Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

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.

Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China.

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 µg/mL with a mean of 0.207 ± 0.0053 µg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

Sensitivity to 12 Fungicides and Resistance Mechanism to Trifloxystrobin, Carbendazim, and Succinate Dehydrogenase Inhibitors in Cucumber Corynespora Leaf Spot (Corynespora cassiicola).

Corynespora cassiicola is the causal agent of cucumber Corynespora leaf spot, which affects many economically important plant species. Chemical control of this disease is hampered by the common development of fungicide resistance. In this study, 100 isolates from Liaoning Province were collected, and their sensitivity to 12 fungicides was determined. All the isolates (100%) were resistant to trifloxystrobin and carbendazim, and 98% were resistant to fluopyram, boscalid, pydiflumetofen, isopyrazam, and fluxapyroxad. However, none were resistant to propiconazole, prochloraz, tebuconazole, difenoconazole, and fludioxonil. The Cytb gene of trifloxystrobin-resistant isolates encoded the G143A mutation, whereas the ß-tubulin gene of carbendazim-resistant isolates encoded the E198A and E198A and M163I mutations. Mutations in SdhB-I280V, SdhC-S73P, SdhC-H134R, SdhD-D95E, and SdhD-G109V were associated with resistance to the succinate dehydrogenase inhibitors (SDHIs). Trifloxystrobin, carbendazim, and fluopyram were barely effective on the resistant isolates, whereas fludioxonil and prochloraz were effective on the isolates that were resistant to the quinone outside inhibitors (QoIs), SDHIs, and benzimidazoles. Ultimately, this study demonstrates that fungicide resistance seriously threatens the effective control of Corynespora leaf spot.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China.

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Occurrence and mechanism of target-site resistance to bensulfuron-methyl in Monochoria korsakowii from China.

Monochoria korsakowii is an increasingly significant threat to rice production across China, particularly in Liaoning province. Few studies have reported herbicide resistance in M. korsakowii, and resistance status and mechanisms are poorly understood. Here, thirty field populations of M. korsakowii were collected from 11 rice-growing regions of Liaoning, and 97% of populations had evolved resistance to bensulfuron-methyl (BM), with majority (24 of 28) showing high resistance levels (RI > 10). The first in-depth analysis of molecular features of AHAS1 and AHAS2 in BM-resistant populations showed that four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified. Notably, novel double Pro197Ser mutations co-occurred in both AHAS1 and AHAS2 in the most resistant line LN-20. Furthermore, resistant mutants were used to investigate the effect of Pro197 mutations on AHAS functionality, binding modes, gene expression and cross-resistance in M. korsakowii. All the detected Pro197 mutations considerably reduced in vitro AHAS sensitivity to BM by weakening hydrogen bonds and hydrophobic interactions in the predicted BM-AHAS complexes, especially the double Pro197Ser mutations. This novel resistance mutation combination slightly impacted the extractable AHAS activity, and increased the affinity and catalytic rate of pyruvate. Also, the AHAS expression level was significantly up-regulated. Moreover, all mutations provided resistance only to other sulfonylureas herbicides but not triazolopyrimidine or pyrimidinyl-benzoates herbicides. In conclusion, bensulfuron-methyl resistance in M. korsakowii was grim in Liaoning, China, and amino acid mutations on AHAS isozymes were the primary resistance mechanism. Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. Thus, this work deepens our understanding of resistance status and mechanisms of M. korsakowii.

Different Geographic Strains of Dinoflagellate Karlodinium veneficum Host Highly Diverse Fungal Community and Potentially Serve as Possible Niche for Colonization of Fungal Endophytes.

In numerous studies, researchers have explored the interactions between fungi and their hosting biota in terrestrial systems, while much less attention has been paid to the counterpart interactions in aquatic, and particularly marine, ecosystems. Despite the growing recognition of the potential functions of fungi in structuring phytoplankton communities, the current insights were mostly derived from phytoplankton hosts, such as diatoms, green microalgae, and cyanobacteria. Dinoflagellates are the second most abundant group of phytoplankton in coastal marine ecosystems, and they are notorious for causing harmful algal blooms (HABs). In this study, we used high-throughput amplicon sequencing to capture global snapshots of specific fungal assemblages associated with laboratory-cultured marine dinoflagellate. We investigated a total of 13 clonal cultures of the dinoflagellate Karlodinium veneficum that were previously isolated from 5 geographic origins and have been maintained in our laboratory from several months to more than 14 years. The total recovered fungal microbiome, which consisted of 349 ASVs (amplicon sequencing variants, sequences clustered at a 100% sequence identity), could be assigned to 4 phyla, 18 classes, 37 orders, 65 families, 97 genera, and 131 species. The fungal consortium displayed high diversity and was dominated by filamentous fungi and ascomycetous and basidiomycetous yeasts. A core set of three genera among all the detected fungi was constitutively present in the K. veneficum strains isolated from geographically distant regions, with the top two most abundant genera, Thyridium and Pseudeurotium, capable of using hydrocarbons as the sole or major source of carbon and energy. In addition, fungal taxa previously documented as endophytes in other hosts were also found in all tested strains of K. veneficum. Because host-endophyte interactions are highly variable and strongly case-dependent, these fungal taxa were not necessarily genuine endosymbionts of K. veneficum; instead, it raised the possibility that dinoflagellates could potentially serve as an alternative ecological niche for the colonization of fungal endophytes. Our findings lay the foundation for further investigations into the potential roles or functions of fungi in the regulation of the growth dynamics and HABs of marine dinoflagellates in the field.

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.

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.

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.

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.

Development of a LAMP method for detecting F129L mutant in azoxystrobin-resistant Pyricularia oryzae.

Azoxystrobin has been widely used since 1996 to control rice blast caused by Pyricularia oryzae. Azoxystrobin resistance related to mutations at the P. oryzae target protein (F129L of Cytb) has been reported worldwide. To quickly identify and detect resistant strains in the field, this research established a rapid loop-mediated isothermal amplification (LAMP) detection system for the F129L mutation. The system could detect the P. oryzae F129L (TTC-TTA) mutation at 62 °C within 60 min, with a detection limit of 100 fg/µL, which is 10 times higher than for conventional PCR. The method had high specificity and repeatability and could detect the F129L (TTC-TTA) mutation in plant tissues within 3 h. The LAMP method established in this study will be useful to detect azoxystrobin-resistant P. oryzae F129L mutant strains and generate significant data for the management of resistant P. oryzae isolates.

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.