The neurotoxin β-N-methylamino-L-alanine (BMAA) : Sources, bioaccumulation and extraction procedures

β-methylamino-L-alanine (BMAA) is a neurotoxin linked to neurodegeneration, which is manifested in the devastating human diseases amyotrophic lateral sclerosis, Alzheimer’s and Parkinson’s disease. This neurotoxin is known to be produced by almost all tested species within the cyanobacterial phylum including free living as well as the symbiotic strains. The global distribution of the BMAA producers ranges from a terrestrial ecosystem on the Island of Guam in the Pacific Ocean to an aquatic ecosystem in Northern Europe, the Baltic Sea, where annually massive surface blooms occur. BMAA had been shown to accumulate in the Baltic Sea food web, with highest levels in the bottom dwelling fish-species as well as in mollusks. One of the aims of this thesis was to test the bottom-dwelling bioaccumulation hypothesis by using a larger number of samples allowing a statistical evaluation. Hence, a large set of fish individuals from the lake Finjasjön, were caught and the BMAA concentrations in different tissues were related to the season of catching, fish gender, total weight and species. The results reveal that fish total weight and fish species were positively correlated with BMAA concentration in the fish brain. Therefore, significantly higher concentrations of BMAA in the brain were detected in plankti-benthivorous fish species and heavier (potentially older) individuals. Another goal was to investigate the potential production of BMAA by other phytoplankton organisms. Therefore, diatom cultures were investigated and confirmed to produce BMAA, even in higher concentrations than cyanobacteria. All diatom cultures studied during this thesis work were show to contain BMAA, as well as one dinoflagellate species. This might imply that the environmental spread of BMAA in aquatic ecosystems is even higher than previously thought. Earlier reports on the concentration of BMAA in different organisms have shown highly variable results and the methods used for quantification have been intensively discussed in the scientific community. In the most recent studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the instrument of choice, due to its high sensitivity and selectivity. Even so, different studies show quite variable concentrations of BMAA. In this thesis, three of the most common BMAA extraction protocols were evaluated in order to find out if the extraction could be one of the sources of variability. It was found that the method involving precipitation of proteins using trichloroacetic acid gave the best performance, complying with all in-house validation criteria. However, extractions of diatom and cyanobacteria cultures with this validated method and quantified using LC-MS/MS still resulted in variable BMAA concentrations, which suggest that also biological reasons contribute to the discrepancies. The current knowledge on the environmental factors that can induce or reduce BMAA production is still limited. In cyanobacteria, production of BMAA was earlier shown to be negative correlated with nitrogen availability – both in laboratory cultures as well as in natural populations. Based on this observation, it was suggested that in unicellular non-diazotrophic cyanobacteria, BMAA might take part in nitrogen metabolism. In order to find out if BMAA has a similar role in diatoms, BMAA was added to two diatom species in culture, in concentrations corresponding to those earlier found in the diatoms. The results suggest that BMAA might induce a nitrogen starvation signal in diatoms, as was earlier observed in cyanobacteria. However, diatoms recover shortly by the extracellular presence of excreted ammonia. Thus, also in diatoms, BMAA might be involved in the nitrogen balance in the cell.

Studies on Bio-Geochemistry, Bio-Optical Properties and Satellite Validation of Coastal Waters of South Eastern Arabian Sea

In situ methods used for water quality assessment have both physical and time constraints. Just a limited number of sampling points can be performed due to this, making it difficult to capture the range and variability of coastal processes and constituents. In addition, the mixing between fresh and oceanic water creates complex physical, chemical and biological environment that are difficult to understand, causing the existing measurement methodologies to have significant logistical, technical, and economic challenges and constraints. Remote sensing of ocean colour makes it possible to acquire information on the distribution of chlorophyll and other constituents over large areas of the oceans in short periods. There are many potential applications of ocean colour data. Satellite-derived products are a key data source to study the distribution pattern of organisms and nutrients (Guillaud et al. 2008) and fishery research (Pillai and Nair 2010; Solanki et al. 2001. Also, the study of spatial and temporal variability of phytoplankton blooms, red tide identification or harmful algal blooms monitoring (Sarangi et al. 2001; Sarangi et al. 2004; Sarangi et al. 2005; Bhagirathan et al., 2014), river plume or upwelling assessments (Doxaran et al. 2002; Sravanthi et al. 2013), global productivity analyses (Platt et al. 1988; Sathyendranath et al. 1995; IOCCG2006) and oil spill detection (Maianti et al. 2014). For remote sensing to be accurate in the complex coastal waters, it has to be validated with the in situ measured values. In this thesis an attempt to study, measure and validate the complex waters with the help of satellite data has been done. Monitoring of coastal ecosystem health of Arabian Sea in a synoptic way requires an intense, extensive and continuous monitoring of the water quality indicators. Phytoplankton determined from chl-a concentration, is considered as an indicator of the state of the coastal ecosystems. Currently, satellite sensors provide the most effective means for frequent, synoptic, water-quality observations over large areas and represent a potential tool to effectively assess chl-a concentration over coastal and oceanic waters; however, algorithms designed to estimate chl-a at global scales have been shown to be less accurate in Case 2 waters, due to the presence of water constituents other than phytoplankton which do not co-vary with the phytoplankton. The constituents of Arabian Sea coastal waters are region-specific because of the inherent variability of these optically-active substances affected by factors such as riverine input (e.g. suspended matter type and grain size, CDOM) and phytoplankton composition associated with seasonal changes.

Assessing the resilience of water supply systems in Oman

Water systems in the Sultanate of Oman are inevitably exposed to varied threats and hazards due to both natural and man-made hazards. Natural disasters, especially tropical cyclone Gonu in 2007, cause immense damage to water supply systems in Oman. At the same time water loss from leaks is a major operational problem. This research developed an integrated approach to identify and rank the risks to the water sources, transmission pipelines and distribution networks in Oman and suggests appropriate mitigation measures. The system resilience was evaluated and an emergency response plan for the water supplies developed. The methodology involved mining the data held by the water supply utility for risk and resilience determination and operational data to support calculations of non-revenue water. Risk factors were identified, ranked and scored at a stakeholder workshop and the operational information required was principally gathered from interviews. Finally, an emergency response plan was developed by evaluating the risk and resilience factors. The risk analysis and assessment used a Coarse Risk Analysis (CRA) approach and risk scores were generated using a simple risk matrix based on WHO recommendations. The likelihoods and consequences of a wide range of hazardous events were identified through a key workshop and subsequent questionnaires. The thesis proposes a method of translating the detailed risk evaluations into resilience scores through a methodology used in transportation networks. A water audit indicated that the percentage of NRW in Oman is greater than 35% which is similar to other Gulf countries but high internationally. The principal strategy for managing NRW used in the research was the AWWA water audit method which includes free to use software and was found to be easy to apply in Oman. The research showed that risks to the main desalination processes can be controlled but the risk due to feed water quality might remain high even after implementing mitigation measures because the intake is close to an oil port with a significant risk of oil contamination and algal blooms. The most severe risks to transmission mains were found to be associated with pipe rather than pump failure. The systems in Oman were found to be moderately resilient, the resilience of desalination plants reasonably high but the transmission mains and pumping stations are very vulnerable. The integrated strategy developed in this study has a wide applicability, particularly in the Gulf area, which may have risks from exceptional events and will be experiencing NRW. Other developing countries may also experience such risks but with different magnitudes and the risk evaluation tables could provide a useful format for further work.

Biogeochemical response of Tagus Estuary to climate change : a modelling study

Estuaries are highly dynamic systems which may be modified in a climate change context. These changes can affect the biogeochemical cycles. Among the major impacts of climate change, the increasing rainfall events and sea level rise can be considered. This study aims to research the impact of those events in biogeochemical dynamics in the Tagus Estuary, which is the largest and most important estuary along the Portuguese coast. In this context a 2D biophysical model (MOHID) was implemented, validated and explored, through comparison with in-situ data. In order to study the impact of extreme rainfall events, which can be characterized by an high increase in freshwater inflow, three scenarios were set by changing the inputs from the main tributaries, Tagus and Sorraia Rivers. A realistic scenario considering one day of Tagus and Sorraia River extreme discharge, a scenario considering one day of single extreme discharge of the Tagus River and finally one considering the extreme runoff just from Sorraia River. For the mean sea level rise, two scenarios were also established. The first with the actual mean sea level value and the second considering an increase of 0.42 m. For the extreme rainfall events simulations, the results suggest that the biogeochemical characteristics of the Tagus Estuary are mainly influenced by Tagus River discharge. For sea level rise scenario, the results suggest a dilution in nutrient concentrations and an increase in Chl-a in specific areas.For both scenarios, the suggested increase in Chl-a concentration for specific estuarine areas, under the tested scenarios, can lead to events that promote an abnormal growth of phytoplankton (blooms) causing the water quality to drop and the estuary to face severe quality issues risking all the activities that depend on it.

Cycles saisonniers, reproduction et qualite des ovocytes chez Pecten maximus en rade de Brest

This work is part of the program, "recruitment determinism in scallops" initiated in 1992 by the "Contrat de Baie". The aim of this research was to explain interannual abundance fluctuations observed for Pecten maximus, in the Bay of Brest. To this end an analytical approach to the life cycle of the species was undertaken to determine its critical phases. A phase may be considered as "critical" if the mortality rate at its level varies from one year to the other. Using data from 1989 to 1994, a precise time-table of the "physiological year" of the scallop in the Bay of Brest was determined. Fecundity and gamete production kinetics were estimated from the annual variation in the weight indices of the gonad. In the laboratory, egg quality was estimated following stimulated spawning of newly caught individuals. The digestive activity was estimated by amylase activity measurements during a three year period. Spawning lasts 6 months in the Bay of Brest from March to October. Spawning, muscle and digestive gland growths, are intimately linked to phytoplancton blooms. Gonad production shows a maximum in April-May, and two relative minima in Match and November. These results, which provide detailed understanding of the adult's annual cycle in the Bay of Brest, will be used to interpret the relationship between the annual cycle and larval and post larval abundance and survival. Already, these results are being used to improve spat production in hatcheries.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Composição do fitoplâncton e cianotoxinas em um reservatório utilizado para o abastecimento público: o efeito do tratamento de água convencional.

Florações de cianobactérias nocivas ocorrem frequentemente em reservatórios brasileiros, devido ao incremento de nutrientes pela eutrofização e pelas mudanças climáticas, como o aquecimento global. Estas florações alteram a qualidade dos corpos hídricos, produzindo compostos de gosto e odor e cianotoxinas, que representam um problema para as Estações de Tratamento de Água (ETAs). Estes compostos, quando dissolvidos na água dificultam os tratamentos convencionais. Além das cianobacérias, um dinoflagelado exótico tem ocorrido em águas doces brasileiras, incluindo reservatórios utilizados para o abastecimento público. Os reservatórios de Caxias do Sul (RS – Brasil) são gerenciados pelo Serviço Autônomo Municipal de Água e Esgoto (SAMAE) e apresentam um histórico de florações de cianobactérias nocivas, como Dolichospermum Bory de Saint-Vincent ex Bornet & Flahault e Microcystis (Kützing) ex Lemmermann, dentre outras. Além disso, desde 2012, tem ocorrido nestes reservatórios florações de Ceratium furcoides. Este organismo quando em extensas florações tem sido relacionado à perda da qualidade dos corpos hídricos. O reservatório Maestra foi construído entre os anos de 1965-1970 e abastece 22% da população de Caxias do Sul. Este reservatório fornece água para a ETA Celeste Gobatto, que utiliza o método convencional de tratamento da água. Este trabalho esta estruturado em três capítulos. O primeiro consiste de uma revisão bibliográfica de assuntos relavantes acerca do histórico do monitoramento da qualidade dos reservatórios no Brasil, da biologia de algas e cianobactérias, e as principais cianotixinas e acidentes devido à intoxicação no Brasil. Além disso, é feita uma breve revisão sobre o tratamento convencional da água, mostrando a importância de cada etapa para a remoção das impurezas, de acordo com os padrões de potabilidade da Portaria 2914. O segundo capítulo é manuscrito na forma de artigo científico intítulado “Composição de algas, cianobactérias e cianotoxinas no reservatório Maestra – Caxias do Sul, RS – Brasil”. Este estudo foi realizado entre janeiro de 2012 a abril de 2013. O terceiro capítulo consta de um manuscrito na forma de artigo científico, intitulado “Efeito do tratamento de água convencional na remoção de algas, cianobactérias e cianotoxinas em uma Estação de Tratamento de Água Convencional”. A eficiência de remoção foi avaliada em escala piloto, em uma Estação de Tratamento de Água de Caxias do Sul – RS a qual utiliza o método convencional de tratamento.

The state and known impacts of fish species transferred to or within continental Africa

Fish introductions have been made from small fish ponds to the largest lakes in Africa. The primary intent of these introductions has been to sustain or increase fish production, although some introductions have been made to develop sport fisheries and to control unwanted organisms. Some of these introductions have fulfilled their objective in the short term, but several of these "successful" introductions have created uncertainties about their long term sustainability. Lates niloticus, Oreochromis niloticus, O. leucostictus, Tilapia melanopleura and T. zilli were introduced into lakes Victoria and Kyoga in 1950s and early 1960s. By the 1980s O. niloticus and O. niloticus dominated the fisheries of these lakes, virtually eliminating a number of endemic fish species. The loss of genetic diversity of the fish in the worlds second largest lake has also been accompanied by a loss of trophic diversity. The transformation of the fish community has, in Lake Victoria coincided with a profound eutrophication (algal blooms, fish kills, hypolimnetic anoxia) which might be related to alterations of the lake's food-web structure. In contrast, the introduction of a planktivore, Limnothrissa miodon into Lake Kivu and the Kariba reservoir has established highly successful fisheries with little documented effect on the pre-existing fish community or trophic ecology of the lakes. The highly endemised species-rich African Great lakes may be particularly sensitive to species introductions and require special consideration and caution when introductions are contemplated because species extinctions, introgressive hybridization and ecosystem alterations may occur following fish introductions.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

Development of error correction techniques for nitrate-N load estimation models

Excess nutrient loads carried by streams and rivers are a great concern for environmental resource managers. In agricultural regions, excess loads are transported downstream to receiving water bodies, potentially causing algal blooms, which could lead to numerous ecological problems. To better understand nutrient load transport, and to develop appropriate water management plans, it is important to have accurate estimates of annual nutrient loads. This study used a Monte Carlo sub-sampling method and error-corrected statistical models to estimate annual nitrate-N loads from two watersheds in central Illinois. The performance of three load estimation methods (the seven-parameter log-linear model, the ratio estimator, and the flow-weighted averaging estimator) applied at one-, two-, four-, six-, and eight-week sampling frequencies were compared. Five error correction techniques; the existing composite method, and four new error correction techniques developed in this study; were applied to each combination of sampling frequency and load estimation method. On average, the most accurate error reduction technique, (proportional rectangular) resulted in 15% and 30% more accurate load estimates when compared to the most accurate uncorrected load estimation method (ratio estimator) for the two watersheds. Using error correction methods, it is possible to design more cost-effective monitoring plans by achieving the same load estimation accuracy with fewer observations. Finally, the optimum combinations of monitoring threshold and sampling frequency that minimizes the number of samples required to achieve specified levels of accuracy in load estimation were determined. For one- to three-weeks sampling frequencies, combined threshold/fixed-interval monitoring approaches produced the best outcomes, while fixed-interval-only approaches produced the most accurate results for four- to eight-weeks sampling frequencies.

Avaliação da toxicidade de florações naturais e de cultura de cianobactérias: efeitos sobre Ceriodaphnia silvestri (Crustacea, Cladocera)

The incidence of toxic cyanobacterial blooms is one of the important consequences of eutrophication in aquatic ecosystems. It is a very common phenomenon in reservoirs and shrimp ponds in the State of Rio Grande do Norte (RN), Brazil. Cyanobacterias produce toxins which can affect aquatic organisms and men trough the food chain. Aiming to contribute to the studies of cyanobacterias in RN, we propose: a) to evaluate the toxicity of isolated cyanobacterias in important fresh-water environments; and b) to verify the effects of both natural and cultured blooms occurred in reservoirs for human supply and in the cladoceran Ceriodaphnia silvestrii. This study was carried out using samples of natural blooms occurred between March and October of 2004 in Gargalheiras Dam (08º L e 39º W), in July of 2004 in Armando Ribeiro Gonçalves Dam (06o S e 37o W) and in commercial shrimp ponds (Litopenaeus vannamei) located in fresh-water environments. The samples were collected with plankton net (20µm.) for identification, isolation and obtaining of phytoplanktonic biomass for liophilization and later toxicity bioassays. The toxicity of cultured samples and natural blooms was investigated through bioassays in Swiss mice. Quantification of cyanobacteria in samples was conducted following the Ütermol method, with 300mL samples fixed with lugol. The toxicity test with Ceriodaphnia silvestrii followed ABNT, 2001 recommendations, and were accomplished with natural hepatotoxic bloom s samples and cultured samples of both non-toxic and neurotoxic C. raciborskii. In this test, five newborns, aged between 6 and 24 hours, were exposed to different concentrations (0 a 800 mg.L-1) of crude cyanobacterial extracts during 24 and 48 hours. Three replicates were used per treatment. The pH, temperature and dissolved oxygen at the beginning and after 24 and 48hours from the test were measured. We estimated the CL50 through the Trimmed Spearman-Karber method. The blooms were constituted by Microcystis panniformis, M. aeruginosa, Anabaena circinalis, Cylindrospermopsis raciborskii and Planktothrix agardhii, producers of mycrocistin-LR confirmed with HPLC analysis. Samples of hepatotoxic blooms registered toxinogenic potential for C. silvestrii, with CL50-24h value of 47.48 mg.L-1 and CL5048h of 38.15 mg.L-1 for GARG samples in march/2005; CL50-24h of 113,13 mg.L-1 and CL5048h of 88,24 mg.L-1 for ARG July/2004; CL50-24h of 300.39 mg.L-1 and CL50-48h of 149.89 mg.L-1 for GARG October/2005. For cultured samples, values of CL50-24h and CL50-48h for C. raciborskii toxic strains were 228.05 and 120.28 mg.L-1, respectively. There was no mortality of C. silvestrii during the tests with non-toxic C. raciborskii strain. The toxicity test with C. silvestrii presented good sensitivity degree to cyanotoxins. The toxicity of natural hepatotoxic blooms samples (microcystins) and cultured neurotoxic saxitoxins producer samples analyzed in this study give us strong indications of that toxin s influence on the zooplanktonic community structure in tropical aquatic environments. Eleven cyanobacteria strains were isolated, representing 6 species: Anabaenopsis sp., Cylindrospermopsis raciborskii, Chroococcus sp., Microcystis panniformis, Geitlerinema unigranulatum e Planktothrix agardhii. None presented toxicity in Swiss mice. The strains were catalogued and deposited in the Laboratório de Ecologia e Toxicologia de Organismos Aquáticos (LETMA), in UFRN, and will be utilized in ecotoxicológical and ecophysiological studies, aiming to clarify the causes and control of cyanobacterial blooms in aquatic environments in RN. This state s reservoirs must receive broader attention from the authorities, considering the constant blooms occurring in waters used for human consumption

Avaliação transcricional de Glutationas -transferases em PEIXE-ZEBRA (Danio rerio) e alterações causadas pela exposição á Microcistina-LR

As Microcistinas são heptapeptídios cíclicos produzidos como metabólitos secundários por diferentes espécies de cianobactérias, sendo relevantes pelo seu potencial hepatotóxico. Peixes apresentam estratégias bioquímicas para detoxificar contaminantes ambientais, incluindo a ativação de enzimas de fase II de biotransformação, que incluem as isoformas de glutationa S-transferase (GST). As GST catalizam a conjugação de glutationa reduzida (GSH) com uma variedade de xenobióticos, incluindo as microcistinas. O presente estudo avaliou os níveis transcricionais de quinze isoformas de GST a fim de identificar isoformas possivelmente envolvidas na detoxificação de contaminantes ambientais como a microcistina-LR (MC-LR) em Danio rerio. A técnica de PCR em tempo real (RT-qPCR) foi utilizada para avaliação dos níveis transcricionais, permitindo análise das GST em diferentes órgãos, abundância e a ativação/repressão das isoformas de GST pela exposição à MC-LR. Foram avaliados os possíveis efeitos causados em brânquia e fígado após exposição por 24 hs às concentrações de 5 µg.L-1 e 50 µg.L-1 de MC-LR. Baseado nos scores de estabilidade para oito genes normalizadores, foram selecionados glicose-6-fosfato desidrogenase (g6pdh), β-actina1 e beta-2-microglobulina (b2m); b2m, alfa-tubulina 1 (tuba) e β- actin1; e tuba, b2m e g6pdh, para normalização dos níveis trancricionais de GST para distribuição órgão-específica, abundância e efeito da MC-LR em brânquia e fígado, respectivamente. A avaliação transcricional da distribuição órgão-específica revelou níveis significativos de gstal e gstk1.1 no fígado; gstp1 e gstp2 em brânquia; mgst3a, gstr1, gstm2, gstm33, gstp1, gstp2 e gstk1.1 no intestino; gstm2, gstm3 e gstal no olho e gstt1a e gsta2.1 no cérebro. Considerando os níveis de transcritos para um dado órgão, gstk1.1, gstal, gstp1 e gstt2 foram mais abundantes nos órgãos de detoxificação, tais como o fígado, brânquias e intestino, enquanto gstt1a e gsta2.1 foram mais abundantes no rim. Em brânquia, gsta2.1 e gstt1b foram reprimidas por 5 µg.L-1 de MC-LR e mgst1.1 foi reprimida em 50 µg.L-1 de MC-LR. No fígado, as isoformas gst2.2 e gstp2 foram reprimidas em ambas as concentrações, gstal foi reprimida em 5 µg.L-1, e gstt1a e gstk1.1 foram reprimidas em 50 µg.L-1 de MC-LR. As isoformas gstal, gstr1, gstp1, mgst3a, gstm1, gstm2 e gstm3 não foram alteradas pela exposição a MC-LR. Os resultados obtidos fornecem informações para a escolha de isoformas específicas de GST possivelmente envolvidas na detoxificação/toxicidade de MC-LR, a serem melhores caracterizadas ao nível protéico e também contribui para a escolha de genes normalizadores a serem utilizados em outros estudos da mesma natureza

Toxin and Growth Responses of the Neurotoxic Dinoflagellate Vulcanodinium rugosum to Varying Temperature and Salinity

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m−2 s−1. V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d−1) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts.

Field study and numerical simulation of developing red tide in the northern Strait of Hormuz

The catastrophic event of red tide has happened in the Strait of Hormuz, the Persian Gulf and Gulf of Oman from late summer 2008 to spring 2009. With its devastating effects, the phenomenon shocked all the countries located in the margin of the Persian Gulf and the Gulf of Oman and caused considerable losses to fishery industries, tourism, and tourist and trade economy of the region. In the maritime cruise carried out by the Persian Gulf and Gulf of Oman Ecological Research Institute, field data, including temperature, salinity, chlorophyll-a, dissolved oxygen and algal density were obtained for this research. Satellite information was received from MODIS and MERIS and SeaWiFS sensors. Temperature and surface chlorophyll images were obtained and compared with the field data and data of PROBE model. The results obtained from the present research indicated that with the occurrence of harmful algal blooms (HAB), the Chlorophyll-a and the dissolved oxygen contents increased in the surface water. Maximum algal density was seen in the northern coasts of the Strait of Hormuz. Less concentration of algal density was detected in deep and surface offshore water. Our results show that the occurred algal bloom was the result of seawater temperature drop, water circulation and the adverse environmental pollutions caused by industrial and urban sewages entering the coastal waters in this region of the Persian Gulf ,This red tide phenomenon was started in the Strait of Hormuz and eventually covered about 140,000 km2 of the Persian Gulf and total area of Strait of Hormuz and it survived for 10 months which is a record amongst the occurred algal blooms across the world. Temperature and chlorophyll satellite images were proportionate to the measured values obtained by the field method. This indicates that satellite measurements have acceptable precisions and they can be used in sea monitoring and modeling.

Nitrate metabolism in the dinoflagellate Lingulodinium polyedrum

Les dinoflagellés sont des eucaryotes unicellulaires retrouvés dans la plupart des écosystèmes aquatiques du globe. Ces organismes amènent une contribution substantielle à la production primaire des océans, soit en tant que membre du phytoplancton, soit en tant que symbiontes des anthozoaires formant les récifs coralliens. Malheureusement, ce rôle écologique majeur est souvent négligé face à la capacité de certaines espèces de dinoflagellés à former des fleurs d'eau, parfois d'étendue et de durée spectaculaires. Ces floraisons d'algues, communément appelées "marées rouges", peuvent avoir de graves conséquences sur les écosystèmes côtiers, sur les industries de la pêche et du tourisme, ainsi que sur la santé humaine. Un des facteurs souvent corrélé avec la formation des fleurs d'eau est une augmentation dans la concentration de nutriments, notamment l’azote et le phosphore. Le nitrate est un des composants principaux retrouvés dans les eaux de ruissellement agricoles, mais également la forme d'azote bioaccessible la plus abondante dans les écosystèmes marins. Ainsi, l'agriculture humaine a contribué à magnifier significativement les problèmes associés aux marées rouges au niveau mondial. Cependant, la pollution ne peut pas expliquer à elle seule la formation et la persistance des fleurs d'eau, qui impliquent plusieurs facteurs biotiques et abiotiques. Il est particulièrement difficile d'évaluer l'importance relative qu'ont les ajouts de nitrate par rapport à ces autres facteurs, parce que le métabolisme du nitrate chez les dinoflagellés est largement méconnu. Le but principal de cette thèse vise à remédier à cette lacune. J'ai choisi Lingulodinium polyedrum comme modèle pour l'étude du métabolisme du nitrate, parce que ce dinoflagellé est facilement cultivable en laboratoire et qu'une étude transcriptomique a récemment fourni une liste de gènes pratiquement complète pour cette espèce. Il est également intéressant que certaines composantes moléculaires de la voie du nitrate chez cet organisme soient sous contrôle circadien. Ainsi, dans ce projet, j'ai utilisé des analyses physiologiques, biochimiques, transcriptomiques et bioinformatiques pour enrichir nos connaissances sur le métabolisme du nitrate des dinoflagellés et nous permettre de mieux apprécier le rôle de l'horloge circadienne dans la régulation de cette importante voie métabolique primaire. Je me suis tout d'abord penché sur les cas particuliers où des floraisons de dinoflagellés sont observées dans des conditions de carence en azote. Cette idée peut sembler contreintuitive, parce que l'ajout de nitrate plutôt que son épuisement dans le milieu est généralement associé aux floraisons d'algues. Cependant, j’ai découvert que lorsque du nitrate était ajouté à des cultures initialement carencées ou enrichies en azote, celles qui s'étaient acclimatées au stress d'azote arrivaient à survivre près de deux mois à haute densité cellulaire, alors que les cellules qui n'étaient pas acclimatées mourraient après deux semaines. En condition de carence d'azote sévère, les cellules arrivaient à survivre un peu plus de deux semaines et ce, en arrêtant leur cycle cellulaire et en diminuant leur activité photosynthétique. L’incapacité pour ces cellules carencées à synthétiser de nouveaux acides aminés dans un contexte où la photosynthèse était toujours active a mené à l’accumulation de carbone réduit sous forme de granules d’amidon et corps lipidiques. Curieusement, ces deux réserves de carbone se trouvaient à des pôles opposés de la cellule, suggérant un rôle fonctionnel à cette polarisation. La deuxième contribution de ma thèse fut d’identifier et de caractériser les premiers transporteurs de nitrate chez les dinoflagellés. J'ai découvert que Lingulodinium ne possédait que très peu de transporteurs comparativement à ce qui est observé chez les plantes et j'ai suggéré que seuls les membres de la famille des transporteurs de nitrate de haute affinité 2 (NRT2) étaient réellement impliqués dans le transport du nitrate. Le principal transporteur chez Lingulodinium était exprimé constitutivement, suggérant que l’acquisition du nitrate chez ce dinoflagellé se fondait majoritairement sur un système constitutif plutôt qu’inductible. Enfin, j'ai démontré que l'acquisition du nitrate chez Lingulodinium était régulée par la lumière et non par l'horloge circadienne, tel qu'il avait été proposé dans une étude antérieure. Finalement, j’ai utilisé une approche RNA-seq pour vérifier si certains transcrits de composantes impliquées dans le métabolisme du nitrate de Lingulodinium étaient sous contrôle circadien. Non seulement ai-je découvert qu’il n’y avait aucune variation journalière dans les niveaux des transcrits impliqués dans le métabolisme du nitrate, j’ai aussi constaté qu’il n’y avait aucune variation journalière pour n’importe quel ARN du transcriptome de Lingulodinium. Cette découverte a démontré que l’horloge de ce dinoflagellé n'avait pas besoin de transcription rythmique pour générer des rythmes physiologiques comme observé chez les autres eukaryotes.