Analysis of factors affecting microbial degradation of cyanobacterial toxins based on theoretical calculations.

Cyanobacterial toxins are the most common algal toxins, which are highly toxic and can persist in the aquatic environment without easy degradation, posing risks to the ecosystem and human health that cannot be ignored. Although microbiological methods for the removal of cyanobacterial toxins from aqueous environments are highly efficient, their degradation efficiency is susceptible to many abiotic environmental factors. In this paper, Microcystin-LR (MC-LR) and its microbial degrading enzymes were selected to study the effects of common environmental factors (temperature (T), NO3-, NH4+, Cu2+, Zn2+) and their levels during microbial degradation of cyanobacterial toxins in aqueous environments by using molecular docking, molecular dynamics simulation, analytical factor design, and the combined toxicokinetics of TOPKAT (toxicity prediction). It was found that the addition of T, NO3- and Cu2+ to the aqueous environment promoted the microbial degradation of MC-LR, while the addition of NH4+ and Zn2+ inhibited the degradation; The level effect study showed that the microbial degradation of MC-LR was promoted by increasing levels of added T and NO3- in the aqueous environment, whereas it was inhibited and then promoted by increasing levels of NH4+, Cu2+ and Zn2+. In addition, the predicted toxicity of common Microcystins (MCs) showed that MC-LR, Microcystin-RR (MC-RR) and Microcystin-YR (MC-YR) were not carcinogenic, developmentally toxic, mutagenic or ocular irritants in humans. MC-LR and MC-RR are mild skin irritants and MC-YR is not a skin irritant. MC-YR has a higher chronic and acute toxicity in humans than MC-LR and MC-RR. Acute/chronic toxicity intensity for aquatic animals: MC-YR > MC-LR > MC-RR and for aquatic plants: MC-LR > MC-YR > MC-RR. This suggests that MC-YR also has a high environmental health risk. This paper provides theoretical support for optimizing the environmental conditions for microbial degradation of cyanobacterial toxins by studying the effects of common environmental factors and their level effects in the aquatic environment.

Molecular forecasting of domoic acid during a pervasive toxic diatom bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms.

Biogenic ROS mediated degradation mechanism of marine toxin domoic acid.

Domoic acid (DA) is a compound generated as a secondary metabolite during harmful algal blooms, has historically received attention as the potent neurotoxicity in marine environment. However, the aerobic degradation mechanism of DA and the DA-degrader remain largely unknown. Here, we revealed the mechanism of aerobic degradation of DA by a ubiquitous marine Pseudoalteromonas sp., and more importantly, we confirmed that the degradation of DA is mediated by biogenic reactive oxygen species (ROS), rather than direct enzyme-mediated as traditionally conceived. Results indicated that DA degradation was caused by biogenic O2- and OH, where DA underwent reactions of decarboxylation, hydroxylation, and oxidation to yield the detoxification terminal product. Besides, whole genome sequencing and RT-qPCR analysis revealed that the genes conferring to encoding leucine dehydrogenase (ldh) and Na+-translocated NADH-quinone oxidoreductase (nqrA, nqrF) are responsible for biogenic ROS production. Finally, we found through comparative proteomic analysis that biogenic ROS mediated the DA degradation may be prevalent in the environment. Overall, this work not only reveals aerobic biotransformation mechanism of DA, but also identifies a novel mechanism of DA degradation, which provides new perspective into the environmental fate of DA and the artificial bioremediation of DA.

The queen scallop Aequipecten opercularis: A slow domoic acid depurator?

Domoic acid (DA) is a dangerous phycotoxin produced by several strains of diatoms of the genus Pseudo-nitzschia, and responsible for Amnesic Shellfish Poisoning (ASP) in humans. The increasingly intense ASP-outbreaks along the English Channel over the last three decades have forced persistent harvest closures of economically important and highly contaminated bivalve stocks exhibiting slow DA-depuration rates, like the king scallop Pecten maximus. Under this scenario, other pectinid species, such as the queen scallop Aequipecten opercularis have been empirically proposed as alternative resources to redress the high economic losses due to the banning of the exploitation of P. maximus. Nevertheless, the kinetics of DA depuration in A. opercularis have not been assessed so far, and its direct extraction after ASP-episodes could represent a serious threat to public health. Hence, the main objective of this work was to estimate the DA-depuration rate in the digestive gland (DG) of naturally contaminated scallops A. opercularis after a toxic Pseudo-nitzschia australis bloom subjected to experimental depuration in the laboratory for 30 days. This study also intended to go further in the knowledge about the anatomical distribution of DA in scallop tissues, and corroborate the implications of autophagy in DA-sequestration in the DG of this species as recently hypothesized. In the DG, the DA-depuration rate (0.018 day-1) suggested that even with toxin burdens as low as 40 mg⋅kg-1 in the DG, queen scallops may remain contaminated for about 70 days, thus longer under intensely contamination scenarios. The subcellular analyses corroborated DA-sequestration mainly through late-autophagy within residual bodies in the DG, without differences in the frequencies of anti-DA labeled residual bodies across the entire depuration process. These results revealed that A. opercularis cannot be considered a fast DA-depurator, and represent a baseline knowledge for decision-making about harvesting natural beds of queen scallops after toxic Pseudo-nitzschia blooms. The findings of this work also represent a cornerstone for further research to accelerate DA-depuration in this species.

Allelochemicals determine competition and grazing control in Alexandrium catenella.

The production of allelochemicals by the toxigenic dinoflagellate Alexandrium catenella is one of the suggested mechanisms to facilitate its bloom formation and persistence by outcompeting other phototrophic protists and reducing grazing pressure. In Southern California, toxic events caused by A. catenella and paralytic shellfish toxins (PSTs) regularly impact coastal ecosystems; however, the trophic interactions and mechanisms promoting this species in a food web context are still not fully understood. In the present study, we combined a dynamical mathematical model with laboratory experiments to investigate potential toxic and allelochemical effects of an A. catenella strain isolated off the coast of Los Angeles, Southern California, on competitors and a common zooplankton consumer. Experiments were conducted using three toxigenic strains of A. catenella, comparing the new Californian isolate (Alex Cal) to two strains previously described from the North Sea, a lytic (Alex2) and non-lytic (Alex5) strain, testing for donor density-dependent effects on two phytoplankton species (Rhodomonas salina, Tetraselmis sp.) and on the rotifer Brachionus plicatilis. Bioassays revealed a steep decline in competitor and consumer populations with increasing Alex Cal concentrations, indicating an intermediate lytic activity compared to the North Sea strains (lytic Alex2 and non-lytic Alex5). The rotifer fed and grew well on the PST- toxic, but non-lytic Alex5 strain, while its survival significantly decreased with increasing concentrations of the two lytic strains Alex Cal and Alex 2, indicating that negative effects on the rotifer were mediated by allelochemicals rather than PST-toxins. Mixed culture experiments including both competitors and consumers demonstrated that the intensity of allelochemical effects not only depended on the A. catenella density but also on the target density. Negative effects on grazers were alleviated by co-occurring competitors with a lower sensitivity to allelochemicals, thus reducing harmful compounds and allowing grazing control on the dinoflagellate to come into effect again. Results from mixed culture experiments were supported by the mathematical approach used in this study which was calibrated with data from simple monoculture growth, pairwise competition and predator-prey experiments, demonstrating the applicability of this model approach to predict the outcome of more complex food web dynamics at the community level.

Sterolysin from a 1950s culture of Karlodinium veneficum (aka Gymnodinium veneficum Ballantine) forms lethal sterol dependent membrane pores.

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957.

Saturated constructed wetlands for the remediation of cylindrospermopsin and microcystin-LR: Plants, microbes, and biodegradation pathways.

Harmful cyanobacterial blooms will be more intense and frequent in the future, contaminating surface waters with cyanotoxins and posing a threat to communities heavily reliant on surface water usage for crop irrigation. Constructed wetlands (CWs) are proposed to ensure safe crop irrigation, but more research is needed before implementation. The present study operated 28 mesocosms in continuous mode mimicking horizontal sub-surface flow CWs. Mesocosms were fed with synthetic lake water and spiked periodically with two cyanotoxins, microcystin-LR (MC-LR) and cylindrospermopsin (CYN), at environmentally relevant cyanotoxins concentrations (10 µg L-1). The influence of various design factors, including plant species, porous media, and seasonality, was explored. The mesocosms achieved maximum MC-LR and CYN mass removal rates of 95 % and 98 %, respectively. CYN removal is reported for the first time in CWs mimicking horizontal sub-surface flow CWs. Planted mesocosms consistently outperformed unplanted mesocosms, with Phragmites australis exhibiting superior cyanotoxin mass removal compared to Juncus effusus. Considering evapotranspiration, J. effusus yielded the least cyanotoxin-concentrated effluent due to the lower water losses in comparison with P. australis. Using the P-kC* model, different scaling-up scenarios for future piloting were calculated and discussed. Additionally, bacterial community structure was analyzed through correlation matrices and differential taxa analyses, offering valuable insights into their removal of cyanotoxins. Nevertheless, attempts to validate microcystin-LR biotransformation via the known mlrA gene degradation pathway were unfruitful, indicating alternative enzymatic degradation pathways occurring in such complex CW systems. Further investigation into the precise molecular mechanisms of removal and the identification of transformation products is needed for the comprehensive understanding of cyanotoxin mitigation in CW. This study points towards the feasibility of horizontal sub-surface flow CWs to be employed to control cyanotoxins in irrigation or recreational waters.

First report of PST-producing Microseira wollei from China reveals its novel toxin profile.

Microseira wollei, a globally distributed freshwater bloom-forming benthic cyanobacterium, is known for its production of cyanotoxins and taste and odor (T&O). While CYN (Cylindrospermopsin)-producing populations of M. wollei are confined to Australia, PST (Paralytic shellfish toxins)-producing populations have been exclusively documented in North America. In this study, four benthic cyanobacterial strains, isolated from West Lake in China, were identified as M. wollei based on morphological and phylogenetic analyses. Detection of sxtA gene and UPLC-MS/MS analysis conclusively confirmed the PST-producing capability of M. wollei CHAB5998. In the phylogenetic tree of 16S rDNA, M. wollei strains formed a monophyletic group with two subclades. Notably, non-PST-producing Chinese strains clustered with Australian strains in Clade II, while all other strains, including PST-producing ones, clustered in Clade I. Additionally, CHAB5998 contains ten PST variants, of which STX, NEO, GTX2, GTX3, GTX5 and C1 were identified for the first time in M. wollei. Sequence analysis of PST biosynthetic gene cluster (sxt) genes indicated potential base variations, gene rearrangements, insertions, and deletions in the strain CHAB5998. Also, sxt gene has a longer evolutionary history in M. wollei than that in cyanobacteria from Nostocales. Multiple recombination breakpoints detected in sxt genes and the inconsistency in the topology of the phylogenetic trees between sxt and 16S rDNA suggested that multiple horizontal gene transfers (HGT) have occurred. Overall, the present study marks the first documented occurrence of PST-producing M. wollei outside of North America and identifies it as the first toxic freshwater benthic cyanobacterium in China. This revelation implies that benthic cyanobacteria may pose a higher environmental risk in China than previously acknowledged.

Role of turbulence in Dinophysis spp. growth, feeding, and toxin leakage in culture.

Dinophysis, a mixotrophic dinoflagellate that is known to prey on the ciliate Mesodinium rubrum, and retain its chloroplasts, is responsible for diarrhetic shellfish poisoning (DSP) in humans and has been identified on all U.S. coasts. Monocultures of Dinophysis have been used to investigate the growth of Dinophysis species in response to variations in environmental conditions, however, little is known about the roles of system stability (turbulence) and mixotrophy in the growth and toxicity of Dinophysis species in the U.S.. To begin to address this gap in knowledge, culturing experiments were conducted with three species (four strains) of Dinophysis, that included predator-prey co-incubation (Dinophysis spp.+ M. rubrum) and prey-only (M. rubrum) flasks. Cultures were investigated for effects of low or high turbulence on Dinophysis spp. growth, feeding, and amounts of intra- and extracellular toxins: okadaic acid and derivatives (diarrhetic shellfish toxins, DSTs) and pectenotoxins (PTXs). Turbulence did not have a measurable effect on the rates of ingestion of M. rubrum prey by Dinophysis spp. for any of the four strains, however, effects on growth and particulate and dissolved toxins were observed. High turbulence (ε = 10-2 m2s-3) significantly slowed growth of both D. acuminata and D. ovum relative to still controls, but significantly stimulated growth of the D. caudata strain. Increasing turbulence also resulted in significantly higher intracellular toxin content in D. acuminata cultures (DSTs and PTXs), but significantly reduced intracellular toxin content (PTXs) in those of D. caudata. An increase in turbulence appeared to promote toxin leakage, as D. ovum had significantly more extracellular DSTs found in the medium under high turbulence when compared to the still control. Overall, significant responses to turbulence were observed, whereby the three strains from the "Dinophysis acuminata complex" displayed a stress response to turbulence, i.e., decreasing growth, increasing intracellular toxin content and/or increasing toxin leakage, while the D. caudata strain had an opposite response, appearing stimulated by, or more tolerant of, high turbulence.

First evidence of the induction of domoic acid production in Pseudo-nitzschia australis by the copepod Temora longicornis from the French coast.

Diatoms of the genus Pseudo-nitzschia are widespread in marine waters. Some of them can produce the toxin domoic acid (DA) which can be responsible for amnesic shellfish poisoning (ASP) when transferred into the food web. These ASP events are of major concern, due to their ecological and socio-economic repercussions, particularly on the shellfish industry. Many studies have focused on the influence of abiotic factors on DA induction, less on the role of biotic interactions. Recently, the presence of predators has been shown to increase DA production in several Pseudo-nitzschia species, in particular in Arctic areas. In order to investigate the relationship between Pseudo-nitzschia species and grazers from the French coast, exposures between one strain of three species (P. australis, P. pungens, P. fraudulenta) and the copepod Temora longicornis were conducted for 5 days. Cellular and dissolved DA content were enhanced by 1,203 % and 1,556 % respectively after the 5-days exposure of P.australis whereas no DA induction was observed in P. pungens and P. fraudulenta. T. longicornis consumed all three Pseudo-nitzschia species. The copepod survival was not related to DA content. This study is an essential first step to better understanding the interactions between planktonic species from the French coast and highlights the potential key role of copepods in the Pseudo-nitzschia bloom events in the temperate ecosystems.

Applying metabolic modeling and multi-omics to elucidate the biotransformation mechanisms of marine algal toxin domoic acid (DA) in sediments.

Domoic acid (DA)-producing algal blooms are a global marine environmental issue. However, there has been no previous research addressing the question regarding the fate of DA in marine benthic environments. In this work, we investigated the DA fate in the water-sediment microcosm via the integrative analysis of a top-down metabolic model, metagenome, and metabolome. Results demonstrated that biodegradation is the leading mechanism for the nonconservative attenuation of DA. Specifically, DA degradation was prominently completed by the sediment aerobic community, with a degradation rate of 0.0681 ± 0.00954 d-1. The DA degradation pathway included hydration, dehydrogenation, hydrolysis, decarboxylation, automatic ring opening of hydration, and ß oxidation reactions. Moreover, the reverse ecological analysis demonstrated that the microbial community transitioned from nutrient competition to metabolic cross-feeding during DA degradation, further enhancing the cooperation between DA degraders and other taxa. Finally, we reconstructed the metabolic process of microbial communities during DA degradation and confirmed that the metabolism of amino acid and organic acid drove the degradation of DA. Overall, our work not only elucidated the fate of DA in marine environments but also provided crucial insights for applying metabolic models and multi-omics to investigate the biotransformation of other contaminants.

Discovery of a series of portimine-A fatty acid esters in mussels.

Vulcanodinium rugosum is a benthic dinoflagellate known for producing pinnatoxins, pteriatoxins, portimines and kabirimine. In this study, we aimed to identify unknown analogs of these emerging toxins in mussels collected in the Ingril lagoon, France. First, untargeted data acquisitions were conducted by means of liquid chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Data processing involved a molecular networking approach, and a workflow dedicated to the identification of biotransformed metabolites. Additionally, targeted analyses by liquid chromatography coupled to triple quadrupole mass spectrometry were also implemented to further investigate and confirm the identification of new compounds. For the first time, a series of 13-O-acyl esters of portimine-A (n = 13) were identified, with fatty acid chains ranging between C12:0 and C22:6. The profile was dominated by the palmitic acid conjugation. This discovery was supported by fractionation experiments combined with the implementation of a hydrolysis reaction, providing further evidence of the metabolite identities. Furthermore, several analogs were semi-synthesized, definitively confirming the discovery of these metabolization products. A new analog of pinnatoxin, with a molecular formula of C42H65NO9, was also identified across the year 2018, with the highest concentration observed in August (4.5 µg/kg). The MS/MS data collected for this compound exhibited strong structural similarities with PnTX-A and PnTX-G, likely indicating a substituent C2H5O2 in the side chain at C33. The discovery of these new analogs will contribute to deeper knowledge of the chemodiversity of toxins produced by V. rugosum or resulting from shellfish metabolism, thereby improving our ability to characterize the risks associated with these emerging toxins.

Fungal removal of cyanotoxins in constructed wetlands: The forgotten degraders.

Harmful cyanobacterial blooms have increased globally, releasing hazardous cyanotoxins that threaten the safety of water resources. Constructed wetlands (CWs) are a nature-based and low-cost solution to purify and remove cyanotoxins from water. However, bio-mechanistic understanding of the biotransformation processes expected to drive cyanotoxin removal in such systems is poor, and primarily focused on bacteria. Thus, the present study aimed at exploring the fungal contribution to microcystin-LR and cylindrospermopsin biodegradation in CWs. Based on CW mesocosms, two experimental approaches were taken: a) amplicon sequencing studies were conducted to investigate the involvement of the fungal community; and b) CW fungal isolates were tested for their microcystin-LR and cylindrospermopsin degradation capabilities. The data uncovered effects of seasonality (spring or summer), cyanotoxin exposure, vegetation (unplanted, Juncus effusus or Phragmites australis) and substratum (sand or gravel) on the fungal community structure. Additionally, the arbuscular mycorrhizal fungus Rhizophagus and the endophyte Myrmecridium showed positive correlations with cyanotoxin removal. Fungal isolates revealed microcystin-LR-removal potentials of approximately 25 % in in vitro biodegradation experiments, while the extracellular chemical fingerprint of the cultures suggested a potential intracellular metabolization. The results from this study may help us understand the fungal contribution to cyanotoxin removal, as well as their ecology in CWs.

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

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

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

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

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

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

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

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

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

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

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

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

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

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