Dinoflagellate Karlodinium Veneficum Research Articles

Accumulation and Depuration of 4,5-dihydro-KmTx2 from Karlodinium veneficum in the bivalves, Mercenaria mercenaria and Sinonovacula constricta

Bivalves (e.g., clams, oysters, mussels, scallops) are a significant part of the global diet and are harvested for their nutritional value, but as filter feeders they are susceptible to the accumulation of toxins produced by certain species of phytoplankton. Karlotoxins (KmTxs) are a class of amphidinol-like compounds with hemolytic, ichthyotoxic, and cytotoxic properties that have been associated with harmful algal blooms, and the dinoflagellate Karlodinium veneficum uses KmTxs to facilitate prey capture and deter grazing by other organisms. In this work, we examined the accumulation and depuration of 4,5-dihydro-KmTx2, a karlotoxin previously isolated from K. veneficum, in two bivalves with different life habits, i.e., Sinonovacula constricta Lamarck and Mercenaria mercenaria Linnaeus. After both bivalves received K. veneficum GM5 in their feed for a long period of 10 days, 4,5-dihydro-KmTx2 was accumulated considerably in the visceral mass, but there was almost no toxin accumulation in the muscle. The accumulation was more severe for M. mercenaria than S. constricta. The toxin was cleared almost completely from the bivalves 5 days after K. veneficum GM5 was removed from the feed. For both bivalves, the bait microalgae I. galbana slowed both the accumulation and the depuration of the toxin.

Short-term and long-term exposure to combined elevated temperature and CO2 leads to differential growth, toxicity, and fatty acid profiles in the harmful dinoflagellate Karlodinium veneficum

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.

Molecular Insights into the Synergistic Effects of Putrescine and Ammonium on Dinoflagellates.

Ammonium and polyamines are essential nitrogen metabolites in all living organisms. Crosstalk between ammonium and polyamines through their metabolic pathways has been demonstrated in plants and animals, while no research has been directed to explore this relationship in algae or to investigate the underlying molecular mechanisms. Previous research demonstrated that high concentrations of ammonium and putrescine were among the active substances in bacteria-derived algicide targeting dinoflagellates, suggesting that the biochemical inter-connection and/or interaction of these nitrogen compounds play an essential role in controlling these ecologically important algal species. In this research, putrescine, ammonium, or a combination of putrescine and ammonium was added to cultures of three dinoflagellate species to explore their effects. The results demonstrated the dose-dependent and species-specific synergistic effects of putrescine and ammonium on these species. To further explore the molecular mechanisms behind the synergistic effects, transcriptome analysis was conducted on dinoflagellate Karlodinium veneficum treated with putrescine or ammonium vs. a combination of putrescine and ammonium. The results suggested that the synergistic effects of putrescine and ammonium disrupted polyamine homeostasis and reduced ammonium tolerance, which may have contributed to the cell death of K. veneficum. There was also transcriptomic evidence of damage to chloroplasts and impaired photosynthesis of K. veneficum. This research illustrates the molecular mechanisms underlying the synergistic effects of the major nitrogen metabolites, ammonium and putrescine, in dinoflagellates and provides direction for future studies on polyamine biology in algal species.

Division time (td) for in situ growth measurements demonstrates thermal ecotypes of Karlodinium veneficum

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 (td) in four geographically distinct laboratory strains of K. veneficum at three different environmentally relevant temperatures. The results demonstrated that the td 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 td spread across four geographically distinct laboratory strains and a nearly fourfold range of growth rates implies that 1.6 h represents the td value of K. veneficum. Additionally, temperature conditions yielding this value for td 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 td 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.

Transcriptome profiling reveals a global response in harmful dinoflagellate Karlodinium veneficum to naturally-occurring bacterial algicides

Dinoflagellates are among the most toxigenic phytoplankton that cause harmful algal blooms; they can produce toxins that accumulate through the aquatic food chains to cause illness and even death in marine animals and humans. Shewanella sp. IRI-160 is a naturally-occurring bacterium that secretes a suite of algicidal compounds (collectively designated as IRI-160AA) specifically targeting dinoflagellates. Studies revealed IRI-160AA inhibited photosynthesis, while inducing cell cycle arrest, DNA damage, and reactive oxygen species (ROS) production, as well as other markers associated with programmed cell death (PCD). Recent research indicated that IRI-160AA contains ammonium and other compounds as active ingredients for its algicidal activity, while impacts by ammonium differed from the algicide with respect to photobiology. Here, transcriptomic analysis was conducted on the toxic dinoflagellate Karlodinium veneficum exposed to IRI-160AA to investigate the effects of this algicide at the molecular level. Transcriptomic analysis was also conducted on K. veneficum treated with ammonium to further differentiate its effects from IRI-160AA. Results demonstrated differential impacts by IRI-160AA and ammonium on K. veneficum at the molecular level and revealed a global response of K. veneficum to algicide exposure, supported by the enriched biological processes involved in regulating gene expression, protein activity, and morphology. Differentially expressed genes associated with stress and ROS response, DNA damage response, cell cycle checkpoint activation, and PCD were also identified in K. veneficum exposed to IRI-160AA. The potential involvement of a recovery mechanism from photodamage in K. veneficum induced by IRI-160AA was identified. Overall, results of this study further differentiated the impacts of ammonium from IRI-160AA on K. veneficum and illustrated the cellular mechanisms behind the algicidal effect. This research provided insights on algal response to bacterial derivatives in nature.

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

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

Identification and implications of a core bacterial microbiome in 19 clonal cultures laboratory-reared for months to years of the cosmopolitan dinoflagellate Karlodinium veneficum

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

Possible functions of CobW domain-containing (CBWD) genes in dinoflagellates using Karlodinium veneficum as a representative

Since > 91% of dinoflagellates are proven auxotrophs of vitamin B12 and the cobalamin synthetase W (CobW) is a key gene involved in vitamin B12 synthesis pathway, a number of CobW domain-containing (CBWD) genes in dinoflagellates (DinoCBWDs) were surprisedly found from our transcriptomic and meta-transcriptomic studies. A total of 88 DinoCBWD genes were identified from the genomes and transcriptomes of four dinoflagellates, with five being cloned for full-lengths and characterized using the cosmopolitan and ecologically-important dinoflagellates Karlodinium veneficum and Scrippsiella trochoidea (synonym of Scrippsiella acuminata). DinoCBWDs were verified being irrelevant to vitamin B12 biosynthesis due to their transcriptions irresponsive to vitamin B12 levels and their phylogenetic positions. A comprehensive phylogenetic analysis demonstrated 75 out of the 88 DinoCBWD genes identified belong to three subfamilies of COG0523 protein family, of which most prokaryotic members are reported to be metallochaperones and the eukaryotic members are ubiquitously found but mostly unknown for their functions. Our results from K. veneficum demonstrated DinoCBWDs are associated with metal homeostasis and other divergent functions, with four KvCBWDs involving in zinc homeostasis and KvCBWD1 likely functioning as Fe-type nitrile hydratase activator. In addition, conserved motif analysis revealed the structural foundation of KvCBWD proteins that are consistent with previously described CBWD proteins with GTPase activity and metal binding. Our results provide a stepping-stone toward better understanding the functions of DinoCBWDs and the COG0523 family.

Metabolomic Insights of the Effects of Bacterial Algicide IRI-160AA on Dinoflagellate Karlodinium veneficum.

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.

Karlodinium veneficum: Growth optimization, metabolite characterization and biotechnological potential survey.

The major aim of this work was to consistently optimize the production of biomass of the dinoflagellate Karlodinium veneficum and evaluate its extracts biotechnological potential application towards food, nutraceutical or/and pharmaceutical industries. A successful approach of biomass production of K. veneficum CCMP 2936 was optimized along with the chemical characterization of its metabolite profile. Several temperatures (12, 16, 20, 25, 30°C), L1 nutrient concentrations (0.5×, 2×, 2.5×, 3×) and NaCl concentrations (20, 25, 30, 40gL-1 ) were tested. The growth rate was maximum at 16°C, 2× nutrient concentration and 40gL-1 of NaCl; hence, these conditions were chosen for bulk production of biomass. Methanolic extracts were prepared, and pigments, lipids and phenolic compounds were assessed; complemented by antioxidant and anti-inflammatory capacities, and cytotoxicity. Fucoxanthin and derivatives accounted for 0.06% of dry weight, and up to 60% (w/w) of all quantified metabolites were lipids. Said extracts displayed high antioxidant capacity, as towards assessed via the NO•- and ABTS•+ assays (IC50 =109.09±6.73 and 266.46±2.25µgE ml-1 , respectively), unlike observed via the O2 •- assay (IC25 reaching 56.06±5.56µgE ml-1 ). No signs of cytotoxicity were observed. Karlodinium veneficum biomass production was consistently optimized in terms of temperature, L1 nutrient concentrations and NaCl concentration. In addition, this strain appears promising for eventual biotechnological exploitation. This work provides fundamental insights about the growth and potential of value-added compounds of dinoflagellate K. veneficum. Dinoflagellates, as K. veneficum are poorly studied regarding its biomass production and added-value compounds for potential biotechnological exploitation. These organisms are difficult to maintain and grow in the laboratory. Thus, any fundamental contribution is relevant to share with the scientific community.

A three-dimensional mixotrophic model of Karlodinium veneficum blooms for a eutrophic estuary

Blooms of dinoflagellate Karlodinium veneficum are widely distributed in estuarine and coastal waters and have been found to cause fish kills worldwide. K. veneficum has a mixed nutritional mode and relies on both photosynthesis and phagotrophy for growth; it is a mixotroph. Here, a model of mixotrophic growth of K. veneficum (MIXO) was developed, calibrated with previously-reported laboratory physiological data, and subsequently embedded in a 3D-coupled hydrodynamic (ROMS)-biogeochemical (RCA) model of eutrophic Chesapeake Bay, USA. The resulting ROMS-RCA-MIXO model was applied in hindcast mode to investigate seasonal and spatial distributions. Simulations showed that K. veneficum blooms occurred during June-August and were confined to the upper and middle Bay, consistent with long-term field observations. Autotrophic growth dominated in spring but heterotrophic growth dominated during the summer. The number of prey ingested by K. veneficum varied from 0.1 to 0.6 day−1 and the food vacuole content reached up to 50% of the core mixotroph biomass. The ingestion rate increased with prey density and also when P:N ratio fell below ∼0.03 (N:P ∼ 33), indicating that K. veneficum only switched to mixotrophic feeding in P-deficient waters when sufficient prey were available; this occurred during the summer months. The digestion rate increased with both the food vacuole content and temperature. The modeling analysis affirms K. veneficum as a phagotrophic ‘alga’ which is primarily photosynthetic but switches to mixotrophic feeding under nutrient deficient conditions.

Applying Surfactin in the Removal of Blooms of Karlodinium veneficum Increases the Toxic Potential

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.

Application of clay minerals to remove extracellular ichthyotoxins produced by the dinoflagellates Karlodinium veneficum and Karenia mikimotoi

Mitigation of fish-killing algal toxins by clay minerals offers great promise as an emergency strategy for fish farms threatened by harmful algal blooms, but its efficiency is highly clay and algal species (i.e. ichthyotoxin) specific. We here screened several different clay types (kaolin, zeolite, Korean loess and six bentonites) for their adsorptive capacity of extracellular Karlodinium veneficum and Karenia mikimotoi ichthyotoxins as quantified with the rainbow trout RTgill-W1 cell line assay. Treatment with Korean loess, zeolite (0–0.5 g L − 1), polyaluminium chloride (0–0.1 g L − 1) and clays modified with this flocculant (0–0.25 g L − 1) could not significantly improve gill cell viability compared to toxic controls. Kaolin only demonstrated effective removal in case of K. mikimotoi, but concentrations required for complete removal of cytotoxicity were at least 2 x those required for bentonite. Bentonites of high swelling capacity and ideally small particle size (<2 µm) proved best suited for ichthyotoxin removal against both algal species (100% removal at concentrations as low as 0.1 g L − 1). Complete elimination of K. veneficum and K. mikimotoi toxicity towards the rainbow trout gill cell line was achieved by bentonite clay, demonstrating the potential to control ichthyotoxicity in an aquaculture setting through targeted clay application.

Mixotrophy upgrades food quality for marine calanoid copepods

AbstractInorganic nutrient limitation affects the stoichiometry and nutritional quality of marine phytoplankton. Mixoplankton, able to photosynthesize and feed simultaneously in the one cell, can compensate shortage of nutrients by phagotrophy, theoretically upgrading their nutritional quality for their predators: the zooplankton. Yet, the additional value that phagotrophy in mixoplankton may provide to support zooplankton growth and recruitment has been poorly explored. Therefore, we investigated the feeding and reproductive performances of the copepods Paracartia grani and Centropages typicus on mono‐diets of the dinoflagellate Karlodinium veneficum grown as strict autotroph and as mixotroph, both under N and P depletion, and in nutrient‐balanced conditions (f/2 medium; only as autotroph). Feeding and reproduction outputs were generally higher in P. grani than in C. typicus. Both copepod species ingested the mixotrophic K. veneficum at similar rates than the autotrophic ones in either nutrient‐limited scenario. However, egg production and recruitment rates generally increased when feeding on mixotrophs, in P. grani on both N‐ and P‐limited diets, and in C. typicus under P limitation. In general, P limitation influenced copepod physiology more than N depletion. Our results show that phagotrophy upgrades nutritional quality in nutrient‐limited mixotrophs as prey for copepods compared to the strict autotrophic ones. These findings are among the first reported cases of copepod ingestion in the laboratory on actively feeding mixoplankton, and they highlight the importance of considering the trophic mode of the protist and the response by various zooplanktonic predators when attempting to understand the functioning of marine planktonic food webs.

Quantitative nuclear DNA content and cell cycle analysis of a mixotrophic dinoflagellate by image cytometry

AbstractThe goal of this work was to develop and demonstrate the utility of microscope‐based image cytometry (ICM) as a method for quantifying nuclear DNA content and cell cycle phase distribution in microalgae both in culture and in natural blooms, as an alternative to flow cytometry (FCM). To do so, aliquots from the same samples of the dinoflagellate Karlodinium veneficum were examined using both ICM and FCM. ICM specimen preparation and data acquisition methods were optimized to improve precision and agreement between the two techniques. Accuracy and precision of DNA measurements by ICM were significantly higher using the DNA fluorophore DAPI compared to SYBR® Green I. Milli‐Q H2O was found to be superior to Tris‐EDTA as a staining and slide preparation solution for ICM analyses. Lower‐powered objective magnification (10x, 20x) in image acquisition for ICM produced higher precision in nuclear DNA measurements. Overall precision of ICM analysis of DAPI‐stained K. veneficum cells was comparable to FCM, with respective 1C DNA peak coefficients of variation as low as 6.2%. Cell cycle distributions of mid‐log culture samples analyzed by both ICM and FCM were in agreement (two‐way ANOVA; p = 0.93); while distributions analyzed in a field sample were similar but not identical (Z‐test; p &lt; 0.001). Overall, the results show the feasibility of ICM as a useful tool for microalgal cell cycle analysis, with the potential for more flexible application to mixotrophic/phagotrophic species and complex field populations.

A preliminary study on the allelopathy and toxicity of the dinoflagellate Karlodinium veneficum

The dinoflagellate Karlodinium veneficum has worldwide distribution and is associated with harmful algal blooms through the production of karlotoxins. We investigated the allelopathy and toxicity to explore the potential ecological implications. Prorocentrum donghaiense was inhibited significantly when grown either in co-cultures or in culture filtrate of K. veneficum. In addition, the effect of the co-occurring microalga species (P. donghaiense) on the hemolytic activity of K. veneficum was also evaluated. P. donghaiense did not inhibit the growth of K. veneficum but increased the hemolytic activity. The culture of K. veneficum was loaded onto an RP-C18 column and eluted with different percentages of aqueous methanol solution. 80% methanol fraction not only inhibited the growth of P. donghaiense by allelopathy but also exhibited strong hemolytic activity, indicating that the allelochemicals and toxins of K. veneficum might be the same components. Furthermore, KmTx 3 (C68H124O24) was identified using HPLC-HRMS from this fraction.

Functional trait thermal acclimation differs across three species of mid-Atlantic harmful algae

Characterizing the thermal niche of harmful algae is crucial for understanding and projecting the effects of future climate change on harmful algal blooms. The effects of 6 different temperatures (18–32 °C) on the growth, photophysiology, and toxicity were examined in the dinoflagellate Karlodinium veneficum, and the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa from the Delaware Inland Bays (DIB). K. veneficum and H. akashiwo had skewed unimodal growth patterns, with temperature optima (Topt) at 28.6 and 27.3 °C respectively and an upper thermal niche limit of 32 °C. In contrast, C. subsalsa growth increased linearly with temperature, suggesting Topt and upper thermal boundaries >32 °C. K. veneficum photosystem II (PSII) photochemical efficiency remained stable across all temperatures, while H. akashiwo PSII efficiency declined at higher temperature and C. subsalsa was susceptible to low temperature (~18 °C) photoinactivation. Cell toxicity thermal response was species-specific such that K. veneficum toxicity increased with temperature above Topt. Raphidophyte toxicity peaked at 25–28 °C and was in close agreement with Topt for growth in H. akashiwo but below C. subsalsa maximal growth. The mode of toxicity was markedly different between the dinoflagellate and the raphidophytes such that K. veneficum had greater hemolytic activity while the raphidophytes had pronounced fish gill cell toxicity. These results and patterns of natural abundance for these algae in the DIB suggest that continued ocean warming may contribute to C. subsalsa bloom formation while possibly promoting highly toxic blooms of K. veneficum.

Immobilization of algicidal bacterium Shewanella sp. IRI-160 and its application to control harmful dinoflagellates

Shewanella sp. IRI-160 is an algicidal bacterium isolated from Delaware Inland Bays. It secretes water-soluble compounds that inhibit the growth of dinoflagellates. Previous research indicated that this bacterium does not have a negative impact on other algal species. In this research, Shewanella sp. IRI-160 was immobilized to different porous matrices, including agarose, alginate hydrogel, cellulosic sponge, and polyester foam. The retention of Shewanella sp. IRI-160 on or within these matrices was examined at 4 and 25 °C for 12 days. Results indicated that alginate was superior in terms of cell retention, with >99% of Shewanella cells retained in the matrix after 12 days. Shewanella sp. IRI-160 cells were then immobilized within alginate beads to evaluate algicidal effects on harmful dinoflagellates Karlodinium veneficum and Prorocentrum minimum at bacterial concentrations of 106 to 108 cells mL−1. The effects on dinoflagellates were compared to non-harmful cryptophyte Rhodomonas sp., as well as the effects of free-living bacteria on these species. Results indicated that immobilized Shewanella sp. IRI-160 in alginate beads were as effective as the free-living bacteria to control the growth of K. veneficum and P. minimum, while no negative impacts of immobilized Shewanella sp. IRI-160 on the non-harmful control species Rhodomonas sp. were observed. Overall, this study suggests that immobilized Shewanella sp. IRI-160 may be used as an environmentally friendly approach to prevent or mitigate the blooms of harmful dinoflagellates and provides insight and directions for future studies.

Evidence for resting cyst production in the cosmopolitan toxic dinoflagellate Karlodinium veneficum and the cyst distribution in the China seas

The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species that frequently forms harmful algal blooms (HABs) in coastal waters. This species has been intensively studied from multiple aspects including toxicology, toxins, nutrition mode (e.g., mixotrophy, phagotrophy, etc.), blooming dynamics, allelopathy, and behavior, while the mechanisms accounting for its global distribution and possible invasion to new regions have not been investigated. Since the first report of a bloom of this species from the South China Sea in 2003, K. veneficum has been frequently detected in coastal waters of China. While resting cyst has been well documented to play vital roles in the initiation and decline of HABs and in facilitating geographical expansion of HABs species, whether or not K. veneficum forms resting cyst remains an open question. Here, we provide proofs for the resting cyst formation in K. veneficum based on both the observations on the life history of clonal cultures and cyst detections from field sediment. We microscopically observed the mating gametes, gametes in fusion, planozygotes (judged from the two longitudinal flagella and cell morphology such as a larger size), dark brown, thick-walled cysts with smooth surface, and cyst germination. The resting cyst was produced homothallically (i.e. from single clonal culture). We also determined the diploidity of cysts via measuring the copy numbers of the large subunit (LSU) rRNA gene in resting cysts and vegetative cells. The presence of K. veneficum cysts in field sediments was detected via fluorescence in situ hybridization (FISH) using species-specific probes, and further confirmed by single-cell PCR sequencing for the FISH-detected cysts. The distribution and abundance of K. veneficum cysts in the China Seas (Bohai Sea, Yellow Sea, East China Sea, and South China Sea) were mapped using a combined approach of real-time PCR and FISH, and quantified after measuring and taking into account the copy numbers of LSU rRNA gene in vegetative cells and cysts. We found a wide distribution of resting cysts of this organism in the seas of China, but generally with a low abundance in most of the samples (0 to 15 cysts per 32 g of wet sediment for FISH method; 0 to 25 cysts per 32 g of wet sediment for qPCR method). The confirmation of resting cyst production from both the laboratory cultures and field sediments and detection of a wide distribution of cysts in the China coasts in this study provide a possible mechanistic explanation for the frequent recurrences of blooms and the cosmopolitan distribution of K. veneficum. Our work also necessitates both a more intensive investigation on the life history (e.g. germination potential of cysts in the field) and an extensive cyst monitoring in coastal sediments, in order to better understand the general ecology and the bloom dynamics specific to this important species.

A strain of the toxic dinoflagellate Karlodinium veneficum isolated from the East China Sea is an omnivorous phagotroph

Karlodinium veneficum is a cosmopolitan, toxic, and harmful algal bloom-forming dinoflagellate, of which the mixotrophy has been suggested to be a key factor in the formation and maintaining of HABs and thus deserves more intensive explorations. Here, we report an investigation on the phagotrophy of K. veneficum using a clonal culture isolated from the coastal water of East China Sea. We found K. veneficum is an omnivorous phagotroph feeding on both live and dead bodies/cells of a fish (Oryzias melastigma), brine shrimp (Artemia salina), rotifer (Brachionus plicatilis), co-cultivated microalgae Akashiwo sanguinea, Margalefidinium polykrikoides, Alexandrium leei, Rhodomonas salina, Isochrysis galbana, and its own species. Karlodinium veneficum extracted the cell contents of all species provided through either a peduncle (i.e. myzocytosis) or by engulfing the whole cell of small preys (i.e. phagotrophy sensu stricto). Karlodinium veneficum preferred to ingest non-motile or newly dead preys, no matter whether they were fish, zooplankton, or phytoplankton. Importantly, K. veneficum exhibited micropredation on animals with sizes much larger than itself (fish, rotifer, and brine shrimp), especially when they were injured or newly dead. The LysoSensor- and LysoTracker-stained lysosomes or/and phagolysosomes of K. veneficum increased when preys were added. Cannibalism in K. veneficum, i.e. a cell feeds on other unhealthy or dead cells of the same species, was observed as the first time in the study, which can help the growth and elongated maintaining of the population under nutrient deficiency (i.e. the culture maintained viable in culture plates without nutrient supplement up to a year). The growth rate of K. veneficum exhibited significant positive correlation with ingestion rate, which differed among prey species, and the highest growth rate was observed when feeding on R. salina. The ingest ability of K. veneficum was triggered by nutrient deficiency. In conclusion, the omnivorous mixotrophy is proposed to be a key autecological mechanism for K. veneficum to widen its ecological niche and succeed in forming a cosmopolitan distribution and frequent blooms.