MORPHOLOGICAL,TOXICOLOGICAL, AND GENETIC DIFFERENCES AMONG PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) SPECIES IN INLAND EMBAYMENTS AND OUTER COASTAL WATERS OF WASHINGTON STATE,USA1

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.     

Influence of sudden salinity variation on the physiology and domoic acid production by two strains of Pseudo‐nitzschia australis

Several coastal countries including France have experienced serious and increasing problems related to Pseudo‐nitzschia toxic blooms. These toxic blooms occur in estuarine and coastal waters potentially subject to fluctuations in salinity. In this study, we document for the first time the viability, growth, photosynthetic efficiency, and toxin production of two strains of Pseudo‐nitzschia australis grown under conditions with sudden salinity changes. Following salinity variation, the two strains survived over a restricted salinity range of 30–35, with favorable physiological responses, as the growth, effective quantum yield and toxin content were high compared to the other conditions. In addition, high cellular quotas of domoic acid (DA) were observed at a salinity of 40 for the strain IFR‐PAU‐16.1 in comparison with the other strain IFR‐PAU‐16.2 where the cell DA content was directly released into the medium. On the other hand, the osmotic stress imposed at lower salinities, 20 and 10, resulted in cell lysis and a sudden DA leakage in the medium. Intra‐specific variability was observed in growth and toxin production, with the strain IFR‐PAU‐16.1 apparently able to withstand higher salinities than the strain IFR‐PAU‐16.2. On the whole, DA does not appear to act as an osmolyte in response to sudden salinity changes. Since most of the shellfish harvesting areas of bivalve molluscs in France are located in areas where the salinity generally varies between 30 and 35, Pseudo‐nitzschia australis blooms might potentially impact public health and commercial shellfish resources in these places.     

CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) ISOLATED FROM IRISH WATERS1

A nonaxenic isolate of the potentially toxic diatom Pseudo‐nitzschia australis (Frenguelli) from Irish waters was tested in two separate batch culture experiments. When grown under a low irradiance (～12 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 16:8‐h light:dark cycle) for up to 40 days, the culture produced only trace amounts of the neurotoxin domoic acid (DA) during late stationary phase. Growth at a higher irradiance (～115 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 12:12‐h light:dark cycle) resulted in DA production starting during late exponential phase and reaching a maximum concentration of 26 pg DA·cell ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
during late stationary phase. Liquid chromatography coupled to mass spectrometry was used to confirm the identity of DA in the culture. Irradiance and photoperiod could be important factors that contribute directly or indirectly to the control of DA production in P. australis. This is the first record of a DA‐producing diatom in Irish waters, and results indicate P. australis may have been the source of DA that has recently contaminated shellfisheries in this area.     

MICROSATELLITE MARKER DEVELOPMENT AND GENETIC VARIATION IN THE TOXIC MARINE DIATOM PSEUDO‐NITZSCHIA MULTISERIES (BACILLARIOPHYCEAE)1

The genetic structure of phytoplankton populations is largely unknown. In this study we developed nine polymorphic microsatellite markers for the domoic acid–producing marine diatom Pseudo‐nitzschia multiseries (Hasle) Hasle. We then used them in the genotyping of 25 physiologically diverse field isolates and six of their descendants: 22 field isolates originated from eastern Canadian waters, two from European waters, and one from Russian waters. The numbers of alleles per locus ranged from three to seven and the observed heterozygosities from 0.39 to 0.70. A substantial degree of genetic variation was observed within the field isolates, with 23 different genotypes detected. The Russian isolate was the most genetically distinct, although there was also evidence of genetic differentiation at a more local scale. Mating experiments demonstrated that alleles were inherited in a Mendelian manner. Pseudo‐nitzschia multiseries primer pairs were tested on DNA from four congeners: P. calliantha Lundholm, Moestrup et Hasle; P. fraudulenta (P. T. Cleve) Hasle; P. pungens (Grunow ex P. T. Cleve) Hasle; and P. seriata (P. T. Cleve) H. Peragallo. Cross‐reactivity was only observed in P. pungens. Our results are a first step in understanding the genetic variation present at the Pseudo‐nitzschia“species” level and in determining the true biogeographic extent of Pseudo‐nitzschia species.     

IDENTIFICATION AND ASSESSMENT OF DOMOIC ACID PRODUCTION IN OCEANIC PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) FROM IRON‐LIMITED WATERS IN THE NORTHEAST SUBARCTIC PACIFIC1

We identified and investigated the potential toxicity of oceanic Pseudo‐nitzschia species from Ocean Station Papa (OSP), located in a high‐nitrate, low‐chlorophyll (HNLC) region of the northeast (NE) subarctic Pacific Ocean. Despite their relatively low abundances in the indigenous phytoplankton assemblage, Pseudo‐nitzschia species richness is high. The morphometric characteristics of five oceanic Pseudo‐nitzschia isolates from at least four species are described using SEM and TEM. The species identified are Pseudo‐nitzschia dolorosa Lundholm et Moestrup, P. granii Hasle, P. heimii Manguin, and P. cf. turgidula (Hust.) Hasle. Additional support for the taxonomic classifications based on frustule morphology is provided through the sequencing of the internal transcribed spacer 1 (ITS1) rDNA. Pseudo‐nitzschia species identification was also assessed by the construction of ITS1 clone libraries and using automated ribosomal intergenic spacer analysis (ARISA) for environmental samples collected during the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES), conducted in close proximity to OSP in July of 2002. Based on ITS1 sequences, the presence of P. granii, P. heimii, P. cf. turgidula, and at least five other putative, unidentified Pseudo‐nitzschia ITS1 variants was confirmed within iron‐enriched phytoplankton assemblages at OSP. None of the oceanic isolates produced detectable levels of particulate domoic acid (DA) when in prolonged stationary phase due to silicic acid starvation. The lack of detectable concentrations of DA suggests that either these strains produce very little or no toxin, or that the physiological conditions required to promote particulate DA production were not met and thus differ from their coastal, toxigenic congeners.     

Differential Influence of Life Cycle on Growth and Toxin Production of three Pseudo‐nitzschia Species (Bacillariophyceae)

We used a multistrain approach to study the intra‐ and interspecific variability of the growth rates of three Pseudo‐nitzschia species – P. australis, P. fraudulenta, and P. pungens – and of their domoic acid (DA) production. We carried out mating and batch experiments to investigate the respective effects of strain age and cell size, and thus the influence of their life cycle on the physiology of these species. The cell size – life cycle relationship was characteristic of each species. The influence of age and cell size on the intraspecific variability of growth rates suggests that these characteristics should be considered cautiously for the strains used in physiological studies on Pseudo‐nitzschia species. The results from all three species do not support the hypothesis of a decrease in DA production with time since isolation from natural populations. In P. australis, the cellular DA content was rather a function of cell size. More particularly, cells at the gametangia stage of their life cycle contained up to six times more DA than smaller or larger cells incapable of sexual reproduction. These findings reveal a link between P. australis life cycle and cell toxicity. This suggest that life cycle dynamics in Pseudo‐nitzschia natural populations may influence bloom toxicity.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.     

EFFECT OF SALINITY ON PSEUDO‐NITZSCHIA SPECIES (BACILLARIOPHYCEAE) GROWTH AND DISTRIBUTION1

Salinity varies widely in coastal areas that often have a high abundance of Pseudo‐nitzschia H. Peragallo. Pseudo‐nitzschia is abundant in Louisiana waters, and high cellular domoic acid has been observed in natural samples but no human illness has been reported. To assess the threat of amnesic shellfish poisoning (ASP), we examined the effect of salinity on Pseudo‐nitzschia occurrence in the field and growth in the laboratory with special emphasis on the salinity range where oysters are harvested (10–20 psu). In Louisiana coastal waters, Pseudo‐nitzschia spp. occurred over a salinity range of 1 to >35 psu, but they occurred more frequently at higher rather than lower salinities. Seven species were identified, including toxigenic species occurring at low salinities. In culture studies, seven clones of three species grew over a salinity range of 15 to 40 psu, some grew at salinities down to 6.25 psu, and most grew at salinities up to 45 psu. Tolerance of low salinities decreased from Pseudo‐nitzschia delicatissima (Cleve) Heiden to P. multiseries (Hasle) Hasle to P. pseudodelicatissima (Hasle) Hasle emend. Lundholm, Hasle et Moestrup. In conclusion, although Pseudo‐nitzschia was more prevalent in the field and grew better in the laboratory at higher salinities, it grew and has been observed at low salinities. Therefore, the probability of ASP from consumption of oysters harvested from the low salinity estuaries of the northern Gulf of Mexico is low but not zero; animal mortality events from toxin vectors other than oysters at higher salinity on the shelf are more likely.     

GENETIC POPULATION STRUCTURE OF PSEUDO‐NITZSCHIA PUNGENS (BACILLARIOPHYCEAE) FROM THE PACIFIC NORTHWEST AND THE NORTH SEA1

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, F_ST) 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 a P. 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 Pseudo‐nitzschia is important for bloom prediction and aiding the identification of molecular markers that can be used for rapid detection assay development.     

CRYPTIC AND PSEUDO‐CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONS OF PSEUDO‐NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

A high degree of pseudo‐cryptic diversity was reported in the well‐studied diatom genus Pseudo‐nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo‐nitzschia. Forty‐one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1–D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.     

GROWTH AND DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA SERIATA (BACILLARIOPHYCEAE) UNDER PHOSPHATE AND SILICATE LIMITATION1

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.     

The diatom genus Pseudo‐nitzschia (Bacillariophyceae) in New South Wales,Australia: morphotaxonomy,molecular phylogeny,toxicity, and distribution

Species belonging to the potentially harmful diatom genus Pseudo‐nitzschia, isolated from 16 localities (31 sampling events) in the coastal waters of south‐eastern Australia, were examined. Clonal isolates were characterized by (i) light and transmission electron microscopy; (ii) phylogenies, based on sequencing of nuclear‐encoded ribosomal deoxyribonucleic acid (rDNA) regions and, (iii) domoic acid (DA) production as measured by liquid chromatography–mass spectrometry (LC‐MS/MS). Ten taxa were unequivocally confirmed as Pseudo‐nitzschia americana, P. arenysensis, P. calliantha, P. cuspidata, P. fraudulenta, P. hasleana, P. micropora, P. multiseries, P. multistriata, and P. pungens. An updated taxonomic key for south‐eastern Australian Pseudo‐nitzschia is presented. The occurrence of two toxigenic species, P. multistriata (maximum concentration 11 pg DA per cell) and P. cuspidata (25.4 pg DA per cell), was documented for the first time in Australia. The Australian strains of P. multiseries, a consistent producer of DA in strains throughout the world, were nontoxic. Data from 5,888 water samples, collected from 31 oyster‐growing estuaries (2,000 km coastline) from 2005 to 2009, revealed 310 regulatory exceedances for “Total Pseudo‐nitzschia,” resulting in six toxic episodes. Further examination of high‐risk estuaries revealed that the “P. seriata group” had highest cell densities in the austral summer, autumn, or spring (species dependent), and lowest cell densities in the austral winter, while the “P. delicatissima group” had highest in winter and spring.     

First reports of Pseudo‐nitzschia micropora and P. hasleana (Bacillariaceae) from the Southern Hemisphere: Morphological,molecular and toxicological characterization

Pseudo‐nitzschia H. Peragallo is a marine diatom genus found worldwide in polar, temperate, subtropical and tropical waters. It includes toxigenic representatives that produce domoic acid (DA), a neurotoxin responsible for Amnesic Shellfish Poisoning. In this study we characterized two species of Pseudo‐nitzschia collected from Port Stephens and the Hawkesbury River (south eastern Australia) previously unreported from Australian waters. Clonal isolates were sub‐sampled for (i) light and transmission electron microscopy; (ii) DNA sequencing, based on the nuclear‐encoded partial large subunit ribosomal RNA gene and internal transcribed spacer (ITS)‐ITS1, 5.8S and ITS2 rDNA regions and, (iii) DA production as measured by liquid chromatography‐mass spectrometry. Morphological and molecular data unambiguously revealed the species to be Pseudo‐nitzschia micropora Priisholm, Moestrup & Lundholm (Port Stephens) and Pseudo‐nitzschia hasleana Lundholm (Hawkesbury River). This is the first report of the occurrence of these species from the Southern Hemisphere and the first report of P. micropora in warm‐temperate waters. Cultures of P. micropora, tested for DA production for the first time, proved to be non‐toxic. Similarly, no detectable toxin concentrations were observed for P. hasleana. Species resolution and knowledge on the toxicity of local Pseudo‐nitzschia species has important implications for harmful algal bloom monitoring and management.     

LIFE CYCLE,SIZE REDUCTION PATTERNS,AND ULTRASTRUCTURE OF THE PENNATE PLANKTONIC DIATOM PSEUDO‐NITZSCHIA DELICATISSIMA (BACILLARIOPHYCEAE)1

Pseudo‐nitzschia delicatissima (Cleve) Heiden is a very common pennate planktonic diatom found in temperate marine waters, where it is often responsible for blooms. Recently, three distinct internal transcribed spacer types have been recorded during a P. delicatissima bloom in the Gulf of Naples (Mediterranean Sea, Italy), which suggests the existence of cryptic diversity. We carried out mating experiments with clonal strains belonging to the most abundant internal transcribed spacer type. Pseudo‐nitzschia delicatissima is heterothallic and produces two functional anisogametes per gametangium. The elongated auxospore possesses a transverse and a longitudinal perizonium. The sexual phase was observed to occur over a wide size spectrum, spanning 19–80 μm and corresponding to almost the whole range of cell length observed for P. delicatissima. We also investigated cell morphology, valve ultrastructure and morphometry of parental, F1‐generation strains, and the progeny of crosses between parental and F1 strains. Although ultrastructural features match those described for P. delicatissima, variability in cell shape was recorded in the largest cells of the F1 generation as well as in valves with an abnormal arrangement of poroids. As many other diatoms, P. delicatissima undergoes size reduction over its life cycle, and cells of different size showed differences in growth rates and the amount of size reduction per cell cycle. Cells between 60 and 30 μm in length showed the fastest growth and the slowest rates of size reduction per generation. In culture, P. delicatissima cells can decrease to 8 μm in length; however, such small cells (≤30 μm) are not recorded in the sea, and this raises interesting questions about the factors that control their survival in the natural environment.     

UV‐EXCITED BLUE AUTOFLUORESCENCE OF PSEUDO‐NITZSCHIA MULTISERIES (BACILLARIOPHYCEAE)1

A flow cytometer coupled to a scanning monochromator and a fluorescence microscope were used to characterize the fluorescence spectrum of Pseudo‐nitzschia multiseries (Hasle) Hasle, a pennate diatom that produces the neurotoxin domoic acid, a lethal amnesic. In this research, we characterize the fluorescence spectrum of P. multiseries in vivo over the wavelength range of 360 to 850 nm and show that this diatom autofluoresces blue when excited with UV light (350–365 nm). The autofluorescence characterization of Pseudo‐nitzschia may provide new methods for rapid in situ monitoring of diatom populations and reiterates the usefulness of flow cytometry in the analysis and study of marine phytoplankton.     

First report of the potentially toxic marine diatom Pseudo‐nitzschia simulans (Bacillariophyceae) from the East Australian Current

Certain species of the marine diatom genus Pseudo‐nitzschia are responsible for the production of the domoic acid (DA), a neurotoxin that can bioaccumulate in the food chain and cause amnesic shellfish poisoning (ASP) in animals and humans. This study extends our knowledge by reporting on the first observation of the potentially toxic species Pseudo‐nitzschia simulans from this region. One clonal strain of P. simulans was isolated from the East Australian Current and characterized using light and transmission electron microscopy, and phylogenetic analyses based on regions of the internal transcribed spacer (ITS) and the D1–D3 region of the large subunit (LSU) of the nuclear‐encoded ribosomal deoxyribonucleic acid (rDNA), as well as examined for DA production as measured by liquid chromatography–mass spectrometry. Although this strain was non‐toxic under the defined growth conditions, the results unambiguously confirmed that this isolate is the potentially toxic species P. simulans – the first report of this species from the Southern Hemisphere.