Effects of salinity, light and inorganic nitrogen on growth and toxigenicity of the marine dinoflagellate Alexandrium tamarense from northeastern Canada

Growth and toxin production of a highly toxic clone of the marine dinoflagellate Alexandrium tamarense, isolated from the lower St Lawrence estuary (Quebec) in eastern Canada, were studied in unialgal batch cultures under different conditions. Controlled experiments were conducted on the production of paralytic shellfish poisoning (PSP) toxins under conditions of varying light (40, 60, 150, 230 and 470 mol m^-21 s^-1), salinity (10, 15, 20, 25 and 30) and nitrate concentrations (0, 88, 364, 528 and 880 mol l^-1). The effects of variable environmental factors on both toxin composition (% molar) and cell toxicity [pg STXeq (saxitoxin equivalents) cell^-1] were determined through the culture cycle. The toxin profile (% molar; mean SD), determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), remained stable and was consistently dominated by the low-potency N-sulfocarbamoyl toxins C1/C2 (64.0 ± 3%). There were also substantial relative amounts of the high-toxicity carbamate derivatives gonyautoxin 1-4 (GTX1-4) (1.7 ± 0.5%), neosaxitoxin (NEO) (16.2 ± 2%) ans saxitoxin (STX) (17.8 ± 2%). The cellular toxicity (mean ± SD: 58.8 ± 7 pg STXeq cell^-1) was essentially independent of light, salinity and nitrate concentration throughout the exponential growth phase, but varied over the growth stages in cultures. A positive correlation was observed between cellular toxicity and salinity-dependent growth rate, indicating that cell toxin quota may be affected by extrinsic factors, but it is not always a direct functional response to specific environmental stress.      

Sterols of the syndinian dinoflagellate Amoebophrya sp., a parasite of the dinoflagellate Alexandrium tamarense (Dinophyceae)

Several harmful photosynthetic dinoflagellates have been examined over past decades for unique chemical biomarker sterols. Little emphasis has been placed on important heterotrophic genera, such as Amoebophrya, an obligate, intracellular parasite of other, often harmful, dinoflagellates with the ability to control host populations naturally. Therefore, the sterol composition of Amoebophrya was examined throughout the course of an infective cycle within its host dinoflagellate, Alexandrium tamarense, with the primary intent of identifying potential sterol biomarkers. Amoebophrya possessed two primary C(27) sterols, cholesterol and cholesta-5,22Z-dien-3beta-ol (cis-22-dehydrocholesterol), which are not unique to this genus, but were found in high relative percentages that are uncommon to other genera of dinoflagellates. Because the host also possesses cholesterol as one of its major sterols, carbon-stable isotope ratio characterization of cholesterol was performed in order to determine whether it was produced by Amoebophrya or derived intact from the host. Results indicated that cholesterol was not derived intact from the host. A comparison of the sterol profile of Amoebophrya to published sterol profiles of phylogenetic relatives revealed that its sterol profile most closely resembles that of the (proto)dinoflagellate Oxyrrhis marina rather than other extant genera.     

The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

Occurrence of dinoflagellate Alexandrium tamarense, a causative organism of paralytic shellfish poisoning in Chinhae Bay, Korea

A paralytic shellfish poisoning (PSP) incident caused by consumptionof the mussel Mytilus edulis occurred for the first time inKorea in April 1986. Weekly water samplings were carried Outduring the period from 7 March to 21 April 1989 in Chinhae Bay,Korea, in order to identify the causative organism. The temperaturecharacteristics of the water column indicated three differenthydrological regimes: well mixed (up to 7 March), weakly stratified(17–31 March) and stratified (7–21 April). Toxicityof the phytoplankton was detected during the weakly stratifiedperiod, but only in the 10–50 p.m phytoplankton size fraction.This study presents the occurrence of the toxigenic dinoflagellateAlexandrium tamarense, which is a causative organism of PSP,in Korean coastal waters. Its biomass varied at different depthsin the water column, ranging from 200 to 8000 cells 1^–1in the water column. The weekly fluctuation of A.tamarense toxicitywas similar to that of mussel toxicity. ¹ Present address: Department of Biology, College of NaturalSciences, Hanyang University, Seoul 133-791, Korea     

Relative importance of nitrogen sources,algal alarm cues and grazer exposure to toxin production of the marine dinoflagellate Alexandrium catenella

Dinoflagellate paralytic shellfish toxin (PST) production is mediated by several abiotic and biotic factors. This study compared the relative importance of nitrogen source and concentration, prey alarm cues and grazer presence on toxin production of the marine dinoflagellate Alexandrium catenella (Group I, strain BF-5). In separate assays run under either nutrient-replete (F/2 medium) or nutrient-depleted (filtered seawater) conditions, PST production of A. catenella was measured as a function of varying concentrations of added nitrogen sources (ammonium and urea), alarm cues from lysed conspecific (A. catenella Group I strains) and interspecific (the diatom, Thalassiosira weissflogii, and the green flagellate, Tetraselmis sp.) algae, and the presence of a grazer (the copepod Acartia hudsonica). Results showed that addition of ammonium or urea did not increase PST production. Unexpectedly, interspecific alarm cues increased toxin production but conspecific ones did not. Grazer presence dramatically induced PST production in A. catenella, irrespective of nutrient conditions, and this effect was an order of magnitude greater than any of the other variables tested. These results corroborate previous studies on grazer-induced PST production, and support the hypothesis that grazer-induced toxin production is not an experimental artifact, but rather a prey defense mechanism.     

The effect of a toxic dinoflagellate (Alexandrium tamarense) on the oxygen uptake of juvenile filter-feeding bivalve molluscs

• 1.1. Oxygen uptake and grazing rates of juvenile bivalve molluscs Mytilus edulis, Mya arenaria, Geukensia demissa, Placopecten magellanicus and Crassostrea virginica were measured following 1 hr exposure to bloom concentrations (10⁶ cells/1) of the toxic dinoflagellate Alexandrium tamarense (GT429) using a non-toxic clone of the same species (PLY 173) as control.
• 2.2. For all bivalves, prefeeding estimates of V̇O₂ were similar to postfeeding values and values recorded 24 hr after exposure to bloom conditions.
• 3.3. V̇O₂ was similar for bivalves fed on both the toxic and non-toxic strains of A. tamarense suggesting that there were no adverse effects on V̇O₂ following 1 hr exposure to toxic GT429.
• 4.4. Bivalves differed in their rates of grazing between toxic GT429 and non-toxic PLY 173. Similar grazing rates were recorded for M. edulis and G. demissa. For P. magellanicus and M. arenaria reduced rates of clearance were recorded in GT429 compared with the non-toxic strain.

     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Modelling the population dynamics of the toxic dinoflagellate Alexandrium tamarense in Hiroshima Bay, Japan. II. Sensitivity to physical and biological parameters

Using the numerical model developed in a previous paper [Yamamotoet al. (2002c) J. Plankton Res., 24, 33–47], the sensitivityof population dynamics of Alexandrium tamarense to physicaland biological parameters was analysed. Horizontal and verticaldiffusions led to the dispersion of dense A. tamarense populationsin the surface layer of the innermost portion of the bay. Temperatureand salinity influenced the timing of the A. tamarense bloomdue to its stenothermal and stenohaline characteristics. Althoughincreasing the light intensity caused the bloom of A. tamarenseto begin earlier, it lowered the cell density at the bloom peakas a result of phosphate depletion in the ambient water. Bothincreasing the cyst density and the excystment rate had littleinfluence on the population dynamics of A. tamarense vegetativecells. Increasing the phosphate concentration led to increasesin cell density of A. tamarense, indicating that growth is phosphate-limited.Oysters, which are cultured intensively in this bay, appearto stimulate the bloom of A. tamarense through the regenerationof phosphorus from their faeces/pseudofaeces. The phosphorusreduction measure that has been taken since 1980 and the recentconstruction of a large dam are discussed as important factorsthat may influence the population dynamics of A. tamarense.     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon,southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

Life history,excystment features,and growth characteristics of the Mediterranean harmful dinoflagellate Alexandrium pseudogonyaulax

Studies considering the biology and ecology of the toxic bloom‐forming species, Alexandrium pseudogonyaulax, are rare. Our results highlight five features not described before in A. pseudogonyaulax life cycle: (i) A. pseudogonyaulax gametes showed two modes of conjugation, anisogamy and isogamy, (ii) sexual conjugation occurs either in the dark or in the light phase by engulfment or a fusion process, (iii) the presence of planozygote and newly formed cysts in monoclonal culture suggests homothallism, (iv) newly formed cysts have very dark vesicular content and are mostly unparatabulated when observed under light microscope and (v) natural resting cysts are able to give either a planomeiocyte or two vegetative cells. Cyst viability was enhanced after 5 months of cold storage (4°C), with excystment rate reaching 97% after 3 d of incubation. Excystment rate was highest (43%–79%) in Enriched Natural Sea Water diluted culture medium, whereas few germling cells were able to survive without the culture medium (0%–13%). Salinity‐irradiance experiments revealed that the highest cell concentrations occur at high irradiances for all the tested salinities. Vegetative growth rates generally increased with increasing irradiance, and were less dependent on salinity variations. The relatively low growth rate, low cell densities in the laboratory, and the notable capacity of producing cysts along growth phases of A. pseudogonyaulax could explain the occurrence of high resting cysts densities in the sediment of Bizerte lagoon and the relatively low abundances of vegetative cells in the water column.     

Identification and characterization of a "biomarker of toxicity" from the proteome of the paralytic shellfish toxin-producing dinoflagellate Alexandrium tamarense (Dinophyceae)

The objective of this study was to identify and characterize a "biomarker of toxicity" from the proteome of Alexandrium tamarense, a paralytic shellfish toxin (PST)-producing dinoflagellate. A combination of 2-DE and MS approaches was employed to identify proteins of interest in the vegetative cells of several strains of A. tamarense with different toxin compositions and from different geographical locations. The electrophoretic analysis of the total water-soluble proteins from these toxic strains by 2-DE showed that several abundant proteins, namely AT-T1, AT-T2 and AT-T3, differing slightly in apparent Mr and pIs, were consistently present in all toxic strains of A. tamarense. Further analysis by MALDI-TOF MS and N-terminal amino acid sequencing revealed that they are isoforms of the same protein. Even more intriguing is that these proteins in A. tamarense have similar amino acid sequences and are closely related to a "biomarker of toxicity" previously reported in A. minutum. Unambiguous and highly species-specific identification was later achieved by comparing the PMFs of proteins in these two species. An initial attempt to characterize these proteins by generation of murine polyclonal antibodies against the AT-T1 protein was successful. Western blot analysis using the murine AT-T1-polycolonal antibodies identified all the toxic strains of A. tamarense and A. minutum, but not the nontoxic strain of A. tamarense. These results indicate that these protein characteristics for toxic strains are species-specific and that they are stable properties of the tested algae which are clearly distinguishable irrespective of geographical location and toxin composition. To our knowledge, this is the first study to demonstrate the use of polyclonal antibodies against marker proteins purified from 2-DE gels to distinguish different strains and species of the PST-producing dinoflagellate Alexandrium. It provides the basis for the production of monoclonal antibody probes against the "biomarkers of toxicity" for those dinoflagellates whose genome is incompletely characterized. Potentially, immunoassays could be developed to detect the presence of toxic algae in routine monitoring programs as well as to predict bloom development and movement.     

有机碳源和氮源对三角褐指藻生长的影响

研究了8种有机碳源和4种氮源对三角褐指藻(Phaeodactylum tricornutum)生长的影响.结果表明,三角褐指藻具有兼养生长的能力,碳浓度为5mmol/L和50mmol/L时,葡萄糖、果糖、乙酸钠和甘油对其生长有明显的促进作用,乳酸钠和乙醇抑制藻细胞生长,半乳糖和柠檬酸钠对其生长随有机碳浓度而异.甘油、葡萄糖和乙酸钠的适宜浓度范围分别为5 -800mmol/L、100 -400mmol/L和50 -300mmol/L.培养液中分别加入50mmol/L甘油、400mmol/L葡萄糖和150mmol/L乙酸钠,培养第14天,三角褐指藻的最大生物量分别为对照的1.45倍、1.25倍和1.11倍.甘油兼养生长的最优氮源是尿素,适宜浓度范围为0.88-8.8mmol/L.当尿素浓度为4.4mmol/L时,最高生物量可达1.31g/L.     

Influence of carbon and nitrogen sources on growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus

The effects of different carbon and nitrogen sources on the growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus were investigated. The amino acids asparagine, aspartic acid, and glutamic acid affected microbial growth and nitrogenase activity. Several enzymatic activities involved in the tricarboxylic acid cycle were affected by the carbon source used. In addition, glucose and gluconate significantly increased the oxygen consumption (respiration rate) of whole cells of G. diazotrophicus grown under aerobic conditions. Enzymes responsible for direct oxidation of glucose and gluconate were especially active in cells grown with sucrose and gluconate. The presence of amino acids in the apoplastic and symplastic sap of sugarcane stems suggests that these compounds might be of importance in the regulation of growth and nitrogenase activity during the symbiotic association. The information obtained from the plant-bacterium association together with the results of other biochemical studies could contribute to the development of biotechnological applications of G. diazotrophicus.     

Role of carbon and nitrogen sources in bacterial growth and sporulation

[This corrects the article on p. 131 in vol. 22.].     

Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

The growth and photosynthesis of Alexandrium tamarense (Lebour) Balech in different nutrient conditions were investigated. Low nitrate level (0.0882 mmol/L) resulted in the highest average growth rate from day 0 to day 10 (4.58 × 10² cells mL^?1 d^?1), but the lowest cell yield (5420 cells mL^?1) in three nitrate level cultures. High nitrate‐grown cells showed lower levels of chlorophyll a‐specific and cell‐specific light‐saturated photosynthetic rate (P_m^{chl a} and P_m^cell), dark respiration rate (R_d^chla and R_d^cell) and chlorophyll a‐specific apparent photosynthetic efficiency (α^chla) than was seen for low nitrate‐grown cells; whereas the cells became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at 0.7938 mmol/L in late exponential growth phase, or with nitrate at 0.7938 mmol/L and phosphate at 0.072 mmol/L in stationary growth phase, the cell yield was drastically enhanced, a 7–9 times increase compared with non‐supplemented control culture, achieving 43 540 cells mL^?1 and 52 300 cells mL^?1, respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the cell yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of A. tamarense.     

Sea ice bacterial growth rate, growth efficiency and preference for inorganic nitrogen sources in the Baltic Sea

Seasonal Baltic Sea ice is structurally similar to polar sea ice and provides habitats for diverse ice organism assemblages that are integral to the biogeochemistry and ecology of the sea during winter. Temperature and inorganic nitrogen sources have been suggested to control bacterial growth, with increasing dependence on ammonium at low temperatures. To study the bacterial growth and preference for the nitrogen source, we conducted experiments at 0 and 4°C, using ammonium and nitrate as nitrogen sources at two coastal fast-ice stations in the Gulf of Finland and in the Gulf of Bothnia during three successive winters. The two study sites differ markedly in relation to the allochthonous dissolved organic matter supply from the catchment area. High levels of bacterial growth were recorded at both study sites, with community generation times of 15–37 h. The measured bacterial growth efficiencies of 20–58% suggest that the Baltic sea ice brines provide a rich medium for bacterial growth and efficient functioning of bacteria-based food webs. Our experiments with sea ice samples showed a preference for ammonium at both temperatures and high potential growth in both types of nitrogen supplies. No major differences in phosphorus depletion rates were found at the two temperatures, but rates were always highest when ammonium was added to the experiments. These experiments point out that ice maturity, presumably through changes in bacterial community structure, impacts nitrogen processes and that these processes are pronounced prior to melting of the ice.     

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.     

Comparing diatom and Alexandrium catenella/tamarense blooms in Thau lagoon: Importance of dissolved organic nitrogen in seasonally N-limited systems

Diatom blooms in Thau lagoon are always related to rain events leading to inputs of inorganic nutrients such as phosphate, ammonium and nitrate through the watershed with time lags of about 1 week. In contrast, blooms of Alexandrium catenella/tamarense can occur following periods of 3 weeks without precipitation and no significant input of conventional nutrients such as nitrate and phosphate. Field results also indicate a significant drop (from 22–25 to 15–16 μM over 3 days) in dissolved organic nitrogen (DON) at the bloom peak, as well as a significant inverse relationship between A. catenella/tamarense cell density and DON concentrations that is not apparent for diatom blooms. Such dinoflagellate blooms are also associated with elevated (6–9 μM) ammonium concentrations, a curious feature also observed by other investigators, possibly the results of ammonium excretion by this organism during urea or other organic nitrogen assimilation.The potential use of DON by this organism represents short cuts in the nitrogen cycle between plants and nutrients and requires a new model for phytoplankton growth that is different from the classical diatom bloom model. In contrast to such diatom blooms that are due to conventional (nitrate, phosphate) nutrient pulses, Alexandrium catenella/tamarense blooms on the monthly time scale are due to organic nutrient enrichment, a feature that allows net growth rates of about 1.3 d⁻¹, a value higher than that generally attributed to such organisms.