Domoic Acid Isomers Research Articles

Comparative study of domoic acid accumulation, isomer content and associated digestive subcellular processes in five marine invertebrate species

Despite the deleterious effects of the phycotoxin domoic acid (DA) on human health, and the permanent threat of blooms of the toxic Pseudo-nitzschia sp. over commercially important fishery-resources, knowledge regarding the physiological mechanisms behind the profound differences in accumulation and depuration of this toxin in contaminated invertebrates remain very scarce. In this work, a comparative analysis of accumulation, isomer content, and subcellular localization of DA in different invertebrate species was performed. Samples of scallops Pecten maximus and Aequipecten opercularis, clams Donax trunculus, slippersnails Crepidula fornicata, and seasquirts Asterocarpa sp. were collected after blooms of the same concentration of toxic Pseudo-nitzschia australis. Differences (P < 0.05) in DA accumulation were found, wherein P. maximus showed up to 20-fold more DA in the digestive gland than the other species. Similar profiles of DA isomers were found between P. maximus and A. opercularis, whereas C. fornicata was the species with the highest biotransformation rate (∼10 %) and D. trunculus the lowest (∼4 %). DA localization by immunohistochemical analysis revealed differences (P < 0.05) between species: in P. maximus, DA was detected mainly within autophagosome-like vesicles in the cytoplasm of digestive cells, while in A. opercularis and C. fornicata significant DA immunoreactivity was found in post-autophagy residual bodies. A slight DA staining was found free within the cytoplasm of the digestive cells of D. trunculus and Asterocarpa sp. The Principal Component Analysis revealed similarities between pectinids, and a clear distinction of the rest of the species based on their capabilities to accumulate, biotransform, and distribute the toxin within their tissues. These findings contribute to improve the understanding of the inter-specific differences concerning the contamination-decontamination kinetics and the fate of DA in invertebrate species.

Residue Analysis and Assessment of the Risk of Dietary Exposure to Domoic Acid in Shellfish from the Coastal Areas of China.

Harmful algal blooms in Chinese waters have caused serious domoic acid (DA) contamination in shellfish. Although shellfish are at particular risk of dietary exposure to DA, there have been no systematic DA risk assessments in Chinese coastal waters. A total of 451 shellfish samples were collected from March to November 2020. The presence of DA and four of its isomers were detected using liquid chromatography-tandem mass spectrometry. The spatial-temporal distribution of DA occurrence and its potential health risks were examined. DA was detected in 198 shellfish samples (43.90%), with a maximum level of 942.86 μg/kg. DA was recorded in all 14 shellfish species tested and Pacific oysters (Crassostrea gigas) showed the highest average DA concentration (82.36 μg/kg). The DA concentrations in shellfish showed distinct spatial-temporal variations, with significantly higher levels of occurrence in autumn than in summer and spring (p < 0.01), and particularly high occurrence in Guangdong and Fujian Provinces. The detection rates and maximum concentrations of the four DA isomers were low. While C. gigas from Guangdong Province in September showed the highest levels of DA contamination, the risk to human consumers was low. This study improves our understanding of the potential risk of shellfish exposure to DA-residues.

Update of the Planktonic Diatom Genus Pseudo-nitzschia in Aotearoa New Zealand Coastal Waters: Genetic Diversity and Toxin Production.

Domoic acid (DA) is produced by almost half of the species belonging to the diatom genus Pseudo-nitzschia and causes amnesic shellfish poisoning (ASP). It is, therefore, important to investigate the diversity and toxin production of Pseudo-nitzschia species for ASP risk assessments. Between 2018 and 2020, seawater samples were collected from various sites around Aotearoa New Zealand, and 130 clonal isolates of Pseudo-nitzschia were established. Molecular phylogenetic analysis of partial large subunit ribosomal DNA and/or internal transcribed spacer regions revealed that the isolates were divided into 14 species (Pseudo-nitzschia americana, Pseudo-nitzschia arenysensis, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia cuspidata, Pseudo-nitzschia delicatissima, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia galaxiae, Pseudo-nitzschia hasleana, Pseudo-nitzschia multiseries, Pseudo-nitzschia multistriata, Pseudo-nitzschia plurisecta, Pseudo-nitzschia pungens, and Pseudo-nitzschia cf. subpacifica). The P. delicatissima and P. hasleana strains were further divided into two clades/subclades (I and II). Liquid chromatography-tandem mass spectrometry was used to assess the production of DA and DA isomers by 73 representative strains. The analyses revealed that two (P. australis and P. multiseries) of the 14 species produced DA as a primary analogue, along with several DA isomers. This study is the first geographical distribution record of P. arenysensis, P. cuspidata, P. galaxiae, and P. hasleana in New Zealand coastal waters.

Direct evidence for toxin production by Pseudo-nitzschia plurisecta (Bacillariophyceae) and extension of its distribution area

ABSTRACTWe confirm an amnesic shellfish poisoning risk associated with domoic acid production by the species Pseudo-nitzschia plurisecta and determine the toxin profile, which includes three domoic acid isomers. Although the species was reported once in European waters, along the Spanish Atlantic coast, here we describe and characterize P. plurisecta collected during the first occurrence in French Atlantic waters. Three strains (IFR-PPL-15.1, 15.2 and 15.3) were isolated from French coastal water samples in June 2015. These strains were identified and characterized by microscopic observation, molecular (ITS region sequencing and phylogenetic analysis) and metabolite (toxins and pigments) analyses. The cell shape is lanceolate and the valve contains a central interspace with uniseriate stria of oval-square poroids, each divided into 3–10 sectors. The ITS gene sequence is homologous to the original sequence available for the Spanish strain P. plurisecta. The pigment profile contains chl a, c3, c2, fucoxanthin, diatoxanthin, diadinoxanthin and traces of β,β-carotene. Results on morphology, phylogeny and pigments led to the identification of P. plurisecta and confirmed its first occurrence in French waters.

Pseudo-nitzschia, Nitzschia, and domoic acid: New research since 2011

Some diatoms of the genera Pseudo-nitzschia and Nitzschia produce the neurotoxin domoic acid (DA), a compound that caused amnesic shellfish poisoning (ASP) in humans just over 30 years ago (December 1987) in eastern Canada. This review covers new information since two previous reviews in 2012. Nitzschia bizertensis was subsequently discovered to be toxigenic in Tunisian waters. The known distribution of N. navis-varingica has expanded from Vietnam to Malaysia, Indonesia, the Philippines and Australia. Furthermore, 15 new species (and one new variety) of Pseudo-nitzschia have been discovered, bringing the total to 52. Seven new species were found to produce DA, bringing the total of toxigenic species to 26.We list all Pseudo-nitzschia species, their ability to produce DA, and show their global distribution. A consequence of the extended distribution and increased number of toxigenic species worldwide is that DA is now found more pervasively in the food web, contaminating new marine organisms (especially marine mammals), affecting their physiology and disrupting ecosystems.Recent findings highlight how zooplankton grazers can induce DA production in Pseudo-nitzschia and how bacteria interact with Pseudo-nitzschia. Since 2012, new discoveries have been reported on physiological controls of Pseudo-nitzschia growth and DA production, its sexual reproduction, and infection by an oomycete parasitoid. Many advances are the result of applying molecular approaches to discovering new species, and to understanding the population genetic structure of Pseudo-nitzschia and mechanisms used to cope with iron limitation. The availability of genomes from three Pseudo-nitzschia species, coupled with a comparative transcriptomic approach, has allowed advances in our understanding of the sexual reproduction of Pseudo-nitzschia, its signaling pathways, its interactions with bacteria, and genes involved in iron and vitamin B12 and B7 metabolism.Although there have been no new confirmed cases of ASP since 1987 because of monitoring efforts, new blooms have occurred. A massive toxic Pseudo-nitzschia bloom affected the entire west coast of North America during 2015–2016, and was linked to a ‘warm blob’ of ocean water. Other smaller toxic blooms occurred in the Gulf of Mexico and east coast of North America. Knowledge gaps remain, including how and why DA and its isomers are produced, the world distribution of potentially toxigenic Nitzschia species, the prevalence of DA isomers, and molecular markers to discriminate between toxigenic and non-toxigenic species and to discover sexually reproducing populations in the field.

A PRESENCE OF A SUBSTANCE BINDING WITH THE SPECIFIC ANTIBODY AGAINST DOMOIC ACID IN THE THORNY OYSTER SPONDYLUS VERSICOLOR

Domoic acid (DA) extracts from 10 specimens of a thorny oyster, Spondylus versicolor collected at Nha Phu bay, Khanh Hoa province, Vietnam in August, 2010 was analyzed individually by High Performance Liquid Chromatography (HPLC) and Indirect Enzyme Linked Immunosorbent Assay (ELISA). Domoic acid levels in the extracts detected by ELISA were always higher than those by HPLC, significantly. In the HPLC chromatogram, the peak of an unknown compound with retention time very close to DA, but different from the known DA isomers was always observed. This substance reacted well with DA-specific antibody in Western Blot experiment, but not corresponding to any standard protein. Our result indicates the occurrence of a certain non-protein substance, which was co-presented with DA in S. versicolor. Further investigation on chemical feature of this substance is under progress.

Photocatalytic properties of nanocrystalline titanium dioxide films in the degradation of domoic acid in aqueous solution: potential for use in molluscan shellfish biotoxin depuration facilities

Domoic acid (DA) is a water-soluble marine neurotoxin produced and released by certain species of the diatom genus Pseudo-nitzschia. Present in coastal waters, it can be a threat to public health and marine life, and can result in severe economic losses to the molluscan shellfish and crustacean harvesting industries. Here we report on the efficiency of nanocrystalline (NC) titania (TiO2) thin films used as a photocatalyst in the ultraviolet light photodegradation of DA. Titanium dioxide thin films produced by a sol-gel dip-coating method in the presence of polyethylene glycol of different molecular weights (200, 400 and 600) were deposited on glass substrates and crystallised at 90°C. The films were characterised using spectroscopic ellipsometry, Fourier transform infrared spectrometer (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Raman spectroscopy. The photocatalytic activity measurements were carried out by immersing the NC TiO2 films in a DA solution (2500 ng ml−1) and then exposing them for various times at room temperature to UVA irradiation (λ = approximately 350 nm). The degradation of DA, quantified by HPLC analysis, was not significant when using daylight or ultraviolet light irradiation alone, whereas the NC TiO2 films prepared at low temperature proved to be a very efficient photocatalyst when used in conjunction with UVA light. The effectiveness of the photodegradation was improved by increasing molecular weight of polyethylene glycol, which increased the thickness of the film. The presence, transformation and degradation of three DA isomers were observed. The approach may eventually be practical for destroying DA in seawater used by aquaculture industry depuration facilities.

Comparative toxicity to mice of domoic acid and isodomoic acids A, B and C

Seafood in many parts of the world may become contaminated with high levels of domoic acid and domoic acid isomers, and such seafood has been shown to cause toxic effects in humans and in marine animals. Domoic acid itself has been held responsible for the observed effects, although the possible contribution of the isomers to toxicity has not been investigated. In the present study, the acute intraperitoneal toxicity of isodomoic acid C in mice was found to be lower than that of domoic acid. Furthermore, the severities of the behavioural changes induced by isodomoic acids A, B and C were all much lower than that of domoic acid itself, suggesting that these substances pose relatively little risk to human or animal health.

Photocatalytic degradation of domoic acid using nanocrystalline TiO 2 thin films

Domoic acid (DA) is a water-soluble marine neurotoxin produced and released by Pseudo-nitzschia species. It is remarkably pervasive in North American coastal waters, where it is a threat to public health and some marine life, and has resulted in severe economic losses in the shellfish and crustacean harvesting industry. In this paper we report on the development of nanocrystalline titania thin films that were used as photocatalyst in the UV photodegradation of DA. TiO 2 thin films produced by a sol–gel dip-coating method in the presence of polyethylene glycol (PEG) of different molecular weights (200, 400 and 600), were deposited on glass and fused quartz substrates, and crystallized at different temperatures (90, 500 and 900 °C). The films were characterized using UV–vis, FTIR, SEM, XRD, and Raman spectroscopy. For the photocatalytic activity measurements, the TiO 2 films were immersed in a DA solution (2500 ng/mL) and exposed for various times at room temperature to UV irradiation ( λ ∼ 350 nm). DA analysis was carried out by HPLC, and the photocatalytic activity was quantified as the rate of disappearance of DA. The photocatalytic activity of the TiO 2 thin films was studied as a function of their crystallization temperature, type of PEG content, and thickness. The films prepared at low temperature proved to be very efficient photocatalysts. They showed a higher photocatalytic activity than those produced at high temperature. Also, using PEG of higher molecular weight in the TiO 2 coating solutions increased the effectiveness of the degradation. When using UV irradiation alone, without photocatalyst, the degradation of DA was not significant. The presence, transformation, and degradation of various DA isomers were observed during the photodegradation process, and a photodegradation mechanism is proposed.

Accumulation, transformation and tissue distribution of domoic acid, the amnesic shellfish poisoning toxin, in the common cuttlefish, Sepia officinalis

Domoic acid (DA) is a phycotoxin produced by some diatoms, mainly from the Pseudo-nitzschia genus, and has been detected throughout the marine food web. Although DA has been frequently found in cephalopod prey such as crustaceans and fish, little is known about DA accumulation in these molluscs. This study presents the first data showing relevant concentrations of DA detected in the common cuttlefish, Sepia officinalis, which is one of the most studied cephalopod species in the world. Domoic acid was consistently found throughout 2003 and 2004 in the digestive gland of cuttlefish reaching concentrations of 241.7 μg DA g −1. The highest DA values were detected during spring and summer months, periods when Pseudo-nitzschia occur in the plankton. In fact, Pseudo-nitzschia blooms preceded the highest DA concentrations in cuttlefish. Evaluation of DA tissue distribution showed elevated DA concentrations in the digestive gland and branchial hearts. Further, DA isomers comprised a relevant percentage of the toxin profile, indicating degradation and biotransformation of the toxin in the branchial hearts. The common cuttlefish, like other cephalopod species, plays a central position in the food web and might be a new DA vector to top predators like marine mammals. Human intoxications are not expected since DA was only seldom detected in the mantle and even then in very low levels (max 0.7 μg DA g −1). However, in some countries whole juvenile animals are consumed (i.e. without evisceration) and in this case they might represent a risk to human health. This study contributes to understanding the occurrence of phycotoxins in cephalopods and reveals a new member of the marine food web able to accumulate DA.

Production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay

For production of monoclonal antibodies against domoic acid, a causative agent of amnesic shellfish poisoning, three immunogens, domoic acid conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and human gamma globulin (HGG), were prepared. The antiserum obtained from BALB/c mice immunized with domoic acid–BSA showed the highest affinity for domoic acid. The monoclonal antibody, DA-3, obtained from the mice was highly specific for domoic acid and showed a minor cross-reactivity with the isomers of domoic acid (isodomoic acids B, E, F, G and H), except for isodomoic acid A. Using DA-3 antibody, an indirect competitive enzyme immunoassay (idc-EIA) was developed for measurement of domoic acid. The working range for quantitative measurement of domoic acid and the quantification limit for domoic acid in shellfish were estimated to be 0.15–10 ng/ml and less than 0.04 μg/g, respectively. The mean recovery of domoic acid added to extracts of shellfish at toxin levels of 0.02 to 0.2 μg/ml was 103% with a coefficient of variation of 4.5%. The newly developed idc-EIA seems to be a useful method for monitoring domoic acid in shellfish.

Two new isomers of domoic acid from a red alga, Chondria armata

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata, collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be ( E, E) and ( Z, E) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro-lidineacetic acid, based on electrospray ionization mass and [ 1H]nuclear magnetic resonance spectral analyses including [ 1H- 1H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.