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.

Ichthyotoxic Karlodinium veneficum (Ballantine) J Larsen in the Upper Swan River Estuary (Western Australia): Ecological conditions leading to a fish kill.

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.

Effects of polystyrene nanoplastics on growth and hemolysin production of microalgae Karlodinium veneficum.

There 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.

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.

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.

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.

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

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.

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.

Rapid and sensitive detection of Karlodinium veneficum by a novel double-nick rolling circle amplification.

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.

Oyster hatchery breakthrough of two HABs and potential effects on larval eastern oysters (Crassostrea virginica).

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.

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.

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.

Easy detection of karlodinium veneficum using PCR-based dot chromatography strip.

In this study, a novel detection method by PCR-based dot chromatography strip (PDCS) is proposed. To investigate the application of PDCS in the detection of harmful microalgae, the internal transcribed spacer sequence of Karlodinium veneficum, one of the most common bloom-forming species, was cloned and sequenced to design and screen specific primers with tag sequences and probes, including gold nanoparticle probe, test probe, and control probe. The PDCS was prepared manually, and PCR amplicons prepared from the genomic DNA of K. veneficum using tagged specific primers were analyzed by PDCS for visual detection of the target species. The resulting test strip showed red spots at the predicted test and control points visible to the naked eyes, showing the successful development of PDCS. This detection technique is independent of expensive experimental equipment (except a DNA thermal cycler for PCR) but requires an aliquot of PCR amplicons mixed with development buffer to apply to the sample pad of PDCS for approximately 10 min to visualize the analytical results. Cross-reactivity test with 21 microalgae, including K. veneficum, showed that the established PDCS technique has excellent specificity. The detection limit of PDCS was 9.13 × 10-2 ng µL-1 for genomic DNA and 5.3 × 105 cells L - 1 for crude DNA extracts of the target alga. In summary, the PDCS with high sensitivity and specificity can be prepared by hand, which is less expensive than traditional strip, thus providing a promising alternative to the detection of K. veneficum in natural samples.

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.

Critical light-related gene expression varies in two different strains of the dinoflagellate Karlodinium veneficum in response to the light spectrum and light intensity.

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.

Characterization of bubble column photobioreactors for shear-sensitive microalgae culture.

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 (EDtop) was one order of magnitude higher than the average energy dissipation in the whole reactor (EDwhole), and the value in the sparger zone (EDspar) was one order of magnitude higher than EDtop. Cultures of K. veneficum were limited by CO2 transfer at low EDwhole and severely stressed above a critical value of EDwhole.

Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick.

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.

Initial evidence of functional siRNA machinery in dinoflagellates.

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 CO2-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.

Distribution of Karlodinium veneficum in the coastal region of Xiangshan Bay in the East China Sea, as detected by a real-time quantitative PCR assay of ribosomal ITS sequence.

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.