48 th jírovec's protozoological days sbornik jpd48.pdf · orewford 27 28 dear...

48th Jírovec's1

Protozoological Days2

Conference Proceedings3

Department of Biology and Ecology4

University of Ostrava, Faculty of Science5

Ostrava 20186

48th Jírovec's7

Protozoological Days8

Conference Proceedings9

Department of Biology and Ecology10

University of Ostrava, Faculty of Science11

Ostrava 201812

48thJírovec's Protozoological Days13

Conference Proceeding14

This publication did not undergone any language (nor misspelling) editing.15

16

c○University of Ostrava, Faculty of Science, Department of Biology and Ecology, 201817

ISBN 978-80-7599-000-618

Content19

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920

Program Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1321

List of Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2122

Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2723

List of Participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9724

Partners of Conference . . . . . . . . . . . . . . . . . . . . . . . . . . . 10425

5

Foreword26

7

Foreword

27

Dear protistologists,28

29

with a great pleasure I welcome you to the 48th Jírovec's Protozoological30

Days, the annual conference organized under the aegis of the Czech Society31

for Parasitology! From year to year, the conference becomes more and more32

international due to the growing number of participants from different countries.33

This has been recognized by the International Society for Protistology, which34

supported our conference this year and therefore enabled inviting five prominent35

scientists from abroad. Those are: Dr. Mark Carrington from the University36

of Cambridge, Dr. Javier del Campo from the Institute of Marine Sciences in37

Barcelona, Dr. Matthias Fisher from Max Planck Institute for Medical Research,38

Dr. David Bass from the Natural History Museum in London and Dr. Luděk39

Kořený from the University of Cambridge. The meeting will be especially useful40

to younger participants, which will have an opportunity to get acquainted with41

advances in various fields of protistology and present their own research to the42

protistological community. I am sure that the appeasing conference venue in43

Kunčice pod Ondřejníkem will ensure friendly and fruitful communication.44

45

Have a pleasant time!46

47

Alexei Kostygov48

9

Program Schedule49

11

Program Schedule

Monday April 30, 2018

15:00 Registration

18:00 Dinner

Tuesday May 1, 2018

7:50 Breakfast

8:50 Conference Opening

Biodiversity

9:00:::::David

::::Bass: (New) Ways of Finding (New) Parasites

9:40Pawe l Ha lakuc, Anna Karnkowska, & Rafa l Milanowski: TracingEvolutionary Changes in rRNA Genes in Euglenozoa

10:00Pavla Hanousková, & Ivan Čepička: Unexpected Diversity of thePeculiar Genus Creneis (Excavata: Heterolobosea)

10:20 Coffee break

10:40Andrzej Kaczanowski: Breeding Strategies and Genome Inte-gration in Tetrahymena

11:00Michael Kotyk, Zuzana Kotyková Varadínová, Pavla Hanousková& Ivan Čepička: Diversity and Host Specificity of ParabasalianSymbionts of Non-Termite Cockroaches

11:20

Tomáš Pánek, Kristýna Záhonová, Naoji Yubuki, Eliška Zadro-bílková, Sebastian Cristian Treitli, Vyacheslav Yurchenko, IvanČepička, & Marek Eliáš: Novel Lineage of Non-PhotosyntheticChlamydomonadales with Peculiar Plastid Genome

11:40

Johana Rotterová, Roxanne Beinart, William Bourland, Petr Tá-borský, Virginia P. Edgcomb, Martin Kolísko, & Ivan Čepička: TheFirst Phylogenomic Analysis of Free-living Anaerobic Ciliateswithin SAL Super-group (Ciliophora)

12:00

Daria Tashyreva, Galina Prokopchuk, Jan Votýpka, Akinori Ya-buki, Aleš Horák, Binnypreet Kaur, Drahomíra Faktorová, & Ju-lius Lukeš: Life Cycle, Ultrastructure and Phylogeny of NewDiplonemids

12:20 Lunch

13

48th Jírovec's Protozoological Days

Molecular Biology

13:20Janaina Nasciemento de Freitas, Steve Kelly, Jack Sunter, &

:::::Mark

::::::::::Carrington: Codon Use is a Major Determinant of mRNA

Levels in Trypanosomes

14:00Diego Henrique Fagundes Macedo, Danyil Grybchuk, AlexeiKostygov, & Vyacheslav Yurchenko: RNA viruses of Blechomo-nadinae

14:20Tereza Faitová, Zoltán Füssy, & Miroslav Oborník: In silico Cha-racterization of The Plastid Proteomes of Chromera velia andVitrella brassicaformis

14:40Ondřej Gahura, Martin G. Montgomery, John E. Walker, & AlenaZíková: The F1-ATPase from Trypanosoma brucei is Elaboratedby Three Copies of an Additional p18-subunit

15:00Ansgar Gruber: Nucleotide Biosynthesis and Transport in Dia-toms

15:20 Coffee break

15:40

Iosif Kaurov, Marie Vancova, Lawrence Rudy Cadena, Jiří Heller,Tomáš Bily, David Potěšil, Zbyněk Zdrahal, Julius Lukeš, &Hassan Hashimi: The Role of Kinetoplastid MICOS Complex inCristae Shaping and Intermembrane Space Import

16:00

Sneha Kulkarni, Helmut Stanzl, Alan Kessler, Eva Heged"usová,Juan D Alfonzo, & Zdeněk Paris: Queuosine: The Role of an Es-sential tRNAModification in Parasitic Protist Trypanosoma bru-cei

16:20Anna M. G. Novák Vanclová, & Vladimír Hampl: Investigating theMolecules, Sequences and Mechanisms Involved in the CrypticPlastid Protein Import of Euglena gracilis

16:40

Julius Lukeš, Drahomíra Faktorová, Olga Flegontova, Aleš Ho-rák, Binnypreet Kaur, Galina Prokopchuk, Ingrid Škodová-Svera-ková, Daria Tashyreva, Kristína Záhonová: Where are we withDiplonemids and where do we want to go?

17:00 Poster Session

18:00 Dinner

19:00 Demonstration of Protists

20:00 CSP meeting

14

Program Schedule

Wednesday May 2, 2018

8:00 Breakfast

Molecular Biology

9:00::::::::Matthias

::G.

::::::Fischer, & Thomas Hackl: Endogenous Virophages in

Marine Heterotrophic Flagellates: Smoking Gun of an AdaptiveDefense System against Giant Viruses?

9:40Marie Jalovecka, David Hartmann, Yukiko Miyamoto, LarsEckmann, Ondrej Hajdusek, Anthony J. O'Donoghue, & DanielSojka: Validation of Babesia Proteasome as a Drug Target

10:00Lucie Podešvová, Natalya Kraeva, & Vyacheslav Yurchenko: Bi-cistronic Protein Expression in Leishmania mexicana

10:20Petr Soukal, Štěpánka Hrdá, Anna Vanclová, Naoji Yubuki, Ma-rek Eliáš, & Vladimír Hampl: Gene Transfer Accompanying theSecondary Endosymbiosis of Euglenid Plastid

10:40 Coffee break

11:00Ingrid Sveráková, Martina Džubanová, Anton Horváth, & JúliusLukeš: Diplonema papillatum – The Master of Adaptation

11:20Vojtěch Vacek, Lukáš V. F. Novák, Sebastian Treitli, Ivan Če-pička, Martin Kolisko, Patrick J. Keeling, & Vladimír Hampl: Fe–SCluster Assembly in Oxymonads and Related Protists

11:40

Kristína Záhonová, Zoltán Füssy, Erik Birčák, Vladimír Klimeš,Matej Vesteg, Juraj Krajčovič, Miroslav Oborník, & Marek Eliáš:Colourless but not Invisible: Stories about the Non-Photosyn-thetic Plastid of Euglena longa

12:00Hana Váchová, Miroslava Šedinová, Glenda Alquicer, & VladimírVarga: Studying the Flagellar Tip of Trypanosoma brucei

12:20 Lunch

13:20 Group Photo

13:50 Trip to Rožnov

19:00 Dinner/Banquet

15


Thursday May 3, 2018

8:00 Breakfast

9:00 :::::Javier

:::del

::::::Campo: The Eukaryotic Microbiomes of Benthic and

Planktonic Marine Animals

Genomics

9:40

Anzhelika Butenko, Olga Flegontova, Aleš Horák, VladimírHampl, Patrick Keeling, Ryan Gawryluk, Denis Tikhonenkov, Pa-vel Flegontov, & Julius Lukeš: Comparative TranscriptomicAnalysis of Euglenozoa: Insights into the Evolution of Meta-bolic Capabilities and Molecular Features

10:00Micha l Karlicki, & Anna Karnkowska: Searching for the PlastidGenomes in the Metagenomic Data

10:20Kacper Maciszewski, & Anna Karnkowska: Overview of the FirstTwo Chloroplast Genomes of Dictyochophyceae (Ochrophyta)

10:40 Coffee break

11:00Anna Nenarokova, Kristína Záhonová, Serafim Nenarokov, Vya-cheslav Yurchenko, & Julius Lukeš: Genomics of Blastocrithidia,the Trypanosomatid with All Three Stop Codons Reassigned

11:20S. Nenarokov, F. Burki, D. J. Richter, M. Kolisko, & P. J. Ke-eling: Detection and Removal of Cross–Contaminations fromTranscriptome Sequencing Projects

11:40Lukáš V. F. Novák, Sebastian C. Treitli, Anna Karnkowska, &Vladimír Hampl: Metabolism and Cell Biology of PreaxostylaFlagellates: A Comparative Genomic Study

12:00

Romana Petrželková, & Marek Eliáš: A Phylogenetically BroadAnalysis of Protist Genomes Unveils the Ancestral Eukaryo-tic Complexity of the Ras Superfamily of GTPases and NovelAspects of Eukaryotic Cell Biology

12:20 Lunch

16

Program Schedule

13:20Abdoallah Sharaf, Kateřina Jiroutová, & Miroslav Oborník: TheEvolution of Aminoacyl-tRNA Synthetases in Chromerids

13:40

Tatiana Yurchenko, Tereza Ševčíková, Pavel Přibyl, Khalid ElKarkouri, Vladimír Klimeš, Raquel Amaral, Eunsoo Kim, & MarekEliáš: A Gene Transfer Event Suggests a Long-Term Partner-ship between Eustigmatophyte Algae and a Novel Lineage ofEndosymbiotic Bacteria

14:00

David Žihala, Martin Kolísko, Serafim Nenarokov, Eleni Gente-kaki, Denis Lynn, Feng Gao, Tomáš Pánek, & Marek Eliáš: How isour Python Code Helping us to Understand Evolution of theGenetic Code

14:20 Coffee break

Cell Biology

14:40:::::Luděk

:::::::Kořený, Konstantin Barylyuk, Kathryn Lilley, & Ross

Waller: High-Throughput Discovery of Novel Conoid-Aassocia-ted Proteins in Toxoplasma gondii

15:20Alena Dohnálková, Tamara Smutná, Róbert Šuťák, & Ivan Hrdý:Cytosolic Hydrogenase in Trichomonas vaginalis Does Exist

15:40

Jennifer M. Holden, Ludek Koreny, Samson Obado, Alexander V.Ratushny, Wei-Ming Chen, Brian T. Chait, John D. Aitchison, Mi-chael P. Rout, & Mark C. Field: A Moonlighting Nuclear PoreGene Controls Gene Expression in African Trypanosomes

16:00Kateřina Kabeláčová, Aleš Tomčala, & Miroslav Oborník: TheFitness of Three Strains of the Alga Chromera velia

16:20 Coffee break

16:40Martina Kornalíková, Sebastian Treitli, & Vladimír Hampl: Ana-lyses of Ploidy and Karyotype of Oxymonads Using FISH

17:00

Jitka Štáfková, Petr Rada, Dionigia Meloni, Vojtěch Žárský, Ta-mara Smutná, Nadine Zimmann, Karel Harant, Petr Pompach, IvanHrdý, & Jan Tachezy: Dynamic Secretome of Trichomonas vagi-nalis: Case Study of β-amylases

17:20Luboš Voleman, Pavla Tůmová, & Pavel Doležal: Dynamics of Gi-ardia intestinalis Mitosomes

17:40 Concluding Remarks

18:00 Dinner

17


Friday May 4, 2018

8:00 Breakfast

9:00 Departure of Participants

50

Speakers' names are underlined. Invited speakers' names are wavy underlined.51

18

Poster Session52

19

Poster Session

Claretta Bianchi, Natalia Kraeva, Alexei Kostygov, Kristína Záho-nová, Lukeš Julius, & Vyacheslav Yurchenko: Catalase in Blastocrithi-

dia spp.

Katarína Bilková, Ľuboš Hudák, Ingrid Sveráková, & Anton Horváth:Termotolerant Trypanosomatides Crithidia thermophila and Leptomo-

nas seymouri

William Bourland, Johana Rotterová, & Ivan Čepička: Morphologicand Molecular Characterization of Brachonella pulchra (Kahl, 1927)comb. nov. (Armophorea, Ciliophora) with Comments on CystStructure and Formation

Ondřej Brzoň, & Vladimír Hampl: Oxymonads in the Gut of Reticuli-termes flavipes

Lawrence Rudy Cadena, Iosif Kaurov, Julius Lukeš, & Hassan Hashimi:Dropping the Mic: Knockdown of MICOS Subunits Yields IntriguingPhenomenon in Trypanosoma brucei

Arzuv Charyyeva, & Vyacheslav Yurchenko: Leishmania Genome as aModel of Gene Conversion

Eva Doleželová, Kunzová M, Panicucci B, & Alena Zíková: Mitochon-drial Metabolic Remodeling During Trypanosoma brucei Develop-mental Differentiation

Michaela Horčičková, Nikola Holubová, Dana Květoňová, Lenka Hlás-ková, John McEvoy, Dušan Rajský, Bohumil Sak, & Martin Kváč: Cryp-tosporidium spp. in Wild Coypu (Myocastor coypus)

Flávia M. Silva, Alexei Kostygov, Viktoria V. Spodareva, AnzhelikaButenko, Regis Tossou, Julius Lukeš, Vyacheslav Yurchenko, & Jo~aoM.P. Alves: A Surprise from the Bacterial Eendosymbiont of Kento-monas sorsogonicus: Loss of the Heme Pathway

Tomáš Kovalinka, Bianka Kováčová, & Anton Horváth: MitochondrialProteases of T. brucei

21


Jana Králová, Jan Votýpka, Julius Lukeš, Alexei Yu Kostygov, Vikto-ria Spodareva, & Vyacheslav Yurchenko: Diversity of Trypanosoma-tids from the Philippines

Alžběta Krupičková, Courtney Stairs, Vladimíra Najdrová, & PavelDoležal: The News about ISC System in the Mitosomes of Giardiaintestinalis

Martin Kváč, Nikola Holubová, Lenka Hlásková, & Bohumil Sak:Susceptibility of Chicken Embryos to Cryptosporidium spp. Infection

Tien Le, Vojtěch Žárský, Eva Nývltová, Eliška Kočířová, Zdeněk Ver-ner, & Jan Tachezy: Anaerobic Peroxisomes in Mastigamoeba balamu-

thi

Martina Lisnerová, & Ivan Fiala: Myxozoa Wherever You Look: Un-covering Myxozoan Species Diversity

Markéta Petrů, Alžběta Krupičková, & Pavel Doležal: Nosema bom-

bycis (Microsporidia), a Model for the Biological Nanotube

Kateřina Poláková, Johana Rotterová, & Ivan Čepička: The Diver-sity of Anaerobic Ciliates (Scuticociliatia, Oligohymenophorea) andTheir Ecologically Important Symbiotic Prokaryotes

Lenka Raabová, & Ľubomir Kováčik: Representants of the Green AlgalGenus Pseudodictyochloris in Arctic – it is possible?

Vendula Rašková, Jan Pyrih, & Julius Lukeš: A Novel Bacterial CellDivision Protein ZapE and its Role in the Mitochondrion of Trypa-nosoma brucei

Viktoria Spodareva, Alexei Kostygov, Hana Pecková, Astrid Holzer,Julius Lukeš, & Vyacheslav Yurchenko: Trypanosomes of FreshwaterFish: Diversity and Specificity

22

Poster Session

Ivana Schneedorferová, Aleš Tomčala, Iva Opekarová, Jaromír Cihlář,& Miroslav Oborník: Tracking Ingest Glycine via Labelled Isotopeand Metabolomics to Show Mixotrophy in Chromera velia, an Api-complexan Cousin

Dominika Vešelényiová, Erik Birčák, & Juraj Krajčovič: Calpains inthe Phylum Euglenozoa

Halszka Wysocka-Korzun, Magdalena P lecha, Anna Karnkowska,Ryan Gawryluk, Patrick J. Keeling, & Rafa l Milanowski: Characteris-tics of Nonconventional Introns in Genomes of Marine Diplonemids

Natalia Wandyszewska, & Pavel Doležal: Are Mitosomes Truly Es-sential?

Anastasiia Grybchuk-Ieremenko, Jan Votýpka, Julius Lukeš, PetrKment, Alexei Yu. Kostygov, & Vyacheslav Yurchenko: Insect Trypa-nosomatids in Papua New Guinea: High Endemism and New Cladeson the Tree

53

The names of the presenters are underlined.54

23

Abstracts55

25

Abstracts

(New) Ways of Finding (New) Parasites56

David Bass57

Centre for Environment, Fisheries, and Aquaculture Research (Cefas), Barrack Road, The Nothe,58

Weymouth, Dorset DT4 8UB, UK59

Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK60

61

The combination of modern molecular biology and sequencing techniques, molecu-62

lar phylogenetics and high resolution molecular taxonomy, and a range of microscopical63

methods offers a powerful and flexible toolbox for discovering and characterizing para-64

sites of animals, plants, and other organisms. These approaches can also be adapted to65

gain insight into parasite lifecycles, ecology, and assessing disease risk. As high throu-66

ghput sequencing studies are demonstrating an ever increasing diversity of microbes67

(eukaryotes, bacteria, viruses) in environmental and other samples, our appreciation of68

the great richness of parasites, pathogens, and symbionts also increases, raising many69

new questions and suggesting new areas of research. One example of this is the current70

interest in the concept of the pathobiome, a departure from the `one–pathogen–one–71

disease' paradigm, which is acknowledged as limiting our understanding of pathogen72

diversity and interaction among themselves and with their hosts. I will present an over-73

view of these new synergistic approaches, explain their strengths and shortcomings, and74

provide a range of current and recent case studies of new protistan parasites/symbionts,75

with particular focus on ascetosporeans (Rhizaria) and Filasterea, and pathobiomes of76

animals farmed for food around the world.77

78

27


Catalase in Blastocrithidia spp.79

Claretta Bianchi1, Natalia Kraeva1, Alexei Kostygov1, 2, Kristína Záhonová3, Lukeš80

Julius3, 4, & Vyacheslav Yurchenko1, 381

1University of Ostrava, Faculty of Science, Life Science Research Centre, Ostrava82

2Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia83

3Biology Centre ASCR, v. v. i., Institute of Parasitology, České Budějovice84

4University of South Bohemia, Faculty of Science, České Budějovice85

86

Catalase is an enzyme able to convert hydrogen peroxide to water and molecu-87

lar oxygen. It plays an important role in oxidative stress protection in virtually all88

extant species. The hydrogen peroxide is not highly reactive by itself, but can be very89

dangerous for the cells when reacting with iron producing hydroxyl radicals. Catalase90

can be considered one of most widely distributed enzymes on Earth, but some orga-91

nisms do not have it. Its evolutionary history is rather complicated and the topology92

of catalase–based trees suggests an unusually high number of horizontal gene transfer93

(HGT) events, especially among bacteria (Faguy and Doolittle, 2000). In the case of94

Leishmaniinae, they received this enzyme by HGT from Brachyspirales (Kraeva et al.,95

2017). Previously, no catalase gene was identified in any species outside of the subfa-96

mily Leishmaniinae, suggesting that its acquisition was a relatively recent evolutionary97

event. Moreover, within this clade, the catalase was restricted to monoxenous genera98

and was apparently secondarily lost from the dixenous Leishmania spp. (Kraeva et al.,99

2017). Blastocrithidia is a genus of Trypanosomatidae characterized by its flabbergas-100

ted genetic code in which all three stop codons do encode amino acids (Záhonová et al,101

2016). Here we report that Blastocrithidia spp. possess catalase. Insects are generally102

resistant to microorganisms (Sant'Anna et al., 2012) and their immune system pro-103

duces Reactive Oxygen Species in response to pathogens. Blastocrithidia parasitizes104

the insect's midgut and we propose that catalase may be essential for survival in this105

environment.106

References:107

Faguy, D.M., Doolittle, W.F., 2000. Horizontal transfer of catalase–peroxidase genes108

between archaea and pathogenic bacteria. Trends Genetic. 16, 196–197.109

Kraeva, N., Horáková, E., Kostygov, A.Y., Kořený, L., Butenko, A., Yurchenko, V.,110

Lukeš, J., 2016. Catalase in Leishmaniinae: with me or against me? Infect. Genet. Evol.111

50, 121–127.112

Sant'Anna, M.R., Darby, A.C., Brazil, R.P., Montoya-Lerma, J., Dillon, V.M., Bates,113

P.A., Dillon, R.J., 2012. Investigation of the bacterial communities associated with114

females of Lutzomyia sand fly species from South America. PLoS One 7, e42531.115

Záhonová, K., Kostygov, A.Y., Ševčíková, T., Yurchenko, V., Eliáš M., 2016. An unprece-116

dented non–canonical nuclear genetic code with all three termination codons reassigned117

as sense codons. Curr. Biol. 26, 2364–2369.118

119

28

Abstracts

Termotolerant Trypanosomatids Crithidia thermophila and120

Leptomonas seymouri121

Katarína Bilková, Ľuboš Hudák, Ingrid Sveráková, & Anton Horváth122

Comenius University, Department of Biochemistry, Faculty of Natural Sciences, Bratislava123

124

Trypanosomatids are obligatory parasites belonging to the class Kinetoplastida125

(Phylum Euglenozoa). According to their lifecycle, they can be classified as dixenic126

(alternate two hosts) or monoxenic (one host). Monoxenic species Crithidia thermo-127

phila and Leptomonas seymouri are capable to grow at 34 ∘C. Therefore they could128

become a good model for studying adaptation to different temperature environments,129

which is a precondition for parasitism in warm-blooded organisms. We have shown130

that C. thermophila and L. seymouri can grow equally well at higher (34 ∘C) and131

lower temperatures (17 ∘C). For both organisms, we studied the impact of the culti-132

vation temperature on selected parameters. Using spectrophotometric measurements133

and detecting activities in native gels, we monitored the activities of oxidative phospho-134

rylation enzyme complexes. We also analyzed the phospholipids, triacylglycerols, and135

fatty acids by different thin-layer chromatography.136

Acknowledgment:137

This work was created with financial support of grant agencies VEGA a APVV within projects138

APVV-0286-12 a VEGA1/0387/17.139

140

29


Morphologic and Molecular Characterization of Bracho-141

nella pulchra (Kahl, 1927) comb. nov. (Armophorea, Cili-142

ophora) with Comments on Cyst Structure and Formation143

William Bourland1, Johana Rotterová2, & Ivan Čepička2144

1Boise State University, Department of Biological Sciences, Boise, Idaho, USA145

2Charles University, Faculty of Science, Department of Zoology, Praha146

147

Despite the description of Metopus es by Müller (1776) nearly 250 years ago,148

efforts to characterize and establish the phylogeny of the free-living Metopida by in-149

tegrating modern morphologic and molecular methods has only recently been initi-150

ated. Previous descriptions and redescriptions of metopid taxa either predated the151

widespread application of PCR and DNA sequencing or focused on only a morphologic152

or, a mainly molecular approach. The non-monophyly of the most species-rich metopid153

genus,Metopus, was suspected early on by Corliss and is now recognized on the basis of154

recent molecular phylogenies. Many taxa from the once species-rich genus Brachonella155

Jankowski, 1964 have been transferred to other genera or synonymized, leaving seven156

nominal species. Combined morphologic and molecular data is available only for the157

type species, Brachonella contorta (Levander, 1894) Jankowski, 1964. In this report158

we provide morphologic and morphometric data and an 18S rRNA gene sequence for159

another member of this genus, Brachonella pulchra (Kahl, 1927) comb. nov. Little is160

known about the cyst life stage and its possible taxonomic significance for metopids in161

particular or armophorean ciliates in general. Here we also provide preliminary data162

on resting cyst structure and formation in this species.163

164

30

Abstracts

Oxymonads in the Gut of Reticulitermes flavipes165

Ondřej Brzoň, & Vladimír Hampl166

Charles University, Faculty of Science, Department of Parasitology, Praha167

168

Oxymonads are a poorly–studied group of anaerobic or microaerophilic protists,169

which are interesting by the total absence of mitochondrial organelle. Majority of oxy-170

monads live in the guts of lower termites or cockroaches, but their role in this ecosys-171

tem is nearly unknown so far. We have investigated endobionts diversity of the eas-172

tern subterranean termite, Reticulitermes flavipes, using metagenomics and electron173

microscopy. For metagenomics analysis, we prepared amplicon libraries of variable re-174

gions of the genes for prokaryotic and eukaryotic SSU ribosomal RNAs from the DNA175

isolated from the termite hindgut and these were subsequently sequenced on Illumina176

MiSeq platform. Reads were clustered on 95% sequence identity. Depending on the177

amplified region and the set of oxymonad–specific primers we found 2–3 OTUs belon-178

ging to the genus Pyrsonympha and 3–10 OTUs belonging to the genus Dinenympha.179

No other oxymonad sequence has been recovered. We also mapped the composition of180

parabasalids and bacterial communities in this environment using universal eukaryotic181

and universal prokaryotic primers.182

183

31


Comparative Transcriptomic Analysis of Euglenozoa: Insi-184

ghts into the Evolution of Metabolic Capabilities and Mo-185

lecular Features186

Anzhelika Butenko1, 2, Olga Flegontova2, Aleš Horák2, Vladimír Hampl3, Patrick187

Keeling4, Ryan Gawryluk5, Denis Tikhonenkov4, 6, Pavel Flegontov1, 2, 7, & Julius188

Lukeš2, 7189

1University of Ostrava, Faculty of Science, Department of Biology and Ecology, Ostrava190


3Charles University, Faculty of Science, Department of Parasitology, Praha192

4Department of Botany, University of British Columbia, V6T 1Z4 Vancouver, British Columbia,193

Canada194

5Department of Biology, University of Victoria, Victoria, BC, Canada195

6Laboratory of Microbiology, Institute for Biology of Inland Waters, Russian Academy of Sciences,196

Yaroslavl Region, Borok 152742, Russian Federation197


199

Phylum Euglenozoa (Excavata) incorporates four main groups of unicellular euka-200

ryotes of widely different lifestyles: i) diplonemids, ii) kinetoplastids, iii) euglenids, and201

iv) postgaardians. The results of recent metabarcoding studies suggest that diplone-202

mids are the most diverse marine planktonic eukaryotes. However, no sequencing data203

is available for these enigmatic protists. Kinetoplastids are widely known for the no-204

torious parasites belonging to the family Trypanosomatidae containing medically and205

veterinary important Trypanosoma and Leishmania spp. The majority of euglenids206

are osmotrophic, and yet the group is known mostly by its photosynthetic members207

(e.g. Euglena gracilis). Postgaardians remain understudied with almost no molecular208

data available.We have composed a dataset incorporating the transcriptomes of three209

diplonemid and three euglenid species, as well as twelve kinetoplastid genomes and210

transcriptomes including those of free-living representatives of the Prokinetoplastina211

clade and bodonids. Our phylogenomic data demonstrates that diplonemids and ki-212

netoplastids represent sister clades. The analysis of metabolic pathways revealed that213

trypanosomatids and bodonids, except for free–living representatives of the Prokine-214

toplastina clade, are characterized by lower metabolic capabilities compared to diplo-215

nemids, euglenids and free-living heterotrophic protists of several other groups. Only216

genomes of ciliates, encoding 771 metabolic enzymes on average, are similar in this217

respect to kinetoplastid genomes containing ∼660 genes encoding metabolic proteins.218

Important enzymes of amino acid biosynthesis and degradation, purine and pyrimidine219

metabolism, vitamins and cofactors' biosynthesis were lost in all kinetoplastids or wi-220

thin the kinetoplastid tree. Using diplonemids as an outgroup also allowed us to shed221

some light on the evolution of several molecular features which are considered unique222

for the family Trypanosomatidae.223

224

32

Abstracts

Dropping the Mic: Knockdown of MICOS Subunits Yields225

Intriguing Phenomenon in Trypanosoma brucei226

Lawrence Rudy Cadena1, 2, Iosif Kaurov1, 2, Julius Lukeš1, 2, 3, & Hassan Hashimi1, 2227



3Canadian Institute for Advanced Research, Toronto, Canada230

231

Belonging to the Kinetoplastida class, Trypanosoma brucei plays an interesting232

role in understanding the evolutionary divergence of the mitochondria and consequently233

the functionality and characterization of its intermembrane space (IMS) proteins. The234

mitochondrial contact site and cristae organization system (MICOS) is a protein com-235

plex that is crucial for the formation and maintenance of cristae. Cristae are invaginati-236

ons of the mitochondrial inner membrane that increase its surface area and thus are237

important for the maintenance of both structure and function of the mitochondria. We238

present novel subunits of this complex that have not been bioinformatically identified in239

Trypanosoma brucei, due to high evolutionary divergence and the lack of mainstream240

interest on MICOS outside of opisthokont models such as humans and yeast. Knoc-241

kdown of individual MICOS subunits via RNA interference results not only in altered242

cristae morphology and cellular growth arrest, but additionally in the downregulation243

of an essential respiration protein needed in the biogenesis of other small IMS proteins.244

These subunits do not only correlate with key metabolic pathways, but also propose the245

existence of a fundamental protein used in the importation of small cysteine–containing246

proteins in the IMS, previously postulated to be absent in Trypanosoma brucei.247

248

33


Codon Use is a Major Determinant of mRNA Levels in249

Trypanosomes250

Janaina Nasciemento de Freitas1, Steve Kelly2, Jack Sunter1, & Mark Carrington1251

1University of Cambridge, Department of Biochemistry252

2University of Oxford, Department of Plant Sciences253

254

Selective transcription of individual protein coding genes does not occur in try-255

panosomes and the cellular copy number of each mRNA must be determined post–256

transcriptionally. Here, we provide evidence that codon choice directs the levels of257

constitutively expressed mRNAs. First, a novel codon usage metric, the gene expres-258

sion codon adaptation index (geCAI), was developed that maximised the relationship259

between codon choice and the measured abundance for a transcriptome. Second, geCAI260

predictions of mRNA levels were tested using differently coded GFP transgenes and261

were successful over a 25-fold range, similar to the variation in endogenous mRNAs.262

Third, translation was necessary for the accelerated mRNA turnover resulting from263

codon choice. Thus, in trypanosomes, the information determining the levels of most264

mRNAs resides in the open reading frame and translation is required to access this265

information.266

267

34

Abstracts

Leishmania Genome as a Model of Gene Conversion268

Arzuv Charyyeva1, & Vyacheslav Yurchenko1, 2269



272

Leishmania species cause a disease with various symptoms, following the transmis-273

sion. Gene conversion is a unidirectional exchange of genetic material between similar274

sequences, which is one sequence is used as a template to repair or correct another275

sequence. Average identity between flanking Short Interspersed DEgenerated Retro-276

posons (SIDERs) is 99% and 100% for 20 randomly chosen 2-clusters from L. major277

and L. mexicana, respectively. The similar situation was observed for 15 randomly cho-278

sen 2-clusters from L. major and L. braziliensis. Average identity between orthologous279

SIDERs is 75–86%. The high similarity of SIDERs in clusters demonstrates that evolu-280

tion of Leishmania genes might have been significantly affected by gene conversion. To281

study this process, we generated Leishmania expressing GFP, which was mutated on282

either C- or N-termini, to eliminate fluorescence. We hypothesize that gene conversion283

may facilitate proper repair of mutated variants and restoring of fluorescence, when284

both copies are present simultaneously.285

Acknowledgment:286

This work was supported by ERD Funds, project OPVVV CZ.02.1.01/0.0/0.0/16 019/0000759287

(Centrum výzkumu patogenity a virulence parazitů).288

289

35


The Eukaryotic Microbiomes of Benthic and Planktonic290

Marine Animals291

Javier del Campo292

Institut de Ciencies del Mat –CSIC, Barcelona, Spain293

294

Microbiomes associated with host organisms have a strong influence on host evo-295

lution, physiology and ecological functions. Unlike the study of bacterial microbiomes,296

the study of the micro-eukaryotes associated with animals has largely been limited297

to visual identification or molecular targeting of particular groups. The application of298

high-throughput sequencing (HTS) approaches, such as those used to look at bacteria,299

has been restricted because the barcoding gene we use to study micro-eukaryotic eco-300

logy and distribution in the environment, the 18S rRNA gene, is also present in the host301

animals. As a consequence, when host-associated microbial eukaryotes are analyzed by302

HTS, the results are dominated by host sequences. Stemming from our work on marine303

animals associated micro-eukaryotes, we successfully developed an approach that avo-304

ids the amplification of metazoan host genes, which allows us to use high-throughput305

methods to study the micro-eukaryotic communities of animals. I am currently stu-306

dying how climate change effects, such as rising temperature and acidity, impact the307

composition of the microbiomes (bacterial and eukaryotic) in corals and copepods, and308

consequently how these changes affect the hosts. It is known that the ongoing climate309

change has strong impacts on free-living marine microbial communities, but its effects310

have not been properly addressed on host-associated microbiomes.311

312

36

Abstracts

Cytosolic Hydrogenase in Trichomonas vaginalis Does313

Exist314

Alena Dohnálková, Tamara Smutná, Róbert Šuťák, & Ivan Hrdý315

BIOCEV – Biotechnology and Biomedicine Center of the Academy of Sciences and Charles Univer-316

sity, Faculty of Science, Department of Parasitology, Vestec317

318

In T. vaginalis genome, up to nine genes encoding Fe-dependent hydrogenase319

homologues have been identified. Six of these homologues have conserved cysteine resi-320

dues known to form the essential H cluster, which is crucial for the proper hydrogenase321

activity. Five of those six hydrogenases have been found in the proteome of hydrogeno-322

some, while the distribution of the other enzymes remains unknown. Hydrogenosomal323

hydrogenases produce molecular hydrogen as one of the end products of the pyruvate324

decarboxylation and are iconic, organelle–defining enzymes of anaerobic eukaryotes.325

Cytosolic localization of hydrogenase in T. vaginalis have never been proposed or tes-326

ted, however, our metabolic studies indicated the presence of hydrogenase in T. va-327

ginalis cytosol. Among the hydrogenase homolouges present in T. vaginalis genome,328

we have identified one gene as the putative cytosolic hydrogenase and overexpressed329

this enzyme in T. vaginalis T1 strain. The enzyme was indeed localized in cytosol and330

biochemical assays constantly detected very high cytosolic hydrogenase activity in the331

overexpressing cells comparing to wild type T. vaginalis. The typical redox partner332

of hydrogenase is ferredoxin, which is localized in the hydrogenosome of T. vaginalis.333

Purified cytosolic hydrogenase is not able to interact with ferredoxin, however, it is334

capable of reducing the cytosolic homologue of cytochrome b5, function of which in335

the metabolism of T. vaginalis remains unknown.336

337

37


Mitochondrial Metabolic Remodeling During Trypano-338

soma brucei Developmental Differentiation339

Eva Doleželová1, Kunzová M1, 2, Panicucci B1, & Alena Zíková1, 2340



343

The Trypanosoma brucei mitochondrion undergoes extensive structural and me-344

tabolic remodeling during the parasite´s life cycle since the insect stage fully relies on345

oxidative phosphorylation (OXPHOS) to produce ATP while the mammalian blood-346

stream stage generates ATP by aerobic glycolysis. This complex developmental diffe-347

rentiation is exemplified during the flagellated protist's migration from the tsetse fly348

midgut to the salivary glands, a process that can now be mimicked in vitro by overex-349

pressing a single RNA binding protein. Here we demonstrate that the mitochondrial350

membrane potential and reactive oxygen species are increased at the early transition351

stages. Meanwhile, respiratory complexes III and IV become reduced and the electron352

flow is redirected from the OXPHOS pathway to an alternative oxidase. This coincides353

with the increased abundance of respiratory complex II and proline degradation enzy-354

mes that may act to provide ATP by substrate phosphorylation. Molecular triggers for355

this metabolic rewiring are being explored.356

357

38

Abstracts

RNA viruses of Blechomonadinae358

Diego Henrique Fagundes Macedo, Danyil Grybchuk, Alexei Kostygov, & Vya-359

cheslav Yurchenko360

University of Ostrava, Faculty of Science, Life Science Research Centre, Ostrava361

362

The monoxenous (= one host) species comprise the majority of the known trypa-363

nosomatid diversity. For a long time they were seen just as dull relatives of their more364

interesting the dixenous (= two hosts) relatives. One of these neglected groups unites365

several parasites of fleas belonging to the genus Blechomonas (subfamily Blechomonadi-366

nae). This genus was shown to be closely related to all other trypanosomatids excluding367

Trypanosomatinae (Trypanosoma spp.) and Paratrypanosomatinae (Paratrypanosoma368

spp.).369

Viruses, and double-stranded RNA (dsRNA) viruses in particular, can be found in370

any cellular life, explaining their immense diversity. They also play an important role371

in regulation of gene expression and general post transcriptional processing of RNA in372

eukaryotic cells. Recently, Leishmania dsRNA viruses have started to attract research373

attention due to their association with pathogenesis of the disease and survival of the374

parasites.375

Previous reports documented presence of the tombus-like viruses, Bunyavirales,376

Narnaviridae, and a unique ostravirus in monoxenous trypanosomatids. Interestingly,377

no relatives of Leishmaniavirus have been found in analyzed flagellates, leading to a378

speculation that LRV1/2 were acquired by an ancestor of modern Leishmania and379

subsequently lost in most extant species.380

In this work, we investigated diversity of viruses infecting Blechomonadinae from381

13 isolates of 11 different species using the DNase I/LiCl method followed by NGS. We382

document presence of three different types of viruses in 5 of these isolates, including383

the first virus related to Leishmaniavirus found outside of Leishmania.384

385

39


In silico Characterization of The Plastid Proteomes of386

Chromera velia and Vitrella brassicaformis387

Tereza Faitová1, Zoltán Füssy2, & Miroslav Oborník1, 2388

1University of South Bohemia, Faculty of Science, Department of Molecular Biology and Genetics,389

České Budějovice390


392

Plastids, organelles of plants and algae, play important role not only in photosyn-393

thesis, but also in several other biochemical processes of the cell, such as biosynthesis of394

amino acids, tetrapyrroles, fatty acids and isoprenoids. Identifying proteins with plas-395

tid targeting pre-sequences allows us to understand more deeply what function plastid396

has in the cellular metabolism in chromerids and possibly in other closely related orga-397

nisms. The plastid proteomes of complex red-derived algae Chromera velia and Vitrella398

brassicaformis have not been thoroughly investigated. Here we study the subcellular399

localization of proteins in chromerid algae. Several prediction tools were used and their400

performance was evaluated on reference datasets of proteins with known localization.401

The best-suited prediction tool for plastid-targeted proteins turned to be ASAFind,402

which was then applied to the entire protein sets to predict subcellular proteomes with403

an emphasis on the plastid; the results are presented here. Putative plastid-targeted404

proteins were further analyzed as for their evolutionary origin.405

406

40

Abstracts

A Moonlighting Nuclear Pore Gene Controls Gene Expres-407

sion in African Trypanosomes408

Jennifer M. Holden1, Luděk Kořený1, Samson Obado2, Alexander V. Ratushny3,409

Wei-Ming Chen3, Brian T. Chait2, John D. Aitchison3, Michael P. Rout2, &410

Mark C. Field1411

1School of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, UK412

2The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA413

3Seattle Biomedical Research Institute and Institute for Systems Biology, Seattle, WA 98109–5234,414

USA.415

416

Components of the nuclear periphery operate in a multitude of processes including417

transport, cell-cycle progression and chromatin organization. Nuclear pore complexes418

are large dynamic structures, comprised of nucleoporins, and mediate nucleocytoplas-419

mic transport as well as coordinating mRNA processing and transcriptional regulation.420

Nucleoporins also define areas of high transcriptional activity both at the nuclear peri-421

phery and nucleoplasm. Lineage-specific features underpin organization and functional422

diversification at the nuclear periphery. For example trypanosomatids branched early423

from animals and fungi and possess unique features within their lamina, kinetochores424

and NPCs. Here we describe TbNup53b, an FG-repeat containing nucleoporin that425

localizes within the nucleoplasm as well as at the NPC. In addition to associating with426

numerous nucleoporins, TbNup53b interacts with a known trans-splicing component,427

TSR1, and has a clear role in the control of the developmentally-regulated RNA PolI-428

-transcribed nucleolar periphery-located procyclin genes. Significantly, though several429

nucleoporins have been implicated as intranuclear transcriptional regulators in meta-430

zoa, TbNup53b is orthologous to metazoan Nup58, a component of the core scaffold431

and for which no intranuclear/transcriptional function is known. These data suggest432

that FG-Nups are frequently co-opted to transcriptional functions during evolution,433

and also extends evidence for control of gene expression by FG-repeat Nups to trypa-434

nosomes.435

436

41


Endogenous Virophages in Marine Heterotrophic Flagella-437

tes: Smoking Gun of an Adaptive Defense System against438

Giant Viruses?439

Matthias G. Fischer1, & Thomas Hackl2440

1Max Planck Institute for Medical Research, Heidelberg, Germany441

2Massachusetts Institute of Technology, Cambridge, USA442

443

One of the major classes of viruses infecting protists are the so-called giant DNA444

viruses, with particle and genome sizes that overlap with those of bacteria. Giant viruses445

of the family Mimiviridae replicate in the host cytoplasm in a replication compartment446

termed the viral factory, which provides many enzymatic functions that are usually re-447

stricted to the nucleus. This feature may have led to the evolutionary adaptation of448

a distinct class of smaller DNA viruses called virophages, which use the cytoplasmic449

transcription machinery of the giant virus instead of the nuclear host equivalent. Viro-450

phages thus strictly depend on a co-infecting giant virus, and they can severely inhibit451

their replication, making virophages \viruses of viruses". This, in turn, benefits the gi-452

ant virus-infected host cell population with considerable ecological consequences. The453

virophage mavirus infects the marine phagotrophic flagellate Cafeteria roenbergensis454

and protects it against infection by the giant virus CroV. Although mavirus does not455

replicate in the absence of CroV, it can integrate its genome into the host genome,456

where the resulting endogenous virophage is transcriptionally silent and maintained457

by the host. Upon CroV infection, the integrated mavirus genome becomes active and458

newly synthesized virophage particles can inhibit CroV in subsequent co-infections. We459

sequenced and assembled the nuclear genomes of four C. roenbergensis strains from460

the Atlantic and Pacific Oceans and found that all of them contained endogenous vi-461

rophages that were related to mavirus. We hypothesize that these viral genomes are462

specific for and may provide defense against different strains of CroV-related giant463

viruses. These findings suggest that virophages may provide long-term protection of464

marine phagotrophic flagellates against lytic giant viruses.465

466

42

Abstracts

The F1-ATPase from Trypanosoma brucei is Elaborated by467

Three Copies of an Additional p18-subunit468

Ondřej Gahura1, Martin G. Montgomery2, John E. Walker2, & Alena Zíková1469


2MRC Mitochondrial Biology Unit, Cambridge, UK471

472

The sophisticated rotational mechanism of ATP synthesis coupled to H+ translo-473

cation across membranes imposes significant constraint to evolutionary diversification474

of bacterial and eukaryotic F-type ATP synthases. The compositional and structural475

variability of ATP synthases is restricted to the regions not engaged in H+ pumping,476

torque transmission, or ATP generation. The matrix-facing catalytic subcomplex, F1-477

-ATPase, has been considered invariant across eukaryotes. However, the F1-ATPase478

purified from Trypanosoma brucei contains an additional essential polypeptide, called479

p18. Quantification of 14C-iodoacetic acid labeling revealed that p18 is present in three480

copies per complex. Using X-ray crystallography we have generated an atomic mo-481

del of F1-ATPase to determine localization of the p18-subunit in the complex and to482

dissect additional structural divergences of the parasite's enzyme. We are also determi-483

ning the structure of the F1-ATPase with its inhibitory protein IF1 by single particle484

cryo-electron microscopy (cryoEM). Atomic details of the F1-IF1 interaction can be485

ultimately exploited in structure-based drug design.486

487

43


Nucleotide Biosynthesis and Transport in Diatoms488

Ansgar Gruber489

Biology Centre ASCR, v. v. i., Institute of Parasitology, České Budějovice490

491

Plastids of diatoms and related algae evolved via eukaryote – eukaryote endo-492

symbiosis, a process that increased the structural complexity of the resulting cell. For493

example, diatom plastids are surrounded by four envelope membranes, two membranes494

more compared to plastids of land plants. These additional membranes are barriers for495

the exchange of metabolites between the plastid and the cytosol or other organelles.496

Furthermore, eukaryote – eukaryote endosymbiosis also altered the metabolic comple-497

xity of the resulting cell. This can be seen in the intracellular distribution of metabolic498

pathways compared to other photosynthetic organisms. For example, diatom plastids499

depend on nucleotide uptake from the cytosol because, unlike in plants, nucleotide500

de novo synthesis exclusively occurs in the cytosol. Diatom genomes encode a higher501

number of nucleotide transporters (NTTs) compared to plants. By exchanging ATP502

against ADP+Pi, land plant NTTs (which are only found in the inner plastid enve-503

lope) provide energy to the plastid without net transport of nucleotides. In contrast504

to this, diatom NTTs are also found in other parts of the cell and show a broader505

range of transport activities. Six different isoforms of diatom NTTs (NTT1, –2 and506

–3 of Thalassiosira pseudonana and NTT1, –2 and –5 of Phaeodactylum tricornutum)507

have meanwhile been characterized by phylogenetic studies, transport assays with the508

recombinant proteins and GFP–based targeting analyses. The results provide evidence509

that diatom NTTs form a specifically adapted system for net nucleotide transport510

between cytosol and plastids in diatoms.511

512

44

Abstracts

Unexpected Diversity of the Peculiar Genus Creneis (Ex-513

cavata: Heterolobosea)514

Pavla Hanousková, & Ivan Čepička515

Charles University, Faculty of Science, Department of Zoology, Praha516

517

Creneis is a recently (2014) discovered genus of marine anaerobic heteroloboseids.518

It comprises only a single species, C. carolina, with unique morphology, ultrastructure,519

and life cycle, quite unusual for Heterolobosea. C. carolina is an amoeboid flagellate520

with a single flagellum and ability to form a fast–swimming flagellate with more than521

ten flagella. In addition, the amoeboid flagellate has a unique structure of the flagellar522

apparatus. Creneis carolina was described on the basis of a single isolate and has never523

been reported since. Here we show that Creneis is, in fact, a widespread and diverse524

lineage of anaerobic protists. We have established 13 marine Creneis strains in culture.525

According to the morphology and SSU rRNA gene sequences, our strains represent526

at least six novel species of Creneis. At least two new species are able to form the527

fast–swimming multiflagellates, which, however, possess only four or five flagella. We528

will present the first ultrastructure data of the unique multiflagellate form.529

530

45


Tracing Evolutionary Changes in rRNA Genes in Eugleno-531

zoa532

Pawe l Ha lakuc, Anna Karnkowska, & Rafa l Milanowski533

University of Warsaw, Faculty of Biology, Department of Molecular Phylogenetics and Evolution,534

Biological and Chemical Research Centre, Warsaw, Poland535

536

Eukaryotic ribosomes are composed of a few dozen proteins and usually four RNA537

molecules. Genes coding three of them, 18S, 5.8S and 28S rRNA are clustered in a538

single operon and transcribed together. They are separated by the internal transcribed539

spacers (ITS1 and ITS2), which are removed in the post-transcriptional processing.540

Surprisingly, the rDNA cistron arrangement is different in the members of Euglenozoa541

group (Excavata).Euglenozoa consists of three major phyla: Euglenida (e.g. secondarily542

photosynthetic Euglenophytes), Kinetoplastea (e.g. bodonids and trypanosomatids)543

and Diplonemea (important marine single-cell predators). The structure of ribosomes544

was examined in several trypanosomatid species and it was revealed to contain more545

than three typical rRNA. The even more complex structure is observed in Euglena546

gracilis (Euglenida). Its large ribosomal subunit consists of 15 separate rRNAs, which547

is caused by the presence of additional ITSs in DNA fragment corresponding to 28S548

rDNA. In our work, we traced the evolution of rDNA within Euglenozoa, focusing549

particularly on the distribution of ITS sequences. We extracted all available complete550

rDNA sequences for euglenids, diplonemids, and non-trypanosomatid kinetoplastids.551

Several trypanosomatid species with known 3D ribosome structure were used as a552

reference for better in silico prediction of rRNA secondary structure. It allowed us553

to predict presence or absence of additional ITSs in analyzed lineages and identify554

evolutionary trends. We found no additional ITSs in all examined diplonemid species555

and confirmed homology of euglenids and kinetoplastids ITSs. Moreover, the number556

of ITSs seems to be conserved in each particular lineage.557

558

46

Abstracts

Cryptosporidium spp. in Wild Coypu (Myocastor coypus)559

Michaela Horčičková1, 2, Nikola Holubová1, 2, Dana Květoňová2, Lenka Hlásková2,560

John McEvoy3, Dušan Rajský4, Bohumil Sak2, & Martin Kváč1, 2561

1University of South Bohemia, Faculty of Agriculture, České Budějovice562


3Veterinary and Microbiological Sciences Department, North Dakota State University, Fargo564

4Faculty of Forestry, Technical University in Zvolen, Zvolen, Slovakia565

566

The results of field studies show a huge variety of parasites from the genus567

Cryptosporidium, particularly in wildlife. Rodents are ubiquitous mammals comprising568

about 40% of the mammalian diversity and occupying a wide range of habitats. Studies569

so far have shown that rodent species are predominantly parasitized with host–specific570

Cryptosporidium species and genotypes. The coypu, also known as the nutria, origi-571

nally native in South America, has since been introduced to North America, Europe,572

Asia, and Africa, primarily by fur ranchers. Cryptosporidium infection in farmed and573

wild coypu is rarely studied. In the present study, 96 faecal samples from wild coypus574

were screened for presence of Cryptosporidium by microscopy (aniline-carbol-methyl575

violet staining) and PCR/sequencing. Cryptosporidium infections were detected in 10576

coypus (10.3%). Phylogenetic analysis of small-subunit rRNA, 70 kDa heat shock pro-577

tein, and actin gene sequences revealed the presence of C. ubiquitum (n=6) and a578

novel Cryptosporidium coypu genotype (n=4) never found before in any host. Oocysts579

of Cryptosporidium coypu genotype are indistinguishable from those of C. parvum and580

are experimentally infectious for adult coypus but not for SCID mice (Mus musculus),581

Mongolian gerbils (Meriones unqutulatus), and chickens (Gallus gallus). The prepatent582

and patent period was 5 and 30 days post infection, respectively. Infection intensity583

was 8,000–65,000 oocysts per gram of faeces. Histology and electron microscopy of584

digestive tract epithelium revealed the presence of developmental stages in the small585

intestine. Experimentally infected coypu showed no clinical signs of cryptosporidiosis.586

Morphological, genetic, and biological data suggest the novel genotype from wild coypu587

is a separate species of the genus Cryptosporidium.588

Acknowledgment:589

This study was funded by the Ministry of Education, Youth and Sports of the Czech Republic590

(LTAUSA 17165), the Grant Agency of the University of South Bohemia (002/2016/Z).591

592

47


Validation of Babesia Proteasome as a Drug Target593

Marie Jalovecka1, 2, David Hartmann1, 2, Yukiko Miyamoto3, Lars Eckmann3, Ondrej594

Hajdusek1, Anthony J. O'Donoghue4, & Daniel Sojka1595



3Department of Medicine, University of California, San Diego, La Jolla, USA598

4Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La599

Jolla, USA600

601

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the602

genus Babesia. Treatment of this emerging malaria-related disease has relied on an-603

timalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent604

of malaria, has recently been validated as a target for anti-malarial drug development605

and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib,606

ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome607

inhibitors on the growth and survival of Babesia. Testing the compounds against Babe-608

sia divergens in vitro revealed suppressive effects on parasite growth with activity that609

was higher than the cytotoxic effects on non-transformed mouse macrophage cell line.610

Furthermore, we showed that the most-effective compound, carfilzomib, significantly611

reduces parasite multiplication in a Babesia microti infected mouse model without no-612

ticeable adverse effects. Overall, our results demonstrate that the Babesia proteasome613

is a valid target for drug development and warrants the design of potent and more614

selective B. divergens proteasome inhibitors for antibabesial treatment.615

616

48

Abstracts

The Fitness of Three Strains of the Alga Chromera velia617

Kateřina Kabeláčová1, Aleš Tomčala2, & Miroslav Oborník1, 2618

1University of South Bohemia, Faculty of Science, Department of Molecular Biology, České Budějo-619

vice620


622

Chromera velia is the closest known relative photosynthetic organism to obligate623

parasites from the group apicomplexa. Parasites during their life cycle have to survive in624

different environments inside hosts as changes in temperature, salinity or pH. Since the625

C. velia is relative to this parasites they should share this ability with apicomplexans.626

Thus the growing ability of three strains of the Chromera velia was tested through627

cultivation in environments with a wide range of salinity and varying pH levels. The628

growing curves were obtained by spectrophotometer TECAN. Results show the ability629

of C. velia to sustain growth almost in all environments. In addition, to assess the rate630

of stress to organism the lipidomic analysis was performed by HPLC ESI MS method.631

The ability to store high amounts of fatty acids makes C. velia a potential candidate632

for biotechnological applications.633

634

49


Breeding Strategies and Genome Integration in Tetrahy-635

mena636

Andrzej Kaczanowski637

University of Warsaw, Faculty of Biology, Warsaw, Poland638

639

Since micronucleus in Tetrahymena is transcriptionaly silent, deletrious micro-640

nuclear mutations and aneuploidy are not expressed until conjugation and development641

of new macronuclei. It may be a ratchet mechanism, leading to a loss of fitness and to642

extinction after development of unfunctional macronucleus. There are three strategies643

to escape this problem: (1) Genomic exclusion (GE) and death of amicronuclear cells644

in T. thermophila. Aneuploid cell does not produce functional pronucleus, but it may645

obtain functional pronucleus from a fertile mate (round I of GE). If segregation of ane-646

uploid chromosomes results in a loss of micronucleus, the amicronuclear cells undergo647

transformation into unviable, crinkled cells, which are removed from the population.648

(2) There are many amicronuclear species of Tetrahymena, which do not mate. Only649

macronuclear mutations and independent assortment of multiple chromosomes provide650

their genetic polymorphism increasing fitness in variable environment, but it is likely651

that it takes a longer time than selection following conjugation and outbreeding. (3)652

Autogamy. We have studied life history of T. rostrata, histophagous ciliate, living in653

renal organs of snails. T. rostrata undergoes encystment induced autogamy, which re-654

sults in whole genome homozygosity, whenever it is starved in external environment.655

There is a question how T. rostrata life history compensates for a lack of polymor-656

phism, induced by heteroparental sex. The cell division rate in T. rostrata declines657

with a number of divisions from the last autogamy. It is likely, that the senescing cells658

are rejuvenated by encystment-induced autogamy and that snails may undergo secon-659

dary self-infections by rejuvenated cells, which outcompete the older ones enhancing660

fixation of new favorable alleles and epigenetic changes.661

662

50

Abstracts

Searching for the Plastid Genomes in the Metagenomic663

Data664

Micha l Karlicki, & Anna Karnkowska665



668

Microbial eukaryotes are essential components of many environments, but they669

are often understudied. Recent advances in the metagenomic approaches opened up670

new possibilities for environmental studies of uncultured organisms. Most of the me-671

tagenomic studies, however, focusing on the prokaryotes because of the smaller size672

of prokaryotic genomes and the abundance of reference genomes already sequenced.673

Recently, projects such as TARA Oceans expeditions have shown a tremendous diver-674

sity of small novel eukaryotic lineages in the oceans, suggesting that new approaches675

for eukaryotic metagenomics are needed. We focused on the plastid genomes in the676

metagenomic data, which are easier to handle than nuclear genomes, and information677

about their presence and abundance is a direct indicator of the photosynthetic activity678

of eukaryotes in the environment.Here we introduce MetaPlastHunter, the first tool for679

fast and memory-efficient identification of reads of chloroplast origin from large me-680

tagenomic datasets. It uses two well-established methods of classifying metagenomic681

sequences: exact k-mer matching and alignment to the reference databases. Accurate682

taxonomic assignment of reads relies on LCA algorithm which is supported by refe-683

rence genome coverage and read–pairing information. Preliminary results have shown684

that reads derived from the chloroplast genomes are abundant in the TARA Oceans685

metagenomic data. Using MetaPlastHunter, we were able to identify dominant photo-686

synthesizing taxa in the sample and track the presence of groups of interest across the687

samples. For the most abundant species, we assembled pieces of plastid genomes up to688

∼100 kB, which might constitute nearly complete plastid genomes. Currently, we are689

analyzing the samples from several locations to perform comparative analyses and to690

test the software limitations.691

692

51


The Role of Kinetoplastid MICOS Complex in Cristae Sha-693

ping and Intermembrane Space Import694

Iosif Kaurov1, 2, Marie Vancova1, 2, Lawrence Rudy Cadena2, Jiří Heller1, Tomáš695

Bily1, 2, David Potěšil3, Zbyněk Zdrahal3, Julius Lukeš1, 2, 4, & Hassan Hashimi1, 2696



3Central European Institute of Technology, Masaryk University, Brno699

4Canadian Institute for Advanced Research, Toronto, Canada700

701

Mitochondrial inner membrane forms internal ridges, named cristae. Cristae702

junctions connect cristae and the mitochondrial inner boundary membrane. The Mi-703

tochondrial Contact Site and Cristae Organization System (MICOS) is responsible for704

proper cristae formation and therefore for the cell viability since oxidative phosphory-705

lation occurs on the cristae membrane. Knowledge about MICOS is limited to opistho-706

kont models, especially yeast. Our highly diverged model species, Trypanosoma brucei,707

shares only one bioinformatically recognizable MICOS subunit with yeast. It is the708

homolog of the core protein Mic10, which was duplicated in most trypanosomatids.709

We have allele tagged both paralogs and have confirmed that they associate with mi-710

tochondrial cristae. We performed immunoprecipitation, using Mic10 paralogs as the711

bait. Among the rest, it pulled down the same set of proteins, which we demonstrate712

are MICOS subunits. They are quite diverged, although they share some domains with713

opisthokont MICOS subunits. RNAi cell lines targeting newly discovered subunits were714

created. Most of them developed growth phenotype both in glucose–poor and glucose–715

rich media. In several cases, significant alterations in cristae shape and structure were716

detected, resembling the deletion phenotype of core yeast MICOS subunits. Depletion717

of TbMICOS subunits is followed by the down–regulation in TbMic10–1. We found that718

silencing of TbMic20, decreases the abundance of TbERV1, a key component of the719

mitochondrial intermembrane space assembly (MIA) pathway. There is an overlap be-720

tween the TbMic20 and TbERV1 depletomes. Furthermore, half of TbMic20 depletome721

consists of intermembrane space proteins, and many of them are annotated as respira-722

tory chain complex assembly factors. We propose that in addition to cristae junction723

formation and cristae shaping, which are conserved MICOS functions, kinetoplastid724

MICOS also takes part in IMS protein import.725

726

52

Abstracts

Analyses of Ploidy and Karyotype of Oxymonads Using727

FISH728

Martina Kornalíková, Sebastian Treitli, & Vladimír Hampl729


731

Oxymonads are a group of flagellate protists living in low oxygen concentration732

environments. They inhabit mainly the gut of insects and vertebrates. In this study733

we focus to analyse the ploidy and karyotype of various species of oxymonads using734

Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and735

telomeric repeats. From the genome of Monocercomonoides exilis we know that oxy-736

monads have the ancestral type of telomeric repeat (TTAGGG). By using a probe737

against these telomeric repeat we tried to estimate the number of chromosomes for738

seven strains (5 species) of Monocercomonoides. With a single exception that average739

number of signal was below 20 indicating low number of chromosome. In the strains740

of M. mercovicensis we observed much higher number of signals which could suggest741

that the cells are polyploid or have really high number of chromosomes. Currently we742

are working to determine the ploidy of these strains by using flow cytometry and pro-743

bes against single copy genes. For the latter we decided to use the SufSU gene which744

encodes an enzyme of the SUF pathway. In oxymonads it is present in a unique fusion745

of SUFS and SUFU parts, which enables its specific amplification. Our preliminary746

results show that all investigated strains are haploid, because most of the cells have a747

single signal in the nucleus, however, further optimization of the method is required to748

get better signal and eliminate the background fluorescence.749

750

53


A Surprise from the Bacterial Eendosymbiont of Kentomo-751

nas sorsogonicus: Loss of the Heme Pathway752

Flávia M. Silva1, Alexei Kostygov2, 3, Viktoria V. Spodareva2, 3, Anzhelika Bu-753

tenko2, 4, Regis Tossou1, Julius Lukeš4, 5, Vyacheslav Yurchenko2, 4, & Jo~ao M.P.754

Alves1755

1Department of Parasitology, Institute of Biomedical Sciences, University of S~ao Paulo, S~ao Paulo,756

Brazil757





762

Trypanosomatids of the genera Angomonas and Strigomonas (subfamily Strigo-763

monadinae) have long been known to contain intracellular beta–proteobacteria, which764

provide them with many important nutrients such as heme, essential amino acids,765

and vitamins. Recently, Kentomonas sorsogonicus, a divergent member of Strigomo-766

nadinae, has been described. Herein, we characterize the genome of its endosymbiont,767

Candidatus Kinetoplastibacterium sorsogonicusi. This genome is completely syntenic768

with those of other known Ca. Kinetoplastibacterium spp., but more reduced in size,769

(∼742 kb, compared to 810–833 kb, respectively). Gene losses are not concentrated in770

any hot spots but are instead distributed throughout the genome. The most conspicu-771

ous loss is that of the heme synthesis pathway. For long, removing hemin from the772

culture medium has been a standard procedure in cultivating trypanosomatids iso-773

lated from insects; continued growth was considered as an evidence of endosymbiont774

presence. However, we demonstrate that, despite bearing the endosymbiont,K. sorsogo-775

nicus cannot grow in culture without heme. Thus, the traditional test cannot be taken776

as a reliable criterion of the absence or presence of endosymbionts in trypanosomatid777

flagellates. It remains unclear why the ability to synthesize such an essential compound778

was lost in Ca. Kinetoplastibacterium sorsogonicusi, whereas all other known bacterial779

endosymbionts of trypanosomatids retain them.780

Acknowledgment:781



784

54

Abstracts

Diversity and Host Specificity of Parabasalian Symbionts of785

Non-Termite Cockroaches786

Michael Kotyk1, Zuzana Kotyková Varadínová1, 2, Pavla Hanousková1 & Ivan Če-787

pička1788


2Department of Zoology, National Museum, Prague, Czech Republic790

791

Hypermastigids, the visually attractive parabasalian endosymbiots of xylophagous792

cockroaches from Cryptocercidae and Isoptera (termites), have been attracting the in-793

terest of biologists for several decades. These symbionts possess large and complex cells794

and have a great ecological significance. They are essential for the cellulose metabo-795

lism of the cockroach and exhibit strict host specificity. Phylogenetic analyses showed796

that hypermasitigids are not monophyletic, but have arisen at least six times indepen-797

dently from the \small" trichomonads. What united them, nonetheless, was the lack798

of knowledge about their small close relatives. At least until now. We have examined799

more than 400 non-termite cockroach individuals from 150 species, covering 24 of 33800

subfamilies of which 53% of species were positive for parabsalid symbionts. We have801

not observed any new hypermastigid forms. We have, however, sequenced SSU rRNA802

gene of more than 130 strains of parabasalids and about half of them nest within Ho-803

nigbergiellida, where they form several clades around the small hypermastigid Cthulhu804

with approximately 20 flagella and trichomonad genera Hexamastix and Cthylla with 6805

flagella. By contrast, our strains have cells possessing three or four flagella. This makes806

Cthulhu the only hypermastigid with known (and numerous) cloud of closely related807

\small" trichomonads. Majority of the rest of our strains belong into the small and808

understudied genus Hypotrichomonas. Surprisingly, they form multiple novel clades809

closely related to previously described lineages from vertebrates, indicating that this810

genus may have originated in cockroaches. Moreover, we show that particular lineages811

of Hypotrichomonas are specific for certain cockroach lineages.812

813

55


Mitochondrial Proteases of T. brucei814

Tomáš Kovalinka, Bianka Kováčová, & Anton Horváth815

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Biochemistry, Bra-816

tislava817

818

Protozoan parasite Trypanosoma brucei (Euglenozoa, Kinetoplastea, Trypano-819

somatida) is the causative agent of sleeping sickness in human and disease Nagano820

in animals. Our research is mainly focused on mitochondrial proteases, specifically on821

FtsH protease, which has not been widely studied among trypanosomatids so far. FtsH822

is a representative of mitochondrial peptidases belonging to the group of metalopro-823

teinases from AAA family, with specific M41 sequence. T. brucei possess six putative824

homolog subunits of FtsH subunits apart from e.g. S. cerevisiae and human where has825

been identified only three homologues. In general subunits can form homo or hetero826

hexameric structures, with central pore, anchored in inner mitochondrial membrane827

with orientation either to matrix or to intermembrane space. We have prepared cell828

lines with inducible RNAi of each subunit and V5 tagged cell lines for localization and829

orientation experiments. We have observed change of growth phenotype and activity of830

oxidative phosphorylation enzymes after RNAi induction. We proposed orientation of831

the individual subunits based on: i) in silico analysis for the presence of transmembrane832

domains of all six homologues; ii) protease assay of isolated mitochondria.833

Acknowledgment:834

This work was created with financial support of grant agencies VEGA a APVV within projects835

APVV-0286-12 a VEGA1/0387/17836

837

56

Abstracts

High-Throughput Discovery of Novel Conoid-Aassociated838

Proteins in Toxoplasma gondii839

Luděk Kořený, Konstantin Barylyuk, Kathryn Lilley, & Ross Waller840

University of Cambridge, Department of Biochemistry, Cambridge, UK841

842

One of the defining features of the group Apicomplexa is the assemblage of structu-843

ral and secretory elements forming the apical complex, which plays pivotal roles in host844

cell invasion and proliferation. The central structures of the apical complex are the api-845

cal polar ring that serves as an organizing center for an array of subpellicular microtu-846

bules, and the mobile conoid that sits within the apical polar ring. The conoid consists847

of tubulin fibers and associated proteins tightly organized into a hollow barrel that848

protrudes during invasion. Other structures closely associated with the conoid are the849

preconoidal rings at its distal tip and two short intraconoid microtubules, which may850

be implicated in the delivery of secretory vesicles from the micronemes and rhoptries851

during cell invasion. Despite extensive characterization of the apical complex through852

ultrastructural studies, our knowledge of its molecular composition and function is limi-853

ted. To address this issue, and shed more light on the function and importance of these854

structures, we selected several protein markers that localize in close proximity to the855

conoid in Toxoplasma and fused them with a promiscuous biotin ligase to use as baits856

for proximity biotinylation assays. Furthermore, we applied a novel spatial proteomics857

technology termed LOPIT (Localisation of Organelle Proteins by Isotopic Tagging) to858

simultaneously map the localization of several thousand proteins on a cell–wide scale.859

Our proteomic data provided new candidate proteins associated with the Toxoplasma860

apex, and several of them were localized to different parts of the conoid by 3D-SIM861

super-resolution microscopy.862

863

57


The News about ISC System in the Mitosomes of Giardia864

intestinalis865

Alžběta Krupičková1, Courtney Stairs2, Vladimíra Najdrová1, & Pavel Doležal1866

1BIOCEV – Biotechnology and Biomedicine Center of the Academy of Sciences and Charles Uni-867

versity, Faculty of Science, Department of Parasitology, Vestec868

2Department of Cell and Molecular Biology, Uppsala University, Sweden869

870

Mitosomes of Giardia intestinalis are the most reduced mitochondria found to871

date. They do not have any DNA and their proteome is extensively reduced – just a872

few tens of proteins are identified as mitosomal. The only known metabolic pathway873

is the synthesis of iron-sulphur clusters (ISC system).There are many unknowns in mi-874

tosome biology mainly concerning its biogenesis, the transport of biomolecules across875

mitosomal membranes and the actual role of mitosomal ISC pathway for the function876

of other cellular compartments. In this project, we tackle these questions by two bi-877

ochemical approaches (i) we attempt to establish affinity purification of the whole878

mitosomes from cell lysate. The techniques is based on specific biotinylation of the879

outer mitosomal membrane proteins. (ii) we also purify individual component of ISC880

pathway to identify missing functional components and the long sought substrate(s) of881

the pathway. Recently, we identified G. intestinalis BolA homologue, which is normally882

present in ISC pathway of aerobic organisms.883

884

58

Abstracts

Diversity of Trypanosomatids from the Philippines885

Jana Králová1, Jan Votýpka2, 3, Julius Lukeš3, 4, Alexei Yu Kostygov1, 5, Viktoria886

Spodareva1, 5, & Vyacheslav Yurchenko1, 3, 6887






6University of Ostrava, Faculty of Science, Institute of Environmental Technologies, Ostrava893

894

The interest in monoxenous (with one host) trypanosomatids has increased in895

recent years due the numerous studies demonstrating that all medically and veteri-896

nary important dixenous (with two hosts) flagellates derived from their monoxenous897

relatives. In this work we summarize results of the biodiversirty assay of monoxenous898

trypanosomatids in Philippines based on the analysis of their 18S rRNA gene sequences.899

Our data revealed phylogenetic affinities of the isolates under study to the previously900

known trypanosomatid clades and allowed to select two potentially interesting groups901

for further investigation. The first one corresponds to the \Clade 2" from Týč et al.,902

2013; the second group unites several putative species of the subfamily Paratrypano-903

somatinae, relatives of Paratrypanosoma confusum.904

905

59


Queuosine: The Role of an Essential tRNA Modification in906

Parasitic Protist Trypanosoma brucei907

Sneha Kulkarni1, 2, Helmut Stanzl1, 2, Alan Kessler3, Eva Heged"usová1, Juan D Al-908

fonzo3, & Zdeněk Paris1, 2909



3Department of Microbiology and The Center for RNA Biology, The Ohio State University, Colum-912

bus, OH 43210, USA913

914

A general feature of tRNAs is a high number of nucleotide modifications that915

are introduced post-transcriptionally. Queuosine (Q), one of the most complex tRNA916

modifications, is found at the first position of the anticodon (wobble base) of several917

tRNAs. Despite its omnipresence in bacteria and eukaryotes, the function of Q is not918

completely clear, although it is proposed to affect the rate and fidelity of translation. As919

eukaryotes cannot synthesize queuine, they rely on their environment or their micro-920

biome. In this study, we have used the protozoan parasite Trypanosoma brucei as a921

model for a comprehensive analysis of the tRNA guanine transglycosylase (TGT), the922

enzyme responsible for Q-tRNA formation. Unlike its bacterial counterpart, in most923

eukaryotes TGT predominantly functions as a heterodimer. We identified two TGT924

subunits in T. brucei, using bioinformatic approaches, TbTGT1 and TbTGT2. Inte-925

restingly, contrary to reports in higher eukaryotes, TbTGT heteromer is localized to926

the nucleus. However, splicing of tRNAs occurs in the cytosol in T. brucei, thus cre-927

ating a requirement for retrograde transport of tRNAs to the nucleus to obtain this928

modification. Hence, this system becomes ideal to study the dynamics of tRNA pro-929

cessing and trafficking. In order to determine the functional significance of Q-tRNA930

modification in trypanosomes, we generated a gene knock-out of the TbTGT2 and per-931

formed additional phenotypic in vivo characterization directly in the bloodstream of932

the mammalian host with the goal to simulate actual conditions associated with para-933

site infection. After infecting mice with the mutant parasites, we observed that it takes934

significantly longer for the trypanosome cells to appear in the blood, and eventually to935

kill the animals, as compared to WT parasites. Our data suggests that queuosine plays936

an important physiological role during survival of the parasites inside the mammalian937

host and may be at the heart of virulence.938

939

60

Abstracts

Susceptibility of Chicken Embryos to Cryptosporidium spp.940

Infection941

Martin Kváč1, 2, Nikola Holubová1, 2, Lenka Hlásková1, & Bohumil Sak1942


2University of South Bohemia, Faculty of Agriculture, České Budějovice944

945

The genus Cryptosporidium comprises species of protist parasites that infect epi-946

thelial cells in the microvillus border of the gastrointestinal tract, lungs and the bursa947

of Fabricius of vertebrate hosts. Most Cryptosporidium species and genotypes have a948

narrow host specificity, and, with the exception of C. parvum, those that infect a broa-949

der host range do not infect different classes of vertebrates. Cryptosporidium parvum950

has been detected in more than 200 species of mammals and birds. Reports of C. par-951

vum in birds have been from field studies and have not been verified experimentally.952

This study examined the infectivity of C. parvum (mammalian species) and C. baileyi953

(bird species) for one-day-old chickens and chicken embryos. Cryptosporidium baileyi954

was infectious for both one-day-old chickens and chicken embryos. Following embryo955

infection, hatched chickens shed oocysts of C. baileyi from the first day after hatching956

with an infection intensity up to 45,000,000 oocysts per gram of faeces (OPG). In com-957

parison, chickens infected at one day old shed a maximum of 150,000 OPG beginning at958

four days post infection (DPI). Cryptosporidium baileyi infection in one-day-old chic-959

kens was localised to the small intestine and trachea, and the infection resolved within960

50 days. In chickens that hatched from infected embryos, all organs were infected with961

C. baileyi and the chicken died within 14 days post hatching. Cryptosporidium par-962

vum was not infectious for one-day old chickens. Chickens that hatched from embryos963

infected with C. parvum shed oocysts from day 0 to day 20 post hatching, with an964

infection intensity of 2,000-50,000 OPG, and C. parvum was found in the small and965

large intestine and trachea.966

Acknowledgment:967

This study was funded by Grant Agency of the Czech Republic (18-12364S), the Grant Agency968

of the University of South Bohemia (002/2016/Z) and supported by MEYS CR (LM2015062969

Czech-BioImaging).970

971

61


Anaerobic Peroxisomes in Mastigamoeba balamuthi972

Tien Le, Vojtěch Žárský, Eva Nývltová, Eliška Kočířová, Zdeněk Verner, & Jan973

Tachezy974


976

Peroxisome is a typical organelle in aerobic eukaryotes, which is involved in various977

metabolic processes, notably in oxygen-dependent metabolism. Consequently, peroxi-978

somes are omnipresent in aerobic organisms to scavenge toxic oxygen compounds such979

as hydrogen peroxide. In contrast, anaerobic parasites such as Entamoeba histolytica,980

Giardia intestinalis, and Trichomonas vaginalis are believed to lack these organelles.981

Unexpectedly, analysis of the genome of Mastigamoeba balamuthi, an anaerobic free-982

-living relative of E. histolytica revealed presence of a complete set of peroxins (Pexes)983

that are responsible for the biogenesis of peroxisomes. Following in silico searches in984

M. balamuthi genome sequence and quantitative mass spectrometry of cellular fracti-985

ons revealed presence of forty-five putative peroxisomal proteins. Interestingly, some986

of them are shared with its pathogenic relative Entamoeba histolytica. Immunoflu-987

orescence microscopy revealed localization of MbPex3, MbPex11, and MbPex14, in988

numerous vesicles that are distinct from other cellular organelles including hydrogeno-989

somes, ER and Golgi apparatus. Heterologous expression of M. balamuthi proteins in990

yeast revealed that MbPexin14 and eight matrix proteins are specifically targeted to991

yeast peroxisomes. Based on the in silico analysis and experimental investigation, a992

putative metabolic map of peroxisomal pathways was constructed, predicting the in-993

volvement of peroxisomes in metabolism of purine, pyrimidine, CoA, and nicotinamide.994

The predicted map also suggested the novel functions in metabolism of pyruvate and995

galactose. In conclusion, we characterized the first peroxisomes functioning in anaerobic996

eukaryote.997

998

62

Abstracts

Myxozoa Wherever You Look: Uncovering Myxozoan Spe-999

cies Diversity1000

Martina Lisnerová1, 2, & Ivan Fiala1, 21001



1004

Myxozoans are microscopic metazoan parasites infecting typically fish as alternate1005

hosts and annelids as definitive hosts. Some myxozoans are economically important pa-1006

rasites causing serious fish diseases. There are about 2,300 described species of these1007

morphologically extremely reduced cnidarian parasites classified in 67 genera. However,1008

myxozoan species diversity still remains highly unrecognized. Some authors estimate1009

that only in neotropical region there might be up to 16,000 of myxosporean species due1010

to high diversity of freshwater fish in Amazon region and host specificity of myxospo-1011

rean species. We performed myxozoan screening in freshwater fish from selected ponds,1012

rivers and dams in south and central Bohemia to assess a myxozoan biodiversity in the1013

region that has a long term history of myxozoan research.We screened 30 fish species1014

(285 fish individuals) from 18 different localities. We performed light microscopy and1015

PCR screening of mostly gills, kidneys and gall bladders, typical sites of myxosporean1016

infection.We revealed 53 different myxozoan species using SSU rDNA sequencing. Se-1017

venteen SSU rDNA sequences were identical with sequences available in GenBank and1018

36 sequences belong to newly identified myxozoan taxa. Interestingly, not only large1019

dams as Římov and Švihov contain high number of myxozoans (e.g. 19 myxosporeans1020

in 8 screened fish species in Švihov) but also small brook Hostačovka is home for 91021

myxosporeans based only on three screened fish species, or six fish species from a very1022

small pond are host for 16 myxosporean species. Rutilus rutilus was the most infected1023

fish species with 8 recognised myxosporeans. Diversity of the Myxozoa is highly unde-1024

restimated even in the areas of long tradition of myxozoan research. We are currently1025

working on environmental DNA sequencing trying to detect DNA of myxozoan spores1026

in the water and water sediments that would enable us more efficiently assess the true1027

myxozoan diversity.1028

1029

63


Where are we with Diplonemids and where do we want to1030

go?1031

Julius Lukeš1, Drahomíra Faktorová, Olga Flegontova, Aleš Horák, Binnypreet1032

Kaur, Galina Prokopchuk, Ingrid Škodová-Sveraková, Daria Tashyreva, Kristína Zá-1033

honová1034


1036

Diplonemids are heterotrophic flagellates recently found to be the most species-1037

-rich group of marine protists. We are interested to shed light on their evolution, diver-1038

sity, life styles, ultrastructure, as well as DNA and RNA blueprints of their nucleus and1039

mitochondrion. Moreover, we also aim to bring them to the attention of a wider commu-1040

nity by establishing protocols for their genetic transformation. In my talk, I will review1041

the data obtained by us so far and outline future research directions, with the hope to1042

instigate a discussion with the community about the most interesting/promising lines1043

of research of these fascinating hyperdiverse marine flagellates.1044

1045

64

Abstracts

Overview of the First Two Chloroplast Genomes of Dicty-1046

ochophyceae (Ochrophyta)1047

Kacper Maciszewski, & Anna Karnkowska1048



1051

Dictyochophyceae (silicoflagellates) are a group of unicellular marine organisms1052

within Ochrophyta, possessing plastids acquired through secondary endosymbiosis. Al-1053

though there have been numerous reports of their presence in marine waters around1054

the world, these organisms have not been common research targets in genomics and1055

thus far, no nuclear or organellar genome of any member of this group has been sequen-1056

ced. Here we present the results of sequencing, assembly and functional annotation of1057

cpGenomes of two silicoflagellate strains: Florenciella parvula (Florenciellaceae) and1058

Pseudopedinella elastica (Pedinellaceae). The cpGenomes of these two species seemed1059

highly similar at first, with genetic content differing only by four protein-coding ge-1060

nes and both genomes possessing a quadripartite structure. A more thorough analysis,1061

however, revealed some interesting features present in both genomes, some of which1062

were unique to each of the investigated strains, e.g., a tic20 -derived pseudogene in both1063

examined genomes or a 150 nt deletion in one of the two 16S rRNA genes in P. elastica.1064

Some of conventional plastid genes were found to be missing from both genomes, from1065

which we deduced that they might have been transferred to the nucleus. By referring1066

to transcriptome of F. parvula available in a public database, we confirmed that most1067

of the missing genes are indeed nucleus-encoded.The next step of our research will be1068

reconstruction of Ochrophyta phylogeny based on plastid-encoded genes. The results1069

presented here will also be a basis for further research in the evolution of cpGenomes1070

of silicoflagellates, including investigation of the genetic background of secondary loss1071

of photosynthesis in species belonging to this group. Moreover, we believe that our1072

data may provide important insight into the evolutionary history of Pelagophyceae (si-1073

ster clade to Dictyochophyceae), as well as haptophytes, which are assumed to possess1074

plastids of silicoflagellate-related origin.1075

1076

65


Detection and Removal of Cross–Contaminations from1077

Transcriptome Sequencing Projects1078

S. Nenarokov1, F. Burki2, D. J. Richter3, M. Kolisko1, & P. J. Keeling41079


2Department of Organismal Biology, Uppsala University, Uppsala, Sweden1081

3Institut de Biologia Evolutiva (CSIC–UPF), Barcelona, Spain1082

4Department of Botany, University of British Columbia, Vancouver, Canada1083

1084

The low cost of next generation sequencing (NGS) allowed affordable and straight1085

forward sequencing of non-model organisms on a large scale. NGS techniques have1086

become a standard for generating transcriptomic and genomic data and it is very1087

common, especially in protistology, for a laboratory to sequence in parallel several1088

different species at a time. Such parallel sequencing commonly leads to a small amount1089

of cross–contamination and even a miniscule contamination is likely to be present in the1090

resulting dataset due to the extremely deep coverage generated by NGS methods. Cross-1091

-contamination can arise from both the research laboratory and the sequencing centre.1092

This is especially problematic for transcriptome sequencing projects in which there is1093

no genomic context to confirm the true origin of each assembled sequence. We have1094

developed a software tool for detecting and removing cross-contaminated contigs from1095

assembled transcriptomes. The program uses BLAST to identify suspicious contigs and1096

RPKM values to sort these as either correct or contamination. Through adjustment of1097

the parameters, it also allows for decontamination of species which are taxonomically1098

close or if they are associated by a predator-prey relationship. To demonstrate the1099

effectiveness of our software, we successfully identified cross–contaminations within1100

the ∼700 transcriptomes generated by the Marine Microbial Eukaryote Transcriptome1101

Sequencing Project (MMETSP) datasets (MOORE foundation) and generated clean1102

datasets.1103

1104

66

Abstracts

Genomics of Blastocrithidia, the Trypanosomatid with All1105

Three Stop Codons Reassigned1106

Anna Nenarokova1, 2, Kristína Záhonová1, 3, 4, Serafim Nenarokov1, Vyacheslav Yur-1107

chenko3, 4, & Julius Lukeš1, 21108





1113

Recently, two groups of protists bewildered molecular biologists: trypanosomatid1114

Blastocrithidia and several ciliate species were shown to reassign all three stop codons1115

for encoding amino acids. In these organisms at least one stop codon has ambiguous1116

meaning: it acts as a sense codon in some cases and as a termination codon in the1117

other. This finding challenges the established view of protein synthesis termination,1118

one of the most basic cellular processes. However, the mechanisms of this reassignment1119

and translation termination in such systems still remain speculative. Blastocrithidia1120

represents an ideal model system for studying this phenomenon. It belongs to kine-1121

toplastids, a well-studied protist group, which include model objects, such as Trypa-1122

nosoma and Leishmania, with available complete genomes and established laboratory1123

methods and techniques. Unlike ciliates, which are well-known for stop codon reassig-1124

nment, all known kinetoplastids aside from Blastocrithidia genus have the canonical1125

nuclear genetic code. Thus, looking to this lineage, we can trace the main steps leading1126

to the emergence of such system. Here, we have sequenced and analyzed genomes of1127

two cultivable Blastocrithidia species and Leptomonas jaculum, the closest relative of1128

Blastocrithidia with the canonical genetic code. We have created a new software for1129

annotation of Blastocrithidia genome, as existing annotation programs are not able to1130

deal with ambiguous stop codons. This allowed us to look at the reassigned stop codons1131

from a wider perspective to see the general trends in their features and distribution.1132

The ultimate goal of our study is to address the following intriguing questions: How1133

does translation termination function without defined stop codons? How do numerous1134

reassigned stop codons influence translation? What are the prerequisites of this lineage1135

that made the reassignment possible? What could be the intermediate steps between1136

a system with a standard genetic code and one with all three stop codons reassigned?1137

1138

67


Metabolism and Cell Biology of Preaxostyla Flagellates: A1139

Comparative Genomic Study1140

Lukáš V. F. Novák1, Sebastian C. Treitli1, Anna Karnkowska2, & Vladimír Hampl11141



2University of Warsaw, Department of Molecular Phylogenetics and Evolution, Warsaw, Poland1144

1145

The least studied of the three major lineages of metamonads, evolutionary and1146

parasitologically important group of anaerobic protists, is Preaxostyla, a taxon which1147

has recently attracted attention of the protistological community when Monocercomo-1148

noides exilis was identified as the first known completely amitochondriate eukaryote.1149

We have sequenced, assembled, and annotated genomes of two other members of Prea-1150

xostyla. Blattamonas nauphoetae is morphologically almost indistinguishable from M.1151

exilis but represents a phylogenetically distinct lineage. It also differs in the lifestyle as1152

M. exilis inhabits guts of rodents, while B. nauphoetae is a symbiont of cockroaches.1153

Paratrimastix pyriformis, a bacteriovorous flagellate with typical excavate morphology,1154

is one of the closest free-living relatives of M. exilis and B. nauphoetae. Unlike them, it1155

still retains a reduced mitochondrion. Comparisons of multiple cellular systems, with1156

emphasis on energy, amino acid, and mitochondrial metabolism, between M. exilis, B.1157

nauphoetae, and P. pyriformis will be presented.1158

1159

68

Abstracts

Investigating the Molecules, Sequences and Mechanisms In-1160

volved in the Cryptic Plastid Protein Import of Euglena1161

gracilis1162

Anna M. G. Novák Vanclová, & Vladimír Hampl1163


1165

Euglena gracilis is a facultatively phototrophic flagellate belonging to Eugleno-1166

zoa within the Excavata paraphylum. It used to be a very popular model organism in1167

the past and it is gaining relevance again today as its capacity to synthesize various1168

chemical compounds usable in biofuel industry or pharmacology is being investiga-1169

ted.Phototrophic euglenids are endowed with secondary plastids derived from those of1170

a Pyramimonas-related endosymbiont. These organelles are enveloped by three mem-1171

branes, as opposed to most other secondary plastids which are generally equipped with1172

four membranes: two of these inherited from the cyanobacteria, one from endosymbio-1173

tic primary alga, and one from the endomembraneous system of the final host. Which1174

one of these membranes is the one missing in euglenid plastid was not confirmed yet,1175

neither was the protein composition or transport system of the remaining three inves-1176

tigated very thoroughly. analyses as well as gene silencing via RNA interference were1177

used to obtain more hints regarding the functions of these candidates and their roles in1178

plastid protein import. A set of protein sequences with highly credible plastidal locali-1179

zation determined by mass spectrometry was used for the analysis of plastid-targeting1180

signals which might be both sequence- and structure-coded, and for the optimization.1181

1182

69


Nosema bombycis (Microsporidia), a Model for the Biolo-1183

gical Nanotube1184

Markéta Petrů, Alžběta Krupičková, & Pavel Doležal1185

Charles University, Faculty of Science, Department of Parasitology, Vestec1186

1187

Microsporidia are obligate intracellular parasites closely relative to fungi. First1188

known as causative agent of pébrine, disease of the silkworms devastating silk industry,1189

but able to infect broad spectrum of animal hosts. Outside the host, the parasite occurs1190

only as very resistant spore whose entire interior is highly adapted to infection. The1191

anchoring disk, polar tube, polaroplast and posterior vacuole are structures within1192

sporoplasm, specific to Microsporidia and their infection process. The polar tube has1193

become the center of our interest. It is a hollow tube, through which sporoplasm passes1194

to the host, a biological nanotube unparalleled in the nature. Even though many ul-1195

trastructure studies do exist, it is still not known, what the building blocks of the tube1196

are and how they combine during spore maturation. We have established life cycle of1197

Nosema bombycis in our laboratory and started with experiments which will hopefully1198

allow us to understand more to polar tube phenomena.1199

1200

70

Abstracts

A Phylogenetically Broad Analysis of Protist Genomes Un-1201

veils the Ancestral Eukaryotic Complexity of the Ras Su-1202

perfamily of GTPases and Novel Aspects of Eukaryotic Cell1203

Biology1204

Romana Petrželková, & Marek Eliáš1205

University of Ostrava, Faculty of Science, Department of Biology and Ecology, Ostrava1206

1207

The highly diversified Ras superfamily of GTPases is one of the central compo-1208

nents of the molecular pathways underpinning the basic logistics in the eukaryotic cell.1209

Different eukaryotic groups may differ substantially in the complexity of their com-1210

plements of Ras superfamily paralogs due to lineage–specific duplications and losses,1211

but it is clear this diversity stems from a certain number of ancestral paralogs that1212

define the core cell biology of a prototypical eukaryotic cell. We have been engaged in1213

a long–term project to reconstruct the evolutionary history of the Ras superfamily in1214

eukaryotes, with a particular aim to define the actual set of paralogs that can be traced1215

to the last eukaryotic common ancestor (LECA). The accumulation of genomic and1216

transcriptomic data from phylogenetically diverse eukaryotes, particularly protists, has1217

now enabled to draw a picture of the LECA's complement of Ras superfamily paralogs.1218

The LECA seems to have been endowed with up to around 60 different proteins of the1219

Ras superfamily, which is a number substantially exceeding previous estimates. Whe-1220

reas some of these ancestral paralogs have ever since remained an essential component1221

of the eukaryotic cell, others have experienced more or less frequent losses. A notable1222

category are paralogs correlated in their distribution with the capability of the orga-1223

nism to build a cilium. It includes not only well established cilium–associated GTPases,1224

but also some paralogs hitherto lacking a clear functional assignment. Our analyses for1225

the first time show wider taxonomic occurrence and apparent ancestral origin of some1226

GTPases so far reported only from metazoans, and unveil novel, functionally uncha-1227

racterized ancestral paralogs with a sporadic distribution avoiding standard model1228

organisms. These GTPases presumably indicate the existence of unknown functional1229

pathways in the prototypical eukaryotic cell, making their study one of the priorities1230

of evolutionary cell biology.1231

1232

71


Bicistronic Protein Expression in Leishmania mexicana1233

Lucie Podešvová1, Natalya Kraeva1, & Vyacheslav Yurchenko1, 21234



1237

The genus Leishmania unites parasitic protists of the family Trypanosomatidae1238

causing leishmaniases, closely related diseases that affect human and animal populati-1239

ons mainly in the tropical and subtropical regions. The clinical manifestations vary1240

from spontaneously healing skin lesions to progressive and possibly fatal visceral in-1241

fections. Leishmaniases represent a global health problem with over 500 million people1242

at risk and an annual incidence rate of 5–10 million worldwide.1243

Several molecular tools have been developed in recent years to study Leishma-1244

nia mexicana, a causative parasite of cutaneous leishmaniasis. These methods have1245

greatly extended knowledge concerning functions of numerous genes and their associ-1246

ation to Leishmania virulence. One of such approaches relies on T7 polymerase-driven,1247

Tetracycline-inducible gene expression (1). The main limitation of this system was its1248

unsuitableness for developmental studies, due to the high impact the untranslated regi-1249

ons (UTRs), flanking both the gene of interest and T7 polymerase, have on the mRNA1250

levels (2).1251

Here, we report a novel system overcoming limitations of using exogenous UTRs.1252

It is based on the 2A self-cleaving peptide, derived from the Porcine teschovirus-1. This1253

approach enables simultaneous production of two separate proteins located upstream1254

and downstream from its sequence. Importantly, protein expression is regulated by1255

endogenous UTRs, thus allowing studying the protein function in cases which require1256

its stable expression, e.g. during the life cycle of Leishmania. It can also be used for1257

investigation of metacyclic- or amastigote-specific proteins.1258

Acknowledgment:1259



References:1262

1. Kraeva, N., Ishemgulova, A., Lukeš, J., Yurchenko, V. Tetracycline-inducible gene ex-1263

pression system in Leishmania mexicana. Mol. Biochem. Parasitol., 2014, 198: 11-13.1264

2. Ishemgulova, A., Kraeva, N., Faktorová, D., Podešvová, L., Lukeš, J., Yurchenko, V.1265

T7 polymerase-driven transcription is downregulated in metacyclic promastigote and1266

amastigote Leishmania mexicana. Folia Parasitol., 2016, 63: 016.1267

1268

72

Abstracts

The Diversity of Anaerobic Ciliates (Scuticociliatia, Oligo-1269

hymenophorea) and Their Ecologically Important Symbio-1270

tic Prokaryotes1271

Kateřina Poláková, Johana Rotterová, & Ivan Čepička1272

Charles University, Faculty of Science, Department of Zoology, Praha1273

1274

Anaerobic ciliates are important protists inhabiting anoxic marine and freshwater1275

sediments, yet we still know a little about their diversity. A common feature of anaerobic1276

ciliates is to host ecto- and endosymbiotic prokaryotes. As a model group for studying1277

mutualistic relationships in anoxic environments, we have chosen anaerobic ciliates from1278

the subclass Scuticociliatia (class Oligohymenophorea). They are common in oxygen-1279

-depleted environments, especially in marine habitats. Yet, present knowledge about the1280

molecular diversity of anaerobic scuticociliates is based almost solely on environmental1281

data. We are successfully maintaining over 30 strains of mostly marine representatives1282

in long-term cultures. By analyzing their 18S rRNA gene sequences, we have shown1283

that anaerobic scuticociliates constitute a novel diverse clade and thus represent an1284

important fraction of the overall diversity of scuticociliates.Although symbioses with1285

prokaryotes are common among anaerobic representatives, we lack information about1286

the true nature of the interactions. Importantly, symbioses with methanogenic Archaea1287

are widespread among our cultured anaerobic scuticociliates. Although methanogenic1288

endosymbionts were described only in a few freshwater species, we confirmed their1289

presence also in our marine strains. In addition, we noticed a common presence of1290

ectosymbiotic prokaryotes living on the host cell surface. Interestingly, it seems that the1291

presence/absence of the ectosymbionts is ciliate-lineage-dependent. According to our1292

preliminary results from CARD-FISH method, the ectosymbionts are sulfate-reducing1293

bacteria. Thanks to maintaining many anaerobic strains in long-term cultures and1294

the persistence of various symbioses in the culture, we have a great opportunity to1295

study the symbioses in detail. Further research can provide a new insight into the1296

evolution of protists living in poorly studied anoxic environments and their symbioses1297

with prokaryotes.1298

1299

73


Novel Lineage of Non-Photosynthetic Chlamydomonadales1300

with Peculiar Plastid Genome1301

Tomáš Pánek1, Kristýna Záhonová1, Naoji Yubuki2, 4, Eliška Zadrobílková2, Sebas-1302

tian Cristian Treitli3, Vyacheslav Yurchenko1, Ivan Čepička2, & Marek Eliáš11303




4Université Paris–Sud, Unité d'Ecologie, Systematique et Evolution, Orsay, France1307

1308

Colourless genera of algal group Chlamydomonadales (Archaeplastida: Chloro-1309

phyta) constitute at least three unrelated lineages representing independent losses of1310

photosynthesis. We investigated two new strains of non-photosynthetic chlamydomo-1311

nadid algae (AMAZONIE, MBURUCU) that were isolated from microoxic freshwater1312

sediments in South America. Phylogenetic analysis of their 18S rRNA gene showed1313

that both strains are closely related. However, they represent a separate deep clade of1314

Chlamydomonadales and the fourth independently arisen non-photosynthetic lineage1315

within the group. Morphological differences together with several compensatory base1316

changes in the ITS2 rDNA region indicate that AMAZONIE and MBURUCU repre-1317

sent two different species of a new genus. Using the Illumina sequencing we obtained1318

genomic and transcriptomic data from the AMAZONIE strain. Searching for plastid1319

genome sequences returned 34 genes encoding plastid proteins, none of them with a1320

photosynthesis-specific function. The genes were distributed on separate contigs with1321

each gene flanked by repetitive non-coding regions. The plastid genome of the AMA-1322

ZONIE strain may thus be organized similarly to the recently characterized inflated1323

repeat-rich plastid genome of Polytoma uvella, or it may alternatively consist of indi-1324

vidual single-gene \minichromosomes" with the terminal repeats serving as telomeres.1325

To distinguish between the two possibilities we generated long-read sequencing data1326

using the Oxford Nanopore technology. The yield of plastid genome-derived data was1327

low due to heavy bacterial contamination of the culture, but enabled us to assemble1328

a contig of 22,875 bp containing two protein-coding genes (rpoC1 and atpB) and two1329

tRNA genes, providing the first evidence that the AMAZONIE plastid genome might1330

be unsegmented. The length of non-coding repeat-rich regions separating the genes1331

suggests that the whole plastid genome may be even more inflated than that of P.1332

uvella.1333

Acknowledgment:1334



1337

74

Abstracts

Representants of the Green Algal Genus Pseudodictyochlo-1338

ris in Arctic – it is possible?1339

Lenka Raabová1, & Ľubomir Kováčik21340

1University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Bio-1341

logy, Trnava1342

2University of Comenius in Bratislava, Faculty of Natural Sciences, Department of Botany, Bratislava1343

1344

Genus Pseudodictyochloris was originally described by Vinatzer in 1975 from Ti-1345

rolia in Austria based on morphological properties. This genus contains two species P.1346

dissecta Vinatzer (type species) and P. multinucleata (Broady) Ettl & Gärtner. The1347

morphology of the both species is very similar with solitary, oval, round or lemon–like1348

cells with pale green color. Cells are multinucleate and reproduce by motile zoospores.1349

Nowadays, this genus is usually recorded from soils samples in Antarctic and occasio-1350

nally from Bulgaria and Russia as well. In our study we isolated from Arctic samples1351

the tree strains of green algae, which according the morphological features are fit to1352

description of P. multinucleata. Our strains form the compact of bright green clusters1353

on agar plate and in liquid medium. Cells were studied by fluorescens microscopy also1354

and the nucleus visualized by DAPI. The strains were analyzed by molecular methods1355

also. Based on 18S rRNA gene a phylogenetic tree was constructed, which showed a1356

similarity with the genus Chloromonas. Genus Pseudodictyochloris weren't study by1357

molecular tools yet, so the sequences of this genus can't be find in GenBank. Based on1358

our analyses, we expect, that it is possible, that Arctic strains of Pseudodictyochloris1359

can be in fact the Chloromonas species. Further analysis of their taxonomic position1360

is in progress.1361

1362

75


Dynamic Secretome of Trichomonas vaginalis: Case Study1363

of β-amylases1364

Jitka Štáfková1, Petr Rada1, Dionigia Meloni1, Vojtěch Žárský1, Tamara Smutná1,1365

Nadine Zimmann1, Karel Harant1, Petr Pompach2, 3, Ivan Hrdý1, & Jan Tachezy11366




versity, Institute of Biotechnology, Vestec1370

3Charles University, Faculty of Science, Department of Biochemistry, Praha1371

1372

Glucose is an essential nutrient for human parasite Trichomonas vaginalis to gene-1373

rate ATP via anaerobic fermentation in the cytosol and extended glycolytic pathways1374

in hydrogenosomes. In vaginal fluids, the main source of glucose is likely free glyco-1375

gen derived from vaginal epithelial cells. To be utilized by T. vaginalis, glycogen and1376

glucose-containing polymers needs to be extracellularly digested to monomeric glucose1377

that is transported into the cells. Glycogen hydrolysis is catalyzed by various enzymes1378

of which exo-acting β-amylases hydrolyzes α-1,4-linkages of glycogen from the non-redu-1379

cing end liberating β-maltose. Next α-glucosidase activity of T. vaginalis can hydrolyze1380

maltose to glucose. To get more insight into β-amylases distribution, we search for β-1381

-amylase coding genes across eukaryotic supergroups. In addition to T. vaginalis, in1382

which we identified 4 genes for β-amylase (BA1–4), we found orthologous genes in re-1383

lated bovine pathogen Tritrichomonas foetus, and in Nagleria gruberi. β-amylases are1384

also common in land plants, and Amoebozoa group, whereas animals and fungi seem to1385

be devoid of β-amylases. Next we were interested whether T. vaginalis β-amalyses are1386

secreted by classical or non-classical secretory pathway. The co-expression of ER-lo-1387

calized biotine ligase (BirA) and acceptor peptide tagged BA1-4 revealed that BA1–31388

pass via classical secretory pathway and they are release to the cell environment. This1389

process is inhibited by brefeldin A. In ER, BA-1 appeared to be heavily glycosylated1390

with Asn-linked GlcNAc2Man5. Interestingly, BA4 is trapped in ER and is not secre-1391

ted. Incubation of T. vaginalis under various environmental conditions revealed that1392

presence of glycogen and iron regulate β-amylase gene expression. Our data indicate1393

that β-amylases are novel important members of T. vaginalis secretome.1394

1395

76

Abstracts

A Novel Bacterial Cell Division Protein ZapE and its Role1396

in the Mitochondrion of Trypanosoma brucei1397

Vendula Rašková1, 2, Jan Pyrih1, & Julius Lukeš1, 21398



1401

Trypanosoma brucei is an important model organism, especially thanks to1402

unique features, such as kinetoplast DNA, trans-splicing and RNA editing. Re-1403

cently, we have identified in its genomes two genes encoding proteins homologous to1404

ZapE/AFG-1/LACE1. This protein is likely involved in the division of bacteria and1405

may participate in maintaining the mitochondrial integrity, as its inactivation leads1406

to an elongated phenotype. Moreover, it was demonstrated that this protein is part1407

of the FtsZ ring (the so-called Z-ring), which facilitates the cell division in bacteria.1408

The overall presence of ZapE homologs in mitochondria of eukaryotes is surprising,1409

since other components of the FtsZ ring are absent with only a handful of exceptions.1410

We assume that these proteins can possibly represent the link between bacterial and1411

mitochondrial type of division. Using in situ tagging, we found that both proteins are1412

localized in the mitochondrion of T. brucei. We also created single RNAi knock-downs1413

for both genes which, however, are so far without a notable phenotype. Since they may1414

substitute each other, we are in process of generating a double RNAi knock-down cell1415

line.1416

1417

77


The First Phylogenomic Analysis of Free-living Anaerobic1418

Ciliates within SAL Super-group (Ciliophora)1419

Johana Rotterová1, Roxanne Beinart2, William Bourland3, Petr Táborský4, Virgi-1420

nia P. Edgcomb5, Martin Kolísko4, & Ivan Čepička11421


2University of Rhode Island, Rhode Island, USA1423

3Boise State University, Boise, Idaho, USA1424


5Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA1426

1427

Ciliates, possibly the most studied group of protists, are well known for their1428

various symbiotic relationships as either symbionts or hosts, as well as their ability1429

to inhabit the most diverse environments, including extreme biotopes such as ano-1430

xic sediments. And yet, there are lineages that have been heavily overlooked despite1431

their ecological importance and cosmopolite distribution. While mapping the diver-1432

sity of anaerobic ciliates within the SAL (Spirotrichea, Armophorea, and Litostoma-1433

tea) group, we have enriched the known diversity of Armophorea and discovered two1434

new deep lineages of marine anaerobic ciliates. Here, we present the first phylogeno-1435

mic analysis of anaerobic ciliates within the SAL group. In the previous studies, the1436

only phylogenomic data available for anaerobic cili ates wit hin SAL group were from1437

the endobiotic Nyctotherus ovalis. We have sequenced metagenomes of four species1438

of Metopida (Armophorea) and one species of the novel marine lineage, as well as a1439

transcriptome of one species of the other novel ciliate lineage. We have performed pilot1440

studies on the energy metabolism and characterization of metabolic pathways from the1441

transcriptome. Furthermore, we studied the MROs (mitochondrion related organelles)1442

present in these organisms and found that the MROs of several studied lineages ap-1443

pear to have a genome. In addition, we studied various prokaryotic symbionts, hosted1444

by the vast majority of the studied ciliate taxa, using autofluorescence, FISH, and1445

CARD – FISH methods, and we discovered that at least some of the endosymbionts as1446

well as ectosymbionts are methanogenic Archaea, while others host sulphate-reducing1447

deltaproteobacteria as ectosymbionts.1448

1449

78

Abstracts

The Evolution of Aminoacyl-tRNA Synthetases in Chrome-1450

rids1451

Abdoallah Sharaf1, 2, Kateřina Jiroutová1, & Miroslav Oborník1, 31452


2Genetic Dept. Faculty of Agriculture, Ain Shams University, Cairo, 11241, Egypt1454

3University of South Bohemia, České Budějovice1455

1456

Aminoacyl–tRNA synthetases (aaRSs) are enzymes that catalyze the ligation of1457

tRNAs to their cognate amino acids. There are aaRSs specific to each of the 20 stan-1458

dard amino acids. These enzymes are divided into two classes, class I and class II,1459

which are unrelated in both sequence and structure. aaRSs can function in multiple1460

sub–cellular compartments due to the phenomenon of dual targeting. We searched1461

the total predicted proteins of Chromera velia and Vitrella brassicaformis for aaRSs1462

proteins. Localizations prediction of the identified genes were performed to test its mul-1463

tiple targeting hypothesis. In order to examine the complex evolutionary path of the1464

aminoacyl–tRNA synthetases, Phylogenetic analyses of all available 21 aaRSs sequences1465

were performed using maximum likelihood and Bayesian inference. Computer predicti-1466

ons of the intracellular location of the identified enzymes were performed to test the1467

multiple targeting hypothesis. Forty–eight genes encoding aaRS were identified in C.1468

velia, while only 39 aaRSs were found in V. brassicaformis. Forty-five percent of C.1469

velia's aaRss are encoded by three distinct loci, whereas 45% of aaRSs are encoded1470

by two distinct loci. Interestingly, gluRS is encoded by only one locus. In contrast,1471

85% of the V. brassicaformis aaRSs are encoded by just two distinct loci and only1472

pheRS is encoded by three distinct loci. Most of the molecular phylogenies of aaRSs1473

indicate that for each aaRS the evolutionary pattern is different and eukaryotic genes1474

are usually retained. Targeting predictions show that particular enzymes are not often1475

used in the compartments where they originate. The results of this study provide the1476

first report of aaRSs, its multiple targeting and evolution in chromerids, As a first step1477

toward a more nuanced understanding of protein targeting in these complex algae.1478

1479

79


Gene Transfer Accompanying the Secondary Endosymbiosis1480

of Euglenid Plastid1481

Petr Soukal1, Štěpánka Hrdá1, Anna Vanclová1, Naoji Yubuki1, Marek Eliáš2, &1482

Vladimír Hampl11483



1486

Autotrophic euglenids (Euglenophyta) form a monophyletic group with secondary1487

green plastids, which were most probably acquired by their common ancestor. However,1488

the acquisition of the plastid earlier in the evolution of euglenids (plastid-early hypo-1489

thesis) cannot be ruled out. The process of organelle acquisition is accompanied by the1490

transfer of genes from the endosymbiont to host (EGT), the presence of such genes1491

could indicate past endosymbiosis. To test the plastid-early hypothesis and to learn1492

more about the contribution of EGT to euglenid genome, we have analyzed transcrip-1493

tomes of 5 euglenids (2 osmotrophic, 3 autotrophic) using an automatic pipeline, which1494

enabled us to select genes related to algae. The contribution of algal genes in autot-1495

rophic euglenids (around 2% of genes) is higher than in primary osmotrophs (around1496

0.07%) supporting the plastid-late hypothesis. Surprisingly, we observed a higher num-1497

ber of genes related to secondary red algal groups than green algae.1498

1499

80

Abstracts

Trypanosomes of Freshwater Fish: Diversity and Specificity1500

Viktoria Spodareva1, 2, Alexei Kostygov1, 2, Hana Pecková3, Astrid Holzer3, Julius1501

Lukeš3, 4, & Vyacheslav Yurchenko1, 3, 51502






1508

Trypanosomes of freshwater fishes is a poorly studied group. About 200 species1509

were described, but it was mostly in the pre–molecular era when discrimination of spe-1510

cies was based on hosts and rough morphology seen with light microscope. However,1511

the morphology of trypanosomes is variable due to pleomorphism (changes during the1512

life cycle). Thus, morphological differences between previously described species can1513

be misleading. Cross–infection experiments demonstrated that one species of trypano-1514

somes is capable of infecting several species of fishes. Therefore, classification based1515

on host specificity is also unreliable. Recent attempts to probe the diversity of fish1516

trypanosomes showed that one host species and even one individual could be infec-1517

ted with more than one species of trypanosomes. We analyzed the collection of fish1518

trypanosome cultures (97 strains from 17 fish species) isolated from ponds and rivers1519

of Central Europe and preserved in the Institute of Parasitology (České Budějovice).1520

Direct sequencing of 18S rRNA gene showed that there are only five species present1521

in different proportions. The number of trypanosomes species infecting freshwater fish1522

in the studied region is quite modest. We identified only five and some of them were1523

found in many unrelated hosts. However, in two trypanosome species we observed limi-1524

ted host range, which points to some specificity, probably related to hosts physiology.1525

All samples that were experimentally passed through goldfish upon their isolation from1526

various hosts revealed to be one species. This may be indicative of selective pressure1527

in goldfish. In addition, the extensive sampling allowed us to find a rare species, which1528

was found only in one isolate and had never been documented before.1529

Acknowledgment:1530



1533

81


Diplonema papillatum – The Master of Adaptation1534

Ingrid Sveráková1, 2, Martina Džubanová1, Anton Horváth1, & Július Lukeš21535

1Comenius University, Faculty of Natural Sciences, Bratislava1536


1538

Diplonema papillatum is the most studied species of diplonemids, which are among1539

the most abundant and diverse heterotrophic eukaryotes in the world's ocean. Diplo-1540

nema is capable to survive under very diverse conditions. Amino acids are preferred1541

carbon source of energy even if glucose is present, what reminds the situation in pro-1542

cyclic form of Trypanosoma brucei, the kinetoplastid parasite belonging to the sister1543

group of diplonemids. Cultures cultivated in rich media show only small rate of oxygen1544

consumption and low activities of enzymes of respiratory chain. On the other hand,1545

short-term starvation accelerates the oxygen consumption and it is accompanied by1546

increasing activity of all respiratory enzymes. Switching cultivation from rich to mini-1547

mal medium containing sea salt only, does not change the motility of cells. The only1548

visual change is depletion of some vesicles from cytoplasm. Our data suggest that the1549

vanishing vesicles may represent the storage polysaccharides, which are degraded under1550

conditions of low carbon source.1551

1552

82

Abstracts

Life Cycle, Ultrastructure and Phylogeny of New Diplone-1553

mids1554

Daria Tashyreva1, Galina Prokopchuk1, Jan Votýpka1, 2, Akinori Yabuki3, Aleš Ho-1555

rák1, 4, Binnypreet Kaur1, 4, Drahomíra Faktorová1, 4, & Julius Lukeš1, 41556



3Department of Marine Diversity, Japan Agency for Marine-Earth Science and Technology, Yoko-1559

suka, Japan1560


1562

Diplonemids are colorless heterotrophic, predominantly marine flagellates belon-1563

ging to Euglenozoa. For a long time, they were considered as a small and rare group of1564

protists. However, recent global-scale metabarcoding survey revealed that, with over1565

45,000 OTUs, diplonemids qualify as the most species-rich marine planktonic eukaryo-1566

tes. Until recently, only four diplonemid species were sequenced and formally described,1567

whereas nearly all other representatives are known only by a short V9 region of their 18S1568

rRNA gene. In the frame of our project, we isolated into axenic cultures and described1569

seven new species of diplonemids, four of which fell within Diplonema and Rhyncho-1570

pus genera, while for three other novel species, we established new genera Flectonema,1571

Lacrimia and Sulcionema. The newly described species display striking resemblance of1572

morphological and ultrastructural traits with previously known diplonemids. Yet, some1573

new diplonemids contain several unique features such as complex life cycle consisting1574

of trophic and swimming stages, which dramatically differ in motility and structure of1575

their flagellum and the presence of tubular extrusomes. In addition, new representati-1576

ves of the genus Diplonema established endosymbiosis with bacteria, a rare occurrence1577

among Euglenozoa and the first report from diplonemids. These bacteria constitute a1578

novel branch within Holosporales (α-proteobacteria), common endosymbionts of cilia-1579

tes and amoebas. Remarkably, endosymbionts reside both in the cytoplasm and the1580

mitochondrion, which is an extremely rare case.1581

1582

83


Tracking Ingest Glycine via Labelled Isotope and Metabo-1583

lomics to Show Mixotrophy in Chromera velia, an Apicom-1584

plexan Cousin1585

Ivana Schneedorferová1, 2, Aleš Tomčala1, Iva Opekarová3, 4, Jaromír Cihlář1, 2, &1586

Miroslav Oborník1, 2, 51587

1Biology Centre ASCR, v. v. i., Institute of Parasitology, Laboratory of Evolutionary Protistology,1588

České Budějovice1589


3Biology Centre ASCR, v.v.i., Institute of Entomology, Laboratory of Analytical Biochemistry, České1591

Budějovice1592

4University of Chemistry and Technology, Faculty of Food and Biochemical Technology, Department1593

of Chemistry of Natural Compounds, Praha1594

5ASCR, v. v. i., Institute of Microbiology, Třeboň1595

1596

Chromera velia is nowadays a well-known alga even it was isolated only ten years1597

ago. The popularity of this alga is caused mainly by its unique phylogenetic position1598

showing C. velia as the most related photosynthetic organism to parasitic phylum1599

Apicomplexa. The easy and rapid culturing of C. velia makes this alga great model for1600

studying elementary biochemical principals and helps to understand the evolutionary1601

shift from photosynthesis to parasitism. The means of liquid and gas chromatography1602

and mass spectrometry were used to the revealed essence of mixotrophy in C. velia.1603

Chemical analytical techniques were used for tracking the catabolism of glycine-1-13C1604

labelled glycine. The methodology describing the ratio of 13C incorporation to final1605

metabolic products was proposed and the speed of labelled glycine consumption and1606

13C fate were investigated. The catabolic biochemical pathway based on Kegg database1607

was proposed and the BLAST technique for the presence of particular genes responsible1608

for the involved enzymes was performed. Then the analyses were focused on the product1609

of the hem pathway – chlorophyll, free fatty acids, lipids and monosaccharides. The1610

experiment shows that labelled glycine was almost catabolized during 15 hours after1611

administration. The primary target of 13C was other amino acids and lipids, where1612

artificial 13C occurred within 7 hours after administration. Incorporation of 13C to the1613

chlorophyll was significantly recorded after 48 hours. Detail investigation of detected1614

monosaccharides revealed no presence of the artificial 13C atoms.1615

Acknowledgment:1616

This work was supported by Czech Science Foundation (P501-12-G055).1617

1618

84

Abstracts

Studying the Flagellar Tip of Trypanosoma brucei1619

Hana Váchová1, 2, Miroslava Šedinová1, Glenda Alquicer1, & Vladimír Varga11620

1ASCR, v. v. i., Institute of Molecular Genetics, Department of Cell Motility, Praha1621

2Charles University, Faculty of Science, Department of Developmental and Cell Biology, Praha1622

1623

Flagella are found on the surface of many eukaryotic cells. Their structure is very1624

complex and highly evolutionary conserved from protists to mammals. Various flagellar1625

structures, such as the basal bodies, the transition zone and the axoneme, have already1626

been studied in various organisms. However, there is a dearth of information on the1627

flagellar tip where the axonemal assembly takes place. We study the flagellar tip in1628

the protozoan Trypanosoma brucei, a causative agent of African sleeping sickness. T.1629

brucei is a highly experimentally tractable organism with the flagellum essential to1630

its life for movement, morphogenesis, cellular division and attachment to the tsetse1631

fly salivary gland epithelium. In addition, T. brucei offers an opportunity to study a1632

nascent and existing flagellum within one cell. To identify flagellum tip localizing pro-1633

teins, we combine the biochemical structure immunoprecipitation approach with data1634

from the TrypTag project (TrypTag.org), which aims to localize all proteins encoded1635

in the trypanosome genome. So far, we have identified over 20 previously unknown1636

tip localizing proteins. Interestingly, proteins with various tip localizations were found.1637

These include proteins localizing to the tips of both flagella, or the tip of the nascent1638

or the old flagellum, respectively. Moreover, different signal patterns such as a dot, a1639

rod, a horseshoe and a comet tail exist. We study whether the individual proteins are1640

structural or detergent–soluble, we characterize their turnover and phenotypes asso-1641

ciated with their depletion. With only a single structural tip protein characterized in1642

other eukaryotes, T. brucei appears as an ideal model to understand structures and1643

processes at the flagellar tip.1644

1645

85


Fe–S Cluster Assembly in Oxymonads and Related Protists1646

Vojtěch Vacek1, Lukáš V. F. Novák1, Sebastian Treitli1, Ivan Čepička2, Martin Ko-1647

lisko3, Patrick J. Keeling4, & Vladimír Hampl11648




4University of British Columbia, Department of Botany, Vancouver, Canada1652

1653

Oxymonad Monocercomonoides exilis was reported as the first eukaryote, which1654

has completely disposed mitochondrial compartment. It was proposed that an impor-1655

tant prerequisite for such a radical evolutionary step had been the acquisition of Fe-S1656

cluster assembly pathway SUF from prokaryotes. We have investigated available geno-1657

mic a transcriptomic data of six oxymonad species and their relatives composing the1658

group Preaxostyla (Metamonada, Excavata) for the presence and absence of enzymes1659

involved in Fe-S cluster biosynthesis. None possesses enzymes of mitochondrial ISC1660

pathway and all apparently possess the SUF pathway composed of SufB, C, D, S and1661

U proteins. This and the fact that the Preaxostyla proteins form a clade on the tree1662

suggests that the pathway was acquired in their common ancestor. The donor of genes1663

cannot be discerned by phylogenetic analyses but the presence of SufU suggests that1664

the pathway was probably acquired from a Gram–positive bacterium. Interestingly,1665

the proteins SufD, S and U tend to fuse in Preaxostyla in the sequential order, which1666

corresponds to the organization of the SUF operon in Firmucutes. Specifically, we ob-1667

served the SufDSU fusion in all three oxymonads with genomic data available, and the1668

SufSU fusion in Paratrimastix pyriformis. The inventory of the CIA pathway enzymes1669

is consistent with the rest of Metamonada indicating that the switch from ICS to SUF1670

did not markedly affect the downstream process of maturation of cytosolic and nuclear1671

Fe-S proteins.1672

1673

86

Abstracts

Calpains in the Phylum Euglenozoa1674

Dominika Vešelényiová1, Erik Birčák2, & Juraj Krajčovič11675

1University of ss. Cyril and Metodius, Faculty of Natural Sciences, Department of Biology, Trnava,1676

Slovakia1677

2Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava1678

1679

Calpain family is relatively large and contains cysteine proteases that are activa-1680

ted by calcium ions. Calpains are well evolutionary conserved. They have been found in1681

wide range of organisms, from bacteria, unicellular eukaryotes, to mammals, including1682

humans. Although calpains are well conserved, their distribution among organisms is1683

uneven. There have been 16 calpain–coding genes found in humans, in contrast to1684

plants, where only a single calpain have been found. Also, proteins that belong to1685

calpain family differ in many aspects, including their localization, domain structure,1686

concentration of calcium needed for their activation, and function. Calpains are essen-1687

tial for many processes, and their dysfunction leads to pathologies such as diabetes1688

type II, muscular dystrophy and many others. Our research focuses on calpains in1689

organisms from the phylum Euglenozoa (supergroup Excavata). Recent studies and1690

sequencing data predict, that the number of calpain–coding genes in organisms from1691

the phylum Euglenozoa is even higher than in mammals. Although the exact number,1692

structure or function of calpains in these single–celled organisms is still unclear. Using1693

hidden Markov models, we identified proteins that belong to calpain family. We built1694

our model based on the presence of calpain catalytic domain (CysPc), which is typical1695

for calpains, and it is responsible for their activity. By further in silico analysis we were1696

able to identify domains associated with CysPc domain. We identified great number of1697

domains, six of which have not been associated with calpain proteins before. To further1698

prove the presence of calpains in Euglenozoa, we conducted in vitro experiments, using1699

our model organisms Euglena gracilis and Euglena longa. We amplified the sequences1700

of catalytic domain, as well as complete open reading frames of selected calpains by1701

PCR. PCR products have been purified and sequenced.1702

1703

87


Dynamics of Giardia intestinalis Mitosomes1704

Luboš Voleman1, Pavla Tůmová2, & Pavel Doležal11705

1Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University, Vestec1706

2Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University and Ge-1707

neral University Hospital, Prague1708

1709

Mitosomes are the smallest evolutionary forms of mitochondria that evolved in1710

eukaryotes adapted to anaerobic environments. This adaptation manifests as the ab-1711

sence of the mitochondrial genome and vast majority of the mitochondrial proteome,1712

including the components of the mitochondrial division machinery. Here, we studied the1713

dynamics of mitosomes in the human parasite Giardia intestinalis during interphase1714

and mitosis and during differentiation into the cyst stage. We found that mitosomal1715

division is restricted to mitosis, when both central and peripheral organelles divide in1716

a unique and synchronized manner. During the segregation of the divided mitosomes,1717

the subset of the organelles between two G. intestinalis nuclei had a prominent role.1718

Surprisingly, despite the absence of the ERMES components, the division involves the1719

association of mitosomes with the endoplasmic reticulum, a relationship commonly seen1720

during the division of mammalian and fungal mitochondria. Moreover, the mitosome–1721

ER interface is occupied by lipid metabolism enzyme long chain acyl–CoA synthetase1722

4.1723

1724

88

Abstracts

Characteristics of Nonconventional Introns in Genomes of1725

Marine Diplonemids1726

Halszka Wysocka-Korzun1, Magdalena P lecha1, Anna Karnkowska1, Ryan Gawry-1727

luk2, Patrick J. Keeling2, & Rafa l Milanowski11728

1University of Warsaw, Faculty of Biology, Department of Molecular Phylogenetics and Evolution,1729


2University of British Columbia, Department of Botany, Vancouver, Canada1731

1732

Diplonemids are a group of flagellates within the Euglenozoa phylum. Marine di-1733

plonemids are abundant in the ocean waters, but they have been poorly investigated.1734

Recently, some light has been shed on their genetics as 10 diplonemid cells belonging1735

to 10 different, previously undocumented species were isolated from waters around the1736

coasts of California (USA). Single–cell sequencing and subsequent analysis of their ge-1737

nomes revealed that their genes contain nonconventional introns. Additionally, it has1738

been discovered that one of the introns in the tubA gene (in one of those diplone-1739

mids' cells) contains an open reading frame coding a protein with reverse transcriptase1740

domain, which may be responsible for the spread of the nonconventional introns throu-1741

ghout the genomes. In this study, we perform an in–depth analysis of genomic data of1742

these marine diplonemids, aiming to characterize the nonconventional introns. So far,1743

we have analyzed the distribution of introns in seven genes, examined the secondary1744

structure of introns and searched for open reading frames within these introns. 4001745

introns in 144 contigs were analyzed. Most of the introns formed a stable secondary1746

structure bringing together their ends, although they mostly did not conform to the1747

model of euglenids (close relatives of diplonemids) nonconventional introns. After closer1748

inspection, neither similarities in their sequences nor structure could be found. Most of1749

them were observed at the unique positions (heterologous), with only 37% sharing an1750

intron insertion site with other sequences (homologous), suggesting that most of them1751

are a new in occurrence. 2,411 contigs containing the sequence encoding RT domain1752

were found. The blastx search has shown that most of them contained at the same time1753

only the RT sequence and no other gene sequences. Most of the introns characterized1754

do not contain the RT coding sequence. Due to that we can conclude that the presence1755

of RT coding sequence in the tubA introns is rather an effect of retrotransposon inser-1756

tions and is not a remnant of the intron spread system, but given the incompleteness1757

of data, that possibility cannot be fully rejected.1758

1759

89


Are Mitosomes Truly Essential?1760

Natalia Wandyszewska, & Pavel Doležal1761


1763

Mitosomes are highly reduced forms of mitochondria, which evolved independently1764

during the evolution of several groups of organisms like diplomonads, amoebas and1765

microsporidia. Mitosomes are much smaller than mitochondria; do not possess respi-1766

ratory chain complex or Krebs cycle. The only pathway known to be still present in1767

mitosomes is the iron–sulfur cluster assembly pathway (ISC) and it has likely been1768

the main reason for preserving the organelles. Interestingly, recently an organism that1769

completely lost any form of mitochondria was discovered and it has been hypothesi-1770

zed that it was only possible due to the acquisition of alternative cytosolic iron–sulfur1771

cluster assembly pathway [Karnkowska et. al. 2016]. Therefore, we asked ourselves a1772

fundamental question: whether mitosomes are truly essential for Giardia.The growing1773

number of successful gene interruption in diverse protist groups using Cas9–based sys-1774

tems encouraged us to test the method in Giardia. Furthermore, recent introduction1775

of Cre/loxP system into Giardia genetic manipulation [Wampfler et.al. 2014] drama-1776

tically changes the toolbox available for research of this organism. In order to answer1777

the vital question of mitosomal necessity, we are to perform series of allele knock–ins1778

and knock–outs of three giardial mitosomal proteins: IscU, Tom40 and MPP using no-1779

vel Cre/loxP system and possibly combine it with Cas9. The Cre/loxP method enables1780

\recycling" of antibiotic resistance gene, thus allowing deletion of one allele at the time.1781

Perceived as an advantage, the method allows us to investigate not only the knock–out1782

phenotype but also establish what the minimal gene dosage is for tetraploid Giardia1783

necessary for the uncompromised growth.1784

1785

90

Abstracts

Insect Trypanosomatids in Papua New Guinea: High Ende-1786

mism and New Clades on the Tree1787

Anastasiia Grybchuk-Ieremenko1, Jan Votýpka2, 3, Julius Lukeš2, 4, Petr Kment5,1788

Alexei Yu. Kostygov1, & Vyacheslav Yurchenko1, 21789




4University of South Bohemia, České Budějovice1793

5National Museum, Department of Entomology, Prague1794

1795

The biologically extremely diverse archipelagos of Wallacea and Melanesia have1796

long stimulated ecologists and evolutionary biologists. However, the parasitic protists1797

in this geographic area remained neglected and no molecular analyses have been carried1798

out to understand the evolutionary patterns and relationship with their hosts. Papua1799

New Guinea (PNG) is a biodiversity hotspot containing over 5% of the world's bi-1800

odiversity in less than 1% of the total land area. In the current work, we analyzed1801

insect heteropteran hosts collected in PNG for the presence of trypanosomatid parasi-1802

tes. It is the first time, when the insect flagellates' diversity was analyzed East of the1803

Wallace's line, one of the most widely known biogeographic boundaries of the world.1804

Out of 907 specimens (23 different heteropteran families from eight localities) dissected1805

and analyzed, 137 (15% overall prevalence) were found to be infected by at least one1806

trypanosomatid species. High species diversity of captured Heteropteran hosts (1301807

species) correlates well with high diversity of the trypanosomatids. Of 58 trypanoso-1808

matid Typing Units (TUs) found in PNG insect hosts, only 9 were widespread, while1809

49 (84%) have never been documented elsewhere. The widespread TUs were found in1810

both widely distributed and endemic/sub–endemic insects. About half of the endemic1811

TUs were found in widespread host species and the rest – in endemic and sub-ende-1812

mic insects. However, we did not find any case of endemic TU being hosted by an1813

endemic family-group host taxon. The TUs from PNG form clades with conspicuous1814

host–parasite coevolution pattern, as well as those with remarkable lack of this trait. In1815

addition, our analysis revealed several new clades within the subfamily Leishmaniinae1816

and generic groups \jaculum" and Blastocrithidia.1817

1818

91


A Gene Transfer Event Suggests a Long-Term Partnership1819

between Eustigmatophyte Algae and a Novel Lineage of En-1820

dosymbiotic Bacteria1821

Tatiana Yurchenko1, 2, Tereza Ševčíková1, Pavel Přibyl3, Khalid El Karkouri4, Vla-1822

dimír Klimeš1, Raquel Amaral5, , Eunsoo Kim6, 7, & Marek Eliáš1, 21823

1University of Ostrava, Faculty of Science, Department of Biology and Ecology, Life Science Research1824

Centre, Ostrava1825


3Centre for Phycology and Biorefinery Research Centre of Competence, Institute of Botany CAS,1827

Dukelská 135, CZ–379 82 Třeboň1828

4Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63,1829

CNRS7278, IRD198, INSERMU1095, Institut Hospitalo–Universitaire Méditerranée–Infection, Aix–1830

Marseille Université, Faculté de Médecine, 27 boulevard Jean Moulin, 13385 Marseille cedex 5, France1831

5Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, 3000–4561832

Coimbra, Portugal1833

6Sackler Institute for Comparative Genomics, American Museum of Natural History, Central Park1834

West at 79th Street, New York, New York, 10024, USA1835

7Division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th1836

Street, New York, New York, 10024, USA1837

1838

Rickettsiales are obligate intracellular bacteria originally found in metazoans, but1839

more recently recognized as widespread endosymbionts of various protists. One genus1840

was detected also in several green algae, but reports on rickettsialean endosymbionts1841

in other algal groups are lacking. Here we show that several distantly related eustig-1842

matophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by1843

Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The1844

genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Tra-1845

chydiscus minutus CCALA 838, revealed genomic features (size, GC content, number1846

of genes) typical for other Rickettsiales, but some unusual aspects of the gene content1847

were noted. Specifically, Phycorickettsia lacks genes for several components of the respi-1848

ration chain, haem biosynthesis pathway, or c-di-GMP–based signalling. On the other1849

hand, it uniquely harbours a six-gene operon of enigmatic function that we recently1850

reported from plastid genomes of two distantly related eustigmatophytes and from1851

various non–rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely1852

related to the one from Phycorickettsia, suggesting a gene transfer event between the1853

endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize1854

an important role of the operon in the physiology of Phycorickettsia infection and a1855

long-term eustigmatophyte–Phycorickettsia coexistence.1856

1857

92

Abstracts

Colourless but not Invisible: Stories about the Non-Photo-1858

synthetic Plastid of Euglena longa1859

Kristína Záhonová1, #, Zoltán Füssy2, #, Erik Birčák3, Vladimír Klimeš1, Matej Ves-1860

teg4, Juraj Krajčovič5, Miroslav Oborník2, 6, & Marek Eliáš11861

1University of Ostrava, Faculty of Science, Department of Biology and Ecology and Institute of1862

Environmental Technologies, Life Science Research Centre, Ostrava1863


3Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava1865

4Matej Bel University, Faculty of Natural Sciences, Department of Biology and Ecology, Banská1866

Bystrica1867

5University of ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Biology,1868

Trnava1869


# These authors contributed equally to this work.1871

1872

Plastid endosymbiosis brought many eukaryotic groups the privilege to harvest the1873

energy of sunlight, but photosynthesis comes with high costs and numerous lineages of1874

eukaryotes gave up on this capacity. Strikingly, these secondary heterotrophs frequently1875

keep their non-photosynthetic plastids (leucoplasts), pointing out that other processes1876

in these organelles are equally essential to the cell as photosynthesis. Both the known1877

model Euglena gracilis and its colourless sibling E. longa contain secondary plastids1878

of green descent. Through comparative transcriptomic analyses we have identified pro-1879

teins that supposedly participate in their plastids' maintenance. While searching for the1880

role of the E. longa leucoplast, we revealed other biological features worth discourse.1881

First, the plastid translation apparatus of euglenids employs a horizontally acquired1882

bacterial termination factor, Rho. Second, plastid targeting in euglenids holds several1883

differences compared to red alga-derived plastids, in terms of both the structure of1884

targeting presequences and the architecture of the protein translocon channels. The1885

targeting presequences are conserved for photosynthesis-related and non-photosynthe-1886

tic proteins, and remain highly similar in E. longa and E. gracilis. Many of the key1887

translocon components are missing, suggesting their extreme divergence or a use of1888

alternative channels. Similarly, the plastid division mechanism remains elusive, as we1889

have not identified any of the known conserved division components. Third, several1890

plastid-targeted proteins in Euglena are encoded as translational fusions. Adding to1891

a handful of known fusion proteins, we have found new ones including cases where1892

the two encoded mature proteins are functionally unrelated. Fusion of proteins may1893

facilitate effective transport, but at the same requires linker peptide recognition and1894

cleavage. All these features of euglenophyte plastids qualify them as interesting models1895

for molecular biologists.1896

1897

93


How is our Python Code Helping us to Understand Evolu-1898

tion of the Genetic Code1899

David Žihala1, Martin Kolísko2, Serafim Nenarokov2, Eleni Gentekaki3, Denis Lynn4,1900

Feng Gao5, Tomáš Pánek1, & Marek Eliáš11901



3Mae Fah Luang University, School of Science, Chiang Rai, Thailand1904

4University of Guelph, Department of Integrative Biology, Guelph, Canada1905

5Ocean University of China, Institute of Evolution, and Marine Biodiversity, Quingdao, China1906

1907

Our lab has started to work in the field of alternative genetic codes relatively1908

recently. Nonetheless, we have already contributed to this field by two important dis-1909

coveries: (1) organisms can use all three stop codons as sense codons in a context–1910

dependent manner; and (2) there is probably no evolutionary constraint that would1911

restrict UAA and UAG to always mean a stop codon or to encode the same amino1912

acid. Ciliates are undoubtedly the most interesting group of organisms when alterna-1913

tive genetic codes are concerned. To date, seven different genetic code variants were1914

found in Ciliophora, including the standard genetic code. However, a comprehensive1915

phylogenetic study is necessary to achieve a better understanding of the evolution of1916

the genetic code in this group . Unfortunately, a number of wrongly determined genetic1917

codes in different ciliate taxa can be found in the NCBI database or in the literature.1918

Another complication is that tools for protein predictions usually have a limited set1919

of predefined genetic code alternatives that can be used or they require a training da-1920

taset based usually on transcriptomic data. To address these complications we have1921

developed a tool for a quick genetic code determination, and additionally a tool that1922

predicts conserved parts of proteins for phylogenetic analysis from a genome sequence.1923

This talk will be about our recent discoveries in the field of alternative genetic codes1924

and will also briefly introduce our new tools, which will become open source soon.1925

Acknowledgment:1926



1929

94

List of Participants1930

95

List of Participants

name e-mail

Bianchi Claretta [email protected]

Bilková Katarína [email protected]

Bourland William [email protected]

Brdičková Klára [email protected]

Brzoň Ondřej [email protected]

Butenko Anzhelika [email protected]

Cadena Lawrence Rudy [email protected]

Charyyeva Arzuv [email protected]

Čepička Ivan [email protected]

Dohnálek Vít [email protected]

Dohnálková Alena [email protected]

Doležal Pavel [email protected]

Doleželová Eva [email protected]

Eliáš Marek [email protected]

Fagundes Macedo Diego Henrique [email protected]

Faitová Tereza [email protected]

Fiala Ivan [email protected]

Field Mark [email protected]

Flegontov Pavel [email protected]

Füssy Zoltán [email protected]

Gahura Ondřej [email protected]

Gruber Ansgar [email protected]

Hampl Vladimír [email protected]

Hanousková Pavla [email protected]

Ha lakuc Pawe l [email protected]

Heged"usová Eva [email protected]

97


name e-mail

Horvath Anton [email protected]

Horčičková Michaela [email protected]

Jalovecka Marie [email protected]

Juraj Krajčovič [email protected]

Kabeláčová Kateřina [email protected]

Kaczanowski Andrzej [email protected]

Karlicki Micha l [email protected]

Karnkowska Anna [email protected]

Kaurov Iosif [email protected]

Kolisko Martin [email protected]

Kornalíková Martina [email protected]

Kostygov Alexei [email protected]

Kotyk Michael [email protected]

Kovalinka Tomáš [email protected]

Kořený Luděk [email protected]

Krupičková Alžběta [email protected]

Králová Jana [email protected]

Kubánková Aneta [email protected]

Kulkarni Sneha [email protected]

Kváč Martin [email protected]

Le Tien [email protected]

Lisnerová Martina [email protected]

Lukeš Julius [email protected]

Maciszewski Kacper [email protected]

Mishra Rahul [email protected]

Nenarokov Serafim [email protected]

98

List of Participants

name e-mail

Nenarokova Anna [email protected]

Novák Lukáš [email protected]

Novák Vanclová Anna [email protected]

Oborník Miroslav [email protected]

Pánek Tomáš [email protected]

Papežík Petr [email protected]

Petrů Markéta [email protected]

Petrželková Romana [email protected]

Pe lesz Agnieszka [email protected]

Podešvová Lucie [email protected]

Poláková Kateřina [email protected]

Pružincová Martina [email protected]

Raabova Lenka [email protected]

Rada Petr [email protected]

Rašková Vendula [email protected]

Rotterová Johana [email protected]

Sharaf Abdoallah [email protected]

Soukal Petr [email protected]

Spodareva Viktoria [email protected]

Sveráková Ingrid [email protected]

Švagrová Eva [email protected]

Tripathi Pragya [email protected]

Tashyreva Daria [email protected]

Tomčala Aleš [email protected]

Vacek Vojtěch [email protected]

Varga Vladimír [email protected]

99


name e-mail

Vešelényiová Dominika [email protected]

Voleman Luboš [email protected]

Votýpka Jan [email protected]

Váchová Hana [email protected]

Walkiewicz Halszka [email protected]

Wandyszewska Natalia [email protected]

Wisniewska Monika [email protected]

Yurchenko Tatiana [email protected]

Yurchenko Vyacheslav [email protected]

Zelená Marie [email protected]

Záhonová Kristína [email protected]

Žihala David [email protected]

1931

The organizer of the next Protodny is highlighted.1932

100

Partners of Conference1933

101

Image of trypanosome ultrastructure is based on the original drawing by Prof. K. Vic-1934

kerman (courtesy of Dr. Richard Wheeler, University of Oxford).1935

Title: 48th Jírovec's Protozoological Days1936

Subtitle: Conference Proceedings1937

Redaction: Alexei Kostygov (University of Ostrava, Faculty of Science, Department of Biology1938

and Ecology, Ostrava)1939

Editor: Petr Soukal (Charles University, Faculty of Science, Department of Parasitology, Praha)1940

Publisher: University of Ostrava, Faculty of Science, Department of Biology and Ecology1941

Place and Year of Publication: Ostrava, 20181942

First Edition1943

Number of Pages: 1081944

Permanent Link:1945

http://www.parazitologie.cz/protozoologie/Protodny2018/JPD_sbornik_2018.pdf1946

Circulation: 1001947

Exposure and Print: POINT CZ, s.r.o., Milady Horákové 20, Brno 602 001948

This publication did not undergone any language (nor misspelling) editing.1949

Not for sale.1950

http://www.parazitologie.cz/protozoologie/Protodny2018/JPD_sbornik_2018.pdf

48 th jírovec's protozoological days sbornik jpd48.pdf · orewford 27 28 dear...

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