Genes and Genomes in Ciliates: • Gene expression analysis in Tetrahymena thermophila under stress conditions • Approach to the characterization of the germline (micronuclear) genome in Euplotes crassus

I carried out my PhD work at the University of Camerino in the molecular and cellular biology laboratory under the supervision of prof. Cristina Miceli and prof. Sandra Pucciarelli. The research activities of the laboratory are mainly focused on the study of molecular and cellular adaptations and responsive mechanisms of eukaryotic microorganisms to environmental changes and stressed conditions. The research approach includes the study of genome organization and control of gene expression. Ciliated protozoa, such as the freshwater Tetrahymena thermophila (Order Hymenostomatida) and marine Euplotes species (Order Hypotrichida), are used as model organisms. General objectives of my thesis were to demonstrate the applicability of transcriptional profiling, for elucidating the mechanisms of toxic action of engineered nanomaterials in the ciliate Tetrahymena thermophila, endorsing it as a valid model organism for the freshwater environmental pollution studies and to pave the way to the use of marine Euplotes species as additional valid models for studies on the marine environment. Despite their genetic diversity, all ciliates are characterized by two common features: the possession of complexes of cilia, used for swimming or crawling and for phagocytic food capture, and the presence of nuclear dimorphism. Each ciliate contains a germ line diploid micronucleus (mic), inactive in vegetative cells, but transcriptionally active in mating cells and responsible for the genetic continuity during sexual reproduction, and a larger polyploid somatic macronucleus (MAC), the site of transcriptional activity in the vegetative growing cell. Different ciliate species can contain different numbers of micronuclei: Tetrahymena and Euplotes species have one mic, which divides mitotically during vegetative growth. Tetrahymena species have a single ovoid MAC. Euplotes species contain a single, highly elongated MAC (Prescott 1994)[...]. When two starved cells of different mating type undergo conjugation, they exchange haploid mic and develop a new MAC from the mic. The developing MAC is called the Anlagen. During its differentiation several programmed DNA rearrangements occur (M.-C. Yao and Chao 2005): 1) the deletion of segments of the mic genome known as internally eliminated sequences (IESs); 2) the site-specific fragmentation of the chromosomes (Fan and Yao 2000); 3) the addition of a telomere to each new end (Yu and Blackburn 1991) generating the MAC chromosomes (Eisen et al. 2006) , and 4) their amplification (M. C. Yao, Yao, and Monks 1990). In Tetrahymena approximately 6,000 IESs are removed, resulting in the MAC genome being 10% to 20% smaller than that of the mic (M. C. Yao et al. 1984). Hypotrichs undergo the same three global genomic changes of elimination, fragmentation, and amplification during MAC development, but the phenomena are more extreme and entail the elimination of 96% of the mic genomic DNA complexity in Oxytricha species and 98% in Stylonychia lemnae (Prescott 1994), generating very small chromosomes called nanochromosomes that in most cases contain a single genetic unit. Conjugation results in complete genome replacement in each exconjugant and genetic identity of both exconjugants in some species (Orias, Cervantes, and Hamilton 2011). T. thermophila has normal dimensions of about 50 μm in length and 20 μm in width. It swims in temperate freshwater environments like streams, lakes and ponds, and it tolerates a wide range of temperatures, from 12°C up to about 41°C (Frankel 1999). T. thermophila cell is covered by multi-layer cortex that is semi-rigid and arranged into 18-21 longitudinal rows (ciliary rows) of cortical units containing basal bodies mostly accompanied by the cilia. Nutrients are taken up via pinocytosis and phagocytosis, the latter is the main feeding mechanism of Tetrahymena. Food vacuoles are formed in an oral apparatus made up of four compound ciliary elements (hence Tetra-hymena) embedded within fibrillar structures located near the anterior end of the cell. Near the posterior end of the cell is located cytoproct where undigested food particles are excreted from the cell. An osmoregulatory organelle, the contractile vacuole, accumulates and releases collected fluid through contractile vacuole (CV) pores [...] (Wloga and Frankel 2012; Nusblat, Bright, and Turkewitz 2012; Frankel J. 2007). Having a doubling time of less than 3 h, T. thermophila is considered one of the fastest growing eukaryotes. It can readily grow to a high density on a wide range of media. Its life cycle (Figure 3) allows the use of conventional tools of genetic analysis, and molecular genetic tools suitable for gene function analysis. In addition, although it is unicellular, it possesses most of the conserved cell structures and molecular processes found in multicellular eukaryotes. Fundamental discoveries of molecular biology were made in this ciliate protozoan and it has been the first member of the phylum Ciliophora to have its complete somatic (MAC) genome sequenced (Eisen et al. 2006). The amplified MAC genome consists of about 104 Mb, that contain more than 27,000 protein coding genes. The Tetrahymena Functional Genomics Database (TetraFGD) is available (Xiong et al. 2013) and it facilitates the study of the molecular bases of environmental responses, since data obtained in different environmental conditions can be easily compared. The research work of my thesis is divided in three parts: two extensive Chapters and one Additional Project section as an appendix. In Chapter 1, I reported a study on the potential toxic effect of silver nanoparticles (AgNPs) accumulation in the environment, using T. thermophila as a model organism. This was started by a trilateral collaboration project including Estonia and China in the frame of the European Cooperation in Science and Technology (COST) action BM1102 entitled “Ciliates as model systems to study genome evolution, mechanisms of non-Mendelian inheritance, and their roles in environmental adaptation” and coordinated by our laboratory. Since the nano-industry has grown incredibly fast in the last few years (Ivask et al. 2014) and AgNPs are the most widely commercialized NPs that are used as antimicrobials in various consumer products (Bondarenko, Juganson, et al. 2013), concentrations of Ag ions are increasing in soil and water (Aueviriyavit, Phummiratch, and Maniratanachote 2014). The collateral dispersion of AgNPs in the environment may pose a threat to non-target organisms (Ivask et al. 2014). Therefore, to understand their toxicity mechanisms is essential for the design of more efficient nano-antimicrobials and for the design of biologically and environmentally benign nanomaterials. In a previous study, only a few genes known to be involved in detoxification by oxidative stress were analysed by qPCR, after exposure of T. thermophila to AgNPs compared with the effect of a soluble silver salt, AgNO3, to evaluate the contribution of the dissolved silver to the overall toxic effect of AgNPs (Juganson et al. 2017). This study showed that Ag-ions play a major role in the toxicity of AgNPs in T. thermophila. However, although oxidative stress related genes were overexpressed in AgNP-exposed Tetrahymena, the intracellular ROS level was not elevated, possibly due to Tetrahymena's very efficient antioxidant defense mechanisms that certainly involves other not yet investigated genes. The study also highlighted the relevance of the AgNPs toxicity for environmentally abundant organisms and still left open the question of which other genes are involved in the Tetrahymena response to AgNPs. Does the organization in AgNPs produce a different effect on the total gene expression regulation with respect to the effect induced by the silver ions released by the salt AgNO3? Therefore, to answer to these open questions and to obtain a global vision of the changes occurring in T. thermophila gene expression, we decided to perform an RNA sequencing analysis to compare the cell’s exposure to AgNO3 and to AgNPs. For this purpose, I went to the The National Institute of Chemical Physics and Biophysics (NICPB) of Tallinn (Estonia) to repeat the toxicity test and to learn how to handle AgNPs. Then I joined the Protozoan Functional Genomics laboratory at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences (CAS) in Wuhan (China). In this laboratory coordinated by Dr. Wei Miao, a deep RNA-sequencing study of T. thermophila during the three major stages of the life cycle (growth, starvation and conjugation) was performed and a Tetrahymena functional database (TetraFGD) was developed (Xiong et al. 2013). There, as a pilot experiment, I exposed T. thermophila to two sub-lethal concentrations of silver compounds and collected the cells after 2 and 24 h to perform RNA isolation and RNA-sequencing. Differential gene expression was analyzed. The experimental sequences were compared with the controls to evaluate quantitatively the inhibition or increase of gene expression due to NPs or Ag ions. Then, I performed gene set enrichment analyses. According to these results we decided to investigate further the 24 h exposure condition, which was considered more consistent for the biological model, also considering T. thermophila doubling time. Later, at the University of Camerino, I performed a biological replica of the 24 h exposure experiment to obtain statistically significant data. I also validated my data with qPCR and all this work is reported in detail in Chapter 1 of my thesis. The results presented in Chapter 1 confirmed that T. thermophila is a valid freshwater model organism to study stress response mechanisms. No similar models are provided so far from the marine environment. In order to propose a seawater counterpart, my attention was focused on Euplotes marine species. These species still require analysis of the genomes to have a reference for the environmental response studies. Some steps in this direction have already been done in our laboratory in collaborations, through a first assembly of MAC genomes of Euplotes crassus and E. focardii that revealed important aspects of the translation machinery that frequently uses frameshifting (Lobanov et al. 2017). This analysis was followed by a more deeply annotation of the Euplotes MAC to which I am currently contributing (data not yet published). However, Euplotes mic sequences are still unknown as well as the details on the DNA rearrangements to produce the MAC genome by transpositions and DNA elimination. This gap is significant for our research because the mechanisms of the stress response are most probably influenced by the DNA rearrangements and transpositions. To address this gap in research, I developed a technique to isolate E. crassus micronuclear chromosomes, as reported in Chapter 2 of this thesis. This work was supported by The Marine Microbiology Initiative (MMI, funded by Gordon and Betty Moore foundation) and developed in collaboration with the University of Bern and the University of Connecticut. Finally, in order to generate real valid genetic models with Euplotes species, comparable with the Tetrahymena model, it would be necessary to establish genetic manipulation techniques for functional studies and further applications. In the Additional Project section of this thesis are listed different experimental approaches for Euplotes transfection. This study was also supported by MMI which aims to accelerate development of genetic tools to enable development of experimental model systems in marine microbial ecology. The MMI also co-funded my PhD scholarship. The three parts of my thesis are written to be used for three independent publications. I apologize for repetitions mainly present in the three introductions.