Paralytic Shellfish Toxins Research Articles

The Contents and Composition of Tetrodotoxin and Paralytic Shellfish Poisoning Toxins in Marine Pufferfish Canthigaster rivulata.

The contents and composition of tetrodotoxin (TTX) and paralytic shellfish toxins (PSTs) in skin, muscle, and internal organs of two samples of marine puffer fish Canthigaster rivulata from Wakayama prefecture, Japan, were analyzed. Liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography with post-column derivatization and fluorescence detection (LC-FLD) were used for the analysis of TTX and PSTs, respectively. For both samples, TTX and two analogues of PSTs, saxitoxin (STX) and decarbamoyl STX (dcSTX), were detected at levels over the limit of quantization (LOQ) only in the skin. These toxins in the muscle and internal organs were at trace levels, or not detected (ND). TTX contents were 11,000 and 13,000 ng/g (or 35 and 41 nmol/g), while PSTs contents were 168 and 460 ng/g (or 0.63 and 1.72 nmol/g) in the two skin specimens. The compositions of total toxin content were 98.2 and 96.0 mol% TTX and 1.8 and 4.0 mol% PSTs, respectively. Thus, the main contributor to toxin content in C. rivulata skin was TTX and the levels of PSTs toxicity in C. rivulata were very low. When the PSTs contents were converted into mouse unit score from the LC-FLD results, the resulting values of 1.0 and 2.8 MU/g of PSTs in C. rivulata skin were similar to those in Takifugu poecilonotus and Takifugu vermicularis in Japan, as determined in previous studies.

Determination of Gonyautoxin-4 in Echinoderms and Gastropod Matrices by Conversion to Neosaxitoxin Using 2-Mercaptoethanol and Post-Column Oxidation Liquid Chromatography with Fluorescence Detection.

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step.

Quantity of the dinoflagellate sxtA4 gene and cell density correlates with paralytic shellfish toxin production in Alexandrium ostenfeldii blooms

Many marine dinoflagellates, including several species of the genus Alexandrium, Gymnodinium catenatum, and Pyrodinium bahamense are known for their capability to produce paralytic shellfish toxins (PST), which can cause severe, most often food-related poisoning. The recent discovery of the first PST biosynthesis genes has laid the foundation for the development of molecular detection methods for monitoring and study of PST-producing dinoflagellates. In this study, a probe-based qPCR method for the detection and quantification of the sxtA4 gene present in Alexandrium spp. and Gymnodinium catenatum was designed. The focus was on Alexandrium ostenfeldii, a species which recurrently forms dense toxic blooms in areas within the Baltic Sea. A consistent, positive correlation between the presence of sxtA4 and PST biosynthesis was observed, and the species was found to maintain PST production with an average of 6 genomic copies of sxtA4. In August 2014, A. ostenfeldii populations were studied for cell densities, PST production, as well as sxtA4 and species-specific LSU copy numbers in Föglö, Åland, Finland, where an exceptionally dense bloom, consisting of 6.3×106cellsL−1, was observed. Cell concentrations, and copy numbers of both of the target genes were positively correlated with total STX, GTX2, and GTX3 concentrations in the environment, the cell density predicting toxin concentrations with the best accuracy (Spearman's ρ=0.93, p<0.01). The results indicated that all A. ostenfeldii cells in the blooms harbored the genetic capability of PST production, making the detection of sxtA4 a good indicator of toxicity.

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n=22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large sub-unit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.

Depuration of paralytic shellfish toxins in Japanese scallop (Patinopecten yessoensis) in natural environment

To study the paralytic shellfish toxins (PSTs) depuration in Japanese scallop Patinopecten yessoensis in natural environment, Japanese scallops naturally contaminated with paralytic shellfish poisoning (PSP) toxins in the Dayao Bay in the northern Huanghai Sea are transited to Qipanmo waters in the Bohai Sea of no reported PSTs incidents. The levels and profile of PSTs during 30-day depuration are detected by the high performance liquid chromatography with fluorescence detection (HPLC-FLD). The results show that the toxicity of the PSTs in soft tissues decreases to a relatively low level at Day 9. Moreover, the depurated ratio at the early stage of the PSTs depuration is higher than that at the later stage. The toxicity analysis of dissected organs reveals that the digestive gland is the most contaminated PSTs part, which is of important implication for the human health and scallop aquiculture. The mortality of Japanese scallops during PSTs depuration experiment is relevant to PSTs level in the soft tissue.

Effect of the endoparasite Amoebophrya sp. on toxin content and composition in the paralytic shellfish poisoning dinoflagellate Alexandrium fundyense (Dinophyceae)

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing “edible food” for consumers of the marine microbial food-web during toxic algal bloom event.

ITRAQ-based quantitative proteomic analysis of a toxigenic dinoflagellate Alexandrium catenella and its non-toxic mutant.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. The PST biosynthesis gene cluster and several toxin-related proteins have been unveiled in cyanobacteria, yet little is known about dinoflagellates. Here, we compared the protein profiles of a toxin-producing dinoflagellate Alexandrium catenella (ACHK-T) and its non-toxic mutant (ACHK-NT), and characterized differentially displayed proteins using a combination of the iTRAQ-based proteomic approach and the transcriptomic database. Totally 3488 proteins were identified from A. catenella, and proteins involved in carbohydrate, amino acid and energy metabolism were the most abundant. Among them, 185 proteins were differentially displayed: proteins involved in amino acid biosynthesis, protein and carbohydrate metabolism and bioluminescence were more abundant in ACHK-T, while proteins participating in photosynthesis, fatty acid biosynthesis, and the processes occurring in peroxisome displayed higher abundances in ACHK-NT. Seven toxin-related proteins were identified but they varied insignificantly between the two strains. Different carbon and energy utilization strategies were potentially related to the toxin producing ability, and the regulation mechanism of PST biosynthesis was more complex in dinoflagellates. Our study provides the first comprehensive dataset on the dinoflagellate proteome and lays the groundwork for future proteomic study.

Changes in gene expression and histological injuries as a result of exposure ofCrassostrea gigasto the toxic dinoflagellateGymnodinium catenatum

The harmful alga Gymnodinium catenatum is one of the species that produces the neurotoxic, hydrophilic, tetrahydropurine derivatives known as paralytic shellfish toxins (PST). Studies have shown that several classes of marine toxins have genotoxic effects on bivalves. However, there are few studies on PST and their genotoxic effects on bivalves such as oysters. Experiments were performed to assess the effects of the toxic dinoflagellate G. catenatum on changes in the transcription level of the genes involved in cellcycle regulation (p21, p53, cafp55) and initial inflammatory (casp1) processes and injuries in tissues of juvenile Crassostrea gigas. Our results reveal an effect at the level of transcription. The high transcription levels of the p21 and p53 genes found in oysters exposed to dinoflagellate diets suggest an activation of the cell-cycle checkpoints by DNA damage induction. At the histological level, the oysters exposed to the toxic dinoflagellate showed exfoliation, epithelial rupture and inflammation, which correlate with the observed changes in expression level of casp1 that is known to play a role in initial inflammatory responses. Our results provide molecular data suggesting probable DNA damage through expression modulation of the analysed key genes, in addition to histological evidence of tissue injuries due to PST dinoflagellate exposure.

Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including Alexandrium minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 105cells/L within 30min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research.

Elevated Na(+) and pH influence the production and transport of saxitoxin in the cyanobacteria Anabaena circinalis AWQC131C and Cylindrospermopsis raciborskii T3.

Saxitoxins (STX), neurotoxic alkaloids, fall under the umbrella of paralytic shellfish toxins produced by marine dinoflagellates and freshwater cyanobacteria. The genes responsible for the production of STX have been proposed, but factors that influence their expression and induce toxin efflux remain unclear. Here we characterize the putative STX NorM-like MATE transporters SxtF and SxtM. Complementation of the antibiotic-sensitive strain Escherichia coli KAM32 with these transporters decreased fluoroquinolone sensitivity, indicating that while becoming evolutionary specialized for STX transport these transporters retain relaxed specificity typical of this class. The transcriptional response of STX biosynthesis (sxtA) along with that of the STX transporters (sxtM and sxtF from Cylindrospermopsis raciborskii T3, and sxtM from Anabaena circinalis AWQC131C) were assessed in response to ionic stress. These data, coupled with a measure of toxin intracellular to extracellular ratios, provide an insight into the physiology of STX export. Cylindrospermopsis raciborskii and Anabaena circinalis exhibited opposing responses under conditions of ionic stress. High Na(+) (10 mM) induced moderate alterations of transcription and STX localization, whereas high pH (pH 9) stimulated the greatest physiological response. Saxitoxin production and cellular localization are responsive to ionic strength, indicating a role of this molecule in the maintenance of cellular homeostasis.

An innovative method based on quick, easy, cheap, effective, rugged, and safe extraction coupled to desorption electrospray ionization-high resolution mass spectrometry for screening the presence of paralytic shellfish toxins in clams

The capabilities of desorption electrospray ionization-high resolution mass spectrometry (DESI-HRMS) were tested for screening the presence of some paralytic shellfish toxins in clams. A quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach is proposed for sample clean-up. QuEChERS extraction was optimized by using a full factorial design followed by the multicriteria method of the desirability functions. Quantitation limits in the microgram per kilogram range proved reliability of the method for the detection of the investigated toxins in accordance to the rules laid down by European legislation. The optimized QuEChERS-DESI-HRMS based-method allowed for a rapid reliable screening of the investigated compounds at levels of interest, thus being useful for high-throughput analyses.

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C9H16N6O2. This structure differed from that of decarbamoyl STX (dcSTX; C9H16N6O3) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.

Fish gill damage by the dinoflagellate Alexandrium catenella from Chilean fjords: Synergistic action of ROS and PUFA

Outbreaks of the dinoflagellate Alexandrium catenella have caused significant economic losses to the salmon industry in the south of Chile. However, the precise ichthyotoxic mechanism by this paralytic shellfish toxin (PST) – producing dinoflagellate remains poorly understood. The rainbow trout cell line RTgill-W1 assay was used to investigate fish gill damage by multiple A. catenella strains under different environmental conditions, and potency of whole cells, lysed cells and culture medium were compared. Cytotoxic potency was highly variable among strains and strongly correlated to dinoflagellate cell-abundance. Lysed cells produced more gill damage than intact cells and supernatant, reducing gill cell viability down to 20% of controls at 4000cellsmL−1. However, gill cells exposed to pure PST fractions (C1&C2, STX, GTX 1&4) exhibited only very limited loss of viability (<30%), even at cell concentrations equivalent to exceeding those detected in southern Chilean fjords. A. catenella lytic compounds rapidly (1–4 days) degraded in the light, but were pH (7–9) and temperature stable (17–85°C), thus arguing against the involvement of proteinaceous compounds. Superoxide production by Chilean dinoflagellate strains was as high as by the raphidophyte Chattonella marina (max. 8.67±0.14pmol O2−cell−1h−1), variable among strains and enhanced by cell disruption and environmental stress (low nutrients, low salinity, low or high pH). Fatty acid profiles included high concentrations of the long-chain (≥C20) polyunsaturated fatty acids (PUFA) – docosahexaenoic acid (DHA, 22:6ω3; 16–20% of total fatty acids) in addition to several other PUFA, which were highly cytotoxic against gill cells as a purified fraction (LC50 1.30μgmL−1). In addition, potency was enhanced 9-fold in combination with superoxide anion (LC50 0.15μgmL−1). In conclusion, the present study demonstrates that fish gill damage during A. catenella fish-kill events in Chilean fjords cannot be explained by PST toxins, but can to a large extent be accounted for by the synergistic interaction between ROS (superoxide anion) and DHA, and potentially other PUFA, notably under conditions that promote cell lysis.

Analysis of paralytic shellfish toxins, potential chemical threat agents, in food using hydrophilic interaction liquid chromatography–mass spectrometry

A novel method for determining paralytic shellfish toxin (PST) profiles in food was developed using a combination of silica and strong cation exchange (SCX) solid phase extraction (SPE) coupled to hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). Besides the risk for natural contamination of seafood and drinking water, PSTs also pose potent threats through intentional contamination of food, due to their high toxicity and the wide distributions of toxin-producing algae. The new preparation method aim to maintain the samples’ original toxin profiles by avoiding conditions known to induce interconversion or degradation of the PSTs. The method was evaluated for PST extraction from water, milk, orange juice, apple purée, baby food, and blue mussels (Mytilus edulis). The extracts were found to produce reproducible retention times in HILIC–MS/MS analysis. When an authentic toxic mussel sample was analyzed using the novel method, saxitoxin and gonyautoxin-3 were identified, in agreement with data acquired using the Lawrence pre-column oxidation high-performance liquid chromatography–fluorescence detection (HPLC–FLD) method. Overall recoveries of the PSTs from tested foods by the novel method ranged from 36% to 111%.

Southern right whale (Eubalaena australis) calf mortality at Península Valdés, Argentina: Are harmful algal blooms to blame?

AbstractPenínsula Valdés (PV) in Argentina is an important calving ground for southern right whales (SRWs, Eubalaena australis). Since 2005, right whale mortality has increased at PV, with most of the deaths (~90%) being calves &lt;3 mo old. We investigated the potential involvement of harmful algal blooms (HABs) in these deaths by examining data that include: timing of the SRW deaths, biotoxins in samples from dead SRWs, abundances of the diatom, Pseudo‐nitzschia spp., and the dinoflagellate, Alexandrium tamarense, shellfish harvesting closure dates, seasonal availability of whale prey at PV and satellite chlorophyll data. Evidence of the whales' exposure to HAB toxins includes trace levels of paralytic shellfish toxins (PSTs) and domoic acid (DA) in tissues of some dead whales, and fragments of Pseudo‐nitzschia spp. frustules in whale feces. Additionally, whales are present at PV during both closures of the shellfish industry (due to high levels of PSTs) and periods with high levels of Pseudo‐nitzschia spp. and A. tamarense. There is a positive statistical relationship between monthly Pseudo‐nitzschia densities (but not A. tamarense) and calf deaths in both gulfs of PV.

A comparison of the physiological responses, behaviour and biotransformation of paralytic shellfish poisoning toxins in a surf-clam (Paphies donacina) and the green-lipped mussel (Perna canaliculus)

The accumulation of paralytic shellfish toxins (PSTs) in bivalves is species specific. We compared the physiological responses and the toxin profiles in tissues of the burrowing surf clam, Paphies donacina, and the green-lipped mussel, Perna canaliculus, exposed to the toxic dinoflagellate Alexandrium tamarense. Bivalves were supplied with the toxic algae for 10 days, then allowed a detoxification period of 8 days. Clearance rates of mussels and clams were similar when fed either with toxic A. tamarense or non-toxic A. margalefi. Byssus production in the mussel was inhibited and exhalent siphon activity in clams was erratic following exposure to A. tamarense. There were considerable differences in the toxic profile between the dinoflagellate A. tamarense, and tissues of the mussel and the surf clam, indicating that bioconversion of the PSTs had taken place. Toxin profiles of the tissues were both species and tissue specific. Following an 8-day detoxification period, total PSTs in mussels had fallen to safe concentrations below 50µg per 100g, whereas concentrations in clams remained high, with an average value greater than 600µg STX di-HCL equivalents per 100g. The results confirmed that mussels and clams are important monitoring organisms for toxic algal blooms and can be used to minimise the health risk of PSTs to humans.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Scientific questionParalytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. Technical significanceHigh-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. Scientific significanceThis review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms.

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation.

Paralytic shellfish toxins (PSTs) are originated from cyanobacteria and dinoflagellates, including Alexandrium tamarense, the common dinoflagellate species. In this study, a toxic dinoflagellate strain of A. tamarense CI01 was selected for studying the PSTs' concentration and the related protein variation during the whole cell cycle under different nutrient conditions. High-performance liquid chromatography, 2-D DIGE and Western blotting were used collectively for protein profiling and identification. Results showed that the toxin content was suppressed under nitrogen limiting condition, but enhanced in phosphorous limiting medium. Based on the results of proteomics analysis, 7 proteins were discovered to be related to the PSTs biosynthesis of A. tamarense CI01, including S-adenosylhomocysteine hydrolase, ornithine cyclodeaminase, argininosuccinate synthase, methyluridine methyltransferase cystine ABC transporter, phosphoserine phosphatase, argininosuccinate synthase and acyl-CoA dehydrogenase, which corresponds to the metabolism of the methionine, cysteine, ornithine, arginine and proline. Moreover, some photosynthesis relating proteins also increased their expression during PST synthesis period in A. tamarense CI01, such as phosphoenolpyruvate carboxylase, chloroplast phosphoglycerate kinase, peridinin-chlorophyll α-binding protein, Mg(2+) transporter protein and chloroplast phosphoglycerate kinase. The above findings are in support of our hypothesis that these proteins are involved in toxin biosynthesis of A. tamarense CI01, but cause-and-effect mechanisms need to be investigated in further studies.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

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).