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Metagenomics and single‐cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single‐virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high‐quality genomic data. We report a sequencing dataset of viral single‐amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole‐genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase‐polymerase features, and WGA‐X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology. This work has been supported by Gordon and Betty Moore Foundation (grant 5334) and Spanish Ministry of Economy and Competitiveness (refs CGL2013‐40564‐R, RTI2018‐094248‐B‐I00 and SAF2013‐49267‐EXP). Work at CRG, BIST and UPF was in part funded by the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013‐2017’ and the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Maria de Maeztu 2016‐2019’.

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

Viruses are extremely diverse and modulate important biological and ecological processes globally. However, much of viral diversity remains uncultured and yet to be discovered. Several powerful culture-independent tools, in particular metagenomics, have substantially advanced virus discovery. Among those tools is single-virus genomics, which yields sequenced reference genomes from individual sorted virus particles without the need for cultivation. This new method complements virus culturing and metagenomic approaches and its advantages include targeted investigation of specific virus groups and investigation of genomic microdiversity within viral populations. In this Review, we provide a brief history of single-virus genomics, outline how this emergent method has facilitated advances in virus ecology and discuss its current limitations and future potential. Finally, we address how this method may synergistically intersect with other single-virus and single-cell approaches. This work was supported by the Gordon and Betty Moore Foundation (grant 5334), the US National Science Foundation (NSF- OPP 1644155, NSF- OCE 1933289), the Spanish Ministry of Economy and Competitiveness (CGL2013-40564- R, RTI2018-094248- B- I00 and SAF2013-49267- EXP) and Generalitat Valenciana (ACOM/2015/133 and ACIF/2015/332).

The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270–14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison. This work has been supported by Spanish Ministry of Economy and Competitiveness (Ref. RTI2018-094248-B-I00), Generalitat Valenciana (Ref. ACOM/2015/133 and ACIF/2015/332), and Gordon and Betty Moore Foundation (Grant 5334).

The coccolithophore Emiliania huxleyi forms some of the largest phytoplankton blooms in the ocean. The rapid demise of these blooms has been linked to viral infections. E. huxleyi abundance, distribution, and nutritional status make them an important food source for the heterotrophic protists which are classified as microzooplankton in marine food webs. In this study we investigated the fate of E. huxleyi (CCMP 374) infected with virus strain EhV-86 in a simple predator-prey interaction. The ingestion rates of Oxyrrhis marina were significantly lower (between 26.9 and 50.4%) when fed virus-infected E. huxleyi cells compared to non-infected cells. Despite the lower ingestion rates, O. marina showed significantly higher growth rates (between 30 and 91.3%) when fed infected E. huxleyi cells, suggesting higher nutritional value and/or greater assimilation of infected E. huxleyi cells. No significant differences were found in O. marina cell volumes or fatty acids profiles. These results show that virally infected E. huxleyi support higher growth rates of single celled heterotrophs and in addition to the “viral shunt” hypothesis, viral infections may also divert more carbon to mesozooplankton grazers.

The coccolithophore Emiliania huxleyi forms some of the largest phytoplankton blooms in the ocean. The rapid demise of these blooms has been linked to viral infections. E. huxleyi abundance, distribution, and nutritional status make them an important food source for the heterotrophic protists which are classified as microzooplankton in marine food webs. In this study we investigated the fate of E. huxleyi (CCMP 374) infected with virus strain EhV-86 in a simple predator-prey interaction. The ingestion rates of Oxyrrhis marina were significantly lower (between 26.9 and 50.4%) when fed virus-infected E. huxleyi cells compared to non-infected cells. Despite the lower ingestion rates, O. marina showed significantly higher growth rates (between 30 and 91.3%) when fed infected E. huxleyi cells, suggesting higher nutritional value and/or greater assimilation of infected E. huxleyi cells. No significant differences were found in O. marina cell volumes or fatty acids profiles. These results show that virally infected E. huxleyi support higher growth rates of single celled heterotrophs and in addition to the “viral shunt” hypothesis, viral infections may also divert more carbon to mesozooplankton grazers.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on ‘giant viruses’ that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

Emiliania huxleyi (Lohm.) Hay and Mohler is a ubiquitous unicellular marine alga surrounded by an elaborate covering of calcite platelets called coccoliths. It is an important primary producer involved in oceanic biogeochemistry and climate regulation. Currently, E. huxleyi is separated into five morphotypes based on morphometric, physiological, biochemical, and immunological differences. However, a genetic marker has yet to be found to characterize these morphotypes. With the use of sequence analysis and denaturing gradient gel electrophoresis, we discovered a genetic marker that correlates significantly with the separation of the most widely recognized A and B morphotypes. Furthermore, we reveal that the A morphotype is composed of a number of distinct genotypes. This marker lies within the 3' untranslated region of a coccolith associated protein mRNA, which is implicated in regulating coccolith calcification. Consequently, we tentatively termed this marker the coccolith morphology motif.

International audience; During viral infection of Emiliania huxleyi, laboratory studies have shown that photo system (PS) II efficiency declines during the days post-infection and is thought to be associated with viral-induced interruption of electron transport rates between photosystems. However, measuring the impact of viral infection on PSII function in E. huxleyi populations from natural, taxonomically diverse phytoplankton communities is difficult, and whether this phenomenon occurs in nature is presently unknown. Here, chlorophyll fluorescence analysis was used to assess changes in PSII efficiency throughout an E. huxleyi bloom during a mesocosm experiment off the coast of Norway. Specifically, we aimed to determine whether a measurable suppression of the efficiency of PSII photochemistry could be observed due to viral infection of the natural E. huxleyi populations. During the major infection period prior to bloom collapse, there was a significant reduction in PSII efficiency with an average decrease in maximum PSII photochemical efficiency (F-v/F-m) of 17% and a corresponding 75% increase in maximum PSII effective absorption cross-section (sigma(PSII)); this was concurrent with a significant decrease in E. huxleyi growth rates and an increase in E. huxleyi virus (EhV) production. As E. huxleyi populations dominated the phytoplankton community and potentially contributed up to 100% of the chlorophyll a pool, we believe that the variable chlorophyll fluorescence signal measured during this period was derived predominantly from E. huxleyi and, thus, reflects changes occurring within E. huxleyi cells. This is the first demonstration of suppression of PSII photochemistry occurring during viral infection of natural coccolithophore populations.

Metagenomics has opened new windows on investigating viral diversity and functions. Viromic studies typically require large sample volumes and filtration through 0.2 μm pore-size filters, consequently excluding or under-sampling tailed and very large viruses. We have optimized a targeted viromic approach that employs fluorescence-activated sorting and whole genome amplification to produce dsDNA-enriched libraries from discrete viral populations from a 1-ml water sample. Using this approach on an environmental sample from the Patagonian Shelf, we produced three distinct libraries. One of the virus libraries was dominated (79.65% of sequences with known viral homology) by giant viruses from the Mimiviridae and Phycodnaviridae families, while the two other viromes were dominated by smaller phycodnaviruses, cyanophages and other bacteriophages. The estimated genotypic richness and diversity in our sorted viromes, with 52–163 estimated genotypes, was much lower than in previous virome reports. Fragment recruitment of metagenome reads to selected reference viral genomes yields high genome coverage, suggesting little amplification and sequencing bias against some genomic regions. These results underscore the value of our approach as an effective way to target and investigate specific virus groups. In particular, it will help reveal the diversity and abundance of giant viruses in marine ecosystems.

Micro-organisms are vital for the functioning of all food webs and are the major drivers of the global biogeochemical cycles. The microbial community compositions and physicochemical conditions of the different water masses in the North Sea, a biologically productive sea on the northwestern European continental shelf, were studied during two summer cruises, in order to provide detailed baseline data for this region and examine its microbial biogeography. For each cruise the stations were clustered according to their physicochemical characteristics and their microbial community composition. The largest cluster, which covered most of the central and northern North Sea, consisted of stations that were characterized by a thermally stratified water column and had low chlorophyll a autofluorescence and generally low microbial abundances. The second main cluster contained stations that were dominated by picoeukaryotes and showed the influence of influxes of North Atlantic water via the English Channel and south of the Shetland Islands. The third main cluster was formed by stations that were dominated by cyanobacteria and nanoeukaryotes in the reduced salinity Norwegian Coastal and Skagerrak waters, while the fourth cluster represented the German Bight, a region with strong riverine input, high nutrient concentrations, and consequently high heterotrophic bacterial and viral abundances. Despite the complex and dynamic hydrographic nature of the North Sea, the consistent distinctions in microbiology between these different hydrographic regions during both cruises illustrate the strong links between the microbial community and its environment, as well as the possibility to use microorganisms for long-term monitoring of environmental change.

We studied the temporal succession of vertical profiles of Emiliania huxleyi and their specific viruses (EhVs) during the progression of a natural phytoplankton bloom in the North Sea in June 1999. Genotypic richness was assessed by exploiting the variations in a gene encoding a protein with calcium-binding motifs (GPA) for E. huxleyi and in the viral major capsid protein gene for EhVs. Using denaturing gradient gel electrophoresis and sequencing analysis, we showed at least three different E. huxleyi and EhV genotypic profiles during the period of study, revealing a complex, and changing assemblage at the molecular level. Our results also indicate that the dynamics of EhV genotypes reflect fluctuations in abundance of potential E. huxleyi host cells. The presence and concentration of specific EhVs in the area prior to the bloom, or EhVs transported into the area by different water masses, are significant factors affecting the structure and intraspecific succession of E. huxleyi during the phytoplankton bloom.

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi -specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi . Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi -dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.

The spatial distribution of an uncultured clade of marine diazotrophic γ-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these γ-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.

Despite the global oceanic distribution and recognised biogeochemical impact of coccolithoviruses (EhV), their diversity remains poorly understood. Here we employed a metagenomic approach to study the occurrence and progression of natural EhV community genomic variability. Analysis of EhV metagenomes from the early and late stages of an induced bloom led to three main discoveries. First, we observed resilient and specific genomic signatures in the EhV community associated with the Norwegian coast, which reinforce the existence of limitations to the capacity of dispersal and genomic exchange among EhV populations. Second, we identified a hyper-variable region (approximately 21kbp long) in the coccolithovirus genome. Third, we observed a clear trend for EhV relative amino-acid diversity to reduce from early to late stages of the bloom. This study validated two new methodological combinations, and proved very useful in the discovery of new genomic features associated with coccolithovirus natural communities.