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.

Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA.

A reagent-less DNA sensor has been developed exploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87 nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10 min, in both cases. Amplification time was further optimized to be 40 min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.

Direct electrochemical detection of enzyme labelled, isothermally amplified DNA.

Genosensors for the detection of DNA via hybridisation normally require post-amplification processing such as the generation of single-stranded DNA and pre-detection labelling, complicating and lengthening the assay. A straightforward electrochemical genosensor, for the direct detection of isothermally generated nucleic acid amplicons via hybridisation is reported. The detection of Karlodinium armiger, responsible for harmful algae blooms was used as a model system to demonstrate the proof of concept. The approach exploits the use of specifically modified primers designed to generate amplicons with a central duplex flanked by a single-stranded tail at one end of the duplex and a horse-radish peroxidase on the other end. Individual gold electrodes of an array were functionalised with self-assembled monolayers of short thiolated DNA probes, designed to hybridise with the single-stranded tailed amplicon with the reporter enzyme label incorporated. The optimum amplification time was determined to be 60 min, at a fixed temperature of 37 °C. The hybridisation time to the enzyme labelled amplicon was optimised to be 10 min, but 2 min hybridisation time was also adequate. In this first example of using horse radish peroxidase-labelled primer in solution-phase recombinase polymerase amplification for subsequent detection via solid-phase hybridisation, the detection limit achieved was 0.4 fM, equivalent to 27622 cells/L, and the developed genosensor was applied to the detection of synthetic as well as genomic DNA, which had been extracted from a seawater sample.

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.

Distinctive chemotactic responses of three marine herbivore protists to DMSP and related compounds.

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

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.

Changes in biochemical metabolites in manila clam after a temporary culture with high-quality microalgal feed mixed with the dinoflagellate species Karlodinium veneficum and K. zhouanum.

Phytoplankton composition is an important factor affecting the growth and physiological biochemical characteristics of filter-feeding bivalves. With the increasing trend in dinoflagellate biomass and blooms in mariculture areas, how the physio-biochemical traits and seafood quality of the mariculture organism are affected by the dinoflagellates, especially those at nonfatal levels, is not well understood. Different densities of two Karlodinium species, namely K. veneficum (KV) and K. zhouanum (KZ), mixed with high quality microalgal food Isochrysis galbana was applied in feeding manila clam Ruditapes philippinarum in a 14-day temporary culture, to comparatively study how the critical biochemical metabolites such as glycogen, free amino acids (FAAs), fatty acids (FAs), volatile organic compounds (VOCs) in the clam were affected. The survival rate of the clam showed dinoflagellate density and species specificity. The high-density KV group inhibited survival to 32% lower than that of the pure I. galbana control, respectively, while KZ at low concentrations did not significantly affect the survival compared with the control. In the high-density KV group, the glycogen and FAA contents decreased (p < 0.05), indicating that energy and protein metabolism were significantly affected. Amount of carnosine (49.91 ± 14.64 to 84.74 ± 8.59 µg/g of muscle wet weight) was detected in all the dinoflagellate-mixed groups, while it was not present in the field samples or in the pure I. galbana control, showing that carnosine participated in the anti-stress activities when the clam was exposed to the dinoflagellates. The global composition of FAs did not significantly vary among the groups. However, contents of the endogenous C18 PUFA precursors linoleic acid and α-linolenic acid significantly decreased in the high-density KV group compared to all the other groups, indicating that high density of KV affected the metabolisms of fatty acids. From the results of the changed VOC composition, oxidation of fatty acids and degradation of free amino acids might occur in the clams exposed to dinoflagellates. The increased VOCs, such as aldehydes, and decreased 1-octen-3-ol probably produced a more fishy taste and reduced food flavor quality when the clam was exposed to the dinoflagellates. This present study demonstrated that the biochemical metabolism and seafood qulity of the clam were affected. However, KZ with moderate density in the feed seemed to be beneficial in aquaculture for increasing the content of carnosine, a high-valued substance with multiple bioactivities.

A dinoflagellate bloom caused by multiple species of Kareniaceae in the coastal waters of Fujian in June 2022 and its adverse impacts on Brachionus plicatilis and Artemia salina.

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

A 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.

Morphological variation and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an intensive cold-water algal bloom in eastern Hokkaido, Japan.

Harmful algal blooms responsible for mass mortalities of marine organisms have been rare in Hokkaido, northern Japan, although fish-killing blooms have been frequently reported from western Japanese coasts. In September-November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurring unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1-D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis, with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of Kr. selliformis in the bloom were discoid, dorsoventrally flattened, and 35.3-43.6 (39.4  ±  2.1) µm in length, which was larger than the cell sizes in previous reports. Transparent cells of Kr. selliformis, lacking chloroplasts or having a few shrunken chloroplasts and oil droplets, were also found. Cells of Kr. selliformis showed morphological variation, but the species could be distinguished from other co-occurring Karenia species by the nucleus positioned in the hypocone and chloroplasts numerous (46-105) in number and small (2.9-4.6 µm) in diameter. Cell density of Kr. selliformis exceeding 100 cells mL-1 was recorded in the temperature range of 9.8-17.6 °C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from the bloom in Kamchatka, Russia in 2020.

Emerging harmful algal bloom species over the last four decades in China.

The first recorded micro-algae bloom in Chinese coastal waters dates back to 1933 and was caused by a mixture of Noctiluca scintillans and Skeletonema costatum sensu lato along the Zhejiang coast (the East China Sea). While well-documented harmful algal blooms (HABs) appeared to be extremely scarce from the 1950s to 1990, both the frequency and intensity have been reportedly increasing since 1990. Among them, the fish-killing HABs, mainly caused by Karenia mikimotoi, Karlodinium digitatum, Karlodinium veneficum, Margalefidinium polykrikoides, and Heterocapsa spp., have intensified. Karenia mikimotoi was responsible for at least two extremely serious events in the Pearl River Estuary in 1998 and the Taiwan Strait (in the East China Sea) in 2012, which appeared to be associated with abnormal climate conditions and excessive nutrients loading. Other major toxic algal blooms have been caused by the species responsible for paralytic shellfish poisoning (including Alexandrium catenella, Alexandrium pacificum, Gymnodinium catenatum) and diarrhetic shellfish poisoning (including Dinophysis spp., and a couple of benthic dinoflagellates). Consequent closures of shellfish farms have resulted in enormous economic losses, while consumption of contaminated shellfish has led to occasional human mortality in the Bohai Sea and the East China Sea. Expansions of these HABs species along the coastline of China have occurred over the last four decades and, due to the projected global changes in the climate and marine environments and other anthropological activities, there is potential for the emergence of new types of HABs in China in the future. This literature review aimed to present an updated overview of HABs species over the last four decades in China.

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.

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.

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.

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.

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.