Toxic Alexandrium minutum (Dinophyceae) from Vietnam with new gonyautoxin analogue

Clonal cultures of Alexandrium species collected from a shrimp pond on the northern coast of Vietnam were established and morphologically identified as Alexandrium minutum. Nucleotide sequences of domains 1 and 2 of the large subunit ribosomal (LSU) rRNA gene showed high sequence similarity to A. minutum isolates from Malaysia. Paralytic shellfish toxin profile of the clones was characterized by the dominance of GTX4, GTX1, and NEO. GTX3, GTX2, and dcSTX were also present in trace amount. Toxin content varied among the strains and growth stages, ranged from 3.0 to 12.5 fmol cell⁻¹. In addition to these known toxin components, a new gonyautoxin derivative was detected by HPLC, eluting between GTX4 and GTX1. The peak of this compound disappeared under non-oxidizing HPLC condition but unchanged either after treated with 0.05 M ammonium phosphate/10% mercaptoethanol or 0.1N HCl hydrolysis. LCMS ion scanning showed a parental ion of [M + H]⁺ at m/z 396, [M − SO₃]⁺ at m/z 316, and [M − SO₄]⁺ at m/z 298. Based on these results, the derivative was identified as deoxy-GTX4-12ol, and this represents the first report of this toxin analogue.     

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.     

Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists

The marine dinoflagellate genus Alexandrium (Halim) Balech contains members that produce highly potent phycotoxins (PSP toxins or spirolides) as well as lytic substances and other allelochemicals of unknown structure and ecological significance. One isolate each of six Alexandrium species (A. tamarense, A. ostenfeldii, A. lusitanicum, A. minutum, A. catenella, A. taylori), of the closely related gonyaulacoid dinoflagellate Fragilidium subglobosum, and of the peridinioid Scrippsiella trochoidea were tested in 24 h co-incubation experiments for their short-term deleterious effects on a diversity of marine protists. Both autotrophs (Rhodomonas salina, Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum) were included as target species. All donor isolates except S. trochoidea exhibited lytic effects on at least some target species. Lytic effects were observed with all Alexandrium species, for both whole cell samples and culture filtrate (<10 μm and <0.2 μm). Antibiotic treated cultures with drastically reduced bacterial numbers did not show any general reduction in lytic capacity, therefore direct involvement of extracellular bacteria in allelochemical production is unlikely. Values of EC₅₀, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, differed by two orders of magnitude depending on the donor/target combination, from 3.1 × 10³ cells ml⁻¹ (A. minutum/O. marina) down to 0.02 × 10³ cells ml⁻¹ (A. catenella/D. salina). Within the array of nine donor Alexandrium/target combinations, variable ratios in EC₅₀ values between donor/target combination cannot be explained by quantitative differences in allelochemical production, but rather indicate qualitative differences in the composition of compounds produced by different Alexandrium strains. In conclusion, our study confirms the widespread lytic capacity within the genus Alexandrium, although allelochemical effects are not restricted to this genus. Allelochemical interactions mediated by such lytic substances may be significant in explaining the formation and maintenance of Alexandrium blooms through direct destructive effects on competing algae or unicellular grazers.     

Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea

Nutritional and environmental conditions were characterized for a batch culture of the marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea for its growth (cells ml⁻¹), cellular toxin content (Qt in fmol cell⁻¹) and toxin composition (mol%). Under a nutrient replete condition, Qt increased with cell growth and peaked at the late stationary phase. Toxin content increased with the nitrate concentration in the culture while it reached a maximum at 5 μM phosphate. When nitrate was replaced with ammonia, Qt decreased by 4.5-fold. Salinity and light intensity were important factors affecting Qt. The latter increased two-fold over the range of salinity from 15 to 30‰, while decreased 38% as light intensity increased from 80 to 220 μE m⁻² s⁻¹. Toxin composition varied with growth phase and culture conditions. In nutrient replete cultures, toxin composition varied greatly in the early growth phase (first 3 days) and then C1/C2, C3/C4 and GTX1 remained relatively constant while GTX4 increased from 32 to 46% and GTX5 decreased from 28 to 15%. In general, the composition of GTXs was affected in a much greater extent than C toxins by changes in nutrient conditions, salinity and light intensity. This is especially true with GTX4 and GTX5. These data indicate that the cellular toxin content and toxin composition of A. tamarense HK9301 are not constant, but that they vary with growth phase and culture conditions. Use of toxin composition to identify a toxigenic marine dinoflagellate is not always valid. The data also reveal that high salinity and low light intensity, together with high nitrate and low phosphate concentrations, would favor toxin production by this species.     

The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup

The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m⁻² s⁻¹), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day⁻¹ with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m⁻² s⁻¹ for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day⁻¹ versus 0.2–1.0 div day⁻¹). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m⁻² s⁻¹ versus 65 μmol m⁻² s⁻¹), and light compensation (20–30 μmol m⁻² s⁻¹1). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.     

Toxicity of Dinophysis spp. in relation to population density and environmental conditions on the Swedish west coast

The aim of this study in the field was to investigate whether there are differences between the outer archipelago (Gullmar Fjord) and a semi-enclosed fjord system (Koljö Fjord) in occurrences of D. acuta and D. acuminata as well as in their content of diarrheic shellfish toxin (DST) per cell. When all data pairs of cell toxicity of D. acuminata and the corresponding number of cells l⁻¹ from the two sites were tested in a regression analysis, a statistically significant negative correlation became evident and was apparent as a straight line on a log–log plot (p < 0.0001). Obviously, there was an overall inverse relationship between the population density of D. acuminata and the toxin content per cell. Plotted on a linear scale, all data-pairs of cell toxicity and cell number made up a parabolic curve. On this curve the data-pairs could be separated into three groups: (i) D. acuminata occurring in numbers of fewer than approximately 100 cells l⁻¹, and with a toxin content per cell above 5 ρg cell⁻¹; (ii) cell numbers between 100 and approximately 250 cells l⁻¹ with a cell toxin content from 5 to 2 ρg cell⁻¹; (iii) when the population became greater than 250 cells l⁻¹, the toxicity, with few exceptions, was less than 2 ρg cell⁻¹. By applying this subdivision, some clear patterns of the distribution of the differently toxic D. acuminata became evident. When comparing the cell toxicity of the two sites, it was obvious that the D. acuminata cells from all depths from the Gullmar Fjord as a mean were significantly more toxic compared to the Koljö Fjord samples. The results have demonstrated that approximately 100 high-toxicity cells in a low-density population at surface may lead to the same accumulation of DST in a mussel as the ingestion of 1500 low-toxicity cells from a high-density pycnocline population.     

Growth, uptake, and assimilation of ammonium, nitrate, and urea, by three strains of Karenia brevis grown under low light

Observations of near-bottom populations of Karenia brevis suggest that these cells may derive nutrients from the sediment–water interface. Cells undergoing a metabolic-mediated migration may be in close proximity to enhanced concentrations of nutrients associated with the sediment during at least a fraction of their diel cycle. In this study, the growth, uptake and assimilation rates of ammonium, nitrate, and urea by K. brevis were examined on a diel basis to better understand the potential role of these nutrients in the near-bottom ecology of this species. Three strains of K. brevis, C6, C3, and CCMP 2229, were grown under 12:12 light dark cycle under 30 μmol photons m⁻² s⁻¹ delivered to the surface plain of batch cultures. Nitrogen uptake was evaluated using ¹⁵N tracer techniques and trichloroacetic acid extraction was used to evaluate the quantity of nitrogen (N) assimilated into cell protein. Growth rates ranged from a low of 0.12 divisions day⁻¹ for C6 and C3 grown on nitrate to a high of 0.18 divisions day⁻¹ for C3 grown on urea. Diurnal maximum uptake rates, ρ_max, varied from 0.41 pmol-N cell⁻¹ h⁻¹ for CCMP 2229 grown on nitrate, to 1.29 pmol-N cell⁻¹ h⁻¹ for CCMP 2229 grown on urea. Average nocturnal uptake rates were 29% of diurnal rates for nitrate, 103% of diurnal uptake rates for ammonium and 56% of diurnal uptake rates for urea. Uptake kinetic parameters varied between substrates, between strains and between day and night measurements. Highest maximum uptake rates were found for urea for strains CCMP2229 and C3 and for ammonium for strain C6. Rates of asmilation into protein also varied day and night, but overall were highest for urea. The comparison of maximal uptake rates as well as assimilation efficiencies indicate that ammonium and urea are utilized (taken up and assimilated) more than twice was fast as nitrate on a diel basis.     

Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

Nutrition and growth kinetics in nitrogen- and phosphorus-limited cultures of the novel red tide flagellate Chattonella ovata (Raphidophyceae)

In the summer of 2004, a harmful bloom caused by Chattonella ovata (Raphidophyceae) occurred over almost the entire area of the Seto Inland Sea and caused fishery damages. This incident was the first record of a bloom and damage to the fisheries caused by this species in Japanese waters. In order to elucidate the mechanism of the bloom outbreak, we examined the nutrition and the growth kinetics in nitrogen (N)- and phosphorus (P)-limited semi-continuous cultures of this species. Inorganic N compounds, such as nitrate, nitrite, and ammonium, were found to be good nitrogen sources for the growth of C. ovata, while organic nitrogen (urea and uric acid) was not utilized. This species was capable of using ATP, ADP and inorganic phosphorus compounds, but did not utilize phosphate monoesters as a sole P source. Under both N-limited and P-limited steady state conditions, the growth rate as a function of cell nitrogen and phosphorus quota, respectively, followed the Droop equation. Kinetic parameters μ_m (maximum growth rate) and k_q (minimum cell quota) obtained for N- and P-limited cultures were 0.79 day⁻¹ and 5.5 pmol N cell⁻¹ and 0.86 day⁻¹ and 0.48 pmol P cell⁻¹, respectively. The minimum cell quotas were 23–30% lower than those of C. antiqua. The nutrient availability and kinetic parameters of C. ovata are compared with other harmful algae and the ecological implications of these characteristics discussed.     

Can soil Chytridiomycota survive and grow in different osmotic potentials?

Twenty isolates from soil in the orders Spizellomycetales, Blastocladiales and Chytridiales (Chytridiomycota) grew on complex solid media supplemented with 10 g l⁻¹ sodium chloride. In a synthetic liquid medium, 4.4 g l⁻¹ sodium chloride strongly inhibited growth in three of the five isolates, possibly because of the effect of the ions or osmolarity of the solution. The maximum concentration for growth in synthetic liquid medium with different osmotic potentials using polyethylene glycol (PEG) varied considerably amongst the isolates. Three patterns of growth with increasing concentrations of PEG were evident among isolates within the genus Rhizophydium. Up to the concentration where growth ceased, the dry weight of each isolate either decreased, remained constant, or in one case, increased. Most of the fungi survived when incubated at room temperature for 7 d in complex liquid media supplemented with 35 g l⁻¹ sodium chloride or 300 g l⁻¹ PEG. These data indicate that soil Chytridiomycota can survive various osmotic potentials that may occur during the wetting and drying phases in soils.     

Entropy decrease associated to solute compartmentalization in the cell

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately −14.4 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−0.7 J K⁻¹ L⁻¹) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately −49.6 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−1.0 J K⁻¹ L⁻¹) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.     

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.     

Use of androgenesis for estimating maternal and mitochondrial genome effects on development and oxygen consumption in rainbow trout, Oncorhynchus mykiss

Chromosome set manipulation was used to produce rainbow trout, Oncorhynchus mykiss, with identical nuclear backgrounds, but different maternal backgrounds to determine mitochondrial effects on development rate and oxygen consumption. Significant differences in development rate and oxygen consumption were observed between groups from different females. Development rates ranged from a mean of 317.97 degree days (°d) to 335.25 °d in progeny from different females. Mean oxygen consumption rates ranged from 3.31 μmol O₂ g^− 1 wet mass h^− 1 to 9.66 μmol O₂ g^− 1 wet mass h^− 1. Oxygen consumption and development rate analysis revealed the two slowest developing groups had the highest oxygen consumption rates. Development rate differences between second generation clonal females indicate that mitochondrial genomes play a significant role on early development and are comparable to development rate differences between clonal lines of rainbow trout. These results indicate that selection for mitochondrial genomes could increase growth rates and possibly food conversion ratios in aquaculture species.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters

The diversity of Alexandrium spp. in Irish coastal waters was investigated through the morphological examination of resting cysts and vegetative cells, the determination of PSP toxin and spirolide profiles and the sequence analysis of rDNA genes. Six morphospecies were characterised: A. tamarense, A. minutum, A. ostenfeldii, A. peruvianum, A. tamutum and A. andersoni. Both PSP toxin producing and non-toxic strains of A. tamarense and A. minutum were observed. The average toxicities of toxic strains for both cultured species were respectively 11.3 (8.6 S.D.) and 2.3 (0.5 S.D.) pg STX equiv. cell⁻¹. Alexandrium ostenfeldii and A. peruvianum did not synthesise PSP toxins but HPLC–MS analysis of two strains showed distinct spirolide profiles. A cyst-derived culture of A. peruvianum from Lough Swilly mainly produced spirolides 13 desmethyl-C and 13 desmethyl-D whereas one of A. ostenfeldii, from Bantry Bay, produced spirolides C and D. Species identification was confirmed through the analyses of SSU, ITS1-5.8S-ITS2 and LSU rDNA genes. Some nucleotide variability was observed among clones of toxic strains of A. tamarense, which all clustered within the North American clade. However, rDNA sequencing did not allow discrimination between the toxic and non-toxic forms of A. minutum. Phylogenetic analysis also permitted the differentiation of A. ostenfeldii from A. peruvianum. Resting cysts of PSP toxin producing Alexandrium species were found in Cork Harbour and Belfast Lough, locations where shellfish contamination events have occurred in the past, highlighting the potential for the initiation of harmful blooms from cyst beds. The finding of supposedly non-toxic and biotoxin-producing Alexandrium species near aquaculture production sites will necessitate the use of reliable discriminative methods in phytoplankton monitoring.     

Copepod feeding response to varying Alexandrium spp. cellular toxicity and cell concentration among natural plankton samples

We challenged four species of copepod grazers (Acartia hudsonica, Centropages hamatus, Eurytemora herdmani, Calanus finmarchicus) with natural water samples containing non-toxic algae mixed with one of three clones of Alexandrium spp.—A. tamarense GTCN16 (non-toxic), A. fundyense GTCA28 (moderate toxicity), and A. fundyense BC1 (higher toxicity), each at relatively high (10⁵ cells L⁻¹) and low (10⁴ cells L⁻¹) concentrations. Within any one copepod species, significant differences existed in copepod clearance rates and total food ingested between high and low Alexandrium cell concentrations, and between levels of toxicity, but feeding response did not follow a predictable relationship proportional to toxin levels—rather, the presence or absence of toxin was more important than the level of toxicity. C. finmarchicus behaved differently from the smaller coastal copepods, showing less selectivity and greater concentration dependence. In low Alexandrium concentration treatments, copepod clearance rates on Alexandrium were usually higher, and electivity indices for Alexandrium less negative (indicating less avoidance), compared to high concentration treatments. In high toxicity (BC1) treatments of all copepod species (except C. finmarchicus), total food consumption was consistently less at high Alexandrium concentrations compared to low concentrations, suggesting that high toxicity and concentration suppress overall feeding, while in non-toxic (GTCN 16) treatments total consumption was always higher at high Alexandrium concentrations. Copepod grazers do not follow predictable feeding rules throughout a continuum of conditions, but become more predictable at extremes of concentration and toxicity of prey, consistent with the conclusion that both factors are important. Results support the hypothesis that grazer deterrence imparted by toxicity is only effective at high cell concentrations, but even then will not protect against all grazers.     

Blooms of Pseudo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003–2004

Abundances of Pseudo-nitzschia spp. and concentrations of particulate domoic acid (DA) were determined in the Southern California Bight (SCB) along the coasts of Los Angeles and Orange Counties during spring and summer of 2003 and 2004. At least 1500 km² were affected by a toxic event in May/June of 2003 when some of the highest particulate DA concentrations reported for US coastal waters were measured inside the Los Angeles harbor (12.7 μg DA L⁻¹). Particulate DA levels were an order of magnitude lower in spring of 2004 (February and March), but DA concentrations per cell at several sampling stations during 2004 exceeded previously reported maxima for natural populations of Pseudo-nitzschia (mean = 24 pg DA cell⁻¹, range = 0–117 pg DA cell⁻¹). Pseudo-nitzschia australis dominated the Pseudo-nitzschia assemblage in spring 2004. Overall, DA-poisoning was implicated in >1400 mammal stranding incidents within the SCB during 2003 and 2004. Ancillary physical and chemical data obtained during our regional surveys in 2004 revealed that Pseudo-nitzschia abundances, particulate DA and cellular DA concentrations were inversely correlated with concentrations of silicic acid, nitrogen and phosphate, and to specific nutrient ratios. Particulate DA was detected in sediment traps deployed at 550 and 800 m depth during spring of 2004 (0.29–7.6 μg DA (g sediment dry weight)⁻¹). The highest DA concentration in the traps was measured within 1 week of dramatic decreases in the abundances of Pseudo-nitzschia in surface waters. To our knowledge these are the deepest sediment trap collections from which DA has been detected. Sinking of the spring Pseudo-nitzschia bloom may constitute a potentially important link between DA production in surface waters and benthic communities in the coastal ocean near Los Angeles. Our study indicates that toxic blooms of Pseudo-nitzschia are a recurring phenomenon along one of the most densely populated coastal stretches of the SCB and that the severity and magnitude of these events can be comparable to or greater than these events in other geographical regions affected by domoic acid.