DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.

A combined approach detected novel species diversity and distribution of dinoflagellate cysts in the Yellow Sea, China.

Resting cysts of dinoflagellates seed harmful algal blooms (HABs) and their geographic expansion, which makes it fundamentally important to obtain comprehensive inventories of dinoflagellate resting cysts in HABs-prone regions. The Yellow Sea (YS) of China has observed numerous outbreaks of dinoflagellate HABs with some novel species recorded recently indicating an underestimated HABs-causing species diversity. We report our investigation of dinoflagellate cysts of YS via an approach combining metabarcoding sequencing and single-cyst morpho-molecular identification, which identified many novel cyst species and a significant controlling effect of the Yellow Sea Cold Water Mass on cyst composition. The metabarcoding and single cyst-based sequencing detected 11 cyst species never being unambiguously reported in China, 10 never reported as cyst producers, and 3 HABs-causing species never reported from YS. Our detections of many potentially toxic or HABs-causative, particularly novel, cysts and distribution pattern provide important insights into the risks and ecology of dinoflagellate HABs.

Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements.

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent. In this study, using the cosmopolitan HABs-causing species Scrippsiella acuminata as a representative, we measured the transcriptional activity of the most efficient pathway of the energy catabolism tricarboxylic acid (TCA) cycle, cell viability (via neutral red staining), and the cellular ATP content of resting cysts under a set of mock conditions in marine sediments (e.g., 4 °C, darkness, and anoxia) for a maximum period of one year. Based on the correlation analyses among the expression levels of genes, cyst viability, and ATP content, we revealed that the TCA cycle was still a crucial pathway of energetic catabolism for resting cysts under aerobic conditions, and its expression was elevated at higher temperatures, light irradiation, and the early stage of dormancy. Under anaerobic conditions, however, the TCA cycle pathway ceased expression in resting cysts, as also supported by ATP measurements. Our results have laid a cornerstone for the comprehensive revelation of the energetic metabolism and biochemical processes of dormancy of resting cysts in marine sediments.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation.

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 µM; P∼ 0.0362 µM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.

Possible functions of CobW domain-containing (CBWD) genes in dinoflagellates using Karlodinium veneficum as a representative.

Since > 91% of dinoflagellates are proven auxotrophs of vitamin B12 and the cobalamin synthetase W (CobW) is a key gene involved in vitamin B12 synthesis pathway, a number of CobW domain-containing (CBWD) genes in dinoflagellates (DinoCBWDs) were surprisedly found from our transcriptomic and meta-transcriptomic studies. A total of 88 DinoCBWD genes were identified from the genomes and transcriptomes of four dinoflagellates, with five being cloned for full-lengths and characterized using the cosmopolitan and ecologically-important dinoflagellates Karlodinium veneficum and Scrippsiella trochoidea (synonym of Scrippsiella acuminata). DinoCBWDs were verified being irrelevant to vitamin B12 biosynthesis due to their transcriptions irresponsive to vitamin B12 levels and their phylogenetic positions. A comprehensive phylogenetic analysis demonstrated 75 out of the 88 DinoCBWD genes identified belong to three subfamilies of COG0523 protein family, of which most prokaryotic members are reported to be metallochaperones and the eukaryotic members are ubiquitously found but mostly unknown for their functions. Our results from K. veneficum demonstrated DinoCBWDs are associated with metal homeostasis and other divergent functions, with four KvCBWDs involving in zinc homeostasis and KvCBWD1 likely functioning as Fe-type nitrile hydratase activator. In addition, conserved motif analysis revealed the structural foundation of KvCBWD proteins that are consistent with previously described CBWD proteins with GTPase activity and metal binding. Our results provide a stepping-stone toward better understanding the functions of DinoCBWDs and the COG0523 family.

Identification and implications of a core bacterial microbiome in 19 clonal cultures laboratory-reared for months to years of the cosmopolitan dinoflagellate Karlodinium veneficum.

Identification of a core microbiome (a group of taxa commonly present and consistently abundant in most samples of host populations) is important to capture the key microbes closely associated with a host population, as this process may potentially contribute to further revealing their spatial distribution, temporal stability, ecological influence, and even impacts on their host's functions and fitness. The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species, which is also notorious in forming harmful algal blooms (HABs) and causing massive fish-kills. Here we reported the core microbiome tightly associated with 19 strains of K. veneficum that were originally isolated from 6 geographic locations along the coast of China and from an estuary of Chesapeake Bay, United States, and have been maintained in the laboratory for several months to over 14 years. Using high-throughput metabarcoding of the partial 16S rRNA gene amplicons, a total of 1,417 prokaryotic features were detected in the entire bacterial microbiome, which were assigned to 17 phyla, 35 classes, 90 orders, 273 families, and 716 genera. Although the bacterial communities associated with K. veneficum cultures displayed heterogeneity in feature (sequences clustered at 100% sequence similarity) composition among strains, a core set of 6 genera were found persistent in their phycospheres, which could contribute up to 74.54% of the whole bacterial microbiome. Three γ-proteobacteria members of the "core," namely, Alteromonas, Marinobacter, and Methylophaga, were the predominant core genera and made up 83.25% of the core bacterial microbiome. The other 3 core genera, Alcanivorax, Thalassospira, and Ponticoccus, are reported to preferably utilize hydrocarbons as sole or major source of carbon and energy, and two of which (Alcanivorax and Ponticoccus) are recognized as obligate hydrocarbonoclastic bacteria (OHCB). Since OHCB generally present in extremely low abundance in marine water and elevate their abundance mostly in petroleum-impacted water, our detection in K. veneficum cultures suggests that the occurrence of obligate and generalist hydrocarbon-degrading bacteria living with dinoflagellates may be more frequent in nature. Our work identified a core microbiome with stable association with the harmful alga K. veneficum and opened a window for further characterization of the physiological mechanisms and ecological implications for the dinoflagellate-bacteria association.

Measuring viability of dinoflagellate cysts and diatoms with stains to test the efficiency of facsimile treatments possibly applicable to ships' ballast water and sediment.

Expansion of harmful algal bloom (HAB) species through ships' ballast water and sediment has been an increasing concern. Determining whether a microalgal cell, particularly for the toxic and HAB-forming species, is "viable" or "dead" is fundamental to understanding the effectiveness of the many ballast-water treatments that have been considered. To this end, we screened a variety of stains to assess the viability of dinoflagellate (Gymnodinium catenatum, GC) cysts and diatom (Corethron hystrix) vegetative cells to test the efficiency of ballast water treatments. Results showed that the stains fluorescing red or green are not sound candidates for viability measurements due to the interference of chlorophyll-induced red fluorescence or cytosolic green autofluorescence, while the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is limited by its toxicity, pseudo-positive judgment and the consequent confusion between cysts and vegetative cells. We further demonstrated that the stain Neutral Red (NR) is a sound candidate as the "vital stain" and can be easily applied for functionally defining the viability of both dinoflagellate cysts and diatoms. Another stain, the Evans Blue (EB), could be used as a "mortal stain" for the vegetative diatom cells but not a sensitive indicator of viability for GC cysts. The NR staining for GC cysts generally needs a higher dosage (0.005%) and longer staining time (24 h) than that were used for staining zooplankton, diatoms, and vegetative cells of dinoflagellates. In all cases, EB staining defined a "percentage of viable cells" significantly higher than that defined by NR. We conclude that the viability of a population is highly dependent on the species of stains used thus must be referred as a method-defined indicator.

Abundant Species Diversity and Essential Functions of Bacterial Communities Associated with Dinoflagellates as Revealed from Metabarcoding Sequencing for Laboratory-Raised Clonal Cultures.

Interactions between algae and bacteria represent an important inter-organism association in aquatic environments, which often have cascading bottom-up influences on ecosystem-scale processes. Despite the increasing recognition of linkages between bacterioplankton and dynamics of dinoflagellate blooms in the field, knowledge about the forms and functions of dinoflagellate-bacteria associations remains elusive, mainly due to the ephemeral and variable conditions in the field. In this study, we characterized the bacterial community associated with laboratory cultures of 144 harmful algal strains, including 130 dinoflagellates (covering all major taxonomic orders of dinoflagellates) and 14 non-dinoflagellates, via high-throughput sequencing for 16S rRNA gene amplicons. A total of 4577 features belonging to bacteria kingdom comprising of 24 phyla, 55 classes, 134 orders, 273 families, 716 genera, and 1104 species were recovered from the algal culture collection, and 3 phyla (Proteobacteria, Bacteroidetes, and Firmicutes) were universally present in all the culture samples. Bacterial communities in dinoflagellates cultures exhibited remarkable conservation across different algal strains, which were dominated by a relatively small number of taxa, most notably the γ-proteobacteria Methylophaga, Marinobacter and Alteromonas. Although the bacterial community composition between dinoflagellates and non-dinoflagellate groups did not show significant difference in general, dinoflagellates harbored a large number of unique features (up to 3811) with relatively low individual abundance and enriched in the potential methylotrophs Methylophaga. While the bacterial assemblages associated with thecate and athecate dinoflagellates displayed no general difference in species composition and functional groups, athecate dinoflagellates appeared to accommodate more aerobic cellulolytic members of Actinobacteria, implying a more possible reliance on cellulose utilization as energy source. The extensive co-occurrence discovered here implied that the relationships between these algal species and the bacterial consortia could be viewed as either bilaterally beneficial (i.e., mutualism) or unilaterally beneficial at least to one party but virtually harmless to the other party (i.e., commensalism), whereas both scenarios support a long-term and stable co-existence rather than an exclusion of one or the other. Our results demonstrated that dinoflagellates-associated bacterial communities were similar in composition, with enrichment of potential uncultured methylotrophs to one-carbon compounds. This work enriches the knowledge about the fundamental functions of bacteria consortia associated with the phycospheres of dinoflagellates and other HABs-forming microalgae.

Emerging harmful algal bloom species over the last four decades in China.

The first recorded micro-algae bloom in Chinese coastal waters dates back to 1933 and was caused by a mixture of Noctiluca scintillans and Skeletonema costatum sensu lato along the Zhejiang coast (the East China Sea). While well-documented harmful algal blooms (HABs) appeared to be extremely scarce from the 1950s to 1990, both the frequency and intensity have been reportedly increasing since 1990. Among them, the fish-killing HABs, mainly caused by Karenia mikimotoi, Karlodinium digitatum, Karlodinium veneficum, Margalefidinium polykrikoides, and Heterocapsa spp., have intensified. Karenia mikimotoi was responsible for at least two extremely serious events in the Pearl River Estuary in 1998 and the Taiwan Strait (in the East China Sea) in 2012, which appeared to be associated with abnormal climate conditions and excessive nutrients loading. Other major toxic algal blooms have been caused by the species responsible for paralytic shellfish poisoning (including Alexandrium catenella, Alexandrium pacificum, Gymnodinium catenatum) and diarrhetic shellfish poisoning (including Dinophysis spp., and a couple of benthic dinoflagellates). Consequent closures of shellfish farms have resulted in enormous economic losses, while consumption of contaminated shellfish has led to occasional human mortality in the Bohai Sea and the East China Sea. Expansions of these HABs species along the coastline of China have occurred over the last four decades and, due to the projected global changes in the climate and marine environments and other anthropological activities, there is potential for the emergence of new types of HABs in China in the future. This literature review aimed to present an updated overview of HABs species over the last four decades in China.

Geographic distribution and historical presence of the resting cysts of Karenia mikimotoi in the seas of China.

The toxic dinoflagellate Karenia mikimotoi frequently forms harmful algal blooms (HABs) and thus causes massive kills of fish and shellfish in worldwide coastal waters, which has led to intensive investigations on multiple facets of the species. Following our recent discovery of K. mikimotoi forming resting cyst, a very possible mechanism for the inoculation of blooms and geographic expansion for this and many other HABs-causing species, here we report our detection of K. mikimotoi resting cysts in 125 surface sediment samples collected from the coastal waters (covering a latitude range from 18.29°N to 39.85°N) and 3 sediment cores (accumulated in 70‒100 years) collected from the East China Sea where are adjacent to the frequent blooming areas of K. mikimotoi. Via applications of quantitative real-time PCR (LSU rDNA-targeted), species-specific fluorescence in situ hybridization (FISH), and nested-PCR-and-sequencing to both types of the sediment samples that were pretreated with sodium polytungstate solution (SPT), we demonstrated that 1) K. mikimotoi cysts are widely present in surface sediments of the China seas (Bohai Sea (BS), Yellow Sea (YS), East China Sea (ECS), and South China Sea (SCS)), 2) the abundance of cysts is generally low (0 to 33 cysts in 32 g wet sediment), with that in the ECS and the SCS being higher than that in the YS and the BS, and the highest abundance was observed in sites of the ECS (e.g., Ningde, Fujian province) where the blooms of the species occurred frequently, as quantified by both methods, and 3) the cysts of K. mikimotoi have been present in the sediments of the ECS since 1970s, a short time prior to the first recorded bloom of K. mikimotoi in the SCS at 1980s. Our results not only demonstrated the wide geographic distribution of resting cyst of K. mikimotoi along the coast of China, but also proved a 50 years preservation of the cysts in the sediments of coastal area prone to forming frequent blooms. We consider our results have provided critical insights into the mechanisms of frequent bloom outbreaks and global distribution of K. mikimotoi in general, and particularly into the historical origin of K. mikimotoi in China. Further investigations are suggested to focus on on-site surveys for the cyst production and germination rates.

Dependence of genome size and copy number of rRNA gene on cell volume in dinoflagellates.

Dinoflagellates are an ecologically important group of protists in aquatic environment and have evolved many unusual and enigmatic genomic features such as immense genome sizes, high repeated genes, and a large portion of hydroxymethyluracil in DNA. Although previous studies have observed positive correlations between the large subunit (LSU) rRNA gene copy number and genome size of a variety of eukaryotic organisms (e.g. higher plants and animals), or between cell volume and LSU rRNA gene copy number, and/or between genome size and cell size, which suggests a possible co-evolution among these three features in different lineages of life, it remains an open question regarding the relationships among these three parameters in dinoflagellates. For the first time, we estimated the copy numbers of the LSU rRNA gene, the genome sizes, and cell volumes within a broad range of dinoflagellates (covering 15 species of 11 genera) using single-cell qPCR-based assay (determining LSU rRNA gene copy number), FlowCAM (cell volume measurement), and ultraviolet spectrophotometry (genome size estimation). The measured copy number of LSU rRNA gene ranged from 398 ± 184 (Prorocentrum minimum) to 152,078 ± 33,555 copies•cell-1 (Alexandrium pacificum), while the genome size and the cell volume ranged from 5.6 ± 0.2 (Karlodinium veneficum) to 853 ± 19.9 pg•cell-1 (Pseliodinium pirum), and from 1,070 ± 225 (Kar. veneficum) to 168,474 ± 124,180 µm3 (Ps. pirum), respectively. Together with the three parameters measured in literature, there are significant positive linear correlations between LSU rRNA gene copy numbers and genome sizes, cell volumes and LSU rRNA gene copy numbers, and between genome sizes and cell volumes via comparisons of multi-model regression analyses, suggesting a dependence of genome size and rRNA gene copy number on the cell volumes of dinoflagellates. Validation of the measurement methods was conducted via comparisons between reported data in the literature and that predicted using the linear equations we obtained, and between genome size measured by flow cytometry (FCM) and ultraviolet spectrophotometry (Nanodrop). These results provide insightful understandings of dinoflagellate evolution in terms of the relationships among genomes, gene copy number, and cell volume, and of rRNA gene-based studies in intra-populational and intra-individual genetic diversity, taxonomy, and diversity assessment in the environment of dinoflagellates. The results also provide a dataset useful for reads calibration in environmental metabarcoding studies of dinoflagellates and selection of candidate species for whole genome sequencing.

Expression Patterns of the Heat Shock Protein 90 (Hsp90) Gene Suggest Its Possible Involvement in Maintaining the Dormancy of Dinoflagellate Resting Cysts.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone functioning in cellular structural folding and conformational integrity maintenance and thus plays vital roles in a variety of biological processes. However, many aspects of these functions and processes remain to be fully elucidated, particularly for non-model organisms. Dinoflagellates are a group of eukaryotes that are exceedingly important in primary production and are responsible for the most harmful algal blooms (HABs) in aquatic ecosystems. The success of dinoflagellates in dominating the plankton community is undoubtedly pertinent to their remarkable adaptive strategies, characteristic of resting cyst production and broad tolerance to stresses of temperature and others. Therefore, this study was conducted to examine the putative roles of Hsp90 in the acclimation to temperature stress and life stage alterations of dinoflagellates. Firstly, we isolated the full-length cDNA of an Hsp90 gene (StHsp90) via RACE from the cosmopolitan HAB species Scrippsiella trochoidea and tracked its transcriptions in response to varied scenarios via real-time qPCR. The results indicated that StHsp90 displayed significant mRNA augment patterns, escalating during 180-min treatments, when the cells were exposed to elevated and lowered temperatures. Secondly, we observed prominently elevated StHsp90 transcriptions in the cysts that were stored at the cold and dark conditions compared to those in newly formed resting cysts and vegetative cells. Finally, and perhaps most importantly, we identified 29 entries of Hsp90-encoding genes with complete coding regions from a dinoflagellate-specific environmental cDNA library generated from marine sediment assemblages. The observed active transcription of these genes in sediment-buried resting cysts was fully supported by the qPCR results for the cold-stored resting cysts of S. trochoidea. Hsp90s expressions in both laboratory-raised and field-collected cysts collectively highlighted the possible involvement and engagement of Hsp90 chaperones in the resting stage persistence of dinoflagellates.

Morpho-molecular description of a new HAB species, Pseudocochlodinium profundisulcus gen. et sp. nov., and its LSU rRNA gene based genetic diversity and geographical distribution.

Harmful algal blooms (HABs) caused by an unknown dinoflagellate species have frequently occurred in the Pearl River Estuary, China Since 2006. These blooms were associated with severe water discoloration and economic losses, ranging from several km2 to 300 km2 with the maximum recorded cell density being 2.77 × 107 cells⋅L-1. This unknown dinoflagellate species was initially identified as Cochlodinium geminatum and subsequently reclassified as Polykrikos geminatus. However, after reviewing the original descriptions for Cochlodinium geminatum sensu Schütt (1895) and the genus Polykrikos, we considered this species is incongruent with their original descriptions. Further morphological examinations and particularly phylogenetic analyses based on the SSU and partial LSU rRNA genes of isolates and resting cysts from China and Japan prompted us to consider it a new species of a new genus. This new species was proposed to be Pseudocochlodinium profundisulcus gen. et sp. nov., based on its open comma-shaped apical structure complex (ASC), cingulum encircling the cell less than one and a half turns, a deep sulcus with a torsion of a half turn, either single cell or cell chain consisting of two cells with the same number of nuclei and zooids, the resting cyst bearing lobed ornaments, and the evolutionary distances from Polykrikos (and others) on the phylogenetic trees constructed using the concatenated SSU and partial LSU rRNA gene sequences. Metabarcoding investigation of surface sediment samples collected in China revealed that the species to be widely present along the entire Chinese coast with the highest abundance in the South China Sea. Further re-analysis of the Tara Oceans metabarcoding dataset targeting the SSU rRNA gene V9 domain suggested a global distribution of this new genus. Phylogenetic analyses on 46 OTUs (average length: ∼552 bases) of its LSU rRNA gene sequences (mainly D1-D2 domains) obtained from surface sediment samples revealed intraspecific genetic diversity of this species. Interestingly, based on the different distributions and the abundance of these OTUs along the coast of China, this species appeared to have expanded its distribution from the South China Sea to the northern Yellow Sea, or preferred a warm water habitat. We consider that the present work improves the taxonomy and provides important insights into the biogeography of Pseudocochlodinium profundisulcus.⋅.

Exploration of resting cysts (stages) and their relevance for possibly HABs-causing species in China.

The studies on the species diversity, distribution, environmental implications, and molecular basis of resting cysts (stages) of dinoflagellates and a few species of other groups conducted in China during the last three decades are reviewed. The major achievements are summarized as the following five aspects: 1) The continual efforts in detecting the species diversity of resting cysts (spores) in dinoflagellates and other classes using either morphological or molecular approaches, or both, in the four seas of China, which led to identifications of 106 species of dinoflagellate resting cysts and 4 species of resting stages from other groups of microalgae, with a total of 64 species of dinoflagellate cysts and the resting stage of the brown tide-causing Aureococcus anophagefferens being unequivocally identified via molecular approaches from the sediments of Chinese coastal waters; 2) The well-known toxic and HABs-causing dinoflagellates Karenia mikimotoi, Karlodinium veneficum, Akashiwo sanguinea and the pelagophyte A. anophagefferens were proven to be resting cyst (stage) producers via laboratory studies on their life cycles and field detections of resting cysts (resting stage cells). And, via germination experiment and subsequent characterization of vegetative cells, numerous dinoflagellate species that had never been described or found to form cysts were discovered and characterized; 3) The distributions of the resting cysts of Alexandrium catenella, A. pacificum, Gymnodinium catenatum, K. mikimotoi, K. veneficum and Azadinium poporum and the resting stage cells of A. anophagefferens were morphologically and molecularly mapped in all four seas of China, with A. anophagefferens proven to have been present in the Bohai Sea for at least 1,500 years; 4) Obtaining important insights into the 'indicator' values of the dinoflagellate cyst assemblages in sediment cores for tracking eutrophication, environmental pollution and other anthropological influences in coastal waters; 5) Studies on the cyst-pertinent processes and genetic basis (transcriptomics together with physiological and chemical measurements) of resting cyst dormancy not only revealed the regulating patterns of some environmental factors in cyst formation and germination, but also identified many characteristically active or inactive metabolic pathways, differentially expressed genes, and the possibly vital regulating function of the phytohormone abscisic acid and a group of molecular chaperones in resting cysts. We also identified seven issues and three themes that should be addressed and explored by Chinese scientists working in the area in the future.

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the ß subunit of ATP synthase gene (ß-F1-ATPase), a key indicator for the status of cell's energetic metabolism. The full-length cDNA of ß-F1-ATPase gene from S.trochoidea (Stß-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stß-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stß-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stß-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays.

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24 h exposed to the live culture (2000-6710 cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48 h when exposed to 500-6000 cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100 cells·mL-1 within 24 h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.

Pseudocochlodinium profundisulcus Resting Cysts Detected in the Ballast Tank Sediment of Ships Arriving in the Ports of China and North America and the Implications in the Species' Geographic Distribution and Possible Invasion.

Over the past several decades, much attention has been focused on the dispersal of aquatic nonindigenous species via ballast tanks of shipping vessels worldwide. The recently reclassified dinoflagellate Pseudocochlodinium profundisulcus (previously identified as Cochlodinium sp., Cochlodinium geminatum, or Polykrikos geminatus) was not reported in China until 2006. However, algal blooming events caused by this organism have been reported almost every year since then in the Pearl River Estuary and its adjacent areas in China. Whether P. profundisulcus is an indigenous or an invasive species has thus become an ecological question of great scientific and practical significance. In this study, we collected the sediments from ballast tanks of ships arriving in the ports of China and North America and characterized dinoflagellate resting cysts via a combined approach. We germinated two dark brownish cysts from the tank of an international ship (Vessel A) arriving at the Jiangyin Port (China) into vegetative cells and identified them as P. profundisulcus by light and scanning electron microscopy and phylogenetic analyses for partial LSU rDNA sequences. We also identified P. profundisulcus cyst from the ballast tank sediment of a ship (Vessel B) arriving in the port of North America via single-cyst PCR and cloning sequencing, which indicated that this species could be transported as resting cyst via ship. Since phylogenetic analyses based on partial LSU rDNA sequences could not differentiate all sequences among our cysts from those deposited in the NCBI database into sub-groups, all populations from China, Australia, Japan, and the original sources from which the cysts in the two vessels arrived in China and North America were carried over appeared to share a very recent common ancestor, and the species may have experienced a worldwide expansion recently. These results indicate that P. profundisulcus cysts may have been extensively transferred to many regions of the world via ships' ballast tank sediments. While our work provides an exemplary case for both the feasibility and complexity (in tracking the source) of the bio-invasion risk via the transport of live resting cysts by ship's ballast tanks, it also points out an orientation for future investigation.

Contact micropredation may play a more important role than exotoxicity does in the lethal effects of Karlodinium australe blooms: Evidence from laboratory bioassays.

Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe, while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L-1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L-1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general.

Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga Scrippsiella trochoidea (Dinophyceae).

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.

An assessment on the intrapopulational and intraindividual genetic diversity in LSU rDNA in the harmful algal blooms-forming dinoflagellate Margalefidinium (= Cochlodinium) fulvescens based on clonal cultures and bloom samples from Jiaozhou Bay, China.

Large subunit ribosomal DNA (LSU rDNA) sequences have been increasingly used to infer the phylogeny and species identity of organisms, a few previous studies, however, have observed high intraspecific and even intraindividual variability in LSU rDNA in some dinoflagellate species due to, assumably, large copy numbers of rDNA in dinoflagellates. Since the copy number of LSU rDNA varies tremendously among dinoflagellate species, the intraspecific and intraindividual diversity for a species of particular interest thus needs to be investigated individually. As a toxic and HABs-forming dinoflagellate, Margalefidinium (= Cochlodinium) fulvescens has been observed to approach blooming density in Jiaozhou Bay, China since 2015 after numerous blooms having been reported from other countries. In trying to identify the source of this newly observed HABs-forming species in China by sequencing the LSU rDNA for both field samples and clonal cultures, we noticed and thus further investigated high intrapopulational and intraindividual genetic diversities of the dinoflagellate. The D1-D6 region of the LSU rDNA (1,435 bases) was amplified from 7 field samples (pooled cells) and 11 clonal cultures, cloned, sequenced, and analyzed phylogenetically for 2,341 sequences obtained. All the numbers of sequences obtained from each clonal culture were far less than the estimated rDNA copy number in M. fulvescens. In the clone library, only one unique sequence was contained in all samples as the most dominant sequence. We found high intrapopulational and intraindividual genetic diversity in M. fulvescens as reflected in the number of polymorphic sites and unique sequences in the clone library for different field samples and clonal cultures in comparison to other species. The mean number of nucleotide differences of each sequence from different field samples and clonal cultures were 6.43 and 4.42 bases, respectively, with the highest being 132 bases, nearly 10%. The sequences with highest variability may be easily annotated as different species if they were obtained from environmental genomic studies because sequence-based species identification in meta-barcoding studies often use "97% identity" threshold. Based on that the mean and overall intrapopulational genetic diversity calculated for 7 field samples was equivalent to the mean and overall intraindividual variability for 11 clonal cultures in indices of genetic diversity, together with the result of AMOVA analysis, we infer that the variability within individual cells (i.e. variability among LSU rDNA polymorphic copies) caused both the intraindividual and intrapopulational genetic diversities observed in the M. fulvescens population, and a higher interpopulational diversity may exist among different geographic populations. The results provide an insightful basis for such a comprehensive interpopulational comparison and important implications for identifying species and establishing new taxa based on the similarity comparison to reference sequences deposited in databases.