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.     

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.     

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.     

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.     

DEVELOPMENT OF IMMUNOASSAYS FOR THE IRON‐REGULATED PROTEINS FERREDOXIN AND FLAVODOXIN IN POLAR MICROALGAE1

While the growth of Southern Ocean phytoplankton is often limited by iron availability, there are no comparable experiments on sea‐ice algae. Here we assess the use of ferredoxin and flavodoxin to investigate the iron nutritional status of sea‐ice algae and describe the development of a quantitative immunoassay for both proteins in marine diatoms. High‐affinity monoclonal antibodies toward both proteins were produced from Cylindrotheca closterium (Ehrenb.) J. M. Lewin et Reimann, and these were used to develop Western blots. Western blots run on whole protein extracts detected both proteins with little cross‐reactivity toward other proteins. The two proteins could be successfully quantitated when applied to gels at between 5 and 50 ng in a volume of 25 μL (0.2–2 μg · mL^?1). Flavodoxin and ferrodoxin expression was examined in the Antarctic diatoms Entomoneis kjellmannii (Cleve) Poulin et Cardinal, Navicula directa (W. Sm.) Ralfs, Fragilariopsis curta (Van Heurck) Hust., Pseudo‐nitzschia sp., Porosira glacialis (Grunow) E. G. Jørg., Fragilariopsis cylindrus (Grunow) Willi Krieg., Fragilariopsis sublinearis (Van Heurck) Heiden et Kolbe, C. closterium, Nitzschia lecointei Van Heurck, and the dinoflagellate Polarella glacialis Montresor, Procaccini et Stoecker. Two Arctic isolates were also examined, Nitzschia frigida (Grunow) and Fragilariopsis oceanica (Cleve) Hasle. Significant heterogeneity of protein expression was observed despite all cultures being grown in iron‐replete f/2 medium. Only one species, F. cylindrus, displayed the expected expression of ferredoxin only in iron‐replete medium. Four were observed to produce both proteins under iron‐replete conditions. Ferredoxin was not detected at all in F. curta and Pseudo‐nitzschia sp., but distinct flavodoxin bands were observed in both of these organisms. All species examined were observed to express either flavodoxin or ferredoxin or both of the proteins as determined by Western immunoblotting.     

Interspecific plastidial recombination in the diatom genus Pseudo‐nitzschia

Plastids are usually uni‐parentally inherited and genetic recombination between these organelles is seldom observed. The genus Pseudo‐nitzschia, a globally relevant marine diatom, features bi‐parental plastid inheritance in the course of sexual reproduction. This observation inspired the recombination detection we pursued in this paper over a ~1,400‐nucleotide‐long region of the plastidial rbcL, a marker used in both molecular taxonomy and phylogenetic studies in diatoms. Among all the rbcL‐sequences available in web‐databases for Pseudo‐nitzschia, 42 haplotypes were identified and grouped in five clusters by Bayesian phylogeny. Signs of hybridization were evident in four of five clusters, at both intra‐ and interspecific levels, suggesting that, in diatoms, (i) plastidial recombination is not absent and (ii) hybridization can play a role in speciation of Pseudo‐nitzschia spp.     

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.     

Taxonomical diversity of Pseudo‐nitzschia from the Central Adriatic Sea

The aim of our research was to study the composition of Pseudo‐nitzschia species during a period when neurotoxin domoic acid (DA) was present in shellfish. Sampling was conducted in Ka?tela Bay (Central Adriatic Sea), between November 2015 and January 2016. Concentrations of DA analyzed in various shellfish species were low, below the regulatory limit, while the highest abundance of Pseudo‐nitzschia spp. reached 1.85 × 10⁵ cells L^?1 in the surface layer, at the beginning of November. Within the temperature and salinity range recorded during the investigated period, a positive correlation of Pseudo‐nitzschia spp. abundance was recorded with temperature. Morphological analyses by scanning electron microscopy revealed the presence of five Pseudo‐nitzschia species that had already been reported in the Adriatic Sea – P. calliantha, P. delicatissima, P. fraudulenta, P. pseudodelicatissima /P. cuspidata and P. subfraudulenta – as well as an unknown Pseudo‐nitzschia sp. The composition of the Pseudo‐nitzschia assemblage changed over the investigated period. The species P. pseudodelicatissima/P.cuspidata was found throughout the entire period and the highest diversity was noticed in January, when all six observed species were recorded. These results represent the first taxonomical investigation of the genus Pseudo‐nitzschia in Ka?tela Bay, as well as the first report of DA in shellfish from this area.     

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.     

The first report of the potentially harmful diatom Pseudo‐nitzschia caciantha from Australian coastal waters

The diatom Pseudo‐nitzschia is a significant component of coastal waters worldwide and a producer of the potent neurotoxin, domoic acid. Sixteen species belonging to this genus have been reported from Australian waters, but the potentially toxic species P. caciantha has not been previously known from this region. Two clonal strains of P. caciantha were isolated from Coogee Beach, south‐east Australia, and the morphological, molecular and toxicological evidence for this species delineation were examined using light and transmission electron microscopy, phylogenetic analysis based on sequences of the second internal transcribed spacer and domoic acid production as measured by liquid chromatography–mass spectrometry. The results unambiguously confirmed that these isolates are the potentially toxic species P. caciantha , being only the second report of this species in the Southern Hemisphere. The potential for further hidden Pseudo‐nitzschia diversity in these waters is considerable.     

AUXOSPORE FORMATION BY THE SILICA‐SINKING,OCEANIC DIATOM FRAGILARIOPSIS KERGUELENSIS (BACILLARIOPHYCEAE)1

Size restoration by the auxospore that develops from the zygote is a crucial stage in diatom life cycles. However, information on sexual events in pelagic diatom species is very limited. We report for the first time auxospore formation by the pennate diatom Fragilariopsis kerguelensis (O'Hara) Hustedt during an iron‐induced bloom in the Southern Ocean (EIFEX, European Iron Fertilization EXperiment). Auxospores of F. kerguelensis resembled those described for Pseudo‐nitzschia species. The auxospore was characterized by an outer coating, the perizonium; two caps, one at each distal end; and four chloroplasts, one at each end and two in the central part. Different stages of auxospore elongation were recorded, with a length of 24–91 μm, but only the largest auxospores contained the initial cell, whose apical axis ranged between 76 and 90 μm. Gametangial cell walls were often attached to the auxospores and ranged from 10 to 31 μm in length. Auxospore abundances were consistently higher in the fertilized patch, where an increase in the F. kerguelensis population was observed, as compared with surrounding waters.     

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.     

Southern right whale (Eubalaena australis) calf mortality at Península Valdés,Argentina: Are harmful algal blooms to blame?

Península Valdés (PV) in Argentina is an important calving ground for southern right whales (SRWs, Eubalaena australis). Since 2005, right whale mortality has increased at PV, with most of the deaths (~90%) being calves <3 mo old. We investigated the potential involvement of harmful algal blooms (HABs) in these deaths by examining data that include: timing of the SRW deaths, biotoxins in samples from dead SRWs, abundances of the diatom, Pseudo‐nitzschia spp., and the dinoflagellate, Alexandrium tamarense, shellfish harvesting closure dates, seasonal availability of whale prey at PV and satellite chlorophyll data. Evidence of the whales' exposure to HAB toxins includes trace levels of paralytic shellfish toxins (PSTs) and domoic acid (DA) in tissues of some dead whales, and fragments of Pseudo‐nitzschia spp. frustules in whale feces. Additionally, whales are present at PV during both closures of the shellfish industry (due to high levels of PSTs) and periods with high levels of Pseudo‐nitzschia spp. and A. tamarense. There is a positive statistical relationship between monthly Pseudo‐nitzschia densities (but not A. tamarense) and calf deaths in both gulfs of PV.     

INTER‐ AND INTRASPECIFIC COMMUNITY STRUCTURE WITHIN THE DIATOM GENUS PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE)1

Pseudo‐nitzschia‐specific PCR primers (PnAll F/R) were designed to amplify a polymorphic region of the internal transcribed spacer 1 (ITS1) from at least 11 Pseudo‐nitzschia species. The primers were used to generate environmental clone libraries from Puget Sound, Washington, and Vancouver Island, British Columbia, to confirm that the primers were specific for Pseudo‐nitzschia and to determine the extent of ITS1 sequence diversity within individual species. All environmental ITS1 sequences generated with PnAll primers displayed the greatest similarity to known Pseudo‐nitzschia ITS1 sequences. The length of cloned ITS1 fragments differed among species but was conserved within a species. Intraspecific genotypes exhibited <3% sequence divergence for seven of the 10 species detected in clone libraries. Several ITS1 genotypes unique to the Pacific Northwest were identified in environmental samples, and other genotypes were more broadly distributed. The Pseudo‐nitzschia primers were also used to develop an automated ribosomal intergenic spacer analysis (ARISA) to rapidly identify Pseudo‐nitzschia species in environmental samples based on species‐specific variation in the length of the targeted ITS1 region. The ARISA peaks were then associated with the environmental clone sequences for Pseudo‐nitzschia species. Surveying the genetic composition of communities at both the inter‐ and intraspecific levels will enhance our understanding of Pseudo‐nitzschia bloom dynamics.     

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c₂ and c₃.     

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.