Interactions between marine picoeukaryotes and their viruses one cell at a time Interacciones entre picoeucariotas marinos y sus virus célula a célula Yaiza M. Castillo de la Peña Aquesta tesi doctoral està subjecta a la llicència Reconeixement 4.0. Espanya de Creative Commons . Esta tesis doctoral está sujeta a la licencia Reconocimiento 4.0. España de Creative Commons . This doctoral thesis is licensed under the Creative Commons Attribution 4.0. Spain License . Interactions between marine picoeukaryotes and their viruses one cell at a time (Interacciones entre picoeucariotas marinos y sus virus célula a célula) Yaiza M. Castillo de la Peña Tesis doctoral presentada por Dª Yaiza M. Castillo de la Peña para obtener el grado de Doctora por la Universitat de Barcelona y el Institut de Ciències del Mar, programa de doctorado en Biotecnología, Facultat de Farmàcia i Ciències de l’Alimentació . Directoras: Dra. Mª Dolors Vaqué Vidal y Dra. Marta Sebastián Caumel Tutora: Dra. Josefa Badía Palacín Universitat de Barcelona (UB) Institut de Ciències del Mar (ICM-CSIC) La doctoranda La directora La co -directora La tutora Yaiza M. Castillo Dolors Vaqué Marta Sebastián Josefa Badía En Barcelona, a 25 de noviembre de 2019 Cover design and images: © Yaiza M. Castillo. TEM images from Derelle et al ., 2008 This thesis has been funded by the Spanish Ministry of Economy and competitivity (MINECO) through a PhD fellowship to Yaiza M. Castillo de la Peña (BES-2014- 067849), under the program “Formación de Personal Investigador (FPI)”, and adscribed to the project: “Impact of viruses on marine microbial communities using virus-host models and metagenomic analyzes.” MEFISTO (Ref. CTM2013‐43767‐P, P.I.Dr. Dolors Vaqué). Other projects that contributed partially to the completion of this thesis were: ALLFLAGS (CTM2016‐75083‐R, MINECO, P.I. Dr. Ramon Massana), INTERACTOMICS (CTM2015‐69936‐P, MINECO, Dr. Ramiro Logares), the EU project SINGEK (H2020‐ MSCAITN‐2015‐675752, P.I. Dr. Ramon Massana) and Tara Oceans Expedition (http://www.embl.de/tara-ocean s/). A mis padres y a mis abuelos ACKNOWLEDGEMENTS Primero de todo quiero agradecer a mis directoras de tesis Dolors Vaqué y Marta Sebastián por apoyarme y guiarme a lo largo de estos años de doctorado. Por su paciencia, motivación y conocimientos. Sin ellas esta tesis no habría sido posible. Tampoco habría sido posible sin la inmensa ayuda de Irene Forn y Jeff Mangot, los cuales han trabajado conmigo codo con codo para poder sacar adelante este trabajo. A todas mis compañeras del ICM que me han acompañado durante estos años y han hecho que la experiencia de la tesis tuviera un toque de luz especial. Gracias a Mariri Vicioso, Marta Masdeu, Idaira Santos, Carolina Marín, Elisabet Sà, Isabel Sanz, Anna Arias, Sergio González, Caterina Rodríguez, Dorleta Orue-Echevarría, Mireia Mestre, Ramiro Logares, Xavi Leal, Estela Romero, Isabel Marín, Néstor Arandia, Sheree Yau, Fran Aparicio y Sdena Nunes. Espero no haberme dejado a nadie, si es así, espero que me disculpéis. Por último, gracias a mi familia por su apoyo incondicional. A mis padres, por todo el amor que me han dado y todo lo que han hecho por mí. A mis abuelos, por haber creído siempre en mí y haber estado tan orgullosos de su nieta, siempre estaréis en mi memoria. Os quiero. CONTENTS Abstract/Resumen/Resum ................................................................ 11 General introduction and aim of the thesis ........................................ 17 General Introduction ............................................................................... 19 Aim of the thesis ..................................................................................... 33 Chapter 1 ................................ ................................ .......................... 39 Visualization of viral infection dynamics in a unicellular eukaryote and quantification of viral production using VirusFISH. Abstract ................................................................................................... 41 1.1. Introduction ..................................................................................... 42 1.2. Materials and methods .................................................................... 43 1.3. Results .............................................................................................. 48 1.4. Discussion ......................................................................................... 55 References ............................................................................................... 62 Supplementary information .................................................................... 65 Chapter 2 ................................ ................................ .......................... 73 Seasonal dynamics of Ostreococcus spp. viral infection at the single cell level using VirusFISH Abstract ................................................................................................... 75 2.1. Introduction ..................................................................................... 76 2.2. Materials and methods .................................................................... 79 2.3. Results .............................................................................................. 83 2.4. Discussion ......................................................................................... 92 References ............................................................................................... 96 Supplementary information ................................................................... 100 9 Chapter 3 ................................ ................................ ........................ 109 Assessing the viral content of uncultured picoeukaryotes in the global‐ocean by single cell genomics Abstract ................................................................................................. 111 3.1. Introduction ................................................................................... 112 3.2. Materials and methods .................................................................. 115 3.3. Results ............................................................................................ 120 3.4. Discussion ....................................................................................... 136 References ............................................................................................. 142 Supplementary information ................................................................... 150 Comments and future perspectives ................................ ................. 191 General conclusions ................................ ................................ ........ 199 10 Abstract/Resumen/Resum ABSTRACT Marine viruses are key components of marine microbial communities, as they influence the cellular abundances and the community structure of microbes, participate in their genetic exchange, and intervene in the ocean biogeochemical cycles. Most studies dealing with the role of viruses in the marine environment have been done from a bulk community point of view, but going from the bu lk community perspective to specific virus ─host relationships is essential in order to understand the role of viruses in shaping a determined host community, in modifying host genomes, and ultimately in the release of organic compounds from the lysed cells. For this reason, in this thesis we implemented and applied different methodologies that are able to detect, visualize and quantify virus ─host interactions in marine eukaryotes at the single cell level. We focused on picoeukaryotes (cells <3 µm) because they play crucial roles in marine food webs and biogeochemical cycles, and virus ─host interactions in natural populations of these minute eukaryotes are largely unknown. In the first chapter we combined previously developed techniques, used to assess prokaryotic host ─phage interactions, to implement VirusFISH for detecting specific virus ─host dynamics, using as a model system the photosynthetic picoeukaryote Ostreoccocus tauri and its virus OtV5. With the VirusFISH technique, we could also monitor the infection, as well as quantify the free viruses produced during the lysis of the host in a non-axenic culture, which allowed the calculation of the burst size. This study set the ground for the application of the VirusFISH technique to natural samples. In the second chapter of this thesis, we applied VirusFISH to seawater samples from the Bay of Biscay (Cantabrian Sea) to study the dynamics of viral infection in natural populations of Ostreococcus along a seasonal cycle. We were able to quantify the percentage of cells infected over time, and compared these results with the transcriptional viral and host activities derived from metatranscriptomic 11 Abstract/Resumen/Resum data. This constitutes the first study where a specific viral ─host interaction has been visualized and monitored over time in a natural system. Picoeukaryotes in the ocean are prevalently uncultured, and thus, in the third chapter of this thesis we went an step further to unveil novel viral ─host relationships in eukaryotic uncultured hosts. For this purpose, we mined single amplified genomes (SAGs) of picoeukaryotes obtained during the Tara Oceans expedition for viral signatures. We found that almost 60% of the cells analyzed presented an associated virus with narrow host specificity. Some of the viral sequences were widely distributed and some geographically constrained, and they were preferentially