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Saxitoxin (STX) represents a marine toxin of significant concern due to its deleterious implications for aquatic ecosystems and public food safety. As a potent paralytic agent, the role of STX in obstructing voltage-gated sodium channels (VGSCs) is well-characterized. Yet, the mechanistic details underlying its low-dose toxicity remain largely enigmatic. In the current study, zebrafish embryos and larvae were subjected to subchronic exposure of graded STX concentrations (0, 1, 10, and 100 µg/L) until the 7th day post-fertilization. A tactile stimulus-based assay was employed to evaluate potential behavioral perturbations resulting from STX exposure. Both behavioral and transcription level analyses unveiled a compromised tactile response, which was found to be associated with a notable upregulation in the mRNA of two distinct VGSC isoforms, specifically the scn8aa/ab and scn1Laa/ab transcripts, even at the minimal STX dose. Notably, exposure to this lowest STX concentration also resulted in alterations in the transcriptional patterns of pivotal genes for cholinergic and GABAergic pathways, including ache and gabra1. Furthermore, STX induced a marked decrease in the levels of the neurotransmitter GABA. Our findings underscore that prolonged low-dose STX exposure during early development can significantly compromise the tactile response behavior in zebrafish. This study reveals that chronic low-dose STX exposure of developing zebrafish alters neurotransmission pathways that converge on altered tactile behavior.

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Alexandrium tamiyavanichii is a marine dinoflagellate known to produce Paralytic Shellfish Poisoning (PSP) toxin. Thus, a strain was isolated from La Paz Bay, Baja California Sur, Mexico and used to explore whether stress conditions, such as phosphorus limitation (PL) and nitrogen enrichment (NE) modulate population growth and PSP toxin production in the GSe medium. Growth kinetics showed that the PL treatment produced a 3.4-fold increase in cell density versus control at day 30 of the culture cycle. The highest PSP concentration was found in the control culture (309 fmol cell-1) on day 21. Saxitoxin (STX) was the main analog in all the treatments (> 40 % mol). In conclusion, PL and NE treatments promoted growth kinetics in the species studied but did not affect the PSP toxin production. For the first time, the present research describes A. tamiyavanichii high toxicity strain isolated from Mexican coasts relative to the South-Atlantic strains.

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Harmful algal blooms (HABs) can produce a variety of noxious effects and, in some cases, the massive mortality of wild and farmed marine organisms. Some HAB species produce toxins that are released into seawater or transferred via food webs (particulate toxin fraction). The objective of the present study was to identify the toxicological effects of subacute exposure to saxitoxin (STX) during embryonic and early larval stages in Seriola rivoliana. Eggs were exposed to dissolved 19 STX (100 µg L-1). The toxic effects of STX were evaluated via the hatching percentage, the activity of three enzymes (protein and alkaline phosphatases and peroxidase), and the expression of four genes (HSF2, Nav1.4b, PPRC1, and DUSP8). A low hatching percentage (less than 5%) was observed in 44 hpf (hours post fertilization) embryos exposed to STX compared to 71% in the unexposed control. At this STX concentration, no oxidative stress in the embryos was evident. However, STX induced the expression of the NaV1.4 channel α-subunit (NaV1.4b), which is the primary target of this toxin. Our results revealed the overexpression of all four candidate genes in STX-intoxicated lecithotrophic larvae, reflecting the activation of diverse cellular processes involved in stress responses (HSF2), lipid metabolism (PPRC1), and MAP kinase signaling pathways associated with cell proliferation and differentiation (DUSP8). The effects of STX were more pronounced in young larvae than in embryos, indicating a stage-specific sensitivity to the toxin.

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The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1-0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

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In September and November 2016, eight marine sampling sites along the coast of the southeastern Gulf of Mexico were monitored for the presence of lipophilic and hydrophilic toxins. Water temperature, salinity, hydrogen potential, dissolved oxygen saturation, inorganic nutrients and phytoplankton abundance were also determined. Two samples filtered through glass fiber filters were used for the extraction and analysis of paralytic shellfish toxins (PSTs) by lateral flow immunochromatography (IFL), HPLC with post-column oxidation and fluorescent detection (FLD) and UHPLC coupled to tandem mass spectrometry (UHPLC-MS/MS). Elevated nutrient contents were associated with the sites of rainwater discharge or those near anthropogenic activities. A predominance of the dinoflagellate Pyrodinium bahamense was found with abundances of up to 104 cells L-1. Identification of the dinoflagellate was corroborated by light and scanning electron microscopy. Samples for toxins were positive by IFL, and the analogs NeoSTX and STX were identified and quantified by HPLC-FLD and UHPLC-MS/MS, with a total PST concentration of 6.5 pg cell-1. This study is the first report that confirms the presence of PSTs in P. bahamense in Mexican waters of the Gulf of Mexico.

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Marine phycotoxins are organic compounds synthesized by some species of microalgae, which accumulate in the tissues of filter-feeder organisms such as bivalve mollusks. These toxins can cause acute intoxication episodes in humans, a severe threat to aquaculture and fisheries. In the State of Pará, Brazil, oyster farming has community, artisanal and sustainable bases, using mangroves as cultivation environment and seed banks. In small-scale production, there are often no established methods of safeguarding the health of consumers elevating the potential risks of shellfish poisoning outbreaks. Our study evaluated the presence of phycotoxins in oysters cultivated in five municipalities in the region of the Atlantic Amazon (Pará, Brazil) assessing the quality of the final product. We further evaluated the microalgae, water quality, and the spatio-temporal variation of physicochemical factors in the same area. Diatoms dominated the microalgae composition, followed by dinoflagellates, some of which are reported to be potentially toxic and producers of paralytic shellfish toxins. For the first time, we describe the occurrence of the potentially toxic dinoflagellate Ostreopsis sp. in the Amazon region. Furthermore, for the first time, toxins were detected in oyster farming in the northeast of the State of Pará, namely GTX2,3, STX, and dc-STX nevertheless, with nontoxic values. The identified toxins represent a potential threat to shellfish consumers.

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Saxitoxin (STX) group toxins consist of a set of analogues which are produced by harmful algal blooms (HABs). During a HAB, filter-feeding marine organisms accumulate the dinoflagellates and concentrate the toxins in the tissues. In this study, we analyze the changes in antioxidant enzymes and oxidative damage in the bivalves Mytilus chilensis and Ameghinomya antiqua, and the gastropod Concholepas concholepas during a bloom of Alexandrium pacificum. The results show that during the exponential phase of the bloom bivalves show an increase in toxicity and activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathinoe reductase, p < 0.05), while in the gastropods, increased activity of antioxidant enzymes was associated with the bioaccumulation of toxins through the diet. At the end of the bloom, decreased activity of antioxidant enzymes in the visceral and non-visceral tissues was detected in the bivalves, with an increase in oxidative damage (p < 0.05), in which the latter is correlated with the detection of the most toxic analogues of the STX-group (r = 0.988). In conclusion, in areas with high incidence of blooms, shellfish show a high activity of antioxidants, however, during the stages involving the distribution and bioconversion of toxins, there is decreased activity of antioxidant enzymes resulting in oxidative damage.

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Toxic cyanobacteria blooms are a frequent problem in subtropical reservoirs and freshwater systems. The purpose of this study was to investigate the occurrence of potentially toxic cyanobacteria and the environmental conditions associated with the presence of cyanotoxins in a Brazilian subtropical reservoir. Five collections were carried out at seven sampling locations in the reservoir, during the rainy and dry seasons, between the years 2016 and 2017. There was permanent occurrence of Raphidiopsis raciborskii (Woloszynska) Aguilera, Berrendero Gómez, Kastovsky, Echenique & Salerno (Phycologia 57(2):130-146, 2018), ranging between dominant and abundant, with an average biomass of 38.8 ± 29.9 mg L-1. Also abundant were Dolichospermum solitarium, D. planctonicum, Planktothrix isothrix, and Aphanizomenon gracile. Saxitoxin (STX) was detected in all the collected samples (0.11 ± 0.05 µg L-1). Microcystin (MC) was also detected, but at lower concentrations (0.01 ± 0.0 µg L-1). Low availability of NO3- and phosphorus limitation had significant effects on the R. raciborskii biomass and the levels of STX and MC. It was observed that R. raciborskii was sensitive to thermal stratification, at the same time that STX levels were higher. This suggested that STX was produced under conditions that restricted the growth of R. raciborskii. These are important findings, because they add information about the permanent occurrence of STX and R. raciborskii in an aquatic ecosystem limited by phosphorus, vulnerable to climatic variations, and polluted by domestic effluents.

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Saxitoxin is a cyanotoxin which is very harmful to human health; the concentration limit in drinking water is only 3 µg/L. Therefore, a simple, fast, sensitive, low-cost, and specific method for its detection, quantification, and monitoring in water bodies is needed to avoid adverse effects on animal and human health. In this work, we developed an electrochemical impedimetric biosensor using a specific aptamer as recognition element for saxitoxin detection. This method allies the superior sensing characteristics of aptamers with the nondestructive, label-free, and easy working principles of the electrochemical impedance technique. The device presented sensitivity for detecting saxitoxin concentrations above 0.3 µg/L, with high selectivity in negative control experiments, demonstrating a promising alternative for water toxin detection.

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We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

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