Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

First report of Alexandrium taylori and Alexandrium peruvianum (Dinophyceae) in Malaysia waters

The occurrence of Alexandrium taylori and Alexandrium peruvianum is reported for the first time in Malaysia waters. The Malaysian A. taylori isolates were pyriform in shape with a transdiameter range of 36–40 μm and a cell length range of 33–37 μm. The first apical plate (1′) was pentagonal with two distinctive anterior margins. No direct connection between 1′ and the apical pore complex was observed. The posterior sulcal plate (S.p.) was large, elongated and oblique to the right with anterior projections. The ventral pore (vp) was relatively large and situated at a confluence point of 1′, the second apical (2′) and the fourth apical (4′) plates. Cells of A. peruvianum were slightly anteriorly and posteriorly compressed. S.p. had an irregular pentagonal shape, with the anterior margin divided into 2 portions. 1′ was boomerang-shaped with a large and truncated ventral pore in the middle right margin. The anterior right margin of 1′ was straight. The sixth precingular plate (6″) was wider than long. The anterior sulcal plate (S.a.) was triangular and lacked a left portion extension. In laboratory cultures, both A. taylori and A. peruvianum produced paralytic shellfish toxins, with GTX4 and GTX6 as the predominant toxin, respectively. This is the first report of PSP toxins production for both species as well as the occurrences in Malaysia waters.     

Domoic acid production by Pseudo-nitzschia calliantha Lundholm, Moestrup et Hasle (bacillariophyta) isolated from the Black Sea

A species of Pseudo-nitzschia isolated from Sevastopol Bay, Black Sea, was examined for its toxicity. The species was identified as P. calliantha Lundholm, Moestrup et Hasle based on SEM and TEM examination. Domoic acid (DA) was detected in batch culture throughout the growth cycle of P. calliantha. The production of DA by this diatom species was confirmed by fluorenylmethoxycarbonyl (FMOC) derivatization and HPLC-fluorescence method. The cellular DA level was higher in the early exponential phase, with the maximum value of 0.95 pg DA cell⁻¹. In the stationary phase, the cellular DA levels declined. This is the first record of a DA producing diatom isolated from the Black Sea.     

Nitrogenous preference of toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from field and laboratory experiments

Field and laboratory experiments were designed to determine the differential growth and toxin response to inorganic and organic nitrogen additions in Pseudo-nitzschia spp. Nitrogen enrichments of 50 μM nitrate (KNO₃), 10 μM ammonium (NH₄Cl), 20 μM urea and a control (no addition) were carried out in separate carboys with seawater collected from the mouth of the San Francisco Bay (Bolinas Bay), an area characterized by high concentrations of macronutrients and iron. All treatments showed significant increases in biomass, with chlorophyll a peaking on days 4–5 for all treatments except urea, which maintained exponential growth through the termination of the experiment. Pseudo-nitzschia australis Frenguelli abundance was 10³ cells l⁻¹ at the start of the experiment and increased by an order of magnitude by day 2. Particulate domoic acid (pDA) was initially low but detectable (0.15 μg l⁻¹), and increased throughout exponential and stationary phases across all treatments. At the termination of the experiment, the urea treatment produced more than double the amount of pDA (9.39 μg l⁻¹) than that produced by the nitrate treatment (4.26 μg l⁻¹) and triple that of the control and ammonium treatments (1.36 μg l⁻¹ and 2.64 μg l⁻¹, respectively). The mean specific growth rates, calculated from increases in chlorophyll a and from cellular abundance of P. australis, were statistically similar across all treatments.These field results confirmed laboratory experiments conducted with a P. australis strain isolated from Monterey Bay, CA (isolate AU221-a) grown in artificial seawater enriched with 50 μM nitrate, 50 μM ammonium or 25 μM of urea as the sole nitrogen source. The exponential growth rate of P. australis was significantly slower for cells grown on urea (ca. 0.5 day⁻¹) compared to the cells grown on either nitrate or ammonium (ca. 0.9 day⁻¹). However the urea-grown cells produced more particulate and dissolved domoic acid (DA) than the ammonium- or nitrate-grown cells. The field and laboratory experiments demonstrate that P. australis is able to grow effectively on urea as the primary source of nitrogen and produced more pDA when grown on urea in both natural assemblages and unialgal cultures. These results suggest that the influence of urea from coastal runoff may prove to be more important in the development or maintenance of toxic blooms than previously thought, and that the source of nitrogen may be a determining factor in the relative toxicity of west coast blooms of P. australis.     

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.     

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.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Intercalibration of classical and molecular techniques for identification of Alexandrium fundyense (Dinophyceae) and estimation of cell densities

A workshop with the aim to compare classical and molecular techniques for phytoplankton enumeration took place at Kristineberg Marine Research Station, Sweden, in August 2005. Seventeen different techniques – nine classical microscopic-based and eight molecular methods – were compared. Alexandrium fundyense was the target organism in four experiments. Experiment 1 was designed to determine the range of cell densities over which the methods were applicable. Experiment 2 tested the species specificity of the methods by adding Alexandrium ostenfeldii, to samples containing A. fundyense. Experiments 3 and 4 tested the ability of the methods to detect the target organism within a natural phytoplankton community. Most of the methods could detect cells at the lowest concentration tested, 100 cells L⁻¹, but the variance was high for methods using small volumes, such as counting chambers and slides. In general, the precision and reproducibility of the investigated methods increased with increased target cell concentration. Particularly molecular methods were exceptions in that their relative standard deviation did not vary with target cell concentration. Only two of the microscopic methods and three of the molecular methods had a significant linear relationship between their cell count estimates and the A. fundyense concentration in experiment 2, where the objective was to discriminate that species from a morphologically similar and genetically closely related species. None of the investigated methods were affected by the addition of a natural plankton community background matrix in experiment 3. The results of this study are discussed in the context of previous intercomparisons and the difficulties in defining the absolute, true target cell concentration.     

Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes

To improve labeling-intensity of whole-cell fluorescence in situ hybridization (FISH) in the molecular identification of toxic Alexandrium tamiyavanichii, two DNA probes (TAMID2 plus TAMIS1 designed from the LSU and SSU rDNA regions, respectively) were used to test the labeling intensity of targeted cultured A. tamiyavanichii cells. The cross-reactivity of the DNA probe to natural seawater samples and six Alexandrium species: A. affine, A. catenella, A. fraterculus, A. insuetum, A. pseudogonyaulax and A. tamarense, was also tested. The labeling intensity of the DNA probe TAMID2S1, a combination of two separate probes that target different regions of the rRNA, was 1.7–2.7 times higher than that of the single DNA probe TAMID2. With cultured A. tamiyavanichii cells in the dead growth phase at 30 days, the TAMID2S1 intensity was 1.9 times higher than that of TAMID2. During a 30-day culture, the labeling intensity of A. tamiyavanichii cells hybridized with TAMID2S1 decreased to 49.4% of the original intensity. No cross-reactivity to various microorganisms in natural seawater samples was found. The two DNA probes together, designated as TAMID2S1, readily detected A. tamiyavanichii added to natural seawater samples, even aged cultured cells.     

Phylogenetic relationship of Alexandrium monilatum (Dinophyceae) to other Alexandrium species based on 18S ribosomal RNA gene sequences

The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rDNA sequences. Maximum likelihood phylogenetic analysis of the combined rDNA sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the first of the Alexandrium taxa to diverge, followed by Alexandrium margalefii. All three are members of the Alexandrium subgenus Gessnerium Halim nov. comb.     

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.     

Genetic variability among Alexandrium tamarense and Alexandrium minutum strains studied by RAPD banding pattern analysis

We performed random amplification of polymorphic DNA (RAPD) analysis on five strains of Alexandrium tamarense and nine strains of Alexandrium minutum. Arbitrary 10-mer oligonucleotides were used as primers for the PCR. Electrophoresis on denaturing acrylamide gels improved RAPD reproducibility and increased the band number. Eight of the 20 primers assayed gave reproducible results and the band profiles generated by them were used for constructing a similarity matrix. Analyses were performed independently for the strains of each species and jointly for all the strains of both species. Results for A. tamarense showed the highest similarity for two distinct clones isolated from the same water sample in the Baltic Sea during a bloom (KAC01 and KAC02). The highest similarity among A. minutum clones was found for three strains (AL1V, AL2V and AL3V) isolated in the Ria de Vigo in NW Spain. The results show a high genetic diversity within a single species. We have shown the potential of the RAPD technique to discriminate between two conspecific strains, as well as for establishing similarities that are related to the biogeographic origin of the strains.     

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.     

Alexandrium camurascutulum sp. nov. (Dinophyceae): a new dinoflagellate species from New Zealand

A new species, Alexandrium camurascutulum sp. nov. MacKenzie et Todd, is described from specimens collected from Tasman Bay and the Marlborough Sounds New Zealand. These small (26–28 μm long × 21–24 μm wide) cells can be discriminated from other species in the Alexandrium minutum group by three distinctive morphological features. The sixth pre-cingular plate (6′′) is up to 1.6 times wider than high and the left side of the plate is concave resulting in a markedly ‘hooked’ appearance. In all specimens observed, the first apical plate (1′) does not directly connect with the apical pore plate (Po) and the posterior sulcal plate (S.p.) is markedly different from the usual A. minutum form and may contain a posterior attachment pore (pap) connected to the right side plate margin. The cells may or may not have an anterior attachment pore (aap) in the apical pore plate (Po). The cells display a prominent list along the left sulcal margin and the thecal surface is perforated with numerous areolated pores. A. camurascutulum sp. nov. has been observed occasionally over a number of years in coastal waters of the northern South Island of New Zealand. There is circumstantial evidence that suggests it is not toxic.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

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.     

Grazing on toxic Alexandrium fundyense resting cysts and vegetative cells by the eastern oyster (Crassostrea virginica)

In laboratory experiments, oysters (Crassostrea virginica) were fed Alexandrium fundyense (strain CB501) vegetative cells or resting cysts (from strains CB501 and GMT25) produced from laboratory cultures. The toxicity per cyst was 1.7 pg STXequiv/cyst and for vegetative cells 3.9 pg STXequiv/cell. The toxic, resting cysts and vegetative cells were removed from suspension in the experimental containers within about 4 h. Oysters fed toxic vegetative cells digested 72% of cells ingested, and 28% survived gut passage by forming temporary cysts. Toxin levels of oysters fed vegetative cells averaged 27 μg STXequiv/100 g meat. Resting cysts added to the experimental containers adhered to the walls so that only 40% of the cysts added were available to the oysters during the experiment. Of the cysts that were ingested, approximately 59% were digested, and oysters accumulated toxins (an average of 1.2 μg STXequiv/100 g meat), showing that consumption of resting cysts can cause toxicity in oysters. Direct consumption of resting cysts, thus, may explain shellfish toxicity in areas without known blooms, but with toxic resting cysts in the sediment. These results suggest a possible role of toxic cysts in mediating time-lags between surface blooms and appearance of toxicity in benthic grazers, and the possible role of benthic grazers in controlling seed populations, except in anoxic areas, which can serve as cyst “refuges” from grazing mortality.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

Use of PCR and partial sequencing of the large-subunit rRNA gene to identify Alexandrium catenella (Dinophyceae) from the South of Chile

A fragment of the large-subunit ribosomal DNA gene (LSU rDNA) from Chilean Alexandrium catenella clones isolated from two different geographic regions (XI and XII) was amplified by PCR and the products cloned and sequenced. Based on the analysis of the PCR products it is possible to distinguish two strains of A. catenella, denominated strain type 1 (a single PCR product band) and strain type 2 (two PCR product bands). These two strains proliferate in both, the XI and XII regions. Only in the XI region, there is evidence that they bloom simultaneously. The LSU rDNA sequence analysis indicate that the Chilean A. catenella isolated clones are more related to the North American ribotype-Western subribotype.