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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,
