Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish.

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Marine Toxins as Pharmaceutical Treasure Troves: A Focus on Saxitoxin Derivatives from a Computational Point of View.

This work highlights the significant potential of marine toxins, particularly saxitoxin (STX) and its derivatives, in the exploration of novel pharmaceuticals. These toxins, produced by aquatic microorganisms and collected by bivalve mollusks and other filter-feeding organisms, offer a vast reservoir of chemical and biological diversity. They interact with sodium channels in physiological processes, affecting various functions in organisms. Exposure to these toxins can lead to symptoms ranging from tingling sensations to respiratory failure and cardiovascular shock, with STX being one of the most potent. The structural diversity of STX derivatives, categorized into carbamate, N-sulfocarbamoyl, decarbamoyl, and deoxydecarbamoyl toxins, offers potential for drug development. The research described in this work aimed to computationally characterize 18 STX derivatives, exploring their reactivity properties within marine sponges using conceptual density functional theory (CDFT) techniques. Additionally, their pharmacokinetic properties, bioavailability, and drug-likeness scores were assessed. The outcomes of this research were the chemical reactivity parameters calculated via CDFT as well as the estimated pharmacokinetic and ADME properties derived using computational tools. While they may not align directly, the integration of these distinct datasets enriches our comprehensive understanding of the compound's properties and potential applications. Thus, this study holds promise for uncovering new pharmaceutical candidates from the considered marine toxins.

The role of physico-chemical interactions in the seasonality of toxic dinoflagellate cyst assemblages: The case of the NW Patagonian fjords system.

Harmful algal blooms (HABs) are recurrent in the NW Patagonia fjords system and their frequency has increased over the last few decades. Outbreaks of HAB species such as Alexandrium catenella, a causal agent of paralytic shellfish poisoning, and Protoceratium reticulatum, a yessotoxins producer, have raised considerable concern due to their adverse socioeconomic consequences. Monitoring programs have mainly focused on their planktonic stages, but since these species produce benthic resting cysts, the factors influencing cyst distributions are increasingly gaining recognition as potentially important to HAB recurrence in some regions. Still, a holistic understanding of the physico-chemical conditions influencing cyst distribution in this region is lacking, especially as it relates to seasonal changes in drivers of cyst distributions, as the characteristics that favor cyst preservation in the sediment may change through the seasons. In this study, we analyzed the physico-chemical properties of the sediment (temperature, pH, redox potential) and measured the bottom dissolved oxygen levels in a "hotspot" area of southern Chile, sampling during the spring and summer as well as the fall and winter, to determine the role these factors may play as modulators of dinoflagellate cyst distribution, and specifically for the cysts of A. catenella and P. reticulatum. A permutational analysis of variance (PERMANOVA) showed the significant effect of sediment redox conditions in explaining the differences in the cyst assemblages between spring-summer and fall-winter periods (seasonality). In a generalized linear model (GLM), sediment redox potential and pH were associated with the highest abundances of A. catenella resting cysts in the spring-summer, however it was sediment temperature that most explained the distribution of A. catenella in the fall-winter. For P. reticulatum, only spring-summer sediment redox potential and temperature explained the variation in cyst abundances. The implications of environmental (physico-chemical) seasonality for the resting cysts dynamics of both species are discussed.

Effects of homeopathic preparations on raphidiopsis raciborskii (Cyanobacteria) toxicity in artemia salina mode

Cyanobacteria are microorganisms found in different parts of the world. Some genera are cyanotoxins producers a sodium channel blockingneurotoxin (saxitoxins). Some homeopathic preparations have been identified as remedial action on toxicity models in Artemia salina. This study aimed to observe whether homeopathic products influence the toxicity ofR.raciborskiiextract onA.salinaby inducing cyst hatching arrest, anembryo bioresiliencemodel previously developed in our laboratory (Pinto et al., 2021; Mohammad et al., 2022). Thus, previous toxicity testswere carried out on cysts in 96-well plates, using different concentrations of the extract obtained from regular cultivation of R. raciborskii in HCl 0.05M, whose strain, named T3, is kept in the laboratory of Cyanobacteria at FURGS, Brazil.The standardization of toxin concentration was based on an established scale developed at FURGS, in which the number of T3 filaments is associated withspecific saxitoxin concentrationsdefined by chromatography. The concentration of 2.6 µg/L was chosen since it reducedthe cysthatching rate by 30%, the ideal level to observe embryo bioresilience. Then, a screeningstudy with 22 homeopathic preparations was tested blind in three experimental series, in duplicate,against threecontrols (unchallenged, water,and succussed water)for possible toxicity attenuationon Artemia salinacysts hatching rate. Homeopathic medicines were prepared in pure,sterile water from a stock homeopathic solution, one potency below the working potency. After the 1:100 dilution, 100 succussions were made using a robotic arm (Denise, Autic). The medicines were inserted into the seawater on a 10% basis. Due to the high sensitivity of A. salinato the circalunar variations, all experiments were performed during the first quarter moon. Statistical analysis was performed by two-way ANOVA followed by Tukey, with α=0.05. The most significant results indicative of bioresilience improvement were seen after the treatment with Nitric acidum6 cH, Plumbum metallicum6 cH, isotherapic 200 cH, and hydrochloric acid 1 cH being the last one used as a vehicle of the extracts. Thus, these preparations were chosen to be used in further experiments. In conclusion, the Artemia salinamodel has also beenuseful to study bioresilienceimprovement by homeopathic medicines after intoxication with saxitoxin.

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.

Ecophysiological Aspects and sxt Genes Expression Underlying Induced Chemical Defense in STX-Producing Raphidiopsis raciborskii (Cyanobacteria) against the Zooplankter Daphnia gessneri.

Cyanobacteria stand out among phytoplankton when they form massive blooms and produce toxins. Because cyanotoxin genes date to the origin of metazoans, the hypothesis that cyanotoxins function as a defense against herbivory is still debated. Although their primary cellular function might vary, these metabolites could have evolved as an anti-predator response. Here we evaluated the physiological and molecular responses of a saxitoxin-producing Raphidiopsis raciborskii to infochemicals released by the grazer Daphnia gessneri. Induced chemical defenses were evidenced in R. raciborskii as a significant increase in the transcription level of sxt genes, followed by an increase in saxitoxin content when exposed to predator cues. Moreover, cyanobacterial growth decreased, and no significant effects on photosynthesis or morphology were observed. Overall, the induced defense response was accompanied by a trade-off between toxin production and growth. These results shed light on the mechanisms underlying zooplankton-cyanobacteria interactions in aquatic food webs. The widespread occurrence of the cyanobacterium R. raciborskii in freshwater bodies has been attributed to its phenotypic plasticity. Assessing the potential of this species to thrive over interaction filters such as zooplankton grazing pressure can enhance our understanding of its adaptive success.

Saxitoxins from the freshwater cyanobacterium Raphidiopsis raciborskii can contaminate marine mussels.

Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii) is a freshwater cyanobacterium potentially producing saxitoxins (STX) and cylindrospermopsin. Its ecophysiological versatility enables it to form blooms in the most diverse types of environments, from tropical to temperate, and from relatively pristine to polluted. In Peri Lake, located in the subtropical south of Brazil, growing populations of STX-producing R. raciborskii have been detected since 1994, posing risks to the use of its waters that supply a population of about 100,000 inhabitants. Despite the existence of a monitoring system for the presence and toxicity of cyanobacteria in Peri Lake water, no assessment has been made in the coastal region, downstream of outflowing lake water, thereby potentially making available a toxic biomass to natural and cultivated shellfish populations in the salt water ecosystem. To address this problem, the present study evaluated environmental variables and STX concentration by profiling the outflowing waters between Peri Lake and the adjacent coastal zone. Laboratory experiments were carried out with three strains of R. raciborskii in order to confirm the effect of salinity on STX production and verify if Perna Perna mussels fed with R. raciborskii cultures would absorb and accumulate STX. Results showed that environmental concentrations of STX reach high levels (up to 6.31 µg L-1 STX eq.), especially in the warmer months, reaching the coastal zone. In laboratory tests, it was found that the strains tolerate salinities between 4 and 6 and that salinity influences the production of STX. In addition, mussels fed with R. raciborskii effectively absorb and accumulate STX, even in typically marine salinities (22 to 30), suggesting that R. raciborskii biomass remains available and toxic despite salinity shock. These results draw attention to the ecological and health risk associated with R. raciborskii blooms, both in the lake environment and in the adjacent marine environment, calling attention to the need to improve the monitoring and management systems for water and shellfish toxicity in the region of interest, as well as other places where toxic cyanobacteria of limnic origin can reach the coastal zone.

Effects of saxitoxins exposure on oligodendrocyte development in mouse neonates.

Saxitoxins (STXs) are neurotoxins produced by cyanobacteria and dinoflagellates, and they are primarily known to block voltage-gated sodium channels in neurons. The present study aimed to obtain further information regarding the effects of these toxins on neurodevelopment by investigating the responses of murine subventricular zone (SVZ) neural progenitors to STXs. An in vitro neonatal mouse SVZ explant model was exposed to different concentrations of toxic cyanobacterial extracts to evaluate the migration and differentiation of SVZ-derived progenitor cells. To test the ability of STX to cross the placental barrier, pregnant mice received a single intraperitoneal injection of STXs (7.5 µg/kg body weight) on gestational day fifteen. Immunocytochemistry was performed to detect proliferating and differentiating progenitors, including oligodendrocyte progenitor cells (OPCs). It was found that specific proliferation of OPCs was significantly increased, but there was no corresponding increase in the number of differentiated oligodendrocytes, which may indicate a negative effect on the maturation process of these cells. Additionally, the data showed that STXs crossed the placental barrier. Thus, STXs can be considered a potential risk to fetal neurodevelopment.

Multiple biomarkers response in a Neotropical fish exposed to paralytic shellfish toxins (PSTs).

The Alagados Reservoir (Southern Brazil) is used as water supply, and since 2002 there have been reports with a presence of cyanobacterial blooms and cyanotoxins. In order to assess the water quality and the ecological integrity of the reservoir, we evaluated biochemical, genotoxic and osmoregulatory biomarkers in the freshwater cichlid fish (Geophagus brasiliensis) that were exposed to PSTs. The fish were sampled in the Alagados Reservoir in February 2016 (Summer) and were divided in three groups: 1) Reservoir group (RES): fish were collected immediately after sampling; 2) Depuration group (DEP): fish were submitted to the depuration experiment for 90 days in the laboratory; and 3) Reproduction group (REP): fish were kept in the laboratory until the fertilization and the chemical analyses were performed on the offspring (F1 generation). In the RES and DEP the blood, brain, muscle, liver and gills were collected for biochemical, genotoxic and osmoregulatory biomarkers analysis. Our results showed that the fish from the Alagados Reservoir (RES) presented oxidative stress and DNA damage; and after 90 days (DEP), the antioxidant system and DNA damage were recovered. Although PSTs were considered a risk to the ecological integrity of this water body; PSTs concentrations were not found in the tissues of the F1 generation. In addition, the biomarkers used were useful tools to evaluate the effects of environment contamination. Therefore, it is necessary to develop new technologies and monitoring programs in order to reduce cyanobaterial blooms, cyanotoxins and human activities that cause the contamination in aquatic environments.

Effects of an aluminum-based chemical remediator on the cyanobacteria population: a study in the northeast of Brazil.

Water sources destined to human supply are increasingly threatened worldwide due to various sources of pollution, either point or diffuse. In this sense, the objective of this work was to evaluate the efficiency of an aluminum-based chemical remediator in the Joanes River. An statistical analysis of physical, chemical, biological, and hydraulic monitoring data was performed relying on a 2013-2018 recording period, provided by the local sanitation service provider and the environmental agency. The results showed that even with the use of aluminum-based chemical remediators, the key parameters for controlling flowering events remained high with mean values of 0.18 mg P L-1, 176.155 cells mL-1 of cyanobacteria and peaks of 1.56 µg L-1 and 4.02 µg L-1 for microcystin and saxitoxin, respectively. At the end of this study, it was verified that the aluminum-based chemical remediator showed low effectiveness in the reduction of phosphorus and cyanobacteria, opposing to expectations of the sanitation provider.

Combined Effect of Light and Temperature on the Production of Saxitoxins in Cylindrospermopsis raciborskii Strains.

Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium that can tolerate a wide range of light and temperature. Due to climatic changes, the interaction between light and temperature is studied in aquatic systems, but no study has addressed the effect of both variables on the saxitoxins production. This study evaluated the combined effect of light and temperature on saxitoxins production and cellular quota in C. raciborskii. Experiments were performed with three C. raciborskii strains in batch cultures under six light intensities (10, 40, 60, 100, 150, and 500 µmol of photons m-2 s-1) and four temperatures (15, 20, 25, and 30 °C). The growth of C. raciborskii strains was limited at lower temperatures and the maximum growth rates were obtained under higher light combined with temperatures equal or above 20 °C, depending on the strain. In general, growth was highest at 30 °C at the lower light intensities and equally high at 25 °C and 30 °C under higher light. Highest saxitoxins concentration and cell-quota occurred at 25 °C under high light intensities, but were much lower at 30 °C. Hence, increased temperatures combined with sufficient light will lead to higher C. raciborskii biomass, but blooms could become less toxic in tropical regions.

Single and combined effects of microcystin- and saxitoxin-producing cyanobacteria on the fitness and antioxidant defenses of cladocerans.

Cyanobacteria produce different toxic compounds that affect animal life, among them hepatotoxins and neurotoxins. Because cyanobacteria are able to produce a variety of toxic compounds at the same time, organisms may be, generally, subjected to their combined action. In the present study, we demonstrate the single and combined effects on cladocerans of cyanobacteria that produce microcystins (hepatotoxins) and saxitoxins (neurotoxins). Animals were exposed (either singly or combined) to 2 strains of cyanobacteria isolated from the same environment (Funil Reservoir, Rio de Janeiro, Brazil). The effects on clearance rate, mobility, survivorship, fecundity, population increase rate (r), and the antioxidant enzymes glutathione-S-transferase (GST) and catalase (CAT) were measured. Cladoceran species showed a variety of responses to cyanobacterial exposures, going from no effect to impairment of swimming movement, lower survivorship, fecundity, and general fitness (r). Animals ingested cyanobacteria in all treatments, although at lower rates than good food (green algae). Antioxidant defense responses were in accordance with fitness responses, suggesting that oxidative stress may be related to such effects. The present study emphasizes the need for testing combined actions of different classes of toxins, because this is often, and most likely, the scenario in a more eutrophic world with global climatic changes. Environ Toxicol Chem 2017;36:2689-2697. © 2017 SETAC.

Effects of crude extracts of a saxitoxin-producer strain of the cyanobacterium Cylindrospermopsis raciborskii on the swimming behavior of wild and laboratory reared guppy Poecilia vivipara.

The cyanobacterium Cylindrospermopsis raciborskii is an invasive species in water supply reservoirs worldwide, which can produces cylindrospermopsins and saxitoxins. In the wild, guppy (Poecilia vivipara) can be exposed to cyanotoxins, but those born and reared in laboratory are free of this contact. The aim of this paper was to comparatively measure the locomotor activity of 'wild' and 'lab' P. vivipara before and after exposure to crude extracts of two different cultures of C. raciborskii (CYRF-01), a saxitoxin-procucer strain. The movement of each fish was recorded using an image monitoring system (Videomex V®) before and after 48 h exposure to cyanobacterial extracts. Each experiment was performed during 4 h, with 1 h acclimation and 3 h recording period of the parameters Distance performed (DP), Swimming time (SwT), Stereotypic time (StT), Resting time (RT) and Average speed (AS). The quantification of saxitoxin in the solutions was performed by the enzyme-linked immunosorbent assay (ELISA). The weight or the total length did not influence the locomotor activity of fish in any of the experiments. The saxitoxin value was similar for both cultures (Culture 1: 7.3 µg L-1 and Culture 2: 8.6 µg L-1). However, in experiments with Culture 1 an increased activity in most parameters was observed, while in Culture 2, a decreased activity was observed only in 'lab' fish. Wild fish was less affected, showing higher resistance to both cyanobacterial crude extracts. This study showed that different cultures of the same strain of C. raciborskii and with similar contents of saxitoxin are able to change the locomotor activity of P. vivipara, contributing to the validation of the use of behavioral parameters to the evaluation of sublethal effects of toxic cyanobacteria on fish.

Toxicity Overrides Morphology on Cylindrospermopsis raciborskii Grazing Resistance to the Calanoid Copepod Eudiaptomus gracilis.

Toxicity and morphology may function as defense mechanisms of bloom-forming cyanobacteria against zooplankton grazing. Yet, the relative importance of each of these factors and their plasticity remains poorly known. We tested the effects of chemical and morphological traits of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii on the feeding response of the selective feeder Eudiaptomus gracilis (Calanoida, Copepoda), using a saxitoxin-producing strain (STX+) and a non-saxitoxin (STX-)-producing strain as food. From these two chemotypes, we established cultures of three different morphotypes that differed in filament length (short, medium, and long) by incubating the strains at 17, 25, and 32 °C. We hypothesized that the inhibitory effects of saxitoxins determine the avoidance of C. raciborskii, and that morphology would only become relevant in the absence of saxitoxins. Temperature affected two traits: higher temperature resulted in significantly shorter filaments in both strains and led to much higher toxin contents in the STX+ strain (1.7 µg eq STX L(-1) at 17 °C, 7.9 µg eq STX L(-1) at 25 °C, and 25.1 µg eq STX L(-1) at 32 °C). Copepods strongly reduced the ingestion of the STX+ strain in comparison with STX- cultures, regardless of filament length. Conversely, consumption of shorter filaments was significantly higher in the STX- strain. The great plasticity of morphological and chemical traits of C. raciborskii and their resultant contrasting effects on the feeding behavior of zooplankton might explain the success of this cyanobacterium in a variety of aquatic environments.

Effects of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming behavior of Daphnia (cladocera).

The present study aimed to test the effects of raw water samples from a eutrophic reservoir and of a saxitoxin-producing strain of Cylindrospermopsis raciborskii on the swimming behavior of 2 key herbivore species of Daphnia. Two complementary approaches were used, acute bioassays and behavioral assays using an automated movement tracking system for measuring the following activity parameters: swimming time, resting time, distance traveled, and mean velocity. In both assays, animals were exposed to field samples or to toxic filaments in different concentrations and observed for 2 h to 3 h. In the acute bioassays, there was a decrease in the number of swimming individuals during the exposure period and a recovery following removal from toxic algae. A significant relationship was found between median effective concentration and the saxitoxin content of seston (r(2) = 0.998; p = 0.025) in the acute bioassays with raw water samples. Behavioral assays also showed significant effects in the activity parameters with both field samples and the strain of C. raciborskii, with some recovery during the exposure period. Both approaches corroborated previous research on the effects of neurotoxic C. raciborskii on the swimming activity of Daphnia, and these effects are compatible with the mechanism of action of saxitoxins. The present study showed that activity parameters of aquatic organisms may be a useful tool in the evaluation of sublethal toxicity and detection of neurotoxins in raw water.

Avaliação da remoção de saxitoxinas por meio de técnicas de tratamento das águas de abastecimento / Saxitoxins removal evaluation by means of the drinking-water treatment processes

O objetivo do trabalho é avaliar a eficiência da adsorção com carvão ativado em pó (CAP) e da cloração de cálcio na remoção de saxitoxinas. As saxitoxinas dispersas em água destilada foram produzidas por meio da extração de células viáveis de cianobactérias da espécie Cylindrospermopsis raciborskii. Os ensaios foram realizados em equipamento de jar test adaptado e empregando-se três tipos de CAP e hipoclorito de cálcio. Os tempos de detenção aplicados foram de duas horas para adsorção e 30 e 60 minutos para oxidação. Os resultados evidenciaram que a eficiência de remoção, para a adsorção, está intrinsecamente relacionada ao tipo de carvão e à dosagem empregada, obtendo maior eficiência para o CAP de madeira. Para oxidação, os dois tempos de contato e as dosagens avaliadas apresentaram eficiência praticamente constante, da ordem de 80 por cento, atendendo ao estabelecido pela Portaria 518.

This paper focuses on the evaluation of the saxitoxins removal by means of adsorption and oxidation. These toxins were produced through extraction of viable cells from cyanobacteria Cylindrospermopsis raciborskii. The tests were carried out in the batch scale with three different powdered activated carbons and calcium hypochlorite. The detention times were two hours for adsorption, and 30 and 60 minutes for oxidation tests. The results pointed out that the type and dosage of carbon are strongly related, and the wood carbon presented higher efficiency. The oxidation efficiency was approximately 80 percent for both evaluated detention times and distinct dosages, keeping practically constant for all tests according to the limit established by the Brazilian Drinking-water Legislation 518.