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Exposure to toxigenic harmful algal blooms (HABs) can result in widely recognized acute poisoning in humans. The five most commonly recognized HAB-related illnesses are diarrhetic shellfish poisoning (DSP), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), and ciguatera poisoning (CP). Despite being caused by exposure to various toxins or toxin analogs, these clinical syndromes share numerous similarities. Humans are exposed to these toxins mainly through the consumption of fish and shellfish, which serve as the main biological vectors. However, the risk of human diseases linked to toxigenic HABs is on the rise, corresponding to a dramatic increase in the occurrence, frequency, and intensity of toxigenic HABs in coastal regions worldwide. Although a growing body of studies have focused on the toxicological assessment of HAB-related species and their toxins on aquatic organisms, the organization of this information is lacking. Consequently, a comprehensive review of the adverse effects of HAB-associated species and their toxins on those organisms could deepen our understanding of the mechanisms behind their toxic effects, which is crucial to minimizing the risks of toxigenic HABs to human and public health. To this end, this paper summarizes the effects of the five most common HAB toxins on fish, shellfish, and humans and discusses the possible mechanisms. [ABSTRACT FROM AUTHOR]

Exposure to toxigenic harmful algal blooms (HABs) can result in widely recognized acute poisoning in humans. The five most commonly recognized HAB-related illnesses are diarrhetic shellfish poisoning (DSP), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), and ciguatera poisoning (CP). Despite being caused by exposure to various toxins or toxin analogs, these clinical syndromes share numerous similarities. Humans are exposed to these toxins mainly through the consumption of fish and shellfish, which serve as the main biological vectors. However, the risk of human diseases linked to toxigenic HABs is on the rise, corresponding to a dramatic increase in the occurrence, frequency, and intensity of toxigenic HABs in coastal regions worldwide. Although a growing body of studies have focused on the toxicological assessment of HAB-related species and their toxins on aquatic organisms, the organization of this information is lacking. Consequently, a comprehensive review of the adverse effects of HAB-associated species and their toxins on those organisms could deepen our understanding of the mechanisms behind their toxic effects, which is crucial to minimizing the risks of toxigenic HABs to human and public health. To this end, this paper summarizes the effects of the five most common HAB toxins on fish, shellfish, and humans and discusses the possible mechanisms.

Harmful algal blooms are an increasing worldwide threat to the seafood industry and human health as a consequence of the natural production of biotoxins that can accumulate in shellfish. In the Argentine Sea, this has been identified as an issue for the offshore fisheries of Patagonian scallops (Zygochlamys patagonica), leading to potentially harmful effects on consumers. Here we assess spatial and temporal patterns in marine biotoxin concentrations in Patagonian scallops harvested in Argentinian waters between 2012–2017, based on analyses for paralytic shellfish toxins, lipophilic toxins, and amnesic shellfish toxins. There was no evidence for concentrations of lipophilic or amnesic toxins above regulatory acceptance thresholds, with trace concentrations of pectenotoxin 2, azaspiracid 2 and okadaic acid group toxins confirmed. Conversely, paralytic shellfish toxins were quantified in some scallops. Gonyautoxins 1 and 2 dominated the unusual toxin profiles (91%) in terms of saxitoxin equivalents with maximum concentrations reaching 3985 µg STX eq/kg and with changes in profiles linked in part to seasonal changes. Total toxin concentrations were compared between samples of the adductor muscle and whole tissue, with results showing the absence of toxins in the adductor muscle confirming toxin accumulation in the digestive tracts of the scallops and the absence of a human health threat following the processing of scallop adductor meat. These findings highlight that paralytic shellfish toxins with an unusual toxin profile can occur in relatively high concentrations in whole Patagonian scallops in specific regions and during particular time periods, also showing that the processing of scallops on board factory ships to obtain frozen adductor muscle is an effective management process that minimizes the risk of poisonings from final products destined for human consumption. [ABSTRACT FROM AUTHOR]

Harmful algal blooms are an increasing worldwide threat to the seafood industry and human health as a consequence of the natural production of biotoxins that can accumulate in shellfish. In the Argentine Sea, this has been identified as an issue for the offshore fisheries of Patagonian scallops (Zygochlamys patagonica), leading to potentially harmful effects on consumers. Here we assess spatial and temporal patterns in marine biotoxin concentrations in Patagonian scallops harvested in Argentinian waters between 2012–2017, based on analyses for paralytic shellfish toxins, lipophilic toxins, and amnesic shellfish toxins. There was no evidence for concentrations of lipophilic or amnesic toxins above regulatory acceptance thresholds, with trace concentrations of pectenotoxin 2, azaspiracid 2 and okadaic acid group toxins confirmed. Conversely, paralytic shellfish toxins were quantified in some scallops. Gonyautoxins 1 and 2 dominated the unusual toxin profiles (91%) in terms of saxitoxin equivalents with maximum concentrations reaching 3985 µg STX eq/kg and with changes in profiles linked in part to seasonal changes. Total toxin concentrations were compared between samples of the adductor muscle and whole tissue, with results showing the absence of toxins in the adductor muscle confirming toxin accumulation in the digestive tracts of the scallops and the absence of a human health threat following the processing of scallop adductor meat. These findings highlight that paralytic shellfish toxins with an unusual toxin profile can occur in relatively high concentrations in whole Patagonian scallops in specific regions and during particular time periods, also showing that the processing of scallops on board factory ships to obtain frozen adductor muscle is an effective management process that minimizes the risk of poisonings from final products destined for human consumption.

Domoic acid (DA), the toxin causing amnesic shellfish poisoning (ASP), is produced globally by some diatoms in the genus Pseudo-nitzschia. DA has been detected in several marine mammal species in the Alaskan Arctic, raising health concerns for marine mammals and subsistence communities dependent upon them. Gastrointestinal matrices are routinely used to detect Harmful Algal Bloom (HAB) toxin presence in marine mammals, yet DA stability has only been studied extensively in shellfish-related matrices. To address this knowledge gap, we quantified DA in bowhead whale fecal samples at multiple time points for two groups: (1) 50% methanol extracts from feces, and (2) raw feces stored in several conditions. DA concentrations decreased to 70 ± 7.1% of time zero (T0) in the 50% methanol extracts after 2 weeks, but remained steady until the final time point at 5 weeks (66 ± 5.7% T0). In contrast, DA concentrations were stable or increased in raw fecal material after 8 weeks of freezer storage (−20 °C), at room temperature (RT) in the dark, or refrigerated at 1 °C. DA concentrations in raw feces stored in an incubator (37 °C) or at RT in the light decreased to 77 ± 2.8% and 90 ± 15.0% T0 at 8 weeks, respectively. Evaporation during storage of raw fecal material is a likely cause of the increased DA concentrations observed over time with the highest increase to 126 ± 7.6% T0 after 3.2 years of frozen storage. These results provide valuable information for developing appropriate sample storage procedures for marine mammal fecal samples.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.

Diatoms are abundant and important biological components of the marine environment that biosynthesize diverse natural products. These microalgae are rich in various lipids, carotenoids, sterols and isoprenoids, some of them containing toxins and other metabolites. Several groups of diatom natural products have attracted great interest due to their potential practical application as energy sources (biofuel), valuable food constituents, and prospective materials for nanotechnology. In addition, hydrocarbons, which are used in climate reconstruction, polyamines which participate in biomineralization, new apoptotic agents against tumor cells, attractants and deterrents that regulate the biochemical communications between marine species in seawaters have also been isolated from diatoms. However, chemical studies on these microalgae are complicated by difficulties, connected with obtaining their biomass, and the influence of nutrients and contaminators in their environment as well as by seasonal and climatic factors on the biosynthesis of the corresponding natural products. Overall, the number of chemically studied diatoms is lower than that of other algae, but further studies, particularly those connected with improvements in the isolation and structure elucidation technique as well as the genomics of diatoms, promise both to increase the number of studied species with isolated biologically active natural products and to provide a clearer perception of their biosynthesis.

Diatoms of the genus Pseudo-nitzschia produce domoic acid (DA), a toxin that is vectored in the marine food web, thus causing serious problems for marine organisms and humans. In spite of this, knowledge of interactions between grazing zooplankton and diatoms is restricted. In this study, we examined the interactions between Calanus copepodites and toxin producing Pseudo-nitzschia. The copepodites were fed with different concentrations of toxic P. seriata and a strain of P. obtusa that previously was tested to be non-toxic. The ingestion rates did not differ among the diets (P. seriata, P. obtusa, a mixture of both species), and they accumulated 6%–16% of ingested DA (up to 420 µg per dry weight copepodite). When P. seriata was exposed to the copepodites, either through physical contact with the grazers or separated by a membrane, the toxicity of P. seriata increased (up to 3300%) suggesting the response to be chemically mediated. The induced response was also triggered when copepodites grazed on another diatom, supporting the hypothesis that the cues originate from the copepodite. Neither pH nor nutrient concentrations explained the induced DA production. Unexpectedly, P. obtusa also produced DA when exposed to grazing copepodites, thus representing the second reported toxic polar diatom.

Bacteria are known to influence domoic acid (DA) production by Pseudo-nitzschia spp., but the link between DA production and physiology of diatoms requires more investigation. We compared a toxic P. multiseries to a non-toxic P. delicatissima, investigating links between DA production, physiological parameters, and co-occurring bacteria. Bacterial communities in cultures of both species were reduced by antibiotic treatment, and each of the diatoms was inoculated with the bacterial community of the other species. The physiology of P. delicatissima was minimally affected by the absence of bacteria or the presence of alien bacteria, and no DA was detected. P. multiseries grew faster without bacteria, did not produce a significant amount of DA, and exhibited physiological characteristics of healthy cells. When grown with alien bacteria, P. multiseries did not grow and produced more DA; the physiology of these cells was affected, with decreases in chlorophyll content and photosynthetic efficiency, an increase in esterase activity, and almost 50% mortality of the cells. The alien bacterial community had morphological and cellular characteristics very different from the original bacteria, and the number of free-living bacteria per algal cell was much higher, suggesting the involvement of bacteria in DA production.

Domoic acid epileptic disease is characterized by spontaneous recurrent seizures weeks to months after domoic acid exposure. The potential for this disease was first recognized in a human case study of temporal lobe epilepsy after the 1987 amnesic shellfish-poisoning event in Quebec, and was characterized as a chronic epileptic syndrome in California sea lions through investigation of a series of domoic acid poisoning cases between 1998 and 2006. The sea lion study provided a breadth of insight into clinical presentations, unusual behaviors, brain pathology, and epidemiology. A rat model that replicates key observations of the chronic epileptic syndrome in sea lions has been applied to identify the progression of the epileptic disease state, its relationship to behavioral manifestations, and to define the neural systems involved in these behavioral disorders. Here, we present the concept of domoic acid epileptic disease as a delayed manifestation of domoic acid poisoning and review the state of knowledge for this disease state in affected humans and sea lions. We discuss causative mechanisms and neural underpinnings of disease maturation revealed by the rat model to present the concept for olfactory origin of an epileptic disease; triggered in dendodendritic synapases of the olfactory bulb and maturing in the olfactory cortex. We conclude with updated information on populations at risk, medical diagnosis, treatment, and prognosis.

Currently, animal experiments in rodents are the gold standard for developmental neurotoxicity (DNT) investigations; however, testing guidelines for these experiments are insufficient in terms of animal use, time, and costs. Thus, alternative reliable approaches are needed for predicting DNT. We chose rat neural stem cells (rNSC) as a model system, and used a well-known neurotoxin, domoic acid (DA), as a model test chemical to validate the assay. This assay was used to investigate the potential neurotoxic effects of Ochratoxin A (OTA), of which the main target organ is the kidney. However, limited information is available regarding its neurotoxic effects. The effects of DA and OTA on the cytotoxicity and on the degree of differentiation of rat rNSC into astrocytes, neurons, and oligodendrocytes were monitored using cell-specific immunofluorescence staining for undifferentiated rNSC (nestin), neurospheres (nestin and A2B5), neurons (MAP2 clone M13, MAP2 clone AP18, and Doublecortin), astrocytes (GFAP), and oligodendrocytes (A2B5 and mGalc). In the absence of any chemical exposure, approximately 46% of rNSC differentiated into astrocytes and neurons, while 40% of the rNSC differentiated into oligodendrocytes. Both non-cytotoxic and cytotoxic concentrations of DA and OTA reduced the differentiation of rNSC into astrocytes, neurons, and oligodendrocytes. Furthermore, a non-cytotoxic nanomolar (0.05 µM) concentration of DA and 0.2 µM of OTA reduced the percentage differentiation of rNSC into astrocytes and neurons. Morphometric analysis showed that the highest concentration (10 μM) of DA reduced axonal length. These indicate that low, non-cytotoxic concentrations of DA and OTA can interfere with the differentiation of rNSC.

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (μ = 0.45-0.73 d-1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species. [ABSTRACT FROM AUTHOR]

Phycotoxins, which are produced by harmful microalgae and bioaccumulate in the marine food web, are of growing concern for Australia. These harmful algae pose a threat to ecosystem and human health, as well as constraining the progress of aquaculture, one of the fastest growing food sectors in the world. With better monitoring, advanced analytical skills and an increase in microalgal expertise, many phycotoxins have been identified in Australian coastal waters in recent years. The most concerning of these toxins are ciguatoxin, paralytic shellfish toxins, okadaic acid and domoic acid, with palytoxin and karlotoxin increasing in significance. The potential for tetrodotoxin, maitotoxin and palytoxin to contaminate seafood is also of concern, warranting future investigation. The largest and most significant toxic bloom in Tasmania in 2012 resulted in an estimated total economic loss of ~AUD$23M, indicating that there is an imperative to improve toxin and organism detection methods, clarify the toxin profiles of species of phytoplankton and carry out both intra- and inter-species toxicity comparisons. Future work also includes the application of rapid, real-time molecular assays for the detection of harmful species and toxin genes. This information, in conjunction with a better understanding of the life histories and ecology of harmful bloom species, may lead to more appropriate management of environmental, health and economic resources. [ABSTRACT FROM AUTHOR]

Because domoic acid, a neurotoxic secondary metabolite produced by marine diatoms in the genus Pseudo-nitzschia, is hypothesized to be part of a high affinity iron uptake system, we investigated whether domoic acid could improve the competitive ability of Pseudo-nitzschia delicatissima, and whether the availability of iron changed the outcome of competition experiments. We found that domoic acid had a slight negative effect on growth of the diatom Skeletonema marinoi when it was grown in monocultures. However, when S. marinoi was cultured with P. delicatissima the presence of domoic acid resulted in a reduction of S. marinoi cells by up to 38% and an increase in P. delicatissima cell numbers by up to 17% under iron replete conditions. Similar effects were not observed in low iron treatments. Domoic acid was not taken up by P. delicatissima cells. Overall, our results indicate that domoic acid can improve the competitive ability of Pseudo-nitzschia spp. and that iron is likely to be involved. This study provides an unusual example of indirect inhibition of competitor growth mediated by a secondary metabolite.

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.

Neurotoxicity induced in fish by domoic acid (DA) was assessed with respect to occurrence of neurotoxic signs, lethality, and histopathology by light microscopy. Sparus aurata were exposed to a single dose of DA by intraperitoneal (i.p.) injection of 0, 0.45, 0.9, and 9.0 mg DA kg−1 bw. Mortality (66.67 ± 16.67%) was only observed in dose of 9.0 mg kg−1 bw. Signs of neurological toxicity were detected for the doses of 0.9 and 9.0 mg DA kg−1 bw. Furthermore, the mean concentrations (±SD) of DA detected by HPLC-UV in extracts of brain after exposure to 9.0 mg DA kg−1 bw were 0.61 ± 0.01, 0.96 ± 0.00, and 0.36 ± 0.01 mg DA kg−1 tissue at 1, 2, and 4 hours. The lack of major permanent brain damage in S. aurata, and reversibility of neurotoxic signs, suggest that lower susceptibility to DA or neuronal recovery occurs in affected individuals.

Domoic acid produced by marine algae has been shown to cause acute and chronic neurologic sequelae in Californian sea lions following acute or low-dose exposure. Histological findings in affected animals included a degenerative cardiomyopathy that was hypothesized to be caused by over-excitation of the glutamate receptors (GluRs) speculated to be present in the sea lion heart. Thus tissues from five sea lions without lesions associated with domoic acid toxicity and one animal with domoic acid-induced chronic neurologic sequelae and degenerative cardiomyopathy were examined for the presence of GluRs. Immunohistochemistry localized mGluR 2/3, mGluR 5, GluR 2/3 and NMDAR 1 in structures of the conducting system and blood vessels. NMDAR 1 and GluR 2/3 were the most widespread as immunoreactivity was observed within sea lion conducting system structures. PCR analysis, cloning and subsequent sequencing of the seal lion GluRs showed only 80% homology to those from rats, but more than 95% homologous to those from dogs. The cellular distribution and expression of subtypes of GluRs in the sea lion hearts suggests that exposure to domoic acid may induce cardiac damage and functional disturbances.

The immunotoxic potential of domoic acid (DA), a well-characterized neurotoxin, has not been fully investigated. Phagocytosis and lymphocyte proliferation were evaluated following in vitro and in vivo exposure to assay direct vs indirect effects. Mice were injected intraperitoneally with a single dose of DA (2.5 µg/g b.w.) and sampled after 12, 24, or 48 hr. In a separate experiment, leukocytes and splenocytes were exposed in vitro to 0, 1, 10, or 100 µM DA. In vivo exposure resulted in a significant increase in monocyte phagocytosis (12-hr), a significant decrease in neutrophil phagocytosis (24-hr), a significant decrease in monocyte phagocytosis (48-hr), and a significant reduction in T-cell mitogen-induced lymphocyte proliferation (24-hr). In vitro exposure significantly reduced neutrophil and monocyte phagocytosis at 1 µM. B- and T-cell mitogen-induced lymphocyte proliferation were both significantly increased at 1 and 10 µM, and significantly decreased at 100 µM. Differences between in vitro and in vivo results suggest that DA may exert its immunotoxic effects both directly and indirectly. Modulation of cytosolic calcium suggests that DA exerts its effects through ionotropic glutamate subtype surface receptors at least on monocytes. This study is the first to identify DA as an immunotoxic chemical in a mammalian species.

California sea lions have been a repeated subject of investigation for early life toxicity, which has been documented to occur with increasing frequency from late February through mid-May in association with organochlorine (PCB and DDT) poisoning and infectious disease in the 1970's and domoic acid poisoning in the last decade. The mass early life mortality events result from the concentrated breeding grounds and synchronization of reproduction over a 28 day post partum estrus cycle and 11 month in utero phase. This physiological synchronization is triggered by a decreasing photoperiod of 11.48 h/day that occurs approximately 90 days after conception at the major California breeding grounds. The photoperiod trigger activates implantation of embryos to proceed with development for the next 242 days until birth. Embryonic diapause is a selectable trait thought to optimize timing for food utilization and male migratory patterns; yet from the toxicological perspective presented here also serves to synchronize developmental toxicity of pulsed environmental events such as domoic acid poisoning. Research studies in laboratory animals have defined age-dependent neurotoxic effects during development and windows of susceptibility to domoic acid exposure. This review will evaluate experimental domoic acid neurotoxicity in developing rodents and, aided by comparative allometric projections, will analyze potential prenatal toxicity and exposure susceptibility in the California sea lion. This analysis should provide a useful tool to forecast fetal toxicity and understand the impact of fetal toxicity on adult disease of the California sea lion.

Domoic acid was identified as the toxin responsible for an outbreak of human poisoning that occurred in Canada in 1987 following consumption of contaminated blue mussels [Mytilus edulis]. The poisoning was characterized by a constellation of clinical symptoms and signs. Among the most prominent features described was memory impairment which led to the name Amnesic Shellfish Poisoning [ASP]. Domoic acid is produced by certain marine organisms, such as the red alga Chondria armata and planktonic diatom of the genus Pseudo-nitzschia. Since 1987, monitoring programs have been successful in preventing other human incidents of ASP. However, there are documented cases of domoic acid intoxication in wild animals and outbreaks of coastal water contamination in many regions world-wide. Hence domoic acid continues to pose a global risk to the health and safety of humans and wildlife. Several mechanisms have been implicated as mediators for the effects of domoic acid. Of particular importance is the role played by glutamate receptors as mediators of excitatory neurotransmission and the demonstration of a wide distribution of these receptors outside the central nervous system, prompting the attention to other tissues as potential target sites. The aim of this document is to provide a comprehensive review of ASP, DOM induced pathology including ultrastructural changes associated to subchronic oral exposure, and discussion of key proposed mechanisms of cell/tissue injury involved in DOM induced brain pathology and considerations relevant to food safety and human health.

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1mM] stimulated a statistically significant increase in TNF-α mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF- β1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-α (TNF-α) and matrix metalloproteinases-9 (MMP-9), as well as the anti- inflammatory cytokine transforming growth factor β1 (TGF-β1).

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (μ = 0.45–0.73 d−1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2–4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50–70% reduction in cell density and 70–90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species.

Phycotoxins, which are produced by harmful microalgae and bioaccumulate in the marine food web, are of growing concern for Australia. These harmful algae pose a threat to ecosystem and human health, as well as constraining the progress of aquaculture, one of the fastest growing food sectors in the world. With better monitoring, advanced analytical skills and an increase in microalgal expertise, many phycotoxins have been identified in Australian coastal waters in recent years. The most concerning of these toxins are ciguatoxin, paralytic shellfish toxins, okadaic acid and domoic acid, with palytoxin and karlotoxin increasing in significance. The potential for tetrodotoxin, maitotoxin and palytoxin to contaminate seafood is also of concern, warranting future investigation. The largest and most significant toxic bloom in Tasmania in 2012 resulted in an estimated total economic loss of~AUD$23M, indicating that there is an imperative to improve toxin and organism detection methods, clarify the toxin profiles of species of phytoplankton and carry out both intra‐ and inter‐species toxicity comparisons. Future work also includes the application of rapid, real‐time molecular assays for the detection of harmful species and toxin genes. This information, in conjunction with a better understanding of the life histories and ecology of harmful bloom species, may lead to more appropriate management of environmental, health and economic resources.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment. [ABSTRACT FROM AUTHOR]

Diatoms of the genus Pseudo-nitzschia produce domoic acid (DA), a toxin that is vectored in the marine food web, thus causing serious problems for marine organisms and humans. In spite of this, knowledge of interactions between grazing zooplankton and diatoms is restricted. In this study, we examined the interactions between Calanus copepodites and toxin producing Pseudo-nitzschia. The copepodites were fed with different concentrations of toxic P. seriata and a strain of P. obtusa that previously was tested to be non-toxic. The ingestion rates did not differ among the diets (P. seriata, P. obtusa, a mixture of both species), and they accumulated 6%-16% of ingested DA (up to 420 µg per dry weight copepodite). When P. seriata was exposed to the copepodites, either through physical contact with the grazers or separated by a membrane, the toxicity of P. seriata increased (up to 3300%) suggesting the response to be chemically mediated. The induced response was also triggered when copepodites grazed on another diatom, supporting the hypothesis that the cues originate from the copepodite. Neither pH nor nutrient concentrations explained the induced DA production. Unexpectedly, P. obtusa also produced DA when exposed to grazing copepodites, thus representing the second reported toxic polar diatom. [ABSTRACT FROM AUTHOR]

Diatoms are abundant and important biological components of the marine environment that biosynthesize diverse natural products. These microalgae are rich in various lipids, carotenoids, sterols and isoprenoids, some of them containing toxins and other metabolites. Several groups of diatom natural products have attracted great interest due to their potential practical application as energy sources (biofuel), valuable food constituents, and prospective materials for nanotechnology. In addition, hydrocarbons, which are used in climate reconstruction, polyamines which participate in biomineralization, new apoptotic agents against tumor cells, attractants and deterrents that regulate the biochemical communications between marine species in seawaters have also been isolated from diatoms. However, chemical studies on these microalgae are complicated by difficulties, connected with obtaining their biomass, and the influence of nutrients and contaminators in their environment as well as by seasonal and climatic factors on the biosynthesis of the corresponding natural products. Overall, the number of chemically studied diatoms is lower than that of other algae, but further studies, particularly those connected with improvements in the isolation and structure elucidation technique as well as the genomics of diatoms, promise both to increase the number of studied species with isolated biologically active natural products and to provide a clearer perception of their biosynthesis. [ABSTRACT FROM AUTHOR]

Domoic acid epileptic disease is characterized by spontaneous recurrent seizures weeks to months after domoic acid exposure. The potential for this disease was first recognized in a human case study of temporal lobe epilepsy after the 1987 amnesic shellfish-poisoning event in Quebec, and was characterized as a chronic epileptic syndrome in California sea lions through investigation of a series of domoic acid poisoning cases between 1998 and 2006. The sea lion study provided a breadth of insight into clinical presentations, unusual behaviors, brain pathology, and epidemiology. A rat model that replicates key observations of the chronic epileptic syndrome in sea lions has been applied to identify the progression of the epileptic disease state, its relationship to behavioral manifestations, and to define the neural systems involved in these behavioral disorders. Here, we present the concept of domoic acid epileptic disease as a delayed manifestation of domoic acid poisoning and review the state of knowledge for this disease state in affected humans and sea lions. We discuss causative mechanisms and neural underpinnings of disease maturation revealed by the rat model to present the concept for olfactory origin of an epileptic disease; triggered in dendodendritic synapases of the olfactory bulb and maturing in the olfactory cortex. We conclude with updated information on populations at risk, medical diagnosis, treatment, and prognosis. [ABSTRACT FROM AUTHOR]

Because domoic acid, a neurotoxic secondary metabolite produced by marine diatoms in the genus Pseudo-nitzschia, is hypothesized to be part of a high affinity iron uptake system, we investigated whether domoic acid could improve the competitive ability of Pseudo-nitzschia delicatissima, and whether the availability of iron changed the outcome of competition experiments. We found that domoic acid had a slight negative effect on growth of the diatom Skeletonema marinoi when it was grown in monocultures. However, when S. marinoi was cultured with P. delicatissima the presence of domoic acid resulted in a reduction of S. marinoi cells by up to 38% and an increase in P. delicatissima cell numbers by up to 17% under iron replete conditions. Similar effects were not observed in low iron treatments. Domoic acid was not taken up by P. delicatissima cells. Overall, our results indicate that domoic acid can improve the competitive ability of Pseudo-nitzschia spp. and that iron is likely to be involved. This study provides an unusual example of indirect inhibition of competitor growth mediated by a secondary metabolite. [ABSTRACT FROM AUTHOR]

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins. [ABSTRACT FROM AUTHOR]

Neurotoxicity induced in fish by domoic acid (DA) was assessed with respect to occurrence of neurotoxic signs, lethality, and histopathology by light microscopy. Sparus aurata were exposed to a single dose of DA by intraperitoneal (i.p.) injection of 0, 0.45, 0.9, and 9.0 mg DA kg-1 bw. Mortality (66.67 ± 16.67%) was only observed in dose of 9.0 mg kg-1 bw. Signs of neurological toxicity were detected for the doses of 0.9 and 9.0 mg DA kg-1 bw. Furthermore, the mean concentrations (±SD) of DA detected by HPLC-UV in extracts of brain after exposure to 9.0 mg DA kg-1 bw were 0.61 ± 0.01, 0.96 ± 0.00, and 0.36 ± 0.01 mg DA kg-1 tissue at 1, 2, and 4 hours. The lack of major permanent brain damage in S. aurata, and reversibility of neurotoxic signs, suggest that lower susceptibility to DA or neuronal recovery occurs in affected individuals. [ABSTRACT FROM AUTHOR]

Domoic acid produced by marine algae has been shown to cause acute and chronic neurologic sequelae in Californian sea lions following acute or low-dose exposure. Histological findings in affected animals included a degenerative cardiomyopathy that was hypothesized to be caused by over-excitation of the glutamate receptors (GluRs) speculated to be present in the sea lion heart. Thus tissues from five sea lions without lesions associated with domoic acid toxicity and one animal with domoic acid-induced chronic neurologic sequelae and degenerative cardiomyopathy were examined for the presence of GluRs. Immunohistochemistry localized mGluR 2/3, mGluR 5, GluR 2/3 and NMDAR 1 in structures of the conducting system and blood vessels. NMDAR 1 and GluR 2/3 were the most widespread as immunoreactivity was observed within sea lion conducting system structures. PCR analysis, cloning and subsequent sequencing of the seal lion GluRs showed only 80% homology to those from rats, but more than 95% homologous to those from dogs. The cellular distribution and expression of subtypes of GluRs in the sea lion hearts suggests that exposure to domoic acid may induce cardiac damage and functional disturbances. [ABSTRACT FROM AUTHOR]

Domoic acid (DA), the toxin causing amnesic shellfish poisoning (ASP), is produced globally by some diatoms in the genus Pseudo-nitzschia. DA has been detected in several marine mammal species in the Alaskan Arctic, raising health concerns for marine mammals and subsistence communities dependent upon them. Gastrointestinal matrices are routinely used to detect Harmful Algal Bloom (HAB) toxin presence in marine mammals, yet DA stability has only been studied extensively in shellfish-related matrices. To address this knowledge gap, we quantified DA in bowhead whale fecal samples at multiple time points for two groups: (1) 50% methanol extracts from feces, and (2) raw feces stored in several conditions. DA concentrations decreased to 70 ± 7.1% of time zero (T0) in the 50% methanol extracts after 2 weeks, but remained steady until the final time point at 5 weeks (66 ± 5.7% T0). In contrast, DA concentrations were stable or increased in raw fecal material after 8 weeks of freezer storage (−20 °C), at room temperature (RT) in the dark, or refrigerated at 1 °C. DA concentrations in raw feces stored in an incubator (37 °C) or at RT in the light decreased to 77 ± 2.8% and 90 ± 15.0% T0 at 8 weeks, respectively. Evaporation during storage of raw fecal material is a likely cause of the increased DA concentrations observed over time with the highest increase to 126 ± 7.6% T0 after 3.2 years of frozen storage. These results provide valuable information for developing appropriate sample storage procedures for marine mammal fecal samples. [ABSTRACT FROM AUTHOR]

Currently, animal experiments in rodents are the gold standard for developmental neurotoxicity (DNT) investigations; however, testing guidelines for these experiments are insufficient in terms of animal use, time, and costs. Thus, alternative reliable approaches are needed for predicting DNT. We chose rat neural stem cells (rNSC) as a model system, and used a well-known neurotoxin, domoic acid (DA), as a model test chemical to validate the assay. This assay was used to investigate the potential neurotoxic effects of Ochratoxin A (OTA), of which the main target organ is the kidney. However, limited information is available regarding its neurotoxic effects. The effects of DA and OTA on the cytotoxicity and on the degree of differentiation of rat rNSC into astrocytes, neurons, and oligodendrocytes were monitored using cell-specific immunofluorescence staining for undifferentiated rNSC (nestin), neurospheres (nestin and A2B5), neurons (MAP2 clone M13, MAP2 clone AP18, and Doublecortin), astrocytes (GFAP), and oligodendrocytes (A2B5 and mGalc). In the absence of any chemical exposure, approximately 46% of rNSC differentiated into astrocytes and neurons, while 40% of the rNSC differentiated into oligodendrocytes. Both non-cytotoxic and cytotoxic concentrations of DA and OTA reduced the differentiation of rNSC into astrocytes, neurons, and oligodendrocytes. Furthermore, a non-cytotoxic nanomolar (0.05 µM) concentration of DA and 0.2 µM of OTA reduced the percentage differentiation of rNSC into astrocytes and neurons. Morphometric analysis showed that the highest concentration (10 μM) of DA reduced axonal length. These indicate that low, non-cytotoxic concentrations of DA and OTA can interfere with the differentiation of rNSC. [ABSTRACT FROM AUTHOR]

California sea lions have been a repeated subject of investigation for early life toxicity, which has been documented to occur with increasing frequency from late February through mid-May in association with organochlorine (PCB and DDT) poisoning and infectious disease in the 1970's and domoic acid poisoning in the last decade. The mass early life mortality events result from the concentrated breeding grounds and synchronization of reproduction over a 28 day post partum estrus cycle and 11 month in utero phase. This physiological synchronization is triggered by a decreasing photoperiod of 11.48 h/day that occurs approximately 90 days after conception at the major California breeding grounds. The photoperiod trigger activates implantation of embryos to proceed with development for the next 242 days until birth. Embryonic diapause is a selectable trait thought to optimize timing for food utilization and male migratory patterns; yet from the toxicological perspective presented here also serves to synchronize developmental toxicity of pulsed environmental events such as domoic acid poisoning. Research studies in laboratory animals have defined age-dependent neurotoxic effects during development and windows of susceptibility to domoic acid exposure. This review will evaluate experimental domoic acid neurotoxicity in developing rodents and, aided by comparative allometric projections, will analyze potential prenatal toxicity and exposure susceptibility in the California sea lion. This analysis should provide a useful tool to forecast fetal toxicity and understand the impact of fetal toxicity on adult disease of the California sea lion.

Domoic acid was identified as the toxin responsible for an outbreak of human poisoning that occurred in Canada in 1987 following consumption of contaminated blue mussels [Mytilus edulis]. The poisoning was characterized by a constellation of clinical symptoms and signs. Among the most prominent features described was memory impairment which led to the name Amnesic Shellfish Poisoning [ASP]. Domoic acid is produced by certain marine organisms, such as the red alga Chondria armata and planktonic diatom of the genus Pseudo-nitzschia. Since 1987, monitoring programs have been successful in preventing other human incidents of ASP. However, there are documented cases of domoic acid intoxication in wild animals and outbreaks of coastal water contamination in many regions world-wide. Hence domoic acid continues to pose a global risk to the health and safety of humans and wildlife. Several mechanisms have been implicated as mediators for the effects of domoic acid. Of particular importance is the role played by glutamate receptors as mediators of excitatory neurotransmission and the demonstration of a wide distribution of these receptors outside the central nervous system, prompting the attention to other tissues as potential target sites. The aim of this document is to provide a comprehensive review of ASP, DOM induced pathology including ultrastructural changes associated to subchronic oral exposure, and discussion of key proposed mechanisms of cell/tissue injury involved in DOM induced brain pathology and considerations relevant to food safety and human health.

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-alpha, IL-Beta, IL-6, the small inducible cytokine, interferon protein 10P-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all down-regulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity.

The immunotoxic potential of domoic acid (DA), a well-characterized neurotoxin, has not been fully investigated. Phagocytosis and lymphocyte proliferation were evaluated following in vitro and in vivo exposure to assay direct vs indirect effects. Mice were injected intraperitoneally with a single dose of DA (2.5 microg/g b.w.) and sampled after 12, 24, or 48 hr. In a separate experiment, leukocytes and splenocytes were exposed in vitro to 0, 1, 10, or 100 microM DA. In vivo exposure resulted in a significant increase in monocyte phagocytosis (12-hr), a significant decrease in neutrophil phagocytosis (24-hr), a significant decrease in monocyte phagocytosis (48-hr), and a significant reduction in T-cell mitogen-induced lymphocyte proliferation (24-hr). In vitro exposure significantly reduced neutrophil and monocyte phagocytosis at 1 muM. B- and T-cell mitogen-induced lymphocyte proliferation were both significantly increased at 1 and 10 microM, and significantly decreased at 100 microM. Differences between in vitro and in vivo results suggest that DA may exert its immunotoxic effects both directly and indirectly. Modulation of cytosolic calcium suggests that DA exerts its effects through ionotropic glutamate subtype surface receptors at least on monocytes. This study is the first to identify DA as an immunotoxic chemical in a mammalian species.

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).