Diversity and dynamics of a widespread bloom of the toxic dinoflagellate Alexandrium fundyense

Historically, cosmopolitan phytoplankton species were presumed to represent largely unstructured populations. However, the recent development of molecular tools to examine genetic diversity have revealed differences in phytoplankton taxa across geographic scales and provided insight into the physiology and ecology of blooms. Here we describe the genetic analysis of an extensive bloom of the toxic dinoflagellate Alexandrium fundyense that occurred in the Gulf of Maine in 2005. This bloom was notable for its intensity and duration, covering hundreds of kilometers and persisting for almost two months. Genotypic analyses based on microsatellite marker data indicate that the open waters of the northeastern U.S. harbor a single regional population of A. fundyense comprising two genetically distinct sub-populations. These subpopulations were characteristic of early- and late-bloom samples and were derived from the northern and southern areas of the bloom, respectively. The temporal changes observed during this study provide clear evidence of succession during a continuous bloom and show that selection can act on the timescale of weeks to significantly alter the representation of genotypes within a population. The effects of selection on population composition and turnover would be magnified if sexual reproduction were likewise influenced by environmental conditions. We hypothesize that the combined effects of differential growth and reproduction rates serves to reduce gene flow between the sub-populations, reinforcing population structure while maintaining the diversity of the overall regional population.     

Effects of the toxic dinoflagellate, Alexandrium fundyense on three species of larval fish: a food-chain approach

Sublethal behavioural effects of exposure to paralytic shellfish toxins (PST; saxitoxin and its derivatives) from the toxic dinoflagellate Alexandrium fundyense were investigated in newly settled winter flounder Pseudopleuronectes americanus , larval sheepshead minnow Cyprinodon variegatus and larval mummichog Fundulus heteroclitus through an A. fundyense –copepod–fish food chain. Consumption of as few as six to 12 toxin-containing copepods was lethal to the fishes. After consuming fewer toxin-containing copepods, all three fish species exhibited sublethal effects from vector-mediated exposure. Prey-capture ability of mummichogs was reduced in larvae that had consumed toxic copepods, Coullana canadensis . After consuming toxic C. canadensis or mixed copepods, mummichog larvae had reduced swimming performance. Swimming activity was also significantly reduced in winter flounder after consuming toxic copepods, including time spent in motion and distance travelled. Prey capture and predator avoidance were reduced in first-feeding sheepshead minnow larvae that had consumed toxic dinoflagellate cells. Adverse effects on prey capture or predator avoidance may reduce larval survival and facilitate the transmission of PST through the food web. This study provides baseline information on sublethal effects of PST exposure on fishes using a novel food-chain approach with zooplankton as vectors.     

Differences in swimming pattern between life cycle stages of the toxic dinoflagellate Alexandrium fundyense

Different life stages of Alexandrium fundyense have different swimming behavior; gametes often are said to “swarm” or “dance” before mating. This behavior was studied, and quantitative measures of these motility patterns in two-dimensions were generated using motion-analysis software applied to video records of individual-cell movements. Behavior, swimming patterns, and growth were studied in two strains of A. fundyense and compared in encystment medium and growth medium. Vegetative cells swam straight, rotating around the apical axis until they hit something and then swam straight in a different direction. Gamete swimming behavior was slower and characterized by frequent direction changes and circular motion. Gametes contacted other cells frequently (>5 cell contacts min⁻¹ cell⁻¹). Zygotes swam slowly when newly formed and later became nearly immobile; these cells continued to contact other cells and also surfaces. The results are in accordance with field observations of long swimming distances for vegetative cells, accumulation in thin layers of gametes, and sinking of developing resting cysts attached to marine snow for zygotes.     

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella

The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.     

Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor

The steady rise of observations of harmful or toxic algal blooms throughout the world in the past decades constitute a menace for coastal ecosystems and human interests. As a consequence, a number of programs have been launched to monitor the occurrence of harmful and toxic algae. However, the identification is currently done by microscopic examination, which requires a broad taxonomic knowledge, expensive equipment and is very time consuming. In order to facilitate the identification of toxic algae, an inexpensive and easy-to-handle DNA-biosensor has been adapted for the electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii. The detection of the toxic algae is based on a sandwich hybridisation, which is carried out on a disposable sensor chip. A set of two probes for the species-specific identification of A. ostenfeldii was developed. The specificity of the probes could be shown in dot-blot hybridisations and with the DNA-biosensor. The sensitivity of the DNA-biosensor was optimised with respect to hybridisation temperature and NaCl-concentration and a significant increase of the sensitivity of the DNA-biosensor could be obtained by a fragmentation of the rRNA prior to the hybridisation and by adding a helper oligonucleotide, which binds in close proximity to the probes to the hybridisation.     

Microbial community diversity in the phycosphere of natural populations of the toxic alga, Alexandrium fundyense

The dinoflagellate Alexandrium fundyense is the major causative organism of paralytic shellfish poisoning in the Gulf of Maine. While laboratory studies have shown that A. fundyense population dynamics can be affected dramatically by co-occurring bacteria, little is known about these interactions in nature. Because A. fundyense is typically a minor Gulf of Maine phytoplankton community member, analyses of the bulk community cannot be used to address bacterium-A. fundyense associations. Therefore, an immunomagnetic bead method was used to selectively capture A. fundyense cells, and the bacteria attached to them, from complex natural samples. Bulk particle-associated and free-living bacterial communities were collected simultaneously. DNA was extracted from all sample types and subjected to 16S rRNA gene fragment amplification, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. Ordination analysis of DGGE profiles confirmed that A. fundyense-associated bacteria community profiles were distinct from bulk bacterial community profiles, indicating selection of specific phylotypes in the A. fundyense phycosphere. Phylogenetic analyses confirmed that Alexandrium-associates were distinct from bulk particle-associated bacteria and that they included a greater prevalence and broader diversity of Gammaproteobacteria than previously thought to be associated with toxic algae. Phylogenetic groups known to be associated with dinoflagellates were also found, including members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae.     

No evidence for induction or selection of mutant sodium channel expression in the copepod Acartia husdsonica challenged with the toxic dinoflagellate Alexandrium fundyense

Some species in the dinoflagellate genus Alexandrium spp. produce a suite of neurotoxins that block sodium channels, known as paralytic shellfish toxins (PST), which have deleterious effects on grazers. Populations of the ubiquitous copepod grazer Acartia hudsonica that have co‐occurred with toxic Alexandrium spp. are better adapted than naïve populations. The mechanism of adaptation is currently unknown. We hypothesized that a mutation in the sodium channel could account for the grazer adaptation. We tested two hypotheses: (1) Expression of the mutant sodium channel could be induced by exposure to toxic Alexandrium fundyense; (2) in the absence of induction, selection exerted by toxic A. fundyense would favor copepods that predominantly express the mutant isoform. In the copepod A. hudsonica, both isoforms are expressed in all individuals in varying proportions. Thus, in addition to comparing expression ratios of wild‐type to mutant isoforms for individual copepods, we also partitioned copepods into three groups: those that predominantly express the mutant (PMI) isoform, the wild‐type (PWI) isoform, or both isoforms approximately equally (EI). There were no differences in isoform expression between individuals that were fed toxic and nontoxic food after three and 6 days; induction of mutant isoform expression did not occur. Furthermore, the hypothesis that mutant isoform expression responds to toxic food was also rejected. That is, no consistent evidence showed that the wild‐type to mutant isoform ratios decreased, or that the relative proportion of PMI individuals increased, due to the consumption of toxic food over four generations. However, in the selected line that was continuously exposed to toxic food sources, egg production rate increased, which suggested that adaptation occurred but was unrelated to sodium channel isoform expression.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.     

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.     

Modelling the population dynamics of the toxic dinoflagellate Alexandrium Tamarense in Hiroshima Bay, Japan

In the present study, a numerical model that simulates speciescompetition between the toxic dinoflagellate Alexandrium tamarenseand the non-toxic diatom Skeletonema costatum was constructedusing data from a number of experiments and field observations.In the model, not only vertical migration of swimming cellsbut also the encystment and excystment processes of A. tamarensewere taken into consideration. Sinking of S. costatum cellswas also considered. Both zooplankton and oysters, which areintensively cultured in this bay, feed on the phytoplanktonbut the contribution of grazing to the decrease in the celldensities of both species was small. The model predicts thatwhile sinking is one of the major processes that reduce thecell density of S. costatum, encystment of A. tamarense is acrucial process that determines the cessation of blooms of thisdinoflagellate. Advection and diffusion are also important physicalprocesses that affect, in positive and negative ways, the fluctuationof cell densities.     

Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.