Growth and mortality of aerobic anoxygenic phototrophs in the North Pacific Subtropical Gyre.

Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combined in situ sampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. The in situ decline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75-0.90 d-1. This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d-1. An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure.

Global ocean lipidomes show a universal relationship between temperature and lipid unsaturation.

Global-scale surveys of plankton communities using "omics" techniques have revolutionized our understanding of the ocean. Lipidomics has demonstrated the potential to add further essential insights on ocean ecosystem function but has yet to be applied on a global scale. We analyzed 930 lipid samples across the global ocean using a uniform high-resolution accurate-mass mass spectrometry analytical workflow, revealing previously unknown characteristics of ocean planktonic lipidomes. Focusing on 10 molecularly diverse glycerolipid classes, we identified 1151 distinct lipid species, finding that fatty acid unsaturation (i.e., number of carbon-carbon double bonds) is fundamentally constrained by temperature. We predict substantial declines in the essential fatty acid eicosapentaenoic acid over the next century, which are likely to have serious deleterious effects on economically critical fisheries.

Complex marine microbial communities partition metabolism of scarce resources over the diel cycle.

Complex assemblages of microbes in the surface ocean are responsible for approximately half of global carbon fixation. The persistence of high taxonomic diversity despite competition for a small suite of relatively homogeneously distributed nutrients, that is, 'the paradox of the plankton', represents a long-standing challenge for ecological theory. Here we find evidence consistent with temporal niche partitioning of nitrogen assimilation processes over a diel cycle in the North Pacific Subtropical Gyre. We jointly analysed transcript abundances, lipids and metabolites and discovered that a small number of diel archetypes can explain pervasive periodic dynamics. Metabolic pathway analysis of identified diel signals revealed asynchronous timing in the transcription of nitrogen uptake and assimilation genes among different microbial groups-cyanobacteria, heterotrophic bacteria and eukaryotes. This temporal niche partitioning of nitrogen uptake emerged despite synchronous transcription of photosynthesis and central carbon metabolism genes and associated macromolecular abundances. Temporal niche partitioning may be a mechanism by which microorganisms in the open ocean mitigate competition for scarce resources, supporting community coexistence.

Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes.

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species.

Using High-Sensitivity Lipidomics To Assess Microscale Heterogeneity in Oceanic Sinking Particles and Single Phytoplankton Cells.

Sinking particulate organic matter (POM) is a primary component of the ocean's biological carbon pump that is responsible for carbon export from the surface to the deep sea. Lipids derived from plankton comprise a significant fraction of sinking POM. Our understanding of planktonic lipid biosynthesis and the subsequent degradation of lipids in sinking POM is based on the analysis of bulk samples that combine many millions of plankton cells or dozens of sinking particles, which averages out natural heterogeneity. We developed and applied a nanoflow high-performance liquid-chromatography electrospray-ionization high-resolution accurate-mass mass spectrometry lipidomic method to show that two types of sinking particles─marine snow and fecal pellets─collected in the western North Atlantic Ocean have distinct lipidomes, providing new insights into their sources and degradation that would not be apparent from bulk samples. We pressed the limit of this approach by examining individual diatom cells from a single culture, finding marked lipid heterogeneity, possibly indicative of fundamental mechanisms underlying cell division. These single-cell data confirm that even cultures of phytoplankton cells should be viewed as mixtures of physiologically distinct populations. Overall, this work reveals previously hidden lipidomic heterogeneity in natural POM and phytoplankton cells, which may provide critical new insights into microscale chemical and microbial processes that control the export of sinking POM.

Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic.

Seasonal shifts in phytoplankton accumulation and loss largely follow changes in mixed layer depth, but the impact of mixed layer depth on cell physiology remains unexplored. Here, we investigate the physiological state of phytoplankton populations associated with distinct bloom phases and mixing regimes in the North Atlantic. Stratification and deep mixing alter community physiology and viral production, effectively shaping accumulation rates. Communities in relatively deep, early-spring mixed layers are characterized by low levels of stress and high accumulation rates, while those in the recently shallowed mixed layers in late-spring have high levels of oxidative stress. Prolonged stratification into early autumn manifests in negative accumulation rates, along with pronounced signatures of compromised membranes, death-related protease activity, virus production, nutrient drawdown, and lipid markers indicative of nutrient stress. Positive accumulation renews during mixed layer deepening with transition into winter, concomitant with enhanced nutrient supply and lessened viral pressure.

Production of Two Highly Abundant 2-Methyl-Branched Fatty Acids by Blooms of the Globally Significant Marine Cyanobacteria Trichodesmium erythraeum.

The bloom-forming cyanobacteria Trichodesmium contribute up to 30% to the total fixed nitrogen in the global oceans and thereby drive substantial productivity. On an expedition in the Gulf of Mexico, we observed and sampled surface slicks, some of which included dense blooms of Trichodesmium erythraeum. These bloom samples contained abundant and atypical free fatty acids, identified here as 2-methyldecanoic acid and 2-methyldodecanoic acid. The high abundance and unusual branching pattern of these compounds suggest that they may play a specific role in this globally important organism.

Arsenolipids in Plankton from High- and Low-Nutrient Oceanic Waters Along a Transect in the North Atlantic.

Although the natural occurrence of arsenic-containing lipids (arsenolipids) in marine organisms is now well established, the possible role of these unusual compounds in organisms and in the cycling of arsenic in marine systems remains largely unexplored. We report the finding of arsenolipids in 61 plankton samples collected from surface marine waters of high- and low-nutrient content along a transect spanning the Gulf Stream in the North Atlantic Ocean. Using high-performance liquid chromatography (HPLC) coupled to both elemental and molecular mass spectrometry, we show that all 61 plankton samples contained six identifiable arsenolipids, namely, three arsenosugar phospholipids (AsPL958, 10-13%; AsPL978, 13-25%; and AsPL1006, 7-10% of total arsenolipids), two arsenic-containing hydrocarbons (AsHC332, 4-10% and AsHC360, 1-2%), and a methoxy-sugar arsenolipid that contained phytol (AsSugPhytol, 1-3%). The relative amounts of the six arsenolipids showed clear dependence on the nutrient status of the ambient water with plankton collected from high-nutrient waters having less of the arsenosugar phospholipids and more of the three non-P containing arsenolipids compared to low-nutrient waters. By combining these first field data of arsenolipids in plankton with reported global phytoplankton productivity, we estimate that the oceans' phytoplankton transform per year 50 000-100 000 tons of arsenic into arsenolipids.

Microbial production and consumption of hydrocarbons in the global ocean.

Seeps, spills and other oil pollution introduce hydrocarbons into the ocean. Marine cyanobacteria also produce hydrocarbons from fatty acids, but little is known about the size and turnover of this cyanobacterial hydrocarbon cycle. We report that cyanobacteria in an oligotrophic gyre mainly produce n-pentadecane and that microbial hydrocarbon production exhibits stratification and diel cycling in the sunlit surface ocean. Using chemical and isotopic tracing we find that pentadecane production mainly occurs in the lower euphotic zone. Using a multifaceted approach, we estimate that the global flux of cyanobacteria-produced pentadecane exceeds total oil input in the ocean by 100- to 500-fold. We show that rapid pentadecane consumption sustains a population of pentadecane-degrading bacteria, and possibly archaea. Our findings characterize a microbial hydrocarbon cycle in the open ocean that dwarfs oil input. We hypothesize that cyanobacterial hydrocarbon production selectively primes the ocean's microbiome with long-chain alkanes whereas degradation of other petroleum hydrocarbons is controlled by factors including proximity to petroleum seepage.

Combined pigment and metatranscriptomic analysis reveals highly synchronized diel patterns of phenotypic light response across domains in the open oligotrophic ocean.

Sunlight is the most important environmental control on diel fluctuations in phytoplankton activity, and understanding diel microbial processes is essential to the study of oceanic biogeochemical cycles. Yet, little is known about the in situ temporal dynamics of phytoplankton metabolic activities and their coordination across different populations. We investigated diel orchestration of phytoplankton activity in photosynthesis, photoacclimation, and photoprotection by analyzing pigment and quinone distributions in combination with metatranscriptomes in surface waters of the North Pacific Subtropical Gyre (NPSG). We found diel cycles in pigment abundances resulting from the balance of their synthesis and consumption. These dynamics suggest that night represents a metabolic recovery phase, refilling cellular pigment stores, while photosystems are remodeled towards photoprotection during daytime. Transcript levels of genes involved in photosynthesis and pigment metabolism had synchronized diel expression patterns among all taxa, reflecting the driving force light imparts upon photosynthetic organisms in the ocean, while other environmental factors drive niche differentiation. For instance, observed decoupling of diel oscillations in transcripts and related pigments indicates that pigment abundances are modulated by environmental factors extending beyond gene expression/regulation reinforcing the need to combine metatranscriptomics with proteomics and metabolomics to fully understand the timing of these critical processes in situ.

Nitric oxide mediates oxylipin production and grazing defense in diatoms.

Diatom blooms are important features of productive marine ecosystems and are known to support higher trophic levels. However, when stressed or wounded, diatoms can produce oxylipin molecules known to inhibit the reproduction and development of copepods and decrease microzooplankton growth rates. Using oxylipin chemical treatments, lipidomic analysis and functional genomic approaches, we provide evidence that nitric oxide (NO) and oxylipin signalling pathways in diatoms respond to protist grazers, resulting in increased defence fitness and survival. Exposure of the diatom Phaeodactylum tricornutum to the dinoflagellate Oxyrrhis marina resulted in NO production by P. tricornutum and pronounced change in its dissolved oxylipin profile. Experimentally elevating levels of NO also resulted in increased oxylipin production, and lower overall grazing rates. Furthermore, O. marina preferentially grazed on P. tricornutum prey with lower levels of NO, suggesting that this molecule and its effect on oxylipin pathways play a key role in prey selection. Exposure of O. marina grazing on P. tricornutum to exogenous oxylipins also decreased grazing rates, which is consistent with a grazing deterrence role for these molecules. These results suggest that NO and oxylipin production help to structure diatom communities, in part by modulating interactions with microzooplankton predators.

Coordinated transformation of the gut microbiome and lipidome of bowhead whales provides novel insights into digestion.

Whale digestion plays an integral role in many ocean ecosystems. By digesting enormous quantities of lipid-rich prey, whales support their energy intensive lifestyle, but also excrete nutrients important to ocean biogeochemical cycles. Nevertheless, whale digestion is poorly understood. Gastrointestinal microorganisms play a significant role in vertebrate digestion, but few studies have examined them in whales. To investigate digestion of lipids, and the potential contribution of microbes to lipid digestion in whales, we characterized lipid composition (lipidomes) and bacterial communities (microbiotas) in 126 digesta samples collected throughout the gastrointestinal tracts of 38 bowhead whales (Balaena mysticetus) harvested by Alaskan Eskimos. Lipidomes and microbiotas were strongly correlated throughout the gastrointestinal tract. Lipidomes and microbiotas were most variable in the small intestine and most similar in the large intestine, where microbiota richness was greatest. Our results suggest digestion of wax esters, the primary lipids in B. mysticetus prey representing more than 80% of total dietary lipids, occurred in the mid- to distal small intestine and was correlated with specific microorganisms. Because wax esters are difficult to digest by other marine vertebrates and constitute a large reservoir of carbon in the ocean, our results further elucidate the essential roles that whales and their gastrointestinal microbiotas play in the biogeochemical cycling of carbon and nutrients in high-latitude seas.

Silicon limitation facilitates virus infection and mortality of marine diatoms.

Diatoms are among the most globally distributed and ecologically successful organisms in the modern ocean, contributing upwards of 40% of total marine primary productivity1,2. By converting dissolved silicon into biogenic silica, and photosynthetically fixing carbon dioxide into particulate organic carbon, diatoms effectively couple the silicon (Si) and carbon cycles and ballast substantial vertical flux of carbon out of the euphotic zone into the mesopelagic and deep ocean3-5. Viruses are key players in ocean biogeochemical cycles6,7, yet little is known about how viral infection specifically impacts diatom populations. Here, we show that Si limitation facilitates virus infection and mortality in diatoms in the highly productive coastal waters of the California Current Ecosystem. Using metatranscriptomic analysis of cell-associated diatom viruses and targeted quantification of extracellular viruses, we found a link between Si stress and the early, active and lytic stages of viral infection. This relationship was also observed in cultures of the bloom-forming diatom Chaetoceros tenuissimus, where Si stress accelerated virus-induced mortality. Together, these findings contextualize viruses within the ecophysiological framework of Si availability and diatom-mediated biogeochemical cycling.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi.

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms.

The mutual interplay between calcification and coccolithovirus infection.

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, 'induced media'. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host-virus interactions in the ocean and interpreting their impact on the global carbon cycle.

Diverse diazotrophs are present on sinking particles in the North Pacific Subtropical Gyre.

Sinking particles transport carbon and nutrients from the surface ocean into the deep sea and are considered hot spots for bacterial diversity and activity. In the oligotrophic oceans, nitrogen (N2)-fixing organisms (diazotrophs) are an important source of new N but the extent to which these organisms are present and exported on sinking particles is not well known. Sinking particles were collected every 6 h over a 2-day period using net traps deployed at 150 m in the North Pacific Subtropical Gyre. The bacterial community and composition of diazotrophs associated with individual and bulk sinking particles was assessed using 16S rRNA and nifH gene amplicon sequencing. The bacterial community composition in bulk particles remained remarkably consistent throughout time and space while large variations of individually picked particles were observed. This difference suggests that unique biogeochemical conditions within individual particles may offer distinct ecological niches for specialized bacterial taxa. Compared to surrounding seawater, particle samples were enriched in different size classes of globally significant N2-fixing cyanobacteria including Trichodesmium, symbionts of diatoms, and the unicellular cyanobacteria Crocosphaera and UCYN-A. The particles also contained nifH gene sequences of diverse non-cyanobacterial diazotrophs suggesting that particles could be loci for N2 fixation by heterotrophic bacteria. The results demonstrate that diverse diazotrophs were present on particles and that new N may thereby be directly exported from surface waters on sinking particles.

Daily changes in phytoplankton lipidomes reveal mechanisms of energy storage in the open ocean.

Sunlight is the dominant control on phytoplankton biosynthetic activity, and darkness deprives them of their primary external energy source. Changes in the biochemical composition of phytoplankton communities over diel light cycles and attendant consequences for carbon and energy flux in environments remain poorly elucidated. Here we use lipidomic data from the North Pacific subtropical gyre to show that biosynthesis of energy-rich triacylglycerols (TAGs) by eukaryotic nanophytoplankton during the day and their subsequent consumption at night drives a large and previously uncharacterized daily carbon cycle. Diel oscillations in TAG concentration comprise 23 ± 11% of primary production by eukaryotic nanophytoplankton representing a global flux of about 2.4 Pg C yr-1. Metatranscriptomic analyses of genes required for TAG biosynthesis indicate that haptophytes and dinoflagellates are active members in TAG production. Estimates suggest that these organisms could contain as much as 40% more calories at sunset than at sunrise due to TAG production.

Coccolithovirus facilitation of carbon export in the North Atlantic.

Marine phytoplankton account for approximately half of global primary productivity 1 , making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon 2 , which can stimulate nutrient regeneration, primary production and upper ocean respiration 2 via lytic infection and the 'virus shunt'. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean 2 . Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (~100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in 'early-infected blooms' with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.

Arsenobetaine in Seawater: Depth Profiles from Selected Sites in the North Atlantic.

Arsenic occurs in marine waters, typically at concentrations of 1-2 µg As kg-1, primarily as the inorganic species arsenate. Marine animals, however, contain extremely high levels of arsenic (typically 2000-20 000 µg As kg-1 wet mass), most of which is present as arsenobetaine, an organic form of arsenic that has never been found in seawater. We report a method based on ion-exchange preconcentration and HPLC/mass spectrometry to measure arsenobetaine in seawater, and apply the method to samples of seawater collected at various depths from seven sites in the North Atlantic. Arsenobetaine was detected in most samples at levels ranging from 0.5 to 10 ng As kg-1, and was found at depths down to 4900 m. Furthermore, we report the presence of 15 additional organoarsenicals in seawater, 14 of which had never been detected in marine waters. The arsenobetaine depth profile was related, albeit weakly, to that of chlorophyll; this relationship probably reflects arsenobetaine's release to water from marine animals associated with the euphotic zone rather than its direct biosynthesis by primary producers. Future application of the new method for seawater analysis will shed new light on the biogeochemical cycle of marine arsenic.