Molecular monitoring of Dinophysis species assemblage in mussel farms in the Northwestern Adriatic Sea.

Several Dinophysis species can produce potent lipophilic toxins that pose a risk to human health when contaminated seafood is consumed, especially filter-feeding bivalve mussels. In the mussel farms of the Northwestern Adriatic Sea, seawater and seafood are regularly monitored for the presence of Dinophysis species and their associated toxins, but the current methodological approaches, such as light microscopy determinations, require a long time to make results available to local authorities. A molecular qPCR-based assay can be used to quantify various toxic Dinophysis species in a shorter timeframe. However, this approach is not currently employed in official testing activities. In this study, field samples were collected monthly or bi-weekly over one year from various mussel farms along the Northwestern Adriatic coast. The abundance of Dinophysis species in the seawater was determined using both traditional microscopy and qPCR assays. In addition, the concentration of lipophilic toxins for DSP in mussel flesh was quantified using LC-MS/MS focusing on the okadaic acid group. Dinophysis spp. site-specific single cells were isolated and analysed by qPCR yielding a mean rDNA copy number per cell of 1.21 × 104 ± 1.81 × 103. The qPCR assay gave an efficiency of 98 % and detected up to 10 copies of the rDNA target gene. The qPCR and light microscopy determinations in environmental samples showed a significant positive correlation (Spearman rs = 0.57, p-value < 0.001) with a ratio of 2.24 between the two quantification methods, indicating that light microscopy estimates were generally 44.6 % lower than those obtained by the qPCR assay. The qPCR approach showed several advantages such as rapidity, sensitivity and efficiency over conventional microscopy analysis, showing its potential future role in phytoplankton monitoring under the Official Controls Regulations for shellfish.

Role of turbulence in Dinophysis spp. growth, feeding, and toxin leakage in culture.

Dinophysis, a mixotrophic dinoflagellate that is known to prey on the ciliate Mesodinium rubrum, and retain its chloroplasts, is responsible for diarrhetic shellfish poisoning (DSP) in humans and has been identified on all U.S. coasts. Monocultures of Dinophysis have been used to investigate the growth of Dinophysis species in response to variations in environmental conditions, however, little is known about the roles of system stability (turbulence) and mixotrophy in the growth and toxicity of Dinophysis species in the U.S.. To begin to address this gap in knowledge, culturing experiments were conducted with three species (four strains) of Dinophysis, that included predator-prey co-incubation (Dinophysis spp.+ M. rubrum) and prey-only (M. rubrum) flasks. Cultures were investigated for effects of low or high turbulence on Dinophysis spp. growth, feeding, and amounts of intra- and extracellular toxins: okadaic acid and derivatives (diarrhetic shellfish toxins, DSTs) and pectenotoxins (PTXs). Turbulence did not have a measurable effect on the rates of ingestion of M. rubrum prey by Dinophysis spp. for any of the four strains, however, effects on growth and particulate and dissolved toxins were observed. High turbulence (ε = 10-2 m2s-3) significantly slowed growth of both D. acuminata and D. ovum relative to still controls, but significantly stimulated growth of the D. caudata strain. Increasing turbulence also resulted in significantly higher intracellular toxin content in D. acuminata cultures (DSTs and PTXs), but significantly reduced intracellular toxin content (PTXs) in those of D. caudata. An increase in turbulence appeared to promote toxin leakage, as D. ovum had significantly more extracellular DSTs found in the medium under high turbulence when compared to the still control. Overall, significant responses to turbulence were observed, whereby the three strains from the "Dinophysis acuminata complex" displayed a stress response to turbulence, i.e., decreasing growth, increasing intracellular toxin content and/or increasing toxin leakage, while the D. caudata strain had an opposite response, appearing stimulated by, or more tolerant of, high turbulence.

Impaired photoacclimation in a kleptoplastidic dinoflagellate reveals physiological limits of early stages of endosymbiosis.

Dinophysis dinoflagellates are predators of Mesodinium ciliates, from which they retain only the plastids of cryptophyte origin. The absence of nuclear photosynthetic cryptophyte genes in Dinophysis raises intriguing physiological and evolutionary questions regarding the functional dynamics of these temporary kleptoplastids within a foreign cellular environment. In an experimental setup including two light conditions, the comparative analysis with Mesodinium rubrum and the cryptophyte Teleaulax amphioxeia revealed that Dinophysis acuminata possessed a smaller and less dynamic functional photosynthetic antenna for green light, a function performed by phycoerythrin. We showed that the lack of the cryptophyte nucleus prevented the synthesis of the phycoerythrin α subunit, thereby hindering the formation of a complete phycoerythrin in Dinophysis. In particular, biochemical analyses showed that Dinophysis acuminata synthesized a poorly stable, incomplete phycoerythrin composed of chromophorylated ß subunits, with impaired performance. We show that, consequently, a continuous supply of new plastids is crucial for growth and effective photoacclimation in this organism. Transcriptome analyses revealed that all examined strains of Dinophysis spp. have acquired the cryptophyte pebA and pebB genes through horizontal gene transfer, suggesting a potential ability to synthesize the phycobilin pigments bound to the cryptophyte phycoerythrin. By emphasizing that a potential long-term acquisition of the cryptophyte plastid relies on establishing genetic independence for essential functions such as light harvesting, this study highlights the intricate molecular challenges inherent in the enslavement of organelles and the processes involved in the diversification of photosynthetic organisms through endosymbiosis.

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.

Advances in the Early Warning of Shellfish Toxification by Dinophysis acuminata.

In Western Europe, the incidence of DST is likely the highest globally, posing a significant threat with prolonged bans on shellfish harvesting, mainly caused by species of the dinoflagellate genus Dinophysis. Using a time series from 2014 to 2020, our study aimed (i) to determine the concentration of D. acuminata in water at which shellfish toxin levels could surpass the regulatory limit (160 µg OA equiv kg-1) and (ii) to assess the predictability of toxic events for timely mitigation actions, especially concerning potential harvesting bans. The analysis considered factors such as (i) overdispersion in the data, (ii) distinct periods of presence and absence, (iii) the persistence of cells, and (iv) the temporal lag between cells in the water and toxins in shellfish. Four generalized additive models were tested, with the Tweedie (TW-GAM) model showing superior performance (>85%) and lower complexity. The results suggest existing thresholds currently employed (200 and 500 cells L-1) are well-suited for the Portuguese coast, supported by empirical evidence (54-79% accuracy). The developed algorithm allows for thresholds to be tailored on a case-by-case basis, offering flexibility for regional variations.

Population dynamics of dominant dinoflagellate species in the North Sea: in situ growth rates, photosynthetic potential, and losses due to parasitism.

In the North Sea, Tripos and Dinophysis are commonly occurring mixotrophic planktonic dinoflagellate genera. In order to understand their bloom dynamics, an occurring bloom dominated by T. furca and D. norvegica was followed for several days. High cell abundances of these species were located to estimate: in situ growth rates from cell cycle analyses, depth distributions, growth rates sustained by photosynthesis, and parasite infection prevalence in all T. furca, T. fusus, D. norvegica and D. acuminata. Cell abundances were over 10000 cells L-1 for T. furca and up to 18000 cells L-1 for D. norvegica. Cells accumulated between 15-25 m depth and presented low specific in situ growth rates of 0.04-0.15 d-1 for T. furca and 0.02-0.16 d-1 for D. norvegica. Photosynthesis could sustain growth rates of 0.01-0.18 d-1 for T. furca and 0.02 to 0.14 d-1 for D. norvegica, suggesting that these species were relying mainly on photosynthesis. Parasite infections where generally low, with occasional high prevalence in D. norvegica (by Parvilucifera sp.) and T. fusus (by Amoebophrya sp.), while both parasites showed comparable prevalence in D. acuminata, which could offset in situ growth rates by parasite-induced host mortality. The restructuring effect of parasites on dinoflagellate blooms is often overlooked and this study elucidates their effect to cell abundances and their growth at the final stages of a bloom.

Modeling the dynamics of harmful algal bloom events in two bays from the northern Chilean upwelling system.

The bays of Tongoy and Guanaqueros are located in the Humboldt Current system, where Argopecten purpuratus has been the subject of intense aquaculture development. These bays lie in one of the most productive marine ecosystems on Earth and are dominated by permanent coastal upwelling at Lengua de Vaca Point and Choros Point, one of the three upwelling centers on the Chilean coast. Significantly, this productive system experiences a high recurrence of harmful algal bloom (HAB) events. This paper examines 9-year (2010-2018) samples of three toxic microalgal species collected in different monitoring programs and research projects. During this period, nine HAB events were detected in Guanaqueros Bay and 14 in Tongoy Bay. Among these, three HAB events were produced simultaneously in both bays by Pseudo-nitzschia australis, and two events produced simultaneously were detected in one bay by Alexandrium spp. and the other by Dinophysis acuminata. Before El Niño 2015-16, there were more HAB events of longer duration by the three species. Since El Niño, the number and duration of events were reduced and only produced by P. australis. HAB events were simulated with the FVCOM model and a virtual particle tracker model to evaluate the dynamics of bays and their relationship with HAB events. The results showed retention in bays during the relaxation conditions of upwelling and low connectivity between bays, which explains why almost no simultaneous events were recorded.

Does prey availability influence the detection of Dinophysis spp. by the imaging FlowCytobot?

The Imaging FlowCytobot (IFCB) is a field-deployable imaging-in-flow cytometer that is increasingly being used to monitor harmful algae. The IFCB acquires images of suspended particles based on their chlorophyll-a fluorescence and/or the amount of light they scatter (side scattering). The present study hypothesized that fluorescence-based image acquisition would undercount Dinophysis spp., a genus of non-constitutive mixotrophs, when prey is limited. This is because Dinophysis spp. acquire plastids via ingestion of their ciliate prey Mesodinium spp., and lose photosynthetic capacity and autofluorescence in the absence of prey. Even small blooms of Dinophysis spp. can be highly toxic and result in diarrhetic shellfish poisoning (DSP), highlighting the importance of accurately detecting low abundances. To explore this, laboratory experiments were conducted to determine optimal IFCB settings for a fed culture of Dinophysis acuminata, and an existing time series of IFCB observations collected in Puget Sound (Washington, U.S.A) was used to compare Dinophysis spp. abundance estimates from samples triggered via side scattering versus fluorescence in relation to Mesodinium spp. abundance. This study introduces a quantitative approach for optimizing the detection of target harmful algae which can be repeated across multiple IFCBs and demonstrates the effects of IFCB calibration on Dinophysis spp. detection. The laboratory experiments showed that IFCB settings for fluorescence-based image acquisition need to be fairly sensitive to accurately detect D. acuminata cells. A poorly calibrated IFCB can miss a significant proportion of D. acuminata abundance whatever the method used to trigger the image acquisition. Field results demonstrated that the physiological status of Dinophysis spp. can influence their detection by the IFCB when triggering on fluorescence. This was observed during a 7-day period when the IFCB failed to detect Dinophysis spp. cells when triggering on fluorescence while cells were still detected using the side scattering triggering method as well as observed by microscopy. During this period, Mesodinium spp. was not detected, IFCB-derived autofluorescence level of individual cells of Dinophysis spp. was low, and less than 50 % of Dinophysis spp. cells exhibited autofluorescence under the microscope. Together, this indicates that the unique feeding ecology of Dinophysis spp. may affect their detection by the IFCB when cells are starved.

Operational modelling to assess advective harmful algal bloom development and its potential to impact aquaculture.

A particle tracking model is described and used to explore the role of advection as the source of harmful algal blooms that impact the Shetland Islands, where much of Scotland's aquaculture is located. The movement of particles, representing algal cells, was modelled using surface velocities obtained from the 1.5 km resolution Atlantic Margin Model AMM15. Following validation of model performance against drifter tracks, the model results recreate previously hypothesised onshore advection of harmful algal cells from west of the archipelago during 2006 and 2013, when exceptional Dinophysis spp. abundances were measured at Shetland aquaculture sites. Higher eastward advection of Dinophysis spp. cells was also suggested during 2018. Wind roses explain this higher eastward advection during 2006, 2013 and 2018. The study suggests that the European Slope Current is important for the transport of harmful algal blooms, particularly those composed of dinoflagellates.

Seasonal occurrence of toxic phytoplankton and phycotoxins at a mussel aquaculture site in Nova Scotia, Canada.

A three-year field study at a mussel (Mytilus edulis) aquaculture site in Ship Harbour, Nova Scotia, Canada was carried out between 2004 and 2006 to detect toxic phytoplankton species and dissolved lipophilic phycotoxins and domoic acid. A combination of plankton monitoring and solid phase adsorption toxin tracking (SPATT) techniques were used. Net tow and pipe phytoplankton samples were taken weekly to determine the abundance of potentially toxic species and SPATT samplers were deployed weekly for phycotoxin analysis. Mussels were also collected for toxin analysis in 2005. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the samples for spirolides (SPXs), pectenotoxins (PTXs), okadaic acid group toxins (OA, DTXs) and domoic acid (DA). Phycotoxins were detected with SPATT samplers beginning from the time of deployment until after the producing organisms were no longer observed in pipe samples. Seasonal changes in toxin composition occurred over the sampling period and were related to changes in cell concentrations of Alexandrium Halim, Dinophysis Ehrenberg and Pseudo-nitzschia (Hasle) Hasle. Spirolides peaked in late spring and early summer, followed by DA in mid-July. Okadaic acid, DTX1 and PTXs occurred throughout the field season but peaked in late summer. Concentrations of some phycotoxins detected in SPATT samplers deployed within the area where mussels were suspended on lines were lower than in those deployed outside the mussel farm. The SPATT samplers provided a useful tool to detect the presence of phycotoxins and to establish trends in their appearance in the Ship Harbour estuary.

The Presence of Pseudo-nitzschia australis in North Atlantic Aquaculture Sites, Implications for Monitoring Amnesic Shellfish Toxins.

The farming of shellfish plays an important role in providing sustainable economic growth in coastal, rural communities in Scotland and acts as an anchor industry, supporting a range of ancillary jobs in the processing, distribution and exporting industries. The Scottish Government is encouraging shellfish farmers to double their economic contribution by 2030. These farmers face numerous challenges to reach this goal, among which is the problem caused by toxin-producing microplankton that can contaminate their shellfish, leading to harvesting site closure and the recall of product. Food Standards Scotland, a non-ministerial department of the Scottish Government, carries out a monitoring programme for both the toxin-producing microplankton and the toxins in shellfish flesh, with farms being closed when official thresholds for any toxin are breached. The farm remains closed until testing for the problematic toxin alone, often diarrhetic shellfish toxin (DST), shows the site to have dropped below the regulatory threshold. While this programme has proved to be robust, questions remain regarding the other toxins that may be present at a closed site. In this study, we tested archival material collected during site closures but only tested for DSTs as part of the official control monitoring. We found the presence of amnesic shellfish toxin (AST) in low concentrations in the majority of sites tested. In one case, the level of AST breached the official threshold. This finding has implications for AST monitoring programmes around Europe.

Quantitative Polymerase Chain Reaction for the estimation of toxigenic microalgae abundance in shellfish production waters.

Certain species of marine microalgae produce potent biotoxins that pose a risk to human health if contaminated seafood is consumed, particularly filter feeding bivalve shellfish. In regions where this is likely to occur water and seafood produce are regularly monitored for the presence of harmful algal cells and their associated toxins, but the current approach is flawed by a lengthy delay before results are available to local authorities. Quantitative Polymerase Chain Reaction (qPCR) can be used to measure phytoplankton DNA sequences in a shorter timeframe, however it is not currently used in official testing practices. In this study, samples were collected almost weekly over six months from three sites within a known HAB hotspot, St Austell Bay in Cornwall, England. The abundance of algal cells in water was measured using microscopy and qPCR, and lipophilic toxins were quantified in mussel flesh using LC-MS/MS, focusing on the okadaic acid group. An increase in algal cell abundance occurred alongside an increase in the concentration of okadaic acid group toxins in mussel tissue at all three study sites, during September and October 2021. This event corresponded to an increase in the measured levels of Dinophysis accuminata DNA, measured using qPCR. In the following spring, the qPCR detected an increase in D. accuminata DNA levels in water samples, which was not detected by microscopy. Harmful algal species belonging to Alexandrium spp. and Pseudo-nitzschia spp. were also measured using qPCR, finding a similar increase in abundance in Autumn and Spring. The results are discussed with consideration of the potential merits and limitations of the qPCR technique versus conventional microscopy analysis, and its potential future role in phytoplankton surveillance under the Official Controls Regulations pertaining to shellfish.

Short-Term Interactions of Noctiluca scintillans with the Toxic Dinoflagellates Dinophysis acuminata and Alexandrium minutum: Growth, Toxins and Allelopathic Effects.

The Galician Rías (NW Iberian Peninsula) are an important shellfish aquaculture area periodically affected by toxic episodes often caused by dinoflagellates such as Dinophysis acuminata and Alexandrium minutum, among others. In turn, water discolorations are mostly associated with non-toxic organisms such as the heterotrophic dinoflagellate Noctiluca scintillans, a voracious non-selective predator. The objective of this work was to study the biological interactions among these dinoflagellates and their outcome in terms of survival, growth and toxins content. To that aim, short experiments (4 days) were carried out on mixed cultures with N. scintillans (20 cells mL-1) and (i) one strain of D. acuminata (50, 100 and 500 cells mL-1) and (ii) two strains of A. minutum (100, 500 and 1000 cells mL-1). Cultures of N. scintillans with two A. minutum collapsed by the end of the assays. Both D. acuminata and A. minutum exposed to N. scintillans arrested its growth, though feeding vacuoles in the latter rarely contained any prey. Toxin analyses at the end of the experiment showed an increase in intracellular OA levels in D. acuminata and a significant reduction in PSTs in both A. minutum strains. Neither OA nor PSTs were detected in N. scintillans. Overall, the present study indicated that the interactions among them were ruled by negative allelopathic effects.

Forecasting harmful algae blooms: Application to Dinophysis acuminata in northern Norway.

Dinophysis acuminata produces Diarrhetic Shellfish Toxins (DST) that contaminate natural and farmed shellfish, leading to public health risks and economically impacting mussel farms. For this reason, there is a high interest in understanding and predicting D. acuminata blooms. This study assesses the environmental conditions and develops a sub-seasonal (7 - 28 days) forecast model to predict D. acuminata cells abundance in the Lyngen fjord located in northern Norway. A Support Vector Machine (SVM) model is trained to predict future D. acuminata cells abundance by using the past cell concentration, sea surface temperature (SST), Photosynthetic Active Radiation (PAR), and wind speed. Cells concentration of Dinophysis spp. are measured in-situ from 2006 to 2019, and SST, PAR, and surface wind speed are obtained by satellite remote sensing. D. acuminata only explains 40% of DST variability from 2006 to 2011, but it changes to 65% after 2011 when D. acuta prevalence reduced. The D. acuminata blooms can reach concentration up to 3954 cells l-1 and are restricted to the summer during warmer waters, varying from 7.8 to 12.7 °C. The forecast model predicts with fair accuracy the seasonal development of the blooms and the blooms amplitude, showing a coefficient of determination varying from 0.46 to 0.55. SST has been found to be a useful predictor for the seasonal development of the blooms, while the past cells abundance is needed for updating the current status and adjusting the blooms timing and amplitude. The calibrated model should be tested operationally in the future to provide an early warning of D. acuminata blooms in the Lyngen fjord. The approach can be generalized to other regions by recalibrating the model with local observations of D. acuminata blooms and remote sensing data.

Growth, Toxin Content and Production of Dinophysis Norvegica in Cultured Strains Isolated from Funka Bay (Japan).

The successful cultivation of Dinophysis norvegica Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL-1) for more than 20 months was achieved by feeding them with the ciliate Mesodinium rubrum Lohmann, 1908, along with the addition of the cryptophyte Teleaulax amphioxeia (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL-1 (n = 7), and 0.7 and 3.6 ng per mL-1 (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell-1 (n = 7) and 0.5 to 1.2 pg per cell-1 (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, D. norvegica exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, D. norvegica grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24-27), reaching a maximum concentration of 3000 cells per mL-1 at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL-1 and 154.7 ng per mL-1 of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL-1) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of D. norvegica, as well as insights into the maintenance and culturing of this species.

The niche of a stress-tolerant specialist, Dinophysis acuminata, in a coastal upwelling system.

Dinophysis acuminata, the main cause of shellfish harvesting bans in Europe, blooms in the Galician Rías (NW Spain) throughout the upwelling season (ca. March to September). Here we illustrate rapid changes in vertical and across ría-shelf distributions of diatoms and dinoflagellates (including D. acuminata vegetative and small cells) in Ría de Pontevedra (RP) and Ría de Vigo (RV) during transitions from spin-down to spin-up phases of upwelling cycles. A subniche approach based on a Within Outlying Mean Index (WitOMI) showed that under the transient environmental conditions met during the cruise, both vegetative and small cells of D. acuminata colonized the Ria and Mid-shelf subniches, exhibiting good tolerance and extremely high marginality, in particular the small cells. Bottom-up (abiotic) control overwhelmed biological constraints, and shelf waters became a more favourable environment than the Rías. Contrasting higher biotic constraints inside the Rías were found for the small cells, with a subniche possibly controlled by unsuitable physiological status (notwithstanding the higher density) of the vegetative cell population. Results here on behaviour (vertical positioning) and physiological traits (high tolerance but very specialized niche) of D. acuminata give new insights into the ability of this species to remain in the upwelling circulation system. Higher shelf-ría exchanges in the Ría (RP) with more dense and persistent D. acuminata blooms reveal the relevance of transient event-scales and species- and site-specific characteristics to the fate of these blooms. Earlier statements about simple linear relationships between average upwelling intensities and the recurrence of Harmful algae bloom (HAB) events in the Galician Rías Baixas are questioned.

SoundToxins: A Research and Monitoring Partnership for Harmful Phytoplankton in Washington State.

The more frequent occurrence of marine harmful algal blooms (HABs) and recent problems with newly-described toxins in Puget Sound have increased the risk for illness and have negatively impacted sustainable access to shellfish in Washington State. Marine toxins that affect safe shellfish harvest because of their impact on human health are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP), diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP) and the recent measurement of azaspiracids, known to cause azaspiracid poisoning (AZP), at low concentrations in Puget Sound shellfish. The flagellate, Heterosigma akashiwo, impacts the health and harvestability of aquacultured and wild salmon in Puget Sound. The more recently described flagellates that cause the illness or death of cultivated and wild shellfish, include Protoceratium reticulatum, known to produce yessotoxins, Akashiwo sanguinea and Phaeocystis globosa. This increased incidence of HABs, especially dinoflagellate HABs that are expected in increase with enhanced stratification linked to climate change, has necessitated the partnership of state regulatory programs with SoundToxins, the research, monitoring and early warning program for HABs in Puget Sound, that allows shellfish growers, Native tribes, environmental learning centers and citizens, to be the "eyes on the coast". This partnership enables safe harvest of wholesome seafood for consumption in the region and helps to describe unusual events that impact the health of oceans, wildlife and humans.

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.

Mortality and histopathology in sheepshead minnow (Cyprinodon variegatus) larvae exposed to pectenotoxin-2 and Dinophysis acuminata.

Toxic species of the dinoflagellate genus Dinophysis can produce diarrheic toxins including okadaic acid (OA) and dinophysistoxins (DTXs), and the non-diarrheic pectenotoxins (PTXs). Okadaic acid and DTXs cause diarrheic shellfish poisoning (DSP) in human consumers, and also cause cytotoxic, immunotoxic and genotoxic effects in a variety of mollusks and fishes at different life stages in vitro. The possible effects of co-produced PTXs or live cells of Dinophysis to aquatic organisms, however, are less understood. Effects on an early life stage of sheepshead minnow (Cyprinodon variegatus), a common finfish in eastern USA estuaries, were evaluated using a 96-h toxicity bioassay. Three-week old larvae were exposed to PTX2 concentrations from 50 to 4000 nM, live Dinophysis acuminata culture (strain DAVA01), live cells resuspended in clean medium or culture filtrate. This D. acuminata strain produced mainly intracellular PTX2 (≈ 21 pg cell-1), with much lower levels of OA and dinophysistoxin-1. No mortality or gill damages were observed in larvae exposed to D. acuminata (from 5 to 5500 cells mL-1), resuspended cells and culture filtrate. However, exposure to purified PTX2 at intermediate to high concentrations (from 250 to 4000 nM) resulted in 8 to 100% mortality after 96 h (24-h LC50 of 1231 nM). Histopathology and transmission electron microscopy of fish exposed to intermediate to high PTX2 concentrations revealed important gill damage, including intercellular edema, necrosis and sloughing of gill respiratory epithelia, and damage to the osmoregulatory epithelium, including hypertrophy, proliferation, redistribution and necrosis of chloride cells. Tissue damage in gills is likely caused by the interaction of PTX2 with the actin cytoskeleton of the affected gill epithelia. Overall, the severe gill pathology observed following the PTX2 exposure suggested death was due to loss of respiratory and osmoregulatory functions in C. variegatus larvae.

Sub-monthly prediction of harmful algal blooms based on automated cell imaging.

Harmful algal blooms (HABs) are an increasing threat to global fisheries and human health. The mitigation of HABs requires management strategies to successfully forecast the abundance and distribution of harmful algal taxa. In this study, we attempt to characterize the dynamics of 2 phytoplankton genera (Pseudo-nitzschia spp. and Dinophysis spp.) in Narragansett Bay, Rhode Island, using empirical dynamic modeling. We utilize a high-resolution Imaging FlowCytobot dataset to generate a daily-resolution time series of phytoplankton images and then characterize the sub-monthly (1-30 days) timescales of univariate and multivariate prediction skill for each taxon. Our results suggest that univariate predictability is low overall, different for each taxon and does not significantly vary over sub-monthly timescales. For all univariate predictions, models can rely on the inherent autocorrelation within each time series. When we incorporated multivariate data based on quantifiable image features, we found that predictability increased for both taxa and that this increase was apparent on timescales >7 days. Pseudo-nitzschia spp. has distinctive predictive dynamics that occur on timescales of around 16 and 25 days. Similarly, Dinophysis spp. is most predictable on timescales of 25 days. The timescales of prediction for Pseudo-nitzschia spp. and Dinophysis spp. could be tied to environmental drivers such as tidal cycles, water temperature, wind speed, community biomass, salinity, and pH in Narragansett Bay. For most drivers, there were consistent effects between the environmental variables and the phytoplankton taxon. Our analysis displays the potential of utilizing data from automated cell imagers to forecast and monitor harmful algal blooms.