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A heatwave that blanketed the northeast Pacific Ocean in 2013–2015 had severe impacts on the marine ecosystem through altered species composition and survival. A direct result of this marine heatwave was a sustained, record-setting harmful algal bloom (HAB), caused by the toxigenic diatom, Pseudo-nitzschia, that led to an unprecedented delay in harvest opportunity for commercial Dungeness crab (Metacarcinus magister) and closure of other recreational, commercial and tribal shellfish harvest, including razor clams. Samples collected during a cruise in summer 2015, showed the appearance of a highly toxic “hotspot” between Cape Mendocino, CA and Cape Blanco, OR that was observed again during cruises in the summers of 2016–2018. The transport of toxic cells from this retentive site northward during wind relaxations or reversals associated with storms resulted in economically debilitating delay or closure of Dungeness crab harvest in both northern California and Oregon in 2015–2019. Analyses of historic large-scale Pseudo-nitzschia HABs have shown that these events occur during warm periods such as El Niño, positive phases of the Pacific Decadal Oscillation, or the record-setting marine heatwave. In order to reduce the impacts of large-scale HABs along the west coast of North America, early warning systems have been developed to forewarn coastal managers. These early warning systems include the Pacific Northwest and California HAB Bulletins, both of which have documented elevated domoic acid and increased risk associated with the northern California hotspot. These early warnings enable mitigative actions such as selective opening of safe harvest zones, increased harvest limits during low risk periods, and early harvest in anticipation of impending HAB events. The aims of this study are to show trends in nearshore domoic acid along the US west coast in recent years, including the recent establishment of a new seed bed of highly-toxic Pseudo-nitzschia, and to explore how early warning systems are a useful tool to mitigate the human and environmental health and economic impacts associated with harmful algal blooms.

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 μg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

A coastwide bloom of the toxigenic diatom Pseudo‐nitzschia in spring 2015 resulted in the largest recorded outbreak of the neurotoxin, domoic acid, along the North American west coast. Elevated toxins were measured in numerous stranded marine mammals and resulted in geographically extensive and prolonged closures of razor clam, rock crab, and Dungeness crab fisheries. We demonstrate that this outbreak was initiated by anomalously warm ocean conditions. Pseudo‐nitzschia australis thrived north of its typical range in the warm, nutrient‐poor water that spanned the northeast Pacific in early 2015. The seasonal transition to upwelling provided the nutrients necessary for a large‐scale bloom; a series of spring storms delivered the bloom to the coast. Laboratory and field experiments confirming maximum growth rates with elevated temperatures and enhanced toxin production with nutrient enrichment, together with a retrospective analysis of toxic events, demonstrate the potential for similarly devastating ecological and economic disruptions in the future., Key Points The 2015 U.S. West Coast wide toxic Pseudo‐nitzschia australis bloom was facilitated by anomalous ocean conditionsThe seasonal transition to upwelling provided nutrients for the bloom, and spring storms delivered toxic cells to the nearshore environmentWest Coast toxic Pseudo‐nitzschia events are triggered by warm anomalies associated with El Niño and the Pacific Decadal Oscillation

The Makah Tribe of Neah Bay, Washington, has historically relied on the subsistence harvest of coastal seafood, including shellfish, which remains an important cultural and ceremonial resource. Tribal legend describes visitors from other tribes that died from eating shellfish collected on Makah lands. These deaths were believed to be caused by paralytic shellfish poisoning, a human illness caused by ingestion of shellfish contaminated with saxitoxins, which are produced by toxin-producing marine dinoflagellates on which the shellfish feed. These paralytic shellfish toxins include saxitoxin, a potent Na+ channel antagonist that binds to the pore region of voltage gated Na+ channels. Amino acid mutations in the Na+ channel pore have been demonstrated to confer resistance to saxitoxin in softshell clam populations exposed to paralytic shellfish toxins present in their environment. Because of the notion of resistance to paralytic shellfish toxins, the study aimed to determine if a resistance strategy was possible in humans with historical exposure to toxins in shellfish. We collected, extracted and purified DNA from buccal swabs of 83 volunteer Makah tribal members and sequenced the skeletal muscle Na+ channel (Nav1.4) at nine loci to characterize potential mutations in the relevant saxitoxin binding regions. No mutations of these specific regions were identified after comparison to a reference sequence. This study suggests that any resistance of Makah tribal members to saxitoxin, if present, is not a function of Nav1.4 modification, but may be due to mutations in neuronal or cardiac sodium channels, or some other mechanism unrelated to sodium channel function.

Over the last decade, our understanding of the environmental controls on Pseudo-nitzschia blooms and domoic acid (DA) production has matured. Pseudo-nitzschia have been found along most of the world's coastlines, while the impacts of its toxin, DA, are most persistent and detrimental in upwelling systems. However, Pseudo-nitzschia and DA have recently been detected in the open ocean's high-nitrate, low-chlorophyll regions, in addition to fjords, gulfs and bays, showing their presence in diverse environments. The toxin has been measured in zooplankton, shellfish, crustaceans, echinoderms, worms, marine mammals and birds, as well as in sediments, demonstrating its stable transfer through the marine food web and abiotically to the benthos. The linkage of DA production to nitrogenous nutrient physiology, trace metal acquisition, and even salinity, suggests that the control of toxin production is complex and likely influenced by a suite of environmental factors that may be unique to a particular region. Advances in our knowledge of Pseudo-nitzschia sexual reproduction, also in field populations, illustrate its importance in bloom dynamics and toxicity. The combination of careful taxonomy and powerful new molecular methods now allow for the complete characterization of Pseudo-nitzschia populations and how they respond to environmental changes. Here we summarize research that represents our increased knowledge over the last decade of Pseudo-nitzschia and its production of DA, including changes in worldwide range, phylogeny, physiology, ecology, monitoring and public health impacts.

Several species of the diatom Pseudo-nitzschia produce the neurotoxin domoic acid (DA). Consumption of fish and shellfish that have accumulated this potent excitotoxin has resulted in severe illness and even death in humans, marine mammals, and seabirds. Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle is a cosmopolitan diatom commonly occurring in the waters of the Pacific Northwest (PNW) and the eastern North Atlantic, including the North Sea. However, genetic and physiological relationships among populations throughout this large geographic distribution have not been assessed. Population genetic parameters (e.g., Hardy‐Weinberg equilibrium, linkage equilibrium, FST) calculated for P. pungens collected from the Juan de Fuca eddy region in the PNW indicated the presence of two distinct groups that were more divergent from each other than either was from aP. pungens sample from the North Sea. Geographic heterogeneity was also detected within each of the two PNW groups. These results suggested that the populations of P. pungens recently mixed in the Juan de Fuca eddy region (a seasonally retentive feature off the coasts of Washington State, USA, and Vancouver Island, Canada) but did not exchange genetic material by sexual reproduction. Alternatively, these two groups may be cryptic (morphologically identical, but reproductively isolated) species. Identifying cryptic diversity in Pseudonitzschia is important for bloom prediction and aiding the identification of molecular markers that can be used for rapid detection assay development.

Plankton samples from three inland embayments and several outer coastal sites of Washington State were collected from 1997 through 1999 and were examined for the presence of diatoms of the genus Pseudo-nitzschia and levels of the toxin, domoic acid (DA). Seven species were observed, including Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle, P. multiseries (Hasle) Hasle, P. australis Frenguelli, P. fraudulenta (Cleve) Hasle, P. cf. heimii Manguim, P. pseudodelicatissima (Hasle) Hasle, and P. delicatissima (Cleve) Heiden. The coastal Pseudo-nitzschia species assemblages differed significantly from those observed within embayments. The dominant species observed at coastal sites were P. pseudodelicatissima and P. cf. heimii. Pseudo-nitzschia assemblages found in embayments included one or more of the following species: P. pungens, P. multiseries, P. australis, P. pseudodelicatissima, and P. fraudulenta. The nuclear large subunit rRNA gene was sequenced for six of the seven species identified. This sequence revealed that P. multiseries, P. pungens, P. australis, and P. heimii were genetically similar to those found in California, whereas P. delicatissima and P. pseudodelicatissima were distinct from the California isolates. Although the concentrations of DA in razor clams along Washington State coasts have exceeded regulatory limits several times since 1991, levels of DA in shellfish from Washington State embayments have not yet exceeded regulatory limits. The widespread presence of toxin-producing Pseudo-nitzschia species suggests, however, that toxic blooms are likely to occur within embayments in the future. In conjunction with the monitoring of environmental conditions conducive to toxic bloom formation, the development of species-specific probes for rapid and accurate detection of potentially toxic Pseudo-nitzschia species in this region would enable the forecasting of a toxic event before DA accumulates in shellfish, thereby reducing the impacts to coastal communities.

Sea lion mortalities in central California during May and June 1998 were traced to their ingestion of sardines and anchovies that had accumulated the neurotoxin domoic acid. The detection of toxin in urine, feces, and stomach contents of several sea lions represents the first proven occurrence of domoic acid transfer through the food chain to a marine mammal. The pennate diatoms, Pseudo-nitzschia multiseriesand P. australis, were the dominant, toxinproducing phytoplankton constituting algal blooms near Monterey Bay, Half Moon Bay, and Oceano Dunes, areas where sea lions with neurological symptoms stranded. Toxic Pseudo-nitzschia were also found near Morro Bay, Point Conception, Point Arguello, and Santa Barbara, demonstrating that these species were widespread along the central California coast in June 1998. Measurements of domoic acid during three cruises in early June showed the )†

D-tagatose, a stereoisomer of D-fructose, is a naturally occurring ketohexose proposed for use as a low-calorie bulk sweetener. Ingested D-tagatose appears to be poorly absorbed. The absorbed portion is metabolized in the liver by a pathway similar to that of D-fructose. The main purpose of this study was to determine if acute or repeated oral doses of D-tagatose would cause elevations in plasma uric acid (as is seen with fructose) in normal humans and Type 2 diabetics. In addition, effects of subchronic D-tagatose ingestion on fasting plasma phosphorus, magnesium, lipids, and glucose homeostasis were studied. Eight normal subjects and eight subjects with Type 2 diabetes participated in this two-phase study. Each group was comprised of four males and four females. In the first phase, all subjects were given separate 75 g 3-h oral glucose and D-tagatose tolerance tests. Uric acid, phosphorus, and magnesium were determined in blood samples collected from each subject at 0, 30, 60, 120, and 180 min after dose. In the 8-week phase of the study, the normals were randomly placed into two groups which received 75 g of either D-tagatose or sucrose (25 g with each meal) daily for 8 weeks. The diabetics were randomized into two groups which received either 75 g D-tagatose or no supplements of sugar daily for 8 weeks. Uric acid, phosphorus, magnesium, lipids, glycosylated hemoglobin, glucose, and insulin were determined in fasting blood plasma of all subjects at baseline (time zero) and biweekly over the 8 weeks. The 8-week test did not demonstrate an increase in fasting plasma uric acid in response to the daily intake of D-tagatose. However, a transient increase of plasma uric acid levels was observed after single doses of 75 g of D-tagatose in the tolerance test. Plasma uric acid levels were found to rise and peak at 60 min after such dosing. No clinical relevance was attributed to this treatment-related effect because excursions of plasma uric acid levels above the normal range were small and were of short duration. Consistent with earlier observations on fructose, the increase of plasma uric acid was associated with a slight decrease of plasma phosphorus and a slight increase of magnesium. The daily ingestion of D-tagatose for 8 weeks had no effect on fasting plasma magnesium, phosphorus, cholesterol, triglycerides, glycosylated hemoglobin, glucose, and insulin levels. The ingestion of three 25-g doses per day for a period of 8 weeks resulted in varying amounts of flatulence in seven of the eight subjects, and some degree of diarrhea in six subjects. D-tagatose holds promise as a sweetener with no adverse clinical effects observed in these studies.

A bloom of the pennate diatom Pseudo-nitzschia, several species of which are associated with the production of the potent excitotoxin domoic acid, was observed in a Puget Sound, Washington embayment in July and August of 1997. Penn Cove, which receives nutrients from the nearby Skagit River and abundant sunshine during summer months due to its location in the rain shadow of the Olympic Mountains, is the home of a commercial mussel farm which supplies shellfish to many coastal areas of the USA. Levels of domoic acid in mussels increased to 3 ppm on 6 and 10 July, corresponding to the observation of a brown algal bloom in Penn Cove. Four species of Pseudo-nitzschia (P. pungens, P. multiseries, P. australis, and P. pseudodelicatissima) were present in our samples from the cove, corresponding to levels of domoic acid in seawater ranging from 0.1–0.8 µg l−1 as measured by a receptor binding assay. The highest Pseudo-nitzschia concentration during the time of our sampling was 13 million cells per liter on 28 July. The bloom of Pseudo-nitzschia occurred after a period of strong discharge from the Skagit River and rain accompanied by elevated south and southeasterly winds. Stratification of the cove, providing optimal bloom conditions, was facilitated by weak winds, sunshine, and a freshwater lens at the mouth of the cove. The position of the Pseudo-nitzschia bloom was influenced by buoyancy fronts caused by exchange of water within the cove with that of Saratoga Passage. The decay of this bloom in Penn Cove was accompanied by decreasing nitrate levels at all measured depths. These and future observations aid in the development of a model for prediction of toxic bloom events in the shallow embayments of Puget Sound. Copyright © 1999 John Wiley & Sons, Ltd.

Hybridization between genetically distinguishable taxa provides opportunities for investigating speciation. While hybridization is a common phenomenon in various macro-organisms, natural hybridization among micro-eukaryotes is barely studied. Here we used a nuclear and a chloroplast molecular marker and morphology to demonstrate the presence of natural hybrids between two genetically and morphologically distinct varieties of the marine planktonic diatom Pseudo-nitzschia pungens (vars. pungens and cingulata) in a contact zone in the northeast Pacific. Cloning and sequencing of the rDNA internal transcribed spacer region revealed strains containing ribotypes from both varieties, indicating hybridization. Both varieties were found to also have different chloroplast-encoded rbcL sequences. Hybrid strains were either hetero- or homoplastidial, as demonstrated by denaturing gradient gel electrophoresis, which is in accordance with expectations based on the mode of chloroplast inheritance in Pseudo-nitzschia. While most hybrids are probably first generation, there are also indications for further hybridization. Morphologically, the hybrids resembled var. pungens for most characters rather than having an intermediate morphology. Further research should focus on the hybridization frequency, by assessing the spatial and temporal extent of the contact zone, and hybrid fitness, to determine the amount of gene flow between the two varieties and its evolutionary consequences.

Several diseases are characterized by chronic inflammation, a condition frequently associated with angiogenesis and fibrogenesis that account for the development of granulation tissue. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a crucial modulator of intracellular prosurvival signaling pathways and is implicated in the pathogenesis of inflammatory process. In this study, we have investigated the role of NF-kappaB in the angiogenic and fibrogenic response induced by lambda-carrageenin in a rat model of chronic inflammation at 1, 3, and 5 days. The subcutaneous implant of lambda-carrageenin-soaked sponges in rat induced a time-related increase of granulation tissue formation accompanied by intense neovascularization. These lambda-carrageenin-induced changes were significantly reduced by coinjection of wild-type oligodeoxynucleotide (WT ODN) decoy to NF-kappaB. Molecular, morphological, and ultrastructural analysis performed on whole granulation tissue demonstrated: (1) inhibition of NF-kappaB/DNA binding activity; (2) downregulation of cyclooxygenase-2, matrix metalloproteinase-9, tumor necrosis factor-alpha, and vascular endothelial growth factor; (3) upregulation of thrombospondin (TSP)-1 at 1 day and TSP-2 at 5 days; and (4) increase in Bax to Bcl-2 ratio. Our findings show that the blockade of NF-kappaB activation by WT ODN decoy prevents the development of granulation tissue induced by lambda-carrageenin-soaked sponge implant upregulating Bax as well as TSP-1 and TSP-2 expression.