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Species from the family Kareniaceae (Dinophyceae) frequently cause harmful algal blooms (HABs), with serious ecological impacts and risks to human safety and aquaculture activities in coastal waters worldwide. However, due to their small size, lack of morphological divergence, and low abundance during non-bloom periods, the diversity within this family is not well understood. By comparing the commonly used molecular markers, the Internal Transcribed Spacer (ITS) region was found to have an appropriate mutation rate to distinguish three of the most common genera (Karenia, Karlodinium, and Takayama) within the Kareniaceae family and different geographical strains of Kareniaceae. Specific primers targeting the ITS region of Karenia, and the other primers specific to the genera Karlodinium and Takayama, were designed. Specificity of the primers was tested using 17 strains of Kareniaceae species and 15 non-target species. Representative Kareniaceae species could be successfully detected even at low concentrations of target DNA template with a limit of detection of 3.2 pg. The primers were also assessed using high-throughput sequencing with two environmental samples from the South China Sea (SCS). Analysis of the reads identified as Kareniaceae species revealed a high diversity and the existence of unreported Kareniaceae species in the SCS. In conclusion, the newly developed molecular barcodes specifically detected Kareniaceae species in the field and will provide technical support for the effective warning and monitoring of Kareniaceae HABs.

On May 24–29, 2019, a harmful algal bloom occurred in Pingtan coastal areas, Fujian, southeast China, and caused mass mortality of cage-cultured fish. Two clonal cultures of an unknown naked dinoflagellate were set up from seawater samples taken during the bloom. The cultures were examined for morphological features, ultrastructure characters, photosynthetic pigments, the large subunit (LSU) of the rRNA gene, and the internal transcribed spacer (ITS) sequences, as well as acute toxicity analysis. The cell was unarmored, small-sized, and ovoid, and was characterized by elaborate striations on the epicone and hypocone. The nucleus was large, ellipsoid to oval or kidney-shaped, and centrally located in the cell. A long linear apical groove originated above the sulcus in the ventral epicone and extended to the dorsal side. An elongate, slit-like "ventral pore" was located on the left of the epicone, well away from the apical groove. The chloroplasts were yellowish brown, numerous, band like, and irregularly distributed in the cell periphery. Fucoxanthin was the main accessory pigment composition. Phylogeny topology reconstructed on partial LSU rDNA showed that the unknown dinoflagellate branched as a sister species to Karlodinium sp. (strain IFR981 & IFR797, from France) and Karlodinium corrugatum (strain KDGSO08, from Australia), with genetic divergences of 0.6% and 3.3%, respectively. Based on the morphological and molecular phylogenetic analysis, we describe the novel dinoflagellate as Karlodinium elegans sp. nov. A toxicity assay revealed that the clonal culture of K. elegans (strain PTB601) had no adverse effect on brine shrimp (Artemia salina) and marine medaka (Oryzias melastigma), indicating it may not be a toxic species. [ABSTRACT FROM AUTHOR]

Marine planktonic predator-prey interactions occur in microscale seascapes, where diffusing chemicals may act either as chemotactic cues that enhance or arrest predation, or as elemental resources that are complementary to prey ingestion. The phytoplankton osmolyte dimethylsulfoniopropionate (DMSP) and its degradation products dimethylsulfide (DMS) and acrylate are pervasive compounds with high chemotactic potential, but there is a longstanding controversy over whether they act as grazing enhancers or deterrents. Here, we investigated the chemotactic responses of three herbivorous dinoflagellates to point-sourced, microscale gradients of dissolved DMSP, DMS, and acrylate. We found no evidence for acrylate being a chemotactic repellent and observed a weak attractor role of DMS. DMSP behaved as a strong chemoattractor whose potential for grazing facilitation through effects on swimming patterns and aggregation depends on the grazer's feeding mode and ability to incorporate DMSP. Our study reveals that predation models will fail to predict grazing impacts unless they incorporate chemotaxis-driven searching and finding of prey., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Morphological variation, ultrastructure, pigment composition and phylogeny of an oceanic, star-shaped, unarmored dinoflagellate Asterodinium gracile were examined using a culture established from Nagasaki, Japan, in 2017. Our examinations were based on LM, SEM, TEM, high-performance liquid chromatography (HPLC) and molecular phylogeny inferred from nuclear-encoded internal transcribed spacer (ITS) and 28S rDNA and chloroplast-encoded 16S rDNA sequences. Typical cells were stellate, possessing an apical, two lateral, and two antapical extensions. Cells ranged in size from 62.5 to 105.6 µm long and 21.2 to 39.5 µm in cingular width. The length and orientation of cellular extensions were variable; that is, two lateral extensions were usually symmetrically bent or retracted. Ellipsoidal cells without extensions were also observed. The apical structure complex was straight or slightly sigmoid. A spherical nucleus was located at the left side in the hyposome. The numerous discoid, yellowish-brown chloroplasts were variable in size and distributed also in the cell extensions. Among Kareniaceae, the lack of a pyrenoid in Asterodinium is unique. Nuclear-encoded ITS and 28S phylogenies suggested that A. gracile is closely related to Karenia papilionacea in the Kareniaceae, but its affinity to Brachidinium capitatum, another species with extensions, was unclear. A phylogeny of 16S rDNA showed that chloroplasts of A. gracile are related to other kareniaceans derived from a haptophyte. These chloroplasts were of the fucoxanthin type, composed of pigments similar to those in other Kareniaceae; however, the detection of 19′-hexanoyloxyfucoxanthin as a major carotenoid, the low amount of fucoxanthin, and lack of 19′-butanoyloxyfucoxanthin in A. gracile were unusual for the family. [ABSTRACT FROM AUTHOR]

As the number of mixotrophic protists has been increasingly documented, “mixoplankton”, a third category separated from the traditional categorization of plankton into “phytoplankton” and “zooplankton”, has become a new paradigm and research hotspot in aquatic plankton ecology. While species of dinoflagellates are a dominant group among all recorded members of mixoplankton, the trophic modes of Karlodinium, a genus constituted of cosmopolitan toxic species, were reviewed due to their representative features as mixoplankton and harmful algal blooms (HABs)-causing dinoflagellates. Among at least 15 reported species in the genus, three have been intensively studied for their trophic modes, and all found to be phagotrophic. Their phagotrophy exhibits multiple characteristics: (1) omnivority, i.e., they can ingest a variety of preys in many forms; (2) flexibility in phagotrophic mechanisms, i.e., they can ingest small preys by direct engulfment and much bigger preys by myzocytosis using a peduncle; (3) cannibalism, i.e., species including at least K. veneficum can ingest the dead cells of their own species. However, for some recently described and barely studied species, their tropical modes still need to be investigated further regarding all of the above-mentioned aspects. Mixotrophy of Karlodinium plays a significant role in the population dynamics and the formation of HABs in many ways, which thus deserves further investigation in the aspects of physiological ecology, environmental triggers (e.g., levels of inorganic nutrients and/or presence of preys), energetics, molecular (genes and gene expression regulations) and biochemical (e.g., relevant enzymes and signal molecules) bases, origins, and evaluation of the advantages of being a phagotroph.

Recently, dinoflagellate blooms have frequently occurred in the coastal waters of Fujian, East China Sea. In June 2022, a fish-killing bloom of Kareniaceae species occurred in this region. In this study, four species of Kareniaceae, namely, Karenia longicanalis , K. papilionacea , Karlodinium veneficum , and Karl. digitatum were identified from this bloom event based on the results of single-cell PCR and clone libraries, and intraspecies genetic diversity was found in the Karl. veneficum population. The results of acute toxicity assays of the bloom water to two zooplankton species (Brachionus plicatilis and Artemia salina) demonstrated this bloom event strongly inhibited their swimming capacities and survival. The results of this study suggested that the bloom events caused by multiple species of Kareniaceae in the Fujian coastal waters had adverse impacts on the local fishery resources and zooplankton community. • A fish-killing bloom of Kareniaceae multiple species outbroke in Fujian coast in June 2022. • The bloom was dominated by Karenia longicanalis , K. papilionacea , Karlodinium veneficum , and Karl. digitatum. • Karl. veneficum population has intraspecies genetic diversity. • The bloom strongly inhibited the survival of Brachionus plicatilis and Artemia salina. [ABSTRACT FROM AUTHOR]

SUMMARY An unarmored dinoflagellate, Karlodinium jejuense sp. nov., was isolated from the northern East China Sea. The species was characterized by light, fluorescence, and scanning electron microscopy of laboratory cultures, as well as SSU rDNA, ITS region (ITS1‐5.8S‐ITS2) and LSU rDNA gene sequences. Cells were ellipsoid to oval, small‐sized, 9.9–16.0 μm in length and 6.2–12.7 μm in width. The chloroplasts were 7–10 in number and yellow‐green in color. The central nucleus was large, located in the center of the cell. The linear apical groove with outward thick rolled margins was very short, and there was no ventral pore. Small pores were irregularly distributed on the cell surface. The amphiesmal vesicles were hexagonal and pentagonal. Karlodinium jejuense was morphologically similar to K. ballantinum but differed in cell surface characteristics and swimming pattern. The partial LSU sequence of K. jejuense differed by 3.7–4.1% from that of K. ballantinum. Molecular phylogenetic analyses of multi‐gene sequences revealed that K. jejuense was an independent species nested within the Karlodinium clade. Phylogenetic analysis based on partial LSU rDNA gene sequences indicated that K. jejuense was most closely related to K. gentienii and K. ballantinum. [ABSTRACT FROM AUTHOR]

• Glycogen metabolism was mainly affected by Karlodinium veneficum. • FAAs content decreased in clam feeding on Karlodinium. • Anabolic metabolism of carnosine was initiated by feeding on Karlodinium. • K. veneficum affect the production of endogenous C 18 PUFA precursors. • VOCs composition differed by feeding different microalgae. • High density of Karlodinium affected the taste and odor. Phytoplankton composition is an important factor affecting the growth and physiological biochemical characteristics of filter-feeding bivalves. With the increasing trend in dinoflagellate biomass and blooms in mariculture areas, how the physio-biochemical traits and seafood quality of the mariculture organism are affected by the dinoflagellates, especially those at nonfatal levels, is not well understood. Different densities of two Karlodinium species, namely K. veneficum (KV) and K. zhouanum (KZ), mixed with high quality microalgal food Isochrysis galbana was applied in feeding manila clam Ruditapes philippinarum in a 14-day temporary culture, to comparatively study how the critical biochemical metabolites such as glycogen, free amino acids (FAAs), fatty acids (FAs), volatile organic compounds (VOCs) in the clam were affected. The survival rate of the clam showed dinoflagellate density and species specificity. The high-density KV group inhibited survival to 32% lower than that of the pure I. galbana control, respectively, while KZ at low concentrations did not significantly affect the survival compared with the control. In the high-density KV group, the glycogen and FAA contents decreased (p < 0.05), indicating that energy and protein metabolism were significantly affected. Amount of carnosine (49.91 ± 14.64 to 84.74 ± 8.59 μg/g of muscle wet weight) was detected in all the dinoflagellate-mixed groups, while it was not present in the field samples or in the pure I. galbana control, showing that carnosine participated in the anti-stress activities when the clam was exposed to the dinoflagellates. The global composition of FAs did not significantly vary among the groups. However, contents of the endogenous C 18 PUFA precursors linoleic acid and α-linolenic acid significantly decreased in the high-density KV group compared to all the other groups, indicating that high density of KV affected the metabolisms of fatty acids. From the results of the changed VOC composition, oxidation of fatty acids and degradation of free amino acids might occur in the clams exposed to dinoflagellates. The increased VOCs, such as aldehydes, and decreased 1-octen-3-ol probably produced a more fishy taste and reduced food flavor quality when the clam was exposed to the dinoflagellates. This present study demonstrated that the biochemical metabolism and seafood qulity of the clam were affected. However, KZ with moderate density in the feed seemed to be beneficial in aquaculture for increasing the content of carnosine, a high-valued substance with multiple bioactivities. [ABSTRACT FROM AUTHOR]

The dinoflagellate Karlodinium and the diatom Pseudo-nitzschia are bloom-forming genera frequently present in Alfacs Bay. Both microalgae are associated with toxic events. Therefore, understanding their population dynamics and predict their occurrence in short-term is crucial for an optimal management of toxic events for the local shellfish production and ecosystem managers. Artificial neural networks have been successfully used to model the complex nonlinear dynamics of phytoplankton. In this study, this approach was applied to predict absence-presence and abundance of Karlodinium and Pseudo-nitzschia microalgae in Alfacs Bay (NW Mediterranean) using biological and/or environmental variables. Neural Interpretation Diagram (NID) and Connection Weight Approach (CWA) methodologies were applied to obtain ecological information from the models. The dataset used was long-term (1990–2015) time series of environmental and phytoplankton variables from different monitoring stations established in Alfacs Bay (Ebre Delta), meteorological data and Ebre River flow rates. Several models were presented. The best ones were achieved for one-week ahead procedures performed with environmental and biological variables using all the available data. A sensitivity analysis showed the larger the data set used, the better the models obtained. However, Karlodinium absence-presence models developed with five years of data present high accuracy. The size of the neural networks denotes complex relationships between environmental and phytoplankton variables. The environmental variables had stronger influence on the abundance models while biological variables had more importance in the absence-presence models. These results highlight a complex ecosystem in Alfacs Bay involving anthropogenic, climatic and hydrologic factors forcing phytoplankton dynamics. In addition, a change in the ecosystem dynamics regarding Karlodinium is detected. The configuration and the accuracy achieved with the models allow their use in different real-world applications as automated systems and/or monitoring programs. [ABSTRACT FROM AUTHOR]

Harmful algal blooms can adversely affect fish communities, though their impacts are highly context-dependent and typically differ between fish species. Various approaches, comprising univariate and multivariate analyses and multimetric Fish Community Indices (FCI), were employed to characterise the perceived impacts of a Karlodinium veneficum bloom on the fish communities and ecological condition of the Swan Canning Estuary, Western Australia. The combined evidence suggests that a large proportion of the more mobile fish species in the offshore waters of the bloom-affected area relocated to other regions during the bloom. This was indicated by marked declines in mean species richness, catch rates and FCI scores in the bloom region but concomitant increases in these characteristics in more distal regions, and by pronounced and atypical shifts in the pattern of inter-regional similarities in fish community composition during the bloom. The lack of any significant changes among the nearshore fish communities revealed that bloom impacts were less severe there than in deeper, offshore waters. Nearshore habitats, which generally are in better ecological condition than adjacent offshore waters in this system, may provide refuges for fish during algal blooms and other perturbations, mirroring similar observations of fish avoidance responses to such stressors in estuaries worldwide. [ABSTRACT FROM AUTHOR]

• An intensive algal bloom along the eastern Hokkaido coasts was dominated by Karenia selliformis. • Cells of Kr. selliformis in the bloom were large and possessing numerous small chloroplasts. • Kr. selliformis cells showed morphological variation, including transparent cells without chloroplasts. • Co-occurred kareniaceans were Karenia longicanalis, Karenia mikimotoi, Takayama spp., etc. • Partial rDNAs of Kr. selliformis (group I) from Hokkaido 2021 and Kamchatka 2020 were identical. Harmful algal blooms responsible for mass mortalities of marine organisms have been rare in Hokkaido, northern Japan, although fish-killing blooms have been frequently reported from western Japanese coasts. In September–November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurring unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1–D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis , with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of Kr. selliformis in the bloom were discoid, dorsoventrally flattened, and 35.3–43.6 (39.4 ± 2.1) µm in length, which was larger than the cell sizes in previous reports. Transparent cells of Kr. selliformis , lacking chloroplasts or having a few shrunken chloroplasts and oil droplets, were also found. Cells of Kr. selliformis showed morphological variation, but the species could be distinguished from other co-occurring Karenia species by the nucleus positioned in the hypocone and chloroplasts numerous (46–105) in number and small (2.9–4.6 µm) in diameter. Cell density of Kr. selliformis exceeding 100 cells mL−1 was recorded in the temperature range of 9.8–17.6 °C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from the bloom in Kamchatka, Russia in 2020. [ABSTRACT FROM AUTHOR]

Abstract: Three near-shore type harmful dinoflagellates, Prorocentrum minimum, Prorocentrum donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monocultures and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N):phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algae, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected by the addition of potential prey. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. K. veneficum overgrew P. minimum in the presence of Rhodomonas, a potential nutrient source. The growth rates of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. In addition to nutrient competition, mixotrophy and allelopathy were likely mechanisms in determining the dominant species. [Copyright &y& Elsevier]

Most photosynthetic dinoflagellates harbour a red alga-derived secondary plastid. In the dinoflagellate Karlodinium micrum, this plastid was replaced by a subsequent endosymbiosis, resulting in a tertiary plastid derived from a haptophyte. Evolution of endosymbionts entails substantial relocation of endosymbiont genes to the host nucleus: a process called endosymbiotic gene transfer (EGT). In K. micrum, numerous plastid genes from the haptophyte nucleus are found in the host nucleus, providing evidence for EGT in this system. In other cases of endosymbiosis, notably ancient primary endosymbiotic events, EGT has been inferred to contribute to remodeling of other cell functions by expression of proteins in compartments other than the endosymbiont from which they derived. K. micrum provides a more recently derived endosymbiotic system to test for evidence of EGT and gain of function in non-plastid compartments. In this study, we test for gain of haptophyte-derived proteins for mitochondrial function in K. micrum. Using molecular phylogenies we have analysed whether nucleus-encoded mitochondrial proteins were inherited by EGT from the haptophyte endosymbiont, or vertically inherited from the dinoflagellate host lineage. From this dataset we found no evidence of haptophyte-derived mitochondrial genes, and the only cases of non-vertical inheritance were genes derived from lateral gene transfer events. [Copyright &y& Elsevier]

Abstract: Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution. [Copyright &y& Elsevier]

Abstract: The difficulties encountered in attempts to differentiate between dinoflagellate species of the genera Alexandrium and Karlodinium using morphological characteristics are well-known. For this reason, species of these genera were analyzed by flow cytometry to determine whether haploid DNA content served as a valid criterion for species identification. The DNA content of species often confused with each other due to their overlapping size and geographical occurrence, such as Alexandrium ostenfeldii and the complexes Alexandrium catenella, Alexandrium tamarense, Alexandrium minutum and Alexandrium tamutum, and Karlodinium veneficum and Karlodinium armiger were analyzed. These species differed greatly in DNA content, which provided a means of distinguishing among them. The only cases of DNA overlap involved A. ostenfeldii with Alexandrium peruvianum, and A. catenella with A. tamarense, two groups not yet clearly established either morphologically or genetically. Variability in intraspecies DNA content was observed only in the species K. veneficum. Significant differences between the two A. tamarense strains analyzed were not detected, and the haploid DNA content (63pgcell−1) was very different from the one reported previously for this species (103.5pgcell−1), suggesting cryptic speciation within this group. Flow-cytometric analysis of field samples identified K. veneficum as the causative species of a bloom, suggesting this method as a tool to readily identify species responsible for natural blooms. Additionally, after clonal cultures had been established, cytometric analyses corroborated the variability in the haploid DNA content of this species. [Copyright &y& Elsevier]

Abstract: The population dynamics of potentially harmful microalgae was investigated in the semi-enclosed shallow Gulf of Kalloni, Greece (Aegean Sea, Eastern Mediterranean), during a 2-year period from August 2004 to March 2006. A total of 21 potentially harmful microalgae (bloom-forming and/or toxic) were identified including 3 diatoms and 18 dinoflagellates. The densities of each species were analyzed in time and space and in relation to environmental parameters. Some species such as Alexandrium insuetum, Heterocapsa circularisquama, Karlodinium veneficum, Scrippsiella trochoidea, and Ceratium spp. developed high cell concentrations, particularly during a Pseudo-nitzschia calliantha winter bloom. Other species such as Dinophysis caudata, Ostreopsis ovata, Prorocentrum minimum, and Protoperidinium crassipes were rare or appeared in small numbers. Densities of the most abundant species were closely associated with freshwater nutrient-rich inputs during winter, being negatively correlated with temperature and salinity and positively correlated with nitrogen. The spatial distribution of the abundant species exhibited a marked increase towards the inner part of the gulf, close to the main freshwater inputs, whereas some species were mainly concentrated in the dilute surface layer (1m depth). Examination of the abundance–occupancy relationship revealed that the species more prone to bloom are those with wide spatial distribution and frequent presence throughout the year such as the diatom P. calliantha. Although blooms of cyst-forming species are rarer, an increased risk can be foreseen under favorable resource supply and environmental conditions during winter. [Copyright &y& Elsevier]

We examined the sterol profile of Karlodinium veneficum (D. Ballant.) J. Larsen, Akashiwo sanguinea (Hiraska) Ge. Hansen et Moestrup, Alexandrium tamarense (M. Lebour) Balech, Alexandrium affine (H. Inoue et Fukuyo) Balech, Gonyaulax polygramma F. Stein , and Gymnodinium instriatum (Freud. et J. J. Lee) Coats , along with their Amoebophyra parasites. There were no consistent sterol profiles that characterized the genus Amoebophyra. Instead, in five out of six comparisons, the host and parasite sterol profiles where highly correlated. The one exception, Amoebophyra sp. ex Alex.  tamarense, was least like its host in sterol profile and also possessed the widest host range for infection. There was little correlation between host and parasite in fatty acid profiles, with the parasite being deficient in fatty acids characteristic of the plastid [e.g., 18:5(n-3) associated with galactolipids of the thylakoids, as previously published by Adolf et al. (2007)]. Those hosts and parasites with sterol profiles dominated by desmethyl sterols were most sensitive to karlotoxin toxicity. In the host-parasite pairs most sensitive to karlotoxin addition, recovery of the intact karlotoxin molecule was poorest. Given the sensitivity to karlotoxin, some species of Amoebophyra may avoid infection of K. veneficum. [ABSTRACT FROM AUTHOR]

Karlodinium veneficum (D. Ballant.) J. Larsen strains, 16 from the U.S. Atlantic eastern seaboard and two from New Zealand (CAWD66 and CAWD83), were used to characterize toxin profiles during batch culture. All 18 strains were determined as the same species based on ITS sequence analyses, a positive signal in a chloroplast real-time PCR assay and pigment composition. Five karlotoxin 1 (KmTx 1) containing strains were analyzed from the Chesapeake Bay, and 10 karlotoxin 2 (KmTx 2) strains were analyzed from Florida to North Carolina. One strain (MD5) from the Chesapeake Bay produced no detectable toxin. The two cultures from New Zealand contained both novel karlotoxins with lower masses and earlier elution times. Toxin type did not change during batch culture, although the KmTx phenotype did change in some strains under extensive (months) phototrophic growth in replete media. KmTx cell quota did not change during batch culture for most strains. The mass spectrum for every KmTx examined showed a pattern of multiple coeluting congeners within each HPLC peak, with masses typically differing by 16 amu. KmTx congeners tested showed nearly a 500-fold range in specific hemolytic activity, with KmTx 1 (typically occurring at lower cellular levels) most hemolytic and CAWD66 toxin least hemolytic, while KmTx 2 and the CAWD83 toxin had similar intermediate specific activity. Despite morphological, genetic, and photopigment indicators consistent with species homogeneity among the 18 strains of K. veneficum, the high degree of toxin variability suggests different functional roles among strains that likely coexist in situ. [ABSTRACT FROM AUTHOR]

The unarmoured dinoflagellate assemblage of the Gulf of Naples has been investigated in the frame of a 1-year sampling of natural surface samples collected weekly at a coastal station c. 2 miles offshore. Twenty-six strains of unarmoured dinoflagellates were brought into culture by means of serial dilution. Observations at both light and scanning electron microscopy (SEM) together with molecular phylogenetic analyses allowed identification of eight dinoflagellate species. Gymodinium aureolum, Karlodinium veneficum, Protodinium simplex and Takayama acrotrocha were previously reported in the Mediterranean; whereas, Karlodinium ballantinum and Lepidodinium viride together with a taxon identified as Karenia cf. longicanalis are recorded in the basin for the first time. A new thin-walled dinoflagellate is described as Woloszynskia cincta sp. nov. on the basis of morphological results. Molecular analysis showed that Woloszynskia cincta is closely related to W. halophila and W. pseudopalustris and only distantly related to W. pascheri. In Protodinium simplex, a straight acrobase and an arrangement of the amphiesmal vesicles in latitudinal series were revealed by SEM observations, demonstrating the relatedness of this species with woloszynskioid dinoflagellates. Based on new morphological information on Karlodinium species, Gyrodinium corsicum is transferred to Karlodinium corsicum. New morphological features are identified for the recognition of Takayama acrotrocha, such as the arrangement of the acrobase and the presence of a pore on the ventral side of the cell. The latter species was found to be genetically distinct from other species in the genus Takayama, which is monophyletic. [ABSTRACT FROM AUTHOR]

Abstract: Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (102 to 103 mL−1), but capable of forming blooms of 104 to 105 cellsmL−1 under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421μm3 per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability. [Copyright &y& Elsevier]

Six new species of unarmored dinoflagellates in the family Kareniaceae were isolated from the Australian sector of the Southern Ocean in March 2006: Takayama tuberculata de Salas sp. nov, Karlodinium antarcticum de Salas sp. nov., Karl. ballantinum de Salas sp. nov., Karl. conicum de Salas sp. nov., Karl. corrugatum de Salas sp. nov., and Karl. decipiens de Salas et Laza-Martínez sp. nov. These new taxa were characterized using light and electron microscopy and sequencing of the LSU rDNA and are well supported based either on their morphology or molecular phylogeny. Takayama tuberculata, isolated just north of the polar front (55°–57° S), is genetically close to T. tasmanica, but smaller, with a significantly reduced number of amphiesmal vesicles. Medium-sized Karl. antarcticum, also isolated from near the polar front, is characterized by its long ovoid cell outline and very long apical groove. The small Karl. ballantinum has a very short apical groove. The large Karl. conicum has a distinct conical epicone and spherical posterior nucleus. The small Karl. corrugatum, from just south of the polar front, has distinctive parallel striations on the epicone surface and a distinctively shaped and placed ventral pore. The large and widespread Karl. decipiens, distributed through Southern Ocean waters from the polar front to Tasmanian coastal waters, and coastal Spain, has a helicoidal chloroplast arrangement and a large central nucleus. This study represents the first description of species in the potentially ichthyotoxic family Kareniaceae recorded from the Southern Ocean. [ABSTRACT FROM AUTHOR]

We examined whether fatty acid (FA) composition changed when Karlodinium veneficum (D. Ballantine) J. Larsen (Dinophyceae) was grown phototrophically or mixotrophically on Storeatula major Butcher ex D. R. A. Hill (Cryptophyceae). We hypothesized that the FA composition of mixotrophic K. veneficum would not change relative to the FA composition of phototrophic K. veneficum. As in other phototrophic dinoflagellates, octadecapentaenoic acid (18:5n3) represented 9% to 20% of total FA in K. veneficum and was enriched within chloroplast-associated galactolipid classes. The 18:5n3 content showed a highly significant positive correlation ( r2 = 0.95) with chl a content and a highly significant negative correlation with growth rate ( r2 = 0.88). A previously undescribed chloroplast galactolipid molecular species, digalactosyldiacylglycerol (DGDG; 18:5n3/18:5n3), was a dominant structural lipid in K. veneficum. Docosahexaenoic acid (22:6n3) represented 14% to 19% of total K. veneficum FA and was enriched within phospholipids. In the prey S. major, 18:5n3 was not present, but octadecatetraenoic acid (18:4n3) and α-linolenic acid (18:3n3) represented approximately 50% of total FA and were enriched within chloroplast-associated galactolipid classes. Eicosapentaenoic acid (20:5n3) and 22:6n3 represented approximately 18% of total FA in S. major and were enriched within phospholipids. The FA profile of mixotrophic K. veneficum, compared to phototrophic K. veneficum, showed elevated levels of 18:3n3, 18:4n3, and 20:5n3, and lower but persistent levels of 18:5n3. Production to ingestion ( P: I) ratios >1 for major polyunsaturated fatty acids (PUFAs) indicated that direct assimilation from prey under balanced growth could not support rates of PUFA production in mixotrophic K. veneficum. These data suggest that the plastid plays a continuing and essential role in lipid metabolism during mixotrophic growth. [ABSTRACT FROM AUTHOR]

Abstract: Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (104–105 K. veneficum ml−1), grazing rates (cells ingested per Oxyrrhis h−1) on toxic K. veneficum strain CCMP 2064 were ∼55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<103 cellsml−1), grazing rates (cells ingested per Oxyrrhis h−1) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100ngml−1) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ. [Copyright &y& Elsevier]

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug-like structures in amphiesma and a possible layer of semi-opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low- ( K. veneficum) and high-light ( K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates. [ABSTRACT FROM AUTHOR]

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S )-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (ED) and its 27-nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis . They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis , K. mikimotoi , and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates. [ABSTRACT FROM AUTHOR]

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S )-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (ED) and its 27-nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis . They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis , K. mikimotoi , and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates. [ABSTRACT FROM AUTHOR]

As the number of mixotrophic protists has been increasingly documented, "mixoplankton", a third category separated from the traditional categorization of plankton into "phytoplankton" and "zooplankton", has become a new paradigm and research hotspot in aquatic plankton ecology. While species of dinoflagellates are a dominant group among all recorded members of mixoplankton, the trophic modes of Karlodinium, a genus constituted of cosmopolitan toxic species, were reviewed due to their representative features as mixoplankton and harmful algal blooms (HABs)-causing dinoflagellates. Among at least 15 reported species in the genus, three have been intensively studied for their trophic modes, and all found to be phagotrophic. Their phagotrophy exhibits multiple characteristics: (1) omnivority, i.e., they can ingest a variety of preys in many forms; (2) flexibility in phagotrophic mechanisms, i.e., they can ingest small preys by direct engulfment and much bigger preys by myzocytosis using a peduncle; (3) cannibalism, i.e., species including at least K. veneficum can ingest the dead cells of their own species. However, for some recently described and barely studied species, their tropical modes still need to be investigated further regarding all of the above-mentioned aspects. Mixotrophy of Karlodinium plays a significant role in the population dynamics and the formation of HABs in many ways, which thus deserves further investigation in the aspects of physiological ecology, environmental triggers (e.g., levels of inorganic nutrients and/or presence of preys), energetics, molecular (genes and gene expression regulations) and biochemical (e.g., relevant enzymes and signal molecules) bases, origins, and evaluation of the advantages of being a phagotroph. [ABSTRACT FROM AUTHOR]