Domoic Acid Production Research Articles

Effects of turbulence on diatoms of the genus Pseudo-nitzschia spp. and associated bacteria.

Turbulence is one of the least investigated environmental factors impacting the ecophysiology of phytoplankton, both at the community and individual species level. Here, we investigated, for the first time, the effect of a turbulence gradient (Reynolds number, from Reλ = 0 to Reλ = 360) on two species of the marine diatom Pseudo-nitzschia and their associated bacterial communities under laboratory conditions. Cell abundance, domoic acid (DA) production, chain formation, and Chl a content of P. fraudulenta and P. multiseries were higher for intermediate turbulence (Reλ = 160 or 240). DA was detectable only in P. multiseries samples. These observations were supported by transcriptomic analyses results, which suggested the turbulence related induction of the expression of the DA production locus, with a linkage to an increased photosynthetic activity of the total metatranscriptome. This study also highlighted a higher richness of the bacterial community associated with the nontoxic strain of P. fraudulenta in comparison to the toxic strain of P. multiseries. Bacillus was an important genus in P. multiseries cultures (relative abundance 15.5%) and its highest abundances coincided with the highest DA levels. However, associated bacterial communities of both Pseudo-nitzschia species did not show clear patterns relative to turbulence intensity.

Analysis of Amphora ostrearia var. minor Grunow, a blooming marine diatom (Bacillariophyceae) in French Atlantic coastal waters and its transfer to the genus Tetramphora

A marine amphoroid diatom, responsible for blooms in 2011 and 2019 in French Atlantic waters was studied using morphology and molecular phylogeny. The highest cell concentrations were reached in the region of Marennes Oléron on 19 August 2011 and were related to a greyish discolouration of gills of oysters and mussels. It was identified as Amphora ostrearia var. minor Grunow, based on morphological characteristics. To ensure the correct identification, type material was re-investigated, using the type slide n° 142 (Cleve & Möller 1878. Diatoms. Part II. Esatas Edquists Boktryckeri, Upsala: 49–108) that enabled us to establish the identity of the taxon. Plastid morphology corresponded to Mereschkowsky’s type 8 amphoroid chloroplast, a feature typical for the genus Tetramphora Mereschkowsky. Results of a phylogenetic analysis inferred from a concatenated alignment including the nuclear marker SSU and the chloroplast rbcL indicated that A. ostrearia var. minor was positioned inside the monophyletic clade Tetramphora (Mereschkowsky) Stepanek & Kociolek. This, together with the morphological data, necessitated a transfer of A. ostrearia var. minor Grunow to the genus Tetramphora (Mereschkowsky) Stepanek & Kociolek. In addition, the genetic distances (p-values) from SSU–rbcL sequences were estimated among the species of Tetramphora including T. ostrearia, the generitype. These revealed that the divergence between Tetramphora ostrearia (Brébisson) Mereschkowsky and its variety minor was higher than the mean value within species of the genus. As a consequence, the studied taxon is raised to the species level as Tetramphora minor (Grunow) Nézan, comb. et stat. nov. Toxin analysis of four strains of T. minor revealed the absence of domoic acid production. Pigment analysis of T. ostrearia revealed a high fucoxanthin production, similar to other amphoroid species.

First evidence of the induction of domoic acid production in Pseudo-nitzschia australis by the copepod Temora longicornis from the French coast

Diatoms of the genus Pseudo-nitzschia are widespread in marine waters. Some of them can produce the toxin domoic acid (DA) which can be responsible for amnesic shellfish poisoning (ASP) when transferred into the food web. These ASP events are of major concern, due to their ecological and socio-economic repercussions, particularly on the shellfish industry. Many studies have focused on the influence of abiotic factors on DA induction, less on the role of biotic interactions. Recently, the presence of predators has been shown to increase DA production in several Pseudo-nitzschia species, in particular in Arctic areas. In order to investigate the relationship between Pseudo-nitzschia species and grazers from the French coast, exposures between one strain of three species (P. australis, P. pungens, P. fraudulenta) and the copepod Temora longicornis were conducted for 5 days. Cellular and dissolved DA content were enhanced by 1,203 % and 1,556 % respectively after the 5-days exposure of P.australis whereas no DA induction was observed in P. pungens and P. fraudulenta. T. longicornis consumed all three Pseudo-nitzschia species. The copepod survival was not related to DA content. This study is an essential first step to better understanding the interactions between planktonic species from the French coast and highlights the potential key role of copepods in the Pseudo-nitzschia bloom events in the temperate ecosystems.

Emerging phylogeographic perspective on the toxigenic diatom genus Pseudo-nitzschia in coastal northern European waters and gateways to eastern Arctic seas: Causes, ecological consequences and socio-economic impacts

The diatom Pseudo-nitzschia H. Peragallo is perhaps the most intensively researched genus of marine pennate diatoms, with respect to species diversity, life history strategies, toxigenicity, and biogeographical distribution. The global magnitude and consequences of harmful algal blooms (HABs) of Pseudo-nitzschia are particularly significant because of the high socioeconomic impacts and environmental and human health risks associated with the production of the neurotoxin domoic acid (DA) among populations of many (although not all) species. This has led to enhanced monitoring and mitigation strategies for toxigenic Pseudo-nitzschia blooms and their toxins in recent years. Nevertheless, human adaptive actions based on future scenarios of bloom dynamics and proposed shifts in biogeographical distribution under climate-change regimes have not been implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) program these issues were addressed with respect to past, current and anticipated future status of key HAB genera such as Pseudo-nitzschia and expected benefits of enhanced monitoring. Data on the distribution and frequency of Pseudo-nitzschia blooms in relation to DA occurrence and associated amnesic shellfish toxin (AST) events were evaluated in a contemporary and historical context over the past several decades from key northern CoCliME Case Study areas. The regional studies comprised the greater North Sea and adjacent Kattegat-Skagerrak and Norwegian Sea, eastern North Atlantic marginal seas and Arctic gateways, and the Baltic Sea. The first evidence of possible biogeographical expansion of Pseudo-nitzschia taxa into frontier eastern Arctic gateways was provided from DNA barcoding signatures. Key climate change indicators, such as salinity, temperature, and water-column stratification were identified as drivers of upwelling and advection related to the distribution of regional Pseudo-nitzschia blooms. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change indicators may play key roles in selecting for the occurrence and diversity of Pseudo-nitzschia species within the broader microeukaryote communities. Shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for high-magnitude Pseudo-nitzschia blooms, currently absent from this area. Ecological and socioeconomic impacts of Pseudo-nitzschia blooms are evaluated with reference to effects on fisheries and mariculture resources and coastal ecosystem function. Where feasible, effective adaptation strategies are proposed herein as emerging climate services for the northern CoCLiME region.

Potential Domoic Acid (Neurotoxin) Producing Phytoplankton Pseudonitzschia in Indian Coastal Water - do We Need to Care?

Pseudonitzschia species are potential domoic acid producers, a neurotoxin, responsible for the infamous human HAB intoxication at Prince Edward Island, Canada in 1987, costing human lives. Global warming has widened the reach of these phytoplankton species and it is being reported in Indian waters. We report the occurrence of ten Pseudonitzschia species in the northwestern coastal waters of India, out of which, seven are potential domoic acid producers. The question arises, are we vulnerable to HAB (Domoic Acid) toxicity? In light of the observation that Pseudonitzschia dominates the coastal waters of Veraval and its abundance is increasing with time, the present study briefly synthesizes the available information on the ecology, metabolism, and other relevant knowledge related to the domoic acid production by Pseudonitzschia and assesses the risk of human intoxication through trend and forecast analysis with the possible preventive measures required.

Simulated upwelling and marine heatwave events promote similar growth rates but differential domoic acid toxicity in Pseudo-nitzschia australis

Along the west coast of the United States, highly toxic Pseudo-nitzschia blooms have been associated with two contrasting regional phenomena: seasonal upwelling and marine heatwaves. While upwelling delivers cool water rich in pCO2 and an abundance of macronutrients to the upper water column, marine heatwaves instead lead to warmer surface waters, low pCO2, and reduced nutrient availability. Understanding Pseudo-nitzschia dynamics under these two conditions is important for bloom forecasting and coastal management, yet the mechanisms driving toxic bloom formation during contrasting upwelling vs. heatwave conditions remain poorly understood. To gain a better understanding of what drives Pseudo-nitzschia australis growth and toxicity during these events, multiple-driver scenario or ‘cluster’ experiments were conducted using temperature, pCO2, and nutrient levels reflecting conditions during upwelling (13 °C, 900 ppm pCO2, replete nutrients) and two intensities of marine heatwaves (19 °C or 20.5 °C, 250 ppm pCO2, reduced macronutrients). While P. australis grew equally well under both heatwave and upwelling conditions, similar to what has been observed in the natural environment, cells were only toxic in the upwelling treatment. We also conducted single-driver experiments to gain a mechanistic understanding of which drivers most impact P. australis growth and toxicity. These experiments indicated that nitrogen concentration and N:P ratio were likely the drivers that most influenced domoic acid production, while the impacts of temperature or pCO2 concentration were less pronounced. Together, these experiments may help to provide both mechanistic and holistic perspectives on toxic P. australis blooms in the dynamic and changing coastal ocean, where cells interact simultaneously with multiple altered environmental variables.

Annual variation in domoic acid in phytoplankton and shellfish samples from Daya Bay of the South China Sea

Domoic acid (DA) is a well-known phycotoxin that causes amnesic shellfish poisoning (ASP) and is mainly produced by diatom species belonging to the genus Pseudo-nitzschia. An annual survey was conducted monthly over the period of September 2020 to August 2021 in Daya Bay of the South China Sea to investigate the dynamics of particulate and shellfish DA and their relationships with the abundance of Pseudo-nitzschia spp. and environmental parameters. Pseudo-nitzschia spp. was one of the most dominant phytoplankton taxa, and a Pseudo-nitzschia bloom occurred during the survey with the highest abundance of 1.91 × 106 cells L−1. DA was detected in almost all plankton samples with the highest value of 120.7 ng L−1, and high DA concentrations coincided with the abundant presence of Pseudo-nitzschia. DA is prevalent in Daya Bay throughout the year, with detection rates of 98.3%, 82.6%, and 82.6% in plankton samples, in-situ and purchased shellfish, respectively. Higher DA concentrations were detected in the scallop (Chamys nobilis), with the highest concentration of 5.34 µg g−1. High water temperature and low DSi:DIN ratio promoted the growth of Pseudo-nitzschia and DA production. The results suggest that the increasing nitrogen loading and silicate limitation during Pseudo-nitzschia blooms together with the increase in water temperature may increase the risk of DA contamination in Daya Bay.

Pseudo-nitzschia species, toxicity, and dynamics in the southern Indian River Lagoon, FL

The Indian River Lagoon (IRL) spans approximately one-third of the east coast of Florida and, in recent years, has faced frequent harmful algal blooms (HABs). Blooms of the potentially toxic diatom, Pseudo-nitzschia, occur throughout the lagoon and were reported primarily from the northern IRL. The goal of this study was to identify species of Pseudo-nitzschia and characterize their bloom dynamics in the southern IRL system where monitoring has been less frequent. Surface water samples collected from five locations between October 2018 and May 2020 had Pseudo-nitzschia spp. present in 87% of samples at cell concentrations up to 1.9×103 cells mL−1. Concurrent environmental data showed Pseudo-nitzschia spp. were associated with relatively high salinity waters and cool temperatures. Six species of Pseudo-nitzschia were isolated, cultured, and characterized through 18S Sanger sequencing and scanning electron microscopy. All isolates demonstrated toxicity and domoic acid (DA) was present in 47% of surface water samples. We report the first known occurrence of P. micropora and P. fraudulenta in the IRL, and the first known DA production from P. micropora.

Domoic acid production by Pseudo-nitzschia australis: Re-evaluating the role of macronutrient limitation on toxigenicity

The toxigenic diatom Pseudo-nitzschia australis (Frenguelli), isolated from the California Current System (CCS), was examined in unialgal laboratory cultures to evaluate domoic acid (DA) production and cellular growth as a response to macronutrient limitation. Toxic blooms of P. australis are common in the coastal waters of eastern boundary upwelling systems (EBUS), including those of the CCS off the west coast of the United States where limitation by macronutrients, specifically silicon as silicic acid [Si(OH)4], or phosphorus as phosphate [PO43-], has been suggested to increase the production of DA by these diatoms. This study used batch cultures grown under conditions of macronutrient sufficiency and limitation, expected during and after a natural upwelling event, to determine whether PO43- or Si(OH)4 deficiency enhances the production of DA and the expected risk of DA toxicity in natural coastal ecosystems. These controlled lab studies demonstrate that despite increases in cell-specific DA concentrations found during the nutrient-limited stationary phase, DA production rates did not increase due to either PO43- or Si(OH)4 limitation, and total DA production rates were statistically greater during the nutrient-replete, exponential growth phase compared to the nutrient-limited, stationary phase. In addition, the relative contribution of particulate DA (pDA) and dissolved DA (dDA) varied markedly with growth phase, where the contribution of pDA to total DA (pDA + dDA) declined from an average of 70% under P- and Si-replete conditions to 49% under P-limited conditions and 39% under Si-limited conditions. These laboratory results demonstrate that macronutrient sufficiency does not regulate the biosynthetic production of DA by this strain of P. australis. This finding, together with a comparative analysis of the various equations employed to estimate DA production, suggests that the current paradigm of increased toxigenicity due to macronutrient limitation be carefully re-examined, particularly when attempting to forecast the toxic threat of DA to coastal ecosystems as a function of macronutrient availability.

Progresses of the Influencing Factors and Detection Methods of Domoic Acid

Domoic acid (DA) is a neurotoxin mainly produced by Pseudo-nitzschia diatom, which belongs to the genera Rhomboida. It can combine with the receptors of glutamate of neurotransmitters, then affecting the normal nerve signal transmission of the organism and causing nervous system disorders. However, as a natural marine drug, DA can also be used for pest prevention and control. Although the distribution of DA in the world has already been reported in the previous reviews, the time and location of its first discovery and the specific information are not complete. Therefore, the review systematically summarizes the first reported situation of DA in various countries (including species, discovery time, and collection location). Furthermore, we update and analyze the factors affecting DA production, including phytoplankton species, growth stages, bacteria, nutrient availability, trace metals, and so on. These factors may indirectly affect the growth environment or directly affect the physiological activities of the cells, then affect the production of DA. Given that DA is widely distributed in the environment, we summarize the main technical methods for the determination of DA, such as bioassay, high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), biosensor, and so on, as well as the advantages and disadvantages of each method used so far, which adds more new knowledge in the literature about DA until now. Finally, the DA research forecast and its industrial applications were prospected to prevent its harm and fully explore its potential value.

Physiological conditions favorable to domoic acid production by three Pseudo-nitzschia species

Twenty-six species of the cosmopolitan genus Pseudo-nitzschia can produce domoic acid (DA), a neurotoxin responsible for amnesic shellfish poisoning (ASP). To improve knowledge on this issue, we studied the physiological conditions favorable to DA production and accumulation by three Pseudo-nitzschia species from French coastal waters: P. australis, P. pungens, and P. fraudulenta. They were grown in batch cultures under silicate limitation to characterize their physiological traits and calculate their DA production rates. Three strains were studied per species to consider intraspecific diversity and better characterize interspecific differences. DA production was not influenced by growth or silicate limitation in any of the three species. In contrast, silicate limitation in the stationary phase led to DA accumulation by inhibiting cell division, while DA production was still active. The maximum cellular DA (cDA) production rate was 2.95 pg cell−1 d−1 for P. australis, 0.07 pg cell−1 d−1 for P. pungens, and 0.03 pg cell−1 d−1 for P. fraudulenta. The physiological conditions favorable to cDA production and accumulation by P. australis and P. pungens differed. The three species presented similar growth rates, but P. australis had higher photosynthetic capacities that could partly explain its higher DA production potential. The cDA production and the net dissolved DA (dDA) production in the culture medium were favored by different growth conditions. The cDA production to net dDA production ratio was species specific, with P. pungens excreting proportionally more of its produced DA. These laboratory results on cultures imply that cDA production and net dDA production can occur during all phases of P. australis, P. pungens, and P. fraudulenta blooms. The interactions between the species composition of the bloom, the species-specific capacity for DA production, and the effect of silicate limitation – among other factors – on DA cell quotas drive the toxigenicity of Pseudo-nitzschia blooms.

Development, calibration, and evaluation of a model of Pseudo-nitzschia and domoic acid production for regional ocean modeling studies

Pseudo-nitzschia species are one of the leading causes of harmful algal blooms (HABs) along the western coast of the United States. Approximately half of known Pseudo-nitzschia strains can produce domoic acid (DA), a neurotoxin that can negatively impact wildlife and fisheries and put human life at risk through amnesic shellfish poisoning. Production and accumulation of DA, a secondary metabolite synthesized during periods of low primary metabolism, is triggered by environmental stressors such as nutrient limitation. To quantify and estimate the feedbacks between DA production and environmental conditions, we designed a simple mechanistic model of Pseudo-nitzschia and domoic acid dynamics, which we validate against batch and chemostat experiments. Our results suggest that, as nutrients other than nitrogen (i.e., silicon, phosphorus, and potentially iron) become limiting, DA production increases. Under Si limitation, we found an approximate doubling in DA production relative to N limitation. Additionally, our model indicates a positive relationship between light and DA production. These results support the idea that the relationship with nutrient limitation and light is based on direct impacts on Pseudo-nitzschia biosynthesis and biomass accumulation. Because it can easily be embedded within existing coupled physical-ecosystem models, our model represents a step forward toward modeling the occurrence of Pseudo-nitzschia HABs and DA across the U.S. West Coast.

A Decade of Time Series Sampling Reveals Thermal Variation and Shifts in Pseudo-nitzschia Species Composition That Contribute to Harmful Algal Blooms in an Eastern US Estuary

In 2016-17, shellfish harvesting closed for the first time in Narragansett Bay, Rhode Island, USA, from domoic acid (DA), a neurotoxin produced by diatoms of the Pseudo-nitzschia genus. Pseudo-nitzschia have occurred frequently for over 60 years in Narragansett Bay’s Long-Term Plankton Time Series (NBPTS), therefore it is surprising that the first closure only recently occurred. Pseudo-nitzschia species are known to vary in their toxin production, thus species identification is critical for understanding the underlying ecological causes of these harmful algal blooms (HABs). DNA in plankton biomass can be preserved for many years, so molecular barcoding of archived samples is useful for delineation of taxa over time. This study used amplification of the Pseudo-nitzschia-specific 18S-5.8S rDNA internal transcribed spacer region 1 (ITS1) in plankton samples and high throughput sequencing to characterize Pseudo-nitzschia species composition over a decade in Narragansett Bay, including eight years before the 2016-17 closures and two years following. This metabarcoding method can discriminate nearly all known Pseudo-nitzschia species. Several species recur as year-round residents in Narragansett Bay (P. pungens var. pungens, P. americana, P. multiseries, and P. calliantha). Various other species increased in frequency after 2015, and some appeared for the first time during the closure period. Notably, P. australis, a species prevalent in US West Coast HABs and known for high DA production, was not observed in Narragansett Bay until the 2017 closure but has been present in several years after the closures. Annual differences in Pseudo-nitzschia composition were correlated with physical and chemical conditions, predominantly water temperature. The long-term composition trends of Pseudo-nitzschia in Narragansett Bay serve as a baseline for identifying the introduction of new species, understanding shifting assemblages that contributed to the 2016-17 closures, and monitoring species that may be cause for future concern.

Comparative Analysis of Pseudo-nitzschia Chloroplast Genomes Revealed Extensive Inverted Region Variation and Pseudo-nitzschia Speciation

Pseudo-nitzschia is a species-rich genus where many species can induce harmful algae blooms (HABs) associated with the toxin domoic acid (DA) production. Despite the importance of Pseudo-nitzschia species to coastal environments, their genomic information is rather limited, hindering research on biodiversity and evolutionary analysis. In this study, we report full-length chloroplast genomes (cpDNAs) of nine Pseudo‐nitzschia, among which cpDNAs of eight Pseudo-nitzschia species were reported for the first time. The sizes of these Pseudo-nitzschia cpDNAs, which showed typical quadripartite structures, varied substantially, ranging from 116,546 bp to 158,840 bp in size. Comparative analysis revealed the loss of photosynthesis-related gene psaE in cpDNAs of all Pseudo-nitzschia species except that of P. americana, and the selective loss of rpl36 in P. hainanensis. Phylogenetic analysis showed that all Pseudo-nitzschia strains were grouped into two clades, with clade 1 containing cpDNAs of P. multiseries, P. pungens, P. multistriata, and P. americana, and clade 2 containing cpDNAs of P. hainanensis, P. cuspidata, Pseudo-nitzschia sp. CNS00097, P. delicatissima, and P. micropora. The small size of the P. americana cpDNA was primarily due to its shortened inverted repeat (IR) regions. While psaA and psaB were found in the IR regions of cpDNAs of other eight species, these two genes were found outside of the IR regions of P. americana cpDNA. In contrast, P. hainanensis had the largest size because of expansion of IR regions with each IR region containing 15 protein-coding genes (PCGs). Eleven genetic regions of these Pseudo-nitzschia cpDNAs exhibited high nucleotide diversity (Pi) values, suggesting that these regions may be used as molecular markers for distinguishing different Pseudo-nitzschia species with high resolution and high specificity. Phylogenetic analysis of the divergence of nine Pseudo-nitzschia species indicated that these species appeared at approximately 41 Mya. This study provides critical cpDNA resources for future research on the biodiversity and speciation of Pseudo-nitzschia species.

Costs and benefits of predator-induced defence in a toxic diatom.

Phytoplankton employ a variety of defence mechanisms against predation, including production of toxins. Domoic acid (DA) production by the diatom Pseudo-nitzschia spp. is induced by the presence of predators and is considered to provide defence benefits, but the evidence is circumstantial. We exposed eight different strains of P. seriata to chemical cues from copepods and examined the costs and the benefits of toxin production. The magnitude of the induced toxin response was highly variable among strains, while the costs in terms of growth reduction per DA cell quota were similar and the trade-off thus consistent. We found two components of the defence in induced cells: (i) a 'private good' in terms of elevated rejection of captured cells and (ii) a 'public good' facilitated by a reduction in copepod feeding activity. Induced cells were more frequently rejected by copepods and rejections were directly correlated with DA cell quota and independent of access to other food items. By contrast, the public-good effect was diminished by the presence of alternative prey suggesting that it does not play a major role in bloom formation and that its evolution is closely associated with the grazing-deterrent private good.

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea.

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

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.

Effects of ocean acidification on the growth, photosynthetic performance, and domoic acid production of the diatom Pseudo-nitzschia australis from the California Current System

Pseudo-nitzschia australis (Frenguelli), a toxigenic pennate diatom capable of producing the neurotoxin domoic acid (DA), was examined in unialgal laboratory cultures to quantify its physiological response to ocean acidification (OA) - the decline in pH resulting from increasing partial pressure of CO2 (pCO2) in the oceans. Toxic blooms of P. australis are common in the coastal waters of eastern boundary upwelling systems (EBUS), including those of the California Current System (CCS) off the west coast of the United States where increased pCO2 and decreased seawater pH are well-known. This study determined the production of dissolved (dDA) and particulate DA (pDA), the rates of growth and nutrient (nitrate, silicate and phosphate) utilization, cellular elemental ratios of carbon and nitrogen, and the photosynthetic response to declining pH during the exponential and stationary growth phases of a strain of P. australis isolated during a massive toxic bloom that persisted for months along much of the U.S. west coast during 2015. Our controlled lab studies showed that DA production significantly increased as pCO2 increased, and total DA (pDA + dDA) normalized to cell density was 2.7 fold greater at pH 7.8 compared to pH 8.1 (control) during nutrient-limited stationary growth. However, exponential growth rates did not increase with declining pH, but remained constant until pH of 7.8 was reached, and then specific growth rates declined by ca. 30%. The toxin results demonstrate that despite minimal effects of OA observed during the nutrient-replete exponential growth phase, the enhancement of DA production, notably the 3-fold increase in particulate DA per cell, with declining pH from 8.1 to 7.8 during the nutrient-depleted stationary phase, supports the hypothesis that increasing pCO2 will result in greater toxic risk to coastal ecosystems from elevated ambient concentrations of particulate DA. The ecological consequences of decreasing silicate uptake rates and increasing cellular carbon quotas with declining pH may potentially ameliorate some negative impacts of OA on Pseudo-nitzschia growth in natural systems.

First Report of Domoic Acid Production from Pseudo-nitzschia multistriata in Paracas Bay (Peru).

The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell−1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.