uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records
Bournemouth Univ, Fac Sci & Technol, Dept Life & Environm Sci, Poole BH12 5BB, Dorset, England;Florida Atlantic Univ, Harbor Branch Oceanog Inst, Ft Pierce, FL 34946 USA.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-6898-6377
Bournemouth Univ, Fac Sci & Technol, Dept Life & Environm Sci, Poole BH12 5BB, Dorset, England.
2018 (English)In: Protist, ISSN 1434-4610, E-ISSN 1618-0941, Vol. 169, no 6, p. 875-886Article in journal (Refereed) Published
Abstract [en]

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600 km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1 mm in size, could have a global biogeographical distribution.

Place, publisher, year, edition, pages
2018. Vol. 169, no 6, p. 875-886
Keywords [en]
Protist, stop codon, RNA-seq, anaerobic respiration, symbiotic algae, Heterotrich
National Category
Ecology
Identifiers
URN: urn:nbn:se:uu:diva-372698DOI: 10.1016/j.protis.2018.08.001ISI: 000452432800005PubMedID: 30447617OAI: oai:DiVA.org:uu-372698DiVA, id: diva2:1277099
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research Swedish Research Council, 2015-04959
Note

Hunter N. Hines and Henning Onsbring contributed equally to this work.

Available from: 2019-01-09 Created: 2019-01-09 Last updated: 2019-09-07Bibliographically approved
In thesis
1. Novel insights into protist diversity and niche adaptation using single cell transcriptomics
Open this publication in new window or tab >>Novel insights into protist diversity and niche adaptation using single cell transcriptomics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protists are a polyphyletic group of microbes that represents the vast majority of eukaryotic diversity. Despite this, most sequencing efforts targeting eukaryotes have been focused on animals, fungi and plants. The sequencing bias towards multicellular organisms can partially be explained by the difficulty in cultivating protists, which is needed in traditional sequencing workflows. In this thesis, single-cell RNA sequencing has been used to generate transcriptome data from environmental protists, without being dependent on establishing a culture. These transcriptome data have been used to discover novel protist diversity, as well as exploring the cell biology of two ciliates.

In the first chapter, transcriptomes of cell fragments were generated for the ciliate Stentor. This ciliate is well-known for its ability to repair drastic cellular wounds, and the transcriptomes uncovered genes involved in processes such as cell cycle, signaling and microtubule-based movement to be activated during Stentor regeneration.

Spirostomum semivirescens is another ciliate, whose transcriptome was generated using single-cell RNA sequencing. The transcriptome data suggest that S. semivirescens is using rhodoquinol-dependent fumarate reduction for respiration in environments with low levels of oxygen.

Single-cell RNA sequencing was further used to target cells smaller than Stentor and Spirostomum. By generating 124 transcriptomes of environmental protists, a high number of novel lineages could be identified. The generated transcriptome data included free-living prokinetoplastids, non-photosynthetic euglenids, metamonads and katablepharids.

A few modifications to the single-cell RNA sequencing protocol Smart-seq2 were necessary to generate the 124 transcriptomes of small protists cells. The impact of these modifications to Smart-seq2 was benchmarked using Giardia intestinalis. The generated single-cell transcriptomes revealed that addition of freeze-thaw cycles to Smart-seq2 improved transcript recovery. Finally, we propose a protocol that allows identification of failed cDNA reactions, based only on measuring DNA concentration, without compromising on transcript recovery. Reducing the dependency on quality control will be important if single-cell RNA sequencing would be done in a high-throughput workflow.

In conclusion, single-cell RNA sequencing can be a powerful tool for studying protist diversity and biology. In particular, it has the potential to efficiently uncover protist diversity, provided that a robust and efficient method to isolate single cells from the environment is established.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 52
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1853
Keywords
Protists, microbial eukaryotes, cultivation-independent methods, single-cell RNA sequencing, phylogenomics
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-392618 (URN)978-91-513-0747-3 (ISBN)
Public defence
2019-10-25, B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-10-01 Created: 2019-09-07 Last updated: 2019-10-15

Open Access in DiVA

fulltext(1737 kB)112 downloads
File information
File name FULLTEXT01.pdfFile size 1737 kBChecksum SHA-512
417506c6ca58bb7036444c231e9eb917d27b8e97caf055fa3709c3efb52375263b15847307d1e1e8fd62747377e1a7e0c61fa2e1986d397fb3550a1e85b53b7f
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Authority records BETA

Onsbring Gustafson, HenningEttema, Thijs J. G.

Search in DiVA

By author/editor
Onsbring Gustafson, HenningEttema, Thijs J. G.
By organisation
Molecular EvolutionScience for Life Laboratory, SciLifeLab
In the same journal
Protist
Ecology

Search outside of DiVA

GoogleGoogle Scholar
Total: 112 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 121 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf