La diatomée Pseudo-nitzschia comprend certaines espèces toxiques capables de produire de l'acide domoïque (AD), une neurotoxine amnésiante. La prolifération de ces espèces toxiques présente de nombreux risques allant de graves dommages écologiques et économiques à des menaces pour la santé publique. Les problèmes liés à cette prolifération se multiplient dans le monde entier et le besoin de la société scientifique de mieux comprendre les facteurs environnementaux influant sur l’apparition de ces blooms est plus nécessaire que jamais. Dans ce contexte, plusieurs espèces et souches de Pseudo-nitzschia ont été étudiées afin de mieux comprendre et prédire leurs réponses physiologiques face à de futurs changements climatiques. En particulier, les expérimentations effectuées ont permis d’explorer l’impact de la variation de la salinité et de l’acidification de l’océan (diminution de pH) sur la croissance et la production d’AD chez les espèces de Pseudo-nitzschia australis et P. fraudulenta. Les résultats obtenus ont montré que les espèces de Pseudo-nitzschia sont capables de s'adapter, proliférer et produire des toxines dans des conditions variables de salinité et de pH, malgré une importante diversité de réponses en fonction des souches ou des espèces étudiées. Ces résultats ont permis de déterminer l’influence et la capacité d’acclimatation des cellules de Pseudo-nitzschia face aux futures conditions environnementales (salinité, pH). Les travaux réalisés représentent une contribution à l’avancée des connaissances sur l’écophysiologie et la production toxinique par les espèces de Pseudo-nitzschia. / The diatom Pseudo-nitzschia includes certain toxic species capable of producing domoic acid (DA), an amnesic neurotoxin. The proliferation of these toxic species presents many risks ranging from serious ecological and economic damage to threats to public health. Problems related to these proliferations are increasing worldwide and the need for scientist to better understand the environmental factors influencing the appearance of these blooms is more necessary than ever. In this context, several species and strains of Pseudo-nitzschia have been studied to better understand and predict their physiological responses to future climate change. In particular, the experiments carried out allowed to explore the impact of salinity variations and ocean acidification (decrease of pH) on the growth, photosynthesis and DA production by several Pseudo-nitzschia species and strains. The results obtained showed that Pseudo-nitzschia species are flexible, able to adapt, proliferate and produce toxins under varying conditions of salinity and pH, despite the large diversity of responses depending on the strains or species studied. These results made it possible to determine the influence and acclimation capacities of Pseudo-nitzschia cells facing future environmental conditions (pH, salinity). Thus, the work carried out represents a contribution to the advancement of knowledge on the ecophysiology and toxin production by Pseudo-nitzschia species.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 145-160).

The Indian River Lagoon (IRL) spans approximately one-third of the east coast of Florida and faces numerous harmful algal blooms. The potentially toxic diatom, Pseudonitzschia, has been observed in many locations of the IRL. The goal of this study was to obtain a better understanding of the factors contributing to population dynamics of Pseudo-nitzschia in the southern IRL system. Bi-monthly surface water samples were collected for 18 months from five locations. Cell counts enumerated all microphytoplankton, and environmental data was collected at sampling sites throughout the study by the Indian River Lagoon Observatory Network. Six species of Pseudonitzschia were isolated and characterized through 18S Sanger sequencing and scanning electron microscopy, all showed toxicity. Surface water samples also showed domoic acid (DA) presence. We report the first known occurrence of Pseudo-nitzschia micropora in the IRL and the first known DA production from this taxon. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Chez les diatomées du genre Pseudo-nitzschia, certaines espèces produisent une neurotoxine, l’acide domoïque responsable chez l’homme d’intoxications amnésiantes suite à la consommation de mollusques contaminés. En Baie de Seine, la variabilité interannuelle de ces efflorescences toxiques, qui provoquent certaines années des fermetures de zones de pêche à la coquille Saint-Jacques, est liée à des changements de diversité spécifique au sein des communautés de Pseudo-nitzschia. Cette thèse avait pour objectif de caractériser la diversité écophysiologique au sein du genre Pseudo-nitzschia afin de mieux comprendre les facteurs qui influencent les différences de toxicité et de phénologies des efflorescences des principales espèces de Pseudo-nitzschia de Baie de Seine. Différents paramètres physiologiques, tels que la croissance, la production d’acide domoïque, les quotas élémentaires, les paramètres photosynthétiques, ainsi que les paramètres de cinétiques d’absorption du nitrate, du silicate et du phosphate, ont été étudiés chez quatre espèces : P. australis, P. pungens, P. fraudulenta et P. delicatissima. De plus, la variabilité intraspécifique a été évalué en étudiant de nombreuses souches par espèce et en estimant l’influence des variations de la taille cellulaire liées au cycle de vie de Pseudo-nitzschia sur les paramètres physiologiques. Les résultats montrent une importante diversité intraspécifique de la physiologie des quatre espèces, en partie liée à la taille cellulaire et parfois au cycle de vie. La connaissance de cette diversité intraspécifique a permis de caractériser différents traits physiologiques pour chaque espèce. Les résultats apportent des éléments de compréhension sur les conditions physiologiques favorables à la production d’acide domoïque et le rôle des éléments nutritifs dans le déterminisme des efflorescences des espèces de Pseudo-nitzschia. / In the diatom Pseudo-nitzschia, some species produce a neurotoxin called domoic acid responsible for amnesic shellfish poisoning in humans following the consumption of contaminated molluscs. In the Bay of Seine (English Channel), the variability of bloom toxicity is linked to differences in the Pseudo-nitzschia species diversity. The aim of this thesis was to characterize the ecophysiological diversity within the genus Pseudo-nitzschia in order to better understand the physiological parameters influencing differences in toxicity and bloom phenology among the main Pseudo-nitzschia species of the Bay of Seine. Growth, domoic acid production, elemental intracellular quotas, photosynthetic parameters and kinetic parameters for nitrate, phosphate, and silicic acid uptake were studied in four Pseudo-nitzschia species: P. australis, P. pungens, P. fraudulenta et P. delicatissima. The intraspecific variability was characterized with a multi-strain approach and also considering the effect of cell size on the studied physiological parameters. The results highlight the importance of the intraspecific variability in the physiology of the four species, in part due to cell size reduction linked to Pseudo-nitzschia life cycle. Delineate the intraspecific diversity allowed to characterize different physiological traits for each species. This work brings new information on the physiological conditions favorable to domoic acid production and on the role of nutrients in controlling Pseudo-nitzschia species diversity during blooms.

This work investigates, for the first time, the Pseudo-nitzschia (PN) dynamics in the western English Channel (L4) and the environmental factors impacting on domoic acid (DA) production in these waters. This is combined with laboratory studies examining key environmental factors and the multifactorial impact of multiple macronutrient and micronutrient availability on PN growth and DA production. An LC-MS method was established, optimised, and compared with ELISA for the accurate and reproducible extraction and determination of particulate and dissolved DA. The method was used to measure the seasonal variation in DA at L4 during 2009 and this was compared to PN seasonal abundance and diversity. Three groups a P. delicatissima-group, a P. seriata-group, and a P. pungens/multiseries-group were identified and were found to have different ecological distributions with the latter two groups significantly correlating with DA concentration. Macronutrients, in combination with other environmental factors, were found to influence PN populations at L4. Multifactorial laboratory culture experiments investigating the availability of nitrate, phosphate, and silicate, confirmed that the interrelatedness of all these nutrients significantly affected the growth, decline, and DA production of P. multiseries, and highlight the importance of both phosphate and silicate availability for DA production. When the impacts of both macronutrient (phosphate and silicate) and micronutrient (iron and copper) availability were investigated, limited growth and DA production was observed in P. multiseries cultures. Results revealed the complexity and interrelationship of factors affecting both PN growth and DA production. Furthermore, molecular methods were developed to elucidate the PN species present from 2009 Lugol’s-preserved L4 samples. DNA was successfully extracted and amplified from these samples which had been stored for up to 2 years. Initial sequence analysis identified the rbcL DNA marker as an informative site for future work with a number of L4 sequences closely relating to different Pseudo-nitzschia spp.

Domoic acid (DA) is a neurotoxin produced by the harmful algae Pseudo-nitzschia that has been directly linked to mass stranding events of the California Sea Lion (CSL). The purpose of this paper is to review the anthropogenic influence on the production of this neurotoxin and examine how human activities are impacting this marine mammal species. A comprehensive and interdisciplinary literature review was conducted to evaluate the future sustainability of the CSL population. It was found that while Pseudo-nitzschia bloom developments are vulnerable to anthropogenic influences, the incontestable existence of natural contributing factors adds a certain complexity to the determination of causalities and the development of solutions. Strong evidence exists to show that DA can cause major and irreversible neurological damage in CSLs. Rehabilitation of DA-impacted CSLs is a polarizing issue in the U.S. and presents interesting implications for sustainable development. While the CSL population is currently healthy and plenteous, the strong abundance of future uncertainties warrants concern. A balance must be found between the involving social, economic, and environmental factors to ensure a promising future for the CSL species.

The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose- 1,5-biphosphate carboxylase/oxygenase small subunit (rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (including four P. multiseries) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r2 ! 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 107 cells/L to 103 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 x 104 to 5.35 x 104, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 84-88). Description: x, 88 leaves : ill. ; 29 cm. / Along the Oregon coast blooms of Pseudo-nitzschia spp. have been an almost annual occurrence since monitoring began in the late 80's. This study looks at the distribution of Pseudo-nitzschia spp., in relation to meteorological and oceanographic conditions. A series of transects (1995-98) were done in Coos Bay and over the adjacent continental shelf under a variety of oceanographic conditions. An intense sampling program was undertaken in June 1998, with simultaneous nearshore and in bay (Coos Bay) sampling, including a nearshore grid, 24 hour anchor station in the bay, and an in bay incoming tide drift study. The results suggest that blooms develop nearshore after upwelling events. In conditions of relaxed upwelling highest cell concentrations were found immediately adjacent to the coast in surface and mid-depth waters. At stations in Coos Bay, Pseudonitzschia spp. concentrations increased with the incoming tide. Nearshore phytoplankton populations were transported into the bay, initially in the more saline bottom waters, but were rapidly mixed throughout the water column by the turbulent flow in the channels. On ebbing tides there was a reduced number of phytoplankton in the water column, possibly indicating consumption by in bay filter feeders or sinking and deposition on the bottom.

Diatoms play a fundamental role in marine food webs, and significantly contribute to global primary production and carbon sequestration into the deep ocean. In many offshore areas of the open ocean, iron (Fe) input is low, and its availability often limits phytoplankton biomass. Recently, gene sequences encoding ferritin, a nearly ubiquitous iron storage and detoxifying protein, have been identified in pennate diatoms such as Pseudo-nitzschia, but not in other Stramenopiles (which include centric diatoms, brown algae and some protist plant parasites) or Cryptophyte relatives. Members of this genus readily bloom upon addition of iron to Fe-limited waters, and are known to produce the neurotoxin domoic acid. Until now, the reason for the success of pennate diatoms in the open ocean was uncertain; however, expressing ferritin would allow pennate species to store Fe after a transient input, using it to dominate Fe stimulated algal blooms. Here, the ferritin gene was cloned from the coastal pennate diatom Pseudonitzschia multiseries, overexpressed in Escherichia coli, and purified using liquid chromatography. The ferritin protein sequence appears to encode a non-heme, ferritinlike di-iron carboxylate protein, while gel filtration chromatography and SDS-PAGE indicate that this ferritin is part of the 24 subunit maxi-ferritins. Spectroscopically monitoring the addition of Fe(II) to a buffered ferritin solution shows that the P. multiseries protein demonstrates ferroxidase activity, binding iron and storing it as Fe(III) in excess of 600 equivalents per protein shell. In keeping with the typical stoichiometry of the ferroxidase reaction, oxygen (O₂) is consumed in a 2 Fe:O₂ratio while hydrogen peroxide is produced concurrently. iii Diatoms evolved from secondary endosymbiosis involving eukaryotic red algae; however, a broad phylogenetic comparison suggests that P. multiseries ferritin was likely acquired via lateral gene transfer from cyanobacteria – not from its ancestral endosymbionts. Until recently, no other ferritins have been identified in diatoms, and the protein characterized here is unique in that it seems to be derived from a prokaryotic organism yet it occurs in a marine eukaryote. These findings have direct implications for the success of pennate diatoms in both Fe rich coastal waters and upon Fe addition in the open ocean.

Diatoms play a fundamental role in marine food webs, and significantly contribute to global primary production and carbon sequestration into the deep ocean. In many offshore areas of the open ocean, iron (Fe) input is low, and its availability often limits phytoplankton biomass. Recently, gene sequences encoding ferritin, a nearly ubiquitous iron storage and detoxifying protein, have been identified in pennate diatoms such as Pseudo-nitzschia, but not in other Stramenopiles (which include centric diatoms, brown algae and some protist plant parasites) or Cryptophyte relatives. Members of this genus readily bloom upon addition of iron to Fe-limited waters, and are known to produce the neurotoxin domoic acid. Until now, the reason for the success of pennate diatoms in the open ocean was uncertain; however, expressing ferritin would allow pennate species to store Fe after a transient input, using it to dominate Fe stimulated algal blooms. Here, the ferritin gene was cloned from the coastal pennate diatom Pseudonitzschia multiseries, overexpressed in Escherichia coli, and purified using liquid chromatography. The ferritin protein sequence appears to encode a non-heme, ferritinlike di-iron carboxylate protein, while gel filtration chromatography and SDS-PAGE indicate that this ferritin is part of the 24 subunit maxi-ferritins. Spectroscopically monitoring the addition of Fe(II) to a buffered ferritin solution shows that the P. multiseries protein demonstrates ferroxidase activity, binding iron and storing it as Fe(III) in excess of 600 equivalents per protein shell. In keeping with the typical stoichiometry of the ferroxidase reaction, oxygen (O₂) is consumed in a 2 Fe:O₂ratio while hydrogen peroxide is produced concurrently. iii Diatoms evolved from secondary endosymbiosis involving eukaryotic red algae; however, a broad phylogenetic comparison suggests that P. multiseries ferritin was likely acquired via lateral gene transfer from cyanobacteria – not from its ancestral endosymbionts. Until recently, no other ferritins have been identified in diatoms, and the protein characterized here is unique in that it seems to be derived from a prokaryotic organism yet it occurs in a marine eukaryote. These findings have direct implications for the success of pennate diatoms in both Fe rich coastal waters and upon Fe addition in the open ocean.