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Protistology ■ 67
School of Medicine, The University of Tokyo 3 - School of Tropical Medicine and Global Health, Nagasaki University hzsakamoto@hotmail.co.jp
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 Dayana.Salas@dal.ca
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
eric.salomaki@gmail.com
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
68 • "PROTIST—2016
evolution as an organism gives up autotrophy in favor of moving onto its relatives' couch and raiding their refrigerator. We sequenced draft transcripto-mes for the parasitic red alga Choreocolax polysi-phoniae and its free-living host Vertebrata lanosa. These data, in combination with genomic DNA sequence data for these taxa was compared with other published red algal genomes and transcripto-mes to investigate the early consequences of tran-sitioning from autotrophy to parasitism. Choreocolax polysiphoniae appears to have lost genes involved in plastid maintenance and photosynthetic processes. Investigation ofthese data and their implications for the evolution of parasitism remains ongoing.
MOLECULAR DIVERSITY AND PHYLO-GENY OF OLIGOTRICHIA AND CHOREO-TRICHIA (CILIOPHORA, SPIROTRICHEA) Santoferrara L.F., McManus G.B. Department of Marine Sciences, University of Connecticut
luciana.santoferrara@uconn.edu We revise and expand the molecular data on the subclasses Oligotrichia and Choreotrichia in terms of both genetic markers and taxonomic sampling. Two approached are used, one based on sequences from morphologically-characterized specimens, and another one including also the unidentified environmental sequences available in NCBI Gen Bank. First, concatenated sequences of ribosomal DNA markers (18S rDNA, 28S rDNA and ITS regions) from seventeen families and four clades of the paraphyletic genus Tintintinnopsis were analyzed, including two families newly studied by single cell sequencing and six genera newly sequenced for at least one marker. Second, all the 18S rDNA sequences available in GenBank for these subclasses were retrieved and curated as part of the EukRef initiative, resulting in almost 3,200 sequences and 1,000 OTUs (operational taxonomic units clustered at 99% similarity). The concatenated dataset allowed confirming or discovering novel phylogenetic relationships within Choreotrichia. The 18S rDNA dataset allowed rough comparisons of molecular diversity between the two subclasses. Because Choreotrichia has been easier to barcode (mainly for the loricate Tintinnida), analyses have not revealed novel diversity, but exposed known and new misidentifications and classification inconsistencies. In contrast, three clades within Oligotrichia included only environmental sequences, thus highlighting a high proportion of partially-characterized or novel diversity in this subclass. These analyses help solving classification conflicts and will provide a reference for studying the diversity and distribution
of these abundant and ecologically relevant ciliates in marine plankton.
TAXONOMIC COMPOSITION OF PROKA-RYOTES-ASSOCIATES OF HALOPHILIC PRO-TISTS
Selivanova Olga1, Gogoleva Natalya2, Khlopko Yuri1, Plotnikov Andrey1
1 - Institute for Cellular and Intracellular Symbiosis UB RAS, Orenburg, Russia
2 - Kazan Institute ofBiochemistry and Biophysics KSC RAS, Kazan, Russia selivanova-81@mail.ru
As known, taxonomic structure of communities in hypersaline environments is simple. It includes prokaryotes, photosynthetic and heterotrophic protists, fungi, and crustaceans. Under extreme salinity the community is simplified up to only microorganisms, where phototrophic protists are the main producers of organic matter, and heterotrophic protists are the only consumers. Symbiotic associations with halophilic phototrophic protists are well studied for green alga Dunaliella and its prokaryotic associates, while heterotrophic protists and their role in forming of halophilic microbial community are studied poorly. Aim of the study was to estimate taxonomic composition of prokaryotes — associates of autotrophic and heterotrophic protists isolated from salt water bodies in Salt-Iletsk and Elton regions. The method of 16S metagenomic sequencing was used. 111 OTUs were found in association with heterotrophic flagellate Pleurostomum salinum. The genera of archaea Natronomonas sp., Haloarcula sp., Haloplanus sp., Halorubrum sp. and bacteria Halovibrio sp., uncultured bacterium (Bacteroide-tes) were predominant. Heterotrophic flagellate Tulamoeba bucina had very similar taxonomic composition of prokaryotes. There were 129 OTUs including uncultured bacterium (Bacteroidetes), Natronomonas sp., Halovibrio sp., Haloferax sp., Halorubrum sp. as predominant genera. There were 32 and 41 OTUs in associations with Pharyngomonas sp. and Heterolobosea aff. Euplaesiobystra respectively. Bacteria Idiomarina sp., Halomonas sp., Gracilimonas sp. were predominant in both cultures. Phototrophic flagellate Dunaliella parva had 19 OTUs of prokaryotes — associates. Bacteria Halovibrio sp. and archaea Halorubrum sp. were the most abundant. A crucial factor influencing the taxonomic composition of prokaryotes in the associations with halophilic protists was level of medium mineralization. Taxonomic diversity was higher in associations with heterotrophic protists than in associations with autotrophs. The highest
