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66 • "PROTIST—2016
LYING ON THE SURFACE: NEW DATA ON THE EPISYMBIONTS OF CILIATES Sabaneyeva E.1, Lanzoni O.2, Lebedeva N.3, Benken K.4, Potekhin A.5, Petroni G.2
1 - Department of Cytology and Histology, Saint-Petersburg State University, Saint-Petersburg, Russia
2 - Department of Zoology, University of Pisa, Pisa, Italy
3 - Core Facility Center for Cultivation of Microorganisms, Saint-Petersburg State University, Saint-Petersburg, Russia
4 - Core Facility Center for Microscopy and Microanalysis, Saint-Petersburg State University, Saint-Petersburg, Russia
5 - Department ofMicrobiology, Saint-Petersburg State University, Saint-Petersburg, Russia [email protected]
Protists often form symbiotic associations with prokaryotes. Ciliates are especially well known for harbouring endosymbiotic and ectosymbiotic bacteria, the relationship between the partners ranging from mutualism to parasitism. Epibionts can be found both in anaerobic and in free living aerobic ciliates, however, so far they have never been reported in paramecia. Here we present morphological (DIC, AFM, CLSM, TEM) and molecular characterization of the bacterium fouling cells of Paramecium primaurelia strain isolated from nature in Cyprus. At the last stage of infection, epibionts formed a thick coat on the cortex of the ciliate causing complete loss of cilia, changes of the cell shape, dwarfing, and finally, the host death. Fluctuations of the ciliate population density of the infected strain registered throughout long term observations of the laboratory culture imply parasitoid nature of the epibiont. In preliminary FISH experiments, epibionts were easily revealed with the universal eubacterial probe Eub 338, however, they never showed positive signal when hybridized with probes specific for Alpha-, Beta-, Gamma- or Deltaproteobacteria. Molecular characterization was done following the full-cycle rRNA approach and association of the epibionts with the host was confirmed by FISH experiments with newly designed species-specific probes. The obtained 16S rDNA sequence showed a similarity of circa 82% with Alphaproteobacteria class, thus suggesting that epibionts are extremely unusual novel organisms. Phylogenetic analysis, which is under way, will enlighten the evolutionary position of this enigmatic organism, presumably, either a basal alphaproteobacterium or a fast-evolving line within the order Rickettsiales.
BIODIVERSITY OF BENTHIC DINOFLA-GELLATES ALONG THE SOUTHERN COAST OF OMAN WITH EMPHASIS ON POTENTIALLY TOXIC SPECIES Saburova M.
Kuwait Institute for Scientific Research, P.O. BOX 1638, Salmiya 22017, Kuwait; long-term consultancy [email protected]
Benthic dinoflagellates have attracted considerable research attention during the last decades due to their morphological and taxonomic diversity, ecological significance in marine benthic ecosystems, and their capability to produce toxins and cause toxic blooms in the coastal areas. A pilot taxonomic survey of the benthic dinoflagellates was performed for the first time at the Arabian Sea coast along Dhofar Governorate of the Sultanate of Oman in the vicinity of Salalah City at five sampling sites on February 2014 and 2016. It has been shown that the bottom sediments and the surface of brown and red macroalgae along the southern Omani coast were inhabited by taxonomically diverse and abundant assemblages of sand-dwelling and epiphytic dinoflagellates. A total of 38 dinoflagellate species belonging to five orders were recorded in this study including Dinophysiales, Gymnodiniales, Gonyaulacales, Peridiniales, and Prorocentrales. High diversity of the sand-dwelling dinoflagellates was supported mainly by gymnodinioid and peri-dinioid taxa. In contrast, members of Coolia, Gambierdiscus, Ostreopsis, and Prorocentrum were among the most abundant and diverse epiphytic dinoflagellates. A number of known toxin producers were observed including the ichthyotoxic dinoflagellates of the genus Amphidinium, the okadaic acid producing Prorocentrum species, the cooliatoxin producing Coolia, the palytoxin producer Ostreopsis, and the ciguatera-related Gambierdiscus species. The presence of known toxic dinoflagellate species may indicate a potential risk of toxicity in the marine environment of the southern Omani coast, and underscores the need for further studies on taxonomy, ecology and toxicology of benthic dinoflagellates in Oman.
DEVELOPMENT OF A MULTIPLE-DRUG SELECTION SYSTEM FOR DUAL-TRANSFORMATION OF THE OYSTER PARASITE PERKINSUS MARINUS
Sakamoto Hirokazu1, Hirakawa Yoshihisa1, Ishida Ken-ichiro1, Kita Kiyoshi23, Matsuzaki Motomichi2
1 - Faculty of Life and Environmental Sciences, University of Tsukuba
2 - Department of Biomedical Chemistry, Graduate
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School of Medicine, The University of Tokyo 3 - School of Tropical Medicine and Global Health, Nagasaki University [email protected]
Plastids in apicomplexan parasites are highly degenerated. The organelle is nevertheless essential for completion of the parasite life cycle. Interestingly, an oyster parasite Perkinsus marinus, which is sister to dinoflagellates and close to Apicomplexa, also has a DNA-lacking, extremely degenerated plastid. Functional analysis of the cryptic organelle is attracting and required to understand the relationship between the organelle degeneration and parasitism. The transgenic technique is a convincing approach for the analyses ofproteins ofinterest and is practicable in P. marinus. However, each transfected cell must be isolated from untransfected cells by hand labor using a micromanipulator multiple times to obtain any transfected cell lines. This is because drug selection system has not been established. Here, we identified two drugs that are available for selection of transfected P. marinus cells. Firstly, we screened antibiotics shown utility in apicomplexan parasites and determined that blasticidin S, bleo-mycin and puromycin effectively inhibited the parasite growth. Then, their resistance genes were fused downstream of gfp or mCherry gene, and each construct was transfected to the parasite. After two months, the fluorescent signals were observed in almost all cells cultured with bleomycin or puromycin. Furthermore, dual transfected cells were selected by using the two drugs, which enables us to examine colocalization of plastid proteins. We believe that this system provides new opportunities for functional analyses ofthe plastids in the parasite.
A DRAFT GENOME OF THE ANAEROBIC FLAGELLATE CARPEDIEMONAS MEMBRA-NIFERA, A FREE-LIVING RELATIVE OF METAMONAD PARASITES Salas Leiva D.E.1, Kolisko M.2, Curtis B.1, Eme L.1, Kamikawa R.3, Roger A.1
1 - Centre for Comparative Genomics and Evolutionary Bioinformatics (CGEB), Department ofBiochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada, B3H4R2
2 - Beatty Biodiversity Centre, Dept. Botany, University ofBritish Columbia
3 - Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Nihonmatsu cho, Kyoto 606-8501, Japan [email protected]
Carpediemonas membranifera is a free living flagellated metamonad related to diplomonad parasites such as Giardia intestinalis and Spironucleus
salmonicida. We are interested in elucidating the evolutionary transitions to anaerobiosis and parasitism within metamonada, and sequenced the genome of C. membranifera. The genome assembly is 22.4 Mb long with 11328 predicted protein-coding genes, 41% of those have introns. Automatic annotation was carried out by searching against the Interpro, PFAM, Prosite, TIGR databases to identify domains, assign putative functions and predict metabolic pathways. Expert manual annotation is underway for genes encoding proteins functioning in DNA repair, mRNA degradation, mitochondrion-related organelles, cell surface or external cellular processes involved in host tissue adhesion, immune evasion, pathogenicity, nutrient acquisition, metabolite transport and environmental sensing, among others. We have completed analyses ofthe DNA repair pathways. Those can drive sexual/ parasexual pathways, antigen diversification and copy number variation, and are of great importance for adaptive evolution. C. membranifera possesses a complete system for excision repair, and the double strand break repair machinery including 1) a homologous recombination pathway and 2) microhomology-mediated end joining and singlestrand annealing. Also, it has several gene family expansions, as well as, a complete repertoire of cell cycle checkpoints and sex-related proteins. G. intestinalis and S. salmonicida have minimalistic and slightly different versions ofthe pathways found in C. membranifera suggesting that there have been some secondary losses and modifications in diplomonads as a result of their parasitic lifestyle.
THE GENOMIC COST OF BECOMING A RED ALGAL FREELOADER Salomaki E.D., Lane C.E.
DepartmentofBiologicalSciences, University ofRho-de Island
An abundance of genomic and transcriptomic data have been gathered over the past decade providing a wealth of knowledge about what it takes to be a successful parasite. Genomes of highly derived eukaryotic parasites have been sequenced including those from formerly photosynthetic lineages including apicomplexans. These data have revealed fascinating innovations that evolved over hundreds of millions of years, enabling parasites to infect and evade their hosts. Unlike highly derived lineages of eukaryotic parasites, red algae appear to be fertile ground for adopting a parasitic life strategy as seen by numerous recent and independent evolutions of parasitic taxa. Red algal parasites provide a great system to investigate the early stages of genome
