Is photosynthesis a requirement for paralytic shellfish toxin production in the dinoflagellate <Emphasis Type="Italic">Alexandrium minutum</Emphasis> algal–bacterial consortium?

While there is increasing evidence that marine bacteria are involved in the production of paralytic shellfish toxins in algal blooms, the exact roles of the bacteria and microalgae have proved elusive. A novel experimental approach to this problem involved incubating parallel cultures of toxin producing Alexandrium minutum Anokoha A in the dark and in a natural daylight cycle. High-performance liquid chromatography was used to measure paralytic shellfish toxins while bacterial growth was monitored by culture on high- and low-nutrient agar media. After a 22-day incubation period in the dark, A. minutum Anokoha A failed to produce saxitoxin while parallel light-grown cultures produced 1.17 μg per 10,000 algal cells. However, both dark- and light-grown cultures showed comparable gonyautoxin production. Copiotrophic and oligotrophic algal-associated bacteria showed similar growth patterns over the incubation period except that a dip in bacterial numbers corresponded to a peak in algal numbers in the light-grown cultures. It appears that inhibition of photosynthesis resulted in changes in the toxin profile of A. minutum Anokoha A. When used with other methods, this approach may help to elucidate the algal–bacterial-toxin connection.     

Detection and quantification of cultured marine Alexandrium species by real-time PCR

The occurrence of harmful algal blooms (HABs) throughout the world has increased and poses a large threat to human health, fishery resources and tourism industries. The genus Alexandrium includes a number of toxic species associated with HABs. Therefore, it is very important to rapidly detect and monitor the harmful algae, such as Alexandrium genus. In this study, a standard curve of plasmid containing 18S rDNA-28S rDNA region from Alexandrium catenella was constructed and 5.8S rDNA sequence served as the primer of the real-time PCR. Cultured A. catenella, Alexandrium affine, Alexandrium lusitanicum and Alexandrium minutum samples were analyzed by real-time PCR using the same set of primers simultaneously. Using microscopy cells counts, 5.8S rDNA copies per cell and total DNA per cell were estimated. This assay method is promising for rapid detection of large number of Alexandrium samples.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

Characterization of Nontoxic and Toxin-Producing Strains of Alexandrium minutum (Dinophyceae) in Irish Coastal Waters

A comparative analysis of the morphology, toxin composition, and ribosomal DNA (rDNA) sequences was performed on a suite of clonal cultures of the potentially toxic dinoflagellate Alexandrium minutum Halim. These were established from resting cysts or vegetative cells isolated from sediment and water samples taken from the south and west coasts of Ireland. Results revealed that strains were indistinguishable, both morphologically and through the sequencing of the D1-D2 domain of the large subunit and the ITS1-5.8S-ITS2 regions of the rDNA. High-performance liquid chromatography fluorescence detection analysis, however, showed that only strains derived from retentive inlets on the southern Irish coast synthesized paralytic shellfish poisoning (PSP) toxins (GTX2 and GTX3), whereas all strains of A. minutum isolated from the west coast were nontoxic. Toxin analysis of net hauls, taken when A. minutum vegetative cells were in the water column, revealed no PSP toxins in samples from Killary Harbor (western coast), whereas GTX2 and GTX3 were detected in samples from Cork Harbor (southern coast). These results confirm the identity of A. minutum as the most probable causative organism for historical occurrences of contamination of shellfish with PSP toxins in Cork Harbor. Finally, random amplification of polymorphic DNA was carried out to determine the degree of polymorphism among strains. The analysis showed that all toxic strains from Cork Harbor clustered together and that a separate cluster grouped all nontoxic strains from the western coast.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

Description of the new phototrophic dinoflagellate Alexandrium pohangense sp. nov. from Korean coastal waters

The planktonic phototrophic dinoflagellate Alexandrium pohangense sp. nov. isolated from the coastal waters off Korea is described from living and fixed cells by light and scanning electron microscopy (SEM). DNA sequence data were collected from the small subunit (SSU), the large subunit (LSU), internal transcribed spacer regions (ITS1 and ITS2), and 5.8S of the ribosomal DNA (rDNA). The SSU and LSU rDNA sequences of the new dinoflagellate were 4–7% and 14–17%, respectively, different from those of Alexandrium minutum, Alexandrium ostenfeldii, Alexandrium tamutum, Alexandrium margalefii, and Alexandrium pseudogonyaulax, the most closely related species. In addition, the 5.8S rDNA sequence of the new dinoflagellate was also 12% different from those of A. minutum, A. ostenfeldii, A. tamutum, and Alexandrium peruvianum. In a phylogenetic tree based on LSU rDNA sequences, A. pohangense formed a clade with A. margalefii, and this clade was clearly distinct from the clade of A. minutum, Alexandrium diversaporum, A. tamutum, Alexandrium leei, A. ostenfeldii, and Alexandirum andersoni. Moreover, in a phylogenetic tree based on SSU rDNA sequences, A. pohangense was positioned at the base of the clade containing A. leei and A. diversaporum. Morphological analysis showed that A. pohangense has a Kofoidian plate formula of Po, 4′, 6′′, 6c, 8s, 5′′′, and 2′′′′, which confirmed its assignment to the genus Alexandrium. This dinoflagellate has a wide rectangular 1′ plate, the upper left side of which is slightly bent, protruding, and touching the 2′ plate, unlike A. margalefii, which has a wide rectangular 1′ plate that does not touch the 2′ plate, or A. pseudogonyaulax and Alexandrium camurascutulum, which have a narrower elongated pentagonal 1′ plate that touches the 2′ plate. Furthermore, the 1′ plate of A. pohangense meets the 1′′ plate as a straight vertical line, whereas that of A. camurascutulum meets the 1′′ plate as an inclined line because it is lifted by the intrusion of the 1′′ plate. In addition, A. pohangense had a relatively small ventral pore whose majority was located on the 4′ plate, unlike A. margalefii or A. pseudogonyaulax, which have a relatively large ventral pore whose majority is located on the 1′ plate. Furthermore, A. pohangense had pores of two different sizes on the cell surface, unlike A. margalefii and A. pseudogonyaulax, which have similar pores of only one size. On the basis of morphological and phylogenetic criteria, it is proposed that this is a new species of the genus Alexandrium.     

ALEXANDRIUM TAMUTUM SP. NOV. (DINOPHYCEAE): A NEW NONTOXIC SPECIES IN THE GENUS ALEXANDRIUM1

A new species of the dinoflagellate genus Alexandrium, A. tamutum sp. nov., is described based on the results of morphological and phylogenetic studies carried out on strains isolated from two sites in the Mediterranean Sea: the Gulf of Trieste (northern Adriatic Sea) and the Gulf of Naples (central Tyrrhenian Sea). Vegetative cells were examined in LM and SEM, and resting cysts were obtained by crossing strains of opposite mating type. Alexandrium tamutum is a small‐sized species, resembling A. minutum in its small size, the rounded‐elliptical shape and the morphology of its cyst. The main diagnostic character of the new species is a relatively wide and large sixth precingular plate (6″), whereas that of A. minutum is much narrower and smaller. Contrary to A. minutum, A. tamutum strains did not produce paralytic shellfish poisoning toxins. Phylogenies inferred from the nuclear small subunit rDNA and the D1/D2 domains of the large subunit nuclear rDNA of five strains of A. tamutum and numerous strains of other Alexandrium species showed that A. tamutum strains clustered in a well‐supported clade, distinct from A. minutum.     

TOXIN PROFILE,PIGMENT COMPOSITION,AND LARGE SUBUNIT RDNA PHYLOGENETIC ANALYSIS OF AN ALEXANDRIUM MINUTUM (DINOPHYCEAE) STRAIN ISOLATED FROM THE FLEET LAGOON,UNITED KINGDOM1

Paralytic shellfish toxins, pigment composition, and large subunit (LSU) rDNA sequence were analyzed for a clonal culture of Alexandrium minutum Halim isolated in 2000 from the coastal Fleet Lagoon, Dorset, United Kingdom. The HPLC pigment analysis revealed the presence of chl a, peridinin, and diadinoxanthin as major pigments and chl c₁+c₂ and c₃, diatoxanthin, and β‐carotene as minor components. The toxins responsible for paralytic shellfish poisoning were analyzed by HPLC with postcolumn derivatization and fluorescence detection. The paralytic shellfish poisoning toxin profile of the Fleet Lagoon strain of A. minutum in exponential growth phase was dominated by gonyautoxin‐3 up to 54%, whereas gonyautoxin‐2 made up 10% and saxitoxin (STX) 36%. The average toxicity of the culture was 3.8 pg STX Eq·cell^?1, and total toxin content varied from 5.6 fmol·cell^?1 on day 1 to a maximum of 16.8 fmol·cell^?1 during the early stationary phase. Sequence analysis of the LSU rDNA revealed the strain to be closely related to several European strains of A. minutum and one isolated from Australian waters, although most of these do not produce STX. The shallow Fleet Lagoon may provide a favorable environment for A. minutum to bloom, and the presence of highly potent saxitoxins in this strain indicates potential for future shellfish contamination.     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

Induction of toxin production in dinoflagellates: the grazer makes a difference

The dinoflagellate Alexandrium minutum has previously been shown to produce paralytic shellfish toxins (PST) in response to waterborne cues from the copepod Acartia tonsa. In order to investigate if grazer-induced toxin production is a general or grazer-specific response of A. minutum to calanoid copepods, we exposed two strains of A. minutum to waterborne cues from three other species of calanoid copepods, Acartia clausi, Centropages typicus and Pseudocalanus sp. Both A. minutum strains responded to waterborne cues from Centropages and Acartia with significantly increased cell-specific toxicity. Waterborne cues from Centropages caused the strongest response in the A. minutum cells, with 5 to >20 times higher toxin concentrations compared to controls. In contrast, neither of the A. minutum strains responded with significantly increased toxicity to waterborne cues from Pseudocalanus. The absolute increase in PST content was proportional to the intrinsic toxicity of the different A. minutum strains that were used. The results show that grazer-induced PST production is a grazer-specific response in A. minutum, and its potential ecological importance will thus depend on the composition of the zooplankton community, as well as the intrinsic toxin-producing properties of the A. minutum population.     

A review of the global distribution of Alexandrium minutum (Dinophyceae) and comments on ecology and associated paralytic shellfish toxin profiles,with a focus on Northern Europe

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that are known to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human and ecosystem health. This review highlights that A. minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A. minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A. minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have a range of toxin profile clusters and this area appears to have the most diverse set of PST producing A. minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A. minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due to oceanic circulation and a high level of shipping and recreational boating. Further research into the interrelationships between the A. minutum populations in this global region would be of value.     

The Inhibitory Effects of Garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) and Diallyl Trisulfide on <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> and other Harmful Algal Species

Using cell suspension ability as an indicator, we studied the inhibitory effect of garlic (Allium sativum) and diallyl trisulfide on six species of red tide causing algae. This included: the inhibition by 0.08% garlic solution of five algal species — Alexandrium tamarense, Scrippsiella trochoidea, Alexandrium catenella, Alexandrium minutum and Alexandrium satoanum; the effects of garlic concentration on the inhibition of A. tamarense, S. trochoidea and Chaetoceros sp.; the effects of inhibitory time on the rejuvenation of algal cells; and the effects of heating and preservation time on algal inhibition by garlic solution. In addition, whether or not the ingredients of garlic solution had a possible algicidal effect was studied by comparing inhibition of A. tamarense by garlic solution and man-made diallyl trisulfide. The results showed that 1) inhibition by garlic solution was significant on A. tamarense, A. satoanum, A. catenella and S. trochoidea, and the least effective was a concentration of 0.04% on A. tamarense and S. trochoidea. Moreover, the higher the concentration, the stronger was the inhibition, and a high inhibitory rate (IR) could be maintained for at least three days when the garlic concentration was above 0.04%. For A. tamarense, it was also found that the longer the inhibitory time and the higher the concentration, the lower was the rate of resumed cell activity. On the contrary, garlic solution could not inhibit A. minutum or Chaetoceros sp.; 2) The IR to A. tamarense was reduced slightly as the heating time of the garlic solution was lengthened, but the average IR was still above 80%. There was no significant difference between the IR of the supernatant and sediment of the garlic solution. Furthermore, no change of algal inhibition was found when the garlic solution was preserved at 20°C for several days; 3) As with garlic solution, diallyl trisulfide inhibited A. tamarense strongly; the IR was above 93% and was maintained for at least three days, as long as the concentration was 3.2–10.0 mg L⁻¹. Thus, diallyl trisulfide may have been the major ingredient in garlic solution which inhibited the algae but, in addition, more than one ingredient may have been inhibiting the algae. In conclusion, garlic was a good algal inhibitor with many advantages, such as being common, cheap, non toxic and with high efficiency, and diallyl trisulfide, one of the components of garlic, was similarly effective in algal inhibition. It would be useful, therefore, to further study garlic as an environmentally friendly algal inhibitor.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

Toxic mucus traps: A novel mechanism that mediates prey uptake in the mixotrophic dinoflagellate Alexandrium pseudogonyaulax

The functional role of harmful substances (i.e. toxins) produced by marine planktonic algae is still, in many cases, unknown. This study describes a novel mechanism by which the phototrophic dinoflagellate Alexandrium pseudogonyaulax secretes a toxic mucus trap where prey items are caught and immobilized prior to ingestion. Prey cells remain entrapped and immobile in the mucus trap, but most stay intact, readily available as whole-cell prey. It is shown that food uptake by A. pseudogonyaulax increases its growth rate considerably even in nutrient-replete, high-light conditions. The increase in growth rate was more enhanced in light-limited treatments and A. pseudogonyaulax grew significantly faster when fed Heterocapsa rotundata, than when fed Teleaulax acuta under both light conditions. For comparison, strains of Alexandrium catenella and Alexandrium minutum were studied for their mixotrophic capabilities. None of these strains were mixotrophic under the conditions provided. In addition, the toxic effects on various protistan targets of these Alexandrium strains as well as Alexandrium tamarense and Alexandrium ostenfeldii were compared to that of A. pseudogonyaulax. A. tamarense and A. catenella did immobilize and lyse target cells through substances leaked directly into the water, differing from all the strains of A. pseudogonyaulax studied. Results show that the toxic effect of A. pseudogonyaulax is non-specific causing nearly 100% immobilization of a variety of protistan targets at relatively low cell concentrations (500 cells ml⁻¹ of donor cell). A critical donor cell density was not required as only one A. pseudogonyaulax cell was able to cause immobilization of target cells. For the first time, the connection between excreted toxins and phagotrophy is evident in an Alexandrium species and this particular strategy has the potential to severely impact competing phytoplankton communities.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.     

Growth- and nutrient-dependent gene expression in the toxigenic marine dinoflagellate Alexandrium minutum

The toxigenic marine dinoflagellate Alexandrium minutum forms toxic blooms causing paralytic shellfish poisoning (PSP), primarily in coastal waters, throughout the world. We examined effects on physiology and gene expression patterns associated with growth and nutrient starvation in a toxic strain of A. minutum. Bloom-relevant factors, including growth rate, intracellular toxin content, allelochemical activity and nutrient status were investigated in A. minutum cultures grown under different environmental regimes. Allelochemical activity of A. minutum cultures, quantified with a cryptomonad Rhodomonas bioassay, increased with age but was independent of nutrient status.The phenotypic data were integrated and compared with gene expression in cell samples taken at selected points along the growth curve. We observed 489 genes consistently differentially expressed between exponentially growing and growth-limited cultures. The expression pattern of stationary-phase cultures was characterized by conspicuous down-regulation of translation-associated genes, up-regulation of sequences involved in intracellular signalling and some indications of increased activity of selfish genetic elements such as transposons. Treatment-specific patterns included five genes regulated in parallel in all nutrient-limited cultures. The conspicuous decrease in photosynthetic performance identified in N-starved cultures was paralleled by down-regulation of chloroplast-associated genes.The particular gene expression patterns we identified as specifically linked with exponential growth, cessation of growth or nutrient limitation may be suitable biomarkers for indicating the beginning of growth limitation in field- or mesocosm studies.     

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.