Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel

This study used analytical flow cytometry (AFC) to monitor the abundance of phytoplankton, coccoliths, bacteria and viruses in a transect that crossed a high reflectance area in the western English Channel. The high reflectance area, observed by satellite, was caused by the demise of an Emiliania huxleyi bloom. Water samples were collected from depth profiles at four stations, one station outside and three stations inside the high reflectance area. Plots of transect data revealed very obvious differences between Station 1, outside, and Stations 2–4, inside the high reflectance area. Inside, concentrations of viruses were higher; E. huxleyi cells were lower; coccoliths were higher; bacteria were higher and virus:bacteria ratio was lower than at Station 1, outside the high reflectance area. This data can simply be interpreted as virus-induced lysis of E. huxleyi cells in the bloom causing large concentrations of coccoliths to detach, resulting in the high reflectance observed by satellite imagery. This interpretation was supported by the isolation of two viruses, EhV84 and EhV86, from the high reflectance area that lysed cultures of E. huxleyi host strain CCMP1516. Basic characterization revealed that they were lytic viruses approximately 170 nm–190 nm in diameter with an icosahedral symmetry. Taken together, transect and isolation data suggest that viruses were the major contributor to the demise of the E. huxleyi population in the high reflectance area. Close coupling between microalgae, bacteria and viruses contributed to a large organic carbon input. Consequent cycling influenced the succession of an E. huxleyi-dominated population to a more characteristic mixed summer phytoplankton community

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light synchronised axenic Prochlorococcus (PCC9511 strain) culture and 35S-methionine and 3H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >104 times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G2 cell cycle stage were consistently 2.2 times higher than those of cells at the G1 stage. Furthermore, S+G2 cells up-regulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday, and up to 42% at dusk, of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations

Ecophysiological basis of spatiotemporal patterns in picophytoplankton pigments in the global ocean

Information on the intracellular content and functional diversity of phytoplankton pigments can provide valuable insight on the ecophysiological state of primary producers and the flow of energy within aquatic ecosystems. Combined global datasets of analytical flow cytometry (AFC) cell counts and High-Performance Liquid Chromatography (HPLC) pigment concentrations were used to examine vertical and seasonal variability in the ratios of phytoplankton pigments in relation to indices of cellular photoacclimation. Across all open ocean datasets, the weight-to-weight ratio of photoprotective to photosynthetic pigments showed a strong depth dependence that tracked the vertical decline in the relative availability of light. The Bermuda Atlantic Time-series Study (BATS) dataset revealed a general increase in surface values of the relative concentrations of photoprotective carotenoids from the winter-spring phytoplankton communities dominated by low-light acclimated eukaryotic microalgae to the summer and early autumn communities dominated by high-light acclimated picocyanobacteria. In Prochlorococcus-dominated waters, the vertical decline in the relative contribution of photoprotective pigments to total pigment concentration could be attributed in large part to changes in the cellular content of photosynthetic pigments (PSP) rather than photoprotective pigments (PPP), as evidenced by a depth-dependent increase of the intracellular concentration of the divinyl chlorophyll-a (DVChl-a) whilst the intracellular concentration of the PPP zeaxanthin remained relatively uniform with depth. The ability of Prochlorococcus cells to adjust their DVChl-a cell-1 over a large gradient in light intensity was reflected in more highly variable estimates of carbon-to-Chl-a ratio compared to those reported for other phytoplankton groups. This cellular property is likely the combined result of photoacclimatory changes at the cellular level and a shift in dominant ecotypes. Developing a mechanistic understanding of sources of variability in pigmentation of picocyanobacteria is critical if the pigment markers and bio-optical properties of these cells are to be used to map their biogeography and serve as indicators of photoacclimatory state of subtropical phytoplankton communities more broadly. It would also allow better assessment of effects on, and adaptability of phytoplankton communities in the tropical/subtropical ocean due to climate change

Determining drivers of phytoplankton carbon to chlorophyll ratio at Atlantic Basin scale

We demonstrate the ability of flow cytometry to determine species specific cellular carbon and chlorophyll content in vivo by using laboratory cultures of phytoplankton encompassing a wide range of cell sizes. When applied to the large Atlantic Meridional Transect flow cytometry dataset, we reveal patterns in the species-specific phytoplankton carbon (C), chlorophyll (Chl) and C:Chl ratio. For Prochlorococcus the range of C:Chl is between 2 – 604; for Synechococcus 0.5 – 558. Using a Random Forest machine learning approach, we show that predictability of phytoplankton C:Chl, dominated by the prevalence of Prochlorococcus, is largely driven by silicate and nitrite concentration in the Atlantic Ocean

Comparison of in vitro and in situ plankton production determinations

Plankton production was measured using 8 techniques at 4 stations in the Celtic Sea, North Atlantic Ocean, in April 2002. Primary production (PP) was derived from 14C incorporation into particulate carbon after 24 h simulated in situ, PP(14CSIS), and 2 h photosynthesis-irradiance incubations, PP(14CPUR), and from 2 published satellite algorithms, PP(VGPM) and PP (M91). Gross production (GP) was calculated from O2 evolution, GP(O2), and 18O enrichment of dissolved O 2, GP(18O), after 24 h simulated in situ incubations, and from in situ active fluorescence measured by fast repetition rate fluorometry (FRRF). Net community production (NCP) was determined from changes in in situ dissolved oxygen, NCP(?O2), and from changes in oxygen during 24 h simulated in situ incubations, NCP(O2). Dark community respiration (DCR) was derived from changes in oxygen during a 24 h dark incubation, DCR(O2), and daily oxygen uptake, DOU(18O, O2), was calculated from the difference between GP(18O) and NCP(O2). Three stations were dominated by picoautotrophs and the fourth station was dominated by diatoms. While most of the comparisons between techniques fell within previously published ranges, 2 anomalies occurred only at the diatom-dominated station. Rates of PP(14CPUR) were oxygen uptake in the dark. The low rates of PP( 14CPUR) in relation to PP(14CSIS) may have resulted from the heterogeneous nature of the bloom and differences in sampling time. However, it is also possible that dissolved organic material (DOM) released by the stressed diatom population restricted the diffusion of 14C into the cells, thereby causing a greater underestimate of PP by techniques using short incubations. The significantly higher rates of oxygen uptake in the light are difficult to reconcile, and we do not know whether the light enhanced oxygen uptake was directly linked to carbon fixation. However, the release of DOM may also have provided substrate for enhanced respiration in the light. These anomalies were only revealed through the concurrent measurement of plankton production by this wide range of techniques. Further investigation of DOM excretion and light-enhanced respiration during diatom blooms is warranted

Seasonal phosphorus and carbon dynamics in a temperate shelf sea (Celtic Sea)

The seasonal cycle of resource availability in shelf seas has a strong selective pressure on phytoplankton diversity and the biogeochemical cycling of key elements, such as carbon (C) and phosphorus (P). Shifts in carbon consumption relative to P availability, via changes in cellular stoichiometry for example, can lead to an apparent ‘excess’ of carbon production. We made measurements of inorganic P (Pi) uptake, in parallel to C-fixation, by plankton communities in the Celtic Sea (NW European Shelf) in spring (April 2015), summer (July 2015) and autumn (November 2014). Short-term (<8 h) Pi-uptake coupled with dissolved organic phosphorus (DOP) release, in parallel to net (24 h) primary production (NPP), were all measured across an irradiance gradient designed to typify vertically and seasonally varying light conditions. Rates of Pi-uptake were highest during spring and lowest in the low light conditions of autumn, although biomass-normalised Pi-uptake was highest in the summer. The release of DOP was highest in November and declined to low levels in July, indicative of efficient utilization and recycling of the low levels of Pi available. Examination of daily turnover times of the different particulate pools, including estimates of phytoplankton and bacterial carbon, indicated a differing seasonal influence of autotrophs and heterotrophs in P-dynamics, with summer conditions associated with a strong bacterial influence and the early spring period with fast growing phytoplankton. These seasonal changes in autotrophic and heterotrophic influence, coupled with changes in resource availability (Pi, light) resulted in seasonal changes in the stoichiometry of NPP to daily Pi-uptake (C:P ratio); from relatively C-rich uptake in November and late April, to P-rich uptake in early April and July. Overall, these results highlight the seasonally varying influence of both autotrophic and heterotrophic components of shelf sea ecosystems on the relative uptake of C and P

Plankton community respiration and bacterial metabolism in a North Atlantic Shelf Sea during spring bloom development (April 2015)

Spring phytoplankton blooms are important events in Shelf Sea pelagic systems as the increase in carbon production results in increased food availability for higher trophic levels and the export of carbon to deeper waters and the sea-floor. It is usually accepted that the increase in phytoplankton abundance and production is followed by an increase in plankton respiration. However, this expectation is derived from field studies with a low temporal sampling resolution (5–15 days). In this study we have measured the time course of plankton abundance, gross primary production, plankton community respiration, respiration of the plankton size classes (>0.8 µm and 0.2–0.8 µm) and bacterial production at ≤5 day intervals during April 2015 in order to examine the phasing of plankton autotrophic and heterotrophic processes. Euphotic depth-integrated plankton community respiration increased five-fold (from 22 ± 4 mmol O2 m−2 d−1 on 4th April to 119 ± 4 mmol O2 m−2 d−1 on 15th April) at the same time as gross primary production also increased five-fold, (from 114 ± 5 to 613 ± 28 mmol C m−2 d−1). Bacterial production began to increase during the development of the bloom, but did not reach its maximum until 5 days after the peak in primary production and plankton respiration. The increase in plankton community respiration was driven by an increase in the respiration attributable to the >0.8 µm size fraction of the plankton community (which would include phytoplankton, microzooplankton and particle attached bacteria). Euphotic depth-integrated respiration of the 0.2–0.8 µm size fraction (predominantly free living bacteria) decreased and then remained relatively constant (16 ± 3 – 11 ± 1 mmol O2 m−2 d−1) between the first day of sampling (4th April) and the days following the peak in chlorophyll-a (20th and 25th April). Recent locally synthesized organic carbon was more than sufficient to fulfil the bacterial carbon requirement in the euphotic zone during this productive period. Changes in bacterial growth efficiencies (BGE, the ratio of bacterial production to bacterial carbon demand) were driven by changes in bacterial production rates increasing from 0.8 µm during the development of the spring bloom, followed 5 days later by a peak in bacterial production. In addition, the size fractionated respiration rates and high growth efficiencies suggest that free living bacteria are not the major producers of CO2 before, during and a few days after this shelf sea spring phytoplankton bloom.The Leverhulme Trust | Ref. RPG-2017-089UK Natural Environment Research Council (NERC) | Ref. NE/K00168X/1UK Natural Environment Research Council (NERC) | Ref. NE/ K001884/1UK Natural Environment Research Council (NERC) | Ref. NE/K002058/1UK Natural Environment Research Council (NERC) | Ref. NE/K001701/

Microbial uptake dynamics of choline and glycine betaine in coastal seawater

Choline and glycine betaine (GBT) are utilized as osmolytes to counteract osmotic stress, but also constitute important nutrient sources for many marine microbes. Bacterial catabolism of these substrates can then lead to the production of climate active trace gases such as methylamine and methane. Using radiotracers, we investigated prokaryotic choline/GBT uptake and determined biotic and abiotic factors driving these processes in the Western English Channel, UK. Kinetic uptake parameters indicated high affinity (nM range) for both osmolytes and showed a seasonal pattern for choline uptake. Generalized linear modeling of uptake parameters suggested a significant influence of sea surface temperature and salinity on prokaryotic uptake of both osmolytes. The presence of diatoms significantly influenced prokaryotic choline/GBT uptake dynamics. Choline uptake was further related to the occurrence of Phaeocystis spp., which were highly abundant in the phytoplankton community during spring, and dinoflagellates abundance during summer. While Rhodobacteraceae were the most important bacterial drivers for prokaryotic choline uptake, prokaryotic GBT uptake was associated with various groups such as SAR11 (Pelagibacterales) and Gammaproteobacteria, suggesting a wider capacity for GBT catabolism than previously recognized. Furthermore, using a newly developed approach we determined the first available data for dissolved GBT concentrations in seawater and found both osmolytes to be at the sub-nanomolar range. Together, this study improves our understanding of the biogeochemical cycling of these environmentally important osmolytes and highlights how their cycles may be affected by a changing climate

Assessing phytoplankton community composition in the Atlantic Ocean from in situ and satellite observations

The Atlantic Meridional Transect (AMT) program (www.amt-uk.org) provides the perfect opportunity to observe the phytoplankton community size structure over a long latitudinal transect 50oN to 50oS, thereby covering the most important latitude-related basin-scale environmental gradients of the Atlantic Ocean. This work presents cell abundance data of phytoplankton taxa recently collected during cruises AMT28 and 29 (in 2018 and 2019, respectively) using flow cytometer and microscope observations, as well as the pigment composition of the community, to assess the abundance and spatial distribution of taxonomic groups across the Atlantic. The community size structure showed a clear consistency between cruises at large spatial scale, with a dominance of picoplanktonic Cyanobacteria in oceanic gyres, an increase in all groups in the equatorial upwelling region, and high biomass of microplankton size class at higher latitudes. Phytoplankton carbon biomass for oceanographic provinces, ranged from median values of 10 to 47 mg Carbon m-3, for the oligotrophic gyres, and South Atlantic (45°S-50oS), respectively. Satellite images of total chlorophyll a (as a proxy for phytoplankton biomass) as well as the relative contribution of the three phytoplankton size classes were produced for both cruises, and despite the small number of matchups, statistically agreed well with in situ size classes estimated as carbon biomass, constituting the first attempt in the literature to match satellite size classes with in situ data derived from cell abundance. The comparison of community structure between recent cruises (2019, 2018, 2015) and earlier ones (1995-1998) indicates a decrease in the number of diatom-bloom forming species, and an increase in Dinoflagellates, whereas nitrogen-fixing Trichodesmium abundance in tropical Atlantic remains constant. Within the recent period, a relative increase in the median values of picoplankton fraction was seen in SATL region, counterbalanced by a decrease in both nano- and microplankton fractions. Additionally, this study includes a database of species identified by microscopy, which had been interrupted for 20 years, providing a basis for long-term series of phytoplankton species

Abundance of a chlorophyll a precursor and the oxidation product hydroxychlorophyll a during seasonal phytoplankton community progression in the Western English Channel

This study presents the first in-situ measurements of the chlorophyll a oxidation product, hydroxychlorophyll a as well as the chlorophyll a precursor, chlorophyll aP276 conducted over an annual cycle. Chlorophyll a oxidation products, such as hydroxychlorophyll a may be associated with the decline of algal populations and can act as an initial step in the degradation of chlorophyll a into products which can be found in the geochemical record, important for studying past climate change events. Here, hydroxychlorophyll a and chlorophyll aP276 were measured at the long-term monitoring station L4, Western Channel Observatory (UK, www.westernchannelobservatory.org) over an annual cycle (2012). Weekly measurements of phytoplankton species composition and abundance enabled detailed analysis of possible sources of hydroxychlorophyll a. Dinoflagellates, 2 diatom species, the prymnesiophyte Phaeocystis spp. and the coccolithophorid Emiliania huxleyi were all associated with hydroxychlorophyll a occurrence. However, during alternate peaks in abundance of the diatoms, no association with hydroxychlorophyll a occurred, indicating that the oxidation of chlorophyll a was dependant not only on species but also on additional factors such as the mode of mortality, growth limiting factor (i.e. nutrient concentration) or phenotypic plasticity. Surface sediment samples contained 10 times more hydroxychlorophyll a (relative to chlorophyll a) than pelagic particulate samples, indicating that more chlorophyll a oxidation occurred during sedimentation or at the sediment-water interface, than in the pelagic environment. In addition, chlorophyll aP276 correlated with chl-a concentration, thus supporting its assignment as a chl-a precursor