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gene transcription level. In the present investigation, we studied the acute toxicity of heavy metal -cadmium and the transcriptional changes of hsp70, metallothionein and glutathione peroxidise gene in spirotrich ciliates. The effect of cadmium on the expression of these gene transcripts was investigated by real-time qPCR. The gene expression increased significantly in cadmium treated cells against the control cells indicating that these genes may be involved in cellular defence mechanisms by elevating their gene expression against heavy metal-induced stress.
EVALUATION OF ACUTE TOXICITY OF HEAVY METALS AND THE ACTIVITY OF ANTIOXIDANT ENZYMES IN FRESHWATER CILIATES
Somasundaram S.1, Abraham J.S.1, Toteja R.1, Makhija S.1, Gupta R.2
1 - Acharya Narendra Dev College, University ofDelhi, Delhi, India
2 - Maitreyi College, University ofDelhi, Delhi, India [email protected]; [email protected] Heavy metal concentration in the environment is increasing world-wide due to anthropogenic activities and India is no exception to this. Metals are biologically important though increase in the threshold concentration of heavy metal in the environment may interfere with the metabolic activity of organisms. As studies on the cellular level could detect stress reactions earlier in time compared to common physiological methods, and since ciliates offer higher sensitivity towards heavy metals as compared to other microorganisms mainly due to lack of cell wall, ciliates were used as model organism in this study. In the present investigation, the toxicity of heavy metals, viz., Cadmium and Copper was assessed in freshwater ciliates; Tetmemena sp. and Euplotes sp. It was noted that growth rate exhibited inverse relationship with the heavy metal concentration. Mortality rate and various doses of heavy metals (control, LC30, LC50 & LC70) were determined in the ciliates. Generation of reactive oxygen species (ROS) was determined in these ciliates by using specific fluorescent dye in the presence of heavy metals. Increase in ROS production indicated that heavy metal stress induced ROS in living cells. Since it is known that antioxidant enzymes get activated to detoxify ROS, activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined in ciliates at various doses of heavy metals. The levels of SOD and GPx were found to be significantly higher in the presence of heavy metals. This study indicates that antioxidant enzymes can be used as an effective
biomarker to evaluate cytotoxic effect of heavy metals.
GENOMIC AND TRANSCRIPTOMIC SURVEY OF OSMOTROPHIC EUGLENID RHAB-DOMONAS COSTATA
Soukal P.1, Hrda S.1, Karnkowska A.1, Strnad H.2, Hroudova M.2, Vlcek C.2, Hampl V.1
1 - Department of Parasitology, Charles University in Prague, Czech Republic
2 - Institute of Molecular Genetics, Academy of Sciences, Czech Republic [email protected].
Euglenids represent a diverse group of protists with diverse modes offeeding including heterotrophy and photosynthesis, e. g. in Euglena gracilis. Until today, there is not available any complete genome sequence of euglenids and this probably represents the biggest gap in the sampling of eukaryotic genomes. We are running genomic and transcriptomic projects on a primary osmotroph Rhabdomonas costata. To avoid bacterial contamination, the DNA sample was prepared from cells picked using laser microdissection and amplified by GenomePlex®. Almost 1GB of reads was generated in four runs on Illumina MiSeq platform. The current assembly is too fragmented to be used for genome annotation, nevertheless the comparison of transcriptomic and genomic data allow us to estimate gene density and the features of introns. Annotation of the mitochondrial backbone metabolism provides the first data on Rhabdomonas mitochondrion.
GENE TRANSFER ACCOMPANYING THE SECONDARY ENDOSYMBIOSIS OF EUGLENID PLASTID
Soukal P.1, Hrda S.1, Karnkowska A.1, Yubuki N.12, Hroudova M.3, Vlcek C.3, Elias M.4, Hampl V.1
1 - Charles University in Prague, Faculty of Science, Department of Parasitology, Prague, Czech Republic
2 - Charles University in Prague, Faculty of Science, Department of Zoology, Prague, Czech Republic
3 - ASCR, v. v. i., Institute of Molecular Genetics, Department ofGenomics andBioinformatics, Prague, Czech Republic
4 - University of Ostrava, Faculty of Science, Life Science Research Centre, Ostrava, Czech Republic [email protected].
Autotrophic euglenids (Euglenophyta) form a monophyletic group with secondary green plastids, which were most probably acquired by their common ancestor. However, the acquisition of the plastid earlier in the evolution of euglenids (plastid-early hypothesis) cannot be ruled out. The process of organelle acquisition is accompanied by
78 • "PROTIST—2016
the transfer of genes from the endosymbiont to host (EGT), the presence of such genes indicates past endosymbiosis. To test the plastid-early hypothesis and to learn more about the contribution of EGT to euglenid genome, we have analyzed transcriptomes of 5 euglenids (2 osmotrophic, 3 autotrophic) using a pipeline, which enabled us to select genes related to algae. The contribution of algal genes in autotrophic euglenids (around 2 % of genes) is higher than in primary osmotrophs (around 0.07 %) supporting the plastid-late hypothesis. Surprisingly, we observed a high number of genes related to other algal groups than green algae.
CHARACTERIZATION OF A NOVEL PARAMECIUM ENDOSYMBIONT AND A CRITICAL REVISION OF "BASAL RICKETTSIALES" Szokoli F.12, Schrallhammer M.3, Sabaneyeva E.4, Castelli M.2, Krenek S.1, Doak T.G.56, Verni F.2, Berendonk T.U.1, Petroni G.2
1 - Institut für Hydrobiologie, Technische Universität Dresden, Germany
2 - Dipartimento di Biologia, Université di Pisa, Italy
3 - Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-UniversitätFreiburg, Germany
4 - Department of Cytology and Histology, St. Petersburg State University, Russia
5 - Biology, Indiana University, IN, USA
6 - National Center for Genome Analysis Support, IN, USA
[email protected] In the past ten years, the number of newly described endosymbionts within the bacterial order Rickettsiales has constantly grown, and 18 novel Rickettsiales genera inhabiting protists—such as ciliates or amoeba—have been described since 2006. Half of these new species belong to the recently proposed family " Candidatus Midichloriaceae", whereas five novel bacterial symbionts were found belonging to Rickettsiaceae and six belonging to Holosporaceae and other Rickettsiales with uncertain position ("basal Rickettsiales"). In this work, we provide molecular and ultrastructural data on a novel bacterial endosymbiont inhabiting a Paramecium biaurelia isolate collected near Bloomington, Indiana (USA). We propose the name " Candidatus Jurandia parameciophila" in honor of Dr. Artur Jurand. The Gram-negative bacteria (1.5-2.0 * 0.35-0.38 ^m in size) occupies the host cytoplasm and is devoid of flagella. For molecular characterization, the SSU rRNA gene was sequenced and used for taxonomic assignment and the design of species-specific oligonuc-leotide probes. Phylogenetic analyses indicate that the endosymbiont belongs to "basal Ricket-
tsiales" (Alphaproteobacteria), clustering with the recently proposed family " Candidatus Paracaedi-bacteraceae". We obtained tree topologies showing a strong separation of Rickettsiales into at least two groups; one represented by the families Rickettsiaceae, Anaplasmataceae, and "Candidatus Midichloriaceae", the so-called RAM clade, and the other group represented by "basal Rickettsiales" including " Candidatus Jurandia parameciophila". Therefore, and in accordance with other recent publications, we propose to limit the order Rickettsiales to the RAM clade and rise "basal Rickettsiales" to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, presently comprising four clades at the family level.
'ANAERAMOEBA' - NEW LINEAGE WITHIN METAMONADA
Taborskv P.1, Panek T.12, Kolisko M.3, Cepicka I.1
1 - Department of Zoology, Charles University in Prague, Prague, Czech Republic
2 - Department of Biology and Ecology Faculty of Science University ofOstrava Chittussiho 10, Ostrava, 710 00
3 - Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada [email protected]
We have isolated and cultured nine strains of anaerobic amoebae ('Anaeramoeba') from marine coastal hypoxic sediments worldwide. 'Anaeramoeba' cells were fan-shaped, with extremely flattened pseudopodia and trailing uroidal filaments, and morphologically belonging to flamellian or flabellate morphotype. We distinguished six morphospecies that differed in a number of features including cell size and nuclear morphology. ' Anaer-amoeba' species shared a unique combination of morphological features. All three strains examined by TEM possessed double membrane-bound organelles with no cristae, presumably hydrogenosomes, associated with prokaryotes (probably methanogens). Cytoplasm contained a large acentriolar centrosome, and no basal bodies were observed. Peculiar isokont flagellates with two or four flagella were rarely observed in two strains representing different species. Single- and also five-gene analyses showed that 'Anaeramoeba' clade was monophyletic, but were completely unable to assess its phylogenetic position. We analysed transcriptomic data of two 'Anaeramoeba'species. Phylogenomic analysis based on 160 protein-coding genes surprisingly showed that 'Anaeramoeba' instead ofbeing a member ofAmoebozoa, represents a novel deep lineage within Metamonada (Eukaryota: Excavata).
