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Special issue on Oxygen Minimum Zone Microbiology.-- 11 pages, 5 figures, 3 tables, supporting information https://doi.org/10.1111/1462-2920.15504.-- This is the pre-peer reviewed version of the following article: Yaiza M. Castillo, Irene Forn, Sheree Yau, Xosé Anxelu G. Morán, Laura Alonso-Sáez, Néstor Arandia-Gorostidi, Dolors Vaqué, Marta Sebastián; Seasonal dynamics of natural Ostreococcus viral infection at the single cell level using VirusFISH; Environmental Microbiology 23(6): 3009-3019 (2021), which has been published in final form at https://doi.org/10.1111/1462-2920.15504. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions, Ostreococcus is a cosmopolitan marine genus of phytoplankton found in mesotrophic and oligotrophic waters, and the smallest free-living eukaryotes known to date, with a cell diameter close to 1 μm. Ostreococcus has been extensively studied as a model system to investigate viral–host dynamics in culture, yet the impact of viruses in naturally occurring populations is largely unknown. Here, we used Virus Fluorescence in situ Hybridization (VirusFISH) to visualize and quantify viral-host dynamics in natural populations of Ostreococcus during a seasonal cycle in the central Cantabrian Sea (Southern Bay of Biscay). Ostreococcus were predominantly found during summer and autumn at surface and 50 m depth, in coastal, mid-shelf and shelf waters, representing up to 21% of the picoeukaryotic communities. Viral infection was only detected in surface waters, and its impact was variable but highest from May to July and November to December, when up to half of the population was infected. Metatranscriptomic data available from the mid-shelf station unveiled that the Ostreococcus population was dominated by the species O. lucimarinus. This work represents a proof of concept that the VirusFISH technique can be used to quantify the impact of viruses on targeted populations of key microbes from complex natural communities, This work was supported by the Spanish projects MEFISTO (CTM2013-43767-P, MINECO) and ALLFLAGS (CTM2016-75083-R, MINECO). YMC was supported by a FPI Spanish fellowship (BES-2014-067849), SY was supported by a Juan de la Cierva fellowship (IJCI-2017-34245) and LAS was supported by a Juan de la Cierva and Ramon y Cajal contract (RYC-2012-11404). MS was supported by a Viera y Clavijo contract funded by the ACIISI and the ULPGC. NAG was supported by Basque Government's PhD-program fellowship, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Ostreococcus tauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13–22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >104 transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20–40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.

Prasinoviruses are large dsDNA viruses commonly found in aquatic systems worldwide, where they can infect and lyse unicellular prasinophyte algae such as Ostreococcus. Host susceptibility is virus strain-specific, but resistance of susceptible Ostreococcus tauri strains to a virulent virus arises frequently. In clonal resistant lines that re-grow, viruses are usually present for many generations, and genes clustered on chromosome 19 show physical rearrangements and differential expression. Here, we investigated changes occurring during the first two weeks after inoculation of the prasinovirus OtV5. By serial dilutions of cultures at the time of inoculation, we estimated the frequency of resistant cells arising in virus-challenged O. tauri cultures to be 10&minus, 3&ndash, 10&minus, 4 of the inoculated population. Re-growing resistant cells were detectable by flow cytometry 3 days post-inoculation (dpi), visible re-greening of cultures occurred by 6 dpi, and karyotypic changes were visually detectable at 8 dpi. Resistant cell lines showed a modified spectrum of host-virus specificities and much lower levels of OtV5 adsorption.

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analyzed the life cycle of Ostreococcus tauri virus 5 (OtV5), a lytic virus, using transcriptome sequencing (RNA-Seq) from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 h, but during the night, viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the dark appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4% of the host's genes. Some host cells underwent lysis progressively during the night, but a larger proportion were lysed the following morning. Our data suggest that the life cycles of algal viruses mirror the diurnal rhythms of their hosts. IMPORTANCE Prasinoviruses are common in marine environments, and although several complete genomes of these viruses and their hosts have been characterized, little is known about their life cycles. Here we analyze in detail the transcriptional changes occurring over a 27-h-long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime, when the most energy (from light) is available, but during the night viral transcription activates, accompanied by expression of a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

Ostreococcus tauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13−22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >104 transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20−40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of world's oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae., Author Summary We propose that chromosome 19 of O. tauri is specialized in defence against viral attack, a constant threat for all planktonic life, and that the most likely cause of resistance is the over-expression of numerous predicted glycosyltransferase genes. O. tauri thus provides an amenable model for molecular analysis of genome evolution under environmental stress and for investigating glycan-mediated host-virus interactions, such as those seen in herpes, influenza, HIV, PBCV and mimivirus.

In surveys of marine phytoplankton diversity, prasinophyte algae are commonly found and are often a major component, especially in coastal areas. They form a paraphyletic assemblage of at least nine clades, with the Mamiellophyceae (prasinophyte clade II) being a major group. In the order Mamiellales, the three genera Bathycoccus, Micromonas and Ostreococcus are the dominant picoeukaryotic organisms in many different oceanic areas, as witnessed by their abundance in analyses of environmental DNA samples. Each of the six complete genomes analyzed from cultured representatives of this group has revealed densely packed coding sequences, with strong evolutionary divergence from its nearest phylogenetically defined neighbors. These species lie at the base of the green lineage, but various metabolic processes reflect their marine lifestyles and distinguish them from land plants, including a high proportion of selenoprotein enzymes and C4 photosynthesis. They all possess two unusual chromosomes with lower GC content and atypical gene content, whose function so far remains enigmatic.