Protists are defined as eukaryotic organisms that are not animals, land plants, or fungi and make up the vast majority of eukaryotic diversity. Unfortunately, they are often overlooked and under sampled. Some rationales behind this are that they are often microscopic, most rarely the cause of human disease, and are often difficult to get into culture. Molecular phylogenetics has revolutionized our understanding of the evolutionary history of eukaryotes and without a clear picture of protist evolution our understanding would be severely hindered. Phylofisher is an easy-to-use phylogenomic software package, with an accompanying database and various utilities, intended for deep phylogenomic analyses. Here I present thorough documentation for the use of PhyloFisher. I also developed a PhyloFisher utility, nucl_matrix_constructor.py, to allow the software package to build nucleotide phylogenomic matrices. This will allow users to address more shallow evolutionary questions. I developed a metabarcoding pipeline based on Oxford Nanopore Technology’s long read sequencing platform to explore the diversity of eukaryotic organisms in environmental samples. I characterized a novel discosean amoeba. Lastly, I predicted the proteomes from publicly available, but non-annotated genomes for 19 Acanthamoeba spp. and two Balamuthia spp. and used the PhyloFisher tools documented and developed to gain a better understanding into the evolutionary history of the clade as well as provide key resources in the future research of the clade.

Entamoeba histolytica is an important human intestinal parasite that has a major impact on human health and is responsible for approximately 100,000 deaths each year. Entamoeba histolytica is one of several known eukaryotes that harbour strongly reduced mitochondria, called mitosomes, which have lost the vast majority of mitochondrial pathways as well as their organellar genome. While the occurrence and function of mitosomes have been well studied, little is known about their inheritance and division. Mitochondrial division in all studied eukaryotes relies on the participation of dynamin proteins for membrane scission. The central aim of this study was to characterize the dynamin protein family in Entamoeba histolytica and to analyze if they participate in mitosomal division. In relation to this work we studied the occurrence of mitosomes in the distantly related reptilian parasite Entamoeba invadens and revisited the phylogenetic relationships among mitosomal Hsp70, a protein we used for mitosomal localization experiments. Our studies revealed that Entamoeba histolytica contains two classical and two strongly derived members of the dynamin protein family, which we called Drp1, Drp2, Drp3 and Drp4. Drp1 and Drp2 exhibit the classical dynamin protein structure with a GTPase, middle and GTPase effector domain, while Drp3 and Drp4 only appear to contain the dynamin GTPase domain. Using phylogenetic reconstructions we could not identify closely, and thus functionally related, dynamins for Drp1 and Drp2 within the eukaryotic tree of life including the mitochondria‐associated amoebozoan dynamins DymA and DymB. The structurally derived dynamins however, were closely related to amoebozoan and archaeplastidan proteins involved in cytokinesis and chloroplast division. All Entamoeba dynamins are differentially expressed in trophozoites with EhDrp2 appearing to be most abundant and Drp3 expressed the least. We conducted stage conversion experiments using E. invadens to understand the importance of dynamins during cyst formation. During encystation all dynamin expression levels increased. Interestingly, Drp3 expression is strongly upregulated in the mid cyst stages and Drp4 during the late phase of encystation. Thus, Drp3 and Drp4 appear not to be involved in cytokinesis and possibly evolved a novel function in the cyst formation process. We carried out Drp2 enzymatic characterization and localization experiments as well as complementation studies using the related amoebozoan Dictyostelium discoideum in order to understand the role and function of E. histolytica Drp2 in the cell. We found that its kinetic characteristics are comparable to other members of the eukaryotic dynamin protein family by exhibiting low substrate specificity, the ability to oligomerize to higher structures and a substrate dependent cooperative enzyme activity. Drp2 localized to abundant punctate structures in the cytosol but did not colocalize with mitosomes. In addition, Drp2 was not able to complement D. discoideum DymA. Both findings suggest that Drp2 is not directly involved in mitosomal (or mitochondrial) division. We overexpressed Drp2 in E. histolytica and D. discoideum and found a significant effect on cytoskeletal organization. Both strains showed a strong impairment in amoeboid movement, cell‐surface attachment and cell growth. Additionally, the number of nuclei was increased significantly. Our data imply that Drp2 plays an important role for cytoskeletal organization. Additionally in this study, we show that mitosomes are also abundantly present in E. invadens suggesting that mitosomes are characteristic for all Entamoeba spp. Furthermore, we demonstrate that E. invadens cysts contain mitosomes in high abundance comparable to its vegetative life stage. Our studies verify that mitosomal Hsp70 is part of the amoebozoan protein family and of mitochondrial origin as shown by in silico characterization and localization experiments using the homologous Hsp70 antibody.

Mitochondria are eukaryotic organelles derived in evolution from within the ? subdivision of Proteobacteria. Although mitochondria are structurally and metabolically complex, modern-day mitochondrial genomes (mtDNA) encode only a small number of RNAs and proteins predominantly involved in adenosine triphosphate (ATP) formation through electron transport coupled to oxidative phosphorylation, as well as translation of mtDNA-encoded proteins. In humans, only 13 of the >1000 polypeptides that constitute the complete mitochondrial protein complement (proteome) are encoded in mtDNA; the remainder is encoded by nuclear DNA (nuDNA). It is therefore imperative to comprehensively catalog nuDNA-encoded mitochondrial proteins in order to understand holistically the evolution of mitochondria. Mitochondrial proteome investigations of animals, fungi and land plants have dramatically altered our conception of mitochondrial evolution: in contrast to mtDNA-encoded proteins, few nuDNA-encoded mitochondrial proteins are demonstrably derived from the eubacterial progenitor of mitochondria, and many are found only in eukaryotes. Notably, however, little is known about the mitochondria of eukaryotic microbes (protists), which constitute the bulk of biochemical and genetic diversity within the domain Eucarya. The proteomic characterization of protist mitochondria is therefore crucial to fully elucidating mitochondrial function and evolution. Employing tandem mass spectrometry (MS/MS), I have analyzed highly purified mitochondria from Acanthamoeba castellanii (Amoebozoa). In combination, nearly 750 nuDNA- and mtDNA-encoded proteins were identified. These data were used to catalog metabolic pathways and protein complexes, and to infer functional and evolutionary profiles of A. castellanii mitochondria. My analyses suggest that while A. castellanii mitochondria have many features in common with other eukaryotes, they possess several novel attributes and pronounced metabolic versatility. An analysis of the A. castellanii electron transport chain (ETC) was also performed, utilizing a combination of blue native polyacrylamide gel electrophoresis (BN-PAGE), MS/MS and bioinformatic queries. A significant proportion of A. castellanii ETC proteins was identified, yielding several insights into ETC evolution in eukaryotes. Lastly, I present two unusual cases of ‘split’ mitochondrial proteins: the iron-sulfur subunit SdhB of succinate:ubiquinone oxidoreductase (Complex II), in the phylum Euglenozoa and Cox1 of cytochrome c:O2 oxidoreductase (Complex IV) in various eukaryotes, including A. castellanii. Functional and evolutionary implications of these findings are discussed.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21157号 / 農博第2283号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5131(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 吉村 剛, 教授 髙野 俊幸, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Praha, 2011 1 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...

The aim of this research was to isolate and characterize novel strains of marine protists with potential to commercially produce PUFAs. Twelve trips were made visiting 10 different locations in the Canadian Maritime Provinces. Sixty-nine strains were isolated and screened for biomass and fatty acid production. Those meeting specific criteria were selected for further investigation including characterization by 18S rDNA sequencing. Isolate ONC-KTB-56 produced the greatest amount of biomass (1 807 mg L-1) and fatty acids (24.6% dry weight). Of the total fatty acids, ARA, EPA and DHA comprised 0.89, 1.22 and 4.7 percent, respectively. Isolate ONC-KTB-14 produced 1 704 mg L-1 dry biomass with 5.4 percent fatty acids including 1.44, 1.35 and 37.5 percent, ARA, EPA and DHA, respectively. Through optimization of culture conditions biomass, fatty acid content and the proportions of specific fatty acids can be increased. With such optimization, there is potential for isolates ONC-KTB-14 and ONC-KTB-56 to be grown at a commercial scale for production of PUFAs.

Most living eukaryotes are currently classified into one of five or six ‘supergroups’, which are in turn often divided between two assemblages: ‘unikonts’ and ‘bikonts’. This thesis explores the cytoskeletal morphology and phylogeny of three lineages that do not belong to any supergroup: ancyromonads, apusomonads, and breviates, likely relatives of supergroups Opisthokonta and Amoebozoa. It also investigates the phylogeny of malawimonads (basal members of supergroup Excavata) and collodictyonids (another unaffiliated lineage). Serial-section transmission electron microscopy was used to model the flagellar apparatus cytoskeletons of the ancyromonad Ancyromonas sigmoides, the breviate Breviata anathema, and the apusomonad Thecamonas trahens. Each has two main posterior microtubular roots and at least one anterior root (two in Ancyromonas). All three possess splitting posterior right microtubular roots and supernumerary singlets, features also characteristic of basal members of the supergroup Excavata (‘typical excavates’). One peripheral microtubule system in Ancyromonas, and the ‘right ribbon’ in Thecamonas, are likely homologous to dorsal fans in Breviata and ‘typical excavates’, and to the ‘r2’ root of myxogastrid Amoebozoa. One of the branches of the splitting root in Breviata and Thecamonas joins the right and intermediate roots, similarly to some myxogastrids. This implies that myxogastrids, and not the simpler pelobionts, represent the ancestral state for Amoebozoa. A phylogenomic analysis was performed focussing on apusomonads breviates, ancyromonads, and the problematic ‘typical excavate’ malawimonads, based on new transcriptomic data from Ancyromonas and an undescribed malawimonad. Rapid-site- removal analyses recover the ‘unikont’/‘bikont’ partition, and do not support the previously demonstrated affiliation between breviates and the ‘unikont’ supergroup Amoebozoa. Specifically, they group apusomonads with the ‘unikont’ supergroup Opisthokonta, and ancyromonads with breviates. Taxon-removal analyses group ancyromonads, breviates, and apusomonads together. Most analyses group malawimonads (perhaps with collodictyonids, another problematic group) between ‘unikonts’ and (other) ‘bikonts’, while other excavates are in a basal position amongst other ‘bikonts’. Combining these morphological and phylogenetic results suggests that splitting right roots, supernumerary intermediate singlets, and dorsal fans are found in multiple ‘basal’ lineages in both ‘unikont’ and ‘bikont’ portions of the eukaryotic tree, are likely characters of the last common ancestor of most or all living eukaryotes.

Les symbioses ont un role majeur dans le fonctionnement et l'equilibre des ecosystemes. Dans les oceans, qui couvrent pres de 70 % de la surface de la planete, vivent une multitude d'organismes incapables de lutter contre les courants et la plupart sont microscopiques, il s'agit du plancton. Les organismes du plancton, comme ceux d'autres ecosystemes, entretiennent des symbioses, mais la nature et l'ampleur de ces interactions sont encore mal connues dans le plancton du fait la petite taille de ces organismes et de la difficulte d'echantillonnage des ecosystemes planctoniques, surtout dans les zones les plus eloignees des cotes. Les principaux objectifs de cette these sont de donner un apercu global de la place occupee par ces symbioses dans le plancton et de proposer des methodes originales permettant leur detection. Les travaux presentes dans ce manuscrit s'appuient sur l'analyse des donnees generees lors de l'expedition Tara Oceans (2009-2013) pendant laquelle 210 stations oceaniques ont ete echantillonnees a travers le monde. Ils concernent plus precisement le jeu de donnees environnemental obtenu grace au sequencage a haut debit (Illumina) de la region hypervariable V9 (130 nucleotides) de la sousunite 18S de l'ADN ribosomique des organismes eucaryotes (metabarcoding). Dans un premier temps, un etat des lieux de la diversite et de la structure des communautes du pico-nano-micro-mesoplancton (0,8-2000 μm) eucaryote de la zone photique des oceans temperes a tropicaux est realise. Il met en evidence la place importante occupee par les symbiotes au sein de ces communautes. Ensuite, l'etude de deux cas de symbiose (Blastodinium- Copepodes et Symbiodinium-Tiarina) montre les difficultes inherentes a la detection de couples symbiotiques a partir d’un jeu de donnees issue d'etudes par metabarcoding du plancton (flexibilite de la specificite des symbioses dans le plancton), mais aussi la possibilite de distinguer les differentes phases de vie des symbiotes (libres et symbiotiques) lorsque les echantillons etudies ont ete fractionnes. Enfin, un ensemble de methodes est propose afin d'ameliorer l'efficacite de la detection de symbioses dans le cadre d'etudes par reseau de cooccurrences des communautes planctoniques. L'analyse de la distribution des metabarcodes le long des fractions de taille (piconano- (0.8-5 μm), nano- (5-20 μm), micro- (20-180 μm), et meso-plancton (180-2000 μm)) permet de differencier ceux provenant d'organismes symbiotiques de ceux d'organismes libres, sans a priori. De plus la comparaison de l'abondance de groupes genetiques definis a differents niveaux de resolution permet de detecter des associations symbiotiques peu specifiques et d'apprecier leur niveau de specificite. / The oceans, which cover nearly 70 % of the earth's surface, is host to a myriad of mostly microscopic organisms that drift with the currents and are collectively called plankton. As in other ecosystems, symbioses play a major role in the functioning and equilibrium of the plankton. But the exact nature and strength of those symbiotic interactions are still poorly known, not only due to the small size of most planktonic organisms, but also because of the inherent difficulty of sampling planktonic ecosystems, especially in the high-seas. The main goals of this thesis are to give a global view of the importance of planktonic symbioses and to propose novel methods for their detection. The work presented in this manuscript is based on analyses of data generated during the Tara Oceans expedition (2009-2013), during which sea water was collected and size fractionated by filtration at 210 sampling locations distributed across the world's oceans. The data analyses presented herein mostly focus on an environmental metabarcoding dataset obtained from next-generation sequencing (Illumina) of the V9 hypervariable region (~130 nucleotides long) of the 18S small ribosomal subunit of eukaryotic organisms. We begin by assessing the diversity and structure of pico-, nano-, micro and meso-planktonic eukaryotic communities (0.8-2000 μm) in the photic zone of tropical to temperate sea regions. Then, we present two cases of symbioses (Blastodinium-Copepods and Symbiodinium-Tiarina) to illustrate both the difficulties encountered when trying to detect symbiotic relationships using metabarcoding data due to varying specificities of symbiotic relationships, but also the potential solutions offered by size-fractionated sampling to distinguish between the different stages of the life cycle of symbiotic organisms (free living and symbiotic stages). Finally, we propose a set of methods to improve the detection of symbioses by studying the co-occurrence of organisms in planktonic communities: we use the distribution of metabarcodes along size fractions ((piconano- (0.8-5 μm), nano- (5-20 μm), micro- (20-180 μm), and meso-plancton (180-2000 μm)) to distinguish likely free living organisms from those that have a symbiotic life style, and we compare the abundance of genetic groups constructed by clustering metabarcodes at different resolution levels, which allows us to detect interactions occurring above the species level and to evaluate their level of specificity.

Unicellular, eukaryotic organisms - known as protists - form the base of all aquatic food webs. Frequently, marine protists are divided into either phytoplankton or (proto)zooplankton. Phytoplankton use phototrophy to acquire their energy from light to fix carbon dioxide into organic carbon, while protozooplankton use phagotrophy to directly acquire organic carbon from their prey. Mixoplankton that employ mixotrophy, i.e. the combination of phototrophy and phagotrophy within one cell, are often neglected. However, many marine protists are mixoplankton and they are ubiquitous in the worlds’ oceans. In oligotrophic oceans, mixoplankton are the base of food webs and many harmful algal blooms are formed by mixoplankton. Yet, the concept of mixoplankton is slow to mature within coastal water management. This thesis hypothesizes that the whole protist community, including mixoplankton, needs to be taken into account to understand and predict the effect of anthropogenic pressures on coastal systems. This thesis is a cumulative summary of three papers that employ data analysis, model developments and modelling scenarios to test this hypothesis. As a study area the Southern North Sea was chosen as it is an exceptionally well sampled coastal sea that is forecast to be heavily modified in the future. In a first step, routine monitoring data from the Southern North Sea were analyzed. The data analysis showed that the relative occurrence of mixoplankton was highest in seasonally stratified, clear, dissolved inorganic nutrient depleted environments. In a second step, a mathematical model, called PROTIST, was developed with the aim to reproduce the trophic composition of protist communities across abiotic gradients. Not only was PROTIST capable of reproducing the trophic composition of protist communities in the Southern North Sea, a sensitivity analysis conducted on the model results also showed that the occurrence of mixoplankton in the Southern North Sea is driven mainly by the availability of dissolved inorganic phosphate and silica and not by the availability of light. In a third step, PROTIST was used in a 3D model scenario of the North Sea to research whether the planned intensification of seaweed aquaculture affects the composition of protist communities. Preliminary 3Dmodel results show that seaweed aquaculture in the Southern North Sea could decrease nutrient concentrations in winter and lead to an increase in mixoplankton biomass. Pooling the information gained from the different approaches, this thesis concludes that coastal zone management should take mixoplankton into account to understand and predict the effect of future anthropogenic pressures on coastalecosystems. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished