Phaeoviruses Present in Cultured and Natural Kelp Species, Saccharina latissima and Laminaria hyperborea (Phaeophyceae, Laminariales), in Norway.

Phaeoviruses (Phycodnaviridae) are large icosahedral viruses in the phylum Nucleocytoviricota with dsDNA genomes ranging from 160 to 560 kb, infecting multicellular brown algae (Phaeophyceae). The phaeoviral host range is broader than expected, not only infecting algae from the Ectocarpales but also from the Laminariales order. However, despite phaeoviral infections being reported globally, Norwegian kelp species have not been screened. A molecular analysis of cultured and wild samples of two economically important kelp species in Norway (Saccharina latissima and Laminaria hyperborea) revealed that phaeoviruses are recurrently present along the Norwegian coast. We found the viral prevalence in S. latissima to be significantly higher at the present time compared to four years ago. We also observed regional differences within older samples, in which infections were significantly lower in northern areas than in the south or the fjords. Moreover, up to three different viral sequences were found in the same algal individual, one of which does not belong to the Phaeovirus genus and has never been reported before. This master variant therefore represents a putative new member of an unclassified phycodnavirus genus.

Differential toxicity of bioorthogonal non-canonical amino acids (BONCAT) in Escherichia coli.

Single-cell methods allow studying the activity of single bacterial cells, potentially shedding light on regulatory mechanisms involved in services like biochemical cycling. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising method for studying bacterial activity in natural communities, using the methionine analogues L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) to track protein production in single cells. Both AHA and HPG have been deemed non-toxic, but recent findings suggest that HPG affects bacterial metabolism. In this study we examined the effect of AHA and HPG on Escherichia coli with respect to acute toxicity and growth. E. coli exposed to 5.6-90 µM HPG showed no growth, and the growth rate was significantly reduced at >0.35 µM HPG, compared to the HPG-free control. In contrast, E. coli showed growth at concentrations up to 9 mM AHA. In assays where AHA or HPG were added during the exponential growth phase, the growth sustained but the growth rate was immediately reduced at the highest concentrations (90 µM HPG and 10 mM AHA). Prolonged incubations (20h) with apparently non-toxic concentrations suggest that the cells incorporating NCAAs fail to divide and do not contribute to the next generation resulting in the relative abundance of labelled cells to decrease over time. These results show that HPG and AHA have different impact on the growth of E. coli. Both concentration and incubation time affect the results and need to be considered when designing BONCAT experiments and evaluating results. Time course incubations are suggested as a possible way to obtain more reliable results.

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms.

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2-4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms.

Multiomics in the central Arctic Ocean for benchmarking biodiversity change.

Multiomics approaches need to be applied in the central Arctic Ocean to benchmark biodiversity change and to identify novel species and their genes. As part of MOSAiC, EcoOmics will therefore be essential for conservation and sustainable bioprospecting in one of the least explored ecosystems on Earth.

Flow Cytometric Analysis of Bacterial Protein Synthesis: Monitoring Vitality After Water Treatment.

Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT-SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.

How Microbial Food Web Interactions Shape the Arctic Ocean Bacterial Community Revealed by Size Fractionation Experiments.

In the Arctic, seasonal changes are substantial, and as a result, the marine bacterial community composition and functions differ greatly between the dark winter and light-intensive summer. While light availability is, overall, the external driver of the seasonal changes, several internal biological interactions structure the bacterial community during shorter timescales. These include specific phytoplankton-bacteria associations, viral infections and other top-down controls. Here, we uncover these microbial interactions and their effects on the bacterial community composition during a full annual cycle by manipulating the microbial food web using size fractionation. The most profound community changes were detected during the spring, with 'mutualistic phytoplankton'-Gammaproteobacteria interactions dominating in the pre-bloom phase and 'substrate-dependent phytoplankton'-Flavobacteria interactions during blooming conditions. Bacterivores had an overall limited effect on the bacterial community composition most of the year. However, in the late summer, grazing was the main factor shaping the community composition and transferring carbon to higher trophic levels. Identifying these small-scale interactions improves our understanding of the Arctic marine microbial food web and its dynamics.

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms.

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

High CO2 concentration and iron availability determine the metabolic inventory in an Emiliania huxleyi-dominated phytoplankton community.

Ocean acidification (OA), a consequence of anthropogenic carbon dioxide (CO2 ) emissions, strongly impacts marine ecosystems. OA also influences iron (Fe) solubility, affecting biogeochemical and ecological processes. We investigated the interactive effects of CO2 and Fe availability on the metabolome response of a natural phytoplankton community. Using mesocosms we exposed phytoplankton to ambient (390 µatm) or future CO2 levels predicted for the year 2100 (900 µatm), combined with ambient (4.5 nM) or high (12 nM) dissolved iron (dFe). By integrating over the whole phytoplankton community, we assigned functional changes based on altered metabolite concentrations. Our study revealed the complexity of phytoplankton metabolism. Metabolic profiles showed three stages in response to treatments and phytoplankton dynamics. Metabolome changes were related to the plankton group contributing respective metabolites, explaining bloom decline and community succession. CO2 and Fe affected metabolic profiles. Most saccharides, fatty acids, amino acids and many sterols significantly correlated with the high dFe treatment at ambient pCO2 . High CO2 lowered the abundance of many metabolites irrespective of Fe. However, sugar alcohols accumulated, indicating potential stress. We demonstrate that not only altered species composition but also changes in the metabolic landscape affecting the plankton community may change as a consequence of future high-CO2 oceans.

Incubation in light versus dark affects the vitality of UV-irradiated Tetraselmis suecica differently: A flow cytometric study.

In this study, we used flow cytometry to examine how incubation in dark versus light affects the vitality and viability of UV-irradiated Tetraselmis suecica. High UV doses (300 and 400 mJ/cm2) affected the esterase activity, membrane permeability, and chlorophyll content more when the subsequent incubation took place in light. For non- or low UV dose (100 and 200 mJ/cm2)-treated cells, incubation in light resulted in cell regrowth as compared to incubation in dark. Damaged cells (enzymatically active but with permeable membranes) did not recover when incubated under light or dark conditions. Exposure to light reduces the evaluation time of any given ballast water treatment, as viable cells will be detected at an earlier stage and the vitality is more affected. When evaluating the performance of UV-based ballast water treatment systems (BWTS), these results can be useful for type approval using T. suecica as a test organism in the test regime.

The potential of sedimentary ancient DNA for reconstructing past sea ice evolution.

Sea ice is a crucial component of the Arctic climate system, yet the tools to document the evolution of sea ice conditions on historical and geological time scales are few and have limitations. Such records are essential for documenting and understanding the natural variations in Arctic sea ice extent. Here we explore sedimentary ancient DNA (aDNA), as a novel tool that unlocks and exploits the genetic (eukaryote) biodiversity preserved in marine sediments specifically for past sea ice reconstructions. Although use of sedimentary aDNA in paleoceanographic and paleoclimatic studies is still in its infancy, we use here metabarcoding and single-species quantitative DNA detection methods to document the sea ice conditions in a Greenland Sea marine sediment core. Metabarcoding has allowed identifying biodiversity changes in the geological record back to almost ~100,000 years ago that were related to changing sea ice conditions. Detailed bioinformatic analyses on the metabarcoding data revealed several sea-ice-associated taxa, most of which previously unknown from the fossil record. Finally, we quantitatively traced one known sea ice dinoflagellate in the sediment core. We show that aDNA can be recovered from deep-ocean sediments with generally oxic bottom waters and that past sea ice conditions can be documented beyond instrumental time scales. Our results corroborate sea ice reconstructions made by traditional tools, and thus demonstrate the potential of sedimentary aDNA, focusing primarily on microbial eukaryotes, as a new tool to better understand sea ice evolution in the climate system.

Seasonality Drives Microbial Community Structure, Shaping both Eukaryotic and Prokaryotic Host⁻Viral Relationships in an Arctic Marine Ecosystem.

The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from the surface to 1000 m in depth. We employed metabarcoding analysis of major capsid protein g23 and mcp genes in order to investigate T4-like myoviruses and large dsDNA viruses infecting prokaryotic and eukaryotic picophytoplankton, respectively. Microbial abundances were assessed using flow cytometry. Metabarcoding results demonstrated that seasonality was the key mediator shaping virus communities, whereas depth exerted a diversifying effect within seasonal virus assemblages. Viral diversity and virus-to-prokaryote ratios (VPRs) dropped sharply at the commencement of the spring bloom but increased across the season, ultimately achieving the highest levels during the winter season. These findings suggest that viral lysis may be an important process during the polar winter, when productivity is low. Furthermore, winter viral communities consisted of Operational Taxonomic Units (OTUs) distinct from those present during the spring-summer season. Our data provided a first insight into the diversity of viruses in a hitherto undescribed marine habitat characterized by extremes in light and productivity.

Simple models combining competition, defence and resource availability have broad implications in pelagic microbial food webs.

In food webs, interactions between competition and defence control the partitioning of limiting resources. As a result, simple models of these interactions contain links between biogeochemistry, diversity, food web structure and ecosystem function. Working at hierarchical levels, these mechanisms also produce self-similarity and therefore suggest how complexity can be generated from repeated application of simple underlying principles. Reviewing theoretical and experimental literature relevant to the marine photic zone, we argue that there is a wide spectrum of phenomena, including single cell activity of prokaryotes, microbial biodiversity at different levels of resolution, ecosystem functioning, regional biogeochemical features and evolution at different timescales; that all can be understood as variations over a common principle, summarised in what has been termed the 'Killing-the-Winner' (KtW) motif. Considering food webs as assemblages of such motifs may thus allow for a more integrated approach to aquatic microbial ecology.

Bacterial community composition responds to changes in copepod abundance and alters ecosystem function in an Arctic mesocosm study.

Combining a minimum food web model with Arctic microbial community dynamics, we have suggested that top-down control by copepods can affect the food web down to bacterial consumption of organic carbon. Pursuing this hypothesis further, we used the minimum model to design and analyse a mesocosm experiment, studying the effect of high (+Z) and low (-Z) copepod density on resource allocation, along an organic-C addition gradient. In the Arctic, both effects are plausible due to changes in advection patterns (affecting copepods) and meltwater inputs (affecting carbon). The model predicts a trophic cascade from copepods via ciliates to flagellates, which was confirmed experimentally. Auto- and heterotrophic flagellates affect bacterial growth rate and abundance via competition for mineral nutrients and predation, respectively. In +Z, the model predicts low bacterial abundance and activity, and little response to glucose; as opposed to clear glucose consumption effects in -Z. We observed a more resilient bacterial response to high copepods and demonstrate this was due to changes in bacterial community equitability. Species able to use glucose to improve their competitive and/or defensive properties, became predominant. The observed shift from a SAR11-to a Psychromonodaceae - dominated community suggests the latter was pivotal in this modification of ecosystem function. We argue that this group used glucose to improve its defensive or its competitive abilities (or both). Adding such flexibility in bacterial traits to the model, we show how it creates the observed resilience to top-down manipulations observed in our experiment.

The Response of Heterotrophic Prokaryote and Viral Communities to Labile Organic Carbon Inputs Is Controlled by the Predator Food Chain Structure.

Factors controlling the community composition of marine heterotrophic prokaryotes include organic-C, mineral nutrients, predation, and viral lysis. Two mesocosm experiments, performed at an Arctic location and bottom-up manipulated with organic-C, had very different results in community composition for both prokaryotes and viruses. Previously, we showed how a simple mathematical model could reproduce food web level dynamics observed in these mesocosms, demonstrating strong top-down control through the predator chain from copepods via ciliates and heterotrophic nanoflagellates. Here, we use a steady-state analysis to connect ciliate biomass to bacterial carbon demand. This gives a coupling of top-down and bottom-up factors whereby low initial densities of ciliates are associated with mineral nutrient-limited heterotrophic prokaryotes that do not respond to external supply of labile organic-C. In contrast, high initial densities of ciliates give carbon-limited growth and high responsiveness to organic-C. The differences observed in ciliate abundance, and in prokaryote abundance and community composition in the two experiments were in accordance with these predictions. Responsiveness in the viral community followed a pattern similar to that of prokaryotes. Our study provides a unique link between the structure of the predator chain in the microbial food web and viral abundance and diversity.

Correction: Ruiz, E. et al. Emerging Interaction Patterns in the Emiliania Huxleyi-EhV System. Viruses 2016, 9, 61.

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Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship.

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

Emerging Interaction Patterns in the Emiliania huxleyi-EhV System.

Viruses are thought to be fundamental in driving microbial diversity in the oceanic planktonic realm. That role and associated emerging infection patterns remain particularly elusive for eukaryotic phytoplankton and their viruses. Here we used a vast number of strains from the model system Emiliania huxleyi/Emiliania huxleyi Virus to quantify parameters such as growth rate (µ), resistance (R), and viral production (Vp) capacities. Algal and viral abundances were monitored by flow cytometry during 72-h incubation experiments. The results pointed out higher viral production capacity in generalist EhV strains, and the virus-host infection network showed a strong co-evolution pattern between E. huxleyi and EhV populations. The existence of a trade-off between resistance and growth capacities was not confirmed.

Metabarcoding and metabolome analyses of copepod grazing reveal feeding preference and linkage to metabolite classes in dynamic microbial plankton communities.

In order to characterize copepod feeding in relation to microbial plankton community dynamics, we combined metabarcoding and metabolome analyses during a 22-day seawater mesocosm experiment. Nutrient amendment of mesocosms promoted the development of haptophyte (Phaeocystis pouchetii)- and diatom (Skeletonema marinoi)-dominated plankton communities in mesocosms, in which Calanus sp. copepods were incubated for 24 h in flow-through chambers to allow access to prey particles (<500 µm). Copepods and mesocosm water sampled six times spanning the experiment were analysed using metabarcoding, while intracellular metabolite profiles of mesocosm plankton communities were generated for all experimental days. Taxon-specific metabarcoding ratios (ratio of consumed prey to available prey in the surrounding seawater) revealed diverse and dynamic copepod feeding selection, with positive selection on large diatoms, heterotrophic nanoflagellates and fungi, while smaller phytoplankton, including P. pouchetii, were passively consumed or even negatively selected according to our indicator. Our analysis of the relationship between Calanus grazing ratios and intracellular metabolite profiles indicates the importance of carbohydrates and lipids in plankton succession and copepod-prey interactions. This molecular characterization of Calanus sp. grazing therefore provides new evidence for selective feeding in mixed plankton assemblages and corroborates previous findings that copepod grazing may be coupled to the developmental and metabolic stage of the entire prey community rather than to individual prey abundances.

Ocean acidification reduces transfer of essential biomolecules in a natural plankton community.

Ocean acidification (OA), a process of increasing seawater acidity caused by the uptake of anthropogenic carbon dioxide (CO2) by the ocean, is expected to change surface ocean pH to levels unprecedented for millions of years, affecting marine food web structures and trophic interactions. Using an in situ mesocosm approach we investigated effects of OA on community composition and trophic transfer of essential fatty acids (FA) in a natural plankton assemblage. Elevated pCO2 favored the smallest phytoplankton size class in terms of biomass, primarily picoeukaryotes, at the expense of chlorophyta and haptophyta in the nano-plankton size range. This shift in community composition and size structure was accompanied by a decline in the proportion of polyunsaturated FA (PUFA) to total FA content in the nano- and picophytoplankton size fractions. This decline was mirrored in a continuing reduction in the relative PUFA content of the dominant copepod, Calanus finmarchicus, which primarily fed on the nano-size class. Our results demonstrate that a shift in phytoplankton community composition and biochemical composition in response to rising CO2 can affect the transfer of essential compounds to higher trophic levels, which rely on their prey as a source for essential macromolecules.

Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry.

This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400 mJ/cm(2) rendered inactivation after 1 day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200 mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm(2) and ≤200 mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.