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The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC–MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26–C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC–MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90–92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC–MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26–C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC–MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90–92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

Azaspiracids (AZAs) are a group of biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. Of the 60 AZAs identified, levels of AZA1, AZA2, and AZA3 are regulated in shellfish as a food safety measure based on occurrence and toxicity. Information about the metabolism of AZAs in shellfish is limited. Therefore, a fraction of blue mussel hepatopancreas was made to study the metabolism of AZA1–3 in vitro. A range of AZA metabolites were detected by liquid chromatography–high-resolution tandem mass spectrometry analysis, most notably the novel 22α-hydroxymethylAZAs AZA65 and AZA66, which were also detected in naturally contaminated mussels. These appear to be the first intermediates in the metabolic conversion of AZA1 and AZA2 to their corresponding 22α-carboxyAZAs (AZA17 and AZA19). α-Hydroxylation at C-23 was also a prominent metabolic pathway, producing AZA8, AZA12, and AZA5 as major metabolites of AZA1–3, respectively, and AZA67 and AZA68 as minor metabolites via double-hydroxylation of AZA1 and AZA2, but only low levels of 3β-hydroxylation were observed in this study. In vitro generation of algal toxin metabolites, such as AZA3, AZA5, AZA6, AZA8, AZA12, AZA17, AZA19, AZA65, and AZA66 that would otherwise have to be laboriously purified from shellfish, has the potential to be used for the production of standards for analytical and toxicological studies.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin 2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL−1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L−1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Boronates bind reversibly to vic-diols, a common structural feature of algal toxins. This boronate–diol interaction can be exploited for selective toxin clean-up and concentration. Boric acid gel (BAG) solid phase extraction (SPE) was recently shown to eliminate interferences and matrix effects in LC-MS analyses of azaspiracids (AZAs) in mussel extracts. Here, we report a modified approach for cleanup of tetrodotoxin (TTX) and many of its congeners, which also contain vic-diols. The reaction between TTX and boronic acids was first investigated in solution to optimize conditions for TTX binding. Then, TTX-contaminated mussel extracts were applied to BAG SPE columns. TTXs were selectively bound and released from the BAG to yield very clean extracts containing TTX analogues and very little else. Potential interferences in LC-MS analyses, such as arginine and other amino acids, were completely eliminated, making this a promising approach for analytical sample preparation.

The purpose of this work is to review all the historical monitoring data gathered by the Marine Institute, the national reference laboratory for marine biotoxins in Ireland, including all the biological and chemical data from 2005 to 2017, in relation to diarrheic shellfish poisoning (DSP) toxicity in shellfish production. The data reviewed comprises over 25,595 water samples, which were preserved in Lugol’s iodine and analysed for the abundance and composition of marine microalgae by light microscopy, and 18,166 records of shellfish flesh samples, which were analysed using LC-MS/MS for the presence and concentration of the compounds okadaic acid (OA), dinophysistoxins-1 (DTX-1), dinophysistoxins-2 (DTX-2) and their hydrolysed esters, as well as pectenotoxins (PTXs). The results of this review suggest that DSP toxicity events around the coast of Ireland occur annually. According to the data reviewed, there has not been an increase in the periodicity or intensity of such events during the study period. Although the diversity of the Dinophysis species on the coast of Ireland is large, with 10 species recorded, the two main species associated with DSP events in Ireland are D. acuta and D. acuminata. Moreover, the main toxic compounds associated with these species are OA and DTX-2, but concentrations of the hydrolysed esters are generally found in higher amounts than the parent compounds in the shellfish samples. When D. acuta is dominant in the water samples, the DSP toxicity increases in intensity, and DTX-2 becomes the prevalent toxin. Pectenotoxins have only been analysed and reported since 2012, and these compounds had not been associated with toxic events in Ireland; however, in 2014, concentrations of these compounds were quantitated for the first time, and the data suggest that this toxic event was associated with an unusually high number of observations of D. tripos that year. The areas of the country most affected by DSP outbreaks are those engaging in long-line mussel (Mytilus edulis) aquaculture.

Dinoflagellate species of Dinophysis are obligate mixotrophs that require light, nutrients, and prey for sustained growth. Information about their nitrogenous nutrient preferences and their uptake kinetics are scarce. This study aimed to determine the preferred nitrogen sources in cultures of D. acuminata and D. acuta strains from the Galician Rías Baixas (NW Spain) and to compare their uptake kinetics. Well-fed versus starved cultures of D. acuminata and D. acuta were supplied with N15 labeled inorganic (nitrate, ammonium) and organic (urea) nutrients. Both species showed a preference for ammonium and urea whereas uptake of nitrate was negligible. Uptake rates by well-fed cells of D. acuminata and D. acuta were 200% and 50% higher, respectively, than by starved cells. Uptake of urea by D. acuminata was significantly higher than that of ammonium in both nutritional conditions. In contrast, similar uptake rates of both compounds were observed in D. acuta. The apparent inability of Dinophysis to take up nitrate suggests the existence of incomplete nitrate-reducing and assimilatory pathways, in line with the paucity of nitrate transporter homologs in the D. acuminata reference transcriptome. Results derived from this study will contribute to understand Harmful Algal Blooms succession and differences in the spatio-temporal distribution of the two Dinophysis species when they co-occur in stratified scenarios.

Dinoflagellate species of Dinophysis, in particular D. acuminata and D. acuta, produce lipophilic toxins that pose a threat to human health when concentrated in shellfish and jeopardize shellfish exploitations in western Europe. In northwestern Iberia, D. acuminata has a long growing season, from spring to early autumn, and populations develop as soon as shallow stratification forms when the upwelling season begins. In contrast, D. acuta blooms in late summer, when the depth of the pycnocline is maximal and upwelling pulses are moderate. In situ observations on the hydrodynamic regimes during the two windows of opportunity for Dinophysis species led us to hypothesize that D. acuta should be more sensitive to turbulence than D. acuminata. To test this hypothesis, we studied the response of D. acuminata and D. acuta to three realistic turbulence levels elow (LT), ε ≈ 10 −6 m 2 s-3 ; medium (MT), ε ≈ 10-5 m 2 s-3 and high (HT), ε ≈ 10-4 m 2 s-3 egenerated by Turbogen, a highly reproducible, computer-controlled system. Cells of both species exposed to LT and MT grew at rates similar to the controls. Marked differences were found in the response to HT: D. acuminata grew slowly after an initial lag phase, whereas D. acuta cell numbers declined. Results from this study support the hypothesis that turbulence may play a role in shaping the spatio-temporal distribution of individual species of Dinophysis. We also hypothesize that, in addition to cell disturbance affecting division, sustained high shear generated by microturbulence may cause a decline in Dinophysis numbers due to decreased densities of ciliate prey.

Azaspiracids (AZAs) are microalgal toxins that can accumulate in shellfish and lead to human intoxications. To facilitate their study and subsequent biomonitoring, purification from microalgae rather than shellfish is preferable; however, challenges remain with respect to maximizing toxin yields. The impacts of temperature, growth media, and photoperiod on cell densities and toxin production in Azadinium spinosum were investigated. Final cell densities were similar at 10 and 18 °C, while toxin cell quotas were higher (~3.5-fold) at 10 °C. A comparison of culture media showed higher cell densities and AZA cell quotas (2.5–5-fold) in f10k compared to f/2 and L1 media. Photoperiod also showed differences, with lower cell densities in the 8:16 L:D treatment, while toxin cell quotas were similar for 12:12 and 8:16 L:D treatments, but slightly lower for the 16:8 L:D treatment. AZA1, -2, and -33 were detected during the exponential phase, while some known and new AZAs were only detected once the stationary phase was reached. These compounds were additionally detected in field water samples during an AZA event.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 μg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels (Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20–C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

Azaspiracids (AZAs) are a group of biotoxins that appear periodically in shellfish and can cause food poisoning in humans. Current methods for quantifying the regulated AZAs are restricted to LC-MS but are not well suited to detecting novel and unregulated AZAs. An ELISA method for total AZAs in shellfish was reported recently, but unfortunately, it used relatively large amounts of the AZA-1-containing plate-coating conjugate, consuming significant amounts of pure AZA-1 per assay. Therefore, a new plate-coater, OVA–cdiAZA1 was produced, resulting in an ELISA with a working range of 0.30–4.1 ng/mL and a limit of quantification of 37 μg/kg for AZA-1 in shellfish. This ELISA was nearly twice as sensitive as the previous ELISA while using 5-fold less plate-coater. The new ELISA displayed broad cross-reactivity toward AZAs, detecting all available quantitative AZA reference materials as well as the precursors to AZA-3 and AZA-6, and results from shellfish analyzed with the new ELISA showed excellent correlation (R2 = 0.99) with total AZA-1–10 by LC-MS. The results suggest that the new ELISA is suitable for screening samples for total AZAs, even in cases where novel AZAs are present and regulated AZAs are absent, such as was reported recently from Puget Sound and the Bay of Naples.

Photosynthetic species of the genus Dinophysis are obligate mixotrophs with temporary plastids (kleptoplastids) that are acquired from the ciliate Mesodinium rubrum, which feeds on cryptophytes of the Teleaulax-Plagioselmis-Geminigera clade. A metabolomic study of the three-species food chain Dinophysis-Mesodinium-Teleaulax was carried out using mass spectrometric analysis of extracts of batch-cultured cells of each level of that food chain. The main goal was to compare the metabolomic expression of Galician strains of Dinophysis acuminata and D. acuta that were subjected to different feeding regimes (well-fed and prey-limited) and feeding on two Mesodinium (Spanish and Danish) strains. Both Dinophysis species were able to grow while feeding on both Mesodinium strains, although differences in growth rates were observed. Toxin and metabolomic profiles of the two Dinophysis species were significantly different, and also varied between different feeding regimes and different prey organisms. Furthermore, significantly different metabolomes were expressed by a strain of D. acuminata that was feeding on different strains of the ciliate Mesodinium rubrum. Both species-specific metabolites and those common to D. acuminata and D. acuta were tentatively identified by screening of METLIN and Marine Natural Products Dictionary databases. This first metabolomic study applied to Dinophysis acuminata and D.acuta in culture establishes a basis for the chemical inventory of these species.

Kleptoplastic mixotrophic species of the genus Dinophysis are cultured by feeding with the ciliate Mesodinium rubrum, itself a kleptoplastic mixotroph, that in turn feeds on cryptophytes of the Teleaulax/Plagioselmis/Geminigera (TPG) clade. Optimal culture media for phototrophic growth of D. acuminata and D. acuta from the Galician Rías (northwest Spain) and culture media and cryptophyte prey for M. rubrum from Huelva (southwest Spain) used to feed Dinophysis, were investigated. Phototrophic growth rates and yields were maximal when D. acuminata and D. acuta were grown in ammonia-containing K(-Si) medium versus f/2(-Si) or L1(-Si) media. Dinophysis acuminata cultures were scaled up to 18 L in a photobioreactor. Large differences in cell toxin quota were observed in the same Dinophysis strains under different experimental conditions. Yields and duration of exponential growth were maximal for M. rubrum from Huelva when fed Teleaulax amphioxeia from the same region, versus T. amphioxeia from the Galician Rías or T. minuta and Plagioselmis prolonga. Limitations for mass cultivation of northern Dinophysis strains with southern M. rubrum were overcome using more favorable (1:20) Dinophysis: Mesodinium ratios. These subtleties highlight the ciliate strain-specific response to prey and its importance to mass production of M. rubrum and Dinophysis cultures. Key Contribution: Phototrophic growth of D. acuminata and D. acuta was maximal in ammonia-containing K media; ciliate M. rubrum and its cryptophyte prey had higher growth rate and yields with f/2(-Si). Sustained growth and yields in mixotrophic cultures of Dinophysis were maximal when M. rubrum was grown with the cryptophyte T. amphioxeia from the same location as prey; additionally, high growth rates were achieved with high ratios of prey (1:20, Dinophysis: M. rubrum) grown with T. minuta and P. prolonga.

Dinoflagellates of the genus Dinophysis are the most persistent producers of lipophilic shellfish toxins in western Europe. Their mixotrophic nutrition requires a food‐chain of cryptophytes and plastid‐bearing ciliates for sustained growth and photosynthesis. In this study, cultures of D. acuminata and D. acuta, their ciliate prey Mesodinium rubrum and the cryptophyte, Teleaulax amphioxeia, were subject to three experimental settings to study their physiological response to different combinations of light intensity and quality. Growth rates, pigment analyses (HPLC), photosynthetic parameters (PAM‐fluorometry) and cellular toxin content (LC‐MS) were determined. Specific differences in photosynthetic parameters were observed in Dinophysis exposed to different photon fluxes (10‐650 μmol photons · m⁻² · s⁻¹), light quality (white, blue and green) and shifts in light regime. Dinophysis acuta was more susceptible to photodamage under high light intensities (370‐650 μmol photons · m⁻²· s⁻¹) than D. acuminata but survived better with low light (10 μmol photons · m⁻² · s⁻¹) and to a prolonged period (28 d) of darkness. Mesodinium rubrum and T. amphioxeia showed their maximal growth rate and yield under white and high light whereas Dinophysis seemed better adapted to grow under green and blue. Toxin analyses in Dinophysis showed maximal toxin per cell under high light after prey depletion at the late exponential‐plateau phase. Changes observed in photosynthetic light curves of D. acuminata cultures after shifting light conditions from low intensity‐blue light to high intensity‐white light seemed compatible with photoacclimation in this species. Results obtained here are discussed in relation to different spatio‐temporal distributions observed in field populations of D. acuminata and D. acuta in northwestern Iberia. This article is protected by copyright. All rights reserved.

Immunoaffinity columns are widely used in sample preparation for the analysis of mycotoxins. However, despite the availability of antibodies with broad specificity to many families of algal toxins, little use has been made of these antibodies in immunoaffinity columns. We have developed immunoaffinity columns using antibodies originally produced for ELISA at the Norwegian Veterinary Institute. Columns targeting the microcystin/nodularin, okadaic acid, azaspiracid, yessotoxin, and pinnatoxin/spirolide families were produced and then tested on a range of sample matrices including algal blooms, cyanobacterial “health food” supplements, algal cultures and shellfish. Results showed high recoveries with remarkable clean-up of samples, removing contaminants from the sample matrix and reducing the matrix effects in subsequent LC-MS analyses. The columns will make it possible to improve analytical capacity in labs without the most expensive LC-MS-equipment. They could also simplify semitargeted metabolomics studies of various types of samples. As an example, the microcystin-antibodies resulted in columns capturing standards of MC-LR, MC-RR, MC-YR, MC-LA, MC-LY , MC-LW, MC-LF, [D-Asp3]MC-LR, [D-Asp3]MC-RR and NOD-R with recoveries of 78-83% eluted within the first 3 mL and 92-104% within 9 mL. The total capacity was ~700 ng for a column with ~1.4 mL gel and preliminary attempts suggest the columns to be reusable with similar recovery at least five times. These columns were highly effective for cleaning up natural bloom, culture, and food supplement samples for LC-MS analysis.

This file contains color photographs of fractions from the three boric acid gel columns, a molecular model of a tetrahedral phenylboronate complex with a 22-desmethyl-23-hydroxyazaspiracid, tabulated amounts of azaspiracid analogues from the fractionated IRNO LRM extract, and graphs showing matrix effects for LC–MS analysis of AZA2 and AZA3 in spiked extracts of mussels with and without boric acid gel fractionation.

The aim was to study the in vitro metabolism of selected algal toxins using enzyme reparations from blue mussels (Mytilus edulis) and Wistar rats.

Azaspiracids (AZAs) belong to a family of more than 50 polyether toxins originating from marine dinoflagellates such as Azadinium spinosum. All of the AZAs reported thus far contain a 21,22-dihydroxy group. Boric acid gel (BAG) can bind selectively to compounds containing vic-diols or α-hydroxycarboxylic acids via formation of reversible boronate complexes. Here we report use of BAG to selectively capture and release AZAs from extracts of blue mussels. Analysis of the extracts and BAG fractions by LC–MS showed that this procedure resulted in an excellent clean-up of the AZAs in the extract. Analysis by ELISA and LC–MS indicated that most AZA analogues were recovered in good yield by this procedure. The capacity of BAG for AZAs was at least 50 μg/g, making this procedure suitable for use in the early stages of preparative purification of AZAs. In addition to its potential for concentration of dilute samples, the extensive clean-up provided by BAG fractionation of AZAs in mussel samples almost eliminated matrix effects during subsequent LC–MS, and could be expected to reduce matrix effects during ELISA analysis. The method may therefore prove useful for quantitative analysis of AZAs as part of monitoring programs. Although LC-MS data showed that okadaic acid analogues also bound to BAG, this was much less efficient than for AZAs under the conditions used. The BAG methodology is potentially applicable to other important groups of natural toxins containing diols, including ciguatoxins, palytoxins, pectenotoxins,karlotoxins, tetrodotoxin, trichothecenes, and toxin glycosides.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved via a late stage NHK coupling to form the C21‒C22 bond with the C20 configuration unambiguously established from L-(+)-tartaric acid. Post-coupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R, 19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned (20R)-epimer did not.

Sandvik M, Rundberget T, Fæste CK, Samdal IA, Miles CO, Petersen D. In vitro biotransformation of algal toxins. 5th International Conference on Molluscan Shellfish Safety, 14.-18. Juni 2004, Galway, Irland.

Sandvik M, Rundberget T, Fæste CK, Miles CO, Petersen D. In vitro biotransformasjon av algetoksiner isolert fra sjøvann, algekulturer og skjell. Havbruk 2004, 23.-24. Mars, Gardemoen, Norway.