Domoic Acid Cell Research Articles

Physiological conditions favorable to domoic acid production by three Pseudo-nitzschia species

Twenty-six species of the cosmopolitan genus Pseudo-nitzschia can produce domoic acid (DA), a neurotoxin responsible for amnesic shellfish poisoning (ASP). To improve knowledge on this issue, we studied the physiological conditions favorable to DA production and accumulation by three Pseudo-nitzschia species from French coastal waters: P. australis, P. pungens, and P. fraudulenta. They were grown in batch cultures under silicate limitation to characterize their physiological traits and calculate their DA production rates. Three strains were studied per species to consider intraspecific diversity and better characterize interspecific differences. DA production was not influenced by growth or silicate limitation in any of the three species. In contrast, silicate limitation in the stationary phase led to DA accumulation by inhibiting cell division, while DA production was still active. The maximum cellular DA (cDA) production rate was 2.95 pg cell−1 d−1 for P. australis, 0.07 pg cell−1 d−1 for P. pungens, and 0.03 pg cell−1 d−1 for P. fraudulenta. The physiological conditions favorable to cDA production and accumulation by P. australis and P. pungens differed. The three species presented similar growth rates, but P. australis had higher photosynthetic capacities that could partly explain its higher DA production potential. The cDA production and the net dissolved DA (dDA) production in the culture medium were favored by different growth conditions. The cDA production to net dDA production ratio was species specific, with P. pungens excreting proportionally more of its produced DA. These laboratory results on cultures imply that cDA production and net dDA production can occur during all phases of P. australis, P. pungens, and P. fraudulenta blooms. The interactions between the species composition of the bloom, the species-specific capacity for DA production, and the effect of silicate limitation – among other factors – on DA cell quotas drive the toxigenicity of Pseudo-nitzschia blooms.

Costs and benefits of predator-induced defence in a toxic diatom.

Phytoplankton employ a variety of defence mechanisms against predation, including production of toxins. Domoic acid (DA) production by the diatom Pseudo-nitzschia spp. is induced by the presence of predators and is considered to provide defence benefits, but the evidence is circumstantial. We exposed eight different strains of P. seriata to chemical cues from copepods and examined the costs and the benefits of toxin production. The magnitude of the induced toxin response was highly variable among strains, while the costs in terms of growth reduction per DA cell quota were similar and the trade-off thus consistent. We found two components of the defence in induced cells: (i) a 'private good' in terms of elevated rejection of captured cells and (ii) a 'public good' facilitated by a reduction in copepod feeding activity. Induced cells were more frequently rejected by copepods and rejections were directly correlated with DA cell quota and independent of access to other food items. By contrast, the public-good effect was diminished by the presence of alternative prey suggesting that it does not play a major role in bloom formation and that its evolution is closely associated with the grazing-deterrent private good.

Organic polymer consumption facilitates domoic acid entry into the marine food web without direct ingestion of Pseudo-nitzschia

Domoic acid (DA) is a neurotoxin produced by diatoms from the genera Pseudo-nitzschia and Nitzschia. DA is transferred through the food web when consumed by organisms such as copepods (e.g., Acartia tonsa). DA bioaccumulates in higher trophic levels and poses a threat to human health through amnesic shellfish poisoning. Laboratory experiments using a DA reference standard demonstrated that mild turbulence facilitates formation of organic polymer aggregates >0.6 µm in-vivo that can scavenge dissolved DA (dDA). Using A. tonsa, we demonstrate that DA can be assimilated through consumption of these organic polymers which scavenged dDA —a pathway which does not require direct ingestion of the toxin-producer Pseudo-nitzschia. In filtered seawater with spiked DA, copepods accumulated 24.8 ± 4.7 pg DA copepod−1 (2.1 ppm) on average by consuming organic polymers. This was validated in one out of five experiments using ambient DA concentrations. Copepods were suspended in particle-free seawater and accumulated 14.4 ± 3.8 pg DA copepod−1 (1.20 ppm), and in particle-concentrated seawater they accumulated 40.9 ± 3.8 pg DA copepod−1 (3.42 ppm). Data from this experiment suggests that ~34% of the total assimilated DA entered via an organic polymer-bound DA pathway. This experiment had the highest Pseudo-nitzschia spp. abundance (~225,000 cells L − 1) and cellular toxin quota, up to 0.88 pg DA cell−1, relative to the other four ambient DA experiments. These results demonstrate the potential for DA to enter the marine food web through an alternate pathway and may have considerable implications to understanding the flow of DA through marine food webs, and how we monitor DA and its potential vectors into the food web.

Evaluation of intracellular and extracellular domoic acid content in Pseudo-nitzschia multiseries cell cultures under different light regimes

Pseudo-nitzschia multiseries is a diatom species associated with the production of domoic acid (DA), a water soluble neurotoxin that is easily transferred up in the food web, causing devastating effects on top marine organisms and humans. Despite studies on Pseudo-nitzschia are relevant to human health safety, partitioning of marine toxins between intracellular and extracellular fractions are poorly documented. This study aimed to determine the growth rates and DA content, both intracellular and extracellular, of Pseudo-nitzschia multiseries cultures at three different light settings (15, 120 and 560 μmol m−2 s−1). The optimal conditions for cell growth were observed at 120 and 560 μmol m−2 s−1, whereas DA production was observed in P. multiseries at 15 and 120 μmol m−2 s−1, ranging between 0.18-2.56 and 0.16–3.5 pg DA cell−1, respectively. Higher intracellular DA concentrations were found during the senescence phase at low light intensity and during the exponential phase at medium light intensity, while higher concentrations of dissolved DA were found at low and medium light intensities in the senescence phase reaching 3 and 10 ng DA mL−1 respectively. The amount of toxin released into the culture medium represents the most important fraction ranging between 63 and 98% during the exponential phase and nearly 99% during the senescence phase. In contrast, under low light intensity, dissolved DA was detected in the culture medium only during the senescence phase. This study confirms the importance of light intensity on DA production and clearly shows that dissolved domoic acid is an important fraction in Pseudo-nitzschia cultures, suggesting with the careful assumptions of results from static cultures extrapolated to bloom situations that waterborne exposure of marine organism should be considered during blooms of Pseudo-nitzschia multiseries.

Morphological, molecular and toxigenic characteristics of Namibian Pseudo-nitzschia species – including Pseudo-nitzschia bucculenta sp. nov.

A field study was undertaken to investigate the occurrence and toxin production of species in the diatom genus Pseudo-nitzschia in Namibian waters, in the extremely productive Benguela upwelling system. From surveys conducted on the R/V Mirabilis and the R/V !Anichab, 52 strains were morphologically determined to species level, supported by nuclear ITS rDNA data. Seven species were identified; P. australis, P. decipiens, P. dolorosa, P. fraudulenta, P. plurisecta, P. pungens var. cingulata, and the new species P. bucculenta F. Gai, C. K. Hedemand, N. Lundholm & Ø. Moestrup sp. nov.Molecular and morphological diversity of the Namibian Pseudo-nitzschia species is discussed. Most importantly, P. bucculenta is both morphologically and phylogenetically most similar to P. dolorosa differing mainly in valve width and densities of striae, poroids and band striae as well as by four hemi-compensatory base changes in the ITS2. Morphological and molecular differences among the strains of P. decipiens suggest a temperate and a warm water subdivision. The geographical and toxigenic characteristics of the identified Pseudo-nitzschia species are described and compared to previous studies. Initial tests of toxin production in all seven species revealed production of domoic acid (DA) in two species: one strain of P. australis (0.074 pg DA cell−1) and two strains of P. plurisecta (0.338 pg DA cell−1 and 0.385 pg DA cell−1).

Inter and intra-specific growth and domoic acid production in relation to nutrient ratios and concentrations in Pseudo-nitzschia: phosphate an important factor

The factors responsible for inducing the synthesis of toxins and responses from toxic phytoplankton blooms remain unclear. In this study we compare the influence of genotypic (at both the intra and interspecific levels) and environmental factors (nutrient concentration and ratio) on growth (in terms of cell densities) and domoic acid (DA) production in three Pseudo-nitzschia species: P. australis, P.pungens and P.fradulenta. A strong phosphate effect was detected. More precisely, a low initial concentration in phosphate, even at high initial nitrogen and silicate concentrations, induced the highest DA concentrations and the lowest cell densities in all strains/species studied. In contrast, a low initial concentration of nitrogen and silicate combined, with a higher phosphate concentration resulted in low cell densities, but without high DA production. Inter-species effects were also observed in DA production, where P. australis represented the most toxigenic species of all. Intra-specific variations were only moderate, except for a recently isolated P. australis strain, suggesting the influence of time since isolation on the physiology and DA production of Pseudo-nitzschia species. Overall, the lack of strong interaction between environmental and genotypic factors showed that the various genotypes investigated did not extensively diverge in their ability to respond (in terms of DA production and cell densities) to contrasting nutrient supply.

Detection of domoic acid in Mytilus galloprovincialis and Ostrea edulis linked to the presence of Nitzschia bizertensis in Bizerte Lagoon (SW Mediterranean)

Seasonal variation of DA in bivalves was monitored from August 2008 to March 2009 at a shellfish station in Bizerte Lagoon, one of the most important shellfish areas in Tunisia. At the same time, dynamics of potentially toxic diatoms was studied at six stations in the lagoon. Several diatom strains were isolated, maintained in culture and their identification as well as the toxicity was examined.During the study period, domoic acid (DA) was detected in Tunisia, in both mussels (Mytilus galloprovincialis: 0.13–0.86 μg DA g−1 tissue) and oysters (Ostrea edulis: 0.42–1.04 μg DA g−1 tissue). The amount of DA in the two species was below the EU guideline limits for shellfish (20 μg DA g−1).Within the phytoplankton community, potentially toxic diatoms were observed in high densities (104–106 cells l−1). Among the 9 established cultured strains, seven were identified as the recently described species Nitzschia bizertensis sp. nov, which is apparently a commonly occurring species in the Bizerte Lagoon. Among tested five strains of N. bizertensis, four were confirmed to be toxin producers, containing 2–7 × 10−3 pg DA cell−1 during stationary growth phase.Our results indicate for the first time a Nitzschia species, here Nitzschia bizertensis, could be in relation to accumulation of DA in shellfish. Monitoring of DA in phytoplankton and shellfish should be considered for Bizerte Lagoon in order to gain knowledge about the ecology and toxin-production of N. bizertensis and to explore the risk of DA accumulation in the local shellfish industry caused by this species.

Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California, Mexico, cultured under batch conditions at different temperatures and two Si:NO 3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day −1) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO 3 (2.5) or low Si:NO 3 (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell −1) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m −2 s −1) and low light (LL; 30 μmol quanta m −2 s −1) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell −1). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.

Crassostrea virginica grazing on toxic and non-toxic diatoms

Despite high abundances of toxic Pseudo-nitzschia spp. over Louisiana oyster beds ( Crassostrea virginica; eastern oyster) there have been no documented cases of amnesic shellfish poisoning (ASP) in the state. Two possible explanations are that oysters do not readily feed on long pointed chains of Pseudo-nitzschia cells or they discriminate against toxic cells while grazing. To test these hypotheses, short-term grazing experiments were conducted with several diatoms, including the domoic acid (DA)-producing Pseudo-nitzschia multiseries (1.31 ± 0.057 pg DA cell −1) and the non-toxic Pseudo-nitzschia delicatissima, Thalassiosira weissflogii, and Ditylum brightwellii. Grazing rates on the small centric species T. weissflogii were significantly higher than on the larger and pointier D. brightwellii and either Pseudo-nitzschia species. Grazing on toxic P. multiseries and non-toxic P. delicatissima was not significantly different. Pseudofeces production was higher and feces production was occasionally lower in oysters fed Pseudo-nitzschia spp. than in oysters fed the other two diatoms. Our data demonstrate lower filtration rates of C. virginica on Pseudo-nitzschia spp. relative to the other diatoms tested and comparable filtration on toxic and non-toxic Pseudo-nitzschia spp. These findings suggest that eastern oysters do not discriminate amongst food types due to DA content.

Distribution, Abundance and Domoic Acid Analysis of the Toxic Diatom Genus Pseudo-nitzschia from the Chesapeake Bay

Very little research has been conducted on mid-Atlantic estuarine populations of the diatom Pseudo-nitzschia despite recent evidence of toxicity in regional isolates. We collected field samples from the Chesapeake Bay region from 2002 to 2007 for Pseudo-nitzschia enumeration and toxin analysis. Abundances of Pseudo-nitzschia were highest in the winter and spring at ∼103 cells ml−1. Domoic acid (DA) was detectable in 42% of samples tested, but concentrations were generally low, ranging from 0 to 1,037 pg DA ml−1 (mean 176 pg DA ml−1), and DA cell quota ranged from <0.1 to 49.4 pg DA per cell (mean 1.4 pg DA per cell). Although Pseudo-nitzschia populations were observed year round when salinity was ≥5, populations were highest from February to May when temperatures were low (2–15°C) and salinity relatively high (≥10). Six species of Pseudo-nitzschia were identified via transmission electron microscopy of the samples: P. pungens (Grunow ex Cleve) Hasle, P. calliantha Lundholm, Moestrup et Hasle, P. subpacifica (Hasle) Hasle, P. cuspidata (Hasle) Hasle emend. Lundholm, Moestrup et Hasle, P. fraudulenta (Cleve) Hasle and P. multiseries (Hasle) Hasle. P. calliantha was the most common and not previously reported from the Chesapeake Bay. Of these species, P. pungens, P. calliantha, P. cuspidata, P. fraudulenta and P. multiseries are known to produce domoic acid.

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.

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 km 2 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.

The effect of Fe and Cu on growth and domoic acid production by Pseudo-nitzschiamultiseries and Pseudo‐nitzschia australis

Toxigenic pennate diatoms of the genus Pseudo‐nitzschia produce domoic acid (DA), the neurotoxin linked to amnesic shellfish poisoning. We investigated how Fe and Cu affect growth and DA production by P. multiseries and P. australis clones isolated from Monterey Bay, California. Growth rates of these species could be inhibited by both Fe limitation (pFe 20.5, µ = 50% ³max) and Cu toxicity (pCu 10.5, 00B5 =5 30−50% ³max). The rate of DA production during the exponential phase was a significant inverse function of cellular growth rates (P &lt; 0.02). Voltammetric measurements of dissolved DA‐equivalents in short‐term experiments indicated that the cellular production of DA increased from ~5 amol DA cell−1 h−1 under optimal growth conditions to ~40 amol DA cell−1 h−1 under Fe‐deficient conditions and ~105 amol DA cell−1 h−1 under Cu‐stressed conditions. The DA was released to the medium under metal stress conditions, with intracellular DA concentrations decreasing relative to nonstressed cells. Fe uptake rates by P. multiseries were slow compared to other marine diatoms in the absence of dissolved DA but were enhanced threefold (P &lt; 0.03) by adding DA to the medium. DA addition also partially alleviated toxic Cu conditions. Our findings suggest that DA production during exponential growth of these two toxigenic Pseudo‐nitzschia species is directly induced by Fe‐deficient or Cu stress conditions and that 95% of this DA is actively released into the medium. Changing trace metal conditions in coastal waters therefore may have a profound effect upon intracellular DA concentrations and thereby influence the toxic effect of these harmful bloom events.

Pseudo-nitzschia sp. cf. pseudodelicatissima - a confirmed producer of domoic acid from the northern Gulf of Mexico

Domoic acid (DA), a potent neurotoxin, is synthesized by certain members of the ubiq- uitous marine diatom genus Pseudo-nitzschia. We recently detected elevated concentrations of DA in phytoplankton field samples from the northern Gulf of Mexico. In searching for a possible source of the toxin, we used a receptor-binding assay to detect DA activity in cultures of P. sp. cf. pseudodeli- catissima (Hasle) isolated from this region and confirmed its presence in 2 of 7 clones using liquid chromatography coupled with tandem mass-spectrometric detection (LC-MS/MS). Unlike other toxic Pseudo-nitzschia species examined previously (e.g., P. multiseries, P. australis), cellular levels and net production of DA in these clones were highest in the early exponential phase, while the population growth rate was high and cell concentration was low. There was a negative correlation between cel- lular DA and cell concentration. The maximum cellular DA activity in cultures was 36 fg DA equiv. cell -1 . No net toxin production was evident in the stationary phase, yet extracellular DA levels increased markedly during this period to as much as 88% of the total DA in the cultures. Interest- ingly, these 2 toxic clones were able to enlarge their cell size after the apical axes declined to 15 to 25 µm, and these larger cells had considerably higher levels of DA than the original small cells. This study unequivocally establishes P. sp. cf. pseudodelicatissima as a source of DA in the northern Gulf of Mexico. Moreover, our work suggests that rapidly growing, rather than nutrient-limited, popula- tions of this diatom should yield maximum net DA production rates and DA cell quotas. Thus, the presence of P. sp. cf. pseudodelicatissima cells, even at the low levels of early, rapidly growing bloom stages, can potentially lead to toxic events.