Abstract

Flow cytometric sorting, based on cellular optical properties and macromolecule content, has been successfully employed to taxonomically affiliate bacterioplankton. However, this approach has not been much used for eukaryotic plankton. To redress this imbalance, we identified a conspicuous group of red autofluorescent picoplankton in surface waters of the South Atlantic Ocean. Using catalysed reporter deposition fluorescence in situ hybridization, virtually, all cells sorted from that group were affiliated with the Mamiellales clade II (84 ± 4%, division Chlorophyta) with a size of 1.6 ± 0.03 μm. Based on electron microscopy, the Mamiellales clade II–sorted cells have a simple morphology with apparently no scales, flagella or surface features. Their latitudinal distribution resembled the distribution of Synechococcus with very low concentrations in the surface waters of the Southern subtropical gyre (0.6–1.6 × 103 cells mL−1) and increased concentrations in the Southern temperate waters 8.3 × 103 cells mL−1. Identification of the flow cytometric group as Mamiellales clade II allowed us to characterize the morphology of these enigmatic uncultured picoplanktonic cells by electron microscopy and to determine their apparent preference for temperate rather than subtropical oceanic photic waters.

Introduction

Flow cytometry (FCM) is routinely used for the analysis of prokaryotes and eukaryotes. Cytometric groups can be discriminated and quantified based on cell size, pigments (Chisholm et al., 1988; Li, 1994) or DNA content after staining with DNA dyes (Marie et al., 1997; Zubkov et al., 2000). The latter approach has enabled the discrimination of prokaryotic cells with high nucleic acid from those with low nucleic acid content (Gasol et al., 1999; Lebaron et al., 2001). Using fluorescence in situ hybridization on sorted cells, these groups could be assigned to almost single taxonomic groups, at the genus level (Zubkov et al., 2001; Mary et al., 2006) with little overlap between them (Schattenhofer et al., 2011). This approach, however, has been sparingly used for studying marine picoeukaryotes (Jardillier et al., 2010; Grob et al., 2011).

Picoeukaryotes play a major role in CO2 fixation in the ocean (Li, 1994; Jardillier et al., 2010; Massana, 2011); however, their diversity and distribution patterns remain poorly understood (Worden & Not, 2008), partly because of methodological constraints. Their abundance is orders of magnitude lower than prokaryotes; therefore, their efficient recovery by filtration is difficult (Cuvelier et al., 2010). Flow sorting allows cell separation and enrichment for studying populations present in low numbers. Sorting of picoeukaryotes (< 3 μm) based on pigments and size has been successful in enriching plastidic (chloroplast containing) organisms (Shi et al., 2009; Cuvelier et al., 2010; Jardillier et al., 2010; Grob et al., 2011; Balzano et al., 2012). However, the diversity and taxonomic composition of the sorted cells remains unclear. In coastal waters, there is evidence that flow cytometric clusters of plastidic picoeukaryotic can be comprised by a single family (Marie et al., 2010; Balzano et al., 2012), while in the open ocean flow, cytometric clusters are usually comprised of a highly variable assemblage of classes and families (Shi et al., 2009; Jardillier et al., 2010; Grob et al., 2011), and a high proportion of cells in clusters remains unidentified (Grob et al., 2011). In the studies cited above, both from coastal areas and the open ocean, broad sorting gates, which include all cells that could belong to the target group, were defined guided by chlorophyll a cellular red autofluorescence.

We hypothesized that if tight FCM gates, which include only cells that belong to the target cluster, are defined based on cellular pigment content, DNA content and size, the sorted cells could be assigned to a taxonomic clade, similar to what has been reported for prokaryotes. The objective of the present study was to characterize a group of picoplanktonic red autofluorescent cells, with DNA content higher than Synechococcus cyanobacteria but lower than the majority of eukaryotes. The group was pronounced in the surface mixed layer in the temperate region of the South Atlantic Ocean, while in adjacent surface waters of the southern subtropical gyre (SG) group, concentrations were considerably lower. Here, we compare the taxonomic composition and distribution of the group in the two regions.

Materials and methods

On the 20th Atlantic Meridional Transect (AMT) cruise on board the Royal Research Ship (R.R.S) James Cook (cruise number JC053, October-November 2010), samples were collected from surface down to a depth of 100 m using a rosette of 20-L Niskin bottles mounted on a conductivity-temperature-depth profiler. Taxonomic and morphological characterization of the group of small red autofluorescent cells was carried out in the surface mixed layer of the SG (three stations) and southern temperate (ST, one station) waters of the Atlantic Ocean (Fig. 1). Samples for cell sorting were collected from 20 m depth as a representative depth of the mixed layer, chosen as the shallowest depth unaffected by the ship's presence. Using a peristaltic pump, microbial cells were concentrated by a CellTrap™ (MEMTEQ Ventures Ltd, UK) into a volume of 1.5 mL from an approximately 2-L sample of seawater, pre-filtered through 10 μm and supplemented with Pluronic F86 (Sigma Aldrich, Dorset, UK) 0.05% w/v final concentration to prevent the formation of cell aggregates. Samples were fixed with 1% paraformaldehyde (PFA, final concentration, v/v) or 1% glutaraldehyde (final concentration, v/v) for approximately 1 h at 4 °C and subsequently flash-frozen in liquid nitrogen and stored at −80 °C. Samples were thawed on ice and stained with SYBR Green I DNA dye (Marie et al., 1997; Zubkov et al., 2000) and flow-sorted on a MoFlo flow cytometer (Beckman Coulter), equipped with an Argon ion laser (Innova-A300) tuned to 488 nm. Microbial cells were visualized on a bivariate plot (Summit, Dako Cytomation) of green fluorescence (530 ± 40 nm filter) of stained cells vs. red fluorescence (670 ± 40 nm filter) to register extra red fluorescence of chlorophyll-containing cells. Synechococcus were discriminated from other cells using their characteristic orange phycoerythrin (570 ± 40 nm filter) autofluorescence. A logical gate, which is a region drawn around the cluster of interest and used to isolate that cluster (Ormerod, 2010), for sorting the target group was created to omit Synechococcus. Particle-free, autoclaved (< 0.1 μM) 0.1% NaCl (w/v) solution was used as a sheath fluid. To achieve the highest purity, the sort mode ‘single one drop’ was used, which was calibrated by sorting multifluorescence 3-μm beads (Fluoresbrite Microparticles, Polysciences, Warrington, PA) onto glass slides followed by counting the sorted beads under an epifluorescence microscope. Between 2000 and 4000, cells were sorted and immediately filtered onto 3-mm-diameter polycarbonate filters for catalysed amplification reporter deposition fluorescence in situ hybridization (CARD-FISH) and scanning electron microscopy (SEM) analysis. CARD-FISH was performed on sorted cells as described before (Pernthaler et al., 2004). Briefly, filters were hybridized for 3 h at 46 °C with horseradish peroxidase-labelled oligonucleotide probes (Biomers, Ulm, Germany) at varying formamide concentrations depending on the probe used (Table 1). The probe-delivered horseradish peroxidase was detected with fluorescently labelled tyramide Alexa 488 at a ratio of 2 : 1000 in amplification buffer. Cells were manually enumerated (between 300 and 500 cells) on an Axioplan II epifluorescence microscope (Zeiss, Jena, Germany) equipped with filter sets for Alexa 488 and DAPI. Triplicate hybridizations were performed for all probes.

Eukaryotic FISH oligonucleotide probes used in this study

Probe Target Sequence (5′–3′) % FA (46 °C) Reference 
EUK516 Domain Eukarya ACCAGACTTGCCCTCC Amann et al. (1990
CHLO02 Phylum Chlorophyta CTTCGAGCCCCCAACTTT 20 Simon et al. (2000
PRAS04 Mamiellales clade II (Class Prasinophyceae; Family MamiellaceaCGTAAGCCCGCTTTGAAC 20 Not et al. (2004
MICRO01 Micromonas pusilla AATGGAACACCGCCGGCG 20 Not et al. (2004
OSTREO01 Ostreococcus CCTCCTCACCAGGAAGCT 20 Not et al. (2004
BATHY01 Bathylococcus prasinos ACTCCATGTCTCAGCGTT 20 Not et al. (2004
Probe Target Sequence (5′–3′) % FA (46 °C) Reference 
EUK516 Domain Eukarya ACCAGACTTGCCCTCC Amann et al. (1990
CHLO02 Phylum Chlorophyta CTTCGAGCCCCCAACTTT 20 Simon et al. (2000
PRAS04 Mamiellales clade II (Class Prasinophyceae; Family MamiellaceaCGTAAGCCCGCTTTGAAC 20 Not et al. (2004
MICRO01 Micromonas pusilla AATGGAACACCGCCGGCG 20 Not et al. (2004
OSTREO01 Ostreococcus CCTCCTCACCAGGAAGCT 20 Not et al. (2004
BATHY01 Bathylococcus prasinos ACTCCATGTCTCAGCGTT 20 Not et al. (2004

FA, formamide concentration (v/v) in the hybridization buffer, hybridizations done at 46 °C.

Map showing (a) the study area in the South Atlantic Ocean and (b) detailed view of the sampling stations. Black symbols indicate the stations where cell sorting followed by taxonomic and morphological characterization, and FCM counts were performed while grey symbols indicate stations where only the latter analysis was done. (c) Latitudinal distribution of Synechococcus and Prochlorococcus, which were used to identify the boundaries of the two oceanic regions: Dashed grey line indicates oceanic regions in (b) and (c).

Map showing (a) the study area in the South Atlantic Ocean and (b) detailed view of the sampling stations. Black symbols indicate the stations where cell sorting followed by taxonomic and morphological characterization, and FCM counts were performed while grey symbols indicate stations where only the latter analysis was done. (c) Latitudinal distribution of Synechococcus and Prochlorococcus, which were used to identify the boundaries of the two oceanic regions: Dashed grey line indicates oceanic regions in (b) and (c).

For SEM filters with deposited flow-sorted cells, which had been fixed with 1% glutaraldehyde, were freeze-dried using an Edwards Freeze Dryer (Edwards High Vacuum International, Sussex, UK). Dried filters were then placed on carbon-coated self-adhesive stubs and sputter coated (Cressington Sputter Coater 208HR, Watford, UK) with a 5-nm layer of gold palladium and stored in a dessicator. A Zeiss Ultra Plus Field Emission Scanning Electron Microscope (Zeiss) was used to image the cells using an accelerating voltage of 2 kV a working distance of 2 mm, and the in-lens secondary electron detector. Approximately 18 × 103 cells, fixed with 1% glutaraldehyde, were sorted as described above and subsequently prepared for transmission electron microscopy (TEM). Sorted cells were embedded in ultra low gelling temperature agarose (Sigma Aldrich, Dorset, UK) and processed as previously described (Glauert & Lewis, 1998) but with longer dehydration times. Cells were embedded in Spurr resin (Spurr, 1969) (TAAB Ltd, Berks, UK), and ultrathin, gold sections cut on a Reichert Ultracut S ultramicrotome (Leica, Vienna, Austria). Sections were stained in uranyl acetate and lead citrate (Frasca & Parks, 1965) and observed on a Hitachi H-7100 TEM at 100 kV (Hitachi Ltd, Tokyo, Japan).

Picoeukaryote populations were enumerated with a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK) in 15 stations, including those where cell sorting and identification was done, from surface waters down to 100 m. Samples of 1.6 mL were fixed with 1% PFA and stained as described above and analysed on board. Yellow-green 1-μm reference beads (Fluoresbrite Microparticles, Polysciences) were used in all analyses as an internal standard for both fluorescence and flow rates. The absolute concentration of beads in the stock solution was determined using syringe pump FCM (Zubkov & Burkill, 2006).

Results and discussion

Flow cytometric analyses of marine plankton revealed discrete populations of protists, based on their stained nucleic acid content (green fluorescence, Fig. 2), relative size indicator (90° light scatter, Fig. 2a and b) and relative chlorophyll content (red fluorescence, Fig. 2c and d; Zubkov et al., 2007, Christaki et al., 2011). Typically, three major populations could be differentiated: populations of smaller and larger plastidic (chloroplast containing) protists (Plast-S, ~ 2 μm & Plast-L, ~ 3 μm, respectively) and a population of aplastidic protists (without chloroplast, Aplast, ~ 3 um, Hartmann et al., 2012). However, at the bottom of the Plast-S general population, a pronounced subgroup of plastidic extra small cells (Plast-XS, average size 1.59 ± 0.39 μm, Table 2), with DNA content lower than most protists but higher than Synechoccocus cyanobacteria, were observed in the ST water as well as in the SG. These Plast-XS cells were flow-sorted for identification by CARD-FISH (Fig. 2e and f).

Average size of Plast-XS-sorted cells

Region Latitude (̊ N) Size (μm) 
SG −33 1.60 ± 0.36 
ST −36 1.62 ± 0.39 
ST −39 1.55 ± 0.39 
ST −42 1.60 ± 0.41 
 Average 1.59 ± 0.39 
Region Latitude (̊ N) Size (μm) 
SG −33 1.60 ± 0.36 
ST −36 1.62 ± 0.39 
ST −39 1.55 ± 0.39 
ST −42 1.60 ± 0.41 
 Average 1.59 ± 0.39 

Average of 150 DAPI stained cells measured manually by epifluorescence microscopy.

Flow cytometric density plot signatures of SYBR Green – DNA-stained picoeukaryotes. Axes represent SYBR Green DNA-stained green fluorescence and 90° light scatter in (a) SG and (b) ST. SYBR Green DNA-stained green fluorescence and red autofluorescence from chlorophyll a, indicating picoeukaryotic groups and the plastidic population (Plast-XS, red letters) sorted in this study in (c) SG and (d) ST, and actual flow-sorted group sorted in (e) SG and (f) ST. One micrometre reference beads shown.

Flow cytometric density plot signatures of SYBR Green – DNA-stained picoeukaryotes. Axes represent SYBR Green DNA-stained green fluorescence and 90° light scatter in (a) SG and (b) ST. SYBR Green DNA-stained green fluorescence and red autofluorescence from chlorophyll a, indicating picoeukaryotic groups and the plastidic population (Plast-XS, red letters) sorted in this study in (c) SG and (d) ST, and actual flow-sorted group sorted in (e) SG and (f) ST. One micrometre reference beads shown.

Once cells were sorted, we applied a suite of nested probes specific for different eukaryotic taxonomic levels. On average, 91 ± 5% of the Plast-XS cells hybridized with the general eukaryotic probe EUK516, which targets the domain Eukaryota (Amann et al., 1990; Figs 3 and 4a), which confirmed their identity as eukaryotes despite of their small size. However, when the tight sorting gate around the core of the Plast-XS cells, which included only cells that belong to the cluster, was broadened to include Plast-S and other cells around the core that could belong to the target cluster, only 68 ± 6% of cells, sorted using the broad gate, hybridized with the EUK516 probe (n = 5). It suggests that particles outside the core but within the broad gate could include bacterial and archaeal cells, or eukaryotic cells with degraded ribosomes. Therefore, the tight gate was used for routine sorting of the Plast-XS cells.

Relative abundance (% DAPI counts) of Eukarya,Chlorophyta and Mamiellales clade II in the Plast-XS-sorted group in the different stations of the South Atlantic determined with probes listed in Table 1.

Relative abundance (% DAPI counts) of Eukarya,Chlorophyta and Mamiellales clade II in the Plast-XS-sorted group in the different stations of the South Atlantic determined with probes listed in Table 1.

(a–c) Epifluorescence micrographs of Plast-XS-sorted cells. Blue: DAPI signals of DNA-containing cells and green signals of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH)-positive cells using probes targeting (a) Eukarya, (b) Chlorophyta and (c) Mamiellales clade II from the ST. (d–e) Scanning electron micrographs of Plast-XS-sorted cells from (d) SG and (e) ST and (f) transmission electron micrograph of Plast-XS-sorted cells from the ST, inset shows one cell at a higher magnification. Scale bars: (a–c) 2 μm, (d–e) 1 μm and (f) 100 and 50 nm.

(a–c) Epifluorescence micrographs of Plast-XS-sorted cells. Blue: DAPI signals of DNA-containing cells and green signals of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH)-positive cells using probes targeting (a) Eukarya, (b) Chlorophyta and (c) Mamiellales clade II from the ST. (d–e) Scanning electron micrographs of Plast-XS-sorted cells from (d) SG and (e) ST and (f) transmission electron micrograph of Plast-XS-sorted cells from the ST, inset shows one cell at a higher magnification. Scale bars: (a–c) 2 μm, (d–e) 1 μm and (f) 100 and 50 nm.

The majority of the Plast-XS-sorted cells were identified as members of the division Chlorophyta. On average, 86 ± 8% of cells hybridized with the CHLO02 probe (Simon et al., 2000; Figs 3 and 4b). Within the Chlorophyta division, the Plast-XS cells were identified as members of the class Prasinophyceae, more specifically, the cells were affiliated with the family Mamiellop-hyceae (Marin & Melkonian, 2010), which comprised the Mamiellales clade II (named according to (Guillou et al., 2004; Marin & Melkonian, 2010). The majority of the Plast-XS cells hybridized with the probe PRAS04 (Not et al., 2004), 83 ± 4% of total DAPI signals (Figs 3 and 4c), which specifically targets the Mamiellales clade II. The specificity of the probe PRASO04 was evaluated using a comprehensive and quality-checked rRNA gene sequence database that contains 62 587 Eukaryotic sequences (Silva release 108, Pruesse et al., 2007). The probe PRAS04 targets the family Mamiellophyceae, which comprises the orders Bathycoccus,Crustomastix,Dolichomastix,Mamiella,Mantoniella,Micromonas,Monomastix and Ostreococcus. Virtually all sorted cells were hybridized with the set of nested probes, while other probes such as CHRYSO1037 (Chrysophyceae, Fuller et al., 2006; Jardillier et al., 2010), CRYPT13 (Cryptophyceae, Lepere et al., 2008), PRYM02 and PELA01 (Prymnesiophyceae and Pelagophyceae respectively, Simon et al., 2000) did not hybridize with the sorted cells. Moreover, in the Plast-S fraction, only few cells hybridized with the probe PRAS04 were observed. Therefore, we concluded that the Plast-XS population in the SG and ST is composed only of cells affiliated with the Mamiellales clade II. Probes targeting genera within the clade (Table 1), however, did not confer positive signals with the sorted Plast-XS cells. Similarly to our results from the South Atlantic, the majority of Mamiellales inhabiting the northern North Pacific as well as areas influenced by Atlantic currents in the Norwegian Sea, could neither be assigned to Micromonas,Bathycoccus or Ostreococcus (Not et al., 2005; Balzano et al., 2012).

The Mamiellales clade II cells in the SG and in ST were small, spherical and lacked flagella, scales or any other surface features and hence resembled prokaryotic cells (Fig. 4d–f). However, we do not exclude a possibility that their flagella could be lost during sorting and routine SEM preparation. Their morphology, in agreement with CARD-FISH, supports the notion that they are not Micromonas, which have flagella, or Bathycoccus, which have scales. The uniform group of tiny eukaryotic sorted cells presented a very simple morphology and lacked extracellular structures similar to the apparent overall simplicity of Ostreococcus, which has been referred as the ‘bare limits’ of a photosynthetic organism (Derelle et al., 2006). We cannot rule out that the Plast-XS cells are affiliated with the genus Ostreococcus despite the fact that they did not hybridize with the Ostreococcus-specific probe. Negative FISH results with the probe OSTREO01 have been attributed to the small size of oceanic Ostreococcus cells (Demir-Hilton et al., 2011) or to poor accessibility of the probed site of 18S ribosomal RNA. The Plast-XS cells, however, hybridized with other probes (Figs 3 and 4) that suggest that they more likely belong to another subclade of Mamiellales than to Ostreococcus.

Being able to identify a flow cytometric population of Plast-XS as a single taxonomic clade allowed us to measure concentrations of Mamiellales clade II and to assess its vertical and spatial distribution to deduce its possible ecological niche. The abundance of Mamiellales clade II in the oligotrophic SG was lower (between 0.6 and 1.6 × 103 cells mL−1), accounting for 38 ± 8% of total picoeukaryotes than in the mesotrophic ST, where they peaked at 8.3 × 103 cells mL−1 (40 ± 16% of total picoeukaryotes). Mamiellales clade II were present in the photic layer down to 100 m (< 0.9 × 103 cells mL−1), suggesting tolerance to low light. In general, the latitudinal distribution of Mamiellales clade II resembled the distribution of Synechococcus (Fig. 5b), indicating a preference for mesotrophic environments by the former as it has been reported for the latter (i.e. Zubkov et al., 1998, 2000; Heywood et al., 2006).

Latitudinal depth distribution of (a) Mamiellales clade II and (b) Synechococcus. Dots represent the sampling depths.

Latitudinal depth distribution of (a) Mamiellales clade II and (b) Synechococcus. Dots represent the sampling depths.

Given the small size of the Plast-XS Mamiellales clade II cells (1.6 μm), they have a relatively large surface area to volume ratio (4 : 1), larger than most eukaryotes and comparable to prokaryotes. This could make them efficient osmotrophs, that is, able to acquire dissolved nutrients including organic molecules, similar to cyanobacteria. For example, cyanobacteria could effectively compete with other prokaryotes for organic molecules (Paerl, 1991; Zubkov et al., 2003) including in the mesotrophic ST region (Zubkov & Tarran, 2005). Besides, the genomic study of two Ostreococcus strains suggests that they have developed competitive strategies for nutrient acquisition uncommon among other eukaryotic phototrophic cells (Derelle et al., 2006). Mamiellales cells could be efficiently competing with cyanobacteria in the ST in terms of their biomass. The abundance of Mamiellales clade II was approximately one-tenth the abundance of Synechococcus cyanobacteria (Fig. 5). However, if an approximate tenfold difference in their cell size is taken into account, then their biomasses in the ST region become comparable. Consequently, miniaturization of Mamiellales cells could be sufficient for effective nutrient competition with cyanobacterial cells in temperate waters.

Concluding remarks

Flow sorting of a population of picoplanktonic chlorophyll-containing cells (tightly gated including only cells that belong to the targeted cluster, based on their red fluorescence, relative size and DNA content) allowed taxonomic affiliation of virtually all sorted cells with a single eukaryotic clade – the Mamiellales clade II. These cells have a simple morphology with apparently no scales, flagella or surface features. The successful taxonomic identification of a flow cytometric population permitted a comparison of the latitudinal distributions of Mamiellales and Synechococcus in the photic layer, which revealed a similar distribution pattern. The abundance of Mamiellales clade II increased in the ST Atlantic Ocean, indicating their preference for mesotrophic photic waters – a first insight into the ecological niche of these enigmatic, uncultured smallest eukaryotic cells.

Acknowledgements

We thank the captain, officers and crew aboard the RRS James Cook for their help during both cruises. We are grateful to D. Scanlan for helpful discussions, to J. Wulf for help with the flow cytometric sorting and to R. Holland for flow cytometry analysis. This study was supported by the UK Natural Environment Research Council through Research Grants NE/H010572/1, the Oceans 2025 Core Programme of the National Oceanography Centre and Plymouth Marine Laboratory. This is Atlantic Meridional Transect Publication no. 223. There is no conflict of interest among the authors.

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Author notes

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Editor: Julian Marchesi