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Development of an Alexandrium minutum Halim bloom affecting a Mediterranean harbor was monitored in detail using a multidisciplinary approach. A. minutum was by far the most abundant species at and near the bloom maximum, but always coexisted with members of three additional dinoflagellate genera and prasinophytes. Bloom initiation (early February) occurred during prolonged influences of sunny weather conditions, when day length exceeded 10.5 h and water temperatures reached 10.2°C. Subsequent development toward its maximum (end of March) also relied on good weather conditions, with specific wind directions favoring accumulation of cells. Arrival of rainy weather, associated with frontal boundaries of large-scale low-atmospheric-pressure systems and characterized by reduced solar irradiance (heavy cloud coverage), opposite wind directions, and enhanced wind speeds, always caused temporal declines of the bloom. These declines were attributed to dispersal or displacement of algae, but a vertical migration of A. minutum cells toward the sediment was not excluded. Delayed inflows of excess terrestrial rainwater along the inner harbor wall strongly reduced salinity and prolonged a temporal decline far beyond influences of bad weather. The associated nutrient supply favored development of the phytoplankton population but reduced the toxin production of A. minutum cells. The HPLC-determined Gonyautoxin (GTX) 1 + 4/GTX 2 + 3 ratio strongly increased toward the bloom maximum. This ratio was influenced by nutrient status and cell density and has a potential value for monitoring developmental stages of blooms. Prolonged bad weather conditions eventually hindered continuation of bloom development, and subsequent declines of algal biomass were attributed to grazing

We characterised the spatial and temporal variation in the bacterioplankton assemblage composition during bloom events of different Alexandrium species (Dinophyceae) in the littoral of the NW Mediterranean Sea by means of catalysed reporter deposition fluorescence in situ hybridisation with oligonucleotide probes (CARD FISH). We studied several Alexandrium blooms through their seasonal development (at La Fosca beach) or in their spatial variability (in Arenys Harbour and Olbia Bay), and we complemented these observations by describing the composition of the bacterial assemblage associated with cultures of Alexandrium species isolated from the same sites. Our studies on natural bacterioplankton assemblages identified the Bacteroidetes lineage and the Alphaproteobacteria as the dominating components during the studied blooms of Alexandrium. Alphaproteobacteria dominated in the La Fosca and Olbia blooms, while bacteria belonging to the Bacteroidetes were abundant in the development phase of the La Fosca beach bloom and in the winter Arenys bloom. Gammaproteobacteria contributed in low proportions without significant changes through the different bloom phases at La Fosca beach and in Olbia Bay, but were more abundant in Arenys Harbour. While the absolute bacterial abundances in the spatial study of Olbia Bay covaried with the Alexandrium densities, there were no spatial changes in the bacterioplankton assemblage composition. Alteromonas-like organisms were never an important fraction of the assemblage, but Roseobacter dominated Alphaproteobacteria in Arenys Harbour. Furthermore, the bacterioplankton assemblages associated with Alexandrium spp. cultures were very different from the natural bacterial assemblages during blooms of the same species. W conclude that the presence of a given harmful algal bloom species during a bloom will not always necessarily be accompanied by the same bacterial assemblage structure, and studies done with dinoflagellate cultures may only refl

A filter system for collecting phytoplankton cells coupled to a target species-specific polymerase chain reaction (PCR) assay was performed on a spatial and temporal series of net and seawater samples as part of the Mediterranean Sea EU project Strategy (EVK-CT-2001-00046). The application of PCR allowed rapid detection of several harmful dinoflagellate species and genera, including Alexandrium spp. In this study, oligonucleotide primers targeting the 5.8S rDNA and ITS regions of the genus Alexandrium and the species A. minutum were developed. Field samples were concentrated onto filter membranes, total DNA was extracted from mixed phytoplankton populations and PCR assays were carried out with specific primers. Qualitative PCR results were compared with light and epifluorescence microscopic examinations. Results indicated that the molecular assay was able to detect harmful target cells at concentrations undetectable by microscopy. Application of this filter PCR assay to seawater samples revealed it to be a sensitive and rapid procedure for routine monitoring of field samples. Copyright © NISC Pty Ltd.

Alexandrium catenella is a cyst-forming dinoflagellate causative of paralytic shellfish poisoning. Strain and nutritional factors affecting cyst formation and germination in this species were studied in cultures of several clonal crosses. Sexual reproduction was monitored and the effect of nutrients on sexual stages was proved because planozygotes isolated in medium with no phosphates added encysted in lower percentages than those placed in media with no nitrates added or in replete conditions, where the highest encystment was achieved. However, other unknown factors prevented encystment, because, even in replete conditions, some planozygotes were unable to encyst. Dormancy period of cysts ranged from 5 to 65 days at 24°C. A progressive germination inside this time interval was recorded for cysts from all six clonal crosses employed, and therefore, identified as a species characteristic. This gradual germination was modulated by nutrient levels in both encystment and excystment media: (1) cysts formed in conditions where nitrates and phosphates were added needed more time to excyst than those from media lacking one of these nutrients and (2) cysts from any encystment treatment germinated faster, and in higher percentages in poor external conditions compared to those placed in replete L1 medium. More than 90% of the cysts isolated to seawater germinated in less than 20 days, whereas those isolated to L1 medium had not begun to excyst, and, after 60 days, the percentage of germinated cysts in replete conditions remained below 40%. However, ultimate germination frequencies and postmeiotic viability of cysts might depend on genetic characteristics because cysts from the clonal crosses of A. catenella employed showed significant differences. Ecdysal cysts were mainly observed under the treatments in which the smallest number of resting cysts were produced. In 65% of the cases germination of these cysts under replete conditions occurred within 15 days.

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug-like structures in amphiesma and a possible layer of semi-opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low- (K. veneficum) and high-light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates. © 2006 by the Phycological Society of America

HAB occurrence is becoming more frequent and problematic in marine recreational waters. However, the exploitation of the coastal area for recreational use is promoting the necessary conditions for the HAB increase. In terms of the harmful effects, we can consider two types of causative organism: the toxic producers and the high-biomass producers. Toxic events can be produced by a very low concentration of the causative organism. This characteristic implies a difficulty for the monitoring programs in relation to human health. It is important to point out in the context of human health and HAB events, that in some coastal regions (e.g. the Mediterranean basin) HABs are an emerging problem. In these regions, the local population and visitors may face a health risk that is difficult to measure. The monitoring of toxic species has mainly been associated -with shellfish farming. However, the risk of intoxication could become even greater in areas not subject to legislation of aquaculture activities

The taxonomic structure of phytoplankton populations in two Mediterranean coastal lagoons were compared with those of nearby marine waters (external waters). Mediterranean confined lagoons remain isolated for most the year and concentrate phytoplankton to a very high biomass. Coastal lagoons on the Mediterranean may, therefore, act as accumulators of neritic phytoplankton (including species related to harmful algal blooms). We examined whether coastal lagoons act as concentrators of marine toxic dinoflagellates during confinement periods, and the common environmental factors that favour growth of specific harmful species in the two ecosystems considered: coastal lagoons and external waters. An alternation between the dominance of diatoms and dinoflagellates was observed, coinciding with that described in Margalef's mandala, occurring in external waters as well as in coastal lagoons. Moreover, the temporal patter was different in the two ecosystems. Dinoflagellate species composition and their bloom period were highly variable in time and space, thus, species had to be analysed individually. Most of the dinoflagellate species found in this study were potentially harmful and high biomass producers. Harmful dinoflagellate species performed well in both, external waters and lagoons, but the specific species-dependent affinity to each of these environments determined which organisms bloom there. Thus, expansion of harmful algal blooms (HAB) to inland waters is not likely and some environmental factors such as the oxidised state of available nitrogen, became determinant to the success and bloom of a species in the coastal lagoon ecosystem

Los problemas relacionados con las proliferaciones de microalgas nocivas (PAN) en el Mediterráneo no están únicamente relacionados con la salud alimentaria y la acuicultura. [...]

El concepto de calidad en el agua litoral está muy ligado a su contenido en nutrientes hasta el punto que eutrofización se suele usar como sinónimo de degradación. [...]

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate. © 2005 Elsevier B.V. All rights reserved

The presence of the paralytic shellfish poisoning (PSP) dinoflagellate Alexandrium catenella in the north western (NW) Mediterranean Sea has been known since 1983. From this date on, the species has spread along the Spanish and Italian coastlines. Information concerning A. catenella isolates in the NW Mediterranean Sea was gained through phylogenetic studies. Twenty established toxic cultures of A. catenella taken from various NW Mediterranean Sea locations were analysed by nucleotide sequencing of the 5.8S rDNA and internal transcribed spacer regions. These rDNA ribosomal markers resulted useful in delineating the phylogenetic position of this species in the genus Alexandrium as well as in determining relationships between A. catenella isolates from different geographic areas. The phylogenetic position of the Mediterranean A. catenella ribotype, when compared to the >Alexandrium tamarense/catenella/ fundyense species complex>, fits this species complex well. All the Mediterranean A. catenella isolates were constituted by only one genetic ribotype. By comparing the isolate sequences with those of other geographic areas, it revealed that the Mediterranean A. catenella ribotype was closely related to the A. catenella from Japan, Western Pacific Ocean. It was also evident that in temperate Japanese waters, a genetic variability was detected within A. catenella isolates; in fact, all strains resulted divergent showing as many as 15 mutational steps. The possibility that A. catenella has been recently introduced into the Mediterranean basin from temperate Asian areas is discussed. © Springer-Verlag 2005

Diatoms of the genus Pseudonitzschia are widely distributed in seas and oceans. They attract world wide interest since they were found to produce the lethal neurotoxin domoic acid (DA). Back in 1987, a member of this genus (P. multiseries) caused amnesic shellfish poisoning (ASP) in eastern Canada [1]. Production of DA is reported for at least ten species of Pseudo-nitzschia [2], and their blooms result in marked toxin accumulation through the food chains, with subsequent impacts on individual marine organisms, total ecosystems, humans, and even the economy [3]. In the Mediterranean, both Pseudonitzschia galaxiae and P. multistriata were shown to be toxic [4]. [...]

The word SEED is not an acronym or abbreviation; it directly refers to seed as produced by plants. However, the indication ‘SEED’ refers to a new project entitled Life cycle transformations among HAB species, and the environmental and physiological factors that regulate them, for which we do not want an acronym. [...]

Alexandrium catenella is a cyst-forming dinoflagellate causative of paralytic shellfish poisoning. Strain and nutritional factors affecting cyst formation and germination in this species were studied in cultures of several clonal crosses. Sexual reproduction was monitored and the effect of nutrients on sexual stages was proved because planozygotes isolated in medium with no phosphates added encysted in lower percentages than those placed in media with no nitrates added or in replete conditions, where the highest encystment was achieved. However, other unknown factors prevented encystment, because, even in replete conditions, some planozygotes were unable to encyst. Dormancy period of cysts ranged from 5 to 65 days at 24°C. A progressive germination inside this time interval was recorded for cysts from all six clonal crosses employed, and therefore, identified as a species characteristic. This gradual germination was modulated by nutrient levels in both encystment and excystment media: (1) cysts formed in conditions where nitrates and phosphates were added needed more time to excyst than those from media lacking one of these nutrients and (2) cysts from any encystment treatment germinated faster, and in higher percentages in poor external conditions compared to those placed in replete L1 medium. More than 90% of the cysts isolated to seawater germinated in less than 20 days, whereas those isolated to L1 medium had not begun to excyst, and, after 60 days, the percentage of germinated cysts in replete conditions remained below 40%. However, ultimate germination frequencies and postmeiotic viability of cysts might depend on genetic characteristics because cysts from the clonal crosses of A. catenella employed showed significant differences. Ecdysal cysts were mainly observed under the treatments in which the smallest number of resting cysts were produced. In 65% of the cases germination of these cysts under replete conditions occurred within 15 days

Los estudios de biología, autoecología y toxinología de especies del género Dinophysisse han visto muy limitados por la normalescasez de estas especies, la falta de cultivos estables de las mismas, y las limitaciones de las técnicas analíticas al uso, que requieren muestras de varios cientos de células. En el transcurso del proyecto DINOPHYSIS 2000 se han realizado importantesavances tecnológicos que abren el camino para futuros muestreos, más sistemáticos, sobre parámetros fisiológicos y dinámicos de las poblaciones naturales de Dinophysis. La aplicación de las nuevas técnicas permitió obtener resultados novedosos sobre el ciclo celular, ciclo de vida, toxinología y dinámica de poblaciones de las principales especies del género Dinophysisagentes de episodios diarreogénicos en Galicia. Se resumen los objetivos del proyecto DINOPHYSS 2000, métodos aplicados y principales resultados alcanzados.

A recurrent Alexandrium minutum bloom in the Arenys de Mar harbour (Catalan coast, North Western Mediterranean) was monitored in order to establish the relationship between vegetative cells and cyst production. The bloom lasted from January 21 to February 24, 2002 and reached cell concentrations of up to 47 x106 cell L-1. Two aspects related to the resting cysts deposition were studied: (i) production of resting cysts during the bloom period (by means of sediment traps) and (ii) distribution of resting cysts in the sediment after the bloom (May 2002). Cyst formation in Arenys clearly started in a period with high vegetative cell densities in the water column. Once production was initiated encystment fluxes remained constant for two weeks, and covering the periods of maintenance and decline of the bloom. High cyst fluxes (up to 6000 cysts cm-2 day-1) were quantified as a result of the high vegetative cell concentration. Moreover, encystment occurring in less than 1% of the total population indicates that most of the cells are not involved in resting cysts formation. A comparison of the resting cyst flux values obtained from the sediment traps and the resting cyst concentrations in surface sediment (628–3270 cysts cm-3) three months later, revealed that the number of cysts in the sediment decreased during that time. The studies of excystment showed a high germination percentage (91%) and germling viability (100%). These data, together with the resting cyst distribution in the sediment, are important in assessing the role of resting cysts in the bloom dynamics of A. minutum in confined waters.

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detectio

The toxic dinoflagellate Alexandrium catenella is usually known as a cold waters species; however, during the summer of 1996 a bloom was observed in the Barcelona harbour (water temperatures above 20°C). This was the first time that an A. catenella bloom occurred in Catalan waters. Recurrent blooms have been observed annually from 1996 to 1999 during the warm season in this harbour. Since its first detection A. catenella has been recorded at an increasing number of stations along the coast, suggesting a progressive areal expansion. Two toxic, widespread events of this species occurred in non-confined coastal waters along 100 km of coastline during the summers of 1998 and 1999. We argue that the newly constructed harbours, serving as confined habitats for the development and maintenance of A. catenella, have contributed to the expansion of the dinoflagellate on the NW Mediterranean coast

The in situ growth rate of phytoplankton is defined as the potential growth rate without adjustment for losses due to cell mortality when it is measured by the cell cycle approach. In contrast, the method that assesses the cell number variation of the population in diffusion chambers takes cell mortality into account. We compare the 2 methods for natural blooms of dinoflagellates (Alexandrium minutum, Alexandrium taylori, Ceratium furca, Dinophysis sacculus, Gymnodinium pulchellum and Gyrodinium corsicum) grown in various situations and locations and data reported elsewhere. The in situ potential growth rates showed great intraspecific variability. The use of the differences between the 2 methods as a measure of in situ cell lysis is discussed