Biodiversity of Philippine marine fishes: A DNA barcode reference library based on voucher specimens.

Accurate identification of fishes is essential for understanding their biology and to ensure food safety for consumers. DNA barcoding is an important tool because it can verify identifications of both whole and processed fishes that have had key morphological characters removed (e.g., filets, fish meal); however, DNA reference libraries are incomplete, and public repositories for sequence data contain incorrectly identified sequences. During a nine-year sampling program in the Philippines, a global biodiversity hotspot for marine fishes, we developed a verified reference library of cytochrome c oxidase subunit I (COI) sequences for 2,525 specimens representing 984 species. Specimens were primarily purchased from markets, with additional diversity collected using rotenone or fishing gear. Species identifications were verified based on taxonomic, phenotypic, and genotypic data, and sequences are associated with voucher specimens, live-color photographs, and genetic samples catalogued at Smithsonian Institution, National Museum of Natural History. The Biodiversity of Philippine Marine Fishes dataset is released herein to increase knowledge of species diversity and distributions and to facilitate accurate identification of market fishes.

Structure and toxicity of AZA-59, an azaspiracid shellfish poisoning toxin produced by Azadinium poporum (Dinophyceae).

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish.

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States.

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.

Using low-coverage whole genome sequencing (genome skimming) to delineate three introgressed species of buffalofish (Ictiobus).

Consumption of buffalofish has been sporadically associated with Haff disease-like illnesses involving sudden onset muscle pain and weakness due to skeletal muscle rhabdomyolysis, but determination of precisely which species are associated with these illnesses has been impeded by a lack of species-specific DNA-based markers. Here, three closely related species of buffalofish native to the Mississippi River Basin (Ictiobus bubalus, Ictiobus cyprinellus and Ictiobus niger) that have previously proven genetically indistinguishable using both mitochondrial and nuclear single-locus sequencing were reliably discriminated using low-coverage whole genome sequencing ('genome skimming'). Using 44 specimens representing the three species collected from the mid/upper (Missouri) and lower (Louisiana) regions of the species' native ranges, the SISRS (Site Identification from Short Read Sequences) bioinformatics pipeline was adapted to (1) identify over 620Mbp of putatively homologous nuclear sequence data and (2) isolate over 140,000 single-nucleotide polymorphisms (SNPs) that supported accurate species delimitation, all without the use of a reference genome or annotation data. These sites were used to classify Ictiobus spp. samples with genome-skim data, along with a larger set (n = 67) where ultraconserved elements (UCEs) were sequenced. Analyses of whole mitochondrial data revealed more limited signal. Nearly all samples matched their purported species based on morphologic identification, but two Missouri samples morphologically identified as I. niger grouped with samples of I. bubalus, albeit with significant enrichment of I. niger SNPs. To our knowledge this is the first report of a DNA-based tool to reliably discriminate these three morphologically distinct species.

Haff disease associated with consumption of buffalofish (Ictiobus spp.) in the United States, 2010-2020, with confirmation of the causative species.

BACKGROUND: In the United States, buffalofish (Ictiobus spp.) are sporadically associated with sudden onset muscle pain and weakness due to rhabdomyolysis within 24 h of fish consumption (Haff disease). Previous genetic analyses of case-associated samples were unable to distinguish the three species of buffalofish that occur in the US, Ictiobus cyprinellus (bigmouth buffalo), Ictiobus bubalus (smallmouth buffalo), and Ictiobus niger (black buffalo). METHODS: Ten events were investigated between 2010 and 2020 and demographic and clinical information was collected for 24 individuals. Meal remnants were collected from 5 of 10 events with additional associated samples (n = 24) collected from another five of 10 events. Low-coverage whole-genome sequencing (genome skimming) was used to identify meal remnants. RESULTS: Patients (26-75 years of age) ranged from 1-4 per event, with 90% involving ≥2 individuals. Reported symptoms included muscle tenderness and weakness, nausea/vomiting, and brown/tea-colored urine. Median incubation period was 8 h. Ninety-six percent of cases were hospitalized with a median duration of four days. The most commonly reported laboratory finding was elevated creatine phosphokinase and liver transaminases. Treatment was supportive including intravenous fluids to prevent renal failure. Events occurred in California (1), Illinois (2), Louisiana (1), New York (1), Mississippi (1), Missouri (2), New Jersey (1), and Texas (1) with location of harvest, when known, being Illinois, Louisiana, Mississippi, Missouri, Texas, and Wisconsin. Meal remnants were identified as I. bubalus (n = 4) and I. niger (n = 1). Associated samples were identified as I. bubalus (n = 16), I. cyprinellus (n = 5), and I. niger (n = 3). DISCUSSION: Time course, presentation of illness, and clinical findings were all consistent with previous domestic cases of buffalofish-associated Haff disease. In contrast to previous reports that I. cyprinellus is the causative species in US cases, data indicate that all three buffalofish species are harvested but I. bubalus is most often associated with illness.

Ciguatoxin in Hawai'i: Fisheries forecasting using geospatial and environmental analyses for the invasive Cephalopholis argus (Epinephelidae).

Invasive species can precede far-reaching environmental and economic consequences. In the Hawai'ian Archipelago Cephalopholis argus (family Serranidae) is an established invasive species, now recognized as the dominant local reef predator, negatively impacting the native ecosystem and local fishery. In this region, no official C. argus fishery exists, due to its association with Ciguatera seafood poisoning (CP); a severe intoxication in humans occurring after eating (primarily) fish contaminated with ciguatoxins (CTXs). Pre-harvest prediction of CP is currently not possible; partly due to the ubiquitous nature of the microalgae producing CTXs and the diverse bioaccumulation pathways of the toxins. This study investigated the perceived risk of CP in two geographically discrete regions (Leeward and Windward) around the main island of Hawai'i, guided by local fishers. C. argus was collected and investigated for CTXs using the U.S. Food and Drug Administration (FDA) CTX testing protocol (in vitro neuroblastoma N2a-assay and LC-MS/MS). Overall, 76% of fish (87/113) exceeded the FDA guidance value for CTX1B (0.01 ng g-1 tissue equivalents); determined by the N2a-assay. Maximum CTX levels were ≅2× higher at the Leeward vs Windward location and, respectively, 95% (64/67) and 54% (25/46) of fish were positive for CTX-like activity. Fisher persons and environmental understandings, regarding the existence of a geographic predictor (Leeward vs Windward) for harvest, were found to be (mostly) accurate as CTXs were detected in both locations and the local designation of C. argus as a risk for CP was confirmed. This study provides additional evidence that supports the previous conclusions that this species is a severe CP risk in the coastal food web of Hawai'i, and that ocean exposure (wave power) may be a prominent factor influencing the CTX content in fish within a hyperendemic region for CP.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

A case of long-term neurological and respiratory sequelae of inhalational exposure to palytoxin.

Palytoxin has been found in several soft coral species which are popular for in-home reef aquariums. Although a limited number of case reports describing acute respiratory distress after inhalational exposure to palytoxin have been reported, there have been no reports regarding the potential long-term effects of inhalational exposure to palytoxin in the literature. This case follows an aquatic specialist in the U.S. over a period of seven years after a single intense occupational exposure to the aerosolized toxin from cleaning of a residential aquarium.

Microcystin Toxins at Potentially Hazardous Levels in Algal Dietary Supplements Revealed by a Combination of Bioassay, Immunoassay, and Mass Spectrometric Methods.

Microcystins (MCs) are hepatotoxic heptapeptides produced by cyanobacteria and are potent inhibitors of protein phosphatases in eukaryotic cells. Algae for dietary supplements are harvested from outdoor environments and can be contaminated with MCs. Monitoring of MCs in these products is necessary but is complicated by their structural diversity (>250 congeners). We used a combination of protein phosphatase inhibition assay (PPIA), ELISA, LC-MS/MS, and nontargeted LC-high-resolution MS (LC-HRMS) with thiol derivatization to characterize the total MCs in 18 algal dietary supplements. LC-MS/MS revealed that some products contained >40 times the maximum acceptable concentration (MAC) of 1 µg/g MCs, but ELISA and PPIA showed up to 50-60 times the MAC. LC-HRMS identified all congeners targeted by LC-MS/MS plus MC-(H4)YR contributing up to 18% of total MCs, along with numerous minor MCs. Recommended dosages of the products greater than the MAC would result in 2.6-75 times the tolerable daily intake, presenting a risk to consumers. This study confirms the need for monitoring these products and presents strategies to fully describe the total MC pool in environmental samples and algal products.

Characterization of Dinophysis spp. (Dinophyceae, Dinophysiales) from the mid-Atlantic region of the United States1.

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a "small Dinophysis sp." that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.

Evaluation of variation within the barcode region of Cytochrome c Oxidase I (COI) for the detection of commercial Callinectes sapidus Rathbun, 1896 (blue crab) products of non-US origin.

Callinectes sapidus Rathbun, 1896 is a western Atlantic species with a disjointed natural geographic range from Massachusetts, USA to Venezuela (distribution area 1) and from Alagoas, Brazil to northern Argentina (distribution area 2). It is the only species of portunid crab commercially harvested in the continental United States but is also imported into the US from several Latin American countries, Venezuela and Mexico in particular. In the United States, crab products labeled as "blue crab" and "Product of the USA" may not legally contain other species of crab or C. sapidus not harvested in the United States. The present study documents nucleotide variation within the barcode region of cytochrome c oxidase I (COI) in 417 reference specimens of C. sapidus collected from throughout its natural range. The goal of this study is to determine if this variation can be utilized to detect mislabeled C. sapidus products sold in interstate commerce by comparing genetic signatures in reference specimens to those observed in commercial crabmeat labeled as "Product of the USA" and "Product of Venezuela." In reference specimens, we observed high levels of genetic variation in the barcode region. However, three lineages were consistently observed with significant pairwise F st values between the lineages. Lineage 1 was observed throughout the natural geographic range but predominated in the continental US and was the only lineage observed in the major crabmeat-producing states (MD, LA, VA, NC). Lineage 2 primarily occurred in the Caribbean region of distribution area 1 but was also infrequently encountered in the South Atlantic Bight region of the US coast. Finally, Lineage 3 was only observed in Brazilian waters and had the lowest haplotype and nucleotide diversity values. Lineages 1 and 2 were separated by a mean pairwise distance (p-distance) of 3.15%, whereas Lineage 3 had a mean p-distance of 2.55% and 1.35% to Lineages 1 and 2, respectively. Within lineage mean p-distances were 0.45%, 0.19%, and 0.07% for Lineages 1, 2, and 3, respectively. Among all vouchered reference specimens collected from the continental United States, Mexico, Puerto Rico, and Venezuela, we identified 22 phylogenetically informative sites that drive observed lineage divergences. Haplotypes identified from barcode COI sequences from commercial C. sapidus products labeled as originating from the US all aligned with haplotypes from Lineage 1 reference specimens and haplotypes from commercial products labeled as originating from Venezuela all aligned with Lineage 2, suggesting that these lineages may be useful for indicating whether products originate from the continental US or are imported when package labeling is in question.

Resolution of the Portunusgladiator species complex: taxonomic status and identity of Monomiagladiator (Fabricius, 1798) and Monomiahaanii (Stimpson, 1858) (Brachyura, Decapoda, Portunidae).

The United States Food and Drug Administration (FDA) has recently adopted DNA barcoding for the purpose of determining the species identity of commercial seafood products. This effort has revealed instances of incongruence between current scientifically accepted taxon names and those utilized by the seafood industry in product labelling. One such case is that of "Portunushaanii", a name utilized by the seafood industry to label commercial products under the market name "red swimming crab." However, carcinologists currently regard P.haanii as synonym of Portunusgladiator Fabricius, 1798, which itself is the subject of debate over whether it is a secondary homonym of Cancer gladiator Fabricius, 1793. Further complicating matters, DNA barcode sequences from commercial products match GenBank sequences identified as Portunuspseudoargentatus Stephenson, 1961. Here the complicated taxonomic history of the Portunusgladiator complex is reviewed and a resolution proposed based on combined morphological descriptions and molecular phylogenetic analyses. It is demonstrated that, given the provisions of the International Code of Zoological Nomenclature and the current elevation of Monomia Gistel, 1848, to full genus rank, its type species, Portunusgladiator Fabricius, 1798, should be treated as a valid and available taxon name. It is also shown, upon examination and comparison of types and topotypic material that Monomiahaanii (Stimpson, 1858) is a distinct taxon from M.gladiator, and Portunuspseudoargentatus Stephenson, 1961, is a junior subjective synonym of M.haanii (Stimpson, 1858). Furthermore, it is shown that crab meat sold in the US currently labeled as "Portunushaanii" and/or "red swimming crab" is in fact M.haanii using comparative analysis of DNA barcode sequences between museum-vouchered reference specimens, whole crabs provided directly by a seafood importer, and processed commercial products purchased at retail.

Elucidation and partial NMR assignment of monosulfated maitotoxins from the Caribbean.

Compounds similar to maitotoxin (MTX) have been isolated from several laboratory strains of the dinoflagellate Gambierdiscus spp. from the Caribbean. Mass spectral results suggest that these compounds differ from MTX by the loss of one sulfate group and, in some cases, the loss of one methyl group with the addition of one degree of unsaturation. NMR experiments, using approximately 50 nmol of one of these compounds, have demonstrated that the 9-sulfo group of MTX is still present, suggesting that these compounds are 40-desulfo congeners of MTX.

Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation.

Species delimitation is a major quest in biology and is essential for adequate management of the organismal diversity. A challenging example comprises the fish species of red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). Recent genetic studies using mitochondrial markers, however, failed to delineate these nominal species, leading to the current lumping of the northern and southern populations into a single species ( L. campechanus). This decision carries broad implications for conservation and management as red snappers have been commercially over-exploited across the Western Atlantic and are currently listed as vulnerable. To address this conflict, we examine genome-wide data collected throughout the range of the two species. Population genomics, phylogenetic and coalescent analyses favour the existence of two independent evolutionary lineages, a result that confirms the morphology-based delimitation scenario in agreement with conventional taxonomy. Despite finding evidence of introgression in geographically neighbouring populations in northern South America, our genomic analyses strongly support isolation and differentiation of these species, suggesting that the northern and southern red snappers should be treated as distinct taxonomic entities.

Saxitoxin Poisoning in Green Turtles (Chelonia mydas) Linked to Scavenging on Mass Mortality of Caribbean Sharpnose Puffer Fish (Canthigaster rostrata-Tetraodontidae).

Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale puffer fish mortality events. In 2013, 2014, and 2017, puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the fish (0.5-0.7 µg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole fish (16.6-47.5 µg STX-eq/g) exceeded the 0.8 µg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested fish), brain, lung, kidney, and serum from three affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.

Toxicity and pathophysiology of palytoxin congeners after intraperitoneal and aerosol administration in rats.

Preparations of palytoxin (PLTX, derived from Japanese Palythoa tuberculosa) and the congeners 42-OH-PLTX (from Hawaiian P. toxica) and ovatoxin-a (isolated from a Japanese strain of Ostreopsis ovata), as well as a 50:50 mixture of PLTX and 42-OH-PLTX derived from Hawaiian P. tuberculosa were characterized as to their concentration, composition, in-vitro potency and interaction with an anti-PLTX monoclonal antibody (mAb), after which they were evaluated for lethality and tissue histopathology after intraperitoneal (IP) and aerosol administration to rats. Once each preparation was characterized as to its toxin composition by LC-HRMS and normalized to a total PLTX/OVTX concentration using HPLC-UV, all four preparations showed similar potency towards mouse erythrocytes in the erythrocyte hemolysis assay and interactions with the anti-PLTX mAb. The IP LD50 values derived from these experiments (0.92, 1.93, 1.81 and 3.26 µg/kg, for the 50:50 mix, 42-OH-PLTX, PLTX, and ovatoxin-a, respectively) were consistent with published values, although some differences from the published literature were seen. The aerosol LD50 values (0.063, 0.045, 0.041, and 0.031 µg/kg for the 50:50 mix, 42-OH PLTX, PLTX, and ovatoxin-a, respectively) confirmed the exquisite potency of PLTX suggested by the literature. The tissue histopathology of the different toxin preparations by IP and aerosol administration were similar, albeit with some differences. Most commonly affected tissues were the lungs, liver, heart, salivary glands, and adrenal glands. Despite some differences, these results suggest commonalities in potency and mechanism of action among these PLTX congeners.

Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit.

The cyanobacterium Aphanizomenon flos-aquae (AFA), from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA) dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 µg/g microcystin (MC) for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP) regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 µg/g. The objectives of this study were (1) identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2) use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA), based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51) distinct AFA-BGA products had MC ≥0.25 µg/g (the detection limit of the kit), 10 products had MC concentrations between 0.5 and 1.0 µg/g, and 4 products exceeded the limit (1.1-2.8 µg/g). LC-MS/MS confirmed PPIA results ≥0.5 µg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 µg/g MC, most were at or below 1.0 µg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC contamination found.

Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms.

Marine biotoxin-contaminated seafood has caused thousands of poisonings worldwide this century. Given these threats, there is an increasing need for improved technologies that can be easily integrated into coastal monitoring programs. This study evaluates approaches for monitoring toxins associated with recurrent toxin-producing Alexandrium and Dinophysis blooms on Long Island, NY, USA, which cause paralytic and diarrhetic shellfish poisoning (PSP and DSP), respectively. Within contrasting locations, the dynamics of pelagic Alexandrium and Dinophysis cell densities, toxins in plankton, and toxins in deployed blue mussels (Mytilus edulis) were compared with passive solid-phase adsorption toxin tracking (SPATT) samplers filled with two types of resin, HP20 and XAD-2. Multiple species of wild shellfish were also collected during Dinophysis blooms and used to compare toxin content using two different extraction techniques (single dispersive and double exhaustive) and two different toxin analysis assays (liquid chromatography/mass spectrometry and the protein phosphatase inhibition assay (PP2A)) for the measurement of DSP toxins. DSP toxins measured in the HP20 resin were significantly correlated (R² = 0.7-0.9, p < 0.001) with total DSP toxins in shellfish, but were detected more than three weeks prior to detection in deployed mussels. Both resins adsorbed measurable levels of PSP toxins, but neither quantitatively tracked Alexandrium cell densities, toxicity in plankton or toxins in shellfish. DSP extraction and toxin analysis methods did not differ significantly (p > 0.05), were highly correlated (R² = 0.98-0.99; p < 0.001) and provided complete recovery of DSP toxins from standard reference materials. Blue mussels (Mytilus edulis) and ribbed mussels (Geukensia demissa) were found to accumulate DSP toxins above federal and international standards (160 ng g-1) during Dinophysis blooms while Eastern oysters (Crassostrea virginica) and soft shell clams (Mya arenaria) did not. This study demonstrated that SPATT samplers using HP20 resin coupled with PP2A technology could be used to provide early warning of DSP, but not PSP, events for shellfish management.

Identification of Azadinium species and a new azaspiracid from Azadinium poporum in Puget Sound, Washington State, USA.

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC-MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized.

Suspected Palytoxin Inhalation Exposures Associated with Zoanthid Corals in Aquarium Shops and Homes - Alaska, 2012-2014.

On August 12, 2014, an Anchorage hospital notified the Alaska Section of Epidemiology (SOE) that a middle-aged male resident of Anchorage (patient A) had arrived in the emergency department with possible palytoxin exposure. Patient A complained of a bitter metallic taste, fever, weakness, cough, and muscle pain 7-8 hours after introduction of live zoanthid coral into his home aquarium. Palytoxin, a potent toxin known to produce the reported effects, is contained in zoanthid marine corals.