Toxin Cell Quota Research Articles

Dynamic variations in profile of intra- and extracellular paralytic shellfish toxins during the growth cycle of Alexandrium species and their accumulation in mussels

The toxigenic Alexandrium species can secrete paralytic shellfish toxins (PSTs) into seawater during their growth, potentially threatening marine organisms and human health. The release of intracellular toxins during the growth cycle of Alexandrium spp. and the uptake of dissolved PSTs by shellfish are still not clear. Three strains of A. minutum GY-H46, A. minutum ATDH and A. pacificum ATHK were used to explore the dynamic variations in profile of intra- and extracellular toxins during their growth cycles. And a comparative study was carried out on the accumulation and tissue distribution of PSTs in mussels Mytilus unguiculatus exposed to the dissolved and particulate toxins during a 5-days period. Results showed that the toxin cell quotas increased rapidly during the early exponential growth period, then decreased gradually and plateaued at the stationary growth period for ATHK and ATDH, while gradually increased during the stationary growth period for GY-H46. The toxin cell quotas for GY-H46, ATDH and ATHK ranged from 8.3 to 21.5, 5.3 to 11.7 and 7.4 to 9.3 fmol cell−1, respectively, during the whole growth cycle. The contents of extracellular toxins increased gradually throughout the growth of GY-H46, ATDH and ATHK, and the molar mass percentage of extracellular toxins ranged from 53% to 72%, 20% to 46% and 36% to 68%, respectively. Mussels can actively uptake the dissolved PSTs from seawater medium, and mainly accumulate toxins in the digestive glands. The accumulation level of PSTs from the dissolved phase by mussels was about 1% of that from feeding on microalgal cells. The results of this study will contribute to a comprehensive understanding of the dynamic release of intracellular PSTs during microalgal growth and the accumulation of dissolved toxins by shellfish.

Novel insights into toxin changes associated with the growth of Alexandrium pacificum: Revealing active toxin-secretion ability and toxin cell quota variation

Paralytic shellfish toxins (PSTs) are widely distributed globally and are produced by Alexandrium pacificum in marine system. However, the characteristics of toxins producing and secreting associated with growth phases are still unclear, especially whether A. pacificum has the ability to actively secrete PSTs is controversial. In this study, variation characteristics of intracellular and extracellular PSTs contents associated with A. pacificum growth phases were investigated thoroughly. The results showed that intracellular and extracellular PSTs contents increased sharply during the exponential phase. But during the stationary phase, the intracellular PSTs content increased by only 26 %, and the extracellular PSTs content did not increase significantly. Since the increase in extracellular PSTs content mainly occurred at the exponential phase, when most cells were living, we speculated that active PSTs secretion of living cells might be an important production pathway of extracellular toxins besides leakage from dead cells. Furthermore, toxin cell quota variation associated with the growth phase was analysed. In the exponential phase, the toxin cell quota first increased and then decreased, with a maximum of 19.02 ± 1.80 fmol/cell at 6 d. However, after entering the stationary phase, this value slowly increased again, suggesting that vigilance should be raised for the plateau of Alexandrium blooms. In addition, cells in the exponential phase mainly produced O-sulfated components such as GTX1&4, cells in the stationary phase mainly produced O-sulfate-free components such as GTX5. In this study, the toxigenic rules of A. pacificum were comprehensively uncovered, which provided theoretical guidance for the prevention and mitigation of A. pacificum blooms.

Asynchrony of Gambierdiscus spp. Abundance and Toxicity in the U.S. Virgin Islands: Implications for Monitoring and Management of Ciguatera.

Ciguatera poisoning (CP) poses a significant threat to ecosystem services and fishery resources in coastal communities. The CP-causative ciguatoxins (CTXs) are produced by benthic dinoflagellates including Gambierdiscus and Fukuyoa spp., and enter reef food webs via grazing on macroalgal substrates. In this study, we report on a 3-year monthly time series in St. Thomas, US Virgin Islands where Gambierdiscus spp. abundance and Caribbean-CTX toxicity in benthic samples were compared to key environmental factors, including temperature, salinity, nutrients, benthic cover, and physical data. We found that peak Gambierdiscus abundance occurred in summer while CTX-specific toxicity peaked in cooler months (February–May) when the mean water temperatures were approximately 26–28 °C. These trends were most evident at deeper offshore sites where macroalgal cover was highest year-round. Other environmental parameters were not correlated with the CTX variability observed over time. The asynchrony between Gambierdiscus spp. abundance and toxicity reflects potential differences in toxin cell quotas among Gambierdiscus species with concomitant variability in their abundances throughout the year. These results have significant implications for monitoring and management of benthic harmful algal blooms and highlights potential seasonal and highly-localized pulses in reef toxin loads that may be transferred to higher trophic levels.

Effects of Polystyrene Microplastics on Growth and Toxin Production of Alexandrium pacificum.

Microplastics (MP) widely distributed in aquatic environments have adverse effects on aquatic organisms. Currently, the impact of MP on toxigenic red tide microalgae is poorly understood. In this study, the strain of Alexandrium pacificum ATHK, typically producing paralytic shellfish toxins (PST), was selected as the target. Effects of 1 and 0.1 μm polystyrene MP with three concentration gradients (5 mg L−1, 25 mg L−1 and 100 mg L−1) on the growth, chlorophyll a (Chl a), photosynthetic activity (Fv/Fm) and PST production of ATHK were explored. Results showed that the high concentration (100 mg L−1) of 1 μm and 0.1 μm MP significantly inhibited the growth of ATHK, and the inhibition depended on the size and concentration of MP. Contents of Chl a showed an increase with various degrees after MP exposure in all cases. The photosynthesis indicator Fv/Fm of ATHK was significantly inhibited in the first 11 days, then gradually returned to the level of control group at day 13, and finally was gradually inhibited in the 1 μm MP treatments, and promotion or inhibition to some degree also occurred at different periods after exposure to 0.1 μm MP. Overall, both particle sizes of MP at 5 and 25 mg L−1 had no significant effect on cell toxin quota, and the high concentration 100 mg L−1 significantly promoted the PST biosynthesis on the day 7, 11 and 15. No significant difference occurred in the cell toxin quota and the total toxin content in all treatments at the end of the experiment (day 21). All MP treatments did not change the toxin profiles of ATHK, nor did the relative molar percentage of main PST components. The growth of ATHK, Chl a content, Fv/Fm and toxin production were not affected by MP shading. This is the first report on the effects of MP on the PST-producing microalgae, which will improve the understanding of the adverse impact of MP on the growth and toxin production of A. pacificum.

Morphological changes and genome evolution in Raphidiopsis raciborskii CS-506 after 23 years in living culture

ABSTRACT Microscopic organisms maintained in long-term living monocultures undergo evolution, adapting to the stable laboratory environment. Culture adaptation can result in isolates no longer being representative of their native environmental niche. This study characterized the morphology and compared the full genomes of four daughter cultures of the toxic cyanobacterium Raphidiopsis raciborskii CS-506 maintained in living culture for 23 years. The four daughter cultures were split from the parent culture between 2 and 10 years ago. The morphotypes of two cultures had changed from coiled to straight (CS-506A, B), while CS-506C, D had smaller cells and shorter coiled trichomes. The toxin cell quota had increased for cultures CS-506A-C but decreased in CS-506D compared to 2012 measurements. Culture CS-506C was unable to produce heterocysts or fix atmospheric nitrogen. The core genome, comprising 78% of all genes and all primary metabolic functions, was unchanged. The variable genome, ~12%, showed difference in gene presence/absence, and ~1% of each genome was unique proteins, with evidence of horizontal gene transfer from contaminating bacteria and multiple gene loss events, and an overall strategy of genome reduction. The changes in morphology, nitrogen fixation and toxin cell quota were not supported by identifiable genome changes, with 100% synteny across nifHDK and cyr gene clusters. The results suggest that R. raciborskii CS-506 has a stable core genome, heritable phenotypes, and a variable genome generated through horizontal gene transfer and gene loss.

Risk Management of Cyanotoxins in Singapore

Cyanotoxins produced by cyanobacteria pose significant challenges to water resource management due to the potential impacts they have on human health. Cylindrospermopsin (CYN) and microcystins (MC) are the more commonly detected cyanotoxins in Singapore’s reservoirs. Among the MC congeners monitored locally, the most frequently detected variants are MC-RR (37.6%), followed by MC-LR (25.6%). MC-LA and MC-YR are the least frequently detected variants (7.1%). No cyanotoxins have been detected in Singapore’s treated drinking water. Singapore’s National Water Agency (PUB) and the National Environment Agency (NEA) developed recreational water quality guidelines using Chl a concentrations of 50 μg/L. In local surface waters, long-term data showed that at 50 μg/L of Chl a, MC-LR concentrations ranged from a concentrations, Microcystis cell counts in reservoir water have also been used to manage cyanotoxin risk in drinking water. Specifically, routinely monitored data from all 17 Singapore reservoirs indicated that to keep MC-LR concentrations below the WHO provisional guideline of 1 μg/L in drinking water, Microcystis cell counts needed to be Microcystis isolates showed M. aeruginosa produced the most MC compared to M. ichthyoblabe, M. flos-aquae, and M. viridis. Based on the maximum toxin cell quota equivalent to the WHO provisional guideline for MC-LR of 1 μg/L in drinking water, a 5000 cells/ml cell count guideline was derived for M. aeruginosa. This cell count has also been incorporated into Singapore’s cyanotoxin risk management framework for reservoirs.

The effect of temperature and salinity on growth rate and azaspiracid cell quotas in two strains of Azadinium poporum (Dinophyceae) from Puget Sound, Washington State

Azaspiracids (AZA) are novel lipophilic polyether marine biotoxins associated with azaspiracid shellfish poisoning (AZP). Azaspiracid-59 (AZA-59) is a new AZA that was recently detected in strains of Azadinium poporum from Puget Sound, Washington State. In order to understand how environmental factors affect AZA abundances in Puget Sound, a laboratory experiment was conducted with two local strains of A. poporum to estimate the growth rate and AZA-59 (both intra- and extracellular) cell quotas along temperature and salinity gradients. Both strains of A. poporum grew across a wide range of temperatures (6.7 °C to 25.0 °C), and salinities (15 to 35). Growth rates increased with increasing temperature up to 20.0 °C, with a range from 0.10 d−1 to 0.42 d−1. Both strains of A. poporum showed variable growth rates from 0.26 d−1 to 0.38 d−1 at salinities from 15 to 35. The percentage of intracellular AZA-59 in both strains was generally higher in exponential than in stationary phase along temperature and salinity gradients, indicating higher retention of toxin in actively growing cells. Cellular toxin quotas varied by strain in both the temperature and salinity treatments but were highest at the lowest growth rates, especially for the faster growing strain, NWFSC1011.Consistent with laboratory experiments, field investigations in Sequim Bay, WA, during 2016–2018 showed that A. poporum was detected when salinity and temperature became favorable to higher growth rates in June and July. Although current field data of A. poporum in Puget Sound indicate a generally low abundance, the potential of local A. poporum to adapt to and grow in a wide range of temperature and salinity may open future windows for blooms. Although increased temperatures, anticipated for the Puget Sound region over the next decades, will enhance the growth of A. poporum, these higher temperatures will not necessarily support higher toxin cell quotas. Additional sampling and assessment of the total toxicity of AZA-59 will provide the basis for a more accurate estimation of risk for azaspiracid poisoning in Puget Sound shellfish.

Effects of Temperature, Growth Media, and Photoperiod on Growth and Toxin Production of Azadinium spinosum.

Azaspiracids (AZAs) are microalgal toxins that can accumulate in shellfish and lead to human intoxications. To facilitate their study and subsequent biomonitoring, purification from microalgae rather than shellfish is preferable; however, challenges remain with respect to maximizing toxin yields. The impacts of temperature, growth media, and photoperiod on cell densities and toxin production in Azadinium spinosum were investigated. Final cell densities were similar at 10 and 18 °C, while toxin cell quotas were higher (~3.5-fold) at 10 °C. A comparison of culture media showed higher cell densities and AZA cell quotas (2.5–5-fold) in f10k compared to f/2 and L1 media. Photoperiod also showed differences, with lower cell densities in the 8:16 L:D treatment, while toxin cell quotas were similar for 12:12 and 8:16 L:D treatments but slightly lower for the 16:8 L:D treatment. AZA1, -2, and -33 were detected during the exponential phase, while some known and new AZAs were only detected once the stationary phase was reached. These compounds were additionally detected in field water samples during an AZA event.

Dinoflagelados productores de yessotoxinas en el Mar argentino

Las yessotoxinas (YTXs) son un grupo de ficotoxinas descrito recientemente, asociado a toxicidad en moluscos y a cardiotoxicidad en ratones de laboratorio. Hasta el momento se conocen tres especies de dinoflagelados productoras de YTXs a nivel mundial: Protoceratium reticulatum, Gonyaulax spinifera y Lingulodinium polyedra. En este estudio se analizó la presencia de YTXs y de sus potenciales productores en cuatro campañas oceanográficas realizadas en diferentes estaciones del año entre los 38 y 55ºS. La detección de toxinas se realizó mediante cromatografía líquida de alta eficiencia acoplada con espectrometría de masas en tándem. Los análisis morfológicos incluyeron observaciones mediante microscopía óptica y electrónica de barrido. Se encontraron dos potenciales productores de YTXs, Protoceratium reticulatum y Gonyaulax spinifera, presentando densidades máximas de 660 y 140 cél. L-1 respectivamente durante las campañas de otoño y primavera, en las cuales se detectó YTX en muestras del Golfo San Jorge y del talud. Los datos obtenidos sugieren que la especie P. reticulatum es la principal productora de YTXs en el Mar Argentino presentando cuotas celulares entre 2,2 y 12,5 pg YTX cél.-1, mientras que G. spinifera representa un productor potencial en la región que no ha mostrado una asociación clara con la detección de YTX.

Notes on the Cultivation of Two Mixotrophic Dinophysis Species and Their Ciliate Prey Mesodinium rubrum.

Kleptoplastic mixotrophic species of the genus Dinophysis are cultured by feeding with the ciliate Mesodinium rubrum, itself a kleptoplastic mixotroph, that in turn feeds on cryptophytes of the Teleaulax/Plagioselmis/Geminigera (TPG) clade. Optimal culture media for phototrophic growth of D. acuminata and D. acuta from the Galician Rías (northwest Spain) and culture media and cryptophyte prey for M. rubrum from Huelva (southwest Spain) used to feed Dinophysis, were investigated. Phototrophic growth rates and yields were maximal when D. acuminata and D. acuta were grown in ammonia-containing K(-Si) medium versus f/2(-Si) or L1(-Si) media. Dinophysis acuminata cultures were scaled up to 18 L in a photobioreactor. Large differences in cell toxin quota were observed in the same Dinophysis strains under different experimental conditions. Yields and duration of exponential growth were maximal for M. rubrum from Huelva when fed Teleaulax amphioxeia from the same region, versus T. amphioxeia from the Galician Rías or T. minuta and Plagioselmis prolonga. Limitations for mass cultivation of northern Dinophysis strains with southern M. rubrum were overcome using more favorable (1:20) Dinophysis: Mesodinium ratios. These subtleties highlight the ciliate strain-specific response to prey and its importance to mass production of M. rubrum and Dinophysis cultures.

Effects of salinity variation on growth and yessotoxin composition in the marine dinoflagellate Lingulodinium polyedra from a Skagerrak fjord system (western Sweden)

The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throughout the world. Three cultured isolates of L. polyedra from a fjord system on the Skagerrak coast of Sweden were analyzed for their growth characteristics and to determine the effects of a strong salinity gradient on toxin cell quotas and composition. The cell quota of yessotoxin (YTX) analogs, as determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), ranged widely among strains. For two strains, the total toxin content remained constant over time in culture, but for the third strain, the YTX cell quota significantly decreased (by 32%) during stationary growth phase. The toxin profiles of the three strains differed markedly and none produced YTX. The analog 41a-homo-YTX (m/z 1155), its putative methylated derivative 9-Me-41a-homo-YTX (m/z 1169) and an unspecified keto-YTX (m/z 1047) were detected in strain LP29-10H, whereas strain LP30-7B contained nor-YTX (m/z 1101), and two unspecified YTX analogs at m/z 1159 and m/z 1061. The toxin profile of strain LP30-8D comprised two unspecified YTX analogs at m/z 1061 and m/z 991 and carboxy-YTX (m/z 1173). Strain LP30-7B cultured at multiple salinities (10, 16, 22, 28 and 34) did not tolerate the lowest salinity (10), but there was a statistically significant decrease (by 21%) in toxin cell quota between growth at the highest versus lower permissible salinities. The toxin profile for strain LP30-7B remained constant over time for a given salinity. At lower salinities, however, the proportion of the unspecified YTX analog (m/z 1061) was significantly higher, especially with respect to nor-YTX (m/z 1101). This study shows high intra-specific variability in yessotoxin composition among strains from the same geographical region and inconsistency in toxin cell quota under different environmental regimes and growth stages in culture. This variation has important implications for the kinetics of YTX production and food web transfer in natural bloom populations from diverse geographical regions.

Genome variation in nine co-occurring toxic Cylindrospermopsis raciborskii strains

Cyanobacteria form harmful algal blooms and are highly adapted to a range of habitats, in part due to their phenotype plasticity. This plasticity is partially the result of co-existence of multiple strains within a single population. The toxic cyanobacterium Cylindrospermopsis raciborskii has remarkable phenotypic plasticity, strain variation and environmental adaptation resulting in an expansion of its global range. To understand the genetic basis of the high level of plasticity within a C. raciborskii population, the genomes of nine co-occurring strains were compared. The strains differed in morphology, toxin cell quotas and physiology, despite being obtained from a single water sample. Comparative genomics showed that three coiled strains were 3.9 Mbp in size, with 3544 ± 11 genes, while straight strains were 3.8 Mbp in size, with 3485 ± 20 genes. The core proteome comprised 86% of the genome and consisted of 2891 orthologous groups (OGs), whereas the variable genome comprised ∼14% (847 OGs), and the strain specific genome only ∼1% (433 OGs).There was a high proportion of variable strain-specific genes for the very closely related strains, which may underpin strain differentiation. The variable genes were associated with environmental responses and adaptation, particularly phage defence, DNA repair, membrane transport, and stress, illustrative of the adaptability of the strains in response to environmental and biological stressors. This study shows that high genomic variability exists between co-occurring strains and may be the basis of strain phenotypic differences and plasticity of populations. Therefore management and prediction of blooms of this harmful species requires different approaches to capture this strain variability.

Salinity effects on growth and toxin production in anAlexandrium ostenfeldii(Dinophyceae) isolate from The Netherlands

Alexandrium ostenfeldii is among the most intensely studied marine planktonic dinophytes and in the last few years blooms have become a recurrent phenomenon mainly in brackish coastal waters. Since 2012, A. ostenfeldii recurs annually in the Ouwerkerkse Kreek, a Dutch brackish water creek discharging into an estuary with large stocks of mussels, oysters and cockles. The creek is characterized by highly dynamic abiotic conditions, notably salinity. Here, we investigated the impacts of salinities ranging from 3 to 34 on growth and toxin content of an A. ostenfeldii isolate from the creek. Our results demonstrate a broad salinity tolerance of the Dutch A. ostenfeldii population, with growth rates from 0.13 to 0.2 d−1 over a salinity range from 6 to 34. Highest paralytic shellfish toxin and cyclic imine toxin cell quotas were observed for the lowest and highest salinities, and were associated with increases in cell size. Lytic activity was highest at the lowest salinity, and was 5-fold higher in the cell-free supernatants compared to cell extracts. Together our results demonstrate a tight coupling between salinity and A. ostenfeldii growth rate, cell size and toxin synthesis, which may have consequences for the seasonal dynamics of bloom toxicity.

Intraspecific variation in growth, morphology and toxin quotas for the cyanobacterium, Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom forming cyanobacterium with complex population dynamics and toxicity. In January of 2013 a single sample was collected from surface waters in Lake Wivenhoe, Australia, and twenty-four individual trichomes were isolated. Each isolate exhibited differences in growth rate, toxin cell quota and morphology, in the absence of phylogenetic heterogeneity. This study demonstrates substantial intraspecific isolate variation within a small sample and this has implications for understanding the population dynamics of this species.

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6°C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27°C and 30°C), as well as projected mean (33°C) and maximum temperatures (36°C) based on tropical climate change estimates of +6°C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33°C and M. flos-aquae at 30°C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33°C were found to boost total MC cell quota for three Microcystis strains although further increase to 36°C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.

Protoceratium reticulatum (Dinophyceae) in the austral Southwestern Atlantic and the first report on YTX-production in shelf waters of Argentina

Protoceratium reticulatum is a dinoflagellate with a life cycle that includes a motile planktonic stage and a resting cyst stage in benthic habitat, both with a wide geographical distribution, including southern South America. P. reticulatum produces yessotoxins (YTX) – these can be accumulated in shellfish and show potent cytotoxicity, posing a risk to human health if contaminated shellfish is consumed. YTX have been reported from coastal shellfish of many localities, but until now it was unknown if they were present in the austral Southwestern Atlantic and also if local populations of P. reticulatum have the ability to produce these toxins. In this study we report the presence of YTX in plankton samples and its production in culture by two P. reticulatum strains isolated from the San Jorge Gulf (SJG). In addition, we describe the geographical distribution and seasonal abundance of this species based on data collected over the past two decades. The YTX cell quotas calculated from net hauls (∼10pgcell−1) are in the same range as the toxin cell quotas observed in these two isolates. The phylogenetic analysis of sequences of the hypervariable region of the large subunit (LSU) 28S rDNA showed that the two clonal strains from the SJG were part of a monophyletic clade that subdivides P. reticulatum into two well-supported, divergent sub-clades. The sequences of the two strains of P. reticulatum from the SJG fell in the same clade as the majority of sequences of P. reticulatum, which belong to a geographically widely distributed evolutionary clade. P. reticulatum was occasionally observed from about 35°S in Uruguayan shelf waters up to 53°S on the Patagonian shelf and north of Tierra del Fuego, and it was present from coastal areas up to the shelf break zone. We recorded P. reticulatum in plankton samples during spring, summer and autumn but invariably in low abundance (maximum: 560cellsL−1). Viable cysts of the species in surface sediments also showed a wide geographical distribution. Together, the high total abundances and high relative numerical contribution to planktonic dinoflagellate assemblages near frontal areas, emphasize the necessity to pay attention to the dynamics of this species in areas of potential risk of harmful algal bloom development.

Influence of predator-prey evolutionary history, chemical alarm-cues, and feeding selection on induction of toxin production in a marine dinoflagellate

The dinoflagellate, Alexandrium fundyense, produces paralytic shellfish toxins and co-occurs with populations of the copepod, Acartia hudsonica, from Maine, but not New Jersey. The hypothesis that history of co-occurrence between predator and prey effects the ability of prey to recognize and respond to predators with increased toxin production was tested for this copepod-alga interaction. When A. fundyense was exposed to waterborne cues released by copepods from Maine (indirect exposure) that were either starved or fed toxic cells, cell toxin quota increased by 35% compared to unexposed controls. The induced response was significantly less for cells exposed to waterborne cues of copepods from New Jersey, and induction (20%) was only elicited by this population when fed toxic cells. These results suggest that A. fundyense responded to a kairomone from copepods from Maine, but required a feeding cue from copepods from New Jersey. An increase of approximately 300% in cell toxin quota, however, occurred when cells were directly exposed to grazing, and was independent of copepod population. Evolutionary history, therefore, had no apparent effect when induction was underlain by feeding cues. In assays with a mixture of toxic and nontoxic cells, selection for the latter was evident, and also independent of copepod population. Selectivity for nontoxic cells, however, could not account for changes in cell toxin content in the mixture experiments. When A. fundyense was exposed to extracts of toxic or nontoxic Alexandrium, toxin production increased significantly (23%), suggesting modest induction by an alga-to-alga alarm signal.

Stoichiometric regulation of phytoplankton toxins

Ecological Stoichiometry theory predicts that the production, elemental structure and cellular content of biomolecules should depend on the relative availability of resources and the elemental composition of their producer organism. We review the extent to which carbon- and nitrogen-rich phytoplankton toxins are regulated by nutrient limitation and cellular stoichiometry. Consistent with theory, we show that nitrogen limitation causes a reduction in the cellular quota of nitrogen-rich toxins, while phosphorus limitation causes an increase in the most nitrogen-rich paralytic shellfish poisoning toxin. In addition, we show that the cellular content of nitrogen-rich toxins increases with increasing cellular N : P ratios. Also consistent with theory, limitation by either nitrogen or phosphorus promotes the C-rich toxin cell quota or toxicity of phytoplankton cells. These observed relationships may assist in predicting and managing toxin-producing phytoplankton blooms. Such a stoichiometric regulation of toxins is likely not restricted to phytoplankton, and may well apply to carbon- and nitrogen-rich secondary metabolites produced by bacteria, fungi and plants.

Diarrheic toxins in field-sampled and cultivated Dinophysis spp. cells from southern Brazil

In southern Brazil, mixotrophic dinoflagellates belonging to the Dinophysis acuminata complex have recently been involved in diarrheic shellfish poisoning episodes through the production of lipophilic toxins such as okadaic acid (OA) and dinophysistoxin-1 (DTX-1). The present investigation used a combination of laboratory cultures and field surveys at three large estuarine systems in that region to examine toxin retention in Dinophysis spp. cells under optimum or growth-limiting conditions. This study represents the first successful culture of a Dinophysis isolate from the Atlantic South America region. Starved D. acuminata complex cells reached 5.6-fold higher cellular OA quotas (up to 18 pg cell−1) than Mesodinium rubrum-fed cultures 20 days after inoculation in the laboratory. Moreover, in field samples, light-limited cells at the bottom of a stratified water column were less abundant, yet 6.6- to 11-fold more toxic (up to 26.4 pg OA and 1.7 pg DTX-1 cell−1) than those located at the illuminated surface. This phenomenon of toxin retention by slow-dividing cells may partially explain the enormous variation in cell toxin quota found within Dinophysis spp. populations from a single location, and it may have serious implications for cell count-based monitoring program in bivalve aquaculture areas. In fact, only low to moderate OA levels were detected in the digestive glands of oysters Crassostrea spp. (up to 17.8 ng g−1) and the guts and livers of filter-feeding fish (44.7 ng g−1) during the present study, despite the relatively high Dinophysis cell densities (up to 19,500 cells L−1) found in the field.

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota.Azadinium spinosum grew in a wide range of conditions; from 10̊C to 26̊C and salinities from 30 to 40, under irradiances ranging from 50μmolm−2s−1 to 250μmolm−2s−1, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18̊C and 22̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10̊C (220fgcell−1) than at temperatures between 18̊C and 26̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth.These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.