Alexandrium catenella (Group I) and A. pacificum (Group IV) cyst germination, distribution, and toxicity in Jinhae-Masan Bay, Korea.

To better understand the outbreaks of paralytic shellfish poisoning and bloom dynamics caused by Alexandrium species in Jinhae-Masan Bay, Korea, the germination and distributions of ellipsoidal Alexandrium cysts were investigated, and paralytic shellfish toxins (PSTs) profiles and contents were determined using strains established from germling cells. The phylogeny and morphological observations revealed that the germinated vegetative cells from ellipsoidal cysts collected from the surface sediments in Jinhae-Masan Bay belong to Alexandrium catenella (Group I) and A. pacificum (Group IV) nested within A. tamarense species complex. Cyst germinations of A. catenella (Group I) were observed at only 10 °C, whereas cysts of A. pacificum (Group IV) could germinate at temperature ranges of 10 to 25 °C. Maximum germination success (85%) for isolated cysts occurred at 15 °C, and the germling cells were A. pacificum (Group IV). The results indicate that the variation in water temperature in Jinhae-Masan Bay can control the seasonal variations in germination of cysts of A. catenella (Group I) and A. pacificum (Group IV). The germination rates of ellipsoidal Alexandrium cysts were different among sampling sites in Jinhae-Masan Bay, probably because of differences in distribution and abundance of A. catenella (Group I) and A. pacificum (Group IV) in the sediments. The ellipsoidal Alexandrium cyst concentrations were much higher in February than in August, however the distributions were similar. Gonyautoxins 3 and 4 (GTX-3 and GTX-4) contributed a large proportion (>90%) of the toxins produced by strains A. catenella (Group I) and A. pacificum (Group IV) established from germling cells, and the total cellular contents were higher in A. catenella (Group I) than in A. pacificum (Group IV).

The harmful raphidophyte Chattonella (Raphidophyceae) in Western Pacific: Its red tides and associated fisheries damage over the past 50 years (1969-2019).

Red tides and associated fisheries damage caused by the harmful raphidophyte Chattonella were reassessed based on the documented local records for 50 years to understand the distribution and economic impacts of the harmful species in the Western Pacific. Blooms of Chattonella with fisheries damage have been recorded in East Asia since 1969, whereas they have been only recorded in Southeast Asia since the 1980s. Occurrences of Chattonella have been documented from six Southeast Asian countries, Indonesia, Malaysia, Philippines, Singapore, Thailand and Viet Nam, with mass mortalities mainly of farmed shrimp in 1980-1990s, and farmed fish in 2000-2010s. These occurrences have been reported with the names of C. antiqua, C. marina, C. ovata, C. subsalsa and Chattonella sp., owing to the difficulty of microscopic species identification, and many were not supported with molecular data. To determine the distribution of C. marina complex and C. subsalsa in Southeast Asia, molecular phylogeny and microscopic observation were also carried out for cultures obtained from Indonesia, Malaysia, Japan, Philippines, Russia, Singapore and Thailand. The results revealed that only the genotype of C. marina complex has been detected from East Asia (China, Japan, Korea and Russia), whereas both C. marina complex (Indonesia and Malaysia) and C. subsalsa (Philippines, Singapore and Thailand) were found in Southeast Asia. Ejection of mucocysts has been recognized as a diagnostic character of C. subsalsa, but it was also observed in our cultures of C. marina isolated from Indonesia, Malaysia, Japan, and Russia. Meanwhile, the co-occurrences of the two harmful Chattonella species in Southeast Asia, which are difficult to distinguish solely based on their morphology, suggest the importance of molecular identification of Chattonella genotypes for further understanding of their distribution and negative impacts.

Harmful algal blooms and associated fisheries damage in East Asia: Current status and trends in China, Japan, Korea and Russia.

Occurrences of harmful algal blooms (HABs) and associated fisheries damage have been continuously monitored since the 1970s along the coasts of East Asia. Fisheries damage comprises mass mortalities of fish and shellfish mainly by harmful dinoflagellates and raphidophytes (e.g., Chattonella antiqua/marina, Cochlodinium polykrikoides and Karenia mikimotoi), and contamination of algal toxins in shellfish in particular Diarrhetic Shellfish Toxins by Dinophysis spp. and Paralytic Shellfish Toxins by Alexandrium spp. Shellfish mass mortalities due to Heterocapsa circularisquama in Hong Kong and western Japan, and fish kills by Karlodinium digitatum are unique incidents for this region, whereas C. antiqua/marina, C. polykrikoides and K. mikimotoi are common also in other regions. Time series data showed that the highest bloom numbers were recorded in 1980 (Japan), in 1998 (Korea) and in 2003 (China), followed by decreasing trends in these countries. These data suggest a shift in microalgal species composition, from dominance by diatoms to dinoflagellates after 1980s in Korea, and from diatoms to small haptophytes and cyanobacteria after 2013 in eastern Russia. HAB species composition and the changes were compared among countries, for better understanding on current status and trend of HAB species in East Asia.

Centrodinium punctatum (Dinophyceae) produces significant levels of saxitoxin and related analogs.

Centrodinium punctatum is a fusiform dinoflagellate with a global marine distribution. Due to a close phylogenetic relationship of one C. punctatum strain to Alexandrium species, toxin production of this C. punctatum strain was assessed using liquid chromatography coupled to tandem mass spectrometry. The paralytic shellfish toxin (PST) profile of C. punctatum was dominated by six analogs, i.e. STX (30%), GTX-1 (20%) and neoSTX (24%), followed by GTX-2 (9%), GTX-4 (9%) and GTX-3 (8%); deoxy-STX was also putatively identified while no gymnodimines, spirolides or goniodomins were detected. This is the first record of C. punctatum producing saxitoxins. The estimated cellular toxicity was rather elevated, between 91 and 212 pg cell-1 (or 259 and 601 fmol cell-1). When considering the toxicity equivalent factors, results suggest that this species can produce high cellular toxicity compared to other STX-producing dinoflagellates. Morphological details of the sulcal area and the hypotheca of Centrodinium punctatum were re-examined by scanning electron microscopy (SEM); this revealed that in the sulcal area, the left posterior sulcal plate (Ssp) is larger and longer than the left posterior sulcal plate and extended into the hypotheca. Based on the morphological observation, a revised interpretation of the sulcus and hypotheca is proposed.

Conditions of nutrients and dissolved organic matter for the outbreaks of Paralytic Shellfish Poisoning (PSP) in Jinhae Bay, Korea.

We measured the concentrations of nutrients, fluorescent dissolved organic matter (FDOM), and photosynthetic pigments in seawater during the springs of 2018 and 2019 in Jinhae Bay, Korea. The samplings were carried out during the severe and weak outbreaks of paralytic shellfish poisoning (PSP) in April 2018 and March 2019, respectively. The additional sampling campaigns were carried out before and after the PSP outbreak for the comparison. During the severe PSP outbreak, lower salinities, higher organic and total nutrients, and higher humic-like FDOM were observed. Although the environmental condition of April 2018 is favorable for the growth of dinoflagellates, the lowest peridinin (dinoflagellate index) and highest fucoxanthin (diatom index) concentrations were observed amongst all sampling periods. Thus, our results suggest that PSP could be more effectively produced by dinoflagellates in the course of the ecological shift by interspecific competition under the environmental condition favorable for dinoflagellates.

Comparative transcriptomics of toxin synthesis genes between the non-toxin producing dinoflagellate Cochlodinium polykrikoides and toxigenic Alexandrium pacificum.

In the present study, we extensively characterized potential toxin-related genes, including polyketide synthase (PKS), saxitoxin (STX) and fatty acid synthase (FAS) from the non-toxin producing marine dinoflagellate Cochlodinium polykrikoides, comparing to those of a toxigenic dinoflagellate Alexandrium pacificum. RNA sequencing revealed 50 and 271 PKS contigs from C. polykrikoides and A. pacificum, respectively. According to domain constitute and amino acid alteration, we further classified the dinoflagellate type I PKS genes into 4 sub-groups. Type III PKS was first identified in C. polykrikoides. Interestingly, we detected a large number (242 and 288) of homologs of 18 sxt genes from two studied dinoflagellates. Most of the eight key genes (sxtA, sxtB, sxtD, sxtG, sxtH/T, sxtI, sxtS and sxtU) for STX synthesis were detected in both dinoflatellates, whereas a core STX biosynthesis gene sxtG was not detected in C. polykrikoides. This may partially explain the absence of saxitoxin production in C. polykrikoides. In addition, we identified several type I and type II FAS genes, including FabD, FabF, FabG, FabH, FabI, and FabZ, whereas FabB was not found in C. polykrikoides. Overall, the numbers of the toxin-related genes in C. polykrikoides were less than that of A. pacificum. Phylogenetic analyses showed that type I PKS/FASs of dinoflagellates had close relationships with apicomplexans and bacteria. These suggest that the toxin-related PKS and sxt genes are commonly present in toxigenic and non-toxin producing dinoflagellates, and may be involved not only in the toxin synthesis, but also in other related molecular metabolic functions.

Prorocentrum shikokuense Hada and P. donghaiense Lu are junior synonyms of P. obtusidens Schiller, but not of P. dentatum Stein (Prorocentrales, Dinophyceae).

In Japanese, Chinese and Korean coastal waters, recurrent blooms of a small, elongate Prorocentrum species have been observed in recent years. In previous studies, this species has been respectively identified as P. shikokuense, P. donghaiense and P. dentatum, despite morphological similarity and identical rDNA sequences. To resolve the confusion, morphological features, including the architectural details of the periflagellar area, were examined and ribosomal DNA (rDNA) sequences were obtained from specimens collected from the East China Sea and Korean coast, and a strain established in the waters off the Canary Islands of Spain. In addition, the descriptions of the three species and allied species were reviewed. Morphological observations and a phylogeny based on the SSU, ITS region and LSU rDNA sequences revealed that the previously confused species and our studied strains are conspecific and that the morphology of strains identified as P. dentatum in the phylogenetic trees does not coincide with P. dentatum sensu stricto. The confusion can be traced back to Dodge (1975, p. 116), who considered P. veloi, P. monacense and P. obtusidens as junior heterotypic synonyms of P. dentatum. However, Dodge's P. dentatum are closer to P. obtusidens sensu stricto, rather than P. dentatum sensu stricto. P. obtusidens sensu stricto can be distinguished from P. dentatum sensu stricto by its relatively small size, parallel sides towards the anterior and a blunt anterior extension on one side. This indicates that P. obtusidens should not be considered a synonym of P. dentatum sensu stricto. In addition, a comparison of the original descriptions of P. obtusidens and allied species allowed to conclude that small, elongate Prorocentrum from Japanese, Chinese and Korean coastal waters previously identified as P. shikokuense, P. donghaiense, P. dentatum sensu Yoo and Lee (1986) and the specimens studied herein, which share identical rDNA sequences, morphologically coincide with P. obtusidens. Therefore, it is proposed that P. shikokuense and P. donghaiense should be regarded as junior synonyms of P. obtusidens.

Tracing the sources of nutrients fueling dinoflagellate red tides occurring along the coast of Korea using radium isotopes.

It is a well held concept that the magnitude of red-tide occurrence is dependent on the amount of nutrient supply if the conditions are same for temperature, salinity, light, interspecific competition, etc. However, nutrient sources fueling dinoflagellate red-tides are difficult to identify since red tides usually occur under very low inorganic-nutrient conditions. In this study, we used short-lived Ra isotopes (223Ra and 224Ra) to trace the nutrient sources fueling initiation and spread of Cochlodinium polykrikoides blooms along the coast of Korea during the summers of 2014, 2016, and 2017. Horizontal and vertical distributions of nutrient concentrations correlated well with 224Ra activities in nutrient-source waters. The offshore red-tide areas showed high 224Ra activities and low-inorganic and high-organic nutrient concentrations, which are favorable for blooming C. polykrikoides in competition with diatoms. Based on Ra isotopes, the nutrients fueling red-tide initiation (southern coast of Korea) are found to be transported horizontally from inner-shore waters. However, the nutrients in the spread region (eastern coast of Korea), approximately 200 km from the initiation region, are supplied continuously from the subsurface layer by vertical mixing or upwelling. Our study highlights that short-lived Ra isotopes are excellent tracers of nutrients fueling harmful algal blooms in coastal waters.

Yellow clay modulates carbohydrate and glutathione responses in the harmful dinoflagellate Cochlodinium polykrikoides and leads to sedimentation.

The marine dinoflagellate Cochlodinium polykrikoides is a harmful algal bloom (HAB) species that severely impacts the environment and causes huge economic losses. Yellow clay (YC), considered to be a non-toxic and naturally-occurring material, represents an important step towards the direct control of HABs. In the present study, we evaluated the physiological and biochemical effects of YC on C. polykrikoides after exposures of up to 72 h. We observed little physiological changes in growth rate, chlorophyll a, lipid peroxidation, antioxidant enzymatic activities of superoxide dismutase and catalase, and activity of alkaline phosphatase after exposure to YC. Interestingly, YC significantly increased total carbohydrate and glutathione levels, affecting the physiology of the cells. These results indicate that total carbohydrate content may play an important role in cell-clay aggregation and it could be the main underlying mechanism that mitigates HAB cells via sedimentation.

Molecular cloning and oxidative-stress responses of a novel manganese superoxide dismutase (MnSOD) gene in the dinoflagellate Prorocentrum minimum.

Dinoflagellate algae are microeukaryotes that have distinct genomes and gene regulation systems, making them an interesting model for studying protist evolution and genomics. In the present study, we discovered a novel manganese superoxide dismutase (PmMnSOD) gene from the marine dinoflagellate Prorocentrum minimum, examined its molecular characteristics, and evaluated its transcriptional responses to the oxidative stress-inducing contaminants, CuSO4 and NaOCl. Its cDNA was 1238 bp and contained a dinoflagellate spliced leader sequence, a 906 bp open reading frame (301 amino acids), and a poly (A) tail. The gene was coded on the nuclear genome with one 174 bp intron; signal peptide analysis showed that it might be localized to the mitochondria. Real-time PCR analysis revealed an increase in gene expression of MnSOD and SOD activity when P. minimum cells were separately exposed to CuSO4 and NaOCl. In addition, both contaminants considerably decreased chlorophyll autofluorescence, and increased intracellular reactive oxygen species. These results suggest that dinoflagellate MnSOD may be involved in protecting cells against oxidative damage.

Significant and conservative long-range transport of dissolved organic nutrients in the Changjiang diluted water.

The Changjiang River is one of the main nutrient sources in the northwestern Pacific marginal seas. However, most of the previous studies have neglected the long-range transport (>200 km) of riverine nutrients since they are rapidly consumed. In this study, we examined the long-range transport (200-800 km) of nutrients in the surface layer during the summer of 2017. The plots of nutrients against salinity display that dissolved organic nitrogen (DON) was conservative over ~800 km, while more than 99% of the dissolved inorganic nitrogen (DIN) was removed within 200 km. As a result, in the study region, DON concentrations (avg. 7.0 ± 1.3 µM), which are minor in the river water, were much higher than DIN concentrations (avg. 0.28 ± 0.26 µM). Both nutrients, N and P, showed a similar pattern. Our results suggest that dissolved organic nutrients play a critical role on the long-range transport of riverine nutrients in surface waters and subsequent ecosystem changes.

CpMCA, a novel metacaspase gene from the harmful dinoflagellate Cochlodinium polykrikoides and its expression during cell death.

Metacaspases (MCAs) are cysteine proteases that share sequence homology with caspases, and may play roles in programmed cell death (PCD). In the present study, we identified a novel MCA gene (CpMCA) from the red tide dinoflagellate Cochlodinium polykrikoides, and examined its molecular characteristics and gene expression in response to algicide-induced cell death. CpMCA cDNA is 1164 bp in length, containing a dinoflagellate spliced leader sequence (dinoSL), an 879-bp open reading frame (ORF), which codes for a 293-aa protein, and a poly (A) tail. Multi-sequence comparison indicated that CpMCA belongs to type I MCA, but it has a different structure at the N-terminal. Phylogenetic analysis showed that C. polykrikoides may have acquired the MCA gene from bacteria by means of horizontal gene transfer (HGT). In addition, expressions of CpMCA significantly increased following exposure to the common algicides copper sulfate and oxidizing chlorine, which trigger cell death in dinoflagellates, suggesting that CpMCA may be involved in cell death.

Which species, Alexandrium catenella (Group I) or A. pacificum (Group IV), is really responsible for past paralytic shellfish poisoning outbreaks in Jinhae-Masan Bay, Korea?

Paralytic shellfish poisoning (PSP) caused the deaths of four people in coastal area of Korea, mainly Jinhae-Masan Bay and adjacent areas, in April 1986 and in 1996. The PSP outbreaks were caused by the consumption of mussels, Mytilus edulis. The organism that caused PSP was identified, from morphological data only, as Alexandrium tamarense which is recently renamed as A. catenella, however recent studies have shown that the morphological diagnostic characteristics used to identify Alexandrium species have uncertainties and molecular tools and other criteria should be considered as well. The organism that caused past PSP outbreaks and incidents in Korea therefore need to be carefully reconsidered. The aim of this study was to re-evaluate the species really responsible for past outbreaks of PSP in Jinhae-Masan Bay, Korea. The temporal production and fluxes of the resting cysts of Alexandrium species were investigated for one year (from March 2011 to February 2012) using a sediment trap, and the morphology and phylogeny of vegetative cells germinated from the resting cysts were analysed. The production of Alexandrium species peaked in August and November, when temporal discrepancies were found in the water temperature (22.4 and 22.7°C in August, 19.1 and 19.6°C in November) and salinity (29.5 and 26.1 psu in August, 30.5 and 31.8 psu in November). The morphological data revealed that Alexandrium species germinated from resting cysts collected in August have a ventral pore on the 1' plate, whereas the 1' plate in Alexandrium species germinated from resting cysts collected in November lacks a ventral pore. Molecular phylogenetic data for the vegetative cells from the germination experiments allowed the August and November peaks to be assigned to Alexandrium catenella (Group I) and A. pacificum (Group IV), respectively. This indicates that the production of resting cysts of A. catenella can be enhanced by relatively high water temperature. This result is not consistent with those of previous studies that A. catenella responsible for PSP outbreaks was found at relatively low water temperature. In addition, large subunit ribosomal sequences data revealed that A. pacificum isolates from Korea were closely related to those from Australia, Japan and New Zealand where the PSP toxicity of shellfish and blooms occurred in the 1990s, indicating that the introduction of toxic dinoflagellates were related to ballast water from bulk-cargo shipping. Based on these results, we concluded that past PSP outbreaks in Jinhae-Masan Bay of Korea could have been caused by A. pacificum rather than by A. catenella.

Genome-wide analysis of transcription and photosynthesis inhibition in the harmful dinoflagellate Prorocentrum minimum in response to the biocide copper sulfate.

Copper is an essential trace metal for organisms; however, excess copper may damage cellular processes. Their efficiency and physiological effects of biocides have been well documented; however, molecular transcriptome responses to biocides are insufficiently studied. In the present study, a 6.0K oligonucleotide chip was developed to investigate the molecular responses of the harmful dinoflagellate Prorocentrum minimum to copper sulfate (CuSO4) treatment. The results revealed that 515 genes (approximately 8.6%) responded to CuSO4, defined as being within a 2-fold change. Further, KEGG pathway analysis showed that differentially expressed genes (DEGs) were involved in ribosomal function, RNA transport, carbon metabolism, biosynthesis of amino acids, photosystem maintenance, and other cellular processes. Among the DEGs, 49 genes were related to chloroplasts and mitochondria. Furthermore, the genes involved in the RAS signaling pathway, MAPK signaling pathway, and transport pathways were identified. An additional experiment showed that the photosynthesis efficiency decreased considerably, and reactive oxygen species (ROS) production increased in P. minimum after CuSO4 exposure. These results suggest that CuSO4 caused cellular oxidative stress in P. minimum, affecting the ribosome and mitochondria, and severely damaged the photosystem. These effects may potentially lead to cell death, although the dinoflagellate has developed a complex signal transduction process to combat copper toxicity.

Survival and growth of Cochlodinium polykrikoides red tide after addition of yellow loess.

We examined the survival rate of Cochlodinium polykrikoides after yellow loess addition and conducted culture experiments to investigate the possibility that red tides maybe caused by C. polykrikoides individuals that are precipitated when loess is added. At least 15% of the C. polykrikoides cells that precipitated to the bottom layer either by the addition of loess or no addition survived for 1 week at all growth phases, rather than disappearing immediately after precipitating. However no live cells were observed after 20 days, regardless of phase or loess addition. In the exponential phase, the number of C. polykrikoides cells increased to >2886 cells ml(-1) after loess was added. However in the stationary phase, the number of cells did not increase until 18 days. In the exponential phase, those C. polykrikoides that survived precipitation caused by scattering loess on cultures did not appear to have the ability to cause red tides again because of the short red tide periods in the field, long lag time after loess addition, and low survival rate after loess addition.