Zinc stimulation of coastal productivity in low carbon dioxide environments.

Zinc (Zn) is a key micronutrient used by phytoplankton for carbon (C) acquisition, yet there have been few observations of its influence on natural oceanic phytoplankton populations. In this study, we observed Zn limitation of growth in the natural phytoplankton community of Terra Nova Bay, Antarctica, due to low (~220 µatm) pCO2 conditions, in addition to primary iron (Fe) limitation. Shipboard incubation experiments amended with Zn and Fe resulted in significantly higher chlorophyll a content and dissolved inorganic carbon drawdown compared to Fe addition alone. Zn and Fe response proteins detected in incubation and environmental biomass provided independent verification of algal co-stress for these micronutrients. These observations of Zn limitation under low pCO2 conditions demonstrate Zn can influence coastal primary productivity. Yet, as surface ocean pCO2 rises with continued anthropogenic emissions, the occurrence of Zn/C co-limitation will become rarer, impacting the biogeochemical cycling of Zn and other trace metal micronutrients.

Adaptive responses of marine diatoms to zinc scarcity and ecological implications.

Scarce dissolved surface ocean concentrations of the essential algal micronutrient zinc suggest that Zn may influence the growth of phytoplankton such as diatoms, which are major contributors to marine primary productivity. However, the specific mechanisms by which diatoms acclimate to Zn deficiency are poorly understood. Using global proteomic analysis, we identified two proteins (ZCRP-A/B, Zn/Co Responsive Protein A/B) among four diatom species that became abundant under Zn/Co limitation. Characterization using reverse genetic techniques and homology data suggests putative Zn/Co chaperone and membrane-bound transport complex component roles for ZCRP-A (a COG0523 domain protein) and ZCRP-B, respectively. Metaproteomic detection of ZCRPs along a Pacific Ocean transect revealed increased abundances at the surface (<200 m) where dZn and dCo were scarcest, implying Zn nutritional stress in marine algae is more prevalent than previously recognized. These results demonstrate multiple adaptive responses to Zn scarcity in marine diatoms that are deployed in low Zn regions of the Pacific Ocean.

Dinoflagellates alter their carbon and nutrient metabolic strategies across environmental gradients in the central Pacific Ocean.

Marine microeukaryotes play a fundamental role in biogeochemical cycling through the transfer of energy to higher trophic levels and vertical carbon transport. Despite their global importance, microeukaryote physiology, nutrient metabolism and contributions to carbon cycling across offshore ecosystems are poorly characterized. Here, we observed the prevalence of dinoflagellates along a 4,600-km meridional transect extending across the central Pacific Ocean, where oligotrophic gyres meet equatorial upwelling waters rich in macronutrients yet low in dissolved iron. A combined multi-omics and geochemical analysis provided a window into dinoflagellate metabolism across the transect, indicating a continuous taxonomic dinoflagellate community that shifted its functional transcriptome and proteome as it extended from the euphotic to the mesopelagic zone. In euphotic waters, multi-omics data suggested that a combination of trophic modes were utilized, while mesopelagic metabolism was marked by cytoskeletal investments and nutrient recycling. Rearrangement in nutrient metabolism was evident in response to variable nitrogen and iron regimes across the gradient, with no associated change in community assemblage. Total dinoflagellate proteins scaled with particulate carbon export, with both elevated in equatorial waters, suggesting a link between dinoflagellate abundance and total carbon flux. Dinoflagellates employ numerous metabolic strategies that enable broad occupation of central Pacific ecosystems and play a dual role in carbon transformation through both photosynthetic fixation in the euphotic zone and remineralization in the mesopelagic zone.

Minimal cobalt metabolism in the marine cyanobacterium Prochlorococcus.

Despite very low concentrations of cobalt in marine waters, cyanobacteria in the genus Prochlorococcus retain the genetic machinery for the synthesis and use of cobalt-bearing cofactors (cobalamins) in their genomes. We explore cobalt metabolism in a Prochlorococcus isolate from the equatorial Pacific Ocean (strain MIT9215) through a series of growth experiments under iron- and cobalt-limiting conditions. Metal uptake rates, quantitative proteomic measurements of cobalamin-dependent enzymes, and theoretical calculations all indicate that Prochlorococcus MIT9215 can sustain growth with less than 50 cobalt atoms per cell, ∼100-fold lower than minimum iron requirements for these cells (∼5,100 atoms per cell). Quantitative descriptions of Prochlorococcus cobalt limitation are used to interpret the cobalt distribution in the equatorial Pacific Ocean, where surface concentrations are among the lowest measured globally but Prochlorococcus biomass is high. A low minimum cobalt quota ensures that other nutrients, notably iron, will be exhausted before cobalt can be fully depleted, helping to explain the persistence of cobalt-dependent metabolism in marine cyanobacteria.

Rebound of shelf water salinity in the Ross Sea.

Antarctic Bottom Water (AABW) supplies the lower limb of the global overturning circulation and ventilates the abyssal ocean. In recent decades, AABW has warmed, freshened and reduced in volume. Ross Sea Bottom Water (RSBW), the second largest source of AABW, has experienced the largest freshening. Here we use 23 years of summer measurements to document temporal variability in the salinity of the Ross Sea High Salinity Shelf Water (HSSW), a precursor to RSBW. HSSW salinity decreased between 1995 and 2014, consistent with freshening observed between 1958 and 2008. However, HSSW salinity rebounded sharply after 2014, with values in 2018 similar to those observed in the mid-late 1990s. Near-synchronous interannual fluctuations in salinity observed at five locations on the continental shelf suggest that upstream preconditioning and large-scale forcing influence HSSW salinity. The rate, magnitude and duration of the recent salinity increase are unusual in the context of the (sparse) observational record.

Phaeocystis antarctica unusual summer bloom in stratified antarctic coastal waters (Terra Nova Bay, Ross Sea).

This study focuses on the potential explanations for a Phaeocystis antarctica summer bloom occurred in stratified waters of Terra Nova Bay (TNB) - which is part of the Antarctic Special Protected Area (n.161) in the Ross Sea - trough a multi-parameter correlative approach. Many previous studies have highlighted that water column stratification typically favors diatom dominance compared to the colonial haptophyte P. antarctica, in the Ross Sea, and this correlation has often been used to explain the historic dominance of diatoms in TNB. To explore the spatial and temporal progression of P. antarctica bloom in coastal waters, four stations were sampled three times each between December 31, 2009 and January 13, 2010. Taxonomic and pigment composition of phytoplankton communities, macro-nutrient concentrations and various different indices, all indicated the relative dominance of P. antarctica. Cell abundances revealed that P. antarctica contributed 79% of total cell counts in the upper 25 m and 93% in the lower photic zone. Similarly, a strong correlation was observed between Chl-a and the Hex:Fuco pigment ratio, corroborating the microscopic analyses. Recent studies have shown that iron can trigger colonial P. antarctica blooms. Based on the Hex:Chl-c3 proxy for iron limitation in P. antarctica, we hypothesize that anomalously higher iron fluxes were responsible for the unusual bloom of colonial P. antarctica observed in TNB.

Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP.

Emiliania huxleyi is a bloom-forming microalga that affects the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide. Top-down regulation of E. huxleyi blooms has been attributed to viruses and grazers; however, the possible involvement of algicidal bacteria in bloom demise has remained elusive. We demonstrate that a Roseobacter strain, Sulfitobacter D7, that we isolated from a North Atlantic E. huxleyi bloom, exhibited algicidal effects against E. huxleyi upon coculturing. Both the alga and the bacterium were found to co-occur during a natural E. huxleyi bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying algal-bacterial interactions in the oceans. During interaction, Sulfitobacter D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which E. huxleyi strains that exuded higher amounts of DMSP were more susceptible to Sulfitobacter D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in an algal strain typically resistant to the bacterial pathogen. This enhanced virulence was highly specific to DMSP compared to addition of propionate and glycerol which had no effect on bacterial virulence. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions among marine organisms, as a mediator of bacterial virulence that may regulate E. huxleyi blooms.

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.

Differences in pigmentation between life cycle stages in Scrippsiella lachrymosa (dinophyceae).

Various life cycle stages of cyst-producing dinoflagellates often appear differently colored under the microscope; gametes appear paler while zygotes are darker in comparison to vegetative cells. To compare physiological and photochemical competency, the pigment composition of discrete life cycle stages was determined for the common resting cyst-producing dinoflagellate Scrippsiella lachrymosa. Vegetative cells had the highest cellular pigment content (25.2 ± 0.5 pg · cell(-1) ), whereas gamete pigment content was 22% lower. The pigment content of zygotes was 82% lower than vegetative cells, even though they appeared darker under the microscope. Zygotes of S. lachrymosa contained significantly higher cellular concentrations of ß-carotene (0.65 ± 0.15 pg · cell(-1) ) than all other life stages. Photoprotective pigments and the de-epoxidation ratio of xanthophylls-cycle pigments in S. lachrymosa were significantly elevated in zygotes and cysts compared to other stages. This suggests a role for accessory pigments in combating intracellular oxidative stress during sexual reproduction or encystment. Resting cysts contained some pigments even though chloroplasts were not visible, suggesting that the brightly colored accumulation body contained photosynthetic pigments. The differences in pigmentation between life stages have implications for interpretation of pigment data from field samples when sampled during dinoflagellate blooms.

Needles in the blue sea: sub-species specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome.

Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8 ± 1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13 ± 15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ∼4 × 10(7) , 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples.

Temperature-induced viral resistance in Emiliania huxleyi (Prymnesiophyceae).

Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi's susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.

Multiple nutrient stresses at intersecting Pacific Ocean biomes detected by protein biomarkers.

Marine primary productivity is strongly influenced by the scarcity of required nutrients, yet our understanding of these nutrient limitations is informed by experimental observations with sparse geographical coverage and methodological limitations. We developed a quantitative proteomic method to directly assess nutrient stress in high-light ecotypes of the abundant cyanobacterium Prochlorococcus across a meridional transect in the central Pacific Ocean. Multiple peptide biomarkers detected widespread and overlapping regions of nutritional stress for nitrogen and phosphorus in the North Pacific Subtropical Gyre and iron in the equatorial Pacific. Quantitative protein analyses demonstrated simultaneous stress for these nutrients at biome interfaces. This application of proteomic biomarkers to diagnose ocean metabolism demonstrated Prochlorococcus actively and simultaneously deploying multiple biochemical strategies for low-nutrient conditions in the oceans.

Decoupling physical from biological processes to assess the impact of viruses on a mesoscale algal bloom.

Phytoplankton blooms are ephemeral events of exceptionally high primary productivity that regulate the flux of carbon across marine food webs [1-3]. Quantification of bloom turnover [4] is limited by a fundamental difficulty to decouple between physical and biological processes as observed by ocean color satellite data. This limitation hinders the quantification of bloom demise and its regulation by biological processes [5, 6], which has important consequences on the efficiency of the biological pump of carbon to the deep ocean [7-9]. Here, we address this challenge and quantify algal blooms' turnover using a combination of satellite and in situ data, which allows identification of a relatively stable oceanic patch that is subject to little mixing with its surroundings. Using a newly developed multisatellite Lagrangian diagnostic, we decouple the contributions of physical and biological processes, allowing quantification of a complete life cycle of a mesoscale (∼10-100 km) bloom of coccolithophores in the North Atlantic, from exponential growth to its rapid demise. We estimate the amount of organic carbon produced during the bloom to be in the order of 24,000 tons, of which two-thirds were turned over within 1 week. Complimentary in situ measurements of the same patch area revealed high levels of specific viruses infecting coccolithophore cells, therefore pointing at the importance of viral infection as a possible mortality agent. Application of the newly developed satellite-based approaches opens the way for large-scale quantification of the impact of diverse environmental stresses on the fate of phytoplankton blooms and derived carbon in the ocean.

Novel molecular determinants of viral susceptibility and resistance in the lipidome of Emiliania huxleyi.

Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E. huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E. huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E. huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E. huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E. huxleyi-EhV system.

Identification of isethionic acid and other small molecule metabolites of Fragilariopsis cylindrus with nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain metabolic profiles of the polar diatom Fragilariopsis cylindrus, leading to the identification of a novel metabolite in this organism. Initial results from an ongoing metabolomics study have led to the discovery of isethionic acid (2-hydroxyethanesulfonic acid, CAS: 107-36-8) as a major metabolite in F. cylindrus. This compound is being produced by the organism under normal culture conditions. This finding is the first report of a diatom producing isethionic acid. In addition to isethionic acid, four other metabolites, dimethylsulfoniopropionate (DMSP), betaine, homarine, and proline were present and may serve as osmoprotectants in F. cylindrus. NMR-based metabolite profiles of F. cylindrus were obtained along a growth curve of the organism. The relative concentration levels of the five metabolites were monitored over a growth period of F. cylindrus from 18 to 25 days. All showed an increase in relative concentration with time, except for proline, which began to decrease after day 21.

Proteomic analysis of a sea-ice diatom: salinity acclimation provides new insight into the dimethylsulfoniopropionate production pathway.

Dimethylsulfoniopropionate (DMSP) plays important roles in oceanic carbon and sulfur cycling and may significantly impact climate. It is a biomolecule synthesized from the methionine (Met) pathway and proposed to serve various physiological functions to aid in environmental stress adaptation through its compatible solute, cryoprotectant, and antioxidant properties. Yet, the enzymes and mechanisms regulating DMSP production are poorly understood. This study utilized a proteomics approach to investigate protein changes associated with salinity-induced DMSP increases in the model sea-ice diatom Fragilariopsis cylindrus (CCMP 1102). We hypothesized proteins associated with the Met-DMSP biosynthesis pathway would increase in relative abundance when challenged with elevated salinity. To test this hypothesis axenic log-phase cultures initially grown at a salinity of 35 were gradually shifted to a final salinity of 70 over a 24-h period. Intracellular DMSP was measured and two-dimensional gel electrophoresis was used to identify protein changes at 48 h after the shift. Intracellular DMSP increased by approximately 85% in the hypersaline cultures. One-third of the proteins increased under high salinity were associated with amino acid pathways. Three protein isoforms of S-adenosylhomo-cysteine hydrolase, which synthesizes a Met precursor, increased 1.8- to 2.1-fold, two isoforms of S-adenosyl Met synthetase increased 1.9- to 2.5-fold, and S-adenosyl Met methyltransferase increased by 2.8-fold, suggesting active methyl cycle proteins are recruited in the synthesis of DMSP. Proteins from the four enzyme classes of the proposed algal Met transaminase DMSP pathway were among the elevated proteins, supporting our hypothesis and providing candidate genes for future characterization studies.

Iron limitation of a springtime bacterial and phytoplankton community in the ross sea: implications for vitamin b(12) nutrition.

The Ross Sea is home to some of the largest phytoplankton blooms in the Southern Ocean. Primary production in this system has previously been shown to be iron limited in the summer and periodically iron and vitamin B(12) colimited. In this study, we examined trace metal limitation of biological activity in the Ross Sea in the austral spring and considered possible implications for vitamin B(12) nutrition. Bottle incubation experiments demonstrated that iron limited phytoplankton growth in the austral spring while B(12), cobalt, and zinc did not. This is the first demonstration of iron limitation in a Phaeocystis antarctica-dominated, early season Ross Sea phytoplankton community. The lack of B(12) limitation in this location is consistent with previous Ross Sea studies in the austral summer, wherein vitamin additions did not stimulate P. antarctica growth and B(12) was limiting only when bacterial abundance was low. Bottle incubation experiments and a bacterial regrowth experiment also revealed that iron addition directly enhanced bacterial growth. B(12) uptake measurements in natural water samples and in an iron fertilized bottle incubation demonstrated that bacteria serve not only as a source for vitamin B(12), but also as a significant sink, and that iron additions enhanced B(12) uptake rates in phytoplankton but not bacteria. Additionally, vitamin uptake rates did not become saturated upon the addition of up to 95 pM B(12). A rapid B(12) uptake rate was observed after 13 min, which then decreased to a slower constant uptake rate over the next 52 h. Results from this study highlight the importance of iron availability in limiting early season Ross Sea phytoplankton growth and suggest that rates of vitamin B(12) production and consumption may be impacted by iron availability.