenbogenforelle Oncorhynchus mykiss verursachte Morbidität und Mortalität wurde bisher in Deutschland noch nicht gründlich untersucht. Ich habe diese Parasiten mittels SEM & TEM charakterisiert und wurde die Art Spironucleus salmonis bestimmt. Erstmals konnten die caudale Projektion, sich entleerende Vakuolen und verformbare Kernloben nachgewiesen werden. Ich untersuchte Mikrohabitatpräferenz von des Parasiten sowie pH-Profile in vier Darmabschnitten der Fische. Das Vorkommen und die Dichte von S. salmonis war in der Pylorusregion wesentlich höher als in anderen Bereichen. Das pH-Profil war bei infizierten und nicht infizierten Fischen gleich. Der optimale pH-Wert für S. salmonis war 7,1-7,5. Ich habe die Lebenszyklus der Diplomonaden mittels LM & SEM unter Kulturbedingungen untersucht. Die Enzystierung begann mit der Anheftung von Trophozoiten mit der Spitze der adhäsiven hinteren Flagellen aneinander oder an Fremdkörper. Die pyriformen Trophozoiten wurden kugelförmig, und die vorderen Flagellen inaktiv. Oberflächenbläschen produzierten eine lichtbrechende Zystenwand. Dies ist der erste Beschreibung der Multifunktionalität von Flagellen bei Diplomonaden. Ich untersuchte pathogene Mechanismen und sezierte die Pylorusregion sowie die Leber mittels H&E, PAS/AB. Bei infizierten Fischen trat eine signifikante Hypertrophie der Becherzellen auf. Zu erkennen war eine Hyperaktivität der Becherzellen, jedoch keine Hyperplasie. Ich entwickelte einen in vitro  Plasma-Inkubationstest zur Bestimmung der Suszeptibilität von Regenbogenforelle, Karpfen und Störe. Die unterschiedliche Resistenz von Stör, Karpfen und Regenbogenforelle gegen S. salmonis entsprechend zu den epizootiologischen Daten. Meine Untersuchungen führten zu einem neuen diagnostischen Hilfsmittel, Vorschlägen für neue Behandlungsmethoden, zu verbesserten in vitro-Kulturbedingungen und einem Modellsystem für die Multifunktionalität von Flagellen und flagellaren Signaltransduktion.
Parasitic diseases pose a significant threat to aquaculture. Diplomonad flagellates in rainbow trout Oncorhynchus mykiss are associated with morbidity and mortality; but in Germany has not been thoroughly studied. I characterised the species by SEM & TEM, which revealed Spironucleus salmonis, allowed its complete description including newly showing the caudal projection, discharging vacuoles, and deformable nuclear lobes; diagnostic keys were improved. The microhabitat preference of diplomonads was tested by recording occurrence and density of infection, and pH profile in 4 intestinal regions in fish. Occurrence and density of S. salmonis were significant higher in the pyloric region than elsewhere. The pH profile in uninfected and infected fish was similar; a causal relationship between microhabitat preference and pH was unlikely, and the optimal pH was between 7.1 – 7.5. I described life cycle and encystment using light and SEM. Encystment in culture began by trophozoites attaching at tip of adhesive posterior flagella to each other/debris. Pyriform trophozoites became sub-spherical, anterior flagella inactive, surface blebs produced a refractile cyst wall. Cysts clusters may exceed minimum infective dose for new infection; suggesting new treatment target. This is the first report of multi-functionality of flagella in diplomonads. I investigated pathogenic mechanism of diplomonads by sectioning and staining the pyloric region of the intestine and liver with H&E, and PAS/AB. There was significant hypertrophy of goblet cells in infected fish. The hyperactivity of goblet cells was seen, but no hyperplasia. This hyper-production of mucus may decrease nutrient absorption, underlying impaired growth in S. salmonis infected fish. I developed an in vitro plasma incubation test to predict host susceptibility of rainbow trout, carp, and sturgeon. The test showed the hierarchy of resistance of S. salmonis in sturgeon > carp > rainbow trout; this parallels epizootiological data. My research yielded new diagnostic tool, suggested new treatment target, improved in vitro conditions, and new model system for multi-functionality of flagella and flagellar signalling.

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.

As sequencing technologies advance genome studies are becoming a basic tool for studying an organism, and with more genomes available comparative genomics is maturing into a powerful tool for biological research. This thesis demonstrates the strength of a comparative genomics approach on a group of understudied eukaryotes, the diplomonads. Diplomonads are a group of single cell eukaryotic flagellates living in oxygen-poor environments. Most diplomonads are intestinal parasites, like the well-studied human parasite Giardia intestinalis. There are seven different G. intestinalis assemblages (genotypes) affecting different hosts, and it’s under debate whether these are one species. A genome-wide study of three G. intestinalis genomes from different assemblages reveals little inter-assemblage sexual recombination, supporting that the different G. intestinalis assemblages are genetically isolated and thus different species. A genomic comparison between the fish parasite S. salmonicida and G. intestinalis reveals genetic differences reflecting differences in their parasitic lifestyles. There is a tighter transcriptional regulation and a larger metabolic reservoir in S. salmonicida, likely adaptations to the fluctuating environments it encounters during its systemic infection compared to G. intestinalis which is a strict intestinal parasite. The S. salmonicida genome analysis also discovers genes involved in energy metabolism. Some of these are experimentally shown to localize to mitochondrion-related organelles in S. salmonicida, indicating that they possess energy-producing organelles that should be classified as hydrogenosomes, as opposed to the mitosomes in G. intestinalis. A transcriptome analysis of the free-living Trepomonas is compared with genomic data from the two parasitic diplomonads. The majority of the genes associated with a free-living lifestyle, like phagocytosis and a larger metabolic capacity, are of prokaryotic origin. This suggests that the ancestor of the free-living diplomonad was likely host-associated and that the free-living lifestyle is a secondary adaptation acquired through horizontal gene transfers.  In conclusion, this thesis uses different comparative genomics approaches to broaden the knowledge on diplomonad diversity and to provide more insight into how the lifestyle differences are reflected on the genetic level. The bioinformatics pipelines and expertise gained in these studies will be useful in other projects in diplomonads and other organismal groups.

This thesis describes the attempt to implement the CRISPR-Cas9 system in the two diplomonad pathogens: Giardia intestinalis and Spironucleus salmonicida. Two approaches of the implementation were done: first the Cas9 was expressed, followed by expression of the guide RNA. Additionally, to extend the understanding of how small nuclear RNAs are processed in Giardia intestinalis, two putative RNA processing enzymes were expressed and localized within the cells. The results show expression of Cas9 in both G. intestinalis and S .salmonicida. However, the Cas9 does not translocate to the nuclei, which is crucial for a functional gene knockout system. The approach where the guide RNA is expressed show a successful expression. In the experiments with the two putative RNA processing enzymes expression is observed in both cases, where one of the enzymes is localized to the nuclei.

For many years hexamitids, Hexamita spp. and Spironucleus spp., have frequently been reported in vertebrates, particularly in fish. This suggests a potentially important role of these parasites in the fish culture industry. Though the majority of hexamitids are not known to cause disease in their vertebrate host, those that have been documented as associated with disease are still in need of further investigation into their geographical distribution, host range, life cycle, host-parasite relationship, pathogenicity, diagnosis, prevention, treatment, and control. Spironucleus vortens is a hexamitid recently described from angelfish (Pterophyllum scalare). Although the structure of this parasite has been investigated using the electron microscope (Poynton et al., 1995), other information on this organism is poorly understood. Thus, the purpose of this research was to study the nature of S. vortens in TYI-S-33 culture medium and in the angelfish host. The optimal environmental conditions for S. vortens growth were investigated using variations of temperature, pH, and bile concentrations. This study is useful in helping to understand the locations and environmental conditions in the host that are suitable for the growth of S. vortens. Treatment of S. vortens, using seven chemotherapeutic agents; dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole, and magnesium sulfate was evaluated. The pathogenicity of S. vortens in angelfish was investigated in fish experimentally inoculated with trophozoites. This study provided information to help understand the pathogenesis of the parasites in the host. Finally, to examine the protective defense mechanisms, the presence of anti-S. vortens antibodies in angelfish serum were evaluated along with the presence of immune cells (lymphocytes, macrophages, eosinophilic granular cells, neutrophils, and plasma cells) at invaded sites of the intestine and other internal organs in response to an experimental Spironucleus vortens infection. The results of this research provide information on this parasite's effect on the fish host which may be useful in understanding the nature of other hexamitids. A few published reports have suggested the in vitro growth requirement of fish Spironucleus (Poynton et al., 1995; Sterud, 1998), but none have examined the optimal conditions required for growth and the pathogenicity of S. vortens. The first study was to examine the optimal requirements for the in vitro growth of the parasite. The organisms were cultivated in either an artificial medium (TYI-S-33) at different temperatures or various pH conditions, or in medium supplemented with different bile concentrations at 25°C. Criteria used to justify the optimal conditions were average cell number ml-1, growth rate, survival time, and cell conditions (motility and morphology). The organisms survived longest at 22°C, and had the highest average cell number ml-1 at 25°, 28° and 31°C. At 25°C the parasites were highly active and survived up to 6 days. The organisms cultivated at pH 6.5, 7.0 and 7.5 yielded the highest average cell number ml-1 with survival periods up to 13-14 days. Most of the organisms cultivated at a pH lower than 6.0 or a pH higher than 7.5 were suppressed and killed within 5-6 days of cultivation. All cultures supplemented with bovine or fish bile yielded lower maximal numbers of parasites than cultures with no bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25°C and pH 6.5 to 7.5 without supplementation with bile. In order to treat spironucleosis, the efficacy of various chemotherapeutic agents on the growth of S. vortens was examined in vitro. In this study nitroimidazoles and benzimidazoles, formerly reported as drugs of choice for the treatment of diplomonads, pyrimethamine and magnesium sulfate (Epsom salt) were evaluated at different concentrations on the growth of S. vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth at concentrations of 1 μg ml-1 or higher. Albendazole and fenbendazole suppressed the growth of parasites at concentrations of 1.0 μg ml-1 or higher after 24 h exposure. Mebendazole was the most effective agent of the benzimidazole group; and inhibited the parasite's growth at concentrations of 0.5 μg ml-1 or higher. Pyrimethamine at concentrations of 1-10 μg ml-1 failed to inhibit the parasite's growth. Magnesium sulfate inhibited the growth of the parasites only at high concentrations (70 mg ml-1 or higher) . This study indicates that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens. To investigate the pathogenesis of spironucleosis, angelfish were orally (PO) or intraperitoneally (IP) inoculated with S. vortens. Control angelfish which were orally gavaged or intraperitoneally injected with PBS were in normal body condition and had no morbidity or mortality. Compared to the control angelfish, PO-infected angelfish were inappetent with no other clinical signs, while IP-infected angelfish showed clinical signs of inappetite, weakness, respiratory distress, and laying on their sides. Twenty percent of the IP-infected angelfish died within the first three weeks after infection. In PO-infected angelfish, the organisms were located only in the intestinal lumen. In IP-infected angelfish, S. vortens were found in the blood, stomach, intestine, and other internal organs (spleen, gall bladder, and ovary). However, no parasites were observed within the intestinal mucosa of either PO- or IP-infected fish. Histopathologic examination of the intestines revealed mild to moderate multifocal enteritis in both PO- and IP-infected angelfish. The mucosal epithelium appeared undamaged although the parasite was closely located and appeared attached to the intestinal mucosa. The results suggest that S. vortens normally causes mild to moderate multifocal enteritis with no morbidity. However, the parasites can cause granulomatous inflammation in a wide variety of host tissues, and may be lethal if they enter the abdominal cavity and disseminate to other organs via the blood circulation. Immunity, both cell mediated and humoral, against S. vortens was investigated in this study. Histopathologic examination revealed a response from inflammatory cells infiltrated and localized in the affected tissues. Macrophages, lymphocytes, and plasma cells were the most common cell types found in the internal organs. Macrophages were active in the affected tissues where the parasites lived in situ. However, in vitro studies indicated that there were no differences in a production of H2O2 or in phagocytosis between macrophages of control and infected angelfish regardless of inoculum dosage and administration route. A preliminary study of humoral antibody indicated that angelfish did not develop anti-S. vortens antibody after they were orally or intraperitoneally infected with either a low or a high number of the organisms. It is suggested that localized leucocyte response may be an important mechanism against Spironucleus vortens infection in angelfish. This research has indicated some of the important environmental factors affecting the parasite's growth, and has provided some initial information on the pathogenicity of S. vortens. In addition, preliminary information on the host's protective immune systems, humoral and cell-mediated immunity, against the parasite have been documented. The results from this research will be useful for aquaculture, particularly of tropical freshwater angelfish, and may help to provide an understanding of the biological roles of other hexamitids.
Ph. D.

Resolving the phylogenetic tree of eukaryotes is an ongoing challenge for evolutionary biologists. One of the most intriguing questions is the phylogenetic status of Excavata, a group that is well supported by morphological evidence, yet usually not recovered as a clade in molecular phylogenies. The most problematic group of excavates are diplomonads (e.g., Giardia), which tend to have very highly divergent gene sequences, making any phylogenetic analyses that include these protists very susceptible to long branch attraction artifact. This thesis first explores which organisms are most closely related to diplomonads. Phylogenies of three marker genes demonstrate that enteromonads, formerly considered a possible sister group to diplomonads, are a polyphyletic group within diplomonads, suggesting complex evolution of cell morphology in this lineage. However, a large diversity of Carpediemonas-like organisms (CLOs) was discovered from marine/saline samples. Most of the major clades of CLOs had not been detected by previous environmental PCR studies. SSU rRNA gene phylogenies show that CLOs form a series of relatively short branches at the base of diplomonads. Phylogenomic analysis of eukaryotes (161 genes), incorporating EST data from 5 excavates, including 3 CLOs, shows that the non-monophyly of Excavata in phylogenomic studies is likely caused by long branch attraction artifact, since most of the methods used to suppress long branch attraction significantly weaken support for this topology. Furthermore, the shorter-branching CLOs represent valuable replacements for the long branching diplomonads; we recovered a robustly supported monophyletic Excavata, when long branches, including diplomonads (and parabasalids), were removed from the analysis. Subsequently, comparative analysis of the putative proteomes of three CLO isolates, the retortamonad Chilomastix, diplomonads and parabasalids was performed. Several putative evolutionary steps leading to the extremely reduced mitochondrial organelle of diplomonads were derived through the comparative analysis of predicted organellar proteomes. This thesis shows the importance of taxon sampling for inferring deep eukaryotic evolution. The more robust understanding of the phylogeny of Excavata, especially diplomonads and parabasalids, and the new availability of a number of deep branching relatives of diplomonads, provide a framework for comparative analyses exploring the evolution of anaerobic organelles or parasitism.