Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France)

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta) with high pDA concentrations (∼1400 ng l⁻¹) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N < 1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios.     

A toxic Pseudo-nitzschia bloom in Todos Santos Bay,northwestern Baja California,Mexico

A toxic Pseudo-nitzschia spp. bloom in the Todos Santos Bay area (31.8°N), Mexico, is described. This is the southernmost report of the presence of domoic acid (DA) in the California Current System and it is also the first report of the distribution of toxic Pseudo-nitzschia species and DA on the Baja California west coast. The maximum cell abundance of Pseudo-nitzschia was 3.02 × 10⁵ cells L^?1 and the maximum concentration of DA in particulate matter (pDA) was 0.86 μg L^?1. P. australis constituted the major proportion of cells identified as Pseudo-nitzschia. The environmental conditions associated with wind-driven upwelling were the cause for the accumulation of toxic cells. Maximum pDA and cell concentration were detected around 14 °C. The ratio of the concentration of macronutrients seemed to be the important factor for the accumulation of P. australis. The highest cell abundance was detected in areas with a high Si(OH)₄ to N ratio in the entire water column. Therefore, the relative increase of silicate concentration related to upwelling conditions was the probable cause for the accumulation of P. australis. Maximum photosystem II (PSII) quantum efficiency of charge separation (F_v/F_m) was negatively correlated to the pDA to fucoxanthin ratio. This ratio was used in this work as an index of cellular DA content. Therefore, the photosynthetic competence of the cells might be an important factor that affected their DA cellular content.     

Characterization of a toxic Pseudo-nitzschia spp. bloom in the Northern Gulf of Mexico associated with domoic acid accumulation in fish

A toxic bloom of Pseudo-nitzschia spp. was observed in the Alabama coastal waters of the northern Gulf of Mexico (NGOM) in June 2009 that resulted in the accumulation of domoic acid (DA) in fish. The bloom initiated following a large storm event that likely caused increased groundwater discharge 16–20 days prior to peak densities. Eleven sites, located in littoral shoreline waters and inshore embayments spanning the entire Alabama NGOM coastline, were sampled during peak densities to assess Pseudo-nitzschia species composition and toxicity, and associated water-quality parameters. Small fish (0.27–11.9 g body weight) were collected at six of these sites for analysis of DA content. High Pseudo-nitzschia spp. densities (8.27 × 10⁴–5.05 × 10⁶ cell l⁻¹) were detected at eight sites located in the littoral shoreline and particulate DA was detected at six of these littoral sites (48.0–540 pg ml⁻¹). The bloom consisted primarily (>90%) of Pseudo-nitzschia subfraudulenta, a species previously characterized as forming only a minor component of Pseudo-nitzschia assemblages and not known to produce DA. Pseudo-nitzschia spp. were at low densities or not detected at the inshore sites and DA was detected at these sites. Pseudo-nitzschia spp. density varied along an estuarine gradient, with greater densities occurring in the most saline, clear, and nutrient-poor waters. Cell density was strongly and negatively correlated with silicate (Si) concentrations and the ratios of silicate to dissolved inorganic nitrogen and phosphate (Si:DIN and Si:PO₄). Cell toxin quota was negatively correlated with phosphate, and strongly and positively correlated with the ratio of total nitrogen to total phosphorus (TN:TP). These relationships are consistent with previous observations that indicate Pseudo-nitzschia spp. density and toxicity are likely to be greater in high salinity, high irradiance, and nutrient-poor waters. DA was detected in 128 of 131 (98%) of the fish collected, which included seven primary and secondary consumer species. This is the first demonstration of trophic transfer of DA in this region of the NGOM, indicating that toxic blooms of Pseudo-nitzschia spp. in Alabama coastal waters have the potential to transfer DA to recreationally and commercially important fish species.     

Understanding the blob bloom: Warming increases toxicity and abundance of the harmful bloom diatom Pseudo-nitzschia in California coastal waters

The toxic diatom genus Pseudo-nitzschia produces environmentally damaging harmful algal blooms (HABs) along the U.S. west coast and elsewhere, and a recent ocean warming event coincided with toxic blooms of record extent. This study examined the effects of temperature on growth, domoic acid toxin production, and competitive dominance of two Pseudo-nitzschia species from Southern California. Growth rates of cultured P. australis were maximal at 23 °C (∼0.8 d⁻¹), similar to the maximum temperature recorded during the 2014–2015 warming anomaly, and decreased to ∼0.1 d⁻¹ by 30 °C. In contrast, cellular domoic acid concentrations only became detectable at 23 °C, and increased to maximum levels at 30 °C. In two incubation experiments using natural Southern California phytoplankton communities, warming also increased the relative abundance of another potentially toxic local species, P. delicatissima. These results suggest that both the toxicity and the competitive success of particular Pseudo-nitzschia spp. can be positively correlated with temperature, and therefore there is a need to determine whether harmful blooms of this diatom genus may be increasingly prevalent in a warmer future coastal ocean.     

Domoic acid in a marine pelagic food web: Exposure of southern right whales Eubalaena australis to domoic acid on the Península Valdés calving ground,Argentina

The gulfs that surround Península Valdés (PV), Golfo Nuevo and Golfo San José in Argentina, are important calving grounds for the southern right whale Eubalaena australis. However, high calf mortality events in recent years could be associated with phycotoxin exposure. The present study evaluated the transfer of domoic acid (DA) from Pseudo-nitzschia spp., potential producers of DA, to living and dead right whales via zooplanktonic vectors, while the whales are on their calving ground at PV. Phytoplankton and mesozooplankton (primary prey of the right whales at PV and potential grazers of Pseudo-nitzschia cells) were collected during the 2015 whale season and analyzed for species composition and abundance. DA was measured in plankton and fecal whale samples (collected during whale seasons 2013, 2014 and 2015) using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The genus Pseudo-nitzschia was present in both gulfs with abundances ranging from 4.4 × 10² and 4.56 × 10⁵ cell l⁻¹. Pseudo-nitzschia australis had the highest abundance with up to 4.56 × 10⁵ cell l⁻¹. DA in phytoplankton was generally low, with the exception of samples collected during a P. australis bloom. No clear correlation was found between DA in phytoplankton and mesozooplankton samples. The predominance of copepods in mesozooplankton samples indicates that they were the primary vector for the transfer of DA from Pseudo-nitzschia spp. to higher trophic levels. High levels of DA were detected in four whale fecal samples (ranging from 0.30 to 710 μg g⁻¹ dry weight of fecal sample or from 0.05 and 113.6 μg g⁻¹ wet weight assuming a mean water content of 84%). The maximum level of DA detected in fecal samples (710 μg DA g⁻¹ dry weight of fecal sample) is the highest reported in southern right whales to date. The current findings demonstrate for the first time that southern right whales, E. australis, are exposed to DA via copepods as vectors during their calving season in the gulfs of PV.     

Marine snow formation by the toxin-producing diatom,Pseudo-nitzschia australis

The formation of marine snow (MS) by the toxic diatom Pseudo-nitschia australis was simulated using a roller table experiment. Concentrations of particulate and dissolved domoic acid (pDA and dDA) differed significantly among exponential phase and MS formation under simulated near surface conditions (16 °C/12:12-dark:light cycle) and also differed compared to subsequent particle decomposition at 4 °C in the dark, mimicking conditions in deeper waters. Particulate DA was first detected at the onset of exponential growth, reached maximum levels associated with MS aggregates (1.21 ± 0.24 ng mL⁻¹) and declined at an average loss rate of ∼1.2% pDA day⁻¹ during particle decomposition. Dissolved DA concentrations increased throughout the experiment and reached a maximum of ∼20 ng mL⁻¹ at final sampling on day 88. The succession by P. australis from active growth to aggregation resulted in increasing MS toxicity and based on DA loading of particles and known in situ sinking speeds, a significant amount of toxin could have easily reached the deeper ocean or seafloor. MS formation was further associated with significant dDA accumulation at a ratio of pDA: dDA: cumulative dDA of approximately 1:10:100. Overall, this study confirms that MS functions as a major vector for toxin flux to depth, that Pseudo-nitzschia-derived aggregates should be considered ‘toxic snow’ for MS-associated organisms, and that effects of MS toxicity on interactions with aggregate-associated microbes and zooplankton consumers warrant further consideration.     

Variation in the abundance of Pseudo-nitzschia and domoic acid with surf zone type

Most harmful algal blooms (HAB) originate away from the shore and, for them to endanger human health, they must be first transported to shore after which they must enter the surf zone where they can be feed upon by filter feeders. The last step in this sequence, entrance into the surf zone, depends on surf zone hydrodynamics. During two 30-day periods, we sampled Pseudo-nitzschia and particulate domoic acid (pDA) in and offshore of a more dissipative surf zone at Sand City, California (2010) and sampled Pseudo-nitzschia in and out of reflective surf zones at a beach and rocky shores at Carmel River State Beach, California (2011). At Sand City, we measured domoic acid in sand crabs, Emerita analoga. In the more dissipative surf zone, concentrations of Pseudo-nitzschia and pDA were an order of magnitude higher in samples from a rip current than in samples collected just seaward of the surf zone and were 1000 times more abundant than in samples from the shoals separating rip currents. Domoic acid was present in all the Emerita samples and varied directly with the concentration of pDA and Pseudo-nitzschia in the rip current. In the more reflective surf zones, Pseudo-nitzschia concentrations were 1–2 orders of magnitude lower than in samples from 125 and 20 m from shore. Surf zone hydrodynamics affects the ingress of Pseudo-nitzschia into surf zones and the exposure of intertidal organisms to HABs on the inner shelf.     

An assessment of Pseudo-nitzschia population dynamics and domoic acid production in coastal Louisiana

Over 1200 samples were collected from Louisiana estuarine and coastal shelf waters between 1989 and 2002, and analyzed to examine the population dynamics of Pseudo-nitzschia and to assess the potential threat posed by domoic acid (DA), a potent neurotoxin produced by some members within this toxigenic diatom genus. Results demonstrated that three species in this region (Pseudo-nitzschia multiseries, P. pseudodelicatissima complex, P. delicatissima) produce DA, and that particulate toxin levels were highest (up to 3.05 μg L⁻¹) during the spring bloom, while cellular concentrations were highest in the winter/early spring when P. multiseries was most abundant (up to 30 pg cell⁻¹). These particulate toxin levels are comparable to those seen in other regions (e.g., United States west coast) where DA poisoning events have occurred in the past. Pseudo-nitzschia were most abundant under dissolved inorganic nitrogen-replete conditions coupled with lower silicate and/or phosphate concentrations, and in the early spring months when temperatures were cooler. Pseudo-nitzschia were occasionally well-represented in the phytoplankton assemblage (≥10⁶ cells L⁻¹ in 14% of samples, over 50% of total phytoplankton in 5% of samples), indicating that planktivores (e.g., Gulf menhaden, Brevoortia patronus) may have little choice but to consume Pseudo-nitzschia cells, thereby providing potential vectors for DA transfer to higher trophic levels. By comparison, eastern oysters (Crassostrea virginica) present in estuarine waters may be more exposed to this toxin when Pseudo-nitzschia cells are part of a mixed assemblage, reducing selective grazing by these bivalves. C. virginica may thus represent the most effective vector for DA exposure in humans.     

First record of the dynamics of domoic acid producing Pseudo-nitzschia spp. in Indonesian waters as a function of environmental variability

Within the past few decades, harmful algal blooms (HABs) have occurred frequently in Indonesian waters, resulting in environmental degradation, economic loss and human health problems. So far, HAB related studies mainly addressed ecological traits and species distribution, yet toxin measurements were virtually absent for Indonesian waters. The aim of the present study was to explore variability of the potentially toxic marine diatom genus Pseudo-nitzschia, as well as its neurotoxin domoic acid as a function of environmental conditions in Ambon Bay, eastern Indonesia. Weekly phytoplankton samples, oceanographic (CTD, nutrients) and meteorological (precipitation, wind) parameters were analyzed at 5 stations in the bay during the dry and wet seasons of 2018. Liquid chromatography – tandem mass spectrometry (LC–MS/MS) was used to detect particulate DA (pDA). Vegetative cells of Pseudo-nitzschia spp. and pDA were found in 98.6% and 51.4% of the samples, respectively. pDA levels were low, yet detected throughout the campaign, implying that Ambon Bay might potentially be subject to amnesic shellfish poisoning. The highest levels of both Pseudo-nitzschia spp. cell abundance and pDA were found in the wet season, showing a strong positive correlation between both parameters, compared to the dry season, (r = 0.87 and r = 0.66 (p < 0.01), respectively). Statistical analyses revealed that temperature and mixed layer depth positively correlated with Pseudo-nitzschia spp. and pDA during the dry season, while ammonium showed positive correlations in both seasons. This study represents the first successful investigation of the presence and variability of Pseudo-nitzschia spp. and its neurotoxin DA in Indonesian waters.     

The relationship between Pseudo-nitzschia (Peragallo) and domoic acid in Scottish shellfish

The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 μg g⁻¹ (now expressed as mg kg⁻¹) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 μg g⁻¹ (now mg kg⁻¹) to <20 μg g⁻¹ (now mg kg^‐1). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation.     

The influence of the Pseudo-nitzschia toxin,domoic acid,on microzooplankton grazing and growth: A field and laboratory assessment

We conducted field and laboratory experiments to determine whether the Pseudo-nitzschia-derived metabolite, domoic acid (DA), functions as a microzooplankton grazing suppressant. Using the seawater dilution technique in natural plankton communities along the Pacific Northwest coast, we found no significant relationship between dissolved DA and microzooplankton grazing rate on Pseudo-nitzschia spp. Dilution experiments amended with either 50 or 80 nM dissolved DA also showed no evidence that microzooplankton community grazing was affected by DA. The relationship between Pseudo-nitzschia spp. intracellular DA and microzooplankton grazing was less clear. On a subset of data where small Pseudo-nitzschia spp. cells dominated community composition, an apparent negative relationship between intracellular DA and microzooplankton grazing was observed. However, we provide evidence that this relationship is a microzooplankton response to Pseudo-nitzschia spp. growth rate, rather than cellular DA. In laboratory experiments, two diatom-consuming dinoflagellates, Protoperidinium excentricum and P. pellucidum, were fed single and mixed diets of a toxic and non-toxic Pseudo-nitzschia species and an optimal prey, Ditylum brightwellii. P. excentricum did not grow or ingest either the toxic or non-toxic Pseudo-nitzschia. However, P. pellucidum grew as well on the toxic Pseudo-nitzschia multiseries as it did on D. brightwellii, but did not grow on the non-toxic Pseudo-nitzschia pungens. Both dinoflagellates were capable of growing if Pseudo-nitzschia spp. diets were mixed with D. brightwellii. Addition of dissolved DA also had no negative effect on dinoflagellate growth when fed the optimal diatom diet. We conclude that domoic acid has no functional role in deterring microzooplankton grazing or growth rates. Further, our findings highlight the difficulty of defining the complex mechanisms that regulate predator and prey interactions within microplankton food webs.     

Remote,subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

Estimating spatial variation in the abundance of potential microcystin-producing Microcystis spp. using real-time PCR during summer bloom in Lake Taihu

Lake Taihu, which is the third largest freshwater lakes in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, quantitative real-time PCR assays targeting on phycocyanin intergenic spacer (PC-IGS) and a microcystin synthetase gene mcyD were established, respectively. Water samples collected from eight sampling sites (including Zhushan Bay (N5), Meiliang Bay (N2), Gonghu Bay (N4), West lake areas (W2 and W4), south-middle lake areas (S2, S4 and S5)) in August of 2009 and 2010 were analyzed using real time PCR for the distribution and abundance of toxic and total Microcystis populations. The results showed that Microcystis exists as a mixed population of potential toxic and non-toxic genotypes, and there was significant spatial changes in the abundance of potential toxic Microcystis on the basis of quantification by quantitative real-time PCR analysis: the abundance of toxic Microcystis population in 2009 and 2010 varied from 4.08 × 10⁴ to 8.28 × 10⁶ copies mL^?1, from 4.45 × 10⁵ to 5.22 × 10⁷ copies mL^?1, respectively. Meanwhile the ratio of the mcyD subpopulation to the total Microcystis varied considerably, from 5.7% to 41.1% in 2009 and from 10.3% to 65.8% in 2010 in all sampling sites, and the value is high in Zhushan Bay and Meiliang Bay with the high level of eutrophication. Correlation analysis showed the abundance of toxic and total Microcystis being strongly related (P < 0.01). However, there is different effects of environmental factors on the abundance of toxic and non-toxic Microcystis populations. The abundance of toxic and total Microcystis populations were positively correlated with chlorophyll-a (Chl-a) concentration (P < 0.01) suggesting that Microcystis is dominated genera of cyanobacterial bloom in Lake Taihu. It was also found that the abundance of toxic Microcystis and the proportion of toxic subpopulation to the total Microcystis were positively correlated with total phosphorus and orthophosphate concentrations (P < 0.01), whereas there was no significant correlation with total nitrogen and nitrate concentration (P > 0.05). All data suggest that phosphorus concentration is a critical factor for determining the abundance of toxic Microcystis population.     

Can cryptophyte abundance trigger toxic Karlodinium veneficum blooms in eutrophic estuaries?

Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (10² to 10³ mL⁻¹), but capable of forming blooms of 10⁴ to 10⁵ cells mL⁻¹ under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421 μm³ per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability.     

Changes in abundances of Alexandrium tamarense resting cysts after the tsunami caused by the Great East Japan Earthquake in Funka Bay,Hokkaido, Japan

The 2011 Great East Japan Earthquake and the subsequent huge tsunami greatly affected both human activity and the coastal marine ecosystem along the Pacific coast of Japan. The tsunami also reached Funka Bay in northern Japan and caused serious damage to the scallop cultures there, and this tsunami was believed to have affected the coastal environments in the bay. Therefore, we investigated the changes in the spatial abundance and distribution of the toxic dinoflagellates Alexandrium tamarense cysts before the tsunami (August 2010) and after the tsunami (May 2011, August 2011, May 2012 and August 2012) in the bay. Further, monthly sampling was conducted after the tsunami to identify seasonal changes of Alexandrium catenella/tamarense cysts and vegetative cells. Significant increases were observed in the populations of A. catenella/tamarense cysts, comparing the abundances before the tsunami (in August 2010; 70 ± 61 cysts g⁻¹ wet sediment) to those just after it (in May 2011; 108 ± 84 cysts g⁻¹ wet sediment), and both A. tamarense bloom (a maximum density was 1.3 × 10³ cells L⁻¹) and PSP (Paralytic Shellfish Poisoning) toxin contamination of scallops (9.4 mouse unit g⁻¹ was recorded) occurred in the bay. Seasonal sampling also revealed that the encystment of A. tamarense and the supply of the cysts to bottom sediments did not occur in the bay from September to April. These results strongly suggested that the mixing of the bottom sediments by the tsunami caused the accumulation of the toxic A. tamarense cysts in the surface of bottom sediment through the process of redeposition in Funka Bay. Moreover, this cyst deposition may have contributed to the toxic bloom formation as a seed population in the spring of 2011.     

Toxicity studies of Trichodesmium erythraeum (Ehrenberg, 1830) bloom extracts,from Phoenix Bay,Port Blair,Andamans

Occurrence of toxic cyanobacterial blooms has become a worldwide problem, increasing the risk of human poisoning due to consumption of seafood contaminated with cyanotoxins. Though no such cases of human intoxication due to toxic blooms have been reported so far from India, most of the studies related to blooms have been restricted to reporting of a bloom and/or antimicrobial activity of its extract. Detailed toxicity study of cyanobacterial blooms are lacking. A study on the toxicity of a dense bloom (14.56 × 10⁶ trichomes L⁻¹) of the marine diazotrophic cyanobacteria, Trichodesmium erythraeum, observed in the coastal waters of Phoenix Bay, Port Blair, Andamans was undertaken. The significance of this bloom is that it was a single species and had conspicuously inhibited the growth of other phytoplankton and complete exclusion of zooplankton from the bloom region, intimating the involvement of toxins in the bloom. The cyanobacterial extracts showed prominent antimicrobial activity against certain human pathogenic bacteria and fungi. Studies on the toxicity of the cyanobacterial extracts was carried out using brine shrimp bioassay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and comet assay. The cyanobacterial extract exhibited toxic effect to Artemia salina causing mortality of up to 40% after 48 h at a concentration of 1 mg mL⁻¹, while it induced cytotoxicity in cell lines (HepG2 and HaCat) and caused DNA damage in human lymphocytes in vitro.     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

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:NO3 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:NO₃ (2.5) or low Si:NO₃ (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.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Seasonal dynamics and spatial distribution of epiphytic dinoflagellates in Peter the Great Bay (Sea of Japan) with special emphasis on Ostreopsis species

Studies of epiphytic dinoflagellates in Peter the Great Bay, Sea of Japan in 2008–2011 revealed the presence of 13 species. Five of the species are known as potentially toxic: Amphidinium carterae, A. operculatum, Ostreopsis cf. ovata, O. cf. siamensis and Prorocentrum lima. The maximum species richness and abundance of epiphytic dinoflagellates were observed in autumn (from September to October). Ostreopsis spp. were most widely distributed and predominated, amounting to 99% of the total density of dinoflagellates. Multi-year seasonal dynamics of Ostreopsis spp. in Peter the Great Bay showed that these cells appear as epiphyton in August after maximum warming of surface waters (22–24 °С) and disappear in early November, when the water temperature decreases below 7 °С. Ostreopsis spp. proliferation occurred in September, when the water temperature was 17.2–21.0 °C. The highest densities of Ostreopsis spp. were recorded on September 9, 2010 on the rhodophyte Neorhodomela aculeata – 230 × 10³ cells g⁻¹ DW or 52 × 10³ cells g⁻¹ FW. The spatial distribution of epiphytic dinoflagellates was investigated in the near-shore areas of Peter the Great Bay during the second half of September 2010 to evaluate the role of hydrodynamic conditions. Epiphytic dinoflagellates were not found in sheltered sites having weak mixing hydrodynamics. However, the abundances of Ostreopsis spp. were significantly higher at sites having moderate turbulence compared to biotopes experiencing strong wave action. Densities of Ostreopsis spp. were not significantly different on macrophytes with branched thallus of all taxonomic divisions. However, the average cell densities of Ostreopsis spp. on green algae with branched thallus were significantly higher than on green algae having laminar thallus.