A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States.

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.

Marine harmful algal blooms (HABs) in the United States: History, current status and future trends.

Harmful algal blooms (HABs) are diverse phenomena involving multiple. species and classes of algae that occupy a broad range of habitats from lakes to oceans and produce a multiplicity of toxins or bioactive compounds that impact many different resources. Here, a review of the status of this complex array of marine HAB problems in the U.S. is presented, providing historical information and trends as well as future perspectives. The study relies on thirty years (1990-2019) of data in HAEDAT - the IOC-ICES-PICES Harmful Algal Event database, but also includes many other reports. At a qualitative level, the U.S. national HAB problem is far more extensive than was the case decades ago, with more toxic species and toxins to monitor, as well as a larger range of impacted resources and areas affected. Quantitatively, no significant trend is seen for paralytic shellfish toxin (PST) events over the study interval, though there is clear evidence of the expansion of the problem into new regions and the emergence of a species that produces PSTs in Florida - Pyrodinium bahamense. Amnesic shellfish toxin (AST) events have significantly increased in the U.S., with an overall pattern of frequent outbreaks on the West Coast, emerging, recurring outbreaks on the East Coast, and sporadic incidents in the Gulf of Mexico. Despite the long historical record of neurotoxic shellfish toxin (NST) events, no significant trend is observed over the past 30 years. The recent emergence of diarrhetic shellfish toxins (DSTs) in the U.S. began along the Gulf Coast in 2008 and expanded to the West and East Coasts, though no significant trend through time is seen since then. Ciguatoxin (CTX) events caused by Gambierdiscus dinoflagellates have long impacted tropical and subtropical locations in the U.S., but due to a lack of monitoring programs as well as under-reporting of illnesses, data on these events are not available for time series analysis. Geographic expansion of Gambierdiscus into temperate and non-endemic areas (e.g., northern Gulf of Mexico) is apparent, and fostered by ocean warming. HAB-related marine wildlife morbidity and mortality events appear to be increasing, with statistically significant increasing trends observed in marine mammal poisonings caused by ASTs along the coast of California and NSTs in Florida. Since their first occurrence in 1985 in New York, brown tides resulting from high-density blooms of Aureococcus have spread south to Delaware, Maryland, and Virginia, while those caused by Aureoumbra have spread from the Gulf Coast to the east coast of Florida. Blooms of Margalefidinium polykrikoides occurred in four locations in the U.S. from 1921-2001 but have appeared in more than 15  U.S. estuaries since then, with ocean warming implicated as a causative factor. Numerous blooms of toxic cyanobacteria have been documented in all 50  U.S. states and the transport of cyanotoxins from freshwater systems into marine coastal waters is a recently identified and potentially significant threat to public and ecosystem health. Taken together, there is a significant increasing trend in all HAB events in HAEDAT over the 30-year study interval. Part of this observed HAB expansion simply reflects a better realization of the true or historic scale of the problem, long obscured by inadequate monitoring. Other contributing factors include the dispersion of species to new areas, the discovery of new HAB poisoning syndromes or impacts, and the stimulatory effects of human activities like nutrient pollution, aquaculture expansion, and ocean warming, among others. One result of this multifaceted expansion is that many regions of the U.S. now face a daunting diversity of species and toxins, representing a significant and growing challenge to resource managers and public health officials in terms of toxins, regions, and time intervals to monitor, and necessitating new approaches to monitoring and management. Mobilization of funding and resources for research, monitoring and management of HABs requires accurate information on the scale and nature of the national problem. HAEDAT and other databases can be of great value in this regard but efforts are needed to expand and sustain the collection of data regionally and nationally.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Behavioral and mechanistic characteristics of the predator-prey interaction between the dinoflagellate Dinophysis acuminata and the ciliate Mesodinium rubrum.

Predator-prey interactions of planktonic protists are fundamental to plankton dynamics and include prey selection, detection, and capture as well as predator detection and avoidance. Propulsive, morphology-specific behaviors modulate these interactions and therefore bloom dynamics. Here, interactions between the mixotrophic, harmful algal bloom (HAB) dinoflagellate Dinophysis acuminata and its ciliate prey Mesodinium rubrum were investigated through quantitative microvideography using a high-speed microscale imaging system (HSMIS). The dinoflagellate D. acuminata is shown to detect its M. rubrum prey via chemoreception while M. rubrum is alerted to D. acuminata via mechanoreception at much shorter distances (89 ±â€¯39 µm versus 41 ±â€¯32 µm). On detection, D. acuminata approaches M. rubrum with reduced speed. The ciliate M. rubrum responds through escape jumps that are long enough to detach its chemical trail from its surface, thereby disorienting the predator. To prevail, D. acuminata uses capture filaments and/or releases mucus to slow and eventually immobilize M. rubrum cells for easier capture. Mechanistically, results support the notion that the desmokont flagellar arrangement of D. acuminata lends itself to phagotrophy. In particular, the longitudinal flagellum plays a dominant role in generating thrust for the cell to swim forward, while at other times, it beats to supply a tethering or anchoring force to aid the generation of a posteriorly-directed, cone-shaped scanning current by the transverse flagellum. The latter is strategically positioned to generate flow for enhanced chemoreception and hydrodynamic camouflage, such that D. acuminata can detect and stealthily approach resting M. rubrum cells in the water column.

Microbial Community Structure and Associations During a Marine Dinoflagellate Bloom.

Interactions between microorganisms and algae during bloom events significantly impacts their physiology, alters ambient chemistry, and shapes ecosystem diversity. The potential role these interactions have in bloom development and decline are also of particular interest given the ecosystem impacts of algal blooms. We hypothesized that microbial community structure and succession is linked to specific bloom stages, and reflects complex interactions among taxa comprising the phycosphere environment. This investigation used pyrosequencing and correlation approaches to assess patterns and associations among bacteria, archaea, and microeukaryotes during a spring bloom of the dinoflagellate Alexandrium catenella. Within the bacterial community, Gammaproteobacteria and Bacteroidetes were predominant during the initial bloom stage, while Alphaproteobacteria, Cyanobacteria, and Actinobacteria were the most abundant taxa present during bloom onset and termination. In the archaea biosphere, methanogenic members were present during the early bloom period while the majority of species identified in the late bloom stage were ammonia-oxidizing archaea and Halobacteriales. Dinoflagellates were the major eukaryotic group present during most stages of the bloom, whereas a mixed assemblage comprising diatoms, green-algae, rotifera, and other microzooplankton were present during bloom termination. Temperature and salinity were key environmental factors associated with changes in bacterial and archaeal community structure, respectively, whereas inorganic nitrogen and inorganic phosphate were associated with eukaryotic variation. The relative contribution of environmental parameters measured during the bloom to variability among samples was 35.3%. Interaction analysis showed that Maxillopoda, Spirotrichea, Dinoflagellata, and Halobacteria were keystone taxa within the positive-correlation network, while Halobacteria, Dictyochophyceae, Mamiellophyceae, and Gammaproteobacteria were the main contributors to the negative-correlation network. The positive and negative relationships were the primary drivers of mutualist and competitive interactions that impacted algal bloom fate, respectively. Functional predictions showed that blooms enhance microbial carbohydrate and energy metabolism, and alter the sulfur cycle. Our results suggest that microbial community structure is strongly linked to bloom progression, although specific drivers of community interactions and responses are not well understood. The importance of considering biotic interactions (e.g., competition, symbiosis, and predation) when investigating the link between microbial ecological behavior and an algal bloom's trajectory is also highlighted.

Characterization and comparison of toxin-producing isolates of Dinophysis acuminata from New England and Canada.

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01-1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub-specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.

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.

In situ dynamics of cyst and vegetative cell populations of the toxic dinoflagellate Alexandrium catenella in Ago Bay, central Japan.

Temporal changes in the in situ germination flux of cysts and the abundance of vegetative cells of the toxic dinoflagellate Alexandrium catenella were investigated in Ago Bay, central Japan from July 2003 to December 2004. The in situ germination flux (cells m-2 day-1) was measured using 'plankton emergence trap/chambers (PET chambers)'. Germination of the cysts in the sediments occurred continuously during the study, ranging from 52 to 1753 cells m-2 day-1, with no temporal trend. This germination pattern appeared to be promoted by a short mandatory dormancy period for newly formed cysts coupled with a broad temperature window for germination. For the vegetative populations, high abundances (>105 cells m-2) were recorded in the water column from spring to summer and from autumn to early winter. The size of the vegetative populations did not correlate with the cyst germination flux but rather larger vegetative populations were often observed when the water temperature was around 20°C, indicating that bloom development was mainly regulated by the temperature. Nonetheless, the continuous germination pattern of A. catenella is advantageous enabling the germinated cells to immediately exploit favorable bloom conditions.

Evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates.

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence.

BACKGROUND: Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by their massive genome sizes that are often several times that in humans. However, modern transcriptomic methods offer promising approaches to tackle this challenging system. Here, we used massively parallel signature sequencing (MPSS) to understand global transcriptional regulation patterns in Alexandrium tamarense cultures that were grown under four different conditions. METHODOLOGY/PRINCIPAL FINDINGS: We generated more than 40,000 unique short expression signatures gathered from the four conditions. Of these, about 11,000 signatures did not display detectable differential expression patterns. At a p-value < 1E-10, 1,124 signatures were differentially expressed in the three treatments, xenic, nitrogen-limited, and phosphorus-limited, compared to the nutrient-replete control, with the presence of bacteria explaining the largest set of these differentially expressed signatures. CONCLUSIONS/SIGNIFICANCE: Among microbial eukaryotes, dinoflagellates contain the largest number of genes in their nuclear genomes. These genes occur in complex families, many of which have evolved via recent gene duplication events. Our expression data suggest that about 73% of the Alexandrium transcriptome shows no significant change in gene expression under the experimental conditions used here and may comprise a "core" component for this species. We report a fundamental shift in expression patterns in response to the presence of bacteria, highlighting the impact of biotic interaction on gene expression in dinoflagellates.

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.

Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species.

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 mum) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.