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Protistology ■ 55
SINGLE CELL RNA SEQUENCING, AN EFFECTIVE APPROACH FOR ANALYZING THE GENOME CONTENT AND EVOLUTION OF NON-CULTIVATABLE MICROBIAL EUKARYOTES
Onsbring H.1, Divne A.M.2, Ettema T.1
1 - Department ofCell and Molecular Biology, Uppsala University, Sweden
2 - Microbial Single Cell Genomics, SciLifeLab henning.onsbring@icm.uu.se
Technical advances in culture-independent techniques have significantly contributed to the discovery of novel microbial lineages. Both metageno-mics and single cell genomics have been shown to be useful tools for studying the genomes ofprokaryotes. However, microbial eukaryotes generally have a complex genome structure, which leads to that these techniques tend to perform poorly. If it would be possible to apply single cell RNA sequencing, issues related to data assembly can potentially be avoided. With a method adapted from Smart-seq2, where template switching is used to amplify cDNA, transcriptomes have been generated for several single protist cells with close to full coverage of the coding potential. In Smart-seq2, transcriptomes can be generated in a 384 well format, which gives the potential for protist transcriptomes to be generated with high throughput. In transcriptomics data, the highly expressed house keeping genes are among the most likely genes to have high coverage and full length. Those genes are also suitable for constructing phylogenies that aim to resolve deep branches in the eukaryotic tree oflife. Therefore, RNA sequencing of many cells in parallel has the potential to effectively generate sequence data for novel or poorly studied protist lineages, and to increase our understanding of their biology and evolution.
ULTRASTRUCTURAL AND TRANSCRIP-TOMIC STUDIES OF KLEPTOCHLORO-PLASTIDIC DINOFLAGELLATE NUSUTTO-DINIUM AERUGINOSUM (DINOPHYCEAE) Onuma R.1, Horiguchi T.2, Miyagishima S.1
1 - Division of Symbiosis and Cell Evolution, Department of Cell Genetics, National Institute of Genetics
2 - Department of Biological Sciences, Faculty of Science, Hokkaido University ronuma@nig.ac.jp
Unarmored dinoflagellates Nusuttodinium spp. possess kleptochloroplasts which are ingested from cryptomonads and retained in the host cell for certain periods. Our previous studies revealed that N. poecilochroum digests cryptomonad nucleus
and never enlarges chloroplast. By contrast, N. aeruginosum enlarges single chloroplast throughout the cell and divides nucleomorph, retaining a cryptomonad nucleus. These differences are able to be interpreted as different evolutionary stages toward acquisition of 'true chloroplast' within the same linage. It is, therefore, clear that these dinoflagellates are interesting materials to investigate evolutionary transitions toward establishment of endosymbiosis. To reveal fate of the cryptomonad organelles in Nusuttodinium aeruginosum after host cell divisions, we have further observed all daughter cells with LM and single-cell TEM methods. These observations showed that cryptomonad karyokinesis did not occur and that only one of the daughter cells inherited a cryptomonad nucleus. Among all daughter cells originating from a single cell through five generations, the cell that inherited the cryptomonad nucleus consistently possessed the largest kleptochloroplast. Therefore, this study suggests that the cryptomonad nucleus carries important information for the enlargement of the kleptochloroplast. These results suggesting cryptomonad nucleus remains transcriptionally-active in the host cell and we are examining changes in transcriptome of dinoflagellate nucleus, cryptomonad nucleus and nucleomorph during the course of transition in the kleptochloroplast development. In this presentation, methods and progresses of transcriptome analyses are discussed in addition to the results of morphological observation.
RETENTION OF BACTERIVORY IN THE DO-MINANTLY PHOTOAUTOTROPHIC GREEN ALGA CYMBOMONAS TETRAMITIFORMIS IS INFLUENCED BY PHOSPHATE LIMITATION
Paasch A.E., Burns J.A., Kim E. American Museum of Natural History apaasch@amnh.org
The lineage Chloroplastida (green algae and land plants) is defined by the presence of a primary plastid, which was acquired through the ingestion of a photosynthetic bacterium presumably during the early- or mid-Proterozoic eon. However, members of Chloroplastida are dominantly photo-autotrophic and only a few members of the early-diverging class, Prasinophyceae, retain the ability to ingest bacteria. It is unclear why bacterivory is restricted to the prasinophytes. The prasinophyte Cymbomonas tetramitiformis was definitively confirmed through transmission electron microscopy to ingest bacteria into a large food vacuole. Recently,
56 • "PROTIST—2016
this phagomixotroph's genome was found to retain a unique combination of genes not present in obligate photoautotrophs or heterotrophs. Additional prasinophytes have been found to ingest fluorescently-tagged bacteria and synthetic particles. To investigate drivers ofbacterivory in Cymbomonas, cultures of the alga were grown under limited N, P and light regimes and fed bacteria as a rescue source of nutrients. The Cymbomonas genome was also mined for metabolic genes related to nutrient uptake and assimilation. Surprisingly, bacteria only rescued Cymbomonas growth under phosphate-limited conditions, but not when nitrogen or light-limited. The genome contains genes related to phosphate metabolism that are not present in other Chloroplastida. A full GS-GOGAT pathway is present and no unique nitrogen-related genes were found. These results suggest that Cymbomonas retains the ability to extract phosphorous from prey, but relies on photoautotrophic pathways for nitrogen and carbon. This trait gives Cymbomonas a competitive advantage in P-limited cultures and may drive retention of bacterivory in this species.
SUPPLIMENTING SYMBIONTS: PATHWAY RESTORATION IN A LONG TIME PARASITE Paight C.J.1, Muñoz-Gómez S.A.2, Saffo M.B.1, Slamovits C.2, Lane C.E.1
1 - Department of Biological Sciences, University of Rhode Island, Kingston
2 - Department ofBiochemistry and Molecular Biology, Dalhousie University cpaight12@gmail.com
Apicomplexans are highly successful parasites, infecting every major metazoan lineage. The genus Nephromyces has recently been described as having a mutualistic relationship to its host Molgula tunicates (Saffo et al., 2010), making Nephromyces the only reported mutualistic apicomplexan genus. Apicomplexans have reduced genomes and have lost the ability to make many essential metabolites. These essential metabolites are instead scavenged from their host. Species of Nephromyces are known to have three different bacterial endosymbionts. Our data show that the bacterial endosymbionts encode a number of essential pathways lost in Apicomplexans. Here we describe insights from the transcriptome from Nephromyces, all three bacterial endosymbi-onts and the tunicate host. These data gives us a glimpse of the complex metabolic relationships and intertwined pathways of hosts and endosymbionts, with a particular focus on the biosynthesis of amino acids and vitamins.
TWO NEW NON-CANONICAL NUCLEAR GENETIC CODES FROM A RHIZARIAN AND A FORNICATE WITH UAG, BUT NOT UAA, AS A SENSE CODON
Pánek T.1, Sokol M.1, Zihala D.1, Derelle R.2, Zadrobílková E.3, Cepicka I.3, Eliás M.1
1 - Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Ostrava, Czech Republic
2 - Unité d'Ecologie, Systématique et Evolution, Centre National de la Recherche Scientifique UMR 8079, Université Paris-Sud, 91405, Orsay, France
3 - Department ofZoology, Faculty ofScience, Charles University in Prague, Vinicna 7,12844Prague, Czech Republic
mistrpanek@sez.nam.cz,
The original presumption that all organisms use the same (standard) genetic code for translation of mRNA sequences into proteins has been challenged by discoveries of deviations ofthis universal language in both prokaryotes and eukaryotes. In eukaryotes the nuclear genetic code has proven to be much more conservative than that ofmitochondria, and plastids; just a few its variants are known. Generally, we can sort them into 3 groups: (1) UGA serves as a sense codon; (2) UAA and UAG simultaneously serve as sense codons; (3) CUG encodes serine or alanine (rather than leucine). We analyzed transcriptomic data from two unrelated protists and found out that these organisms, as only eukaryotes known so far, use UAG as a sense codon in nuclear genetic code while retaining UAA as a termination codon. One of these organisms uses UAG as codon for leucine, similarly to a code variant described from certain mitochondria. The other one instead uses UAG to encode glutamine, resembling thus the non-canonical genetic code of several eukaryotic groups including many ciliates, hexamitin diplomonads, some oxymonads, and some ulvophytes; however, all these taxa have at the same time reassigned also the UAA codon. Phylogenetic analyses place the first organism into the rhizarian lineage Sainouroidea, whereas the second one represents an undescribed lineage of " Carpediemonas-like organisms" in Fornicata (Metamonada). Our findings thus once again show protists as an inexhaustible resource of peculiar departures from the "standard" biology.
AGAMOCOCCIDIANS: COCCIDIANS OR GREGARINES? NEW SPECIES AND NEW DATA ON THE PHYLOGENETIC POSITION OF THE GROUP
Panfilkina Tatiana S.1, Simdyanov Timur G.2, Aleoshin Vladimir V.3, Paskerova Gita G.1
