The Effecting Mechanisms of 100 nm Sized Polystyrene Nanoplastics on the Typical Coastal Alexandrium tamarense.

Due to the increase in nanoplastics (NPs) abundance in aquatic environments, their effects on phytoplankton have aroused large research attention. In this study, 100 nm sized polystyrene NPs were chosen to investigate their effecting performance and mechanisms on a typical dinoflagellates Alexandrium tamarense. The results indicated the population growth and photosynthetic efficiencies of A. tamarense were significantly inhibited by NPs exposure, as well as the increase in cellular total carotenoids and paralytic shellfish toxins (PSTs). Meanwhile, the cellar ROS levels increased, corresponding to the increased activities or contents of multiple antioxidant components, including SOD, CAT, GPX, GR, GSH and GSSG. The transcriptional results support the physiological-biochemical results and further revealed the down-regulation of genes encoding the light reaction centers (PSI and PSII) and up-regulation of genes encoding the antioxidant components. Up-regulation of genes encoding key enzymes of the Calvin cycle and glycolytic pathway together with the TCA cycle could accelerate organic carbon and ATP production for A. tamarense cells resistant to NPs stress. Finally, more Glu and acetyl-CoA produced by the enhanced GSH cycle and the glycolytic pathway, respectively, accompanied by the up-regulation of Glu and Arg biosynthesis genes supported the increase in the PST contents under NPs exposure. This study established a data set involving physiological-biochemical changes and gene information about marine dinoflagellates responding to NPs, providing a data basis for further evaluating the ecological risk of NPs in marine environments.

Toxin production and transcriptomic response to nitrate concentrations in the toxic dinoflagellate Alexandrium tamarense.

The bloom-forming dinoflagellate Alexandrium tamarense is one of the most important producers of paralytic shellfish poisoning toxins. Annually recurrent blooms of this dinoflagellate species is associated with the incremental nitrogen influx, especially excessive nitrate input. However, limited studies have been conducted on the toxin production and underlying molecular regulation mechanisms of A. tamarense under various nitrate (N) conditions. Therefore, toxin production and transcriptomic responses of this species were investigated. The toxin profile of A. tamarense was consistently dominated by the C2-toxins, and the cellular toxicity increased with N concentrations peaking at 9.23 ± 0.03 fmol/cell in the 883 µM N-added group. Under lower N conditions, expressions of two STX-core genes, sxtA and sxtG, were significantly down-regulated, suggesting that N regulated sxt expression and triggered responses related to toxin biosynthesis. Results of this study provided valuable insights into the ecophysiology of A. tamarense, enhancing our understanding of the occurrence of toxification events in natural environments.

Allelochemicals of Alexandrium tamarense and its algicidal mechanism for Prorocentrum donghaiense and Heterosigma akashiwo.

The effects of culture filtrate of Alexandrium tamarense on Prorocentrum donghaiense and Heterosigma akashiwo were investigated, including determination of algal density, photosynthesis, intracellular enzyme content and activity. The filtrate of A. tamarense had a stronger inhibitory effect on P. donghaiense than H. akashiwo, and the inhibitory effect decreased with higher temperature treatment of the filtrate. Instantaneous fluorescence (Ft) and maximum quantum yield of photosystem II (Fv/Fm) values of both kinds of target algae were reduced as exposed to the filtrate of A. tamarense, which proved that allelopathy could inhibit the normal operation of photosynthetic system. The increase of Malondialdehyde (MDA) content of the two kinds of target algae indicated that the cell membrane was seriously damaged by allelochemicals released by A. tamarense. The different responses of Superoxide Dismutase (SOD) and Catalase (CAT) activity in two kinds of target algae demonstrated the complexity and diversity of allelopathic mechanism. The filtrate of A. tamarense also influenced the metabolic function (ATPases) of P. donghaiense and H. akashiwo, and the influence on P. donghaiense was greater. Liquid-liquid extraction was used to extract and isolate allelochemicals from the filtrate of A. tamarense. It was found that only component I with molecular weight of 424.2573 and 434.2857 could inhibit the growth of P. donghaiense by HPLC-MS.

ROS meditated paralytic shellfish toxins production changes of Alexandrium tamarense caused by microplastic particles.

A variety of studies have investigated the toxic effects of microplastics (MPs) on microalgae, but few of them considered their influence on dinoflagellate toxins production, which could cause significant ecological safety concerns in coastal areas. This research investigated the impacts of 5 µg L-1 and 5 mg L-1 polystyrene (PS) MPs on the changes of paralytic shellfish toxins (PSTs) production and their relationship with cellular oxidative stress of Alexandrium tamarense, a common harmful algal blooms causative dinoflagellate. The results showed elevation of reactive oxygen species (ROS) levels, activation of antioxidant system and overproduction of PSTs were positively correlated under PS MPs exposure (especially under 5 mg L-1 PS MPs), and the PSTs changes were eliminated by the ROS inhibitor. Further transcriptomic analysis revealed that ROS could enhance biosynthesis of glutamate, providing raw materials for PSTs precursor arginine, accompanied with enhanced acetyl-CoA and ATP production, finally leading to the overproduction of PSTs. Moreover, the oxidative intracellular environments might block the reduction process from STX to C1&C2, leading to the increase of STX and decrease of C1&C2 proportions. This work brings the first evidence that ROS could mediate PSTs production and compositions of Alexandrium under MPs exposure, with important scientific and ecological significance.

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.

The effects of two sized polystyrene nanoplastics on the growth, physiological functions, and toxin production of Alexandrium tamarense.

Micro- and nano-plastics (MNPs) are increasingly prevalent pollutants in marine ecosystems and result in various deleterious effects on marine organisms. There have been studies evaluated the toxic effects of MNPs on marine microalgae, but few of them focused on the effects of MNPs on dinoflagellate species and their toxins production, which could have significant implications on human health and ecological safety in coastal areas. In this study, the common harmful algal blooms-causing dinoflagellate Alexandrium tamarense was exposed to 0.1 and 1 µm sized polystyrene nanoplastics (NPs) to investigate the responding patterns of population growth, multiple physiological functions, as well as the intracellular paralytic shellfish toxins (PSTs) productions. The results indicated the population growth, photosynthetic parameters, nutrients (nitrate and phosphate) uptake rates and extracellular carbonic anhydrase activities (CAext) were all inhibited by the two sized NPs, accompanied by the prolonged and more aggregated microalgal cells under the observation of scanning electron microscope (SEM), and the inhibition effects were more severe under 1 µm sized NPs than 0.1 µm sized NPs. Finally, we found the intracellular PSTs contents increased 73.59% exposed to 0.1 µm sized NPs while decreased 85.50% exposed to 1 µm sized NPs comparing the controls at 96 h, without significant changes of relative compositions. These results provided evidence that MNPs were toxic to A. tamarense and affected their intracellular PSTs productions within 96 h, which is critical to consider when evaluating the potential risks of MNPs in marine ecosystems.

Comparison between mouse bioassay and HILIC-MS/MS for quantification of paralytic shellfish toxin in Japanese basket clams and mussels caught off coastal Osaka Bay in Japan.

The content and composition of paralytic shellfish toxins (PSTs) in Japanese basket clam (Corbicula japonica) and mussels (Mytilus galloprovincialis) from Osaka Bay, Japan, were investigated using a mouse bioassay (MBA) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), and the association between toxicity values of MBA and HILIC-MS/MS was verified based on research data. The overall toxicity in Japanese basket clam was lower than that in the mussel. The PSTs of Japanese basket clam and mussel consisted mainly of C1, C2, and gonyautoxins 1-4 (GTX1-4) taking toxins compositional differences as mol%. When multiplying the content of different toxins by the toxic equivalent factor (TEF), C2 and GTX1-4 accounted for more than 90% of total toxicity (MU TEF/g) based on the MU TEF score converted by TEF for the two species. The total content of C2 and GTX1-4 converted to toxicity was significantly correlated with the toxicity determined by MBA for the two species (r2 > 0.983). This study provides a suitable and ethical monitoring method to investigate toxicity in bivalves contaminated with A. tamarense by analysis of only predominant toxins, along with reducing use of MBA.

Nitratireductor alexandrii sp. nov., from phycosphere microbiota of toxic marine dinoflagellate Alexandrium tamarense.

A taxonomic study was carried out on a novel algae-associated bacterial strain Z3-1T, which was isolated from phycosphere microbiota of toxic marine dinoflagellate Alexandrium tamarense 880. Cells of strain Z3-1T were Gram-stain-negative, rod-shaped and strictly aerobic and were motile by means of flagella. Strain Z3-1T grew at 25-42 °C, pH 5.0-10.0 and 1.0-5.0 % (w/v) NaCl. Strain Z3-1T reduced nitrate to nitrite, but did not reduce nitrite to nitrogen gas. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Z3-1T belongs to the genus Nitratireductor showing the highest sequence similarity (97.0 %) to Nitratireductor basaltis JCM 14935T. The average nucleotide identity and digital DNA-DNA hybridization relatedness between strain Z3-1T and type strains of genus Nitratireductor with available genome sequences were in the ranges of 72.4-74.4 % and 22.7-23.3 %, respectively. The major fatty acids were summed in feature 8 (C18:1 ω7c and/or C18:1 ω6c), C19:0 ω8c cyclo, C18:1 ω7c 11-metyl and iso-C17:0. The major polar lipids were determined as diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids and four unidentified polar lipids. The genomic DNA G+C content calculated from genome sequence was 65.6 mol%. Based on genotypic, chemotaxonomic and phenotypic data obtained, strain Z3-1T represents a novel species of the genus Nitratireductor, for which the name Nitratireductor alexandrii sp. nov. is proposed with the type strain Z3-1T (=KCTC 62458T=CCTCC AB 2017227T).

Temporal Variation of the Profile and Concentrations of Paralytic Shellfish Toxins and Tetrodotoxin in the Scallop, Patinopecten yessoensis, Cultured in a Bay of East Japan.

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, Patinopecten yessoensis, in 1993; however, no new data have emerged since then. In this study, we simultaneously analyzed PSTs and TTX in scallops cultured in a bay of east Japan using hydrophilic interaction chromatography (HILIC)-MS/MS. These scallops were temporally collected from April to December 2017. The highest concentration of PSTs (182 µmol/kg, total congeners) in the hepatopancreas was detected in samples collected on May 23, lined to the cell density of the dinoflagellate, Alexandrium tamarense, in seawater around the scallops, whereas the highest concentration of TTX (421 nmol/kg) was detected in samples collected on August 22. Contrary to the previous report, temporal variation of the PSTs and TTX concentrations did not coincide. The highest concentration of TTX in the entire edible tissues was 7.3 µg/kg (23 nmol/kg) in samples obtained on August 22, which was lower than the European Food Safety Authority (EFSA)-proposed threshold, 44 µg TTX equivalents/kg shellfish meat. In addition, 12ß-deoxygonyautoxin 3 was firstly identified in scallops.

Optimized culturing conditions for an algicidal bacterium Pseudoalteromonas sp. SP48 on harmful algal blooms caused by Alexandrium tamarense.

Bacteria play an important role in preventing algal blooms and reducing their harm to the environment. To improve the algicidal activity of Pseudoalteromonas SP48 which had an inhibition effect on dinoflagellate Alexandrium tamarense, its growth medium and fermentation conditions were optimized for this bacterium. In this study, we used two steps to establish the optimum conditions. First, the proper proportion of medium was selected based on an orthogonal design. Then, the fermentation conditions were further optimized through uniform design in an enlarged 5L bioreactor. To test the algicidal ability of Pseudoalteromonas SP48 under the optimum conditions, algal cell morphology was observed by transmission electron microscopy (TEM). After the orthogonal design, we found that the optimum medium was [0.7% (m/v) tryptone, 0.2% (m/v) soluble starch, 0.2% (m/v) sucrose, 0.1% (m/v) FeSO4 , and 1.2% (m/v) K2 HPO4 ] for Pseudoalteromonas SP48 growth. Based on these results, optimum fermentation conditions were further explored in a 5L fermentation cylinder using a uniform design; the influence of variables such as incubation time, carbon type, and rotation speed were tested. The optimal fermentation conditions were fermentation time (42 hr), tryptone (1.1%), seeding volume (1.4 × 1013  cells), and rotation speed (250 r/min). Under these established optimum conditions, the biomass of strain SP48 increased by 79.2% and its lethal dose 50% (LD50 ) decreased by 54.0%, respectively. The TEM results showed that compared with the control group, the cell wall and cell membrane of A. tamarense were significantly damaged, and the structure and shape of the organelles were destroyed by algicidal bacteria of Pseudoalteromonas SP48. Overall, our results demonstrate that the optimized culture conditions could significantly enhance the algicidal activity of Pseudoalteromonas SP48 against a harmful dinoflagellate, such as A. tamarense. It will effectively provide a scientific foundation for both production of algicidal substances and HABs control.

Interactive effect of nitrogen source and high CO2 concentration on the growth of the dinoflagellate Alexandrium tamarense and its toxicity to zebrafish (Danio rerio) embryos.

The effects and interactive effects of different nitrogen (N) sources (ammonium, nitrate, and urea) and carbon dioxide (CO2) concentrations were investigated on Alexandrium tamarense, a harmful marine dinoflagellate, by measuring its growth (µ), extracellular carbonic anhydrase (CA), and its toxicity to zebrafish (Danio rerio) embryo. The µ and CA were influenced more strongly by CO2 concentrations rather than by N sources; significant effects of CO2 on µ and CA were observed under low CO2 concentration (LC) conditions compared to high CO2 concentration (HC) conditions. The ammonium and nitrate media under LC conditions had the maximum µ and CA, which was inhibited under HC conditions. The embryotoxic effects were influenced more strongly by the N sources than by CO2 concentrations, thus excluding the lower deformation in urea under HC conditions. Moreover, the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST), and catalase (CAT) were detected in normal (untreated) zebrafish embryos, and among them, the level of SOD was the highest. In summary, this study provides a clear insight for understanding the effects and interactive effects of N sources and CO2 concentrations on the growth and toxicity of harmful dinoflagellates.

Screening of cyclic imine and paralytic shellfish toxins in isolates of the genus Alexandrium (Dinophyceae) from Atlantic Canada.

The dinoflagellate genus Alexandrium Halim has frequently been associated with harmful algal blooms. Although a number of species from this genus are known to produce paralytic shellfish toxins (PST) and/or cyclic imines (CI), studies on comprehensive toxin profiling using techniques capable of detecting the full range of PST and CI analogues are limited. Isolates of Alexandrium spp. from Atlantic Canada were analyzed by targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS). Results showed a number of distinct profiles and wide ranging cell quotas of PST and spirolides (SPX) in both A. catenella (Whedon & Kofoid) Balech and A. ostenfedii (Paulsen) Balech & Tangen. The concentration of PST in A. catenella ranged from 0.0029 to 54 fmol cell-1 with the major components being C2 and GTX4. In addition, putative PST metabolites were confirmed for the first time in A. catenella by high resolution MS/MS. By comparison, A. ostenfeldii isolates showed much lower concentrations of PST (

Dynamics of paralytic shellfish toxins and their metabolites during timecourse exposure of scallops Chlamys farreri and mussels Mytilus galloprovincialis to Alexandrium pacificum.

New C-11 hydroxyl metabolites of paralytic shellfish toxins (PSTs) have been reported in shellfish. To gain further information on these metabolites, as well as the potential for formation of phase-II metabolites and acyl esters of PSTs, bivalves were fed with the PSTs-producing dinoflagellate Alexandrium pacificum (strain ATHK). Through independent experiments, scallops (Chlamys farreri) were fed for 9 days and mussels (Mytilus galloprovincialis) for 5 days plus an additional 5 days of depuration, with representative samples taken throughout. Several common PSTs (C1-4, GTX1-6 and NEO) and metabolites including M1, M3, M5, M7, M9, M2 and M8 were detected in the hepatopancreas of scallops during toxin accumulation and in the hepatopancreas of mussels during both toxin accumulation and elimination periods. The relative molar ratio of metabolites to precursor molecules was used to estimate relative metabolic conversion rates. Conversion rates of C1/2 and GTX2/3 were higher than those of C3/4 and GTX1/4, in scallops and mussels. The first metabolites observed in both bivalve species investigated were M1/3, which are formed from C1/2. However, the conversion of GTX2/3 to M2 was more complete than other biotransformation reactions in both mussels and scallops. In general, metabolic conversion of PSTs was observed after a shorter time and to a greater extent in mussels than in scallops in the exposure period. No acyl esters or conjugation products of PSTs with glucuronic acid, glutathione, cysteine and taurine were detected by liquid chromatography with high resolution tandem mass spectrometry in the samples investigated. Additionally, only GTX1/4 and GTX2/3 were detected in the kidney of scallops, which demonstrates that PSTs are mainly metabolized through the hepatic metabolism pathway in bivalves. This work improves the understanding of PST metabolism during toxin accumulation and depuration in commercially harvested shellfish.

Effect of CO2 on growth and toxicity of Alexandrium tamarense from the East China Sea, a major producer of paralytic shellfish toxins.

In recent decades, the frequency and intensity of harmful algal blooms (HABs), as well as a profusion of toxic phytoplankton species, have significantly increased in coastal regions of China. Researchers attribute this to environmental changes such as rising atmospheric CO2 levels. Such addition of carbon into the ocean ecosystem can lead to increased growth, enhanced metabolism, and altered toxicity of toxic phytoplankton communities resulting in serious human health concerns. In this study, the effects of elevated partial pressure of CO2 (pCO2) on the growth and toxicity of a strain of Alexandrium tamarense (ATDH) widespread in the East and South China Seas were investigated. Results of these studies showed a higher specific growth rate (0.31±0.05day-1) when exposed to 1000µatm CO2, (experimental), with a corresponding density of (2.02±0.19)×107cellsL-1, that was significantly larger than cells under 395µatm CO2(control). These data also revealed that elevated pCO2 primarily affected the photosynthetic properties of cells in the exponential growth phase. Interestingly, measurement of the total toxin content per cell was reduced by half under elevated CO2 conditions. The following individual toxins were measured in this study: C1, C2, GTX1, GTX2, GTX3, GTX4, GTX5, STX, dcGTX2, dcGTX3, and dcSTX. Cells grown in 1000µatm CO2 showed an overall decrease in the cellular concentrations of C1, C2, GTX2, GTX3, GTX5, STX, dcGTX2, dcGTX3, and dcSTX, but an increase in GTX1 and GTX4. Total cellular toxicity per cell was measured revealing an increase of nearly 60% toxicity in the presence of elevated CO2 compared to controls. This unusual result was attributed to a significant increase in the cellular concentrations of the more toxic derivatives, GTX1 and GTX4.Taken together; these findings indicate that the A. tamarense strain ATDH isolated from the East China Sea significantly increased in growth and cellular toxicity under elevated pCO2 levels. These data may provide vital information regarding future HABs and the corresponding harmful effects as a result of increasing atmospheric CO2.

Proposed Biotransformation Pathways for New Metabolites of Paralytic Shellfish Toxins Based on Field and Experimental Mussel Samples.

A seafood poisoning event occurred in Qinhuangdao, China, in April 2016. Subsequently, the causative mussels (Mytilus galloprovincialis) were harvested and analyzed to reveal a high concentration [∼10 758 µg of saxitoxin (STX) equiv kg-1] of paralytic shellfish toxins (PSTs), including gonyautoxin (GTX)1/4 and GTX2/3, as well as new metabolites 11-hydroxy-STX (M2), 11,11-dihydroxy-STX (M4), open-ring 11,11-dihydroxy-STX (M6), 11-hydroxy-neosaxitoxin (NEO) (M8), and 11,11-dihydroxy-NEO (M10). To understand the origin and biotransformation pathways of these new metabolites, uncontaminated mussels (M. galloprovincialis) were fed with either of two Alexandrium tamarense strains (ATHK and TIO108) under laboratory conditions. Similar PST metabolites were also detected in mussels from both feeding experiments. Results supposed that 11-hydroxy-C2 toxin (M1) and 11,11-dihydroxy-C2 (M3) are transformed from C2, while 11-hydroxy-C4 toxin (M7) and 11,11-dihydroxy-C4 (M9) are converted from C4. In addition, the metabolites M2, M4, and M6 appear to be products of GTX2/3, and the metabolites M8 and M10 are likely derived from GTX1/4.

The physiological adaptations and toxin profiles of the toxic Alexandrium fundyense on the eastern Bering Sea and Chukchi Sea shelves.

Abundant cyst distributions of the toxic dinoflagellate Alexandrium fundyense (previous A. tamarense north American clade) were recently observed on the north Chukchi Sea shelf and on the eastern Bering Sea shelf, suggesting that A. fundyense is both highly adapted to the local environments in the high latitude areas and might cause toxin contamination of plankton feeders. However, little is known about the physiological characteristics and toxin profiles of A. fundyense in these areas, which are characterized by low water temperatures, weak sunlight, and more or less permanent ice cover during winter. To clarify the physiological characteristics of A. fundyense, the effects of water temperature and light intensity on the vegetative growth and toxin profiles of this species were examined using A. fundyense strains isolated from one sediment sample collected from each area. Using the same sediments samples, seasonal changes of the cyst germination in different water temperatures were investigated. Vegetative cells grew at temperatures as low as 5°C and survived at 1°C under relatively low light intensity. They also grew at moderate water temperatures (10-15°C). Their cysts could germinate at low temperatures (1°C) and have an endogenous dormancy period from late summer to early spring, and warmer water temperatures (5-15°C) increased germination success. These physiological characteristics suggest that A. fundyense in the Chukchi Sea and eastern Bering Sea is adapted to the environments of high latitude areas. In addition, the results suggest that in the study areas A. fundyense has the potential to germinate and grow when water temperatures increase. Cellular toxin amounts of A. fundyense strains from the eastern Bering Sea and Chukchi Sea were ranged from 7.2 to 38.2 fmol cell-1. These toxin amounts are comparable with A. fundyense strains isolated from other areas where PSP toxin contamination of bivalves occurs. The dominant toxin of the strains isolated from the Chukchi Sea was saxitoxin, while most A. fundyense strains from the eastern Bering Sea are dominated by the C2 toxin. Toxin profiles similar to those detected in Chukchi Sea have not been reported by any previous research. The dominance of a highly toxic PST variant in Chukchi A. fundyense suggests that presence of the species at low cell concentrations may cause toxin contamination of predators. This study revealed that abundant A. fundyense cysts deposited on the eastern Bering Sea and Chukchi Sea shelves potentially germinate and grow with PSP toxin contents in the local environments. In conclusion, a high risk of PSP occurrences exists on the eastern Bering Sea and Chukchi Sea shelves.

Comparative performance of four immunological test kits for the detection of Paralytic Shellfish Toxins in Tasmanian shellfish.

Blooms of the toxic dinoflagellate Alexandrium tamarense (Group 1) seriously impacted the Tasmanian shellfish industry during 2012 and 2015, necessitating product recalls and intensive paralytic shellfish toxin (PST) product testing. The performance of four commercial PST test kits, Abraxis™, Europroxima™, Scotia™ and Neogen™, was compared with the official AOAC LC-FLD method for contaminated mussels and oysters. Abraxis and Europroxima kits underestimated PST in 35-100% of samples when using standard protocols but quantification improved when concentrated extracts were further diluted (underestimation ≤18%). The Scotia kit (cut off 0.2-0.7 mg STX-diHCl eq/kg) delivered 0% false negatives, but 27% false positives. Neogen produced 5% false negatives and 13% false positives when the cut off was altered to 0.5-0.6 mg STX-diHCl eq/kg, the introduction of a conversion step eliminated false negatives. Based on their sensitivity, ease of use and performance, the Neogen kit proved the most suitable kit for use with Tasmanian mussels and oysters. Once formally validated for regulatory purposes, the Neogen kit could provide shellfish growers with a rapid tool for harvesting decisions at the farm gate. Effective rapid screening preventing compliant samples undergoing testing using the more expensive and time consuming LC-FLD method will result in significant savings in analytical costs.

qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays.

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

Column switching combined with hydrophilic interaction chromatography-tandem mass spectrometry for the analysis of saxitoxin analogues, and their biosynthetic intermediates in dinoflagellates.

Hydrophilic-interaction chromatography (HILIC) is reportedly useful for the analysis of saxitoxin (STX) analogues, collectively known as paralytic shellfish toxins. Column switching and two-step gradient elution using HILIC combined with mass spectrometry enabled the simultaneous analysis of the 15 primary STX analogues and their biosynthetic intermediates, arginine, Int-A', and Int-C'2, and the shunt product, Cyclic-C'. Crude extracts of toxin-producing dinoflagellates can be injected without any treatment except filtration. Enrichment of the compounds using this method was highly reproducible with respect to retention times (% RSD was under 1%) and highly sensitive (limits of detection (LODs) were in the range 0.9 (Int-C'2) - 116 (C3) µM) in terms of avoiding matrix effects associated with co-eluting substances. Validation studies demonstrated acceptable performance of this method for specificity, repeatability, linearity and recovery. A comparison of the quantitative results for STX analogues in Alexandrium tamarense using HPLC with post-column fluorescent derivatization and the column-switching HILIC-MS method revealed good agreement. The presence of Int-A', Int-C'2, and Cyclic-C' in toxic dinoflagellate species with different toxin profiles was confirmed using this method. Our data support the hypothesis that the early stages of the STX biosynthesis and shunt pathways are the same in dinoflagellates and cyanobacteria.

Molecularly imprinted polymer as efficient sorbent of solid-phase extraction for determination of gonyautoxin 1,4 in seawater followed by high-performance liquid chromatography-fluorescence detection.

A kind of new molecularly imprinted polymer (MIP) was synthesized by bulk polymerization using guanosine as dummy template molecule, α-methacrylic acid as functional monomer and ethylene glycol dimethyl acrylic ester as crosslinker. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) showed that the MIP had homogenous and uniform-sized cavities. It was confirmed that the MIP had higher binding affinity and selectivity towards gonyautoxins 1,4 (GTX 1,4) than the non-imprinted polymer (NIP) according to the static equilibrium adsorption. An off-line molecularly imprinted solid-phase extraction (MISPE) method followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was established for the analysis of GTX 1,4. 0.1 mol/L acetic acid and 95:5 (v:v) methanol/water were optimized as the washing and elution solutions, respectively. The recoveries of spiked cultured seawater samples were satisfactory, as high as 88 %. Using this method, the concentrations of GTX 1,4 from cultured seawater samples of Alexandrium minutum and Alexandrium tamarense were detected to be 1.10 µg/L and 0.99 µg/L, respectively. Graphical Abstract The synthesis of molecularly imprinted polymer and molecularly imprinted solid-phase extraction analysis for gonyautoxin 1,4.