Alexandrium Species Research Articles

Unraveling the risks of nAl2O3 on harmful algal blooms: Insights from paralytic shellfish toxins production of Alexandrium tamarense

As one of the commonly used and cost-effective nanomaterials, nanosized aluminum oxide (nAl2O3) posses unique properties and chemical stability. However, its extensive use and resultant dissemination into aquatic ecosystems prompt concerns over the proliferation and repercussions of harmful algal blooms, particularly those caused by dinoflagellates producing toxins. This study investigated the sub-chronic effects of nAl2O3 on growth, physiological activities, and paralytic shellfish toxins (PSTs) production in Alexandrium tamarense. Results showed dose-dependent inhibition in growth (EC50 = 20.6 mg L−1), esterase activity, and photosynthetic efficiency (Fv/Fm) during the sub-chronic exposure (13-day). The internalization of nAl2O3 in microalgal cells and the significant decrease in the total cellular PSTs content were observed under high nAl2O3 concentrations (>40 mg L−1). The study also demonstrated a clear decrease in the content of some derivatives of PSTs (GTX5, C1/2, and GTX2/3) with the increase in nAl2O3 concentrations, accompanied by the induction of an unknown derivative. Excessive ROS production, dissolved Al, and physical inhibition were suggested as potential mechanisms for nAl2O3 toxicity and changes in PSTs toxin profile. Overall, this research enhances our understanding of the potentiated risks and threats on the possible concurrent events of toxic dinoflagellate, such as Alexandrium species and nanoparticles in aquatic environments.

Investigating the role of allelochemicals in the interaction between Alexandrium monilatum and other phytoplankton species

Species of the dinoflagellate genus Alexandrium can release bioactive extracellular compounds with allelopathic effects (e.g., immobilization, inhibition of growth, photosynthesis or lysis) towards other phytoplanktonic organisms. In the lower Chesapeake Bay, Virginia, US, succession or co-occurrence of blooms of Akashiwo sanguinea, Margalefidinium polykrikoides and the goniodomin-producing A. monilatum tend to be common during summer months, however the allelopathic potential of A. monilatum, and how it may affect bloom dynamics, has not be studied. We used a rapid fluorescence-based bioassay as well as flow cytometry and an assessment of immobilization to determine the potential effects of A. monilatum culture supernatants on M. polykrikoides, A. sanguinea and the diatom Chaetoceros muelleri (included as a reference strain). In addition, we also investigated the effects of standards of known A. monilatum toxins goniodomin A (GDA) and GDA seco-acid (GDA-sa). Exposure of C. muelleri to culture supernatants from two different strains of A. monilatum resulted in an inhibition of the maximum quantum yield of the photosystem II (Fv/Fm) and cell lysis of the diatom, with strain-specific variation observed, highlighted by a 30-fold difference in the effective concentration resulting in 10 % inhibition (EC10) and 2-fold difference in the lethal concentration resulting in 50 % mortality (LC50) between the two A. monilatum strains tested. Exposure of M. polykrikoides to A. monilatum culture supernatants resulted in a decrease in motility (at ≥ 500 eq. cells mL-1) along with a decrease in Fv/Fm (at ≥ 1,500 cells mL-1) and altered cellular morphology (at ≥ 3,500 eq. cells mL-1). No effect was observed when A. sanguinea was exposed to A. monilatum culture supernatants (at the concentrations tested in these studies). When M. polykrikoides was exposed to the GDA standard, its Fv/Fm (at ≥ 1,951 nM) and motility (at ≥ 390 nM) decreased, and its morphology (at ≥ 975 nM) was modified. The study of supernatant time- and temperature-stability, and the absence of a relationship between observed effects and goniodomin concentrations suggested the presence of additional unknown allelochemicals distinct from goniodomins, as has been observed for other Alexandrium species. Immobilization of M. polykrikoides by A. monilatum culture supernatants and GDA standard in laboratory-based exposure experiments may indicate that A. monilatum has a competitive advantage when both species co-occur in the Chesapeake Bay and this warrants further testing.

Biogeochemical conditions controlling the intensity of paralytic shellfish poisoning (PSP) outbreak caused by Alexandrium blooms: Results from 6-year field observations in Jinhae-Masan Bay, Korea

Previous field observations from 2018 to 2019 revealed that paralytic shellfish poisoning (PSP) caused by the blooms of toxic dinoflagellate Alexandrium species occurred under low concentrations of dissolved inorganic nitrogen (DIN) and high concentrations of dissolved organic nitrogen (DON) and humic-like fluorescent dissolved organic matter (FDOMH) in Jinhae-Masan Bay, Korea. In this study, we obtained more data for DIN, DON, FDOMH, and Alexandrium cell density from 2020 to 2023 to further validate environmental conditions for the PSP outbreak. We also measured total hydrolyzed amino acids (THAA) to determine the bioavailability of DON fueling the PSP outbreak. Over the 6-year observations, there was a consistent pattern of low DIN concentrations and high DON and FDOMH concentrations during the PSP outbreak periods. The Alexandrium cell densities, together with the PSP toxin concentrations, increased rapidly under this environmental condition. The PSP outbreak occurs when a large amount of DIN originating from the stream waters near the upstream sites is transformed into DON by biological production before entering the PSP outbreak area. The produced DON is characterized by high bioavailability based on the various AA-derived indices (enantiomeric ratio, degradation index, non-protein AA mole%, and nitrogen-normalized AA yield). In addition, the intensities of PSP outbreaks are mainly dependent on the conversion stage of DIN to DON and enhanced FDOMH. We found that the strong PSP outbreak occurred consistently under a low level of DIN (<1.0 μM) and high levels of DON (>9.0 μM) and FDOMH (>1.5 R.U.). Thus, our results suggest that the monitoring data of environmental conditions can be used to predict the PSP outbreak in the coastal oceans.

Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web

The dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed.

Variability in Paralytic Shellfish Toxin Profiles and Dinoflagellate Diversity in Mussels and Seawater Collected during Spring in Korean Coastal Seawater.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain microalgae, particularly dinoflagellates, and they can accumulate in shellfish in coastal seawater and thus pose significant health risks to humans. To explore the relationship between toxicity and PST profiles in seawater and mussels, the spatiotemporal variations in PST concentrations and profiles were investigated along the southern coast of Korea under peak PST levels during spring. Seawater and mussel samples were collected biweekly from multiple stations, and the toxin concentrations in the samples were measured. Moreover, the dinoflagellate community composition was analyzed using next-generation sequencing to identify potential PST-producing species. The PST concentrations and toxin profiles showed substantial spatiotemporal variability, with GTX1 and GTX4 representing the dominant toxins in both samples, and C1/2 tending to be higher in seawater. Alexandrium species were identified as the primary sources of PSTs. Environmental factors such as water temperature and salinity influenced PST production. This study demonstrates that variability in the amount and composition of PSTs is due to intricate ecological interactions. To mitigate shellfish poisoning, continuous monitoring must be conducted to gain a deeper understanding of these interactions.

Why cysts of Alexandrium catenella and/or A. pacificum (Gonyaulacales, Dinophyceae) do not remain in sediments as fossils?

The dinoflagellate genus Alexandrium contains a number of species that produce paralytic shellfish toxins and have been the focus of attention as toxic plankton for harmless algal studies. Among Alexandrium species, A. catenella and A. pacificum form ellipsoidal-shaped resting cysts, which are preserved in marine sediments, and have attracted attention as potential seeds for future proliferation after favorable environmental conditions environmental improvement. However, although these cysts are preserved in surface of marine sediments, there is no record of their occurrence from solidified sediments as fossils. In order to clarify the reason for this, we investigated the differences in the chemical composition of cyst walls between colorless cyst of Alexandrium catenella/pacificum and Lingulodinium machaerophorum, Polysphaeridium zoharyi, Spiniferites spp. by measuring the thickness of cyst walls and using Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy. The results showed that the cyst wall of A. catenella/pacificum and L. machaerophorum were all composed of cellulosic organic matter. However, A. catenella/pacificum have the higher percentage of α−/β-glucosidic linkages and that the thickness of their cyst walls is about one-third of that of L. machaerophorum. Therefore, these are reasons for the cysts of A. catenella/pacificum being more easily degraded in the sediment.

Dynamic variations in profile of intra- and extracellular paralytic shellfish toxins during the growth cycle of Alexandrium species and their accumulation in mussels

The toxigenic Alexandrium species can secrete paralytic shellfish toxins (PSTs) into seawater during their growth, potentially threatening marine organisms and human health. The release of intracellular toxins during the growth cycle of Alexandrium spp. and the uptake of dissolved PSTs by shellfish are still not clear. Three strains of A. minutum GY-H46, A. minutum ATDH and A. pacificum ATHK were used to explore the dynamic variations in profile of intra- and extracellular toxins during their growth cycles. And a comparative study was carried out on the accumulation and tissue distribution of PSTs in mussels Mytilus unguiculatus exposed to the dissolved and particulate toxins during a 5-days period. Results showed that the toxin cell quotas increased rapidly during the early exponential growth period, then decreased gradually and plateaued at the stationary growth period for ATHK and ATDH, while gradually increased during the stationary growth period for GY-H46. The toxin cell quotas for GY-H46, ATDH and ATHK ranged from 8.3 to 21.5, 5.3 to 11.7 and 7.4 to 9.3 fmol cell−1, respectively, during the whole growth cycle. The contents of extracellular toxins increased gradually throughout the growth of GY-H46, ATDH and ATHK, and the molar mass percentage of extracellular toxins ranged from 53% to 72%, 20% to 46% and 36% to 68%, respectively. Mussels can actively uptake the dissolved PSTs from seawater medium, and mainly accumulate toxins in the digestive glands. The accumulation level of PSTs from the dissolved phase by mussels was about 1% of that from feeding on microalgal cells. The results of this study will contribute to a comprehensive understanding of the dynamic release of intracellular PSTs during microalgal growth and the accumulation of dissolved toxins by shellfish.

Grazing impact of the calanoid copepods Acartia spp. on the toxic dinoflagellate Alexandrium pseudogonyaulax in the western coastal waters of Korea.

Marine dinoflagellate species in the genus Alexandrium are well known to produce paralytic shellfish poison as well as common coastal species with cosmopolitan distribution. However, few studies on the feeding of copepods on Alexandrium species have been conducted. The toxic dinoflagellate Alexandrium pseudogonyaulax contains goniodomin A and causes red tides in many countries. To investigate the relationship between the toxic dinoflagellate A. pseudogonyaulax and the calanoid copepods Acartia spp., we quantified the ingestion rates of Acartia spp. feeding on A. pseudogonyaulax as a function of prey concentration. Additionally, we estimated grazing coefficients by integrating data from field observations of Acartia spp. and coexisting A. pseudogonyaulax with laboratory measurements of ingestion rates obtained during this investigation. Furthermore, we compared the ingestion rates of Acartia spp. and other predators feeding on Alexandrium species as previously reported. The ingestion rates of Acartia spp. on A. pseudogonyaulax increased continuously with increasing mean prey concentration. The highest values among the ingestion rate of Acartia spp. feeding on A. pseudogonyaulax was 3,407 cells predator-1 d-1 (4,872 ng C predator-1 d-1) at the given prey concentration. The calculated grazing coefficients for Acartia spp. on A. pseudogonyaulax in Shiwha Bay, Korea, were up to 0.073 d-1. The results of this study suggest that A. pseudogonyaulax may decrease or maintain the population of Acartia spp. in marine food webs.

Identification of bacteria in potential mutualism with toxic Alexandrium catenella in Chilean Patagonian fjords by in vitro and field monitoring.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms, repeatedly causing damage to Chilean coastal waters. The causes and behavior of algal blooms are complex and vary across different regions. As bacterial interactions with algal species are increasingly recognized as a key factor driving algal blooms, the present study identifies several bacterial candidates potentially associated with Chilean Alexandrium catenella. This research narrowed down the selection of bacteria from the Chilean A. catenella culture using antibiotic treatment and 16S rRNA metabarcoding analysis. Subsequently, seawater from two Chilean coastal stations, Isla Julia and Isla San Pedro, was monitored for two years to detect Alexandrium species and the selected bacteria, utilizing 16S and 18S rRNA gene metabarcoding analyses. The results suggested a potential association between Alexandrium species and Spongiibacteraceae at both stations. The proposed candidate bacteria within the Spongiibacteraceae family, potentially engaging in mutualistic relationships with Alexandrium species, included the genus of BD1-7 clade, Spongiibbacter, and Zhongshania.

An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

The toxigenic red-tide-forming dinoflagellates Alexandrium leei and Alexandrium catenella differ in terms of the sensitivity to strong light and low temperature of their photosynthetic machinery

Alexandrium leei and Alexandrium catenella are noxious dinoflagellates that form red tides but the two species differ substantially in optimal growth conditions. For formation of blooms in natural environments, A. leei favors moderate temperatures (around 20 °C) while A. catenella favors much lower temperatures (around 10 °C). We examined the effects of temperature and light on cell proliferation and photosynthesis in cell cultures of the two species. Under light at 100 μmol photons m−2 s−1, the growth rate of both species was highest at 20 °C. At 25 °C, A. leei continued to proliferate but A. catenella did not survive. Conversely, at lower temperatures, namely, 12 °C and 15 °C, the growth rate of A. leei was very low while that of A. catenella remained high. The net photosynthetic activity, as determined in terms of oxygen evolution, was highest at 20 °C in A. leei. By contrast, it was highest at 15 °C in A. catenella. Illumination of cells with strong light at 1000 μmol photons m−2 s−1 decreased the activity of photosystem II (PSII), as determined in terms of Fv/Fm, in both species. In A. leei, the activity of PSII declined more rapidly at 12 °C and 15 °C than at 20 °C and 25 °C under strong light. In A. catenella, the activity of PSII declined more rapidly at 20 °C than at 12 °C and 15 °C. Thus, A. catenella appeared to be more resistant to photoinhibition of PSII at low temperatures than A. leei. In addition, the ability to repair photodamaged PSII at 12 °C and 15 °C was greater in A. catenella than that in A. leei. Thus, the tolerance of PSII to strong light and low temperatures might determine the range of temperatures that supports the formation of blooms of the two Alexandrium species.

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.

Propidium Monoazide based selective iDNA monitoring method improves eDNA monitoring for harmful algal bloom Alexandrium species

eDNA, also known as environmental DNA, has garnered significant attention due to its potential applications in various fields such as biodiversity assessment, species distribution monitoring, ecological interaction analysis, and quantitative analysis. However, the presence of non-selective DNA signals in eDNA samples poses challenges in accurately detecting species, assessing biodiversity, and conducting quantitative analysis. To address these limitations, this study developed a novel method for selectively detecting iDNA from specific species in eDNA samples. The method involved the application of PMA treatment to Alexandrium spp. effectively preventing the detection of non-selective exDNA signals. Additionally, by optimizing the filter size used in the sampling process, the researchers were able to selectively collect and analyze iDNA from species of interest, particularly Alexandrium spp. Furthermore, the study successfully demonstrated the selective collection and analysis of iDNA from Alexandrium spp. cysts present in the sediment layer, further strengthening the findings. The results indicated that the combined use of PMA treatment and filter size optimization significantly enhanced the selective detection capability of iDNA. The successful selective detection of iDNA from eDNA in the sediment layer highlights the practical applicability of the developed method. This study holds promise for advancing eDNA monitoring technology by providing a selective iDNA detection method utilizing PMA. Moreover, these findings lay the foundation for effectively utilizing iDNA in environmental conservation, monitoring, and ecological research.

Toxic Algal Bloom Recurrence in the Era of Global Change: Lessons from the Chilean Patagonian Fjords.

Toxic and harmful algal blooms (HABs) are a global problem affecting human health, marine ecosystems, and coastal economies, the latter through their impact on aquaculture, fisheries, and tourism. As our knowledge and the techniques to study HABs advance, so do international monitoring efforts, which have led to a large increase in the total number of reported cases. However, in addition to increased detections, environmental factors associated with global change, mainly high nutrient levels and warming temperatures, are responsible for the increased occurrence, persistence, and geographical expansion of HABs. The Chilean Patagonian fjords provide an "open-air laboratory" for the study of climate change, including its impact on the blooms of several toxic microalgal species, which, in recent years, have undergone increases in their geographical range as well as their virulence and recurrence (the species Alexandrium catenella, Pseudochattonella verruculosa, and Heterosigma akashiwo, and others of the genera Dinophysis and Pseudo-nitzschia). Here, we review the evolution of HABs in the Chilean Patagonian fjords, with a focus on the established connections between key features of HABs (expansion, recurrence, and persistence) and their interaction with current and predicted global climate-change-related factors. We conclude that large-scale climatic anomalies such as the lack of rain and heat waves, events intensified by climate change, promote the massive proliferation of these species by creating ideal conditions for their growth and persistence, as they affect water-column stratification, nutrient inputs, and reproductive rates.

Genomic copy number variability at the genus, species and population levels impacts in situ ecological analyses of dinoflagellates and harmful algal blooms

The application of meta-barcoding, qPCR, and metagenomics to aquatic eukaryotic microbial communities requires knowledge of genomic copy number variability (CNV). CNV may be particularly relevant to functional genes, impacting dosage and expression, yet little is known of the scale and role of CNV in microbial eukaryotes. Here, we quantify CNV of rRNA and a gene involved in Paralytic Shellfish Toxin (PST) synthesis (sxtA4), in 51 strains of 4 Alexandrium (Dinophyceae) species. Genomes varied up to threefold within species and ~7-fold amongst species, with the largest (A. pacificum, 130 ± 1.3 pg cell−1 /~127 Gbp) in the largest size category of any eukaryote. Genomic copy numbers (GCN) of rRNA varied by 6 orders of magnitude amongst Alexandrium (102– 108 copies cell−1) and were significantly related to genome size. Within the population CNV of rRNA was 2 orders of magnitude (105 – 107 cell−1) in 15 isolates from one population, demonstrating that quantitative data based on rRNA genes needs considerable caution in interpretation, even if validated against locally isolated strains. Despite up to 30 years in laboratory culture, rRNA CNV and genome size variability were not correlated with time in culture. Cell volume was only weakly associated with rRNA GCN (20–22% variance explained across dinoflagellates, 4% in Gonyaulacales). GCN of sxtA4 varied from 0–102 copies cell−1, was significantly related to PSTs (ng cell−1), displaying a gene dosage effect modulating PST production. Our data indicate that in dinoflagellates, a major marine eukaryotic group, low-copy functional genes are more reliable and informative targets for quantification of ecological processes than unstable rRNA genes.

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2ʹ/4ʹ suture length, narrower plates 1ʹ and 6ʹʹ, with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1ʹ having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6–14 pg cell−1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4–7.3 pg cell−1). None of the strains contained detectable levels of PSTs and cycloimines.

Combining Nanopore Sequencing with Recombinase Polymerase Amplification Enables Identification of Dinoflagellates from the Alexandrium Genus, Providing a Rapid, Field Deployable Tool.

The armoured dinoflagellate Alexandrium can be found throughout many of the world's temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of Alexandrium cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve Alexandrium to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the Alexandrium genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different Alexandrium species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of A. minutum in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the A. catenella, A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic A. catenella species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (p ≤ 0.05). Furthermore, a McNemar's paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of Alexandrium by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls.

Mass spectrometric characterization of the seco acid formed by cleavage of the macrolide ring of the algal metabolite goniodomin A

Goniodomin A (GDA) is a polyketide macrolide produced by multiple species of the marine dinoflagellate genus Alexandrium. GDA is unusual in that it undergoes cleavage of the ester linkage under mild conditions to give mixtures of seco acids (GDA-sa). Ring-opening occurs even in pure water although the rate of cleavage accelerates with increasing pH. The seco acids exist as a dynamic mixture of structural and stereo isomers which is only partially separable by chromatography. Freshly prepared seco acids show only end absorption in the UV spectrum but a gradual bathochromic change occurs, which is consistent with formation of α,β-unsaturated ketones. Use of NMR and crystallography is precluded for structure elucidation. Nevertheless, structural assignments can be made by mass spectrometric techniques. Retro-Diels-Alder fragmentation has been of value for independently characterizing the head and tail regions of the seco acids. The chemical transformations of GDA revealed in the current studies help clarify observations made on laboratory cultures and in the natural environment. GDA has been found to reside mainly within the algal cells while the seco acids are mainly external with the transformation of GDA to the seco acids occurring largely outside the cells. This relationship, plus the fact that GDA is short-lived in growth medium whereas GDA-sa is long-lived, suggests that the toxicological properties of GDA-sa in its natural environment are more important for the survival of the Alexandrium spp. than those of GDA. The structural similarity of GDA-sa to that of monensin is noted. Monensin has strong antimicrobial properties, attributed to its ability to transport sodium ions across cell membranes. We propose that toxic properties of GDA may primarily be due to the ability of GDA-sa to mediate metal ion transport across cell membranes of predator organisms.

Seasonal change and subniche dynamics of three Alexandrium species in the Korea Strait

Some members of the dinoflagellate genus Alexandrium produce toxins responsible for paralytic shellfish poisoning, which causes environmental impacts and large economic losses worldwide. The Outlying Mean Index (OMI) and the Within Outlying Mean Index (WitOMI) were used to examine the ecological niches of three Alexandrium species identifying factors affecting their population dynamics in the Korea Strait (KS). Species niches were divided into seasonal subniches based on species’ temporal and spatial patterns, with A. catenella being highest in the spring, A. pacificum in the summer, and A. affine in the autumn. These shifts in abundance are likely due to changes in their habitat preferences and resource availability, as well as the effects of biological constraints. A subniche-based approach, which considers the interactions between the environment and the biological characteristics of a species, was useful in understanding the factors shaping the population dynamics of the individual species. Additionally, a species distribution model was used to predict the phenology and biogeography of the three Alexandrium species in the KS and their thermal niches on a larger scale. The model predicted that, in the KS, A. catenella exists on the warm side of the thermal niche, while A. pacificum and A. affine exist on the cold side, indicating that these species may respond differently to increases in water temperature. However, the predicted phenology was incongruent with the abundance of the species as measured by droplet digital PCR. Overall, the WitOMI analysis and species distribution model can provide valuable insights into how population dynamics are influenced by the integrated interplay of biotic and abiotic processes.

Distribution of dinoflagellate cyst assemblages in surface sediments of Magellan fjords and channels (Patagonia, Chile) with a focus on harmful species: An overview on environmental scenario

Several dinoflagellate species form benthic resting cysts in their life cycle. In order to study the composition, abundance and distribution of cyst assemblages, with a focus on harmful species, thirty-one surface sediment samples from the Magellan fjords and channels system were analysed. In situ (temperature, salinity, inorganic nutrients, dissolved oxygen, organic matter, and chlorophyll-a) and five-year (salinity, temperature, dissolved oxygen, and chlorophyll-a) measurement data were analysed to obtain an overview on environmental scenario. A total of 56 cyst morphotypes were recorded, some of which were identified for the first time for the Chilean coast (Dactylodinium cf. arachnoides, Impagidinium cf. velorum, Archaeperidinium sp., Protoperidinium haizhouense, Protoperidinium sinuosum, Protoperidinium tricingulatum, and Dubridinium cf. ulsterum). In vitro germination of cysts allowed the vegetative form of the species of the order Suessiales to be determined. Total cyst concentrations were highly variable, ranging from 145 to 5453 cysts mL−1 of wet sediment (mean 1246 ± 1061 cysts mL−1), with the lowest cyst values in the area adjacent to the Southern Patagonian Ice Field. Pentapharsodinium dalei and Protoperidinium conicoides generally dominated (%) the cyst assemblages. The harmful species Alexandrium catenella and Alexandrium ostenfeldii were not abundant and sparsely distributed, while Protoceratium reticulatum was widespread and highly abundant with Gonyaulax spinifera in Inutil Bay (Magellan Strait). Cluster and nMDS analyses performed with cyst concentrations of the 56 taxa in the 31 stations formed seven cyst assemblage clusters, reflecting high environmental variability. Canonical correspondence analyses performed with cyst concentrations and in situ and long-term environmental data showed different ecological signals, demonstrating that in situ parameters must be used carefully when studying cysts in surface sediments.