Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Chemodiversity of Brevetoxins and Other Potentially Toxic Metabolites Produced by Karenia spp. and Their Metabolic Products in Marine Organisms.

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Profiling of Brevetoxin Metabolites Produced by Karenia brevis 165 Based on Liquid Chromatography-Mass Spectrometry.

In this study, Karenia brevis 165 (K. brevis 165), a Chinese strain, was used to research brevetoxin (BTX) metabolites. The sample pretreatment method for the enrichment of BTX metabolites in an algal culture medium was improved here. The method for screening and identifying intracellular and extracellular BTX metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The results show that the recovery rates for BTX toxins enriched by a hydrophilic-lipophilic balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTX metabolites at different growth stages of K. brevis 165. This is the first time a Chinese strain of K. brevis has been reported that can produce toxic BTX metabolites. Five and eight kinds of BTX toxin metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites. The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages show that the total toxic BTX metabolite contents in single cells ranged between 6.78 and 21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27 and 449.11 µg/L. There were significant differences in the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

UPLC-ESI-Q-TOF/MS Based Metabolic Profiling of Protosappanin B in Rat Plasma, Bile, Feces, Urine and Intestinal Bacteria Samples.

BACKGROUND: Caesalpinia sappan L. is a traditional medicinal plant that is used to promote blood circulation and treat stroke in China. Protosappanin B (PTB) is a unique homoisoflavone compound isolated from Sappan Lignum (the heartwood of Caesalpinia sappan L). In a previous study, the metabolic fate of PTB remained unknown. OBJECTIVE: To explore whether PTB is extensively metabolized, the metabolites of PTB in bile, plasma, urine, feces, and intestinal bacteria samples in rats were investigated. METHODS: The biosamples were investigated by ultraperformance liquid chromatography combined with time-offlight mass spectrometry (UPLC-TOF-MS/MS) with MetabolitePilot software. RESULTS: 28 metabolites were identified in the biosamples: 18 metabolites in rat bile, 8 in plasma, 20 in feces, 7 in urine and 2 in intestinal bacteria samples. Both phase I and phase II metabolites were observed. Metabolite conversion occurred via 9 proposed pathways: sulfate conjugation, glucuronide conjugation, bis-glucuronide conjugation, glucose conjugation, dehydration, oxidation, hydrolysis, methylation and hydroxymethylene loss. The metabolic pathways differed among biosamples and exhibited different distributions. Among these pathways, the most important was sulfate and glucuronide conjugation. CONCLUSION: The results showed that the small intestinal and biliary routes play an important role in the clearance and excretion of PTB. The main sites of metabolism in the PTB chemical structure were the phenolic hydroxyl and the side-chains on the eight-element ring.

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2-3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants.

Ladder-Shaped Ion Channel Ligands: Current State of Knowledge.

Ciguatoxins (CTX) and brevetoxins (BTX) are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of Gambierdiscus and Karenia genera, correspondingly. Ciguatera, evoked by CTXs, is a type of ichthyosarcotoxism, which involves a variety of gastrointestinal and neurological symptoms, while BTXs cause so-called neurotoxic shellfish poisoning. Both types of toxins are reviewed together because of similar mechanisms of their action. These are the only molecules known to activate voltage-sensitive Na⁺-channels in mammals through a specific interaction with site 5 of its α-subunit and may compete for it, which results in an increase in neuronal excitability, neurotransmitter release and impairment of synaptic vesicle recycling. Most marine ciguatoxins potentiate Nav channels, but a considerable number of them, such as gambierol and maitotoxin, have been shown to affect another ion channel. Although the extrinsic function of these toxins is probably associated with the function of a feeding deterrent, it was suggested that their intrinsic function is coupled with the regulation of photosynthesis via light-harvesting complex II and thioredoxin. Antagonistic effects of BTXs and brevenal may provide evidence of their participation as positive and negative regulators of this mechanism.

A new species of Gambierdiscus (Dinophyceae) from the south-west Pacific: Gambierdiscus honu sp. nov.

Two isolates of a new tropical, epiphytic dinoflagellate species, Gambierdiscus honu sp. nov., were obtained from macroalgae sampled in Rarotonga, Cook Islands, and from North Meyer Island, Kermadec Islands. Gambierdiscus honu sp. nov. had the common Gambierdiscus Kofoidian plate formula: Po, 3', 6″, 6C?, 6 or 7S, 5‴, 1p and 2⁗. The characteristic morphological features of this species were its relatively small short dorsoventral length and width and the shape of individual plates, in particular the combination of the hatchet-shaped 2' and pentagonal 3' plates and the length to width ratio of the antapical 1p plate. The combination of these characteristics plus the smooth thecal surface and equal sized 1⁗ and 2⁗ plates differentiated this species from other Gambierdiscus species. The phylogenetic analyses supported the unique description. Both isolates of G. honu produced the putative maitotoxin (MTX)-3 analogue, but neither produced ciguatoxin (CTX) or MTX. Extracts of G. honu were shown to be highly toxic to mice by intraperitoneal injection (0.2mg/kg), although less toxic by gavage. It is possible that toxins other than putative MTX-3 are produced.

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales, Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia).

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food-borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8-D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography-Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3', 0a, 7″, 6c, 7-8s, 5‴, 0p, 2″″ and distinctive by size and hatchet-shaped 2' plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal-derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.

Gonyaulax taylorii, a new yessotoxins-producer dinoflagellate species from Chilean waters.

In summer 2009, during a survey in Bahía Mejillones, a dense bloom of a dinoflagellate from the genus Gonyaulax was detected, as well as the presence of yessotoxin. Phytoplankton samples were analyzed in detail by light and scanning electron microscopy (SEM), revealing the presence of Gonyaulax taylorii. Morphological examination showed that the cells in the bloom fit in Gonyaulax jollifei Murray et Whitting sensu Dodge, subsequently classified as Gonyaulax taylorii by Carbonell-Moore. In this context, some inconsistencies have been found in regard to the holotype; the plate 1"' appears as two plates, 1‴ and 2‴, showing a suture that does not exist in Dodge's figure of G. jollifei, from where the holotype was drawn, nor within the samples collected. Therefore, this plate has been originally described erroneously as two plates named 1"' and 2"' instead of only one named 1‴. After this correction, this species has five instead of six postcingular plates. For this reason, the description of this species must be emended. Phytoplankton net samples were found to contain yessotoxin and homoyessotoxin, with concentrations below 1pgcell-1. The present study identifies, therefore, the dinoflagellate G. taylorii as a new source of yessotoxins.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

Progress in Understanding Algal Bloom-Mediated Fish Kills: The Role of Superoxide Radicals, Phycotoxins and Fatty Acids.

Quantification of the role of reactive oxygen species, phycotoxins and fatty acids in fish toxicity by harmful marine microalgae remains inconclusive. An in vitro fish gill (from rainbow trout Oncorhynchus mykiss) assay was used to simultaneously assess the effect in superoxide dismutase, catalase and lactate dehydrogenase enzymatic activities caused by seven species of ichthyotoxic microalgae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, Alexandrium catenella, Karlodinium veneficum, Prymnesium parvum). Quantification of superoxide production by these algae was also performed. The effect of purified phycotoxins and crude extracts was compared, and the effect of fatty acids is discussed. The raphidophyte Chattonella was the most ichthyotoxic (gill cell viability down to 35%) and also the major producer of superoxide radicals (14 pmol cell-1 hr-1) especially after cell lysis. The raphidophyte Heterosigma and dinoflagellate Alexandrium were the least toxic and had low superoxide production, except when A. catenella was lysed (5.6 pmol cell-1 hr-1). Catalase showed no changes in activity in all the treatments. Superoxide dismutase (SOD) and lactate dehydrogenase exhibited significant activity increases of ≤23% and 51.2% TCC (total cellular content), respectively, after exposure to C. marina, but SOD showed insignificant changes with remaining algal species. A strong relationship between gill cell viability and superoxide production or superoxide dismutase was not observed. Purified brevetoxins PbTx-2 and -3 (from Karenia brevis, LC50 of 22.1 versus 35.2 µg mL-1) and karlotoxin KmTx-2 (from Karlodinium; LC50 = 380 ng mL-1) could almost entirely account for the fish killing activity by those two dinoflagellates. However, the paralytic shellfish toxins (PST) GTX1&4, C1&C2, and STX did not account for Alexandrium ichthyotoxicity. Only aqueous extracts of Alexandrium were cytotoxic (≤65% decrease of viability), whereas crude methanol and acetone extracts of Chattonella, Fibrocapsa, Heterosigma, Karlodinium and Prymnesium decreased cell viability down to 0%. These and our previous findings involving the role of fatty acids confirm that superoxide radicals are only partially involved in ichthyotoxicity and point to a highly variable contribution by other compounds such as lipid peroxidation products (e.g. aldehydes).

Polyketide synthesis genes associated with toxin production in two species of Gambierdiscus (Dinophyceae).

BACKGROUND: Marine microbial protists, in particular, dinoflagellates, produce polyketide toxins with ecosystem-wide and human health impacts. Species of Gambierdiscus produce the polyether ladder compounds ciguatoxins and maitotoxins, which can lead to ciguatera fish poisoning, a serious human illness associated with reef fish consumption. Genes associated with the biosynthesis of polyether ladder compounds are yet to be elucidated, however, stable isotope feeding studies of such compounds consistently support their polyketide origin indicating that polyketide synthases are involved in their biosynthesis. RESULTS: Here, we report the toxicity, genome size, gene content and transcriptome of Gambierdiscus australes and G. belizeanus. G. australes produced maitotoxin-1 and maitotoxin-3, while G. belizeanus produced maitotoxin-3, for which cell extracts were toxic to mice by IP injection (LD50 = 3.8 mg kg(-1)). The gene catalogues comprised 83,353 and 84,870 unique contigs, with genome sizes of 32.5 ± 3.7 Gbp and 35 ± 0.88 Gbp, respectively, and are amongst the most comprehensive yet reported from a dinoflagellate. We found three hundred and six genes involved in polyketide biosynthesis, including one hundred and ninety-two ketoacyl synthase transcripts, which formed five unique phylogenetic clusters. CONCLUSIONS: Two clusters were unique to these maitotoxin-producing dinoflagellate species, suggesting that they may be associated with maitotoxin biosynthesis. This work represents a significant step forward in our understanding of the genetic basis of polyketide production in dinoflagellates, in particular, species responsible for ciguatera fish poisoning.

An improved functional assay for rapid detection of marine toxins, saxitoxin and brevetoxin using a portable cardiomyocyte-based potential biosensor.

Saxitoxin (STX) and brevetoxin (PbTX-2), which are produced by marine dinoflagellates, are highly-toxic marine toxins targeting separate sites of the α subunit of voltage-dependent sodium channels (VDSCs). In this work, a portable cardiomyocyte-based potential biosensor is designed for rapid detection of STX and PbTX-2. This potential biosensor is constructed by cardiomyocyte and microelectrode array (MEA) with a label-free and real-time wireless 8-channel recording system which can dynamically monitor the multisite electrical activity of cardiomyocyte network. The recording signal parameters, spike amplitude, firing rate and 50% of spike potential duration (SPD50) extracted from extracelluar field potential (EFP) signals of the potential biosensor is analyzed to quantitatively evaluate toxicological risk of STX and PbTX-2. Firing rate of biosensor signals presents high sensitivity to STX with the detection limit of 0.35 ng/ml within 5 min. SPD50 shows high sensitivity to PbTX-2 with the detection limit of 1.55 ng/ml within 5 min. Based on the multi-parameter analysis, cardiomyocyte-based potential biosensor will be a promising tool for rapid detection of these two toxins.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light-Harvesting Complex II (LHCII) of Photosystem II.

The brevetoxins are neurotoxins that are produced by the "Florida red tide" dinoflagellate Karenia brevis. They bind to and activate the voltage-gated sodium channels in higher organisms, specifically the Nav 1.4 and Nav 1.5 channel subtypes. However, the native physiological function that the brevetoxins perform for K. brevis is unknown. By using fluorescent and photoactivatable derivatives, brevetoxin was shown to localize to the chloroplast of K. brevis where it binds to the light-harvesting complex II (LHCII) and thioredoxin. The LHCII is essential to non-photochemical quenching (NPQ), whereas thioredoxins are critical to the maintenance of redox homeostasis within the chloroplast and contribute to the scavenging of reactive oxygen. A culture of K. brevis producing low levels of toxin was shown to be deficient in NPQ and produced reactive oxygen species at twice the rate of the toxic culture, implicating a role in NPQ for the brevetoxins.

Susceptibility of gametes and embryos of the eastern oyster, Crassostrea virginica, to Karenia brevis and its toxins.

The bivalve mollusc, Crassostrea virginica, is frequently exposed to blooms of Karenia brevis along the west coast of Florida during periods of spawning and early larval development. A continuous 4-day exposure of gametes and 2-4 cell stage embryos of C. virginica to whole-cell and culture filtrate of K. brevis at 500 and 5000 cells mL(-1), was followed by a 4-day 'recovery' period. Larval growth, percent of normal, abnormal and dead larvae, and the presence of food in the larval gut were measured throughout the exposure period. Results suggest that negative effects mainly occur during embryogenesis and early development. Damage to feeding apparatus/gut may occur during embryonic development or exposure to toxins may act as a feeding deterrent on non-toxic algae. Following 2-h in vitro exposure of gametes, differences in oocyte and sperm cell parameters were investigated using flow cytometry. The reduced sperm viability in the whole-cell 5000 cells mL(-1) treatment suggests the involvement of extracellular brevetoxins (PbTx) and perhaps other harmful, uncharacterized compounds associated with the K. brevis cell membrane. The cumulative effects of reduced sperm viability, fertilization success, embryonic and larval survival, and the near-annual exposure to blooms of K. brevis could cause significant bottlenecks on oyster recruitment.

Uptake and elimination of brevetoxin in the invasive green mussel, Perna viridis, during natural Karenia brevis blooms in southwest Florida.

Perna viridis is a recently introduced species to US coastal waters and have vigorously spread throughout the southeastern seaboard since their invasion. Little information regarding their response to local environmental factors has been reported including responses to the local HAB species, Karenia brevis. This study monitored the tissue toxin concentration of brevetoxins in P. viridis from existing populations throughout two consecutive natural K. brevis blooms. The results showed P. viridis to rapidly accumulate PbTx upon exposure to the bloom, far exceeding the peak tissue concentrations of oysters, Crassostrea virginica, sampled during the same period, 57,653 ± 15,937 and 33,462 ± 10,391 ng g(-1) PbTx-3 equivalent, respectively. Further, P. viridis retained high PbTx concentrations in their tissues post bloom remaining above the regulatory limit for human consumption for 4-5 months, significantly longer than the depuration time of 2-8 weeks for native oyster and clam species. In the second year, the bloom persisted at high cell concentrations resulting in prolonged exposure and higher PbTx tissue concentrations indicating increased bioaccumulation in green mussels. While this species is not currently harvested for human consumption, the threat for post bloom trophic transfer could pose negative impacts on other important fisheries and higher food web implications.

De novo assembly and characterization of the transcriptome of the toxic dinoflagellate Karenia brevis.

BACKGROUND: Karenia brevis is a harmful algal species that blooms in the Gulf of Mexico and produces brevetoxins that cause neurotoxic shellfish poisoning. Elevated brevetoxin levels in K. brevis cells have been measured during laboratory hypo-osmotic stress treatments. To investigate mechanisms underlying K. brevis osmoacclimation and osmoregulation and establish a valuable resource for gene discovery, we assembled reference transcriptomes for three clones: Wilson-CCFWC268, SP3, and SP1 (a low-toxin producing variant). K. brevis transcriptomes were annotated with gene ontology terms and searched for putative transmembrane proteins that may elucidate cellular responses to hypo-osmotic stress. An analysis of single nucleotide polymorphisms among clones was used to characterize genetic divergence. RESULTS: K. brevis reference transcriptomes were assembled with 58.5 (Wilson), 78.0 (SP1), and 51.4 million (SP3) paired reads. Transcriptomes contained 86,580 (Wilson), 93,668 (SP1), and 84,309 (SP3) predicted transcripts. Approximately 40% of the transcripts were homologous to proteins in the BLAST nr database with an E value ≤ 1.00E-6. Greater than 80% of the highly conserved CEGMA core eukaryotic genes were identified in each transcriptome, which supports assembly completeness. Seven putative voltage-gated Na+ or Ca2+ channels, two aquaporin-like proteins, and twelve putative VATPase subunits were discovered in all clones using multiple bioinformatics approaches. Furthermore, 45% (Wilson) and 43% (SP1 and SP3) of the K. brevis putative peptides > 100 amino acids long produced significant hits to a sequence in the NCBI nr protein database. Of these, 77% (Wilson and SP1) and 73% (SP3) were successfully assigned gene ontology functional terms. The predicted single nucleotide polymorphism (SNP) frequencies between clones were 0.0028 (Wilson to SP1), 0.0030 (Wilson to SP3), and 0.0028 (SP1 to SP3). CONCLUSIONS: The K. brevis transcriptomes assembled here provide a foundational resource for gene discovery and future RNA-seq experiments. The identification of ion channels, VATPases, and aquaporins in all three transcriptomes indicates that K. brevis regulates cellular ion and water concentrations via transmembrane proteins. Additionally, > 40,000 unannotated loci may include potentially novel K. brevis genes. Ultimately, the SNPs identified among the three ecologically diverse clones with different toxin profiles may help to elucidate variations in K. brevis brevetoxin production.

Further insights into brevetoxin metabolism by de novo radiolabeling.

The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-¹4C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors.

Osmotic stress does not trigger brevetoxin production in the dinoflagellate Karenia brevis.

With the global proliferation of toxic harmful algal bloom species, there is a need to identify the environmental and biological factors that regulate toxin production. One such species, Karenia brevis, forms nearly annual blooms that threaten coastal regions throughout the Gulf of Mexico. This dinoflagellate produces brevetoxins, which are potent neurotoxins that cause neurotoxic shellfish poisoning and respiratory illness in humans, as well as massive fish kills. A recent publication reported that a rapid decrease in salinity increased cellular toxin quotas in K. brevis and hypothesized that brevetoxins serve a role in osmoregulation. This finding implied that salinity shifts could significantly alter the toxic effects of blooms. We repeated the original experiments separately in three different laboratories and found no evidence for increased brevetoxin production in response to low-salinity stress in any of the eight K. brevis strains we tested, including three used in the original study. Thus, we find no support for an osmoregulatory function of brevetoxins. The original publication also stated that there was no known cellular function for brevetoxins. However, there is increasing evidence that brevetoxins promote survival of the dinoflagellates by deterring grazing by zooplankton. Whether they have other as-yet-unidentified cellular functions is currently unknown.