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.

INFLUENCE OF MIXOTROPHY ON CELL CYCLE PHASE DURATION AND CORRELATION OF KARLOTOXIN SYNTHESIS WITH LIGHT AND G1 PHASE IN KARLODINIUM VENEFICUM: Influence of mixotrophy on cell cycle and toxicity in Karlodinium veneficum

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

Sterolysin from a 1950s culture of Karlodinium veneficum (aka Gymnodinium veneficum Ballantine) forms lethal sterol dependent membrane pores

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957.

Allelopathy of extracellular chemicals released by Karlodinium veneficum on photosynthesis of Prorocentrum donghaiense

Dinoflagellates Prorocentrum donghaiense and Karlodinium veneficum are the dominant species of harmful algal blooms in the East China Sea. The role of their allelopathy on the succession of marine phytoplankton populations is a subject of ongoing debate, particularly concerning the formation of blooms. To explore the allelopathy of K. veneficum on P. donghaiense, an investigation was conducted into photosynthetic performance (including PSII functional activities, photosynthetic electron transport chain, energy flux, photosynthetic different genes and photosynthetic performance) and photosynthetic damage-induced oxidative stress (MDA, SOD, and CAT activity). The growth of P. donghaiense was strongly restrained during the initial four days (1–6 folds, CK/CP), but the cells gradually resumed activity at low filtrate concentrations from the eighth day. On the fourth day of the strongest inhibition, allelochemicals reduced representative photosynthetic performance parameters PI and ΦPSII, disrupted related processes of photosynthesis, and elevated the levels of MDA content in P. donghaiense. Simultaneously, P. donghaiense repairs these impairments by up-regulating the expression of 13 photosynthetic genes, modifying photosynthetic processes, and activating antioxidant enzyme activities from the eighth day onward. Overall, this study provides an in-depth overview of allelopathic photosynthetic damage, the relationship between genes and photosynthesis, and the causes of oxidative damage induced by photosynthesis. Environmental implicationsAs a typical HAB species, Karlodinium veneficum is associated with numerous fish poisoning events, which have negative impacts on aquatic ecosystems and human health. Allelochemicals produced by K. veneficum can provide a competitive advantage by interfering with the survival, reproduction and growth of competing species. This study primarily investigated the effects of K. veneficum allelochemicals on the photosynthesis and photosynthetic genes of Prorocentrum donghaiense. Grasping the mechanism of allelochemicals inhibiting microalgae is helpful to better understand the succession process of algal blooms and provide a new scientific basis for effective prevention and control of harmful algal blooms.

Metabarcoding analysis of potential HABs in the Maowei Sea, China: A baseline information for the “Pinglu Canal Waterway” mega-waterway project

Maowei Sea is one of the cleanest sea areas in China, with relatively little anthropogenic activity. The Maowei Sea is rich in biodiversity and is the largest natural ground for the Hong Kong oyster Crassostrea hongkongensis seed collection. In recent years, the Chinese government has launched a mega project called “Pinglu Canal Waterway”, which will connect the main river system to the Maowei Sea. This project is expected to alter the physical and chemical properties of the Maowei Sea and affect the structure of the phytoplankton community. This study was conducted to screen potential harmful algal bloom species (HABs) in Maowei Sea using metabarcoding technique. In general, nine potential HABs were identified, including six toxin producers (Chattonella spp., Heterosigma akashiwo, Heterocapsa rotundata, Heterocapsa pygmaea, Karenia mikimotoi and Karlodinium veneficum) and three non-toxin producers (Gymnodinium impudicum, Skeletonema costatum and Ulva prolifera). The occurrence and relative abundance of these potential HABs increases with increasing water salinity. The findings of this study not only provide information on the occurrence of potential HABs in Maowei Sea, but also provide valuable information that contributes to the development of aquaculture and fishery management plans to be implemented in Maowei Sea.

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.

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 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.

Mapping harmful microalgal species by eDNA monitoring: A large-scale survey across the southwestern South China Sea

A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.

Thermal niche of the dinoflagellate Karlodinium veneficum across different salinity and light levels

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

Gymnodinialimonas phycosphaerae sp. nov., a phycosphere bacterium isolated from Karlodinium veneficum.

A facultative anaerobic, Gram-negative, non-motile, rod-shaped bacterial strain, designated N5T, was obtained from the phycosphere microbiota of the marine planktonic dinoflagellate, Karlodinium veneficum. Strain N5T showed growth on marine agar at 25 °C, pH 7 and 1 % (w/v) NaCl and produced a yellow colour. According to a phylogenetic study based on 16S rRNA gene sequences, strain N5T has a lineage within the genus Gymnodinialimonas. The G+C content in the genome of strain N5T is 62.9 mol% with a total length of 4 324 088 bp. The NCBI Prokaryotic Genome Annotation Pipeline revealed that the N5T genome contained 4230 protein-coding genes and 48 RNA genes, including a 5S rRNA, 16S rRNA, 23S rRNA, 42 tRNA, and three ncRNAs. Genome-based calculations (genome-to-genome distance, average nucleotide identity and DNA G+C content) clearly indicated that the isolate represents a novel species within the genus Gymnodinialimonas. The predominant fatty acids were C19 : 0 cyclo ω8c and feature 8 (comprising C18 : 1 ω6c and/or C18 : 1 ω7c). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The main respiratory quinone was Q-10. Based on its phenotypic, phylogenetic, genomic and chemotaxonomic features, strain N5T represents a novel species of the genus Gymnodinialimonas, for which the name Gymnodinialimonas phycosphaerae sp. nov. is proposed. The type strain is N5T (=KCTC 82362T=NBRC 114899T).

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.

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.

Phytoplankton community structure in the Western Subarctic Gyre of the Pacific Ocean during summer determined by a combined approach of HPLC-pigment CHEMTAX and metabarcoding sequencing

The Western Subarctic Gyre (WSG) is a cyclonic upwelling gyre in the northwest subarctic Pacific, which is a region with a high concentration of nutrients but low chlorophyll. We investigated the community structure and spatial distribution of phytoplankton in this area by using HPLC-pigment CHEMTAX (a chemotaxonomy program) and metabarcoding sequencing during the summer of 2021. The phytoplankton community showed significant differences between the two methods. The CHEMTAX analyses identified eight major marine phytoplankton assemblages. Cryptophytes were the major contributors (24.96%) to the total Chl a, followed by pelagophytes, prymnesiophytes, diatoms, and chlorophytes. The eukaryotic phytoplankton OTUs obtained by metabarcoding were categorized into 149 species in 96 genera of 6 major groups (diatoms, prymnesiophytes, pelagophytes, chlorophytes, cryptophytes, and dinoflagellates). Dinoflagellates were the most abundant group, accounting for 44.74% of the total OTUs obtained, followed by cryptophytes and pelagophytes. Sixteen out of the 97 identified species were annotated as harmful algal species, and Heterocapsa rotundata, Karlodinium veneficum, and Aureococcus anophagefferens were assigned to the abundant group (i.e., at least 0.1% of the total reads). Nutrients were more important in shaping the phytoplankton community than temperature and salinity. The 24 stations were divided into southern and northern regions along 44°N according to the k-means method, with the former being dominated by high Chl a and low nutrients. Although different phytoplankton assemblages analyzed by the two methods showed various relationships with environmental factors, a common feature was that the dinoflagellate proportion showed a significantly negative correlation with low nutrients and a positive correlation with Chl a.

A Novel C-Type Lectin and Its Potential Role in Feeding and Feed Selection in Ruditapes philippinarum

In recent years, the role of lectins in the feed selection of bivalve has become hot research topic. Manila clam Ruditapes philippinarum is a species of marine bivalve with important economic value. A new C-type lectin (Rpcl) from the clam was obtained and its potential role in feeding and feed selection was studied. Rpcl cDNA was 929 bp in length and had 720 bp of open reading frame. Rpcl encoded 235 amino acids, comprising a carbohydrate recognition domain (CRD) as well as an N-terminal signal peptide. Rpcl contained a conserved CRD disulfide bond including five cysteine residues (Cys125, Cys142, Cys213, Cys219, and Cys237) and the QPN motif (GLN204-PRO205-ASN206). Phylogenetic analysis revealed that the amino acid sequence of Rpcl was closely related to that of Vpclec-1 from R. philippinarum and C-type lectin from Mercenaria. The qPCR analysis indicated that Rpcl expression was observed in all examined tissues and was the highest in gills followed by in the hepatopancreas, and to a lesser extent in the mantle and lip. The in vitro agglutination experiments showed that, the purified Rpcl protein could selectively agglutinate with different microalgae. The strongest agglutinating effect with Chlorella sp. was observed, followed by Karlodinium veneficum and Chaetoceros debilis Cleve. No agglutination with Prorocentrum minimum was observed. In the feeding experiment, compared with that of the starvation group, Rpcl expression in the lip and gill of the clam fed with C. debilis and K. veneficum showed a significant upward trend with the change of time. In addition, it was found that the changes in the expression of the Rpcl gene in the gill and lip, the main feeding tissues, were consistent with the slope of the decrease in the number of algal cells in the water body. In summary, the structure of a new C-type lectin (Rpcl) was reported in this study and its correlation with the feeding and feed selection in R. philippinarum was confirmed.

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.

Sublethal effect of the toxic dinoflagellate Karlodinium veneficum on early life stages of zebrafish (Danio rerio).

Dinoflagellates of the genus Karlodinium are ichthyotoxic species that produce toxins including karlotoxins and karmitoxins. Karlotoxins show hemolytic and cytotoxic activities and have been associated with fish mortality. This study evaluated the effect of toxins released into the environment of Karlodinium veneficum strain K10 (Ebro Delta, NW Mediterranean) on the early stages of Danio rerio (zebrafish). Extracts of the supernatant of K10 contained the mono-sulfated KmTx-10, KmTx-11, KmTx-12, KmTx-13, and a di-sulfated form of KmTx-10. Total egg mortality was observed for karlotoxin concentration higher than 2.69μg L-1. For 1.35μg L-1, 87% of development anomalies were evidenced (all concentrations were expressed as KmTx-2 equivalent). Larvae of 8days postfertilization exposed to 1.35µg L-1 presented epithelial damage with 80% of cells in the early apoptotic stage. Our results indicate that supernatants with low concentration of KmTxs produce both lethal and sublethal effects in early fish stages. Moreover, apoptosis was induced at concentrations as low as 0.01μg L-1. This is of great relevance since detrimental long-term effects due to exposure to low concentrations of these substances could affect wild and cultured fish.

Spatiotemporal distribution of size-fractioned phytoplankton in the Yalu River Estuary, China

ABSTRACT The grain size structure of phytoplankton has great influence on shellfish culture. The present study aimed to assess the spatial and temporal variation in the phytoplankton community structure in the Yalu River Estuary and to explore the relationship between the phytoplankton community structure and various environmental parameters in 2020. High-throughput sequencing was used in this study. The results showed that nanophytoplankton, especially Karlodinium veneficum, dominated the estuary throughout the year. The biomass ratio of picophytoplankton, nanophytoplankton, and microphytoplankton were 20:63:17 in spring, 30:44:26 in summer, 1:38:61 in autumn, and 2:45:53 in winter, respectively. Meanwhile, Dinophyta had the greatest biomass throughout the year, followed by Bacillariophyta. On the spatial dimension (Station average), COD, T, SST had a positive impact on total phytoplankton communities, and Dep had a negative impact. In the time dimension (Monthly average), the environmental factor that significantly controlled the phytoplankton community structure were NO2 and SST.