Assessing how blooms of the Dinoflagellate Prorocentrum donghaiense affect the wider Dinoflagellate community in the East China Sea.

Harmful Algal Blooms have been increasing globally in extension and impacts on public health, aquaculture industry, fisheries, and ecosystems such as oxygen depletion, reduction in water quality. Among all Harmful Algal Bloom-causing species, Dinoflagellates are the most important contributors, as about 75% of reported Harmful Algal Blooms are caused by Dinoflagellates. Dinoflagellates have a number of characteristic features and are one of the most important primary producers and a vital component of Coral Reef symbiotic system. While Harmful Algal Bloom events may be caused by a variety of environmental and autecological factors such as illumination, water temperature, nutrients availability, growth rate, vertical migration, and special life history, as feedbacks, Harmful Algal Blooms may cause many negative effects on the ecosystems that can be viewed from different levels (from ecosystem, community to sub-cellular and molecular levels) and aspects (physical, chemical, biological, public health, and economic). In general, previous studies on the negative effects of Harmful Algal Blooms have been mainly focused on fisheries, aquaculture, and Human public health, relatively fewer studies, however, have investigated the effects of Harmful Algal Blooms on the level of community, and even fewer using high throughput metagenomic approach, such as phytoplankton community diversity, community structure, function and stability. This oddness was at least partly due to the limitations in how to obtain comprehensive lists of species and identify species of small sizes, simple or similar morphologies, low abundances, and to process numerous samples efficiently. Conventional methods for identifying and quantifying phytoplankton species from field samples generally involved in the use of light microscopy, and sometimes were aided with flow cytometry and alike, pigment analysis, however they all have limitations in identifying and quantifying those species of highly small sizes, simple or similar morphologies, low abundances, and novel taxa that have not been described. With the development of molecular approaches, high-throughput gene sequencing (e.g., 18S and 28S rRNA genes) have recently been increasingly applied to environmental samples to conquer these limitations. The well-developed, high-throughput sequencing allows us to deeply sequence environmental samples and to sensitively and accurately identify species, and thus detect slight changes at the community level.

In a paper published in the journal Acta Oceanologica Sinica in April 2020, Huan Wang of the Key Laboratory of Marine Ecology and Environmental Sciences at the Institute of Oceanology of the Chinese Academy of Sciences, and the University of Chinese Academy of Sciences, Zhangxi Hu, Zhaoyang Chai, and Yunyan Deng, of the Key Laboratory of Marine Ecology and Environmental Sciences at the Institute of Oceanology of the Chinese Academy of Sciences, the Laboratory for Marine Ecology and Environmental Science at the Pilot National Laboratory for Marine Science and Technology, and the Center for Ocean Mega-Science of the Chinese Academy of Sciences, Zifeng Zhan of the Department of Marine Organism Taxonomy and Phylogeny at the Institute of Oceanology of the Chinese Academy of Sciences, and Ying Zhong Tang, also of the Key Laboratory of Marine Ecology and Environmental Sciences at the Institute of Oceanology of the Chinese Academy of Sciences, the Laboratory for Marine Ecology and Environmental Science at the Pilot National Laboratory for Marine Science and Technology, and the Center for Ocean Mega-Science of the Chinese Academy of Sciences, present the results of a study which investigated the effects of blooms of a common Harmful Algal Bloom-causing Dinoflagellate in China, Prorocentrum donghaiense, on the Dinoflagellate sub-community level, which the bloom species belongs to, in terms of species richness and other biodiversity indices by applying a high-throughput amplicon sequencing approach.

Wang et al. applied a pair of particularly designed primers targeting the large subunit rRNA gene to sequencing the samples taken before, during, and after Prorocentrum donghaiense blooming from the Sansha Bay in Fujian Province, China. They also measured other variables including cell density of Prorocentrum donghaiense, chlorophyll content, nutrients (total nitrogen, nitrate, nitrite, ammonium , total phosphorus, phosphate, and silicate), salinity, and temperature to examine the interactions among these variables,Prorocentrum donghaiense blooms, and the Dinoflagellate community succession.

The study area, Sansha Bay, is located to the northeast of the city of Ningde, one of Fujian Province’s major aquaculture centres on the East China Sea, where there have been observed highly frequent Harmful Algal Blooms caused by Prorocentrum donghaiense, Karenia mikimotoi, and, occasionally, other species. There were about 161 Harmful Algal Bloom incidents during 2001–2010 and 65 events between 2011 and 2015, with Prorocentrum donghaiense being the main causative species.

Locations of sampling sites in the Sansha Bay, Ningde, China. Wang et al. (2020).

From March to July, 2016, Wang et al. conducted six cruises and collected a total of 50 samples, which covered pre-, during, and post-bloom periods. Four or five sampling sites were selected in the study area. 31 March was prior to the bloom, 22 April, 3 May, and 13 May were during the bloom period (based on cell counts of Prorocentrum donghaiense), with 3 May coinciding the peak of a bloom, and 31 May 31 and 19 July were being post-bloom. 

 

Wang et al define a bloom as to chlorophyll a concentration higher than 5 μg per litre of water  and a dominant specie concentration of over 20 000 cells per millilitre; this being an arbitrarily chosen measure in the absence of any commonly accepted standard of cell density to define a bloom. 

Water temperature and salinity were measured on site using a hand-held thermometer and a refractometer. Water samples were taken from 0.5 m below the surface and transferred into 5 L polyethylene bucket. Plankton samples for DNA extraction were collected by filtering 1.5 L water through a hydrophilic polycarbonate membrane (47 mm diameter, 0.4 μm pore size) with duplicates, put into an icebox and then cooled to –20°C immediately after arriving the laboratory and then stored at –80°C until DNA extraction. Water samples were also fixed with Lugol’s iodine solution (final concentration, 2%) for counting cells of Prorocentrum donghaiense using plankton counting chamber under an inverted light microscope. 

The Dinoflagellate Prorocentrum donghaiense, was the dominant species in Sansha Bay during the 2016 bloom season. Lu et al. (2005).

During the six cruises from March to July of 2016, Prorocentrum donghaiense reached the maximum cell density of 430 000 cells per millilitre on 3 May. The cell density of Prorocentrum donghaiense was 270 cells per millilitre on 31 March (pre-blooms) and the lowest cell density of 83 cells per millilitre was on 19 July (after the blooms). During the bloom period of late April to early May, Prorocentrum donghaiense abundance ranged from 300 to 430 000 cells per millilitre. However, among the sampling sites, Prorocentrum donghaiense cell density varied significantly, with Site B or Site C having significantly higher abundance than that of Site A. 

The chlorophyll a level ranged from 0.3 to 26.8 μgper litre, with the highest observed at Site D on 3 May, coninciding with an observed bloom of Prorocentrum donghaiense was (with a cell density of 50 000 cells per millilitre). There existed a significant positive correlation between Prorocentrum donghaiense cell density and chlorophyll a concentration, indicating chlorophyll was one of, but not the only, major contributors of phytoplankton biomass. Strikingly, it is noteworthy that for the sample taken from site B on 3 May, there was a discrepancy between chlorophyll a and the cell abundance of Prorocentrum donghaiense, which Wang et al. think was possibly due to a lower chlorophyll a content per cell for Prorocentrum donghaiense relative to that of other phytoplankton species such as Diatoms and Green Microalgae because of the very small-sized cells and pigment composition of Prorocentrum donghaiense. In addition, the extremely high abundance of Prorocentrum donghaiense during the blooming period (e.g., early May) also led to a decrease in the abundance of other phytoplankton with higher chlorophyll  a contents per cell.

Water temperature ranged from 13.9°C to 29.5°C during the sampling period. No significant correlation was observed between water temperature and chlorophyll a concentration, nor between temperature and Prorocentrum donghaiense cell density. Wang et al. observed no correlation between Prorocentrum donghaiense cell density and ammonium or phosphate concentrations, but Prorocentrum donghaiense cell density correnlated negatively with nitrate and nitrite (i.e. the concentrations of these nutrients dropped during the bloom peaks), and positively with total nitrogen, total phosphorus and silicate (the concentrations of which went up during peak blooms), suggesting that nitrogen and phosphorus are driving factors for Prorocentrum donghaiense blooms. The ratios of dissolved inorganic nitrogen (nitrate, nitrite, and ammonia) to dissolved inorganic phosphorus (phosphate) in the surface water tended to decrease along with the development and maintenance of bloom. On 13 May this ratio reached the minimum observed. 

A total of 800 185 valid sequence reads of Dinoflagellates with an average length of about 400 bp were generated from the 50 samples collected. By clustering the unique sequences at 97% similarity level, these dinoflagellate sequences were grouped into 560 Operational Taxonomic Units (clusters of organisms, grouped by DNA sequence similarity of a specific taxonomic marker gene; a pragmatic proxy for species in the absence of a full taxonomic study), with the number of Operational Taxonomic Units ranging from 39 to 304 per sample. The highest richness was observed in a sample taken at site C on 19 July 2016 (after bloom) and the lowest richness was observed at site A on 3 May 2016 (during bloom). Operational Taxonomic Unit richness decreased during the blooming period from 22 April to 13 May, and then increased with the disappearance of bloom from 31 May to 17 July. 

Metagenomic analysis revealed changes in the abundance of Operational Taxonomic Units classifiable to various taxonomic levels, including shifts in dominant genera and species on date basis. The top 20 most abundant genera and species of each sample showed that 26 of the 50 samples were dominated by Prorocentrum (76.6%–99.6%), while during the before-blooming period, the Dinoflagellate community was dominated by an Operational Taxonomic Unit that could not be well identified to any currently accepted genus of Dinoflagellates (4.9%–79.6% dominance). All samples taken on 22 April, 3 May, and 13 May except for two taken on 13 May at site A were from the blooming area and dominated by Prorocentrum donghaiense. The samples from 13 May at site A were froma  non-blooming area and dominated by Levanderina fissa. After the blooming period, most samples taken on 19 July were dominated by Levanderina fissa (25.0%–69.3% dominance).

The Dinoflagellate Levanderina fissa was the dominant species in Sansha Bay in 2016 after the end of the bloom season. Moestrup et al. (2014).

Wang et al.'s study demonstrated that the bloom of Prorocentrum donghaiense affected the structure of Dinoflagellate sub-community of the total phytoplankton by reducing the species richness and diversity. The Dinoflagellate community during the blooming period differed significantly from those before and after blooming periods. The species composition of Dinoflagellate community changed with transition stages of the Prorocentrum donghaiense bloom. For instance, the Dinoflagellate community was dominated by a species that has not been well described, Prorocentrum donghaiense, and Levanderina fissa for the periods of before, during, and after blooming, respectively. These results supported Wang et al.'s hypothesis that Prorocentrum donghaiense blooms would reduce the diversity of the Dinoflagellate community and alter the community structure.

Investigations on the effect of Harmful Algal Blooms on species diversity and community succession have been comparatively rare, particularly so for that using high throughput metagenomic approach. An earlier study investigated abundance and composition of phytoplankton populations during different bloom stages of Gymnodinium breve off the North Carolina coast, and found that total phytoplankton abundance increased regardless of Gymnodinium breve abundance. Further, that study discovered that the cell densities of some groups increased but others decreased, which is in contrast to Wang et al.'s results, possibly because the Gymnodinium breve bloom was not monospecific. About 127 phytoplankton species were identified microscopically from all water samples from North Carolina, which was a relatively low number in comparison to Wang et al.'s work targeting on Dinoflagellates only. 

 

However, a more recent study, using high-throughput pyrosequencing approach also but targeting on a broader spectrum of microorganisms, demonstrated that microbial community structure is strongly linked to the bloom progression of Alexandrium catenella in the Nauset Marsh System on Cape Cod, Massachusetts. Multiple aspects of that study are consistent with Wang et al.'s results, such as that a decrease in diversity of the entire community of plankton during the bloom of Alexandrium catenella and reflects complex interactions among taxa comprising the phycosphere environment. 

 

An earlier study on freshwater and brackish water ecosystems in in Fenno–Scandia demonstrated that the diversity of phytoplankton communities is the best predictor for resource use efficiency (e.g., nutrients) of phytoplankton and factors reducing phytoplankton diversity may have direct detrimental effects on the amount and predictability of aquatic primary production. 

 

While environmental variables such as temperature, turbulence, and nutrient levels are generally the primary forces shaping the community structure and driving Harmful Algal Blooms (see the discussion below), a bloom can be a vital driving force by its own for the transition of phytoplankton community structure due to the biological features of the blooming species. For example, most Harmful Algal Bloom-causing species have been demonstrated to be allelopathic (harmfull) to other co-occurring phytoplankton species via releasing allelochemicals which surpress their growth. A blooming species generally can squeeze the living space of other species via fast growth, which will consequently reduce the nutrient and space availability to competitors.

Wang et al.'s results showed that Prorocentrum donghaiense abundance, nitrate and silicate concentrations were the three most important environmental factors affecting the Dinoflagellate community. Prorocentrum donghaiense abundance was correlated negatively to nitrate, nitrite, phosphate, ammonium, temperature and salinity, but positively to total nitrogen, total phosphorus, chlorophyl a and silicate. Although Dinoflagellates do not need silicate for growth, Wang et al.'s results showed that it appeared to be one of those important factors in shaping the Dinoflagellate community, which might be indirectly caused via the effects of on the transition of Diatom community during the sampling period. The ratio of dissolved inorganic nitrogen to dissolved inorganic phosphorus tended to decrease along with the development and maintenance of blooms, and increase along with disappearance of blooms. At the beginning of the survey (31 March), the cell density of Prorocentrum donghaiense was comparatively low (270 cells per millilitre), and the dissolved inorganic nitrogen to dissolved inorganic phosphorus ratio was 18–22, which was more suitable for the growth of Prorocentrum donghaiense, while, during the blooming period, the ratio showed a downward trend in general, possibly due to the different absorption rates for different nutrients by the bloom-forming organism. This trend indicates a faster absorption rate of dissolved inorganic nitrogen by Prorocentrum donghaiense and consequently a larger effect of dissolved inorganic nitrogen on the growth of Prorocentrum donghaiense, compared to phosphate. On 13 May, the ratio reached the minimum, indicating a limiting level of dissolved inorganic nitrogen to Prorocentrum donghaiense growth. Furthermore, it was observed there were significant negative correlations between total phosphorus and diversity, indicating that total phosphorus also stimulated the growth or bloom of Prorocentrum donghaiense. However, phosphate did not exhibit a significant correlation with diversity, indicating the utilisation or uptake of phosphorus by Prorocentrum donghaiense was not linearly correlated with the ambient concentration of phosphate 

 

Wang et al.'s analysis revealed that, in addition to nutrients, temperature and salinity also made contributions to the transition of the Dinoflagellate community, which is in contrast to earlier studies where temperature and salinity were two key environmental factors associated with changes in Bacterial and Archaeal community structure but not with variations in Eukaryotic community. While it is well understandable that temperature acted as an important factor, the apparent correlation between salinity and Prorocentrum donghaiense and the Dinoflagellate community might be a good indication of nutrient input from freshwater runoff.

In summary, Wang et al.'s investigation observed that blooms of Prorocentrum donghaiense negatively affected biodiversity in the Dinoflagellate sub-community. The results showed that the Dinoflagellate community during the blooming period of Prorocentrum donghaiense differed significantly from the community before and after the blooming period. Wang et al.'s analyses indicated that Prorocentrum donghaiense abundance was the most important factor affecting the Dinoflagellate community, which strongly indicates that the bloom of Prorocentrum donghaiense played a vital role in shaping the Dinoflagellate community structure, possibly via processes such as allelopathy, nutrient and space competition, and fast growth itself. Although these results were not beyond their anticipation, Wang et al. believe the work provides meaningful and solid evidence for the negative effects of Harmful Algal Blooms on the plankton community and coastal ecosystem based on a comprehensive series of field sampling and high throughput pyrosequencing.

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Emiliania huxleyi: Modelling how an Atlantic Coccolithophore is invading the Arctic Ocean.

The European Arctic Corridor is the main gateway to the Arctic Ocean, where 80% of both in- and outflow takes place. One of the most prominent feature of the European Arctic Corridor is the northward flowing North Atlantic Waters entering the Arctic Ocean through two main branches, which separate around 70°N. One branch keeps flowing northward toward Fram Strait while the second one turns eastward into the Barents Sea. Over the last three decades, in a context of amplified warming and sea-ice loss, substantial changes have been documented in the North Atlantic Water inflow with a twofold increase of its volume occupation and a northward shift of the polar front structure in the Barents Sea. The North Atlantic Water inflow largely controls the physical and sea-ice conditions of the region In addition, almost 50% of the total Arctic primary production takes place in the European Arctic Corridor, which is of major importance for fisheries. Recent warming of the European Arctic Corridor related to the North Atlantic Water inflow has been suspected to trigger poleward intrusions of temperate phytoplankton and species from higher trophic levels. By carrying biomass and nutrients produced elsewhere, bioadvection has recently been proposed as an 'essential mechanism' for ecosystem dynamics in the Arctic Ocean. Although the actual role of advection has already been identified as a potential driver altering zooplankton dynamics, it has never been assessed quantitatively from observations for phytoplankton in the European Arctic Corridor, or more generally in the Arctic Ocean.

In a paper published in the journal Nature Communications on 10 April 2020, Laurent Oziel of the Ocean and Ecosystem Sciences Division at Fisheries and Oceans Canada, the joint Takuvik International Research Laboratory of Université Laval and the Centre National de la Recherche Scientifique, and the Laboratoire d’Océanographie de Villefranche-sur-Mer of Sorbonne Université, Alberto Baudena, also of the Laboratoire d’Océanographie de Villefranche-sur-Mer, Mathieu Ardyna also of the Laboratoire d’Océanographie de Villefranche-sur-Mer, and of the Department of Earth System Science at Stanford University, Philippe Massicotte and Achim Randelhoff, also of the Takuvik International Research Laboratory, Jean-Baptiste Sallée, again of Sorbonne Université, Randi Ingvaldsen of the Institute of Marine Research, Emmanuel Devred, also of the Ocean and Ecosystem Sciences Division at Fisheries and Oceans Canada, and Marcel Babin, again of the Takuvik International Research Laboratory, present the results of an investigation into how ocean currents control the spatial dynamics of a specific Coccolithophore bloom-forming species, Emiliania huxleyi.  

The European Arctic Corridor. Bathymetry and surface circulation. The Atlantic currents are in red, the Arctic or Polar Waters are in blue and the Coastal Waters are in green. The southern Barents Sea Polar Front is illustrated in black dashed line and separates the Atlantic Waters from the colder and fresher waters from the North. Oziel et al. (2020).

Emiliania huxleyi is usually associated with the surface layer of the temperate North Atlantic Waters in summer, typically in a post-spring bloom context characterized by low nutrients, low light and strong stratification. Since Emiliania huxleyi does not form winter resting spores, this tracer of Atlantic ecosystem is generally considered to be a summer visitor in the Barents Sea, unlike neritic Diatoms species. A combination of bottom-up (i.e. winter darkness, cold temperatures and intense vertical mixing) and top-down controls (i.e. zooplankton grazing, viral lysis) prevent Emiliania huxleyi from year-to-year survival in the north-easternmost parts of the Barents Sea. Because of their high abundance in Emiliania huxleyi (115 000 000 cells per litre), coastal regions and fjords of the Norwegian Sea have been suspected to be the source of Emiliania huxleyi for the whole European Arctic Corridor.

Using a Lagrangian tracking approach based on satellite derived current fields, Oziel et al. explored how the advection of Emiliania huxleyi cells from these upstream coastal temperate regions shapes the distribution of the massive Emiliania huxleyi blooms in the Barents Sea (via the 'seeding effect'). By combining satellite-derived altimetry with ocean-color Particulate Inorganic Carbon (a Coccolithophore biomass proxy) estimates, Oziel et al. demonstrate a major role of bio-advection in phytoplankton transport along the European Arctic Corridor.

An individual Emiliania huxleyi cell. Stig Haugen/Institute of Marine Research.

To document the interannual and decadal variability of surface North Atlantic Water currents, Oziel et al. first performed an statistical analysis of Sea Level Anomalies from 1993 to 2016, on which the seasonal signal was removed. The analysis showed that the two variables accounted for more than 80% of the non-seasonal variability. A dipole structure emerged, centered on the North Atlantic Waters path between the Barents and Norwegian Sea shelves (east of 5°E) and the center of the Norwegian Sea (west of 5°E). The associated time-series showed a linear positive trend over the last 24 years with high energy at the interannual and decadal time scale. This trend toward a more positive phase corresponds to an increase of the sea level gradient across the North Atlantic Waters path, hence intensifying North Atlantic Waters currents, while negative phases are associated with weaker North Atlantic Waters currents.

Statistical analysis of Monthly averaged Sea Level Anomalies in the European Arctic Corridor. The sum of the first two modes account for more than 80% of the total variability. Oziel et al. (2020).

To reveal the impact of changing sea level on surface velocity fields, absolute surface geostrophic velocities, derived from Absolute Dynamic Topography fields were mapped during the extreme negative (1993) and positive (2015) phases of the analysis. These two contrasting years illustrate the ongoing strengthening of the ocean surface circulation in the European Arctic Corridor associated with the Atlantic inflow. Surface geostrophic current linear trends showed that surface currents changed in most of the Atlantic pathway In the Norwegian Sea, the increase in the Atlantic surface current speed reached 2 mm per second per year, corresponding to a relative increase ranging from 30% on the shelf to 100% in the basin. The surface currents in the eastern Barents Sea-Atlantic corridor also significantly increased by about 6 mm per second per year, an increase in flow rate of about 14%. The positive phase associated with stronger north-eastward North Atlantic Waters surface currents since year 2000 reflects an increase in advection at or near the surface. This result is consistent with the recent trends in North Atlantic Waters current velocities observed upstream in the Nordic Seas or modelled in the Barents Sea.

Surface absolute geostrophic velocities during the extremums of the timeseries which are, respectively, reached in (a) December 1993 (minimum) and (b) 2015 (maximum) with (c|) the corresponding absolute linear trend of the entire time-series (all months) over the 1993–2016 period. Areas covered by sea ice (sea-ice concentration over 15%) or with insufficient data coverage for the trend (less than 50%) are in dark gray. Oziel et al. (2020).

The interannual fluctuations as well as the long-term trend observed in the time-series of surface geostrophic velocities are mainly attributed to the dynamics of the North Atlantic Subpolar Gyre, and to the atmospheric forcing of the North Atlantic Oscillation, which are both tightly coupled. This suggests that the increase in surface advection in the European Arctic Corridor is likely due to a natural multidecadal oscillation related to the upstream synoptic oceanic circulation and atmospheric forcing, which could, in turn, drive long-term climatic change in the Barents Sea.

The first hypothesis that ocean currents control the summer spatial distribution of Emiliania huxleyi in the Barents Sea was tested using a Lagrangian model. Oziel et al. advected virtual particles (considered as the inoculum of Emiliania huxleyi cells), using observations of surface geostrophic velocities, from their expected overwintering location in March (defined by sea surface temperatures greater than 4°C and distance from coast no more than 180 km). This model revealed that the spatial distribution of the virtual particles at the end of the advection period matched the extent of Emiliania huxleyi blooms (evidenced by satellite-derived Particulate Inorganic Concentration), with 80% of tracked particles ending up within 50 km of an Emiliania huxleyi blooming location. The year-to-year robustness of the matchup between virtual particles and Particulate Inorganic Concentration clearly supports the fact that the North Atlantic Waters surface currents shape the location and extent of Emiliania huxleyi blooms in the European Arctic Corridor.

Poleward expansion of Emiliania huxleyi (EHux) in the European Arctic Corridor. Comparison between 1998 (a) and 2015 (b). The initialisation (inoculum) of virtual particles in March are illustrated by brown dots. During 6 months, particles drift with the Norwegian Atlantic Current (red arrows) as the ocean seasonally warms as illustrated by the northward expansion of the 4 °C isotherm. In August, the particles end up in positions indicated by the red dots. In the background, remotely sensed Particulate Inorganic Concentration indicating Coccolithophore biomass in summer (July–August–September) is shown in blue colors. Areas with no data are in dark gray. Oziel et al. (2020).

It is noteworthy that the particles reached further north and east in the European Arctic Corridor in 2015 than in 1998, in agreement with Emiliania huxleyi blooms. The north-eastward expansion of the Emiliania huxleyi bloom, expressed here as the distance reached by the leading-edge increased on average by 325 km during the last 19 years as indicated by ocean-color observations, in close agreement with previous estimations (324 km, 40–50°E, 1989–2016) and with the estimates from Oziel et al.'s model.

The 4 °C surface isotherm is considered for Emiliania huxleyi as the lowest temperature required for sufficient growth to allow bloom formation. However, the Emiliania huxleyi blooms do not seem to follow this isotherm in summer and appear to be constrained by another factor. For example, the north-eastward expansion of the winter 4°C isotherm, which delimits the areal extent of the inoculum in Oziel et al.'s model, could contribute to reduce the distance between the Emiliania huxleyi winter location and the Arctic domain. To test this hypothesis, Oziel et al created two additional models to examine both the role of currents and winter temperature on the poleward expansion of Emiliania huxleyi. In the second model Oziel et al. constantly initialised the virtual particles at the same inoculation region for all years, using a climatological mean temperature field to determine the inoculation region. Hence, the second model exclusively reflected the role of currents on the interannual variability of the advected particles. In contrast, in the third model, the constant inoculation region varied from one year to another, but Oziel et al used a climatological mean current to advect the particles. In this way, the interannual variability of the advected particles due to interannual variability of the inoculation region was quantified. The interannual Particulate Inorganic Concentration leading-edge location was highly correlated with the leading-edge from the second model, suggesting a stronger control of the bloom expansion by currents than by winter temperature. On the decadal scale, currents were found responsible for the 56% (240 km) increase in the long-term leading-edge expansion against 44% (186 km) for winter temperature, when compared to the first model. This significant increasing trend in current velocities was also revealed by the greater distance covered by the virtual particles reaching the Barents Sea, which increased by 110 km on average since 1993.

Shifting position of the leading-edge Emiliania huxleyi bloom distribution. Shifting position from ocean-colour Particulate Inorganic Concentration (a), and the three models (b)–(d) for the last 19-years (1998–2016). The comparison between the first model (EXP1), (b) with the second (EXP2), (c) and the third (EXP3), (d) aims at estimating the relative contribution of currents (EXP2, constant temperature) vs. temperature (EXP3, constant currents) on the total Emiliania huxleyi poleward expansion (EXP1, varying temperature and currents). The right panel is a schematic illustration of the poleward expansion of the Emiliania huxleyi with the winter 4°C isotherm (lowest temperature for a ‘regular’ Emiliania huxleyi growth) in blue and the summer bloom position (northern boundary) in red. The two extreme years 1998 (dashed) and 2015 (solid) are represented. Arrows indicate the contribution from temperature and/or currents keeping the same color code. Oziel et al. (2020).

Increasing water temperature has previously been assumed to be the main driver of the spatial distribution of Emiliania huxleyi blooms in the Barents Sea. Oziel et al.'s results demonstrate that the primary driver of the Emiliania huxleyi dynamics (i.e., spatial distribution and timing) could be, in fact, stronger surface currents, which in turn intrinsically shape the temperature field and frontal structures. Oziel et al. show that oceanic currents (i.e. their intensity and fluctuations) drive the spatial distribution of the bloom, its interannual variability and more than 50% of the long-term poleward expansion of Emiliania huxleyi bloom in the Barents Sea. More importantly, from 2006 and onward, the contribution of water temperature to the expansion of Emiliania huxleyi blooms becomes negligible, and its poleward expansion is entirely due to the accelerating currents.

Emiliania huxleyi is largely studied for its significant role in marine geochemical cycles, as illustrated by its sensitivity to ocean acidification, its effect on carbon dioxide pressure and carbon dioxide uptake, and its role on carbon export by providing calcite ballast effect. In Oziel et al.'s study Emiliania huxleyi was used as an indicator of Atlantic ecosystems. By expanding poleward and doubling its areal extent in the Barents Sea, Emiliania huxleyi attests to the ongoing 'Atlantification' of the Arctic Ocean. Both arctic and Atlantic domains have distinct ecological signatures, and the latter is undeniably 'invading' the former. Advected with the surface currents, Emiliania huxleyi will have to survive during the travel (for example avoiding grazing by zooplankton, and subduction under the polar mixed layer in the Fram Strait) until finding more favourable blooming conditions in the Barents Sea in summer. The fate of Emiliania huxleyi in the Barents Sea is therefore of major importance as it determines the potential 'seeding effect' of Emiliania huxleyi in the Arctic regions. Emiliania huxleyi seems to be adapted to the low light, low nutrient, oligotrophic and highly stratified conditions of the North Atlantic Waters in summer such that its expansion, growth and blooming in the Arctic Ocean will be limited at some point by those constraints (bottom-up) but also by the grazing pressure (top-down).

Despite its adaptation to low light conditions, Emiliania huxleyi still requires sufficient light levels to sustain the energy-demanding calcification of its coccoliths. Such conditions are met in highly stratified oceans where Emiliania huxleyi can accumulate in the surface layer. At high latitudes (more than 81°N), even with sea surface temperature above 3 or 4 °C, the survival of Emiliania huxleyi would require adaptation to rapidly decreasing solar radiations in late summer. Emiliania huxleyi's fate thus mainly relies on its ability to drift, with the appropriate timing, to highly stratified and temperate areas that allow it to stay in the surface euphotic layer. These conditions would likely be met in the Eurasian interior shelves of the Arctic Ocean (i.e. the Kara, Laptev, and Siberian seas) where surface waters are warming and freshening. If the increase in advection along the shelf slope continues in the future, Oziel et al. expect Emiliania huxleyi to become a summer resident of the newly 'Atlantified; Eurasian interior shelves, as previously revealed during the last interglacial.

By driving such a poleward expansion, advective processes could affect the entire marine ecosystems by shifting species distribution and modifying interactions at higher trophic levels. The concomitant decline of silicate concentrations in North Atlantic Waters may also contribute to the success of non-silicifying and small phytoplankton such as Emiliania huxleyi. n addition, a change toward temperate pelagic species could have an impact on energy transfer to higher trophic levels, including Marine Mammals and commercial Fish stocks. Considering the role of 'bio-advection' in ecological models (i.e., trait-based and niche-based approaches) must improve predictions of community shifts. The comparable increase in poleward advection of Pacific waters occurring in the Bering Strait suggests that the shrinking polar domain of the Arctic Ocean may be prone to intrusions of temperate species at a pan-Arctic scale.

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Large numbers of Cannonball Jellyfish stranding on Hilton Head Island, South Carolina.

Residents of Hilton Head Island in South Carolina have reported seeing large numbers of Cannonball Jellyfish, Stomolophus meleagris, stranding on beaches on the northern part of the island this week. The Jellyfish started to appear on Friday 10 April 2020, with thousands being seen in some areas. Whilst such large-scale strandings are potentially alarming, they are quite common in the area at this time of year, as packages of warm, nutrient-poor, water from the Gulf Stream begin to come closer to the shore, encountering the cooler, nutrient-rich water of the inshore environment, causing the Jellyfish to undergo population explosions called blooms. Large numbers of Jellyfish in the water can very quickly turn into large numbers of Jellyfish on a beach, as these animals are poor swimmers, and tend to drift where currents and sea breezes take them, with the effect that if conditions change and drive them onshore, they have no way to escape.

Cannonball Jellyfish, Stomolophus meleagris, stranded on a beach on Hilton Head Island, South Carolina, on Friday 10 April 2020. Mandy Matney/FitsNews.

Cannonball Jellyfish are common in the Gulf of Mexico and western Atlantic from the southeaster coast of the US south to Brazil, as well as on both the east and west coasts of the Pacific, from California south to Ecuador, and in the South China Sea and Sea of Japan. The Atlantic population is generally brown in colour, while the Pacific population is bluish, but they are considered to be the same species. These Jellyfish reach about 25 cm in diameter, with a distinctive 'cannonball' shaped bell, and a cluster of short oral arms around the mouth. They excrete toxin from the nematocysts (stinging cells) into the water, stunning small prey items and discouraging predators, but are not harmful to Humans, and are widely eaten in parts of Asia, with Jellyfish being exported from the US to Japan, China, and Thailand for consumption.

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Using High-Throughput Sequencing to understand the composition of marine unicellular Eukaryote assemblages in Thessaloniki Bay.

During the past decade, the advancement of High-Throughput Sequencing technologies have provided unprecedented insights into the diversity, spatial community composition, temporal patterns, and functional and ecological processes affected by marine unicellular Eukaryotes. A recent study attempted to link the taxonomic diversity of Protistan taxa detected with High-Throughput Sequencing with 30 different traits in samples from ecosystems across the coasts of North Atlantic Ocean, suggesting that small taxa are characterized by broader taxonomic and functional diversity. Furthermore, previously underestimated trophic strategies, such as parasitism and decomposition, have been recognized as central to marine planktonic assemblage structure and ecosystem function. In addition, the environmental effects and biotic relationships in unicellular Eukaryotic communities have been explored, using classical and modern ecological tools. It is evident that the plethora of the genetic and ecological information produced provide a better understanding of marine global ecosystem processes. In particular, coastal areas are systems with high risk of pronounced modification in view of climate change and coastal urbanization leading to increased eutrophication. Thus, there is an increase in the attention of marine microbiologists thanks to the development of High-Throughput Sequencing tools. Either by focusing on specific taxonomic groups, such as the parasitoids Perkinsozoa and Labyrinthulomycetes, the cosmopolitan and conspicuous Tintinnid Ciliates, the newly described Vampyrellida (Rhizaria), or by investigating the overall marine protistan diversity, temporal dynamics, function, biotic interactions, relationships with abiotic variables, effects of climate change, and potential activity, marine microbiologists have attempted to answer the classical questions of who, when, why, and where in coastal systems. However, few High-Throughput Sequencing-based studies have investigated marine unicellular Eukaryotic assemblages in degraded eutrophic coastal areas, with high anthropogenic impacts. These studies, albeit scarce, have revealed high and novel diversity, and have attempted to provide insights of the trophic relationships between known groups and previously overlooked links.

Aplanochytrium sp., a parasitic marine Protist in the Class Labyrinthulomycetes, noted for  the production of an extension of the plasma membrane and the ectoplasm called the ectoplasmic net.  Aplanochytrium cells are not embedded in the ectoplasmic net but can move by gliding on ectoplasmic threads. Celeste Leander/Tree of Life Project/Wikimedia Commons.

In a paper published in the journal Diversity on 21 March 2020, Savvas Genitsaris of the School of Science and Technology at the International Hellenic University, and the Department of Botany at the Aristotle University of Thessaloniki, Natassa Stefanidou and Maria Moustaka-Gouni, also of the Department of Botany at the Aristotle University of Thessaloniki, Ulrich Sommer of the Geomar Helmholtz Centre for Ocean Research Kiel, and George Tsipas, also of the School of Science and Technology at the International Hellenic University, present the results of a study which used High-Throughput Sequencing to investigate the marine unicellular eukaryotic diversity and spatial distribution throughout a year of frequent samplings in Thessaloniki Bay, a coastal eutrophic Mediterranean urban area.

The High-Throughput Sequencing study was used as a follow-up to a previous project examining via classical microscopy tools the microbial plankton communities in the same area and the same sampling period, which was characterized by marked harmful Algal blooms, red tides, and mucilage aggregate incidents. The Thessaloniki Bay is the inner part of Thermaikos Gulf, located in northern Greece, and has been accepting for decades a large volume of domestic and industrial wastes from the one million residents of the city of Thessaloniki. Although wastewater treatment has been implemented, decreasing the effects of anthropogenic eutrophication, the restricted water circulation and shallowness of the Bay in combination with high nutrient inputs, lead to harmful Algal blooms, apparent red tides, and mucilage aggregates, leading to growing concerns of the citizens and authorities on the water quality of the Bay and particularly of the urban front. Data on the abundance and dynamics of plankton community (both Phyto- and Protozooplankton) in the urban part of the Gulf obtained by classical microscopy, have attempted to identify and characterize the marine Protist abettors and perpetrators responsible for these phenomena. These studies have shown the contribution of taxa known to thrive due to eutrophication and to cause harmful events, such as the Diatoms Cylindrotheca closterium, Skeletonema costatum, Chaetoceros spp., and the Dinoflagellates Noctiluca scintillans and Dinophysis acuminata. Taking into consideration the continuous increase of eutrophication in coastal areas worldwide, there is a need to identify the causative organisms of these events and implement strategies to mitigate their severe impacts to ecosystem and public health. High-Throughput Sequencing-based studies investigating the unicellular Eukaryotic diversity, dynamics, spatial distributions, and environmental effects in coastal eutrophic areas could contribute towards the early and accurate characterization of the microbes responsible for harmful bloom events and red tides.

The Diatom Cylindrotheca closterium, a solitary, motile species which forms mucilaginous aggregates under high salinity conditions and produces  allelopathic chemicals that suppress growth of other species. Gert Hansen/Scandinavian Culture Collection of Algae and Protozoa/University of Copenhagen/Nordic Microalgae and Aquatic Protozoa.

The aim of Genitsaris et al.'s study was to investigate and describe the overall marine unicellular Eukaryotic assemblages of different sites of the urban front of Thessaloniki throughout a year in short sampling intervals via High-Throughput Sequencing tools, following a previous study concerning the same area and sampling period, where different periods of plankton blooms were identified and analysis of the environmental variables was presented. The sites are located in close proximity, thus allowing us to examine the variability of unicellular Eukaryotic diversity and dynamics in small temporal and spatial scales. The main questions were: (i) Do the assemblages include previously undetected taxa in the area? (ii) How do these taxa contribute to the phenomena of blooms, red tides, and mucilage aggregates? (iii) What are the temporal and spatial distributions of the unicellular eukaryotic assemblages? (iv) Are they affected by nutrients and how?

A 'red tide' of toxic unicellular Algae covering the entire waterfront of Thessaloniki in June 2017. Greek Reporter.

Samples were collected weekly from March 2017 to February 2018 at an urban site in Thessaloniki Bay (Thermaikos Gulf), namely the White Tower station. Simultaneously, samples were taken from White Tower and three other adjacent sites (Aretsou Beach, Music Hall Coast, and Harbour) every four weeks. The stations were located along the coastline with a distance of about 3 Km between the closest consecutive ones and 10 Km between the most distant ones, which were located at the edges of the sampling area. All sampling sites had a maximum depth of 4 m. In total, 83 water samples were collected.

For all samples, in situ measurements of water temperature and salinity were made Water samples of 2 L were collected from the surface layer of 1 m, and subsamples of 100–250 mL (depending on plankton and particulate matter density) were immediately filtered onto 0.7 μm prewashed filters, and the filters were stored in −20 °C for particulate organic nutrient and Chlorophyll a measurements. Moreover, subsamples of 50 mL were filtered through 0.2 μm cellulose acetate filters and were kept in −20 °C for dissolved inorganic nutrient measurements. Finally, the rest of the water volume was prefiltered through a 200 μm sterile mesh, and subsamples of 100–200 mL from each water sample were subsequently filtered through 0.2 μm nucleopore filters and similarly were kept in −20 °C for molecular analysis.

Study area in Thermaikos Bay, indicating the location of the four sampling sites (*). WT: White Tower; AR: Aretsou; MH: Music Hall; HB: Harbour. Genitsaris et al. (2020).

The seawater temperature during the year of the study ranged from 9.6 °C on 24 January 2018 at the White Tower site, to 29.7 °C on 09 August 2017 at the same site. Salinity varied and was higher during the cold months with highest value recorded on 15 March 2017 at White Tower (38.8 g/L), and the lowest on 19 July 2017, also at White Tower (35.3 g/L). Nutrients, i.e., silicate, phosphate, nitrate, nitrite, ammonia, and particulate organic phosphorus, fluctuated considerably throughout the sampling period, and few extreme values were measured (e.g., on 22 March 2017, at White Tower). High values were also recorded for all nutrients on 28 July 2017 at the White Tower and Music Hall sites, 20 September 2017 in all sites, and 10 January 2018 in White Tower. Chlorophyll a, also showed marked variability, ranging from 0.27 μg L−1 on 27 December 2017 at White Tower, to 17.28 μg L−1 on 13 December 2017 also at White Tower.

Several conspicuous plankton blooms were recoreded in the study area during the period of the study. A bloom from March to June 2017 was found to be comprised largely of the Cocolithaphores Leptocylindrus danicus, and Leptocylindrus minimus. A red tide from  22 March to 29 March 2017 was caused by the Dinoflagellate Noctiluca scintillans (sometimes known as the Sea Sparkle as it is bioluminescent when disturbed). Noctiluca scintillans caused further red tides on 12 April 2017 and 14–21 June 2017. From 28 June to 4 July 2017 the area suffered a mucilage aggregates phenomenon (massive accumulation of gelatinous material at and below the water surface). From July to September 2017 the area had a bloom of the Diatoms Chaetoceros spp., Cylindrotheca closterium, and Skeletonema costatum. In November 2017 there was a bloom of the Dinoflagellate Dinophysis cf. acuminata. From December 2017 to 17 January 2018 there was a bloom of the Ciliate Mesodinium rubrum. Finally in January–February 2018 there was another bloom of the Diatoms Cylindrotheca closterium, and Skeletonema costatum.

 

 

The Sea Sparkle, Noctiluca scintillans, a bioluminescent Dinoflagellate which caused several red tides in Thessaloniki Bay during the summer of 2017. Maria Antónia Sampayo/Instituto de Oceanografia/Universidade de Lisboa/Plankton Net/Wikimedia Commons.

In the 47 samples from the White Tower site, a total of 2256 Operational Taxonomic Units (clusters of organisms, grouped by DNA sequence similarity of a specific taxonomic marker gene; a pragmatic proxy for species in the absence of a full taxonomic study) were recovered. The highest number of Operational Taxonomic Units (378) was detected on 23 August and the lowest (44) on 22 March. Alpha-diversity estimators fluctuated during the study, with extremely low values on 22 March 2017 at the White Tower site, indicating high dominance by one (or few) taxa, and high variation between taxa abundances within the community. Indeed, Operational Taxonomic Unit 2, closely related to the Dinoflagellate Noctiluca scintillans, comprised of over 99% of the total number of reads in that sample.

The cumulative number of Operational Taxonomic Units rose steadily throughout the study. On average, 45.9 new Operational Taxonomic Units were introduced in each sample from White Tower. The most prominent introductions of newly detected White Tower were recorded at 29 March 2017 with 152 newly introduced Operational Taxonomic Units, at 4 July 2017 (99 Operational Taxonomic Units), at 27 September 2017 (78 Operational Taxonomic Units), and at 17 January 2018 (180 Operational Taxonomic Units). These spikes of newly introduced Operational Taxonomic Units coincided with the ending of two red tides (29 March 2017 and 17 January 2018, the end of the conspicuous mucilaginous aggregate of late June 2017, and the end of a summer Diatom bloom (September 2017). Overall, the most diverse groups exhibited temporal variations in abundance. Even so, Dinoflagellata in all cases comprised more than 30% of the number of reads per sample, reaching almost 99% of the number of reads on 22 March 2017 at White Tower, when Operational Taxonomic Unit 2, closely affiliated to Noctiluca scintillans, was over-dominant.

The proportions of each group of unicellular Eukaryotes recovered over the course of the study. Genitsaris et al. (2020).

During the monthly sampling, common to the four sites, the total number of Operational Taxonomic Units corresponding to 12 samplings for each site, was 1153, 1195, 1362, and 1071 for White Tower, Aretsou Beach, Music Hall Coast, and Harbour, respectively. The highest average number of Operational Taxonomic Units was calculated at the Music Hall Coast site (262.4), and the lowest at the Harbour site (210.3). Overall, 560 Operational Taxonomic Units were found to be common in all four sampling sites. Concerning each site individually, 172, 231, 351, and 178 unique Operational Taxonomic Units were detected at White Tower, Aretsou Beach, Music Hall Coast, and Harbour, respectively. The average values of the a-diversity estimators were generally similar between all four sites. 

Dinoflagellata in all cases comprised over 30% of the number of reads per sample, while during the warm months of summer, Bacillariophyta (Diatoms) showed higher relative abundance reaching over 50% of the total number of reads at all sites except Harbour. Furthermore, over 40% of the total number of reads were affiliated to Bacillariophyta in November 2017 and February 2018 at White Tower and in November 2017 at Music Hall Coast, while also Ciliophora-related Operational Taxonomic Units (Ciliates) were recorded in high relative abundances (over 40% of the total number of reads) in January 2018, again at Music Hall Coast.

Overall, 36 Operational Taxonomic Units were detected having relative abundance of more than 1% of the total number of reads in each sampling site (instantly abundant), representing the most dominant Operational Taxonomic Units during the study. The majority of these (17/36) belonged to the Dinoflagellata, followed by Ciliophora (6/36) and Bacillariophyta (5/36). Additionally, present among the most dominant OTUs were the groups were Chlorophyta, Marine Stramenopiles, Cercozoa, Acantharia, and Cryptophyta. The closest relatives of these Operational Taxonomic Units were known marine taxa, including the Dinoflagellates Scrippsiella trochoidea, Noctiluca scintillans,
Gonyaulax fragilis, Gymnodinium aureolum, Alexandrium margaelefii, and Peridinium quinquecorne, the Diatoms Chaetoceros tenuissimus, Thalassiosira sp., and Skeletonema pseudocostatum, the Cryptophyte Teleaulax sp., and the Cliates Strobilidium sp., Strombidium biarmatum, and Tintinnopsis sp., with cosmopolitan distribution, but also previously undetected in the area taxa.

The Dinoflagellates Scrippsiella trochoidea, widely distributed bloom-forming species present in Thessaloniki Bay at a high abundance during the study period. Phyto'pedia.

Among the above taxa, Operational Taxonomic Unit 1, Operational Taxonomic Unit 2, Operational Taxonomic Unit 4, Operational Taxonomic Unit 5, Operational Taxonomic Unit 6, and Operational Taxonomic Unit 8 exhibited relative abundances of over 10% of a sample’s total number of reads, representing arbitrarily bloom forming Operational Taxonomic Units, in at least one sample in all four sites. In particular, Operational Taxonomic Unit 1, with closest relative the Dinoflagellate Scrippsiella trochoidea, followed by Operational Taxonomic Unit 2 (the Dinoflagellate Noctiluca scintillans), were detected in relative abundances of over 10% of a sample’s total number of reads for long periods during the study, including before and during the mucilage aggregate event, especially in White Tower for Operational Taxonomic Unit 2. It is noteworthy that Operational Taxonomic Unit 4, closely affiliated to the Diatom Chaetoceros tenuissimus, was frequently detected during the warm months and was strongly positively related to the water temperature, while the Operational Taxonomic Unit 5 (the Dinoflagellate Gonyaulax fragilis) had high abundances during the mucilaginous aggregate phenomenon in June 2017. Other dominant Operational Taxonomic Units were observed in peak abundances in individual samples throughout the study. On the other hand, Operational Taxonomic Units closely related to known harmful species (such as the Dinoflagellate Dinophysis cf. acuminata, which was found as a rare overall Operational Taxonomic Unit) were found in low abundance (number of reads), suggesting the presence of taxa with potential negative impacts on human health.

The Diatom Chaetoceros tenuissimus, was frequently detected during the warm months and was strongly positively related to the water temperature in Thessaloniki Bay during the study period. BioMarks Data Portal.

The study area is considered a heavily modified marine water body according to the EU Water Framework Directive, one of the most polluted coastal areas of Greece, which is influenced by both anthropogenic activity and freshwater inputs. During the study period nitrogen and phosphorus concentrations were among the highest recorded for eutrophic coastal areas of the Mediterranean Sea. Moreover, environmental variability between the closely located sampling stations was reported as nonsignificant for the majority of the parameters examined.

In this nutrient-rich coastal environment, high biodiversity and abundance of marine unicellular Eukaryotes has been previously reported based on classical microscopy observations. The most abundant and diverse planktonic groups in these observations comprised of Bacillariophyta (Diatoms) and Dinoflagellata, while less diverse groups included Haptophyta, Cryptophyta, Chlorophyta, Euglenozoa, etc. Evidenced by High-Throughput Sequencing, the dominant taxonomic groups of unicellular Eukaryotes belonged to the 'usual suspects', frequently found in the area, i.e., Dinoflagella, Bacillariophyta, and Ciliophora, but also previously undetected groups were revealed, belonging to Cercozoa, the Marine Alveolates group, the Marine Stramenopiles group, Labyrinthulea, Picozoa, Oomycota, Fungi, and other marine unicellular Eukaryotes. At the White Tower site, with higher sampling frequency (weekly samplings) a higher diversity was detected, in comparison to the monthly samplings. It is reasonable to assume that with higher sampling efforts, diversity estimates will increase, as rarely occurring taxa have better chances to be detected. Moreover, because of the short generation times and high turnover rates of marine microbes, rare taxa may have low residence and detection times in the marine environment. Accordingly, frequent temporal (e.g., one week sampling interval) and highly resolved spatial samplings can provide with higher diversity coverage for planktonic organisms.

High-Throughput Sequencing tools have been increasingly used in investigations of marine microbial diversity and have similarly revealed high novel and undetected diversity in coastal areas including the Eukaryotic taxonomic groups detected in Genitsaris et al.'s dataset. Previous studies using High-Throughput Sequencing tools have reported 'new players in the succession scene' of the coast of eastern English Channel, and were associated with Marine Alveolates, Marine Stramenopiles, and Cercozoa, which were also detected in Genitsaris et al.'s study. These new players were suggested to participate in complex interactions, shaping the spatial, and temporal unicellular Eukaryote assemblages in the area. The rare microbial fraction of these assemblages appeared to be particularly active and play significant ecological roles.

During the period of the study, conspicuous, frequent, persistent, and successive Phytoplankton blooms were alternated with red tides and mucilage aggregates. These phenomena were detected by means of microscopy almost continuously throughout the study period and were of great ecological importance for the coastal system. They, also, had significant socio-economic impact to the residents of the city of Thessaloniki, as in several occasions the red tides and harmful Algal blooms were macroscopically visible impacting the touristic urban front of the city centre. The key plankton abettors and perpetrators contributing to the formation of the Phytoplankton blooms were the known mucilage-producing Diatoms Cylindrotheca closterium, Leptocylindrus spp., Skeletonema costatum, Chaetoceros spp., and the Dinoflagellate Gonyaulax cf. fragilis, while the species responsible for the development of the red tides were the Dinoflagellates Noctiluca scintillans and its close relative Spatulodinium pseudonoctiluca, and the photosynthetic Ciliate Mesodinium rubrum. The characterisation of the unicellular Eukaryotic communities with High-Throughput Sequencing tools showed that the dominant biosphere, taking over a high percentage of the reads, was closely related to the abovementioned species. In particular, Operational Taxonomic Units closely related to the red tide forming Noctiluca scintillans, the bloom forming Chaetoceros spp., Skeletonema pseudocostatum, Gymnodinium aureolum and Peridinium quinquecorne, and the mucilage aggregates abettor Gonyaulax fragilis were among the most abundant Operational Taxonomic Units in all samples. Indeed, these Operational Taxonomic Units were detected in high relative number of reads during the same periods that their closest relatives were identified by microscopy.

The photosynthetic Ciliate Mesodinium rubrum, one of  the species responsible for the development of the red tides in Thessaloniki Bay during the study period. Phyto'pedia.

Furthermore, during these phenomena the system was taken over by the dominant taxa, as evidenced by the hampered rate of newly introduced  Operational Taxonomic Units in the system during these periods compared to the nonbloom periods. Among these  Operational Taxonomic Units, previously undetected taxa in the area were observed, namely the Dinoflagellates Adenoides eludens and Islandinium tricingulatum, the Haptophyte Haptolina sp., taxa belonging to the Marine Stramenopiles, and the Ciliate Parundella sp., all of which have unusual and complex life cycles and trophic preferences, influential in global processes, and suggesting alternating trophic states and carbon paths. In addition to microscopic data, Operational Taxonomic Units closely related to known harmful species were opportunistically found with high read numbers during bloom/red tide phenomena, such as parasitic Marine Alveolates and Cercozoa, revealing a (previously undetected) significant biodiversity with potentially negative impacts for the unicellular Eukaryotic community and the ecosystem. Marine Alveolates have been detected in high abundances in virtually all studies of marine unicellular Eukaryotes, suggesting their ubiquitous role as parasites of various marine organisms and their significant part as a trophic link in marine systems.

The Haptophyte Haptolina sp.,  previously undetected in the study area. Roscoff Culture Collection.

Temperature and salinity were the two environmental variables that seemed to influence most the overall unicellular Eukaryotic communities, but also individually the majority of the dominant  Operational Taxonomic Units, as expected. Both water temperature and salinity are among the key components of climate change, which are projected to directly affect marine microbial diversity and functions. Temperature has been found to affect the size, metabolic rate, and activity of microbes, thus influencing marine protistan community and food web structure. Previous studies using High-Throughput Sequencing tools in mesocosm salinity manipulations of Thessaloniki Bay unicellular Eukaryotic assemblages detected a number of Operational Taxonomic Units associated with euryhaline taxa, also found in Genitsaris et al.'s dataset (e.g., Skeletonema, Chaetoceros, Prorocentrum, Gyrodinium, and Gymnodinium-related Operational Taxonomic Units). This has led to the conclusion that in this Mediterranean environment, the standing biodiversity can act as a buffer against environmental fluctuations. Beyond the water temperature and salinity nutrient concentrations were only partly associated with specific Operational Taxonomic Units. In particular, nitrogen was positively correlated with the Operational Taxonomic Unit closely related to the Dinoflagellate Noctiluca scintillans, which was identified as the chief responsible species for the observed red tides during the study. Noctiluca scintillans is among the most frequently observed red tide forming Dinoflagellates worldwide in eutrophic systems, and at water temperatures ranging from 10 to 25 °C, and salinity ranges from 28 to 36 g/L, similar to Thessaloniki Bay. The positive connection between Noctiluca scintillans read abundance and ammonia in Genitsaris et al.'s study area agrees with the reported correlation of its cell abundance and ammonia with microscopy data in the same samples, and might indicate nutrient regeneration by this heterotrophic Dinoflagellate and contribution to the local nutrient pool. In addition, the role of Noctiluca scintillans as a recycler of nitrogen has been previously linked to high concentrations of nitrogen in its cells. Even though microscopy data of the same samples showed strong connections of most nutrients measured in Genitsaris et al.'s study, metabarcoding data did not reveal similar trends with the exception of the positive correlation of ammonia and the Operational Taxonomic Unit closely related to Noctiluca scintillans.

The High-Throughput Sequencing tools implemented in Genitsaris et al.'s study showed a high diversity of known, frequently occurring Protists, also detected with microscopy in previous studies of the area. It further revealed several taxa previously undetected in the area, with seemingly important roles in the structure and functioning of the entire unicellular Eukaryotic communities, and key contribution to the phenomena of red tides, harmful Algal blooms, and mucilage aggregates observed during the study. The unicellular Eukaryotic assemblages showed temporal patterns rather than small-scale spatial separation, possibly responding to the water temperature and salinity seasonal variations. The rich species pool along with the eutrophic status of the area facilitates the unicellular Eukaryotic succession, mainly favoring the taxa affected by the established cycles of temperature and salinity. Overall, environmental pressures (eutrophication, nitrogen pollution) under seasonal changes of water temperature and salinity (circulation pattern), are suggested to be the major drivers of the composition changes and the appearance of blooms and red tides in the study area.

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