Harmful phytoplankton diversity and dynamics in an upwelling region (Sagres,SW Portugal) revealed by ribosomal RNA microarray combined with microscopy

The study region in Sagres, SW Portugal, is subject to natural eutrophication of coastal waters by wind-driven upwelling, which stimulates high primary productivity facilitating the recent economic expansion of bivalve aquaculture in the region. However, this economic activity is threatened by harmful algal blooms (HAB) caused by the diatoms Pseudo-nitzschia spp., Dinophysis spp. and other HAB dinoflagellates, all of which can produce toxins, that can induce Amnesic Shellfish Poisoning (ASP), Diarrhetic Shellfish Poisoning (DSP) and Paralytic Shellfish Poisoning (PSP). This study couples traditional microscopy with 18S/28S rRNA microarray to improve the detection of HAB species and investigates the relation between HAB and the specific oceanographic conditions in the region. Good agreement was obtained between microscopy and microarray data for diatoms of genus Pseudo-nitzschia and dinoflagellates Dinophysis spp., Gymnodinium catenatum and raphidophyte Heterosigma akashiwo, with less effective results for Prorocentrum. Microarray provided detection of flagellates Prymnesium spp., Pseudochattonella spp., Chloromorum toxicum and the important HAB dinoflagellates of the genera Alexandrium and Azadinium, with the latter being one of the first records from the study region. Seasonality and upwelling induced by northerly winds were found to be the driving forces of HAB development, with Pseudo-nitzschia spp. causing the risk of ASP during spring and summer upwelling season, and dinoflagellates causing the risk of DSP and PSP during upwelling relaxation, mainly in summer and autumn. The findings were in agreement with the results from toxicity monitoring of shellfish by the Portuguese Institute for Sea and Atmosphere and confirm the suitability of the RNA microarray method for HABs detection and aquaculture management applications.     

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.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

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.     

Noctiluca scintillans may act as a vector of toxigenic microalgae

Noctiluca scintillans food vacuoles containing toxigenic microalgae of the genera Dinophysis and Pseudo-nitzschia are reported in samples from the Galician Rías Baixas (NW Spain). N. scintillans may play an important role in the population dynamics of toxigenic microalgae blooms, and may act as a vector of phycotoxins to higher trophic levels. The harmful effects of Noctiluca in natural shellfish beds and/or aquaculture sites may be magnified if cells are loaded with toxigenic microalgal prey.     

Distribution of Dinophysis species and their association with lipophilic phycotoxins in plankton from the Argentine Sea

Dinophysis is a cosmopolitan genus of marine dinoflagellates, considered as the major proximal source of diarrheic shellfish toxins and the only producer of pectenotoxins (PTX). From three oceanographic expeditions carried out during autumn, spring and late summer along the Argentine Sea (∼38–56°S), lipophilic phycotoxins were determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in size-fractionated plankton samples. Lipophilic toxin profiles were associated with species composition by microscopic analyses of toxigenic phytoplankton. Pectenotoxin-2 and PTX-11 were frequently found together with the presence of Dinophysis acuminata and Dinophysis tripos. By contrast, okadaic acid was rarely detected and only in trace concentrations, and dinophysistoxins were not found. The clear predominance of PTX over other lipophilic toxins in Dinophysis species from the Argentine Sea is in accordance with previous results obtained from north Patagonian Gulfs of the Argentine Sea, and from coastal waters of New Zealand, Chile, Denmark and United States. Dinophysis caudata was rarely found and it was confined to the north of the sampling area. Because of low cell densities, neither D. caudata nor Dinophysis norvegica could be biogeographically related to lipophilic toxins in this study. Nevertheless, the current identification of D. norvegica in the southern Argentine Sea is the first record for the southwestern Atlantic Ocean. Given the typical toxigenicity of this species on a global scale, this represents an important finding for future surveillance of plankton-toxin associations.     

The influence of salinity in the domoic acid effect on estuarine phytoplankton communities

Toxic species of the diatom genus Pseudo-nitzschia, observed worldwide from coastal waters to the open ocean, produce the neurotoxin domoic acid (DA). DA is an important environmental and economic hazard due to shellfish contamination with subsequent effects on higher trophic levels. Previous research has demonstrated that, among other environmental factors, salinity influences the abundance and toxicity of Pseudo-nitzschia. In this study, the environmental factors driving the growth of Pseudo-nitzschia and the production of dissolved DA (dDA) in North Inlet estuary were examined. The effect of salinity on the growth inhibition of phytoplankton induced by the initial presence as well as by an addition of dDA was also assessed. Initially, the diatom abundance was negatively correlated with the abundance of Pseudo-nitzschia and with the concentration of dDA. With the addition of a concentrated solution of dDA, the percent inhibition of cryptophytes and diatoms was significantly correlated with salinity and suggested a higher sensitivity to dDA at extreme salinities. These results emphasize the importance of salinity in assessing the properties of DA and potentially of other phycotoxins on phytoplankton.     

Toxic HAB species from the Sea of Okhotsk detected by a metagenetic approach,seasonality and environmental drivers

During recent decades, the distribution of harmful algal bloom (HAB) species has expanded worldwide together with the increase of blooms and toxicity events. In this study, the presence of toxic HAB species in the Sea of Okhotsk was investigated based on metagenetic data collected during 6 years of weekly monitoring. Operational taxonomic units (OTUs) associated with the toxic HAB species were detected based on amplifying 18S V7-V9 and 28S D1 rRNA gene regions. In total, 43 unique OTUs associated with toxic HAB species were revealed, with 26 of those previously not reported from the Sea of Okhotsk. More OTUs belonging to dinoflagellates were detected by 18S, whereas a similar number of OTUs associated with dinoflagellates and diatoms were detected by targeting the 28S region. Species belonging to genera Alexandrium, Karenia and Karlodinium were mainly associated with OTUs under Dinophyceae, whereas Bacillariophyceae was represented by the species belonging to genus Pseudo-nitzschia. From the detected OTUs, 22 showed a clear seasonal pattern with the majority of those appearing during summer-autumn. For Alexandrium pacificum, Aureococcus anophagefferens, and Pseudo-nitzschia pungens, the seasonal pattern was detected based on both rRNA regions. Additionally, 14 OTUs were detected during all seasons and two OTUs appeared sporadically. OTUs associated with the toxic species had low relative read abundances, which together with other factors such as similar and variable morphology as well as usage of fixatives, may explain why those species have previously not been detected by light microscopy. Environmental parameters, especially water temperature, significantly (<0.05) influenced the variability in OTU relative abundances and displayed significant (<0.05) correlations with the unique OTUs. The results of this study demonstrate the usefulness of the metagenetic approach for phytoplankton monitoring, which is especially relevant for detecting toxic HAB species.     

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.     

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell⁻¹. This is the first toxigenic diatom species reported from Chinese waters.     

Specificity of Lipoxygenase Pathways Supports Species Delineation in the Marine Diatom Genus Pseudo-nitzschia

Oxylipins are low-molecular weight secondary metabolites derived from the incorporation of oxygen into the carbon chains of polyunsaturated fatty acids (PUFAs). Oxylipins are produced in many prokaryotic and eukaryotic lineages where they are involved in a broad spectrum of actions spanning from stress and defense responses, regulation of growth and development, signaling, and innate immunity. We explored the diversity in oxylipin patterns in the marine planktonic diatom Pseudo-nitzschia. This genus includes several species only distinguishable with the aid of molecular markers. Oxylipin profiles of cultured strains were obtained by reverse phase column on a liquid chromatograph equipped with UV photodiode detector and q-ToF mass spectrometer. Lipoxygenase compounds were mapped on phylogenies of the genus Pseudo-nitzschia inferred from the nuclear encoded hyper-variable region of the LSU rDNA and the plastid encoded rbcL. Results showed that the genus Pseudo-nitzschia exhibits a rich and varied lipoxygenase metabolism of eicosapentaenoic acid (EPA), with a high level of specificity for oxylipin markers that generally corroborated the genotypic delineation, even among genetically closely related cryptic species. These results suggest that oxylipin profiles constitute additional identification tools for Pseudo-nitzschia species providing a functional support to species delineation obtained with molecular markers and morphological traits. The exploration of the diversity, patterns and plasticity of oxylipin production across diatom species and genera will also provide insights on the ecological functions of these secondary metabolites and on the selective pressures driving their diversification.     

The copepod Calanus finmarchicus: A potential vector for trophic transfer of the marine algal biotoxin, domoic acid

The marine algal biotoxin, domoic acid (DA), is produced by certain members of the diatom genus Pseudo-nitzschia. This neurotoxin has been responsible for several mass mortality events involving marine birds and mammals. In all cases, the toxin was transferred from its algal producers through marine food webs by one or more intermediate vectors. The ability of some copepod taxa to serve as vectors for DA has been demonstrated; however, the role played in DA trophic transfer by Calanus finmarchicus, which often dominates N. Atlantic zooplankton assemblages and is a primary dietary component of the highly endangered N. Atlantic right whale (Eubalaena glacialis), has been uncertain. In the present study, we examined the ability of C. finmarchicus to consume DA-producing algae and retain the toxin. Results of grazing and toxin accumulation/depuration experiments showed that C. finmarchicus consumed DA-producing Pseudo-nitzschia multiseries regardless of the presence or absence of morphologically similar, but non-toxic, P. pungens, across initial cell concentrations ranging from 1000-4000 cells mL^− 1. Furthermore, C. finmarchicus did not appear to preferentially consume or avoid either Pseudo-nitzschia species tested. After ingestion of P. multiseries, copepods accumulated DA and retained it for up to 48 h post-removal of the toxin source. These findings provide evidence for the potential of C. finmarchicus to facilitate DA trophic transfer in marine food webs where toxic Pseudo-nitzschia is present.     

Host-specific adaptation governs the interaction of the marine diatom,Pseudo-nitzschia and their microbiota

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia–microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom–hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.     

Pseudo-nitzschia physiological ecology,phylogeny, toxicity,monitoring and impacts on ecosystem health

Over the last decade, our understanding of the environmental controls on Pseudo-nitzschia blooms and domoic acid (DA) production has matured. Pseudo-nitzschia have been found along most of the world's coastlines, while the impacts of its toxin, DA, are most persistent and detrimental in upwelling systems. However, Pseudo-nitzschia and DA have recently been detected in the open ocean's high-nitrate, low-chlorophyll regions, in addition to fjords, gulfs and bays, showing their presence in diverse environments. The toxin has been measured in zooplankton, shellfish, crustaceans, echinoderms, worms, marine mammals and birds, as well as in sediments, demonstrating its stable transfer through the marine food web and abiotically to the benthos. The linkage of DA production to nitrogenous nutrient physiology, trace metal acquisition, and even salinity, suggests that the control of toxin production is complex and likely influenced by a suite of environmental factors that may be unique to a particular region. Advances in our knowledge of Pseudo-nitzschia sexual reproduction, also in field populations, illustrate its importance in bloom dynamics and toxicity. The combination of careful taxonomy and powerful new molecular methods now allow for the complete characterization of Pseudo-nitzschia populations and how they respond to environmental changes. Here we summarize research that represents our increased knowledge over the last decade of Pseudo-nitzschia and its production of DA, including changes in worldwide range, phylogeny, physiology, ecology, monitoring and public health impacts.     

Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.     

Insights into the harmful algal flora in northwestern Mediterranean coastal lagoons revealed by pyrosequencing metabarcodes of the 28S rRNA gene

This study investigated the genetic diversity of phytoplankton communities in six shallow lagoons located on the French coast of the northwestern Mediterranean Sea that represented a trophic gradient ranging from oligotrophic to hypereutrophic. The phytoplankton communities were sampled once a month from spring (May) to the beginning of autumn (September/early October) in 2012 and fractionated by size. Metabarcodes were generated from cDNAs by targeting the D1-D2 region of the 28S rRNA gene and pyrosequenced using Roche 454 technology. Examination of the annotated barcodes revealed harmful algal species not previously documented in these lagoons. Three ichthyotoxic species belonging to Pfiesteriaceae were detected: Luciella masanensis was relatively widespread and abundant in many samples, whereas Pfiesteria piscicida and Stoeckeria changwonensis were found as single barcode sequences. Furthermore, a phylogenetic analysis of barcodes annotated as belonging to Pfiesteriaceae suggested the existence of two previously undescribed clades. The other toxic or potentially harmful dinoflagellates detected through rare barcodes were Dinophysis acuminata, Vulcanodinium rugosum, Alexandrium andersonii and A. ostenfeldii. The two most abundant dinoflagellate taxa were Gymnodinium litoralis and Akashiwo sanguinea with respect to sequence numbers. Four diatom species from the genus Pseudo-nitzschia that potentially produce domoic acid were identified (P. galaxiae, P. delicatissima, P. brasiliana and P. calliantha). These observations are discussed in terms of the literature and monitoring records related to the identified taxa in this Mediterranean area.     

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.     

Resting stages of microalgae in recent marine sediments of Peter the Great Bay,Sea of Japan

Data on the qualitative and quantitative composition of resting stages of planktonic microalgae in recent marine sediments of Peter the Great Bay (Sea of Japan) over the period 2000–2007 are presented. A total of sixty one morphological forms of resting stages represented by dinoflagellate and raphidophyte cysts and diatom spores and resting cells were recorded in the sediment samples. This study revealed cysts of the potentially toxic species Alexandrium tamarense, A. cf. minutum, Alexandrium sp., Gymnodinium catenatum (PSP toxin producers), and Protoceratium reticulatum (yessotoxin producer); resting cells of Pseudo-nitzschia sp. (potential producer of domoic acid); and cysts of bloom-forming species Cochlodinium cf. polykrikoides and Heterosigma cf. akashiwo.     

Effects of nutrient-limiting supply ratios on toxin content of Karenia brevis grown in continuous culture

Toxins produced as secondary metabolites can play important roles in phytoplankton communities and contribute to the ecological success of harmful algal bloom (HAB) taxa. Toxin composition and content in phytoplankton are affected by a suite of environmental factors, including nutrient availability. Changes in nutrient availability can increase or decrease toxin content and alter toxin composition, depending on toxin stoichiometry and the mechanisms by which nutrient limitation affects toxin production. The studies that have assessed the effects of nutrient availability on brevetoxin content of the HAB species Karenia brevis have reported contradictory results, although there is growing support that nutrient limitation increases brevetoxin content. In this study, we assessed the effects of decreased nitrogen (N) and phosphorus (P) availability on brevetoxin content and composition of K. brevis grown in chemostats at steady state by altering the nutrient supply ratios of incoming media from the Redfield Ratio. Overall, brevetoxin content was greatest in cultures grown at the lowest rate, regardless of the nutrient supply ratio (i.e., under both Redfield and N-limiting supply ratios). Compared to cultures grown at 0.2 d⁻¹, cultures grown at 0.1 d⁻¹ exhibited 5-fold increases in intracellular toxin content. In contrast, at constant growth rates, N-limiting supply ratios decreased intracellular brevetoxin content by approximately one-third, although this result was significant only in cultures growing at the fastest rate of 0.23 d⁻¹. P-limiting supply ratios had no effect on brevetoxin content or composition. In addition, when cultures grown at rates of 0.2 d⁻¹ were supplied with balanced/Redfield N:P supply ratios, but different absolute nutrient concentrations, toxin content was greater under greater nutrient concentrations. These findings suggest that when growth rate is not nutrient limited, there is a positive relationship between nutrient availability and brevetoxin content. This work contributes to previous studies by demonstrating strong growth rates effects on brevetoxin content and that growth rate and nutrient availability can independently or together affect toxin content of K. brevis. Moreover, our work underscores the value of the chemostat as a tool to elucidate the mechanisms by which nutrient availability and growth rate affect toxin production and content of HAB species.