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Ocean warming and acidification may significantly alter the distribution and intensity of harmful algal blooms as well as their effects on marine food webs. Estimating such effects rely, in part, on understanding the physiological response of individual algal species to controlled laboratory simulations of climate change conditions. Here we report the physiological response of the harmful dinoflagellate Karlodinium veneficum to the combined effects of elevated temperature and CO2 (29°C/1000 ppm CO2). We first examined these effects by comparing ambient control (25°C/441 ppm CO2) and elevated conditions under short-term (~20 generations) growth. Next, we compared the short-term elevated condition to a longer-term (~200 generations) growth scenario under the same elevated temperature and CO2. Under the short-term elevated conditions, K. veneficum growth declined, cell toxicity increased, and saturated and mono-unsaturated fatty acid (FA) composition varied significantly from ambient conditions. Meanwhile, after ~ 200 generations of growth under elevated temperature and CO2, K. veneficum carbon assimilation, growth, and cell toxicity were significantly higher than the short-term elevated treatment. Further, while total saturated FA declined, essential fatty acids increased and likely represented an adaptive temporal response to long-term exposure to high temperature and CO2. Such shifts in FA profiles and cell toxicity may possibly alter K. veneficum nutritional quality as prey and its mixotrophic behavior, thereby affecting the energy and mass transfer through the marine food webs as well as bloom dynamics. [ABSTRACT FROM AUTHOR]

Ocean warming and acidification may significantly alter the distribution and intensity of harmful algal blooms as well as their effects on marine food webs. Estimating such effects rely, in part, on understanding the physiological response of individual algal species to controlled laboratory simulations of climate change conditions. Here we report the physiological response of the harmful dinoflagellate Karlodinium veneficum to the combined effects of elevated temperature and CO2 (29°C/1000 ppm CO2). We first examined these effects by comparing ambient control (25°C/441 ppm CO2) and elevated conditions under short-term (~20 generations) growth. Next, we compared the short-term elevated condition to a longer-term (~200 generations) growth scenario under the same elevated temperature and CO2. Under the short-term elevated conditions, K. veneficum growth declined, cell toxicity increased, and saturated and mono-unsaturated fatty acid (FA) composition varied significantly from ambient conditions. Meanwhile, after ~ 200 generations of growth under elevated temperature and CO2, K. veneficum carbon assimilation, growth, and cell toxicity were significantly higher than the short-term elevated treatment. Further, while total saturated FA declined, essential fatty acids increased and likely represented an adaptive temporal response to long-term exposure to high temperature and CO2. Such shifts in FA profiles and cell toxicity may possibly alter K. veneficum nutritional quality as prey and its mixotrophic behavior, thereby affecting the energy and mass transfer through the marine food webs as well as bloom dynamics.

The interactive effects of temperature (15–30°C), salinity (5–30) and light (low-100 and high-300 μmol photons m−2 s−1) on growth, thermal niche properties and cellular carbon (C) and nitrogen (N) of the toxic dinoflagellate, Karlodinium veneficum , were studied to understand its potential for change under future climate conditions in the eutrophic Chesapeake Bay. Cell growth was highest under conditions of 25–28°C, salinity 10–20 and high light, which represented the preferred physical niche for bloom formation in the present day. In the Chesapeake Bay, blooms generally occur at 25–29°C and salinity 10–14, while low-biomass occurrences have been found at salinities 15–29, consistent with the laboratory findings. High light increased the thermal sensitivity of K. veneficum and lowered the thermal optima for growth. Under conditions of low light, and salinity 10–20, cells exhibited the highest thermal optima for growth. The highest upper thermal maxima were observed at salinity 30, suggesting that cells in the lower estuary would be more thermally resistant than those in upper and mid-estuarine regions, and therefore these higher salinity regions may provide over-summering habitats for K. veneficum. Cellular C and N were highly varied at the preferred salinity and temperature niche and C:N ratios showed decreasing trends with temperature. [ABSTRACT FROM AUTHOR]

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

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments. [ABSTRACT FROM AUTHOR]

• Mixotrophic cultures of Karlodinium veneficum retained diel synchrony when provided cryptophyte prey. • Increased growth rates of mixotrophic cultures were attributed to the shortened duration of G1 phase. • Karlotoxin synthesis occured only during light periods and was positively correlated with proportion of cells in G1 phase. Karlodinium veneficum forms fish killing blooms in estuaries worldwide. The toxicity of these blooms is variable and thought to be connected to bloom stage and in situ growth rates. Methods for measuring in situ growth rates rely on the assumption that cell cycle progression is phased to the diel photocycle, which is true for phototrophically-growing K. veneficum cultures where G1 phase occurs during light and S and G2 + M phases occur during darkness. However, K. veneficum is a facultative mixotroph that also phagocytizes microalgal prey, and the effects of this mixotrophy on its cell cycle synchrony are unknown. Furthermore, toxicity in laboratory cultures is inversely related to growth rate and is light dependent, suggesting synchrony between the cell cycle (G1 phase) and karlotoxin synthesis. To test this, the cell cycle phase distribution and cellular toxin content for phototrophic and mixotrophic cultures of K. veneficum were monitored hourly for a full diel cycle. The results demonstrated that mixotrophic cultures maintained a synchronized cell cycle, despite increased growth rates. The faster growth rates were attributed to a shortened duration of G1 phase in mixotrophic cultures compared to phototrophic cultures (30.8 ± 9.2 h vs 69.4 ± 21.5 h, respectively). Meanwhile, toxin production was observed only during light hours, consistent with synthesis initiating with the photorespiratory byproduct glycolate. Cellular toxin content had a significant positive correlation with the percentage of G1 phase cells and a significant negative correlation with the percentage of S phase cells. These results indicate a clear role in mixotrophy increasing growth rates of K. veneficum and of the diel photocycle in synchronizing the cell and karlotoxin synthetic cycles. [Display omitted] [ABSTRACT FROM AUTHOR]

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

Shewanella sp. IRI-160 is an algicidal bacterium that secretes an algicide, IRI-160AA. This algicide specifically targets dinoflagellates, while having no adverse effects on other algal species tested. Dinoflagellates exposed to IRI-160AA exhibited increased production of reactive oxygen species (ROS), DNA damage, and cell cycle arrest, implying a programmed pathway leading to cell death (PCD). Here, a metabolomic analysis was conducted on dinoflagellate Karlodinium veneficum and a control cryptophyte species Rhodomonas exposed to IRI-160AA to investigate the cellular mechanisms behind the physiological effects and the specificity of this algicide. Results of this research supported previous observations about physiological responses to the algicide. A suite of metabolites was identified that increased in the cell pellets of K. veneficum but not in Rhodomonas, including oxidative stress biomarkers, antioxidants, and compounds involved in DNA damage and PCD. Overall, the results of this study illustrated the metabolomic mechanisms underlying the algicidal effects of IRI-160AA on dinoflagellates. This research also provided insights and future directions for studies on the cellular response of dinoflagellates exposed to antagonistic bacteria in the environment. [ABSTRACT FROM AUTHOR]

Harmful algal blooms caused by Karlodinium veneficum recently occurred with high incidence, posing a serious threat to the marine ecological environment, public health, and mariculture. It is therefore rather vital to establish a method for rapid detection of K. veneficum. In this study, the D1–D2 region of the large subunit rDNA (LSU rDNA D1–D2) of K. veneficum was cloned and sequenced to design the specific probes and primers. A novel method referred to as double-nick rolling circle amplification (dn-RCA) based on the designed probes and primers was initially established. The optimal reaction conditions for dn-RCA were as follows: probe concentration, 200 pM; ligation temperature, 57 °C; ligation time, 50 min; amplification temperature, 60 °C; and amplification time, 60 min. Furthermore, lateral flow dipstick (LFD) was employed instead of agarose gel electrophoresis to analyze dn-RCA products, which can simplify the detection procedure and reduce the operation time. The sensitivity of dn-RCA-LFD was tested with the genomic DNA, the recombinant plasmid containing the inserted LSU rDNA D1–D2, and the DNA crude extract of K. veneficum. The results showed that the sensitivity of dn-RCA-LFD was 10 times higher than that of conventional PCR; the detection limit of dn-RCA-LFD was 1.1 × 10−4 ng μL−1 for the genomic DNA, 360 copies μL−1 for the recombinant plasmid, and 5.3 cells mL−1 for DNA crude extract. The results of the cross-reactivity test with 22 control microalgal species showed that the dn-RCA-LFD had high specificity for K. veneficum. The stability of dn-RCA-LFD was tested by mixing the interfering genomic DNA with the target genomic DNA, which can be expected to simulate the natural samples containing different ratios of interfering cells to target cells. The results indicated that the performance of dn-RCA-LFD was immune to the DNA concentration of the interfering species. Finally, the practicability of dn-RCA-LFD was further confirmed by the test with field samples collected from the East China Sea. In conclusion, the established dn-RCA-LFD has advantages of high sensitivity, strong specificity, and stable performance, and is therefore promising for rapid detection of K. veneficum. [ABSTRACT FROM AUTHOR]

Shewanella sp. IRI-160 is an algicidal bacterium that secretes an algicide, IRI-160AA. This algicide specifically targets dinoflagellates, while having no adverse effects on other algal species tested. Dinoflagellates exposed to IRI-160AA exhibited increased production of reactive oxygen species (ROS), DNA damage, and cell cycle arrest, implying a programmed pathway leading to cell death (PCD). Here, a metabolomic analysis was conducted on dinoflagellate Karlodinium veneficum and a control cryptophyte species Rhodomonas exposed to IRI-160AA to investigate the cellular mechanisms behind the physiological effects and the specificity of this algicide. Results of this research supported previous observations about physiological responses to the algicide. A suite of metabolites was identified that increased in the cell pellets of K. veneficum but not in Rhodomonas, including oxidative stress biomarkers, antioxidants, and compounds involved in DNA damage and PCD. Overall, the results of this study illustrated the metabolomic mechanisms underlying the algicidal effects of IRI-160AA on dinoflagellates. This research also provided insights and future directions for studies on the cellular response of dinoflagellates exposed to antagonistic bacteria in the environment.

Biosurfactant has potential application value in the removal of microalgal blooms, but the ecological risks require more research. In this paper, the effects of surfactin on the toxic dinoflagellate Karlodinium veneficum were studied. The coaction of surfactin and K. veneficum was also evaluated through toxicological experiments on Artemia and juvenile clams. The results showed that: (1) in the concentration range of 0–10 mg/L, surfactin significantly killed algal cells in a dose-dependent manner within 48 h; the 24 h EC50 was 3.065 mg/L; (2) K. veneficum had the ability to restore population growth after stress reduction and the restored proliferation was positively correlated with the initial surfactin concentration; (3) the ability to restore population growth was associated with protection afforded by the promotion of antioxidant enzymes, including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), whose increase was positively correlated with the surfactin concentration; (4) the toxicity of the coculture of surfactin and K. veneficum was significantly greater than that of the K. veneficum culture or surfactin alone and was dose and time dependent. The potential ecological risks should be considered when applying biosurfactants, such as surfactin, in the removal of harmful algal blooms.

Harmful algal blooms (HABs) caused by microalgae pose a great threat to human health, marine ecosystems, tourism, and aquaculture worldwide. Efficient, sensitive, and simple methods for the detection of harmful microalgal species must be developed to reduce the economic losses and damages caused by HABs. This study successfully established a novel method for the detection of the globally distributed HAB-forming species Karlodinium veneficum. The developed method was based on exponential rolling circle amplification (E-RCA) coupled with lateral flow dipstick (LFD) strips. A special oligonucleotide sequence-padlock probe (PLP) was designed on basis of the molecular cloning and subsequent sequencing of the D1–D2 region of the large subunit ribosomal DNA (LSU rDNA) of K. veneficum. The E-RCA reaction system was then established. E-RCA products could be visually analyzed through 2% agarose gel electrophoresis and the LFD assay. The optimized E-RCA conditions were as follows: PLP concentration of 20 pM, ligation cycle number of 12, ligation temperature of 56 °C, amplification duration of 45 min, and amplification temperature of 61 °C. The developed E-RCA-LFD assay was specific for K. veneficum and did not display cross-reactivity with other microalgal species. The E-RCA-LFD assay was 100-fold more sensitive than conventional polymerase chain reaction (PCR) and had detection limits of 8.0 × 101 ag μL−1 for the genomic DNA of K. veneficum and 5.0 × 101 ag μL−1 (approximately 13 copies μL−1) for recombinant plasmids containing the LSU rDNA D1–D2 regions of K. veneficum. Simulative tests indicated that the E-RCA-LFD assay demonstrated considerably higher sensitivity than conventional PCR and a detection limit of 0.01 cell mL−1. The practicability of the E-RCA-LFD assay was confirmed through tests with field samples. In conclusion, the highly sensitive, specific, and facile E-RCA-LFD assay established in this work may enable the field monitoring of natural samples containing K. veneficum. [ABSTRACT FROM AUTHOR]

The toxic dinoflagellate Karlodinium veneficum is widely distributed in cosmopolitan estuaries and is responsible for massive fish mortality worldwide. Intraspecific biodiversity is important for the spread to various habitats, interspecific competition to dominate a population, and bloom formation and density maintenance. Strategies for light adaptation may help determine the ecological niches of different ecotypes. However, the mechanism of phenotypic biodiversity is still unclear. In this study, intraspecific differences in genetic regulatory mechanisms in response to varied light intensities and qualities were comparatively researched on two different strains isolated from coastal areas of the East China Sea, namely, GM2 and GM3. In GM2, the expression of genes in the Calvin cycle, namely, rbcL and SBPase , and a light-related gene that correlated with cellular motility, rhodopsin , were significantly inhibited under high light intensities. Thus, this strain was adapted to low light. In contrast, the gene expression levels were promoted by high light conditions in GM3. These upregulated genes in the GM3 strain probably compensated for the negative effects on the maximum quantum yields of PSII (Fv/Fm) under high light stress, which inhibited both strains, enabling GM3 to maintain a constant growth rate. Thus, this strain was adapted to high light. Compared with white light, monochromatic blue light had negative effects on Fv/Fm and the relative electron transfer rate (ETR) in both strains. Under blue light, gene expression levels of rbcL and SBPase in GM2 were inhibited; in contrast, the levels of these genes, especially rbcL , were promoted in GM3. rbcL was significantly upregulated in the blue light groups. Monochromatic red light promoted rhodopsin gene expression in the two strains in a similar manner. These intraspecific diverse responses to light play important roles in the motor characteristics, diel vertical migration, interspecific relationships and photosynthetic or phagotrophic activities of K. veneficum and can determine the population distribution, population maintenance and bloom formation. • Different intraspecific responses to light were observed in the ecotype strains. • Light-related gene regulation can show completely opposite intraspecific patterns. • Upregulation of photosynthetic genes may compensate for photochemical loss. • Red light promoted gene expression of rhodopsin in K. veneficum. • K. veneficum has high-light and low-light ecotypes. [ABSTRACT FROM AUTHOR]

• The RPA primers and LFD probe were designed to target the ITS of K. veneficum. • The RPA-LFD was specific for K. veneficum. • The RPA-LFD was 100-fold more sensitive than PCR. • The practicability of RPA-LFD was confirmed by test with the spiked field samples. The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 °C–45 °C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPA-LFD was 10 ng μL−1 of genomic DNA and 0.1 cell mL−1, which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future. [ABSTRACT FROM AUTHOR]

• The duration of cytokinesis in Karlodinium veneficum was determined for four geographically distinct strains and is approximately 1.6 hours. • Uniformity of the duration of cytokinesis across a fourfold range of optimal growth rates for four geographically distinct strains indicates a genetically conserved t d of 1.6 hours. • Differences in growth responses to temperature indicate that the strains used represent three thermal ecotypes. The toxic dinoflagellate Karlodinium veneficum forms fish killing blooms in temperate estuaries worldwide. These blooms have variable toxicity which may be related to bloom stage and in situ growth rates of the constituent K. veneficum cells. Measurement of in situ growth rates is challenging and methods such as the mitotic index technique require knowledge of the dynamics of cell division. In order to better understand these dynamics, we determined the duration of cell division (t d) in four geographically distinct laboratory strains of K. veneficum at three different environmentally relevant temperatures. The results demonstrated that the t d value for each strain, growing at strain-specific optimal temperatures, was 1.6 ± 0.1 h. This value corresponded to a range of growth rates from 0.17 ± 0.08 d−1 to 0.62 ± 0.07 d−1. Equivalent values of t d spread across four geographically distinct laboratory strains and a nearly fourfold range of growth rates implies that 1.6 h represents the t d value of K. veneficum. Additionally, temperature conditions yielding this value for t d and the highest growth rates varied among strains, indicating cold-adapted (Norway), warm-adapted (Florida, USA), and eurythermally-adapted (Maryland, USA) strains. These differences have been apparently retained in culture over many years, indicating a conserved genetic basis that suggests distinct thermal ecotypes of the morphospecies K. veneficum. This knowledge together with the first estimate of t d for K. veneficum will be useful in future field studies aimed at correlating bloom toxicity with in situ growth rate using the mitotic index technique. [ABSTRACT FROM AUTHOR]

• Polystyrene nanoplastics inhibit the growth of Karlodinium veneficum in a concentration and time-dependent manner. • The exposure of polystyrene nanoplastics enhances the hemolysin production of Karlodinium veneficum. • The toxicity mechanisms of polystyrene nanoplastics to Karlodinium veneficum are attributed to oxidative stress, energy metabolism imbalance, physical damage and photosynthesis inhibition. T here are few studies on the effects of nanoplastics on growth and hemolysin production of harmful algal bloom species at present. In this study, Karlodinium veneficum was exposed to different concentrations (0, 5, 25, 50, 75 mg/L) of polystyrene nanoplastics (PS-NPs, 100 nm) for 96 h. The effects of PS-NPs on growth of K. veneficum were investigated by measuring algal cell abundance, growth inhibition rate (IR), total protein (TP), malondialdehyde (MDA), glutathione reductase (GSH), superoxide dismutase (SOD), ATPase activity (Na+/K+ ATPase and Ca2+/Mg2+ ATPase). Scanning electron microscope and transmission electron microscope (SEM and TEM) images of microalgae with or without nanoplastics were also observed. The effects of PS-NPs on hemolysin production of K. veneficum were studied by measuring the changes of hemolytic toxin production of K. veneficum exposed to PS-NPs on 1, 3, 5 and 7 days. High concentrations (50 and 75 mg/L) of PS-NPs seriously affected the growth of K. veneficum and different degrees of damage to cell morphology and ultrastructure were found. Excessive free radicals and other oxidants were produced in the cells, which disrupted the intracellular redox balance state and caused oxidative damage to the cells, and the basic activities such as photosynthesis and energy metabolism were weakened. The athletic ability of K. veneficum was decreased, but the ability to produce hemolysin was enhanced. It was suggested that the presence of nanoplastics in seawater may strengthen the threat of harmful algal bloom species to aquatic ecosystems and human health. [Display omitted] [ABSTRACT FROM AUTHOR]

The ichthyotoxic dinoflagellate Karlodinium veneficum has a worldwide distribution and produces highly potent lytic toxins (karlotoxins) that have been associated with massive fish kill events in coastal environments. The capacity of K. veneficum to gain energy from photosynthesis as well as phagotrophy enables cellular maintenance, growth and dispersal under a broad range of environmental conditions. Coastal ecosystems are highly dynamic in light of the prevailing physicochemical conditions, such as seawater carbonate speciation (CO2, HCO3−, and CO32−) and pH. Here, we monitored the growth rate and ichthyotoxicity of K. veneficum in response to a seawater pH gradient. K. veneficum exhibited a significant linear reduction in growth rate with elevated seawater acidity [pH(totalscale) from 8.05 to 7.50]. Ichthyotoxicity was assessed by exposing fish gill cells to K. veneficum extracts and subsequent quantification of gill cell viability via resorufin fluorescence. Extracts of K. veneficum indicated increased toxicity when derived from elevated pH treatments. The variation in growth rate and toxin production per cell in regard to seawater pH implies that (1) future alteration of seawater carbonate speciation, due to anthropogenic ocean acidification, may negatively influence physiological performance and ecosystem interactions of K. veneficum and (2) elevated seawater pH values (>8.0) represent favorable conditions for K. veneficum growth and toxicity. This suggests that prey of K. veneficum may be exposed to increased karlotoxin concentrations at conditions when nutrients are scarce and seawater pH has been elevated due to high photosynthetic activity from prior autotrophic phytoplankton blooms.

Results from a dynamic mathematical model are presented simulating the growth of the harmful algal bloom (HAB) mixotrophic dinoflagellate Karlodinium veneficum and its algal prey, Rhodomonas salina. The model describes carbon-nitrogen-phosphorus-based interactions within the mixotroph, interlinking autotrophic and phagotrophic nutrition. The model was tuned to experimental data from these species grown under autotrophic conditions and in mixed batch cultures in which nitrogen:phosphorus stoichiometry (input molar N:P of 4, 16, and 32) of both predator and prey varied. A good fit was attained to all experimentally derived carbon biomass data. The potential effects of temperature and nutrient changes on promoting growth of prey and thus K. veneficum bloom formation were explored using this simulation platform. The simulated biomass of K. veneficum was highest when they were functioning as mixotrophs and when they consumed prey under elevated N:P conditions. The scenarios under low N:P responded differently, with simulations showing larger deviation between mixotrophic and autotrophic growth, depending on temperature. When inorganic nutrients were in balanced proportions, lower biomass of the mixotroph was attained at all temperatures in the simulations, suggesting that natural systems might be more resilient against Karlodinium HAB development in warming conditions if nutrients were available in balanced proportions. These simulations underscore the need for models of HAB dynamics to include consideration of prey; modeling HAB as autotrophs is insufficient. The simulations also imply that warmer, wetter springs that may bring more N with lower N:P, such as predicted under climate change scenarios for Chesapeake Bay, may be more conducive to development of these HABs. Prey availability may also increase with temperature due to differential growth temperature responses of K. veneficum and its prey.

Graphical abstract Highlights • Froude number correlates gas holdup and mass transfer in the bubble column. • Energy dissipation rate can be described as a function of Froude number. • Average energy dissipated correlates the growth of K. veneficum. • CO 2 mass transfer limits the growth of K. veneficum at low energy dissipation. • A model for the influence of dissipated energy on cell growth is proposed. Abstract The shear-sensitive marine algal dinoflagellate Karlodinium veneficum was grown in a cylindrical bubble column photobioreactor with an internal diameter of 0.044 m. Initial liquid height varied from 0.5 to 1.75 m, superficial gas velocities from 0.0014 to 0.0057 ms−1, and nozzle diameter from 1 to 2.5 mm. Computational fluid dynamics was used to characterize the flow hydrodynamics and energy dissipation rates. Experimental gas holdup and volumetric mass transfer coefficient strongly depended on the liquid height and correlated well with the Froude number. Energy dissipation near the head space (ED top) was one order of magnitude higher than the average energy dissipation in the whole reactor (ED whole), and the value in the sparger zone (ED spar) was one order of magnitude higher than ED top. Cultures of K. veneficum were limited by CO 2 transfer at low ED whole and severely stressed above a critical value of ED whole. [ABSTRACT FROM AUTHOR]

Grazing studies were conducted on the mixotrophic dinoflagellate, Karlodinium veneficum, in the presence of single prey species, the cryptophyte Rhodomonas salina, and a phycocyanin-containing strain of the cyanobacterium, Synechococcus sp. and when prey were mixed in varying proportions. A multiwavelength PAM fluorometer was used for non-invasive biomass estimates and to detect changes in photophysiology. Growth and grazing rates of K. veneficum increased as the function of increasing prey concentrations of R. salina, regardless of whether the prey were provided as a single prey item or in a mixed prey assemblages. With Synechococcus as the prey, in single or mixed prey assemblages, it was poorly grazed and growth rates of the mixotroph declined. The maximal quantum yields of PSII fluorescence (F v/ F m) of the mixotroph declined when Synechococcus was the prey but remained high when fed on R. salina. The F v/ F m of R. salina declined by about 60% as it was consumed, while that of Synechococcus sp. increased as mixotroph growth declined. Robust relationships were established between flow cytometry-based cell counts and PAM fluorometry-based chlorophyll measurements, validating the usefulness of this rapid and non-intrusive quantification approach for measuring mixotrophy in predator–prey interactions among multiple differently pigmented species. [ABSTRACT FROM AUTHOR]

The dinoflagellate Karlodinium veneficum blooms kill fish by the production of karlotoxins. We investigated the allelopathy (Akashiwo sanguinea as the target species) and toxicity (Oryzias melastigma, Artemia salina and Brachionus plicatilis as test animals) of four strains of K. veneficum isolated from coastal waters of China and the USA to explore the ecological implications of allelopathy and toxicity. In general, all four strains of K. veneficum at high cell densities (>100 000 cells·mL−1) significantly inhibited the growth of A. sanguinea to different extents, but they exhibited either positive or negative allelopathy at lower densities. The toxicity of the four strains showed a decreasing order coinciding with their culture histories in the laboratory, suggesting K. veneficum may have gradually lost toxicity during laboratory culturing. The allelopathic and toxic potencies among K. veneficum strains were not perfectly parallel, indicating that either the allelochemicals and toxins of K. veneficum may be different chemicals, or the same chemicals functioned in different modes. Moreover, we found A. salina avoided feeding on more toxic strains. These results together suggest that toxins provide an advantage for K. veneficum via avoiding predation, but allelopathy may not play an important role in initiating blooms of K. veneficum. [ABSTRACT FROM AUTHOR]

Highlights • Real-time qPCR is an efficient tool to quantifiy K. veneficum at relatively low abundance of field samples. • Three genotypes of K. veneficum were verified in the coastal area of Xiangshan Bay area in the East China Sea (ECS) based on comparing with the documented strain. • Despite of relatively low cell abundance, distribution of K. veneficum in this area was remarkably extensive. • Abundance of K. veneficum varied in correspondence with critical environmental factors such as temperature, salinity and water depth. • Toxic dinoflagellate K. veneficum can grow in muddy seawater of low light transparency. Abstract Athecate dinoflagellate Karlodinium veneficum is a universal toxic species possessing karlotoxins recognized especially as ichthyotoxic as well as cytotoxic and hemolytic. Blooms of K. veneficum , both single-species or accompanied with other species, occurred more frequently worldwide in recent years, including the coastal region of China. Normally, K. veneficum present in relatively low abundance in phytoplankton communities in estuary regions. Being small and difficult to identify with light microscopy, it has been ignored for a long time till its blooming and toxins being confirmed. How it presents in background level and what is its relationship with critical geological and hydrological environment factors are basically not clear. In this study, the paper reports the application of a real-time quantitative PCR (qPCR) method to investigate the abundance and distribution of K. veneficum in the coastal waters of Xiangshan Bay in the East China Sea (ECS), a typical bay area of harmful algae blooms and heavily affected by anthropogenic activities. The real-time qPCR assay came out being an efficient method at detecting even low cell densities of K. veneficum of different genotypes. A total of 38 field samples of surface (0.5 m) and bottom water (9–100 m in depth) were analyzed and 12 samples were found positive for K. veneficum. At least 3 genotypes of K. veneficum present in this region. Temperatures in sites of K. veneficum positive ranged from 21.7 to 23.4 °C, and salinity levels were between 21.1 and 26.3. The K. veneficum distributed quite extensively in the waters of Xiangshan Bay, cell abundance varied from a low of 4 cells/L to a maximum of 170 cells/L. Most of the samples containing K. veneficum were collected from bottom water in different sites. At three of the 19 sampling sites, K. veneficum was detected in both surface and bottom water samples. Especially at sampling site near Beilun port, where the water is typically muddy with low transparency, relative high cell numbers of K. veneficum were found in both surface and bottom waters. Mixotrophy and vertical migration of K. veneficum could be important eco-physiological factors to consider in terms of understanding these distribution characteristics. The ideal conditions for K. veneficum growth and aggregation in this area still needs further study. [ABSTRACT FROM AUTHOR]

Highlights • This is the first time that RNA interference (RNAi) was applied to dinoflagellates. • Silencing RNA (siRNA) was used to target rhodopsin and Rubisco in two species. • siRNA treatment caused decreases in growth rate in both species. • siRNA treatment resulted in suppression of both genes in both species. Abstract Dinoflagellates are a major group of protists widely distributed in the aquatic environments. Many species in this lineage are able to form harmful algal blooms (HAB), some even producing toxins, making this phylum the most important contributors of HAB in the marine ecosystem. Despite the ecological importance, the molecular mechanisms underpinning the basic biology and HAB formation of dinoflagellates are poorly understood. While the high-throughput sequencing studies have documented a large and growing number of genes in dinoflagellates, their functions remained to be experimentally proven using a functional genetic tool. Unfortunately, no such tool is yet available. This study was aimed to adopt the RNA interference (RNAi) gene-silencing tool for dinoflagellate research, and to investigate the potential effects of RNAi-based silencing of proton-pump rhodopsin and CO 2 -fixing enzyme Rubisco encoding genes in dinoflagellates. It was found that RNAi treatment caused a significant decrease in growth rate in both species. Compared with the non- endogenous target (GFP-siRNA) and the blank control, RNAi treatments also suppressed the expression of the target genes. These results constitute the first experimental evidence of the existence and operation of siRNA in two species of dinoflagellates, present initial evidence that dinoflagellate rhodopsins are functional as a supplemental energy acquisition mechanism, and provide technical information for future functional genetic research on dinoflagellates. [ABSTRACT FROM AUTHOR]

Abstract Karlodinium is a dinoflagellate responsible for fish-killing events worldwide. In Alfacs Bay (NW Mediterranean Sea), the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. This work presents a method that combines recombinase polymerase amplification (RPA) with an enzyme-linked oligonucleotide assay (ELONA) to identify, discriminate and quantify these two species. The system was characterised using synthetic DNA and genomic DNA, and the specificity was confirmed by cross-reactivity experiments. Calibration curves were constructed using 10-fold dilutions of cultured cells, attaining a limit of detection of around 50,000 cells/L, far below the Karlodinium spp. alert threshold (200,000 cells/L). Finally, the assay was applied to spiked seawater samples, showing an excellent correlation with the spiking levels and light microscopy counts. This approach is more rapid, specific and user-friendly than traditional microscopy techniques, and shows great promise for the surveillance and management of harmful algal blooms. Graphical abstract Image 1 Highlights • An assay for the detection of two Karlodinium species has been developed. • The assay is based on isothermal DNA amplification and a sandwich hybridisation. • The assay discriminates between Karlodinium veneficum and Karlodinium armiger. • Seawater samples spiked with Karlodinium cells have been analysed. • Excellent correlations between the assay and light microscopy have been obtained. [ABSTRACT FROM AUTHOR]

Typical harmful micro-algal species constantly occurred in high density in marine aquaculture ponds in Xiangshan and Sanmen Bay, Zhejiang Province. Fates of the microalgal cells influenced by activity of the cultured animals largely determined the effects of the harmful microalgae. However, it is difficult to detect the in situ process. In this paper, toxic activities of three harmful microalga, namely Prymnesium parvum, Pleurochrysis elongata, Karlodinium veneficum, which were isolated from the local ponds, were comparatively studied based on brine shrimp toxic bioassays. Different lethal activities of live cells, cell debris, cellular extracts, and cell free mediums prepared by different process were analyzed. The results showed that, (1) all of the three microalgal species had density and time dependent lethal effects on Artermia nauplii, while P. parvum was the most toxic one and had acute lethal effects in 5 h. No such acute lethal effects were observed in P. elongata or K. veneficum; (2) live cells, cell debris and cellular extracts of P. parvum had the same lethal pattern. Prymnesins, toxin from P. parvum, is probably not exotoxic active; For P. elongata, toxic activity mainly came from live cells and cell debris; For K. veneficum, toxic activity was relatively lower compared with the other two species. However, Karlotoxin, toxin from K. veneficum, is exotoxic active. Physical disturbance triggered K. veneficum cells actively releasing toxins, which made it an active predator. [ABSTRACT FROM AUTHOR]

Karlodinium is a dinoflagellate genus responsible for massive fish mortality events worldwide. It is commonly found in Alfacs Bay (NW Mediterranean Sea), where the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. Microscopy analysis is not able to differentiate between these two species. Therefore, new and rapid methods that accurately and specifically detect and differentiate these two species are crucial to facilitate routine monitoring, to provide early warnings and to study population dynamics. In this work, a quantitative real-time PCR (qPCR) method to detect and enumerate K. veneficum and K. armiger is presented. The ITS1 region of the ribosomal DNA was used to design species-specific primers. The specificity of the primers together with the melting curve profile provided a reliable qualitative identification and discrimination between the two Karlodinium species. Additionally, a simple and rapid DNA extraction method was used. Standard curves were constructed from 10-fold dilutions of cultured microalgae cells. Finally, the applicability of the assay was tested with field samples collected from Alfacs Bay. Results showed a significant correlation between qPCR determinations and light microscopy counts (y = 2.838 x + 564; R2 = 0.936). Overall, the qPCR method developed herein is specific, rapid, accurate, and promising for the detection of these two Karlodinium species in environmental samples. [ABSTRACT FROM AUTHOR]

Blooms of ichthyotoxic microalgae pose a great challenge to the aquaculture industry world-wide, and there is a need for fast and specific methods for their detection and quantification in monitoring programs. In this study, quantitative real-time PCR (qPCR) assays for the detection and enumeration of three ichthyotoxic flagellates: the dinoflagellate Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup and the two raphidophytes Heterosigma akashiwo (Hada) Hada ex Hara & Chihara and Fibrocapsa japonica Toriumi & Takano were developed. Further, a previously published qPCR assay for the dinoflagellate Karlodinium veneficum (Ballantine) Larsen was used. Monthly samples collected for three years (Aug 2009–Jun 2012) in outer Oslofjorden, Norway were analysed, and the results compared with light microscopy cell counts. The results indicate a higher sensitivity and a lower detection limit (down to 1 cell L −1 ) for both qPCR assays. Qualitative and semi-quantitative results were further compared with those obtained by environmental 454 high throughput sequencing (HTS, metabarcoding) and scanning electron microscopy (SEM) examination from the same samplings. All four species were detected by qPCR and HTS and/or SEM in outer Oslofjorden (Aug 2009–Jun 2012); Karlodinium veneficum was present year-round, whereas Karenia mikimotoi, Heterosigma akashiwo and Fibrocapsa japonica appeared mainly during the autumn in all three years. This is the first observation of Fibrocapsa japonica in Norwegian coastal waters. This species has previously been recorded off the Swedish west coast and German Bight, which may suggest a northward dispersal. [ABSTRACT FROM AUTHOR]

A pilot-scale bioprocess was developed for the production of karlotoxin-enriched extracts of the marine algal dinoflagellate Karlodinium veneficum . A bubble column and a flat-panel photobioreactors (80–281 L) were used for comparative assessment of growth. Flow hydrodynamics and energy dissipation rates (EDR) in the bioreactors were characterized through robust computational fluid dynamic simulations. All cultures were conducted monoseptically outdoors. Bubble column (maximum cell productivity in semicontinuous operation of 58 × 10 3  cell mL −1  day −1 ) proved to be a better culture system for this alga. In both reactors, the local EDR near the headspace, and in the sparger zone, were more than one order of magnitude higher than the average value in the whole reactor (=4 × 10 −3  W kg −1 ). Extraction of the culture and further purification resulted in the desired KTXs extracts. Apparently, the alga produced three congeners KTXs: KmTx-10 and its sulfated derivative (sulfo-KmTx-10) and KmTx-12. All congeners possessed hemolytic activity. [ABSTRACT FROM AUTHOR]

The harmful dinoflagellate, Karlodnium veneficum , has been implicated in fish-kill and other toxic, harmful algal bloom (HAB) events in waters worldwide. Blooms of K. veneficum are known to be related to coastal nutrient enrichment but the relationship is complex because this HAB taxon relies not only on dissolved nutrients but also particulate prey, both of which have also changed over time. Here, applying cross-correlations of climate-related physical factors, nutrients and prey, with abundance of K. veneficum over a 10-year (2002–2011) period, a synthesis of the interactive effects of multiple factors on this species was developed for Chesapeake Bay, where blooms of the HAB have been increasing. Significant upward trends in the time series of K. veneficum were observed in the mesohaline stations of the Bay, but not in oligohaline tributary stations. For the mesohaline regions, riverine sources of nutrients with seasonal lags, together with particulate prey with zero lag, explained 15%–46% of the variation in the K. veneficum time series. For the oligohaline regions, nutrients and particulate prey generally showed significant decreasing trends with time, likely a reflection of nutrient reduction efforts. A conceptual model of mid-Bay blooms is presented, in which K. veneficum, derived from the oceanic end member of the Bay, may experience enhanced growth if it encounters prey originating from the tributaries with different patterns of nutrient loading and which are enriched in nitrogen. For all correlation models developed herein, prey abundance was a primary factor in predicting K. veneficum abundance. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4 pg/μL (approximately 6.5 cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assay’s accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum. [ABSTRACT FROM AUTHOR]

Production of biomass of the shear-sensitive marine algal dinoflagellate Karlodinium veneficum was successfully scaled-up to 80 L using a bubble column photobioreactor. The scale factor exceeded 28,500. Light-emission diodes were used as the light source. The diel irradiance profile mimicked the outdoor profile of natural sunlight. The final cell concentration in the absence of nutrient limitation in the scaled-up photobioreactor was nearly 12 × 10 5 cells mL −1 , or the same as in laboratory culture systems. The pH-controlled culture (pH = 8.5) was always carbon-sufficient. The culture was mixed pneumatically by using a superficial air velocity of 1.9 × 10 −3 m s −1 and the temperature was controlled at 21 ± 1 °C. [ABSTRACT FROM AUTHOR]

The dinoflagellate Karlodinium veneficum is a harmful algal bloom species with a worldwide distribution. This small athecate dinoflagellate makes a family of polyketide toxins that are hemolytic, cytotoxic and ichthyotoxic. The first chemical structure for karlotoxins from East China Sea (ECS) is reported here. The two new karlotoxins, namely 4,5-dihydro-KmTx 2 (compound 1 ) and 4,5-dihydro-dechloro-KmTx 2 (compound 2 ), were isolated and purified from monoalgal cultures of K. veneficum strain GM2. Their structures were determined by spectroscopic analysis, including tandem mass spectrometry as well as 1D and 2D NMR experiments. These new karlotoxin congeners feature a saturated polyol arm different from previously reported for KmTx 2 that appears to increase hemolytic activity. [ABSTRACT FROM AUTHOR]

A feed-forward back-propagation neural network (FBN) was used as a tool for predicting the growth dynamics of the microalga Karlodinium veneficum in a culture medium with any specified concentrations of the key nutrients. A 3-layered FBN configuration of 27-25-1 nodes was used. This FBN satisfactorily represented the nonlinear interactions among all the nutrients of a culture medium containing up to 25 different components. The FBN model was trained using the growth dynamics data from more than 420 batch culture experiments involving different media compositions. The relative impact of individual nutrients, the initial cell concentration and the culture duration on growth profiles were determined through a systematic analysis of the partitioning of the FBN connection weights. Microelements and vitamins together had a higher relative impact on growth compared to the impact of the macronutrients. The trained FBN successfully predicted the cell concentration dynamics in cultures with previously untested initial conditions. The FBN proved to be an excellent tool for predicting the growth curves in the range of culture conditions that were relevant to this study. [ABSTRACT FROM AUTHOR]

The dinoflagellate Karlodinium veneficum was grown in bubble column photobioreactors and a genetic algorithm-based stochastic search strategy used to find optimal values for the culture parameters gas flow rate, culture height, and nozzle sparger diameter. Cell production, concentration of reactive oxygen species (ROS), membrane fluidity and photosynthetic efficiency were studied throughout the culture period. Gas-flow rates below 0.26 L min −1 , culture heights over 1.25 m and a nozzle diameter of 1.5 mm were found to provide the optimal conditions for cell growth, with an increase of 60% in cell production with respect to the control culture. Non-optimal conditions produced a sufficiently high shear stress to negatively affect cell growth and even produce cell death. Cell physiology was also severely affected in stressed cultures. The production of ROS increased by up to 200%, whereas cell membrane fluidity decreased by 60% relative to control cultures. Photosynthetic efficiency decreased concomitantly with membrane fluidity. [ABSTRACT FROM AUTHOR]

The dinoflagellate Prorocentrum donghaiense is a dominant harmful algal bloom (HAB) species on the East China Sea (ECS) coast. The co-occurrence of Karlodinium veneficum with P. donghaiense is often observed and can later develop into dense blooms. However, the role of K. veneficum in P. donghaiense population dynamics is unknown. In the current study, three K. veneficum (GM1, GM2, and GM3) strains were isolated from the ECS with one (GM1) from a mixed, dense bloom of P. donghaiense and other HAB species. All three isolates had identical ITS sequences that were concordant with the species designation. Unique karlotoxin congeners were isolated from one strain (GM2). The sterol compositions of P. donghaiense and K. veneficum were consistent with sensitivity to karlotoxin in the former and insensitivity in the latter. Additional experimentation showed that: (1) in monocultures, higher growth rate of P. donghaiense than K. veneficum is observed in nutrient-enriched and nutrient-depleted media. In co-cultures, the growth of P. donghaiense is inhibited; (2) feeding on P. donghaiense by K. veneficum is clearly demonstrated by fluorescent dye tracking; and (3) the isolated karlotoxin is lethal to P. donghaiense in a concentration-dependent manner. From these studies we propose that K. veneficum may play a negative role in P. donghaiense bloom maintenance and that P. donghaiense may in turn be a bloom initiator as a prey item for K. veneficum . [ABSTRACT FROM AUTHOR]

Ichthyotoxic Karlodinium veneficum has become a persistent problem in the eutrophic Swan River Estuary (SRE) near Perth, Western Australia. Karlotoxin (KmTx) concentrations and K. veneficum were sampled from March to July 2005, spanning a bloom confirmed by microscopy and genetics (ITS sequence), and a fish kill coincident with end of the bloom. The objective of this study was to investigate K. veneficum cell and toxin dynamics, and water quality conditions, leading up to the bloom and fish kill in this estuarine system. Abundance of K. veneficum increased as diatom abundance decreased over a 3-month period (Jan–Mar) preceding the bloom. Low freshwater flow to the SRE characterized the bloom initiation period, while elevated seasonal flows altered water quality and preceded the end of the bloom and fish kill. The bloom of K. veneficum was localized over a bottom layer of hypoxic water in a stratified water column. Low nitrate levels, DIN:DIP (mol) near unity, and particulate C:N:P of K. veneficum -rich water samples were consistent with nitrogen limitation of phytoplankton. A KmTx 2 congener was present in the concentration range 0–1052 ng KmTx mL −1 , levels that were sufficient to kill larval fish in the laboratory within 4 h. A KmTx cell quota of 2.8 pg KmTx cell −1 was estimated for the bloom, which is moderately high for the species. Gill histopathology of fish from this fish kill showed signs of damage similar to those caused by KmTx in the lab. Results from this study suggest that conditions in the SRE, including elevated K. veneficum abundance and KmTx cell quotas, as well as hypoxia in the upper SRE, likely contribute to seasonal fish kills observed in this system. [ABSTRACT FROM AUTHOR]

In addressing a potential bioprocess for bioactives production from microalgae, the choice of a proper culture medium is a key factor. In the present work, an optimal culture medium for producing the marine dinoflagellate microalga Karlodinium veneficum was formulated using a stochastic search strategy based on genetic algorithm. The optimized formulation had 25 components including the macronutrients, trace elements and vitamins. With this medium, the final attainable cell concentration during batch growth was enhanced by 120% relative to the control culture grown in the L1 medium. The final titer of the bioactive karlotoxins measured using the hemolytic activity of the culture filtrate was enhanced by 190% relative to the L1 control. These results are a promising basis for further development of photobioreactor culture strategies for microalgal dinoflagellates. [ABSTRACT FROM AUTHOR]

• DinoCBWD s are not linked to vitamin B 12 biosynthesis. • DinoCBWDs mainly belong to three subfamilies of the COG0523 protein family of GTPases. • DinoCBWD s are associated with metal homeostasis and other divergent functions, with some DinoCBWDs involving in zinc or iron homeostasis. Since > 91% of dinoflagellates are proven auxotrophs of vitamin B 12 and the cobalamin synthetase W (CobW) is a key gene involved in vitamin B 12 synthesis pathway, a number of CobW domain-containing (CBWD) genes in dinoflagellates (DinoCBWD s) were surprisedly found from our transcriptomic and meta-transcriptomic studies. A total of 88 DinoCBWD genes were identified from the genomes and transcriptomes of four dinoflagellates, with five being cloned for full-lengths and characterized using the cosmopolitan and ecologically-important dinoflagellates Karlodinium veneficum and Scrippsiella trochoidea (synonym of Scrippsiella acuminata). DinoCBWD s were verified being irrelevant to vitamin B 12 biosynthesis due to their transcriptions irresponsive to vitamin B 12 levels and their phylogenetic positions. A comprehensive phylogenetic analysis demonstrated 75 out of the 88 DinoCBWD genes identified belong to three subfamilies of COG0523 protein family, of which most prokaryotic members are reported to be metallochaperones and the eukaryotic members are ubiquitously found but mostly unknown for their functions. Our results from K. veneficum demonstrated DinoCBWD s are associated with metal homeostasis and other divergent functions, with four KvCBWDs involving in zinc homeostasis and KvCBWD1 likely functioning as Fe-type nitrile hydratase activator. In addition, conserved motif analysis revealed the structural foundation of KvCBWD proteins that are consistent with previously described CBWD proteins with GTPase activity and metal binding. Our results provide a stepping-stone toward better understanding the functions of DinoCBWDs and the COG0523 family. [ABSTRACT FROM AUTHOR]

Karlodinium veneficum is a cosmopolitan species, but has been poorly recorded in the East China Sea (ECS) due to its small size and difficulty in identification. The bloom dynamics of this species is not well understood globally. In this study, we examined its morphological characteristics that suggest the K. veneficum is the co-occurring bloom causative species of large scale Prorocentrum donghaiense blooms in spring 2011. The epicone of K. veneficum recorded in the investigated area is conical or rounded, and the hypocone is hemispherically rounded. The ventral pore is located at the left side of the apical groove. Nucleus is positioned centrally within the hypocone. Four large irregular chloroplasts are equally distributed in the epicone and hypocone. The mean length of cultured cells was 13.6±1.2μm (range 11.0–15.8μm) and the mean width was 10.0±1.1μm (range 8.0–12μm) (n=50). Cell abundance of K. veneficum population was low, in the region 1000–1600cellsL−1, along a transect in the East China Sea on April 19, 2011, when the water column was not distinctly stratified. Cell densities reached 3×107 cellsL−1 along the same transect on May 13 2011 when the bloom occurred in the 10m layer surface and the water column was distinctly stratified. Cell abundances therefore appear closely related to water column stratification. [ABSTRACT FROM AUTHOR]

Harmful algal blooms often occurred in the East China Sea (ECS) and the German coastal waters of the North Sea but Karlodinium veneficum had not been taxonomically reported. Two strains of Karlodinium (LAMB090611 and LAMB010601) were isolated from the two areas in 2009. The morphological characteristics and molecular phylogeny of two strains are compared on the basis of observation of a light microscope, a scanning electron microscope (SEM), a laser scanning microscope (LSM) and an internal transcribed spacer (ITS) sequence data. The mean cell length of strain LAMB090611 is (14.2±1.8) µm (range 11.1-18.7 µm) and mean width is (10.8±1.5) µm (range 8.2-14.7 µm). The mean cell length of strain LAMB010601 is (15.1±1.2) µm (range 12.7-17.9 µm) and the mean width is (11.4±1.1) µm (range 9.1-14.7 µm), respectively. The two strains are similar in morphological characteristics, including a straight apical groove, distinct ventral pore, sulcal extension, cingulum displacement, two or four irregular shaped chloroplasts within the cell and almost equal sized epicone and hypocone. The large and round nucleus is located at the center or at the hypocone of the cell. The sequence length of the ECS strain LAMB090611 and the German strain LAMB010601 is 640 and 646 bp, respectively. The GC content is 49%. The nucleotide similarity of the two strains is 98.1%. The sequence divergence is 0.003. Both strains are confirmed as Karlodinium veneficum (D. Ballantine) J. Larsen and this is the first taxonomic report from China and Germany coastal waters. The population dynamics of this toxic species in the ECS and German coastal waters needs to be investigated in the near future. [ABSTRACT FROM AUTHOR]

Abstract: We isolated eleven strains of the harmful algal bloom (HAB)-forming dinoflagellate Karlodinium veneficum during a bloom event in the NW Mediterranean coastal waters and we studied the inter-strain variability in several of their physiological and biochemical traits. These included autotrophic growth parameters, feeding capabilities (mixotrophy), lipid composition, and, in some cases, their responses to biotic and abiotic factors. The strains were found to differ in their growth rates (0.27–0.53d−1) and in the maximum cell concentrations achieved during stationary phase (6.1×104–8.6×104 cellsmL−1). Their ingestion performance, when offered Rhodomonas salina as prey, was also diverse (0.22–1.3 cells per K. veneficum per day; 8–52% of their daily ration). At least two strains survived for several months under strict heterotrophic conditions (no light, low inorganic nutrients availability, and R. salina as food source). These strains also showed very distinct fatty acid compositions, with very low contents of monounsaturated and polyunsaturated fatty acids. According to a Bray Curtis similarity analysis, three or four strain groups able to perform different roles in bloom development were identified. We further analyzed one strain from each of the two most distinct groups with respect to prey concentration, light intensity, nutrient availability, and we determined the functional responses (growth and feeding rates) to food concentration. Taken together, the results served to highlight the role of mixotrophy and clone variability in the formation of HABs. [Copyright &y& Elsevier]

Abstract: Fish-killing microalgae can cause substantial mortalities of cultured finfish, but their killing mechanisms are not completely understood. Since use of cell lines offers significant advantages compared to working with whole organisms, a simple in vitro assay for microalgal ichthyotoxicity is described using the rainbow trout cell line RTgill-W1. We describe the application of a microplate based assay for testing the toxicity of fatty acids to gill cells in a time-dependent manner. Additionally, a modification of this assay using an insert support with permeable membranes is presented to test the toxicity of living marine microalgae. The endpoint is measurement of cell viability using alamarBlue as an indicator dye. Gill cells remained 100% viable for 60h in the modified version of the basal growth medium, L-15/ex, without fetal bovine serum and with 1% methanol (in which fatty acids were dissolved). In contrast, gill cells survived for 3h in microplates with insert supports upon exposure to algal seawater-based media. Toxicity of palmitic acid was less than 10%, as observed by loss of gill cell viability, during the first 12h at all concentrations. However, the highest toxicity was observed at 48–60h with a reduction of cell viability of 50% after exposure to 140mgL−1. The ichthyotoxic raphidophyte Chattonella marina and dinoflagellate Karlodinium veneficum had a negative effect on gill cells, causing up to 70 and 38% loss of viability, respectively, when exposed to high algal concentrations for 2h. These two simple and reproducible screening tests represent a sensitive and suitable alternative to whole fish ichthyotoxic bioassays for fish-killing marine microalgae. [Copyright &y& Elsevier]

Dinoflagellates form an important component of marine and freshwater phytoplankton. They are a remarkably diverse and complex group with various modes of nutrition and ability to produce toxins, and are major contributors to harmful algal blooms. In the present work, we identify the presence of Karlodinium veneficum in India. Its detection in the coastal waters of India was possible because of live sample analysis. This species has both toxic and nontoxic strains and due to its bloom-forming capability, it is known to influence the coexisting phytoplankton community structure. In this communication we provide a description of this species, possible methods for its identification in preserved samples and effect of its extracellular and intracellular extract on the growth of Skeletonema costatum, an important and abundant diatom in the phytoplankton community in the Indian waters. [ABSTRACT FROM AUTHOR]

Microalgae are microscopic heterotrophic–autotrophic photosynthesizing organisms with enormous potential as a source of biofuel. Dinoflagellates, a class of microalgae, contain large amounts of high-quality lipids, the principal component of fatty acid methyl esters. The biotic characteristics of the dinoflagellate species Karlodinium veneficum include a growth rate of 0.14 day−1, a wet biomass of 16.4 g/L, a growth period of approximately 30 days, and an approximate 97% increase in fatty acid content during the transition from exponential phase to stationary phase. These parameters make K. veneficum a suitable choice as a bioresource for biodiesel production. Similarly, two other species were also determined to be appropriate for biodiesel production: the Dinophyceae Alexandrium andersoni and the Raphidophyte Heterosigma akashiwo. [ABSTRACT FROM AUTHOR]

We examined the influence of N or P depletion, alternate N- or P-sources, salinity, and temperature on karlotoxin (KmTx) production in strains of Karlodinium veneficum (D. Ballant.) J. Larsen, an ichthyotoxic dinoflagellate that shows a high degree of variability of toxicity in situ. The six strains examined represented KmTx 1 (CCMP 1974, MD 2) and KmTx 2 (CCMP 2064, CCMP 2283, MBM1) producers, and one strain that did not produce detectable karlotoxin under nutrient-replete growth conditions (MD 5). We hypothesized that growth-limiting conditions would result in higher cell quotas of karlotoxin. KmTx was present in toxic strains during all growth phases and increased in stationary and senescent phase cultures under low N or P, generally 2- to 5-fold but with some observations in the 10- to 15-fold range. No karlotoxin was observed under low-N or low-P conditions in the nontoxic strain MD 5. Nutrient-quality (NO3, NH4, urea, and glycerophosphate) did not affect growth rate, but growth on NH4 produced 2- to 3-fold higher cellular toxicity and a 50% higher ratio of KmTx 1-1:KmTx 1-3 in CCMP 1974. CCMP 1974 showed higher cellular toxicity at low salinity (≤5 ppt) and high temperature (25°C). Our results suggested that given the presence of a toxic strain of K. veneficum in situ, the existence of environmental conditions that favor cellular accumulation of karlotoxin is likely a significant factor underlying K. veneficum–related fish kills that require both high cell densities (104 · mL−1) and high cellular toxin quotas relative to those generally observed in nutrient-replete cultures. [ABSTRACT FROM AUTHOR]

Conventional methods to identify the fish-killing dinoflagellate Karlodinium veneficum rely on rDNA sequence and scanning electron microscopy analyses that are not suitable for processing a large number of samples. To overcome this difficulty, SYTO9 and TaqMan format real-time PCR probes were developed for the internal transcribed spacer rDNA region for rapid detection and quantification of K. veneficum in the marine environment. Assay specificity, sensitivity, and accuracy were confirmed by testing against related organisms, sequencing PCR amplicons from field samples, and comparing real-time PCR results with microscopic counts. The SYTO9-based assay produced highly reproducible DNA melting curves over a broad range of starting DNA template. When the SYTO9- and TaqMan-based assays were compared in the quantitative measurements, they showed comparable results and reasonable reproducibility. The K. veneficum-specific assays were used in conjunction with Pseudopfiesteria shumwayae (Dinophyceae)-, Pfiesteria piscicida (Dinophyceae)-, and Cryptoperidiniopsis brodyi (Dinophyceae)-specific real-time PCR probes to investigate temporal changes in abundances of these four species in Chinhae Bay, South Korea. The field survey revealed that K. veneficum was dominant (maximum 970 cells ml21) among those species; whereas, Pfiesteria species rarely occurred. These findings suggest that the SYTO9- and TaqMan-based assays are specific and sensitive for detecting and quantifying K. veneficum in the environment. The wide distribution of K. veneficum also indicates potential fish kills by this organism in Chinhae Bay. [ABSTRACT FROM AUTHOR]