School of Doctoral Studies in Biological Sciences UNIVERSITY OF SOUTH BOHEMIA, FACULTY OF SCIENCE Insight into insect trypanosomatid biology via whole genome sequencing Ph.D. Thesis RNDr. Tomáš Skalický Supervisor: Prof. RNDr. Julius Lukeš, CSc. Biology Centre CAS, Institute of Parasitology České Budějovice 2017 This thesis should be cited as: Skalický T (2017) Insight to insect trypanosomatid diversity via whole genome sequencing. Ph. D. Thesis. Uniccersity of South Bohemia, Faculty of Science, School of Doctoral Studies in Biological Science, České Budějovice, Czech Republic, 94 pp. Annotation This thesis is composed of two topics both concerning diverse and obligatory trypanosomatid parasites. First part deals with identification of new Trypanosoma species identified in blood meal of tsetse flies caught in Dzanga-Sangha Protected Areas, Central African Republic, and identification of feeding preferences of tsetse flies. The second part concerns extraordinary monoxenous trypanosomatid Paratrypanosoma confusum which constitutes the most basal branch between free-living Bodo saltans and parasitic trypanosomatids. This thesis helped to elucidate morphology and biology of this deep branching trypanosomatid. Using genome and transcriptome sequencing and comparative bioinformatics approaches enabled search for ancestral genes shared with free-living bodonids and confirmed genome streamlining in trypanosomatids. Declaration [in Czech] Prohlašuji, že svoji disertační práci jsem vypracoval samostatně pouze s použitím pramenů a literatury uvedených v seznamu citované literatury. Prohlašuji, že v souladu s § 47b zákona č. 111/1998 Sb. v platném znění souhlasím se zveřejněním své disertační práce, a to v nezkrácené podobě elektronickou cestou ve veřejně přístupné části databáze STAG provozované Jihočeskou univerzitou v Českých Budějovicích na jejích internetových stránkách, a to se zachováním mého autorského práva k odevzdanému textu této kvalifikační práce. Souhlasím dále s tím, aby toutéž elektronickou cestou byly v souladu s uvedeným ustanovením zákona č. 111/1998 Sb. zveřejněny posudky školitele a oponentů práce i záznam o průběhu a výsledku obhajoby kvalifikační práce. Rovněž souhlasím s porovnáním textu mé kvalifikační práce s databází kvalifikačních prací Theses.cz provozovanou Národním registrem vysokoškolských kvalifikačních prací a systémem na odhalování plagiátů. České Budějovice, 06.07.2017 ............................................... Tomáš Skalický This thesis originated from a partnership of Faculty of Science, University of South Bohemia, and Institute of Parasitology, Biology Centre of the CAS, supporting doctoral studies in the Molecular and Cell Biology and Genetics study programme. Financial support This work was supported by: ERC CZ, grant LL1205 The Czech Science Foundation, grant 14-23986S, The Praemium Academiae 2009 award to Julius Lukeš The Grant Agency of the University of South Bohemia, grant 108/2013/P Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisor Julius Lukeš for giving me the opportunity to join his team, his patient guidance, motivation and continuous support of my research during all the years. His enthusiasm and passion for science was always contagious and brought me where I stand now. I would also like to thank all the people from the institutes that I had the opportunity to work with during the years, especially those from Jula’s, Alča’s and Parda’s lab, both former and present. They have created a pleasant and stimulating working environment throughout my PhD studies. I would also like to thank David Wildridge and Carmen Priscila Pena Diaz for proofreading. In addition, my thanks go to all my friends, whom I had the chance to get to know over the years spent in South Bohemia. Thanks to all of them, my studies on Faculty of Sciences were both happy and pleasurable. Finally, my special thanks go to my beloved Zuzka and my family for their never-ending support, help and constant faith in me. List of papers and author’s contribution The thesis is based on the following papers (listed chronologically): I. Flegontov P*, Votýpka J*, Skalický T*, Logacheva MD, Penin AA, Tanifuji G, Onodera NT, Kondrashov AS, Volf P, Archibald JM, Lukeš J (2013) Paratrypanosoma is a novel early-branching trypanosomatid. Current Biology 23(18):1787-93 (IF = 9.733). Tomáš Skalický participated in study design, experimental data generation, data analysis and interpretation and draft editing of the manuscript. II. Lukeš J, Skalický T, Týč J, Votýpka J, Yurchenko V (2014) Evolution of parasitism in kinetoplastid flagellates. Molecular and Biochemical Parasitology 195(2):115- 122 (IF = 2.068). Tomáš Skalický performed analysis and interpretation of the data, prepared figures and contributed to writing of the manuscript. III. Votýpka J*, Rádrová J*, Skalický T*, Jirků M, Jirsová D, Mihalca AD, D'Amico G, Petrželková KJ, Modrý D, Lukeš J (2015) A tsetse and tabanid fly survey of African great apes habitats reveals the presence of a novel trypanosome lineage but the absence of Trypanosoma brucei. International Journal of Parasitology 45(12):741-8 (IF = 4.242). Tomáš Skalický participated in experimental procedures, designed, prepared and sequenced amplicon DNA library, analyzed the results, performed recombinant detection and contributed to writing of the manuscript. IV. Skalický T*, Dobáková E*, Wheeler R*, Tesařová M, Flegontov P, Jirsová D, Votýpka J, Yurchenko V, Ayala FJ, Lukeš J (submitted manuscript) Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early- branching trypanosomatid. Tomáš Skalický participated in study design, experimental data generation, genome assembly and annotation, data analysis and interpretation and contributed to writing of the manuscript. * These authors contributed equally Co-author agreement Julius Lukeš, the supervisor of this Ph.D. thesis and co-author of all presented papers, fully acknowledges the contribution of Tomáš Skalický. …………………………………………. Prof. RNDr. Julius Lukeš, CSc. Contents 1 Introduction.................................................................................................................... 1 1.1 Kinetoplastea .......................................................................................................... 1 1.2 Trypanosomatida .................................................................................................... 2 1.2.1 Trypanosomatid systematics and phylogeny .................................................. 3 1.2.2 Trypanosomatid diversity ................................................................................ 5 1.2.3 Evolution of parasitism in trypanosomatids.................................................. 10 1.2.4 Trypanosomatid nuclear genomes ................................................................ 13 1.2.5 RNA interference ........................................................................................... 16 2 Research objectives ...................................................................................................... 19 3 Summary of results and discussion .............................................................................. 19 3.1 Survey of new trypanosomes transmitted by tsetse fly ....................................... 20 3.2 Paratrypanosoma confusum, a unique trypanosomatid ...................................... 21 4 REFERENCES ................................................................................................................. 27 5 Original publications .................................................................................................... 36 5.1 PAPER I .................................................................................................................... 36 5.2 PAPER II ................................................................................................................... 55 5.3 PAPER III .................................................................................................................. 64 5.4 MANUSCRIPT I ........................................................................................................... 73 6 Curriculum vitae ........................................................................................................... 89 1 Introduction 1.1 Kinetoplastea Kinetoplastids represent one of the putatively most ancestral eukaryotic lineages (Cavalier-Smith, 2010). This widespread and extremely speciose group of eukaryotic protists, sometimes called microeukaryotes, belongs to the Euglenozoa, supergroup Excavata (Adl et al., 2012). Together with three sister groups Euglenida, Symbiontida and Diplonemea, Kinetoplastid flagellates are characterized by specific features including polycistronic transcription and trans-splicing of nuclear genes or a large mitochondrion with discoidal cristae containing a prominent mitochondrial DNA termed kinetoplast, which gave the group its name. Phylogenetic analyses support division of the class Kinetoplastea into subclasses Prokinetoplastina and Metakinetoplastina (Adl et al., 2012; d'Avila-Levy et al., 2015), with the former clade containing only two known representatives: Ichthyobodo – an ectoparasite of fish with two flagella and Perkinsela – an aflagellar endosymbiont of amoebozoans of the genus Paramoeba parasitizing on gills of fish (Dykova et al., 2008; Young et al., 2014). Perkinsela, which according to the Giemsa staining has more DNA in its mitochondrion than in its nucleus, is