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42 • "PROTIST—2016
roles of PDI on Toxoplasma gondii-host interplay, in the context of acute/chronic infection, and evaluating their usefulness as drug-targets using antihuman PDI commercial MAbs. The potential cross-reactions of PDI anti-human MAbs with T. gondii, confirming their usefulness for immunolocalization of PDI not only in human fibroblasts (HFF) target cells, but also in Toxoplasma tachyzoites/cysts were verified. In order to validate this hypothesis a bioinformatics analysis was conducted in the Toxoplasma genome database to search for homologous regions: of the antigens' complete amino acid sequence used in the manufacturing processes of the MAbs utilized in this study; and/or of their more predicted immunogenic regions in case of antigens with longer sequences. Confirmation was obtained by reverse search on human genome for the predicted T. gondii antigens/peptides sequences. Results from indirect immunolabeling assays using immunofluorescence techniques with antihuman PDI MAbs: PDI, PDIA3(ErP57), PDIA6, Calnexin(CNX), glucose-regulated proteins/immu-noglobulin heavy-chain binding protein (GRP78/ BiP) and GRP94 suggest both cross-reaction with target cell-lines and Toxoplasma, being suitable for their profile identification and to evaluate PDI usefulness as a drug-target against T. gondii infection. Different patterns of immunolabeling were observed in Toxoplasma infected and non-infected HFF cells according to the MAb used. The functional confirmation of the characterized PDI involved in the host-pathogen interaction, in the presence of PDI inhibitors, is in progress. Further analyses will be performed by RNA silencing to get complementary evidence of these specific proteins in Toxoplasma-host interactions. Acknowledgments: Supported by FCT ref:VIH/ SAU/0019/2011.
ANALYSIS OF THREE SINGLE-CELL AMPLIFIED GENOMES OF THE CHOANO-FLAGELLATE MONOSIGA BREVICOLLIS REVEALS SINGLE-CELL GENOMICS AS A LIMITED APPROACH FOR EVOLUTIONARY PURPOSES
Lopez-Escardo D. \ Ruiz-Trillo I.12
1 - Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Maritim de la Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain
2 - Institucio Catalana de Recerca i Estudis Avangats (ICREA), Barcelona, Catalonia, Spain david.lopez.@upf.edu
Environmental metabarcoding data is providing a new view of the real protist diversity. This new diversity often occupies key phylogenetic positions
which can led to important insights in the evolution of different eukaryotic lineages. This is the case of the Opisthokonta, in which the identification of new unicellular lineages close to animals has the potential to improve our understanding of the transition towards animal multicellularity. However, isolating these new organisms is a challenge. Instead, single-cell genomics (SCG) seems a prominsing approach to obtain directly the genomes from the environment. However, SCG technologies, which have mainly been employed in prokaryotes, are affected by important biases, specially during the whole genome amplification step. The potential of SGC for eukaryotes remains unclear. To test the potential of this technique for evolutionary studies in eukaryotes, we here analyze three environmental single amplified genomes (SAGs) from the choanoflagellate Monosiga brevicollis, whose genome is already sequenced. We show the genome recovery obtained from our SAGs was low (between 30-5%, around 3,684 out of 9,175 genes). Interestingly, the percentatge of pylogenomic markers that our SAGs contains is good (almost 100% for some datasets). We also explored different techniques to improve the quality and the recovery of the assemblies obtained. We found that genome recovery increased up to 48% by pooling the data from the three different SAGs. Current SCG technologies seem a limited approach for adressing some questions but has the potential to provide enough data for phylogenomic analyses.
DIPLONEMIDS - NEW KIDS ON THE BLOCK Lukes Julius12, Flegontova Olga12, Flegontov Pavel123, Faktorovâ Drahomira12, Kaur Binny-preet12, Votypka Jan124, Tashyreva Dana12, Yabuki Akinori5, Malviya Shruti6, de Vargas Colomban78, Bowler Chris6, Burger Gertraud9, Horâk Ales12
1 - Institute of Parasitology, Biology Centre, Czech Academy of Sciences
2 - Faculty of Science, University of South Bohemia, Ceské Budëjovice
3 - Faculty ofScience, University ofOstrava, Ostrava, Czech Republic
4 - Faculty of Science, Charles University, Prague, Czech Republic
5 - Japan Agency for Marine-Earth Science and Technology, Kanagawa, Japan
6 - Ecole Normale Supérieure, PSL Research University, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Paris, France
7 - Station Biologique de Roscojf, Roscoff, France
8 - Sorbonne Universités, Paris, France
9 - Université de Montréal, Montreal, Canada jula@paru.cas.cz.
Protistology ■ 43
Results from the Tara Oceans metabarcoding study revealed that a significant fraction of the plankton in the sunlit ocean is constituted by diplonemid flagellates, a poorly known group of heterotrophic excavate protists. In an analysis of V9 18S rDNA metabarcodes from planktonic communities collected in 123 globally distributed stations, diplonemids emerge as a highly abundant and extremely diverse group that lacks geographic structuring but displays strong vertical separation. Uniquely among planktonic eukaryotes, taxonomic identification of diplonemids is far from saturation. They are more abundant in polar regions and deeper waters, yet their biological function in the ocean — the largest ecosystem of our biosphere - remains unknown. We are attempting to isolate and culture new marine diplonemids in order to sequence their genomes, analyze their ultrastructure and establish protocols for their genetic manipulation. By this combined approach, we hope to shed more light on these „new kids on the block".
THE SMALL SUBUNIT rRNA GENE SEQUENCE OF THE CHONOTRICH CHILODO-CHONA CARCINI JANKOWSKI, 2007 CONFIRMS CHONOTRICHS AS A DYSTERIID-DERIVED CLADE (PHYLLOPHARYNGEA, CILIOPHORA) Lynn D.H.
Department of Integrative Biology, University of Guelph, Guelph, ON Canada N1G 2W1 lynn@zoology.ubc.ca
The chonotrichs are sessile ciliated protozoa ectosymbiotic on the body parts of a variety of crustaceans. They have long been considered a separate group because their sessile habit has resulted in the evolution of a very divergent body form and reproductive strategy compared to free-living ciliates. In the mid-20th Century, the free-living dysteriid cyrtophorian ciliates were proposed as a potential sister clade because the chonotrich bud or daughter cell showed similarities during division morphogenesis (i.e. ontogeny) to these free-living dysteriids. A single small subunit (SSU) rRNA gene sequence is available for the chonotrich Isochona sp. However, its authenticity has been questioned in the recent literature, and the placement of this sequence within the dysteriid clade has added to this controversy. In this report, the SSUrRNA gene sequence of the chonotrich Chilodochona carcini ectosymbiotic on the green crab Carcinus maenas is provided. Topology testing of the SSUrRNA gene phylogeny constructed by Bayesian Inference robustly supports the sister-group relationship of Isochona sp. and C. carcini, the monophyly of
these two chonotrichs, and the divergence of the chonotrich clade within the dysteriid clade.
MAPPING THE HIDDEN DIVERSITY OF FREE LIVING FRESH WATER CILIATES FROM DELHI REGION, INDIA Makhija S.1, Abraham J.S.1, Somasundaram S.1, Toteja R.1, Gupta R.2
1 - Acharya Narendra Dev College, University ofDelhi, Delhi, India
2 - Maitreyi College, University ofDelhi, Delhi, India seemamakhija@andc.du.ac.in
India, with 2.4% of the world's area, has over 8% of the world's total biodiversity, making it one ofthe 12 mega diversity countries in the world. Despite this richness, the data concerning ciliate diversity from India is rather scarce. We at University of Delhi have been involved in the morphological and molecular taxonomy of ciliates from freshwater bodies namely from Okhla Bird Sanctuary, Sanjay Lake, Pond at Rajghat in Delhi region. A large number of species from different classes have been observed from these water samples namely from genus Tetmemena, Stylonychia, Laurentiella, Euplotes, Aspidisca, Paraurostyla, Gastrostyla, Pseudourostyla, Urostyla, Oxytricha and Uroleptus from class Spirotrichea; Blepharisma, Spirostomum and Stentor from class Heterotrichea; Paramecium, Frontonia, Vorticella and Cyclidium from class Oligohymenophorea; Litonotus, Dileptus, Didinium and Lacrymaria from class Litostomatea; Colpoda from class Colpodea; Chilodonella from class Phyllopharyngea; Loxodes from class Karyorelectia; and Coleps from class Prostomatea have been identified by live cell observations, protargol staining, silver staining and morphometric measurements. In the present study, we describe in detail few species from the subclass Stichotrichia and Hypotrichea. There is difficulty in understanding the phylogenetic relationship between different species of these group based solely on morphological markers. Therefore, we are trying to look into both morphological and multiple molecular markers like SSU- rRNA, ITS 1, ITS2 , hsp70 and histone genes and we could depict morphological and molecular congruence in the species from subclass Stichotrichia and Hypotrichea.
GLOBAL SHIFTS IN GENE EXPRESSION PROFILES ACCOMPANIED WITH ENVIRONMENTAL CHANGES IN CNIDARIAN-DINOFLAGELLATE ENDOSYMBIOSIS Maruyama S.1, Aihara Y.2, Yamaguchi T.3, Yama-guchi K.4, Shigenobu S.4, Takahashi H.3, Kawata M.1, Ueno N.3, Minagawa J.2
