Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

Occurrence and Seasonal Monitoring of Domoic Acid in Three Shellfish Species from the Northern Adriatic Sea

As filter feeders, bivalves and ascidians can accumulate contaminants present in the environment and pass them on to higher food chain levels as vectors. The consumption of bivalves contaminated with the potent neurotoxin domoic acid (DA) can cause amnesic shellfish poisoning in humans. The aim of this study was to determine seasonal differences in occurrence and accumulation of this phycotoxin in European oysters (Ostrea edulis Linnaeus, 1758) (n = 46), Queen scallops (Aequipecten opercularis Linnaeus, 1758) (n = 53), and edible ascidians of the Microcosmus spp. (n = 107), originating from the same harvesting area in the Northern Adriatic Sea. The quantification was performed using ultra-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) preceded by derivatization with dansyl chloride. DA was found in very low concentrations throughout the year, with a maximum value of 810 μg/kg in Queen scallops. This study reveals differences in the occurrence and accumulation of DA between Queen scallops and the other two investigated species (oysters and ascidians) and the highest concentrations during the colder part of the year. Even though DA was detected in all of them, Queen scallops showed higher DA accumulation compared to the other two (p < 0.001), hence representing a sentinel species suitable for the monitoring of DA level in seafood.

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains from Southern Chile

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5–55° S) 50 years ago and is responsible for most of the area’s cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell−1), with the more toxic strains occurring in the north (−40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000–1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.

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.

A Generic LC-HRMS Screening Method for Marine and Freshwater Phycotoxins in Fish, Shellfish, Water, and Supplements

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains From Southern Chile

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in Southern Chile (39.5&ndash;55&deg;S) 50 years ago and is responsible for most of the area&rsquo;s cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n=45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Although the toxin profiles were similar for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell&minus;1), with the more toxic strains occurring in the north (&minus;40.6&deg;S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000&ndash;1650km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.