Paralytic Shellfish Poisoning Research Articles

Environmental Factors Modulate Saxitoxins (STXs) Production in Toxic Dinoflagellate Alexandrium: An Updated Review of STXs and Synthesis Gene Aspects.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms (HABs) and produces saxitoxin (STX) and its derivatives (STXs) that cause paralytic shellfish poisoning (PSP) in humans. Cell growth and cellular metabolism are affected by environmental conditions, including nutrients, temperature, light, and the salinity of aquatic systems. Abiotic factors not only engage in photosynthesis, but also modulate the production of toxic secondary metabolites, such as STXs, in dinoflagellates. STXs production is influenced by a variety of abiotic factors; however, the relationship between the regulation of these abiotic variables and STXs accumulation seems not to be consistent, and sometimes it is controversial. Few studies have suggested that abiotic factors may influence toxicity and STXs-biosynthesis gene (sxt) regulation in toxic Alexandrium, particularly in A. catenella, A. minutum, and A. pacificum. Hence, in this review, we focused on STXs production in toxic Alexandrium with respect to the major abiotic factors, such as temperature, salinity, nutrients, and light intensity. This review informs future research on more sxt genes involved in STXs production in relation to the abiotic factors in toxic dinoflagellates.

A case of paralytic shellfish poisoning caused by consumption of visceral balls from geoduck Panopea japonica in Japan

In the end of March 2018, an unprecedented food poisoning incident due to ingestion of the visceral balls of geoduck Panopea japonica occurred in Japan. The patient, presented with symptoms of numbness on the lips and general weakness, was diagnosed as paralytic shellfish poisoning (PSP). The patient immediately treated with the mechanical ventilation recovered and left the hospital after 3 days treatment. Saxitoxins (STXs) in the plasma and urinary samples collected from the patient on the first and second day after hospitalization were analyzed by ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) and liquid chromatography with post-column fluorescent detection (LC/FLD). The STXs levels of 499.1 and 6.0 μg/L of STX dihydrochloride equivalent (STX·2HCl eq.) were quantitated by LC/FLD in the urinary samples on the first and second day, respectively. In addition, geoducks harvested from the same areas of the PSP causative specimens after the incident were analyzed by LC/FLD, and the results showed the level of STXs in their whole bodies of the geoducks exceeding 0.8 mg STX·2HCl eq./kg which is the maximum levels of STX in CODEX STAN 292–2008. Prominent toxins in STXs that detected in urinary and geoduck samples and identified by UHPLC/MS/MS and LC/FLD were gonyautoxin-1+4 (GTX1+4). These results concluded that the incident was the food poisoning due to STXs accumulated in the geoducks. This is the first PSP case caused by consumption of geoducks in Japan. This is also the first PSP case that causative toxins are detected in urinary samples of patients involved in PSP in Japan.

Expansion of Toxic Algal Blooms in Coastal and Marine Areas in the Philippines and Malaysia: Is It Climate Change Related?

This paper provides a review of toxic algal blooms in the Philippine and Malaysian coastal and marine systems, considering relevant available knowledge, including climate change dimension/s in the assessment of their recorded recent expansion. The first record of human toxicity in the Philippines associated with HABs/toxic algal blooms specifically was during the bloom of Pyrodinium bahamense in the Sorsogon, Samar, and Leyte waters in 1983. Since then, the species has been identified to occur and cause blooms in about 44 sites/areas in the country. Recent government reports, i.e., 2021, 2022, and 2023, have also identified other paralytic shellfish poisoning (PSP) causative organisms (Gymnodinium catenatum, Alexandrium spp.) in the country. New records indicate that the presence of PSP causative species has been reported almost year-round in the Philippines. In Malaysia, PSP caused by P. bahamense was initially confined in 1981 to the state of Sabah, Malaysia Borneo, but since then, blooms of this species have been reported almost annually at different scales across the coastal waters of Sabah. P. bahamense and other cyst-forming dinoflagellates could be transported naturally or through human activities. Other eco-physiological and environment factors from the field and the laboratory have been used to study the bloom dynamics and transport of PSP causative species in several areas in the Philippines and Malaysia. More recently, plastics and other marine litter have been considered potential vectors of invasion/transport or expansion of dinoflagellates with other microorganisms. ENSO events have been observed to be stronger since 1950 compared with those recorded from 1850 to 1950. The extreme phases of the ENSO phenomenon have a strong modulating effect based on seasonal rainfall in the Philippines, with extreme ENSO warm events (El Niño) often associated with drought and stresses on water resources and agriculture/aquaculture. In contrast, cold events (La Niña) often result in excessive rainfall. The La Nina Advisories from 2021 to 2023 (18 advisories) showed the persistence of this part of ENSO, particularly in regions with recurrent and new records of HABs/toxic algal blooms. More studies and monitoring of another type of toxic algal bloom, Ciguatera Fish Poisoning (CFP), are recommended in tropical countries such as the Philippines and Malaysia, which have extensive reef areas that harvest and culture marine fish for local and export purposes, as accelerating reports of this type of poisoning have apparently increased and causative organisms have been identified in several areas. There is an urgent need to enhance HAB/toxic algal bloom research and monitoring, particularly those related to climate change, which has apparently impacted these blooms/occurrences directly or indirectly. Local researchers and managers should be made aware of the knowledge and tools already available for their utilization and enhancement to meet local conditions and challenges for potential recurrence and expansion of HABs/toxic algal blooms. Regional and international HAB research and collaboration should be further advanced for the protection of public health and marine resources.

Harmful blooms caused by dinoflagellates in the Pacific of Guatemala (2019–2022)

Plankton studies in Guatemala are associated with the history of harmful algal blooms (HAB). An important event was observed in 1987, with 193 human poisonings due to shellfish consumption, of which 22 were lethal. The causative organism was Pyrodinium bahamense var. compressum. This species was reported again in November 2019 in the coastal Guatemalan Pacific. The species reached an abundance of 1 × 104 cells/L. In September 2020, the abundance of the Margalefidinium polykrikoides vegetative stage reached 1.24 × 106 cells/L, and its cysts 1.5 × 106 cells/L. Noctiluca scintillans proliferated (the first quantitatively estimated bloom of this species in Guatemala) in November 2020 and March 2021 (up to 1.2 × 106 cells/L). From January to December 2021, monthly monitoring was carried out in front of the Port of Quetzal at three sites (bottle and 25 μm mesh net samples). In April 2022, 11 cases of humans presenting symptoms of paralytic shellfish poisoning (PSP) were recorded in the western sector of the Guatemalan Pacific, in particular, in the municipality of Tiquisate in the department of Escuintla. From May 3 to 27, 2022, a HAB event caused the death of 4 and the poisoning of 34 humans in the departments near Mexico: Retalhuleu (Champerico), San Marcos (Tilapa) and Escuintla (Tiquisate, Buena Vista and Iztapa). The maximum saxitoxin (STX) concentration was determined in Retalhuleu (14,099 MU/100 g); until 7 July 2022, STX continued to be detected in Tilapa and Tiquisate (1,021 MU/100 g). The P. bahamense cells were observed in the stomach contents of the clam Tagelus sp. (Bivalvia: Solecurtidae).

A dual-mode optical sensor for sensitive detection of saxitoxin in shellfish based on three-in-one functional nanozymes

Saxitoxin (STX), one of paralytic shellfish poison neurotoxins with high toxicity, posing a significant threat to seafood safety. Therefore, it is of great need to protect human health by screening STX contaminated seafood samples. In this study, to achieve specificity for STX, molecularly imprinted polymers (MIP) were fabricated on Au-Pt nanoparticles modified Fe3O4 magnetic nanozymes (MS@Au-Pt NZs) with numerous memory cavities (MS@Au-Pt/MIP), which catalyzed the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). STX will occupy the recognition cavity of MIP and obstruct the catalytic site of MS@Au-Pt, thus hindering the color development reaction, and the oxidized TMB (oxTMB) can be measured by Raman spectroscopy and recorded with Raman signals. Under the optimal conditions, the MIDMs method obtained a wide linear range for colorimetric assay (0.01 μM to 100 μM) and Raman detection (0.1 nM to 100 nM), with limits of detection (3σ/S, n=3) of 3.1 nM and 0.03 nM, respectively. As a proof-of-concept, the approach showed acceptable recoveries of 78.0∼96.2% in real samples of mussel and clam. The dual-mode optical sensor, therefore, is hopeful to be a potential candidate for sensitive and accurate detection of STX in seafood.

Determination of hydrophilic marine biotoxins in bivalve molluscs by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

This work describes a method for the determination of paralytic shellfish toxins in mussels (M. galloprovincialis), oysters (Crassostrea gigas), and clams (Ruditapes decussatus) samples. The acetic acid extraction method is used for simultaneous analysis of Paralytic Shellfish Poisoning (PSP) and the separation of these compounds was achieved on ethylene bridged hybrid (BEH) Amide column with gradient mobile phase composed of acetonitrile (MeCN) and water (as eluent A and eluent B) both containing 2 mM ammonium formate and 50 mM formic acid. The method specificity, linearity, sensitivity, precision, and accuracy were successfully evaluated towards the detection of 13 hydrophilic marine biotoxins analyzed together (single run) in different sample matrixes such as mussels. oysters. and clams. Calibration curves were found to be linear (R2 ≥ 0.98). average Recovery% of 98.5–99.9%. limits of detection (LODs): 0.16–1.92 µg·kg−1; limits of quantitation (LOQs): 0.54–6.41 µg·kg−1) and the relative standard deviation (RSD%) of the precision was ≤ 2.8%. The method was applied for the determination of PSP in shellfish collected from several Tunisian locations. All LC-MS/MS results of analyzed molluscs collected from different Tunisia locations, reveal no PSP toxin group contamination and that the amount found does not exceeding the threshold of 800 µg STXequ·kg−1.

A demographic model to forecast Dinophysis acuminata harmful algal blooms

Harmful algal blooms (HABs) in marine environments have significant adverse effects on public health, aquaculture and recreational activities. Surges of certain phytoplanktonic toxin-producing microalgae (mostly dinoflagellates or diatoms species) can induce Amnesic, Diarrhetic or Paralytic Shellfish Poisoning (ASP, DSP and PSP). Among HAB species, the genus Dinophysis leads to DSP in human consumers; this being the most recurrent problem in the Iberian Peninsula with the biggest economic impact on clam production and harvesting. While complete elimination of HABs is not feasible, timely implementation of appropriate measures can prevent their negative consequences. This is critical for aquaculture. Research on D. acuminata (dominant Dinophysis species in the North Atlantic) has been focused on ecophysiology and population dynamics, although with few modelling attempts. Weekly monitoring along the Portuguese coast since 2006 has revealed that D. acuminata thrives under spring/summer photosynthetically active radiation (PAR) coupled with water temperatures below 20°C, which typically coincide with the local upwelling regime. In order to advance this knowledge numerically, we developed a demographic model linking D. acuminata growth rate to PAR and sea surface temperature (SST). The 13-year (1-Jan-2006 to 31-Dec-2018) time-series of observations was closely fit by model forecasts. However, the model demonstrated limitations in issuing timely warnings of harmful proliferation of D. acuminata, failing to do so in 50% of cases, and issuing incorrect warnings in 5% of the cases. Furthermore, improving the odds of emitting timely warnings always worsened the odds of emitting false warnings, and vice-versa. To simultaneously improve both aspects, the modelling results clearly indicated the need of implementing both census/projection intervals smaller than 7 days and a laboratory detection limit below 20 cell/L. The time resolution of the census and of the model proved to be the most limiting factor that must be addressed in order to improve numerical forecasting of HABs.

Evaluation of biotoxins and toxic metal risks in mussels from the Sea of Marmara following marine mucilage

The mucilage phenomenon observed in the Sea of Marmara in 2021, has raised public concern about seafood safety. Mediterranean mussels serve as a vehicle in food chain, enabling the transfer of pollutants. Farmed and wild mussels were collected from 4 different stations throughout the fishing season. Biotoxins causing amnesic, paralytic, or diarrhetic shellfish poisonings (ASP, PSP, or DSP) were examined during monthly samplings. Potential health risks posed by cadmium, lead and arsenic were assessed. Health risks were evaluated considering 150 g/week mussel consumption, accounting for the different age groups of consumers (50, 60, 70 kg). Estimated Weekly Intake calculations of metals were determined to be lower than Provisional Tolerable Weekly Intake at all age groups throughout the sampling period in all stations. Target Hazard QuotientCd of mussels captured from Istanbul Strait was always determined <1, while it was equal to 1 for 50 kg individuals in Gelibolu samples. All THQAs were >1. Target carcinogenic Risk was evaluated for Pb and iAs, which were found to be negligible and acceptable, respectively. No biotoxins responsible for ASP, PSP, or DSP were detected. Hg levels were under detectable limits. Excluding Cd, the results did not reveal any risks associated with mussel consumption during mucilage.

Differential Proteomic Analysis of Low-Dose Chronic Paralytic Shellfish Poisoning.

Shellfish poisoning is a common food poisoning. To comprehensively characterize proteome changes in the whole brain due to shellfish poisoning, Tandem mass tag (TMT)-based differential proteomic analysis was performed with a low-dose chronic shellfish poisoning model in mice. A total of 6798 proteins were confidently identified, among which 123 proteins showed significant changes (fold changes of >1.2 or <0.83, p < 0.05). In positive regulation of synaptic transmission, proteins assigned to a presynaptic membrane (e.g., Grik2) and synaptic transmission (e.g., Fmr1) changed. In addition, altered proteins in nervous system development were observed, suggesting that mice suffered nerve damage due to the nervous system being activated. Ion transport in model mice was demonstrated by a decrease in key enzymes (e.g., Kcnj11) in voltage-gated ion channel activity and solute carrier family (e.g., Slc38a3). Meanwhile, alterations in transferase activity proteins were observed. In conclusion, these modifications observed in brain proteins between the model and control mice provide valuable insights into understanding the functional mechanisms underlying shellfish poisoning.

Development of saxitoxin biosynthesis gene sxtB-targeted qPCR assay for the quantification of toxic dinoflagellates Alexandrium catenella (group I) and A. pacificum (group IV) occurring in the Korean coast

Toxic dinoflagellate Alexandrium can produce saxitoxins (STXs) and cause paralytic shellfish poisoning (PSP), and thus they are monitored for environmental safety management. Microscopic discrimination of dinoflagellates is difficult to distinguish between toxic and non-toxic species due to their similar morphology. Meanwhile, an alternative quantitative PCR (qPCR) assay is sensitive, rapid, and cost-effective for harmful species monitoring. Herein, we developed a novel qPCR assay to detect the STXs biosynthesis gene sxtB of Alexandrium catenella and A. pacificum, the leading cause of PSP outbreaks in Asian coasts and worldwide. The newly designed sxtB TaqMan probes target the species without any positive signal in other relative dinoflagellates. Deming regression analysis revealed that the sxtB copy number of A. catenella and A. pacificum was 3.6 and 4.1 copies per cell, respectively. During the blooming periods (April 13th–14th, 2020), only A. catenella cells were detected through the qPCR assay, ranging from 5.0 × 10 to 2.5 × 104 eq cells L−1. In addition, sxtB qPCR quantified more accurately compared to large subunit (LSU) rRNA targeting qPCR assay that overestimate cell density. Besides, the sensitivity of sxtB was higher compared to the microscope when the species were rarely present (5.0 × 102 cells L−1). These suggest that the sxtB qPCR assay can be applied to toxic Alexandrium monitoring in the Korean coast, even in the early stage of bloomings.

Spatial and Temporal Variability of Saxitoxin-Producing Cyanobacteria in U.S. Urban Lakes.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

A fast and reliable LC-MS-MS method for the quantification of saxitoxin in blood plasma samples.

Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.

Development of fluorometric detection for saxitoxin with its specific binding peptide

A fluorometric sensor for marine saxitoxin was developed using its specific binding peptide based on the graphene oxide-mediated quenching phenomenon.

Growth and paralytic shellfish poisoning toxin production by a Mexican dinoflagellate strain of Alexandrium tamiyavanichii Balech (1994) under different nutrient conditions

Alexandrium tamiyavanichii is a marine dinoflagellate known to produce Paralytic Shellfish Poisoning (PSP) toxin. Thus, a strain was isolated from La Paz Bay, Baja California Sur, Mexico and used to explore whether stress conditions, such as phosphorus limitation (PL) and nitrogen enrichment (NE) modulate population growth and PSP toxin production in the GSe medium. Growth kinetics showed that the PL treatment produced a 3.4-fold increase in cell density versus control at day 30 of the culture cycle. The highest PSP concentration was found in the control culture (309 fmol cell−1) on day 21. Saxitoxin (STX) was the main analog in all the treatments (> 40 % mol). In conclusion, PL and NE treatments promoted growth kinetics in the species studied but did not affect the PSP toxin production. For the first time, the present research describes A. tamiyavanichii high toxicity strain isolated from Mexican coasts relative to the South-Atlantic strains.

A Microfabricated, Flow-Driven Grinding Mill for Mechanical Cell Lysing.

This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, Alexandrium, a neurotoxin-producing genus of algae that is responsible for red tide and paralytic shellfish poisoning. The mill consists of a high-speed, hydrodynamically driven microrotor coupled to a micro grinding mill that lyses robust algal cells by mechanical abrasion with single-pass efficiencies as high as 97%. These efficiencies are comparable to, or better than, current mechanical and chemical lysing methods without adding complications associated with harsh chemical additives that can interfere with subsequent downstream bioanalysis. Release of cytoplasm from lysed algae was confirmed using polymerase chain reaction (PCR) amplification of Alexandrium DNA using dinoflagellate primers.

Diversity and distribution of species of the planktonic dinoflagellate genus Alexandrium (Dinophyta) from the tropical and subtropical Mexican Pacific Ocean

Abstract Species of the dinoflagellate genus Alexandrium are marine and planktonic forms, widely distributed, and some are recognized to form harmful algal blooms and to produce saxitoxins causing Paralytic Shellfish Poisoning in humans, and other toxins. We studied the species composition of Alexandrium in tropical and subtropical coastal areas of the Mexican Pacific: from the southern Gulf of California to the Gulf of Tehuantepec. Eleven Alexandrium species were identified, described, and illustrated using light microscopy, and occasionally scanning electron microscopy for certain species. Additionally, the genetic characterization of seven strains and four species, was performed, using sequences of the D1/D2 LSU rDNA and ITS regions. We identified the species (morphospecies): Alexandrium affine, A. gaarderae, A. globosum, A. leei, A. margalefii, A. minutum, A. monilatum, A. pseudogonyaulax, A. tamarense, A. tamiyavanichii, and A. tropicale. Of these, A. affine, A. leei, A. minutum, A. monilatum, A. pseudogonyaulax, A. tamarense and A. tamiyavanichii have been widely recognized as harmful algae. Alexandrium gaarderae, A. globosum and A. tropicale are new records for the Mexican Pacific. This is the first morphological documentation of A. pseudogonyaulax. Future studies of the genus might increase its species richness if more cultures are established and metabarcoding approach is used.

Saxitoxin hazard! Detection of toxic algae in molluscs breeding areas in North-Western Adriatic Sea

Abstract Background Saxitoxin (SXT) can cause the paralytic shellfish poisoning (PSP) with severe impacts on both human health and marine ecosystems. Dinoflagellates are the major marine producers of STX and other variety of secondary toxic metabolites that can gather in molluscs becoming a risk for consumers’ poisoning. The aim of this work was to investigate the presence of the main algal species producing SXT in an area intended for molluscs breeding. Methods During 2020-2023, 40 seawater samples were collected from two macro-areas in the Adriatic Sea (North-Western) at different distances from the coast. After water filtration, DNA was extracted (DNeasy PLANT Mini Kit) and analysed with Real Time PCR for Alexandrium minutum and Gymnodinium catenatum detection. The possible ability to produce SXT was confirmed by sxt4A gene detection. Molecular results were compared with the microscopy gold-standard method. Results A. minutum was detected in 62.5% (CI 95% 0.47-0.75) of the samples, with 84% (CI 95% 0.65-0.94) of them possibly producing SXT. G. catenatum was found in 95% (CI 95% 0.83-0.99) of samples, with 73.7% (CI 95% 0.58-0.85) of them presenting STX gene. By microscopy, only Alexandrium spp. were identified without successful species identification. No other saxitoxin-producing genera or species were observed. Conclusions Our results underlined that A. minutum and G. catenatum can play an important role in the production of SXT, confirming their harmful presence in the areas intended for molluscs breeding. Microscopic identification can lead to an underestimation of the presence of potentially toxic algal species, stressing the relevance of improving molecular tools also in this field. These findings highlighted the importance of reporting and monitoring toxic algae presence, given the saxitoxin impact for food safety, and its significance for public health. Key messages • Saxitoxin-producing algae detection through molecular methods in molluscs breeding areas may represent an early warning useful to improve food safety and human health. • Harmful algal monitoring can improve water management in a One Health perspective.

Feeding Behavior Responses of the Small Copepod, Paracalanus parvus, to Toxic Algae at Different Concentrations.

The feeding relationship between copepods and phytoplankton has immense ecological significance. This study investigated the feeding behavior of copepods by studying the feeding selectivity of Paracalanus parvus, a key small copepod species, using a high-speed camera. The feeding behavior of P. parvus separately fed on three algae, Prorocentrum minimum, Alexandrium minutum, and Thalassiosira weissflogii, was studied at five different concentrations. The factors characterizing feeding behavior, including the beating frequency (BF), beating time (BT), and rejection behavior, were analyzed. The average BT and BF of P. parvus fed on toxic algae were significantly lower than those of copepods fed on nontoxic algae, indicating that the toxic algae negatively affected their feeding behavior. There were no significant differences in feed rejection among the three algae during the short period of experimentation, indicating that the rejection behavior was insignificant in the early period (within 20 min) of feeding on toxic algae. The feeding behavior was inhibited when the concentration reached 250 cells/mL. The BT was initially affected at increasing concentrations followed by the BF, and P. minimum and A. minutum reduced the BF at concentrations of 250 and 1000 cells/mL, respectively. Analysis of the average BFs revealed that P. parvus was more significantly affected by P. minimum containing diarrheal shellfish poison than by A. minutum containing paralytic shellfish poison. The BF of copepods fed on P. minimum was significantly lower than that of copepods fed on A. minutum at 250-500 cells/mL but was not significantly different from that at 1000 cells/mL. This indicated that the inhibitory effect of P. minimum on the feeding behavior was more significant at concentrations observed at the onset of red tide blooms (0.25-0.5 × 102 cells/mL), but insignificant at concentrations reaching those in advanced red tides (>103 cells/mL). This study demonstrates that toxic dinoflagellates alter the feeding behavior of copepods and describes the variations in their feeding response to different algal species and concentrations. The findings provide crucial insights for further studies on the feeding relationship between copepods and phytoplankton and on functional assessment of plankton ecosystems.

Harmful algae diversity from a coastal upwelling system detected by high-throughput sequencing

IntroductionIn recent years, environmental DNA (eDNA) amplicon sequencing has been used to unveil plankton diversity in the field. Nevertheless, molecular methods, such as this, are rarely used in harmful algal bloom (HAB) monitoring programs, which mainly rely on morphological identification by conventional light microscopy.MethodsThe present study focused on a shallow marine environment (Ría de Vigo, Northwest Spain), where sediment and plankton samples were collected from 2016 to 2018.ResultsThe application of eDNA amplicon sequencing allowed us to simultaneously detect 25 potential harmful species (mainly diatoms and dinoflagellates) included in the IOC-UNESCO Taxonomic Reference List of Harmful Microalgae. Among these, causative agents of amnesic shellfish poisoning (Pseudo-nitzschia spp.) paralytic shellfish poisoning (Gymnodinium catenatum and Alexandrium minutum), azaspiracid producers (Azadinium poporum) and ichthyotoxic haptophytes (Chrysochromulina leadbeateri), were identified. Some toxic microalgae were better represented in sediment (e.g., Pseudo-nitzschia pungens, Gymnodinium catenatum) or planktonic fractions (e.g., Pseudo-nitzschia, Gymnodinium smaydae), confirming the importance of including both sediment and plankton fractions in eDNA monitoring studies. Despite the limitations of sequencing short amplicons, it was possible to discern in this study six Pseudo-nitzschia species and associate each of them with each seasonal peak produced in summer periods. Furthermore, several species previously unreported in Ría de Vigo (Pseudo-nitzschia turgidula, Chrysochromulina leadbeateri, Azadinium poporum) could be detected.DiscusionThese results point out the application of eDNA amplicon sequencing to expand our knowledge about harmful species in HAB monitoring programs and early warning systems for low abundant and rare taxa.

Effects of nitrate on the saxitoxins biosynthesis revealed by sxt genes in the toxic dinoflagellate Alexandrium pacificum (group IV)

The dinoflagellate Alexandrium pacificum (group IV) is of particular interest because of its involvement in harmful algal blooms and production of saxitoxin (STX), which causes paralytic shellfish poisoning. The toxicity from STX and its analogues (STXs) is suspected to be affected by nitrogen (N) availability. However, the toxicity-associated behavior and STX-biosynthesis gene responses of the toxic A. pacificum under N fluctuations have not been sufficiently investigated. In the present study, we identified the sxtI gene involved in sxt biosynthesis pathway and evaluated the effects of nitrate (NO3−) on STXs production and the expression of four sxt core genes (sxtA4, sxtG, sxtB, and sxtI). Quantification of total STXs levels in the cultures under different NO3− regimes showed that NO3− concentration influenced STXs production. In addition, the proportion and concentration of STXs varied depending on the NO3− concentration. Core sxt transcript abundance was also influenced by available NO3− in a time-dependent manner. Expressional levels and patterns of sxtI were correlated with those of sxtA and sxtB. The relationship between the toxins and sxt responses in A. pacificum under various NO3− regimes suggests the direct involvement of N in the STXs biosynthesis pathway. Understanding this link would provide a tool to understand the toxin dynamics of dinoflagellates following N shifts in marine environments.