Antibiotic treatment enhances C2 toxin production by Alexandrium tamarense in batch cultures

The effects of a mixture of penicillin G and streptomycin on the growth and C2 toxin production of a marine dinoflagellate, Alexandrium tamarense CI01, were investigated to determine if antibiotic treatment would increase the toxin yield of the cultured algae in batch cultures. Algal growth and toxin production were both enhanced markedly when the culture was supplemented with the antibiotics, each at an initial concentration of 100 unit ml⁻¹ in medium,² but were severely inhibited when the concentration was 500 unit ml⁻¹ or higher. Short-term pretreatment of algal inocula with the antibiotics at 100, 500, and 1000 unit ml⁻¹ all produced the enhancing effects on the algal cultures in an autoclaved medium. A prolonged antibiotic pretreatment of the algal culture followed by repeated sterile cultivation resulted in an algal culture free of cultivable bacteria. This “drug-treated” culture became more resistant to the toxicity and more responsive to the enhancing effects of the antibiotics. Our results indicated that the antibiotics can enhance growth and C2 toxin productivity not only through their inhibition of the growth of bacteria that compete for nutrients with the coexisting algae, but also through their direct effects on the physiology of the algae. Supplementation of the two antibiotics therefore is an efficient way to increase the yield of C2 toxin in the production cultures of A. tamarense CI01.     

Probable origin and toxin profile of Alexandrium tamarense (Lebour) Balech from southern Brazil

The distribution of the toxic dinoflagellate Alexandrium tamarense Lebour has apparently expanded within the southern hemisphere during the last 2 decades. Toxic blooms of A. tamarense were recorded in Argentinean coastal waters since 1980; however, the first documented bloom in southern Brazil was in 1996. In this study, 13 strains of A. tamarense from southern Brazil were isolated and kept in culture. Phylogenetic analysis using RFLP and DNA sequences of the D1–D2 region of large subunit ribosomal DNA (rDNA) clearly indicates that Brazilian strains are most closely related to other South American strains. The strains from South America are placed firmly within a phylogenetic clade which contains strains from North America, northern Europe and northern Asia, previously called the North American clade. Possible dispersal hypotheses are discussed. The cultures were also analyzed for saxitoxin and its derivatives by high performance liquid chromatography (HPLC). The main saxitoxin groups found were the low toxicity N-sulfocarbamoyl group, C1, 2 (30–84%), followed by the high potency carbamate toxins, gonyautoxins 1, 4 (6.6–55%), gonyautoxins 2, 3 (0.3–29%), neosaxitoxin (1.4–24%) and saxitoxin (0–4.4%). The toxin composition is similar to that of other strains from South America, supporting a close relationship between A. tamarense from southern Brazil and other areas of South America. Toxicity values were variable (7.07–65.92 pg STX cell⁻¹), with the higher range falling among the most toxic values recorded for cultures of A. tamarense, indicating the significant risk for shellfish contamination and human intoxication during blooms of this species along the southern Brazilian coast.     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Non-toxic and toxic subclones obtained from a toxic clonal culture of Alexandrium tamarense (Dinophyceae): Toxicity and molecular biological feature

Alexandrium tamarense (Lebour) Taylor strain OF935-AT6 is a rare strain of paralytic shellfish toxin (PST)-producing dinoflagellate, in which non-toxic and toxic cells are found in an approximately 1:1 ratio, isolated in Japan. The non-toxic characteristics of UAT-014-009, an axenic non-toxic subclone of OF935-AT6, have been confirmed at the attomole per cell level. Three out of nine toxic subclones of OF935-AT6 became non-toxic over a relatively short period of time (4–6 years), while the other toxic subclones retained their toxicity and the non-toxic subclones retained to be non-toxic. Two axenic subclones from OF935-AT6, UAT-014-009 (non-toxic) and Axat-2 (toxic) are indistinguishable from one another, and from popularly known A. tamarense by rDNA sequence analysis. The most significant difference identified by subtractive hybridization of cDNA pertains to gene fragments homologous with mitochondrial cytochrome c oxidase polypeptide three (cox3) and cytochrome b (cob). Thus, the polymorphism targeting these regions was investigated by comparison of the gene length amplified by PCR using total DNA from other subclones with a range of toxicities. No direct correlation between any allele and toxicity was observed in this study.     

The toxigenic marine dinoflagellate Alexandrium tamarense as the probable cause of mortality of caged salmon in Nova Scotia

The toxins associated with paralytic shellfish poisoning (PSP) are potent neurotoxins produced by natural populations of the marine dinoflagellate Alexandrium tamarense. In early June 2000, a massive bloom (>7×10⁵ cells l⁻¹) of this dinoflagellate coincided with an unusually high mortality of farmed salmon in sea cages in southeastern Nova Scotia. Conditions in the water column in the harbour were characterised by the establishment of a sharp pycnocline after salinity stratification due to abundant freshwater runoff. In situ fluorescence revealed a high sub-surface (2–4 m depth) chlorophyll peak related to the plankton bloom. The intense bloom was virtually monospecific and toxicity was clearly related to the concentration of Alexandrium cells in plankton size fractions. Cultured clonal isolates of A. tamarense from the aquaculture sites were very toxic on a per cell basis and yielded a diversity of PSP toxin profiles, some of which were similar to those from plankton concentrates from the natural bloom population. The toxin profile of plankton concentrates from the 21–56 μm size fraction was complex, dominated by the N-sulfocarbamoyl derivative C2, with levels of other PSP toxins GTX4, NEO, GTX5 (=B1), GTX3, GTX1, STX, C1, and GTX2, in decreasing order of relative abundance. Although no PSP toxin was found systemically in the fish tissues (liver, digestive tract) from this salmon kill event, the detection of Alexandrium cells and low levels of PSP toxins in salmon gills provide evidence that the enhanced mortalities were caused by direct exposure to toxic Alexandrium cells and/or to soluble toxins released during the bloom.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

不同pH和盐度下海洋细菌对赤潮藻生长和产毒的影响

研究了在可控生态条件下。一株分离自厦门西海域沉积物的海洋细菌S10在不同pH和盐度条件下对赤潮原因种塔玛亚历山大藻(Alexandrium tamarense)生长和产毒的影响．结果表明。实验用藻株适宜生长pH为6～8,适宜盐度为20～34;该藻株在不同pH及不同盐度条件下,藻细胞毒力差异显著,且随着pH升高而下降。随着盐度增加而加大,到盐度为30时达到最高值。然后逐渐下降;菌株Sl0(1．02×10^l0cells·ml^-1)在pH7～9和盐度15～34下均能有效抑藻生长和产毒。且在pH7、盐度34时其抑藻生长作用最强;在pH7时抑藻产毒效果较好,且其抑藻产毒作用强度不随盐度变化而异．     

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.     

Growth rates and elemental composition of Alexandrium monilatum, a red-tide dinoflagellate

The combined effects of temperature and salinity on growth of Alexandrium monilatum were studied in laboratory cultures. This toxic, red-tide dinoflagellate grew faster with higher temperatures, up to a maximum of approximately 1 division per day at 31 °C. Salinities above 15 psu had a lesser effect on growth rate, as might be expected for an estuarine species. Growth rates of cultures exposed to natural light and temperature fluctuations were comparable to laboratory cultures. The minimum N cell quota suggested that high N flux would be required to support bloom development. A literature survey of documented A. monilatum blooms indicated that within US waters, blooms occur in July–September in nearshore or estuarine regions of the Gulf of Mexico and the Florida Atlantic coast. Temperature and salinity measured during blooms correspond to the optimal growth conditions of the laboratory cultures. Nevertheless, the occurrence of A. monilatum blooms is sporadic compared to the occurrence of seemingly optimal growth conditions. Laboratory growth experiments predict when blooms of this species are unlikely due to low growth rates, but so far cannot predict individual blooms.     

Temperature as a factor regulating growth and toxin content in the dinoflagellate Alexandrium catenella

Controlled laboratory culture of Alexandrium catenella was used to determine the effects of a range of temperatures between 10 and 16 °C on the growth and saxitoxin content of this dinoflagellate, using strain ACC02 isolated from seawater at Aysen, XI Region, Southern Chile. Cell cultures were made using L1 culture medium at 30‰ salinity, and a photon flux density of 59.53 μmol m² s⁻¹. The results showed that the duration of the exponential growth phase was determined by the experimental temperature, with maximum cell concentrations obtained at 12 °C; significantly lower cell concentrations and growth rates were obtained at 16 °C. Cell dry weight and chlorophyll a values followed cell growth trends. The toxicity of A. catenella was variable at all the experimental temperatures, with a tendency towards having an inverse relation to temperature, with the highest values occurring at 10 °C and the lowest at 16 °C. The optimal range of temperature for the growth of the Chilean strain of A. catenella differed from rates reported for this species isolated at other latitudes, and was correlated with natural temperature conditions predominant in the environment from which it was isolated. The inverse relation of toxicity with temperature in the laboratory was broadly reflected in observations on the toxicity of this dinoflagellate in the field, and coincided with results from the literature.     

Effect of five chinese traditional medicines on the biological activity of a red-tide causing alga—Alexandrium tamarense

The activity of algal cells plays an important role in the development of a red tide. Thus, when mass sedimentation of algae occurs, the scale of the red tide is reduced to some degree. Using cell suspension ability as an indicator, we studied the inhibitory effect of five Chinese traditional medicines/Chinese herbs on the activity of the red tide causing alga, Alexandrium tamarense, strain DH01. The results showed that among the five herbs, golden thread (Rhizoma coptidis Huang Lian) and areca seed (Semen arecae Bing Lang) had the best inhibitory effect, followed by indigowoad (Isatis tinctoria L.), and the least effect was found in lightyellow sophora (Sophora flavescens Ait.) and chameleon plant (Houttuynia cordata Thunb.). The 1hLC₅₀ of golden thread and areca seed was 0.054% (w/v) and 0.074% (w/v), respectively. Moreover, the higher the concentration of the herbs, the higher the inhibitory rate (IR), and the shorter reaction time needed. Combination of two herbs improved, to some extent, the inhibitory effect on the algae. A mixture of golden thread and areca seed was the most effective treatment against cell mobility of this red tide alga, and the 1 h inhibitory rate was 70.7%. Next most effective was the combination of indigowoad root and lightyellow sophora. The best inhibition results in the experiments using absorbents were golden thread and indigowoad with aloe (Aloe vera L. var. chinensis (Haw.) Berg.); followed by areca seed with aloe, and golden thread and areca seed with kelp (Laminaria japonica). In general, the zeolite based algal inhibitors were less effective. The inhibitory rates of the algal inhibitors LA, LI, KG, KA and KG were all above 90%, and remained high for 72 h. Our research results showed that the red tide inhibitors made with Chinese herbs, such as golden thread and areca seed, had the merits of low concentration, fast reaction time, and demonstrated a promising future for use in the control of red tides.     

Isolation and characterization of a marine algicidal bacterium against the toxic dinoflagellate Alexandrium tamarense

Interactions between bacteria and harmful algal bloom (HAB) species have been acknowledged as an important factor regulating both the population dynamics and toxin production of these algae. A marine bacterium SP48 with algicidal activity to the toxic dinoflagellate, Alexandrium tamarense, was isolated from the Donghai Sea area, China. Genetic identification was achieved by polymerase chain reaction amplification and sequence analysis of 16S rDNA. Sequence analysis showed that the most probable affiliation of SP48 was to the γ-proteobacteria subclass and the genus Pseudoalteromonas. Bacterial isolate SP48 showed algicidal activity through an indirect attack. Additional organic nutrients but not algal-derived DOM was necessary for the synthesis of unidentified algicidal compounds but β-glucosidase was not responsible for the algicidal activity. The algicidal compounds produced by bacterium SP48 were heat tolerant, unstable in acidic condition and could be easily synthesized regardless of variation in temperature, salinity or initial pH for bacterial growth. This is the first report of a bacterium algicidal to the toxic dinoflagellate A. tamarense and the findings increase our knowledge of bacterial–algal interactions and the role of bacteria during the population dynamics of HABs.     

An autecological study of the potentially toxic dinoflagellate Alexandrium affine isolated from Vietnamese waters

The potentially toxic dinoflagellate species Alexandrium affine isolated from Ha Long Bay (Tonkin Gulf), Vietnam was cultured and maintained for morphological, physiological and toxicological studies. Classical morphological examinations including plate pattern were in good agreement with the international nomenclature of the species. The fine structure of A. affine, including morphology of its developmental stages during vegetative and sexual reproduction was found to be typical of other species in the genus. Two general trends in growth of A. Affine from Vietnamese waters were apparent: (1) growth rates were low at low salinities (10 and 15 psu) in all experimental temperatures (21–27 °C); (2) growth rates were high at salinities 25, 30, and 35 psu in all temperatures. There were no significant differences in growth rates at different salinities at low temperature (21 °C), and the most significant difference in growth rate was between high temperature–high salinity and high temperature–low salinity. The optimum temperature and salinity for growth were 24 °C and 30 psu. Maximum division rates per day (0.5–0.7) were at salinities 30 and 35 psu and at temperatures 24 and 27 °C. But the best conditions for division rate were 21 and 24 °C at salinities 30 and 35 psu. Toxicity analyses indicated A. affine to be both toxic and non-toxic at certain times. In the former case, toxicity was very low, 2.28 fmol  per cell; the toxicity component of A. affine was compared with that of A. leei and the mussel Perna viridis including neoSTX, STX, and GTX₁–GTX₄.     

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels (Mytilus galloprovincialis) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense, and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense, mussels, and seawater were the N-sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense, mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.     

Effects of winds, tides and river water runoff on the formation and disappearance of the Alexandrium tamarense bloom in Hiroshima Bay, Japan

Effects of winds, tides and river water runoff on the formation and disappearance of Alexandrium tamarense blooms in Hiroshima Bay, Japan were investigated using data from March to June of 1992–1998. The north wind at the initial growth phase of A. tamarense appeared to have prevented bloom formation by dispersing the organism offshore and/or through turbulent mixing. The decrease in the cell density at the end of the blooms was significantly affected by tidal mixing, indicating that the turbulent mixing induced by tidal excursions may be one of the factors terminating the bloom. Box model analyses applied to the data collected from the observations in 1996 and 1997 showed that river water runoff apparently dispersed the bloom, implying that stratification of the water column due to river water runoff is not necessary for the bloom formation. In conclusion, calm conditions with less wind and tidal mixing along with less river water runoff are considered to be important for the formation of the A. tamarense bloom in Hiroshima Bay, Japan.     

Variety of PSP toxin profiles in various culture strains of Alexandrium tamarense and change of toxin profile in natural A. tamarense population

Paralytic shellfish poisoning (PSP) toxin profiles were compared between clonal and axenic culture strains of Alexandrium tamarense prepared from cysts. The cysts were collected from two stations in northern Japan. The major toxin components of A. tamarense were C2 and GTX4, however, the proportions of C2 and GTX4 varied largely 0.7-78.8 mol% and 79.4-8.5 mol%, respectively. Some culture strains contained significantly higher proportion of neoSTX than other strains. These results indicate that strains with various toxin profiles exist in the same region, and suggest that the comparison of the toxin profiles between strains at different localities is considerably difficult. A drastic change of the toxin profile was observed in natural planktonic populations containing A. tamarense. This may be explained by the presence of a lot of planktonic populations with various toxin profiles growing around the sea area.     

Intra-population clonal variability in allelochemical potency of the toxigenic dinoflagellate Alexandrium tamarense

Clonal variability in exponential growth rate and production of secondary metabolites was determined from clonal isolates of Alexandrium tamarense originating from a single geographical population from the east coast of Scotland. To assess variability in the selected phenotypic characteristics over a wide spectrum, 10 clones were chosen for experimentation from 67 clonal isolates pre-screened for their lytic capacity in a standardized bioassay with the cryptophyte Rhodomonas salina. Specific growth rates (μ) of the 10 clonal isolates ranged from 0.28 to 0.46 d⁻¹ and were significantly different among clones. Cell content (fmol cell⁻¹) and composition (mol%) of paralytic shellfish toxins (PSTs), analyzed by liquid chromatography with fluorescence detection (LC–FD), varied widely among these isolates, with total PST quotas ranging from 20 to 89 fmol cell⁻¹. Except for strain 3, the toxins C1/C2, neosaxitoxin (NEO), saxitoxin (STX), and gonyautoxins-1 and -4 (GTX1/GTX4), were consistently the most relatively abundant, with lesser amounts of GTX2/GTX3 evident among all isolates. Only clone 3 contained >20 mol% of toxin B1, with C1/C2, GTX2/GTX3 and NEO in almost equimolar ratios.Eight of the 10 clones caused cell lysis of both R. salina and the heterotrophic dinoflagellate Oxyrrhis marina, as quantified from the dose–response curves from short-term (24 h) co-incubation bioassays. For two clones, no significant mortality even at high Alexandrium cell concentrations (ca. 10⁴ mL⁻¹) was observed. Allelochemical activity expressed as EC₅₀ values, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, varied by about an order of magnitude and was significantly different among clones. No correlation was observed between growth rate und allelochemical potency (as EC₅₀) indicating that at least under non-limiting growth conditions no obvious growth reducing costs are associated with the production of allelochemically active secondary metabolites.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.