Flow cytometric detection of saxitoxins using fluorescent voltage-sensitive dyes

By virtue of their ability to block depolarization of nerve cells, the saxitoxins exert the toxic effects associated with paralytic shellfish poisoning and allow for their detection through various methodologies. When veratridine-induced depolarization is followed using voltage-sensitive fluorescent dyes, the presence of these toxic blocking agents can be observed as a decrease in fluorescence of dye-treated nerve cells. Detection using flow cytometry provides for selection of the most responsive population of cultured mouse neuroblastoma (Neuro 2a) cells thereby enhancing assay sensitivity and this approach can be accomplished in real time. The method is demonstrated in preliminary studies using saxitoxin and crude shellfish extracts.     

Cyanotoxins in inland lakes of the United States: Occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007

A large nation-wide survey of cyanotoxins (1161 lakes) in the United States (U.S.) was conducted during the EPA National Lakes Assessment 2007. Cyanotoxin data were compared with cyanobacteria abundance- and chlorophyll-based World Health Organization (WHO) thresholds and mouse toxicity data to evaluate potential recreational risks. Cylindrospermopsins, microcystins, and saxitoxins were detected (ELISA) in 4.0, 32, and 7.7% of samples with mean concentrations of 0.56, 3.0, and 0.061 μg/L, respectively (detections only). Co-occurrence of the three cyanotoxin classes was rare (0.32%) when at least one toxin was detected. Cyanobacteria were present and dominant in 98 and 76% of samples, respectively. Potential anatoxin-, cylindrospermopsin-, microcystin-, and saxitoxin-producing cyanobacteria occurred in 81, 67, 95, and 79% of samples, respectively. Anatoxin-a and nodularin-R were detected (LC/MS/MS) in 15 and 3.7% samples (n = 27). The WHO moderate and high risk thresholds for microcystins, cyanobacteria abundance, and total chlorophyll were exceeded in 1.1, 27, and 44% of samples, respectively. Complete agreement by all three WHO microcystin metrics occurred in 27% of samples. This suggests that WHO microcystin metrics based on total chlorophyll and cyanobacterial abundance can overestimate microcystin risk when compared to WHO microcystin thresholds. The lack of parity among the WHO thresholds was expected since chlorophyll is common amongst all phytoplankton and not all cyanobacteria produce microcystins.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Oxidative stress in rats induced by consumption of saxitoxin contaminated drink water

Saxitoxins (STXs) are neurotoxins produced by cyanobacteria such as Cylindrospermopsis raciborskii. During bloom events, the production of these compounds causes contamination on public water supply sources. STXs block voltage gated sodium channels and can lead to severe poisoning and death of organisms at different trophic levels. Other toxicity mechanism of STX is the generation of reactive oxygen species (ROS). The aim of this study was to investigate the effect of consumption of water contaminated with a C. raciborskii strain (producing variants of Neo-STX and STX) by rats during 30 days through the analysis of oxidative stress biochemical parameters. Total antioxidant capacity (ACAP) and oxidative stress parameters were analyzed at pre-frontal cortex, hippocampus and liver of adult Wistar rats (2–3 months old). Treated animals ingested concentrations of 3 and 9 μg/L of STX equivalents and were compared with a control group (culture medium ASM-1). At the concentration of 3 μg/L, a decrease in ROS production associated with lower ACAP at hippocampus was observed. Furthermore, a decrease of glutamate cysteine ligase (GCL) activity in the cortex and an increase of brain and liver glutathione concentration were also observed. At the highest concentration (9 μg/L), there was an ACAP increase in the hippocampus as well as in the activity GCL and glutathione-S-transferase in the cortex and hippocampus. At both concentrations, lipid peroxidation was registered in the liver. Therefore, chronic ingestion of STXs can alter the antioxidant defenses and induce oxidative stress in brain and liver. The present results point to the values adopted as threshold limit for STXs in potable waters (3 μg/L) shows already significant chronic effects that alter antioxidant defenses and induce oxidative stress at least in two of the organs studied.     

Monitoring a toxic bloom of Alexandrium minutum using novel microarray and multiplex surface plasmon resonance biosensor technology

Blooms of Alexandrium occur annually during the summer months in the North Channel of Cork Harbour on the south coast of Ireland. This study monitored an extensive bloom of the toxin producing Alexandrium minutum during the summer of 2011 with the use of the MIDTAL (Microarrays for the Detection of Toxic Algae) microarray and a prototype multiplex surface plasmon resonance (multi SPR) biosensor. Microarray signal intensities and toxin results from three testing platforms of the prototype multi SPR biosensor, commercial (CER) enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) were compared against light microscopy counts. The main aim was to demonstrate the use of these methodologies to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying the harmful phytoplankton community and their toxins in natural water samples. Both the microarray signals and multi SPR biosensor results followed a significant trend with light microscopy results and both techniques indicated detection limits of <4000 cells of A. minutum in natural seawater samples.     

Environmental drivers of paralytic shellfish toxin producing Alexandrium catenella blooms in a fjord system of northern Southeast Alaska

Paralytic shellfish poisoning (PSP) is a persistent problem that threatens human health and the availability of shellfish resources in Alaska. Regular outbreaks of marine dinoflagellates in the genus Alexandrium produce paralytic shellfish toxins (PSTs) that make shellfish consumption unsafe, and impose economic hardships on Alaska’s shellfish industry. Phytoplankton and environmental monitoring spanning 2008–2016, and a pilot benthic cyst survey in 2016, were focused in the Juneau region of Southeast Alaska to investigate Alexandrium catenella distributions and conditions favorable to bloom development. Overwintering Alexandrium cysts were found in near-shore sediments throughout the study region. Alexandrium catenella cells were present in the water column across a range of sea surface temperatures (7–15 °C) and surface salinities (S = 4–30); however, an optimal temperature/salinity window (10–13 °C, 18–23) supported highest cell concentrations. Measurable levels of PSTs were associated with lower concentrations (100 cells L⁻¹) of A. catenella, indicating high cell densities may not be required for shellfish toxicity to occur. Several interacting local factors were identified to support A. catenella blooms: 1) sea surface temperatures ≥7 °C; 2) increasing air temperature; 3) low to moderate freshwater discharge; and 4) several consecutive days of dry and calm weather. In combination, these bloom favorable conditions coincide with toxic bloom events during May and June in northern Southeast Alaska. These findings highlight how integrated environmental and phytoplankton monitoring can be used to enhance early warning capacity of toxic bloom events, providing more informed guidance to shellfish harvesters and resource managers in Alaska.     

Cyanotoxin production and phylogeny of benthic cyanobacterial strains isolated from the northeast of Brazil

Most of the knowledge about cyanobacteria toxin production is traditionally associated with planktonic cyanobacterial blooms. However, some studies have been showing that benthic cyanobacteria can produce cyanotoxins. According to this, we aimed to evaluate the production of microcystins and saxitoxins in benthic cyanobacteria isolated from aquatic ecosystems in the Northeast of Brazil and to use a polyphasic approach for their identification. Forty-five clonal strains were isolated from rivers and water supply reservoirs, and identified using morphological and molecular phylogenetic characteristics. In order to evaluate the toxins production, the strains were screened for genes involved in the biosynthesis of microcystins and saxitoxins, positive results were confirmed and cyanotoxins quantified using HPLC. Eight species were identified belonging to the Phormidiaceae, Pseudanabaenaceae and Nostocaceae families. This is the first study in Brazil that shows that strains from the Geitlerinema genus correspond to at least three phylogenetic lineages, which possibly correspond to three distinct species to be subsequently reclassified. The strains that showed one of the genes involved in the cyanotoxins production were analyzed by HPLC and Geitlerinema amphibium, Geitlerinema lemmermannii, Cylindrospermum stagnale and Phormidium uncinatum were identified as producing one or more saxitoxins variants. Thus, this is the first report of saxitoxins production for those first three species and the first report in Brazil for P. uncinatum.     

Effects of saxitoxin- and non-saxitoxin-producing strains of the cyanobacterium Cylindrospermopsis raciborskii on the fitness of temperate and tropical cladocerans

Life-table experiments were performed with two strains of Cylindrospermopsis raciborskii, a saxitoxin-producing strain (T3) and a non-saxitoxin-producing strain (NPLP-1) combined with the green algae Ankistrodesmus falcatus, aiming to detect effects on the reproduction of three cladocerans species: Daphnia pulex, Daphnia gessneri and Moina micrura. Survivorship, age at first reproduction, clutch size and population growth rate during 12–15 days were recorded. Cladocerans showed different susceptibility to saxitoxin-producing strain (T3), with D. pulex and M. micrura being negatively affected, followed by and D. gessneri, which did not show any decrease in fitness and performed even better than the controls with this strain. A reverse response was found in experiments with the non-saxitoxin-producing strain (NPLP-1): while D. pulex and M. micrura had an increase, D. gessneri showed a significant decrease in fitness, suggesting that this strain may produce some bioactive compound. The contrasting responses of the cladoceran species to saxitoxins and non-saxitoxin-producing strains of C. raciborskii suggest that the presence of this cyanobacterium may be a selective factor in determining the composition of zooplankton communities.     

Occurrence of saxitoxins and an anatoxin-a(s)-like anticholinesterase in a Brazilian drinking water supply

Blooms of toxic cyanobacteria are very common in Brazilian waterbodies, as a consequence of eutrophication processes. Our investigations were focused on the detection of neurotoxins during a cyanobacterial bloom in Tapacurá reservoir, which serves as a water supply for Recife city in northeastern Brazil. We also investigated the possible presence of neurotoxins in strains of Anabaena spiroides isolated from this environment. Samples were collected from March to May 2002 at the water surface and close to the dam. Limnological parameters (conductivity, pH, inorganic nutrients) and cyanobacterial abundance were measured. The samples were assayed for toxicity by mouse bioassay and acetylcholinesterase-inhibiting activity by a colorimetric method; saxitoxins (paralytic shellfish poisons) were quantified by a HPLC-FLD postcolumn derivatization method. The dominant cyanobacteria during the bloom were found to be A. spiroides, Pseudanabaena sp., Cylindrospermopsis raciborskii and Microcystis aeruginosa. The mouse bioassays showed the presence of neurotoxins during both A. spiroides and C. raciborskii dominance, whereas anticholinesterase activity was only observed during periods of A. spiroides dominance. The A. spiroides strains isolated during the study also exhibited an acetylcholinesterase inhibitor. HPLC-FLD chromatograms of bloom material extracts revealed the presence of saxitoxin, neosaxitoxin and dc-saxitoxin, probably produced by C. raciborskii.