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The photosynthetic and diazotrophic cyanobacteriumTrichodesmiumis a key contributor to marine biogeochemical cycles in the subtropical-oligotrophic oceans.Trichodesmiumforms colonies that harbor a distinct microbial community, which expands their functional potential and is predicted to influence the cycling of carbon, nitrogen, phosphorus and iron (C, N, P, and Fe). To link key traits to taxa and elucidate how community structure influences nutrient cycling, we assessed Red SeaTrichodesmiumcolonies using metagenomics and metaproteomics. This diverse consortium comprises bacteria that typically associate with algae and particles, such as the ubiquitousAlteromonas macleodii,but also lineages specific toTrichodesmium, such as members from the order Balneolales. These bacteria carry functional traits that would influence resource cycling in the consortium, including siderophore biosynthesis, reduced phosphorus metabolism, vitamins, denitrification, and dissimilatory-nitrate-reduction-to-ammonium (DNRA) pathways. Denitrification and DNRA appeared to be modular as bacteria collectively completed the steps for these pathways. The vast majority of associated bacteria were auxotrophic for vitamins, indicating the interdependency of consortium members.Trichodesmiumin turn may rely on associated bacteria to meet its high Fe demand as several lineages can synthesize the photolabile siderophores vibrioferrin, rhizoferrin, and petrobactin, enhancing the bioavailability of particulate-Fe to the entire consortium. Our results highlight thatTrichodesmiumis a hotspot for C, N, P, Fe, and vitamin exchange. The functional redundancy of nutrient cycling in the consortium likely underpins its resilience within an ever-changing global environment.ImportanceColonies of the cyanobacteriaTrichodesmiumact as a biological hotspot for the usage and recycling of key resources such as C, N, P and Fe within an otherwise oligotrophic environment. WhileTrichodesmiumcolonies are known to interact with a unique community of algae and particle-associated microbes, our understanding of the taxa that populate these colonies and the gene functions they encode is still limited. Characterizing the taxa and adaptive strategies that influence consortium physiology and its concomitant biogeochemistry is critical in a future ocean predicted to have increasing particulate fluxes and resource-depleted regions.

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.

The physical and biological dynamics that influence phytoplankton communities in the oligotrophic ocean are complex, changing across broad temporal and spatial scales. Eukaryotic phytoplankton (e.g., diatoms), despite their relatively low abundance in oligotrophic waters, are responsible for a large component of the organic matter flux to the ocean interior. Mesoscale eddies can impact both microbial community structure and function, enhancing primary production and carbon export, but the mechanisms that underpin these dynamics are still poorly understood. Here, mesoscale eddy influences on the taxonomic diversity and expressed functional profiles of surface communities of microeukaryotes and particle-associated heterotrophic bacteria from the North Pacific Subtropical Gyre were assessed over 2 years (spring 2016 and summer 2017). The taxonomic diversity of the microeukaryotes significantly differed by eddy polarity (cyclonic versus anticyclonic) and between sampling seasons/years and was significantly correlated with the taxonomic diversity of particle-associated heterotrophic bacteria. The expressed functional profile of these taxonomically distinct microeukaryotes varied consistently as a function of eddy polarity, with cyclones having a different expression pattern than anticyclones, and between sampling seasons/years. These data suggest that mesoscale forcing, and associated changes in biogeochemistry, could drive specific physiological responses in the resident microeukaryote community, independent of species composition.

The widespread coccolithophore Emiliania huxleyi is an abundant oceanic phytoplankton, impacting the global cycling of carbon through both photosynthesis and calcification. Here, we examined the transcriptional responses of populations of E. huxleyi in the North Pacific Subtropical Gyre to shifts in the nutrient environment. Using a metatranscriptomic approach, nutrient-amended microcosm studies were used to track the global metabolism of E. huxleyi. The addition of nitrate led to significant changes in transcript abundance for gene pathways involved in nitrogen and phosphorus metabolism, with a decrease in the abundance of genes involved in the acquisition of nitrogen (e.g. N-transporters) and an increase in the abundance of genes associated with phosphate acquisition (e.g. phosphatases). Simultaneously, after the addition of nitrate, genes associated with calcification and genes unique to the diploid life stages of E. huxleyi significantly increased. These results suggest that nitrogen is a major driver of the physiological ecology of E. huxleyi in this system and further suggest that the addition of nitrate drives shifts in the dominant life-stage of the population. Together, these results underscore the importance of phenotypic plasticity to the success of E. huxleyi, a characteristic that likely underpins its ability to thrive across a variety of marine environments.

The pelagophyte Aureococcus anophagefferens causes harmful brown tide blooms in marine embayments on three continents. Aureococcus anophagefferens was the first harmful algal bloom species to have its genome sequenced, an advance that evidenced genes important for adaptation to environmental conditions that prevail during brown tides. To expand the genomic tools available for this species, genomes for four strains were assembled, including three newly sequenced strains and one assembled from publicly available data. These genomes ranged from 57.11 to 73.62 Mb, encoding 13,191-17,404 potential proteins. All strains shared ~90% of their encoded proteins as determined by homology searches and shared most functional orthologs as determined by KEGG, although each strain also possessed coding sequences with unique functions. Like the original reference genome, the genomes assembled in this study possessed genes hypothesized to be important in bloom proliferation, including genes involved in organic compound metabolism and growth at low light. Cross-strain informatics and culture experiments suggest that the utilization of purines is a potentially important source of organic nitrogen for brown tides. Analyses of metatranscriptomes from a brown tide event demonstrated that use of a single genome yielded a lower read mapping percentage (~30% of library reads) as compared to a database generated from all available genomes (~43%), suggesting novel information about bloom ecology can be gained from expanding genomic space. This work demonstrates the continued need to sequence ecologically relevant algae to understand the genomic potential and their ecology in the environment.

Heterosigma akashiwo is a raphidophyte known for forming ichthyotoxic blooms. In order to predict the potential impacts of rising CO2 on H. akashiwo it is necessary to understand the factors influencing growth rates over a range of CO2 concentrations. Here we examined the physiology and gene expression response of H. akashiwo to concentrations from 200 to 1000ppm CO2. Growth rate data were combined from this and previous studies and fit with a CO2 limitation-inhibition model that revealed an apparent growth optimum around 600-800ppm CO2. Physiological changes included a significant increase in C:N ratio at ∼800ppm CO2 and a significant decrease in hydrogen peroxide concentration at ∼1000ppm. Whole transcriptome sequencing of H. akashiwo revealed sharp distinctions in metabolic pathway gene expression between ∼600 and ∼800ppm CO2. Hierarchical clustering by co-expression identified groups of genes with significant correlations to CO2 and growth rate. Genes with significant differential expression with CO2 included carbon concentrating mechanism genes such as beta-carbonic anhydrases and a bicarbonate transporter, which may underpin shifts in physiology. Genes involved in cell motility were significantly changed by both elevated CO2 and growth rate, suggesting that future ocean conditions could modify swimming behavior in this species.

1.AbstractMesoscale eddies have been shown to support elevated dinitrogen (N2) fixation rates (NFRs) and abundances of N2-fixing microorganisms (diazotrophs), but the mechanisms underlying these observations are not well understood. We explored relationships among NFRs and cyanobacterial diazotroph abundances in eddy pairs of opposite polarity sampled in the North Pacific Subtropical Gyre and compared our observations to seasonal trends from the Hawaii Ocean Time-series (HOT) program. Consistent with previous reports, we found that NFRs were anomalously high for this region (up to 3.7-fold above previous monthly HOT observations) in the centers of the sampled anticyclones, coinciding with elevated abundances of Crocosphaera in the summertime. We then coupled our field-based observations, together with transcriptomic analyses of nutrient stress marker genes and ecological models, to evaluate potential mechanisms controlling diazotroph abundance and activity at the mesoscale. Specifically, we evaluated the role of biological (via estimates of growth and grazing rates) and physical controls on populations of Crocosphaera, Trichodesmium, and diatom symbionts. Our results suggest that increased Crocosphaera abundances associated with summertime anticyclones resulted from the alleviation of phosphate limitation, allowing cells to grow at rates exceeding grazing losses. In contrast, distributions of larger, buoyant taxa (Trichodesmium and diatom symbionts) appeared less affected by eddy-driven biological controls. Instead, they appeared driven by physical dynamics along frontal boundaries that separate cyclonic and anticyclonic eddies. Together, the interplay of eddy-specific changes in bottom-up control, top-down control, and the physical accumulation of cells likely explains the elevated diazotroph abundances and NFRs associated with anticyclones and eddy fronts.

Pelagophytes are abundant picophytoplankton within open ocean ecosystems and the causative algae of harmful brown tide blooms in estuaries. The physiological capabilities facilitating the ecological success of pelagophytes in these diverse ecosystems remains poorly understood. Here, we investigated the transcriptional response of two coastal pelagophytes (Aureococcus anophagefferens and Aureoumbra lagunensis) and two open ocean pelagophytes (Pelagococcus subviridis and Pelagomonas calceolata) to conditions commonly found within the marine ecosystems where they thrive: low concentrations of nitrogen (N), phosphorus (P), or light. OrthoMCL was used to generate a total of 62,653 orthologous groups (OGs) with only a small fraction of these OGs (2,776 or 4.4%) being shared among all four pelagophytes. Of the commonly shared OGs, 8% were significantly differentially abundant under low N, P, or light with the majority associated with energy and lipid metabolism. Distinct responses among pelagophytes included increased abundance of transcripts encoding phosphate transporters (Aureococcus) and transcripts encoding a pyrophosphatase (Aureococcus and Pelagomonas) under low P, the expression of a suite of organic nitrogen-degrading enzymes under low N (Aureoumbra and Pelagomonas), increased abundance of transcripts encoding flavodoxins relative to ferredoxins (Pelagomonas) and transcripts encoding lysophospholipase (Pelagococcus) under low light, and both increases and decreases in abundance of transcripts encoding selenoproteins in all pelagophytes except Pelagococcus. Collectively, this study provides new information on the expressed gene compliment of these poorly characterized taxa and demonstrates that these pelagophytes possess a combination of shared and unique physiological features that likely facilitate their adaptation to distinct environmental conditions.

SummaryResources such as nitrogen (N) and phosphorus (P) play an important role in primary production and constraining phytoplankton bloom dynamics. Models to predict bloom dynamics require mechanistic knowledge of algal metabolic shifts in response to resource limitation. For well-studied model phytoplankton like diatoms, this information is plentiful. However, for less-studied groups such as the raphidophytes, there remain significant gaps in understanding metabolic changes associated with nutrient limitation.Using a novel combination of metabolomics and transcriptomics, we examined how the harmful algal bloom-forming raphidophyte Heterosigma akashiwo shifts its metabolism under N- and P-stress. We chose H. akashiwo because of its ubiquity within estuarine environments worldwide, where bloom dynamics are influenced by N and P availability.Our results show that each stress phenotype is distinct in both the allocation of carbon and the recycling of macromolecules. Further, we identified biomarkers of N- and P-stress that may be applied in situ to help modelers and stakeholders manage, predict, and prevent future blooms.These findings provide a mechanistic foundation to model the metabolic traits and trade-offs associated with N- and P-stress in H. akashiwo, and evaluate the extent to which these metabolic responses can be inferred in other phytoplankton groups.

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.

Sunlight drives daily rhythms of photosynthesis, growth, and division of photoautotrophs throughout the surface oceans. However, the cascading impacts of oscillatory light input on diverse microbial communities and community-scale metabolism remains unclear. Here we use an unsupervised machine learning approach to show that a small number of diel archetypes can explain pervasive periodic dynamics amongst more than 65,000 distinct time series, including transcriptional activity, macromolecules, lipids, and metabolites from the North Pacific Subtropical Gyre. Overall, we find evidence for synchronous timing of carbon-cycle gene expression that underlie daily oscillations in the concentrations of particulate organic carbon. In contrast, we find evidence of asynchronous timing in gene transcription related to nitrogen metabolism and related metabolic processes consistent with temporal niche partitioning amongst microorganisms in the bacterial and eukaryotic domains.

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 frequency of phytoplankton metabolic activities and their coordination across different populations. We investigated the diel orchestration of phytoplankton activity involved in photosynthesis, photoacclimation, and photoprotection by analyzing the pigment and quinone distribution in combination with metatranscriptomes in the 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. The night represents a metabolic recovery phase to refill cellular pigment stores, while the photosystems are remodeled towards photoprotection during the day. Transcript levels of genes involved in photosynthesis and pigment metabolism had highly synchronized diel expression patterns among all taxa, suggesting that there are similar regulatory mechanisms for light and energy metabolism across domains, and that other environmental factors drive niche differentiation. Observed decoupling of diel oscillations in transcripts and related pigments in the NPSG indicates that pigment abundance is modulated by environmental factors extending beyond gene expression/regulation, showing that metatranscriptomes may provide only limited insights on real-time photophysiological metabolism.

Trichodesmium is a marine, diazotrophic cyanobacterium that plays a central role in the biogeochemical cycling of carbon and nitrogen. Colonies ubiquitously co‐occur with a diverse microbiome of heterotrophic bacteria. We show that manipulation of the microbiome with quorum sensing acyl homoserine lactones (AHLs) significantly modulated rates of N2 fixation by Trichodesmium collected from the western North Atlantic, with positive and negative effects of varied magnitude. Changes in Trichodesmium N2 fixation did not correlate with changes in microbiome composition or geochemistry. With AHL addition, a subset of the significantly differentially expressed genes was related to known quorum sensing responses in model bacteria. However, there was little overlap in specific microbiome transcriptional responses to AHL addition between stations. Overall, these host‐microbiome interactions reflect a complex interplay of biotic and environmental factors that together form an overlooked mechanism modulating Trichodesmium N2 fixation.

N2 fixation by the genus Trichodesmium is predicted to support a large proportion of the primary productivity across the oligotrophic oceans, regions that are considered among the largest biomes on Earth. Many of these environments remain poorly sampled, limiting our understanding of Trichodesmium physiological ecology in these critical oligotrophic regions. Trichodesmium colonies, communities that consist of the Trichodesmium host and their associated microbiome, were collected across the oligotrophic western tropical South Pacific (WTSP). These samples were used to assess host clade distribution, host and microbiome metabolic potential, and functional gene expression, with a focus on identifying Trichodesmium physiological ecology in this region. Genes sets related to phosphorus, iron, and phosphorus–iron co-limitation were dynamically expressed across the WTSP transect, suggestive of the importance of these resources in driving Trichodesmium physiological ecology in this region. A gene cassette for phosphonate biosynthesis was detected in Trichodesmium, the expression of which co-varied with the abundance of Trichodesmium Clade III, which was unusually abundant relative to Clade I in this environment. Coincident with the expression of the gene cassette, phosphate reduction to phosphite and low-molecular-weight phosphonate compounds was measured in Trichodesmium colonies. The expression of genes that enable use of such reduced-phosphorus compounds were also measured in both Trichodesmium and the microbiome. Overall, these results highlight physiological strategies employed by consortia in an undersampled region of the oligotrophic WTSP and reveal the molecular mechanisms underlying previously observed high rates of phosphorus reduction in Trichodesmium colonies.

Prochlorococcus is a globally important marine cyanobacterium that lacks the gene catalase and relies on ‘helper’ bacteria such as Alteromonas to remove reactive oxygen species. Increasing atmospheric CO2 decreases the need for carbon concentrating mechanisms and photorespiration in phytoplankton, potentially altering their metabolism and microbial interactions even when carbon is not limiting growth. Here, Prochlorococcus (VOL4, MIT9312) was co-cultured with Alteromonas (strain EZ55) under ambient (400 p.p.m.) and elevated CO2 (800 p.p.m.). Under elevated CO2, Prochlorococcus had a significantly longer lag phase and greater apparent die-offs after transfers suggesting an increase in oxidative stress. Whole-transcriptome analysis of Prochlorococcus revealed decreased expression of the carbon fixation operon, including carboxysome subunits, corresponding with significantly fewer carboxysome structures observed by electron microscopy. Prochlorococcus co-culture responsive gene 1 had significantly increased expression in elevated CO2, potentially indicating a shift in the microbial interaction. Transcriptome analysis of Alteromonas in co-culture with Prochlorococcus revealed decreased expression of the catalase gene, known to be critical in relieving oxidative stress in Prochlorococcus by removing hydrogen peroxide. The decrease in catalase gene expression was corroborated by a significant ~6-fold decrease in removal rates of hydrogen peroxide from co-cultures. These data suggest Prochlorococcus may be more vulnerable to oxidative stress under elevated CO2 in part from a decrease in ecosystem services provided by heterotrophs like Alteromonas. This work highlights the importance of considering microbial interactions in the context of a changing ocean.

Trichodesmium is a genus of marine diazotrophic colonial cyanobacteria that exerts a profound influence on global biogeochemistry, by injecting 'new' nitrogen into the low nutrient systems where it occurs. Colonies of Trichodesmium ubiquitously contain a diverse assemblage of epibiotic microorganisms, constituting a microbiome on the Trichodesmium host. Metagenome sequences from Trichodesmium colonies were analyzed along a resource gradient in the western North Atlantic to examine microbiome community structure, functional diversity and metabolic contributions to the holobiont. Here we demonstrate the presence of a core Trichodesmium microbiome that is modulated to suit different ocean regions, and contributes over 10 times the metabolic potential of Trichodesmium to the holobiont. Given the ubiquitous nature of epibionts on colonies, the substantial functional diversity within the microbiome is likely an integral facet of Trichodesmium physiological ecology across the oligotrophic oceans where this biogeochemically significant diazotroph thrives.

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth.

Emiliania huxleyi is a calcifying haptophyte, contributing to both the organic and inorganic marine carbon cycles. In marine ecosystems, light is a major driver of phytoplankton physiology and ultimately carbon flow through the ecosystem. Here, we analysed a Lagrangian time-series of metatranscriptomes collected in the North Pacific Subtropical Gyre (NPSG) to examine how in situ populations of E. huxleyi modulate gene expression over day-night transitions. Many E. huxleyi contigs had a diel expression pattern, with 61% of contigs clustering into modules with statistically significant diel periodicity. Contigs involved in processes that build up energy stores, like carbon fixation and lipid synthesis, peaked around dawn. In contrast, contigs involved in processes that released energy stores, like respiration and lipid degradation, peaked mid-day and towards dusk. These patterns suggest an orchestrated cycle of building, then consuming energy stores in E. huxleyi populations in the NPSG. Selected contigs related to the cell cycle also exhibited significant diel periodicity consistent with phased modulations of division observed in culture. Overall, these patterns of gene expression suggest a daily metabolic cascade that could contribute to both organic and inorganic carbon flow in this nutrient depleted ecosystem.

Ocean greening off Hawai'i From June to August 2018, the eruption of Kīlauea volcano triggered a diatom-dominated phytoplankton bloom. Wilson et al. set sail to sample the plume, deploying subsea gliders and using satellite monitoring to measure the dynamics of this rare event in the nutrient-poor Pacific (see the Perspective by Ducklow and Plank). They found subsurface chlorophyll maxima not visible by remote sensing, performed transcriptome and N isotope marker analysis, and measured nutrients, partitioning of biomass into different organisms, and primary production. Much of the data are corroborated by physical modeling of the ocean dynamics. The authors conclude that the plume was fed by the lava heating subsurface water and triggering upwelling of deepwater nutrients to the surface rather than by direct injection of micronutrients from lava. Science , this issue p. 1040 ; see also p. 978

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies.

Populations of nitrogen-fixing cyanobacteria in the genus Trichodesmium are critical to ocean ecosystems, yet predicting patterns of Trichodesmium distribution and their role in ocean biogeochemistry is an ongoing challenge. This may, in part, be due to differences in the physiological ecology of Trichodesmium species, which are not typically considered independently in field studies. In this study, the abundance of the two dominant Trichodesmium clades (Clade I and Clade III) was investigated during a survey at Station ALOHA in the North Pacific Subtropical Gyre (NPSG) using a clade-specific qPCR approach. While Clade I dominated the Trichodesmium community, Clade III abundance was >50% in some NPSG samples, in contrast to the western North Atlantic where Clade III abundance was always 80 m, while Clade III populations were only observed in the mixed layer and found to be significantly correlated with depth and temperature. These data suggest active niche partitioning of Trichodesmium species from different clades, as has been observed in other cyanobacteria. Tracking the distribution and physiology of Trichodesmium spp. would contribute to better predictions of the physiological ecology of this biogeochemically important genus in the present and future ocean.

Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

Complexities associated with dissolved organic matter (DOM) isolation from seawater have hampered compositional characterization of this key component of global carbon and nutrient cycles. DOM isolation efficiency by electrodialysis (ED) from salt-containing waters was optimized and evaluated on samples including coastal ocean seawater, open ocean seawater, artificial seawater from axenic cultures of marine phytoplankton, and artificial seawater samples containing standard compounds of different molecular sizes and charge. ED was performed with a system optimized for processing 2–10 L sample volumes. Additionally, the combination of ED and solid-phase extraction, using Bond Elut PPL exchange resin, was evaluated. Using only ED, the following DOC recoveries were achieved: coastal seawater, 71.3 ± 6.5 %; open ocean, 50.5 ± 3.1 %; phytoplankton cultures, 70.3 ± 12.5 %; glucose, 90.2 ± 2.1 %; EDTA, 67.5 ± 9.9 %; and vitamin B12, 98.3 ± 1.6 %. With the combination of PPL and ED techniques, an average DOC recovery of 76.7 ± 2.6 % was obtained for coastal seawater, but this recovery was not statistically different from seawater recoveries using only ED. Comparison of C/N ratios and fluorescence excitation emission matrices taken at the beginning and end of the recovery process for coastal samples processed using only ED indicated that the final recovered material was representative of the DOM present in the original samples. Typical recoveries using combined PPL and ED exceed those of previous isolation methods.

Summary Nitrogen-fixing cyanobacteria in the genus Trichodesmium play a critical role in the productivity of the tropical and subtropical oligotrophic oceans. The ecological success of these populations is likely associated with the diverse microbial interactions occurring within the Trichodesmium holobiont, especially between Trichodesmium and heterotrophic bacterial epibionts. Yet, the composition of the Trichodesmium holobiont and the processes governing microbial assemblage are not well documented. Here, we used high-resolution 16S rDNA amplicon sequencing to examine the diversity of Trichodesmium and associated epibionts across different ocean regions and colony morphologies (puffs and rafts). Trichodesmium Clade I (i.e., T. thiebautii-like) dominated the colonies in all ocean basins regardless of morphology, although the Trichodesmium community structure significantly varied between morphologies in some regions. On average, Alphaproteobacteria (i.e., Thalassobius), Gammaproteobacteria (i.e., Pseudoalteromonas), Sphingobacteria (i.e., Microscilla and Vibrio) and Flavobacteria dominated the epibiont communities, but community composition and structure significantly differed between regions. Epibionts from the two colony morphologies were taxonomically and functionally distinct in the North Atlantic and North Pacific. These findings suggest that the colony types might define two distinct niches and that epibiont assemblage might be driven in part by selective processes, where epibionts are selected according to their influence on colony metabolism.

The composition of sinking particles and the mechanisms leading to their transport ultimately control how much carbon is naturally sequestered in the deep ocean by the “biological pump.” While detrital particles often contain much of the sinking carbon, sinking of intact phytoplankton cells can also contribute to carbon export, which represents a direct flux of carbon from the atmosphere to the deep ocean by circumventing the surface ocean food web. Phytoplankton that contributed to carbon flux were identified in sinking material collected by short-term sediment trap deployments conducted along a transect off the eastern shore of South America. Particulate organic carbon flux at 125 m depth did not change significantly along the transect. Instead, changes occurred in the composition and association of phytoplankton with detrital particles. The fluxes of diatoms, coccolithophores, dinoflagellates, and nano-sized cells at 125 m were unrelated to the overlying surface population abundances, indicating that functional-group specific transport mechanisms were variable across locations. The dominant export mechanism of phytoplankton at each station was putatively identified by principal component analysis and fell into one of three categories; (1) transport and sinking of individual, viable diatom cells, (2) transport by aggregates and fecal pellets, or (3) enhanced export of coccolithophores through direct settling and/or aggregation.

The N2 fixing cyanobacterium Trichodesmium is a critically important organism in oligotrophic marine ecosystems, supplying "new" nitrogen (N) to the otherwise N-poor tropical and subtropical regions where it occurs. Low concentrations of phosphorus (P) in these regions can constrain Trichodesmium distribution and N2 fixation rates. Physiological characterization of a single species in a mixed community can be challenging, and 'omic approaches are increasingly important tools for tracking nutritional physiology in a taxon-specific manner. As such, studies examining the dynamics of gene and protein markers of physiology (e.g., nutrient stress) are critical for the application and interpretation of such 'omic data in situ. Here we leveraged combined transcriptomics, proteomics, and enzyme activity assays to track the physiological response of Trichodesmium erythraeum IMS101 to P deficiency and subsequent P re-supply over 72 h of sampling. P deficiency resulted in differential gene expression, protein abundance, and enzyme activity that highlighted a synchronous shift in P physiology with increases in the transcripts and corresponding proteins for hydrolyzing organic phosphorus, taking up phosphate with higher affinity, and modulating intracellular P demand. After P deficiency was alleviated, gene expression of these biomarkers was reduced to replete levels within 4 h of P amendment. A number of these gene biomarkers were adjacent to putative pho boxes and their expression patterns were similar to a sphR response regulator. Protein products of the P deficiency biomarkers were slow to decline, with 84% of the original P deficient protein set still significantly differentially expressed after 72 h. Alkaline phosphatase activity tracked with proteins for this enzyme. With the rapid turnover time of transcripts, they appear to be good biomarkers of a P stress phenotype, whereas proteins, with a slower turnover time, may better reflect cellular activities. These results highlight the importance of validating and pairing transcriptome and proteome data that can be applied to physiological studies of key species in situ.

The cyanobacterium Raphidiopsis raciborskii is a nuisance in freshwater ecosystems. Strains vary in their physiological responses to environmental drivers, thus a greater understanding of the magnitude of strain variation is required to characterize the species. In this study, two strains of R. raciborskii isolated from a tropical Australian water reservoir were grown with and without phosphorus (P) to determine any relative response to P stress. The strains had the same growth rates and under P free conditions, cells grew at the same rate as P replete conditions until day 9 when cell growth ceased. There was no difference in the alkaline phosphatase activity per cell for the P replete and P free conditions, but the level of activity per cell was greater in CS-505 than CS-506. P acquisition genes were identified from the sequenced genomes; both strains contained the same genes, but with differences in copy number of phoA (7 and 6), phnK (3 and 1) and phnH (2 and 1) between CS-505 and CS-506 (respectively). The expression of P acquisition genes under P stress was measured throughout the experiment and shown to vary in magnitude and timing across strains, and in P replete versus P free cultures. In strain CS-505, upregulation of the pstS1 and phoA genes occurred late in the growth phase and into senescence. These genes are involved in phosphate uptake and use of various forms of organic P. For strain CS-506, there was upregulation of the phosphate uptake gene, pit, and organic P utilization genes, phoA, phoU, phnD and phnK, commencing late in the growth phase. Our study shows that despite the fact that these two strains were isolated from the same waterbody, they differed markedly in their gene expression response to P free conditions. This capacity of R. raciborskii to vary in strain responses to P conditions gives the organism flexibility in responding to environmental change, particularly P stress conditions.

In the surface ocean, light fuels photosynthetic carbon fixation of phytoplankton, playing a critical role in ecosystem processes including carbon export to the deep sea. In oligotrophic oceans, diatom–diazotroph associations (DDAs) play a keystone role in ecosystem function because diazotrophs can provide otherwise scarce biologically available nitrogen to the diatom host, fueling growth and subsequent carbon sequestration. Despite their importance, relatively little is known about the nature of these associations in situ. Here we used metatranscriptomic sequencing of surface samples from the North Pacific Subtropical Gyre (NPSG) to reconstruct patterns of gene expression for the diazotrophic symbiont Richelia and we examined how these patterns were integrated with those of the diatom host over day–night transitions. Richelia exhibited significant diel signals for genes related to photosynthesis, N(2) fixation, and resource acquisition, among other processes. N(2) fixation genes were significantly co-expressed with host nitrogen uptake and metabolism, as well as potential genes involved in carbon transport, which may underpin the exchange of nitrogen and carbon within this association. Patterns of expression suggested cell division was integrated between the host and symbiont across the diel cycle. Collectively these data suggest that symbiont–host physiological ecology is strongly interconnected in the NPSG.

Heterosigma akashiwo is a raphidophyte known for forming ichthyotoxic blooms. In order to predict the potential impacts of rising CO

N2 fixation by the genus Trichodesmium is predicted to support a large proportion of the primary productivity across the oligotrophic oceans, regions that are considered among the largest biomes on Earth. Many of these environments remain poorly sampled, limiting our understanding of Trichodesmium physiological ecology in these critical oligotrophic regions. Trichodesmium colonies, communities that consisted of the Trichodesmium host and their associated microbiome, were collected across the oligotrophic western tropical South Pacific (WTSP). These samples were used to assess host clade distribution, host and microbiome (holobiont) metabolic potential, and functional gene expression, with a focus on identifying Trichodesmium physiological ecology in this region. Expression dynamics across the WTSP transect indicated potential co-limitation of Trichodesmium for phosphorus and iron. A gene cassette for phosphonate biosynthesis was detected in Trichodesmium, the expression of which co-varied with the abundance of Trichodesmium Clade III, which was unusually abundant relative to Clade I in this environment. Coincident with the expression of the gene cassette, phosphate reduction to phosphite and low molecular weight phosphonate compounds was measured in Trichodesmium colonies as well as genes that enable use of this reduced phosphorus in both Trichodesmium and the microbiome. Overall, these results highlight physiological strategies for survival by the Trichodesmium holobiont in the oligotrophic ocean, revealing mechanisms with the potential to influence the cycling of resources like nitrogen and phosphorus.

The N(2)-fixing cyanobacterium Trichodesmium is intensely studied because of the control this organism exerts over the cycling of carbon and nitrogen in the low nutrient ocean gyres. Although iron (Fe) and phosphorus (P) bioavailability are thought to be major drivers of Trichodesmium distributions and activities, identifying resource controls on Trichodesmium is challenging, as Fe and P are often organically complexed and their bioavailability to a single species in a mixed community is difficult to constrain. Further, Fe and P geochemistries are linked through the activities of metalloenzymes, such as the alkaline phosphatases (APs) PhoX and PhoA, which are used by microbes to access dissolved organic P (DOP). Here we identified significant correlations between Trichodesmium-specific transcriptional patterns in the North Atlantic (NASG) and North Pacific Subtropical Gyres (NPSG) and patterns in Fe and P biogeochemistry, with the relative enrichment of Fe stress markers in the NPSG, and P stress markers in the NASG. We also observed the differential enrichment of Fe-requiring PhoX transcripts in the NASG and Fe-insensitive PhoA transcripts in the NPSG, suggesting that metalloenzyme switching may be used to mitigate Fe limitation of DOP metabolism in Trichodesmium. This trait may underpin Trichodesmium success across disparate ecosystems.

Polyphosphate (polyP) was examined within the upper water column (≤ 150 m) of Station ALOHA (22° 45′N, 158° 00′W) during two cruises conducted in May–June 2013 and September 2013. Phosphorus molar ratios of particulate polyP to total particulate phosphorus (TPP) were relatively low, similar to previously reported values from the temperate western North Atlantic, and did not exhibit strong vertical gradients, reflecting a lack of polyP recycling relative to other forms of TPP with depth. Furthermore, relationships among polyP:TPP, soluble reactive phosphorus (SRP), and alkaline phosphatase activity (APA) were also consistent with previous observations from the Atlantic Ocean. To ascertain potential mechanisms of biological polyP production and utilization, surface seawater was incubated following nutrient additions. Results were consistent with polyP:TPP enrichment under opposite extremes of APA, suggesting diverse polyP accumulation/retention mechanisms. Addition of exogenous polyP (45 ± 5 P atoms) to field incubations did not increase chlorophyll content relative to controls, suggesting that polyP was not bioavailable to phytoplankton at Station ALOHA. To clarify this result, phytoplankton cultures were screened for the ability to utilize exogenous polyP. PolyP bioavailability was variable among model diatoms of the genus Thalassiosira, yet chain length did not influence polyP bioavailability. Thus, microbial community composition may influence polyP dynamics in the ocean, and vice versa.

Nitrogen fixation is an important yet still incompletely constrained component of the marine nitrogen cycle, particularly in the subsurface. A Video Plankton Recorder (VPR) survey in the subtropical North Atlantic found higher than expected Trichodesmium colony abundances at depth, leading to the hypothesis that deep nitrogen fixation in the North Atlantic may have been previously underestimated. Here, Trichodesmium colony abundances and modeled nitrogen fixation from VPR transects completed on two cruises in the tropical and subtropical North Atlantic in fall 2010 and spring 2011 were used to evaluate that hypothesis. A bio-optical model was developed based on carbon-normalized nitrogen fixation rates measured on those cruises. Estimates of colony abundance and nitrogen fixation were similar in magnitude and vertical and geographical distribution to conventional estimates in a recently compiled climatology. Thus, in the mean, VPR-based estimates of volume-specific nitrogen fixation rates at depth in the tropical North Atlantic were not inconsistent with estimates derived from conventional sampling methods. Based on this analysis, if Trichodesmium nitrogen fixation by colonies is underestimated, it is unlikely that it is due to underestimation of deep abundances by conventional sampling methods.