Deciphering changes in the symbiotic microbe community within Corals during bleaching events.

El Niño events have a significant impact on global climate, most notably causing warming events, which affect the stability of marine ecosystems. Widespread bleaching of coral reefs, resulting in high levels of Coral mortality due to heat stress, is now recognized as a global threat to coral. 2015–2017 were the three warmest years in the instrumental record period since 1880 and record high temperatures triggered a pan-tropical Coral bleaching episode. The world’s largest Coral Reef ecosystem, the Great Barrier Reef, experienced the highest temperatures ever recorded and lost nearly 30% of coral cover. Coral is closely associated with a complex group of microorganisms, including Symbiodiniaceae (symbiotic Dinoflagellates), Fungi, Bacteria, Archaea, Endolithic Algae, and Viruses, in a relationship known as Coral  Symbiosis. Coral microbes play an important role in nutrient cycling and antimicrobial protection in Coral Reefs. Therefore, it is important to investigate the effects of bleaching events on the function of Coral microbial communities. Recently, metagenomics has been used to investigate the taxonomic diversity and metabolic capabilities of Coral-associated microbes under thermal stress or bleaching. These studies suggested that microbes can undergo major shifts, from symbionts to opportunistic microbes or potential disease-causing Bacteria, during heat stress or bleaching. In addition to this, the metabolism of the microbial community can shift from autotrophy to heterotrophy, which involves sulphur and nitrogen metabolism, fatty acid and lipid utilisation, and secondary metabolism.

In a paper published in the journal Frontiers in Microbiology on 20 March 2020, Fulin Sun of the State Key Laboratory of Tropical Oceanography and the Daya Bay Marine Biology Research Station of the South China Sea Institute of Oceanology of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory, Hongqiang Yang, also of the State Key Laboratory of Tropical Oceanography and the Key Laboratory of Ocean and Marginal Sea Geology at the South China Sea Institute of Oceanology, and the Nansha Marine Ecological and Environmental Research Station of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory,  and Guan Wang and Qi Shi, again of the Key Laboratory of Ocean and Marginal Sea Geology at the South China Sea Institute of Oceanology of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory, present the results of a study of the microbiome (micro-organism community) of Corals from Xiane Reef in the Nansha Islands of the South China Sea (part of the disputed Spratly Island group), during the 2016 bleaching event.

Traditionally, research into coral bleaching has mainly focused on studying photosynthetic symbiotic Dinoflagellate Algae known as Symbiodiniaceae. Physiological damage and expulsion of Algal symbionts are thought to be the result of the host immune response triggered by reactive oxygen species produced by Coral hosts, Algal symbionts, or both. Research on Symbiodiniaceae has been focused on changes in diversity and density of Symbiodiniaceae, Photosystem II damage in symbiotic Dinoflagellates, thermal tolerance of Symbiodiniaceae, and functional changes in Symbiodiniaceae. However, few studies have reported the functional response of Symbiodiniaceae to bleaching.

Although previous studies had not focused on Eukaryotes, they have hinted that Eukaryotes are the most abundant component of Coral symbionts. Microeukaryotes have been most widely associated with Coral diseases and mortality. To date, studies on other Microeukaryotes associated with Coral have mainly focused on several key populations, including Fungi, Endolithic Microalgae and Protists. The potential diversity of Coral related Fungi suggests a broader role beyond pathogenicity. Metagenomic analysis has revealed that Endolithic Algae can play a key role in the microbial community by driving important chemical processes. When Zooxanthellae are absent, other Microeukaryotes can provide nutrients that increase Coral survival during periods of acute stress. Despite the fact that other Eukaryotes are ubiquitous in Corals, little is known about their diversity and ecological function during Coral bleaching events.

Until now, fundamental gaps have existed in our understanding of the Coral microbiome and its functional contribution to Coral. In 2016, a bleaching event also affected a large area of Coral in the South China Sea, where mass Coral bleaching had not previously been recorded. In Sun et al.'s study, four different Coral species were studied to provide an overview of the metagenomic (DNA) and metatranscriptome (RNA) response of Coral symbionts (Prokaryotes, Symbiodiniaceae, other Endolithic Eukaryotes and the Coral itself) to bleaching and to highlight differences in their functional performance. Coral species were collected at the same location to eliminate any potential external environmental influences. In order to study the different components of Coral symbionts separately, each symbiotic component was separated based on the National Center for Biotechnology Information non-redundant protein database. Combined with Kyoto Encyclopedia of Genes and Genomes database annotation, the corresponding functions of DNA and RNA in different components were explored.

Coral samples were collected at Xiane reef, Nansha Islands, South China Sea, in June 2016. Four Coral species (with unbleached coral and bleached coral being collected from the same Coral colony), Acropora tenuis, Goniastrea minuta, Pocillopora verrucosa, and Pocillopora meandrina, were sampled. Water temperatures in the sampling area ranged from 30.5 to 31° C. Three replicate samples of unbleached and bleached parts of Coral (including tissue, mucus and skeleton) were collected using a hammer and chisel. Once collected, the Coral samples (1 cm x 1 cm) were washed with sterile seawater three times to remove any surface attachments. Each sample was divided into two parts and placed in sterile centrifuge tubes with Sample Protector (Takara, Japan) for DNA and RNA extraction. DNA and RNA isolation of replicate samples was mixed and conducted using DNeasy and RNeasy plant mini kits (Qiagen, Germany) following the manufacturer’s instructions. Coral tissues were removed with an airbrush for the identification of each species. All Coral samples were identified according to their ecological and morphological characteristics.

Location map of sampling sites in the South China Sea during the 2016 El Niño period. Sun et al. (2020).

At the DNA level, the abundance of unbleached Coral genomes accounted for more than 80% of the total number of sequences, but the DNA content had low abundance in bleached Acropora tenuis (48.55%) and Goniastrea minuta (7.90%) Corals. In comparison with unbleached Corals, Bacterial abundance increased sharply in bleached Acropora tenuis (32.53%) and Goniastrea minuta (78.63%) Corals, as did the abundance of Eukaryotes in bleached Acropora tenuis. At the RNA level, bleached Acropora tenuis Coral had a reduced abundance of Symbiodiniaceae compared with unbleached Acropora tenuis Coral. In contrast, bleached Acropora tenuis Coral had a higher abundance of Prokaryotes and Eukaryotes than unbleached Acropora tenuis. Unlike Acropora tenuis Corals, the abundance of Eukaryotes was higher in bleached Pocillopora verrucosa and Pocillopora meandrina in comparison to unbleached Corals. Symbiodiniaceae abundance was significantly lower in bleached Pocillopora verrucosa and Pocillopora meandrina Corals compared with unbleached bleached Pocillopora verrucosa and Pocillopora meandrina Corals.

The field sampling process of Acropora tenuis. (A) Acropora tenuis is in the process of bleaching during the sampling time, the lower left part of its growth base is bleached Favites sp.; (B) This is the red frame part of the (A), Acropora tenuis in the process of bleaching; (C) This is the yellow frame of the part (B); the upper blue frame of part (C) is the collection part of the bleached Coral sample, and the lower part of the green frame is the collection part of unbleached Coral samples. Sun et al. (2020).

The results showed that there were distinct differences in the composition of symbionts among different Corals at RNA and DNA levels. For Acropora tenuis and Goniastrea minuta Corals, metagenomic analysis showed that the most obvious response to bleaching was the increase in abundance of Bacterial taxa, affiliated with Proteobacteria (Alphaproteobacteria, Deltaproteobacteria,
Epsilonproteobacteria, and Gammaproteobacteria), Bacteroidetes (Cytophagia) and Green Suplhur Bacteria (Chlorobia), in addition to a significant decrease in Dinophyceae (Dinoflagellata) abundance. 

A colony of Goniastrea minuta in the Philippines. Charlie Veron/Corals of the World.

Furthermore, distinct differences were observed at the class level between unbleached and bleached Corals. The dominant class of symbionts in Pocillopora verrucosa and Pocillopora meandrina were Dinophyceae, Eurotiomycetes (Ascomycotae Fungi) and Pucciniomycetes (Basidiomycote Fungi), and Bacilli. In the case of both unbleached and bleached Pocillopora verrucosa and Pocillopora meandrina Corals, there was a very low abundance of Prokaryotes compared with other symbiont components. Apart from the decreased abundance of Dinophyceae, another feature of bleachied Pocillopora verrucosa and Pocillopora meandrina Corals was the higher abundance of Eukaryotes, especially Pucciniomycetes, when compared with unbleached Corals. The major genera identified at the RNA and DNA levels varied with samples. For Acropora tenuis and Goniastrea minuta Corals, the most obvious response to bleaching in Coral symbionts was the shift in abundance of Symbiodiniaceae and Bacterial taxa. Bleached Corals exhibited a high diversity and abundance of Bacterial taxa at the DNA and RNA level. These taxa included Chlorobiales (Green Sulphur Bacteria), Rhodobacterales (Alphaproteobacteria), Alteromonadales (Gammaproteobacteria), Oceanospirillales (Gammaproteobacteria), and Vibrionales (Gammaproteobacteria). More Bacterial orders were recorded from Acropora tenuis and Goniastrea minuta than Pocillopora verrucosa and Pocillopora meandrina at the DNA and RNA levels. A higher amount of Campylobacterales (Epsilonproteobacteria), Alteromonadales and Streptomycetale (Actinobacteria) were revealed at the RNA level, in contrast to the results for Rhodobacterales and Vibrionales, which were highly abundant at both the RNA and DNA levels.

A colony of Pocillopora verrucosa at the Birch Aquarium in San Diego. Wikimedia Commons.

For Pocillopora verrucosa and Pocillopora meandrina Corals, the symbiotic component was mainly composed of Microeukaryotes, and there was a distinct difference in the abundance and composition for unbleached and bleached Corals. Most notably, Dinophyceae-like genera had a significantly lower abundance in bleached Corals, including Symbiodiniaceae, Crypthecodinium, Amphidinium, Karlodinium, Heterocapsa, Pfiesteria, and Prorocentrum. The Puccinia Fungus genus (Basidiomycota) was dominant at the RNA level, and was more abundant in bleached than unbleached corals. In unbleached Corals, Dinoflagellates of the orders Gymnodiniales, Cantharellales, Peridiniales, Prorocentrales and Gonyaulacales, all of which are affiliated with Dinophyceae, were observed to be more abundant at the RNA and DNA levels in comparison with bleached Corals.

A colony of the Coral Pocillopora meandrina in American Samoa. Douglas Fenner/NOAA Fisheries.

For Acropora tenuis and Goniastrea minuta Corals, gene composition and transcriptional abundance increased significantly in bleached Corals in comparison with unbleached Corals, including ribosomal, carbon fixation, cofactor and vitamin biosynthesis, and ATP synthesis. Both the metagenomic and metatranscriptome results of Sun et al.'s study indicated that the main contributing prokaryotes for these functions were Proteobacteria, Bacteroidetes, Chlorobi and Actinobacteria, and the abundance of Bacterial orders was significantly correlated with function, especially at the RNA level. Rhodobacterales, Campylobacterales, Vibrionales and Alteromonadales contributed more to gene function at the RNA level, in contrast to the finding that dominant of Flavobacteriales, Oceanospirillales and Cellvibrionales had a slightly higher contribution to gene function at the DNA level. For Pocillopora verrucosa and Pocillopora meandrina Corals, the Bacterial contribution to gene function was very low in both bleached and unbleached Corals. 

The abundance of Prokaryotes was significantly correlated with functional abundance at the RNA and DNA levels. For Acropora tenuis and Goniastrea minuta Corals, carbon fixation pathways, including the reductive citrate cycle (Arnon-Buchanan cycle), the reductive pentose phosphate cycle and the 3-hydroxypropionate bi-cycle, were contributed by Proteobacteria and Bacteroidetes. However, the gene abundance of carbon fixation was very low in unbleached and bleached Pocillopora verrucosa and Pocillopora meandrina Corals. Dissimilatory nitrate reduction was the main pathway of nitrogen metabolism identified. Proteobacteria, Bacteroidetes, Chlorobi and Tectomicrobia mainly contributed to sulphur metabolism in Acropora tenuis and Goniastrea minuta Corals. Two of three high abundance sulphur metabolism pathways, assimilatory sulphate reduction and dissimilatory sulphate reduction, were detected in Acropora tenuis and Goniastrea minuta Corals only. Dominant bacteria, such as Campylobacterales (Arcobacter and Sulfurimonas) mainly contributed to assimilatory sulphate reduction.

Sulfurimonas Bacteria were found to contribute to assimilatory sulphate reduction in bleached Acropora tenuis and Goniastrea minuta Corals. Sikorski et al. (2010).

The results of metagenomic and metatranscriptomic analysis showed that 24 Symbiodiniaceae species were detectable in the Corals. According to the results, all Symbiodiniaceae species decreased in abundance in bleaching corals in comparison with unbleached corals, especially for dominant species Cladocopium and Symbiodinium. 

Of all Symbiodinium types, Cladocopium and Symbiodinium were the two genera with the highest abundance of gene composition and transcription. Among the functions performed by Symbiodiniaceae, photosynthesis and ATP synthesis were the most important functions. The abundance of Symbiodiniaceae was significantly correlated with functional abundance at the RNA and DNA levels. In comparison with unbleached Corals, Symbiodiniaceae were less abundant in almost all functional genes in bleached Corals. Cladocopium and Symbiodinium were the main contributors to photosynthesis. Cladocopium and Symbiodinium minutum were the main contributors to ATP synthesis. At the RNA level, Cladocopium was the main contributor to ATP synthesis in Acropora tenuis Corals, while Cladocopium and Symbiodinium minutum were the main contributors for Pocillopora verrucosa and Pocillopora meandrina Corals. Cladocopium was the only performer for RNA processing and Spliceosome at the RNA level. Spliceosome genes in unbleached Corals had a higher expression of abundance than bleached Corals, the majority of which belonged to genes involving the heat shock gene (HSPA1_8). There was one carbon fixation pathway (Calvin-Benson cycle) detected in the Symbiodiniaceae. It had a high abundance in all unbleached Corals, and exhibited a reduced gene abundance in bleached Corals.

Symbiodinium minutum Dinoflagellates, major contributers to ATP production in the Corals Pocillopora verrucosa and Pocillopora meandrina. LaJeunesse et al. (2012).

The results indicated that most eukaryotes displayed reduced function in bleached Corals compared with unbleached Corals. This decline in function resulted in decreased proteasome function, carbon fixation, ATP synthesis and central carbohydrate metabolism. However, photosynthetic activity was found to increase in all bleached Corals, in particular Acropora tenuis. The main contributors to function are the dominant Dinophyceae (including Gymnodiniales, Peridiniales, Prorocentrales, and Gonyaulacales) and Eurotiales (Ascomycote Fungi). A higher number of Gymnodiniales, Peridiniales, Prorocentrales and Gonyaulacales contributed to function at the RNA level, in contrast to the DNA level, where Eurotiales mostly contributed to the function. It was unexpectedly found that Dinophyceae were the main contributors to all functions except photosynthesis. Interestingly, there was an obvious increase in the abundance of genes involved in photosynthesis in bleached Coral, mainly attributed to Bacillariophyceae (Diatoms), Florideophyceae (Red Algae), and Trebouxiophyceae (Green Algae). The Calvin cycle is the main pathway for carbon fixation of these Eukaryotes. There was a lower abundance of genes involved in carbon fixation in bleached Corals compared with unbleached Corals. Dinophyceae were the main contributor to Spliceosomes, and the abundance of the heat shock 70 gene was significantly reduced in bleached Corals compared with unbleached Corals.

Almost all of the functions of bleached Acropora tenuis Coral were reduced compared with unbleached Corals, in particular cell signaling, ribosomal activity, spliceosome activity, glycan metabolism, RNA processing and ATP synthesis. In contrast to Acropora tenuis Coral, almost all functions of bleached Pocillopora verrucosa and Pocillopora meandrina were increased in comparison with unbleached Corals.

An obvious response to bleaching displayed by Coral symbionts was the shift in abundance of Bacteria. Bleached Corals exhibited a higher diversity and abundance of Bacterial taxa at the DNA and RNA level than unbleached Corals, indicating that Bacteria were easily affected by Coral bleaching. Many studies have shown that potentially opportunistic microbes can sharply increase in abundance and become dominant in bleached Corals, causing the coral to move toward an unstable state, even inducing Coral disease. Coral bleaching can alter the chemical composition of Coral mucus, increase organic matter and mucus production, which induces a shift in the Coral-associated microbial community, The results of the current study indicated that these opportunistic microbes were highly abundant in bleached Coral, and may have resulted in an elevation of bacterial-organic matter coupling.

Almost all functional genes of Prokaryotes were improved in bleached Acropora tenuis and Goniastrea minuta Corals. Previous studies demonstrated that the metabolism of the microbial community could shift from autotrophy to heterotrophy under stress, resulting in an increase in the abundance of microbial genes involved in sulphur and nitrogen metabolism, and secondary metabolism. As the microbial community shifts from autotrophy to heterotrophy, Bacterial consumption of organic matter becomes greatly enhanced, and the contribution of fixed nitrogen and photosynthesis for nitrogen and carbon budgets became less obvious. High abundance sulphur metabolism pathways (assimilatory sulphate reduction and dissimilatory sulphate reduction) were detected in bleaching bleached Acropora tenuis and Goniastrea minuta Corals, increasing the possibility of producing sulphide. High abundance of these heterotrophic Bacteria could deplete nutrients in Coral, and deteriorate the microenvironment, ultimately making Coral bleaching irreversible.

According to the results of Sun et al.'s study, the abundance of all Symbiodiniaceae obviously decreased in all bleached Corals in comparison with unbleached Corals, indicating that the nutrition supplied to Coral by Symbiodiniaceae decreased. Exocytosis or in situ symbiotic degradation during bleaching seemed to be less invasive and cost effective than host cell degradation. Sun et al.'s tudy identified that the four Coral species could simultaneously host a very high diversity of genotypic Symbiodiniaceae phylotypes, more than described by other studies. Cladocopium is often regarded as a sensitive species to temperature or bleaching, and is dominant in Scleractinian Corals in the South China Sea. Cladocopium were the main contributors to photosynthesis and ATP synthesis in Coral.

This result provided direct evidence that bleaching may have important effects on photosynthesis via the inhibition of the Calvin cycle, limiting carbon fixation in Symbiodiniaceae, as described in previous studies. It had been found that carbon fixation via the Calvin cycle is sensitive to heat stress. Previous studies reported that the maximum quantum yield of photosystem II was significantly lower and highly variable in bleached Corals in comparison with healthy Corals. This occurred with a corresponding loss of electron flow supporting carbon fixation. ATP synthesis was essential for repair in photosystem II, therefore the repair rate that this study suggested was decreased through the inhibition of ATP synthesis in bleached Coral. In addition, a low abundance of the heat shock gene indicated that bleaching inhibited the activity of heat-inducible genes and heat acclimation of Symbiodiniaceae resulting in the reduced ability of Symbiodiniaceae to resist thermal stresses.

Another finding of Sun et al.'s study was that, when Corals are bleaching, they not only expel Symbiodiniaceae, but all of the identified Dinophyceae genera, which indicates that these Algae have the same response mechanism to Coral bleaching. Other Eukaryotic Algae also showed distinct changes in bleached Corals compared to unbleached Corals. It has been reported that when Symbiodiniaceae are absent, these Algae could provide an alternative source of photoassimilates, and provide nutrients that increase Coral survival during stress. Sun et al.'s study also suggested that Fungi displayed high abundance of RNA in bleached Pocillopora verrucosa and Pocillopora meandrina Corals, and bleaching stimulated Fungal growth. Some studies have suggested that Fungi were also thought to be opportunistic pathogens, and their abundance depended on Coral health.

The functional genes of other Eukaryote Algae increased in abundance during bleaching, indicating that the photosystems of other Eukaryotes (such as Bacillariophyta, Chlorophyta) used a different mechanism and had higher levels of thermal tolerance compared to Dinophyceae Algae. It was also thought that other endosymbiotic Algae benefit the host Coral during periods of stress. This may be because during bleaching, the shading effect of Symbiodiniaceae was lost, allowing increased light to penetrate the Coral skeleton, possibly resulting in increased photosynthetic activity of these Algae. Given the ability of Microalgae to adapt rapidly to heat stress, these populations might become important as 'secondary' symbionts, and continue to provide nutrients for coral through photosynthesis.

The results of Sun et al.'s study indicated that the Coral symbionts under investigation were at different stages of bleaching. Almost all of the functions of bleached Acropora tenuis and Goniastrea minuta Corals were reduced compared with unbleached Corals, suggesting that the nucleic acid of bleaching Corals was being degraded and was in an apoptotic state. A reduction in mRNA abundance of cytochrome c and ATP synthase, which were central components of the respiratory electron transport chain inhibited the ability of the host to survive or recover from thermal stress. This suggested that bleached Acropora tenuis and Goniastrea minuta Corals lost most of their physiological metabolic activity and function.

However, almost all functions of bleached Pocillopora verrucosa and Pocillopora meandrina Corals were increased in comparison with unbleached Corals. This indicated that Coral was in a temporary stage of transformation from an unbleached to a seriously bleached state. At this stage, Corals exhibit symptoms of bleaching as they have expelled most symbiotic Algae; however, the symbiotic structure in bleached Corals has certain similarities in composition to unbleached corals. The remaining Symbiodiniaceae and other Algae play an important role in bleached Coral, providing nutrition to the host and maintaining Coral activities under stress.

For both unbleached and bleached Coral, it was found that different Coral species have common symbiotic taxa that perform biological functions in vivo. Overall, different Coral species were found to have common characteristics when bleached: a decreased abundance of Symbiodiniaceae and associated function and the exclusion of Dinophyceae-like Eukaryotes. Furthermore, Sun et al.'s study might reflect the different stages of the Coral bleaching process. In the early stages of Coral bleaching, Algae such as Symbiodiniaceae and other Dinophyceae were expelled from Corals. If the Coral microbiome could maintain a level of stability similar to that of an unbleached Coral, the Coral itself could retain its functional activity. Otherwise, the Coral itself gradually decreased in activity due to the lack of nutrients usually provided by Algae. Opportunistic Bacteria then multiply in large numbers, and result in the deterioration of the Coral microenvironment.

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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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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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