Dinoflagellate Alexandrium Catenella Research Articles

Boundary influences on HAB phytoplankton ecology in a stratification-enhanced upwelling shadow

Coastal marine ecosystems are profoundly influenced by processes that originate from their boundaries. These include fluid boundaries—with the atmosphere, oceanic boundary currents and terrestrial aquatic systems, as well as solid boundaries—with the seafloor and coast. Phytoplankton populations transfer complexly interacting boundary influences into the biosphere. In this contribution, we apply data from an ocean observing and modeling system to examine boundary influences driving phytoplankton ecology in Monterey Bay, CA, USA. The study was focused on species that may cause harmful algal blooms (HABs). During September–October 2010, autonomous molecular analytical devices were moored at two locations characterized by different degrees of stratification and exposure to upwelling dynamics. The time-series revealed multiple transitions in local HAB phytoplankton communities, involving diatoms (Pseudo-nitzschia spp.), dinoflagellates (Alexandrium catenella), and raphidophytes (Heterosigma akashiwo). Observational and model results showed that the biological transitions were closely related to environmental changes that resulted from a variety of boundary processes—responses of oceanic circulation to wind forcing, influxes of different water types that originated outside the bay, and emergence of strongly stratified nearshore water into the greater bay. Boundary processes were further implicated at patch scales. High-resolution mapping and sampling of a phytoplankton-enriched patch were conducted in a Lagrangian framework using autonomous underwater vehicles. These highly resolved measurements showed that small-scale spatial patterns in the toxicity of Pseudo-nitzschia populations were related to the coupling of resuspended sediments from the bottom boundary layer to the surface mixed layer.

Impact of the toxic dinoflagellateAlexandrium catenellaon Pacific oyster reproductive output: application of flow cytometry assays on spermatozoa

The toxic dinoflagellate Alexandrium catenella recurrently blooms on the coasts of France and produces Paralytic Shellfish Toxins (PSTs) that accumulate in bivalves. These toxins can affect various physiological functions including reproduction. The present study aims to validate measurements of sperm viability, DNA content and mitochondrial membrane potential in Pacific oyster Crassostrea gigas using flow cytometry coupled with fluorescent markers, and to use these measurements to assess the cellular parameters of sperm from Pacific oysters exposed to A. catenella. These parameters may influence fertilization, embryogenesis and larval development in free-spawning shellfish. Sperm viability and DNA content estimation were assessed using SYBR-14, which only penetrates cells with intact membranes. Cell mortality was measured with propidium iodide (PI), which penetrates cells with membrane damage. Mitochondrial membrane potential, used as an estimate of mitochondrial function, was measured using JC-1 dye, which selectively enters into mitochondria and reversibly changes colour from green to orange as the membrane potential increases. To assess the effect of toxic algae on oyster sperm, broodstock (ripe oysters) were fed toxic (A. catenella) or non toxic (Heterocapsa triquetra) dinoflagellates at 250 cell ml-1 for 9 days. After this exposure period, mature oysters were stripped and cellular responses of sperm analysed. Average DNA staining, as measured by SYBR-14, appeared lower and more variable in gametes from A. catenella-exposed oysters than in those from control oysters fed H. triquetra. Additionally, mitochondrial membrane potential of sperm from A. catenella-exposed oysters was significantly higher (1.5 fold) than that of sperm produced by oysters fed H. triquetra. Both the increase of mitochondrial membrane potential and the modification of DNA structure can be expected to impact spermatozoa ability to fertilize oocytes and could thus impact related reproductive processes.

Paralytic Toxins Accumulation and Tissue Expression of α-Amylase and Lipase Genes in the Pacific Oyster Crassostrea gigas Fed with the Neurotoxic Dinoflagellate Alexandrium catenella

The pacific oyster Crassostrea gigas was experimentally exposed to the neurotoxic Alexandrium catenella and a non-producer of PSTs, Alexandrium tamarense (control algae), at concentrations corresponding to those observed during the blooming period. At fixed time intervals, from 0 to 48 h, we determined the clearance rate, the total filtered cells, the composition of the fecal ribbons, the profile of the PSP toxins and the variation of the expression of two α-amylase and triacylglecerol lipase precursor (TLP) genes through semi-quantitative RT-PCR. The results showed a significant decrease of the clearance rate of C. gigas fed with both Alexandrium species. However, from 29 to 48 h, the clearance rate and cell filtration activity increased only in oysters fed with A. tamarense. The toxin concentrations in the digestive gland rose above the sanitary threshold in less than 48 h of exposure and GTX6, a compound absent in A. catenella cells, accumulated. The α-amylase B gene expression level increased significantly in the time interval from 6 to 48 h in the digestive gland of oysters fed with A. tamarense, whereas the TLP gene transcript was significantly up-regulated in the digestive gland of oysters fed with the neurotoxic A. catenella. All together, these results suggest that the digestion capacity could be affected by PSP toxins.

Toxicity of benz(a)anthracene and fluoranthene to marine phytoplankton in culture: Does cell size really matter?

The toxicity of benz(a)anthracene and fluoranthene (polycyclic aromatic hydrocarbons, PAHs) was evaluated on seven species of marine algae in culture belonging to pico-, nano-, and microphytoplankton, exposed to increasing concentrations of up to 2mgL−1. The short-term (24h) toxicity was assessed using chlorophyll a fluorescence transients, linked to photosynthetic parameters. The maximum quantum yield Fv/Fm was lower at the highest concentrations tested and the toxicity thresholds were species-dependent. For acute effects, fluoranthene was more toxic than benz(a)anthracene, with LOECs of 50.6 and 186μgL−1, respectively. After 72h exposure, there was a dose-dependent decrease in cell density, fluoranthene being more toxic than benz(a)anthracene. The population endpoint at 72h was affected to a greater extent than the photosynthetic endpoint at 24h. EC50 was evaluated using the Hill model, and species sensitivity was negatively correlated to cell biovolume. The largest species tested, the dinoflagellate Alexandrium catenella, was almost insensitive to either PAH. The population endpoint EC50s for fluoranthene varied from 54μgL−1 for the picophytoplankton Picochlorum sp. to 418μgL−1 for the larger diatom Chaetoceros muelleri. The size/sensitivity relationship is proposed as a useful model when there is a lack of ecotoxicological data on hazardous chemicals, especially in marine microorganisms.

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12d−1. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.

Proteomic analysis of a toxic dinoflagellate Alexandrium catenella under different growth phases and conditions

Alexandrium is a widely spread dinoflagellate genus throughout many regions of the world, which not only causes the harmful algal blooms (HABs) but also results in the paralytic shellfish poisoning (PSP) throughout the world. This study compared protein profiles of A. catenella grown under different growth phases and conditions using a proteomic approach, and identified the differentially expressed proteins. The results showed that the expressions of proteins identified in three different regions of the gels, the groups 1, 2 and 3 proteins, varied significantly with the growth phases and conditions. Group 1 proteins and six Group 2 proteins were highly expressed at the initial, exponential and stationary growth phases, eight Group 2 proteins were highly expressed only at the initial phase, and Group 3 proteins were highly expressed at the exponential and/or stationary phases. However, all these proteins were expressed at low levels or were barely visible at the dissipation phase. The expressions of groups 1 and 2 proteins were low or barely visible in various growth conditions except in continuous darkness they were highly expressed. Group 3 proteins, on the other hand, were overexpressed in continuous illumination and expressed at low levels or barely visible in continuous darkness or under nitrate-starvation. The data from MALDI-TOF-TOF mass spectrometry demonstrated that these differentially expressed proteins were associated with macromolecular biosynthesis, photosynthesis, tRNA synthesis and DNA stability, stress response and cell division regulation. Synthetase was the major component of the altered proteins. This is one of the first comprehensive proteomic study of a dinoflagellate, A. catenella, that provides a fundamental understanding of the proteins involved in A. catenella growth and response to environmental stresses, and potential physiological indicator proteins related to growth and environmental stress have been identified.

유독성 와편모류 Alexandrium catenella에 대한 Pseudoalteromonas sp. NH-12의 살조능

BACKGROUND: The aim of this study was to isolate and identify algicidal bacterium that tends to kill the toxic dinoflagellate Alexandrium catenella, and to determine the algicidal activity. METHODS AND RESULTS: Among of four algicidal bacteria isolated in this study, NH-12 isolate was the strongest algicidal activity against A. catenella. NH-12 isolate was identified on the basis of biochemical characteristics and analysis of 16S rRNA gene sequences. The isolate showed 97.67% homology with Pseudoalteromonas prydzensis ACAM (U85855), and was designated Pseudoalteromonas sp. NH-12. The optimal culture conditions of this isolate were , initial pH 8.0, and 3.0% (w/v) NaCl concentration. The algicidal activity of NH-12 was significantly increased to maximum value in the late of logarithmic phase of bacterial culture. As a result of 'cell culture insert' experiment, NH-12 is assumed to produce secondary metabolites, as an indirect attacker. When 10% culture filtrate of NH-12 was applied to A. catenella, over 99% of algal cells were destroyed within 24 h. In addition, the killing effects were increased in dose and time dependent manners. CONCLUSION(S): Taken together, our results suggest that Pseudoalteromonas sp. NH-12 could be a candidate for controlling of toxic algal blooms.

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris–EDTA (TE) buffer, (2) heating at 65°C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP.

Specific detection of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella from single vegetative cells by a loop-mediated isothermal amplification method

In this study, we succeeded in developing a loop-mediated isothermal amplification (LAMP) method that enables sensitive and specific detection of the toxic marine dinoflagellates Alexandrium tamarense and Alexandrium catenella from single cells of both laboratory cultures and naturally blooming cells within 25min, by monitoring the turbidimeter from the start of the LAMP reaction. The fluorescence intensity was strong enough to allow discrimination between positive and negative results by naked eye under a UV lamp, even in amplified samples from a single cell, by using the LAMP method. Unambiguous detection by naked eye was possible even in half the volume of LAMP cocktail recommended by the manufacturer, suggesting the potential to significantly reduce the cost of Alexandrium monitoring. Therefore, we can conclude that this method is one of the most convenient, sensitive, and cost-effective molecular tools for Alexandrium monitoring.

The growth, toxicity and genetic characterization of seven strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated during the 2009 summer outbreak in southern Chile

The dinoflagellate Alexandrium catenella causes recurrent harmful algal blooms in southern Chile. This species belongs to the “ Alexandrium tamarense/ catenella/ fundyense species complex” (the “ tamarensis complex”), defined by morphological attributes. Ribosomal sequences serve to differentiate five evolutionary lineages (clades) in this species complex. These distinctions reflect the geographic distribution and toxicity of the populations rather than their morphological designations. Despite the social and economic impact that harmful blooms produce in Chile, few strains of A. catenella have been isolated. Moreover, physiological and/or genetic studies of the group are scarce. The aim of this work was to examine possible physiological and genetic variability among populations of A. catenella having different geographical origins but isolated from the same toxic event. Seven strains of A. catenella were isolated and established from phytoplankton samples collected in the Aysén and Los Lagos regions of southern Chile during a recent outbreak (February–March 2009). Growth, toxicity, and ITS sequences were compared among these strains. All the strains included in this study were grouped with strains belonging to the previously described “North America” clade. The genetic diversity detected among Chilean strains was 3%, a much higher value than those reported for comparisons among strains from other parts of the world. In addition, a remarkable variability of growth parameters and toxicity was detected among strains. Strain PFB45 showed the highest PSP toxin content, whereas strain PFB41 had the lowest value of this parameter but had the highest maximum cell density. In strains PFB38, PFB42, and PFB37, more than 98% of the total PSP toxin content occurred in the form of gonyautoxins (primarily GTX-4,1 and GTX-3,2). In strains PFB39, PFB36, and PFB45, neoSTX, and STX toxins were detected. These results demonstrated remarkable variability at the genetic and physiological level among strains of A. catenella isolated from the same outbreak. No correlations were found between the phenotypic traits (growth and toxicity) and the genetic affiliation of the strains studied.

Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR.

The presence of neurotoxic species within the genus Alexandrium along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of Alexandrium catenella and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying A. catenella in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter⁻¹, 2 orders of magnitude below threshold abundances that can impact food webs. A. catenella was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter⁻¹). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter⁻¹, whereas the maximum STX concentration of 26 ng liter⁻¹ occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but A. catenella was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of A. catenella at subbloom and bloom abundances.

Short-term effects of different nitrogen substrates on growth and toxin production of dinoflagellate Alexandrium catenella Balech (strain ACDH)

Blooms of Alexandrium catenella were frequently found in sea areas adjacent to the estuary of the Changjiang River in spring of the last several years, often in association with large-scale blooms of Prorocentrum donghaiense. It was considered that the increasing intensity of these dinoflagellate blooms was related to the incremental nutrient influx from the Changjiang River over the last 40 years, especially the “excess” nitrogen (N) input. A nutrient-addition experiment was then designed to study the responses of a strain (ACDH) of A. catenella, isolated from the East China Sea, to the addition of different N substrates, including nitrate, ammonium and urea. It was found that N-starved cells of strain ACDH could use all the three N substrates as the sole N source to grow, with similar specific growth rate and maximum cell density. All the three N substrates could elevate cellular toxin content shortly after the addition of nutrients, but cellular toxin content of the urea group was much lower than the nitrate group and the ammonium group. In the following days, the cellular toxin content of the urea group was even lower than the N-limited control group, but cellular toxin contents of the nitrate group and ammonium group were still comparable to the control. It was presumed, based on these findings, that a relatively slower N uptake and assimilation rate occurred in the urea group, and urea–N adsorbed was preferentially utilized for growth rather than toxin synthesis. Therefore, urea in natural seawater could promote the formation of algal blooms, but decrease the toxicity, of strain ACDH.

Harmful phytoplankton ecology studies using an autonomous molecular analytical and ocean observing network

Using autonomous molecular analytical devices embedded within an ocean observatory, we studied harmful algal bloom (HAB) ecology in the dynamic coastal waters of Monterey Bay, California. During studies in 2007 and 2008, HAB species abundance and toxin concentrations were quantified periodically at two locations by Environmental Sample Processor (ESP) robotic biochemistry systems. Concurrently, environmental variability and processes were characterized by sensors co‐located with ESP network nodes, regional ocean moorings, autonomous underwater vehicle surveys, and satellite remote sensing. The two locations differed in their long‐term average physical and biological conditions and in their degree of exposure to episodic wind‐forced variability. While anomalously weak upwelling and strong stratification during the 2007 study favored toxigenic dinoflagellates (Alexandrium catenella), anomalously strong upwelling during the 2008 study favored toxigenic diatoms (Pseudo‐nitzschia spp.). During both studies, raphidophytes (Heterosigma akashiwo) were detected within a similar range of concentrations, and they reached higher abundances at the relatively sheltered, stratified site. During 2008, cellular domoic acid reached higher concentrations and was far more variable at the shallower ESP node, where phytoplankton populations were influenced by resuspended sediments. Episodic variability caused by wind forcing, lateral mixing, internal waves, and subsurface phytoplankton layers influenced ESP detection patterns. The results illustrate the importance of mobilizing HAB detection on autonomous platforms that can intelligently target sample acquisition as a function of environmental conditions and biological patch encounter.

Past trends and future scenarios for environmental conditions favoring the accumulation of paralytic shellfish toxins in Puget Sound shellfish

The risk of harmful algal blooms (HABs) of the dinoflagellate Alexandrium catenella in Puget Sound, Washington State, can be assessed by identifying and predicting climate and environmental conditions that are favorable for bloom development and the accumulation of paralytic shellfish toxins (PSTs) in shellfish. When these favorable conditions occur in combination, a harmful algal bloom window of opportunity (HAB-WOO) exists for A. catenella. The original HAB-WOO was identified by Moore et al. (2009) for the time period 1993–2007. In general, it showed that warm air and water temperatures, low streamflow, low winds, and small tidal variability precede PST events. Here, we use the HAB-WOO model to examine (i) changes in the annual HAB-WOO duration over the period from 1967 to 2006, and (ii) the potential effect of future climate change on HAB risk through the late 21st century. The annual HAB-WOO duration increased between 1978 and 2006, as did the frequency and geographic extent of PST events. Two step-like changes occurred in 1978 and 1992 with higher annual values attained by the HAB-WOO compared to previous years. The 1978 step change may be related to the 1977 reversal of the Pacific Decadal Oscillation from cool to warm phase. Climate change projections from global climate models and regionally downscaled climate models for the Pacific Northwest are used to evaluate scenarios for the future HAB-WOO. Under a moderate greenhouse gas emissions scenario (i.e., A1B), the annual HAB-WOO for A. catenella in Puget Sound is projected to increase by an average of 13 days by the end of the 21st century. Furthermore, the annual HAB-WOO may begin up to 2 months earlier in the year and persist for up to 1 month later in the year compared to the present day. This research provides managers, health authorities, and shellfish growers in Washington State with critical information for anticipating climate impacts on toxic HABs in the Pacific Northwest now and in a future warmer climate.

Dynamic of intoxication and detoxification in juveniles ofMytilus chilensis(Bivalvia: Mytilidae) exposed to paralytic shellfish toxins

Juveniles of the mussel Mytilus chilensis were exposed to a diet containing paralytic shellfish poisoning (PSP) toxins produced by the dinoflagellate Alexandrium catenella (strain ACC02). The feeding behaviour and the dynamics of intoxication and detoxification were evaluated over an intoxication period of nine days, followed by a detoxification period of eight days. A significant reduction in the feeding activity was measured during the first days of exposure to the PSP toxins (days 0 and 2), followed by a period of recovery observed on days 5 and 9, when the clearance rate of the contaminated mussels significantly increased. During the detoxification period, the contaminated bivalves showed a total recovery of clearance rate, and no significant differences were observed between contaminated and control groups. The intoxication dynamic was characterised by a rapid and sustained increase in the amount of PSP toxins accumulated in the tissues of the bivalves. Due to this increase, the level of PSP toxins critical for human consumption was reached on the first day, and at the end of the intoxication period, the mussels had accumulated 1601 μg STX eq. 100 g-1 tissue. During the detoxification period, a decrease of PSP toxins was observed, down to 319 μg STX eq. 100 g-1 tissue. The lower clearance rates observed over the first days of exposure would produce a decrease in the energy intake and could affect the rate of growth of juveniles. Despite this initial effect, the rapid intoxication capacity of M. chilensis corroborates that this species is a good indicator for the early detection of harmful algal blooms.

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry.

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions.

Germination characteristics of Alexandrium catenella cysts from surface sediments in Quartermaster Harbor, Puget Sound, Washington, USA

The dinoflagellate Alexandrium catenella causes frequent outbreaks of paralytic shellfish toxins (PSTs) in Puget Sound, Washington; however, little is known about its basic biology and ecology. Most of what is known is inferred mainly from shellfish toxin records and recent work on cyst distribution and germination potential. We report on a year-long study of cyst dormancy and germination potential based on experiments using surface sediment collected from a shallow embayment, Quartermaster Harbor, in Puget Sound. Cyst abundance in Quartermaster Harbor was 1550–1750 cysts cm −3 when sediment was collected in mid-October 2006. Germination experiments set up monthly had germination occur in all months when cysts were provided with adequate growth supporting conditions. Germination rates were highest in May and June when 100% germination occurred within 2 days. Longer incubation periods observed for the first two months of the study (November and December) may indicate a mandatory dormancy period of up to 5 months if the majority of cysts were deposited in the sediments following a late summer bloom that apparently occurred in Quartermaster Harbor in 2006 as indicated by Washington State Department of Health's toxin records. The data provide no evidence of an endogenous clock restricting germination to a specific annual timeframe for these shallow water cysts. This contrasts with the presence of a circannual endogenous clock, for deep water cysts of A. fundyense in the Gulf of Maine. The significance of these results is that germination of the A. catenella Puget Sound population appears to be primarily regulated by physiological requirements. However, once physiological requirements are met cysts can germinate rapidly, often within 24 h, when provided with adequate environmental conditions.

An integrative response by Mytilus chilensis to the toxic dinoflagellate Alexandrium catenella

Physiological responses of Mytilus chilensis exposed to the toxic dinoflagellate Alexandrium catenella were measured over 21 days in the laboratory and were compared with control mussels not exposed to the dinoflagellate. Mussels were collected from culturing ropes at Yaldad Bay, southern Chile (43o08′S 73o44′W), in August 2004 and acclimated to laboratory conditions for one week prior to the experiment. After 8 days, the paralytic shellfish poisoning (PSP) toxins (i.e. saxitoxin) in the tissues of exposed mussels exceeded safe levels for human consumption. Clearance rates, ingestion of organic matter, and absorption efficiency of exposed mussels were significantly lower than those of controls on day 0, but this was followed by an increase on day 3. The exposed mussels also increased their excretion rate over time, and this increase was significantly correlated with the accumulation of PSP toxins in their tissues. Oxygen consumption was not affected by the PSP toxins. The scope for growth (SFG) on day 0 was negative in exposed mussels, but it increased during the experiment. Although feeding activity and absorption efficiency were adversely affected during the first few days of exposure to PSP toxins from A. catenella in the laboratory, the M. chilensis cultured in Yaldad Bay may have evolved mechanisms that allow them to exploit the toxic dinoflagellate as a food source.

Abundance of the Toxic Dinoflagellate Alexandrium catenella in Jinhae Bay, Korea as Measured by Specific Real-time PCR Probe

The marine toxic dinoflagellate Alexandrium catenella has been implicated in numerous paralytic shellfish poisoning (PSP) events in many countries. Due to difficulties in rapidly identifying A. catenella, field-based study of this species has been problematic. The present study developed a TaqMan format A. catenella-specific probe for real-time PCR assay (specific to Korean genotype) based on LSU rDNA sequence information for studying geographic and temporal distribution of the species in surface sediments and water columns of Jinhae Bay, Korea. The field survey from 2007 to 2008 revealed that A. catenella occurred in most seasons at low densities, mostly below 1 cell <TEX>$mL^{-1}$</TEX>, and was more abundant in spring (maximum cell density of 2 cells <TEX>$mL^{-1}$</TEX>) when shellfish exceed the quarantine toxin level for PSP toxins in Jinhae Bay.

Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods

A number of species belonging to the genus Alexandrium are among the main toxic microalgae responsible for Harmful Algal Blooms (HABs). The monitoring of coastal waters for the presence of these microalgae is essential to identify correlations between cell abundances and environmental factors that regulate bloom dynamics. In the attempt to improve the monitoring sensitivity and the rapidity at which a large number of field samples can be processed, several molecular methods for the detection of genetically distinct HAB species have been developed during the last years. In particular, real-time PCR has been shown to be a powerful method for quantitative detection of HAB species in environmental samples. When a plasmid is used as a standard, the knowledge of the amount of target gene per cell is essential for the determination of the cell number in the field sample. In this study, we analyzed the rRNA gene content variability in several Alexandrium catenella and Alexandrium taylori strains isolated from the Mediterranean Sea using a real-time PCR-based approach. The rRNA gene content was also analyzed in different growth phases, from early exponential to stationary conditions. The results showed a general variability in the rRNA gene content depending on the strain and, for the species A. taylori, in relation also to the growth phase. These results should be taken into account for the application of the real-time quantitative PCR-based techniques for monitoring purposes in coastal seawaters.