Molecular investigation of the ciliate Spirostomum semivirescens, with first transcriptome and new geographical records

Hunter N. Hines1,3*, Henning Onsbring2*, Thijs J. G. Ettema2 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. Phylogenetic analysis of the rRNA gene cluster revealed both isolates to be identical. 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 1mm in size, could have a global biogeographical distribution

Novel insights into protist diversity and niche adaptation using single cell transcriptomics

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

Novel insights into protist diversity and niche adaptation using single cell transcriptomics

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

Formation of the musculoskeletal system during the craniofacial development of zebrafish

The musculoskeletal system supports the internal structures of the body and consists of bones, ligaments, muscles and tendons. This system forms during early embryonic development, a process where many components today are unknown. In order to get a better understanding for those developmental steps, fluorescent in situ hybridisation has been performed on five genes. All five genes represent different transcription factors. These genes were selected based on the assumption that they could be important for the formation of the musculoskeletal system. After in situ hybridisation was performed, embryos were stained by immunohistochemistry to get a reference signal in the cartilage to enable easier interpretation of the expression pattern. In this study four of the selected transcription factors, Scleraxis a, Scleraxis b, Mohawk a and Mohawk b turned out to be expressed close to points where muscles are attached to the cartilage elements in the zebrafish head. Therefore, these genes are good candidates for future functional studies of muscle attachment development

Formation of the musculoskeletal system during the craniofacial development of zebrafish

The musculoskeletal system supports the internal structures of the body and consists of bones, ligaments, muscles and tendons. This system forms during early embryonic development, a process where many components today are unknown. In order to get a better understanding for those developmental steps, fluorescent in situ hybridisation has been performed on five genes. All five genes represent different transcription factors. These genes were selected based on the assumption that they could be important for the formation of the musculoskeletal system. After in situ hybridisation was performed, embryos were stained by immunohistochemistry to get a reference signal in the cartilage to enable easier interpretation of the expression pattern. In this study four of the selected transcription factors, Scleraxis a, Scleraxis b, Mohawk a and Mohawk b turned out to be expressed close to points where muscles are attached to the cartilage elements in the zebrafish head. Therefore, these genes are good candidates for future functional studies of muscle attachment development

Formation of the musculoskeletal system during the craniofacial development of zebrafish

The musculoskeletal system supports the internal structures of the body and consists of bones, ligaments, muscles and tendons. This system forms during early embryonic development, a process where many components today are unknown. In order to get a better understanding for those developmental steps, fluorescent in situ hybridisation has been performed on five genes. All five genes represent different transcription factors. These genes were selected based on the assumption that they could be important for the formation of the musculoskeletal system. After in situ hybridisation was performed, embryos were stained by immunohistochemistry to get a reference signal in the cartilage to enable easier interpretation of the expression pattern. In this study four of the selected transcription factors, Scleraxis a, Scleraxis b, Mohawk a and Mohawk b turned out to be expressed close to points where muscles are attached to the cartilage elements in the zebrafish head. Therefore, these genes are good candidates for future functional studies of muscle attachment development

RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities. Onsbring et al. sequence transcriptomes of individual bisections of regenerating cells of the giant heterotrichous ciliate Stentor polymorphus. Their differential expression analysis reveals that protein phosporylation, microtubule-based processes, and genes involved in the cell cycle are important for cellular regeneration.</p

Nya sensorsystem för kombinationseffekter av antibiotika

Idag är antibiotikaresistens ett allvarligt problem. En tillfällig lösning på problemet är att utnyttja synergieffekter som kan uppstå mellan olika typer av antibiotika. Det innebär att antibiotika skulle kunna ha effekt mot bakterier som har utvecklat resistens. I denna rapport har flera olika metoder, för att mäta bakterietillväxt, utvärderats. Dessa metoder indikerar om antibiotikakombinationerna har en effekt. Däribland har både kommersiella och nyligen publicerade metoder undersökts. De kommersiella metoderna granskades genom en marknadsanalys. De krav som har satts på systemet är att det ska gå att köra flera prov samtidigt, analysen ska kunna utföras på kort tid, metoden ska inte vara destruktiv mot bakterierna samt att datan bör vara på formen av cellkoncentration levande celler. Bland de kommersiella mätmetoderna valdes tre metoder ut som verkade vara bland de bästa utifrån kraven. Dessa var ATP-analys, LIVE/DEAD® BacLight™ samt SYBR Green I. Från artiklar hittades fyra nyligen publicerade system som var av intresse utifrån kraven. Dessa var BioMEMS (eng. Biomedical Micro-Electro-Mechanical Sensor), odling i slutna mikrokanaler, SMR (sluten mikrokanalresonator) samt mikroflödessystem med pH-sensor. Utifrån vad som finns på marknaden idag och de nyligen publicerade systemen som har undersökts ansåg gruppen att BioMEMS och användande av mikrokanaler är de två bäst lämpade metoderna för ändamålet

Nya sensorsystem för kombinationseffekter av antibiotika

Idag är antibiotikaresistens ett allvarligt problem. En tillfällig lösning på problemet är att utnyttja synergieffekter som kan uppstå mellan olika typer av antibiotika. Det innebär att antibiotika skulle kunna ha effekt mot bakterier som har utvecklat resistens. I denna rapport har flera olika metoder, för att mäta bakterietillväxt, utvärderats. Dessa metoder indikerar om antibiotikakombinationerna har en effekt. Däribland har både kommersiella och nyligen publicerade metoder undersökts. De kommersiella metoderna granskades genom en marknadsanalys. De krav som har satts på systemet är att det ska gå att köra flera prov samtidigt, analysen ska kunna utföras på kort tid, metoden ska inte vara destruktiv mot bakterierna samt att datan bör vara på formen av cellkoncentration levande celler. Bland de kommersiella mätmetoderna valdes tre metoder ut som verkade vara bland de bästa utifrån kraven. Dessa var ATP-analys, LIVE/DEAD® BacLight™ samt SYBR Green I. Från artiklar hittades fyra nyligen publicerade system som var av intresse utifrån kraven. Dessa var BioMEMS (eng. Biomedical Micro-Electro-Mechanical Sensor), odling i slutna mikrokanaler, SMR (sluten mikrokanalresonator) samt mikroflödessystem med pH-sensor. Utifrån vad som finns på marknaden idag och de nyligen publicerade systemen som har undersökts ansåg gruppen att BioMEMS och användande av mikrokanaler är de två bäst lämpade metoderna för ändamålet

Giardia intestinalis Raw sequence reads

Raw sequence reads generated to benchmark different versions of a protocol for generation of cDNA from single protist cell