Protistology An International Journal Vol. 10, Number 2, 2016 ______

CONTENTS

INTERNATIONAL SCIENTIFIC FORUM «PROTIST–2016»

Yuri Mazei (Vice-Chairman) Welcome Address 2

Organizing Committee 3

Organizers and Sponsors 4

Abstracts 5

Author Index 94 Forum “PROTIST-2016” June 6–10, 2016 Moscow, Russia Website: http://onlinereg.ru/protist-2016

WELCOME ADDRESS

Dear colleagues! Republic) entitled “Diplonemids – new kids on the block”. The third lecture will be given by Alexey The Forum “PROTIST–2016” aims at gathering Smirnov (Saint Petersburg State University, Russia): the researchers in all protistological fields, from “Phylogeny, diversity, and evolution of Amoebozoa: molecular biology to ecology, to stimulate cross- new findings and new problems”. Then Sandra disciplinary interactions and establish long-term Baldauf (Uppsala University, Sweden) will make a international scientific cooperation. The conference plenary presentation “The search for the eukaryote will cover a wide range of fundamental and applied root, now you see it now you don’t”, and the fifth topics in Protistology, with the major focus on plenary lecture “Protist-based methods for assessing evolution and phylogeny, taxonomy, systematics and marine water quality” will be made by Alan Warren DNA barcoding, genomics and molecular biology, (Natural History Museum, United Kingdom). cell biology, organismal biology, parasitology, diversity and biogeography, ecology of soil and There will be two symposia sponsored by ISoP: aquatic protists, bioindicators and palaeoecology. “Integrative co-evolution between mitochondria and their hosts” organized by Sergio A. Muñoz- The Forum is organized jointly by the International Gómez, Claudio H. Slamovits, and Andrew J. Society of Protistologists (ISoP), International Roger, and “Protists of Marine Sediments” orga- Society for Evolutionary Protistology (ISEP), nized by Jun Gong and Virginia Edgcomb. Protozoological Society affiliated with Russian Academy of Sciences, and Protist action group within The primary goal of this Forum is to bring together the Global Soil Biodiversity Initiative (GSBI). the globally renowned research leaders in all protistological fields, as well as invite the beginners The Forum is hosted by the leading academic center and young scientists, friends, colleagues and project- of Russian Federation – Lomonosov Moscow State partners from around the world to participate in an University. The University is well-known for its open discussion under the roof of the oldest Russian strong natural sciences tradition: 11 Nobel Prize University, in the wonderful city of Moscow. winners and 6 Fields medalists were alumni or Finally, I can’t but mention genuinely that it has been academics at this University. a privilege and honor for me to serve as an organizer of the Forum PROTIST-2016. In particular, I The program of the Forum is going to be informative, thank all the colleagues who have provided help dynamic and highly interactive. In total, we will and assistance in organizing this conference. Also, have more than 130 oral presentations and about I would like to express my sincere gratitude to all of 100 poster presentations. I believe that our diverse the Forum attendees: you have traveled thousands group of speakers and panelists will provide profound of miles to come and join this conference, and we insight into the various issues in different areas of hope that you will find the meeting informative and Protistology. worthwhile attending. Your presence at the Forum helps to make this event a real success, and your This meeting aims at presenting some cool and enthusiasm and positive spirit make the Forum days intriguing, cutting edge basic research. At the Opening both productive and enjoyable. Plenary Session, Frederick Speigel (University of Arkansas, United States of America) will give a talk Wishing you a successful Forum PROTIST-2016! entitled “Finding a Place for Protistology in Biology, in General”, which will be followed by a lecture of Sincerely, Julius Lukeš (Institute of Parasitology, University Professor Yuri Mazei of South Bohemia, České Budějovice, Czech Vice-Chairman Protistology · 3

Forum “PROTIST-2016” June 6–10, 2016 Moscow, Russia Website: http://onlinereg.ru/protist-2016

PROGRAMME COMMITTEE

Avelina Espinosa ([email protected]) Roger Williams University, USA Alastair Simpson ([email protected]) Dalhousie University, Canada Frederick Spiegel ([email protected]) University of Arkansas, USA Edward A. D. Mitchell ([email protected]) University of Neuchatel, Switzerland Stefan Geisen ([email protected]) Netherlands Institute of Ecology, Netherlands Daniel Lahr ([email protected]) University of Sao Paulo, Brazil Andrew J. Roger ([email protected]) Dalhousie University, Canada Kamran Shalchian-tabrizi ([email protected]) University of Oslo, Norway

ORGANIZING COMMITEE

Mikhail Kirpichnikov, co-chair: Full member of the Russian Academy of Sciences (RAS), Dean of the Faculty of Biology, Moscow State University (MSU) Sergey Shoba, co-chair: Corresponding member of RAS, Dean of the Faculty of Soil Science, MSU Yuri Mazei, vice-chair: Deputy vice-rector for international affairs, MSU Sergei Skarlato, vice-chair: Deputy Director of the Institute of Cytology RAS Andrey Azovsky: Professor, Faculty of Biology, MSU Ludmila Ilyash: Professor, Faculty of Biology, MSU Anatoly Bobrov: Professor, Faculty of Soil Science, MSU Elena Novenko: Leading Scientist, Faculty of Geography, MSU Alexey Tiunov: Head of the Laboratory Severtsov Institute of Ecology and Evolution, RAS Irena Telesh: Principal scientist, Zoological Institute RAS Andrew Goodkov: Leading Scientist, Department of Cytology of Unicellular Organisms, Institute of Cytology RAS

FORUM SECRETARIAT

Denis Tikhonenkov ([email protected]): Leading Scientist, Institute for Biology of Inland Waters of Russian Academy of Science Andrey Tsyganov ([email protected]): Associate professor, Penza State University Ekaterina Nadezhkina ([email protected]) 4 · Organizers and Sponsors

Forum “PROTIST-2016” June 6–10, 2016 Moscow, Russia Website: http://onlinereg.ru/protist-2016

ORGANIZERS AND SPONSORS

Faculty of Biology, Russian Academy of Sciences Faculty of Soil Studies, Lomonosov Moscow Lomonosov Moscow State University State University

Institute for Biology Russian Foundation of Inland Waters RAS for Basic Research

International Society International Society for Protozoological Society of Protistology Evolutionary Protistology affiliated with RAS

The official Service Agency of the International Scientific Forum “PROTIST-2016”: MONOMAX Phone: +7 812 335 20 55 (ext. 205) E-mail: [email protected] Protistology 10 (2), 5–93 (2016) Protistology

Abstracts of the International Scientific Forum “PROTIST-2016”

ULTRASTRUCTURE AND PHYLOGENY OF BIODIVERSITY AND COMMUNITY STRUC- NEW SPECIES OF GLUGEA INFECTING THE TURE OF SOIL CILIATES COLLECTED FROM INTESTINAL WALL OF CEPHALOPHOLIS THREE DIFFERENT SITES OF DELHI, INDIA HEMISTIKTOS IN SAUDI ARABIA TO ASSESS SOIL QUALITY Abdel-Gawwad Abdel-Azeem1,2, Al-Quraishy Abraham J.S.1, Somasundaram S.1, Jangra S.1, Saleh1, Azevedo Carlos3,1 Yadav K.1, Singh S.1, Zutshi S.1, Singh B.1, Dagar 1 - Zoology Department, College of Science, King Saud J.1, Kumar A.1, Goyal A.1, Bhatnagar M.1, Upadhaya University, Riyadh, Saudi Arabia. M.1, Choudhary A.1, Toteja R.1, Gupta R.2, Gambhir 2 - Zoology Department, Faculty of Science, Beni-Suef G.1, Makhija S.1 University, Egypt. 1 - Acharya Narendra Dev College, University of Delhi, 3 - Laboratory of Cell Biology, Institute of Biomedical Delhi, India Sciences, University of Porto, Porto, Portugal 2 - Maitreyi College, University of Delhi, Delhi, India [email protected] [email protected], [email protected] A new microsporidian species of the genus Glugea Soil is a natural body of mineral and organic material Thélohan, 1891 parasitizing the marine teleost fish differentiated into horizons, which differ among Cephalopholis hemistiktos Rüppell, collected from themselves as well as from underlying materials in the Red Sea in Saudi Arabia, is described on the basis their morphology, physical make-up and chemical of microscopic and molecular procedures. Spherical composition. Biological components of the soil can and whitish xenoma were observed adhering to the vary from one site to another site depending upon intestinal wall. The numerous spores contained the inorganic and organic make up of the soil. Soil within these xenoma, were ovoid to pyriform and ciliates are an integral part of the soil community. measured 5.1 (4.3–6.0) µm in length and 2.2 (1.8– They play an important role in nutrient recycling 2.9) µm in width. The spore’s wall was composed by feeding on bacteria. Also, they are known to of two thick layers, which were thinner in the area stimulate ammonification and nitrification that contacting the anchoring disk. The latter appeared can later be used by plants and other members of at the spore’s anterior pole, in an eccentric position the food web. Although they play an influential to the longitudinal axis. A lamellar polaroplast ecological role, information on soil ciliate diversity surrounded the uncoiled portion of the polar is still rudimentary. Particularly from India, filament projected to the basal region of the spore, very few data are available on soil ciliates. In this giving rise to 26–29 turns with winding from the present study, the ciliate diversity was investigated base to the anterior zone of the spore. The posterior from three different areas, that is, from human vacuole, located at the spore’s posterior pole, and inhabitant land [Acharya Narendra Dev College surrounded by the polar filament coils, was irregular (ANDC) campus], an agricultural land (Karnal) and composed of light material. Molecular analysis and a sewage treatment plant (Rithala) within Delhi, of the rRNA genes, including the ITS region, was India. Physicochemical analysis of soil samples was performed using maximum parsimony, neighbour- conducted for its pH, nitrogen content, organic joining and maximum likelihood methods. The carbon content. Ciliate abundance was correlated ultrastructural features observed, combined with the with physicochemical properties. In total ciliates phylogenetic data analyzed, suggest this parasite to belonging to 5 Classes, 7 Orders, 15 genera and be a new species of the genus Glugea. 20 species were found with maximum number

© 2016 The Author(s) Protistology © 2016 Protozoological Society Affiliated with RAS 6 · “PROTIST–2016” belonging to Spirotrichs. Maximum number of Species identification is crucial in biodiversity oxytrichids were found in Rithala sewage site though research (ecology, taxonomy, barcoding etc.). ciliate diversity was less. Maximum ciliate diversity In tintinnids, taxonomy and classification are was found in ANDC whereas ciliate diversity was almost exclusively based on features of their vase- less in Karnal. Colpodids were present in all the shaped loricae (houses). The lorica-based species sites examined but most abundant in sewage site. circumscriptions are, however, problematic as Variation in soil ciliate diversity can be correlated loricae might show a considerable intraspecific with soil quality of studied habitats. variability and interspecific similarity and might be influenced by the cell cycle and physico- SOIL PROTIST BACTERIA CONSUMPTION IS chemical factors. To overcome the difficulties CENTRAL TO NUTRIENT CYCLING with deviating species limitations suggested by Adl S.M. “lumpers” or “splitters” in revisionary treatises, it University of Saskatchewan is recommend to “go back to the roots”, i.e., to use [email protected] exclusively the original descriptions or authoritative Protists are the most abundant consumers of redescriptions for identification. Additionally, bacteria in soils. Their diversity covers most good documentation of the identified loricae by lineages making them the most diversity group of means of illustrations and morphometric data is organisms in soils. Communities assemble along advisable. Depending on the aim of the tintinnid abiotic microgradients, such as pO2, pCO2, pH, study, further procedures are proposed to generate temperature, etc. Species of protist bacterivores at high rates long-lasting high-quality species re-/ are not homologous or interchangeable. Species descriptions and/or DNA barcodes, which are in functional groups have differing ecological essential for reliable phylogenetic analyses and thus preferences, occupying distinct niches. There are for the establishment of a natural tintinnid classifica- significant measurable species specific differences tion. These suggestions represent a compromise regarding prey ingestion rates and prey preferences. between data quality and work effort and can most It has been well recognised in both soil and aquatic easily be followed by collaborations of molecular ecology that ignoring inter-species variability in biologists and morphologic taxonomists. Financially behaviour and consumption rates is problematic. supported by FWF project P28790. The top-down grazing pressure on bacteria was calculated for a variety of species representing a MICROBIAL EUKARYOTES IN OILSANDS- variety of feeding types, from functional response ASSOCIATED ENVIRONMENTS OF NOR- curves. We calculated threshold prey levels that THERN ALBERTA sustained growth and initial rate slopes for ingestion Aguilar M.1, Richardson E.1, Paoli L.2, Nesbo C.3, rates, to compare competitive ability at low food Foght J.3, Dunfield P.F.4, Dacks J.B.1,5 conditions. Together with maximum growth rates 1 - Department of Cell Biology, University of Alberta. and maximum ingestion rates inter-specific compe- Edmonton, Canada titiveness and niche preferences could be distin- 2 - Département de Biologie, Ecole normale supérieure, guished. Data will be presented for several well Paris, France established cercozoa cultures, amoebae cultures, 3 - Department of Biological Sciences, University of ciliates, and other protists. This information helps to Alberta, Edmonton, Canada improve our calculations of global bacteria biomass 4 - Department of Biological Sciences, University of turnover rates in soils. It also helps to understand Calgary, Calgary, Canada how soils can support dozens of bacterivorous 5 - Department of Life Sciences, Natural History species in each gram of soil, in a seemingly similar Museum, London, UK habitat. [email protected] The oil sands in Northern Alberta are the second BIODIVERSITY STUDIES IN LORICATE largest bitumen deposit in the world, and comparable PROTISTS: THE CASE OF TINTINNIDS (ALVE- in magnitude to world’s reserves of conventional OLATA, CILIOPHORA, SPIROTRICHA) petroleum. They have global significance as energy Agatha S.1, Santoferrara L.F.2 reservoirs. However, some aspects of their exploi- 1 - Dept. of Ecology and Evolution, University of tation are also cause for environmental concern. Salzburg, Salzburg, Austria Fluid wastes from bitumen extraction are retained 2 - Dept. Marine Sciences, University of Connecticut, in enormous tailings ‘ponds’ under a policy of no Groton, Connecticut, USA release to the environment. The estimated surface [email protected] area of tailings ponds in Alberta ranges from 77-176 Protistology · 7 km2. Thus, since their commissioning approximately group was stramenopiles. This study contributes five decades ago, the ponds have accumulated to improve our understanding of the diversity of anoxic, brackish, hydrocarbon and heavy metal protist communities living in ancient lakes as well containing sediments overlain by a thinner layer as to comprehend better intra-lake evolutionary of process-affected water that may be oxic near the diversification processes. surface. Microbial processes are anticipated to play Supported by project of RAS # 0345–2014–0003 a major role in remediation of these environments, and, although the prokaryotic communities are MODELLING THE LOCAL-TO-GLOBAL DI- increasingly well-characterized, little is known about VERSITY FOR MICROBES AND MULTI- the microbial eukaryotes present in the oxic and CELLULARS: COULD WE ESTIMATE THE anoxic environments. We have recently reported the NEAR-IMPONDERABLE? first NGS-based exploration of protists in tailings Azovsky A.I. ponds. We found that, despite the anoxic and Lomonosov Moscow State University, Moscow, Russia hydrocarbon-enriched nature of the environment, [email protected] the tailings ponds harbour complex communities of In the current discussion around either “protist microbial eukaryotes indicating that these organisms diversity is different” or not, the ratios of local:global should be taken into account when studying the diversity and relative endemicity are often treated as microbiology of the oil sands. arguments. This approach has, however, been flawed by the fact that actual global diversity of protists is NOVEL PICOPLANKTONIC GROUPS FROM still largely unknown, especially for the rare species. LAKE BAIKAL REVEALED BY MASSIVE SE- I use simple models to simulate distribution of QUENCING species on the 400-cell lattice. Three models have Annenkova N.V.1, Logares R.2 been considered: 1 - Limnological Institute SB RAS, Russia 1) “Everything is everywhere” (equal probability for 2 - Institute of Marine Sciences, CSIC, Spain a particular species to occur at every cell); [email protected] 2) “Environmental selection” (a species can Compared to the ocean, freshwater systems offer occupy only a fixed portion of randomly distributed multiple ecological niches in terms of oxygen and “suitable localities”); and DOC concentrations, light accessibility, temperature 3) “Endemicity” (most of species are restricted in variability. However, we are still limited by the data of its potential distribution to a few regions). freshwater microbes biodiversity. Studies are highly To parameterize the models, several world-wide biased because correspond mainly to Europe and datasets on ciliates, flagellates and harpacticoid North America. In particular, studies of Northern copepods were used. The local:regional and re- Asian protists are very limited, though this region gional:global diversity ratios and percentage of extends on about 8000 km in length and has a big endemics were estimated by simulating the equal- impact on microorganisms’ migrations. Lake Baikal effort sampling across the lattice, with varying the is the oldest and deepest lake in the world, being full number of species (“actual diversity”) and located in Northern Asia. It is cold, oxygen rich, number of samplings per cell (“sampling effort”). and has one of the world richest endemic freshwater All parameters, predicted by every model, strongly biotas, belonging both to very ancient lineages and depended on the sampling effort but were only to recent immigrants. Our goal was to explore small slightly, if ever, influenced by the observed:actual planktonic protists populating the lake. The genetic global diversity ratio. The predictions of the first diversity (V4 region of the 18S rDNA) of planktonic two models were generally close to each other, but microeukaryotes (< 8 µm fraction) was analyzed differed noticeably from those of the third one. in 48 samples using the Illumina MiSeq platform. These results indicate that the above-mentioned We determined 1,461 protist OTUs, with 9.6% of parameters are heavily influenced by undersampling. them having less than 90% similarity with sequences Nevertheless, they can serve as informative cha- from SILVA database. We suggest that they belong racteristics in comparative biodiversity studies, even to endemic Baikal protists. We analyzed the if the true number of species can hardly be estimated phylogenetic relationships of groups such as MAST, overall. Telonemiidae, Perkinsiidae, Chitridiomycota which were not previously described in Lake Baikal. GLOBAL DIVERSITY AND DISTRIBUTION Interestingly, we found Amoebophrya-like 18S OF MARINE BENTHIC HETEROTROPHIC rDNA in Baikal, even though Syndiniales were not FLAGELLATES reported from freshwaters. Overall, the most diverse Azovsky A.I.1, Tikhonenkov D.V.2,3 8 · “PROTIST–2016”

1 - Lomonosov Moscow State University, Moscow, However, its abiotic conditions are characterized by Russia a number of features typical of the Arctic seas. In this 2 - Institute for Biology of Inland Waters, Russian study, the molecular diversity of picoeukaryotes in Academy of Sciences, Borok, Russia July 2014 in Kandalaksha Bay (the White Sea) was 3 - University of British Columbia, Department of examined using conventional filter fractionation and Botany, Vancouver, BC, Canada Illumina sequencing. In < 2 µm size fraction 186 [email protected] taxa were revealed that included some nanoplankton Protists are ubiquitous, but the factors influencing (2–20 µm) and even microplankton (>20 µm) taxa. their diversity and biogeography are poorly un- Plankton assemblage was prevailed by autotrophs derstood. We use a comprehensive database on (>57% of all sequences). Putative picoeukaryotic the marine benthic heterotrophic flagellate (HF) autotrophs were mostly dominated by three genus: morphospecies to explore the worldwide patterns Micromonas sp., Bathycoccus sp. and Ostreococcus in their diversity and distribution in comparison sp. (35% of all sequences). Putative heterotrophic with predictions of the Ubiquity model (UM) and picoeukaryote assemblage was more diverse and Moderate Endemicity model (MEM). Number was presented by marine stramenopiles (MAST, of known HF morphospecies was limited (even if Labyrinthulomycetes), Amoebozoa, Cercozoa considering the rates of descriptions), and local-to- and picozoa. Illumina sequencing revealed some global diversity ratio was relatively high (10-25%). new groups and genus of nano- and microplankton Regional diversity was highly correlated with the organisms which have been never detected in the investigation effort, indicating considerable under- White Sea. Our study revealed that diversity of exploration. Regional endemics were few (not over picoeukaryotes in summer in the sub-Arctic White 19% of total richness), and many morphospecies Sea corresponds to that of the other Arctic seas, such were widespread or even cosmopolitan. No obvious as the Beaufort and Norwegian Seas. latitudinal trend in HF diversity was detected. By species composition, the regions were distinctly NEW PARAMECIUM SPECIES “CANDIDATUS arranged into three groups according to cold, PARAMECIUM OSSIPOVI” temperate and warm waters, but not in accordance Beliavskaia A., Kiselev A., Rautian M. with geographical distances. This distribution Saint Petersburg State University pattern was most likely explained by contemporary [email protected] climate (temperature) but did not suggest clear Among Paramecium species P. putrinum is one of geographical barriers for dispersal. Therefore, the the best identifiable. Most important discriminative HF morphospecies are less concordant with the features are small cell size (70-140 µm), single big MEM predictions but closer to the UM than other micronucleus (MI) and very special contractile (larger) protists. (In)consistency between the vacuole (CV) represented as set of small vacuoles. distributional patterns obtained from genetic- and We have studied several clones from different morphology-based data are briefly discussed. collecting sites that were characterized by all features This study was supported by Russian Foundation for mentioned above but with unusual habitus. Detailed Basic Research (grants № 15-04-02245 to AA and analysis revealed several very specific characteristics № 14-04-00553, 15-29-02518 to DT). of these clones. Usually the cells are bigger up to 120- 140 µm. Their single MI has got “chromosomal” MOLECULAR DIVERSITY OF SUMMER structure. Contractile vacuole differs from typical PLANKTON PICOEUKARYOTES IN THE P. putrinum one. It has got short channels and is WHITE SEA similar to CV of P. nephridiatum, or P. polycarium. Belevich T.A.1, Ilyash L.V.1, Milyutina I.A.2, Holospora-like bacterium found in P. putrinum do Logacheva M.D.2, Goryunov D.V.2, Troitsky A.V.2 not infect “atypical” strains. Finally the sequence of 1 - Lomonosov Moscow State University, Faculty of cytochrome c oxidase subunit I gene has only 86% Biology identity with the one of P. putrinum, which is much 2 - Belozersky Institute of Physico-Chemical Biology, higher than difference between different P. putrinum Lomonosov Moscow State University syngens. Taking in account all above, we propose [email protected] new species “Candidatus Paramecium ossipovi”. Picoeukaryotes (protists < 2 µm) play an important Scientific research was performed at the Center for role in marine ecosystems, although knowledge of Culturing Collection of Microorganisms and Center their diversity and ecosystem functioning is limited. for Molecular and Cell Technologies of Research The White Sea is located in the sub-Arctic zone. park of St. Petersburg State University. Protistology · 9

NUCLEUS-ASSOCIATED ACTIN IN DIFFE- flagellate. The basic culture of P. minimum, isolated RENT STAGES OF AMOEBA PROTEUS CELL from the Black Sea, was grown in 17 psu f/2 medium CYCLE (cultivation conditions described in Pozdnyakov Berdieva M.A., Bogolyubov D.S., Demin S.Y., et al., 2014). Experiments were carried out by Podlipaeva Y.I., Goodkov A.V. inoculation of cells into 4, 8, 35 psu f/2 medium Institute of Cytology of the Russian Academy of and 17 psu as a control for 30 min. Then cells were Sciences, St. Petersburg, Russia returned to the basic medium, incubated for 24 h [email protected] and fixed for flow cytometry or transmission electron Despite of plenty of the reports and reviews there microscopy. We detected DNA concentration value are still a lot of blank spots in the matter of the (1.2 pg/cell) and fine structure of chromosomes organization and functioning of Amoeba proteus nuc- in control series. After inoculation of cells into lear apparatus. According to our 3D-reconstruction 8 psu f/2 medium we observed the highest DNA of its chromatin compartment, it has a complicated concentration (1.5 pg/cell) and the lowest cell and highly dynamic structure (Demin et al., 2016). mortality rates. Chromosomes became more Actin was shown to be a key protein actor in providing condensed, local unwound sites increased in different nuclear processes. Immunocytochemical number and appeared more distinct. Transfer into study has shown an actin meshwork strongly co- the medium with native for these organisms salinity localized with chromatin fibrils. On the other 35 psu (the World ocean level) did not caused any hand, the amoeba nucleus appears embraced by reliable differences in DNA concentration (1,3 pg/ a basket-like structure formed by F-actin that in cell). At the ultrastructural level, we observed total turn is connected with cytoplasmic actin filaments. splitting of chromosomes in most cases. Inoculation 3D-observations of the nucleus during the cell cycle of cells into 4 psu f/2 did not caused any significant indicate a regular process of the chromatin extrusi- changes in DNA concentration value, but led to high on/elimination of the part of the chromatin “excess” cell mortality rates. Our results are in agreement to the cytoplasm. Based on the 3D-reconstruction with the protistan species maximum concept for of phalloidin-stained cells, we suppose the existence the horohalinicum. Funded by the Russian Science of specific chromatin-binding sites interacting Foundation, project 16-14-10116. with actin filaments meshwork. We also propose a hypothesis that the chromatin extrusion is provided FINDING AND ANALYSIS OF AMOEBO- by actin filaments which may pull out the chromatin ZOA-SPECIFIC GENES TO STUDY ENVI- fibrils from the nucleus. Funded by the Russian RONMENTAL DIVERSITY OF AMOEBAE Foundation for Basic Research, projects 15-04- Bondarenko N.I., Smirnov A.V. 03451, 15-04-01857, and the granting program Department of Invertebrate Zoology, Faculty of “Molecular and Cell Biology” of the Presidium of Biology, Saint-Petersburg State University, Saint- RAS. Petersburg, Russia [email protected] INFLUENCE OF SALINITY STRESS ON DNA Amoebozoa is a one of the supergroups of eukaryo- SYNTHESIS AND CHROMOSOME FINE tes, which includes naked and testate lobose STRUCTURE OF DINOFLAGELLATES PRO- amoebae, pelobionts, mycetozoa, and several groups ROCENTRUM MINIMUM (PAVILLARD) of flagellated organisms. In environmental DNA SCHILLER surveys done on traditional DNA barcodes (SSU- Berdieva M.A.1, Filatova N.A.1, Knyazev N.A.1,2, rDNA, Cox I gene) amoebozoan genes normally Skarlato S.O.1 constitutes a minor part of the total gene diversity 1 - Institute of Cytology of the Russian Academy of and represent only the most abundant lineages. Sciences, St. Petersburg, Russia To resolve this problem, we attempted to find 2 - Nanotechnology Research and Education Centre, Amoebozoa-specific genes and gene families with Russian Academy of Sciences, St. Petersburg, Russia low level of paralogy appropriate for the application [email protected] as a DNA barcodes for this group of protists. We Prorocentrum minimum is a common planktonic analyzed the Amoebozoa RNA-Seq data which were potentially toxic bloom-forming dinoflagellate. available from MMETSP (http://www.moore.org/). Being widely distributed, this species demonstrates First, we analyzed assembled transcriptomes from high adaptive capacity to different factors, particu- MMETSP and found transcripts with unusually big larly salinity. We studied cellular and biochemical size (more than 50 000 bp) in several assemblies. This aspects of physiological adaptation of this dino- led us to the decision to repeat data assembly de novo. 10 · “PROTIST–2016”

During this work we developed pipeline, based on Metatranscriptome analysis provided an examina- publically available bioinformatics tools and our own tion of the functional repertoire of deep subseafloor scripts written in Python for transcriptome assembly fungi. Gene expression was assigned to metabolic and annotation. We have found 300 groups of genes, and biosynthetic processes, responses to stress, which not found outside Amoebozoa or were highly cell and membrane functions, conidiogenesis and derived within this group of protists. Among them biosynthesis of secondary metabolites (Pachiadaki we selected 15 groups of genes with low level of et al., in revision). These results all provide further paralogy and performed phylogenetic analysis and support for the notion of fungal presence and activity primers construction. These genes are promising in the deep subseafloor biosphere, with the ability DNA barcodes for studies of environmental diversity to interact with other microbial populations by of Amoebozoa. Supported with MK-4853.2015.4 synthesizing antimicrobial compounds (Navarri et President grant, RFBR 16-34-60111 and SPSU al. 2016). grant 1.38.251.2014. THE GREEN ALGA AND THE SALAMANDER: BURIED BUT NOT DEAD: INSIGHTS INTO A SUFFOCATING LOVE STORY THE DIVERSITY, PHYSIOLOGY, FUNC- Burns J.A.1, Zhang H.2, Hill E.2, Kerney R.2, Kim E.1 TIONS AND ECOLOGICAL ROLES OF DEEP 1 - American Museum of Natural History SUBSEAFLOOR FUNGI USING AN INTE- 2 - Gettysburg College GRATED APPROACH [email protected] Burgaud G.1, Rédou V.1, Pachiadaki M.G2,3, Navarri The recently discovered endosymbiosis between the M.1, Fleury Y.1, Barbier G.1, Edgcomb V.P.2 green alga Oophila amblystomatis and the salaman- 1 - Université de Brest, EA 3882, Laboratoire Uni- der Ambystoma maculatum is a unique relationship versitaire de Biodiversité et Ecologie Microbienne, among the chloroplastida and vertebrates. Using ESIAB, Technopôle de Brest Iroise, 29280 Plouzané, a dual RNA-seq approach, we assembled novel France transcriptomes of these two organisms and identified 2 - Woods Hole Oceanographic Institution, Depart- differentially expressed transcripts between ecto- ment of Geology and Geophysics, Woods Hole, Massa- and endo-symbiotic algae as well as between chusetts, United States of America salamander cells with and without endosymbiotic 3 - Bigelow Laboratories, East Boothbay, Maine, USA algae. The results offer a glimpse at the changes in [email protected] both organisms that take place during this novel Bacteria and Archaea are the most commonly endosymbiosis. We found that the intracellular studied microorganisms in the marine environment, algae downregulate nutrient transporters related and habitats such as deep subseafloor ecosystems to phosphate and nitrogen acquisition from the are no exception. However, recent studies strongly environment. They also exhibit hallmarks of cellular support the idea that deep subseafloor microbial stress, especially related to osmotic stress, sulfur communities include Fungi, which seem to domi- starvation, and hypoxia. Further, the results suggest nate those micro-eukaryotic communities. Using that the alga undergoes a large scale metabolic shift sediment samples from the IODP Expedition 317 from oxidative metabolism to fermentation with the as a model, our aims were (i) to better understand potential evolution of hydrogen gas. The salamander the diversity, physiology and functions of deep cells exhibit milder differences, including changes subseafloor fungi and (ii) to provide clues about in gene expression indicating the initiation of an how they interact with other microbial populations innate immune response to the alga, and alterations in those communities. Using a record depth in nutrient sensing related to insulin sensitivity. The sediment core, fungal molecu-lar signatures and salamander cells do not exhibit large scale stress or fungal cultures were obtained from samples as deep apoptotic responses suggesting that intracellular as 1740mbsf (Rédou et al. 2014) and 1884mbsf algae are not a big drain on the salamander cell’s (Rédou et al. 2015), respectively. In spite of the resources. fact that those complementary approaches revealed low diversity of higher fungal lineages, DNA and GENOME AND TRANSCRIPTOME OF HEMI- rRNA signatures as well as almost 200 cultured STASIA PHAEOCYSTICOLA, A FLAGELLATE isolates provide direct evidence that fungi persist RELATED TO A NOVEL HYPER-DIVERSE in this challenging habitat. Consistent with this CLADE OF MARINE PROTISTS idea, physiological analyses indicate some deep Butenko A.1, Yabuki A.2, Flegontova O.3,4, Horák subseafloor fungal isolates appear well-adapted to A.3, Flegontov P.1,3,5, Lukeš J.3,4,6 in situ conditions. 1 - Life Science Research Centre, Faculty of Science, Protistology · 11

University of Ostrava, 710 00 Ostrava, Czech Republic ral, Lower Volga and Kama 39 species of the 2 - Japan Agency for Marine-Earth Science and mixotrophic ciliates are revealed. Their structure Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, is heterogeneous: there are “epilimnic” aerophylic Kanagawa 237-0061, Japan myxotrophs and microaerophylic or anaerobic 3 - Biology Centre, Institute of Parasitology, Czech myxotrophs. The first group appears in the low Academy of Sciences, 370 05 České Budějovice trophic state reservoirs; reservoirs with unstable (Budweis), Czech Republic stratification; in the epilimnium of the stratified 4 - Faculty of Science, University of South Bohemia, waterbodies; in acid, colored, with level fluctuation 370 05 České Budějovice (Budweis), Czech Republic waterbodies. The second group prefers microaerobic 5 - A.A. Kharkevich Institute for Information Trans- and anaerobic conditions of stratified eutrophic and mission Problems, Russian Academy of Sciences, 127 hypertrophic lakes with high sulfides concentration. 051 Moscow, Russian Federation The absolute maximums of abundance (206540 6 - Canadian Institute for Advanced Research, ind./l) and biomass (7264 mg/m3) of mixotrophs Toronto, Ontario M5G 1Z8, Canada are registered during summer stratification in a [email protected] highly trophic meromictic pond, and the maximum Diplonemea (Euglenozoa) is an understudied group contribution to biomass - in the small polyhumic of heterotrophic flagellates, related to euglenids lakes (98%).The ciliates’ mixotrophic biomass (6,1 and kinetoplastids. Diplonemids are abundant in g/m3) is comparable with the anoxic phototrophic the deep ocean and, surprisingly, represent the bacteria biomass (9,6 g/m3) and phytoflagellates most diverse clade of pelagic eukaryotes, according (2,6 g / m3) in chemocline and adjacent layers. to our metabarcoding data. There are four major Their contribution to the overall “phototrophic” clades within diplonemids: i/ clade of ‘classic’ component biomass reaches about 37%, while diplonemids, Diplonema and Rhynchopus; ii/ small in other waterbodies the total biomass of ciliates clade of Hemistasia; iii/ small clade of environmental several orders of magnitude less than the biomass sequences (DSPD II, deep-sea pelagic diplonemids of other protists and bacteria. The scheme of II); iv/ large clade of environmental sequences different mixotrophs species confinement to (DSPD I). The DSPD I clade accounts for >97% of various waterbodies and ecotopes in gradient of diplonemid abundance and diversity in the oceanic major abiotic factors has been drawn. An inverse plankton, and Hemistasia phaeocysticola, a recently relationship between environmental indicators isolated marine flagellate that feeds on diatoms and saprobity and mixotrophic ciliates percentage dinoflagellates, appears to be the closest relative of was shown on example of oligo-mesotrophic DSPD I according to some phylogenetic analyses. lake. This can be used when assessing the state of It not clear whether H. phaeocysticola is a predator, aquatic ecosystems. It was proved that the species a parasite, or a scavenger of dead cells. An axenic composition and the nature of their distribution are culture was obtained from H. phaeocysticola growing determined by specific abiotic and biotic conditions with Thalassiosira cf. rotula. The genome and and weakly dependent on the zonal factors. transcriptome of H. phaeocysticola were sequenced using 300 and 250 nt paired-end Illumina MiSeq THE ROLE OF PROTISTS IN THE PLANKTON reads, respectively. A draft genome assembly was COMMUNITY OF FRESHWATER LAKE IN obtained using CLC Genomics Workbench v.8.1 and THE PERIOD OF ITS EUTROPHICATION has the length of ~260 Mbp and N50 of 1.5 kbp. The Bykova S.V., Umanskaya M.V., Gorbunov M.U., results suggest that the genome of H. phaeocysticola Tarasova N.G., Zharikov V.V., Muchortova O.V. is similar to that of Diplonema papillatum in size and Institute of ecology of the Volga river basin of RAS has a very high repeat content. Additional genome [email protected] sequencing using PacBio will be performed and the The Lake Kandry-Kul is one of the largest natural metabolism of H. phaeocysticola will be studied using lakes of the Middle Volga basin. It is a popular a transcriptome assembly. resting place and therefore experience significant recreation load. From 2010 to 2012 the trophic MIXOTROPHIC CILIATES AS SPECIAL ECO- level of the lake has increased from a predominantly LOGICAL GROUP IN WATERBODIES OF oligotrophic to mesotrophic. We studied the CENTRAL AND LOWER VOLGA AND KAMA following groups of protists: ciliates, dinoflagellates, Bykova S.V. chrysophytes, cryptophytes and euglenids. Their Institute of ecology of the Volga river basin of RAS biomass in 2012 compared to 2010 has increased [email protected] 2.4 times. Their contribution to the total biomass In plankton of polytypic waterbodies from Cent- of plankton community and to the to-tal unicellular 12 · “PROTIST–2016” plankton biomass has also increased 1.9 and 1.3 dorsal fan; the excavate ventral groove is missing. times, respectively. Along with these changes, a SSU rRNA gene analyses demonstrated that I. redistribution of the total biomass of the plankton spirale branches as a sister lineage to the free-living community has occured between the pelagic and CLO Hicanonectes teleskopos and its relatives, which littoral parts. In the pelagic zone of the lake the means that it represents the third known endobiotic increase was most significant, 3.8 times, and in lineage of Fornicata. the littoral zone it amounted 1.3 times. The share of protists in the unicellular plankton biomass in SPATIOTEMPORAL DISTRIBUTION OF SPE- the pelagic zone remained unchanged, while in CIES DIVERSITY AND COMMUNITY STRUC- the littoral zone it increased 2.3 times. Protist’s TURE OF CILIATES IN INTERTIDAL SANDY contribution into the total plankton biomass in the SEDIMENTS OF HUOKUN’AO BEACH, NANJI pelagic zone increased 1.3 times, and in the littoral ISLANDS (EAST CHINA SEA, CHINA) 3.4 times. These changes were mainly related to the Chen X.1, Chen W.2, Cai H.2, Xu K.1 increased role of the cryptophytes, dinoflagellates 1 - Institute of Oceanology, Chinese Academy of and ciliates, especially mixotrophic. In the context Sciences, Qingdao 266071, China of eutrophication the most significant correlations of 2 - Nanji Islands National Marine Nature Reserve individual groups of protists were registered with the Administration, Pingyang 325401, China concentrations of phosphorus, ammonia nitrogen [email protected] and chlorophyll a. The influence of environmental In intertidal sediments, ciliates are a highly diverse and factors on the peculiarities of protist distribution and quantitatively dominant group, whose community ratios of their groups are discussed. structure, in some sense, may reflect the condition of environmental quality. We investigated the GEN. ET SP. NOV., A IOTANEMA SPIRALE spatiotemporal distribution of species diversity and NEW ENDOBIOTIC LINEAGE OF FORNI- community structure of ciliates in the intertidal CATA WITH STRIKINGLY SIMPLIFIED sandy sediments of Huokun’ao beach, Nanji Islands MORPHOLOGY AND ULTRASTRUCTURE National Marine Natural Reserve based on sample Čepička I.1, Yubuki N.1, Zadrobilkova E.1,2 collections in November 2013 (autumn), February 1 - Department of Zoology, Charles University in (winter), May (spring) and August (summer) 2014. Prague, Vinicna 7, 128 44 Prague 2, Czech Republic The results showed highly diverse and abundant 2 - Centre for Epidemiology and Microbiology, ciliates in the intertidal sediments of the beach, National Institute of Public Health, Srobarova 48, where a total of 148 species representing 19 orders 100 42 Prague, Czech Republic and over 70 genera were identified. The abundance [email protected] Fornicata is a lineage of excavates living in low and species composition of benthic ciliates showed oxygen environments and lacking a conventional significant correlation with seasons and sediment mitochondrion. Three main morphological/eco- depths. Bacterivorous ciliates constituted the logical subgroups of Fornicata are recognized: most abundant group, followed by carnivores free-living Carpediemonas-like organisms (CLOs) and algivores. The predominance of bacterivores from marine habitats and two parasitic/commensal indicated that the ciliate community in the intertidal lineages living in animal hosts, retortamonads and sediment of Huokun’ao was not a typical interstitial diplomonads. The research of Fornicata centers ciliates fauna, but a pattern similar to those in muddy on evolutionary perspectives such as transition sand sediments with high level of organic matter. from free-living lifestyle to parasitic one and This is likely a subsequent effect of long-term human mitochondrion transformation. Nonetheless, activities. In spite of the impact, the high diversity current modest knowledge of the biodiversity of of ciliates indicates that the benthic environment in Fornicata limits our ability to draw the whole the Huokun’ao beach and its surrounding area is in picture of evolutionary history of this group. The fair condition, which is attributed to the biodiversity free-living CLOs are particularly important due to conservation measures adopted, e.g. the relocation the plesiomorphic traits of excavates. In this study, of surrounding residents and facilities. we cultured a novel member of Fornicata, Iotanema spirale gen. et sp. nov., isolated from fresh feces of PROKARYOTIC ENDOCYTOBIONTS OF a gecko. I. spirale exhibits several unique features PELOMYXIDAE not seen among fornicates. It possesses a single Chistyakova L.V.1, Berdieva M.A.2, Kostygov flagellum and a highly reduced cytoskeletal system A.Yu.3,4, Frolov A.O.3 with a single microtubular root, two fibers, and 1 - Centre of Core Facility of SPSU “Culturing Protistology · 13 collections of microorganisms”, St. Petersburg, Russia extremophilic environments (pH1.5~3, 35~55oC). 2 - Institute of Cytology, Russian Academy of Sciences, Cyanidiales species are reported from hot springs St.Petersburg, Russia around world including Yellowstone National Park 3 - Zoological Institute of the Russian Academy of (US), Italy, Iceland, New Zealand, Indonesia, Sciences,St. Petersburg, Russia and Japan. Some Cyanidiales species are also 4 - Life Science Research Centre, Faculty of Science, found in acidic bogs, mesophilic caves, interlithic University of Ostrava or endolithic environments. In addition, they [email protected] show trophic differences being either autotroph or At present the taxonomy of Archamoebae is based mixotroph. From previous researches, the genomes on molecular phylogeny, and it is problematic to of Cyanidiales are highly reduced, but horizontally find non-molecular synapomorphies for different acquired from bacteria to adopt in extreme habitats. lineages within this group. The family Pelomyxidae Here we report two nuclear genomes of Galdieria includes two archamoebal genera: Pelomyxa and maxima and Cyanidium caldarium and conduct Mastigella. All pelomyxae and most mastigellae have comparative genome analysis with three available various prokaryotic endocytobionts. Host-symbiont genomes of Cyanidioschyzon merolae, Galdieria composition could be useful for the phylogeny sulphuraria, and Galdieria phlegrea. We will discuss reconstruction, but information about such asso- gene contents, mutation rates, and unique horizon- ciations is controversial. We analyzed consortia tal gene transfer that related to the environmental of prokaryotic symbionts in Mastigella nitens and adaptation. 11 Pelomyxa spp. There are two kinds of such consortia within pelomyxid cells: binary and ternary EVOLUTION OF HEME BIOSYNTHESIS PATH- ones (with 2 and 3 symbionts correspondingly). WAY IN ALGAE WITH COMPLEX PLASTIDS The composition of these associations revealed to Cihlář J.1,2, Fussy Z.1, Oborník M.1,2 be species-specific. According to morphological 1 - Biology Centre CAS, České Budějovice, Czech characters, autofluorescence ability and results Republic of Gram staining 4 main types of cytobionts 2 - University of South Bohemia, České Budějovice, can be distinguished. One of them is common Czech Republic to all investigated pelomyxae and M. nitens. [email protected] Morphologically similar prokaryotes can be also Tetrapyrroles are organic compounds essential found in the cytoplasm of other Mastigella spp. for life. Organisms are either synthesizing tetra- Distribution of the remaining types of cytobionts pyrroles or they have to obtain them from their among hosts species is not correlated with the environment, host or prey. We showed that the molecular phylogeny of Archamoebae based on composition of the biosynthetic pathway is shaped 18S rRNA gene. We propose the ability to form by passed endosymbiotic evens in eukaryotes. We symbiotic associations with prokaryotes as a shared map the pathway in phototrophic eukaryotes, feature of all Pelomyxidae. particularly in algae with secondary or other This work was supported by the grant RFBR 15-04- advanced plastids, by investigating origins of 00396_a. Scientific research were performed at the involved enzymes and predicting their location Center for Culturing Collection of Microorganisms in the cell of the euglenophyte Euglena gracillis, and “Chromas” Center of Research park of St. the chlorarachniophyte Bigelowiella natans, the Petersburg State University. cryptophyte Guillardia theta, the dinoflagellate with green secondary plastid Lepidodinium chlorophorum, THE GENOME EVOLUTION OF THE THER- and dinoflagellates bearing diatom endosymbiont MOACIDOPHILIC CYANIDALES RED ALGAE (also called “dinotoms”) – Glenodinium foliaceum, Cho C.H.1, Park S.I.1, Ciniglia C.2, Yoon H.S.1 Kryptoperidinum foliaceum and Durinskia baltica. 1 - Department of Biological Sciences, Sungkyunkwan Chlorarachniophytes and euglenophytes still possess University, Korea two independently operating tetrapyrrole pathways 2 - Department of Environmental, Biological and with the first common precursor δ-aminolevulinic Pharmaceutical Science and Technologies, Second acid synthesized either by the C4 pathway in two University of Naples, Italy steps using the mitochondrially located ALA [email protected] synthase (ALAS), or by the plastid located C5 The Cyanidiales is an early diverged red algal group pathway by consecutive enzymes glutamyl-tRNA that comprised of seven species with numerous reductase (GTR) and glutamate-1-semiladehyde cryptic species. They are asexual, unicellular 2,1 aminomutase (GSA-AT). We propose that such photosynthetic eukaryote, which usually thrive in arrangement of the pathway was ancestral for all 14 · “PROTIST–2016” phototrophic eukaryotes. Other algae show partial system. Single ciliates were hand-isolated and (cryptophyte) or total (dinoflagellates) reduction of subjected to whole genome amplification, prior to the redundant mitochondrially-cytosolic pathway. cloning and sequencing. This approach identified In dinotoms, two redundant plastid located path- specific protistan taxa that were likely some of the ways are present suggesting a presence of the two most active members of the deep-sea community of plastids of different origins in the dinotom cell. microbial eukaryotes. Although L. chlorophorum and B. natans contain chlorophyte-derived plastids, enzymes involved FIRST RECORDS OF MOSS CILIATES IN in tetrapyrrole biosynthesis are dominantly of MEXICO rhodophyte origins. This may suggest an early Cruz Jimenez G.1, Mayén-Estrada R.2, González acquisition of the rhodophyte-derived secondary Adame G.1 plastid through the secondary endosymbiotic event 1 - Lab. de Ecología, Instituto de Análisis Ambiental, on the root of SAR group. Universidad de la Sierra Juárez. Av. Universidad, Ixtlán de Juárez, C. P. 68725, Oaxaca, México MICROBIAL EUKARYOTES FROM THE DEEP: 2 - Lab. Protozoología, Depto. Biol. Comparada, REVEALING THE DIVERSITY OF PROTISTS Fac. Ciencias, Universidad Nacional Autónoma de AND THEIR TROPHIC RELATIONSHIPS AT México, Circuito Ext. s/núm. Ciudad Universitaria, HYDROTHERMAL VENTS C. P. 04510, Ciudad de México, Mexico Countway P.D.1, Pasulka A.L.2, Mesrop L.Y.3, Hu [email protected] S.K.3, Heidelberg K.B.3, Coyne K.J.4, Cary S.C.5, Mosses (Bryophyta) are not vascular gregarious Caron D.A.3 plants that require some environmental characte- 1 - Bigelow Laboratory for Ocean Sciences ristics to grow, i. e. the presence of some moisture 2 - California Institute of Technology degree, doing possible the establishment of ciliate 3 - University of Southern California communities like those that ocurre in peatlands. 4 - University of Delaware Mexico has a diversity of 984 species of mosses and 5 - University of Waikato for ciliates about 959 species have been recorded [email protected] in several habitats, however the study of ciliates Protists in the deep-sea are a diverse mixture of taxa associated to these plants remains unknown until that are either transported there by sinking or occur the present. The aim of this work was to record there as endemic species. It is not uncommon for the ciliate community in Campylopus pilifer Brid the closest genetic relatives of deep-sea protists to during the summer of the year 2015 in the Sierra be found in samples from other deep-sea habitats Juárez, Oaxaca. Samples were manually collected thousands of kilometers away. In other cases, and observed alive using bright field and DIC the nearest relatives of deep-sea protists are from microscopy. We also employed silver impregnation equally distant, but shallow-water habitats with techniques for species identification. We recorded similar characteristics to the deep-sea locales (e.g., five species of ciliates: Colpoda inflata, C. aspera, low-oxygen or high-temperature sediments). Many Colpoda sp., Vorticella infusionum and Vorticella of the most conspicuous protists at hydrothermal sp. in C. pilifer belonging to habitats with different vents are ciliates and small flagellates that rely on environmental characteristics. These findings the copious bacterial production occurring in the represent the first moss-ciliate record for Mexico. proximity of vent ecosystems. We investigated the Keywords: Ciliates, Bryophytes, México, Moss, diversity and activity of marine protists from two Oaxaca. deep-sea hydrothermal vent ecosystems including Acknowledgements. To Biól. M. Reyes and M. C. those at Guaymas Basin (Gulf of California, N. Maricela Vicencio, Facultad de Ciencias, UNAM, Pacific) and 9-North (Eastern Tropical N. Pacific). for their support with technical procedures. Complementary molecular techniques were emplo- yed to reveal the full breadth of protistan diversity, POPULATION STRUCTURE OF PLANKTO- the diversity of the active fraction, and the taxono- NIC MICRO-ORGANISMS (TINTINNID CI- mic identities of some of the most conspicuous and LIATES OF THE MICROZOOPLANKTON) IN active deep-sea ciliates. Clone libraries based on THE SOUTH PACIFIC OCEAN: COMPARISON RNA extractions revealed a substantially greater OF A HIGH PRODUCTIVITY BLOOM AND A proportion of the protists that appeared to be the TYPICAL OLIGOTROPHIC SITE most active via microscopy, while libraries based Dolan J.R. on DNA captured the diversity of both endemic CNRS Laboratoire d’Océanographie de Ville- organisms and those that sank into the vent eco- franche-sur-Mer Protistology · 15 [email protected] such a way, the list of specialized troglobiont ciliates We hypothesized that the population structure of must include suctorians Spelaeophrya troglocaridis planktonic grazers in a high productivity site differs from shrimps, Tokophrya bathynellae from syncarids, from that known for typical oligotrophic sites. We Echinophrya stenaselli and T. microcerberi from expected the high productivity site to harbor more subterranean isopods. The analysis of literary and species. We compared a high productivity site with own data on distribution and host prevalence of dense populations of N-fixing Trichodesmuim, suctorian ciliate T. niphargi from amphipods and and a distant oligotrophic site. We examined peritrich ciliate Ballodora marceli from wood louses patterns of species abundance distribution and the permit to define these ciliates to epigean group of abundance distribution of ecological types, that species. As for apostomes Gymnodinioides sp. from is species of similar shell or lorica oral diameter, cavernicolous amphipods and shrimps, status of analogous to gape size. We also examined species these ciliates remains to be seen. packing within ecological types, defined as lorica oral diameter size-classes. Population abundance SPECIES DIVERSITY AND BIOTOPICAL differed between the sites by a factor of 3, and the DISTRIBUTION OF HETEROTROPHIC FLA- productive site population was highly dominated by GELLATES IN SMALL RIVER FLOODPLAIN a single species (not found at the oligotrophic site) Dubrovsky Yu.V.1, Mylnikov A.P.2 but species richness and turnover were very similar. 1 - Institute for Evolutionary Ecology of the Nacional Species abundance distributions for both sites on Academy of Sciences of Ukraine (IEE NAS of Ukra- all dates most closely fit a log-series or lognormal ine), Laboratory of Wildlife Protection and Restoration distribution. Abundance distributions of ecological 2 - Papanin Institute for Biology of Inland Waters, types, forms of distinct lorica oral diameter, were Russian Academy of Sciences, Borok, Russia the typical geometric for the oligotrophic site. In [email protected] contrast, at the high productivity site, lognormal Twenty-seven species and forms of heterotrophic or log-series distributions provided equally good flagellates have been found in the result of analysis of fits. In the oligotrophic site there was a positive 8 water and 4 soil samples (downstream of river Ros’, relationship between the number of individuals in a Ukraine, 49º37´N; 31º30´E) during summer 2014. lorica size-class and the number of species but not Observed species belongs to Opisthokonta (2 spe- in the productive site. Despite large differences in cies), SAR (17), Excavata (8) and incertae sedis population size and dominance, the two populations group (2). The most flagellates were bacterivorous. were surprisingly similar by most measures. Our Allantion tachyploon, Kathablepharis sp., Alphamo- results suggest that overall food web structure is nas edax, Colponema vietnamica, Diphyleia rotans, likely similar as well. Rhynchobodo armata belonged to predators and Goniomonas truncata and Paraphysomonas spp. were ARE SPECIALIZED TROGLOBIONT CILIA- omnivorous. The relationship between locality of TES EXIST? the sample and species composition has not been Dovgal I.V. revealed. This fact indicates the random character Kovalevsky Institute of Marine Biological Research of species distribution. The most abundant (9-13 [email protected] species) were overgrown river floodplain creeks The organisms inhabiting in subterranean waters and ponds, as well as wet soil deciduous forest. can be separated into two groups. One of them The silted ponds and creeks without flow, as well is represented by species, which for any reasons as coastal soils, are characterized by the average being situated in the underground habitats and species richness (6-7 species). River watercourse, have adapted to them. As a rule, these are the same dried puddle, bedrock at a depth of 12 m and the soil species as in epigean waters or soils and possible under field crops possess the lowest species richness has originated from mentioned habitats. Thus, the (2-4 species). The vast majority of species is com- group might denoted as epigean. Another group of mon for all investigated biotopes and occurs both in organisms is represented by specialized troglobiont water and in the soil. Among the 11 species occurring species. The list of ciliated protozoans living in in more than three samples, 7 species are inhabited subterranean waters comprises about 150 species, both water and soil, and 4 species were found only but most of them can be defined as representatives in water samples. The average number of species for of epigean group. Only a few ciliate species, water and soil samples (7 and 6 correspondingly) was which are host-specific commensals or parasites not significantly differed. The specificity of species of subterranean animals (especially crustaceans) composition within habitats is not observed. This possibly, should be considered as troglobionts. In study was supported by the Russian Foundation for 16 · “PROTIST–2016”

Basic Research (grant nos. 14-04-00500, 14-04- morphology alone. We isolated 6 filose amoebae 00554, 15-29-02518). from soils of two European countries and describe a new genus of naked filose amoebae, Kraken gen. THE CHLAMYDOPHRYIDAE REVISITED nov. comprising one new species Kraken carinae WITH SPECIAL FOCUS ON THE GENUS sp. nov. We provide a morphological description LECYTHIUM based on light microscopy and small subunit Dumack Kenneth1, Baumann Christina1, Müller rRNA gene sequences (SSU rDNA). In culture, Marina2, Bonkowski Michael1 Kraken carinae strains were very slow-moving and 1 - University of Cologne preyed on bacteria using a network of filopodia. 2 - Leibniz-Centre for Agricultural Landscape Re- Phylogenetic analyses of SSU sequences reveal that search ZALF Kraken are core Cercozoa, branching weakly at the [email protected] base of the cercomonad radiation, most closely Although testate amoebae have attracted interest of related to Paracercomonas, Metabolomonas, and protistologists for more than 150 years, some groups Brevimastigomonas. Kraken sequences are >99% especially those with a hyaline test are still poorly similar to an environmental sequence obtained known. One of those fairly understudied groups is from a freshwater lake in Antarctica, indicating that the family Chlamydophryidae. They are difficult to Kraken carinae is not exclusively soil dwelling, but culture and therefore only old, sometimes obscure, also inhabits freshwater habitats. species descriptions are available. Further, no molecular data was obtained and therefore in mo- FIRST RECORD OF BROMELIOPHRYA BRA- dern mass sequencing studies they are only recog- SILIENSIS FOISSNER, 2003 (CILIOPHORA: nized as OTUs of unknown affinity. Nevertheless, GLAUCOMIDAE) FROM TANK BROMELIADS they are widely distributed, can occur in high IN MEXICO WITH SOME NOTES ON ITS abundances in freshwater and terrestrial habitats, DISTRIBUTION AND ECOLOGY show interesting feeding behavior and have a Durán-Ramírez C. A.1, Mayén-Estrada R.1, Ciros- high diversity in morphology. This talk will give Pérez J.2 an overview of the up to date knowledge on the 1 - Laboratorio de Protozoología, Departamento Chlamydophryidae and will further focus on one de Biología Comparada, Facultad de Ciencias, of its genera, Lecythium that was first described Universidad Nacional Autónoma de México, Ciudad by Hertwig & Lesser in 1874. We (a) established 7 Universitaria s/n, Coyoacán, 04510 Mexico City cultures of 5 Lecythium species and provide detailed 2 - Proyecto de Investigación en Limnología Tropical. morphological as well as ecological observations UIICSE, FES Iztacala UNAM. Av. de los Barrios No. (b) obtained SSU sequences and conducted phylo- 1, Los Reyes Iztacala, Tlalnepantla 54090, Estado genetic analyses of the Tectofilosida, showing that de México Lecythium is closely related to the Pseudodifflugiidae [email protected] and splits into a terrestrial and freshwater clade Bromeliophrya brasiliensis is a glaucomid bacteri- and (c) performed food choice experiments that vorous ciliate described from terrestrial tank underline the mycophagous and algivorous feeding bromeliads in the Atlantic Coast of Brazil, and behavior of Lecythium. Our talk will show that the recorded in Paraná, Brazil and Ecuador from Chlamydophryidae are a group of protists that are rupicolous bromeliads. The reduction of somatic highly fascinating. ciliature and the presence of two kinetofragments, which migrate to the left margin of the buccal THE BASE OF CERCOMONAD RADIATION IS cavity, are the main cytological characteristics as STILL IN FOR A SURPRISE, KRAKEN GEN. compared with B. quadristicha which presents four NOV. kinetofragments and it has been recorded in the Dumack Kenneth1, Schuster Julia1, Bass David2, Antilles. The objective of this work is to describe the Bonkowski Michael1 ciliate diversity from tank bromeliads in Mexico. We 1 - University of Cologne sampled 30 individuals of epiphytic tank bromeliads 2 - Department of Life Sciences, Natural History including five species of genera Catopsis and Museum London, Cromwell Road, London Tillandsia in three different temperate forest sites [email protected] from Central and Southern Mexico, during the year The term ‘filose amoebae’ describes a highly poly- 2015. The sites were located from 1336 to 2426 m phyletic assemblage of protists whose phylogenetic a.s.l. Observation and identification of B. brasiliensis placement can be unpredictable based on gross was performed using bright and DIC microcopy, Protistology · 17 and with silver impregnation methods. Cellular partitioning. Projects supported by NSF OCE- specific characteristics were analyzed and B. brasi- 0849578, OCE-1061391, and OCE-1336082. liensis was recorded in 23% of the samples with temperature values between 18 to 22 °C, and pH THE MARINE CARAVAN INVADING THE values from 3 to 6, demonstrating a wider range TWO BIG SEAS OF THE RED SEA AND THE of temperature and pH tolerance, and a broad MEDITERRANEAN THROUGH THE MARI- geographic distribution but only in Neotropical tank TIME SUEZ CANAL: I- CILIOPROTISTS bromeliads. This is the first record for the species Elserehy Hamed1,2, Al Quraishi Saleh1, Al-Farraj in these microecosystems at different altitudes in Saleh1 Mexico. Financial support was provided by National 1 - Department of Zoology, College of Science, King Council of Science and Technology (CONACYT) Saud Uuniversity, Riyadh 11451, Saudi Arabia fellowship number 224627. Biol. Margarita Reyes 2 - Department of Marine Science, Faculty of Science, and M.A. Aldi de Oyarzábal from Facultad de Port Said University, Egypt Ciencias, UNAM, are thanked for technical assis- [email protected] tance and scientific illustration respectively. Suez Canal is the main connecting link between the Red Sea in the south and Mediterranean in PROTIST DIVERSITY AND INTERACTIONS the north. It crosses many lakes, which in its turn WITHIN DEEP-OCEAN SEDIMENT AND represent different habitats. 28 plankton ciliate WATER-COLUMN MICROBIAL COMMU- taxa and species were collected from the canal NITIES waters during the present study. Much interest Edgcomb V.P.1, Pachiadaki M.1,2, Taylor C.1, was focused on determining from which end of the Kormas K.Ar.3, Bernhard J.M.1, Taylor G.T.4 canal these organisms were invading the opposite 1 - Woods Hole Oceanographic Institution, Woods sea. Planktonic ciliates appear to enter the Suez Hole, MA, USA Canal from the south via water currents; to do so it 2 - Bigelow Laboratories, East Boothbay, Maine, USA needs to be carried over a distance of 25 km along 3 - University of Thessaly, Greece the canal from the Gulf of Suez into the Bitter 4 - Stony Brook University, Stony Brook, New York, USA Lakes, then pass across the Bitter Lakes before being [email protected] carried a further 15 km along the canal into Lake Protists are integral members of marine food webs and Timsah. Plankton ciliate affinities and differences exhibit complex relationships with other microbial between the eastern Mediterranean and the Red taxa. Phagotrophic protists contribute significantly Sea were discussed in the light of the results of to carbon turnover in the sunlit ocean and evidence distribution studies of the twenty eight species of suggests grazing in the dark ocean can be significant plankton carried out. These observations point to as well. The molecular signatures of parasitic the role of the canal as a selective barrier and/or protists comprise significant fractions of many as a link in the process of marine bio-invasions of high-throughput sequencing datasets, suggesting a planktonic organisms, while cases of invasion from potentially important role in controlling populations one sea to the other are more likely to occur in of their host(s). Prokaryotic symbionts of free-living either direction, those concerning species of indo- protists can be numerous, and, particularly in low- Pacific origin are more successful and numerous oxygen to anoxic marine habitats their collective (23species). Meanwhile, the Suez Canal acts as a metabolisms may contribute significantly to bio- local endemic habitat by itself (5 species). Thus, the geochemical cycling. Particular groups of protists opening of the Suez Canal during the year of 1869 are abundant and common inhabitants of marine caused marine bio-invasions in the Mediterranean sediment and water column oxyclines and very deep Sea and rarely in the Red Sea. Accordingly Red halocline habitats where physical associations with Sea species invaded the Mediterranean ecosystem prokaryotes are frequently observed. Using open and not vice versa. Finally, it would be concluded ocean oxygen minimum zones, deep hypersaline that, a continuous monitoring programme must anoxic basins of East Mediterranean Sea, and the be launched especially after the new expansion of permanently stratified water column of Cariaco the canal and opening the new one to record the Basin, Venezuela as natural laboratories to link recent invasive plankton species and follow up the biogeochemistry and diversity, we report evidence distribution and abundance of those previously of niche partitioning and specialized communities. recorded as invasive or alien species to assess their Sediment communities seem to be dominated impacts on the native biodiversity of the Suez Canal, by different key players but exhibit similar niche Red Sea and Mediterranean. 18 · “PROTIST–2016”

THE STABILITY OF THE MARINE CILIATE clone-recognition experiments in seven Entamoeba COMMUNITY TO ENVIRONMENTAL CHAN- lineages (E. invadens IP-1, E. invadens VK-1:NS, E. GES terrapinae, E. moshkovskii Laredo, E. moshkovskii Esaulov A.S.1, Mazei Yu.A.1,2, Burkovsky I.V.2 Snake, E. histolytica HM-1:IMSS and E. dispar). 1 - Penza State University We demonstrate that amebas themselves can 2 - Lomonosov Moscow State University discriminate self (clone) from different (themselves [email protected] versus other clones). In mix-cell-line cultures A complete study of processes that occur during between closely-related (E. invadens IP-1 versus E. the formation of marine communities and their invadens VK-1:NS) or distant-phylogenetic clones stability to factors of the environment requires (E. terrapinae versus E. moshkovskii Laredo), amebas information for a sufficient period of time that consistently aggregated with same-clone members. would cover a succession of many hundreds or Additionally, we identified six putative cell-signals even thousands of generations of organisms. secreted by the amebas (RasGap/Ankyrin, coronin- Studies like these have been conducted by a team WD40, actin, protein kinases, heat shock 70, and of biologists at the White Sea for a several decades. ubiquitin) and which known functions in Entamoeba In field experiments on the transplantation of frag- spp. included: cell proliferation, cell adhesion, cell ments of natural communities of marine ciliates movement, and stress-induced encystation. Each into an estuary, restoration of the initial or modi- clone was characterized morphometrically (length, fied species structure was observed after a short width, and cell-surface area) and documented how period of adaptation of the organisms. Their they differed statistically from one another (as per responses to new conditions included both spe- single-variable or canonical-discriminant analyses). cies-specific reactions and group reactions (the This is the first multi-clone characterization of synergistic effect). In a group, species are able to Entamoeba spp. aggregative behavior, cell-signaling sustain larger amplitudes of variations in environ- secretion, and morphometrics in the context of mental factors than if they were separate and they clone-recognition. Because unicellular eukaryotes can even occupy extreme biotopes. Under the belong to ancient and highly diverse phylogenetic conditions of the experiment, as well as in nature, a lineages, occupy all environments on Earth, and multitude of structural variants of the psammophile participate in complex interactions with other community (multivariability of structure) were organisms (as hosts, symbionts or parasites), they can created from the united pool of species through be robust model systems to study the implications the recombination of their abundances. In total, of taxa, clone and kin discrimination/recognition the formation and maintenance of communities of in ecological and evolutionary contexts, and with unicellular organisms is determined by a complex emphasis on basic or applied sciences. mechanism that includes physiological (tolerance), population (reproductive properties), cenotic DIVERSITY AND ABUNDANCE OF KINETO- (interspecific interactions), and stochastic (reaction PLASTIDS IN THE WORLD OCEAN to environmental fluctuations) processes; their Flegontova O.1,2, Flegontov P.3,2, Logacheva M.D.4, relative roles depend on the level of organization of Konovalova O.P.4, Malviya S.5, Bowler C.5, Lukeš the community (succession stage) and on the range J.1,2,6, Horák A.1,2 of variations in environmental factors. 1 - Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic AGGREGATIVE BEHAVIOR, CELL SIGNA- 2 - Institute of Parasitology, Biology Centre, Czech LING AND MORPHOMETRICS IN ENTAMO- Academy of Sciences, České Budějovice, Czech Re- EBA DISCRIMINATION STUDIES public Espinosa A.1,2, Paz-y-Mino-C G.2, Hackey M.1, 3 - Faculty of Science, University of Ostrava, Ostrava, Rutherford S.3 Czech Republic 1 - Department of Biology, Roger Williams University 4 - Belozersky Institute of Physico-Chemical Biology, 2 - New England Center for the Public Understanding Lomonosov Moscow State University, Moscow, Russia of Science, Roger Williams University 5 - Ecole Normale Supérieure, PSL Research Univer- 3 - Department of Environmental Science, Roger sity, Institut de Biologie de l’Ecole Normale Supérieure Williams University (IBENS), Paris, France [email protected] 6 - Canadian Institute for Advanced Research, Studies on clone- and kin-discrimination in protists Toronto, Canada have proliferated during the past decade. We report [email protected] Protistology · 19

Kinetoplastids include important protozoan para- eubacterial probes which able to visualize wide rage sites of humans, other mammals and plants. The of most common bacterial endosymbionts are used key genera, Trypanosoma and Leishmania, belong more often. During long time of using (Fokin et al., among the best-studied unicellular eukaryotes. 1996), I have found that sometimes, using the same However, our knowledge of diversity and distribu- protocol, these probes specifically can bind also to tion of basal, mostly free-living heterotrophic several cortical stuctures of ciliated protists, namely: lineages is rather limited, despite their potential kinetosomes, extrusomes, and cortical granules. importance in understanding the evolution of The reason of such unusual FISH results is not parasitism in kinetoplastids. We have analyzed so clear from molecular point of view. However, kinetoplastid 18S rDNA barcodes (V9 region) in this “impregnation technique” could be useful a large global metabarcoding dataset, combining for morphological and taxonomical investigations 123 stations of the Tara Oceans. According to our of ciliates. In many cases (representatives of results, the majority of planktonic kinetoplastids Nassophorea, Plagiopylea, Heterotrichea, and belonged to Neobodonida: about 90% reads and Spirotrichea) such decoration could be find out 70% OTUs. Compared to their sister-clade, diplo- only sporadically, but for Armophorea members nemids, kinetoplastids show similar distribution the cortical granules always impregnated by both patterns: both are more abundant in the mesopelagic oligonucleotide probes. In majority of heterotrichs than in the photic zone, in the piconano-plankton in which presence of cortical granules is very (<5 µm in size), and demonstrate no apparent common feature I could not able to get such a biogeography. Both groups are dominated by a FISH impregnation. Phenomenon discovered few abundant cosmopolitan OTUs: neobodonids not only allows to use FISH technique in some and Bodo sorokini in the case of kinetoplastids. cases for the ciliate’s kinetome investigation, but However, kinetoplastids were much less abundant puts a question about origin of cortical granules in in all depth zones (on average 0.2% of eukaryotic Armophorea and in other ciliate lineages. Unusual reads per sample, and ranging from 0% to 10.8%), labelling are illustrated by FISH images of different and much less diverse (~650 OTUs vs. >50,000 Metopus spp., Brachanella spiralis, Copemetopus sp., OTUs for diplonemids). Also, unlike diplonemids, Blepharisma sp., Euplotes crassus, Sonderia vorax, planktonic kinetoplastids show only weak vertical and Pseudomicrothorax dubius. structure with 23% OTUs present in all sampled depth zones: surface, deep chlorophyll maximum, AN ALVEOLATE ALGA VITRELLA BRASSI- and mesopelagic. Rarefaction curves revealed that CAFORMIS FORMS ZOOSPORES BY BUDD- kinetoplastid diversity was saturated in the whole ING dataset, as well as in separate depth zones, size Füssy Z.1, Kručinská J.1,2, Esson H.J.1, Oborník fractions, and oceanic provinces. Our results suggest M.1,2,3 kinetoplastids are rare but ubiquitous component of 1 - Institute of Parasitology, Biology Centre, Czech the global plankton. Academy of Sciences, České Budějovice 2 - Faculty of Science, University of South Bohemia, FISH TECHNIQUE AS A POSSIBLE TOOL České Budějovice FOR CORTEX INVESTIGATION IN ARMO- 3 - Centre Algatech, Institute of Microbiology, Czech PHOREAN CILIATES (CILIOPHORA, ARMO- Academy of Sciences, Třeboň PHOREA) [email protected] Fokin Sergei I.1,2 The ease of cultivation and availability of genomic 1 - Department of Invertebrate Zoology, St. Petersburg data promoted intense research of free-living State University, 199034 St. Petersburg, Russia relatives of apicomplexans, Chromera and Vitrella, 2 - Department of Biology, Pisa University, 56126 to understand evolutionary processes leading to the Pisa, Italy emergence of parasitism. Recently the ultrastructure [email protected] was revealed of an apically located structure of Fluorescence in situ hybridization (FISH) is now- Chromera velia, supposedly homologous to the adays common molecular technique. One of pos- apical complex essential for host invasion in sible application of this technique is in situ indeti- Apicomplexa. Yet, Chromera and Vitrella differ fication and phylogenetic position of uncultured significantly in their physiology, morphology, bacterial endosymbionts (Amann et al., 1991). For phylogenetic position and genomic features, this purpose rRNA-targeted oligonucleotide probes and Chromera is likely the more evolutionary were developed for different groups of bacteria. In advanced of the couple. Hence, in an expectation protistological studies alphaproteobacterial and of more ancestral features, we inspected in further 20 · “PROTIST–2016” detail the ultrastructure of Vitrella brassicaformis, suggested the tintinnid genus Favella as a model concentrating on the apical region of the flagellar for planktonic ciliates. In the present study on the stage. A previous work described a lifestage, biogeography of Favella panamensis, the species surrounded by roughly structured matter of the records from about one hundred of taxonomical containing sporangium, with an intracytoplasmic and ecological studies were classified according axoneme structure suggested to develop into to their quality: (i) reliable records from the type mature flagellum. Here we describe a novel stage and neo-type localities mentioned in the original with cells developing a flagellum extracellularly. description and authoritative redescription; (ii) more These cells concurrently bud off the center of the or less reliable records supported by descriptions, sporangium, surrounded by smooth matter, in measurements, and/or illustrations that fit the a process reminiscent of ectomerogony as seen original description and redescription; and (iii) in Eimeria. We suggest that the former process unsubstantiated records (mostly simple species lists) generates gamete-like flagellate progeny, while based on uncertain identifications. The comparison the latter process yields zooflagellates. We failed to of the data provided by substantiated records with find apical structure similar to the pseudoconoid the original description revealed false positive and in C. velia. We propose that the common ancestor negative identifications. Since currently the species of Apicomplexa and “chrompodellids” exhibited circumscriptions are uncertain, the usage of the a complex lifecycle, while extant descendants original descriptions or authoritative redescriptions underwent gradual reduction of these processes as for the identification of tintinnid ciliates is strongly forced by their environment. recommended. Only later, when the cell features Acknowledgement: The authors would like to thank and barcodes of the morphotypes are known, we the Czech Science Foundation (JK, HJE, MO) and might be able to perform justified synonymisations. the Czech Academy of Sciences (ZF) for funding. References. Montagnes (2013): Ecophysiology and behavior of tintinnids. In: Dolan J.R., Montagnes DIFFICULTIES IN ASSESSING THE GLOBAL D.J.S., Agatha S., Coats W.D. & Stoecker D.K. DISTRIBUTION OF A MODEL ORGANISM (2013): The Biology and Ecology of Tintinnid - THE BIOGEOGRAPHY OF FAVELLA PANA- Ciliates: Models for Marine Plankton. Wiley- MENSIS (ALVEOLATA, CILIOPHORA) Blackwell: 85-121. Ganser M., Agatha S. University of Salzburg, Dept. Ecology and Evolution THE BEGINNING: INFERENCE OF THE [email protected] TINTINNID ANCESTOR’S MORPHOLOGY Accurate circumscriptions and determinations of (ALVEOLATA, SPIROTRICHA, TINTINNINA) species are essential for biodiversity and biogeo- Ganser M., Agatha S. graphy assessments; traditionally, the morpho- University of Salzburg, Dept. Ecology and Evolution species concept was employed. Tintinnid ciliates [email protected] are exceptional as - in contrast to the vast majority Tintinnids are a speciose group of ciliates, whose of ciliates - the whole taxonomy and classification of taxonomy and systematics are almost exclusively the more than one thousand marine planktonic based on lorica features. Physico-chemical factors species are almost exclusively based on characte- and the cell cycle, however, influence these ristics of their vase-shaped loricae (houses). taxonomic characters, resulting in an artificial Because it is relatively easy to collect, preserve, classification with several non-monophyletic examine, and classify, the lorica has allowed the groupings. The sparse cytological data and the accumulation of invaluable diversity and distribu- more comprehensive molecular phylogenies indi- tion data for more than two centuries. However, cate that the tintinnids developed in the marine lorica-based taxonomy is problematic because of environment and entered freshwater several times the high intraspecific variability and interspecific independently. The tintinnidiid genera Tintinnidium similarity of the loricae; so, the species limitations and Membranicola are typical representatives of the are currently unknown. Besides misidentifications, limnetic plankton and are characterized by a simple the diversity and geographic ranges perceived are somatic ciliary pattern and a strong synapomorphy, especially affected by revisionary taxonomic tre- namely, the two de-novo-originating ventral atises differing in their species circumscriptions organelles. The family is sister to the remaining, from the original descriptions due to synonymisation mainly marine species. As yet, only a single member (range of morphologic features became wider) of the genus Tintinnidium, T. mucicola, is known and splitting (range of features became smaller) of to occur in marine and brackish coastal waters, species. Despite these difficulties, Montagnes (2013) suggesting that it might provide clues pivotal to Protistology · 21 infer the morphology of the tintinnid ancestor’s Tintinnopsis tocantinensis, Rhizodomus tagatzi and morphology. Especially, the ventral organelles of Amphorellopsis acuta replacing the indigenous the Tintinnidiidae require a closer inspection owing species as Codonella lagenula, Helicostomella to their uncommon de-novo-origin which contrasts subulata, T. compressa, T. lobiancoi, T. rossolimi the intrakinetal proliferation of basal bodies in and Metacylis jorgensenii. However, the indigenous the other somatic kineties of the Tintinnidiidae Tintinnopsis campanula, which was not observed and the somatic ciliature in all other tintinnids. during 10 years (since 1999) again, began to be Accordingly, the morphology of lorica and cell of present in the plankton from 2009 constantly. T. mucicola are described based on live observations The finds of alien species are not rare, during and protargol-impregnated material and the cell certain seasons they reach high abundances. For division pattern is analysed with special emphasis example, such species as Eutintinnus lususundae and on the development of the ventral organelles. The E. tubulosa are often dominating in the plankton study indicates that all somatic ciliary rows of T. community. mucicola are exclusively dikinetidal and develop by The reasons for mentioned changes are not known intrakinetal proliferation of basal bodies and thus exactly. It is possible that these related with invasion demonstrate the plesiomorphic character states of ctenophore Mnemiopsis leidyi into the Black Sea, already present in the dorsal rows of the euplotids which caused significant changes in the plankton and hypotrichs as well as in the girdle and ventral community structure. Resulting the current list kineties of the Oligotrichida. The species has two of the Black Sea Tintinnida consist of 23 species obliquely orientated kineties resembling the ventral including invasive. organelles in the other Tintinnidium species and the monotypic genus Membranicola. Hence, the MITOCHONDRIAL TRANSCRIPTOME OF ancestor of the tintinnids apparently had exclusively LEPTOMONAS PYRRHOCORIS H10 - CLOSE dikinetidal somatic kineties all reproducing by MONOXENOUS RELATIVE OF LEISHMANIA intrakinetal proliferation of basal bodies, and the CLADE de-novo-origin of the ventral organelles in the Gerasimov E.S.1, Flegontov P.N.2, Gasparyan remaining Tintinnidiidae necessitates confirmation. A.V.1, Logacheva M.D.3, Kolesnikov A.A.1 The study was financially supported by the Austrian 1 - Department of Biology, M.V. Lomonosov Moscow Science Fund (FWF): Project P28790. State University, 119991, Moscow, Russia 2 - Biology Centre, Institute of Parasitology, Czech THE BLACK SEA TINTINNIDS (SPIROTRI- Academy of Sciences, 370 05 České Budějovice CHEA, CHOREOTRICHIA, TINTINNIDA): (Budweis), Czech Republic CURRENT STATE OF BIODIVERSITY AND 3 - Belozersky Institute of Physico-Chemical Biology, SPECIES COMPOSITION M.V. Lomonosov Moscow State University, 119991, Gavrilova N.A. Moscow, Russia Institute of Marine Biological Researches, Russian [email protected] Academy of Science, Plankton department, Laboratory Mitochondrial genome of trypanosomatids have of microplankton several unusual traits both in it’s structural organi- [email protected] zation and in it’s expression. About 22 mitochon- According to literary and own data considering drial genes are compactly grouped in coding region recent nomenclature revision there were 20 tintinnid of maxicircle - major coding component of the choreotrich species in the Black Sea: Codonella mt-genome. Some transcripts undergo extensive lagenula, Helicostomella subulata, Tintinnopsis RNA-editing process before they become mature campanula, T. compressa, T. lobiancoi, T. rossolimi, mRNAs and this is mediated by small gRNAs T. baltica, T. karajacensis, T. cylindrica, T. tubulosa, coded in minicircles. Biological advantages of T. beroidea, T. meunieri, T. urnula, T. minuta, such complex expression system remains unclear Tintinnidium mucicola, Metacylis jorgensenii, M. but it definitely plays crucial role in mitochondrial mereschkovskii, Favella ehrenbergii, Stenosemella genome expression. Editing is well-studied in ventricosa and S. nivalis. some dixenous species like Trypanosoma brucei It should be noted that against the background of and Leishmania tarentolae but less data exists for stable level of tintinnid diversity we observed the monoxenous species. In this work we present a full significant changes in the species composition of the mitochondrial transcriptome map of Leptomonas group. Thus from 2001 to 2015 we registered 8 inva- pyrrhocoris H10 generated using RNAseq data. sive species: Eutintinnus lususundae, E. tubulosus, The overall transcription profile appeared to E. apertus, Eutintinnus sp., Salpingella decurtata, be similar to profile of Leishmania tarentolae. 22 · “PROTIST–2016”

Optimized U-insertion\deletion search software BIOGEOGRAPHY AND THE GENETIC STRUC- TAligner and usage of NGS technology enabled us TURE OF MORPHOSPECIES IN THE GENUS to focus on molecular intermediates of editing MAYORELLA (AMOEBOZOA, DISCOSEA, process and build detailed editing graphs for all LONGAMOEBIA) maxicircle cryptogenes. Reconstruction of editing Glotova A.A. process in cryptogenes showed that in most cases Department of Invertebrate Zoology, Faculty of Biology, editing can proceed in a variety of alternative ways St. Petersburg State University, Saint Petersburg, Russia yielding ORFs with lots of aminoacid substitutions [email protected] or even with alternative protein product. Our results Due to the lack of biogeographical data for the suggest that editing can be the major driving force majority of naked lobose amoebae genera, the of rapid adaptation to environment changes through genetic structure of amoebae morphospecies have the generation of proteins diversity. been previously observed only in several species from Flabellinia group (Amoebozoa, Discosea). GENETIC DIVERSITY OF CENTROHELID Further extensive study of amoebae biogeography HELIOZOA IN BRACKISH INLAND WATERS including species from different phylogenetic OF RUSSIA lineages is clearly required given the assumption of Gerasimova Elena, Plotnikov Andrey amoebae significance for freshwater microhabitat Institute for Cellular and Intracellular Symbiosis UB function based on their abundance. In this study RAS, Orenburg, Russia Cox I and 18S rDNA genes were used as DNA [email protected] barcodes in order to identify strains of the genus Centrohelid heliozoa are ubiquitous, unicellular, Mayorella (Discosea, Longamoebia) in addition non-ciliate phagoheterotrophs with slender radia- to morphological data, that allowed to elucidate ting axopodia for trapping prey. Centrohelids the morphospecies genetic structure and general have a cosmopolitan distribution; inhabit benthos principles of their distribution in a global scale and and periphyton in both marine and freshwaters to evaluate resolving power of various identification ecosystems, in communities they play role of approaches. The samples were collected from predators grazing on other microorganisms. At distant freshwater habitats of North America, present both genetic and morphological approaches Europe and Russian Far East. Results contributed for their taxonomy and classification are used. to the amoebae morphospecies conception which Morphological and genetic diversity of heliozoa in represents it as a limited set of genetic lineages. environmental saline and brackish waters has been Supported with SPSU project 1.38.251.2014 studied very poorly. Genetic diversity of centrohe- lids with group-specific originally designed primers DEEP SEQUENCING OF ANCIENT 18S RDNA was studied by NGS with MiSeq (Illumina). Fifteen IN SEDIMENT CORES FROM CONTINENTAL samples from brackish inland water bodies of SHELVES OF CHINA Russia with mineralization 2-20‰ were examined. Gong J., Wang Y., Zhu P., Zhang Q. As a result 40 OTUs were obtained. Most of the Yantai Institute of Coastal Zone Research, Chinese environmental OTUs (32) were aligned with Academy of Sciences, Yantai 264003, China Polyplacocystis contractilis (AB196984, NCBI) with [email protected] support 99% and were placed inside Pterocystina Protists/phytoplankton in overlying waters and clade. This fact indicates that the most common terrestrial biomaterials could be transported, habitants in brackish waters are related to this deposited and buried in sediments on continental particular genetic variant of centrohelida. Other shelves of marginal seas. Some of these ancient OTUs with 26% and 28% support grouped with DNA could be preserved, serving as a powerful proxy environmental centrohelidian sequences from for reconstructing past regimes of the ecosystems. hypersaline habitats and occupied a basal position We obtained three sediment cores each about 2.5 in Pterocystina A clade. meters in length from three sites at the Bohai Sea The research was performed in the Center of (the mouth of Yellow River, shorted as YR) and Shared Scientific Equipment «Persistence of Yellow Sea (the northern basin, NYS, and southern microorganisms» of ICIS UB RAS and was suppor- basins, SYS). The DNA was extracted and quanti- ted by RSF (14-14-00515) and RFBR (15-29- fied and subjected to high throughput sequencing 02749, 16-44-560234). of eukaryotic 18S rDNA fragment using Illumina MiSeq. Classification using pipelines indicated the Protistology · 23 presence of molecular signals of eukaryotic groups versity, Daejeon 34134, Korea including Dinophyta, Stramenopiles, Ciliophora [email protected] and Fungi. The relative proportions of these major The genus Undaria consists of large multicellular groups were different among the three sites, with brown algal kelps. In Korea, U. pinnatifida and U. NYS and SYS were more similar to each other than peterseniana are extensively cultivated (~500.000 to YR. Here we will briefly report the community tons/year) for human food and commercial extracts. composition and structure of microbial eukaryotes, Although phylogenetically related, the two species and hope to link these signals in the near future with are ecologically distinct and have strikingly different geochemical information to reflect the ecosystem distributions, morphologies and reproductive conditions and changes of the north China Seas strategies. Spores of U. pinnatifida are produced during the past thousands of years. in a specialized sporophyll that is localized on the lower part of the stipe whereas in U. peterseniana the FEEDING SELECTION OF MIX-TROPHIC sporophylls differentiate directly from the blades, FLAGELLATE, POTERIOOCHROMONAS which is a process similar to that for species of the MALHAMENSIS, ON THE GREEN ALGAE OF genus Saccharina, another common kelp genus. CHLORELLA We produced draft genomes for U. peterseniana and Gong Yingchun1,2, Ma Mingyang1,2, Wei Chaojun1,2 U. pinnatifida; with the previously released draft 1 - Center for Microalgal Biotechnology and Biofuels, genome of Saccharina japonica, they represent the Institute of Hydrobiology, Chinese Academy of only genomic resources available for the kelps. We Sciences, Wuhan, China examined gene content, genome organization, and 2 - Key Laboratory for Algal Biology, Institute of transposable elements dynamics. We will discuss Hydrobiology, Chinese Academy of Sciences, Wuhan, the genomic differences between U. pinnatifida and China U. peterseniana, and we will describe new insights [email protected] into their evolutionary history; most notably, we Poterioochromonas is a very common mix-trophic will comment on the origin of multicellularity in the flagellate in most freshwater environments, which brown algae. Finally, the genome sequences for the can grow in autotrophic way or by feeding bacteria two edible algae should be a new, major resource for and other protists. Several species of algae have kelp crop improvement and biotechnology. been reported to be the prey of Poterioochromonas, however little is known about whether and how NEW GENOMES OF UNICELLULAR HOLO- Poterioochromonas has feeding selection on the prey. ZOANS SHED LIGHT ONTO THE ORIGINS OF In this study, two strains of Chlorella sorokiniana COMPLEX ANIMAL GENE ARCHITECTURE showed significantly different defense ability on the Grau-Bové X.1,2, Ruiz-Trillo I.1,2,3 grazing of Poterioochromonas. Both morphological 1 - Institut de Biologia Evolutiva (UPF-CSIC) and molecular approaches were combined to 2 - Universitat de Barcelona discover the factors which contributed to the defense 3 - Institució Catalana de Recerca i Estudis Avançats ability on grazing. Light microscopic showed that [email protected] the two strains didn’t have much difference on the The origin of animal multicellularity is a major cell size and shape, however transmission electron event in eukaryotic evolution. Metazoans share microscopic observations and protein composition many novelties in genome content and structure analysis indicated that the two strains have different related to their multicellular lifestyle, like gene cell wall composition and biochemical composition. families (transcription factors and specific signaling Our research suggested that probably some protein pathways) and regulatory mechanisms (alternative in the cell wall of Chlorella played an important way splicing or enhancer-enabled introns). Since many to defense the grazing of Poterioochromonas. of these traits predate metazoans, the study of their unicellular holozoan relatives is key to understand THE GENOME OF UNDARIA PINNATIFIDA animal origins. We analyse the evolution of gene AND UNDARIA PETERSENIANA: INSIGHTS architecture using new ichthyosporean genomes, INTO KELP EVOLUTION choanoflagellates and the filasterean Capsaspora Graf L.1, Yang J.H.1, Lee J.M.1, Boo S.M.2, Yoon owczarzaki, plus 40 other eukaryote genomes. H.S.1 This two-fold analysis focuses on the evolution of 1 - Department of Biological Sciences, Sungkyunkwan 1) intron/exon structure and 2) protein domain University, Suwon 16419, Korea architectural rearrangements (which define gene 2 - Department of Biology, Chungnam National Uni- families’ function and diversification). Animals 24 · “PROTIST–2016” have complex gene architectures, intron-rich and representing several Paramecium species. The with high protein domain diversity. We find that results indicated that the strains of the P. aurelia such enrichments occur in differentially-timed species complex and P. caudatum were generally innovation bursts. For example, the most important rather sensitive, while the strains of phylogenetically premetazoan intronization event occurred in the last distant species were almost always resistant. Thus, common ancestor of choanoflagellates and animals Caedibacter might give a significant advantage for (>80% homologous introns when compared to the the host, providing it with a powerful weapon for urme-tazoan). Conversely, animal-specific protein competition with uninfected paramecia normally domain arrangements often appear before, in the present in the same environment. Several strains urholozoan (~12% of novel arrangements, compared of symbiont-free P. biaurelia also appeared to be to frequent contractions in later-branching lineages). resistant, thus allowing to speculate that this species Our results support a scenario of sudden innovation might be genetically adapted to be the host of C. processes within holozoans followed by gradual caryophilus. Supported by RFBR 16-04-01195. simplifications in extant protistan lineages, while Metazoa retain most novelties and develop more of HIDDEN DIVERSITY IN MALLOMONAS MA- their own. This is observed for both intronization and TVIENKOAE SPECIES COMPLEX protein rearrangements. Yet, these aspects of gene Gusev E.S.1, Kapustin D.A.1, Doan Nhu-Hai2, architecture are decoupled in time and mechanis- Nguyen Ngoc-Lam2 tics: non-coding and coding gene elements are under 1 - Papanin’s Institute for Biology of Inland Waters different evolutionary pressures, and innovation Russian Academy of Sciences, Russia, 152742 bursts are identified in different ancestors. Yaroslavl, Nekouz, Borok 2 - Institute of Oceanography, Viet Nam Academy of THE ROLE OF CAEDIBACTER-INDUCED Science and Technology, 01 Cau Da, Vinh Nguyen, KILLER TRAITS IN PARAMECIUM INTER- Nha Trang, Viet Nam STRAIN AND INTERSPECIES COMPETITION [email protected] Grevtseva I.1, Nekrasova I.1, Lanzoni O.2, Lebedeva Mallomonas matvienkoae B. Asmund & Kristi- N.3, Petroni G.2, Potekhin A.1 ansen is considered as a widespread taxon (Kristi- 1 - Faculty of Biology, St Petersburg State University, ansen & Preisig 2007) from the order Synurales, class Saint Petersburg, Russia Chrysophyceae. This species have been described 2 - Department of Biology, University of Pisa, Pisa, from Ukraine under the name Mallomonopsis ellip- Italy tica Matv. (Matvienko 1941) and later redescribed 3 - Centre of Core Facilities “Culture Collections of and transferred into the genus Mallomonas (Asmund Microorganisms”, St Petersburg State University, & Kristiansen 1986). Mallomonas matvienkoae is Saint Petersburg, Russia a polymorphic species; it includes five varieties [email protected] and forms. An investigation of the clonal cultures Some species of Paramecium (Ciliophora) present of M. matvienkoae representing populations from killer traits when inhabited by bacterial endosym- subtropics resulted in descriptions of three new bionts belonging to the genus Caedibacter. These species using both morphological features and bacteria are occasionally released by killer strains to molecular data (Jo et al. 2013). The authors also the environment, get engulfed by other paramecia, described a fossil species of this group. Our studies and secrete a not yet characterized toxin, which kills of this species complex in the tropics (Vietnam and symbiont-free ciliates. We studied three Paramecium Indonesia) revealed at least four new taxa for science biaurelia strains bearing cytoplasmic symbionts confirmed by molecular methods. One of them has Caedibacter caryophilus. The killer strains originated been already described as M. paragrandis E.S. Gusev from St Petersburg region, Russia, and from Sicily, (2015). Additionally, five new morphotypes of scales Italy. All strains were characterized by different rate have been found which can be considered as new and dynamics of killer effect manifestation; sensitive species for science. Three taxa, recently described paramecia displayed various lethal and malfunction by Jo et al. (2013), were also found in Vietnam symptoms. The killer cells were resistant to their own (Mallomonas hexareticulata, M. sorohexareticulata killer trait even after antibiotic-induced removal of and M. pseudomatvienkoae). Thus, Mallomonas symbionts, thus allowing to assume that resistance is matvienkoae species complex is a rather diverse genetically determined. Also killer strains appeared group of pseudocryptic taxa, which needs further to be relatively reciprocally resistant for the other studies using polyphasic approach. This study was killers. We made a series of tests to reveal resistance supported by RFBR grants 15-04-04181 and 16- or sensibility to killer effects for a number of strains 34-60099. Protistology · 25

THE PARASITIC ASSIMILATION FROM DIA- 3 - Friday Harbor Laboratories, University of Wa- TOMS BY APLANOCHYTRIUM (THRAUS- shington, Friday Harbor, WA 98250 USA TOCHYTRIIDAE, LABYRINTHULEA, STRA- 4 - Spanish Bank of Algae – Marine Biotechnology MENOPILES) Center (BEA-CBM), University of Las Palmas de Hamamoto Y.1,2, Honda D.3,2 Gran Canaria, Muelle de Taliarte s/n, 35214, Telde, 1 - Graduate School of Natural Science, Konan Las Palmas, Gran Canaria, Spain University, Japan [email protected] 2 - Institute for Integrative Neurobiology, Konan Modern systematists use molecular markers to University, Japan identify and distinguish species; however, many 3 - Faculty of Science and Engineering, Konan microalgae were described before gene sequencing University, Japan was invented and the holotype was often an ink [email protected] drawing. Cryptic speciation and biogeographic Thraustochytrids inhabit the ocean all over the isolation are potential problems when anchoring world and have been recognized as important an old species name with a modern gene sequence. eukaryotic decomposers in the marine ecosystem. Therefore, to avoid possible confusion caused by The monitoring works have suggested that they a misidentified microalgal culture from a different obtain nutrients from the terrestrial organic matter at locality, the best approach is to sequence genes from estuarine areas. On the other hand, the habitat of the the alga re-collected from the type locality. The thraustochytrids is spread over the coastal areas, in marine alga Sarcinochrysis marina was described which there are almost no influences from terrestrial in 1930 by Geitler from the Canary Islands, Spain; fields. This suggests that the nutrition source of the Geitler used two cultures in his study, but these thraustochytrids in the coastal areas is the detritus cultures no longer exist. Sarcinochrysis marina is of phytoplankton. In this study, we investigated the the oldest genus in the Pelagophyceae. We re- assimilation of the thraustochytrids from diatoms, isolated S. marina from the type locality near Las which are important primary pro-ducers in the Palmas, Gran Canaria. Furthermore, we included coastal areas. First, we examined growth tests of additional Pelagophyceae strains that were obtained the thraustochytrids under the 2-membered culture from culture collections. We produced a total of condition with Skeletonema diatoms. As the result, 109 sequences, representing the nuclear-encoded Aplanochytrium strains have quite higher growth SSU rRNA and the plastid encoded rbcL, psaA, than 8 other genus-level groups of the thraustochy- psaB, psbA, and psbC genes. The sequences were trids. Observation under light microscopy showed used to infer maximum likelihood phylogenetic that the vegetative cells of Aplanochytrium extended trees. We anchored the genus Sarcinochrysis the pseudopod-like ectoplasmic nets to the diatom using the Las Palmas isolate, and we discovered cells, and then chloroplasts of the diatoms shrank and several undescribed genera and species that are bleached. It was suggested that the thraustochytrids morphologically similar to Sarcinochrysis. play the role as not only decomposers but also “predators”. Moreover, Aplanochytrium cells pulled UNEXPECTED DIVERSITY OF THE PECU- the diatom cells and made aggregates, which are LIAR GENUS CRENEIS (EXCAVATA: HETE- suited to be baits for juvenile fishes. It is possible that ROLOBOSEA) the food chain pathway of the Aplanochytrium has a Hanousková P., Čepička Ivan large impact on the marine ecosystem because there Department of Zoology, Charles University in Prague, are few steps in the food chain between the producers Prague, Czech Republic and the higher consumers. [email protected] Creneis is a recently (2014) discovered genus of A RE-INVESTIGATION OF THE PELAGO- marine anaerobic heteroloboseids. Its only species, PHYTE GENUS SARCINOCHRYSIS USING C. carolina, displays several uncommon features MATERIAL COLLECTED FROM THE TYPE including amoeboid flagellates with a single flagel- LOCALITY lum, a multiflagellate form with ca. 14 flagella and Han K.Y.1, Melkonian M.2, Andersen R.A.3, Graf the unique structure of its flagellar apparatus. There- L.1, Pérez Reyes C.4, Yoon H.S.1 fore, its affiliation to Heterolobosea is recognizable 1 - Department of Biological Sciences, Sungkyunkwan only thanks to the results of molecular-phylogenetic University, Suwon 16419, Korea analyses. Creneis was described on the basis of a 2 - Botany Department, Cologne Biocenter, University single isolate and has never been observed again. We of Cologne, Zülpicher Str. 47b, 50674 Cologne, have established six marine Creneis strains in culture. Germany According to the morphology and SSU rRNA gene 26 · “PROTIST–2016” sequences, our strains represent five novel species only of bacteria. Furthermore, it efficiently kills of Creneis. The species morphologically differ from and consumes Dictyostelium discoideum and BGE each other as well as from C. carolina; the diagnostic cells, and ingests material from live Acathamoeba features include the cell size, character of the castellanii. We performed RNAseq of C. owczarzaki flagellum, type and arrangement of pseudopodia, fed by the organisms mentioned before and found and character of the uroid and uroidal adhesive distinct expression profiles for each food type. Our filaments. At least three new species are able to form results strongly suggest that C. owczarzaki is not an the fast-swimming “multiflagellate” form, which, obligate snail symbiont, but rather an opportunistic however, possesses only four or five flagella. Our predatory organism able to feed on a variety of food results show that Creneis is a widespread and diverse types. This research increases the knowledge on the lineage of anaerobic protists. Because six known diversity of lifestyles among unicellular holozoans, species (including C. carolina) are represented by and has implications on our understanding of the only seven strains, its true diversity is certainly much origin of animals. higher. MORPHOLOGY OF TWO SPECIES OF THE PREDATORY CAPABILITIES OF THE FILA- THECATE DINOFLAGELLATE GENUS BLE- STEREAN CAPSASPORA OWCZARZAKI RE- PHAROCYSTA EHRENBERG (DINOPHYTA) VEALS ITS POTENTIAL FOR A FREE-LIVING FROM THE TROPICAL MEXICAN PACIFIC LIFESTYLE Hernández-Becerril David Harcet M.1,2, Lopez-Escardo D.1, Sebe-Pedros A.1,3, Instituto de Ciencias del Mar y Limnología, Uni- Ruiz-Trillo I.1,4,5 versidad Nacional Autónoma de México 1 - Multicellgenome lab, Institut de biologia evolutiva [email protected] (UPF-CSIC) Pssg. de la Barceloneta 37-49. 08003 Species of the thecate dinoflagellate genus Blepharo- Barcelona (Spain) cysta Ehrenberg are usually globose, spherical to 2 - Division of molecular biology, Ruđer Bošković ovoid, and lack a depressed cingulum and sulcus, Institute, Bijenicka cesta 54, 10 000 Zagreb, Croatia typical of most dinoflagellates. The five recognized 3 - Department of Computer Science and Applied Blepharocysta species are truly marine planktonic Mathematics, Weizmann Institute of Science, Rehovot forms and are mainly distributed in tropical and 76100, Israel subtropical areas. During an oceanographic cruise 4 - Institucio Catalaná de Recerca i Estudis Avancats, along tropical coasts of the Mexican Pacific, net Pg Lluis Companys 23, 08010 Barcelona, Spain phytoplankton samples yielded a number of tropical 5 - Departament de Genetica, Universitat de Barcelona, forms of dinoflagellates, including two Blepharocysta 08028 Barcelona, Spain species: Blepharocysta paulseni and B. splendor- [email protected] maris. Both species were studied by LM and SEM Capsaspora owczarzaki is one of only two known and the general morphology previously described members of Filasterea – a group of protists closely in the literature was confirmed, although the theca related to animals. Due to its phylogenetic position, ornamentation was observed in detail. The cell complex life cycle, and wide gene repertoire, it shape, more ovoid in Blepharocysta splendormaris has become an important non-model organism and more spherical in B. paulseni, the development in evolutionary studies, especially on the origin of of sulcal and theca ornamentation lists, with animals. C. owczarzaki was isolated from several Blepharocysta paulseni having more reduced lists and strains of freshwater snail Biomphalaria glabrata. a higher pores density, appear as the morphological However, it remains unclear whether it is an obligate characters separating both species. Additionally, symbiont or it can have a free-living lifestyle. In order Blepharocysta splendormaris has been found not to answer this question, we tested the interaction of only in tropical areas, but also in Antarctic waters. C. owczarzaki with bacteria (Enterobacter aerogenes), These two species (and probably all Blepharocysta with other unicellular eukaryotes (Dyctiostelium species) showed no chloroplasts and are supposedly discoideum and Acanthamoeba castellani), and with heterotrophic. Comparison among all species of the B. glabrata embryonic (BGE) cells. The interactions genus was made, emphasizing the shape and relative were observed and documented by microscopy. In size of the cells, the development and ornamentation addition, we followed the growth of C. owczarzaki of the sulcal lists, tabulation and ornamentation of in the presence of these organisms. We found that the theca, and the general distribution. C. owczarzaki is able to grow on a diet consisting Protistology · 27

TWO NEW SPECIES OF THE DINOFLAGEL- a relict nucleus of the endosymbiont, the so-called LATE GENUS PHALACROMA STEIN (DINO- nucleomorph. Therefore, chlorarachniophyte PHYTA) FROM THE TROPICAL MEXICAN cells possess three different endosymbiotically- PACIFIC derived genomes in the plastid, mitochondrion, Hernández-Becerril David1,2, Esqueda-Lara Karina3 and nucleomorph. These organelle genomes do 1 - Instituto de Ciencias del Mar y Limnología not encode essential DNA replication components 2 - Universidad Nacional Autónoma de México (i.e., DNA polymerases), and it remains unclear 3 - Centro del Cambio Global y la Sustentabilidad del whether nucleus-encoded counterparts are impor- Sureste (CCGSS) ted into respective organelles. To gain insight into [email protected] the evolution of complex plastids, we investigated Species of the thecate dinoflagellate genus Phala- nucleus-encoded DNA polymerases related to croma Stein are common in the marine phytoplank- the chlorarachniophyte organelles. We found ton all over the world, but especially diverse in tropi- that chlorarachniophytes evolved two phyloge- cal areas. Some species are considered to produce netically distinct DNA polymerases for the plastid Okadaic Acid or Dinophysistoxin, which cause and mitochondrion, although a single DNA poly- diarrhetic shellfish poisoning (DSP). The genus merase is dually targeted to both organelles in Phalacroma includes mixotrophic and heterotrophic Archaeplastida. Our phylogenetic analyses suggest species with an elevated epitheca, visible in lateral that the mitochondrion-targeted one is originated view and narrow horizontally projected cingular by the host, and the plastid-targeted one seems to lists. During surveys of phytoplankton from the be derived from a red algal lineage via lateral gene tropical Mexican Pacific, two undescribed species transfer. We also discovered a nucleomorph-tar- of Phalacroma were found, and in this paper they geted DNA polymerase that was phylogenetically are depicted following detailed observations by related to viral sequences. Our data imply that LM and SEM. Phalacroma ornamentatum sp. nov. organelle DNA polymerases of chlorarachniophytes is a new species, superficially similar to species have been replaced by a counterpart of other orga- of the Phalacroma rotundatum (Claparède et nisms during the evolution. Lachmann) Kofoid et Michener group, but its main distinctive characteristics are: (1) thick theca, (2) PRIMARY AND SECONDARY ENDOSYM- ornamentation of the theca and sulcal lists, and (3) BIOTIC GENE TRANSFER OF BACTERIAL relatively narrow sulcal lists and junction close to RECA IN EUKARYOTES the cingulum. The other new species is Phalacroma Hofstatter P.G.1, Lahr D.J.G.1, Brown M.2 palmatum sp. nov., which is relatively similar to 1 - University of São Paulo Phalacroma bipartitum Kofoid et Skogsberg, but is 2 - Mississippi State University characterized by (1) shape and extension of the left [email protected] sulcal list to the posterior part of cell, (2) absence of Recombinases promote DNA repair by homologous R2 in the left sulcal list, (3) sulcal lists joined at the recombination and these genes occur in all known cingulum level, and (4) ornamentation of the theca domains of life: virus and bacteria (RecA), Archaea and both sulcal lists. Phalacroma ornamentatum was (RADA, RADB) and Eukaryotes (RAD51X, DMC1). fairly common in the Gulf of California, whereas The bacterial recombinases (RecA) are present Phalacroma palmatum was very rare. not only among bacteria, but also in several eukaryotes: Chlorophyta, Rhodophyta, Amoebozoa, ORGANELLE DNA REPLICATION IN CHLOR- Peronosporomycetes (Oomycetes), Bacillariophyta ARACHNIOPHYTE ALGAE and other SAR group lineages. The eukaryotic recA Hirakawa Y.1, Watanabe A.2, Suzuki S.2, Ishida K.1 genes have two different origins, a mitochondrial 1 - Faculty of Life and Environmental Sciences, and a chloroplastic. The acquisition of recA genes University of Tsukuba by eukaryotes was possible by means of endosym- 2 - Graduate School of Life and Environmental biotic gene transfer (EGT) in the form of primary Sciences, University of Tsukuba endosymbiosis (bacteria-eukaryote) and secondary [email protected] endosymbiotic gene transfers (eukaryote-euka- Plastids evolved by multiple endosymbiotic events ryote). The RecA proteins present a transit pep- where photosynthetic organisms became fully tide and are imported by the organelles, where they integrated with host eukaryotic cells. Chlorara- act in homologous recombination. Several major chniophyte algae possess complex plastids acquired eukaryotic lines lost the bacterial recombinases, by the uptake of a green alga, and the plastids harbor as seen in Opisthokonta, where the bacterial ho- 28 · “PROTIST–2016” mologues were probably replaced by eukaryotic 2 - Dept. de Biologie, Université Paris-Sud 11 specific homologue or in the secondarily amito- 3 - Dept. of Biology and Ecology, University of Ostrava chondriate groups where it is not necessary any- 4 - Dept. of Zoology, Charles University in Prague more. In the other hand, recA genes were subject 5 - Weill Cornell Medical College, Cornell University to further duplication events in green plants, where 6 - Biozentrum der Universität Basel they present several forms. The reconstruction 7 - Institut Pasteur of the RecA phylogeny with its EGT events re- [email protected] tells the very evolutionary history of the euka- The bacterial origin of mitochondria has been ryotes and also bacteria and enables a further evidenced by a number of shared features with cur- understanding of endosymbiosis. We showed a rent bacteria, including some of the protein trans- group-wide presence of mitochondrial recA genes port components. However, most of the original in Amoebozoa, Oomycetes, green plants and bacterial protein transport pathways have been lost minor groups giving support to an ancestral EGT from the mitochondria and replaced by the protein acquisition of mitochondrial recA prior to eukaryotic import apparatus. To some detail, mitochondria diversification, as well as chloroplastic recA in all of Discoba represent an evolutionary intermediate major photosynthesizing groups. stage as they carry the largest mitochondrial genomes encoding bacterial SecY and TAT trans- UNVEILING SSU RDNA INTRAGENOMIC locases. By a multi-phylome approach we have POLYMORPHISM IN DEEP-SEA FORAMI- analyzed eukaryotic proteomes for nuclear encoded NIFERA USING SINGLE-CELL HIGH THRO- genes, which are exclusive to Discoba. We show UGHPUT SEQUENCING that their nuclei encode for about forty genes not Holzmann Maria1, Apothelot-Perret-Gentil Laure1, found in other eukaryotic lineages. These include Voltsky Ivan1,2, Lejzerowicz Franck1 eight components of bacterial type II secretion 1 - University of Geneva system (T2SS). We show that mitochondria of 2 - Ben Gurion University of the Negev Discoba express minimalist T2SS, which includes [email protected] the pore forming secretin in the outer mitochondrial The SSU rRNA genes are commonly used for the membrane and pseudopilin in the intermembrane assessment of protist diversity. It is assumed that space. Using the bacterial and yeast two hybrid the rDNA copies are identical within the individual assays, we are currently looking for the putative cells. However, previous studies, using cloning and substrate of the ancient mitochondrial protein Sanger sequencing approach showed a high level secretion pathway. of intra-individual polymorphism in shallow water benthic foraminifera. Here, we present the results of OCHER-COLORED GRANULES IN THE single cell high-throughput analysis of SSU rDNA CILIATE CYCLOTRICHIUM SP. ARE EVI- in about 200 specimens of abyssal foraminifera. DENTLY DIATOM CHLOROPLASTS Our data confirm the presence of intragenomic Hoshina Ryo1, Suzaki Toshinobu2, Kusuoka Yasushi3 polymorphism in foraminifera but levels of intra- 1 - Nagahama Institute of Bioscience and Technology individual divergence are different among taxono- 2 - Kobe University mic groups. Our approach further allows us a quick 3 - Lake Biwa Museum sorting of foraminifera for barcoding purposes by [email protected] distinguishing unknown foraminiferal sequences As a crucial part of our exploration of the diversity of from those that are already present in our database. animal protists associated with algal endosymbionts, In the case of unknown sequences the longer we have examined the ultrastructure and molecular SSU fragment is Sanger sequenced afterwards to characteristics of a ciliate of the genus Cyclotrichium phylogenetically characterize the putative new collected from Lake Biwa, the largest and oldest lake species. in Japan. The ciliates, which are nearly spherical and about 100 µm in diameter, are filled with ANCIENT MITOCHONDRIAL PROTEIN SE- hundreds of 2–3 µm ocher-colored granules that CRETION impart their color to the whole cell. The internal Horváthová L.1, Žárský V.1, Derrelle R.2, Krupičková structure of these granules is indistinct by light A.1, Klápšťová V.1, Voleman L.1, Petrů M.1, Eliáš microscopy, but examination by SEM shows them to M.3, Pánek T.3, Čepička I.4, Huysmans G.5, Chami be chloroplasts consisting of three-layered thylakoid M.6, Francetic O.7, Doležal P.1 membranes. Many of the chloroplasts seem to be at 1 - Dept. of Parasitology, Charles University in Prague different phases of digestion in food vacuoles (from Protistology · 29 intact-looking to half-digested), but there are also CONDITIONAL EXPRESSION SYSTEM IS some naked chloroplasts with three surrounding NOT SUITABLE FOR DEVELOPMENTAL membranes in the ciliate cytoplasm. To identify STUDIES IN LEISHMANIA the origin of these chloroplasts, we attempted to Ishemgulova A.1, Kraeva N.1, Faktorová D.2, analyze PCR products obtained from isolated whole Podešvová L.1, Lukeš J.2,3,4, Yurchenko V.1,2,5 ciliate cells using various specific primers. The 1 - Life Science Research Centre, Faculty of Science, diatom-targeted primers yielded a probable nuclear University of Ostrava, 710 00 Ostrava, Czech Republic SSU rDNA, and phylogenetic analyses based 2 - Czech Academy of Sciences, 370 05 České on Neighbor Joining showed that this sequence Budějovice (Budweis), Czech Republic pertains to the centric diatom genus Discostella 3 - Faculty of Sciences, University of South Bohemia, (Stephanodiscaceae). It was especially close to 370 05 České Budějovice (Budweis), Czech Republic that of the diatom symbiont of the dinoflagellate 4 - Canadian Institute for Advanced Research, Peridiniopsis penardii. We are now carrying out a Toronto, ON M5G 1Z8, Canada detailed study of the ciliate and its symbiont from 5 - Department of Pathology, Albert Einstein College taxonomic and life history points of view. of Medicine, Bronx, NY, 10461, USA [email protected] EVOLUTION OF THE MITOCHONDRIAL The genus Leishmania unites parasitic protists of the PROTEOME, FROM THE LARGE-SCALE family Trypanosomatidae causing leishmaniases, PATTERNS TO THE NITTY-GRITTY DETAILS several closely related diseases that affect human Huynen M.A., Duarte I., Szklarczyk R., Elurbe D. and animal populations mainly in the tropical and CMBI, Radboud University Medical Centre, Nijme- subtropical regions. The clinical manifestations gen, Netherlands vary from spontaneously healing skin lesions to [email protected] progressive and potentially fatal visceral infections. I will review what has been inferred about the chan- Leishmaniases represent a global health problem ges at the level of the proteome that accompanied with over 350 million people at risk and an annu- the evolution of the mitochondrion from an al incidence rate of 2–10 million worldwide. Alphaproteobacterium, quantifying the amount of Conventional and conditional systems allow for a gene loss, gene replacement and gene gain. Most controlled activation or repression of gene expres- of the loss and replacements that separate current sion in time and space. Such systems are nowadays day mitochondria from Alphaproteobacteria took widely used to analyse a variety of cellular processes place before the radiation of the eukaryotes. Detailed in numerous parasites including Leishmania. analyses of the evolution of the mitochondrial A T7-driven, tetracycline-inducible system for complexes like the ribosome and oxidative phos- protein expression was established in a human phorylation show that also the acquisition of new pathogen Leishmania mexicana. The gene expres- proteins occurred mainly before the radiation of sion in this strain is tightly regulated and dose- and the eukaryotes, supporting an early acquisition of time dependent. We believe that it can be widely mitochondria in eukaryotic evolution. Secondly I used by the parasitology community to analyse will analyze the accumulation of new supernumerary effects of genes of interest on biology, physiology subunits and assembly factors from pre-existing and virulence of parasites causing cutaneous protein families for one protein complex in detail: leishmaniases. This system was used to analyse gene complex I. There is a spectrum of protein function expression profiles during L. mexicana differentia- conservation between the complex I representatives tion (procyclics, metacyclics, and amastigotes). The and their non-complex I homologs. In general, the transcription/translation of the gene of interest was new complex I proteins appear to have been recruited severely decreased upon Leishmania differentiation from proteins that are active in mitochondria: into metacyclic and amastigotes. However, the proteins one expects to be expressed when and same expression profile was documented for the T7 where complex I is active. Within complex I polymerase. The expression was demonstrated to be and its assembly there are many cases of neo- not locus-specific but dependent on untranslated functionalization after gene duplication, one case regions flanking open reading frames of studied of sub-functionalization, and one case in which a genes. We concluded that the previously established complex I protein itself appears to have been the conventional gene expression systems might have source of the evolution of a new protein in another certain limitations in their common applications. complex. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution. 30 · “PROTIST–2016”

OBSERVATION OF ZOOSPORE SETTLE- the endoplasmic reticulum to the bothrosome and MENT FOCUSING ON THE DEVELOPMENT stretching the membrane invagination along EN. OF ACTIN FILAMENTS (LABYRINTHULEA, STRAMENOPILES) MULTIGENE PHYLOGENY OF SYNURA Iwata I.1,2, Kimura K.3, Tomaru Y.4, Motomura T.5, (SYNUROPHYCEAE) AND DESCRIPTIONS Koike Kana6, Koike Kazu7, Honda D.2,8 OF FOUR NEW SPECIES BASED ON MOR- 1 - Graduate School of Natural Science, Konan PHOLOGICAL AND DNA EVIDENCE University, Japan Jo B.Y.1, Kim J.I.1, Škaloud P.2, Siver P.A.3, Shin 2 - Institute for Integrative Neurobiology, Konan W.1 University, Japan 1 - Department of Biology, Chungnam National 3 - Institute of Lowland and Marine Research, Saga University, Daejeon 305-764, Korea University, Japan 2 - Department of Botany, Faculty of Science, Charles 4 - National Research Institute of Fisheries and University, Benátská 2, Prague 2, CZ-12801, Czech Environment of Inland Sea, Japan Fisheries Research Republic and Education Agency, Japan 3 - Department of Botany, Connecticut College, New 5 - Muroran Marine Station, Field Science Center London, Connecticut 06320, USA for Northern Biosphere, Hokkaido University, Japan [email protected] 6 - Natural Science Center for Basic Research and We used phylogenetic analyses based on multiple Development, Hiroshima University, Japan gene sequences (partial nr SSU and LSU rDNA, 7 - Graduate School of Biosphere Science, Hiroshima partial pt LSU rDNA, psaA, and rbcL) from 148 University, Japan strains (including three outgroups) and scale 8 - Faculty of Science and Engineering, Konan ultrastructure to examine phylogenetic relationships University, Japan among species of the colonial genera Synura and [email protected] Tessellaria. The phylogenetic tree based on the The Labyrinthulea is characterized by an ectoplas- combined dataset was congruent with ultrastructural mic net (EN) system. The nets are superficially characteristics of the scales. Synura was divided into similar to narrow pseudopods of many protists but three major clades, two including species in section different in the following points: their origin from a Synura, and one representing section Peterseniae. unique organelle named the bothrosome, membrane One clade, consisting of seven strains of S. uvella invagination along EN, and absence of mitochon- (section Synura), diverged at the base of the genus. dria and ribosomes. The bothrosome is located The second clade consisted of the remaining species near the cell surface and is a complex of the electron belonging to the section Synura. The third clade, dense material and the endoplasmic reticulum. containing organisms in the section Peterseniae Previously we observed, by immunofluorescence and characterized by scales possessing a keel, was staining, that the actin filaments are located in monophyletic with strong support values. Based EN. In this study, we investigated the process from on our findings, S. uvella needs to be in a separate a zoospore to a vegetative cell of Schizochytrium section from other spine-bearing species, and we aggregatum, especially focusing on the development therefore propose new sectional ranks; Synura, of the bothrosome and behavior of actin filaments. Peterseniae, Curtispinae (presence of body scales After the flagella were drawn into the cell, the with slender spines, tubular scales, and caudal bothrosome appeared de novo at the anterior-ventral scales). We further propose four new species region of the cell. After the zoospore settled and based on phylogenetic analyses and unique scale rounded, the actin appeared as a spot in the same characters; S. longitubularis sp. nov., S. sungminbooi position of the newly appeared bothrosome by sp. nov., S. soroconopea sp. nov., and S. lanceolata immunofluorescence staining. It was expected that sp. nov. the electron dense material of the bothrosome is the organizing center of the actin filaments. However, CORRELATION BETWEEN FRESH WATER under immunoelectron microscopy, reaction by CILIATED PROTIST COMMUNITIES AND the anti-actin antibody was observed in EN and THEIR MICRO-ECOLOGY cytoplasm around the bothrosome, not in the Kamra Komal1, Kaur Harpreet1, Tripathi Charu2, electron dense material of the bothrosome. It was Kaur Surinder1, Narula Laxmi1, Lal Rup2, Warren suggested that the function of the bothrosome is not Alan3 nucleation of actin filaments. We expect that actin 1 - Ciliate Biology Lab, SGTB Khalsa College, relates to morphogenesis of EN, that is, the actin University of Delhi, Delhi 110007 filaments probably play an important role of pulling 2 - Department of Zoology, University of Delhi, Delhi Protistology · 31

110007 packed helical glycostyles, ca. 220 nm in thickness 3 - Natural History Museum, London, SW7 5BD, UK in P. levis and ca. 520 nm in P. valamo. Our study [email protected] reveals that during formation of the food vacuole P. River Yamuna maintains a reasonably good quality valamo resorbs cell coat at the area of contact with from its origin in the Himalayas to its entry into the food object, thus avoiding digestion of glycocalyx National Capital Region (NCR) of Delhi, India. elements. The similar feeding mode is known 18 sewage drains carrying industrial and domestic in other thick-coated amoebae, like Pellita and wastes flow into the river in the 22 km stretch (1.6% Dermamoeba. This observation confirms our earlier of total length) as it flows through the NCR. 80% hypothesis, stating that thick-coated amoebae have of the pollution in the river’s total length of 1375 to adopt special ways to avoid energetically costly km occurs as it passes through the NCR, severely digestion and further reconstruction of glycocalyx compromising its water quality. As free-living elements. We provide the first description of ciliated protist species differ widely in tolerance the cytoplasmic microtubule-organising centres to pollutants, the present study was conducted to (MTOCs) associated with dictyosomes of the Golgi catalogue ciliate communities (with appropriate complex in genus Paradermamoeba. This finding morphological, morphometric and morphogenetic confirms the hypothesis that presence of cytoplas- descriptions) at two selected locations along the mic MTOCs is a synapomorphy of the phylogenetic river, namely the point where the river enters the lineages forming the subclass Longamoebia. De- NCR (S1) and 500 m downstream of the site where tailed study of the ultrastructure of P. levis reveals the largest sewage drain empties into the river (S2). that so-called “supernumerary nucleus” of P. levis Physico-chemical parameters including heavy metal noted in the initial description is not an individual concentrations were analyzed in order to assess the structure but an outgrowth of the cell nucleus water quality at these two locations. Compared to S1: containing its own nucleolus. Supported with RSF the Biochemical Oxygen Demand and phosphate grant 14-14-00474. concentrations increased 5X at S2; total hardness, total dissolved solids, conductivity, turbidity and ISOLATION OF FREE-LIVING AMOEBAE nitrates doubled at S2; total coliforms and faecal FROM SOUTHERN HAN-LIVER IN KOREA coliform increased 50X at S2; lead concentration Kang H.K.1,2, Seong G.S.1,2, Sohn H.J.1,2, Jung S.Y.3, increased 2X times and arsenic 3X at S2. There was a Lee S.E.4, Park M.Y.4, Shin H.J.1,2 significant fall in the species richness and abundance 1 - Department of Microbiology, Ajou University School of ciliated protists at S2. Of the hypotrichous ciliates, of Medicine there was a significant loss of members of the family 2 - Department of Biomedical Science, Graduate Oxytrichidae, whereas euplotid populations were School of Ajou University, Suwon 443-721, Republic maintained. The changes in ciliate community will of Korea be discussed with respect to water quality. 3 - Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, School of NEW DATA ON THE ULTRASTRUCTURE Health and Medicine, Namseoul University, Cheonan OF THE GENUS PARADERMAMOEBA: UN- 31020, Republic of Korea USUAL FEEDING MODE AND PRESENCE 4 - Division of Malaria and Parasitic Disease, Korea OF CYTOPLASMIC MTOCS National Institute of Health, Osong 363-951, Republic Kamyshatskaya O.G., Smirnov A.V. of Korea Department of Invertebrate Zoology, Faculty of Biology, [email protected] St. Petersburg State University, St. Petersburg, Russia Pathogenic Naegleria fowleri and Acanthamoeba [email protected] spp., free-living amoebae exist in the natural Naked amoebae engulf food objects via phago- environment, are causing agents of an acute and cytosis, and during this process the portion of cell lethal primary amoebic meningoencephalitis membrane covered with the glycocalyx appears (PAM) and amoebic keratitis (AK) in humans, inside newly formed food vacuoles. Further cell respectively. To ascertain the existence of free- has to restore membrane and reconstruct respective living amoebae in Korea, in late August 2015, water portion of the glycocalyx. It may be an energetically samples of eight sites were collected in Korean costly process for species possessing thick and highly hydrosphere where water skiing and recreation have differentiated glycocalyx. Genus Paradermamoeba been actively performed, and then the non-nutrient (phylum Amoebozoa, class Discosea) includes agar culture and PCR-based detection technique two species – P. levis and P. valamo. Both possess were carried out. The surface waters were collected highly differentiated cell coat composed of tightly and filtered, and then final samples were cultured 32 · “PROTIST–2016” on non-nutrient agar medium with inactivated Paraphelidium tribonemae. Molecular phylogenetic E. coli and subjected to PCR with various primer analysis of 18S rRNA gene sequences of this parasite pairs (amplify mainly the 18S-small ribosomal indicates that P. tribonemae defines a distinct cluster RNA). Free-living amoebae were intensively which is only distantly related to Amoebaphelidium detected by PCR in two collection regions (Yeoju and Aphelidium, what confirms the wide genetic and Yangpeong city around Southern Han-Liver). diversity of the aphelids. P. tribonemae has amoeboid PCR products obtained from water samples of opisthokont zoospores which are twice smaller than Yeoju and Yangpeong city were subjected to morphologically similar zoospores of Aphelidium aff. gene sequencing. The similarity of 18S-rRNA melosirae. Nonetheless, although the morphology of sequences were compared with various reference Paraphelidium species is generally similar to that of amoebae in GeneBank, and they showed 86-99% Aphelidium representatives, molecular phylogenetic homology with N. gruberi, N. philippinensis, N. analyses unambiguously show, that the two clades clarki, Acanthamoeba polyphaga and Vermamoeba of strains are distantly related and must define two vermiformis. A Korean isolate (confirmed by PCR distinct genera. as A. polyphaga) was isolated from Yeoju sample and We thank RFBR grant No. 15-29-02734 for financial have been subcultured in Nelson’s and PYG liquid support, and for cultivation of strains and access to medium with 10% FBS at 30 ˚C incubator. In the the EM facilities the Center for Culturing Collection in vitro cytotoxicity test, Korean isolate (tentative of Microorganisms and the Research Resource A. polyphaga) showed high cytotoxicity as much as Center for Molecular and Cell Technologies (RRC reference amoebae, A. polyphaga and A. castellanii. MCT) of Research park of St. Petersburg State This study will be useful, in the further study, for the University correspondingly. detailed seasonal detection of free-living amoebae from Korean hydrosphere. AN EVOLUTIONARY TRANSITION OF CHLOROPLAST DEGRADATION IN EU- MORPHOLOGICAL AND GENETIC DIVER- GLENOIDS: HETEROTROPHIC DIGESTION SITY OF OPISTHOSPORIDIA: NEW APHELID TO SECONDARY PLASTID SENESCENCE PARAPHELIDIUM TRIBONEMAE GEN. ET SP. Kashiyama Y.1,2, Kawahara J.1, Maruyama M.1, NOV Kayama M.1, Nakazawa M.3, Tanifuji G.4, Yoko- Karpov S.A.1,2, Tcvetkova V.S.2, Mamkaeva M.A.2, yama A.5, Ishikawa T.6, Tamiaki H.2, Suzaki T.7 Torruella G.3, Timpano H.3, Moreira D.3, López- 1 - Fukui University of Technology García P.3 2 - Ritsumeikan University 1 - Zoological Institute, Russian Academy of Sciences, 3 - Osaka Prefecture University Universitetskaya nab. 1, St. Petersburg 199034, 4 - National Museum of Nature and Science Russian Federation 5 - National Institute for Environmental Studies 2 - St. Petersburg State University, Universitetskaya 6 - Shimane University nab. 7/9, St. Petersburg 199034, Russian Federation 7 - Kobe University 3 - Unité d’Ecologie, Systématique et Evolution, UMR [email protected] CNRS 8079, Université Paris-Sud, 91405 Orsay Establishments of regulatory mechanisms for cedex, France the integrated endosymbiont must have been [email protected] key steps in organelle acquisitions, where both Aphelids are a poorly known group of parasitoids of endosymbiont and host processes cooperated to algae that have raised considerable interest due to evolve new functions. Specifically important for the their pivotal phylogenetic position. Together with chloroplast is to establish regulatory mechanisms Cryptomycota and the highly derived Microspo- for chlorophyll-related metabolisms since the ridia, they have recently re-classified as the Opistho- phototoxicity of chlorophyll would cause fatal sporidia, which constitute the sister group to the fungi damages. In particular, regulated degradation within the Holomycota. Despite their huge diversity, of chloroplasts or chloroplast dismantlement is as revealed by molecular environmental studies, and critical not only for recycling nutrients but also their phylogenetic interest, only three genera have for disposing the phototoxic chlorophylls safely. been described (Aphelidium, Amoeboaphelidium, Unlike land plants, little has been understood for and Pseudaphelidium), from which 18S rRNA gene algae on chloroplast dismantlement and associated sequences exist only for Amoeboaphelidium and chlorophyll catabolism. We previously reported Aphelidium species. Here, we present the molecular that the phototrophic euglenoid accumulates phylogeny, life cycle and ultrastructure of new 132,173-cyclopheophorbide enols (CPEs) within freshwater aphelid, which is a new genus and species their cells; CPEs are non-phototoxic catabolites Protistology · 33 of chlorophylls. A phototrophic euglenoid Euglena measured. Sampling was carried out weekly for 3 gracilis exhibited that chloroplasts underwent weeks during the pre-mon-soon season. Compared dismantled when incubated in the dark, leaving to D1, substantially higher levels of free CO2, brown-colored granules. The brown granules were chloride, phosphates and hardness were observed massive structures of membranes generated after in D2 indicating the water here is more polluted. degraded thylakoids. Chlorophylls are converted The ciliate communities at the two sites differed to CPEs in an early stage of dismantling, and significantly. The results of detailed analyses, proteins were removed during the dismantling. showing correlations between physico-chemical Macroautophagy of chloroplasts was not apparent, parameters (including heavy metals), and ciliate and the dismantling chloroplasts seem not to communities, will be presented. The significance undergo acidification; instead, large acidic vacu- of such data collected over a period of time along oles commonly formed beside the dismantling the drains, the river, and at water treatment plants, chloroplasts. This would be comparable to digestion shall benefit ecologists in developing strategies to of chloroplasts by phycophagic euglenoids, where help mitigate river pollution as ciliates are known also accumulated CPEs. However, the digestive to clarify waste water and act as bio-indicators of degradation proceeds within acidified phagosome specific pollutants. hence being apparently non-homologous to the dismantlement in the phototrophs. We infer that OLIGOTROPHIC LAGOONS OF THE SOUTH the CPE-metabolism, which has not been reported PACIFIC OCEAN ARE HOME TO A SUR- for green algae, might have been inherited from PRISING NUMBER OF NOVEL EUKARYOTIC ancestral heterotrophs to phototrophs, and is now MICROORGANISMS adapted into a novel mechanism to dismantle the Kim E.1, Sprung B.2, Duhamel S.3, Filardi C.1, Shin secondary chloroplasts. M.4 1 - American Museum of Natural History COMPARISON OF FRESH WATER CILIATED 2 - University of Pennsylvania PROTIST COMMUNITIES FROM TWO LO- 3 - Lamont-Doherty Earth Observatory, Columbia CATIONS ALONG NAJAFGARH DRAIN, University DELHI, INDIA, AND THEIR CORRELATION 4 - University of Ulsan WITH PHYSIO-CHEMICAL PARAMETERS [email protected] Kaur Harpreet1, Rani Pooja2, Kaur Surinder1, The diversity of microbial eukaryotes was surveyed Narula Laxmi1, Kamra Komal1, Lal Rup2, Warren by metagenomic sequencing from tropical lagoon Alan3 sites of the South Pacific, collected through the 1 - Ciliate Biology Lab, SGTB Khalsa College, American Museum of Natural History (AMNH)’s University of Delhi, Delhi 110007 Explore21 expedition to the Solomon Islands in 2 - Department of Zoology, University of Delhi, Delhi September 2013. The sampled lagoons presented 110007 low nutrient concentrations typical of oligotrophic 3 - Natural History Museum, London, SW7 5BD, UK waters, but contained levels of chlorophyll a, a [email protected] proxy for phytoplankton biomass, characteristic of There are 18 sewage drains traversing the National meso- to eutrophic waters. Two 18S rDNA sites that Capital Region (NCR) of Delhi, India, that flow into include the V4 and V8-V9 regions were amplified the River Yamuna. Najafgarh Drain is the largest from the total of eight lagoon samples and sequenced with a catchment area of around 400 km2. It picks on the MiSeq system. After assembly, clustering up domestic and industrial wastes from 38 secondary at 97% similarity, and removal of singletons and drains coming from over 2/3 of the city. It carries a chimeras, a total of 2,741 (V4) and 2,606 (V8- total flow of over 2100 million litres per day of which V9) operational taxonomic units (OTUs) were 30% is treated. Sewage treatment plants and effluent identified. Taxonomic annotation of these reads, treatment plants discharging into the drain are including phylogeny, was based on a combination of inadequate and not working to full capacity. Ciliated automated pipeline and manual inspection. 18.4% protist communities were assessed (with appropriate (V4) and 13.8% (V8-V9) of the OTUs could not be morphological, morphometric and morphogenetic assigned to any of the known eukaryotic groups. Of descriptions) at two locations along the drain: the these, we focused on OTUs that were not divergent mid-point of the drain (D1) and at the end of the and possessed multiple sources of evidence for drain before it empties into the river Yamuna (D2). their existence. Phylogenetic analyses of these Various physico-chemical parameters were also sequences revealed more than ten branches that 34 · “PROTIST–2016” might represent new deeply-branching lineages of A new soil urostylid ciliate, Birojimia soyaensis nov. microbial eukaryotes, currently without any cultu- spec., was discovered from Soya Island, Incheon, red representatives or morphological information. South Korea. It is described the species based on live and stained specimen observation, and 18S THE PLASTID GENOMES OF THE CRYPTO- ribosomal RNA gene sequence analyses. Birojimia MONAD ALGAE REVEAL LINEAGE-SPECI- soyaensis nov. spec. is characterized by the following FIC GENE LOSS features: body slender elongate and somewhat Kim J.I.1, Yi G.2, Yoon H.S.3, Shin W.1 twisted; 170–200 µm × 40–50 µm body size in vivo; 1 - Department of Biology, Chungnam National contractile vacuole located at middle of left cell University, Daejeon, Korea margin; cortical granules present; 37–48 adoral 2 - Department of Computer Science, Gangneung- membranelles; 3 frontal and 2 frontoterminal cirri Wonju National University, Wonju, Korea present; III/2 and buccal cirrus present; midventral 3 - Department of Biological Sciences, Sungkyunkwan pairs only; pretransverse ventral and transverse cirri University, Suwon, Korea present; 1 left and 4 right marginal rows, including [email protected] 3 compound rows; 5 complete dorsal kineties with Cryptophytes are an enigmatic group of photo- additional 3 shortened kineties which are anteriorly synthetic unicellular eukaryotes with plastids derived of compound rows; 8–11 caudal cirri; 53–69 from red alga through secondary endosymbiosis macronuclear nodules; and 2 or 3 micronuclei. that contains four different genomes (host nuclear, Birojimia soyaensis nov. spec. is similar to B. ter- mitochondrial, plastid, and nucleomorph) in a ricola, but is distinguished by cortical granules cell. They are comprised of brown-, red-, or blue/ (present vs. absent), number of caudal cirri (8–11 green photosynthetic species, as well as colorless vs. 2–7) and number of dorsal bristle rows (8 vs. 6, nonphotosynthetic species. Although its complex on average). Birojimia muscorum is separated from evolutionary history, the origins of the host and B. terricola and B. soyaensis n. sp. by midventral row endosymbiont components in cryptophyte algae are present (vs. absent, midventral pairs only) which is poorly understood. Here, we report a comparative an important key of the Bakuellidae. Furthermore, analysis of plastid genomes from six cryptophyte in phylogenetic tree, Birojimia soyaensis has widely genera. Three newly determiend cryptomonads distance from the bakuellids. Thus, we agree with plastid genomes of Chroomonas, Cryptomonas, and Berger (2006) that the new genus needs to establish Storeatula share a number of features including for B. muscorum. Phylogenetic tree suggests this gene arrangement and gene content with previously new species is the most closely related to the genus reported ones of Rhodomonas, Teleaulax and Guil- Hemicycliostyla. lardia. The plastid genomes of cryptomonads reveal lineage-specific gene loss and intron insertion in FIRST MARINE PHOTOSYNTHETIC TES- eight significant gene syntenic positions. The chlL/ TATE AMOEBA CONTAINING THE CHRO- chlN/chlB genes, which is the light-independent MATOPHORE: PAULINELLA LONGICHRO- (dark active) protochlorophyllide oxidoreductase MATOPHORA (LIPOR), show the evolutionary changes across the Kim S.1, Park M.G.2 cryptomonad lineages. We will discuss phylogenetic 1 - Department of Oceanography, Pukyong National relationships of the red-derived lineages using 93 University, Busan, Republic of Korea plastid genes showing a strong monophyletic group 2 - Department of Oceanography, Chonnam National of the cryptophytes and haptophytes. University, Gwangju, Republic of Korea [email protected] A NEW SOIL CILIATE, BIROJIMIA SOYAEN- Of 10 formally described species in the genus, all SIS NOV. SPEC. (CILIOPHORA: UROSTYLI- species are marine heterotrophic species, except for DA) FROM SOUTH KOREA one freshwater species, Paulinella chromatophora Kim Kang-San1, Jung Jae-Ho2, Min Gi-Sik3 which is the sole species to have plastids termed the 1 - Department of Biological Sciences, Inha University, “chromatophores” of a Synechococcus/Prochloro- 100 Inha-ro, Nam-gu, Incheon 22212 coccus-like cyanobacterial origin. Here, we report 2 - Department of Biology, Gangneung-Wonju the first marine phototrophic species, Paulinella National University, Jukheon-gil, Gangneung-si, longichromatophora sp. nov., using light and electron Gangwon-do, 25457, South Korea microscopy and molecular data. This new species 3 - Department of Biological Sciences, Inha University, contains two blue-green U-shaped plastids reaching 100 Inha-ro, Nam-gu, Incheon 22212, South Korea up to 40 µm in length. Further, the new Paulinella [email protected] species is characterized by having five oral scales Protistology · 35 surrounding the pseudostomal aperture. All trees separately offered a variety of prey including dino- generated using three nuclear rDNA datasets (18S flagellates, raphidophytes, cryptophytes, and a ci- rDNA, 28S rDNA, and the concatenated 18S liate. In addition, F. mexicanum displayed different + 28S rDNA) demonstrated that three photo- levels of specificity for species of Alexandrium. While synthetic Paulinella species congruently formed F. mexicanum consistently fed on A. fundyense and a monophyletic group with robust bootstrap and A. pacificum, feeding on A. affine was observed Bayesian supports (≥99% RAxML and 1.0 Baye- only once. F. mexicanum ingested prey by direct sian support), but their relationships remained engulfment through the sulcus, after capturing unresolved within the clade in all trees. The P. the prey by a tow filament. Phylogenetic analyses longichromatophora, nevertheless, clustered consis- of 18S and 28S rDNA datasets demonstrated that tently together with Paulinella strain FK01, but Fragilidium sequences formed a monophyletic with very poor supported. Phylogenetic analyses group with high statistical supports and diverged inferred from plastid-encoded 16S rDNA and into four distinct clades. The first clade consisted the concatenated dataset of plastid 16S+23S of seven F. cf. duplocampanaeforme strains, F. rDNA demonstrated that chromatophores of all subglobosum from New Zealand, and an unidentified photosynthetic Paulinella species formed a mono- Fragilidium sp. from Florida, USA. The second clade phyly and fell within cyanobacteria clade with a close branched as a single sequence for F. subglobosum relationship to α-cyanobacterial clade containing from Denmark and formed a sister lineage to F. Prochlorococcus and Synechococcus species with cf. duplocampanaeforme, with weak statistical very robust supports of 100% bootstraps and 1.0 support. The third clade included Fragilidium sp. Bayesian posterior probabilities. Additionally, EUSK D from Angola, as well as Korean strains of phylogenetic analyses of nuclear 18S rDNA and F. mexicanum and F. fissile, and was very strongly plastid 16S rDNA showed divergent evolution within supported. The last clade contained the five most the photosynthetic Paulinella population after a divergent sequences of F. subglobosum strains. single acquisition of the chromatophore. After the Further, phylogenetic analyses revealed that the single acquisition of the chromatophore, ancestral genera Fragilidium and Pyrophacus were sister to a photosynthetic Paulinella appears to diverge into at clade that included Alexandrium and Goniodoma. least two distinct clades, one containing marine P. Pyrophacus was a sister to a clade containing mem- longichromatophora and freshwater Paulinella strain bers of the genus Fragilidium. FK01, the other P. chromatophora CCAC 0185. TWO ENIGMATIC GENERA ARE ONE? COM- PREY SPECIFICITY AND MOLECULAR PHY- PARISON OF BELONOCYSTIS MARINA KLI- LOGENY OF THE THECATE MIXOTROPHIC MOV, ZLATOGURSKY, 2016 WITH LUFFIS- DINOFLAGELLATE FRAGILIDIUM MEXI- PHAERA SPP CANUM Klimov V.I. Kim S.1, Park M.G.2 St.Petersburg State University, Faculty of Biology, 1 - Department of Oceanography, Pukyong National Department of Invertebrate Zoology University, Busan, Republic of Korea [email protected] 2 - Department of Oceanography, Chonnam National Belonocystis Rainer, 1968 and Luffisphaera Belcher University, Gwangju, Republic of Korea and Swale, 1975 are enigmatic genera, which [email protected] currently lack supergroup affiliation. The genus Feeding mechanism and prey specificity of the Belonocystis was studied mainly on the light- mixotrophic thecate dinoflagellate Fragilidium microscopy level, while the genus Luffisphaera was mexicanum (strain Fm-LOHABE01) were exami- studied only with electron microscopy. Recently ned using the culture isolated from Masan Bay, provided ultrastructural data on Belonocystis gave Korea in 2011 during summer blooms of the toxic an opportunity to compare these taxa using one and dinoflagellate Alexandrium pacificum. We also used the same method. This analysis discovered many novel 18S and 28S rDNA sequences for F. mexicanum similarities in the organization of the cell structure to explore inter-species relationships within the as well as in morphology of the coverings. It was genus Fragilidium and to examine its phylogenetic clearly shown that surface structures of Belonocystis relationships with morphologically similar species represent not a solid capsule, but giant scales, which (Alexandrium, Goniodoma, and Pyrophacus). F. me- are similar to those of Luffisphaera. Comparison xicanum (strain Fm-LOHABE01) fed on species of the scales has revealed that some Belonocystis belonging to three dinoflagellate genera (i.e. species were similar to Luffisphaera species more Alexandrium, Ceratium, and Heterocapsa) when than inside the genus. Each scale of B. marina was 36 · “PROTIST–2016” associated with a short cytoplasmic outgrowth and chloroplasts within cytoplasm. These results strongly the same was shown for all of Luffisphaera spp., indicate that high Arctic benthic foraminifera are studied for that matter. All organells, described not dormant during the polar night, and they access for Luffisphaera were found in Belonocystis and algal diet. shown to have almost identical organization. Both Supported by Research Council of Norway grant have tubular mitochondrial cristae and vacuoles 226417/E10 and RFBR grant 14-04-93083. associated with stacks of rough endoplasmic reticulum cisternae and neither of them was shown TIM17 FAMILY PROTEIN IN THE MITO- to have kinetosomes. B. marina was the first record SOMES OF GIARDIA INTESTINALIS of marine species for Belonocystis, which again Kolisko M.1, Martincová E.2, Voleman L.2, Roger put it closer to Luffisphaera, initially recorded in A.J.3,4, Doležal P.2 marine and freshwater habitats. It’s possible that 1 - Canadian Institute for Advanced Research, the subsequent study of Luffisphaera and extra Department of Botany, University of British Columbia, Belonocystis isolates in future may lead to a fusion Vancouver, Canada of both or to transfer some species from one genus 2 - BIOCEV – Biotechnology and Biomedicine Center to another. Study was supported with RFBR grants of the Academy of Sciences and Charles University in 15-04-18101_a, 15-29-02749-ofi_m. Vestec and Department of Parasitology, Faculty of Science, Charles University in Prague, Czech Republic HIGH ARCTIC BENTHIC FORAMINIFE- 3 - Centre for Comparative Genomics and Evolutionary RANS DURING THE POLAR NIGHT: DOR- Bioinformatics, Department of Biochemistry and MANCY OR ACTIVE FEEDING? Molecular Biology, Dalhousie University, Halifax, Knyazeva O., Korsun S. Nova Scotia, Canada Dept Invertebrate Zool., Biol Faculty, St-Petersburg 4 - Program in Integrated Microbial Biodiversity, Univ, Russia Canadian Institute for Advanced Research, Halifax, [email protected] Nova Scotia, Canada Benthic foraminiferans are the vast meiobenthic [email protected] group of protists widely represented in all marine Protein transport across the mitochondrial membra- habitats including the high Arctic. Although nes is mediated by the TIM, TOM and SAM some foraminifera species are known to feed on complexes. These complexes are common to all bacteria, the majority of these protists keep to an supergroups of eukaryotes suggesting that they algal diet, preying on diatoms and dinofllagelates. were already present in the last eukaryotic common Some species are also capable of capturing prey ancestor. However, mitochondria-related organel- chloroplasts and maintaining them functionally les of Giardia intestinalis known as mitosomes active, thus performing kleptoplasty, which also were thought to lack both the SAM (a beta barrel results in a change of the host cytoplasm coloration. assembly complex in the outer membrane) and the Given the seasonal transitions to long periods of TIM (the translocase of the inner membranes) their darkness performed in high latitudes and leading membranes. The question was, how proteins pass to scarceness of primary production, one would the inner mitosomal membrane. Here, we present expect foraminiferans to face absence of prey the identification of Tim17 family protein in giardia algae and starvation, the latter resulting in either mitosomes, which represents a core channel forming death or dormancy of the most part of population. subunit of the TIM complex. The bioinformatic Nevertheless, our results contradict such assump- identification of this highly diverged subunit tions. In January 2015 and 2016, during the was only possible by including newly obtained Marine Night cruise with RV Helmer Hanssen, we orthologous metamonad sequences in the sequence collected live specimens in the Kongfjorden area, profiles. We demonstrate that giardia Tim17 is Svalbard. All major species (Nonionella labradorica, specifically targeted to mitosomes, where it interacts Islandiella helenae, Cassidulina reniforme, Elphidium with other proteins involved in the protein transport excavatum, Elphidium bartletti) had brightly species- and the iron-sulfur cluster assembly. specifically colored cytoplasm implying they did not starve but had access to algal food. Transmission HETEROTROPHIC NANOFLAGELLATES IN electron microscopy revealed that all specimens THE PLANKTON OF LAPTEV SEA had well-developed mitochondria, Golgi apparatus Kopylov A.I.1, Zabotkina E.A.1, Kosolapova N.G.1, and endoplasmic reticulum, therefore being meta- Romanenko A.V.1, Sazhin A.F.2 bolically active throughout the winter season. 1 - Papanin` Institute for Biology of Inland Waters, Moreover, some foraminiferans possessed intact Russian Academy of Sciences, Russia Protistology · 37

2 - Shirshov` Institute of Oceanology, Russian Academy slightly elongated body completely covered with of Sciences, Russia cilia forming dense longitudinal rows. In the [email protected] anterior part of the body, there is an opening The results of the species composition, abundance of vestibulum. Such features are clearly visible and biomass of heterotrophic nanoflagellates under light micro-scope. Because balantidia have (HNF) were obtained for the first time in the very few other morphological characters suitable Laptev Sea in August-September 2014-2015. An for taxonomy, their species identity is usually attempt was made to assess the role of HNF in determined using morphometric data. Comparative grazing productions of bacterioplankton (PB) and analysis of several described Balantidium spp. virioplankton (PV). 24 species and forms of HNF indicated that many of these specific names 9 taxa and groups of uncertain systematic position might be synonyms. All balantidia inhabiting the were found. Kinetoplastea Honigberg, 1963 were intestine of mammals and birds were recognized characterized by the highest species diversity. The to belong to a single species – Balantidium coli, HNF community dominated by bacteriophages recently moved to the genus Balantioides Alexeieff, (eighteen species), also found four omnivorous and 1931. We supposed that some species from amphibi- two carnivorous species. NHNF ranged from 108-651 ans may be synonyms (balantidia are polymorphic) cells/ml, the biomass — 0.3-3.5 mg C/m3. NHNF and some of them may belong to different genera. was three orders of magnitude lower than NB, and For solving this issue, we used modern electron

BHNF was 1.6-63.3% (mean 24.4±4.0%) biomass microscopic techniques and molecular phylogenetic of bacterioplankton. Between NHNF and NB were analysis. We studied the morphology of Balantidium detected high positive correlation (R=0.63, p=0.05). entozoon and B. elongatum from the gut of European The virus particles (diameter of 200-531 nm) po- common frog Rana temporaria Linnaeus, 1758 using tentially can be edible objects for HNF. However, light and electron microscopy. The distinguishing the ratio of the number of large viruses at NHNF was features of B. elongatum are the long thin fusiform low - 42-417. HNF could consume a significant body and relatively short vestibulum. However, amount of virus attached to the walls and inside other traits (number of kinetes and the distance bacteria. The number of bacteria with attached between them) are indistinguishable between two viruses was 8.7-26.5% NB. On bacteria it was up to 11 species. Their intracellular structures also showed virus particles. The number of bacteriophage-viruses no differences. The sequences of 18S rRNA gene in infected cells reached 111 particle/cell. Guided by of both species were identical to each other and literature data on clearance rates of water by Arctic to that of B. entozoon from the GenBank. Thus B. HNF (Sherr et al., 1997), calculated that in the water elongatum must be considered as a younger synonym column of the Laptev sea the HNF community of B. entozoon. consumed 1.1-30.4% (average 8.3%) daily PB and

1.3-77.2% (average 14.9%) the daily PV. The research BRIDGING THE GAP BETWEEN TRADI- supported by the RFBR grant 14-04-00130. TIONAL TAXONOMY AND TRANSCRIP- TOMICS IN ARCELLINIDS (AMOEBOZOA) THE STATUS OF THE SPECIES BALANTI- Kosakyan Anush1, Brown Matthew W.2,3, Lara DIUM ELONGATUM FROM THE GUT OF Enrique4, Mitchell Edward A.D.4,5, Lahr Daniel EUROPEAN COMMON FROG J.G.1 Kornilova O.A.1, Chistyakova L.V.2, Kostygov A. 1 - Laboratory of Evolutionary Protistology, Institute Yu.3,4 of Biosciences, University of São Paulo, Matão, 1 - Herzen State Pedagogical University of Russia, St. Travessa 14,Cidade Universitária, 05508-090 - São Petersburg Paulo, SP, Brasil 2 - Centre of Core Facility of SPSU “Culturing 2 - Department of Biological Sciences, Mississippi State collections of microorganisms”, St. Petersburg, Russia University, Mississippi State, 39762, Mississippi, USA 3 - Zoological Institute of the Russian Academy of 3 - Institute for Genomics, Biocomputing and Bio- Sciences technology, Mississippi State University, Mississippi 4 - Life Science Research Centre, Faculty of Science, State, 39762, Mississippi, USA University of Ostrava 4 - Laboratory of Soil Biology, University of Neuchâtel, [email protected] Rue Emile-Argand 1, 2000 Neuchâtel, Switzerland The representatives of the genus Balantidium 5 - Jardin Botanique de Neuchâtel, Chemin du Claparède et Lachmann, 1858 have several Perthuis-du-Sault 58, 2000 Neuchâtel, Switzerland. common morphological features: sacciform or [email protected] 38 · “PROTIST–2016”

Arcellinid testate amoebae are a diverse group of values of the abundance and biomass of benthic terrestrial and freshwater microbial Eukaryotes. heterotrophic flagellates were 238 and 259 times, Described by naturalists since the eighteenth cen- respectively higher than in plankton. The biomass tury, Arcellinids are frequently used in biomoni- of flagellates constituted on average 11.2% of the toring and paleo-ecological reconstruction of past bacterial biomass in the water column and only climates. They are supposed to include the oldest 0.8% in bottom sediments. The estimation of undisputed eukaryotic fossils, which vests them with the role of flagellates in the consumption of the a particular importance in the study of basal nodes bacterial production demonstrated that HNFs of eukaryotic tree. However, difficulties in culturing were an important factor which regulated the these organisms as well as in the application of bacterioplankton development grazing on average molecular biology protocols have limited the study 32.3% of its daily production whereas their effect on of their diversity and evolutionary history. Our aim in bacteriobenthos was insignificant as they consumed this study was to investigate arcellinid (Amoebozoa, only 2.0% of its production. The moderate positive Tubulinea) diversity, and to recon-struct a solid and correlation (r = 0.33) and (r = 0.52), respectively precise phylogeny combining comprehensive taxon was found between the abundance and biomass of sampling, multiple genes and detailed morpholo- bacteria both in plankton and benthos. gical characterisation. We obtained transcriptomes for twenty-one testate amoeba species, for most MINIONS OF GREAT CTHULHU AWAKE- of which no molecular data was yet available. Our NING – A NEW INSIGHT INTO THE DIVER- phylogenetic reconstruction of Arcellinids is based SITY OF PARABASALID SYMBIONTS OF on the combination of morphological data and COCKROACHES molecular data on about 350 genes and 100 000 Kotyk Michael, Varadínová Zuzana, Čepička Ivan amino acid positions. Department of Zoology, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic HETEROTROPHIC FLAGELLATES IN THE [email protected] WATER COLUMN AND BOTTOM SEDIMENTS Parabasalian symbiots of xylophagous cockroaches IN THE RYBINSK RESERVOIR from families Cryptocercidae and Isoptera (ter- Kosolapova N.G., Kopylov A.I., Kosolapov D.B. mites) have been attracting the interest of biologists Papanin Institute for Biology of Inland Waters, for several decades; they often possess large, comp- Russian Academy of Sciences, Borok, Yaroslavl obl., lex, and visually attractive cells and are of great 152742 Russia ecological significance. Phylogenetic analyses [email protected] showed that the complex forms of parabasalians A total of 56 heterotrophic flagellates (HNF) from (=hypermastigotes) have arisen several times 15 large taxa were identified in water and bottom independently. On the other hand, only a little sediments of the large floodplain mesoeutrophic attention has been paid to endosymbionts of Rybinsk Reservoir during the summer period. “normal” cockroaches, although these insects show Among them 35 species were detected in plankton a great diversity in morphology and lifestyles. We and 45 species were found in benthos. Twenty- have examined the intestines of 250 cockroaches four species of HNF were common for these two belonging to 100 species and 20 subfamilies (out of habitats that constituted 44% of their composition. 33), established 50 stable cultures of trichomonads, Ten species were recorded only in plankton and sequenced their SSU rRNA gene, and studied 20 species were recorded only in benthos. The orders the morphology of some of them. Approximately Chrysomonadida, Euglenida and Cercomonadida one half of the obtained trichomonads formed differed, to a greater extent, in the species compo- a considerably diversified clade that contained sition in water and bottom sediments. Most of the the recently discovered hypermastigote Cthulhu species of heterotrophic flagellates which were with approximately 20 flagella and trichomonad found in the Rybinsk Reservoir were bacteriotrophs. genera Hexamastix and Cthylla with 6 flagella. By Three predatory species were also found. The contrast, our strains have cells equipped with three abundance and biomass of HNF in the water or four. Thus, Cthulhu and Cthylla are not orphans column averaged 991 ± 326 cells/mL and 41.4 ± anymore, but are surrounded by a cloud of lesser 14.1 mg/m3, respectively in the water column and trichomonads. Most of the remaining strains belong (236 ± 61) × 103 cells/mL and 10.7 ± 4.0 µg/mL, to the understudied genus Hypotrichomonas, where respectively in bottom sediments. The average they represent several novel species. Protistology · 39

LAND USE AND LITTER CHEMISTRY IM- Institute of Monitoring of Climatic and Ecological PACT MICROBIAL DECOMPOSER COMMU- Systems of SB RAS, Tomsk NITIES AND LITTER DECOMPOSITION [email protected] IN TROPICAL LOWLANDS OF SUMATRA, The research is devoted to rhizopod analysis INDONESIA (analysis of the testate amoebae complexes) in Krashevska V.1, Malysheva E.2, Klarner B.1, Mazei peat-lake sediments. The aim is the reconstruction Y.3,2, Maraun M.1, Widyastuti R.4, Scheu S.1 of hydrological conditions in the mire during its 1 - J.F. Blumenbach Institute of Zoology and Anth- development in the late Holocene. The study site ropology, Georg August University Göttingen, Berliner Bezrybnoe Mire lies in Yergaki Natural Park in Straße 28, 37073 Göttingen, Germany the Western Sayan Mts, southern Middle Siberia 2 - Penza State University, Russia (52°51’ N, 93°21’ E, 1300 m a.s.l.). The thickness of 3 - Lomonosov Moscow State University, Russia sediments is 6 m, of which the lower 2 m – gyttja, the 4 - Institut Pertanian Bogor - IPB, Department of Soil upper 4 m - peat composed mainly of remnants of Sciences and Land Resources, Damarga Campus, sphagnum moss. Age of peat at a depth of 4 m is 2,420 Bogor 16680, Indonesia cal yr BP. Total 80 testate amoeba taxa was revealed. [email protected] The dominant species were: Amphitrema wrightianum, We investigated how the conversion of rainforest Centropyxis aculeata, C. gibba, Difflugia globulus, into jungle rubber, intensive rubber and oil palm Euglypha rotunda, Schoenbornia smithi, Trinema plantations affects the decomposer community and lineare, T. enchelys. There are both oligotrophic and decomposition processes. Further, we investigated minerotrophic species. 22 species were met only in the role of litter chemistry in structuring the gyttja. Water table depth (WTD) reconstruction is decomposer system. In total 192 litterbags were made on the basis of species optima obtained in our placed into the field: 3 litter types (forest, rubber, oil study of modern oligotrophic and minerotrophic palm) in 4 land use systems, with 2 harvest times and mire habitats in the south of Western Siberia 8 replicates, each. After 6 and 12 months litterbags using the transfer function (training set includes were collected and litter mass loss was measured, 115 samples). Decrease of WTD in the mire are testate amoebae and microbial communities were observed 2350-2300, 1850-1800, 1650, 1450, 1350 studied, and litter element concentrations were 1100-900, 400-0 cal yr BP and increase – in 2400, measured. The decomposition in converted systems 2000, 1900, 1800-1700, 1600-1500, 1400, 1300, was slower as compared to rainforest; and litter of 800-500 cal yr BP. Minerotrophic testate amoebae oil palms and rubber decomposed faster than that of species were prevailed in the studied sediments. forest. A total of 58 species of testate amoebae (TA) However, there were also oligotrophic species. For colonized the litter. Species number and density the reconstruction of WTD during development of of TA, as well as microbial biomass (MB) were such mires the information is needed about optima high in rainforest and jungle rubber and decreased of both oligotrophic and minerotrophic species. significantly in rubber and oil palm plantations. The work was supported by RFBR grant (16-34- Further, TA density and MB was highest in rubber 60057). litter, whereas TA species number was at a similar levels in rubber and forest litter. Overall, our data BENTHIC CILIATES ON HERON ISLAND indicate negative effects of rainforest conversion REEF, AUSTRALIA on the structure and functioning of decomposer Langlois G.A. community and, thereby litter decomposition. Bryant University, Smithfield, RI USA Further, testate amoebae and microorganisms were [email protected] interactively affected by litter element concent- Field studies conducted at the Heron Island Re- rations. The data suggest that element ratios of search Station, located in the Capricorn region of litter material as basal resource of the decomposer the Great Barrier Reef, revealed complex protistan food web exhibit strong bottom-up control on communities. Heron Reef, home to about two- the structure of decomposer communities and thirds of the coral species found on the GBR, has decomposition processes experienced repeated bleaching episodes in the past decade. This study, with comparisons to similar RHIZOPOD ANALYSIS IN THE PEAT-LAKE observations in 2006, emphasizes the diversity SEDIMENTS OF THE MOUNTAIN MIRE of ciliates and diatoms found in reef sediments. “BEZRYBNOE” IN THE SOUTH OF MIDDLE Observations included the interactive role of the SIBERIA protistan component of a coral reef, in particular Kurina I.V., Blyakharchuk T.A. the dynamics of opportunistic and/or potentially 40 · “PROTIST–2016” pathogenic forms associated with damaged corals presence of flagellum at some stage of the life-cycle. undergoing disease conditions, such as Brown Band Moreover, we performed several trans-infection disease. These altered communities are characte- experiments using different species of newly rized by rapid tissue breakdown in hard corals, characterized “Candidatus Megaira” in order to coupled with high levels of dissolved organic matter. understand how representatives of this genus can Samples were taken by direct capture, observed with be spread in the aquatic environment and how they phase contrast and epifluorescence microscopy, influence the host fitness within the same ciliate recorded by video and photomicrography, and population. We also investigated the interaction fixed for later identification and genomic studies. between “Candidatus Megaira” and other symbiotic The relative abundance of diatoms/flagellates/ bacteria already inhabiting or invading the same host ciliates appears to show a higher proportion of cell. All together, our findings highlight the necessity photosynthetic species as compared to previous of a better understanding of the life-cycles of this observations. Brown Band ciliates were not found widespread, but poorly investigated, endosymbiont. on the corals, in the sediments, on algal surfaces, or in the plankton prior to bleaching in late January, THE GENOMIC STUDY OF AGAR PRODUC- posing the question of their location when not acting ING RED ALGA GRACILARIOPSIS CHORDA as opportunistic pathogens. Lee J.M.1, Yang E.C.2, Graf L.1, Yoon H.S.1 1 - Department of Biological Sciences, Sungkyunkwan “CANDIDATUS MEGAIRA”: AN EMERGING University, Suwon 16419, Korea GENUS OF BACTERIAL ENDOSYMBIONTS 2 - Marine Ecosystem Research Division, Korea WIDESPREAD IN CILIATES AND OTHER Institute of Ocean Science & Technology, Ansan, PROTISTS 15627, Korea Lanzoni O.1, Shaidullina A.2, Sabaneyeva E.2, [email protected] Castelli M.1,3, Lebedeva N.4, Potekhin A.2, Petroni Red algae (Rhodophyta) played an important role G.1 in the evolution of photosynthetic organisms as 1 - Department of Biology, University of Pisa, Pisa, plastid donor(s) in chlorophyll-c containing algae Italy including cryptophytes, haptophytes, stramenopiles, 2 - Faculty of Biology, St. Petersburg State University, and alveolates. Most red algal species are important Saint Petersburg, Russia components in marine ecosystems, serving as a 3 - Department of Veterinary Medicine, University of shelter for diverse marine organisms and contributing Milan, Milan, Italy as primary producers. Many red algal species are 4 - Centre of Core Facilities “Culture Collections of heavily cultivating as food and industrial purposes Microorganisms”, St Petersburg State University, such as agar and carrageenan production. Despite Saint Petersburg, Russia of these importances, only several red algal geno- [email protected] mes have been reported among more than 7,000 “Candidatus Megaira” (Rickettsiaceae) is a recently species. From the major multicellular red algal described genus of obligate intracellular bacteria, group of theFlorideophyceae, only one genome which are phylogenetically closely related to the was fully sequenced to date. To fill the gap of our pathogen Rickettsia. Sequences representatives of understanding on red algae, here we analyzed a genus “Candidatus Megaira” can be split in at least new genome of the multicellular red algal species 3-4 species level sub-clades. These sequences have Gracilariopsis chorda, which is one of major cultivar been found in association to evolutionary far related as food and agar production in Korea. Using the host lineages (e.g. unicellular and multicellular algae, long-read single molecule sequencing (PacBio) corals and hydras). The type species of the genus, platform, we completed whole-genome of 89.6 “Candidatus Megaira polyxenophila”, has been Mbp (49% GC content, 1,098 contigs) that encodes found in several ciliates as well as in other protists. a total of 9,992 predicted proteins. We discuss the In ciliates, “Candidatus Megaira polyxenophila” can interesting story of genome reduction/expansion infect different cell compartments (macronucleus and gene duplication in red algal genomes at this and cytoplasm) according to the host species. meeting. Herein, we provide the characterization of a second “Candidatus Megaira” species inhabiting the AN ANCESTRAL BACTERIAL DIVISION cytoplasm of the green ciliate Paramecium bursaria. SYSTEM IS WIDESPREAD IN EUKARYOTIC Within the same host cell, this novel “Candidatus MITOCHONDRIA Megaira” presents polymorphic features such as the Leger M.M.1, Petrů M.2, Žárský V.2, Eme L.1, Vlček association with endosymbiotic algae, or the possible Č.3, Harding T.1, Lang B.F.4, Eliáš M.5, Doležal P.2, Protistology · 41

Roger A.J.1 2 - Marine Ecosystem Research Division, Korea 1 - Dept. of Biochemistry and Molecular Biology, Institute of Ocean Sciences & Technology, Ansan Dalhousie University, Halifax, Canada 15627, Republic of Korea 2 - BIOCEV Group, Department of Parasitology, 3 - Department of Biology, Chungnam National Faculty of Science, Charles University in Prague University, Daejeon 34134, Republic of Korea 3 - Laboratory of Genomics and Bioinformatics, 4 - Department of Oceanography, Chonnam National Institute of Molecular Genetics, Academy of Sciences University, Gwangju 61186, Republic of Korea of the Czech Republic 5 - Friday Harbor Laboratories, University of Wa- 4 - Robert Cedergren Centre for Bioinformatics and shington, WA, 98250, USA Genomics, Département de Biochimie, Université de [email protected] Montréal, Montréal, Canada The thecate filose amoeba Paulinella chromatophora 5 - Department of Biology and Ecology, Faculty of is a good model organism for understanding plastid Science, University of Ostrava, Czech Republic organellogenesis because its plastid was derived [email protected] from a Synechococcus-Cyanobium type of alpha- Bacterial division initiates at the site of a contractile cyanobacterium. Recent studies have shown spe- Z-ring composed of polymerized FtsZ. The location cies-level of divergence after the acquisition of of the Z-ring in the cell is controlled by a system of the organelle; however, a full investigation has not three mutually antagonistic proteins, MinC, MinD, been conducted for the photosynthetic species. We and MinE. Plastid division is also known to be surveyed the biodiversity of this interesting alga using dependent on homologs of these proteins, derived samples collected from around the world. Using four from the ancestral cyanobacterial endosymbiont that gene markers (18S rRNA, 16S rRNA, dnaK1, psaL), gave rise to plastids. In contrast, the mitochondria two distinct lineages with high genetic variation were of model systems such as Saccharomyces cerevisiae, identified, including one new species candidate (i.e., mammals, and Arabidopsis thaliana seem to have P. microporus). In addition, the chromatophore replaced the ancestral alphaproteobacterial Min- genome was fully sequenced from P. microporus based division machinery with host-derived dyna- strain KR01 and the recently reported marine P. min-related proteins that form outer contractile longichromatophora. Comparative genomic analysis rings. By mining transcriptome and genome data, showed 0.17% of sequence divergence between the we show that the mitochondrial division system of Korean strain KR01 and the Japanese strain FK01. these model organisms is the exception, rather than Among 1,626 variable sites, the divergence was the rule, for eukaryotes. We describe endosymbiont- converged on noncoding regions at a rate seven times derived, bacterial-like division systems comprising higher than for coding regions. The chromatophore FtsZ and Min proteins in diverse less-studied genome of P. longichromatophora, when compared eukaryote protistan lineages, including jakobid to other photosynthetic Paulinella species, showed and heterolobosean excavates, a malawimonad, a higher mutation rate. These results suggest that stramenopiles, amoebozoans, a breviate, and an the diversification of the photosynthetic Paulinella apusomonad. For two of these taxa, the amoebo- species has occurred at a rapid rate and that the zoan Dictyostelium purpureum and the jakobid diversification is still ongoing. Stygiella incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous ROLE OF PROTEIN DISULFIDE ISOMERASE expression in Saccharomyces cerevisiae. The disco- (PDI) ON TOXOPLASMA-HOST INTERPLAY: very of a proteobacterial-like division system in IMMUNOLOCALIZATION ASSAYS USING mitochondria of diverse eukaryotic lineages suggests ANTI-HUMAN PDI MONOCLONAL ANTI- that it was the ancestral feature of all eukaryotic BODIES (MABS) ON IN VITRO CULTURE mitochondria and has been supplanted by a host- SYSTEMS derived system multiple times in distinct eukaryote Lobo Maria Luísa1, Novo Carlos2, Matos Olga1 lineages. 1 - Medical Parasitology Unit, Group of Opportunistic Protozoa/HIV and Other Protozoa, Global Health DIVERSITY OF PHOTOSYNTHETIC PAU- and Tropical Medicine, Instituto de Higiene e Me- LINELLA SPECIES INCLUDING A COM- dicina Tropical (IHMT), Universidade Nova de PARATIVE PLASTID GENOME ANALYSIS Lisboa (UNL) Lhee D.H.1, Yang E.C.2, Kim J.I.3, Kim S.4, Park 2 - Medical Parasitology Unit, IHMT, UNL, Lisboa, M.G.4, Andersen R.A.5, Yoon H.S.1 Portugal. 1 - Department of Biological Science, Sungkyunkwan [email protected] University, Suwon 16419, Republic of Korea The main goal of this study is to address the functional 42 · “PROTIST–2016” roles of PDI on Toxoplasma gondii-host interplay, which can led to important insights in the evolution in the context of acute/chronic infection, and eva- of different eukaryotic lineages. This is the case luating their usefulness as drug-targets using anti- of the Opisthokonta, in which the identification human PDI commercial MAbs. The potential cross- of new unicellular lineages close to animals has reactions of PDI anti-human MAbs with T. gondii, the potential to improve our understanding of the confirming their usefulness for immunolocalization transition towards animal multicellularity. However, of PDI not only in human fibroblasts (HFF) tar- isolating these new organisms is a challenge. Instead, get cells, but also in Toxoplasma tachyzoites/cysts single-cell genomics (SCG) seems a prominsing were verified. In order to validate this hypothesis approach to obtain directly the genomes from the a bioinformatics analysis was conducted in the environment. However, SCG technologies, which Toxoplasma genome database to search for homo- have mainly been employed in prokaryotes, are logous regions: of the antigens’ complete amino affected by important biases, specially during the acid sequence used in the manufacturing processes whole genome amplification step. The potential of the MAbs utilized in this study; and/or of their of SGC for eukaryotes remains unclear. To test more predicted immunogenic regions in case of the potential of this technique for evolutionary antigens with longer sequences. Confirmation was studies in eukaryotes, we here analyze three obtained by reverse search on human genome for environmental single amplified genomes (SAGs) the predicted T. gondii antigens/peptides sequences. from the choanoflagellate Monosiga brevicollis, Results from indirect immunolabeling assays whose genome is already sequenced. We show the using immunofluorescence techniques with anti- genome recovery obtained from our SAGs was low human PDI MAbs: PDI, PDIA3(ErP57), PDIA6, (between 30-5%, around 3,684 out of 9,175 genes). Calnexin(CNX), glucose-regulated proteins/immu- Interestingly, the percentatge of pylogenomic noglobulin heavy-chain binding protein (GRP78/ markers that our SAGs contains is good (almost BiP) and GRP94 suggest both cross-reaction with 100% for some datasets). We also explored different target cell-lines and Toxoplasma, being suitable techniques to improve the quality and the recovery for their profile identification and to evaluate of the assemblies obtained. We found that genome PDI usefulness as a drug-target against T. gondii recovery increased up to 48% by pooling the data infection. Different patterns of immunolabeling from the three different SAGs. Current SCG were observed in Toxoplasma infected and non- technologies seem a limited approach for adressing infected HFF cells according to the MAb used. The some questions but has the potential to provide functional confirmation of the characterized PDI enough data for phylogenomic analyses. involved in the host-pathogen interaction, in the presence of PDI inhibitors, is in progress. Further DIPLONEMIDS – NEW KIDS ON THE BLOCK analyses will be performed by RNA silencing to get Lukeš Julius1,2, Flegontova Olga1,2, Flegontov complementary evidence of these specific proteins Pavel1,2,3, Faktorová Drahomíra1,2, Kaur Binny- in Toxoplasma-host interactions. preet1,2, Votýpka Jan1,2,4, Tashyreva Daria1,2, Yabuki Acknowledgments: Supported by FCT ref:VIH/ Akinori5, Malviya Shruti6, de Vargas Colomban7,8, SAU/0019/2011. Bowler Chris6, Burger Gertraud9, Horák Aleš1,2 1 - Institute of Parasitology, Biology Centre, Czech ANALYSIS OF THREE SINGLE-CELL AM- Academy of Sciences PLIFIED GENOMES OF THE CHOANO- 2 - Faculty of Science, University of South Bohemia, FLAGELLATE MONOSIGA BREVICOLLIS České Budějovice REVEALS SINGLE-CELL GENOMICS AS A 3 - Faculty of Science, University of Ostrava, Ostrava, LIMITED APPROACH FOR EVOLUTIONARY Czech Republic PURPOSES 4 - Faculty of Science, Charles University, Prague, López-Escardó D.1, Ruiz-Trillo I.1,2 Czech Republic 1 - Institut de Biologia Evolutiva (CSIC-Universitat 5 - Japan Agency for Marine-Earth Science and Pompeu Fabra), Passeig Marítim de la Barceloneta Technology, Kanagawa, Japan 37-49, 08003 Barcelona, Catalonia, Spain 6 - Ecole Normale Supérieure, PSL Research Uni- 2 - Institució Catalana de Recerca i Estudis Avançats versity, Institut de Biologie de l’Ecole Normale (ICREA), Barcelona, Catalonia, Spain Supérieure (IBENS), Paris, France [email protected] 7 - Station Biologique de Roscoff, Roscoff, France Environmental metabarcoding data is providing 8 - Sorbonne Universités, Paris, France a new view of the real protist diversity. This new 9 - Université de Montréal, Montreal, Canada diversity often occupies key phylogenetic positions [email protected] Protistology · 43

Results from the Tara Oceans metabarcoding these two chonotrichs, and the divergence of the study revealed that a significant fraction of the chonotrich clade within the dysteriid clade. plankton in the sunlit ocean is constituted by diplonemid flagellates, a poorly known group of MAPPING THE HIDDEN DIVERSITY OF heterotrophic excavate protists. In an analysis FREE LIVING FRESH WATER CILIATES of V9 18S rDNA metabarcodes from planktonic FROM DELHI REGION, INDIA communities collected in 123 globally distributed Makhija S.1, Abraham J.S.1, Somasundaram S.1, stations, diplonemids emerge as a highly abundant Toteja R.1, Gupta R.2 and extremely diverse group that lacks geographic 1 - Acharya Narendra Dev College, University of Delhi, structuring but displays strong vertical separation. Delhi, India Uniquely among planktonic eukaryotes, taxonomic 2 - Maitreyi College, University of Delhi, Delhi, India identification of diplonemids is far from saturation. [email protected] They are more abundant in polar regions and deeper India, with 2.4% of the world’s area, has over 8% of waters, yet their biological function in the ocean – the world’s total biodiversity, making it one of the 12 the largest ecosystem of our biosphere - remains mega diversity countries in the world. Despite this unknown. We are attempting to isolate and culture richness, the data concerning ciliate diversity from new marine diplonemids in order to sequence their India is rather scarce. We at University of Delhi have genomes, analyze their ultrastructure and establish been involved in the morphological and molecular protocols for their genetic manipulation. By this taxonomy of ciliates from freshwater bodies namely combined approach, we hope to shed more light on from Okhla Bird Sanctuary, Sanjay Lake, Pond at these „new kids on the block“. Rajghat in Delhi region. A large number of species from different classes have been observed from THE SMALL SUBUNIT rRNA GENE SEQU- these water samples namely from genus Tetmemena, ENCE OF THE CHONOTRICH CHILODO- Stylonychia, Laurentiella, Euplotes, Aspidisca, CHONA CARCINI JANKOWSKI, 2007 CON- Paraurostyla, Gastrostyla, Pseudourostyla, Urostyla, FIRMS CHONOTRICHS AS A DYSTERIID- Oxytricha and Uroleptus from class Spirotrichea; DERIVED CLADE (PHYLLOPHARYNGEA, Blepharisma, Spirostomum and Stentor from class CILIOPHORA) Heterotrichea; Paramecium, Frontonia, Vorticella Lynn D.H. and Cyclidium from class Oligohymenophorea; Department of Integrative Biology, University of Litonotus, Dileptus, Didinium and Lacrymaria from Guelph, Guelph, ON Canada N1G 2W1 class Litostomatea; Colpoda from class Colpodea; [email protected] Chilodonella from class Phyllopharyngea; Loxodes The chonotrichs are sessile ciliated protozoa from class Karyorelectia; and Coleps from class ectosymbiotic on the body parts of a variety of Prostomatea have been identified by live cell crustaceans. They have long been considered a observations, protargol staining, silver staining and separate group because their sessile habit has resulted morphometric measurements. In the present study, in the evolution of a very divergent body form we describe in detail few species from the subclass and reproductive strategy compared to free-living Stichotrichia and Hypotrichea. There is difficulty ciliates. In the mid-20th Century, the free-living in understanding the phylogenetic relationship dysteriid cyrtophorian ciliates were proposed as a between different species of these group based potential sister clade because the chonotrich bud solely on morphological markers. Therefore, we or daughter cell showed similarities during division are trying to look into both morphological and morphogenesis (i.e. ontogeny) to these free-living multiple molecular markers like SSU- rRNA, ITS dysteriids. A single small subunit (SSU) rRNA gene 1, ITS2 , hsp70 and histone genes and we could sequence is available for the chonotrich Isochona depict morphological and molecular congruence sp. However, its authenticity has been questioned in the species from subclass Stichotrichia and in the recent literature, and the placement of this Hypotrichea. sequence within the dysteriid clade has added to this controversy. In this report, the SSUrRNA gene GLOBAL SHIFTS IN GENE EXPRESSION sequence of the chonotrich Chilodochona carcini PROFILES ACCOMPANIED WITH ENVI- ectosymbiotic on the green crab Carcinus maenas RONMENTAL CHANGES IN CNIDARIAN- is provided. Topology testing of the SSUrRNA DINOFLAGELLATE ENDOSYMBIOSIS gene phylogeny constructed by Bayesian Inference Maruyama S.1, Aihara Y.2, Yamaguchi T.3, Yama- robustly supports the sister-group relationship guchi K.4, Shigenobu S.4, Takahashi H.3, Kawata of Isochona sp. and C. carcini, the monophyly of M.1, Ueno N.3, Minagawa J.2 44 · “PROTIST–2016”

1 - Department of Environmental Life Sciences, 4 - Ritsumeikan University Tohoku University, Japan [email protected] 2 - Division of Environmental Photobiology, National Rapaza viridis is a euglenoid that always possesses Institute for Basic Biology, Japan chloroplasts in cells without exception. R. viridis 3 - Division of Morphogenesis, National Institute for requires not only light but also a specific strain Basic Biology, Japan of green algae (Tetraselmis sp.) as its prey for 4 - Functional Genomics Facility, National Institute their survival/growth, hence apparently being of for Basic Biology, Japan obligate mixotrophy. Nonetheless, the degradative [email protected] process of chloroplasts of the prey was barely Stable endosymbiotic relationship between cnida- observed. Furthermore, only trace amount of rian animal and dinoflagellate Symbiodinium spp. 132,173-cyclopheophorbide enols (CPEs), the is a prerequisite for sustaining coral reef ecosystem. non-phototoxic catabolites of chlorophylls, was Recent studies have shown that elevated seawater produced along with the predation, indicating temperature can cause collapse of endosymbiosis that only a small proportion of chlorophylls was by expulsion of the symbiotic algae from cnidarians, actually discarded from the ingested chloroplasts. which is known as ‘bleaching’, and subsequent We thus investigated cell dynamics regarding in- mass mortality. However, the technical difficulty gested chloroplasts in R. viridis to understand the in maintaining and using corals as material in fate of the chloroplasts and chlorophylls contained laboratory has hampered further understanding therein. Unlike other phycophagic euglenoids, of the molecular biology of the cnidarian-dinofla- decrement of chlorophyll fluorescence from the gellate endosymbiosis. Here we show transcriptomic ingested chloroplasts was rarely observed, and any analyses using the symbiotic sea anemone Exaiptasia evidence for digestive degradation of the chloro- pallida (formerly Aiptasia spp.), an emerging model plasts was not recognized. Furthermore, the chloro- cnidarian, in multiple culture conditions. The plastic eye-spots of Tetraselmis sp. became dispersed genome-wide gene expression profiles were ana- several hours after the ingestion and eventually lyzed by mapping the RNAseq reads from symbiotic disappeared. In the next hours, interestingly, the and experimental apo-symbiotic anemones onto ingested chloroplasts became subdivided into more the host and endosymbiont genome sequences. A than several pieces with various sizes. Within a large portion of the genes differentially expressed day, the unique, rough appearance of chloroplasts in response to light and elevated temperature in of Tetraselmis sp. in the DIC image became the symbiotic and apo-symbiotic anemones was insignificant and changed into a rather smooth not overlapped and, in a number of cases, different appearance that is no more distinguishable from members in a single gene family were activated those originally possessed by R. viridis. Afterward, between the symbiotic and apo-symbiotic anemones. the number of “chloroplasts” per cell began to These suggest that the anemones use distinct gene decrease, suggesting progress of R. viridis cell sets to respond to environmental changes depending divisions at this stage. We discuss the mechanism on the symbiosis states with Symbiodinium. From the for the observed chloroplast division and the origin endosymbiont side, several photosynthesis-related of “chloroplasts” of R. viridis. genes associated with the photosystem II core assembly were detected to be down-regulated under HETEROGENEITY IN NUTRIENT UPTAKE the heat condition in hospite, which potentially BY INDIVIDUAL DINOFLAGELLATE CELLS affect the photodamage-induced stress responses. REVEALED USING NanoSIMS Overall, our data suggest that the endosymbiosis Matantseva O.1, Vogts A.2, Voss M.2, Skarlato S.1 with Symbiodinium substantially affect the host’s 1 - Institute of Cytology RAS, St. Petersburg, Russia transcriptional profiles, potentially leading to the 2 - Institute for Baltic Sea Research Warnemuende, altered ecological contribution to the environment. Germany [email protected] CHLOROPLAST DIVISION OF TETRASEL- Dinoflagellates are one of the most successful MIS SP. IN THE FOOD VACUOLE OF MIXO- groups of marine protists. Many photosynthetic TROPHIC ALGAE RAPAZA VIRIDIS dinoflagellates can utilize not only inorganic, Maruyama M.1, Miyagishima S.2, Suzaki T.3, but also organic compounds as nutrients, which Kashiyama Y.1,4 is advantageous in eutrophied coastal zones. 1 - Fukui University of Technology Therefore, nutritional physiology of these organisms 2 - National Institute of Genetics receives much scientific attention. Usually it is 3 - Kobe University studied by bulk approaches; however, the use of Protistology · 45 modern single-cell tools significantly advances our on endosymbiotically acquired proteins. We here knowledge in this field. We used stable isotope tra- surveyed nucleus-encoded plastidal proteins in cers, isotope ratio mass spectrometry and nanoscale Karlodinium and Lepidodinium, and investigated secondary ion mass spectrometry (NanoSIMS) to the origins of the proteins involved in two metabolic investigate concurrent uptake of nitrate and urea pathways localized in the plastid. Chlorophyll a by dinoflagellates Prorocentrum minimum at the biosynthetic pathway in the two species appeared population and single-cell levels. Although bulk to be reorganized in different ways. The pathway in and averaged single-cell measurements revea- Karlodinium was found to be occupied by proteins led similar relationships between the urea and acquired from the haptophyte endosymbiont, while nitrate uptake, NanoSIMS disclosed significant ‘laterally derived’ proteins, which were acquired heterogeneity in nutritional activity of individual from diverse eukaryotes rather than the green cells. Dinoflagellates readily consumed urea even algal endosymbiont, comprise the pathway in if this nutrient was new to the cells, but some cells Lepidodinium. In contrast, the majority of proteins took it up at a 9-folds higher rate than others. which were detected in isoprene biosynthetic path- Furthermore, sudden urea input resulted in overall way in Karlodinium or Lepidodinium were derived 30-40% suppression of the nitrate uptake. However, from host dinoflagellate, suggesting that switch a closer look with NanoSIMS showed that in 30% of from the canonical to non-canonical plastids barely cells nitrate consumption was completely inhibited, triggered reorganization of this particular pathway whereas in other cells it might be not suppressed. in the two species. In this talk, we discuss biological We conclude that even sporadic inputs of urea to reasons for the marked difference in the impact of coastal ecosystems are likely to have a prominent gene transfer between the two metabolic pathways effect on P. minimum populations. Physiological in Karlodinium and Lepidodinium. heterogeneity among individual dinoflagellate cells can represent a so far overlooked strategy towards LAGENOPHRYS PATINA (CILIOPHORA: ecological success. Funded by the Russian Science PERITRICHIA: LAGENOPHRYIDAE) ATTA- Foundation, project 16-14-10116 (to OM, SS) and CHED TO TWO MEXICAN POPULATIONS Federal Ministry of Education and Research, grant OF HYALELLA AZTECA (CRUSTACEA: AM- BMBF 03F0626A (to AV, MV). PHIPODA): STATISTICAL APPROACH TO PROBE SITE PATTERN PREFERENCE ON TRENDS IN ENDOSYMBIOTIC GENE TRAN- HOST SFER ON PLASTID METABOLIC PATHWAYS Mayén-Estrada R.1, Macip-Ríos R.2, Hermoso- IN DINOFLAGELLATES WITH NON-CANO- Salazar M.3, Romero-Niembro V.M.1 NICAL PLASTIDS 1 - Lab. Protozoología, Depto. Biol. Comparada, Matsuo E.1, Inagaki Y.1,2 Fac. Ciencias, Universidad Nacional Autónoma de 1 - Graduate School of Life and Environmental México, Circuito Ext. s/núm. Ciudad Universitaria, Sciences, University of Tsukuba C. P. 04510, Ciudad de México, Mexico 2 - Center for Computational Sciences, University of 2 - ENES Morelia, Universidad Nacional Autónoma Tsukuba de México, Antigua Carretera a Pátzcuaro No.8701, [email protected] Col. Ex Hacienda de San José de la Huerta, C.P. The major photosynthetic dinoflagellates pos- 58190 Morelia, Michoacán, Mexico sess red algal-derived plastids, but some minor 3 - Departamento de Biología Comparada, Facultad lineages established non-canonical plastids deri- de Ciencias, Universidad Nacional Autónoma de ved from phylogenetically diverse eukaryotic México, Circuito Ext. s/núm. Ciudad Universitaria, algae. Dinoflagellates Karlodinium veneficum and C. P. 04510, Ciudad de México, Mexico Lepidodinium chlorophorum, which bear non-ca- [email protected] nonical plastids derived from haptophyte- and Members of genus Lagenophrys are obligate sym- green algal endosymbionts, respectively, and biotic peritrichs of crustaceans, however the pat- their nuclear genomes contain genes encoding tern of distribution on corporal surface varies ac- plastidal proteins that are likely transferred from the cording to involved species. To demonstrate with genomes of the endosymbiont algae (endosymbiotic statistical tests the preference for an amphipod body gene transfer, EGT). Although EGT is generally region or appendage to attach, two Hyalella azteca considered to be an essential step in transforming populations were collected in two Mexican lakes, an endosymbiotic alga into a plastid, it has yet to be Xochimilco Lake and Cuitzeo Lake, and density fully understood to what extent metabolic functions and prevalence of Lagenophrys patina on host surface in the Karlodinium and Lepidodinium plastids rely were calculated. We used a contingency table and 46 · “PROTIST–2016” correspondance analysis to identify a microhabitat two species that also infect orthopterans belonging preference. Except for the head, pleopods and telson, to the same suborder (Ensifera) but different lagenophryid ciliates were attached to 2-9 amphipod families and infraorder (Gryllidea) and belonging body regions, and greatest prevalence was found on to the Old World. These species of Leidyana also coxae, followed by pereiopods. For Cuitzeo Lake possess epimerites resembling the ones found of individuals the total L. patina loricae abundance Leidyana sp. The three species measurements was from 617-1165, being the coxae with the highest comparison was made using a one-way ANOVA values (626), and for Xochimilco Lake L. patina and a correspondence analysis (n=20 each sample). showed a highly significative preference for coxae We found significative differences in the epimerite for their attachment (X2=1752.01, P<0.0001); we length and width, considered as an important also obtained the same highly significant result for character for the eugregarines taxonomy to genus Cuitzeo Lake symbiotic system (with X2=1317.8, P and species level identification. Additionally we < 0.0001). These results could be explained by the show the first record of a species of genus Leidyana shape and function of appendages of amphipods, parasitizing the gut lumen of an orthopteran of the providing the water flow derived by host movements infraorder Tettigoniidea, family Tettigonidae, as and swimming, carrying food and oxygen for sym- well for Mexico. Key words: eugregarines, Mexico, biotic ciliate. Leidyana. Acknowledgements. To Programa de Apoyo a Acknowledgments: To Biol. Margarita Reyes Santos Proyectos de Investigación e Innovación Tecnoló- for her advice in the stain techniques. gica (PAPIIT-DGAPA-UNAM), for the funds to project IN229811 for Cuitzeo Lake samples SOME NOTES ON THE MORPHOLOGY, GEO- collection. To all participants of PAPIIT IN229811 GRAPHIC DISTRIBUTION AND 18S rRNA project for their support in samples collection, GENE SEQUENCE OF A FRESHWATER OXY- especially to Biól. M. Reyes and M. C. Maricela TRICHID CILIATE (HYPOTRICHIA: OXY- Vicencio, Facultad de Ciencias, UNAM, for TRICHIDAE) FROM MEXICO technical procedures. Méndez-Sánchez Daniel1, Mayén-Estrada Rosa- ura1, Hu Xiaozhong2, Luo Xiaotian2, Song Weibo2 COMPARATIVE MORPHOMETRY OF LEIDY- 1 - Laboratorio de Protozoología, Facultad de Ci- ANA SP. (EUGREGARINORIDA) AND ITS encias, Universidad Nacional Autónoma de México, FIRST RECORD IN CONOCEPHALUS IC- Av. Universidad 3000 Ciudad Universitaria, 04510 TUS (ORTHOPTERA: ENSIFERA: TETTIGO- Coyoacán, Ciudad de México, México NIIDAE) FROM MEXICO 2 - Laboratory of Protozoology, Institute of Evolution Medina-Durán J.H.1, Mayén-Estrada R.1, Mariño- and Marine Biodiversity, Ocean University of China, Pérez R.2, Song H.2 Qingdao 266003, China 1 - Laboratorio de Protozoología, Departamento [email protected] de Biología Comparada, Facultad de Ciencias, Species of genus Oxytricha are characterized by 18 Universidad Nacional Autónoma de México, Circuito frontal-ventral-transverse cirri, one right and one Exterior S/N, Ciudad Universitaria, Av. Universidad left row of marginal cirri, undulating membranes 3000, C.P. 04510, Ciudad de México, México and dorsal kineties usually in Oxytricha-pattern, 2 - Department of Entomology, Texas A&M University, and also the presence of caudal cirri. We collected College Station, Texas, USA some samples from a Mexican freshwater lake [email protected] and we isolated Oxytricha granulifera (Foissner & Leidyana is a genus of apicomplexan eugregarine Adam, 1981). Our individuals presented six dorsal inhabiting the digestive tract of insects, mainly kineties compared with the Austrian population lepidopterans and orthopterans. Its classification (with five dorsal kineties) collected in a terrestrial is mainly based on the shape of their epimerite, environment. All available records of O. granulifera protomerite, deuteromerite and oocyst, and also were plotted and obtained a broad geographic dis- on their gametocyst dehiscence. Nevertheless, tribution. Considering the 18S rRNA gene sequence, morphometrical data of the species belonging to our species grouped with other O. granulifera strains this genus have been also considered in order to and populations available in GenBank. Due to the establish reliable descriptions but is not a main number of dorsal kineties we propose that Mexican criterion for its identification. In the present population could be considered like a subspecies of work we compared the morphometric attributes O. granulifera. of Leidyana sp. a parasite of Conocephalus ictus Acknowledgements: To Posgrado en Ciencias collected in Tlanchinol, Hidalgo, Mexico, with Biológica UNAM and CONACyT for the grant to Protistology · 47

D. Méndez-Sánchez. To IRCN-BC for the grant to [email protected] D. Méndez-Sánchez for a short stay at Laboratory Cryptosporidium spp. display a number of unusual of Protozoology OUC. Special thanks to Miss traits, distinct from other apicomplexans; notably Chundi Wang and Mr. Zhishuai Qu (Laboratory of a smaller genome and the absence of an essential Protozoology, OUC) for their technical assistance organelle, the apicoplast. There are also intra-species in the molecular procedures and staining protocols differences, such as a reduced mitochondrion in respectively. We thankful to people from Tziscao, C. parvum and C. hominis, in size and functions. Chiapas, who allowed us recollected samples. Studying these organisms holds great potential for our understanding of how the apicomplexans DIVERSITY OF THECAMOEBID AMOEBAE diverged. Studies of Cryptosporidium are inhibited (AMOEBOZOA: DISCOSEA: THECAMOE- by a limited selection of in-vitro culturing systems, BIDAE) with short lifespans and production volumes. Using Mezentsev Y.S. a novel cell line for propagation we have seen a Saint-Peterburg State University significant increase in parasite production volume [email protected] as well as a longer lifespan, which have enabled Amoebae of the family Thecamoebidae are widely the development of many new techniques for distributed in the different environments: salt and studying Cryptosporidium and its cell biology and fresh water and soil. These protists are relatively biochemistry. Amongst these new techniques is easy to isolate and cultivate. They are relatively the development of NMR and Mass spectrometry easy determining to genus, or even to species, thus protocols, which have been optimised to examine representing a convenient object for many kinds of the metabolic flux of the parasite, as well as studies. The frequency of occurrence of species is investigating the role of the mitosome. We have very different: there are rather common ones like also acquired many electron microscopy (EM) Thecamoeba quadrilineata, T. similis and T. orbis as images of the parasite during its life cycle, identifying well as numerous species known from few findings details of structures at levels previously unseen in or never re- isolated since initial description. Many the literature. Furthermore, using Immuno-EM of these species were studied only at the light- we have shown the first experimental evidence of microscopic level and require investigation with the function of the mitosome in C. parvum, with modern methods, including electron microscopy the localisation of iron sulphur cluster biosynthesis and molecular studies (this especially concerns the proteins. genus Thecamoeba and genera of unclear systematic position like Pseudothecamoeba and Thecochaos). DIVERSITY AND TEMPORAL DYNAMICS Our studies show that “hotspot” of Thecamoeba OF CRYPTOMYCOTA AND APHELIDA, TWO diversity is terrestrial habitats – soil, grass, dry leaves OVERLOOKED GROUPS OF PARASITES IN and surface of trees. During our studies we isolated FRESHWATER ECOSYSTEMS 20 strains of Thecamoeba; some were identified as Moreira D.1, Jardillier L.1, Bertolino P.1, Karpov known species (Thecamoeba aesculea, T. similis, T. S.A.2, Lopez-Garcia P.1 quadrilineata and T. terricola) and 6 strains, which 1 - Ecologie Systématique Evolution, CNRS - Uni- represent new species of the genus Thecamoeba. We versity Paris-Sud, Orsay, France have found two new strains of amoebae belonging 2 - Zoological Institute, Russian Academy of Sciences, to the genus Sappinia. In contrast, amoebae of the St. Petersburg, Russian Federation genus Stenamoeba were never found in terrestrial [email protected] samples. Our data shows that species diversity Basal-branching fungi, especially chytrids, are of thecamoebid amoebae remains considerably important heterotrophic members of aquatic underexplored. microbial food chains, especially in freshwater. Supported with Russian Science Foundation grant More recently, two additional groups very distantly 14-14-00474. related to fungi, Cryptomycota (or Rozellomycota) and Aphelida, have also been found to be ubiquitous ESTABLISHING CRYPTOSPORIDIUM AS A members of freshwater microbial communities. MODEL FOR STUDYING THE BIOLOGY Together with the Microsporidia, they form the AND EVOLUTION OF APICOMPLEXANS recently described superphylum Opisthosporidia. AND UNIQUE ORGANELLES All known opisthosporidian species are parasites Miller C., Brown I., Howard M., Michaelis M., of very diverse eukaryotic hosts. Remarkably, both Tsaousis A. cryptomycetes and aphelids are able to feed by University of Kent, UK phagotrophy, a chief difference with microsporidia 48 · “PROTIST–2016” and true fungi, all of them osmotrophic. Despite pLATE 31. An expression study was performed in the ubiquity of cryptomycetes and aphelids in different E. coli BL21 (DE3) strains: Star, XJB, RIPL diverse environments, little is known about their and PlysS. These strains were transformed with the abundance and temporal dynamics. We have carried recombinant vector pLATE 31 in order to obtain out a 2-year monthly survey of eukaryotic plankton clones. Three random clones were therefore selected diversity in five contrasted freshwater ecosystems and induced with IPTG. The result of the induction (one brook, one small lake, and three shallow ponds) was observed on a SDS-PAGE electrophoresis. The using massive 18S rRNA gene amplicon sequencing recombinant protein was purified by high-affinity to compare cryptomycetes and aphelids with the chromatography with immobilized nickel ions and much better-known chytrids. OTU analysis reveal subsequently analyzed by ELISA, SDS-PAGE that cryptomycetes and aphelids are less diverse than electrophoresis and quantified by Nanodrop 1000. chytrids (556, 313, and 1274 OTUs, respectively) Preliminary results show that the best E. coli strains although in some moments cryptomycete+aphelid for expression are BL21 (DE3) RIPL and BL21 combined OTU number can exceed that of chytrids. (DE3) PlysS, based on SDS-PAGE analysis. The Cryptomycetes show stable low numbers of sequ- optimization of the ELISA assay is in progress. ences (<1% of total sequences) all along the year, Acknowledgments: Supported partially by FCT in contrast with aphelids, which exhibit a more ref:VIH/SAU/0019/2011. heterogeneous dynamics with recurrent abundance peaks in early autumn (>4% of sequences), when THE RAPUNZEL TINTINNID – REDESC- they become even more abundant than chytrids. RIPTION OF TINTINNOPSIS SUBACUTA These results suggest that cryptomycetes and JÖRGENSEN, 1899 (ALVEOLATA, CILIO- aphelids are important overlooked members of PHORA, SPIROTRICHA) freshwater ecosystems that most likely control Mühlthaler A., Kagerer M., Agatha S. other eukaryotic populations through their parasitic Dept. Ecology and Evolution, University of Salzburg, activity. Salzburg, Austria [email protected] APPLICATION OF RECOMBINANT ANTI- Tintinnids contribute distinctly to the microbial GENS FOR THE SERODIAGNOSIS OF TOXO- biomass in the marine plankton. Since the species PLASMOSIS have specific requirements concerning physico- Mota Cátia, Cardoso Fernando, Matos Olga chemical conditions and food items, reliable Medical Parasitology Unit, Group of Opportunistic identification is indispensable for assessing their Protozoa/HIV and Other Protozoa, Global Health and role in the food web. About one thousand extant Tropical Medicine, Instituto de Higiene e Medicina tintinnid species are known, whose descriptions Tropical, Universidade Nova de Lisboa, Portugal are exclusively based on the features of their [email protected] loricae (houses); merely in about 30 species, cell Toxoplasma gondii is an opportunistic apicomplexan characteristics have been studied. Since lorica shape protozoon that can cause devastating disease and size are affected by environmental conditions in immunosuppressed patients and congenital and might show a polymorphism in the cell cycle, the infection. The diagnosis of toxoplasmosis is usually tintinnid classification is artificial. Investigations of done by observing the parasite in biological samples the cell, especially of the ciliary pattern and nuclear or by the detection of specific IgM and IgG against apparatus (generative micronuclei and somatic T. gondii antigens in the patient’s serum. The macronucleus nodules) are, however, supposed improvement of toxoplasmosis diagnostic techni- to provide features for a natural classification; ques and the differentiation between the infection these characters are revealed by protargol (silver stages can be achieved using recombinant antigen. proteinate) staining. Tintinnopsis subacuta was This study aims to use micronemal protein MIC3 collected from surface waters of the Indiana River (elicits a strong specific host immune response) at the Atlantic coast of Florida (USA) and stained recombinant antigens in the serodiagnosis of with protargol. Cell and lorica morphology were toxoplasmosis. Toxoplasma RNA was isolated using investigated under a compound microscope at up the Tri-Reagent method and a PCR was performed to 1250× magnification. The lorica is 55–119 µm, using primers for MIC3 nucleotide sequence. The on average 79 µm long and consists of a cylindroidal bands corresponding in size to the recombinant collar about 34 µm across and a subspherical bowl plasmid were purified and cloned into the cloning about 45 µm wide. The lorica wall has agglutinated vector pLATE 28 and in the expression vector mainly mineral particles. The contracted cell Protistology · 49 measures 30×28 µm and is attached to the bottom THE SPECIALIZATION OF THE PROTO- of the lorica by a contractile peduncle. The somatic MITOCHONDRION AS A RESPIRATORY ciliary pattern is of the most complex type, i.e., it ORGANELLE comprises a ventral, dorsal, and posterior kinety Muñoz-Gómez S.A., Roger A.J., Slamovits C.H. as well as a right, left, and lateral ciliary field. The Centre for Comparative Genomics and Evolutionary ventral kinety has associated an extraordinary ciliary Bioinformatics, Department of Biochemistry and tuft of cell length that extends outside the lorica Molecular Biology, Dalhousie University, Halifax, posteriorly, resembling the golden hair let down NS B3H 4R2, Canada. from the tower by Rapunzel; T. subacuta is unique [email protected] in this respect. The right and left ciliary fields are The ancestor of mitochondria was an alpha- composed of about 11 ciliary rows each, the lateral proteobacterium whose exact phylogenetic and field consists of invariably 15 rows. While the phenotypic nature remains obscure. Therefore, majority of tintinnids have only two macronucleus the proximate selective force that drove the initial nodules, T. subacuta has 4–34, on average 14 endosymbiosis is unknown, even though the nodules. Financially supported by FWF Project ultimate selective advantage was undoubtedly P28790. greater efficiency in energy production through aerobic respiration. The specialization of the PROTISTAN VERSUS CYANOBACTERIAL proto-mitochondrion as the respiratory organelle PICOPHYTOPLANKTON PRODUCTION of eukaryotes required the host to exert increased AND GRAZING MORTALITY IN SEVASTO- control over the biogenesis of the newly evolving POL BAY AND ADJACENT WATERS (THE organelle. Among the several adaptations that BLACK SEA) transformed the ancestral endosymbiont into a Mukhanov V.S., Rylkova O.A., Sakhon E.G. respiratory organelle, two evolutionary innovations A.O. Kovalevsky Institute of Marine Biological were of major importance. The first major innova- Research, Russian Academy of Sciences, Nakhimov tion was the evolution of mitochondrial cristae av. 2, Sevastopol, 299011, Russia to make respiratory sub-compartments. Cristae [email protected] likely evolved from precursor structures in alpha- Seasonal dynamics of abundance, specific growth proteobacteria. Later molecular innovations fur- rate, daily production and grazing mortality of the ther modified cristae to improve their respiratory major picophytoplankton components, eukaryotic function. This required the expansion of MICOS protists and prokaryotic cyanobacteria, were studied (Mitochondrial contact site and Cristae Organizing at three stations in Sevastopol bay and adjacent System) and the evolution of the capability of the waters (the Black Sea) in 2014 by flow cytometry ATP synthase complex to form multimers. The and dilution method. In the shallow coastal waters, second major innovation was the evolution of the protistan picophytoplankton (PP) dominated (64 ability of the host to control the overall morphology, ± 23 (SD) %, n=26) the community in terms of positioning and distribution of mitochondria within abundance (annual average of 16.3 ± 12.4 × 103 the cell. These adaptations optimized bioenergetic cells ml-1), with the latter increasing along the output in response to host needs. This was made nutrient and pollution gradient from the coastal possible by the origin of mitochondrial fusion, as waters outside the bay (7.3 ± 5.4 × 103 cells ml-1) to well as the establishment of interactions between the eastern corner of the bay (28.7 ± 11.4 × 103 cells mitochondria and diverse endomembranes and ml-1). PP demonstrated significantly lower specific the cytoskeleton. I discuss a detailed evolutionary growth rates (0.20 ± 0.19 d-1) and significantly higher scenario for the evolution of these two major daily grazing mortality (4.0 ± 5.8 µg С l-1 d-1) than adaptations in the context of the co-evolutionary cyanobacterial picophytoplankton (0.70 ± 0.46 integration of mitochondria and their host. d-1 and 1.1 ± 1.1 µg С l-1 d-1, respectively) while the protistan and cyanobacterial daily productions THE ULTRASTRUCTURE OF AMOEBOID did not differ significantly (paired t-test, p>0.05, FLAGELLATES AMASTIGOMONAS (CERCO- n=26). Matter flows through both the community ZOA, RHISARIA) components were comparable to or even exceeded Mylnikov A.A., Mylnikov A.P. their biomass stocks that indicated high biomass Papanin Institute for Biology of Inland Waters, turnover rates. Thus, the protistan component has Russian Academy of Sciences, Borok, Russia been shown to play a major role in the community [email protected] functioning in the Black Sea coastal waters. The cytoskeleton of three amoeboid flagellates Amastigomonas spp. dwelling in freshwater (one 50 · “PROTIST–2016” strain) and marine waters (two strains) has been consists of the muff and cylinder. Osmiophilic considered. The morphology of these strains is bodies of various shapes contain crystalloid inclu- relatively similar. The anterior flagellum lies inside sions. The pseudopodia capturing the bacteria the hollow proboscis. The posterior flagellum goes are inserted ventrally. The groove is armored by along the ventral groove. Two heterodynamic the two longitudinal groups of closely situated flagella are smooth and have not been covered by any microtubules. Microbodies and symbiotic bacteria structures. The transitional zone of the flagella do have not been observed. Th. coloniensis differs from not contain additional elements and are of the usual other Thaumatomonas species by the presence of structure. The microtubule band and anterior rootlet osmiophlic bodies and absence of microbodies. This are inserted from the kinetosome of the anterior study was supported by the Russian Foundation for flagellum, the microtubule right and left rootlets Basic Research (grant nos. 14-04-00500, 14-04- and single rootlet are inserted from the kinetosome 00554, 15-29-02518). of the posterior flagellum. The kinerosomes are located at obtuse angle or antiparallel and connected PLANKTONIC CILIATES OF THE SHEKSNA by the three fibrils and cross-striated structure. The RESERVOIR rhizoplast has not been found. The thickened cell Mylnikova Z.M. coverings consist of plasmalemma and epiplasm. Papanin Institute for Biology of Inland Waters, The margins of the coverings form the folds, the Russian Academy of Sciences, Borok, Russia ventral groove goes between them and is bounded [email protected] only by the plasmalemma. The vesicular nucleus The species composition, abundance, biomass and Golgi apparatus are of the usual structure. and distribution of planktonic ciliates across the The mitochondria contain tubular cristae. The Sheksna Reservoir consisting of three parts have pseudopodia inserting from ventral groove serve to been studied. Fifteen species of ciliates, belonging capture bacteria. Front cytoplasmic outgrowth have to four classes: Spirotrichea - 6, Litostomatea - 4, been found for the first time. The resemblance and Prostomatea - 4, Oligohymenophorea - 1 were differences of given species with other apusomonads recorded during the observation period in the have been shown. This study was supported by the pelagic zone. The maximal number of species (11) Russian Foundation for Basic Research (grant nos. was registered in Beloye Lake, minimal one (6) - in 14-04-00500, 14-04-00554, 15-29-02518). Kovzhinsk part. The following species: Tintinnidium fluviatile, Codonella cratera, Limnostrombidium THE ULTRASTRUCTURE OF AMOEBOID viride, L. pelagica and Rimostrombidium velox FLAGELLATE THAUMATOMONAS COLO- were dominants in the most part of sampling NIENSIS WYLEZICH ET AL. 2007 (CERCO- points. Paradileptus conicus, Monodinium balbiani, ZOA, RHIZARIA) Enchelis pupa and Prorodon ovum were registered Mylnikov A.P., Prokina K.I. less frequently and in small quantities , and has Papanin Institute for Biology of Inland Waters, been recorded for the first time on this site. The Russian Academy of Sciences, Borok, Russia maximal average abundance (2502 × 103 ind./m3) [email protected] and biomass (141 mg/m3) were registered in Beloye The ultrathin structure of amoeboid flagellate Lake. The maximal density (2750-4150 × 103 ind./ Th. coloniensis has been considered. The cell is m3 and 156-352 mg/m3) observed in shallow waters surrounded by somatic scales which forming on the of the western coast, in sampling points Mandoma, surface of the mitochondria. The heterodynamic Kustovo, Kium-Mandoma and Belozersk. Lower flagella emerge from the small flagellar pocket. density (1150-1250 × 103 ind./m3 and 62-90 mg/ Both flagella are covered by the cone–shaped scales m3) observed in sampling points near the center of and thin twisted mastigonemes. The kinetosomes the lake, and Sudovoy Khod station. The average lie parallel to each other. The transitional zone of values of abundance and biomass of Sheksna the flagella contains the thin–walled cylinder. The Reservoir accounted 1875000 ind./m3 and 123 mg/ transversal plate of the flagella is located above m3, respectively. The trophic status of the Sheksna cell surface. The flagellar root system consists of Reservoir during the study period can be described 3 microtubular bands and fibrillar rhizoplast. The as mesosaprobic. vesicular nucleus and Golgi apparatus are of the usual structure. The mitochondria contain the PUF PROTEINS IN GIARDIA INTESTINALIS tubular cristae. The extrusive organelles (kineto- Najdrova V., Dolezal P. cysts) which contain the amorphous material and Department of Parasitology, Faculty of Science, capsule have been found in cytoplasm. The capsule Charles University in Prague , Prague, Czech Republic Protistology · 51 [email protected] tence of different cell types in C. owczarzaki aggre- Giardia intestinalis, an anaerobic protozoan parasite, gates. We also show our advances in developing contains highly compact genome with extremely single-cell transcriptomics methodology in these short untranslated regions (UTRs). The regulation organism to molecularly characterize cell types. The of gene expression during giardia cell- and life-cycle possibility of analyzing differential gene expression has been poorly studied and only a handful of RNA at the single-cell level between diverse cell types binding proteins have been characterized so far. of unicellular holozoans will allow us to better PUF proteins bind 3’ UTRs of cognate mRNAs, understand the molecular mechanisms underlying by which they regulate their stability, translation programs of cell differentiation in the origin of and localization. These eukaryotic proteins are animals. The aggregates of C. owczarzaki offer us an evolutionarily conserved from protists to metazoans. ideal model in which to test this, and provide a better We have identified five PUF genes in the genome of framework to understand the origin of the different G. intestinalis and have initiated studies towards the metazoan cell types. characterization of PUFs in giardia biology. PHYLOGENY AND ECOLOGICAL IMPOR- EXPLORING CELL TYPE DIFFERENTIA- TANCE OF PHAEODARIANS (CERCOZOA, TION IN THE FILASTEREAN CAPSASPORA RHIZARIA) OWCZARZAKI BY SINGLE-CELL RNA-SEQ Nakamura Y.1, Somiya R.2, Suzuki N.3, Hori S. Najle S.R.1, Florenza J.1, Mazutis L.2, Ruiz-Trillo R.4, Tuji A.1 I.1,3,4 1 - Department of Botany, National Museum of Nature 1 - Institut de Biologìa Evolutiva (Univesitat Pompeu and Science Fabra – CSIC). Pg. Maritim de la Barceloneta 37-49, 2 - Graduate School of Fisheries Science and Envi- 08003, Barcelona, Catalonia, Spain ronmental Studies, Nagasaki University 2 - Institute of Biotechnology, Vilnius University. V.A. 3 - Graduate School of Science, Tohoku University Graiciuno 8, LT-02241, Vilnius, Lithuania 4 - Graduate School of Science and Engineering, Ehime 3 - Departament de Genètica, Universitat de Barcelo- University na, Av. Diagonal, 645, Barcelona 08028, Catalonia, [email protected] Spain Phaeodarians are a group of marine protists belon- 4 - Institució Catalana de Recerca i Estudis Avançats ging to the phylum Cercozoa, composing Rhizaria (ICREA), Passeig Lluís Companys, 23, Barcelona (SAR). These unicellular siliceous zooplankton 08010, Catalonia, Spain. occasionally become abundant in the ocean, [email protected] however their ecological importance and phylogeny The origin of multicellular animals from their are still wrapped in mystery. Plankton were sampled unicellular ancestors is one of the most important from several depths at ca. 40 stations in the Northern evolutionary transitions in life’s history. However, hemisphere during 2011–2015. Zooplankton were the specific cellular and genetic changes that led sorted and identified in order to clarify the species to this transition remain unknown. Phylogenomic composition of each sample. Some phaeodarians analyses have shown that animals are closely related were cultured to observe their behavior. The 18S to three unicellular lineages: choanoflagellates, rDNA sequences of phaeodarians were determined filastereans and ichthyosporeans, altogether forming by single-cell PCR method. Two undescribed the Holozoa clade. Recent phylogenomic studies phaeodarians were found in the deep waters in the have shown that those premetazoan taxa already Sea of Japan, and one of the species was abundant had a complex repertoire of genes important for through the year, occupying ca. 22% of the total multicellularity, some of them previously thought zooplankton biomass on average. The abundance to be exclusive of animals. Different versions of of phaeodarians was also seen in the East China “simple multicellularity” are found among the Sea, where two species occupied 10.2–13.9% of unicellular relatives of Metazoa. There is the clonal the zooplankton biomass, suggesting that this group development of colonial choanoflagellates, the is an important component of the zooplankton aggregative behavior of Capsaspora owczarzaki, and community and the material cycle in the ocean. The the coenocytic development of ichthyosporeans. cell division of phaeodarians was observed during the Those colonies and aggregates are assumed to be culture experiment. The species morphologically without cell differentiation. However, there is no identified as phaeodarians formed a single clade molecular data proving that all cells within those together with other cercozoans in the phylogenetic colonies or aggregates or coenocytes are identical. tree, suggesting that almost all phaeodarians belong Here we show microscopic evidence for the coexis- to Cercozoa and that Phaeodaria is a monophyletic 52 · “PROTIST–2016” group. The branching pattern within the phaeoda- Garcia P.3 rian clade did not correspond to the families and the 1 - Institute of Cytology RAS, St. Petersburg, Russia orders of the current classification system, and the 2 - Department of Invertebrate Zoology, Faculty system needs to be reconsidered. of Biology, St. Petersburg State University, St. Petersburg, Russia CYANOBACTERIAL GENES IN THE NUC- 3 - Unité d’Ecologie, Systématique et Evolution, LEAR GENOME OF A DIATOM BEARING CNRS, Université Paris-Sud, 91400 Orsay, France

N2-FIXING CYANOBACTERIAL ENDOSYM- [email protected] BIONTS: POTENTIAL FACTORS INVOLVED Metchnikovellids constitute a group of hyperpara- IN THE HOST-ENDOSYMBIONT PARTNER- sites that infect gregarines living in the gut of SHIP polychaetes and other marine invertebrates. Despite Nakayama T., Inagaki Y. they were described in the late 19th century, Center for Computational Sciences, University of they are poorly known and their phylogenetic Tsukuba affinities have remained elusive for a long time. [email protected] Morphological studies suggested an evolutionary The evolution of mitochondria and plastids from relationship with Microsporidia, a group of highly bacterial endosymbionts were key events in the derived intracellular parasites known for its extreme evolution of eukaryotes. While the ancient nature metabolic and genomic simplification, including e.g. of these organelles preclude understanding the loss of the mitochondrion. Microsporidia together transition from a bacterium to an organelle (organel- with Rozellida (Cryptomycota) and Aphelida logenesis), the study of eukaryotic cells with recently form a monophyletic holomycotan clade, the evolved obligate endosymbiotic bacteria has the superphylum Opisthosporidia. The first molecular potential to provide important insights into the early phylogenetic analyses based on SSU rRNA and events in the organellogenesis. Diatoms belonging beta-tubulin genes of Metchnikovella incurvata, a to the family Rhopalodiaceae and their N2-fixing parasite of the gregarine Polyrhabdina sp. from the cyanobacterial endosymbionts (spheroid bodies) are gut of the polychaete Pygospio elegans, supported a emerging as a useful model system in this regard. The close evolutionary relationship with microsporidia. experimental data accumulated to date suggest that However, unraveling the phylogenetic position the endosymbiont has been already integrated into of these organisms is difficult due to their high the host cell during the endosymbiotic relationship. evolutionary rate. To improve the phylogenetic Our previous study on the genome sequence of the signal and ascertain the phylogenetic position of endosymbiont in a rhopalodiacean diatom provided metchnikovellids, we applied a single-cell genomics insight into its reductive evolution and the metabolic approach to individual gregarine cells infected with dependency on the diatom host. However, it M. incurvata. We generated genome data by multiple has yet to be elucidated how the host control the displacement amplification followed by direct HiSeq endosymbionts. In this study, to tackle this question, 2500 Illumina sequencing. After assembly, we mined we obtained both genome and transcriptomic data the genome dataset in search of conserved genes. of a rhopalodiacean diatom, Epithemia adnata, Preliminary phylogenomic analyses of 31 conserved as well as the genome data of its cyanobacterial genes confirm the phylogenetic placement of endosymbiont. Phylogenetic analyses showed that metchnikovellids at the base of Microsporidia and the nuclear genome encodes protein-coding genes after the divergence of Mitosporidium daphniae, a of cyanobacterial origin, which are not seen in other microsporidia-like mitochondrion-bearing parasite. diatom genomes. Some of these ‘cyanobacterial Further exploration of metchnikovellid genomes genes’ likely encode enzymes involved in the would allow determining the genes and traits metabolism of peptidoglycan wall, which is a feature involved in the evolution of extreme parasitism. exclusively associated with the endosymbiont in the Supported by RFBR 15-04-08870 and ERC 322669. E. adnata cell. We will overview the cyanobacterial genes found in the diatom genome, and discuss their EPIGENETIC INCOMPATIBILITY OF PARA- possible contributions to the host-endosymbiont MECIUM TETRAURELIA STRAINS partnership. Nekrasova I.1, Potekhin A.1, Kvitko J.1, Pellerin G.2, Meyer E.2 PHYLOGENOMIC INSIGHTS ON THE EVO- 1 - Faculty of Biology, St Petersburg State University, LUTION OF METCHNIKOVELLIDS Saint Petersburg, Russia Nassonova E.1,2, Moreira D.3, Torruella G.3, 2 - Institut de Biologie, Ecole Normale Superieure, Timpano H.3, Paskerova G.2, Smirnov A.2, Lopez- Paris, France Protistology · 53 [email protected] actomyosin networks have been reported, actual Two epigenetic phenomena occur in crosses of contributions to the membrane deformation are Paramecium tetraurelia strains 32 and 51. Strain 32 still unclear because of the cellular complexities. is deficient for an IES present in one of the mating Here, in order to simplify the complex system, type genes, mtB, of strain 51. Internal eliminated we attempted to reconstitute a simple model sequences are excised from the developing macro- system, in which lipid monolayer was deformed nuclear genome by a fascinating mechanism of by actomyosin. In living cells, the connection genomic subtraction mediated by scanRNAs. between actomyosin and lipid layer is achieved by However, if an IES is present in genome of one various types of proteins. To simply accomplish the partner but absent in genome of another, then F1 actin-membrane connection in vitro, we adapted hybrids deriving from the latter parent are unable to positively-charged lipid DOTAP (1,2-dioleoyl- excise such IES from developing somatic genome: 3-trimethylammonium-propane), expecting the they can’t produce a certain scanRNA. Moreover, electrostatic adhesion between negatively-charged F2 progeny of such cell will inherit this IES retained actin and DOTAP. We extracted actomyosin from in macronucleus. IES inside a gene disrupts its A. proteus and enclosed actomyosin fraction within a function, thus reminding hybrid dysgenesis known spherical space surrounded by a DOTAP monolayer. for Drosophila. Indeed, in 25% of crosses we As a result, active deformation of the lipid monolayer observed loss of mtB function in F2 progeny derived was yielded. From analyses of the static and dynamic from parent 32. We also found unexpectedly that properties of the deformation, we found that the in 20% of crosses IES in mtB gene was retained in depth and width of the deformation were dependent macronucleus of F2 progeny derived from parent 51, on the curvature radius of the sphere. The observed which normally produces scanRNAs and excises this curvature dependence is explained by the theoretical IES. Analogous phenomenon was reported in cross description including elasticity and contractility of of d12 and d48 deletion mutants of P. tetraurelia the cortex. Our results provide a fundamental insight restoring functional gene of surface antigen A. We into the cellular membrane deformation induced by suggest that its mechanism may be connected with the actomyosin cortex during amoeboid locomotion. hemizygocity state of the deleted locus in F1 hybrids For more details, see Nishigami et al. (Sci. Rep. 6, of such crosses, leading somehow to deviation of 19864, 2016) and Ito, Nishigami et al. (Phys. Rev. such sequence excision despite scanRNAs for it are E 92, 062711, 2015). present. These epigenetic effects may contribute into speciation in ciliates, as occasional hemizygocity NUCLEAR DIVISION PROCESS IN TESTATE may lead to lethality of interstrain hybrids. AMOEBA PAULINELLA CHROMATOPHORA Supported by RFBR 16-04-01710. Nomura M., Ishida K. Faculty of Life and Environmental Sciences, University RECONSTRUCTION OF CELLULAR SHAPE of Tsukuba DEFORMATION THROUGH CONTRAC- [email protected] TION OF CORTEX ACTOMYOSIN Paulinella chromatophora is a euglyphid testate Nishigami Yukinori1, Ito Hiroaki2, Sonobe Seiji3, amoeba (Rhizaria, Cercozoa) living in a shell Ichikawa Masatoshi1 composed of ~50 rectangular siliceous scales. 1 - Department of Physics, Graduate School of Science, In this species, the complex shell construction Kyoto University, Kyoto 606-8502, Japan process appears to be integrated under the cell cycle 2 - Department of Mechanical Engineering, Graduate regulation, since the cell division does not proceed School of Engineering, Osaka University, Osaka 565- without the completion of shell construction. Before 0871, Japan cell division, scales produced inside of mother cell 3 - Department of Life Science, Graduate School of are secreted out from the cell and assembled into Life Science, University of Hyogo, Harima Science a new shell by a specialized thick pseudopodium. Park City, Hyogo 678-1297, Japan Following the completion of shell construction, one [email protected] of daughter cells moves into the new shell. Despite Giant free-living amoebae, Amoeba proteus, actively that knowledge, it is still unknown how the cell deform cellular shape during the locomotion. The division process proceeds in response to the shell deformation is induced by contraction of cortical construction. In this study, we focused on how the actin and myosin (actomyosin). In the process, since nucleus divides along with shell construction process actomyosin is connected to the cellar membrane in P. chromatophora. In an intermediate stage of and transmit the generated force to deform the shell construction, the nucleus in the maternal cell membrane. Although the contractile properties of was in prophase. In this phase, the nucleolus, which 54 · “PROTIST–2016” is prominent in interphase, was disappeared and proteins from a list of the mitosome proteome. We chromosomes were scattered in the nucleus. The experimentally confirmed their localization and newly formed shell was almost or fully constructed integration to mitosome membranes by Percoll- when the nuclear division reaches metaphase. In gradient fractionation, carbonate fractionation, this phase, the spindle body was formed and the immunofluorescence assay, and immunoelectron chromosomes were arranged at the equatorial microscopy. These new class of mitosomal membrane plane randomly. At the time of completion of shell proteins including MBOMP30 likely play unique and construction, the nucleus was observed to be in indispensable roles in Entamoeba mitosomes. We also anaphase, and chromosomes were separated into found that two dynamin-related proteins, DrpA and anterior and posterior side of the nucleus. After DrpB, are involved in mitosome fission. Expression of the migration of a daughter cell into new shell, the a mutant form or gene silencing of these Drps caused nucleus with densely condensed chromosomes was abnormal morphology of mitoses and growth defect, observed to locate at posterior end of each daughter suggesting that mitosome fission is mediated in part cell. by these Drps.

MITOSOMES IN ENTAMOEBA HISTOLYTICA: MORPHOLOGY, PHYLOGENY, AND TRANS- DIFFERENTIATION, METABOLITE TRANS- CRIPTOME DATA OF A NEW ANAEROBIC PORT, AND FISSION METOPUS SPECIES (CILIOPHORA, ARMO- Nozaki T.1,2 PHORIDA) FROM YANTAI, CHINA 1 - National Institute of Infectious Diseases Omar A., Zhang Q., Gong J. 2 - University of Tsukuba Laboratory of Microbial Ecology and Matter Cycles, [email protected] Yantai Institute of Coastal Zone Research, Chinese Hydrogenosomes and mitosomes are mitochon- Academy of Science, Yantai, China drion-related organelles (MROs) in anaerobic/ [email protected] microaerophilic eukaryotes with highly reduced A new anaerobic Metopus species was discovered in and divergent functions. Entamoeba possesses a a soil sample from fruit garden in Yantai, China, and highly divergent MRO known as the mitosome. The investigated using morphological, morphometrical, biological functions and their origin of Entamoeba and molecular methods. The morphology was mitosomes have been a longstanding enigma in studied using in vivo observation and protargol the evolution of mitochondria. We previously impregnation. The main features of the new Metopus demonstrated that sulfate activation, which is not species include: (i) size in vivo 75-105 × 35-55 µm; generally compartmentalized to mitochondria, is (ii) body shape ellipsoidal to pyriform; (iii) nuclear a major function of E. histolytica mitosomes. We apparatus invariably in preoral dome, macronucleus recently purified and identified cholesteryl sulfate reniform, micronucleus globular to ellipsoidal (CS) as a final sulfate activation metabolite. We attached to macronucleus; (iv) cytoplasm studied further identified the gene encoding the cholesteryl with lipid droplets especially in preoral dome; (v) five sulfotransferase responsible for synthesis of CS. perizonal and 18-21 somatic ciliary rows of which Supplementation of CS to the culture increased three extend onto preoral dome (dome kineties); (vi) the number of cysts, while, conversely, chlorate, a three to five distinctly elongated caudal cilia; and selective inhibitor of the first enzyme in the sulfate (vii) adoral zone composed of 21-29 membranelles activation pathway, inhibited cyst formation. These and distinctly shorter than perizonal ciliary stripe results indicate that CS plays an important role in (45% vs. 61% of body length on average). Moreover, differentiation, an essential process for transmis- this species contains numerous, conspicuous sion of Entamoeba between hosts. Furthermore, hydrogenosomes, anaerobically-functioning mito- Mastigamoeba balamuthi, an anaerobic, free-living chondrial-related organelles, as an adaptation for amoebozoan species, also has the sulfate activation the anaerobic lifestyle. SSU rRNA and mRNA pathway in MROs, but does not possess the capacity were obtained using a single cell transcriptome for CS production. Hence, we proposed that a unique protocol, and were sequenced with both Sanger function of MROs in Entamoeba contributes to and MiSeq Illumina technology. The obtained data adaptation of its parasitic life cycle. Understanding were used for phylogenomic analyses and analyzing of metabolite trafficking across the two mitosomal basic metabolic processes of this anerobic ciliate, membranes is important to understand metabolic including searching for genes of putative anaerobic- functions of mitosomes. We recently discovered a novel adapting functions. mitosomal β-barrel outer membrane protein of 30 kDa Supported by the NSFC project 31550110213 and (MBOMP30) and several novel membrane-spanning the CAS project 2015PB040. Protistology · 55

SINGLE CELL RNA SEQUENCING, AN and never enlarges chloroplast. By contrast, N. EFFECTIVE APPROACH FOR ANALYZING aeruginosum enlarges single chloroplast throughout THE GENOME CONTENT AND EVOLUTION the cell and divides nucleomorph, retaining a OF NON-CULTIVATABLE MICROBIAL cryptomonad nucleus. These differences are able to EUKARYOTES be interpreted as different evolutionary stages toward Onsbring H.1, Divne A.M.2, Ettema T.1 acquisition of ‘true chloroplast’ within the same 1 - Department of Cell and Molecular Biology, Uppsala linage. It is, therefore, clear that these dinoflagellates University, Sweden are interesting materials to investigate evolutionary 2 - Microbial Single Cell Genomics, SciLifeLab transitions toward establishment of endosymbiosis. [email protected] To reveal fate of the cryptomonad organelles Technical advances in culture-independent tech- in Nusuttodinium aeruginosum after host cell niques have significantly contributed to the disco- divisions, we have further observed all daughter very of novel microbial lineages. Both metageno- cells with LM and single-cell TEM methods. mics and single cell genomics have been shown to be These observations showed that cryptomonad useful tools for studying the genomes of prokaryotes. karyokinesis did not occur and that only one of the However, microbial eukaryotes generally have a daughter cells inherited a cryptomonad nucleus. complex genome structure, which leads to that Among all daughter cells originating from a single these techniques tend to perform poorly. If it would cell through five generations, the cell that inherited be possible to apply single cell RNA sequencing, the cryptomonad nucleus consistently possessed issues related to data assembly can potentially be the largest kleptochloroplast. Therefore, this study avoided. With a method adapted from Smart-seq2, suggests that the cryptomonad nucleus carries where template switching is used to amplify cDNA, important information for the enlargement of transcriptomes have been generated for several the kleptochloroplast. These results suggesting single protist cells with close to full coverage of the cryptomonad nucleus remains transcriptionally- coding potential. In Smart-seq2, transcriptomes can active in the host cell and we are examining be generated in a 384 well format, which gives the changes in transcriptome of dinoflagellate nucleus, potential for protist transcriptomes to be generated cryptomonad nucleus and nucleomorph during with high throughput. In transcriptomics data, the the course of transition in the kleptochloroplast highly expressed house keeping genes are among development. In this presentation, methods and the most likely genes to have high coverage and full progresses of transcriptome analyses are discussed length. Those genes are also suitable for constructing in addition to the results of morphological observa- phylogenies that aim to resolve deep branches in the tion. eukaryotic tree of life. Therefore, RNA sequencing of many cells in parallel has the potential to effectively RETENTION OF BACTERIVORY IN THE DO- generate sequence data for novel or poorly studied MINANTLY PHOTOAUTOTROPHIC GREEN protist lineages, and to increase our understanding ALGA CYMBOMONAS TETRAMITIFORMIS of their biology and evolution. IS INFLUENCED BY PHOSPHATE LIMI- TATION ULTRASTRUCTURAL AND TRANSCRIP- Paasch A.E., Burns J.A., Kim E. TOMIC STUDIES OF KLEPTOCHLORO- American Museum of Natural History PLASTIDIC DINOFLAGELLATE NUSUTTO- [email protected] DINIUM AERUGINOSUM (DINOPHYCEAE) The lineage Chloroplastida (green algae and land Onuma R.1, Horiguchi T.2, Miyagishima S.1 plants) is defined by the presence of a primary 1 - Division of Symbiosis and Cell Evolution, De- plastid, which was acquired through the ingestion partment of Cell Genetics, National Institute of of a photosynthetic bacterium presumably during Genetics the early- or mid-Proterozoic eon. However, 2 - Department of Biological Sciences, Faculty of members of Chloroplastida are dominantly photo- Science, Hokkaido University autotrophic and only a few members of the early- [email protected] -diverging class, Prasinophyceae, retain the ability Unarmored dinoflagellates Nusuttodinium spp. to ingest bacteria. It is unclear why bacterivory is possess kleptochloroplasts which are ingested from restricted to the prasinophytes. The prasinophyte cryptomonads and retained in the host cell for Cymbomonas tetramitiformis was definitively con- certain periods. Our previous studies revealed that firmed through transmission electron microscopy to N. poecilochroum digests cryptomonad nucleus ingest bacteria into a large food vacuole. Recently, 56 · “PROTIST–2016” this phagomixotroph’s genome was found to retain TWO NEW NON-CANONICAL NUCLEAR a unique combination of genes not present in GENETIC CODES FROM A RHIZARIAN AND obligate photoautotrophs or heterotrophs. Addi- A FORNICATE WITH UAG, BUT NOT UAA, tional prasinophytes have been found to ingest AS A SENSE CODON fluorescently-tagged bacteria and synthetic particles. Pánek T.1, Sokol M.1, Žihala D.1, Derelle R.2, To investigate drivers of bacterivory in Cymbomonas, Zadrobílková E.3, Čepička I.3, Eliáš M.1 cultures of the alga were grown under limited N, 1 - Department of Biology and Ecology, Faculty of P and light regimes and fed bacteria as a rescue Science, University of Ostrava, Chittussiho 10, 710 source of nutrients. The Cymbomonas genome was 00 Ostrava, Czech Republic also mined for metabolic genes related to nutrient 2 - Unité d’Ecologie, Systématique et Evolution, Centre uptake and assimilation. Surprisingly, bacteria only National de la Recherche Scientifique UMR 8079, rescued Cymbomonas growth under phosphate- Université Paris-Sud, 91405, Orsay, France limited conditions, but not when nitrogen or 3 - Department of Zoology, Faculty of Science, Charles light-limited. The genome contains genes related University in Prague, Vinicna 7, 128 44 Prague, Czech to phosphate metabolism that are not present in Republic other Chloroplastida. A full GS-GOGAT pathway [email protected] is present and no unique nitrogen-related genes The original presumption that all organisms use were found. These results suggest that Cymbomonas the same (standard) genetic code for translation of retains the ability to extract phosphorous from mRNA sequences into proteins has been challenged prey, but relies on photoautotrophic pathways for by discoveries of deviations of this universal language nitrogen and carbon. This trait gives Cymbomonas in both prokaryotes and eukaryotes. In eukaryotes a competitive advantage in P-limited cultures and the nuclear genetic code has proven to be much more may drive retention of bacterivory in this species. conservative than that of mitochondria, and plastids; just a few its variants are known. Generally, we can SUPPLIMENTING SYMBIONTS: PATHWAY sort them into 3 groups: (1) UGA serves as a sense RESTORATION IN A LONG TIME PARASITE codon; (2) UAA and UAG simultaneously serve as Paight C.J.1, Muñoz-Gómez S.A.2, Saffo M.B.1, sense codons; (3) CUG encodes serine or alanine Slamovits C.2, Lane C.E.1 (rather than leucine). We analyzed transcriptomic 1 - Department of Biological Sciences, University of data from two unrelated protists and found out that Rhode Island, Kingston these organisms, as only eukaryotes known so far, 2 - Department of Biochemistry and Molecular Biology, use UAG as a sense codon in nuclear genetic code Dalhousie University while retaining UAA as a termination codon. One [email protected] of these organisms uses UAG as codon for leucine, Apicomplexans are highly successful parasites, similarly to a code variant described from certain infecting every major metazoan lineage. The mitochondria. The other one instead uses UAG genus Nephromyces has recently been described as to encode glutamine, resembling thus the non- having a mutualistic relationship to its host Molgula canonical genetic code of several eukaryotic groups tunicates (Saffo et al., 2010), making Nephromyces including many ciliates, hexamitin diplomonads, the only reported mutualistic apicomplexan genus. some oxymonads, and some ulvophytes; however, Apicomplexans have reduced genomes and have all these taxa have at the same time reassigned also lost the ability to make many essential metabolites. the UAA codon. Phylogenetic analyses place the first These essential metabolites are instead scavenged organism into the rhizarian lineage Sainouroidea, from their host. Species of Nephromyces are known whereas the second one represents an undescribed to have three different bacterial endosymbionts. Our lineage of “Carpediemonas-like organisms” in data show that the bacterial endosymbionts encode a Fornicata (Metamonada). Our findings thus once number of essential pathways lost in Apicomplexans. again show protists as an inexhaustible resource of Here we describe insights from the transcriptome peculiar departures from the “standard” biology. from Nephromyces, all three bacterial endosymbi- onts and the tunicate host. These data gives us a AGAMOCOCCIDIANS: COCCIDIANS OR glimpse of the complex metabolic relationships and GREGARINES? NEW SPECIES AND NEW intertwined pathways of hosts and endosymbionts, DATA ON THE PHYLOGENETIC POSITION with a particular focus on the biosynthesis of amino OF THE GROUP acids and vitamins. Panfilkina Tatiana S.1, Simdyanov Timur G.2, Aleoshin Vladimir V.3, Paskerova Gita G.1 Protistology · 57

1 - Department of Invertebrate Zoology, Faculty of CAPSASPORA OWCZARZAKI AS A UNICEL- Biology, Saint-Petersburg State University, Uni- LULAR MODEL TO STUDY CO-OPTION OF versitetskaya emb. 7/9, St. Petersburg, 199034, THE ANCESTRAL INTEGRIN ADHESOME Russian Federation Parra-Acero H.1, Harcet M.1,2, Pérez-Posada A.1, 2 - Department of Invertebrate Zoology, Faculty Brown N.H.3, Ruiz-Trillo I.1,4,5 of Biology, Lomonosov Moscow State University, 1 - Institut de Biologia Evolutiva (CSIC-Universitat Leninskiye Gory 1–12, Moscow, 119234, Russian Pompeu Fabra), Passeig Marítim de la Barceloneta Federation 37-49, 08003 Barcelona, Catalonia, Spain 3 - Belozersky Institute for Physico-Chemical Biology, 2 - Division of molecular biology, Ruđer Bošković Lomonosov Moscow State University, Moscow, Institute, Bijenicka cesta 54, 10 000 Zagreb, Croatia 119991, Russian Federation 3 - Dept of Physiology, Development and Neuro- [email protected] science,University of Cambridge,Downing St, Cam- Agamococcidians (Agamococcidiorida Levine, bridge CB2 3DY, UK 1979) represent an enigmatic group of Apicomplexa. 4 - Departament de Genètica, Universitat de Barcelo- The life cycle of these parasites is characterised na, Barcelona, Catalonia, Spain by presence of oocysts with sporocysts (similar 5 - Institució Catalana de Recerca i Estudis Avançats to coccidian), sporozoites and trophozoites, and (ICREA), Barcelona, Catalonia, Spain by absence of merogony and gamogony. This [email protected] group combines two families Rhytidocystidae Adhesion systems and signaling networks are Levine, 1979, parasites of polychaetes, and both essential in multicellular organisms. Some Gemmocystidae Upton & Peters, 1986, parasites elements of the adhesion and signaling pathways of stony corals. The phylogenetic position of these of metazoans, such as proteins from the integrin organisms is still unclear. Previous phylogenetic adhesome are conserved in their closest unicellular studies brought agamococcidians together with relatives. This means these proteins already existed gregarines and cryptosporidians (Leander, Ramey, in the unicellular ancestor of metazoans and that 2006; Rueckert, Leander, 2009; Kristmundsson they were co-opted for a multicellular lifestyle. To et al., 2011; Cavalier-Smith, 2014). In contrast, understand how the integrin adhesome was co- morphological data (oocysts with sporocysts opted at the onset of Metazoa, we aim to unravel and nonmotile trophozoites located within host its function in a close unicellular relative of animals, epithelial tissues) indicate a relationship of the filasterean Capsaspora owczarzaki. This protist agamococcidians with coccidians. We isolated is the closest unicellular relative to metazoans that two putative new species of Rhytidocystis from contains in its genome the basic core of proteins that polychaetes Pectinaria hyperborea and Ophelia constitutes the integrin adhesome. The expression of limacina collected in the Keret Archipelago of the these proteins is upregulated during the aggregative White Sea, Russia. The SSU rDNA sequences stage in culture conditions. In order to understand its obtained from these new parasites clustered strongly role, we are developing some molecular and genetic with Rhytidocystis cyamus and R. polygordiae tools, such as immunostaining, transfection, and within the rhytidocystid clade. Phylogenies infer- CRISPR/cas9. We will discuss preliminary data on red from these sequences demonstrate a close the localization of several cytoskeletal and adhesion relationship between rhytidocystids and marine proteins of the integrin adhesome in C. owczarzaki, coccidians. Interestingly, some coccidians closely obtained by overexpression and by immunostaining related to rhytidocystids, such as Margolisiella with antibodies raised against our proteins of islandica or Aggregata sp., have all three types of interest. We will also discuss the development of reproduction found among apicomplexans: spo- CRISPR system in this organism with the aim to rogony, merogony and gamogony in their life develop a complete model system to analyze the cycles. Thus, our molecular data agree with known origin of animals. morphological data. We discuss the phylogenetic position and perspectives of further investigations ORAL PROTISTS: IMPORTANCE TO CANINE of agamococcidians for more deep understanding PERIODONTAL DISEASE of Apicomplexa evolution. Patel N.1,2, Holcombe L.1, Andrew P.2 Supported by St. Petersburg State University 1 - Mars Petcare UK projects (1.42.1493.2015, 1.42.1099.2016). 2 - University of Leicester [email protected] 58 · “PROTIST–2016”

Periodontal disease is one of the most important penardi Page, 1972, Thecamoeba sphaeronucleolus health concerns for companion animals. Previous Greef, 1891, Penardia mutabilis Cash, 1904 (have studies have demonstrated that at least half of all been registered by us in the Volyn Polissya waters), dogs will have some form of the disease within their Willaertia sp. and Acanthamoeba sp. (have been lifetime which, without early intervention, can registered by us in the Zhytomyr Polissya waters) lead to painful periodontal ligament destruction, are less distributed. Among the above listed species alveolar bone loss, and eventual loss of teeth. The 3, which belong to genera Vahlkampfia Chatton et recent focus of research into canine periodontitis Lalung-Bonnaire, 1912 and Acanthamoeba Vol- has been the identification and characterisation konsky, 1931 are parasitic. The 14 naked amoebae of the bacterial communities present. However, morphotypes are registered in the different water- other microorganisms are known to inhabit the oral bodies: polytactic, monotactic, flamellian, lens-like, cavity and could also influence the disease process. striate, rugose, lingulate, lanceolate, fan-shaped, Our recent research has identified two oral protists in the different waters mayorellian, dactylopodial, that can inhabit the canine oral periodontium. acanthopodial, branched and eruptive. The amoe- Trichomonas sp. and Entamoeba gingivalis were bae with polytactic and acanthopodial morphotypes detected using PCR and next generation sequencing might be mentioned as the least distributed. and had an overall prevalence of 56.52 % (52/92) and 4.34 % (4/92) respectively in UK dogs. Both were EFFECT OF THE SPECTRUM OF AVAILABLE statistically associated to animals with periodontal NITROGEN SOURCES ON PROROCENT- disease indicating their potential involvement in RUM MINIMUM MORPHOLOGY AND PHY- the periodontal disease process. Further work has SIOLOGY led to the development of a quantitative PCR assay Pechkovskaya S.A.1,2, Matantseva O.V.2, Filatova to measure protist abundance. The qPCR assay has N.A.2, Telesh I.V.3 been utilised on plaque samples, collected over a 1 - Department of microbiology, St. Petersburg State 60 week period from individual teeth of miniature University, Russia schnauzer dogs (n =52), to investigate longitudinal 2 - Institute of Cytology RAS, St. Petersburg, Russia changes in abundance of both Trichomonas sp. and 3 - Zoological Institute RAS, St. Petersburg, Russia E. gingivalis as dogs progress from mild gingivitis to [email protected] early stage periodontitis (<25 % attachment loss). Dinoflagellates are the prosperous group of aquatic These findings provide the first conclusive evidence eukaryotes. The ongoing eutrophication of coastal for the presence of canine oral protozoa in dog areas provides a competitive advantage to these plaque and suggest a possible role for protozoa in organisms due to their ability to assimilate nitrogen the periodontal disease process. from a variety of dissolved organic and inorganic sources. The analysis of genomic and transcriptomic NAKED AMOEBAE OF UKRAINIAN POLIS- databases revealed the presence of transporters and SYA FAUNA enzymes involved in uptake and assimilation of Patsyuk M.K. basic nitrogenous compounds present in seawater in Zhytomyr Ivan Franko State University dinoflagellates. In laboratory experiments with the [email protected] culture of dinoflagellates Prorocentrum minimum, At present, the naked amoebae of the Kyiv, Zhyto- we investigated how the spectrum of available myr and Volyn Polissya fauna are represented by nitrogen sources influences morphological and 47 species, which belong to 5 classes, 11 orders, 15 physiological parameters of cells. We showed that families and 20 species. addition of nitrate, ammonium, urea, glycine According to our research the most distributed in and their combinations to the culture growing on the Ukrainian Polissya waters are: Saccamoeba nitrate causes various physiological cell responses. stagnicola Page, 1974, Korotnevella stella Schaeffer, The incorporation of H3-uridine by dinoflagellate 1926, Vexillifera sp., Vannella (cf) lata Page, 1988, cells revealed an increase in the RNA synthesis Cochliopodium sp.(1), Mayorella cantabrigiensis rate after the addition of supplementary nitrogen Page, 1983, Thecamoeba striata Penard, 1890, sources to the culture. Remarkably, the extent Vahlkampfia sp.(1), Vahlkampfia sp.(2). The such to which RNA synthesis was enhanced differed species as Amoeba proteus Leidy, 1878, Polychaos depending on the available nitrogen sources. For sp., Saccamoeba wakulla Bovee, 1972, Rhizamoeba example, the largest increase in the level of RNA sp. (2), Cochliopodium sp. (2), Pellita digitata (Greef, synthesis was achieved in response to addition of 1866) Smirnov et Kudryavtsev, 2004, Mayorella the ammonium/urea combination. The analysis of Protistology · 59 the natural fluorescence of cellular photosynthetic EXPRESSION AND PURIFICATION OF A pigments demonstrated that the observed increase PNEUMOCYSTIS JIROVECII SYNTHETIC in transcription was not directly linked to their RECOMBINANT ANTIGEN AND APPLICA- concentration in cells. Moreover, this analysis TION IN THE DEVELOPMENT OF A SEROLO- revealed heterogeneity in the pigment fluorescence GICAL RAPID DIAGNOSTIC TEST among distinct cells within the model culture. Pinto Mafalda1,2, Cardoso Fernando2, Gomes Funded in part by RFBR, project 15-29-02706. Inês1,3, Pereira Eulália4, Peixoto Miguel4, Franco Ricardo1, Matos Olga2 THE HIGH COMPLEXITY AND DYNAMIC 1 - UCIBIO, REQUIMTE, Departamento de Química, EVOLUTION OF THE RAS SUPERFAMILY Faculdade de Ciências e Tecnologia, Universidade OF GTPASES IN NAEGLERIA Nova de Lisboa (UNL), Portugal Petrželková R.1, Herman E.K.2, Dacks J.B.2, Eliáš 2 - Medical Parasitology Unit, Group of Opportunistic M.1 Protozoa/HIV and Other Protozoa, GHTM, Instituto 1 - University of Ostrava, Faculty of Science, Depart- de Higiene e Medicina Tropical, UNL, Portugal ment of Biology and Ecology, Ostrava, Czech Republic 3 - Instituto de Medicina Molecular, Faculdade de 2 - Department of Cell Biology, University of Alberta, Medicina da Universidade de Lisboa, 1649-028 Edmonton, Alberta T6G 2H7, Canada Lisboa, Portugal [email protected] 4 - UCIBIO, REQUIMTE, Departamento de Química Ras superfamily GTPases constitute a vast group e Bioquímica, Faculdade de Ciências, Universidade of proteins involved in many eukaryote-specific do Porto, Portugal processes. The last eukaryotic common ancestor [email protected] appears to have possessed at least several tens of Ras Pneumocystis pneumonia (PcP) is an infectious superfamily genes, but gene duplications and losses disease caused by Pneumocystis jirovecii, an atypical in different eukaryotic lineages have modified this fungus. PcP remains a major cause of respiratory ancestral set such that substantially different gene illness among immunosuppressed patients. Current complements may be present in different eukaryote PcP diagnosis is based on the detection of P. jirovecii groups. One extreme are taxa harbouring an exten- in respiratory specimens, obtained by invasive sively expanded Ras superfamily complement, as methods such as bronchoalveolar lavage, followed is the case of a free-living amoeboflagellate Nae- by cytochemical staining, immunofluorescent gleria gruberi (Heterolobosea). Recently, geno- staining with monoclonal antibodies (IF/Mab) me sequences of three strains of Naegleria fowleri, or PCR. Therefore, the possibility of an early a causative agent of primary amebic menin- diagnostic method allowing the use of biological goencephalitis (PAM), became available for analy- specimens obtained non-invasively, is highly sis. In order to assess the differences between the desirable. Rapid diagnostic tests (RDTs) using two species and the three strains, we identified and gold nanoparticles (AuNPs) allow a more sensitive, annotated the Ras superfamily genes in the newly fast and cheap diagnosis, to be used in developing sequenced N. fowleri genomes and reannotated countries. The goal of this work is to develop an the respective gene complement in the previously immunochromatographic RDT for the detection published N. gruberi genome. The sets of Ras of P. jirovecii in non-invasive specimens like serum. superfamily genes turned out to differ substantially In this test, spherical AuNPs are conjugated with between the two species, as N. gruberi harbours a multi-epitope synthetic recombinant antigen over 350 genes, whereas N. fowleri exhibits a much (msr) which will allow the detection of circulating less expanded set with “only” over 200 genes. In anti-P. jirovecii antibodies in sera. In order to contrast, little, if any, differences were found for obtain the highest amount of pure antigen, the the three N. fowleri strains. Phylogenetic analyses expression vector pLATE 31, which contains the revealed both species-specific duplications and coding sequence for the MSG g antigen, was isolated losses as the factors responsible for the different and cloned in E. coli XJb (DE3). Extraction and gene numbers in the two species. The evolution of purification through affinity chromatography with the Ras superfamily in the genus Naegleria is thus immobilized metallic ions, ELISA, SDS-PAGE and surprisingly dynamic and points to a hidden level Western-Blot, were performed with the objective of differentiation in cellular physiology of different to obtain the maximum quantity of antigen and Naegleria species. determine its purity. The antigen was then used to form bionanoconjugates with AuNPs, previously 60 · “PROTIST–2016” functionalized with several types of ligands, which 1 - Unité d’Ecologie, Systématique et Evolution, Centre will be the core of the immunochromatographic National de la Recherche Scientifique UMR 8079, RDT with potential for point-of-care diagnostics. Université Paris-Sud, 91405 Orsay, France Acknowledgments: Partially supported by Gilead 2 - Institut de Génétique et Microbiologie, Centre GÉNESE-PGG/001/2014. National de la Recherche Scientifique UMR 8621, Université Paris-Sud, 91405 Orsay, France DIVERSITY OF PROTISTS IN SALINE AND 3 - IMPMC, Sorbonne Universités, Paris 75005, BRACKISH CONTINENTAL WATER BODIES France REVEALED BY HIGH-THROUGHPUT SE- [email protected] QUENCING Primary plastid-bearing eukaryotes evolved by Plotnikov Andrey1,2, Gerasimova Elena1, Poshvina the endosymbiosis of a cyanobacterium within a Daria1, Gogoleva Natalya3, Khlopko Yuri1 heterotrophic host. This gave rise to the supergroup 1 - Institute for Cellular and Intracellular Symbiosis called Archaeplastida, comprising Viridiplantae UB RAS, Orenburg, Russia (green algae and land plants), Rhodophyta (red 2 - Orenburg State Medical University, Orenburg, algae) and Glaucophyta. Although the monophyly Russia of primary plastids has been extensively recovered, 3 - Kazan Institute of Biochemistry and Biophysics KSC the present-day closest cyanobacterial lineage to the RAS, Kazan, Russia chloroplast ancestor is still debated. We performed [email protected] phylogenetic analyses using two concatenated Modern methods of high-throughput sequencing datasets containing 97 plastid-encoded proteins (NGS) are widely used for characterization of and the plastid 16S+23S rRNA cluster, and found protists biodiversity in fresh and marine waters and in both phylogenetic reconstructions that the often result in new data changing our knowledge ancestor of primary plastids was an early-branching about natural microbial communities. At present cyanobacterium related to Gloeomargarita lithopho- only protistian communities from marine biotops ra, the first cultured member of a recently discovered have been studied with NGS, whereas the data on freshwater cyanobacterial lineage widely present in continental saline water bodies are rare (Heidelberg stromatolites and thermophilic microbial mats. This et al., 2013; Triado-Margarit and Casamayor, 2013). discovery has implications for the environmental The aim of this investigation was characterization of conditions in which the endosymbiosis took place. structure and biodiversity of protistian communities in saline and brackish water bodies of the South THE SPECIAL CASE OF HOLOSPORA CARYO- Urals (Russia) with 18S metagenomic sequencing. PHILA, BACTERIAL SYMBIONT OF CILIATES For this purpose water samples from saline and PARAMECIUM brackish lakes and a brackish river were filtered Potekhin A.1, Schrallhammer M.2, Schweikert M.3, through membranes with diameter of pores 0.45 µm. Nekrasova I.1, Lebedeva N.4, Kaltz O.5, Petroni G.6 Total DNA was isolated from the filters and DNA- 1 - Faculty of Biology, St Petersburg State University, libraries were made by PCR with universal primers Saint Petersburg, Russia for V4 region of the gene 18S. High-throughput 2 - Institute of Biology, University of Freiburg, Freiburg, sequencing was conducted with MiSeq (Illumina). Germany The obtained reads were treated with complex of 3 - Institute of Biology, University of Stuttgart, bioinformatic tools. In the report the first data on the Stuttgart, Germany biodiversity of eukaryotes in the deeply continental 4 - Centre of Core Facilities “Culture Collections of saline and brackish water bodies of the South Urals Microorganisms”, St Petersburg State University, (Russia) will be presented. Saint Petersburg, Russia The research was performed in the Center of 5 - Institut of Evolutionary Science, Montpellier Shared Scientific Equipment «Persistence of University 2, Montpellier, France microorganisms» of ICIS UB RAS and was suppor- 6 - Department of Biology, University of Pisa, Pisa, ted by RFBR (16-44-560234, 15-29-02749, 15-29- Italy 02518). [email protected] Infectious bacterium Holospora caryophila, described AN EARLY-BRANCHING CYANOBACTE- as symbiont of the macronucleus of Paramecium RIUM AT THE ORIGIN OF PRIMARY PHO- biaurelia, is an unconventional Holospora. While TOSYNTHETIC EUKARYOTES other Holospora species are highly selective for the Ponce-Toledo R.I.1, Deschamps P.1, López-García host, H. caryophila has been isolated from nature P.1, Zivanovic Y.2, Benzerara K.3, Moreira D.1 in several species of the P. aurelia complex, and in Protistology · 61

P. caudatum. Infection by H. caryophila sometimes families, including four-domain voltage-gated ion kills paramecia, but stable association may last channels (FDVGIC) that played a crucial role in for many years. These premises led to detailed evolution of exciting membranes of eukaryotes and investigation of 6 isolates of H. caryophila, 4 of them nervous system in metazoans. We revealed a high inhabiting the species of the P. aurelia complex, degree of phylogenetic, structural and functional and 2 found in P. caudatum. Screening of more diversity in FDVGIC of dinoflagellates. We deve- than 70 potential hosts – strains belonging to 10 loped a new method to produce spheroplasts of morphological species of Paramecium – showed armored dinoflagellates and for the first time obta- that H. caryophila can infect majority of species of ined single-channel recordings of their ion channels. the P. aurelia complex, P. jenningsi, P. caudatum, Our method allowed us to detect considerable P. multimicronucleatum, and P. putrinum. Symbiosis diversity of cation channels in Prorocentrum minim- could be formed only in certain combinations of the um at the electrophysiological level: potassium- host and the symbiont, though some strains appeared selective channels, inwardly rectifying cation to be universal recipients for all H. caryophila isolates channels, “fast” and “slow” cation channels, and studied. However, most of the checked strains were nonselective cation channels. Funded by the Rus- never infected. P. caudatum strains often died during sian Science Foundation, project 16-14-10116. infection by H. caryophila isolated from P. aurelia strains. Thus, the chance that symbiont can not PHYLOGENY OF PROTISTAN FOUR-DO- develop in host or kills it is higher than possibility MAIN VOLTAGE-GATED ION CHANNELS of successful infection, explaining why ciliates Pozdnyakov I., Skarlato S. harboring symbiotic bacteria are rather rare in nature. Institute of Cytology RAS, St. Petersburg, Russia Molecular phylogenetic analysis of 16S rDNA [email protected] sequences proved that phylogenetic relationships of Four-domain voltage-gated ion channels (FDV H. caryophila with other Holospora species are quite GIC) drive the initial phase of the action potential distant. Physiological and phylogenetic features propagation in metazoans and many protists. support transfer of H. caryophila to the new genus Therefore, these channels are considered as major Preeria. players in evolution of eukaryotic excitability and Supported by RFBR 16-04-01195. metazoan nervous system. In addition to cellular excitability, they are important for cellular motility, CHASING ION CHANNELS OF DINOFLA- intracellular signaling and regulation of rhythmical GELLATES activity. There are five well studied subfamilies of Pozdnyakov I., Matantseva O., Skarlato S. FDVGIC, and all of them are associated with the Institute of Cytology RAS, St. Petersburg, Russia opisthokont lineage: voltage-gated and voltage- [email protected] insensitive sodium channels (Nav and NALCN, Ion channels are transmembrane protein complexes respectively), as well as voltage-gated calcium permeable for ions and playing a crucial role in channels (LVA-Cav and HVA-Cav) and voltage- cell physiology. Ion channels of animals, plants insensitive calcium channels of fungi (Cch). Using and fungi have been intensively studied for many publicly available genomic, transcriptomic and decades; however, noticeably less information protein databases and blast search, we identified is available concerning ion channels of other 277 members of FDVGIC family from different eukaryotes. This lack of knowledge hampers our eukaryotic groups to reconstruct phylogeny of understanding of both evolution of ion transport this ion channel family employing the maximal and physiology of protists. Dinoflagellates are the likelihood (ML) method and Bayesian analysis group of aquatic unicellular eukaryotes of high (BA). In this work, we demonstrated that most of the ecological relevance, but rather little is known about considered protist groups have their own subfamilies their physiology. At the same time, dinoflagellates of FDVGIC that do not form clades with any known are phylogenetically distant from animals, plants subfamily of FDVGIC (i.e. Nav, NALCN, LVA-Cav, and fungi and thus are attractive objects to study HVA-Cav, and Cch). Moreover, both ML and BA evolution of ion channels. However, investigation approaches showed that similar to metazoans some of dinoflagellate ion channels is complicated by protist groups, such as alveolates and stramenopiles, the lack of sufficient genomic data and obstacles possess high phylogenetic diversity of FDVGIC. in applying electrophysiological techniques to Although obtained phylogenies are not fully resolved dinoflagellates due to their complex cell coverings. due to the limited data on ion channel sequences, We analyzed publicly available transcriptomes of the present study advances our understanding of the ten dinoflagellate species and found 12 ion channel diversity and evolution of FDVGIC family. Funded 62 · “PROTIST–2016” by the Russian Science Foundation, project 16-14- [email protected] 10116. Fecal samples were collected from 120 domestic pigeons to determine the Attributable risk of Ca- HETEROTROPHIC FLAGELLATES OF SPHAG- pillaria spp. The Capillaria spp. was observed in 64 NUM BOGS IN SOUTH PATAGONIA, CHILE out of 120 (51%) pigeons (70 males and 50 females) Prokina K.I.1, Mylnikov A.P.1, Philippov D.A.1,2 under study. A total of 64 (39 males and 25 females) 1 - Papanin Institute for Biology of Inland Waters, were naturally infected with Capillaria spp. with Russian Academy of Sciences, Borok, Nekouzsky 51% and 50% in males and females respectively. District, Yaroslavl Oblast, 152742 Russia Qualitative examinations include the direct micro- 2 - Tyumen State Universsity, Tyumen, 630003 Russia scopy and faecal floatation while quantitative [email protected] examination includes McMaster technique (worms Bogs are habitats of great environment-modify- are calculated per gram of the faeces). Month wise ing value, in particular, play a major role in the Attributable risk showed that eggs of the worms formation of the hydrological regime of the sur- were found to be abundant in the month of July rounding territory. A considerable amount of (60% to 73%) because of high humidity in the data has been accumulated on the heterotrophic month of July during the present study. Very high flagellates associated with wetland ecosystems of and very low temperature is not suitable for the the Northern Hemisphere while the large wetlands proper development of the eggs. Qualitative and of the south part of South America remain almost quantitative examination revealed that Capillaria unexplored. Species composition and morphology spp. was more prevalent in males (51%) than females of heterotrophic flagellates collected from six (50%) but overall there is no significant difference sphagnum bogs in Chilean part of South Patagonia (p>0.05) in the male and female because both of and Tierra del Fuego in October–November 2015 individuals invest equal amount of energy in search are described. Fifty-eight species from 33 genera of food and incubating the eggs. Different breeds and 3 macrotaxa (Opisthokonta, SAR, Excavata) of the pigeons gave different Attributable risk in and heterotrophic flagellates ‘incertae sedis’ have different months during the whole study. Group of been recorded. Most of the recorded species are pigeons from different locations showed variable bacterivorous, 7 species – are omnivorous, and 1 Attributable risk. Areas with high humidity were – is a predator, feeding on other flagellates. In two more suitable for the development of eggs that is the bogs of continental part of Chile we have recorded reason that higher Attributable risk was observed in 38 species and forms (38 species in bog no. 1; 1 shahdara (75%). species in drained bog no. 2), and in four bogs in Tierra del Fuego – we have recorded 39 species NEBELA JIUHUENSIS NOV. SP. (AMOEBO- and forms (15 species in bog no. 3; 8 species in no. ZOA; ARCELLINIDA; HYALOSPHENIIDAE): 4; 13 species in no. 5; 16 species in no. 6). Among A NEW MEMBER OF THE NEBELA SACCI- all aquatic habitats associated with bogs, the greatest FERA - EQUICALCEUS - ANSATA GROUP number of species has been recorded in hollows (35 DESCRIBED FROM SPHAGNUM PEAT- species); bogs streams (25); small boggy lakes (24); LANDS IN SOUTH-CENTRAL CHINA pool near trunks of Nothofagus and peat excavation Qin Yangmin1,2,3, Man Baiying2, Anush Kosakyan4, with cyanobacterial mat at the bottom (5); only 1 Enrique Lara3, Gu Yansheng2, Wang Hongmei2, species has been recorded in a drainage canal. All Zhang Wenwen1,2, Edward A.D. Mitchell5,6 identified flagellates are known from different types 1 - Department of Geography, School of Earth Science, of freshwater habitats of the Northern Hemisphere, China University of Geosciences, Wuhan, 430074, and most of them (31 species and 10 not identified China to species level taxa are known from the bogs of the 2 - State Key Laboratory of Biogeology and Envi- Northern Hemisphere. ronmental Geology, China University of Geosciences, This study was supported by the Russian Science Wuhan, 430074, China Foundation (project no. 14-14-01134). 3 - Laboratory of Soil Biology, University of Neuchâtel, Rue Emile Argand 11, CH-2000 Neuchâtel, Swit- ATTRIBUTABLE RISK OF CAPILLARIA SPE- zerland CIES IN DOMESTIC PIGEONS (COLUMBA 4 - Department of Zoology, Institute of Biosciences, LIVIA DOMESTICA) University of São Paulo, Rua do Matão, travessa 14, Qamar M.F.Q.1,2, Arifa Butt A.B.2 São Paulo, Brazil 1 - University of Veterinary & Animal Sciences, Lahore 5 - Laboratory of Soil Biology, University of Neuchâtel, 2 - Department of Zoology, GC University Lahore Rue Emile Argand 11, CH-2000 Neuchâtel, Swit- Protistology · 63 zerland 1 - Laboratorio de Protozoología, Departamento 6 - Jardin Botanique de Neuchâtel, Chemin du Per- de Biología Comparada, Facultad de Ciencias, thuis-du-Sault 58, CH-2000 Neuchâtel, Switzerland Universidad Nacional Autónoma de México, Mexico [email protected] 2 - Departamento de Zoología, Facultad de Biología, Hyalospheniids are among the most common Universidad Complutense, Madrid, España and conspicuous testate amoebae in high-lati- 3 - Laboratorio de Protozoología, Departamento tude peatlands and forest humus. These testate de Biología Comparada, Facultad de Ciencias, amoebae were widely studied as bioindicators, Universidad Nacional Autónoma de México, México and are increasingly used as models in microbial [email protected] biogeography. However, data on their diversity Several samples collected during years 2014 and and ecology are still very unevenly distributed 2015 contained epibenthonic fauna with the crayfish geographically: notably, data are lacking for low Procambarus (Austrocambarus) sp. Samplings were latitude peatlands. We describe here a new species, performed on the freshwater artificial pond Yaleon, Nebela jiuhuensis, from peatlands near the Middle located in Montebello (Chiapas, SE Mexico), using Yangtze River reach of south central China with a 5 mm opening hand net. The environment was characteristic morphology. The test (shell) has strongly polluted with organic matter (assimilated hollow horn-like lateral extensions also found in to a polysaprobic zone). The crayfish Procambarus N. saccifera, N. equicalceus (=N. hippocrepis) and (Austrocambarus) sp. harbored colonies of a peri- N. ansata, three large species restricted mostly to trich ciliate identified as Epistylis hentscheli Kahl, Sphagnum-peatlands of Eastern North America. 1935. This ciliate formed ramified colonies with Mitochondrial cytochrome oxidase (COI) data a moderate number of zooids (up to 30). Stalks confirm that N. jiuhuensis is closely related to the of the colony were dichotomously branched, morphologically very similar North American and they presented peripherical fibres arranged species N. saccifera and more distantly to N. ansata longitudinally. The zooid was elongated; the form within the N. penardiana group. These species are of body was similar to a bell (170-200 µm in lenght), all found in wet mosses growing in poor fens. Earlier with a C-shaped transversal macronucleus, and a re-ports of morphologically similar specimens found spherical micronucleus near to the macronucleus. in South Korea peatlands suggest that N. jiuhuensis The peristomal lip was very thin. The buccal may be distributed in comparable peatlands in infraciliature had the general pattern of peritrichids Eastern Asia (China and Korea). The discovery and was composed by a haplokinety and a polykinety of such a conspicuous new species in Chinese drawing a spiral with 1.5 rounds inside the buccal peatlands suggests that many new testate amoebae infundibulum. These epibionts were located exclu- species are yet to be discovered, including potential sively on the pereiopods. The goal of this contribu- regional endemics. Furthermore, human activities tion is to provide data about cell/colony structure of (e.g. drainage, agriculture, pollution) have reduced E. hentscheli and also its distribution on the crayfish. the known habitat of N. jiuhuensis, which can thus This is the first record of this epibiont peritrich ciliate be considered as locally endangered. We therefore on crustacea, being previously found on algae and suggest that this very conspicuous microorganism fish. Acknowledgements: to Posgrado en Ciencias with a probably limited geographical distribution Biológicas, Universidad Nacional Autónoma de and specific habitat requirement should be consi- México and CONACyT, for the support necessary for dered as a flagship species for microbial biogeo- carry out this research. Additionally, we are indebted graphy as well as local environmental conservation to Dr. Villalobos-Hiriart (Instituto de Biología, and management. Key words: Arcellinid testate UNAM) for the crustacean identification and to amoebae; biodiversity conservation; biogeography; Biol. Reyes-Santos (Facultad de Ciencias, UNAM) DNA barcoding; mtCOI. for their help with impregnation techniques. We also appreciate the technical support of the Department of Zoology (Universidad Complutense, UCM, España). EPIBIONTIC RELATIONSHIP IN AN AR- TIFICIAL POND OF CHIAPAS (MEXICO): ORIGIN AND EVOLUTION OF PARAME- PROCAMBARUS (AUSTROCAMBARUS) SP. CIUM BURSARIA / CHLORELLA SYMBIOTIC (CRUSTACEA: DECAPODA) AND EPISTYLIS SYSTEM: WHAT CAN ONE SAY ABOUT THE HENTSCHELI (CILIOPHORA: PERITRI- HOST EVOLUTION STUDYING EVOLUTION CHIA) OF SYMBIONT? Ramírez-Ballesteros M.1, Fernandez-Leborans G.2, Rautian M., Beliavskaia A., Kiselev A. Mayén-Estrada R.3 Saint Petersburg State University, Russia 64 · “PROTIST–2016” [email protected] after the loss of photosynthesis. Before the present BACKGROUND. P. bursaria belongs to one of report it was unknown if the absence of genome the best studied Ciliate’s genus Paramecium. This in the Polytomella plastids was a shared condition species diverged basically from other paramecia. with species of the Polytoma genus. We present here They possess symbiotic Chlorella in the cyto- the ptDNA of Polytoma uvella UTEX 964, which plasm. These Chlorella were attributed to three is the largest (circa 230 Kb) reported among non- Chlorella species – Ch. vulgaris, Ch. variabilis and photosynthetic algae. The ptDNA of P. uvella has Micractinum reisseri according to their phylogeny lost all genes related with the photosynthetic function inferred from ITS1-5.8S-ITS2-5’LSU rDNA and its residual coding capacity is similar to ptDNAs sequence and their sensitivity to specific viruses from distantly related colorless chlorophytes, such as (PBCV). Morphospecies P. bursaria consist of the pathogens Prototheca and Helicosporidium. The 5 syngens – reproductively isolated intraspecies gene repertoire of the P. uvella ptDNA comprises groups. Previously we have shown that syngens were mostly transcription and translation players and just represented by different branches on phylogenetic few conserved proteins (e.g., FtsH-like, Ycf1-like trees. The purpose of our investigation was to and ClpP). Regardless of the substantial gene loss, study phylogeny of P. bursaria and their symbiotic the P. uvella ptDNA shows evidence of expansion Chlorella using the same set of strains collected in due to the accumulation of intergenic short repeated different geographic regions. RESULTS. Gene sequences (average length of 2.9 Kb). It is plausible RuBisCo have been used as molecular marker for that recombination-based DNA repair mechanisms Chlorella phylogeny. The tree consists of three are responsible of the genomic expansion of the P. separate branches. Diversity in each branch is uvella ptDNA. negligible. Our data support strong correspondence between P. bursaria syngens and branch of Chlorella, SELECTION AND CHARACTERIZATION while weak correlation between Chlorella type and its OF SINGLE CHAIN VARIABLE FRAGMENT geographic location. CONCLUSIONS. According (SCFV) ANTIBODIES AGAINST PNEUMO- to our results we have concluded that successful CYSTIS JIROVECII FROM PHAGE DISPLAY symbiosis between P. bursaria ancestor and Chlorella LIBRARIES has been established at least three times. The last Ribeiro Marta, Cardoso Fernando, Matos Olga time it had been happened before syngens R1 and R2 Medical Parasitology Unit, Group of Opportunistic have diverged. We propose the strategy of evolution Protozoa/HIV and Other Protozoa, Global Health and and geographic dispersion of this symbiotic system. Tropical Medicine, Instituto de Higiene e Medicina Scientific research was performed at the Center for Tropical, Universidade Nova de Lisboa, Portugal Culturing Collection of Microorganisms and Center [email protected] for Molecular and Cell Technologies of Research Pneumocystis pneumonia (PcP) is an infectious park of St. Petersburg State University. Supported disease caused by Pneumocystis jirovecii, an atypical by RFBR grant 13-04-01714. fungus. PcP remains a major cause of respiratory illness among immunosuppressed patients. Current PcP diagnosis is based on the detection of P. jirovecii THE PLASTID GENOME OF POLYTOMA in respiratory specimens, obtained by invasive UVELLA IS THE LARGEST KNOWN AMONG methods such as bronchoalveolar lavage, followed NON-PHOTOSYNTHETIC ALGAE by cytochemical staining, immunofluorescent Reyes-Prieto A.1,2 staining with monoclonal antibodies (IF/Mab) 1 - Biology Department. University of New Brunswick, or PCR. Therefore, the possibility of an early Fredericton diagnostic method allowing the use of biological 2 - Integrated Microbiology Program. Canadian specimens obtained non-invasively, is highly Institute for Advanced Research. desirable. Rapid diagnostic tests (RDTs) using [email protected] gold nanoparticles (AuNPs) allow a more sensitive, The polyphyletic assembly Polytoma and the fast and cheap diagnosis, to be used in developing species of the genus Polytomella represent different countries. The goal of this work is to develop an lineages within the Order Chlamydomonadales immunochromatographic RDT for the detection (Chlorophyta) that lost photosynthesis indepen- of P. jirovecii in non-invasive specimens like serum. dently. The Polytomella clade is one of the two In this test, spherical AuNPs are conjugated with a known groups of plastid-bearing eukaryotes that multi-epitope synthetic recombinant antigen (msr) have lost completely the plastid genome (ptDNA) which will allow the detection of circulating anti-P. Protistology · 65 jirovecii antibodies in sera. In order to obtain the TERRESTRIAL ALGA KLEBSORMIDIUM highest amount of pure antigen, the expression IN THE LIGHT OF THE HYPOTHESIS vector pLATE 31, which contains the coding „EVERYTHING IS EVERYWHERE, BUT THE sequence for the MSG g antigen, was isolated ENVIRONMENT SELECTS“ and cloned in E. coli XJb (DE3). Extraction and Ryšánek D.1, Rindi F.2, Holzinger A.3, Elster J.4, purification through affinity chromatography with Škaloud P.1 immobilized metallic ions, ELISA, SDS-PAGE and 1 - Department of Botany, Faculty of Science, Charles Western-Blot, were performed with the objective University of Prague, Benatska 2, CZ-12801, Prague, to obtain the maximum quantity of antigen and Czech Republic determine its purity. The antigen was then used to 2 - Dipartimento di Scienze della Vita e dell’Ambiente, form bionanoconjugates with AuNPs, previously Università Politecnica delle Marche, Via Brecce functionalized with several types of ligands, which Bianche, IT-60131, Ancona, Italy will be the core of the immunochromatographic 3 - University of Innsbruck, Institute of Botany, RDT with potential for point-of-care diagnostics. Functional Plant Biology, Sternwartestrase 15, A-6020 Acknowledgments: Partially supported by Gilead Innsbruck, Austria GÉNESE-PGG/001/2014. 4 - Centre for Polar Ecology, Faculty of Science, University of South Bohemia, České Budějovice and Academy CHANGES IN CILIATES COMMUNITY AC- of Sciences of the Czech Republic, Institute of Botany, ROSS AN EUTROPHICATION GRADIENT IN Phycology Centrum, Trebon 37901, Czech Republic A LARGE TEMPERATE ESTUARY [email protected] Rubin E., Santoferrara L., Zhang H., McManus G. Baas Becking’s hypothesis “Everything is every- Department of Marine Sciences, University of where, but the environment selects“ first mentioned Connecticut, Groton, CT, USA in 1934 still contains three current controversial [email protected] issues in protists: disunity in the description of The ciliate community composition in Long Island diversity, distribution patterns, and the ecological Sound, USA, was investigated using high through- preferences. We examined a very common, globally put sequencing (Miseq, Illumina) of variable region distributed terrestrial algal genus Klebsormidium 4 (V4) of the small subunit ribosomal gene (V4- and tested (i) whether there is any biogeographical SSU). Due to its proximity to heavily populated pattern of this microorganism, (ii) if there is a areas, the western Sound (WLIS) is eutrophic, and comparable diversity in the polar and temperate strong gradients have been observed down-estuary regions, and (iii) a potential role of ecological (east to west) in nutrients, chlorophyll, and plankton speciation processes on diversification.Our analyses biomass. We sampled six stations on the long axis revealed the presence of two different distribution of the estuary, at two depths (surface and 6 m), in patterns which are supposed to characterize both the summer of 2015. Analysis of ciliate V4-SSU macroorganisms and protists. We demonstrated sequences grouped into operational taxonomic an unlimited dispersal and intensive gene flow units (OTUs) corresponded to the eutrophication within one of the inferred lineages (superclade gradient, with higher OTU-richness found in WLIS B). However, the majority of Klebsormidium compared to the east. The majority of ciliates were clades showed rather a limited distribution. In classified into Choreotrichia and Oligotrichia, addition, we detected a significant decrease of contributing together 65% to 91% of all ciliate species richness towards the poles, i.e. the macro- OTUs. Litostomatea and Prostomatea contributed ecological pattern typical for macroorganisms. from 4 to over 30% of the OTUs. The relative Species within a single protist genus may thus exhibit proportion of Oligotrichia was higher near the highly contrasting distribution patterns, based surface and increased in the eastern, more pristine on their dispersal capabilities, which are usually stations. The Choreotrichia showed the opposite shaped by both intrinsic and extrinsic factors. In pattern. The relative proportion of Oligotrichs and addition, we determined a distinct ecophysiological Choreotrichs across the eutrophication gradient differentiation among distantly and closely related in Long Island Sound was correlated with water lineages, thereby corroborating our hypothesis that transparency and it could be partially explained the sympatric speciation of terrestrial algae is driven by variation in abundance of major phytoplankton by ecological divergence. We clearly showed that groups in the western, central and eastern Long pH is a critical ecological factor that influences Island Sound. the diversity of autotrophic protists in terrestrial habitats. 66 · “PROTIST–2016”

LYING ON THE SURFACE: NEW DATA ON BIODIVERSITY OF BENTHIC DINOFLA- THE EPISYMBIONTS OF CILIATES GELLATES ALONG THE SOUTHERN Sabaneyeva E.1, Lanzoni O.2, Lebedeva N.3, Benken COAST OF OMAN WITH EMPHASIS ON K.4, Potekhin A.5, Petroni G.2 POTENTIALLY TOXIC SPECIES 1 - Department of Cytology and Histology, Saint- Saburova M. Petersburg State University, Saint-Petersburg, Russia Kuwait Institute for Scientific Research, P.O. BOX 2 - Department of Zoology, University of Pisa, Pisa, 1638, Salmiya 22017, Kuwait; long-term consultancy Italy [email protected] 3 - Core Facility Center for Cultivation of Micro- Benthic dinoflagellates have attracted considerable organisms, Saint-Petersburg State University, Saint- research attention during the last decades due to their Petersburg, Russia morphological and taxonomic diversity, ecological 4 - Core Facility Center for Microscopy and Micro- significance in marine benthic ecosystems, and analysis, Saint-Petersburg State University, Saint- their capability to produce toxins and cause toxic Petersburg, Russia blooms in the coastal areas. A pilot taxonomic 5 - Department of Microbiology, Saint-Petersburg State survey of the benthic dinoflagellates was performed University, Saint-Petersburg, Russia for the first time at the Arabian Sea coast along [email protected] Dhofar Governorate of the Sultanate of Oman in Protists often form symbiotic associations with the vicinity of Salalah City at five sampling sites on prokaryotes. Ciliates are especially well known February 2014 and 2016. It has been shown that for harbouring endosymbiotic and ectosymbiotic the bottom sediments and the surface of brown bacteria, the relationship between the partners and red macroalgae along the southern Omani ranging from mutualism to parasitism. Epibionts coast were inhabited by taxonomically diverse can be found both in anaerobic and in free living and abundant assemblages of sand-dwelling and aerobic ciliates, however, so far they have never epiphytic dinoflagellates. A total of 38 dinoflagellate been reported in paramecia. Here we present species belonging to five orders were recorded in morphological (DIC, AFM, CLSM, TEM) and this study including Dinophysiales, Gymnodiniales, molecular characterization of the bacterium fouling Gonyaulacales, Peridiniales, and Prorocentrales. cells of Paramecium primaurelia strain isolated from High diversity of the sand-dwelling dinoflagellates nature in Cyprus. At the last stage of infection, was supported mainly by gymnodinioid and peri- epibionts formed a thick coat on the cortex of the dinioid taxa. In contrast, members of Coolia, ciliate causing complete loss of cilia, changes of the Gambierdiscus, Ostreopsis, and Prorocentrum were cell shape, dwarfing, and finally, the host death. among the most abundant and diverse epiphytic Fluctuations of the ciliate population density of dinoflagellates. A number of known toxin produ- the infected strain registered throughout long cers were observed including the ichthyotoxic term observations of the laboratory culture imply dinoflagellates of the genus Amphidinium, the parasitoid nature of the epibiont. In preliminary okadaic acid producing Prorocentrum species, FISH experiments, epibionts were easily revealed the cooliatoxin producing Coolia, the palytoxin with the universal eubacterial probe Eub 338, producer Ostreopsis, and the ciguatera-related however, they never showed positive signal when Gambierdiscus species. The presence of known toxic hybridized with probes specific for Alpha-, Beta-, dinoflagellate species may indicate a potential risk of Gamma- or Deltaproteobacteria. Molecular toxicity in the marine environment of the southern characterization was done following the full-cycle Omani coast, and underscores the need for further rRNA approach and association of the epibionts studies on taxonomy, ecology and toxicology of with the host was confirmed by FISH experiments benthic dinoflagellates in Oman. with newly designed species-specific probes. The obtained 16S rDNA sequence showed a similarity DEVELOPMENT OF A MULTIPLE-DRUG of circa 82% with Alphaproteobacteria class, thus SELECTION SYSTEM FOR DUAL-TRANS- suggesting that epibionts are extremely unusual FORMATION OF THE OYSTER PARASITE novel organisms. Phylogenetic analysis, which is PERKINSUS MARINUS under way, will enlighten the evolutionary position Sakamoto Hirokazu1, Hirakawa Yoshihisa1, Ishida of this enigmatic organism, presumably, either a Ken-ichiro1, Kita Kiyoshi2,3, Matsuzaki Motomichi2 basal alphaproteobacterium or a fast-evolving line 1 - Faculty of Life and Environmental Sciences, within the order Rickettsiales. University of Tsukuba 2 - Department of Biomedical Chemistry, Graduate Protistology · 67

School of Medicine, The University of Tokyo salmonicida. We are interested in elucidating the 3 - School of Tropical Medicine and Global Health, evolutionary transitions to anaerobiosis and para- Nagasaki University sitism within metamonada, and sequenced the ge- [email protected] nome of C. membranifera. The genome assembly is Plastids in apicomplexan parasites are highly dege- 22.4 Mb long with 11328 predicted protein-coding nerated. The organelle is nevertheless essential for genes, 41% of those have introns. Automatic completion of the parasite life cycle. Interestingly, annotation was carried out by searching against an oyster parasite Perkinsus marinus, which is the Interpro, PFAM, Prosite, TIGR databases sister to dinoflagellates and close to Apicomplexa, to identify domains, assign putative functions also has a DNA-lacking, extremely degenerated and predict metabolic pathways. Expert manual plastid. Functional analysis of the cryptic organelle annotation is underway for genes encoding proteins is attracting and required to understand the rela- functioning in DNA repair, mRNA degradation, tionship between the organelle degeneration and mitochondrion-related organelles, cell surface or parasitism. The transgenic technique is a convincing external cellular processes involved in host tissue approach for the analyses of proteins of interest and is adhesion, immune evasion, pathogenicity, nutrient practicable in P. marinus. However, each transfected acquisition, metabolite transport and environmental cell must be isolated from untransfected cells by sensing, among others. We have completed analyses hand labor using a micromanipulator multiple times of the DNA repair pathways. Those can drive sexual/ to obtain any transfected cell lines. This is because parasexual pathways, antigen diversification and drug selection system has not been established. copy number variation, and are of great importance Here, we identified two drugs that are available for for adaptive evolution. C. membranifera possesses selection of transfected P. marinus cells. Firstly, we a complete system for excision repair, and the screened antibiotics shown utility in apicomplexan double strand break repair machinery including parasites and determined that blasticidin S, bleo- 1) a homologous recombination pathway and 2) mycin and puromycin effectively inhibited the microhomology-mediated end joining and single- parasite growth. Then, their resistance genes were strand annealing. Also, it has several gene family fused downstream of gfp or mCherry gene, and each expansions, as well as, a complete repertoire of construct was transfected to the parasite. After cell cycle checkpoints and sex-related proteins. G. two months, the fluorescent signals were observed intestinalis and S. salmonicida have minimalistic and in almost all cells cultured with bleomycin or slightly different versions of the pathways found in C. puromycin. Furthermore, dual transfected cells membranifera suggesting that there have been some were selected by using the two drugs, which enables secondary losses and modifications in diplomonads us to examine colocalization of plastid proteins. We as a result of their parasitic lifestyle. believe that this system provides new opportunities for functional analyses of the plastids in the parasite. THE GENOMIC COST OF BECOMING A RED ALGAL FREELOADER A DRAFT GENOME OF THE ANAEROBIC Salomaki E.D., Lane C.E. FLAGELLATE CARPEDIEMONAS MEMBRA- Department of Biological Sciences, University of Rho- NIFERA, A FREE-LIVING RELATIVE OF de Island METAMONAD PARASITES [email protected] Salas Leiva D.E.1, Kolisko M.2, Curtis B.1, Eme L.1, An abundance of genomic and transcriptomic data Kamikawa R.3, Roger A.1 have been gathered over the past decade providing 1 - Centre for Comparative Genomics and Evolutionary a wealth of knowledge about what it takes to be Bioinformatics (CGEB), Department of Biochemistry a successful parasite. Genomes of highly derived and Molecular Biology, Dalhousie University, Halifax, eukaryotic parasites have been sequenced including NS, Canada, B3H 4R2 those from formerly photosynthetic lineages in- 2 - Beatty Biodiversity Centre, Dept. Botany, Univer- cluding apicomplexans. These data have revealed sity of British Columbia fascinating innovations that evolved over hundreds 3 - Graduate School of Human and Environmental of millions of years, enabling parasites to infect and Studies, Kyoto University, Yoshida-Nihonmatsu cho, evade their hosts. Unlike highly derived lineages of Kyoto 606-8501, Japan eukaryotic parasites, red algae appear to be fertile [email protected] ground for adopting a parasitic life strategy as seen Carpediemonas membranifera is a free living fla- by numerous recent and independent evolutions of gellated metamonad related to diplomonad para- parasitic taxa. Red algal parasites provide a great sites such as Giardia intestinalis and Spironucleus system to investigate the early stages of genome 68 · “PROTIST–2016” evolution as an organism gives up autotrophy in fa- of these abundant and ecologically relevant ciliates vor of moving onto its relatives’ couch and raiding in marine plankton. their refrigerator. We sequenced draft transcripto- mes for the parasitic red alga Choreocolax polysi- TAXONOMIC COMPOSITION OF PROKA- phoniae and its free-living host Vertebrata lanosa. RYOTES-ASSOCIATES OF HALOPHILIC PRO- These data, in combination with genomic DNA TISTS sequence data for these taxa was compared with Selivanova Olga1, Gogoleva Natalya2, Khlopko other published red algal genomes and transcripto- Yuri1, Plotnikov Andrey1 mes to investigate the early consequences of tran- 1 - Institute for Cellular and Intracellular Symbiosis sitioning from autotrophy to parasitism. Choreocolax UB RAS, Orenburg, Russia polysiphoniae appears to have lost genes involved in 2 - Kazan Institute of Biochemistry and Biophysics KSC plastid maintenance and photosynthetic processes. RAS, Kazan, Russia Investigation of these data and their implications for [email protected] the evolution of parasitism remains ongoing. As known, taxonomic structure of communities in hypersaline environments is simple. It includes MOLECULAR DIVERSITY AND PHYLO- prokaryotes, photosynthetic and heterotrophic GENY OF OLIGOTRICHIA AND CHOREO- protists, fungi, and crustaceans. Under extreme TRICHIA (CILIOPHORA, SPIROTRICHEA) salinity the community is simplified up to only Santoferrara L.F., McManus G.B. microorganisms, where phototrophic protists Department of Marine Sciences, University of are the main producers of organic matter, and Connecticut heterotrophic protists are the only consumers. [email protected] Symbiotic associations with halophilic phototrophic We revise and expand the molecular data on the protists are well studied for green alga Dunaliella subclasses Oligotrichia and Choreotrichia in terms and its prokaryotic associates, while heterotrophic of both genetic markers and taxonomic sampling. protists and their role in forming of halophilic Two approached are used, one based on sequences microbial community are studied poorly. Aim of from morphologically-characterized specimens, the study was to estimate taxonomic composition and another one including also the unidentified of prokaryotes – associates of autotrophic and environmental sequences available in NCBI Gen heterotrophic protists isolated from salt water Bank. First, concatenated sequences of ribosomal bodies in Salt-Iletsk and Elton regions. The DNA markers (18S rDNA, 28S rDNA and ITS method of 16S metagenomic sequencing was regions) from seventeen families and four clades used. 111 OTUs were found in association with of the paraphyletic genus Tintintinnopsis were ana- heterotrophic flagellate Pleurostomum salinum. The lyzed, including two families newly studied by single genera of archaea Natronomonas sp., Haloarcula cell sequencing and six genera newly sequenced sp., Haloplanus sp., Halorubrum sp. and bacteria for at least one marker. Second, all the 18S rDNA Halovibrio sp., uncultured bacterium (Bacteroide- sequences available in GenBank for these subclasses tes) were predominant. Heterotrophic flagellate were retrieved and curated as part of the EukRef Tulamoeba bucina had very similar taxonomic initiative, resulting in almost 3,200 sequences and composition of prokaryotes. There were 129 OTUs 1,000 OTUs (operational taxonomic units clustered including uncultured bacterium (Bacteroidetes), at 99% similarity). The concatenated dataset allo- Natronomonas sp., Halovibrio sp., Haloferax sp., wed confirming or discovering novel phylogenetic Halorubrum sp. as predominant genera. There were relationships within Choreotrichia. The 18S rDNA 32 and 41 OTUs in associations with Pharyngomonas dataset allowed rough comparisons of molecular sp. and Heterolobosea aff. Euplaesiobystra respec- diversity between the two subclasses. Because tively. Bacteria Idiomarina sp., Halomonas sp., Choreotrichia has been easier to barcode (mainly Gracilimonas sp. were predominant in both cultures. for the loricate Tintinnida), analyses have not re- Phototrophic flagellate Dunaliella parva had vealed novel diversity, but exposed known and new 19 OTUs of prokaryotes – associates. Bacteria misidentifications and classification inconsistencies. Halovibrio sp. and archaea Halorubrum sp. were In contrast, three clades within Oligotrichia inclu- the most abundant. A crucial factor influencing ded only environmental sequences, thus highlight- the taxonomic composition of prokaryotes in the ing a high proportion of partially-characterized or associations with halophilic protists was level of novel diversity in this subclass. These analyses help medium mineralization. Taxonomic diversity was solving classification conflicts and will provide a higher in associations with heterotrophic protists reference for studying the diversity and distribution than in associations with autotrophs. The highest Protistology · 69 species richness of prokaryotes – associates has been These results suggested that monoclonal antibodies registered in the culture Tulamoeba bucina. against rNfCPB and rNfCPBL may be useful for the The research was performed in the Center of further immunological study. Shared Scientific Equipment «Persistence of microorganisms» of ICIS UB RAS and was sup- BENTHIC PROTISTS (CILIOPHORA, GRO- ported by RFBR (16-44-560316, 14-04-01796). MIIDA, FORAMINIFERA) IN THE BLACK SEA: THEIR CONTRIBUTION TO THE BOT- CHARACTERIZATION OF MONOCLONAL TOM COMMUNITIES ANTIBODIES FOR CATHEPSIN B AND CA- Sergeeva N.G. THEPSIN B-LIKE PROTEINS OF NAEGLERIA Institute of Marine Biological Research of Russian FOWLERI Academy of Sciences, 2, Nakhimov ave., Sevastopol, Seong G.S.1,2, Sohn H.J.1,2, Kang H.K.1,2, Shin Russia H.J.1,2 [email protected] 1 - Department of Microbiology, Ajou University School Ciliophora, Gromiida and Foraminifera (hard- shell of Medicine and soft-shell forms) are the significant components 2 - Department of Biomedical Science, Graduate of the benthic communities of the Black Sea. School of Ajou University, Suwon 443-721, Republic These protozoans are of great role in the transfer of of Korea bacterial and algal production to the next trophic [email protected] levels. They themselves are consumers of bottom Naegleria fowleri causes a fatal primary amoebic bacteria, microalgae, pollen terrestrial vegetation meningoencephalitis (PAM) in human and expe- and marine filamentous fungi; as predators, they rimental animals. Cathepsin B (NfCPB) and might prey upon unicellular organisms. In addition, cathepsin B-Like (NfCPBL) gene in N. fowleri, are the representatives of above listed groups are an consists of 1,038bp of DNA (345 amino acid) and important food source for many bottom inver- 939bp of DNA (313 amino acid), and molecular tebrates in different habitats. The changes in weights of recombinant proteins are 38.4 and 34 taxa composition and abundance, and also the kDa, respectively. In the previous study, rNfCPB proportions of these protozoan representatives in and rNfCPB-L refolding protein may play impor- the benthic communities along deep-water oxic/ tant roles in host tissue invasion, immune evasion anoxic interface (75-300m) in the Istanbul Strait’s and nutrient uptake. In this study, we produced (Bosporus) outlet area of the Black Sea and NW anti-NfCPB and anti-NfCPBL monoclonal anti- shelf of Crimea Peninsula were studied. In the body (rNfCPB-McAb and rNfCPBL-McAb) oxic zone of the Black Sea, benthic Protozoa using the cell fusion technique and observed the and Metazoa were studied in area near the Kerch immunological characteristics. Seven hybridoma Strait’s on NE shelf of Crimean Peninsula. Our data cells secreting rNfCPB-McAb (2D6, 2D11, 2A7, allowed concluding about tolerance of many benthic 2B7, 2E2, 2E9, 2C9) and three hybridoma cells protozoans to the hypoxic / anoxic conditions secreting rNfCPB-McAb (1E5, 1C8, 1D9) were and sulfidic pollution of bottom sediments of the produced. Among them, 2C9 (a cell line producing Black Sea and of their significant share in the deep- rNfCPB-McAb) and 1C8 (a cell line producing water benthic communities. Benthic Ciliophora, rNfCPBL-McAb) which showed high antibody Gromiida and Foraminifera are numerous, specific titre, respectively, were selected. 2C9 monoclonal and diverse in these conditions. The contribution of antibody was reacted with N. fowleri whole lysate or these protozoans in the meiobenthos communities rNfCPB fusion protein by western blotting, as which had high values and extremely prone to variability. showed about 28 kDa and 38.4 kDa of band pattern, The spatial-bathymetrical distribution of these respectively. 1C9 monoclonal antibody was reacted protists along with benthic multicellular organisms with N. fowleri whole lysate or rNfCPBL fusion had uneven character in the Black Sea. protein by western blotting, as which showed 24 kDa and 34 kDa of band pattern. 2C9 and 1C8 monoclo- CHARACTERIZATION OF “CANDIDATUS nal antibodies were not reacted with another amoebic GORTZIA SHAHRAZADIS”, A NOVEL ENDO- lysates such as N. gruberi, Acanthamoeba castellanii, SYMBIONT OF PARAMECIUM MULTIMI- A. polyphaga in western blot analysis. In the results CRONUCLEATUM FROM INDIA of the immunocytochemistry analysis, NfCPB and Serra V.1, Fokin S.I.1,2, Castelli M.1, Basuri C.K.3, NfCPB-L protein were mainly detected in cytoplasm Nitla V.M.3, Verni F.1, Sandeep B.V.3,4, Kalavathi and cell membrane, especially pseudopodia, of N. C.3, Petroni G.1 fowleri trophozoites under a confocal microscope. 1 - Department of Biology, University of Pisa, Pisa, 70 · “PROTIST–2016”

Italy Multiple bacterial symbioses are rather common 2 - Department of Invertebrate Zoology, St. Petersburg phenomenon in ciliates. Some intracellular bacte- State University, St. Petersburg, Russia ria, like “Candidatus Megaira polyxenophila”, 3 - Department of Zoology, Andhra University, apparently do not produce any effect on the host, and Visakhapatnam, India their mode of interaction with ciliate is unknown; 4 - Department of Biotechnology, Andhra University, some other, like Holospora, are highly infectious Visakhapatnam, India bacteria actively exploiting the host resources. [email protected] However, little is known about interactions among Ciliates are known since a long time to be potential endosymbiotic bacteria inhabiting the same host hosts for bacteria. Some of the most investigated cell. The simultaneous infection of a ciliate with species belong to the so-called Holospora-like several bacteria has been observed but not studied bacteria (HLB) group, which includes all known in detail. Finally, the ability of a bacterium to Holospora species and “Candidatus Gortzia” genus. enter and associate with the host cell already HLB are obligate endosymbionts, inhabiting the occupied by another symbiont remains unclear; macronucleus or the micronucleus of certain species indeed the possible competition for the same cell of Paramecium and, less frequently, Frontonia. compartment between the “native” bacterium They are Gram-negative, non-motile Alphapro- and an “invader” has never been investigated. teobacteria with a distinctive life cycle involving We analyzed how the host cell reacts to multiple two different morphotypes: the smaller and almost infections, and how different symbiotic bacteria roundish reproductive (RF) form, and the much interact inside the host. For this purpose, four strains more elongated, rod-like infectious form (IF). of Paramecium caudatum harboring “Candidatus In the present work we describe a novel species Megaira polyxenophila“ in macronucleus were belonging to “Ca. Gortzia” genus, detected in experimentally infected with representatives of P. multimicronucleatum, a ciliate in which HLB several Holospora species. During our observations have never been reported before. This novel endo- “Candidatus Megaira polyxenophila“ prevented symbiont shows unusual features respect its relatives, development of macronuclear Holospora species, such as deep variations in IF morphology and thus showing the tendency to be the only occupier of capability to infect occasionally host cytoplasm. It the host macronucleus. On the contrary, “Candida- presents small, roundish RF (2.5-3.3 µm) and rod- tus Megaira polyxenophila“ allowed the entrance of shaped IF. Two morphotypes of IF were detected Holospora undulata in the host micronucleus, even in different periods: a shorter IF at the beginning in presence of other cytoplasmic endosymbionts. (6.9-10.7 µm), and a longer one (8.2–14.7 µm) Results on reciprocal infections (paramecia harbo- with irregular distribution of periplasm after one ring Holospora infected with “Candidatus Megaira year of cultivation. The latest were found together polyxenophila“) also suggested that symbionts with a high number of transient forms (TF), which protect their cell compartment from invasions by appeared long (6.7–15.5 µm), rod-shaped and not other bacteria in order to ensure their persistence yet differentiated in IF at ultrastructural level. All and spreading in the environment. forms show a constant diameter of 0.7-0.8 µm. No Supported by RFBR 16-04-01195. production of “connecting piece” during host cell division was observed. We propose for this novel CANINE BABESIOSIS IN MOSCOW REGION HLB from India the name of “Candidatus Gortzia OF RUSSIA shahrazadis”. Shaitanov V.M., Yastreb V.B. K.I. Skriabin All-Russian Institute for Fundamental INTERACTION OF DIFFERENT SYMBIOTIC and Applied Parasitology of Animals and Plants BACTERIA IN THE CILIATE PARAMECIUM [email protected] Shaidullina A.1, Lanzoni O.2, Lebedeva N.3, The blood smears of dogs were examined in Moscow Potekhin A.1 region under light microscope in 2011-2014. If 1 - Faculty of Biology, St Petersburg State University, Babesia merozoites were detected the samples were Saint Petersburg, Russia examined with IFA-tests SNAP 4Dx against for 2 - Department of Biology, University of Pisa, Pisa, Ehrlichia canis, Anaplasma phagocytophilum, Borrelia Italy burgdorferi s.l., Dirofilaria immitis. The microfilaria 3 - Centre of Core Facilities “Culture Collections of were visualized in samples after concentration. Microorganisms”, St Petersburg State University, Differential staining of acid phosphatases was used Saint Petersburg, Russia to distinguish Dirofilaria microfilaria. Anamnesis of [email protected] infected dogs was analyzed. The ticks were removed Protistology · 71 from dogs inside veterinary clinic or collected from with the transcriptome of acute nitrosative-stres- vegetation in different ecosystems biotopes of sed trophozoites (TEANS). N-acetyl ornithine Moscow region. During the survey 1045 ticks were deacetylase (NAOD) was among the 208 genes collected (Dermacentor reticulatus, Ixodes ricinus, I. that were up-regulated in NAT. NAOD catalyzes persulcatus). In the natural habitats of Moscow region the deacylation of N-acetyl-L-ornithine to yield 480 ticks were collected (I. ricinus, I. persulcatus, D. ornithine and acetate. Overexpression of NAOD reticulatus). It is presumed that only D. reticulatus is resulted in significant overproduction of putrescine a natural agent of babesiosis transmission in Moscow and in a better adaptation to NS. Surprisingly, region. In total 283 cases of babesiosis of dogs were overexpression of a catalytically inactive NAOD reported, with 8,0% of cases reported for dogs never (mNAOD) resulted in a better adaptation to NS leaving city limits. Remaining 92 % of dogs were but did not result in putrescine overexpression. transported from time to time to country side. In the These results suggest that NAOD has moonlighting majority of dogs the babesiosis is developing without activity. The binding of NAOD or mNAOD to severe consequences, and only in 2,5% of cases the glyceraldehyde 3-phosphate dehydrogenase (GAP acute renal failure was diagnosed. In 1,7% of dogs DH) prevents the formation of putatively toxic high autoimmune hemolytic anemia was observed. About molecular weight GAPDH species and the absence 0,7% of studied dogs were seropositive for D. immitis of these species may underlie the parasite’s ability all other pathogens were absent in these animals. to survive NS. In 1,7% of cases the microfilaria of D. repens were detected and in 0,7% of cases the microfilaria of D. DISTRIBUTION AND CRYOCONSERVA- immitis were found. TION OF PROTISTS IN THE ARCTIC SOILS Shatilovich A.V.1, Mylnikov A.P.2, Bykova S.V.3, N-ACETYL ORNITHINE DEACETYLASE Lupachev A.V.1, Gubin S.V.1 IS A MOONLIGHTING PROTEIN AND IS 1 - Institute of Physico-chemical and Biological INVOLVED IN THE ADAPTATION OF ENTA- Problems in Soil Science, RAS, Pushchino, Russia MOEBA HISTOLYTICA TO NITROSATIVE 2 - Institute for Biology of Inland Waters, RAS, Borok, STRESS Russia Shahi P.1, Vanunu S.1, Avrahami A.1, Trebicz- 3 - Institute of Ecology of the Volga River Basin, RAS, Geffen M.1, Nagaraja S.1, Hertz R.1, Alterzon- Togliatti, Russia Baumel S.1, Reiter I.1, Methling K.2, Lalk M.2, [email protected] Mazumder M.3, Samudrala G.3 Previous investigations have shown that resting 1 - Faculty of Medicine, Technion cysts of soil protists can survive in permafrost 2 - Institute of Biochemistry, University of Greifswald for thousands of years at subzero temperatures. 3 - Jawaharlal Nehru University School of Life However, our knowledge about mechanisms of Sciences long-term cryobiosis remains incomplete. One of [email protected] the uncertainties is the mechanism of cysts transition Entamoeba histolytica is a causative protozoan into the frozen deposits. The main goal of the study parasite of amebiasis, a disease which is characte- was to gauge the protists species abundance and rized by acute inflammation of the colon. Adapta- diversity in the profiles of Cryosols - typical arctic tion of the parasite to toxic levels of nitric oxide soil, that are subjected to the different processes (NO) that are produced by phagocytes may be of cryogenic mass-exchange redistributed the essential for the establishment of chronic amebiasis fragments of the uppermost soil horizons with and the parasite’s survival in its host. In order to microorganisms inhabiting this material. Taxono- obtain insight into the mechanism of E. histolytica’s mic analysis of ciliates and heterotrophic flagellates adaptation to NO, E. histolytica trophozoites were isolated from 21 soil samples revealed 40 species progressively adapted to increasing amounts of the and forms of heterotrophic flagellates from 9 NO donor drug, S-nitrosoglutathione (GSNO) up taxonomical groups and 32 species of ciliates from to a concentration of 110 µM. These NO-adapted 7 taxonomical groups. Protists vertical distribution trophozoites (NAT) were more resistant to an in soil profiles allows to select two groups of species: acute exposure of 350 µM GSNO) and activated 1) species occur only in the modern uppermost soil macrophages and were better at invading porcine horizons and 2) part of the protists’ community colon explants than wild-type trophozoites. The (53% and 55% of ciliates and flagellates species transcriptome of NATs was investigated by RNA respectively) that occurs in material of organic sequencing (RNA-seq) and the results of the and organo-mineral soil horizons both a modern analysis revealed the existence of a weak overlap and buried into the middle and lowermost parts of 72 · “PROTIST–2016”

Cryosol profiles by different processes of cryogenic heterogeneous in terms of nutrient distribution mass-exchange. According to our results about half and many microbial species can actively exploit of protists communities in Cryosols have adaptive these nutrient patches. Exudates of microbes act as and protective mechanisms enabling long-term nutrient hotspots and also provide chemical cues cryptobiosis in the inhospitable conditions of to their prey or predators. In the present study we the arctic soils and permafrost. Fragments of the examined the chemotactic response of starved and/ uppermost soil horizons that were cryoturbated, or non-starved Dunaliella salina to the exudates of buried by solifluction and accumulated in the upper ciliate grazers (Euplotes vannus, Euplotes sp., and layer of permafrost appear to be the ecologycal niche Diophrys oligothrix) by using two approaches; 1. Co- in profiles of Turbic Cryosols that can significantly culturing of two species under different illumination sustain viability of protists. conditions to evaluate the growth, ingestion and behavioural response of the phytoplankton and/ GENOME ANNOTATION OF ACRASIS KONA or ciliates and 2. Using microfluidics and image Sheikh Sanea, Fu Chengjie, Baldauf Sandra analyses to evaluate the response of D. salina to Department of Organismal Biology, Uppsala Uni- the exudates of ciliates and related nutrient media. versity, Sweden D. salina showed different active swimming and [email protected] an ‘attack-like’ behaviour towards ciliates under Acrasids are single-celled amoebae that can undergo different illumination conditions, despite the high aggregative multicellularity in response to adverse ciliate grazing rates on them. When exposed to the environmental conditions, similar to the well- patches of ciliate exudates, f/2 growth medium, yeast studied dictyostelid social amoebas. However, extraction and ammonium solution, both starved acrasids are unrelated to dictyostelids (supergroup and non-starved D. salina showed chemotactic Amoebozoa), being instead the only multicellular accumulation on/around the exudate and nutrient lineage in the eukaryotic supergroup Excavata. This patches. In both cases positive chemotaxis indices makes Acrasis an interesting model system to study were found towards ciliate exudates suggesting that parallel evolution of social behavior in microbes D. salina could actively uptake nutrients released by as well as to explore the diversity of eukaryotes its ciliate grazers. This specific behavioural response in general. We have sequenced the genome and however, could be costly to D. salina. We suggest transcriptome of Acrasis kona and are currently that this behaviour could also serve as a defence preparing transcriptomes from the four main stages mechanism thereby increasing the benefits for D. of its life cycle. In initial work, we assembled the salina. complete A. kona mitochondrial genome (mtDNA) and find that it is missing 14 protein genes present PELAGIC PROTISTS FEEDING ON PICO- in the mtDNA of its closest sequenced relative, CYANOBACTERIA AND THEIR CHLORO- Naegleria gruberii. We further identified 11 of these PHYLL CATABOLISMS protein genes in A. kona nuclear DNA and find that Shihongi A.1, Kinoshita Y.2, Ishikawa A.3, Tamiaki they carry mitochondrial important signals (transit H.2, Kashiyama Y.1,2 peptides, Fu et al. 2014). We are now using RNAseq 1 - Fukui University of Technology data and the N. gruberii genome in an annotation 2 - Ritsumeikan University pipeline to create a fully annotated A. kona nuclear 3 - Mie University assembly. The results will be used to investigate [email protected] parallel evolution of simple multicellularity, early Pico-phytoplanktons dominate the primary pro- steps in the evolution of eukaryotes and to aid in duction in pelagic oligotrophic oceans. Because resolution of the eukaryote tree of life by breaking these settings cover roughly half areas of the up some of the longer deep branches. Earth surface, thus relatively dilute inhabitances of pico-phytoplanktons actually represent a con- CHEMOTAXIS RESPONSE OF PHYTOPLAN- siderable proportion of the photosynthetic pri- KTON TO CILIATES mary production on the Earth. Nonetheless, Shen Z.1,2, Vidyarathna N.K.1, Liu H.B.1 ecological/bioenergetic networks extended from 1 - Division of Life Science, The Hong Kong University these microbial phototrophs have been poorly of Science and Technology, Kowloon, Hong Kong understood due to insufficient knowledge on the 2 - Institute of Hydrobiology, Jinan University, prey-predator relationship, where the minute cells Guangzhou 510631, China should evade grazing by filter-feeding zooplanktons. [email protected] We have thus investigated on pelagic protists that Marine environment at the micro scale level is potentially prey on picophytoplanktons based on Protistology · 73 microscopic observations and chemical detections share morphological characteristics, namely discoid of chlorophylls catabolites. Pelagic water samples mitochondrial cristae and parallel basal bodies, from the northwestern Pacific were subdivided into with euglenozoans. Furthermore, the flagellate has hundred milliliter-scale flasks with additions of trace a euglenozoan-like tripartite flagellar root system, medium components and/or separately cultured albeit the ventral root splits into two bands, which is picocyanobacteria as preys and incubated under similar to the R2 of other typical excavates. On the dim light at 5 to 10 °C. In cultures with additions of other hand, the flagellate lacks some englenozoan Acaryochloris marina, a cyanobacterium (φ = 2-3 features, such as pellicle, paraxial rod, non-tubular µm; nearly “pico”-scale) that produces chlorophyll mastigonemes, or feeding apparatus. Based on these d (Chl-d) instead of chlorophyll a (Chl-a) and, morphological and ultrastructural features, the early 132,173-cyclopheophorbide d enol (a “CPE” derived character evolution of Englenozoa, as well as that of from Chl-d; cPPB-dE) was detected from the Discoba as a whole, will be discussed. cultures after a few day of incubation. CPEs such as Chl-a-derived cPPB-aE have been reported as non- VIABLE AMOEBOID PROTISTS FROM THE phototoxic catabolites of chlorophylls and known to ARCTIC PERMAFROST be produced by many varieties of protists. Because Shmakova L.A. Chl-d was not detected from any of those samples Institute of Physicochemical and Biological Problems without addition of A. marina that is allochthonous in Soil Science, Russian Academy of Sciences to the pelagic settings, the occurrence of cPPB-dE in [email protected] these experiments suggests presence of phycophagic Viable amoeboid protists were isolated from the protists that potentially feed on picophytoplanktons. Arctic Late Pleistocene and Holocene perma- Some flagellates and amoeba were actually observed frost buried soils and sediments. A total of 36 to have ingested cells of A. marina from microscopic strains of protists of the phylum Amoebozoa have measurements. been obtained. Most isolates belong to the genera Acanthamoeba and Flamella. We also isolated CHARACTERIZATION OF STRAIN SRT308; Vannella, Cochliopodium, Acramoeba, Phalansterium A NEW HETEROTROPHIC FLAGELLATE genera. Many of them belong to new species. Two BASAL TO EUGLENOZOA species belonging to the genus Flamella have been Shiratori T.1, Yazaki E.2, Inagaki Y.3, Hashimoto described. They were named Flamella pleistocenica T.3, Ishida K.1 n. sp. and Flamella beringiania n. sp. Two new 1 - Faculty of Life and Environmental Sciences, species of giant Acanthamoeba viruses, Pithovirus University of Tsukuba sibericum and Mollivirus sibericum, were isolated 2 - Graduate School of Life and Environmental from buried soil of the age 34 000 years, and Sciences, University of Tsukuba described. These are the first representatives of the 3 - Center for Computational Sciences, University of two new families of Acanthamoeba giant viruses. We Tsukuba isolated amoebas from the permafrost of the age up [email protected] to 50,000 years. The age of viable organisms enclosed We isolated a new heterotrophic flagellate, strain in the samples corresponds to the time of the last SRT308 from marine sediment sample collected in freezing of the strata. Mechanisms allowing amoebas Republic of Palau on October 2013. The flagellate to survive such a long cryptobiosis are poorly is round or oval shape with two long subequal understood. All amoeba species from permafrost flagella and shows unique rotating motion by are cyst-forming. We studied the composition of beating the both flagella synchronously. Since the the water extract of trophozoites, unmature, and morphological combination of the flagellate is mature cysts of Acanthamoeba castellanii recovered unique, the flagellate is apparently a novel lineage from permafrost. It was demonstrated that the of eukaryotes. In molecular phylogenetic analysis accumulation of osmolytes such as trehalose, using small subunit ribosomal RNA gene sequences, glycerol, tyrosine phosphate, alanine, choline, the flagellate shows no strong affinity with major and a-Glycerophosphocholin occurs during en- eukaryotic lineages. Large scale phylogenetic cystation. These substances are known to serve as analysis using 153 protein-coding genes placed cell protectants during desiccation and freezing. the flagellate at the base of Euglenozoa with strong This study directly shows for the first time that statistical support, suggesting that the flagellate is amoeba cysts can be conserved not only for years a previously undescribed member of the Discoba and decades but for many thousand years and then clade. Consistent with the position inferred from the recover, contributing to the formation of an active phylogenomic analysis, the flagellate was found to microbial community. 74 · “PROTIST–2016”

PARATRYPANOSOMA, THE MOST BASAL POTENTIALLY TOXIC MARINE DINOFLA- BRANCH AND ANOTHER MODEL SPECIES GELLATES: CELL AND MOLECULAR ADAP- AMONG TRYPANOSOMATIDS TATIONS TO STRESS Skalický T.1, Dobáková E.1, Flegontov P.1,2,3, Skarlato S.O. Votýpka J.1,4, Yurchenko V.1,2, Lukeš J.1,5,6 Institute of Cytology RAS, St. Petersburg, Russia 1 - Institute of Parasitology, Biology Centre, Czech [email protected] Academy of Sciences, České Budějovice, Czech Potentially toxic marine planktonic dinoflagellates Republic represent an ecologically important group of 2 - Faculty of Science, University of Ostrava, Ostrava, single-cell eukaryotes which show pronounced Czech Republic physiological adaptability to various types of 3 - A.A. Kharkevich Institute for Information Trans- environmental stress. These protists respond to mission Problems, Russian Academy of Sciences, harsh conditions by activation of their defensive Moscow, Russian Federation mechanisms, which can differ from those of 4 - Department of Parasitology, Faculty of Science, large multicellular forms. The dinoflagellates Charles University, Prague, Czech Republic Prorocentrum minimum (or P. cordatum) in the 5 - Faculty of Science, University of South Bohemia, Baltic Sea have recently become good model objects České Budějovice, Czech Republic in the environmental and cell biology studies of 6 - Canadian Institute for Advanced Research, unicellular eukaryotes. The goal of this paper is to Toronto, Canada demonstrate what cell and molecular mechanisms [email protected] underpin the fast and effective adaptations of these Dozens of trypanosomatids (Trypanosomatida, protists in the brackishwater habitats. We focused Kinetoplastea, Euglenozoa) have been studied on cell metabolism, ion channel research, and from the genomics perspective, however there is a on the effects of different levels of salinity and strong bias towards species pathogenic for mam- elevated temperature on cell mortality, cell cycle mals, while numerous monoxenous (single-host) pattern, RNA synthesis, and DNA replication parasites of insects have been neglected. This year in P. minimum. It appears that P. minimum can we have published the first high-quality genome of utilize urea as a source of organic nitrogen but a monoxenous insect trypanosomatid, Leptomonas also as a source of carbon. Moreover, urea inhibits pyrrhocoris. Here we present a comprehensive assimilation of nitrate (the “classic” source of study of another probably monoxenous species, nitrogen) and can be the preferable substrate in Paratrypanosoma confusum, a cosmopolitan para- the coastal brackish waters. Dinoflagellates show site of Culex mosquitoes. This species forms the elevated biosynthetic activity and low cell-death most basal branch of the trypanosomatid clade. level at critical salinity 5-8 ‰. We infer that this Paratrypanosoma produces three distinct forms in potentially toxic, bloom forming dinoflagellate axenic culture: swimming promastigotes, immobile species displays great colonizing ability across new amastigotes, and sessile cells, firmly attached to the ecosystems likely due to high physiological plasticity surface. Using scanning and transmission electron and pronounced adaptation potential at different microscopy, still and video light microscopy, and levels of biological organization, from molecules and fluorescent antibodies we have fully characterized cells to populations. Funded by the Russian Science the ultrasctructure and behavior of the unique sessile Foundation, project 16-14-10116. morphotype. Formation of transient sessile stage is stimulated by basic pH and by biopterin, and GENOMIC ANALYSIS OF NEPHROMYCES sessile cells are attached to the surface with the help SHEDS INTO AN ENIGMATIC SYMBIOTIC of an extracellular ‘glue’ resistant to all enzymatic SYSTEM BETWEEN A TUNICATE AND A treatments. We have performed differential RNAseq DIVERGENT APICOMPLEXAN analysis of the promastigote and sessile cells, and Slamovits Claudio1,2, Muñoz-Gómez S.1,2, Kennedy sequenced the genome of P. confusum using Illumina K.1, Paight C.3, Lane C.3 MiSeq reads of 250 nt (a paired-end and a mate pair 1 - Dalhousie University library). A draft genome assembly has been pro- 2 - Canadian Institute for Advanced Research duced with the following statistics: scaffold N50 3 - University of Rhode Island of 458 kbp, 189 scaffolds, 31.6 Mbp genome size, [email protected] ~8,800 genes. Using the Paratrypanosoma genome Nephromyces is a divergent apicomplexan that as an outgroup, we have illuminated patterns of lives as an endosymbiont in the renal sac of mol- gene family gains and losses in an important genus gulid tunicates. The nature of Nephromyces as Trypanosoma and in trypanosomatids as a whole. an apicomplexan remained enigmatic for long Protistology · 75 time, mostly due to: (i) its peculiar habitat, (ii) the as protostelids are dispersed among different groups. presence of cytoplasmic bacterial endosymbionts, The basic finding was the split of naked amoebae in (iii) a complex life-cycle with unusual morphologies, two lineages, named Tubulinea and Discosea and and (iv) a presumably mutualistic association with the fact that testate lobose amoebae form just a single its animal host. Moreover, several lines of evidence branch within Tubulinea. The basal branching of suggest that the renal sac of an individual tunicate Amoebozoa could not be properly resolved with SSU host harbors a complex community of diverse trees. Multigene data help to clarify many essential Nephromyces lineages: the multiple-infection questions, e.g. they show that Discosea does not hypothesis. We decided to further investigate this appear to be a monophyletic group. However, with hypothesis and elucidate the phylogenetic position the increment of taxon sampling multigene trees of Nephromyces within Apicomplexa by using seem to experience the same problems and artifacts deep sequencing of renal sacs in order to better as SSU trees did. Recent studies of extreme and understand the developmental dynamics of this exotic habitats revealed a number of new species, symbiosis. Our initial analyses revealed the presence especially among the smallest amoebozoans, some of eleven apicoplast genomes in one single renal were found to be key taxa for rooting Amoebozoan sac. Surprisingly, there was considerable sequence tree. Genbank contains numerous unnamed sequ- divergence among the apicoplast genomes, although ences belonging to new amoebozoan lineages. their gene content and order was highly conserved. This indicates that environmental diversity of We then used the phylogenetic information con- Amoebozoa is still in much unexplored. Supported tained within 27 apicoplast proteins to infer Neph- with RSF grant 14-14-00474. romyces’ phylogenetic placement within Apicom- plexa. These preliminary results support the idea TRANSCRIPTOME ANALYSIS OF NAEGLE- that molgulid renal sacs are complex ecosystems RIA FOWLERI CYSTS AND TROPHOZOTIES inhabited by a diverse community of different BY NEXT-GENERATION SEQUECING Nephromyces lineages. In addition, we clarified the Sohn Hae-Jin1, Seong Gi-Sang1, Kang Heekyoung1, phylogenetic position of Nephromyces as sister to Jung Suk-Yul2, Shin Ho-Joon1, Kim Jong-Hyun3 ‘core’ apicomplexans (Hematozoa+Coccidia), and 1 - Department of Microbiology, Ajou University School highlighted the significant intra-clade divergence of Medicine, Republic of Korea among these apicoplasts. Future efforts will focus 2 - Department of Biomedical Laboratory Science, on elucidating the metabolic contribution of each Molecular Diagnostics Research Institute, Namseoul partner to the dynamics of this complex symbiotic University, Republic of Korea system. 3 - Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Republic PHYLOGENY, DIVERSITY AND EVOLUTI- of Korea ON OF AMOEBOZOA: NEW FINDINGS AND [email protected] NEW PROBLEMS Naegleria fowleri, ubiquitous pathogenic amoe- Smirnov Alexey ba causing the fatal primary amoebic meningo- Department of Invertebrate Zoology, Faculty of Biology, encephalitis (PAM) in experimental animal and St. Petersburg State University, St. Petersburg, Russia humans, is predominantly living in the ponds, lakes, [email protected] rivers and swimming pools. N. fowelri trophozoites Amoebozoa is a major group of protists that unifies are encysted under unfavorable conditions such naked and testate lobose amoebae, varioseans, slime as cold temperature, starvation and desiccation. molds and pelobionts. The diversity and systematics However, the information in differential expression of some groups, like slime molds and testate amoe- genes between cysts and trophozoites of N. fowelri bae, possessing stable morphological characters is very limited. In this study, RNA-sequencing - fruiting bodies or tests - was relatively well stu- libraries from N. fowleri cysts and trophozoites died, also some evolutionary traits derived from were investigated by Next-Generation Sequencing morphology were further disproved by molecular (NGS) analysis. In the NGS database, the assembly phylogeny. In contrast, naked amoeboid protists procedure resulted in mean full length of 11, 254 with no constant shape remain relatively poorly nucleotides in total 42,220 transcript contigs known, molecular phylogeny nearly entirely rearran- and 37.21 % of C+G contents. RNA sequencing ged earlier views on their evolutionary relation- indicated that upregulated 143 genes in cysts showed ships. SSU-based phylogeny shows that naked 2 folds expression in comparison with trophozoites amoebae belong to three different amoebozoan and 163 genes were downregulated. These genes lineages and the most of organisms formerly known were found to participate in Kyoto Encyclopedia 76 · “PROTIST–2016” of Genes and Genomes (KEGG) pathway. KEGG Amoebic trophozoites or cysts were exposed to pathway included metabolisms(131), cellular ClO2 gas (0.064ppmv/min) for 12- 48 hr. Amoebae processes(43), environmental Information proces- maintained for 12 - 48 hr without exposure to sing(22), genetic information processing(66) and ClO2 gas were used for the control groups. By organismal systems(20). On the other hands, by microscopic examination, amoebic viability was analysis of 10,713 sequences via the gene ontology assessed, and then growth rates were estimated. The database, their annotations included biological results showed that the viability of amoebae was processes(1,069) which were cellular process(228), effectively inhibited by treatment with ClO2 gas, as metabolic process(214) and single organisms which their viability were assessed by re-cultivation process(193), molecular functions(415) containing with each flesh medium. The growth rates of A. catalytic activity(195) and binding(186) and cellular castellanii and A. polyphaga trophpzoites which components (923) possessing cells(240) and cell were exposed to ClO2 gas for 24 hr were decreased parts(225). Increased differential expression trans- by 50 and 60 % respectively. Whereas, the growth criptome levels in N. fowleri cysts compared to rate of N. fowleri exposed with ClO2 gas for 24 hr trophozoites were mainly categorized as serine/ was completely inhibited. Furthermore, the actin threonine protease, kinase, and lipid metabolisms mRNA levels of amoebae checked by RT-PCR related protein. Finally, this study may provide were significantly reduced by treatment of CO2 new insights into the environmental resistant gas. Similarly, the growth rate of A. castellanii and genes or pathogenic related genes in N. fowleri A. polyphaga cysts exposed with ClO2 gas for 12 hr survival and infectivity. Kewords: Naegleria fowleri, was significantly inhibited. Taken together, it is cyst, trophozoite, Next-Generation Sequencing, suggested that ClO2 gas has amoebicidal effect on A. transcriptome. castellanii, A. polyphaga and N. fowleri. Therefore, the chlorine dioxide is proposed as an effective agent AMOEBICIDAL ACTIVITY OF CHLORINE for the prevention and control of Acanthamoeba DIOXIDE AGAINST PATHOGENIC ACAN- and N. fowleri infections. Keywords: Acanthamoeba THAMOEBA CASTELLANII, A. POLYPHGA castellanii, A. polyphaga, Naegleri fowleri, chlorine AND NAGELERIA FOWLERI dioxide, amoebicidal effect. Sohn Hae-Jin1, Song Kyoung-Ju2, Seong Gi-Sang1, Kang Heekyoung1, Kim Jong-Hyun3, Kim Jong- MOLECULAR DEFENCE MECHANISMS IN Rak2, Shin Ho-Joon1 SPIROTRICH CILIATES TO PREVENT HEA- 1 - Department of Microbiology, Ajou University School VY METAL TOXICITY of Medicine, Republic of Korea Somasundaram S.1, Abraham J.S.1, Toteja R.1, 2 - PurgoFarm Co. Ltd, Republic of Korea Gupta R.2, Makhija S.1 3 - Institute of Animal Medicine, College of Veterinary 1 - Acharya Narendra Dev College, University of Delhi, Medicine, Gyeongsang National University, Republic Delhi, India of Korea 2 - Maitreyi College, University of Delhi, Delhi, India [email protected] [email protected] Pathogenic free-living amoebae, Acanthamoeba Heavy metal pollution of water is of major concern spp. and Naegleria fowleri, are widely distributed these days especially in developing countries in soil and water. Several species of Acanthamoeba like India. Anthropogenic activity elevates the and N. fowleri can cause serious human infections concentration of such elements, mainly in aquatic such as granulomatous amoebic encephalitis (GAE) systems. About 85 percent of the water pollution is or primary amoebic meningoencephalitis (PAM) caused by domestic and industrial sources. A wide which almost results in death. Recently, there range of contaminants are continuously introduced has been an increased incidence of Acanthamoeba into the water and among these contaminants, keratitis (AK), particularly in contact lens wearers. heavy metals due to their toxicity, accumulation

Chlorine dioxide (ClO2), yellow-green gas is a and non-degradable nature, constitute one of the powerful disinfectant which is 2.5 and 500,000 times most dangerous groups. Prolonged exposure to more effective than chlorine-based disinfectants and heavy metals such as cadmium, copper, lead and alcohol, respectively. In this study, we examined the zinc disturb normal biological function and evoke amoebicidal effect of ClO2 gas against A. castellanii, cellular stress response. Due to stress condition, A. polyphaga and N. fowleri trophozoites or cysts. apart from the release of antioxidant enzymes,

ClO2 gas was induced from “Puristic” that it was a other proteins like Heat Shock Proteins (HSPs) and ready-to-use product using a tubing stick without Metallothioneins (Mts) are the major ones that get necessitating the ClO2 gas generation on site. induced and these proteins trigger changes at the Protistology · 77 gene transcription level. In the present investiga- biomarker to evaluate cytotoxic effect of heavy tion, we studied the acute toxicity of heavy metal - metals. cadmium and the transcriptional changes of hsp70, metallothionein and glutathione peroxidise gene in GENOMIC AND TRANSCRIPTOMIC SUR- spirotrich ciliates. The effect of cadmium on the VEY OF OSMOTROPHIC EUGLENID RHAB- expression of these gene transcripts was investigated DOMONAS COSTATA by real-time qPCR. The gene expression increased Soukal P.1, Hrda S.1, Karnkowska A.1, Strnad H.2, significantly in cadmium treated cells against the Hroudova M.2, Vlcek C.2, Hampl V.1 control cells indicating that these genes may be 1 - Department of Parasitology, Charles University in involved in cellular defence mechanisms by eleva- Prague, Czech Republic ting their gene expression against heavy metal- 2 - Institute of Molecular Genetics, Academy of induced stress. Sciences, Czech Republic [email protected] EVALUATION OF ACUTE TOXICITY OF Euglenids represent a diverse group of protists with HEAVY METALS AND THE ACTIVITY OF diverse modes of feeding including heterotrophy and ANTIOXIDANT ENZYMES IN FRESHWATER photosynthesis, e. g. in Euglena gracilis. Until today, CILIATES there is not available any complete genome sequence Somasundaram S.1, Abraham J.S.1, Toteja R.1, of euglenids and this probably represents the biggest Makhija S.1, Gupta R.2 gap in the sampling of eukaryotic genomes. We 1 - Acharya Narendra Dev College, University of Delhi, are running genomic and transcriptomic projects Delhi, India on a primary osmotroph Rhabdomonas costata. To 2 - Maitreyi College, University of Delhi, Delhi, India avoid bacterial contamination, the DNA sample [email protected]; [email protected] was prepared from cells picked using laser micro- Heavy metal concentration in the environment dissection and amplified by GenomePlex®. Almost is increasing world-wide due to anthropogenic 1GB of reads was generated in four runs on Illumi- activities and India is no exception to this. Metals na MiSeq platform. The current assembly is too are biologically important though increase in the fragmented to be used for genome annotation, threshold concentration of heavy metal in the nevertheless the comparison of transcriptomic and environment may interfere with the metabolic genomic data allow us to estimate gene density activity of organisms. As studies on the cellular and the features of introns. Annotation of the level could detect stress reactions earlier in time mitochondrial backbone metabolism provides the compared to common physiological methods, first data on Rhabdomonas mitochondrion. and since ciliates offer higher sensitivity towards heavy metals as compared to other microorganisms GENE TRANSFER ACCOMPANYING THE mainly due to lack of cell wall, ciliates were used SECONDARY ENDOSYMBIOSIS OF EU- as model organism in this study. In the present GLENID PLASTID investigation, the toxicity of heavy metals, viz., Soukal P.1, Hrda S.1, Karnkowska A.1, Yubuki N.1,2, Cadmium and Copper was assessed in freshwater Hroudova M.3, Vlcek C.3, Elias M.4, Hampl V.1 ciliates; Tetmemena sp. and Euplotes sp. It was noted 1 - Charles University in Prague, Faculty of Science, that growth rate exhibited inverse relationship with Department of Parasitology, Prague, Czech Republic the heavy metal concentration. Mortality rate and 2 - Charles University in Prague, Faculty of Science, various doses of heavy metals (control, LC30, LC50 Department of Zoology, Prague, Czech Republic 3 & LC70) were determined in the ciliates. Generation - ASCR, v. v. i., Institute of Molecular Genetics, of reactive oxygen species (ROS) was determined Department of Genomics and Bioinformatics, Prague, in these ciliates by using specific fluorescent dye Czech Republic in the presence of heavy metals. Increase in ROS 4 - University of Ostrava, Faculty of Science, Life production indicated that heavy metal stress induced Science Research Centre, Ostrava, Czech Republic ROS in living cells. Since it is known that antioxi- [email protected] dant enzymes get activated to detoxify ROS, activity Autotrophic euglenids (Euglenophyta) form a of superoxide dismutase (SOD) and glutathione monophyletic group with secondary green plastids, peroxidase (GPx) were determined in ciliates at which were most probably acquired by their various doses of heavy metals. The levels of SOD common ancestor. However, the acquisition of and GPx were found to be significantly higher in the plastid earlier in the evolution of euglenids the presence of heavy metals. This study indicates (plastid-early hypothesis) cannot be ruled out. The that antioxidant enzymes can be used as an effective process of organelle acquisition is accompanied by 78 · “PROTIST–2016” the transfer of genes from the endosymbiont to host tsiales” (Alphaproteobacteria), clustering with the (EGT), the presence of such genes indicates past recently proposed family “Candidatus Paracaedi- endosymbiosis. To test the plastid-early hypothesis bacteraceae”. We obtained tree topologies show- and to learn more about the contribution of EGT to ing a strong separation of Rickettsiales into at euglenid genome, we have analyzed transcriptomes least two groups; one represented by the families of 5 euglenids (2 osmotrophic, 3 autotrophic) using a Rickettsiaceae, Anaplasmataceae, and “Candi- pipeline, which enabled us to select genes related to datus Midichloriaceae”, the so-called RAM clade, algae. The contribution of algal genes in autotrophic and the other group represented by “basal Ri- euglenids (around 2 % of genes) is higher than in ckettsiales” including “Candidatus Jurandia para- primary osmotrophs (around 0.07 %) supporting the meciophila”. Therefore, and in accordance with plastid-late hypothesis. Surprisingly, we observed a other recent publications, we propose to limit high number of genes related to other algal groups the order Rickettsiales to the RAM clade and rise than green algae. “basal Rickettsiales” to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, CHARACTERIZATION OF A NOVEL PARA- presently comprising four clades at the family level. MECIUM ENDOSYMBIONT AND A CRITIC- AL REVISION OF “BASAL RICKETTSIALES” ´ANAERAMOEBA´ - NEW LINEAGE WITHIN Szokoli F.1,2, Schrallhammer M.3, Sabaneyeva E.4, METAMONADA Castelli M.2, Krenek S.1, Doak T.G.5,6, Verni F.2, Táborský P.1, Pánek T.1,2, Kolisko M.3, Čepička I.1 Berendonk T.U.1, Petroni G.2 1 - Department of Zoology, Charles University in 1 - Institut für Hydrobiologie, Technische Universität Prague, Prague, Czech Republic Dresden, Germany 2 - Department of Biology and Ecology Faculty of 2 - Dipartimento di Biologia, Università di Pisa, Italy Science University of Ostrava Chittussiho 10, Ostrava, 3 - Mikrobiologie, Institut für Biologie II, Albert- 710 00 Ludwigs-Universität Freiburg, Germany 3 - Department of Botany, University of British 4 - Department of Cytology and Histology, St. Columbia, Vancouver, British Columbia, Canada Petersburg State University, Russia [email protected] 5 - Biology, Indiana University, IN, USA We have isolated and cultured nine strains of 6 - National Center for Genome Analysis Support, anaerobic amoebae (´Anaeramoeba´) from marine IN, USA coastal hypoxic sediments worldwide. ´Anaer- [email protected] amoeba´ cells were fan-shaped, with extremely In the past ten years, the number of newly des- flattened pseudopodia and trailing uroidal filaments, cribed endosymbionts within the bacterial order and morphologically belonging to flamellian Rickettsiales has constantly grown, and 18 novel or flabellate morphotype. We distinguished six Rickettsiales genera inhabiting protists—such as morphospecies that differed in a number of features ciliates or amoeba—have been described since 2006. including cell size and nuclear morphology. ´Anaer- Half of these new species belong to the recently amoeba´ species shared a unique combination proposed family “Candidatus Midichloriaceae”, of morphological features. All three strains exa- whereas five novel bacterial symbionts were found mined by TEM possessed double membrane- belonging to Rickettsiaceae and six belonging bound organelles with no cristae, presumably to Holosporaceae and other Rickettsiales with hydrogenosomes, associated with prokaryotes uncertain position (“basal Rickettsiales”). In this (probably methanogens). Cytoplasm contained a work, we provide molecular and ultrastructural large acentriolar centrosome, and no basal bodies data on a novel bacterial endosymbiont inhabiting were observed. Peculiar isokont flagellates with two a Paramecium biaurelia isolate collected near or four flagella were rarely observed in two strains Bloomington, Indiana (USA). We propose the representing different species. Single- and also five- name “Candidatus Jurandia parameciophila” in gene analyses showed that ´Anaeramoeba´ clade honor of Dr. Artur Jurand. The Gram-negative was monophyletic, but were completely unable bacteria (1.5-2.0 × 0.35-0.38 µm in size) occupies to assess its phylogenetic position. We analysed the host cytoplasm and is devoid of flagella. For transcriptomic data of two ´Anaeramoeba´species. molecular characterization, the SSU rRNA gene Phylogenomic analysis based on 160 protein-coding was sequenced and used for taxonomic assign- genes surprisingly showed that ´Anaeramoeba´ ment and the design of species-specific oligonuc- instead of being a member of Amoebozoa, represents leotide probes. Phylogenetic analyses indicate a novel deep lineage within Metamonada (Eukaryota: that the endosymbiont belongs to “basal Ricket- Excavata). Protistology · 79

THE DRAFT GENOME OF KIPFERIA BIALATA the algaeflora in plankton of river Zivil and 10 of REVEALS THAT THE GAIN OF FUNCTION its tributaries. In the composition of autotrophic CONTRIBUTES THE MASSIVE REDUCTIVE plankton were recorded 105 species of protists. EVOLUTION IN METAMONADA They accounted for 25% of the total species Tanifuji G.1,2, Takabayashi S.2, Kume K.2, Takagi richness of the algalflora. In the Zivil was met 77 M.T.2, Inagaki Y.I.2, Hashimoto T.2 species of protists, in the rivers – 67. However, 1 - National Museum of Nature and Science the average number of species in one sample in the 2 - University of Tsukuba tributaries was 6,7, and in the Zivil – 2,75. The [email protected] qualitative composition of protists of Zivil and its Metamonada is a unicellular eukaryotic group tributaries are similar. The coefficient of species known to consist of free-living and parasitic similarity of Sorenson calculated for the Zivil and organisms. Almost all of metamonads have adapted its tributaries - 53%. Abundance and biomass of to anaerobic or micro aerobic environments, and the protists was significantly higher in the Zivil, lost the several mitochondrial functions such as the compared to the tribu-taries. The contribution of oxidative phosphorylation. The biological pathways protists to the formation of the total abundance localized in those reduced mitochondria (so-called and biomass of phytoplank-ton was also higher in mitochondrion-related organelles or MROs) vary the river Zivil. Thus, the average specific number of depending on the species. The nuclear genome species of protists in the little river is lower than in of the model parasites such as Giradia intestinalis its tributaries. This is probably due to a much more was also reduced in terms of the genome structure varied conditions. However, in the river compared and the number of coding proteins, presumably to the tributaries, the indices of quantitative deve- throughout the adaptive evolution to the intra- lopment of higher protists. Great is the contribution cellular lifestyle. However, little is known how of protist in the formation of the total abundance and the genome reduction progressed in this parasite. biomass of phytoplankton in the Zivil, compared to Here, we present the draft genome sequence of the the tributaries. free-living Kipferia bialata, which is a phylogenetic relative of G. intestinalis, and compare it to the CHANGE COMMUNITY OF AUTOTROPHIC genomes of the model metamonad parasites, G. PROTISTS FROM THE SOURCE TO THE intestinalis and Trichomonas vaginalis. Our data MOUTH OF A SMALL RIVER (ON THE show that 1) K. bialata possesses two substrate-level EXAMPLE OF THE ZIVIL, THE REPUBLIC phosphorylation pathways—one is homologous to OF CHUVASHIA) that in G. intestinalis and the other is to that in T. Tarasova N.G., Tretyakova T.P. vaginalis, suggesting the once expansion of ATP Institute of ecology of the Volga river basin, Toglyatti, synthesis pathways in the metamonad evolution Toglyatti state University, Russia to reach parasitic life style of G. intestinalis, and 2) [email protected] no variant-specific surface protein (VSP), possibly Zivil river – inland waterway of the republic, an evasion mechanism of the host immunity in G. the right tributary of the Volga. The study of the intestinalis, was detected from K. bialata genome, biota of the river began in 1913. In the summer suggesting that the VSPs were acquired somehow on of 2013 conducted a comprehensive study of the the line leading to G. intestinalis after the divergence reservoir from the headwaters to the mouth. In the of K. bialata. In sum, our results suggested that the composition of algaflora was discovered 77 species of gain of function/protein conversely contributed to protists from divisions Chryzophyta, Cryptophyta, the massive reductive evolution in metamonads. Dinophyta, Euglenophyta and Raphydophyta. This is 25% of the total species richness of the algal AUTOTROPHIC PROTISTS OF THE LITTLE flora. The river experiences significant agricultu- RIVER AND ITS TRIBUTARIES (ON THE ral load, so the maximum diversity differed the EXAMPLE OF THE ZIVIL, THE REPUBLIC division Euglenophyta. The specific number of OF CHUVASHIA) species of protists varied from 3 to 28. Maximum Tarasova N.G.1, Burkova T.N.2. species richness was registered in the region of the 1 - Institute of ecology of the Volga river basin of RAS mouth of the Warm river, experiencing significant 2 - Toglyatti state University, Russia development pressure. In most cases, the increase in [email protected] species richness of protists after the confluence of the Small river is a complex natural object. Natural river tributaries. From its source to the mouth of the and human-induced change affect the health of its river is the growth of the species richness of protists. components. In the summer of 2013 was a study of The population and biomass of protists also increase 80 · “PROTIST–2016” from the headwaters to the mouth. The maximum PROFILING THE PROTOZOAN TAXONO- development of the protists received at the mouth MIC AND FUNCTIONAL DIVERSITY OF AN of Warm Creek. Here the number of protists in ANTARCTIC DRY VALLEY 50, and biomass 25 times higher than the average. Thompson A.R.1, Buelow H.2, Takachs-Vesbach Thus, the protists of a small river with significant C.2, Adams B.J.1 agricultural load, have high species diversity. The 1 - Department of Biology, Brigham Young University trends of increasing species richness, abundance and 2 - Department of Biology, University of New Mexico biomass of protists from the source to the mouth of [email protected] the watercourse. The McMurdo Dry Valleys of Victoria Land Antarctica are some of the harshest terrestrial THE PROTISTAN SPECIES MAXIMUM CON- habitat on earth. At 78°S and 163°E, the average CEPT AND ITS IMPLICATIONS TO INVASIVE temperature of the region is <-20°C, precipitation BIOLOGY is <10 cm yr -1 and the growing period during the Telesh I.V. austral summer lasts only a few months when the Zoological Institute RAS, St. Petersburg, Russia landscape is warmed enough for glacier-fed streams [email protected] to flow and some permafrost to thaw. Life is almost The paper discusses possible implications of the entirely microbial in these valleys and the landscape novel protistan species maximum concept for the is dominated by extremely dry soils that are highly predictive modelling of ecosystem resilience and saline, oligotrophic, basic in pH and subject to vulnerability to alien species invasions. Recent frequent freeze-thaw cycles. This system is a great development of the protistan species maximum outdoor laboratory as the extreme conditions have concept for the challenging zone of critical salinity reduced complexity such that intricate interactions 5-8‰ (horohalinicum), where macrozoobenthos between soil taxa can be unraveled. Our research experience destructive osmotic stress (Telesh et aims to investigate protozoan diversity in these al., 2011), is presented. This concept arose from valleys in order to understand fundamental aspects the discovery that not all brackish waters are poor of protozoan ecology that can be applied broadly. in plankton species, which was exemplified by the However, our understanding of the taxonomic Baltic Sea data. The concept has gradually evolved diversity, distribution and functional roles of these to the understanding that in the environment with organisms in these valleys is still lacking. To begin to sharp salinity fluctuations community regulations address this, a variety of soils were sampled from two and life strategies of small unicellular planktonic valley systems and metagenome and transcriptome organisms differ substantially from those of large datasets were constructed using next generation multicellular bottom-dwellers. Special efforts were sequencing. Our results so far suggest that there undertaken to define the major organismal traits is greater taxonomic and functional diversity than and environmental factors responsible for this was previously thought present, with relatively new peculiar biodiversity pattern. Phytoplankton high representation from many major ciliate and cell size, seasonality in development, and water cercozoan clades. Future sequencing efforts will salinity were tested as key characteristics. A long- undoubtedly shed more light on distribution and term (1972-2006) phytoplankton dataset from the richness of individual OTUs. Baltic Sea (“the sea of invaders”) was analyzed by means of correlation analysis, non-metric multi- MANY PREVIOUSLY INCERTAE SEDIS AMO- dimensional scaling, and rarefaction analysis. EBOZOANS FIND A HOME WITHIN THE Results prove statistically that algal cell size CENTRAMOEBIDA minimum underpins the protistan species maximum Tice A.K.1,2, Shadwick L.L.3, Spiegel F.W.3, Geisen in the horohalinicum. Seasonality in phytoplankton S.4, Fiore-Donno A.M.4, Bonkowski M.4, Dumack development promotes the shift in community K.4, Kang S.1,2, Brown M.W.1,2 composition towards dominance of the small- 1 - Department of Biological Sciences, Mississippi State sized species in the critical salinity regions. The University, Mississippi State, MS, 39762 protistan species maximum in the horohalinicum 2 - Institute for Genomics, Biocomputing and Bio- is largely backed up by the significant between- technology, Mississippi State University, Mississippi sample variation in species richness in the highly State, MS, 39762 changeable brackish-water environment. The work 3 - Department of Biological Sciences, University of was funded in part by the Russian Foundation for Arkansas, Fayetteville, AR, 72701 Basic Research, project 15-29-02706. 4 - University of Cologne, Institute of Zoology, Protistology · 81

Department of Terrestrial Ecology, Cologne, Germany reliable comparators; organisms with ancestral or [email protected] intermediate features that represent sister groups of The Centramoebida is a group of amoebozoans well-known taxa. Recent studies on the diversity of with flattened trophic cells that display finely heterotrophic protists and environmental sequence pointed subpseudopodia and a prominent lamellate data have indicated that many such “sister” lineages microtubular organizing center (MTOC). This group exist but remain insufficiently known primarily of amoebozoans has received considerable attention because they are not available in stable cultures. from the broader scientific community due to the Among heterotrophs, predatory protists are of pathenogenic nature and ecological importance of particular interest because their diversity may some of its members. Here we increase the known be the least understood of all protists: they are diversity of the Centramoebida by demonstrating comparatively rare in natural samples and often through multi and single gene phylogenetics difficult to maintain in the laboratory. However, along with light and electron microscopy that the their ability to feed on protozoa and microalgae is formally incertae sedis amoebozoans “Protostelium” relevant to understanding the role of phagotrophy in pyriformis, “Protostelium” arachisporum, and the origin of mitochondria and plastids, and many Stereomyxa ramosa belong within the Centramoe- represent deep-branching evolutionary lineages that bida. We also describe Vacuolamoeba acanthaformis could help us illuminate the origin of major protist n.g. n.sp. a new centramoebid isolated from soil in supergroups and resolve their relationships. Here we Tibet. Our phylogenetic analyses recover a highly report the establishment of multiple cultures of rare, supported Centramoebida that includes highly free-living predatory flagellates, which occupy basal supported clades comprised of “P.” arachisporum, or intermediate evolutionary positions within major Stereomyxa ramosa and Vacuolamoeba acanthafor- eukaryotic supergroups (Alveolata, Stramenopiles, mis respectively. We also show “P.” pyriformis is Rhizaria, Opisthokonta, Kinetoplastida), or repre- robustly nested within the genus Acanthamoeba sent independent deep-branching eukaryotic making it the first reported protosteloid member lineages. Molecular and morphological data from of the genus. As a result of these analyses, we these isolates reveals ancestral and unique mor- formally transfer “Protostelium” arachisporum phological and ultrastructural features, slowly into the genus Luapelameoba and “Protostelium” evolving nuclear genes, and large, slow-evolving pyriformis into the genus Acanthamoeba establishing mitochondrial gene sets. the combinations Luapeleamoeba arachisporum This study was supported by the Russian Science n. comb. and Acanthamoeba pyriformis n. comb. Foundation (grant no. 14-14-00515). Our results greatly increase the known diversity of this medically and ecologically important group, DEVELOPMENT OF THE INTEGRATED and further demonstrate the widespread nature of INFORMATION WEB-SYSTEM ON A BIODI- sporocarpic fruiting across the tree of Amoebozoa. VERSITY OF PROTISTS “PROTIST.RU” Tikhonenkov D.V.1, Tychkov A.Yu.2, Azovsky A.I.3, THE EVOLUTIONARY IMPORTANCE OF Bobrov A.A.3, Plotnikov A.O.4, Tsyganov A.N.2, PREDATORY FLAGELLATES: NEW DEEP Mylnikov A.P.1 BRANCHES ON THE EUKARYOTIC TREE 1 - Institute for Biology of Inland Waters, Russian OF LIFE Academy of Sciences Tikhonenkov D.V.1, Janouškovec J.2, Hehenberger 2 - Penza State University E.3, Burki F.4, Gawryluk R.3, Karnkowska A.3, 3 - Moscow State University Mylnikov A.P.1, Keeling P.J.3 4 - Institute for Cellular and Intracellular Symbiosis, 1 - Institute for Biology of Inland Waters, Russian Russian Academy of Sciences Academy of Sciences [email protected] 2 - Department of Genetics, Evolution & Environment, Protists are the most heterogeneous at cellular and University College London molecular levels group of eukaryotes. They play 3 - Botany Department, University of British Columbia an important role in ecosystem functioning as a 4 - Systematic Biology Department, Uppsala University part of microbial communities and have a high [email protected] potential for human practical activity as a resource The origin and early radiation of major protist for the development of biotechnologies. Inventory, supergroups are linked to understanding key transiti- systematization, and integration of the data on the ons in the evolution fo eukaryotes: endosymbiosis, diversity of protists are of primary importance. genome evolution, and morphological novelty. This We have made the technical platform and started understanding is often limited by the absence of the development of web-system, which allows 82 · “PROTIST–2016” integrating the information on morphological, development of diverse lobosean assemblages. molecular, and ecological features of different Striking similarity between assemblages of Penard protist species. The content of the web-system in the profundal zone of deep Swiss lakes and the will include morphological descriptions and middle watershed of the Danube are attributed to differential diagnoses of the species, photos of living the above environmental factors. It is still debated, cells and whole mounts, electron-microscopic why majority of the presented species have not illustrations of coverings and cell ultrastructure, been reported out of Europe. Finally, a concise movies of live cells, drawings, sequences of methodological proposal is presented to the de- phylogenetically important genes, molecular tection of these inconspicuous testate amoeba phylogenetic trees, databases on geographic and species. Funding: Hungarian Scientific Research biotopic distribution of the species, protistological Fund (T49632). literature. Now we focus on four groups of free-living protists: heterotrophic flagellates, testate amoebae, RARE CORYTHIONELLA AND CYPHODERIA ciliates, centroheliozoans. The work on interactive SPECIES IN THE HUNGARIAN SECTION OF illustrative keys for some taxonomic groups was THE DANUBE started. Registered on the website users can upload Török J.K. and edit content, create personal profiles, and topics Department of Systematic Zoology and Ecology, on the forum. We invite all protistologists to join Eötvös Loránd University, Budapest our team. The integrated web-system is available [email protected] for observing on the Internet at http://protist.ru/. Sandy sediment and seston were investigated This study was supported by the Russian Foundation in several sites north from Budapest along the for Basic Research (grant 15-29-02518). Danube riparian zone in order to find rare filose testate amoebae. Finally, ca. 20 kilometers north HIDDEN DIVERSITY OF FILOSE TESTATE from the capital Corythionella golemanskyi tests AMOEBAE IN THE MIDDLE WATERSHED were detected in 2015, enough for morphometric OF THE DANUBE analysis. A repeated sampling four months later Török J.K. resulted specimens of Cyphoderia calceolus with Department of Systematic Zoology and Ecology, enormous form variations. Shell structure details Eötvös Loránd University, Budapest and morphometry are given for both species. [email protected] Characteristics of the latter are compared with Diversity of filose testate amoebae in the middle those of Cyphoderia myosurus, detected in 1996–97 watershed of the Danube has been searched for sampling of the Danube in the Szigetköz area. As decades, but still features with new detections. Penard (1908) has stated, Cyphoderia myosurus Corythionella golemanskyi is first time detected in has elongated shell plates. Cyphoderia calceolus, Europe in satisfactory number of individuals for however, comprises non-overlapping plates, often correct identification. Cyphoderia calceolus and kidney shaped, without a regular circular outline. Cyphoderia myosurus specimens found during the last Morphometry of Corythionella golemanskyi shells twenty years make possible to distinguish between leads us to conclude that the formerly found active these two similar and rare taxa. Reviewing all the Corythionella specimens (sampled in Szigetköz rare filose testaceans found in the middle watershed Danube, 1996) belong to another species. The very of River Danube from 1962 and considering their first Corythionella specimens ever found in Hungary sampled microhabitats, we conclude that these were detected as early as 1996 at the same site, but species are generally adapted to low organic material being not yet described, they were preserved as weird content and high oxygen concentration. Testate Cyphoderia laevis specimens. These data suggest that amoeba species lists from Hungary are compared although Corythionella is present in the Danube, its throughout five decades, highlighting habitat shy appearence prevents it from recognition during requirements for rare filoseans. We argue, that water analysis of turbid sediment samples. Funding: flow has an important role in formation of riverine Hungarian Scientific Research Fund (T49632). testate amoeba communities: Space clearing floods open new microhabitats for colonization from local PARVULARIA GEN. NOV. REPRESENTS A refugia. Microhabitats like sandy riparian zone FILOSE AMOEBA RELATED TO NUCLEA- remaining free from intensive organic load are RIIDS, THE EARLIEST-BRANCHING LINE- occasionally supplied with additional fresh ground AGE OF HOLOMYCOTA (OPISTHOKONTA) water, and can maintain favourable environmental Torruella G.1, López-Escardó D.2, Moreira D.1, conditions for filoseans for a long time, while prevent Ruiz-Trillo I.2,3, López-García P.1 Protistology · 83

1 - Unité d’Ecologie, Systématique et Evolution, identified from the water column and sediments. Université Paris-Sud 11, France Twenty-seven species, varieties and forms from the 2 - Institut de Biologia Evolutiva (CSIC-Universitat genera Arcella, Centropyxis, Cyclopyxis, Difflugia and Pompeu Fabra), Catalunya Pyxidicula were found in Vietnam for the first time. 3 - Institució Catalana de Recerca i Estudis Avançats The updated list of testate amoebae in Vietnam now (ICREA) includes 286 taxa. The genera Arcella, Centropyxis and [email protected] Difflugia are characterized by the highest frequency Opisthokonts, one of the largest eukaryotic super- of occurrence. The species diversity of Arcella, groups, is divided into two major clades: the Holozoa, Centropyxis, Difflugia was the highest. Centropyxis encompassing animals and their unicellular relatives, aculeata, Arcella discoides, Difflugia schurmanni, and the Holomycota, including fungi and their Netzelia oviformis and Difflugia limnetica were the unicellular relatives. The earliest-branching lineage most common species. Some taxa have shown in Holomycota, which has received many names restricted distribution, e.g. Lesquereusia modesta (e.g. Nucleariidae, Cristidiscoidia, Discicristata), was found in Bau Trang lake with a high frequency remains poorly studied. This group of phagotrophic of occurrence, but was not observed in Bau Sen lake. filose amoebae bears contrasting features in com- The number of observed species,varieties and forms parison with those of their close relatives, the in each lake is 34. The average number of the species parasitic Opisthosporidia and the osmotrophic fungi. per sample on the shores of the lakes subjected to It originally contained a single genus, Nuclearia, human agricultural activity was significantly higher which includes naked amoebae of ca. 40 µm cell than in the samples from the shores of the lakes diameter that feed on filamentous cyanobacteria in with less anthropogenic disturbance. The curves of freshwater environments. More recently, Fonticula “cumulative species number vs. sampling effort” are alba was included as sister to Nuclearia spp. With well fitted by equations S = 7.60N0.69 for Bau Sen much smaller cell size and bacterivore, this amoeba lake and S = 12.52N0.46 for Bau Trang. The curves presents an aggregative multicellular fruiting body. are unsaturated, which indicates that more intensive Here we describe the new genus Parvularia nov. investigations of testate amoebae should be expected gen., a small filose amoeba formerly called Nuclearia to reveal more species. sp. ATCC50694, and we compare its morphological features with those of the genera Nuclearia and DIVERSITY OF THE GENUS MONOCERCO- Fonticula. We also review the whole nucleariid MONOIDES lineage at the onset of Holomycota, focusing on their Treitli S.C.1, Kotyk M.2, Yubuki N.1, Vlasáková diversity, ecology and evolutionary importance. J.1, Šrámová E.1, Smejkalová P.1, Novotná K.K.1, SSU rRNA-based phylogenetic analyses including Čepička I.2, Hampl V.1 environmental sequences suggest that nucleariids are 1 - Charles University in Prague, Faculty of Science, relatively scarce and thrive exclusively in freshwater Department of Parasitology, Prague, Czech Republic systems. Based on existing transcriptomic data for 2 - Charles University in Prague, Faculty of Science, Parvularia and novel data for a canonical Nuclearia Department of Zoology, Prague, Czech Republic strain, we carried out phylogenomic analyses to [email protected] study the internal phylogeny of the whole group. Monocercomonoides sp. is a genus of small flagellates Nucleariids occupy a key evolutionary position, such belonging to the order Oxymonadida. They live as that mapping phenotypic traits on the phylogeny intestinal endosymbionts of insects, but some of of Opisthokonts will help understanding important them can be found also in the intestine of vertebrates. evolutionary transitions such multicellularity, In this work we sequenced the SSU rRNA of 35 parasitism or osmotrophy. different strains of Monocercomonoides isolated from various insect and vertebrate hosts and from TESTATE AMOEBA IN BAU SEN AND BAU unused cesspit. We have performed phylogenetic TRANG LAKES, BINH THUAN PROVINCE, analysis in order to understand the diversity of VIETNAM this genus. Our preliminary results indicate large Tran Q.H., Nguyen T.H.T. variation among strains at the genetic level with Vietnamese-Russian Tropical Center some strains having a typical 1800 bp SSU and [email protected] some having around 3000 bp SSU sequence. The The species composition and distribution of testate strains formed two clades, potentially distinct amoebae in Bau Sen and Bau Trang lakes (Binh genera, however the position of these clades in the Thuan Province, Vietnam) were investigated. oxymonad trees is unclear and we have so far not Fifty-three species, varieties and forms have been found any good diagnostic feature discriminating 84 · “PROTIST–2016” between them. Morphological analysis of selected 3 - School of Science, Mae Fah Luang University strains from the major clade, which we consider as 4 - University of British Columbia the genus Monocercomonoides, failed to find suitable 5 - School of Biosciences, University of Exeter characters for species delineation and it is also very [email protected] difficult to assign lineages to described species. Based Mitochondria are important organelles in all on the morphology and host origin, we assume that eukaryotes and their protein composition and the lineage containing Chinchilla isolate PA203, function(s) depend on the lifestyle of the organism and isolates from guinea pig and Chameleo cristatus harboring them. While being very well studied, their represent species M. exilis. functional distribution amongst ciliates remains a dark box. The Ciliophora group is currently one of CILIOFAUNA OF NATURAL AND ARTIFI- the most studied lineages of single-cell eukaryotes, CIAL ECOSYSTEMS OF KHABAROVSK TER- mainly due to their abundance in diverse habitats. RITORY Despite this, investigations on the cell biology and Tribun M.M., Zhykov A.V., Nikitina L.I. the functional diversification of their organelles Far Eastern State Transport University have been limited, with the exception of certain [email protected] species, mainly Paramecium, Tetrahymena and Environmental degradation due to pollution Nyctotherus. These organisms have been shown to by industrial, agricultural and municipal waste harbor either typical mitochondria or hydrogen- gave rise to serious problems, associated with the producing mitochondria, while their mitochondrial violation of the ecological balance in ecosystems genome has an unusual codon usage. In this project, and the biosphere. Ciliata - an important object we used the recently acquired transcriptomic and in the model laboratory experiments, as they are genomic data to predict the functional diversity used as an indicator of the degree of human impact of mitochondria and related organelles within the on the environment. Sampling was carried out group of ciliates. Our analyses demonstrate that in small rivers of the Khabarovsk territory (rivers members of the ciliates harbor distinctive classes Krasnaya, Chernaya, Berezovaya) and aeration of mitochondria, with a variety of functions and tanks in Khabarovsk (town Berezovii). During adaptations. Two species have been predicted to inventory ciliofauna has been identified 152 have remnant mitochondria (mitosomes) and species of ciliates. The greatest number of species one of them hydrogen-producing mitochondria represented Oligohymenophorea class - 33 and (hydrogenosomes). Surprisingly, we are currently Vorticellidae family - 12 species. 3 species of ciliates observing the whole spectrum of functional clas- were eurybiontic (Paramecium caudatum, Colpidium sification of mitochondria just within ciliates; campylym, Uronema marinum). Species ciliofauna Ciliophora is an exceptional model group to similarity between small rivers and aeration tanks monitor in real time the functional adaptations of is 30% (according to Sorensen coefficient). Ciliata mitochondria. specific types of small rivers Khabarovsk - 14 (32% of the number of species recorded in small rivers), in TESTATE AMOEBA TRANSFER FUNCTION aeration tanks - 32 (57%). Specific types belonged PERFORMANCE ALONG LOCALISED Armophorea classes - 3 kinds, Litostomatea - 3 kinds, HYDROLOGICAL GRADIENTS Phyllopharyngea - 8 species, Oligohymenophorea Tsyganov A.N.1, Mityaeva O.A.1, Mazei Yu.A.1,2, - 13 species. Thus, faunal analysis of ciliofauna Payne R.J.1,3 aquatic ecosystems Khabarovsk shows how the 1 - Department of Zoology and Ecology, Penza State presence of similar species found in a variety of University, Krasnaya str. 40, Penza, 440026, Russia biological communities and the presence of specific 2 - Department of Hydrobiology, Lomonosov Moscow representatives. In particular, this phenomenon is State University, Leninskiye gory, 1, Moscow, 119991, characteristic of biocenosis aeration. Russia 3 - Environment Department, University of York, OVERVIEW OF THE WHOLE SPECTRUM OF Heslington, York YO10 5DD, United Kingdom MITOCHONDRIAL DIVERSITY WITHIN THE [email protected] CILIATE CLADE Testate amoeba transfer functions are widely used Tsaousis A.D.1,2, Williams L.T.1,2, Gentekaki E.3, for reconstruction of palaeo-hydrological regime in Lynn D.4, van der Geizen M.5, Wass M.N.2 peatlands. However, the limitations of this approach 1 - Laboratory of Molecular and Evolutionary have become apparent with increasing attention Parasitology to validation and assessing sources of uncertainty. 2 - School of Biosciences, University of Kent This paper investigates effects of peatland type and Protistology · 85 sampling depth on the performance of a transfer 18S rRNA gene sequence robustly groups with function using an independent test-set from four Pseudoparamoeba pagei (which lacks both PLO Sphagnum-dominated sites in European Russia and scales) and never branches among those of (Penza Region). We focus on transfer function Korotnevella spp. Thus the situation described above performance along localised hydrological gradients, shows that to the moment the boundary between which is a useful analogue for predictive ability two genera Pseudoparamoeba and Korotnevella can through time. The performance of the transfer be correctly drawn only on the basis of 18S rDNA function with the independent test-set was generally sequence. Our data probably mean that actually the weaker than for the leave-one-out or bootstrap presence of scales is a primitive feature for a whole cross-validations. However, the transfer function clade, which unifies genera Pseudoparamoeba, was robust for the reconstruction of relative changes Korotnevella, Paramoeba and Neoparamoeba. Within in water-table depth, provided the presence of good this group scales retained in some species and lost modern analogues and overlap in water-table depth in others. ranges. When applied to subsurface samples, the performance of the transfer function was reduced ECOLOGICAL STUDY OF THRAUSTOCHY- due to selective decomposition, the presence of TRIDS (LABYRINTHULEA, STRAMENO- deep-dwelling taxa or vertical transfer of shells. PILES) Our results stress the importance of thorough Ueda M.1,2, Doi K.1,2, Nomura Y.3, Nakajima M.4, testing of transfer functions, and highlight the role Honda D.3,2 of taphonomic processes in determining results. 1 - Graduate School of Natural Science, Konan Further studies of stratification, taxonomy and University, Japan taphonomy of testate amoebae will be needed to 2 - Institute for Integrative Neurobiology, Konan improve the robustness of transfer function output. University, Japan This research was funded by the Russian Scientific 3 - Faculty of Science and Engineering, Konan Fund (grant 14-14-00891) and grant of the President University, Japan of Russian Federation (MD-7930.2016.4). 4 - Research Institute of Environment, Agriculture and Fisheries, Osaka Prefecture, Japan CELL COAT EVOLUTION AND PHYLOGENY [email protected] OF DACTYLOPODID AMOEBAE (AMOE- Thraustochytrids have been recognized as im- BOZOA, DISCOSEA) portant decomposers in the marine ecosystem. Udalov Ilya A. However, in many cases, this recognition is based Department of Invertebrate Zoology, Faculty of Biology, on the short investigation of the biomass of whole Saint-Petersburg State University, Universitetskaya thraustochytrids without distinguishing the species. nab. 7/9,199034 St. Petersburg, Russia In this study, we investigated the seasonal changes of [email protected] biomass and species composition of thraustochytrids An amoebae order Dactylopodida was established on by continuous monitoring at river mouths and the base of the results of the molecular phylogenetic coastal areas in Japan for five years. Remarkable analysis. To date it includes genera Vexillifera, peaks of cell numbers of the thraustochytrids Cunea, Pseudoparamoeba, Korotnevella, Paramoeba were observed in early summer and/or summer at and Neoparamoeba. All representatives of this order the river mouths. This phenomenon was named share the ability to form non-furcating finger-shaped “thraustochytrid spikes”. Thraustochytrid biomass subpseudopodia (dactylopodia), which considered didn’t correlate with chlorophyll a, but it seemed as morphological synapomorphy for this group. that the peaks occurred after the decrease in salinity. Up to now all these genera were distinguished by This phenomenon probably suggested that the morhological features: peculiarities of locomotive thraustochytrid biomass is affected by the supply form, presence or absence of kinetoplastid endo- of the terrestrial organic matter from river water. symbiont (Perkinsela amoebae-like organism, or Although thraustochytrid biomass was only 1.59% PLO) and structure of the cell coat. The details of the that of bacterial biomass, the fixed energy (as cell coat evolution in Dactylopodida are still unclear. biomass) transferred directly from thraustochytrids Recent studies shown that situation with generic to zooplankton was estimated to be 15.9% of that distinctions in this group actually is much more transferred from bacterioplankton via phagotrophic complex. One of the studied species formally fits protists. Moreover, a clear seasonal succession of the diagnosis of the genus Korotnevella, because thraustochytrid species was repeatedly observed it has scales and lacks PLO. At the same time its every year. However, species composition differs 86 · “PROTIST–2016” between the river mouths and coastal areas. It is IRON SULFUR CLUSTER ASSEMBLY IN suggested that habitat segregation occurred among AMITOCHONDRIATE OXYMONAD MONO- these species and was caused by the environmental CERCOMONOIDES factors and the differences of utilization of terrestrial Vacek Vojtěch1, Karnkowska Anna2, Čepička or marine organic matter. Ivan3, Novák Lukáš1, Treitli Sebastian1, Zubáčová Zuzana1, Hampl Vladimír1 ANALYSES OF PHOTOSYNTHETIC OXIDA- 1 - Department of Parasitology, Charles University in TIVE STRESS RESPONSES IN HERBIVO- Prague, Faculty of Science, Czech Republic ROUS UNICELLULAR ORGANISMS 2 - Department of Botany, University of British Uzuka Akihiro1,2, Hirooka Shunsuke2, Fujiwara Columbia, Vancouver, V6T 1Z4, Canada Takayuki1,2, Kanesaki Yu3, Yoshikawa Hirofumi3, 3 - Department of Zoology, Charles University in Miyagishima Shin-ya1,2 Prague, Faculty of Science, Czech Republic 1 - The Graduate University For Advanced Studies [email protected] [SOKENDAI] Oxymonads are a group of anaerobic or micro- 2 - National Institute of Genetics aerophilic protists living in guts of insects and 3 - Tokyo University of Agriculture, Japan vertebrates. They are the only group of eukaryotes [email protected] without mitochondrion, however in their closest Photosynthesis generates reactive oxygen species free-living relative Paratrimastix pyriformis have (ROS) which destroy biomolecules. It is known been found organelles which are morphologically that photosynthetic organisms, such as plants and ntsimilar to hydrogenosomes. Concomitantly with algae, have strategies to cope with the photosynthetic the absence of mitochondrion, Monocercomonoides oxidative stress to perform photosynthesis safely. lacks classical mitochondrial ISC system for When unicellular predators, such as amoebae, feed synthesis of Fe-S clusters. Instead, subunits of SUF on photosynthetic organisms under illumination, system were found in genome and transcriptome of they are probably exposed to ROS that are generated Monocercomonoides: SufB, SufC, SufS and SufU. by engulfed prey during digestion. The aim of my All these proteins contain well conserved catalytic study is to examine whether the algal predators sites which are needed for their function in FeS are really exposed to oxidative stress, and if so, to cluster assembly. Heterologous localization of understand how they cope with the photosynthetic SufB and SufC in Trichomonas vaginalis expression oxidative stress. To this end, I have isolated amo- system showed cytosolic localization. We have also ebae that feed on both photosynthetic and non- found subunits of SUF system in transcriptomic photosynthetic bacteria from marsh. From them, data from Paratrimastix pyriformis and two other I chose three amoebae which were evolutionally members of Preaxostyla – oxymonad strain distantly related. These amoebae were co-cultured NAU3 distantly related to Monocercomonoides with the cyanobacteria or E. coli under dark or and isolate MORAITICA, the deepest branching light condition and I measured ROS generated lineage of Preaxostyla available at the moment. by engulfed photosynthetic prey and examined Phylogenetic analyses of SUF subunits showed transcriptome changes of amoeba cell. The results that all preaxostyla SUFs forms single clade, which show that singlet oxygen, which is believed to be the is clearly distinct from clades of other eukaryotes main ROS produced by photosynthesis, is generated – proving that common ancestor of all known in amoeba by engulfed photosynthetic prey under Preaxostyla acquired SUF system by horizontal illumination but not in dark condition or during gene transfer independently from other eukaryotes. predation of non-photosynthetic prey. On the other To prove that SUF subunits are indeed functionally hand, higher level of hydrogen peroxide is produced active in Monocercomonoides we have performed under illumination than under dark condition several complementation experiments in E. coli. regardless of the photosynthetic ability of prey. Even Preliminary experiments with complementation if they are evolutionally distantly related species, proved that SufB of Monocercomonoides can three species of amoebae exhibited similar pattern substitute SufB of E. coli in synthesis of Fe-S cluster of transcriptome changes. Now, I am conducting and therefore SUF system is functionally active in some assays to investigate whether phenomena Fe-S cluster assembly. Heterologous localization of which are predicted based on transcriptome changes SufB and SufC in Trichomonas vaginalis expression are really occur. system showed cytosolic localization. Our results indicate that Monocercomonoides is the first known Protistology · 87 organism, which assemble Fe-S clusters in the MORPHOLOGICAL AND MOLECULAR IN- cytosol by concerted action of SUF and CIA VESTIGATION OF MARINE PARAMOEBI- pathways. DAE (AMOEBOZOA, DACTYLOPODIDA) Volkova E.N., Kudryavtsev A.A. SYNCHRONIZED AND ER-DEPENDENT Saint Petersburg State University, Russia DYNAMICS OF MITOSOMES [email protected] Voleman L.1, Najdrová V.1, Ástvaldsson A.2, We present a revision of marine dactylopodid Tůmová P.3, Einarsson E.2, Švindrych Z.4, Hagen amoebae containing an intracellular eukaryotic G.M.4, Tachezy J.1, Svärd S.G.2, Doležal P.1 symbiont traditionally called ‘parasome’, and 1 - BIOCEV – Biotechnology and Biomedicine Center currently known as ‘Perkinsela-like organism’ of the Academy of Sciences and Charles University in (PLO) related to Kinetoplastida. This group Vestec and Department of Parasitology, Faculty of traditionally consists of two genera: Paramoeba Science, Charles University in Prague, Czech Republic Schaudinn, 1896 and Neoparamoeba Page, 1987 2 - Department of Cell and Molecular Biology, BMC, which differ in their cell coat structure; the former Uppsala University, Uppsala, Sweden being covered with scales, while the latter, with 3 - Department of Tropical Medicine, First Faculty the thin, scaleless glycocalyx. The third PLO- of Medicine, Charles University in Prague, Prague, containing genus, Janickina Chatton, 1953 has no Czech Republic clear taxonomic affinities yet, as no molecular data 4 - Institute of Cellular Biology and Pathology, First are available for its members. We present the results Faculty of Medicine, Charles University in Prague, of investigation of the biodiversity and phylogenetic Czech Republic relationships within the genera Paramoeba and [email protected] Neoparamoeba based on 15 marine and brackish Mitosomes are the smallest evolutionary forms of water strains isolated from a broad range of habitats. mitochondria that evolved in eukaryotes adapted to The conclusions are based on morphological, anaerobic environments. While abandoning many ultrastructural and molecular evidence. The data attributes of the aerobic mitochondria such as the obtained allow us to conclude that (1) Morpho- genome, respiration and the cristae, mitosomes species of Paramoeba and Neoparamoeba show have retained the double membrane and the bare considerable levels of intragenomic and intraspecies bones of the pathways for the protein import and the variability based on the SSU rDNA and ITS synthesis of the iron-sulfur clusters. Here, we studied region sequences; (2) A detailed study combining the dynamics of the mitosomes in the parasitic analysis of light-microscopic data, ultrastructure protist Giardia intestinalis, which belongs to one of and molecular evidence is necessary in most of five supergroups of eukaryotes known as Excavata. cases to discriminate species within this group; (3) We found that mitosomes are extremely steady The cell coats of Paramoeba/Neoparamoeba clade organelles during the interphase undergoing neither have evolved from the scale-bearing ancestral taxa the fission nor the fusion during the interphase, through several independent scale losses in various thus being highly prone to become heterogeneous. lineages of this clade. We also present novel data Surprisingly, the mitosomal division is restricted that contribute to further understanding of the to mitosis, when both central and peripheral co-evolution of amoebae and their intracellular organelles divide in a synchronized manner. The symbionts. mitosomes also divide during the encystation of Partially supported by the RFBR grant 15-29-02749; the parasite, thus preconfigure the cyst for the rapid the study utilized the equipment of the core facility excystation in a new host. Interestingly, the division centers of St. Petersburg State University. involves the association of the mitosomes with the endoplasmic reticulum, a relationship typical for the CILIATES AS BIOINDICATORS OF MARINE mitochondria of Opisthokonta. While several such WATER QUALITY tethering mechanisms, which enable lipid transfer Warren A.1, Xu H.2 between the organelles, have been described for 1 - Department of Life Sciences, Natural History Opisthokonta, none of these have been shown to Museum, London, UK function in other eukaryotic supergroups including 2 - Laboratory of Microbial Ecology, Ocean University Excavata. However, we were able to show that of China, Qingdao, China lipid enzyme long chain acyl-CoA synthetase 4 is [email protected] distributed to the mitosome-ER interface. Although protists, and especially ciliates, have long 88 · “PROTIST–2016” been used as indicators of freshwater quality, they biological meaning of both is still unclear and the are rarely used in this capacity in marine waters. relationship between genetic divergence and the Here I will summarize the results of a series of level of species or populations remains uncertain. In studies carried out in Jiaozhou Bay, on the Yellow order to overcome this conflict, we try to combine Sea coast of NE China, in which we investigate the aspects of morphological variability, genetic relationships between ciliate communities, both diversity and reproduction to achieve an integrative planktonic and periphytic, and certain physico- approach for species delimitation in foraminifera. chemical parameters that varied at different To this end, we carry out breeding experiments sites within the Bay. In each study, ciliates were on benthic foraminifera to observe the mating identified and enumerated by direct microscopy, behavior between genetically divergent lineages and data were analyzed using various statistical to detect the level of divergence that corresponds packages mainly within PRIMER. A main aim to reproductive isolation. In addition, we plan to of this investigation was to develop protocols that observe the mating behavior among genetically maximize the efficiency of sampling and analyses of homogenous populations to examine the existence the ciliate communities. Our main findings were: (1) of different mating types within a population. The the 8-sampling events per year may be an optimal mating system largely influences the generation sampling strategy for planktonic ciliated protozoan of genetic variation and contributes to the process seasonal research in marine ecosystems; (2) 90% of adaptation. Understanding its mechanisms in of the periphytic community could be recovered foraminifera is thus essential to understand the on 10 microscope slide replicates immersed at diversification and evolution of the group. one depth for 3 – 21 days; (3) multivariate (step- best-matching) analysis allows a subset of the most THE EVOLUTION OF MITOCHONDRIAL reliable indicator species to be identified without MEMBRANE CONTACT SITES losing accuracy of water quality prediction; (4) Wideman J.G. applying taxonomic efficiency, i.e., identifying to Department of Biosciences, University of Exeter, United the highest rank without losing critical bioindicator Kingdom information, revealed that genus-level identification [email protected] allows for accurate prediction of water quality; (5) It is commonly accepted that mitochondria evolved efficiencies of sample analyses can be achieved by from an alpha-proteobacterial endosymbiont to omitting ubiquitous groups, and using presence/ become the major energy producing organelle absence of others rather than abundance data. of the eukaryote cell. Accounts describing the integration of the pre-mitochondrial symbiont SPECIES CLASSIFICATION AND MATING into host cell processes often focus on this transfer IN FORAMINIFERA of the control of energy production from the Weiner A.K.M., Tsuchiya M., Toyofuku T., symbiont to the host. However, mitochondria are Kitazato H. more than mere ATP generators and have several JAMSTEC, Japan Agency for Marine Earth Science physical and functional links to various cell systems. and Technology, 2-15 Natsushima-cho, Yokosuka One such link is manifested in the physical and 237-0061, Kanagawa, Japan functional link between mitochondria and the [email protected] endomembrane system in the form of membrane Many groups of foraminifera are characterized by contact sites (MCSs). These MCSs are important the formation of elaborate shells, which provide for non-vesicular lipid transport between apposed detailed morphological features, useful for spe- membranes. Recent progress has identified the cies classification. Since the majority of works protein complexes responsible for maintaining focuses on their fossilized shells, a comprehensive MCSs in Saccharomyces cerevisiae. A surprising morphotaxonomy has been established. Yet, genetic number of functionally overlapping mitochondrial analyses revealed an even higher diversity on the MCS tethering complexes have been described, molecular level, hidden within the traditional but the extent to which MCS tethers are conserved morphospecies. These cryptic species are marked between distant lineages appears to vary. Thus, by large genetic distances and differentiated while being functionally redundant in S. cerevisiae, distribution patterns, implying that cryptic species MCSs appear to have a high degree of evolutionary rather than morphospecies represent the level of plasticity in eukaryotes. Taken together, these data species. As a consequence, today we are facing a suggest that the last eukaryote common ancestor conflict between the morphological species concept had a mitochondrion highly connected to diverse and the interpretation of genetic diversity. The endomembranes, but over the course of eukaryote Protistology · 89 divergence ancient MCS tethers were lost while Ansan 15627, Korea others were gained in a lineage-specific manner. 2 - Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea UNRAVELLING THE DIVERSITY OF DIPLO- [email protected] NEMIDS BY CULTURE-BASED TAXONO- The Florideophyceae (5 subclasses and 31 orders) MIC STUDY and Bangiophyceae (1 subclass and 1 order) Yabuki A.1, Kusaka C.1, Votýpka J.2,3, Horák A.3, distribute in both marine and freshwater habitats, Lukeš J.3, Fujikura K.1 and cover the most described red algal diversity, 1 - Department of Marine Biodiversity Research, Japan ca. 7,100 species (98% of Rhodophyta). The Agency for Marine-Earth Science and Technology monophyly those two classes and relationships (JAMSTEC), Yokosuka, Japan among subclasses were well-supported in many 2 - Department of Parasitology, Faculty of Science, phylogenetic analyses, however, interordinal Charles University, Prague, Czech Republic phylogeny remain largely unknown in the subclass 3 - Biology Centre, Institute of Parasitology, Czech Rhodymeniophycidae. In order to resolve ordinal Academy of Sciences and Faculty of Sciences, relationships and evolution of the subclass, we have University of South Bohemia, České Budějovice determined 11 mitochondrial genomes (mtDNA) (Budweis), Czech Republic from representative species and compared with [email protected] published data. Concatenated phylogeny based on Diplonemids (Diplonemida) belong to the phylum 37 mtDNA (6,345 aminoi acid data) of red algae Euglenozoa and they are unicellular flagellates that showed stronger support among order level than are free-living or parasitize algae and invertebrates. previous studies. The mtDNA synteny comparison Although Diplonemida included for decades showed 47 events of gene evolution (indel and only the genera Diplonema and Rhynchopus yet, rearrangement) among Bangiophyceae, Hiden- the genus Hemistasia was recently transferred to brandiophycidae, Nemaliophycidae, Corallino- Diplonemida based on the phylogenetic analysis. phycidae, Ahnfeltiophycidae, and Rhodymenio- Hemistasia phaeocysticola is a representative of phycidae. We demonstrate that all rhodymeniophy- a large environmental clade that remained to cidan mtDNA have conserved architectures with be identified in Diplonemida. Members of this little variation of contents. Based on the best environmental clade have attracted attention not phylogeny, we inferred the ancestral mtDNA only from taxonomic/phylogenetic perspective, but content, evolution and reduction of protein coding also due to highly unusual RNA processing in their genes in red algae. mitochondria, which is reminiscent of trans-splicing and editing, so far reported only from them. Hence, NEW MITOCHONDRIAL GENOMES AND the studies focusing on H. phaeocysticola and the MOLECULAR MARKERS FOR THE COASTAL other members of this environmental clade may DIATOMS OF KOREA expand our knowledge about the structural diversity Yang E.C., Noh J.H. and evolutionary history of unusual structural genes Korea Institute of Ocean Science & Technology, Ansan in this protist group. We have isolated new marine 15627, Korea diplonemids and keep some of them in culture. [email protected] In the present study, we report their morphology The Bacillariophyceae is the largest class of the and phylogenetic positions in Diplonemida: some stramenopiles (ca. 11,200 species), and one of the cultures branch within the clade that is composed most important and abundant primary producers of environmental sequences and H. phaeocysticola, of the aquatic ecosystem. Diverse diatoms are while others are also shown to be novel lineages occurred in inter-tidal mudflats of the west coast in the clade of Diplonema and Rhynchopus. Our of Korea, the Yellow Sea. In spite of ecological findings significantly expand the known diversity importance of the phytoplankton community, there of diplonemids and it is also suggested that the are a little number of molecular markers have been classification system under Diplonemida should be developed, such as nuclear encoded 18S rRNA revised: the establishment of a novel family is possibly (SSU) and plastid encoded rbcL. In present study, needed for the members of the environmental clade. we determined two new mitochondrial genome (mtDNA) of common tube-dwelling diatoms, RED ALGAL PHYLOGENY AND EVOLUTION Berkeleya fennica TA424 (GenBank accession BASED ON MITOCHONDRIAL GENOME NC_026126, 35.509 bp) and Navicula ramosissima Yang E.C.1, Lee J.2, Yoon H.S.2 TA439 (48,652 bp) isolated from the Taean mudflat 1 - Korea Institute of Ocean Science & Technology, (36º44′14.79″N 126º10′41.89″E), and one mtDNA 90 · “PROTIST–2016” of pelagic diatom, Skeletonema marinoi JK029 encoded by the first gene of the operon, whereas (NC_028615, 38,515 bp) isolated from an interdidal the remaining five proteins could be assigned only site of the Sihwaho (37º18′38.91″N 126º36′16.35″E). to broader enzyme superfamilies. Nevertheless, Complete mtDNA of each species shows typical based on these analyses we speculate that the newly bacillariophycean mitochondrial contents, inclu- detected operon encodes enzymes of a pathway ding of 35-42 coding sequences (CDS), 2 ribosomal synthesizing a prenylated aromatic compound, RNA (rRNA), and 25-26 transfer RNA (tRNA) possibly an antimicrobial or other protective genes. All CDS of mtDNA aligned with all available substance. To our knowledge, this is the first report diatoms data, including Phaeodactylum tricornutum of an expansion of the metabolic capacity of a plastid (NC_016739), Fragilaria acus (NC_013710), and mediated by HGT into the plastome. Thalassiosira pseudonana (NC_007405). Based on the saturation test and preliminary sequencing, we SPECIES DIVERSITY AND MORPHOLOGY suggest mitochondrial markers for diatoms survey OF CENTROHELID HELIOZOANS OF SOME on the west coast of Korea. SITES IN EUROPEAN PART OF RUSSIA AND SOUTH-EAST UKRAINE HORIZONTAL TRANSFER OF A NOVEL Zagumyonnyi D.G. SIX-GENE OPERON FROM A BACTERIUM Voronezh State University, Russia INTO THE PLASTID GENOME OF EUSTIG- [email protected] MATOPHYTE ALGAE The species composition and cytoskeleton morpho- Yurchenko T.1,2, Ševčíková T.1, Strnad H.3, Butenko logy of centrohelid heliozoans from different water A.1, Eliáš M.1,2 biotopes of European Russia and South-East Ukraine 1 - University of Ostrava, Faculty of Science, Depart- have been investigated. 21 species from 6 genera and ment of Biology and Ecology, Life Science Research 3 families have been found. Two forms (Acanthocystis Centre, Ostrava, Czech Republic sp. nov. 1. and Acanthocystis sp. nov. 2.) have 2 - University of Ostrava, Faculty of Science, Institute appeared to be as new species. Following species of Environmental Technologies, Ostrava, Czech Acanthocystis turfacea, Heterophrys myriopoda, Republic Polyplacocystis symmetrica, Polyplacocystis coerulea, 3 - Institute of Molecular Genetics, Czech Academy of Acanthocystis dentate have been marked for South- Sciences, Prague, Czech Republic East Ukraine for the first time. Also Acanthocystis [email protected] dentata, Raineriophrys fortesca, Raineriophrys Genes in plastid genomes (plastomes) have been kilianii, Pterocystis foliaea and Pterocystis pulchra primarily inherited from the cyanobacterial plastid have been found for the Central Russia for the ancestor, but cases of genes acquired by horizontal first time. Pterocystis pulchra has been revealed for gene transfer (HGT) have been occassionally heliozoan fauna of Russia firstly at all. Heterophrys reported from plastomes of different algal lineages. marina has been found in fresh waters of Russia. Here we report an interesting case of HGT- Polyplacocystis symmetrica has been found in slightly mediated gene acquisition revealed by sequencing salted biotopes. The morphological descriptions plastomes of the eustigmatophyte algae Monodopsis of all studied species have been given. New data sp. MarTras21 and Vischeria sp. CAUP 202. While on spicule structures of Acanthocystis dentate have the gene complement of the newly and previously been done. Most frequent species was Acanthocystis sequenced eustigmatophyte plastomes proved to pectinata. This study was supported by the Russian be highly conserved, those of Monodopsis sp. and Scientific Foundation (grant nos. 14-14-00515). Vischeria sp. harbour a cluster of six genes not reported from any plastid genome sequenced so RUBISCO IN NON-PHOTOSYNTHETIC ALGA far. All six genes have homologs in various bacteria, EUGLENA LONGA: DIVERGENT FEATURES, where they are usually organized in the same six-gene TRANSCRIPTOMIC ANALYSIS AND REGU- cluster, i.e. a putative operon. Phylogenetic analyses LATION OF COMPLEX FORMATION showed that the cluster from eustigmatophyte Záhonová K.1, Füssy Z.2, Oborník M.2,3,4, Eliáš M.1, plastomes is nested among sequences from the Yurchenko V.1,2 order Cytophagales (phylum Bacteroidetes), with 1 - Life Science Research Centre, Department of the cluster from Sporocytophaga myxococcoides Biology and Ecology and Institute of Environmental constituting a robustly resolved sister group to the Technologies, Faculty of Science, University of eustigmatophyte clade. Sequence analyses using Ostrava, 701 00 Ostrava, Czech Republic different homology-detection tools failed to detect 2 - Institute of Parasitology, Biology Centre ASCR, 370 functionally characterized homologs of the protein 05 České Budějovice, Czech Republic Protistology · 91

3 - University of South Bohemia, Faculty of Science, in water samples. Parasitic Apicomplexa were 370 05 České Budějovice, Czech Republic found frequently from both water and sediment 4 - Institute of Microbiology ASCR, Centrum Agaltech, samples. Based on terminal-restriction fragment 379 01 Třeboň, Czech Republic length polymorphisms, distinct succession and [email protected] contrasting protist community structure was found Euglena longa, a close relative of the photosynthetic among temporal samples and between planktonic model alga Euglena gracilis, possesses an enigmatic and benthic habitats. Redundancy analysis indicated non-photosynthetic plastid. Its genome has re- that the temprature, concentration of dissolved tained a gene for the large subunit of the enzyme phosphate, and N:Si in surface water were the most RuBisCO (rbcL). Here we provide new data significant abiotic variables shaping the planktonic illuminating the putative role of RuBisCO in E. communities. This study indicates that temprature longa. We demonstrated that the E. longa RBCL and stoichiometric ratios play important roles in protein sequence is extremely divergent compared driving succession of protist communities, and to its homologs from the photosynthetic relatives, parasitic protist could be early examed by molecular suggesting a possible functional shift upon the loss technologies for the farming pools. of photosynthesis. Similarly to E. gracilis, E. longa This work was supported by projects from NSFC harbors a nuclear gene encoding the small subunit (No. 313018680) and Scientific Development of RuBisCO (RBCS) as a precursor polyprotein Program of Yantai (No. 2014ZH073). comprising multiple RBCS repeats, one of which is highly divergent. Both RBCL and the RBCS proteins MAKE PERCISE IDENTIFICAITON USING are synthesized in E. longa, but their abundance DNA TAXONOMY TECHNIQUES FOR CLO- is very low compared to E. gracilis. No RBCS SELY RELATED EUPLOTES CONGENERS monomers could be detected in E. longa, suggesting (PROTOZOA, CILIOPHORA) WITH HIGHLY that processing of the precursor polyprotein is PHENOTYPIC PLASTICITY inefficient in this species. The abundance of RBCS Zhao Y.1, Yi Z.2, Song W.3 is regulated post-transcriptionally. Indeed, blocking 1 - Research Center for Eco-Environmental Sciences, the cytoplasmic translation by cycloheximide Chinese Academy of Sciences, Beijing 100085, China has no immediate effect on the RBCS stability in 2 - Guangzhou Key Laboratory of Subtropical Bio- photosynthetically grown E. gracilis, but in E. longa, diversity and Biomonitor, School of Life Science, South the protein is rapidly degraded. E. longa appears to China Normal University, Guangzhou 510631, China lack the chaperone RBCX essential for assembly 3 - Laboratory of Protozoology, Institute of Evolution of a conventional RuBisCO complex. Altogether, & Marine Biodiversity, Ocean University of China, our results revealed signatures of evolutionary Qingdao 266003, China degradation of RuBisCO in E. longa and suggest that [email protected] its role in this species may be rather unorthodox. Ciliated protists constitute astounding diversity and play crucial role in different biotopes yet PROTIST COMMUNITIES IN WATER AND still incompletely characterized microbes. DNA SEDIMENT OF A SEA CUCUMBER FAR- barcoding is promising to address this dilemma. MING SYSTEM This molecular technique based on COX 1 gene Zhang Q., Tan S., Gong J. has become a routine method for animal organism Laboratory of Microbial Ecology and Matter Cycles, identification and taxonomic clarification. This Yantai Institute of Coastal Zone Research, Chinese common region also can distinguish ciliates species, Academy of Science, Yantai, China but has not displayed universality, and establishing [email protected] a standardized DNA barcoding system for ciliated Little is known about the distinctive communities protists is still confronted with many difficulties of uncultured protsit within the aquaculture ecosys- and challenges. Here, we collected the species-rich tem. In this work, the planktonic and benthic protist taxon Euplotes from brackish and fresh waters. In the communities in a sea cucumber farming system framework of traditional classification system, the were simultaneously investigated on three sampling Euplotes spp. with available morphological features dates. Analyses of SSU rRNA gene libraries of six can provide a useful template against which to test samples revealed 108 eukaryotic taxonomic units, the accuracy of DNA-based taxonomy. Using >30 among which 17.5% were rare. Stramenopiles samples, we have assessed the most common COX and Alveolata are predominant groups in both 1 region and the alternative SSU-V4, SSU-V9, sediment and water samples. Dinophyta, Rhizaria LSU-D1/D2, ITS1, ITS2 markers. And SSU-V9, and parasitic Mesomycetozoa were only detected LSU-D1/D2 and ITS1 could distinguish the most 92 · “PROTIST–2016” closely related species and are proper markers for Biology, Saint-Petersburg State University ciliates barcoding. [email protected] “Heliozoa” is a former taxon of spherical axopodial THE INTRICATE EVOLUTIONARY HISTORY protists. These organisms, being outwardly similar OF RhoBTB PROTEINS have revealed to be not necessarily related. So- Žihala D.1, Klimeš V.1, Rivero F.2, Eliáš M.1 me of lineages are now placed into the proper 1 - University of Ostrava, Faculty of Science, Depart- “supergroups”, while many deviant forms still ment of Biology and Ecology, Chittussiho 10, 710 00 remain “orphans”. The most diverse group – the Ostrava, Czech Republic centrohelids – recently was placed in Haptista 2 - Centre for Cardiovascular and Metabolic Research, along with haptophytes and their internal taxonomy The Hull York Medical School and Department of also was considerably revised showing interesting Biological Sciences, University of Hull, Cottingham examples of parallel evolution. But in comparison Road, Hull HU6 7RX, UK to other groups with a developed taxonomy “Helio- [email protected] zoa” still seem to be undeservingly neglected. Many The Rho family is one of the major subgroups of taxa of flagellates, ciliates and in a less degree of the huge superfamily of Ras-like GTPases. Rho amoebae get much more attention, while heliozoan proteins are known primarily as regulators of various taxonomy suffer from the lack of skilled taxonomists. pathways that are connected to the actin cytoskele- The great amount of their diversity is likely to be ton (cell movement, polarization, morphogenesis unknown. The heliozoans require closer attention, etc.). These proteins usually contain only the Rho- being ubiquitous in freshwater, marine (both type GTPase domain, but some of them possess planktonic and benthic) and soil environments, also a tandem of two so-called BTB domains and important consumers of bacteria, protists and even are called RhoBTB proteins. The BTB domain is micrometazoans. But the taxonomic impediment a protein-protein interaction domain and is often currently prevents them from being incorporated a part of proteins that are connected to protein into ecological and biogeographic studies. Many ubiquitination through Cullin3-dependend E3 heliozoans, especially centrohelids, are easy to ligases. Proteins with the domain architecture maintain in the culture, have a rich morphology and characteristic for RhoBTB proteins have been potentially can serve a good model for addressing reported only from metazoans and dictiosteliid many of fundamental questions of protistology, slime moulds, but this distribution was deduced including a species problem, the endemism vs from a phylognetically very limited survey. We have ubiquity dilemma and the evolution of cell coverings. utilized the currently available wealth of genomic Many of them can be indicators of water quality and and transcritpomic data from diverse eukaryotes, should be included to biodiversity indexes. But first including a wide coverage of protist taxa, and of all, their taxonomy need to be evaluated and the found out that RhoBTB proteins occur in many main proportion of species should be described on additional eukaryotic lineages. Although scattered, the modern level. Study support: RFBR grants 15- the phyletic pattern of RhoBTB genes is compatible 04-18101_a, 15-29-02749-ofi_m. with a hypothesis that a primordial RhoBTB gene was present already in the last eukaryotic common THE RELIABILITY OF PROTOZOANS AS ancestor. Interestingly, RhoBTB proteins from INDICATORS OF AQUATIC ECOSYSTEM some taxa (Amoebozoa, Apusomonadida, and HEALTH Cryptomonadida) proved to possess a RING/U-box Zolotarev V.A. domain inserted into the first BTB domain. RING I.D. Papanin Institute for biology of inland waters and U-box are related domains that constitute a Russian Academy of Sciences (IBIW RAS) class of E3 enzymes, so our findings further support [email protected] the idea that RhoBTB3 proteins ancestrally served Metapopulations of microbial organisms occur as components of ubiquitin-mediated regulation. worldwide wherever their required habitats are These and other findings of our evolutionary realised. This is a consequence of ubiquitous analyses of RhoBTB proteins will be presented and dispersal driven by huge population sizes, and the discussed. consequently low probability of local extinction (Finlay, 2002). Microorganisms should be used in THE STATE OF ART IN THE TAXONOMY OF biomonitoring for several compelling reasons. (1) A “HELIOZOA” cosmopolitan distribution facilitates comparisons of Zlatogursky V.V. test results in geographically different regions. (2) Department of Invertebrate Zoology, Faculty of Problems of scale are diminished. (3) Replicability Protistology · 93 is as good as, or better than, tests with larger of the Russian Academy of Sciences», Kurortnoe, organisms. (4) Environmental realism is higher than Feodosia, Crimean Republic, 298188 Russia in tests using larger organisms. (5) The number of e-mail: [email protected] test species is dramatically increased when using At first glance, according to some morphological microorganisms, thus displaying natural variability and habit features Ardissonea crystallina (C. Agardh) much better than tests with a limited array of larger Grunow should be regarded as belonging in pennate organisms, etc. (Cairns, 2005). Biodiversity of the diatoms. However, molecular phylogeny analysis whole aquatic ecosystem depends on the biodiversity strongly suggests this species to be placed among of the microbial communities (Algae, Protozoa, centric diatoms. It is well known that centric diatoms Bacteria) at the basis of the trophic pyramid. The reveal oogamous mode of sexual reproduction. term ‘indicator species’ is usual in literature on Exceptions are cases of autogamy. We studied clonal bioindication of water quality, but we more tend cultures of A. crystallina and were able to initiate to accept the term ‘indicator communities’, or sexual reproduction in the mixtures of clones. ‘model communities’, that could be robust both Heterothallic sexual reproduction was shown to for biological monitoring and ecotoxicology. be nonoogamous. Gametes produced by male and Diversity and relative abundance of ciliates and female gametangia were more or less equal by size. other protozoans can be used as indicators of toxic Gamete behaviour and morphology were different. pollution and acidification. Multivariate statistics Male gametes had no flagella; at the same time, they were used to design “the scale of toxicity” across a were able to produce thin cytoplasmic projections gradient of toxicant stress and organic compounds. similar to those of some araphid pennates (Tabularia A new index of periphyton flagellates (IPF) as fasciculata, T. tabulata, Ulnaria ulna, etc.). Female indicator of the trophic status of a water-body was gametes were located in the maternal frustules and developed (Zolotarev, 2007). For the foreseeable did not leave them until fertilization. Each male future, protozoans could be robust for ecosensors gamete moved in part amoeboidly to the place and ecorobots. of singamy and penetrated in the gap between slightly opened valves of the maternal frustule. The NONOOGAMOUS SEXUAL REPRODUC- described finding raises a question about possibility TION IN THE POLAR CENTRIC DIATOM of independent evolution of isogamy in polar centric ARDISSONEA CRYSTALLINA diatoms. This study was supported by the Russian Davidovich N.A., Davidovich O.I., Podunay Y. A. Foundation for Basic Research (grant No 15-04- Federal State Budget Scientific Institution «T.I. Vya- 00237 A). zemsky Karadag Scientific Station – Nature Reserve 94 · Author Index

Author Index

A Bobrov A.A. 81 Bogolyubov D.S. 9 Abdel-Gawwad Abdel-Azeem 5 Bondarenko N.I. 9 Abraham J.S. 5, 43, 76, 77 Bonkowski Michael 16, 80 Adams B.J. 80 Boo S.M. 23 Adl S.M. 6 Bowler C. 18, 42 Agatha S. 6, 20, 48 Brown I. 47 Aguilar M. 6 Brown M. 27, 37, 80 Aihara Y. 43 Brown N.H. 57 Aleoshin Vladimir V. 56 Buelow H. 80 Al-Farraj Saleh 17 Burgaud G. 10 Al-Quraishy Saleh 5, 17 Burger Gertraud 42 Alterzon-Baumel S. 71 Burki F. 81 Andersen R.A. 25, 41 Burkova T.N. 79 Andrew P. 57 Burkovsky I.V. 18 Annenkova N.V. 7 Burns J.A. 10, 55 Apothelot-Perret-Gentil Laure 28 Butenko A. 10, 90 Arifa Butt A.B. 62 Bykova S.V. 11, 71 Ástvaldsson A. 87 Avrahami A. 71 C Azevedo Carlos 5 Azovsky A.I. 7, 81 Cai H. 12 Cardoso Fernando 48, 59, 64 B Caron D.A. 14 Cary S.C. 14 Baldauf Sandra 72 Castelli M. 40, 69, 78 Barbier G. 10 Čepička I. 12, 25, 28, 38, 56, 78, 83, 86 Bass David 16 Chami M. 28 Basuri C.K. 69 Chen W. 12 Baumann Christina 16 Chen X. 12 Belevich T.A. 8 Chistyakova L.V. 12, 37 Beliavskaia A. 8, 63 Cho C.H. 13 Benken K. 66 Choudhary A. 5 Benzerara K. 60 Cihlář J. 13 Berdieva M.A. 9, 12 Ciniglia C. 13 Berendonk T.U. 78 Ciros-Pérez J. 16 Bernhard J.M. 17 Countway P.D. 14 Bertolino P. 47 Coyne K.J. 14 Bhatnagar M. 5 Cruz Jimenez G. 14 Blyakharchuk T.A. 39 Curtis B. 67 Protistology · 95

D Fu Chengjie...72 Fujikura K. 89 Dacks J.B. 6, 59 Fujiwara Takayuki 86 Dagar J. 5 Füssy Z. 13, 19, 90 Davidovich N.A. 93 Davidovich O.I. 93 G Demin S.Y. 9 Derelle R. 28, 56 Gambhir G. 5 Deschamps P. 60 Ganser M. 20 Divne A.M. 55 Gasparyan A.V. 21 Doak T.G. 78 Gavrilova N.A. 21 Doan Nhu-Hai 24 Gawryluk R. 81 Dobáková E. 74 Geisen S. 80 Doi K. 85 Geizen van der, M. 84 Dolan J.R. 14 Gentekaki E. 84 Doležal P. 28, 36, 40, 50, 87 Gerasimov E.S. 21 Dovgal I.V. 15 Gerasimova Elena 22, 60 Duarte I. 29 Glotova A.A. 22 Dubrovsky Yu.V. 15 Gogoleva Natalya 60, 68 Duhamel S. 33 Gomes Inês 59 Dumack Kenneth 16, 80 Gong J. 22, 54, 91 Dunfield P.F. 6 Gong Yingchun 23 Durán-Ramírez C.A. 16 González Adame G. 14 Goodkov A.V. 9 E Gorbunov M.U. 11 Goryunov D.V. 8 Edgcomb V.P. 10. 17 Goyal A. 5 Einarsson E. 87 Graf L. 23, 25, 40 Eliáš M. 28, 40, 56, 59, 77, 90, 92 Grau-Bové X. 23 Elserehy Hamed 17 Grevtseva I. 24 Elster J. 65 Gu Yansheng 62 Elurbe D. 29 Gubin S.V. 71 Eme L. 40, 67 Gupta R. 43, 76, 77 Esaulov A.S. 18 Gusev E.S. 24 Espinosa A. 18 Esqueda-Lara Karina 27 H Esson H.J. 19 Ettema T. 55 Hackey M. 18 Hagen G.M. 87 F Hamamoto Y. 25 Hampl V. 77, 83, 86 Faktorová D. 29, 42 Han K.Y. 25 Fernandez-Leborans G. 63 Hanousková P. 25 Filardi C. 33 Harcet M. 26, 57 Filatova N.A. 9, 58 Harding T. 40 Fiore-Donno A.M. 80 Hashimoto T. 73, 79 Flegontov P. 10, 18, 21, 42, 74 Hehenberger E. 81 Flegontova O. 10, 18, 42 Heidelberg K.B. 14 Fleury Y. 10 Herman E.K. 59 Florenza J. 51 Hermoso-Salazar M. 45 Foght J. 6 Hernández-Becerril David 26, 27 Fokin Sergei I. 19, 69 Hertz R. 71 Francetic O. 28 Hill E. 10 Franco Ricardo 59 Hirakawa Y. 27, 66 Frolov A.O. 12 Hirooka Shunsuke 86 96 · Author Index

Hofstatter P.G. 27 Kaur Binnypreet 42 Holcombe L. 57 Kaur Harpreet 30, 33 Holzmann Maria 28 Kaur Surinder 30, 33 Holzinger A. 65 Kawahara J. 32 Honda D. 25, 30, 85 Kawata M. 43 Horák A. 10, 18, 42, 89 Kayama M. 32 Hori S.R. 51 Keeling P.J. 81 Horiguchi T. 55 Kennedy K. 74 Horváthová L. 28 Kerney R. 10 Hoshina Ryo 28 Khlopko Yuri 60, 68 Howard M. 47 Kim E. 10, 33, 55 Hrda S. 77 Kim J.I. 30, 34, 41 Hroudova M. 77 Kim Jong-Hyun 75, 76 Hu S.K. 14 Kim Jong-Rak 76 Hu Xiaozhong 46 Kim Kang-San 34 Huynen M.A. 29 Kim S. 34, 35, 41 Huysmans G. 28 Kimura K. 30 Kinoshita Y. 72 I Kiselev A. 8, 63 Kita Kiyoshi 66 Ichikawa Masatoshi 53 Kitazato H. 88 Ilyash L.V. 8 Klápšťová V. 28 Inagaki Y. 45, 52, 73, 79 Klarner B. 39 Ishemgulova A. 29 Klimeš V. 92 Ishida K. 27, 53, 66, 73 Klimov V.I. 35 Ishikawa A. 72 Knyazev N.A. 9 Ishikawa T. 32 Knyazeva O. 36 Ito Hiroaki 53 Koike Kana 30 Iwata I. 30 Koike Kazu 30 Kolesnikov A.A. 21 J Kolisko M. 36, 67, 78 Konovalova O.P. 18 Jangra S. 5 Kopylov A.I. 36, 38 Janouškovec J. 81 Kormas K.Ar. 17 Jardillier L. 47 Kornilova O.A. 37 Jo B.Y. 30 Korsun S. 36 Jung Jae-Ho 34 Kosakyan Anush 37, 62 Jung S.Y. 31, 75 Kosolapov D.B. 38 Kosolapova N.G. 36, 38 K Kostygov A.Yu. 12, 37 Kotyk Michael 38, 83 Kagerer M. 48 Kraeva N. 29 Kalavathi C. 69 Krashevska V. 39 Kaltz O. 60 Krenek S. 78 Kamikawa R. 67 Kručinská J. 19 Kamra Komal 30, 33 Krupičková A. 28 Kamyshatskaya O.G. 31 Kudryavtsev A.A. 87 Kanesaki Yu. 86 Kumar A. 5 Kang H.K. 31, 69, 75, 76 Kume K. 79 Kang S. 80 Kurina I.V. 39 Kapustin D.A. 24 Kusaka C. 89 Karnkowska A. 77, 81, 86 Kusuoka Yasushi 28 Karpov S.A. 32, 47 Kvitko J. 52 Kashiyama Y. 32, 44, 72 Protistology · 97

L Methling K. 71 Meyer E. 52 Lahr D.J.G. 27, 37 Mezentsev Y.S. 47 Lal Rup 30, 33 Michaelis M. 47 Lalk M. 71 Miller C. 47 Lane C.E. 56, 67, 74 Milyutina I.A. 8 Lang B.F. 40 Min Gi-Sik 34 Langlois G.A. 39 Minagawa J. 43 Lanzoni O. 24, 40, 66, 70 Mitchell Edward A.D. 37, 62 Lara Enrique 37, 62 Mityaeva O.A. 84 Lebedeva N. 24, 40, 60, 66, 70 Miyagishima S. 44, 55, 86 Lee J.M. 23, 40, 89 Moreira D. 32, 47, 52, 60, 82 Lee S.E. 31 Mota Cátia 48 Leger M.M. 40 Motomura T. 30 Lejzerowicz Franck 28 Muchortova O.V. 11 Lhee D.H. 41 Mühlthaler A. 48 Liu H.B. 72 Müller Marina 16 Lobo Maria Luísa 41 Mukhanov V.S. 49 Logacheva M.D. 8, 18, 21 Muñoz-Gómez S.A. 49, 56, 74 Logares R. 7 Mylnikov A.A. 49 Lopez-Escardo D. 26, 42, 82 Mylnikov A.P. 15, 49, 50, 62, 71, 81 López-García P. 32, 47, 52, 60, 83 Mylnikova Z.M. 50 Lukeš J. 10, 18, 29, 42, 74, 89 Luo Xiaotian 46 N Lupachev A.V. 71 Lynn D.H. 43, 84 Nagaraja S. 71 Najdrova V. 50, 87 M Najle S.R. 51 Nakajima M. 85 Ma Mingyang 23 Nakamura Y. 51 Macip-Ríos R. 45 Nakayama T. 52 Makhija S. 5, 43, 76, 77 Nakazawa M. 32 Malviya S. 18, 42 Narula Laxmi 30, 33 Malysheva E. 39 Nassonova E. 52 Mamkaeva M.A. 32 Navarri M. 10 Man Baiying 62 Nekrasova I. 24, 52, 60 Maraun M. 39 Nesbo C. 6 Mariño-Pérez R. 46 Nguyen Ngoc-Lam 24 Martincová E. 36 Nguyen T.H.T. 83 Maruyama M. 32, 44 Nikitina L.I. 84 Maruyama S. 43 Nishigami Yukinori 53 Matantseva O. 44, 58, 61 Nitla V.M. 69 Matos Olga 41, 48, 59, 64 Noh J.H. 89 Matsuo E. 45 Nomura M. 53 Matsuzaki Motomichi 66 Nomura Y. 85 Mayén-Estrada R. 14, 16, 45, 46, 63 Novák Lukáš 86 Mazei Yu.A. 18, 39, 84 Novo Carlos 41 Mazumder M. 71 Novotná K.K. 83 Mazutis L. 51 Nozaki T. 54 McManus G. 65, 68 Medina-Durán J.H. 46 O Melkonian M. 25 Méndez-Sánchez Daniel 46 Oborník M. 13, 19, 90 Mesrop L.Y. 14 Omar A. 54 98 · Author Index

Onsbring H. 55 Reyes-Prieto A. 64 Onuma R. 55 Ribeiro Marta 64 Richardson E. 6 P Rindi F. 65 Rivero F. 92 Paasch A.E. 55 Roger A.J. 36, 49, 67 Pachiadaki M.G. 10, 17 Romanenko A.V. 36 Paight C.J. 56, 74 Romero-Niembro V.M. 45 Pánek T. 28, 56, 78 Rubin E. 65 Panfilkina Tatiana S. 56 Ruiz-Trillo I. 23, 26, 42, 51, 57, 83 Paoli L. 6 Rutherford S. 18 Park M.G. 34, 35, 41 Rylkova O.A. 49 Park M.Y. 31 Ryšánek D. 65 Park S.I. 13 Parra-Acero H. 57 S Paskerova Gita G. 52, 56 Pasulka A.L. 14 Sabaneyeva E. 40, 66, 78 Patel N. 57 Saburova M. 66 Patsyuk M.K. 58 Saffo M.B. 56 Payne R.J. 84 Sakamoto Hirokazu 66 Paz-y-Mino-C.G. 18 Sakhon E.G. 49 Pechkovskaya S.A. 58 Salas Leiva 67 Peixoto Miguel 59 Salomaki E.D. 67 Pellerin G. 52 Samudrala G. 71 Pereira Eulália 59 Sandeep B.V. 69 Pérez-Posada A. 57 Santoferrara L.F. 6, 65, 68 Pérez Reyes C. 25 Sazhin A.F. 36 Petroni G. 24, 40, 60, 66, 69, 78 Scheu S. 39 Petrů M. 28, 40 Schrallhammer M. 60, 78 Petrželková R. 59 Schuster Julia 16 Philippov D.A. 62 Schweikert M. 60 Pinto Mafalda 59 Sebe-Pedros A. 26 Plotnikov Andrey 22, 60, 68, 91 Selivanova Olga 68 Podešvová L. 29 Seong G.S. 31, 69, 75, 76 Podlipaeva Y.I. 9 Sergeeva N.G. 69 Podunay Y.A. 93 Serra V. 69 Ponce-Toledo R.I. 60 Ševčíková T. 90 Poshvina Daria 60 Shadwick L.L. 80 Potekhin A. 24, 40, 52, 60, 66, 70 Shahi P. 71 Pozdnyakov I. 61 Shaidullina A. 40, 70 Prokina K.I. 50, 62 Shaitanov V.M. 70 Shatilovich A.V. 71 Q Sheikh Sanea 72 Shen Z. 72 Qamar M.F.Q. 62 Shigenobu S. 43 Qin Yangmin 62 Shihongi A. 72 Shin H.J. 31, 69, 75, 76 R Shin M. 33 Shin W. 30, 34 Ramírez-Ballesteros M. 63 Shiratori T. 73 Rani Pooja 33 Shmakova L.A. 73 Rautian M. 8, 63 Simdyanov Timur G. 56 Rédou V. 10 Singh B. 5 Reiter I. 71 Singh S. 5 Protistology · 99

Siver P.A. 30 Trebicz-Geffen M. 71 Skalický T. 74 Treitli S.C. 83, 86 Škaloud P. 30, 65 Tretyakova T.P. 79 Skarlato S.O. 9, 44, 61, 74 Tribun M.M. 84 Slamovits C.H. 49, 56, 74 Tripathi Charu 30 Smejkalová P. 83 Troitsky A.V. 8 Smirnov A.V. 9, 31, 52, 75 Tsaousis A.D. 47, 84 Sohn H.J. 31, 69, 75, 76 Tsuchiya M. 88 Sokol M. 56 Tsyganov A.N. 81, 84 Somasundaram S. 5, 43, 76, 77 Tuji A. 51 Somiya R. 51 Tůmová P. 87 Song Kyoung-Ju 76 Tychkov A.Yu. 81 Song H. 46 Song Weibo 46, 91 U Sonobe Seiji 53 Soukal P. 77 Udalov Ilya A. 85 Spiegel F.W. 80 Ueda M. 85 Sprung B. 33 Ueno N. 43 Šrámová E. 83 Umanskaya M.V. 11 Strnad H. 77, 90 Upadhaya M. 5 Suzaki T. 28, 32, 44 Uzuka Akihiro 86 Suzuki N. 51 Suzuki S. 27 V Svärd S.G. 87 Švindrych Z. 87 Vacek Vojtěch 86 Szklarczyk R. 29 Vanunu S. 71 Szokoli F. 78 Varadínová Zuzana 38 Vargas de, Colomban 42 T Verni F. 69, 78 Vidyarathna N. K. 72 Táborský P. 78 Vlasáková J. 83 Tachezy J. 87 Vlček Č. 40, 77 Takabayashi S. 79 Vogts A. 44 Takachs-Vesbach C. 80 Voleman L. 28, 36, 87 Takagi M.T. 79 Volkova E.N. 87 Takahashi H. 43 Voltsky Ivan 28 Tamiaki H. 32, 72 Voss M. 44 Tan S. 91 Votýpka Jan 42, 74, 89 Tanifuji G. 32, 79 Tarasova N.G. 11, 79 W Tashyreva Daria 42 Taylor C. 17 Wang Hongmei 62 Taylor G.T. 17 Wang Y. 22 Tcvetkova V.S. 32 Warren Alan 30, 33, 87 Telesh I.V. 58, 80 Wass M.N. 84 Thompson A.R. 80 Watanabe A. 27 Tice A.K. 80 Wei Chaojun 23 Tikhonenkov D.V. 7, 81 Weiner A.K.M. 88 Timpano H. 32, 52 Wideman J.G. 88 Tomaru Y. 30 Widyastuti R. 39 Török J.K. 82 Williams L.T. 84 Torruella G. 32, 52, 82 Toteja R. 5, 43, 76, 77 X Toyofuku T. 88 Tran Q.H. 83 Xu H. 87 100 · Author Index

Xu K. 12 Z

Y Zabotkina E.A. 36 Zadrobilkova E. 12, 56 Yabuki A. 10, 42, 89 Zagumyonnyi D.G. 90 Yadav K. 5 Záhonová K. 90 Yamaguchi K. 43 Zharikov V.V. 11 Yamaguchi T. 43 Žárský V. 28, 40 Yang E.C. 40, 41, 89 Žihala D. 56, 92 Yang J.H. 23 Zivanovic Y. 60 Yastreb V.B. 70 Zhang H. 10, 65 Yazaki E. 73 Zhang Q. 22, 54, 91 Yi G. 34 Zhang Wenwen 62 Yi Z. 91 Zhao Y. 91 Yokoyama A. 32 Zhu P. 22 Yoon H.S. 13, 23, 25, 34, 40, 41, 89 Zhykov A.V. 84 Yoshikawa Hirofumi 86 Zlatogursky V.V. 92 Yubuki N. 12, 77, 83 Zolotarev V.A. 92 Yurchenko T. 90 Zubáčová Zuzana 86 Yurchenko V. 29, 74, 90 Zutshi S. 5 Protistology · 101

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