Advances in Marine Toxins: Characterization, Analysis and Surveillance

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Marine and Freshwater Toxins".

Deadline for manuscript submissions: closed (15 April 2024) | Viewed by 14340

Special Issue Editor


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Guest Editor
Biotoxin Metrology, National Research Council of Canada, Halifax, NS B3H 3Z1, Canada
Interests: analytical chemistry; marine and freshwater toxins; mass spectrometry; liquid chromatography; electrophoresis; metabolism; toxicology

Special Issue Information

Dear Colleagues,

Seafood poisoning is a global problem affecting thousands of people annually due to the contamination of shellfish and finfish by marine toxins. It was not until the late 1950s that scientists began to identify the origins and identities of these toxic substances. The structure of saxitoxin was first established in 1962, and further discoveries followed, including tetrodotoxin (1965), okadaic acid (1980), brevetoxin (1981), domoic acid (1987), ciguatoxin (1989), spirolide (1995), azaspiracid (1998), and others. Each of these groundbreaking discoveries was facilitated by the development of advanced chemical technologies, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and liquid chromatography (LC). It was crucial that methods for monitoring the presence of these toxins in seafood be developed in order to protect public health. The mouse bioassay, developed in 1955, was successful for many years in detecting certain toxins, but this technique had its limitations. Modern biochemical, immunological, and chemical analytical methods were gradually adapted to better detect and quantify the increasing number of toxins. Advanced analytical methodologies, such as LC-MS, have led the way in the detection and identification of many new structural analogues of the principal toxins, further expanding the challenge of regulatory monitoring.

This Special Issue is open to original research articles and reviews investigating advances in the understanding of marine toxins. The following subjects will be considered:

  • The identification of marine toxins
  • Investigations on the origins of toxins: algal producers and biosynthetic pathways
  • Toxicological studies
  • New assays and chemical analytical methods, including international validation studies
  • The development of reference materials for calibration and quality control
  • The surveillance of toxins in algae and seafood

Prof. Dr. Michael A. Quilliam
Guest Editor

Manuscript Submission Information

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • marine toxins
  • shellfish toxins
  • identification
  • toxicology
  • analytical chemistry
  • reference materials
  • metabolism
  • surveillance

Published Papers (11 papers)

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Research

9 pages, 2030 KiB  
Communication
First Detection of Algal Caribbean Ciguatoxin in Amberjack Causing Ciguatera Poisoning in the Canary Islands (Spain)
by Pablo Estevez, Juan Oses-Prieto, David Castro, Alejandro Penin, Alma Burlingame and Ana Gago-Martinez
Toxins 2024, 16(4), 189; https://doi.org/10.3390/toxins16040189 - 13 Apr 2024
Viewed by 411
Abstract
Ciguatera Poisoning (CP) is an illness associated with the consumption of fish contaminated with potent natural toxins found in the marine environment, commonly known as ciguatoxins (CTXs). The risk characterization of CP has become a worldwide concern due to the widespread expansion of [...] Read more.
Ciguatera Poisoning (CP) is an illness associated with the consumption of fish contaminated with potent natural toxins found in the marine environment, commonly known as ciguatoxins (CTXs). The risk characterization of CP has become a worldwide concern due to the widespread expansion of these natural toxins. The identification of CTXs is hindered by the lack of commercially available reference materials. This limitation impedes progress in developing analytical tools and conducting toxicological studies essential for establishing regulatory levels for control. This study focuses on characterizing the CTX profile of an amberjack responsible for a recent CP case in the Canary Islands (Spain), located on the east Atlantic coast. The exceptional sensitivity offered by Capillary Liquid Chromatography coupled with High-Resolution Mass Spectrometry (cLC-HRMS) enabled the detection, for the first time in fish contaminated in the Canary Islands, of traces of an algal ciguatoxin recently identified in G. silvae and G. caribeaus from the Caribbean Sea. This algal toxin was structurally characterized by cLC-HRMS being initially identified as C-CTX5. The total toxin concentration of CTXs was eight times higher than the guidance level proposed by the Food and Drug Administration (0.1 ng C-CTX1/g fish tissue), with C-CTX1 and 17-hydroxy-C-CTX1 as major CTXs. Full article
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16 pages, 5141 KiB  
Communication
Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan
by Anna E. Vlasenko, Alexandra O. Pereverzeva, Peter V. Velansky and Timur Yu. Magarlamov
Toxins 2024, 16(4), 186; https://doi.org/10.3390/toxins16040186 - 10 Apr 2024
Viewed by 292
Abstract
Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs [...] Read more.
Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC–MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC–MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans. Full article
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14 pages, 2897 KiB  
Article
Tetrodotoxin/Saxitoxin Accumulation Profile in the Euryhaline Marine Pufferfish Chelonodontops patoca
by Hongchen Zhu, Takashi Sakai, Hiroyuki Doi, Kenichi Yamaguchi, Akinori Yamada, Tomohiro Takatani and Osamu Arakawa
Toxins 2024, 16(1), 18; https://doi.org/10.3390/toxins16010018 - 28 Dec 2023
Viewed by 1259
Abstract
Marine Takifugu pufferfish, which naturally possess tetrodotoxins (TTXs), selectively take up and accumulate TTXs, whereas freshwater Pao pufferfish, which naturally possess saxitoxins (STXs), selectively take up and accumulate STXs. To further clarify the TTXs/STXs selectivity in pufferfish, we conducted a TTX/STX administration experiment [...] Read more.
Marine Takifugu pufferfish, which naturally possess tetrodotoxins (TTXs), selectively take up and accumulate TTXs, whereas freshwater Pao pufferfish, which naturally possess saxitoxins (STXs), selectively take up and accumulate STXs. To further clarify the TTXs/STXs selectivity in pufferfish, we conducted a TTX/STX administration experiment using Chelonodontops patoca, a euryhaline marine pufferfish possessing both TTXs and STXs. Forty nontoxic cultured individuals of C. patoca were divided into a seawater group (SW, acclimated/reared at 33‰ salinity; n = 20) and a brackish water group (BW, acclimated/reared at 8‰ salinity; n = 20). An aqueous TTX/STX mixture was intrarectally administered (both at 7.5 nmol/fish), and five individuals/group were analyzed after 1–48 h. Instrumental toxin analyses revealed that both TTX and STX were taken up, transferred, and retained, but more STX than TTX was retained in both groups. TTX gradually decreased and eventually became almost undetectable in the intestinal tissue, while STX was retained at ~5–10% of the dose level, and only STX showed transient transfer in the liver. The BW group showed a faster decrease/disappearance of TTX, greater STX retention in the intestine, and greater STX transient transfer to the liver. Thus, C. patoca appears to more easily accumulate STXs than TTXs, especially under hypoosmotic conditions. Full article
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15 pages, 2809 KiB  
Article
The Temporal Distribution of Cyclic Imines in Shellfish in the Bays of Fangar and Alfacs, Northwestern Mediterranean Region
by Lourdes Barreiro-Crespo, Margarita Fernández-Tejedor, Jorge Diogène and Maria Rambla-Alegre
Toxins 2024, 16(1), 10; https://doi.org/10.3390/toxins16010010 - 23 Dec 2023
Cited by 1 | Viewed by 1068
Abstract
Spirolides (SPXs), gymnodimines (GYMs), and pinnatoxins (PnTXs) have been detected in shellfish from the northwestern Mediterranean coast of Spain. Several samples of bivalves were collected from Fangar Bay and Alfacs Bay in Catalonia over a period of over 7 years (from 2015 to [...] Read more.
Spirolides (SPXs), gymnodimines (GYMs), and pinnatoxins (PnTXs) have been detected in shellfish from the northwestern Mediterranean coast of Spain. Several samples of bivalves were collected from Fangar Bay and Alfacs Bay in Catalonia over a period of over 7 years (from 2015 to 2021). Shellfish samples were analyzed for cyclic imines (CIs) on an LC1200 Agilent and 3200 QTrap triple–quadrupole mass spectrometer. In shellfish, SPX-1 was detected in two cases (of 26.5 µg/kg and 34 µg/kg), and GYM-A was only detected in trace levels in thirteen samples. Pinnatoxin G (PnTX-G) was detected in 44.6% of the samples, with its concentrations ranging from 2 µg/kg to 38.4 µg/kg. Statistical analyses revealed that seawater temperature influenced the presence or absence of these toxins. PnTX-G showed an extremely significant presence/temperature relationship in both bays in comparison to SPX-1 and GYM-A. The prevalence of these toxins in different bivalve mollusks was evaluated. A seasonal pattern was observed, in which the maximum concentrations were found in the winter months for SPX-1 and GYM-A but in the summer months for PnTX-G. The obtained results indicate that it is unlikely that CIs in the studied area pose a potential health risk through the consumption of a seafood diet. However, further toxicological information about CIs is necessary in order to perform a conclusive risk assessment. Full article
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16 pages, 4746 KiB  
Article
Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis
by Juan Blanco, Ángeles Moroño, Fabiola Arévalo, Jorge Correa and Juan Pablo Lamas
Toxins 2023, 15(11), 661; https://doi.org/10.3390/toxins15110661 - 16 Nov 2023
Viewed by 1053
Abstract
The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the [...] Read more.
The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg−1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July–August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed. Full article
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15 pages, 3239 KiB  
Article
Lipophilic Shellfish Poisoning Toxins in Marine Invertebrates from the Galician Coast
by Araceli E. Rossignoli, Begoña Ben-Gigirey, Mónica Cid, Carmen Mariño, Helena Martín, Soledad Garrido, Francisco Rodríguez and Juan Blanco
Toxins 2023, 15(11), 631; https://doi.org/10.3390/toxins15110631 - 27 Oct 2023
Cited by 1 | Viewed by 1407
Abstract
For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic [...] Read more.
For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5–75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg−1) and Tellina donacina (63 µg kg−1), respectively. However, mean concentrations for the bivalve group were shallow (2–6 µg kg−1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6–44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg−−1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions. Full article
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11 pages, 2154 KiB  
Article
Hepatorenal Toxicity after 7-Day Oral Administration of Low-Dose Tetrodotoxin and Its Distribution in the Main Tissues in Mice
by Yaqian Zhong, Xiaojun Zhang, Qiyu Yang and Qianfeng Wang
Toxins 2023, 15(9), 564; https://doi.org/10.3390/toxins15090564 - 08 Sep 2023
Viewed by 1054
Abstract
Tetrodotoxin (TTX) is a highly toxic compound detected in various edible marine animals even in European waters. To characterize the hazard by oral exposure to TTX, its tissue distribution was evaluated after single (75 μg/kg) or 7-day (25–125 μg/kg) oral administration in mice. [...] Read more.
Tetrodotoxin (TTX) is a highly toxic compound detected in various edible marine animals even in European waters. To characterize the hazard by oral exposure to TTX, its tissue distribution was evaluated after single (75 μg/kg) or 7-day (25–125 μg/kg) oral administration in mice. Moreover, TTX liver and renal toxicity was evaluated after 7-day oral administration. The elimination cycle of a single oral dose of TTX (75 µg/kg) was found to be approximately 168 h (7 days). Daily oral administration of TTX at doses of 25, 75, and 125 µg/kg for 7 consecutive days revealed dose-dependent toxic effects on the liver and kidney. Histopathological examination showed increased inflammatory cell infiltration in the liver and kidney with higher TTX doses, along with disorganization of the hepatic cord and renal tubular cell arrangement. The study results indicated that TTX had more hepatotoxicity than nephrotoxicity in mice. These findings provide insights into the unintentional ingestion of a low dose of TTX in mammals, including humans, and emphasize the importance of food safety. Full article
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21 pages, 7583 KiB  
Article
Combining Nanopore Sequencing with Recombinase Polymerase Amplification Enables Identification of Dinoflagellates from the Alexandrium Genus, Providing a Rapid, Field Deployable Tool
by Robert G. Hatfield, David Ryder, Annabel M. Tidy, David M. Hartnell, Karl J. Dean and Frederico M. Batista
Toxins 2023, 15(6), 372; https://doi.org/10.3390/toxins15060372 - 01 Jun 2023
Viewed by 2116
Abstract
The armoured dinoflagellate Alexandrium can be found throughout many of the world’s temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant [...] Read more.
The armoured dinoflagellate Alexandrium can be found throughout many of the world’s temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of Alexandrium cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve Alexandrium to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the Alexandrium genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different Alexandrium species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of A. minutum in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the A. catenella, A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic A. catenella species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (p ≤ 0.05). Furthermore, a McNemar’s paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of Alexandrium by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls. Full article
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12 pages, 12383 KiB  
Article
Acute Toxic Effects of Tetrodotoxin in Mice via Intramuscular Injection and Oral Gavage
by Fan Wang, Fuhai Zhang, Juxingsi Song, Shuaijun Zou, Jie Li, Yichao Huang, Liming Zhang and Qianqian Wang
Toxins 2023, 15(5), 334; https://doi.org/10.3390/toxins15050334 - 13 May 2023
Viewed by 1598
Abstract
Tetrodotoxin (TTX) is a highly fatal marine biotoxin. Constantly increasing intoxications and the lack of specific antitoxic drugs in clinical applications highlight the need for further research into the toxic effects of TTX. Current reports on poisoning cases and the TTX toxicity mechanism [...] Read more.
Tetrodotoxin (TTX) is a highly fatal marine biotoxin. Constantly increasing intoxications and the lack of specific antitoxic drugs in clinical applications highlight the need for further research into the toxic effects of TTX. Current reports on poisoning cases and the TTX toxicity mechanism suggest that the blocking of voltage-gated sodium channels (VGSCs) by TTX is probably reversible, but direct evidence of this is lacking, as far as we are aware. This study explored the acute toxic effects of TTX at sub-lethal doses via different routes, analyzing variations in muscle strength and TTX concentration in the blood in mice. We found that the loss of muscle strength in mice caused by TTX was dose-dependent and reversible, and the death time and muscle strength variations after oral gavage with TTX appeared to occur later and were more variable than those after intramuscular injection. In conclusion, we systematically compared the acute toxic effects of TTX for two different administration routes at sub-lethal doses, directly verifying the reversible reaction of TTX blocking VGSCs and speculating that averting a complete block of VGSCs by TTX could be an effective strategy for preventing death from TTX poisoning. This work may provide data for the diagnosis and treatment of TTX poisoning. Full article
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12 pages, 1622 KiB  
Article
Tetrodotoxin and the Geographic Distribution of the Blue-Lined Octopus Hapalochlaena fasciata on the Korean Coast
by Ji-Hoe Kim, Dong-Wook Kim, Sung-Rae Cho, Ka-Jeong Lee and Jong-Soo Mok
Toxins 2023, 15(4), 279; https://doi.org/10.3390/toxins15040279 - 11 Apr 2023
Viewed by 1878
Abstract
The genus Hapalochlaena, including the blue-lined octopus Hapalochlaena fasciata (H. fasciata), is highly toxic. Venomous, blue-lined octopuses were recently found in Korea, but their toxicity, toxin composition, and distribution remain largely unknown. Here we estimated the geographic distribution of the organisms along the [...] Read more.
The genus Hapalochlaena, including the blue-lined octopus Hapalochlaena fasciata (H. fasciata), is highly toxic. Venomous, blue-lined octopuses were recently found in Korea, but their toxicity, toxin composition, and distribution remain largely unknown. Here we estimated the geographic distribution of the organisms along the Korean coast and clarified their toxicity. Tetrodotoxin (TTX) was present in all three specimens of H. fasciata examined, although the toxicity varied largely between individuals. The mean TTX concentration in the whole body of the three specimens was 6.5 ± 2.2 μg/g (range 3.3–8.5 μg/g). Among the body parts examined, the salivary glands exhibited the highest concentration (22.4 ± 9.7 μg/g). From 2012 to 2021, 26 individuals were obtained nearly every month from different regions of the Korean coast. A non-fatal case of a blue-lined octopus bite was reported along the Korean coast in June 2015. This is the first report on the widespread distribution of blue-lined octopuses on the Korean coast and TTX detection. The widespread distribution of the TTX-bearing H. fasciata along the Korean coast within the temperate zone indicates that the species may soon become a serious health issue in Korea. The toxicity of this species is also a potentially significant human health risk. Full article
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11 pages, 1604 KiB  
Article
Artificial Substrates Coupled with qPCR (AS-qPCR) Assay for the Detection of the Toxic Benthopelagic Dinoflagellate Vulcanodinium rugosum
by Aurélien Bouquet, Christine Felix, Estelle Masseret, Coralie Reymond, Eric Abadie, Mohamed Laabir and Jean Luc Rolland
Toxins 2023, 15(3), 217; https://doi.org/10.3390/toxins15030217 - 11 Mar 2023
Cited by 1 | Viewed by 1426
Abstract
Vulcanodinium rugosum is an emerging benthopelagic neuro-toxic dinoflagellate species responsible for seasonal Pinnatoxins and Portimines contaminations of shellfish and marine animals. This species is challenging to detect in the environment, as it is present in low abundance and difficult to be identified using [...] Read more.
Vulcanodinium rugosum is an emerging benthopelagic neuro-toxic dinoflagellate species responsible for seasonal Pinnatoxins and Portimines contaminations of shellfish and marine animals. This species is challenging to detect in the environment, as it is present in low abundance and difficult to be identified using light microscopy. In this work, we developed a method using artificial substrates coupled with qPCR (AS-qPCR) to detect V. rugosum in a marine environment. This sensitive, specific and easy-to-standardize alternative to current techniques does not require specialized expertise in taxonomy. After determining the limits and specificity of the qPCR, we searched for the presence of V. rugosum in four French Mediterranean lagoons using artificial substrates collected every two weeks for one year. The AS-qPCR method revealed its occurrences in summer 2021 in every studied lagoon and detected cells in more samples than light microscopy. As V. rugosum development induces shellfish contamination even at low microalga densities, the AS-qPCR method is accurate and relevant for monitoring V. rugosum in a marine environment. Full article
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