Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Orchestrated translation specializes dinoflagellate metabolism three times per day.

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

Ocean climate and hydrodynamics drive decadal shifts in Northeast Atlantic dinoflagellates.

The abundance of large marine dinoflagellates has declined in the North Sea since 1958. Although hypotheses have been proposed to explain this diminution (increasing temperature and wind), the mechanisms behind this pattern have thus far remained elusive. In this article, we study the long-term changes in dinoflagellate biomass and biodiversity in relation to hydro-climatic conditions and circulation within the North Atlantic. Our results show that the decline in biomass has paralleled an increase in biodiversity caused by a temperature-induced northward movement of subtropical taxa along the European shelf-edge, and facilitated by changes in oceanic circulation (subpolar gyre contraction). However, major changes in North Atlantic hydrodynamics in the 2010s (subpolar gyre expansion and low-salinity anomaly) stopped this movement, which triggered a biodiversity collapse in the North Sea. Further, North Sea dinoflagellate biomass remained low because of warming. Our results, therefore, reveal that regional climate warming and changes in oceanic circulation strongly influenced shifts in dinoflagellate biomass and biodiversity.

Nutrient depletion and heat stress impair the assimilation of nitrogen compounds in a scleractinian coral.

Concentrations of dissolved nitrogen in seawater can affect the resilience of the cnidarian-dinoflagellate symbiosis to climate change-induced bleaching. However, it is not yet known how the assimilation and translocation of the various nitrogen forms change during heat stress, nor how the symbiosis responds to nutrient depletion, which may occur due to increasing water stratification. Here, the tropical scleractinian coral Stylophora pistillata, in symbiosis with dinoflagellates of the genus Symbiodinium, was grown at different temperatures (26°C, 30°C and 34°C), before being placed in nutrient-replete or -depleted seawater for 24 h. The corals were then incubated with 13C-labelled sodium bicarbonate and different 15N-labelled nitrogen forms (ammonium, urea and dissolved free amino acids) to determine their assimilation rates. We found that nutrient depletion inhibited the assimilation of all nitrogen sources studied and that heat stress reduced the assimilation of ammonium and dissolved free amino acids. However, the host assimilated over 3-fold more urea at 30°C relative to 26°C. Overall, both moderate heat stress (30°C) and nutrient depletion individually decreased the total nitrogen assimilated by the symbiont by 66%, and combined, they decreased assimilation by 79%. This led to the symbiotic algae becoming nitrogen starved, with the C:N ratio increasing by over 3-fold at 34°C, potentially exacerbating the impacts of coral bleaching.

The second most abundant dinophyte in the ponds of a botanical garden is a species new to science.

In the microscopy realm, a large body of dark biodiversity still awaits to be uncovered. Unarmoured dinophytes are particularly neglected here, as they only present inconspicuous traits. In a remote German locality, we collected cells, from which a monoclonal strain was established, to study morphology using light and electron microscopy and to gain DNA sequences from the rRNA operon. In parallel, we detected unicellular eukaryotes in ponds of the Botanical Garden Munich-Nymphenburg by DNA-metabarcoding (V4 region of the 18S rRNA gene), weekly sampled over the course of a year. Strain GeoK*077 turned out to be a new species of Borghiella with a distinct position in molecular phylogenetics and characteristic coccoid cells of ovoid shape as the most important diagnostic trait. Borghiella ovum, sp. nov., was also present in artificial ponds of the Botanical Garden and was the second most abundant dinophyte detected in the samples. More specifically, Borghiella ovum, sp. nov., shows a clear seasonality, with high frequency during winter months and complete absence during summer months. The study underlines the necessity to assess the biodiversity, particularly of the microscopy realm more ambitiously, if even common species such as formerly Borghiella ovum are yet unknown to science.

Morphology, behavior, and phylogenomics of Oxytoxum lohmannii, Dinoflagellata.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

Single-cell DNA from West Greenland marine sediments suggests presence of Protoperidinium tricingulatum in the Arctic.

Spiny brown dinoflagellate cysts are commonly used as sea-ice indicators in the Arctic, but their biological affinities are not well known. We present the first indication of hitherto temperate Protoperidinium tricingulatum in the Arctic based on single-cell LSU rDNA sequencing from sediments of the Disko Bay-Vaigat Sound, West Greenland. The morphological similarity of the sequenced cyst morphotype to the sea-ice indicator Islandinium? cezare morphotype 1 is striking. The morphology of the isolated cysts, as well as those observed in the total cyst assemblage following standard palynological preparation, both resemble either I.? cezare morphotype 1 or P. tricingulatum, suggesting that the specimens may in fact be close morphological variants of the same species. In addition, nine LSU rDNA sequences were obtained from morphological variants assigned to Islandinium minutum s.l.: including both subspecies minutum and subspecies barbatum. The two subspecies could not be differentiated based on partial LSU rDNA sequencing. Overall, Arctic spiny brown dinoflagellate cyst species may be morphologically more diverse and taxonomically more complex than shown earlier and further genetic and morphological studies are needed. Importantly, the value of cysts as palaeoecological indicators depends on a sound understanding of their biological affinity and taxonomy.

DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Molecular Insights into the Synergistic Effects of Putrescine and Ammonium on Dinoflagellates.

Ammonium and polyamines are essential nitrogen metabolites in all living organisms. Crosstalk between ammonium and polyamines through their metabolic pathways has been demonstrated in plants and animals, while no research has been directed to explore this relationship in algae or to investigate the underlying molecular mechanisms. Previous research demonstrated that high concentrations of ammonium and putrescine were among the active substances in bacteria-derived algicide targeting dinoflagellates, suggesting that the biochemical inter-connection and/or interaction of these nitrogen compounds play an essential role in controlling these ecologically important algal species. In this research, putrescine, ammonium, or a combination of putrescine and ammonium was added to cultures of three dinoflagellate species to explore their effects. The results demonstrated the dose-dependent and species-specific synergistic effects of putrescine and ammonium on these species. To further explore the molecular mechanisms behind the synergistic effects, transcriptome analysis was conducted on dinoflagellate Karlodinium veneficum treated with putrescine or ammonium vs. a combination of putrescine and ammonium. The results suggested that the synergistic effects of putrescine and ammonium disrupted polyamine homeostasis and reduced ammonium tolerance, which may have contributed to the cell death of K. veneficum. There was also transcriptomic evidence of damage to chloroplasts and impaired photosynthesis of K. veneficum. This research illustrates the molecular mechanisms underlying the synergistic effects of the major nitrogen metabolites, ammonium and putrescine, in dinoflagellates and provides direction for future studies on polyamine biology in algal species.

Impacts on Human Health Potentially Caused by Exposure to an Unprecedented Ostreopsis spp Bloom in the Bay of Biscay, French Basque Coast.

INTRODUCTION: In recent years, climate change and human activity have modified marine biotopes, including the widening distribution of harmful algal blooms (HABs). Bloom events predominated by microalgae of the genus Ostreopsis have been described on the French Mediterranean coast, but in 2021 an unprecedented bloom occurred on the French Basque coast. The objective of this study is to describe the health impact of the Ostreopsis spp bloom that occurred on the French Basque coast in 2021. METHODS: A historical cohort was conducted, including cases of possible exposure to Ostreopsis spp registered at the Centre Antipoison de Nouvelle-Aquitaine between July 1 and September 30, 2021. RESULTS: Of 674 patients with possible toxicity due to Ostreopsis spp, 96.9% had bathed in contaminated waters. Most of them developed respiratory tract symptoms (64.4% of patients). The time to the onset of symptoms was <6 h for 73.6% of 174 short-term (<24 h) exposed patients. The median duration of symptoms was 7.5 days for occupational (e.g., lifeguards and surfing instructors) and 3 days for recreational exposures. There were no severe cases. In total, 3% of the cases were of moderate severity, and 97% were of minor severity, according to the Poisoning Severity Score. CONCLUSION: Toxic reactions caused by Ostreopsis spp are mostly benign. The clinical picture is similar to that described following exposures to Ostreopsis cf. ovata blooms in the Mediterranean area since the end of the 20th century. Ostreopsis spp are present on the Basque coast. The ecological factors promoting its blooms remain to be clarified.

Identification and genomic analysis of Pseudosulfitobacter koreense sp. nov. isolated from toxin-producing dinoflagellate Alexandrium pacificum.

The bacterial strain AP-MA-4T isolated from the marine dinoflagellate Alexandrium pacificum (KCTC AG60911), was subjected to a taxonomic analysis. Cells of strain AP-MA-4T were Gram-stain-negative, aerobic, rod-shaped, optimum growth at 20 °C, pH 7.0, in the presence of 5% (w/v) NaCl. Strain AP-MA-4T shared the highest 16S rRNA gene sequence similarity to Pseudosulfitobacter pseudonitzschiae DSM 26824T (98.5%), followed by Ascidiaceihabitans donghaensis RSS1-M3T (96.3%), Pseudoseohaeicola caenipelagi BS-W13T (95.7%), and Sulfitobacter pontiacus CHLG 10T (95.3%). Based on 16S rRNA phylogeny, strain AP-MA-4T is phylogenetically closely related to Pseudosulfitobacter pseudonitzschiae (type species of Pseudosulfitobacter) and could be distinguished from the type species based on their phenotypic properties. The genome length of strain AP-MA-4T was 3.48 Mbp with a 62.9% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain AP-MA-4 T and its closely related type strains were 72.2-83.3 and 18.2-27.6%, respectively. Summed feature 8 (C18:1ω7c and/or C18:1ω6c) was identified the major fatty acids (> 10%). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phospholipid (PL) were demonstrated as the major polar lipids. The major respiratory quinone is ubiquinone-10 (Q-10). Based on genotypic and phenotypic features, strain AP-MA-4T (= KCTC 92289T = GDMCC 1.3585T) represents a new Pseudosulfitobacter species, in which the name Pseudosulfitobacter koreense sp. nov. is proposed.

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8 (14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae.

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ8(14) sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3ß-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ8(14) sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3ß-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3ß-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8 (14) sterols previously considered Amphidinium-specific biomarkers.

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ8(14) -nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3ß-ol (C28:1 ) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3ß-ol (amphisterol; C29:2 ) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3ß-ol; C27:1 ), which is found in many other dinoflagellate genera, rather than Δ8(14) sterols. While the Δ8(14) sterols 4α-methyl-5α-cholest-8(14)-en-3ß-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3ß-ol (C30:2 ) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3ß-ol (dinosterol; C30:1 ), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ8(14) sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ8(14) sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.

Sterols of morphologically distinct, okadaic acid-producing Prorocentrum texanum var. texanum and var. cuspidatum isolated from the Gulf of Mexico.

Prorocentrum texanum var. texanum and its morphologically distinct yet genetically identical (as based on the sequences of five genes) variety P. texanum var. cuspidatum represent a species of Prorocentrum recently isolated from the Gulf of Mexico. Together, these two varieties represent a sister species to Prorocentrum micans. P. micans has had its sterols, which are ringed lipids common to eukaryotic cell membranes, shown in some studies to be comprised of cholesterol (cholest-5-en-3ß-ol), 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and 4α,23,24-trimethyl-5α-cholestan-3ß-ol (dinostanol) as major sterols, thus placing it within a previously identified cluster of dinoflagellates characterized by the predominance of cholesterol and dinosterol. In this study we have determined the sterol compositions of these two varieties of P. texanum to be abundant in cholesterol, 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and dinostanol such that the varieties are virtually indistinguishable from each other, making them both in general agreement with the sterols of P. micans, its closest species relative. This expands our knowledge of the sterols of this environmentally important dinoflagellate genus.

Developing model systems for dinoflagellates in the post-genomic era.

Dinoflagellates are a diverse group of eukaryotic microbes that are ubiquitous in aquatic environments. Largely photosynthetic, they encompass symbiotic, parasitic, and free-living lineages with a broad spectrum of trophism. Many free-living taxa can produce bioactive secondary metabolites such as biotoxins, some of which cause harmful algal blooms. In contrast, most symbiotic species are crucial for sustaining coral reef health. The year 2023 marked a decade since the first genome data of dinoflagellates became available. The growing genome-scale resources for these taxa are highlighting their remarkable evolutionary and genomic complexities. Here, we discuss the prospect of developing dinoflagellate models using the criteria of accessibility, tractability, resources, research support, and promise. Moving forward in the post-genomic era, we argue for the development of fit-to-purpose models that tailor to specific biological contexts, and that a one-size-fits-all model is inadequate for encapsulating the complex biology, ecology, and evolutionary history of dinoflagellates.

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates.

Increases of atmospheric CO2 cause ocean acidification (OA) and global warming, the latter of which can stratify the water column and impede nutrient supply from deep water. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although the function of AP is known to require pH > 7, how OA affects AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examined the effects of pH conditions (5.5-11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH > 8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, and Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum and K. mikimotoi had the broadest pH niche for AP (7-10) and F. kawagutii the second (8-10), Am. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of Am. carterae AP to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These results in concert suggest OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected more acidified and nutrient-limited future ocean.

First report of epiphytic dinoflagellate Coolia malayensis (Dinophyceae) in the southeastern Pacific Ocean.

Coolia species are epiphytic and benthic dinoflagellates with a cosmopolitan distribution in tropical and subtropical areas. In the austral summer of 2016, during a survey in Bahía Calderilla, a dinoflagellate of the genus Coolia was detected in macroalgae samples, and a clonal culture was established. Subsequently, the cultured cells were observed by scanning electron microscopy (SEM) and identified as C. malayensis based on their morphological characteristics. Phylogenetic analyses based on the LSU rDNA D1/D2 regions confirmed that strain D005-1 corresponded to C. malayensis and clustered with strains isolated from New Zealand, Mexico, and Asia Pacific countries. Although the strain D005-1 culture did not contain yessotoxin (YTX), cooliatoxin, 44-methyl gambierone, or its analogs in detectable amounts by LC-MS/MS, more research is needed to evaluate its toxicity and to determine the possible impact of C. malayensis in northern Chilean waters.

Analysis of research status and trends on marine benthic dinoflagellate toxins: A bibliometric study based on web of science database and VOSviewer.

Marine benthic dinoflagellate toxins, potent bioactive compounds with wide-ranging presence in marine ecosystems, have surged in response to global climate change and human activities, prompting an urgent and imperative inquiry. This study conducts an in-depth review of contemporary research concerning these toxins, employing meticulous bibliometric analysis. Leveraging a dataset of 736 relevant literatures sourced from the Web of Science (spanning from 2000 to May 2023), our analysis delves comprehensively into the scientific discourse surrounding these toxic compounds. Employing tools such as VOSviewer, co-citation analysis, co-occurrence analysis, and cluster analysis, our study yields nuanced insights into the intricate characteristics and trajectories of the field. The co-citation analysis underscores the pivotal role played by benthic and epiphytic dinoflagellates like Ostreopsis and Gambierdiscus in shaping prevailing research trends. Our study identifies four distinct research directions, encompassing the domains of ecology, toxicology, toxin production, and taxonomy. Moreover, it traces the evolutionary journey of research stages, marking the transition from a focus on taxonomy to an emphasis on unraveling molecular mechanisms. The culmination of our comprehensive analysis yields three pertinent research recommendations: a call for widescale global studies, the advancement of rapid toxin monitoring techniques, and a deeper exploration of the factors influencing toxin synthesis and toxicity. These findings provide invaluable insights to researchers grappling with the complex realm of harmful algal blooms and substantially enrich the understanding of this pivotal and pressing field.

In Vitro Phagocytosis of Different Dinoflagellate Species by Coral Cells.

Coral-dinoflagellate symbiosis is a unique biological phenomenon, in which animal cells engulf single-celled photosynthetic algae and maintain them in their cytoplasm mutualistically. Studies are needed to reveal the complex mechanisms involved in symbiotic processes, but it is difficult to answer these questions using intact corals. To tackle these issues, our previous studies established an in vitro system of symbiosis between cells of the scleractinian coral Acropora tenuis and the dinoflagellate Breviolum minutum, and showed that corals direct phagocytosis, while algae are likely engulfed by coral cells passively. Several genera of the family Symbiodiniaceae can establish symbioses with corals, but the symbiotic ratio differs depending on the dinoflagellate clades involved. To understand possible causes of these differences, this study examined whether cultured coral cells show phagocytotic activity with various dinoflagellate strains similar to those shown by intact A. tenuis. We found that (a) A. tenuis larvae incorporate Symbiodinium and Breviolum, but not Cladocopium, and very few Effrenium, (b) cultured coral cells engulfed all four species but the ratio of engulfment was significantly higher with Symbiodinium and Breviolum than Cladocopium and Effrenium, (c) cultured coral cells also phagocytosed inorganic latex beads differently than they do dinoflagellates . It is likely that cultured coral cells preferentially phagocytose Symbiodinium and Breviolum, suggesting that specific molecular mechanisms involved in initiation of symbiosis should be investigated in the future.