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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)

11 pages, 7 figures, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2020.01559/full#supplementary-material.-- All datasets generated for this study are included in the article/Supplementary Material, One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. To this end, techniques that enable the detection and quantification of virus-host interactions at the single-cell level are essential. With this goal in mind, we implemented virus fluorescence in situ hybridization (VirusFISH) using as a model the marine picoeukaryote Ostreococcus tauri and its virus Ostreococcus tauri virus 5 (OtV5). VirusFISH allowed the visualization and quantification of the proportion of infected cells during an infection cycle in experimental conditions. We were also able to quantify the abundance of free viruses released during cell lysis, discriminating OtV5 from other mid-level fluorescence phages in our non-axenic infected culture that were not easily distinguishable with flow cytometry. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were already produced between 8 and 24 h. With this work, we demonstrate that VirusFISH is a promising technique to study specific virus-host interactions in non-axenic cultures and establish a framework for its application in complex natural communities, YC was supported by Ministerio de Economía, Industria y Competitividad, Gobierno de España (MINECO), grant number: BES-2014-067849 during her PhD. MS was supported by Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI), grant: Viera y Clavijo 2016. Ministerio de Economía, Industria y Competitividad, Gobierno de España (MINECO), grant numbers: CTM2013‐43767‐P and CTM2016‐75083‐R supported the project and paid for the investigation (reagents, material, etc.). NG was supported by Agence Nationale de la Recherche (ANR), grant number: ANR-17-CE02-0012-03., With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

11 pages, 5 figures, 1 table, supplementary materials http://advances.sciencemag.org/cgi/content/full/6/14/eaay2587/DC1.-- Data and materials availability: The genome sequence for O. mediterraneus RCC2590 and OmV2 can be found under GenBank accession numbers WMKK00000000 and MN688676, respectively. The complete gene annotations of RCC2590 can be accessed on ORCAE (https://bioinformatics.psb.ugent.be/orcae/). MMETSP sequence data can be accessed from www.imicrobe.us/#/projects/104. The genomes and their GenBank accession numbers used in this study are as follows: B. prasinos (GCA_002220235.1), M. commoda RCC299 (GCA_000090985.2), M. pusilla CCMP1545 (GCA_000151265.1), O. tauri RCC4221 (CAID01000001.2 to CAID01000020.2, CR954200.2, and CR954199.2), O. lucimarinus CCE9901 (GCA_000092065.1), BpV1 (HM004432.1), BpV2 (HM004430.1), MpV1 (HM004429.1), OtV1 (FN386611.1), OtV2 (FN600414.1), OtV5 (EU304328.2), OtV6 (JN225873.1), OlV1 (HM004431.1), OlV2 (KP874736.1), OlV3 (HQ633060.1), OlV4 (JF974316.1), OlV5 (HQ632827.1), OlV6 (HQ633059.1), OlV7 (KP874737.1), and OmV1 (KP874735.1). Ostreococcus sp. RCC809 is available from the JGI Genome Portal under project ID 16233, Virus-microbe interactions in the ocean are commonly described by “boom and bust”dynamics, whereby a numerically dominant microorganism is lysed and replaced by a virus-resistant one. Here, we isolated a microalga strain and its infective dsDNA virus whose dynamics are characterized instead by parallel growth of both the microalga and the virus. Experimental evolution of clonal lines revealed that this viral production originates from the lysis of a minority of virus-susceptible cells, which are regenerated from resistant cells. Whole-genome sequencing demonstrated that this resistant-susceptible switch involved a large deletion on one chromosome. Mathematical modeling explained how the switch maintains stable microalga-virus population dynamics consistent with their observed growth pattern. Comparative genomics confirmed an ancient origin of this “accordion” chromosome despite a lack of sequence conservation. Together, our results show how dynamic genomic rearrangements may account for a previously overlooked coexistence mechanism in microalgae-virus interactions., This research was funded by the ANR grants REVIREC ANR-12-BSV7-0006-01, DECOVIR ANR-12-BSV7-0009, and PHYTNESS ANR-13-JSV6-0005. E.V. is funded by BOF project GOA 01G01715, and L.F.B. is funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. H2020-MSCA-ITN-2015-675752, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. For this, techniques that enable the detection and quantification of virus–host interactions at the single-cell level are essential. With this goal in mind, we implemented VirusFISH (Virus Fluorescencein situHybridization) using as a model the marine picoeukaryoteOstreococcus tauriand its virus OtV5. VirusFISH allowed the visualization and quantification of the fraction of infected cells during an infection experiment. We were also able to quantify the abundance of free viruses released during cell lysis and assess the burst size of our non-axenic culture, because we could discriminate OtV5 from phages. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were produced between 8 and 24 h, propagating the infection. Nevertheless, the production of viral particles increased drastically after 24 h. The burst size for theO. tauri–OtV5 system was 7±0.4 OtV5 per cell, which was consistent with the estimated amount of viruses inside the cell prior to cell lysis. With this work we demonstrate that VirusFISH is a promising technique to study specific virus–host interactions in non-axenic cultures, and set the ground for its application in complex natural communities.

Members of the class Mamiellophyceae comprise species that can dominate picophytoplankton diversity in polar waters. Yet polar species are often morphologically indistinguishable from temperate species, although clearly separated by molecular features. Here we examine four Mamiellophyceae strains from the Canadian Arctic. The 18S rRNA and Internal Transcribed Spacer 2 (ITS2) gene phylogeny place these strains within the family Mamiellaceae (Mamiellales, Mamiellophyceae) in two separate clades of the genus Mantoniella. ITS2 synapomorphies support their placement as two new species, Mantoniella beaufortii and Mantoniella baffinensis. Both species have round green cells with diameter between 3–5 μm, one long flagellum and a short reduced flagellum (~1 μm) and are covered by spiderweb-like scales, making both species similar to other Mantoniella species. Morphologically, M. beaufortii and M. baffinensis are most similar to the cosmopolitan M. squamata with only minor differences in scale structure distinguishing them. Screening of global marine metabarcoding datasets indicates M. beaufortii has only been recorded in seawater and sea ice samples from the Arctic while no environmental barcode matches M. baffinensis. Like other Mamiellophyceae genera that have distinct polar and temperate species, the polar distribution of these new species suggests they are cold or ice-adapted Mantoniella species.

© The Author(s).-- 16 pages, 6 figures, 1 table, supplementary materials https://figshare.com/s/7ff5a7e14308dea9394f, Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics, This research was funded by the University of Oslo, Faculty of Mathematics and Natural Sciences to SGS and BE, by the Research Council of Norway through grant 190307 HAPTODIV to B.E., E.E., and W.E., by grant 225956/E10 MicroPolar to E.E. and B.E. and through grant 294363 VirVar to R.-A.S. and B.E.

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.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35 °C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

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.

Antarctica is arguably the world's most important continent for influencing the Earth's climate and ocean ecosystem function. The unique physico-chemical properties of the Southern Ocean enable high levels of microbial primary production to occur. This not only forms the base of a significant fraction of the global oceanic food web, but leads to the sequestration of anthropogenic CO2 and its transport to marine sediments, thereby removing it from the atmosphere; the Southern Ocean accounts for ~ 30% of global ocean uptake of CO2 despite representing ~ 10% of the total surface area of the global ocean. The Antarctic continent itself harbors some liquid water, including a remarkably diverse range of surface and subglacial lakes. Being one of the remaining natural frontiers, Antarctica delivers the paradox of needing to be protected from disturbance while requiring scientific endeavor to discover what is indigenous and learn how best to protect it. Moreover, like many natural environments on Earth, in Antarctica, microorganisms dominate the genetic pool and biomass of the colonizable niches and play the key roles in maintaining proper ecosystem function. This review puts into perspective insight that has been and can be gained about Antarctica's aquatic microbiota using molecular biology, and in particular, metagenomic approaches. Antarctica is arguably the world's most important continent for influencing the Earth's climate and ocean ecosystem function, and this review puts into perspective the insight that has been and can be gained about Antarctica's aquatic microbiota by using molecular biology, and in particular, metagenomic approaches. © 2012 Federation of European Microbiological Societies.

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment. © 2013 International Society for Microbial Ecology.

In nature, the complexity and structure of microbial communities varies widely, ranging from a few species to thousands of species, and from highly structured to highly unstructured communities. Here, we describe the identity and functional capacity of microbial populations within distinct layers of a pristine, marine-derived, meromictic (stratified) lake (Ace Lake) in Antarctica. Nine million open reading frames were analyzed, representing microbial samples taken from six depths of the lake size fractionated on sequential 3.0, 0.8 and 0.1 m filters, and including metaproteome data from matching 0.1 m filters. We determine how the interactions of members of this highly structured and moderately complex community define the biogeochemical fluxes throughout the entire lake. Our view is that the health of this delicate ecosystem is dictated by the effects of the polar light cycle on the dominant role of green sulfur bacteria in primary production and nutrient cycling, and the influence of viruses/phage and phage resistance on the cooperation between members of the microbial community right throughout the lake. To test our assertions, and develop a framework applicable to other microbially driven ecosystems, we developed a mathematical model that describes how cooperation within a microbial system is impacted by periodic fluctuations in environmental parameters on key populations of microorganisms. Our study reveals a mutualistic structure within the microbial community throughout the lake that has arisen as the result of mechanistic interactions between the physico-chemical parameters and the selection of individual members of the community. By exhaustively describing and modelling interactions in Ace Lake, we have developed an approach that may be applicable to learning how environmental perturbations affect the microbial dynamics in more complex aquatic systems. © 2011 International Society for Microbial Ecology All rights reserved.

Summary: SHAP (simple high-throughput annotation pipeline) is a lightweight and scalable sequence annotation pipeline capable of supporting research efforts that generate or utilize large volumes of DNA sequence data. The software provides Grid capable analysis, relational storage and Web-based full-text searching of annotation results. Implemented in Java, SHAP recognizes the limited resources of many smaller research groups. Availability: Source code is freely available under GPLv3 at https://sourceforge.net/projects/shap. Contact: matt.demaere@unsw.edu.au; r.cavicchioli@unsw.edu.au

Driven by advances in DNA sequencing technologies, an astounding amount of data is being generated from genetic material sourced directly from the environment, and this exponential growth of data is set to continue. By surmounting the challenges of working with such vast datasets, a whole new level of understanding is being gained about microbial diversity, microbial evolution and whole ecosystem function. For precious, pristine and logistically difficult to obtain Antarctic samples, metagenomic and metaproteomic approaches are providing the basis for fundamental new discoveries about how Antarctic systems function.