Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Effects of the toxic dinoflagellate Protoceratium reticulatum and its yessotoxins on the survival and feed ingestion of Argopecten purpuratus veliger larvae.

The effects of yessotoxins (YTXs) produced by the dinoflagellate Protoceratium reticulatum in the early stages of bivalves have not been studied in detail. The present study evaluates the effects of P. reticulatum and YTXs on the survival and feed ingestion of veliger larvae of Argopecten purpuratus. Larvae were 96 h-exposed to 500, 1000 and 2000 P. reticulatum cells mL-1, and their equivalent YTX extract was prepared in methanol. Results show a survival mean of 82 % at the highest density of dinoflagellate, and 38 % for larvae with the highest amount of YTX extract. Feed ingestion is reduced in the dinoflagellate exposure treatments as a function of cell density. Therefore, the effect of YTXs on A. purpuratus represents a new and important area of study for investigations into the deleterious effects of these toxins in the early stages of the life cycle of this and, potentially, other bivalves.

Tissue-specific antioxidative response and metabolism of paralytic shellfish toxins in scallop (Chlamys farreri) mantle with Alexandrium dinoflagellate exposure.

Bivalves show remarkable capacity to acclimate paralytic shellfish toxins (PSTs) produced by dinoflagellates, severely affecting fishery industry and public health. Here, transcriptomic response to PSTs-producing dinoflagellate (Alexandrium minutum) was investigated in Zhikong scallop (Chlamys farreri) mantle. The PSTs accumulated in C. farreri mantle continually increased during the 15 days exposure, with "oxidation-reduction" genes induced compared to the control group at the 1st and 15th day. Through gene co-expression network analysis, 16 PSTs-responsive modules were enriched with up- or down-regulated genes. The concentration of GTXs, major PSTs in A. minutum and accumulated in scallops, was correlated with the up-regulated magenta module, enriching peroxisome genes as the potential mantle-specific PSTs biomarker. Moreover, Hsp70B2s were inhibited throughout the exposure, which together with the expanded neurotransmitter transporter SLC6As, may play essential roles on neurotransmitter homeostasis in scallop mantle. These results paved the way for a comprehensive understanding of defensive mechanism and homeostatic response in scallop mantle against PSTs.

Screen and Optimization of an Aptamer for Alexandrium tamarense-A Common Toxin-Producing Harmful Alga.

Among all the paralytic shellfish toxins (PSTs)-producing algae, Alexandrium tamarense is one of the most widespread harmful species posing a serious threat to marine resources and human health. Therefore, it is extremely important to establish a rapid and accurate monitoring method for A. tamarense that can provide early warnings of harmful algal blooms (HABs) caused by this alga and limit the contamination due to PSTs. In this study, an ssDNA library was first obtained by whole cell systematic evolution of ligands by exponential enrichment after 18 consecutive rounds of iterative screening. After sequencing in combination with subsequent multiple alignment of sequences and secondary structure simulation, the library could be classified into 2 families, namely, Family1 and Family2, according to sequence similarity. Flow cytometry was used to test the affinity and cross-reactivity of Ata19, Ata6, Ata25 and Ata29 belonging to Family2. Ata19 was selected to be modified by truncation, through which a new resultant aptamer named as Ata19-1-1 was obtained. Ata19-1-1 with a KD of 75.16 ± 11.10 nM displayed a much higher affinity than Ata19. The specificity test showed that Ata19-1-1 has the same discrimination ability as Ata19 and can at least distinguish the target microalga from other microalgae. The observation under a fluorescence microscopy showed that the A. tamarense cells labeled with Ata19-1-1 are exhibiting bright green fluorescence and could be easily identified, factually confirming the binding of the aptamer with target cells. In summary, the aptamer Ata19-1-1 produced in this study may serve as an ideal molecular recognition element for A. tamarense, which has the potential to be developed into a novel detection method for this harmful alga in the future.

Cyanotoxins, biosynthetic gene clusters, and factors modulating cyanotoxin biosynthesis.

Cyanobacterial harmful algal blooms (CHABs) are a global environmental concern that encompasses public health issues, water availability, and water quality owing to the production of various secondary metabolites (SMs), including cyanotoxins in freshwater, brackish water, and marine ecosystems. The frequency, extent, magnitude, and duration of CHABs are increasing globally. Cyanobacterial species traits and changing environmental conditions, including anthropogenic pressure, eutrophication, and global climate change, together allow cyanobacteria to thrive. The cyanotoxins include a diverse range of low molecular weight compounds with varying biochemical properties and modes of action. With the application of modern molecular biology techniques, many important aspects of cyanobacteria are being elucidated, including aspects of their diversity, gene-environment interactions, and genes that express cyanotoxins. The toxicological, environmental, and economic impacts of CHABs strongly advocate the need for continuing, extensive efforts to monitor cyanobacterial growth and to understand the mechanisms regulating species composition and cyanotoxin biosynthesis. In this review, we critically examined the genomic organization of some cyanobacterial species that lead to the production of cyanotoxins and their characteristic properties discovered to date.

Degradation of paralytic shellfish toxins during flocculation of Alexandrium pacificum by an oxidized modified clay: A laboratory experiment.

Paralytic shellfish toxins (PSTs), produced by Alexandrium pacificum in the marine environment, are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells. During the toxigenic A. pacificum blooms outbreaks, PSTs can be accumulated through the food chain and finally enter the human body, posing a significant threat to human health and safety. This study experimented with a novel type of oxidized modified clay, potassium peroxymonosulfate modified clay (PMPS-MC), which could remove A. pacificum cells as well as reduce intracellular and extracellular PSTs toxicity rapidly. For the extracellular PSTs, its content decreased to below the detection limit rapidly through oxidative degradation within 15 min of 10 mg/L PMPS-MC treatment. Whereafter, although the residual cells in water column and some viable cells in flocculated sediment continued to secrete toxins, the extracellular PSTs content and toxicity in the PMPS-MC treatment groups remained significantly lower than those in the control group. For the intracellular PSTs, PMPS-MC might induce the transformation of more toxic GTX1&4 to less toxic GTX2&3 and C1&2, resulting in intracellular PSTs toxicity reduced within 15 min. In addition, intracellular PSTs content and toxicity in the PMPS-MC treatment groups were consistently lower than the control group within 48 h, possibly by inhibiting the A. pacificum cells growth. These results will provide a scientific basis for the field application of modified clay to control A. pacificum blooms.

Microcystin-LR exposure interfered maintenance of colonic microenvironmental homeostasis in rat.

Microcystin-leucine arginine (MCLR) is a phycotoxin produced by cyanobacteria. As a hepatotoxin, increasing evidence suggests that it has some negative effects on the mammal gastrointestinal tract, but further studies are warranted. In this study, we investigated the effects of MCLR on the intestinal epithelial microenvironment by oral administration of MCLR. As expected, MCLR at doses of 200 and 400 µg kg-1 bw showed hepatorenal toxicity in rats but without significant gastrointestinal symptoms. MCLR exposure decreased the thickness of the colonic epithelial mucus layer, and down-regulated the expression of main mucin protein (MUC2), cytoskeletal assembly-related genes (Arpc1a, Enah) and cytoskeletal stability-related genes (Ptk2, Prkca, Actn1, Pxn, Tln1, Cttn, Vcl) in colonic tissue to varying degrees, but did not affect the expression of cell connection-related genes including Zo1, Ocln, Cldn2 and Cdh1. In addition, MCLR exposure had a limited effect on gut bacterial diversity but clearly enriched specific bacteria. Prevotella, which plays a crucial role in balancing health and disease, was inhibited, whereas Muribaculaceae concerning the epithelial barrier, was promoted. Together, our findings demonstrate that MCLR exposure can weaken the colonic epithelial barrier by interfering with the stability of the cytoskeleton, which in turn exacerbates the homeostasis maintenance in the intestinal microenvironment.

Genetic association of toxin production in the dinoflagellate Alexandrium minutum.

Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum. 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG, may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum, suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.

Putative high-level toxicity pathways based on evidence of brevetoxin immunotoxicity in marine fauna.

Red tide events, caused by a toxin producing dinoflagellate, Karenia brevis, occur annually in Florida and Texas. These events lead to health risks for both humans and wildlife that utilize coastal environments. Brevetoxins, potent lipophilic neurotoxins produced by K. brevis, modulate immune responses in laboratory studies with model organisms and in the natural environment in both humans and wildlife. Studies show that brevetoxins activate immune cells, stimulate production of gamma-globulins, cytokines, and neutrophils, modulate lysozyme activity, induce apoptosis, and modulate lymphocyte proliferation in marine species. The objective of this review was to summarize brevetoxin-induced immunotoxicity in marine animals based on available peer-reviewed literature about K. brevis blooms and associated health concerns and propose putative toxicity pathways. This review identifies knowledge gaps within current brevetoxin induced immunotoxicity research, including assessing the long-term impacts of brevetoxin exposure, elucidating the mechanistic linkages between brevetoxins and immune cells, and evaluating repeated and chronic versus acute brevetoxin exposure implications on overall organismal health. The putative immunotoxicity pathways based on evidence from brevetoxin-exposure in marine fauna described in this review represent a useful tool and resource for researchers, wildlife managers, and policy makers. This review and proposed putative immunotoxicity pathways will inform decisions regarding the risks of algal blooms, as it pertains to marine animal health.

The paralytic shellfish toxin effect on bioenergetic constituents of the fishery resource Chorus giganteus (Gastropoda: Muricidae).

Alexandrium catenella, one of the most common harmful microalgae observed in southern Chile, produces paralytic shellfish toxins, which can affect many organisms throughout the trophic chain. This research evaluated how paralytic shellfish toxins affected the principal bioenergetic constituents and fatty acids composition of the carnivorous snail Chorus giganteus. Snails were separated into a "toxic" group that was fed the toxic clam Mulinia edulis (which was previously fed A. catenella), and a "non-toxic" group, fed non-toxic clams. Both groups were kept under these conditions for 63 days. Our results indicated no difference in the ingestion rate of toxic versus non-toxic snails; however, a higher protein level was identified in toxic snails. The total lipid content proved to be no different in toxic versus non-toxic snails; although, an effect of the toxic diet on the fatty acid profile of C. giganteus was observed. High levels of essential polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in toxic snails, were identified. Our results suggest that exposure to paralytic shellfish toxins, through diet, may cause changes in the biochemical composition of C. giganteus, which may have a subsequent impact on its energetic physiology.

Transcriptome Analysis Reveals MAPK/AMPK as a Key Regulator of the Inflammatory Response in PST Detoxification in Mytilus galloprovincialis and Argopecten irradians.

Paralytic shellfish toxins (PSTs) are an increasingly important source of pollution. Bivalves, as the main transmission medium, accumulate and metabolize PSTs while protecting themselves from damage. At present, the resistance mechanism of bivalves to PSTs is unclear. In this study, Mytilus galloprovincialis and Argopecten irradians were used as experimental shellfish species for in situ monitoring. We compared the inflammatory-related gene responses of the two shellfish during PSTs exposure by using transcriptomes. The results showed that the accumulation and metabolism rate of PSTs in M. galloprovincialis was five-fold higher than that in A. irradians. The inflammatory balance mechanism of M. galloprovincialis involved the co-regulation of the MAPK-based and AMPK-based anti-inflammatory pathways. A. irradians bore a higher risk of death because it did not have the balance system, and the regulation of apoptosis-related pathways such as the PI3K-AKT signaling pathway were upregulated. Taken together, the regulation of the inflammatory balance coincides with the ability of bivalves to cope with PSTs. Inflammation is an important factor that affects the metabolic pattern of PSTs in bivalves. This study provides new evidence to support the studies on the resistance mechanism of bivalves to PSTs.

A drug discovery approach based on comparative transcriptomics between two toxin-secreting marine annelids: Glycera alba and Hediste diversicolor.

Most animal toxins evolved to interact with specific molecular targets, which makes them highly-prized bioactives for drug development. Marine toxins, in particular, due to their wide chemical diversity, offer a new range of possibilities, a few of which have already been translated into approved drugs. Glycera alba and Hediste diversicolor are sympatric Polychaeta with distinct ecology and behavior suspected to secrete toxins that evolved to interact with distinct molecular targets, thus with differential selectivity and potential applications in drug discovery. Comparative transcriptomics revealed that while G. alba's venom apparatus is localized in the proboscis and neurotoxins are secreted to overtake prey, H. diversicolor secretes fewer and less specific toxins that are seemingly a defense. Human interactome-directed analysis unraveled novel toxins and other bioactives with potential biomedical applications, like proteins from G. alba's venom that can regulate apoptosis, whereas H. diversicolor yielded proteins that may control inflammation and cell proliferation in humans. Omics and bioinformatics appear to be powerful tools for marine bioprospecting and drug discovery, enabling molecular mining through transcriptomes of non-model organisms and link their ecology and physiology with protein's specificity and bioreactivity. Interactome-directed analysis against the human proteome seems an efficient alternative to the design of synthetic drugs.

Toxic Responses of Different Shellfish Species after Exposure to Prorocentrum lima, a DSP Toxins Producing Dinoflagellate.

Prorocentrum lima is a global benthic dinoflagellate that produces diarrhetic shellfish poisoning (DSP) toxins, which can be ingested by filter-feeding bivalves, and eventually pose a great threat to human health through food chain. After being exposed to P. lima, different bivalves may accumulate various levels of DSP toxins and display different toxic responses. However, the underlying mechanism remains unclear. Here, we found that the content of okadaic acid-equivalents (OA-eq) varied in the digestive glands of the three bivalves including Crassostrea gigas, Mytilus coruscus and Tegillarca granosa after P. lima exposure. The degree of esterification of OA-eq in the three bivalves were opposite to the accumulation of OA-eq. The digestive gland tissues of the three bivalve species were damaged to different degrees. The transcriptional induction of Nrf2 targeted genes such as ABCB1 and GPx indicates the functionality of Nrf2 pathway against DSP toxins in bivalves. The oyster could protect against DSP toxins mainly through ABC transporters and esterification, while the mussel and clam reduce the damage induced by DSP toxins mainly by regulating the expression of antioxidant genes. Our findings may provide some explanations for the difference in toxic response to DSP toxins in different shellfish.

The detoxification activities and mechanisms of microcystinase towards MC-LR.

Microcystins (MCs) are the most common and toxic cyanotoxins that are hazardous to human health and ecosystems. Microcystinase is the enzyme in charge of the initial step in the biodegradation of MCs. The characterization, application conditions, and detoxification mechanisms of microcystinase from an indigenous bacterium Sphingopyxis sp. YF1 towards MC-LR were investigated in the current study. The microcystinase gene of strain YF1 was most similar to Sphingomonas sp. USTB-05 and contained a CAAX-family conversed abortive Infection (ABI) domain. The microcystinase was successful obtained and purified by overexpression in Escherichia coli. The highest degradation rate of MC-LR was 1.0 µg/mL/min under the optimal condition of 30 â„ƒ, pH 7, 20 µg/mL MC-LR, and 400 µg/mL microcystinase. The MC-degrading product was identified as linearized MC-LR, which possessed a much lower inhibitory activity against protein phosphatase 2A than MC-LR. Microcystinase interacted with MC-LR via amino acid residues involved in through the formation of conventional Hydrogen Bond, Pi-Pi T-shapes, Van der Waals force, and so on. The optimal MC-degrading condition of pure microcystinase and its detoxification mechanisms against MC-LR were revealed. The toxicity of purified linearized MC-LR was explored for the first time. These findings suggest that pure microcystinase may efficiently detoxify MCs and it is promising in the bioremediation of MC-polluted environments.

Marine toxin domoic acid induces moderate toxicological response in non-target HepG2 cells.

Domoic acid (DA) is a marine neurotoxin produced as a defence compound by diatom Pseudo-nitzschia. Although its toxicity is well known in marine mammals and fish, data on DA cyto/genotoxicity in human non-target cells is still limited. Hence, we aimed to study the effect of DA (0.001-10 µg/mL) on cell viability and proliferation kinetics of human hepatocellular carcinoma (HepG2) cells as well as DNA damage induction after 4, 24 and 72 h of exposure. The results revealed that DA up to 10 µg/mL did not elicit significant changes in HepG2 cell viability, proliferation and cell cycle at applied conditions. DA did not generate DNA double-strand breaks, while it exhibited significant dose- and time-dependent increase of DNA damage in the form of either DNA single-strand breaks or alkali labile sites. Additionally, increased malondialdehyde level after DA treatment indicated oxidative damage to lipids. Altogether, the results showed that neurotoxin DA induced only minor adverse genotoxic effects in non-target HepG2 cells that most probably occurred resulting from the oxidative stress. However, additional research is needed to further elucidate the mechanisms of DA toxicity, particularly in terms of chronic exposure, as well as to understand its potential influence on human non-target cells.

Salt-alkalization may potentially promote Microcystis aeruginosa blooms and the production of microcystin-LR.

The development of saline-alkali lands has contributed to the increasing discharge of alkaline salt-laden wastewater, which poses a threat to aquatic organisms. However, the comprehensive effect of alkaline salt on Microcystis aeruginosa, a harmful cyanobacterium, remains unclear. In this study, the growth, physiology, cell ultrastructure and production of microcystin-LR (MC-LR) in Microcystis aeruginosa exposed to four levels of alkaline salt stress were evaluated. The growth of Microcystis aeruginosa was stimulated at an electrical conductivity (EC) of 2.5 mS/cm compared to the control, as supported by the increased cell density, photosynthetic pigment and protein contents. Microcystis aeruginosa could tolerate a certain level of alkaline salt (i.e., EC of 5 mS/cm) via increasing photosynthetic pigment contents to protect cells from alkaline salt stress, but the antioxidant defence system and cell ultrastructure were not affected. When EC increased to 7.5 mS/cm, alkaline salt caused oxidative stress and toxicity in Microcystis aeruginosa, as evidenced by analysis of the integrated biomarker response (IBR). Furthermore, the photosynthetic pigment and protein contents decreased, and cell apoptosis associated with ultrastructural changes was observed. Therefore, we propose that EC of 7.5 mS/cm is a threshold for growth of Microcystis aeruginosa. Additionally, the intracellular MC-LR content was stimulated by alkaline salt, and the highest value was observed at EC of 2.5 mS/cm. The extracellular MC-LR content increased with the increasing alkaline salt concentration. When EC was 7.5 mS/cm, the extracellular MC-LR content was significantly higher than in the control and was associated with the upregulated mcyH gene. This study recommends that more attention should be paid to the risk of Microcystis aeruginosa bloom and microcystin-LR pollution in lakes located in salinization regions.

Multi-omics analysis reveals metabolism of okadaic acid in gut lumen of rat.

Okadaic acid (OA) is an important marine lipophilic phycotoxin with various pathological properties, responsible for diarrheal shellfish poisoning events in human beings over the world. However, to date no mechanism can well explain the toxicity and symptom of OA, even diarrhea. Here, to reveal the toxic mechanism of OA to mammals, we analyzed the metabolism of OA in rat and the effects of OA exposure on the composition and function of gut bacteria using a multi-omics strategy and rRNA high-throughput technology. We found that OA exerted great effects on gut bacteria, mainly featured in heavy fluctuation of dominant genera and significant changes in the mapped bacterial function genes, including not only virulence genes of pathogenic bacteria, but also bacterial metabolism genes. In the feces of the OA-exposed group, we detected dinophysistoxin-2 (DTX-2), lespedezaflavanone F and tolytoxin, suggesting that OA could be transformed into other metabolites like DTX-2. Other metabolic biomarkers such as N-Acetyl-a-neuraminic acid, N,N-dihydroxy-L-tyrosine, nalbuphine, and coproporphyrin I and III were also highly correlated with OA content, which made the toxicity of OA more complicated and confusing. Spearman correlation test demonstrated that Bacteroides and Romboutsia were the genera most related to OA transformation, suggesting that Bacteroides and Romboutsia might play a key role in the complicated and confusing toxicity of OA. In this study, we found for the first time that OA may be converted into other metabolites in gut, especially DTX-2. This finding could not only help to reveal the complex toxicity of OA, but also have important significance for clarifying the transportation, metabolism, and environmental fate of OA in the food chain.

Mimics of Allergy and Angioedema: Scombroid, Mast Cell Activation Disorders, and Hereditary Alpha Tryptasemia.

Scombroid poisoning, systemic mastocytosis, and hereditary alpha tryptasemia all present with episodes that resemble allergic reactions. Knowledge regarding systemic mastocytosis and hereditary alpha tryptasemia is quickly evolving. Epidemiology, pathophysiology, and strategies to identify and diagnose are discussed. Evidence-based management in the emergency setting and beyond is also explored and summarized. Key differences are described between these events and allergic reactions.

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.

Unknown Extracellular and Bioactive Metabolites of the Genus Alexandrium: A Review of Overlooked Toxins.

Various species of Alexandrium can produce a number of bioactive compounds, e.g., paralytic shellfish toxins (PSTs), spirolides, gymnodimines, goniodomins, and also uncharacterised bioactive extracellular compounds (BECs). The latter metabolites are released into the environment and affect a large range of organisms (from protists to fishes and mammalian cell lines). These compounds mediate allelochemical interactions, have anti-grazing and anti-parasitic activities, and have a potentially strong structuring role for the dynamic of Alexandrium blooms. In many studies evaluating the effects of Alexandrium on marine organisms, only the classical toxins were reported and the involvement of BECs was not considered. A lack of information on the presence/absence of BECs in experimental strains is likely the cause of contrasting results in the literature that render impossible a distinction between PSTs and BECs effects. We review the knowledge on Alexandrium BEC, (i.e., producing species, target cells, physiological effects, detection methods and molecular candidates). Overall, we highlight the need to identify the nature of Alexandrium BECs and urge further research on the chemical interactions according to their ecological importance in the planktonic chemical warfare and due to their potential collateral damage to a wide range of organisms.