Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic
Mixed layer depth and phytoplankton accumulation dynamics in the North AtlanticThe NAAMES expeditions intensively measured biological, chemical, and physical properties from 4 to 7 locations, or stations, in each bloom phase during November (Winter Transition), March−April (Accumulation), May (Climax; same as Climax Transition22), and September (Decline)22. Stations spanned a broad range in latitude (~37 °N to ~55 °N, Fig. 1a), sub-regional classifications (Gulf Stream and Sargasso Sea, Subtropical, Temperate and Subpolar)24, and MLDs (tens to hundreds of meters) (Fig. 1b and Supplementary Fig. 1). MLDs were calculated using a density difference threshold of 0.03 kg m−3 from the top 10 m25. Field data and associated analyses are derived from phytoplankton 1–20 µm in diameter and their associated communities sampled within the photic zone (40, 20, 1% surface irradiance) and within the mixed layer, unless otherwise noted.Fig. 1: Mixed layer depth and phytoplankton accumulation dynamics.a Locations of sampled stations within subregions of the Northwest Atlantic during the NAAMES expeditions (color coded and shaped by the bloom phase; W. Tran = Winter Transition; Acc = Accumulation; Clim = Climax; Decl = Decline; See key in Panel B). Black rectangle represents the study area of NAAMES and this research. b Mixed layer depths within the NAAMES campaigns (black box in Fig. 1a), calculated from CTD casts at each of the station locations (colored symbols) and Bio-ARGO profiling floats that were deployed at stations and sampled continuously (small circles with separate grey lines for each float). The latter provided a history of mixed layer depths before, during, and after occupation. c Bloom phase distribution of accumulation rates for in situ phytoplankton populations sampled several times per day at 5 m. Each point represents the median accumulation rate of each station. d Bloom phase distribution of phytoplankton cell accumulation rates derived from on-deck incubations of phytoplankton populations at simulated in situ light and temperature conditions (see ‘Methods’). Each point represents a biological replicate. Data in panels (c) and (d) are based on cell concentrations and contoured with ridgeline smoothing to represent the distribution of accumulation rates across stations within a given bloom phase. The size of contour peaks is driven by frequency of observations. e Phytoplankton concentration (taken from 5 m) as a function of water column stratification (expressed as buoyancy frequency; s−1). Higher buoyancy frequencies to the right of the plot represent more stratification. A LOESS line of best fit (shaded area = 95% confidence interval) for data shows the general trend of phytoplankton concentration across all seasonal phases. Different letters denote statistically significant groups (p 0.05, Kruskal−Wallis) between populations collected from 5 m in-line sampling throughout the day (in situ) and contemporaneous incubations of the same phytoplankton populations under simulated in situ irradiance and temperature (incubations; see ‘Methods’) (Fig. 1c, d). Accumulation rates using incubations calculated via cell concentration or via biovolume were not statistically different (Supplementary Fig. 2b).Phytoplankton cell concentration and biovolume generally increased with water column stability (stratification), during the Winter Transition, Accumulation, and Climax phases (Fig. 1e and Supplementary Fig. 2c). Stratification was quantified by the buoyancy frequency averaged over the upper 300 m of the water column (see ‘Methods’). Higher values of buoyancy frequency indicate a more stratified water column where exchange with nutrient-rich water below the surface is reduced. Strongly stratified water columns (buoyancy frequencies above 2 × 10−5 s−1) during the Decline phase were associated with lower cell concentrations (Fig. 1e), consistent with enhanced phytoplankton loss or reduced accumulation. Phytoplankton biovolume and cell size distribution within 1–20 µm-sized phytoplankton cells increased during the Decline phase (Supplementary Fig. 2c–e). These higher biovolumes could have been a result of changes in community composition. They could have also been attributed to aggregation caused by virus infection20,21,28, as virus concentrations were highest during this season (discussed below), or by light stress27, as mixed layer populations were more consistently exposed to daily higher irradiance levels characteristic of shallow mixed layers (Fig. 1e).In situ phytoplankton cell concentrations increased from Winter Transition until the Climax phase, from ~1 × 106 to 2.5 × 107 cells L−1 (Fig. 2a, c, gray boxes). On-deck incubations showed similar trends but had higher overall cell concentrations (Fig. 2a, c, white boxes). The Decline phase was characterized by a 4-fold reduction in median phytoplankton cell concentrations from the peak abundances observed during Climax phase (Fig. 2a, c). The stress markers utilized in this study provided a unique view into the physiological status of communities across these annual bloom phases (Supplementary Table 1). Our ROS and compromised cell membranes biomarkers specifically targeted eukaryotic phytoplankton, given the conditions used for flow cytometry analysis (see ‘Methods’). PCD-related proteases and lipids were extracted from biomass collected onto 1.2 and 0.2 µm diameter membrane filters, respectively. Consequently, these biomarkers could also include eukaryotic heterotrophs and bacteria in the system. Induction of caspase and metacaspase activities have been found in diverse phytoplankton, such as coccolithophores, diatoms, chlorophytes, nitrogen-fixing cyanobacteria, and dinoflagellates cells undergoing stress, senescence, and death29. They have also been reported in stressed or dying grazers30, although no marine species has been explicitly studied. TAGs are found mainly in marine eukaryotic phytoplankton31,33,33 and grazers34. The highly unsaturated fatty acids in the PC and OxPCs detected in our measurements are also indicative of eukaryotic organisms, and not marine cyanobacteria32 or heterotrophic bacteria35.Fig. 2: Seasonal phases have distinct physiological state signatures.a, c Concentration of phytoplankton cells sampled within the mixed layer at depths associated with 40, 20, or 1% surface irradiance during different seasonal phases (W.Tran = Winter Transition; Acc = Accumulation; Clim = Climax; Decl = Decline). Data are shown for in situ water (grey bars) and on-deck incubations (open bars). Population-wide levels of a, b cellular reactive oxygen species (colored by fluorescence fold change from unstained; median per population) and c, d cell death (colored by % compromised membrane). Plots (b) and (d) are contoured with ridgeline smoothing to represent the relative in situ distribution of biomarker levels within each phase. The size of contour peaks is driven by frequency of observations. e, f In situ inventories of live (e; green) and dead (f; red) cells within the mixed layer through the different phases. Individual circles denote biological replicates. Box plots in (a), (c), (e) and (f) represent the median value bounded by the upper and lower quartiles with whiskers representing median + quartile × 1.5. Different letters denote statistically significant groups (p 5 µM; PO4 > 0.4 µM). Notably, nutrient concentrations during the Climax phase were similar or higher than those observed for Accumulation phase samples, which had lower ROS signatures (Fig. 2b).Phytoplankton cells in the Decline and Winter Transition phases had a higher percentage of compromised cell membranes, reaching levels as high as 80% (Fig. 2c, d). Both late stage viral infection and PCD have been linked to high levels of compromised membranes13,29. The percentage of phytoplankton cells with compromised membranes was used to calculate concentrations of live and dead cells within the mixed layer across the bloom phases. Living phytoplankton cell concentrations generally increased from the Winter Transition through the Climax phase (Fig. 2e). The variability of dead cells was highest in the Decline phase, which also had the largest variation in total, living, and dead cell concentrations (Fig. 2c, e, f).Targeted analysis of OxPC, and TAGs in resident phytoplankton communities provided further context of changes in physiological states due to their relevance in cellular stress and loss processes. The seasonal bloom phases were characterized by distinct levels of these lipids (Fig. 3 and Supplementary Fig. 4). OxPC levels were highest in the Climax phase (Fig. 3a), where mixed layers had recently shallowed (Fig. 1b) and were concomitant with high intracellular ROS levels (Fig. 2b). Subcellular environments lacking in adequate antioxidant capacity are expected to accumulate OxPC40 particularly when a shallow mixed-layer enhances UV exposure15. Chlorophyll-normalized TAG was highest in the Decline phase (Fig. 3b), which also had the lowest accumulation rates (Fig. 1c, d). High cellular TAG levels have been observed in senescent41,42 or nutrient limited9 diatoms, and virus infected haptophytes43.Fig. 3: Seasonal phases are characterized by distinct lipid profiles and cell death-associated proteolytic activity.a Oxidized phosphatidylcholine (OxPC40:10, OxPC42:11, OxPC44:12) normalized to total phosphatidylcholine (PC40:10, PC42:11, PC44:12). b Triacylglycerol (TAG; pmol L−1), normalized to ChlA (peak area/L). c (top) The proportion of in situ samples with positive caspase activity (cleavage of IETD-AFC; color shading). (bottom) Caspase-specific activity rates (µmol substrate hydrolyzed h−1 µg protein−1) for in situ populations. d (top) The proportion of in situ samples with positive metacaspase activity (cleavage of VRPR-AMC; color shading). (bottom) Metacaspase-specific activity rates (µmol substrate hydrolyzed h−1 µg protein−1) for in situ populations. All box plots represent the median value bounded by the upper and lower quartiles, with whiskers representing median + quartile × 1.5. Different letters denote statistically significant groups (p More
