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The thecate amoeba Paulinella is a valuable model for understanding plastid organellogenesis because this lineage has independently gained plastids (termed chromatophores) of alpha-cyanobacterial provenance. Plastid primary endosymbiosis in Paulinella occurred relatively recently (90–140 million years ago, Mya), whereas the origin of the canonical Archaeplastida plastid occurred >1,500 Mya. Therefore, these two events provide independent perspectives on plastid formation on vastly different timescales. Here we generated the complete chromatophore genome sequence from P. longichromatophora (979,356 bp, GC-content = 38.8%, 915 predicted genes) and P. micropora NZ27 (977,190 bp, GC-content = 39.9%, 911 predicted genes) and compared these data to that from existing chromatophore genomes. Our analysis suggests that when a basal split occurred among photosynthetic Paulinella species ca. 60 Mya, only 35% of the ancestral orthologous gene families from the cyanobacterial endosymbiont remained in chromatophore DNA. Following major gene losses during the early stages of endosymbiosis, this process slowed down significantly, resulting in a conserved gene content across extant taxa. Chromatophore genes faced relaxed selection when compared to homologs in free-living alpha-cyanobacteria, likely reflecting the homogeneous intracellular environment of the Paulinella host. Comparison of nucleotide substitution and insertion/deletion events among different P. micropora strains demonstrates that increases in AT-content and genome reduction are ongoing and dynamic processes in chromatophore evolution.

While the ecophysiology of planktonic Mesodinium rubrum species complex has been relatively well studied, very little is known about that of benthic Mesodinium species. In this study, we examined the growth response of the benthic ciliate Mesodinium coatsi to different cryptophyte prey using an established culture of this species. M. coatsi was able to ingest all of the offered cryptophyte prey types, but not all cryptophytes supported its positive, sustained growth. While M. coatsi achieved sustained growth on all of the phycocyanin‐containing Chroomonas spp. it was offered, it showed different growth responses to the phycoerythrin‐containing cryptophytes Rhodomonas spp., Storeatula sp., and Teleaulax amphioxeia. M. coatsi was able to easily replace previously ingested prey chloroplasts with newly ingested ones within 4 d, irrespective of prey type, if cryptophyte prey were available. Once retained, the ingested prey chloroplasts seemed to be photosynthetically active. When fed, M. coatsi was capable of heterotrophic growth in darkness, but its growth was enhanced significantly in the light (14:10 h light:dark cycle), suggesting that photosynthesis by ingested prey chloroplast leads to a significant increase in the growth of M. coatsi. Our results expand the knowledge of autecology and ecophysiology of the benthic M. coatsi.

The phylum Perkinsozoa is known as an exclusively parasitic group including the parasites of shellfish, fish, dinoflagellates, cryptophytes, and tadpoles and at present comprises seven genera across three families (Parviluciferaceae, Perkinsidae, and Xcellidae), with the genus Parvilucifera having the most abundant species in the family Parviluciferaceae. During intensive sampling along the Korean coast in August and September 2017, a new species of the genus Parvilucifera was discovered and successfully established in cultures. Morphological and ultrastructural observations revealed that the new parasitoid shares almost all known diagnostic characters with other species of Parvilucifera, except that its sporangium has a higher number of apertures although with smaller diameters than those in P. infectans. Molecular phylogenetic trees based on both nuclear small subunit (SSU) and concatenated SSU and large subunit (LSU) ribosomal DNA (rDNA) sequences revealed that the new parasitoid was nested within the family Parviluciferaceae and had a sister relationship with P. infectans. Based on morphological, ultrastructural, and molecular data, we propose to erect a new species, P. multicavata sp. nov., for the new parasitoid found in this study.

Some marine dinoflagellates form ecdysal cyst (=temporary cysts) as part of their life cycle or under unfavorable growth conditions. Whether the dinoflagellates form ecdysal cysts or not may influence susceptibility to parasitism. In this study, parasite prevalence relative to inoculum size of the parasitoid Parvilucifera infectans zoospores for two dinoflagellate hosts (i.e., Fragilidium duplocampanaeforme and Dinophysis acuminata) , which have different life cycle strategies, was examined. Further, susceptibility of cysts to parasitism, encystment signal, duration of encystments, and effects of induced encystment on diel periodicity, using ecdysal cyst-forming F. duplocampanaeforme were explored. The percent hosts infected by P. infectans plotted as a function of inoculum size showed a sharp increase to a maximum in D. acuminata , but a gradual linear rise in F. duplocampanaeforme : while the parasite prevalence in D. acuminata increased to a maximum of 78.8 (±2.4%) by a zoospore:host ratio of 20:1, it in F. duplocampanaeforme only reached 8.9 (±0.3%), even at a zoospore:host ratio of 120:1. In F. duplocampanaeforme , infections were observed only in the vegetative cells and not observed in ecdysal cysts. When exposed to live, frozen, and sonicated zoospores and zoospore filtrate, F. duplocampanaeforme formed ecdysal cysts only when exposed to live zoospores, suggesting that temporary cyst formation in the dinoflagellate resulted from direct contact with zoospores. When the Parvilucifera zoospores attacked and struggled to penetrate F. duplocampanaeforme through its flagellar pore, the Fragilidium cell shed all thecal plates, forming a ‘thecal cloud layer’, in which the zoospores were caught and immobilized and thus could not penetrate anymore. The duration (35 ± 1.8 h) of ecdysal cysts induced with addition of zoospores was significantly longer than that (15 ± 0.8 h) of normally formed cysts (i.e., without addition of zoospores), thereby resulting in delayed growth as well as influencing the pattern of diel periodicity. The results from this study suggest that in addition to the classical predator-prey interaction and allelopathic interaction, parasitism and its accompanying defense can make the food web dynamics much more complicated than previously thought.

Species belonging to the toxic dinoflagellate genus Ostreopsis are widespread, occurring from tropical to temperate waters. As mainly benthic/epiphytic species, they would be expected to show distinct geographical patterns. In this study, ribosomal DNA (rDNA) sequences from partial nuclear LSU D8-D10, 5.8S, and ITS regions were determined for 169 isolates of Ostreopsis species collected from three coastal sites (i.e., Jeju Island, Chuja Island, and Pohang) within Korea. The phylogenetic tree inferred from the LSU rDNA D8-D10 sequences showed that Korean Ostreopsis species corresponded to either Ostreopsis sp. 1 or sp. 6, with Ostreopsis sp. 1 being relatively predominant regarding their distribution. While Ostreopsis sp. 1 occurred throughout all the three sampling sites within Korea, Ostreopsis sp. 6 was confined to the northern part of Jeju Island. When further investigated, the genetic diversity of Ostreopsis sp. 1 in Korea based on ITS sequences showed a total of 21 haplotypes. The presumed ancestral haplotype H3, was also present in the Japanese and Russian populations of Ostreopsis sp. 1. Although the overall demographic history of all the Korean populations of Ostreopsis sp. 1 could not be clearly identified, probably due to a mixture of different regional demographic patterns within Korea, each Ostreopsis sp. 1 population showed a characteristic demographic pattern at a regional scale. While the Jeju Island Ostreopsis sp. 1 population showed a signal in agreement with population equilibrium, the Chuja Island and Pohang Ostreopsis sp. 1 populations showed distribution patterns that are expected in a sudden population expansion model. The results from this study provide a basis for a better understanding of the distribution and genetic structure of the Asian Ostreopsis sp. 1 populations.

Ciliate Mesodinium species are commonly distributed in diverse aquatic systems worldwide. Among Mesodinium species, M. rubrum is closely associated with microbial food webs and red tide formation and is known to acquire chloroplasts from its cryptophyte prey for use in photosynthesis. For these reasons, Mesodinium has long received much attention in terms of ecophysiology and chloroplast evolution. Mesodinium cells are easily identifiable from other organisms owing to their unique morphology comprising two hemispheres, but a clear distinction among species is difficult under a microscope. Recent taxonomic studies of Mesodinium have been conducted largely in parallel with molecular sequence analysis, and the results have shown that the best-known planktonic M. rubrum in fact comprises eight genetic clades of a M. rubrum/M. major complex. However, unlike the planktonic Mesodinium species, little is known of the genetic diversity of benthic Mesodinium species, and to our knowledge, the present study is the first to explore this. A total of ten genetic clades, including two clades composed of M. chamaeleon and M. coatsi, were found in marine sandy sediments, eight of which were clades newly discovered through this study. We report the updated phylogenetic relationship within the genus Mesodinium comprising heterotrophic/mixotrophic as well as planktonic/benthic species. Furthermore, we unveiled the wide variety of chloroplasts of benthic Mesodinium, which were related to the green cryptophyte Chroomonas/Hemiselmis and the red cryptophyte Rhodomonas/Storeatula/Teleaulax groups.

In planktonic predator-prey interactions, predation pressure could be affected by several factors associated with prey, including cell size, shape, nutritional quality, presence of chemical deterrents, and movement behaviors. In addition, parasitism may also play an important role in predator-prey interaction by infecting one or both partners involved in the biological interaction. In this study, parasite mediation in predator-prey interactions of planktonic protists was addressed using model organisms commonly observed in temperate coastal environments, namely, a phototrophic dinoflagellate Akashiwo sanguinea as a potential host and prey, a heterotrophic dinoflagellate predator Luciella masanensis, and a dinoflagellates-infecting parasitoid Parvilucifera infectans. Parasite mediation permitted L. masanensis to exploit the prey A. sanguinea that the predator was otherwise unable to use or did not prefer. However, parasite-mediated exploitation seems to be dependent on the infection cycle of P. infectans. Although zoospores and mature sporangium produced from infected A. sanguinea were not ingested by the predator L. masanensis, both newly infected (i.e., cells that zoospores had just penetrated) and infected (those containing early to late trophocytes) A. sanguinea cells attracted numerous L. masanensis cells and were rapidly ingested by the predator, leading to the predator's positive growth. The results from mixed culture experiments of the three protists showed that the presence of P. infectans at low density led to co-occurrence of the three protists. Thus, the parasitoid seems to play the role of a mediator in either inedible prey- or predator-dominated environments, leading to co-occurrence of the three protists.

Mesodinium rubrum Lohmann is a mixotrophic ciliate and one of the best studied species exhibiting acquired phototrophy. To investigate the fate of cryptophyte organelles in the ciliate subjected to starvation, we conducted ultrastructural studies of a Korean strain of M. cf. rubrum during a 10 week starvation experiments. Ingested cells of the cryptophyte Teleaulax amphioxeia were first enveloped by ciliate membrane, and then prey organelles, including ejectisomes, flagella, basal bodies and flagellar roots, were digested. Over time, prey nuclei protruded into the cytoplasm of the ciliate, their size and volume increased, and their number decreased, suggesting that the cryptophyte nuclei likely fused with each other in the ciliate cytoplasm. At 4 weeks of starvation, M. cf. rubrum cells without cryptophyte nuclei started to appear. At 10 weeks of starvation, only two M. cf. rubrum cells still possessing a cryptophyte nucleus had relatively intact chloroplast-mitochondria complexes (CMCs), while M. cf. rubrum cells without cryptophyte nuclei had a few damaged CMCs. This is the first ultrastructural study demonstrating that cryptophyte nuclei undergo a dramatic change inside M. cf. rubrum in terms of size, shape, and number following their acquisition.

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing "edible food" for consumers of the marine microbial food-web during toxic algal bloom event.

Perkinsozoa is an exclusively parasitic group within the alveolates and infections have been reported from various organisms, including marine shellfish, marine dinoflagellates, freshwater cryptophytes, and tadpoles. Despite its high abundance and great genetic diversity revealed by recent environmental rDNA sequencing studies, Perkinsozoa biodiversity remains poorly understood. During the intensive samplings in Korean coastal waters during June 2017, a new parasitoid of dinoflagellates was detected and was successfully established in culture. The new parasitoid was most characterized by the presence of two to four dome-shaped, short germ tubes in the sporangium. The opened germ tubes were biconvex lens-shaped in the top view and were characterized by numerous wrinkles around their openings. Phylogenetic analyses based on the concatenated SSU and LSU rDNA sequences revealed that the new parasitoid was included in the family Parviluciferaceae, in which all members were comprised of two separate clades, one containing Parvilucifera species (P. infectans, P. corolla, and P. rostrata), and the other containing Dinovorax pyriformis, Snorkelia spp., and the new parasitoid from this study. Based on morphological, ultrastructural, and molecular data, we propose to erect a new genus and species, Tuberlatum coatsi gen. n., sp. n., from the new parasitoid found in this study. Further, we examined and discussed the validity of some diagnostic characteristics reported for parasitoids in the family Parviluciferaceae at both the genus and species levels.

Phagotrophy of the harmful benthic dinoflagellates of the genus Ostreopsis has long been inferred based on observations of food particles present inside cells, but the prey has not yet been identified. This study aimed to investigate the seasonal dynamics of benthic dinoflagellates Ostreopsis spp. in temperate Korean coastal sites, with special emphasis on their phagotrophy. Further, prey species were identified by extracting the ingested food particles from single Ostreopsis cells and determining their rbcL and psaA gene sequences. High concentration of Ostreopsis cells was observed between June and October at all sites, when the water temperatures were higher than 19°C, exhibiting a marked temporal seasonality. The percentage of Ostreopsis cells containing ingested food particles exhibited large spatial and temporal variations among sampling sites, ranging from undetectable level to 29.5%, and was not always associated with Ostreopsis cell abundance. Phylogenetic analyses performed using both plastid-encoded rbcL and psaA genes revealed that all sequences obtained from the ingested food particles of Ostreopsis cells grouped within the class Florideophyceae, Rhodophyta. Our result clearly demonstrates that Ostreopsis species consume various macroalgae from Rhodophyta, but not protists, which have long been thought to be the potential prey. The results of this study provide a basis for better understanding the mixotrophic behavior and nutritional ecology of the harmful benthic dinoflagellate Ostreopsis species.

The syndinean dinoflagellates are a diverse assemblage of alveolate endoparasites that branch basal to the core dinoflagellates. Because of their phylogenetic position, the syndineans are considered key model microorganisms in understanding early evolution in the dinoflagellates. Closed mitosis with an extranuclear spindle that traverses the nucleus in cytoplasmic grooves or tunnels is viewed as one of the morphological features shared by syndinean and core dinoflagellates. Here we describe nuclear morphology and mitosis in the syndinean dinoflagellate Amoebophrya sp. from Akashiwo sanguinea, a member of the A. ceratii complex, as revealed by protargol silver impregnation, DNA specific fluorochromes, and transmission electron microscopy. Our observations show that not all species classified as dinoflagellates have an extranuclear spindle. In Amoebophrya sp. from A. sanguinea, an extranuclear microtubule cylinder located in a depression in the nuclear surface during interphase moves into the nucleoplasm via sequential membrane fusion events and develops into an entirely intranuclear spindle. Results suggest that the intranuclear spindle of Amoebophrya spp. may have evolved from an ancestral extranuclear spindle and indicate the need for taxonomic revision of the Amoebophryidae.

The marine sand-dwelling dinoflagellate Polykrikos lebourae possesses obvious gold-brown pigmented plastids as well as taeniocyst-nematocyst complex structures. Despite of the presence of the visible plastids, previous attempts to establish this species in culture all failed and thus the unavailability of cultures of this species has posed a major obstacle to further detailed exploration of ecophysiology of the dinoflagellate. Here, we isolated P. lebourae from sandy sediment of an intertidal flat on Korean western coast, successfully established it in culture, and have been maintaining the stock cul ture over the past 3 years. Using this stock culture, we explored phagotrophy and potential prey resources of P. lebourae, growth and grazing responses of P. lebourae to different prey organisms, the effect of prey concentration on growth and grazing rates and gross growth efficiency (GGE) of P. lebourae when fed three different prey organisms, and the growth kinetics of P. lebourae under different light regimes. P. lebourae captured prey cells using a tow filament and then phagocy tized them through the posterior end. The dinoflagellate was capable of ingesting a broad range of prey species varying in size, but not all prey species tested in this study supported its sustained growth. GGE of P. lebourae was extremely high at low prey concentration and moderate or low at high prey concentrations, indicating that P. lebourae grows heterotrophically at high prey concentrations but its growth seems to be more dependent on a certain growth factor or photosynthesis of plastids derived from the prey. In the presence of prey in excess, P. lebourae grew well at moderate light intensity of 40 µmol photons m -2 s -1 , but did not grow at dim and high (10 or 120 µmol photons m -2 s -1 ) light intensities. Our results suggest that the benthic dinoflagellate P. lebourae is an obligate mixotroph, requiring both prey and light for sustained growth and survival.

Phototrophic Dinophysis species are known to acquire plastids of the cryptophyte Teleaulax amphioxeia through feeding on the ciliate Mesodinium rubrum or M . cf. rubrum . In addition, several molecular studies have detected plastid encoding genes of various algal taxa within field populations of Dinophysis species. The trophic pathway by which Dinophysis species acquire plastids from algae other than the cryptophyte genus Teleaulax , however, is unknown. In this study, we examined the fate of prey organelles and plastid genes obtained by Dinophysis caudata through ingestion of Mesodinium coatsi , a benthic ciliate that retains green plastids of Chroomonas sp. Transmission electron microscopy and molecular analysis revealed relatively rapid digestion of prey-derived plastids. Following digestion of M. coatsi , however, photodamaged D . caudata cells having olive-green rather than reddish-brown plastids were able to recover some of their original reddish-brown pigmentation. Results further suggest that plastid genes of various algal taxa detected in field populations of Dinophysis species may reflect prey diversity rather than sequestration of multiple plastid types. Ingestion and digestion of prey other than M. rubrum or M . cf. rubrum may also provide nutritional requirements needed to repair and perhaps maintain sequestered T . amphioxeia plastids.

A mixotrophic dinoflagellate Fragilidium duplocampanaeforme has been recently reported to specifically feed on mixotrophic dinoflagellates Dinophysis spp., which specifically feed on a mixotrophic ciliate Mesodinium rubrum (= Myrionecta rubra ), which specifically feeds on a cryptophyte. Using established cultures, in this study we aimed (1) to examine the pattern of phased cell division of a Korean strain of F . duplocampanaeforme , (2) to explore its mixotrophic characteristics, and (3) to address food web interactions among top three protists within the trophic series (i.e., M . rubrum , Dinophysis spp. and F . duplocampanaeforme ). During phototrophic growth, F . duplocampanaeforme displayed the phased cell division, with cell division starting 4 h after light was on and finished before the light off. Motile vegetative cell stage of F . duplocampanaeforme , which can ingest the prey, persisted over the dark period. When offered two Dinophysis species ( Dinophysis acuminata and Dinophysis caudata ) as prey, Korean F . duplocampanaeforme strain did not display any prey preference. The dinoflagellate F . duplocampanaeforme grew well phagotrophically in the dark if offered with plentiful prey (i.e., a facultative mixotroph). The dinoflagellate F . duplocampanaeforme grew well at growth rates of 0.21–0.26 d −1 when supplied with D . acuminata or D . caudata as prey in the dark, but its growth rates were 2.3–3.0 times slower than those when grown mixotrophically in the light. Nonetheless, the growth rates in the dark were similar to those in purely phototrophic growth. Our results from the mixed cultures of three protists demonstrated that the food web dynamics from cryptophyte through ciliate M. rubrum and dinoflagellate Dinophysis spp. to dinoflagellate F . duplocampanaeforme could be affected by predator–prey interaction as well as allelopathic interaction, depending on Dinophysis species included in the trophic series.

Satellite remote sensing has been used as an effective tool to monitor and manage seagrass beds, but this tool is not appropriate to use in highly turbid waters along the coast of Korea. In this study, satellite data were used to identify the method for detecting seagrass beds in turbid bay waters and describing temporal changes over the past 24 years. Moreover, we found the cause of seagrass die-off phenomenon by observing the long-term satellite images and evaluating the impact of the typhoon on seagrass beds. All seagrass spectra from different sensors (Landsat TM/ETM+, Aster, Spot-4, and Kompsat-2) have low reflectance in green and high in NIR regions, whereas unvegetated seawater has the opposite spectrum features. A training area in the green band and Mahalanobis Distance Classification algorithm was adopted for classification and mapping of seagrass beds. The error matrix using the in situ reference data for Kompsat-2 image classification was 72.9% accurate. The average area of seagrass beds was 4.6 km 2 from May 1990 to January 2012, and it was consistently >4.0 km 2 . A die-off of seagrass beds was observed in September 2012 after Typhoon Bolaven, Tembin, and Sanba consecutively passed the study site. When Typhoon Sanba passed the study site, it had sustained maximum wind speed (147.6 km h −1 ) during low tide, unlike the other typhoons. We found that the water column buffer did not have any effect on resistance of seagrass bed to disturbance, which led to greater damage. Additionally, the three sequential typhoons that passed through during the short time probably produced a cumulative effect. These results allow an understanding of past changes, and reveal the sudden and influential changes in seagrass distribution. As seagrass removal might have negative effect on other associated communities, comprehensive monitoring is required to ensure that disturbed seagrass habitats is recovered naturally.

Mesodinium is a globally distributed ciliate genus forming frequent and recurrent blooms in diverse marine habitats. Here, we describe a new marine species, Mesodinium coatsi n. sp., originally isolated from interstitial water of surface sand samples collected at Mohang Beach, Korea. The species was maintained under a mixotrophic growth condition for longer than 1 yr by providing a cryptomonad, Chroomonas sp., as the sole prey. Cell morphology and subcellular structure were examined by light microscopy, scanning, and transmission electron microscopy, and molecular phylogeny was inferred from nuclear-encoded 18S rDNA sequence data. Like other Mesodinium species, M. coatsi consisted of two hemispheres separated by two types of kinetids, and had tentacles located at the oral end of the cell. Several food vacuoles were observed in the cytoplasm, and partially digested prey cells sometimes existed in food vacuoles. Kinetids and the associated accessory structures were quite similar to those previously reported, but M. coatsi was differentiated from other marine Mesodinium species by ultrastructural characters of the dikinetids, polykinetids, and tentacles. We also provided a detailed illustration of infraciliature. Molecular phylogeny revealed that M. coatsi and Mesodinium chamaeleon were closely related to each other.

Members of Amoebophrya ceratii complex are known to infect a number of free-living dinoflagellates including harmful algal bloom species. In August and October 2012, Amoebophrya infections during two bloom events of the dinoflagellate Cochlodinium polykrikoides were observed along southern coastal waters of Korea. Microscopic observations and molecular data revealed that two different Amoebophrya parasites infected the same host species. In addition, while one developed in the host's nucleus, the other in the host's cytoplasm. Phylogenetic analyses showed that the two parasites were not nested in the previously recognized "Amoebophrya ceratii complex clade", which contained sequences of parasites infecting numerous dinoflagellate species. Instead, they branched as sister taxa to the isolate (possibly Amoebophrya) from radiolarians Hexacontium gigantheum. Our result indicates that the two Amoebophrya parasites infecting C. polykrikoides may be different species from those inside the "complex."

Marine dinoflagellates of the genus Dinophysis are well known for producing diarrhetic shellfish poisoning (DSP) toxins and/or pectenotoxins which have a significant impact on public health as well as on marine aquaculture. Out of more than 80 Dinophysis species recorded so far, D. cf. acuminata is the most commonly observed in coastal areas worldwide. Due to their highly similar morphological features, however, an accurate discrimination of the various D. cf. acuminata species such as D. acuminata, D. ovum, and D. sacculus under light microscopy has proven to be a difficult task to accomplish. Hence, these species have thus far been referred to as the "Dinophysis acuminata complex". Recent studies showed a discrimination between local strains of D. acuminata and D. ovum from Galician, northwestern Spain, using the mitochondrial cox1 gene as a genetic marker in addition to commonly used morphological features such as size and contour of the large hypothecal plates, shape of the small cells formed as part of their polymorphic life-cycle, development of the left sulcal list and ribs, and length of the right sulcal list. In the present study, attempts were made to discriminate between D. acuminata and D. ovum following single-cell isolation of 54 "D. acuminata complex" collected from Korean coastal waters, based on the abovementioned traits. Morphological data showed that all the traits analyzed overlapped between the two species. The mitochondrial cox1 (cytochrome c oxidase subunit I) gene sequences of every isolate were also determined, but a genetic distinction between D. acuminata and D. ovum could not be confirmed, suggesting that the cox1 gene is not a suitable genetic marker for discrimination between the two species. The results of this study suggest that the morphological variations observed within the "D. acuminata complex" may have been caused by several factors (e.g. different geographical locations, seasonal changes, and different environmental conditions), and that D. acuminata and D. ovum may be the same species.

Two parasites of dinoflagellates, the perkinsid Parvilucifera infectans and the parasitic dinoflagellate Amoebophrya ceratii , are known to infect numerous dinoflagellate hosts, including some toxic and harmful algal bloom species. These parasites have received particular attention, due to growing interest in developing “natural” biological approaches for controlling harmful red tides. Nonetheless, few published accounts of dinoflagellate parasitism are available for coastal waters of Asia, although that region is strongly influenced by dinoflagellate red tides. In this paper, our goals were (1) to review available information (i.e. distribution and seasonality) on Amoebophrya that infect dinoflagellates of Korean coastal waters, providing novel observation where possible, (2) set data (i.e. biology and genetic diversity) for Korean Amoebophrya within a global context, and (3) discuss the implications of available findings on the potential use of Amoebophrya as a biological control agent of harmful dinoflagellates.

The gonyaulacalean dinoflagellates Amylax spp. were recently found to contain plastids of the cryptophyte origin, more specifically of Teleaulax amphioxeia. However, not only how the dinoflagellates get the plastids of the cryptophyte origin is unknown but also their ecophysiology, including growth and feeding responses as functions of both light and prey concentration, remain unknown. Here, we report the establishment of Amylax triacantha in culture, its feeding mechanism, and its growth rate using the ciliate prey Mesodinium rubrum (= Myrionecta rubra) in light and dark, and growth and grazing responses to prey concentration and light intensity. The strain established in culture in this study was assigned to A. triacantha, based on morphological characteristics (particularly, a prominent apical horn and three antapical spines) and nuclear SSU and LSU rDNA sequences. Amylax triacantha grew well in laboratory culture when supplied with the marine mixotrophic ciliate M. rubrum as prey, reaching densities of over 7.5 × 10(3) cells/ml. Amylax triacantha captured its prey using a tow filament, and then ingested the whole prey by direct engulfment through the sulcus. The dinoflagellate was able to grow heterotrophically in the dark, but the growth rate was approximately two times lower than in the light. Although mixotrophic growth rates of A. triacantha increased sharply with mean prey concentrations, with maximum growth rate being 0.68/d, phototrophic growth (i.e. growth in the absence of prey) was -0.08/d. The maximum ingestion rate was 2.54 ng C/Amylax/d (5.9 cells/Amylax/d). Growth rate also increased with increasing light intensity, but the effect was evident only when prey was supplied. Increased growth with increasing light intensity was accompanied by a corresponding increase in ingestion. In mixed cultures of two predators, A. triacantha and Dinophysis acuminata, with M. rubrum as prey, A. triacantha outgrew D. acuminata due to its approximately three times higher growth rate, suggesting that it can outcompete D. acuminata. Our results would help better understand the ecophysiology of dinoflagellates retaining foreign plastids.

13 pages, 7 figures, Marine microbial interactions involving eukaryotes and their parasites play an important role in shaping the structure of phytoplankton communities. These interactions may alter population densities of the main host, which in turn may have consequences for the other concurrent species. The effect generalist parasitoids exert on a community is strongly dependent on the degree of host specificity. Parvilucifera sinerae is a generalist parasitoid able to infect a wide range of dinoflagellates, including toxic-bloom-forming species. A density-dependent chemical cue has been identified as the trigger for the activation of the infective stage. Together these traits make Parvilucifera-dinoflagellate hosts a good model to investigate the degree of specificity of a generalist parasitoid, and the potential effects that it could have at the community level. Here, we present for the first time, the strategy by which a generalist dinoflagellate parasitoid seeks out its host and determine whether it exhibits host preferences, highlighting key factors in determining infection. Our results demonstrate that in its infective stage, P. sinerae is able to sense potential hosts, but does not actively select among them. Instead, the parasitoids contact the host at random, governed by the encounter probability rate and once encountered, the chance to penetrate inside the host cell and develop the infection strongly depends on the degree of host susceptibility. As such, their strategy for persistence is more of a game of Russian roulette, where the chance of survival is dependent on the susceptibility of the host. Our study identifies P. sinerae as a potential key player in community ecology, where in mixed dinoflagellate communities consisting of hosts that are highly susceptible to infection, parasitoid preferences may mediate coexistence between host species, reducing the dominance of the superior competitor. Alternatively, it may increase competition, leading to species exclusion. If, however, highly susceptible hosts are absent from the community, the parasitoid population could suffer a dilution effect maintaining a lower parasitoid density. Therefore, both host community structure and host susceptibility will determine infectivity in the field, This study was supported by the Spanish Ministry of Science and Innovation through the project ECOPAR, CSIC Intramural Project (EG) and Mid-career research program NRF-2014R1A2A2A01004586 (MP)

"Phototrophic"Dinophysis Ehrenberg species are well known to have chloroplasts of a cryptophyte origin, more specifically of the cryptophyte genus complex Teleaulax/Geminigera. Nonetheless, whether chloroplasts of "phototrophic"Dinophysis are permanent plastids or periodically derived kleptoplastids (stolen chloroplasts) has not been confirmed. Indeed, molecular sequence data and ultrastructural data lead to contradictory interpretations about the status of Dinophysis plastids. Here, we used established cultures of D. caudata strain DC-LOHABE01 and M. rubrum strain MR-MAL01 to address the status of Dinophysis plastids. Our approach was to experimentally generate D. caudata with "green" plastids and then follow the ingestion and fate of "reddish-brown" prey plastids using light microscopy, time-lapse videography, and single-cell TEM. Our results for D. caudata resolve the apparent discrepancy between morphological and molecular data by showing that plastids acquired when feeding on M. rubrum are structurally modified and retained as stellate compound chloroplasts characteristic of Dinophysis species.

"Phototrophic"Dinophysis Ehrenberg species are well known to have chloroplasts of a cryptophyte origin, more specifically of the cryptophyte genus complex Teleaulax/Geminigera. Nonetheless, whether chloroplasts of "phototrophic"Dinophysis are permanent plastids or periodically derived kleptoplastids (stolen chloroplasts) has not been confirmed. Indeed, molecular sequence data and ultrastructural data lead to contradictory interpretations about the status of Dinophysis plastids. Here, we used established cultures of D. caudata strain DC-LOHABE01 and M. rubrum strain MR-MAL01 to address the status of Dinophysis plastids. Our approach was to experimentally generate D. caudata with "green" plastids and then follow the ingestion and fate of "reddish-brown" prey plastids using light microscopy, time-lapse videography, and single-cell TEM. Our results for D. caudata resolve the apparent discrepancy between morphological and molecular data by showing that plastids acquired when feeding on M. rubrum are structurally modified and retained as stellate compound chloroplasts characteristic of Dinophysis species.

The ‘phototrophic’ Dinophysis Ehrenberg species are well known to have plastids of a cryptophyte origin, more specifically cryptophyte genus complex Teleaulax/Geminigera. However, how often the phototrophic Dinophysis could retain both types of plastids from this genus complex at the same time in a cell and also whether the phototrophic cells could retain plastids of the other algal origins rather than cryptophyte have not been investigated in detail. We isolated a total of 67 phototrophic Dinophysis spp. cells between May 2008 and September 2009 along western and southern coasts of Korea and amplified psbA as a tracer to investigate plastid diversity from the isolated cells. Then, the PCR products were digested with a restriction enzyme, SfaNI, to distinguish between the most common Teleaulax amphioxeia-type and the less common T. acuta-type plastids. During this study, we sometimes encountered ‘green’ Dinophysis acuminata cells, which contained varying degree of red autofluorescencing green plastids inside the cell, along together typical orange autofluorescencing reddish-brown plastids. The RFLP patterns of the PCR products digested by SfaNI revealed that a total of 66 Dinophysis cells analyzed in this study all contained T. amphioxeia-type plastid. Further, approximately two-thirds of the analyzed Dinophysis cells contained both T. amphioxeia-type and T. acuta-type plastids at the same time in a single cell. Interestingly, SfaNI digestion of the products amplified on psbA gene from 10 Dinophysis cells produced a different RFLP pattern: in addition to T. amphioxeia-type and sometimes T. acuta-type plastid, undigested fragments occurred. We cloned the PCR products and determined psbA gene sequences from the cells containing undigested fragments with SfaNI. Surprisingly, these Dinophysis cells contained even three (i.e. cryptophytes T. amphioxeia and T. acuta and raphidophyte Heterosigma akashiwo) or four (i.e. cryptophytes T. amphioxeia and T. acuta, raphidophyte H. akashiwo, and chlorophyte Pyramimonas sp.) different plastid sequences at the same time in a single cell. Our result indicates that besides the sole prey Mesodinium rubrum (=Myrionecta rubra) known until now, there may be other potential prey organisms, presumably the plastid-retaining ciliates, from which phototrophic Dinophysis may acquire plastids of several algal origins other than cryptophyte.

Myrionecta rubra , a cosmopolitan red tide ciliate, is not only a predator on cryptomonads but also is a main prey known so far for survival of harmful algae Dinophysis species. This common mixotrophic ciliate functionally seems to have plastid preference, but this possibility has been rarely studied using genetic analysis combined with growth rates of M. rubra . We investigated dynamics of plastids of starved M. rubra (strain MR-MAL01) with the addition of cryptomonads having different types of plastids as prey. Population growth of ‘2-month starved’ M. rubra strain MR-MAL01 was 0.09 ± 0.014 d −1 when supplied with cryptomonad prey strain CR-MAL03, in sharp contrast to the growth rate of the predator strain MR-MAL01, 0.48 ± 0.059 d −1 with prey strain CR-MAL01. Within 30 min after the addition of cryptomonad prey strain CR-MAL03 to a ‘3-week starved’ M. rubra population, both the ‘CR-MAL03 type’ 1192-bp PCR product and the ‘CR-MAL01 type’ 802-bp PCR product from diagnostic primers, which are specific to plastid 16S rRNA gene, were amplified from the ciliates. Only the 802-bp PCR product was detected in the ciliates supplied only with prey strain CR-MAL01. Both kinds of PCR product from M. rubra were continuously detected for 2 weeks after being washed free of prey in the treatment supplied with cryptomonad prey strain CR-MAL03. The M. rubra strain MR-MAL01 thus can ingest both prey strains, CR-MAL01 and CR-MAL03, and can retain plastids of strain CR-MAL03 for an extended period of time. The differential phototrophic growth of M. rubra after the inoculation of the two different cryptomonad prey species implies that M. rubra sometimes can retain plastids with which it is not normally found in a symbiotic relationship, in spite of a resultant lower growth rate.

We documented the abundance of the heterotrophic dinoflagellate Oxyphysis oxytox- oides Kofoid along the Korean coast, cultured it (food source: the marine ciliate Myrionecta rubra Jankowski (= Mesodinium rubrum Lohmann)), sequenced its nuclear-encoded large-subunit (LSU) rDNA, and resolved its phylogenetic relationship within the Dinophysiales. Abundance was low, usu- ally

We verified an active uptake of kleptoplastids in the toxic and bloom-forming dinoflagellatesof the genus Dinophysis from its preferred prey, the ciliate Myrionecta rubra, using a quantitativereal- ...

Samples of the massive drifting green alga, Ulva linza, were collected from the coastal waters of the Yellow Sea, southwest of Korea, in early July 2009, and cultured under laboratory conditions. The effects of various temperature (10–30°C) and irradiance levels (0–1,000 μmol photons m−2 s−1) on photosynthesis, growth, and tissue nutrient content of U. linza were investigated in laboratory for both individuals of the late-stage vegetation (LSV) and the early-stage vegetation (ESV). After 1 h acclimation to various irradiance and temperature conditions, maximum gross photosynthetic rate of ESV was significantly higher than those of LSV. In the long-term (7-d) acclimation experiments to various irradiance and temperature levels, gross photosynthetic rates of ESV individuals were also significantly higher than those of LSV individuals. High photosynthetic rate of ESV individuals induced increase in mass of about 60% over the growth saturation irradiance (136 μmol photons m−2 s−1) and about 20% under low temperature conditions (10–15°C) during 7-d. The gross photosynthesis of LSV individuals was low when examined under temperature and irradiance conditions that were optimum for ESV growth. Consequently, free-floating U. linza exhibits cellular senescence beginning in early July in the Yellow Sea, and green tides formed by this species cannot be maintained beyond this time in the open sea. However, we expect that U. linza can proliferate quickly after settlement on new coastal habitats of the Yellow Sea because of the high tissue nitrogen utilization for photosynthesis in ESV, which is formed by germination of reproductive cells.

In summer to autumn of 2008, a recently described thecate mixotrophic dinoflagellate, Fragilidium duplocampanaeforme Nezan et Chomerat, occurred in Masan Bay, Korea, where it frequently contained bright-orange fluorescent inclusions. Using cultures of F. duplocampanaeforme isolated from Masan Bay, we investigated feeding, digestion, and prey specificity of this mixotroph. F. duplocampanaeforme fed exclusively on Dinophysis spp. when offered a variety of prey including dinoflagellates, a raphidophyte, a cryptophyte, a ciliate, and diatoms separately. In addition, F. duplocampanaeforme had allelopathic effects on other organisms, including cell immobilization/motility decrease (in Dinophysis acuminata, D. caudata, D. fortii, D. infundibulus, Gonyaulax polygramma, Heterocapsa triquetra, and Prorocentrum triestinum), breaking of cell chains (in Cochlodinium polykrikoides), cell death (in Prorocentrum minimum), and temporary cyst formation (in Scrippsiella trochoidea). F. duplocampanaeforme engulfed whole Dinophysis cells through the sulcus. About 1 h after ingestion, F. duplocampanaeforme became immobile and shed all thecal plates. The ecdysal cyst persisted for ~7 h, during which the ingested prey was gradually digested. These observations suggest that F. duplocampanaeforme may play an important role in the Dinophysis population dynamics in the field.

The marine photosynthetic dinoflagellates Dinophysis Ehrenb. species are obligate mixotrophs that require both light and the ciliate prey Myrionecta rubra (= Mesodinium rubrum) for long-term survival. Despite rapid progress on the study of Dinophysis using laboratory cultures, however, whether it has its own permanent plastids or kleptoplastids (i.e., stolen plastids from its ciliate prey) is not fully resolved. Here, we addressed this issue using established cultures of D. caudata Saville-Kent strain DC-LOHABE01 and cross-feeding/starvation experiments encompassing the prey M. rubra strain MR-MAL01 cultures grown on two different cryptophytes (strains CR-MAL01 and CR-MAL11). To follow the fate of prey plastids, psbA gene as a tracer was amplified from individually isolated D. caudata cells, and the PCR products were digested with a restriction enzyme, SfaNI. The RFLP pattern of the PCR products digested by SfaNI revealed that D. caudata continued to keep CR-MAL01–type plastids, while it lost CR-MAL11–type plastids with increasing starvation time. Our results suggest that Dinophysis treats in different ways plastids taken up from different cryptophytes via its ciliate prey M. rubra. Alternatively, D. caudata may already have its own CR-MAL01–type permanent plastid, with two types of plastids (CR-MAL01 and CR-MAL11) obtained from M. rubra being lost within 1 month. This result highlights the need to identify more accurately the origin of plastids in newly isolated photosynthetic Dinophysis species to resolve the issue of plastid permanence.

To survive, the marine dinoflagellate Dinophysis caudata Saville-Kent must feed on the plastidic ciliate Myrionecta rubra (=Mesodinium rubrum), itself a consumer of cryptophytes. Whether D. caudata has its own permanent chloroplasts or retains plastids from its ciliate prey, however, remains unresolved. Further, how long D. caudata plastids (or kleptoplastids) persist and remain photosynthetically active in the absence of prey remains unknown. We addressed those issues here, using the first established culture of D. caudata. Phylogenetic analyses of the plastid 16S rRNA and psbA gene sequences directly from the three organisms (D. caudata, M. rubra, and a cryptophyte) revealed that the sequences of both genes from the three organisms are almost identical to each other, supporting that the plastids of D. caudata are kleptoplastids. A 3-month starvation experiment revealed that D. caudata can remain photosynthetically active for ∼2 months when not supplied with prey. D. caudata cells starved for more than 2 months continued to keep the plastid 16S rRNA gene but lost the photosynthesis-related genes (i.e., psaA and psbA genes). When the prey was available again, however, D. caudata cells starved for more than 2 months were able to reacquire plastids and slowly resumed photosynthetic activity. Taken all together, the results indicate that the nature of the relationship between D. caudata and its plastids is not that of permanent cellular acquisitions. D. caudata is an intriguing protist that would represent an interesting evolutionary adaptation with regard to photosynthesis as well as help us to better understand plastid evolution in eukaryotes.

Four sesterterpenoids were isolated from AMPK activity-guided fraction of marine sponge Sarcotragus species. Their planar structures were determined from combination of extensive 1D and 2D NMR experiments and MS data, and the configuration at the chiral centers were assigned by comparison with the and chemical shifts of the known compounds. Among four compounds, compound 2 was found to be a new sarcotrine derivative. Though not strong, compounds 1-4 moderately showed AMPK activation effect on L6 myoblast cell through Western Blot analysis.

We determined 18S rRNA gene sequences of Amoebophrya strains infecting the thecate dinoflagellates Alexandrium affine and Gonyaulax polygramma from Korean coastal waters and compared those data with previously reported sequences of Amoebophrya from cultures, infected cells concentrated from field samples, and environmental 18S rRNA gene sequences obtained from a variety of marine environments. Further, we used these data to examine genetic diversity in Amoebophrya strains relative to geographic origin, host phylogeny, site of infection, and host specificity. In our analyses of known dinoflagellate taxa, the 13 available Amoebophrya sequences clustered together within the dinoflagellates as three groups forming a monophyletic group with high bootstrap support (maximum likelihood, ML: 100%) or a posterior probability (PP) of 1. When the Amoebophrya sequences were analyzed along with environmental sequences associated with Marine Alveolate Group II, nine subgroups formed a monophyletic group with high bootstrap support (ML: 100%) and PP of 1. Sequences known to be from Amoebophrya spp. infecting dinoflagellate hosts were distributed in seven of those subgroups. Despite differences in host species and geographic origin (Korea, United States, and Europe), Amoebophrya strains (Group II) from Gymnodinium instriatum, A. affine, Ceratium tripos (AY208892), Prorocentrum micans, and Ceratium lineatum grouped together by all of our tree construction methods, even after adding the environmental sequences. By contrast, strains within Groups I and III divided into several lineages following inclusion of environmental sequences. While Amoebophrya strains within Group II mostly developed within the host cytoplasm, strains in Groups I and III formed infections inside the host nucleus, a trait that appeared across several of the subgroups. Host specificity varied from moderately to extremely species-specific within groups, including Group II. Taken together, our results imply that genetic diversity in Amoebophrya strains does not always reflect parasite biology or biogeography.

Microzooplankton grazing on coastal phytoplankton was determined by the dilution method from May 2002 to April 2003 at a fixed site located in the Jinhae Bay, Korea. During the dilution experiments, our study site exhibited a wide range of chlorophyll a concentrations (0.29–127.42 μg l−1), and the species composition of the phytoplankton community changed dramatically over a year, shifting from the predominance of chain-forming diatoms, particularly Chaetoceros spp., Leptocylindrus danicus, Pseudonitzschia pungens, and Skeletonema costatum, between May and September 2002, to a massive bloom of the dinoflagellates, Alexandrium spp. in October 2002, to a dominance of cryptophytes (Chroomonas sp.) between November 2002 and March 2003, and then again to a prevalence of diatoms toward the end of the experiment. Both nutrients enriched (μn) and in situ phytoplankton growth rates (μ0) showed pronounced seasonal variations, ranging from 0.11 to 2.87 d−1 and from −0.63 to 2.08 d−1, respectively. With regard to both variables, the lowest values were obtained during the fall and winter seasons. The average ratio of μ0/μn was 0.96 (SE = 0.08), thereby indicating that phytoplankton growth in the study site was not nutrient-limited. Microzooplankton grazing rates showed the large fluctuations (0–3.86 d−1) over an annual cycle, with non-significant and/or negative grazing frequently (62% of 29 measurements) detected. Relatively high grazing rates did occur frequently at the times during which a large phytoplankton biomass and/or large-sized phytoplankton dominance were observed. Our results contribute to the growing body of evidence suggesting that microzooplankton are important phytoplankton consumers in communities dominated by large phytoplankton, and also bolster the notion that size-based models of food web relationships may be of limited predictive value. The observed large fluctuations in grazing rates over a year, coupled with frequent non-significant and/or negative grazing, indicate that intensive and long-term measurements of microzooplankton grazing are required for a better understanding of the interaction between phytoplankton and microzooplankton in a highly dynamic coastal ecosystem.

The freshwater testate filose amoeba Paulinella chromatophora is the sole species in the genus to have plastids, usually termed "chromatophores", of a Synechococcus/Prochlorococcus-like cyanobacterial origin. Here, we report a new marine phototrophic species, Paulinella longichromatophora sp. nov., using light and electron microscopy and molecular data. This new species contains two blue-green U-shaped chromatophores reaching up to 40 μm in total length. Further, the new Paulinella species is characterized by having five oral scales surrounding the pseudostomal aperture. All trees generated using three nuclear rDNA datasets (18S rDNA, 28S rDNA, and the concatenated 18S + 28S rDNA) demonstrated that three photosynthetic Paulinella species (two freshwater species, P. chromatophora and Paulinella strain FK01, and one marine species, P. longichromatophora) congruently formed a monophyletic group with strong support (≥ 90% of ML and ≥ 0.90 of PP), but their relationship to each other within the clade remained unresolved in all trees. P. longichromatophora, nevertheless, clustered consistently together with Paulinella strain FK01 with very low support, but the clade received strong support in plastid phylogenies. Phylogenetic analyses inferred from plastid-encoded 16S rDNA and a concatenated dataset of plastid 16S+23S rDNA demonstrated that chromatophores of all photosynthetic Paulinella species were monophyletic. The monophyletic group fell within a cyanobacteria clade having a close relationship to an α-cyanobacterial clade containing Prochlorococcus and Synechococcus species with very robust support (100% of ML and 1.0 of PP). Additionally, phylogenetic analyses of nuclear 18S rDNA and plastid 16S rDNA suggested divergent evolution within the photosynthetic Paulinella population after a single acquisition of the chromatophore. After the single acquisition of the chromatophore, ancestral photosynthetic Paulinella appears to have diverged into at least two distinct clades, one containing the marine P. longichromatophora and freshwater Paulinella strain FK01, the other P. chromatophora CCAC 0185.

The dinoflagellate genus Dinophysis in- cludes several species that cause diarrhetic shellfish poi- soning, none of which have yet been established in cul- ture. We report on the maintenance of Dinophysis acuminata cultures that were established in December 2005 and also on its feeding mechanism, and growth rates when fed the ciliate prey Myrionecta rubra with and without the addition of the cryptophyte Teleaulax sp. D. acuminata grew well (growth rate of 0.95 d -1 ) in laboratory culture when supplied with the marine ciliate M. rubra as prey, reaching a maximum concentration of about 2400 cells ml -1 at the end of the feeding experiment. In contrast, D. acuminata did not show sustained growth in the absence of the ciliate or when provided the cryptophyte Teleaulax sp. as prey (D. acuminata used its peduncle to extract the cell contents of the prey organism, M. rubra). Based on the prey- preda- tor interactions occurring among D. acuminata, M. rubra, and Teleaulax sp. in this study, establishment of perma- nent culture of the dinoflagellate D. acuminata may facil- itate a better understanding of the ecophysiology, biol- ogy, and toxicology of Dinophysis species, as well as the evolution of dinoflagellate plastids.