Toxin Production Rate Research Articles

Photoacclimation and photophysiology of four species of toxigenic Dinophysis

This study aimed to explore the effects of different light intensities on the ecophysiology of eight new Dinophysis isolates comprising four species (D. acuminata, D. ovum, D. fortii, and D. caudata) collected from different geographical regions in the US. After six months of acclimation, the growth rates, photosynthetic efficiency (Fv/Fm ratio), toxin content, and net toxin production rates of the Dinophysis strains were examined. The growth rates of D. acuminata and D. ovum isolates were comparable across light intensities, with the exception of one D. acuminata strain (DANY1) that was unable to grow at the lowest light intensity. However, D. fortii and D. caudata strains were photoinhibited and grew at a slower rate at the highest light intensity, indicating a lower degree of adaptability and tolerance to such conditions. Photosynthetic efficiency was similar for all Dinophysis isolates and negatively correlated with exposure to high light intensities. Multiple toxin metrics, including cellular toxin content and net production rates of DSTs and PTXs, were variable among species and even among isolates of the same species in response to light intensity. A pattern was detected, however, whereby the net production rates of PTXs were significantly lower across all Dinophysis isolates when exposed to the lowest light intensity. These findings provide a basis for understanding the effects of light intensity on the eco-physiological characteristics of Dinophysis species in the US and could be employed to develop integrated physical-biological models for species and strains of interest to predict their population dynamics and mitigate their negative effects.

Diarrhetic shellfish toxins production appears to be driven by photosynthesis and phosphate–revealed by different light-adapted strains of Prorocentrum lima complex and P. caipirginum

IntroductionDiarrhetic shellfish toxins (DST) harm shellfish aquaculture and endanger human health, and include well-known marine dinoflagellate-produced toxins such as okadaic acid (OA) and analogues, such as dinophysistoxin-1 (DTX-1). Toxin-producing species have different toxin profiles and contents, with unclear interactions, with the toxins hypothesized to be produced under stress conditions.MethodsFive morphotypes of Prorocentrum lima complex, a well-studied DST-producing species with remarkable phylogenetic variability, were chosen and exposed to three distinct light conditions (photosynthetic active radiation, PAR; PAR+UVA; PAR+UVA and UVB) for 18 or 24 days.Results and discussionThe studied morphotypes were classified as either light-sensitive (LS) or light-tolerant (LT) types based on their light responses and varying abilities to produce DTX-1 across three orders of magnitude (0.001-1 pg cell-1 d-1, abbreviated as LL, ML, and HL in order of rank). All toxin production rates (Rtox) initially increased and then decreased, with the first peaks varying between days 3 to 12. The results of earlier peaks in LL and ML and a linear correlation between Rtox values for OA and DTX-1 in HL indicated that the two compounds may be competitive. When light conditions initially altered, Rtox was either stagnant or negative under all light treatments in the LS cohort, and with UV addition in LT, which subsequently delayed the early peaks. The Rtox data for DTX-1 all demonstrated earlier and higher peaks with UVA addition in LL and ML. Likewise, their growth was facilitated following the addition of UVA light. Therefore, Rtox of pre-peaks is relevant to photosynthetic status and photoprotection ability. As decreasing Rtox data points corresponded closely with phosphate depletion, the phosphate consumption rate was calculated and shown to be linearly or exponentially associated with all downward Rtox. This study proposes a supply-demand link between photosynthetic products and phosphate with DST biosynthesis, inferring a likely competitive interaction between OA and DTX-1 production.

Interaction between temperature and salinity stress on the physiology of Dinophysis spp. and Alexandrium minutum: implications for niche range and blooming patterns

Abrupt changes in environmental conditions in estuaries and coastal waters have direct (physiological) and indirect (through changes in water column stability) effects on planktonic microalgae. Understanding and quantifying these effects is important to improve harmful algal bloom predictive models. Dinophysis spp. (D. acuminata, D. acuta and D. caudata) and Alexandrium minutum produce toxins that are transferred through the food web (particulate) by filter feeders and also released in the seawater. These dinoflagellates cause lengthy harvesting bans in European aquaculture sites and affect marine life. The 4 species were exposed to different combinations of temperature (T) and salinity (S) to investigate their short-term response (days) to and their recovery (weeks) from T/S stress. Dinophysis species showed varying capacities to deal with sudden changes in S, from the most resilient D. acuminata, equally affected by T and S stress, to the less tolerant D. acuta, and in particular D. caudata, more affected by S than T stress. The euryhaline A. minutum thrived under all the T/S combinations assayed. Further experiments showed a similar rate of okadaic acid toxin production (pg cell-1 d-1) for the 3 Dinophysis species under different T/S conditions. In contrast, a significant increment of gonyautoxin 4 content per cell was observed in A. minutum with decreasing S without significant effects associated with T. Our results highlight the response to environmental (T/S) stress of 3 species of Dinophysis and A. minutum with specific adaptations to thrive in different sub-habitats of the Galician Rías, an estuarine/coastal upwelling system in NW Spain.

Quantification of microcystin production and biodegradation rates in the western basin of Lake Erie.

Cyanobacterial biomass forecasts currently cannot predict the concentrations of microcystin, one of the most ubiquitous cyanotoxins that threaten human and wildlife health globally. Mechanistic insights into how microcystin production and biodegradation by heterotrophic bacteria change spatially and throughout the bloom season can aid in toxin concentration forecasts. We quantified microcystin production and biodegradation during two growth seasons in two western Lake Erie sites with different physicochemical properties commonly plagued by summer Microcystis blooms. Microcystin production rates were greater with elevated nutrients than under ambient conditions and were highest nearshore during the initial phases of the bloom, and production rates were lower in later bloom phases. We examined biodegradation rates of the most common and toxic microcystin by adding extracellular stable isotope‐labeled microcystin‐LR (1 μg L−1), which remained stable in the abiotic treatment (without bacteria) with minimal adsorption onto sediment, but strongly decreased in all unaltered biotic treatments, suggesting biodegradation. Greatest biodegradation rates (highest of −8.76 d−1, equivalent to the removal of 99.98% in 18 h) were observed during peak bloom conditions, while lower rates were observed with lower cyanobacteria biomass. Cell‐specific nitrogen incorporation from microcystin‐LR by nanoscale imaging mass spectrometry showed that a small percentage of the heterotrophic bacterial community actively degraded microcystin‐LR. Microcystin production and biodegradation rates, combined with the microcystin incorporation by single cells, suggest that microcystin predictive models could be improved by incorporating toxin production and biodegradation rates, which are influenced by cyanobacterial bloom stage (early vs. late bloom), nutrient availability, and bacterial community composition.

Atmospheric CO2 availability induces varying responses in net photosynthesis, toxin production and N2 fixation rates in heterocystous filamentous Cyanobacteria (Nostoc and Nodularia)

Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N2 and conduct oxygenic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed. In light of this, the effects of elevated atmospheric CO2 availability on growth, carbon and N2 fixation as well as nodularin production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following:Biomass and volume specific biological nitrogen fixation (BNF) rates were respectively almost six and 17 fold higher in the aquatic Nodularia species compared to the terrestrial Nostoc species tested, under elevated CO2 conditions.There was a direct correlation between elevated CO2 and decreased dry weight specific cellular nodularin content in a diazotrophically grown terrestrial Nostoc species, and the aquatic Nodularia species, regardless of nitrogen availability.Elevated atmospheric CO2 levels were correlated to a reduction in biomass specific BNF rates in non-toxic Nodularia species.Nodularin producers exhibited stronger stimulation of net photosynthesis rates (NP) and growth (more positive Cohen’s d) and less stimulation of dark respiration and BNF per volume compared to non-nodularin producers under elevated CO2 levels.This study is the first to provide information on NP and nodularin production under elevated atmospheric CO2 levels for Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions.

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth, photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.

The cost of toxin production in phytoplankton: the case of PST producing dinoflagellates.

Many species of phytoplankton produce toxins that may provide protection from grazing. In that case one would expect toxin production to be costly; else all species would evolve toxicity. However, experiments have consistently failed to show any costs. Here, we show that costs of toxin production are environment dependent but can be high. We develop a fitness optimization model to estimate rate, costs, and benefits of toxin production, using PST (paralytic shellfish toxin) producing dinoflagellates as an example. Costs include energy and material (nitrogen) costs estimated from well-established biochemistry of PSTs, and benefits are estimated from relationship between toxin content and grazing mortality. The model reproduces all known features of PST production: inducibility in the presence of grazer cues, low toxicity of nitrogen-starved cells, but high toxicity of P-limited and light-limited cells. The model predicts negligible reduction in cell division rate in nitrogen replete cells, consistent with observations, but >20% reduction when nitrogen is limiting and abundance of grazers high. Such situation is characteristic of coastal and oceanic waters during summer when blooms of toxic algae typically develop. The investment in defense is warranted, since the net growth rate is always higher in defended than in undefended cells.

Thermal acclimation affects growth and lipophilic toxin production in a strain of cosmopolitan harmful alga Dinophysis acuminata

Species of the harmful algal bloom (HAB) genera Dinophysis are causative of one of the most widespread and expanding HAB events associated with the human intoxication, diarrheic shellfish poisoning (DSP). The effects of warming temperature on the physiology and toxinology of these mixotrophic species remain intractable due to their low biomass in nature and difficulties in establishing and maintaining them in culture. Hence, the present study investigated the influence of warming temperature, encompassing present and predicted climate scenarios, on growth and toxin production in a strain of the most cosmopolitan DSP-causative species, Dinophysis acuminata. The strain was isolated from western Japan, acclimated, and cultured over extended time spans.The specific growth and toxin production rates were highest at 20–26 °C and 17–29 °C, respectively, and had significant linear relationships during exponential phase. The cellular toxin production of okadaic acid and pectenotoxin-2 were highest during early exponential growth phase at temperatures ≤17 °C but highest during late stationary phase at temperatures ≥20 °C. The cellular toxin production of Dinophysistoxin-1, however, increased from early exponential to late stationary growth phase independently from temperature. The net toxin productions were not affected by acclimation temperature but significantly affected by growth and were highest during early exponential growth phase. Warming water temperatures increase growth and promote toxin production of D. acuminata, potentially increasing incidence of diarrheic shellfish poisoning events and closures of shellfish production. It is likely that D. acuminata is more toxic at low cell densities during bloom initiation in winter, and at high cell densities during bloom termination in spring-autumn. The results of the present research are also of importance for the mass production of D. acuminata for subsequent studies of the toxicological and pharmacological bioactivities of DSTs and PTX2, and the fate of these toxins in the natural environment and the vectoring shellfish molluscs.

Cyanobacterial dynamics and toxins concentrations in Lake Alto Flumendosa, Sardinia, Italy

Seasonal blooms of cyanobacteria (CB) are a typical feature of Lake Alto Flumendosa (Sardinia, Italy). The waters of this lake are used for drinking water supply, for agricultural and industrial uses, and fish farming activities. Since cyanotoxins are not monitored in edible organisms, diet could be a relevant route of human exposure. CB also represent a threat for the health of wild and domestic animals that use lake water for beverage. Therefore, to characterize the CB community and assess the risk for human and animal population, CB dynamic, mcyB+ fraction, and microcystins (MCs) concentration have been followed monthly for 18 months, in three stations. Results confirmed the presence of several toxigenic species. Planktothrix rubescens dominated between August 2011 and April 2012 (3.5×106 cells L-1), alternating with Woronichinia naegeliana (8×106 cells L-1) and Microcystis botrys (9×105 cells L-1). Dolichospermum planctonicum was always present at low densities (104 cells L-1). MCs were detected, at values well below the 1 µg L-1 threshold of WHO for drinking water. The molecular analysis of mcyB gene for P. rubescens indicated the presence of a persistent toxic population (average 0.45 mcyB/16S rDNA). Highly significant linear regressions were found between P. rubescens and the sum of the demethylated MC variants, and between M. botrys and the sum of MC-LR and MC-LA, also when co-occurring, suggesting that these two species were responsible for different MC patterns production. The regression lines indicated a quite stable MC cell quota. However, in some spotted samples very different values were obtained for both MC concentrations and cell quota (from 10-fold lower to 30-40-fold higher than the ‘average’) showing an unexpected significant variability in the rate of toxin production. The relatively low cell densities during the monitoring period is consistent with the low-to absent MC contamination level found in trout muscle; however, the analytical method was affected by low recovery, probably due to MC-protein binding. Our results show that, during the study period, no risk of exposure for the human and animal population occurred. However, the persistence of a complex CB community characterised by a significant toxic fraction suggests the need for periodic monitoring activity. Particularly, the hidden deep summer P. rubescens blooms, located where water is taken for drinking water supply, and M. botrys, able to produce the most toxic MC variants with high cell quota, should be kept under control. The documentation and interpretation of sudden changes in toxins concentrations deserve special attention. This is particularly relevant in proximity of fish farming plants and water catchment sites.

Modelling and analysis of a phytoplankton–zooplankton system with continuous and discrete time

ABSTRACTIn this study, a phytoplankton–zooplankton system has been modelled using a system of differential equations with piecewise constant arguments, which represents a new approach to modelling phytoplankton–zooplankton interaction. To analyse the dynamic behaviour of the model, we consider the solution of the system in a certain subinterval, which yields a system of difference equations. Some theoretical results on the boundedness character and local stability properties for the discrete dynamical system are obtained. In addition, we explain the biological dynamics of the bloom in the plankton model through Neimark–Sacker bifurcation and obtain the threshold values for different parameters that govern the periodic nature of the bloom. We conclude that, while other studies explained that the bloom depended on only one parameter, this study explains that the bloom depended on three different parameters, namely (rate of toxin production per phytoplankton), (zooplankton growth efficiency) and (environmental carrying capacity of phytoplankton).

Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source.

BackgroundNosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters.ResultsFifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7 % of all strains, in selw (92.2 %) and in selx (15.7 %). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates.ConclusionProduction rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0630-x) contains supplementary material, which is available to authorized users.

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata.

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell−1·day−1) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (Rtox) was not affected by temperature. During EG, the average specific toxin production rate (µtox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day−1 at 30 °C, and then decreased. Over the entire growth span, µtox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µtox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.

Deterministic and stochastic nutrient-phytoplankton- zooplankton models with periodic toxin producing phytoplankton

Deterministic and stochastic models of nutrient-phytoplankton-zooplankton interaction are proposed to investigate the impact of toxin producing phytoplankton upon persistence of the populations. The toxin liberation by phytoplankton is modeled periodically in the deterministic system. We derive two thresholds in terms of the parameters for which both plankton populations can persist if these thresholds are positive. We construct stochastic models with Itô differential equations to model variability in the environment. It is concluded that the input nutrient concentration along with the toxin liberation rate play critical roles in the dynamics of the planktonic interaction. In particular, toxin producing phytoplankton can terminate harmful algal blooms and the planktonic interaction is more stable if either the input nutrient concentration is smaller or if the toxin production rate is larger.

Role of dissolved nitrate and phosphate in isolates of Mesodinium rubrum and toxin-producing Dinophysis acuminata.

Dinophysis acuminata, a producer of toxins associated with diarrhetic shellfish poisoning (DSP) and/or pectenotoxins (PTXs), is a mixotrophic species that requires both ciliate prey and light for growth. Linkages have been described in the literature between natural abundances of the predator Dinophysis and its prey, Mesodinium rubrum, and culture experiments have demonstrated that prey, in addition to light, is required for toxin production by Dinophysis acuminata; together these suggest Mesodinium is a critical component for Dinophysis growth and toxicity. However, little is known about the role of dissolved inorganic nutrients on Mesodinium growth or that of toxin-producing Dinophysis. Accordingly, a series of experiments were conducted to investigate the possible uptake of dissolved nitrate and phosphate by 1) Dinophysis starved of prey, 2) Dinophysis feeding on Mesodinium rubrum, and 3) M. rubrum grown in nutritionally-modified media. All single-clone or mixed cultures were monitored for dissolved and particulate nutrient levels over the growth cycle, as well as growth rate, biomass, and toxin production when appropriate. D. acuminata did not utilize dissolved nitrate or phosphate in the medium under any nutrient regime tested, i.e., nutrient-enriched and nutrient-reduced, in the absence or presence of prey, or during any growth phase monitored, i.e., exponential and plateau phases. Changes in particulate phosphorus and nitrogen in D. acuminata, were instead, strongly influenced by the consumption of M. rubrum prey, and these levels quickly stabilized once prey were no longer available. M. rubrum, on the other hand, rapidly assimilated dissolved nitrate and phosphate into its particulate nutrient fraction, with maximum uptake rates of 1.38 pmol N/cell/day and 1.63 pmol P/cell/day. While D. acuminata did not benefit directly from the dissolved nitrate and phosphate, its growth (0.37±0.01 day-1) and toxin production rates for okadaic acid (OA), dinophysistoxin-1 (DTX1) or pectenotoxin-2 (PTX2), 0.1, 0.9 and 2.6 pg /cell/day, respectively, were directly coupled to prey availability. These results suggest that while dissolved nitrate and phosphate do not have a direct effect on toxin production or retention by D. acuminata, these nutrient pools contribute to prey growth and biomass, thereby indirectly influencing D. acuminata blooms and overall toxin in the system.

Bacterial community affects toxin production by Gymnodinium catenatum.

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell−1) was similar to the parent cultures (169–206 fmol STX cell−1), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell−1) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell−1). Specific toxin production rate (fmol STX day−1) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell−1 day−1) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.

Growth dynamics in relation to the production of the main cellular components in the toxic dinoflagellate Ostreopsis cf. ovata

In the last decade Ostreopsis cf. ovata blooms have been among the most intense along the entire Mediterranean coast, leading to ecological and human health problems, that are associated with the toxins (palytoxin-like compounds) produced by these algal cells. These compounds are secondary metabolites, whose rates of synthesis depend on the metabolism of their precursors. In general, growth dynamics and toxicity of dinoflagellates reflect the physiological status of the organism. The aim of the present study was to investigate the cellular production of the main biochemical compounds likely involved in the growth and toxicity dynamics of O. cf. ovata during exponential to the late stationary phase in batch cultures of an Adriatic strain. Removal of major nutrients from the medium was monitored along with concentration, biovolume and production of the main cellular components (e.g. polysaccharides, proteins, lipids and toxins). Nutrient uptake, as well as toxin production rates were calculated in the different growth periods. Nutrients (N and P) were completely depleted when cells entered stationary phase and the greatest net toxin production rate (RTOX) occurred during the first days of growth. The various palytoxins reported a relative abundance quite stable during the different growth phases, while the total toxin cellular amount increased along the growth curve. Total and extracellular released polysaccharides, as well as the lipid content increased greatly during the stationary phase, while proteins were mainly produced by cells during the exponential phase. The continuous release of polysaccharides could facilitate cell aggregation and the formation of the benthic community during algal blooms. The trend of production of the main cellular compounds in O. cf. ovata and the growth dynamics of this species lead us to hypothesize that the fast growth of this dinoflagellate, associated with the rapid use of environmental resources (nutrients, and phosphates in particular), may be an ecological/adaptive strategy which could favor this organism in competition with other species.

Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.

Production and excretion of okadaic acid, pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light, food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.

Effects of inorganic and organic nitrogen and phosphorus on the growth and toxicity of two Alexandrium species from Hong Kong

The effects of three nitrogen (N) and two phosphorus (P) inorganic and organic forms on the growth, toxin content and composition, toxin production, and chemical composition of Alexandrium catenella and Alexandrium tamarense isolated from coastal waters of Hong Kong were determined. The toxin production rate and cellular toxin content for A. catenella were at least 10-fold higher than A. tamarense. The highest net production rate (Rtox) of the two Alexandrium species was generally achieved in the exponential phase. However, the highest cellular toxin content occurred in the stationary phase in all cultures, partly due to the enhancement of cell volume caused by P limitation, except for urea grown cultures where cellular toxin content remained low throughout the growth stage. For A. catenella, NH4 induced the highest growth rate (0.59d−1), toxin production rate (μtox, 1.0μmolL−1d−1; Rtox, 2.5pmolcell−1d−1) and cellular toxin content (2.8pmolcell−1) among the three nitrogen sources regardless of inorganic and organic P. The form of phosphorus had limited effect on A. catenella. In contrast, the response of A. tamarense to different forms of nitrogen and phosphorus was more complex. NH4 induced the highest cellular toxin content (445fmolcell−1), while NO3 yielded the highest toxin production rate (μtox, 0.71nmolL−1d−1; Rtox, 140fmolcell−1d−1) and urea produced the highest growth rate (0.57d−1). For A. tamarense, the highest toxin production rate occurred under organic phosphorus. The relationship between toxin accumulation and the form of nitrogen varied with the phosphorus source. A. catenella cultures grown on NO3 and NH4 have about 80–90% C1/2 toxins and 5–15% GTX 1/4 toxins compared to 65–75% C1/2 toxins and 25–35% GTX 1/4 toxins in cultures grown on urea. Our results suggest that during summer when Alexandrium uses NH4 from local sewage effluent as its preferred nitrogen source, it might become more toxic in combination with episodically occurring P limitation in Hong Kong waters.

Numerical modeling on toxin produced by predominant species of cyanobacteria within the ecosystem of Lake Kasumigaura, Japan

The paper investigates the predominant algal (Microcystis aeruginosa, Microcystis viridis, Microcystis ichthyoblabe, and Mirocystis wasenbergii) bloom, their transition behaviors and toxin produced as Microcystin-Leucine+Arginine (MC-LR), Microcystin-Arginine+Arginine (MC-RR) and Microcystin-Tyosine+Arginine (MC-YR) by cyanobacteria within the ecosystem of Lake Kasumigaura, Japan by using hydrodynamic ecosystem coupled model. Integrating the famous Monod functions’ concept the second mode of toxin production and Grover et al., 2011 river reservoirs toxin modeling theoretical notion assumes that the rate of toxin production by cyanobacteria depends on proportional to blue green algal composition and their abundance. Conceptualizing these two novel idea we have developed a three dimensional numerical model and to elucidate the structure of algal species and prediction of toxin production within the lake ecosystem. The simulation results compare the toxin (MC-LR, MC-RR and MC-YR) production of some stations in the Lake Kasumigaura with the observational data in the month of July, August and September for 2005, 2006 and 2007.The simulation result shows that there are some dominant species (Microcystis aeruginosa and Mycrocystis viridis are highly toxic) with regard to toxin produce in July, August and September in 2005. But in 2006 and 2007 there is no toxin produced by cyanobacteria (dominant species by Microcystis ichthyoblabe is toxic/nontoxic) in the ecosystem of Lake Kasumigaura. Because of timing and duration of the cyanobacteria bloom, making scum or colony and dying depends on the selecting parameters i.e. light intensity, temperature, water depth, wind direction, buoyancy and N: P ratio etc. But in some cases, toxin production depends on the Microcystis species toxic and non-toxic characteristics. The numerical modeling was calibrated by tuning toxin decay coefficient and other parameters for achieving a good agreement between the observations and the predictions.