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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. [ABSTRACT FROM AUTHOR]

HABs pose a threat to coastal ecosystems, the economic sector and human health, and are expanding globally. However, their influence on copepods, a major connector between primary producers and upper trophic levels, remains essentially unknown. Microalgal toxins can eventually control copepod survival and reproduction by deterring grazing and hence reducing food availability. We present several 24-h experiments in which the globally distributed marine copepod, Acartia tonsa, was exposed to different concentrations of the toxic dinoflagellate, Alexandrium minutum, grown under three N:P ratios (4:1, 16:1 and 80:1), with the simultaneous presence of non-toxic food (the dinoflagellate Prorocentrum micans). The different N:P ratios did not affect the toxicity of A. minutum, probably due to the low toxicity of the tested strain. Production of eggs and pellets as well as ingested carbon appeared to be affected by food toxicity. Toxicity levels in A. minutum also had an effect on hatching success and on the toxin excreted in pellets. Overall, A. minutum toxicity affected the reproduction, toxin excretion and, to an extent, the feeding behavior of A. tonsa. This work indicates that even short-term exposure to toxic A. minutum can impact the vital functions of A. tonsa and might ultimately pose serious threats to copepod recruitment and survival. Still, further investigation is required for identifying and understanding, in particular, the long-term effects of harmful microalgae on marine copepods. [ABSTRACT FROM AUTHOR]

Harmful algal blooms (HABs) are becoming widely distributed and more frequent, threatening socioecosystems and human health. We determined species composition, abundance and spatial distribution of dinoflagellate cysts in the upper sediment of the Oualidia Lagoon located on the Atlantic coast of Morocco. Sediment samples were collected in 2017 at 51 stations, and environmental parameters were measured together with microphytoplankton abundance. Sediment characteristics including water percentage, organic matter content and grain size were determined. Fourteen dinoflagellate cyst morphotypes were identified, with Lingulodinium polyedrum (50%) and Gonyaulax spinifera (22%) dominating the assemblages. Total cyst densities ranged from 0 to 293 cysts g−1 dry sediment. Cyst densities were positively correlated with water content and organic matter content and increased with decreasing sediment grain size. We revealed the presence of three neurotoxic dinoflagellate species: Alexandrium minutum, the Alexandrium tamarense species complex, and Gymnodinium catenatum. Numerous cysts had accumulated in the sediment, and, because they are likely responsible for the initiation of HABs in Oualidia Lagoon, they should be monitored. [ABSTRACT FROM AUTHOR]

Marine biotoxins can accumulate in filter-feeders bivalve molluscs, that may represent a source of potential health problems being vectors of toxins, that are transferred to humans through their consumption. Harmful Algal Blooms impact on aquaculture may give also economic losses due to temporary closures of contaminated shellfish harvest and marketing. The presence of toxic algae for Paralytic Shellfish Poisoning (PSP), with recurrent toxic blooms of dinoflagellates, such as several Alexandrium species, been known since 2000 in the waters of an Ionian bay of Sicily, the Syracuse harbour, where shellfish farms are located. Our previous works reported in this area the positivity for PSP toxin in mussels (Mytilus galloprovincialis) with saxitoxin concentrations above the limit of the law and the simultaneous presence of toxic species of the genus Alexandrium in the waters. This work reports new recent episodes of algal blooms of Alexandrium minutum in the waters of the Syracuse harbour and PSP toxin contamination in farmed mussels, with values beyond the limits established by law, with the consequent immediate closure of the production area. PSP toxicity was detected with the MBA (Mouse Bioassay) with the confirm carried out with Lawrence method to quantify the total saxitoxin equivalents and characterize the toxic profile. Regular application of the implemented health plan is very important in order to prevent any risk and protect consumer health. [ABSTRACT FROM AUTHOR]

This study evaluated the performance of an easy-cultivation device for the mass culture of Alexandrium minutum (A. minutum), a dinoflagellate that produces paralytic shellfish toxins (PSTs). Five culture conditions including three different sizes of containers (250 mL conical flask, 500 mL beaker, and 20 L jar) in two different environments (out-incubator and incubator) were compared in terms of growth and PSTs production. Compared with the incubator environment, the out-incubator environment had more fluctuations in temperature and light intensity. Results showed that the cell densities of A. minutum increased in all groups, especially in the conical flask (I, out-incubator, 6.29×106 cells/mL) and the beaker (IV, incubator, 7.28×106 cells/mL). When cultured in the 20 L jar under out-incubator condition, the algae had the lowest cell density (2.82×106 cells/mL) but the highest toxicity (93.42 ± 2.55×10−6 MU/cell). The negative correlation between average growth rate and single-cell toxicity could be explained by biocompatibility, thereby indicating that the low growth rate led to high toxicity. HPLC-FLD showed that the cellular toxicity increased due to the quantitative increase in GTX1/4, which are the more toxic derivatives. The PSTs types consistently contained GTX1/4 and GTX2/3. The differences in algae growth and toxin-production could be due to changes in bacteria (out-incubator) and CO 2 (incubator) with vessel size. The effects of environmental factors on algae are strain specific. The out-incubator device can be applied for large-scale cultivation of A. minutum considering the algae density and toxin-producing ability. Image 1 • The biological adaptation of Alexandrium minutum was confirmed. • The Alexandrium minutum cell toxin quota was negatively related to the growth rate. • The cellular toxicity increased due to the increase of gonyautoxins 1/4 proportion. • Bacteria and CO 2 affected algae at different levels in the two environments. • The out-incubator device can be used for large-scale culture of Alexandrium minutum. [ABSTRACT FROM AUTHOR]

• Production of a nanobody-G-Quadruplex DNAzyme chimera with peroxidase activity. • Validation of the functionality of the immunoreagent. • Selective capture and quantification of the toxic microalga A. minutum. • General approach for preparing hybrid macromolecules between proteins and DNA. Hemin-utilizing G-quadruplex DNAzymes with peroxidase-like (POX) activity are widely used as signal reporters in biosensing technology. However, their application to protein detection has been mostly limited to sandwich-type assays involving streptavidin or nanoparticles as indirect bridging platforms between DNAzymes and antibodies. Herein, we describe the generation of a compact, covalently DNAzyme-labeled nanobody which was successfully tested in a direct enzyme-linked immunosorbent assay (ELISA). The conjugation approach was based on the self-labeling protein tag mVirD2, a truncated bacterial relaxase able to covalently bind DNA with 1:1 stoichiometry at a specific amino acid residue. The hybrid molecule combined the nanobody antigen binding affinity and specificity with the DNAzyme catalytic capability to oxidize peroxidase substrates (e.g. ABTS, H 2 O 2). The proposed strategy is simple and cost-effective, enables development into multiplex formats and provides reagents with hitherto unmet reproducibility in terms of POX activity instrumental for both colorimetric and electrochemical reactions. As a proof-of-concept, it was demonstrated that DNAzyme-nanobody conjugates are convenient immunoreagents for rapid and specific detection of the toxic alga Alexandrium minutum. [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning is a human intoxication syndrome associated with the consumption of seafood that has been contaminated with paralytic shellfish toxins (PSTs), a group of natural neurotoxic alkaloids produced by marine dinoflagellates, including some Alexandrium species. This study presents findings of PSTs in mussels (Mytilus galloprovincialis) during 2018–2019 in several mollusc production areas of Sardinia (Italy, western Mediterranean). Investigations of the presence and abundance of PST-producing microalgal species in marine water and of the toxins associated with shellfish were carried out concomitantly. Overall, the results suggested a spatio-temporal expansion of A lexandrium pacificum and A lexandrium minutum in recent years, with an increasing number of PSTs present in molluscs and increased occurrences of toxicity cases. Liquid chromatography with fluorescence detection determined the toxin profile to be composed primarily of the carbamate gonyautoxin-5 and N-sulphocarbamoyltoxins 1 and 2. The study highlights the potential high risk to consumers of poisoning by bivalve molluscs bred in Sardinia, where shellfish production is a very important industrial sector. For this reason, routine monitoring is strongly recommended in order to mitigate any harm to human health as well as negative socio-economic consequences. • Recent findings of paralytic shellfish toxins (PSTs) in mussels from the Mediterranean area are reported. • A spatio-temporal expansion of Alexandrium species had occurred in recent years. • The toxin profile of Alexandrium pacificum suggested that it was a PST producer. • PSTs over the legal limit in mussels in 2018 posed a crucial health threat. [ABSTRACT FROM AUTHOR]

• Exudates of Z. marina negatively affect A. minutum growth rate. • Z. marina exudates stimulate bacterial growth in A. minutum cultures. • The inhibitory effect is likely associated with substances released by Z. marina. Biotic interactions are a key factor in the development of harmful algal blooms. Recently, a lower abundance of planktonic dinoflagellates has been reported in areas dominated by seagrass beds, suggesting a negative interaction between both groups of organisms. The interaction between planktonic dinoflagellates and marine phanerogams, as well as the way in which bacteria can affect this interaction, was studied in two experiments using a non-axenic culture of the toxic dinoflagellate Alexandrium minutum exposed to increasing additions of eelgrass (Zostera marina) exudates from old and young leaves and to the presence or absence of antibiotics. In these experiments, A. minutum abundance, growth rate and photosynthetic efficiency (Fv/Fm), as well as bacterial abundance, were measured every 48 h. Toxin concentration per cell was determined at the end of both experiments. Our results demonstrated that Z. marina exudates reduced A. minutum growth rate and, in one of the experiments, also the photosynthetic efficiency. These results are not an indirect effect mediated by the bacteria in the culture, although their growth modify the magnitude of the negative impact on the dinoflagellate growth rate. No clear pattern was observed in the variation of toxin production with the treatments. [ABSTRACT FROM AUTHOR]

Discharges of CO 2 and nutrients by anthropogenic activities have notable contributions to CO 2 enrichment and eutrophication in coastal systems. Following our previous study that toxic dinoflagellate Alexandrium minutum will increase their growth rate and cellular toxicity under elevated levels of CO 2 , we further examined the joint effects of CO 2 enrichment and excess nitrogen supply through a 29-day experiment under three CO 2 levels (400, 800 and 1200 ppm) with a high N/P ratio of 80. It was found that the two factors have synergistical effects in promoting the increase of cellular toxicity. There were remarkable increases in toxin biosynthesis of paralytic shellfish toxins when both intracellular and extracellular toxins were considered. Under the joint impacts of CO 2 enrichment and excess nitrogen supply, the apparent transformation from gonyautoxins2/3 to gonyautoxins1/4, with much higher toxicity and lower rate of release, is likely to be another major factor accounting for the increasing toxicity. The increasing growth rate and cellular toxicity of A. minutum under the scenarios with elevated concentrations of both CO 2 and nitrogen in coastal systems in the future will increase the risks associated with such toxic algal blooms. [Display omitted] • CO 2 enrichment and excess nitrogen increased Chl- a content of Alexandrium minutum. • Two factors synergistically enhanced toxin quotas of cellular PSTs by A. minutum. • The combined effects lead to a low release of GTX2/3 from inside to outside of cells. • The joint exposure promoted toxin transformation process from GTX2/3 to GTX1/4. • Cellular toxicity increased through toxin synthesis, transformation and transportation. [ABSTRACT FROM AUTHOR]

Highlights • Mean permeate fluxes ranged from 135 to 187 L h−1 m−2 for the complete sequence of filtrations. • Divalent ions were sparsely removed contrary to monovalent ones. • Up to 57% and 78% (mol/mol) of organic matters and salts were removed respectively. • More than 75% (mol/mol) of C1 and C2 were recovered, only 27–50% for GTX2, GTX3 and STX. • Biggest toxins (C1 and C2) were pre-purified contrary to GTX2, GTX3 and STX. Abstract The Paralytic shellfish neurotoxins (PST) are of increasing interest for biomedical applications. The chemical synthesis is often complex and expensive that's why the purification by membrane filtration of PST from Alexandrium minutum dinoflagellate was investigated. Disrupted micro-alga cells by ultrasonic treatment were diafiltred to let pass toxins through an ultrafiltration membrane. Then, the mean permeate was concentrated and diafiltrated by nanofiltration. Mean permeate fluxes equal to 187, 135 and 135 L h−1 m−2 were obtained during the first diafiltration, the concentration step and the final diafiltration respectively. Up to 57% (mol/mol) and 78% (mol/mol) of organic matters and salts were removed respectively. Divalent ions were sparsely removed contrary to monovalent ones. C1 and C2 toxins were successfully purified since more than 75% (mol/mol) were recovered. However, only 27 to 50% (mol/mol) of GTX2, GTX3 and STX were recovered. [ABSTRACT FROM AUTHOR]

Abstract The saxitoxin-producing dinoflagellate Alexandrium minutum is a well-known microalga that causes paralytic shellfish poisoning (PSP) in many coastal regions of the world. In this study, we measured the valve movements of cultivated mussels Mytilus galloprovincialis feeding on toxic A. minutum (n = 29 mussels, shell length = 67.1 ± 3.2 mm, x̅ ± SD) or a morphologically-similar, but toxin-free phytoplankton, Heterocapsa triquetra (n = 24 mussels, shell length = 68.3 ± 2.9 mm). Phytoplankton inoculations were conducted in three sequential "pulses" intended to increase microalgal cell concentrations in a stepwise manner up to ~5000 cells l−1 over a 9-h period. M. galloprovincialis was connected to a non-invasive valvometry apparatus that concurrently measured the magnitude of valve openness once every 0.1 s. It was found that M. galloprovincialis tended to keep its valves open over the course of the experiment, regardless of the phytoplankton species present in water. Standard valve opening metrics, such as the opening duration and opening amplitude, were not significantly affected by the species of phytoplankton. However, the frequency of brief and partial valve closures (microclosures) was significantly influenced by phytoplankton species (P <.01). M. galloprovincialis subjected to toxic A. minutum exhibited 20.3 ± 0.4 (x̅ ± SEM) microclosures per 3-h pulse period, whereas those exposed to the control H. triquetra exhibited 7.9 ± 0.4 (x̅ ± SEM) microclosures. This response was detectable over the 3 h following the first inoculation pulse that provided a phytoplankton concentration of 1000 cells l−1. Our findings are consistent with growing evidence that bivalves are sensitive to very low concentrations of harmful microalgae. Deploying in situ valvometry sensors with real-time monitoring capabilities may provide an early warning of harmful algal blooms. Highlights • The behavioral response of mussels to toxic Alexandrium minutum was assessed. • Valve microclosures were more frequent in the presence of these toxic cells. • The response was manifested within 3 h at low cell concentrations. • The response might serve as an early warning of toxic A. minutum proliferations. [ABSTRACT FROM AUTHOR]

Abstract As other filter-feeders, Crassostrea gigas can concentrate paralytic shellfish toxins (PST) by consuming dinoflagellate phytoplankton species like Alexandrium minutum. Intake of PST in oyster tissues mainly results from feeding processes, i.e. clearance rate, pre-ingestive sorting and ingestion that are directly influenced by environmental conditions (trophic sources, temperature). This study aimed to develop a mechanistic model coupling the kinetics of PST accumulation and bioenergetics in C. gigas based on Dynamic Energy Budget (DEB) theory. For the first time, the Synthesizing Units (SU) concept was applied to formalize the feeding preference of oysters between non-toxic and toxic microalgae. Toxin intake and accumulation were both dependent on the physiological status of oysters. The accumulation was modelled through the dynamics of two toxin compartments: (1) a compartment of ingested but non-assimilated toxins, with labile toxins within the digestive gland eliminated via faeces production; (2) a compartment of assimilated toxins with a rapid detoxification rate (within a few days). Firstly, the DEB-PST model was calibrated using data from two laboratory experiments where oysters have been exposed to A. minutum. Secondly, it was validated using data from another laboratory experiment and from three field surveys carried out in the Bay of Brest (France) from 2012 to 2014. To account for the variability in PST content of A. minutum cells, the saxitoxin (STX) amount per energy units in a toxic algae (ρ PST) was adjusted for each dataset. Additionally, the effects of PST on the oyster bioenergetics were calibrated during the first laboratory experiment. However, these effects were shown to depend on the strain of A. minutum. Results of this study could be of great importance for monitoring agencies and decision makers to identify risky conditions (e.g. production areas, seawater temperature), to properly assess detoxification step (e.g. duration, modalities) before any commercialization or to improve predictions regarding closing of shellfish areas. Highlights • Link between paralytic shellfish toxin accumulation and bioenergetics was studied. • A model based on dynamic energy budget was developed to simulate toxin accumulation. • The developed model allowed to accurately predict toxin accumulation in oysters. • Bioenergetics costs associated to the toxin in oyster were modelled. • Toxic algae have lesser effects on oysters in the field than in laboratory. [ABSTRACT FROM AUTHOR]

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that are known to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human and ecosystem health. This review highlights that A. minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A. minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A. minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have a range of toxin profile clusters and this area appears to have the most diverse set of PST producing A. minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A. minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due to oceanic circulation and a high level of shipping and recreational boating. Further research into the interrelationships between the A. minutum populations in this global region would be of value. [ABSTRACT FROM AUTHOR]

This study was designed to assess the contribution of feeding behavior to inter-individual variability of paralytic shellfish toxin (PST) accumulation in the Pacific oyster Crassostrea gigas . For this purpose 42 oysters were exposed for 2 days to non-toxic algae and then for 2 other days to the PST producer Alexandrium minutum . Individual clearance rate (CR) of oysters was continuously monitored over the 4 days using an ecophysiological measurement system. Comparison of CR values when exposed to toxic and non toxic algae allowed to estimate a clearance rate inhibition index (CRII). Toxin concentration of oysters was quantified at the end of the experiment. These data allowed to estimate the toxin accumulation efficiency (TAE) as the ratio of toxin accumulated on toxin consumed. Changes of clearance rate during the experiment indicated that all individuals stopped feeding immediately after being exposed to A. minutum for at least 7 h. This fast response likely corresponded to a behavioral mechanism of avoidance rather to a toxin-induced response. Individuals also showed high inter-variability in their recovery of filtration after this period. Most of the inter-individual variability (78%) in PST accumulation in C. gigas could be explained by the consumption of A. minutum cells, thus emphasizing the importance of the feeding behavior in accumulation. Based on the toxin concentration in their tissues, oysters were clustered in 3 groups showing contrasted patterns of PST accumulation: the high accumulation group was characterized by high feeding rates both on non-toxic and toxic diet and subsequently a low CRII and high TAE. Inversely, the low accumulation group was characterized by low filtration rates, high CRII and low TAE. Both filtration capacity and sensitivity of oysters to toxins may account for the differences in their accumulation. The contribution of TAE in PST accumulation is discussed and might result from differences in assimilation and detoxification abilities among individuals. [ABSTRACT FROM AUTHOR]

In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning ( PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A. tamarense. Only weak signals of Group I from the A. tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I ( Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A. minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins ( GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A. ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea. [ABSTRACT FROM AUTHOR]

A multitemplate molecularly imprinted polymer was synthesized using fragments similar to gonyautoxins 1 and 4 including 2,4,6-triaminopyrimidine, 4-hydroxy-2-butanone, and imidazole as dummy templates by bulk polymerization. Scanning electron microscopy and infrared spectroscopy showed a porous structure and the formation of organic groups in the imprinting process of the polymer. One millilitre of 0.1 mol L−1acetic acid and 1 mL of 95% methanol were the optimized washing and eluting solutions during the molecularly imprinted solid-phase extraction. The adsorption capacity of the multitemplate molecularly imprinted polymer was lower than for the monotemplate molecularly imprinted polymer prepared in a previous study in seawater in whichAlexandrium tamarenseandAlexandrium minutumwere cultivated. This result may due to the inappropriate combination of the dummy template fragments in the synthesis resulting in the unfitness of the imprinting sites for gonyautoxins 1 and 4. In general, it is inappropriate to use fragments with the similar parts to the analyte as the dummy templates. However, this work indicated the optimal molecularly imprinted polymer for the selective extraction of gonyautoxins 1 and 4. [ABSTRACT FROM PUBLISHER]

The dinoflagellate Alexandrium minutum is a toxic bloom-forming species distributed worldwide. The mechanisms driving and promoting the species blooms and their toxicity are studied and presented here. Most previously published work focuses on local and/or short-term scales. In this study, a broad temporal and spatial approach is addressed using time series covering several sites over several years and combining environmental variables and A. minutum abundances from the French English Channel − Atlantic coasts. Data were explored by means of phenology and threshold analysis. The A. minutum bloom characteristics are defined. Only one bloom per year is measured and it may reach more than a million of cells L −1 . Bloom period extends from April to October and the bloom length ranges from two weeks to six months. In the ecosystems studied, water temperature and river flow, as regional and local factors respectively, are the main environmental drivers influencing the magnitude, growth rate and length of the blooms. Bloom toxicity is linked to the bloom maximum abundance and river flow. This work provides new knowledge for further managing tools for A. minutum blooms in the ecosystems studied. [ABSTRACT FROM AUTHOR]

Potential allelopathic interactions betweenSkeletonema costatumandAlexandrium minutumwere investigated using mixed cultures and culture filtrate in nutrient-replete medium.A. minutumgrowth was inhibited when grown inS. costatumfiltrate, with the inhibitory effect directly proportional to the percentage of filtrate added. This demonstrates that the release of allelopathic compounds caused the growth inhibition. In contrast, the filtrate ofA. minutumexerted no allelopathic activity onS. costatum. An autoinhibitory compound (15(S)-HEPE) extracted and purified fromS. costatumculture was added to cultures of bothS. costatumandA. mintum. This substance could depressS. costatumgrowth, but showed no significant inhibitory activity onA. minutum. This documented a second type of allelochemical interaction, termed auto-allelopathy, caused by a different compound from the one or ones that affectedA. minutumin the co-cultures with added crude filtrate. Further studies are needed to explore the relative importance of these two types of allelopathy as factors influencing the competition betweenS. costatumandA. minutumin the field. Furthermore, given the observed decrease in diatom dominance relative to dinoflagellates with increasing eutrophication, one can predict that toxic species likeA. minutummight become more prevalent in the future in the East China Sea if the trend of increasing pollution of coastal waters continues. [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (∼150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community. [ABSTRACT FROM AUTHOR]

Dinoflagellates of the genus Alexandrium are a major cause of harmful algal blooms (HABs) that have increasingly disrupted coastal ecosystems for the last several decades. Microalgae from the genus Alexandrium are known to produce paralytic shellfish toxins (PST) but also bioactive extracellular compounds (BEC) that can display cytotoxic, allelopathic, ichtyotoxic or haemolytic effects upon marine organisms. The objective of this experimental study was to assess the effects of PST and BEC produced by A. minutum upon juvenile great scallops Pecten maximus . Scallops were exposed for one week to two different strains of A. minutum , the first producing both PST and BEC and the second producing only BEC. Escape response to starfish, daily shell growth, histological effects, and accumulation of PST were recorded after one week of exposure, and after two subsequent weeks of recovery. Daily shell growth was delayed three days in scallops exposed to the BEC-producing A. minutum strain, probably during the three first days of exposure. An increase of reaction time to predators was observed in scallops exposed to the BEC condition, suggesting that BEC may have altered sensing processes. Scallops exposed to PST displayed a less-efficient escape response and muscular damage which could reflect the effects of paralytic toxins upon the nervous system of scallops. This study demonstrates contrasting effects of the distinct toxic compounds produced by A. minutum upon marine bivalves, thus highlighting the importance to better characterize these extracellular, bioactive compounds to better understand responses of other marine organisms. [ABSTRACT FROM AUTHOR]

• The presence of extracellular vesicles was proven with cryo-electron microscopy in a near-axenic culture of the toxic dinoflagellate Alexandrium minutum. • Five different morphotypes were retrieved from the near-axenic culture (rounded, double, rounded electron-dense, lumen electron-dense and irregular), this plurality of shapes suggests a plurality of functions. • The average size of the extracellular vesicles retrieved from the near-axenic culture was of 0.36 µm, which is larger than the average of 0.1 µm of vesicles clearly belonging to prokaryotes. Extracellular Vesicles (EVs) are likely an important strategy of transport and communication in marine microbial community. Their isolation and characterization from axenic culture of microbial eukaryotes represents a technological challenge not fully solved. Here, for the first time, we isolated EVs from a near-axenic culture of the toxic dinoflagellate Alexandrium minutum. Pictures of the isolated vesicles were done with Cryo TEM (Cryogenic Transmission Electron Microscopy). Based on their morphotype the EVs were clustered in five major groups (rounded, rounded electron-dense, lumen electron-dense, double and irregular) and each EV was measured resulting in an average size of 0.36 µm of diameter. Taking in account that in prokaryotes it has been demonstrated that EVs play an important role in the mechanism of toxicity, this descriptive work aims to be the first step to study the possible role of EVs in the toxicity of dinoflagellates. [ABSTRACT FROM AUTHOR]

Cryptochromes are flavin- and pterin-containing photoreceptors of the cryptochrome/photolyase family. They play critical roles in organisms, among are which light-dependent and light-independent roles in biological rhythms. The present work aimed at describing a cryptochrome gene in the oysterCrassostrea gigasby (i) a characterization and phylogenetic analysis and (ii) by studying its expression in the relationship to rhythmic valve behavior in different entrainment regimes. Cryptochrome expression was focused on the adductor muscle of the oyster, the effector of the valve behavior. The results suggest involvement ofCgcry1in oyster rhythmicity as a sensor of environmental zeitgebers, associated with circadian rhythms and potentially to tidal activity. The characterized gene belongs to type 1 cryptochrome/insect-type cry. Additionally,Cgcry1presented a daily oscillation under L:D entrainment, which disappeared in constant darkness. Transcript expression ofCgcry1also oscillated at tidal frequency under tidal entrainment and in constant darkness. Finally, exposure of tidally entrained oysters to saxitoxin (STX)-producing algaAlexandrium minutuminduced a dose effect response in oysters by first alteringCgcry1expression and then the behavior of oysters with increasing concentrations of toxins. This study initiates the characterization of the molecular clock in the oysterC. gigasand its interactions with environmental zeitgebers. [ABSTRACT FROM PUBLISHER]

A kind of new molecularly imprinted polymer (MIP) was synthesized by bulk polymerization using guanosine as dummy template molecule, α-methacrylic acid as functional monomer and ethylene glycol dimethyl acrylic ester as crosslinker. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) showed that the MIP had homogenous and uniform-sized cavities. It was confirmed that the MIP had higher binding affinity and selectivity towards gonyautoxins 1,4 (GTX 1,4) than the non-imprinted polymer (NIP) according to the static equilibrium adsorption. An off-line molecularly imprinted solid-phase extraction (MISPE) method followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was established for the analysis of GTX 1,4. 0.1 mol/L acetic acid and 95:5 (v:v) methanol/water were optimized as the washing and elution solutions, respectively. The recoveries of spiked cultured seawater samples were satisfactory, as high as 88 %. Using this method, the concentrations of GTX 1,4 from cultured seawater samples of Alexandrium minutum and Alexandrium tamarense were detected to be 1.10 μg/L and 0.99 μg/L, respectively. [ABSTRACT FROM AUTHOR]

An innovative and effective extraction procedure based on molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation of gonyautoxins 2,3 (GTX2,3) from Alexandrium minutum sample. Molecularly imprinted polymer microspheres were prepared by suspension polymerization and and were employed as sorbents for the solid-phase extraction of GTX2,3. An off-line MISPE protocol was optimized. Subsequently, the extract samples from A. minutum were analyzed. The results showed that the interference matrices in the extract were obviously cleaned up by MISPE procedures. This outcome enabled the direct extraction of GTX2,3 in A. minutum samples with extraction efficiency as high as 83 %, rather significantly, without any need for a cleanup step prior to the extraction. Furthermore, computational approach also provided direct evidences of the high selective isolation of GTX2,3 from the microalgal extracts. [ABSTRACT FROM AUTHOR]

Monthly water sampling was conducted in Homa Lagoon situated in Izmir Bay, Turkey, between December 2013 and November 2014 aiming to understand how Alexandrium minutum concentration changes due to Chlorophyll-a, nutrients such as nitrite, nitrate, ammonium, silicate, phosphate, and physical parameters like temperature, salinity, dissolved oxygen and pH. A. minutum concentration was highest in July at Station 4 and it was related to nitrate and phosphate concentrations mostly. No bloom of A. minutum was observed during the study. [ABSTRACT FROM AUTHOR]

This study investigated the effect of gametogenesis pattern and sex on levels of paralytic shellfish toxins (PST) accumulated by triploid oysters Crassostrea gigas exposed to a natural bloom of the toxic dinoflagellate Alexandrium minutum in the Bay of Brest (Western Brittany, France), over the summer 2014. Toxin accumulation in oysters was proposed to be influenced by the proportion of energy allocated to reproduction versus other metabolisms, as proposed by Haberkorn et al. (2010). Thus, we hypothesized that triploid oysters with different gametogenesis patterns (α or β, producing respectively numerous gametes or rare gametes) could result in differences in toxin accumulation. Toxin level could also be different according to the gender of the oysters. To test these hypotheses, PST levels were measured in the digestive gland of oysters using an ELISA method. Sex, gametogenesis stage and pattern (α or β) of the triploid oysters were determined by histology. Males (24%), females (38%) and hermaphrodites (38%), including synchronous or successive hermaphrodites were represented among the sampled oysters. All of them were at mature stage (III) of gametogenesis. Both α (46%) and β (54%) patterns were represented in the sample set. In these oysters, PST levels appeared independent from sex and gametogenesis pattern. These results suggest that, in triploid oysters, PST accumulation is not influenced by energy allocated to reproduction. Statement of relevance This study appears to be the first to investigate toxin levels depending on gametogenesis pattern in triploid oysters. This study highlighted that triploid oysters were divided into two classes, α (triploid oysters that maturate with unlocked germ cells and have a consequent number of gametes) and β (triploid oysters that display strongly reduced number of gametes and locked gonial mitosis), with the latter class subdivided into two categories: β1, which presents no germinal cell lineage, and β2, which presents germinal cell lineage. This study showed that toxin levels in the digestive glands of triploids were not significantly different, neither between gametogenesis patterns nor between sexes. Additionally, despite the different patterns of gametogenesis in the triploid oysters of the present study, no difference in toxin levels accumulated in the digestive glands was detected between α and β oysters, suggesting that reproductive investment in triploid oysters does not influence toxin accumulation. [ABSTRACT FROM AUTHOR]

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including Alexandrium minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 10 5 cells/L within 30 min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research. [ABSTRACT FROM AUTHOR]

• Combined temperature and p CO 2 elevation, were investigated at a different transitional period of A. minutum for its acclimation. • This is the first study to consider depicting conditions of ocean warming and acidification on the element storage and related functional processes modification in A. minutum. • Combined temperature and p CO 2 elevation induced luxurious nitrogen and phosphate contents during the acclimation process. • Both nitrogen and phosphate molecules have unique functions to promote efficient growth and proliferation of A. minutum in the future ocean conditions. Ocean warming and acidification are expected to have profound impacts on the marine ecosystem, although the dinoflagellate Alexandrium minutum is reported to be acclimated to such conditions. However, it is unknown on the transition time scale how this species physiologically adjusts their element accumulation and associated resource allocation for this process. We designed a set of experiments to examine how different culture generations (1st, 5th, and 10th) change their cell physiology, cellular quotas and macromolecular cellular contents related to functional processes in A. minutum grown with future (p CO 2 , 1000 ppm; 25°C) and present (p CO 2 , 400 ppm; 21°C) ocean conditions. The differing cell sizes and storage capacity at different generations confirmed that compared to ancestors (1st generation), acclimation cells (10th generation) gained increases in quota carbon (Q C ; 55%; [ p < 0.05]) and quota phosphate (Q P ; 23% [ p < 0.05]). This variation in C:P and N:P influences was transition-specific and largely determined by phosphate-based molecules. It was observed that A. minutum was initially dependent on P molecules, which help cells act as alternative lipids for quick acclimation until N molecules resume carbon-based lipids for their long-term acclimation. Our study demonstrated that rising temperature and p CO 2 concentrations in ocean may increase A. minutum based on the comprehensive analysis of different physiological modifications, including its growth, element accumulation, transformation, and functional allocation. [ABSTRACT FROM AUTHOR]

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters ( Crassostrea gigas ) exposed to a cultured, toxic strain of A. minutum , a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea ( T. lutea , formerly Isochrysis sp., clone Tahitian ( T. iso )), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea . The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum . GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification. [ABSTRACT FROM AUTHOR]

The dynamics and consequences of host-parasite coevolution depend on the nature of host genotype-by-parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies ('Red Queen' dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps ('arms race' dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host-parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host-parasite system. [ABSTRACT FROM AUTHOR]

This is the first report of a bloom of the toxic marine dinoflagellate Alexandrium minutum in the Azores Archipelago (Northeastern Atlantic Ocean). In September 2013, high cell concentrations (1.3 × 10 7 cells L −1 ) of this species were recorded in Santo Cristo coastal lagoon causing an orange-brown water discoloration, death of small pelagic fish and toxification of shellfish resources ( Ruditapes decussatus ). Levels of Paralytic Shellfish Poisoning toxins in clams exceeded 30 times the regulatory limit, and were associated with the intoxication of four people. [ABSTRACT FROM AUTHOR]

In this paper, a simple detection of a toxic algae, Alexandrium minutum, was developed using highly sensitive quartz crystal microbalance. In terms of performance, compared with other conventional analytical tools, the main interest of our immunosensor is based on a fast and direct detection of these living cells. This system requires the use of one monoclonal antibody directed against the surface antigen of A. minutum. We demonstrate that the whole living and motile algae are caught and detected. The high specificity of the biosensor is also demonstrated by testing several other dinoflagellate species. The frequency shift is correlated to the A. minutum cell concentration. This simple system is potentially promising for environmental monitoring purposes. [ABSTRACT FROM AUTHOR]

Background: The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation. Results: The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens. Conclusion: The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic. [ABSTRACT FROM AUTHOR]

Ocean acidification caused by increasing emission of carbon dioxide (CO 2) is expected to have profound impacts on marine ecological processes, including the formation and evolution of harmful algal blooms (HABs). We designed a set of experiments in the laboratory to examine the effects of increasing CO 2 on the growth and toxicity of a toxic dinoflagellate Alexandrium minutum producing paralytic shellfish toxins (PSTs). It was found that high levels of CO 2 (800 and 1200 ppm) significantly promoted the growth of A. minutum compared to the group (400 ppm) representing the current CO 2 level. The total yields of PSTs by A. minutum , including both intracellular and extracellular toxins, were significantly enhanced, probably due to the induction of core enzyme activity and key amino acids synthesis for PST production. More interestingly, high level of CO 2 promoted the transformation from gonyautoxin2&3 to gonyautoxin1&4 and depressed the release of PSTs from inside to outside of the cells. All these processes collectively led to an apparent increase of A. minutum toxicity. Our study demonstrated that rising CO 2 would increase the risk of toxic A. minutum based on the comprehensive analyses of different processes including algal growth and toxin synthesis, transformation and release. [Display omitted] • High level CO 2 significantly promoted growth of toxic Alexandrium minutum. • Total yields of paralytic shellfish toxins by A. minutum were enhanced by rising CO 2. • Rising CO 2 promoted transformation from GTX2&3 to more potent GTX1&4. • High level CO 2 may depress the release of PSTs from inside to outside of the cells. • All the processes collectively increase the risk of A. minutum under CO 2 enrichment. [ABSTRACT FROM AUTHOR]

• Harmful microalgae A. minutum transiently inhibits king scallop filtration and respiration activities • The A. minutum paralytic shellfish toxin accumulation in king scallop is highly variable between individuals • Proteomic analysis of toxic algae A. minutum effect on king scallop: immune response and detoxication. The king scallop, Pecten maximus is a highly valuable seafood in Europe. Over the last few years, its culture has been threatened by toxic microalgae during harmful algal blooms, inducing public health concerns. Indeed, phycotoxins accumulated in bivalves can be harmful for human, especially paralytic shellfish toxins (PST) synthesized by the microalgae Alexandrium minutum. Deleterious effects of these toxic algae on bivalves have also been reported. However, its impact on bivalves such as king scallop is far from being completely understood. This study combined ecophysiological and proteomic analyzes to investigate the early response of juvenile king scallops to a short term exposure to PST producing A. minutum. Our data showed that all along the 2-days exposure to A. minutum , king scallops exhibited transient lower filtration and respiration rates and accumulated PST. Significant inter-individual variability of toxin accumulation potential was observed among individuals. Furthermore, we found that ingestion of toxic algae, correlated to toxin accumulation was driven by two factors: 1/ the time it takes king scallop to recover from filtration inhibition and starts to filtrate again, 2/ the filtration level to which king scallop starts again to filtrate after inhibition. Furthermore, at the end of the 2-day exposure to A. minutum , proteomic analyzes revealed an increase of the killer cell lectin-like receptor B1, involved in adaptative immune response. Proteins involved in detoxification and in metabolism were found in lower amount in A. minutum exposed king scallops. Proteomic data also showed differential accumulation in several structure proteins such as β-actin, paramyosin and filamin A, suggesting a remodeling of the mantle tissue when king scallops are subjected to an A. minutum exposure. [ABSTRACT FROM AUTHOR]

[Display omitted] • Exceptional bloom of Alexandrium minutum in NW Spain (Canary Current Upwelling System). • Unusual meteorological conditions fostered a massive PSP outbreak and red tide episode. • Seedbeds with high numbers of resting cysts detected several months after the bloom. • Climate trends plus biotic factors could favour PSP episodes in the near future. Alexandrium minutum blooms generally occur in semi-enclosed sites such as estuaries, harbours and lagoons, where enhanced stratification, restricted circulation and accumulation of resting cysts in the sediment set suitable habitat conditions for the proliferation of this paralytic shellfish poisoning toxigenic species. In the Galician Rías Baixas (NW Iberian Peninsula), according to weekly time-series between 1994 and 2020, blooms of A. minutum were recurrent in small, shallow estuarine bays inside the Rías de Vigo and Pontevedra, but rarely detected, and if so at low concentrations, out of these environments. However, from May to July 2018 it developed as usual in the small inner bays but then spread over both Rías (Vigo and Pontevedra) causing discoloured waters during one month and prolonged harvesting closures. Meteorological conditions during that period (rains / runoff higher than climatological averages, sustained temperature increment and oscillating wind pattern –i.e., series of upwelling-relaxation cycles), fostered optimal circumstances for the development of that extensive and massive proliferation: strong vertical stratification and the alternation of retention and dispersion processes. Simulations from a particle tracking model portrayed the observed bloom development phases: onset and development inside a small inner bay; transport within the surface layer, from these sites towards the interior parts of the Ría; and dispersion all over the embayment. Seedbeds with high concentrations of resting cysts were detected several months after the bloom, which may have favoured flourishment of A. minutum in the following two years, markedly in 2020. The present work contributes to the general understanding of the dynamics of harmful algal blooms (HABs), from which surveillance indicators of the state of marine ecosystems and their evolution can be derived. We hypothesize that the intensity and frequency of A. minutum proliferations in the Galician Rías could increase under projected climate trends. [ABSTRACT FROM AUTHOR]

Highlights: [•] Alexandrium minutum and STX can negatively affect immunocompetence of Crassostrea gigas hemocyte. [•] Alexandrium minutum and STX decreased phagocytosis and ROS production of Crassostrea gigas hemocyte. [•] Alexandrium minutum and STX modulated differently defense transcripts of Crassostrea gigas hemocyte. [Copyright &y& Elsevier]

Abstract: A strategy to design DNA-biosensors by grafting probe–target duplex onto a 27MHz quartz crystal microbalance gold surface is presented in this work. The idea that removing the DNA target after duplex grafting yields to a well accessible probe, and therefore to efficient target recognition, is investigated. Unfortunately, hybridization efficiency measured using such a straightaway protocol is equal to 15%, which is feeble by comparison with the 31% hybridization efficiency measured for biosensors designed with single-stranded probe. Successive DNA biosensors are designed in this work, taking into account phenomena that occur specifically in an interfacial environment, like non specific adsorption or exchange reactions between adsorbed molecules. A DNA-biosensor with an optimized biolayer in terms of probe accessibility and probe surface density is in fine designed: a 59% hybridization efficiency, for a complementary target including a 20-base non complementary sequence oriented toward the surface, is obtained for the biosensor designed with target–probe duplex. This hybridization efficiency is 3 times to 18% measured for biosensors designed with single-stranded probe. [Copyright &y& Elsevier]

Abstract: Many blooms of the toxic dinoflagellate Alexandrium minutum have been recorded since 1990 in the Gulf of Gabes (southwestern Mediterranean Sea). To understand the determining factors of bloom formation, we studied the distribution of A. minutum in relation to environmental factors in samples taken at shallow sandy (<1 m) beach stations and a lagoon between 1997 and 2006. This was accompanied by laboratory experiments to identify A. minutum. The species forms harmful algal blooms (HABs) in stations subjected to anthropogenic eutrophication and in confined lagoons, living under conditions of varying salinity which gives it a unique opportunity to broaden its physiological tolerance and increase its colonisation potential. Increases in phosphorus appear to be more important than nitrogen or temperature in the control of A. minutum. The stations sampled all along the coast present specific hydrographic properties (shallowness, turbulence) suggesting that factors other than temperature and nutrients influence Alexandrium distribution, the exception being Boughrara Lagoon where the species developed in accordance with published data. Our findings and their interpretations indicate that the mechanism of the sudden A. minutum blooms along the nearshore of the Gulf of Gabes was complex and differed from that of true coastal ecosystems. [Copyright &y& Elsevier]

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ('Global clade') of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ('Pacific clade'). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs. [ABSTRACT FROM AUTHOR]

Abstract: The toxigenic marine dinoflagellate Alexandrium minutum forms toxic blooms causing paralytic shellfish poisoning (PSP), primarily in coastal waters, throughout the world. We examined effects on physiology and gene expression patterns associated with growth and nutrient starvation in a toxic strain of A. minutum. Bloom-relevant factors, including growth rate, intracellular toxin content, allelochemical activity and nutrient status were investigated in A. minutum cultures grown under different environmental regimes. Allelochemical activity of A. minutum cultures, quantified with a cryptomonad Rhodomonas bioassay, increased with age but was independent of nutrient status. The phenotypic data were integrated and compared with gene expression in cell samples taken at selected points along the growth curve. We observed 489 genes consistently differentially expressed between exponentially growing and growth-limited cultures. The expression pattern of stationary-phase cultures was characterized by conspicuous down-regulation of translation-associated genes, up-regulation of sequences involved in intracellular signalling and some indications of increased activity of selfish genetic elements such as transposons. Treatment-specific patterns included five genes regulated in parallel in all nutrient-limited cultures. The conspicuous decrease in photosynthetic performance identified in N-starved cultures was paralleled by down-regulation of chloroplast-associated genes. The particular gene expression patterns we identified as specifically linked with exponential growth, cessation of growth or nutrient limitation may be suitable biomarkers for indicating the beginning of growth limitation in field- or mesocosm studies. [Copyright &y& Elsevier]

Abstract: Some marine dinoflagellates produce important amounts of dimethylsulfoniopropionate (DMSP), a common compatible solute, and its cleavage product dimethylsulfide (DMS), a climatically active trace gas. In the field, dinoflagellate proliferations appear to be favored by calm weather and water column stability; indeed, small-scale turbulence is a physical factor that directly affects ecophysiological aspects of this phytoplankton group, including toxin production. Here we report the effect of experimentally generated turbulence on DMSP production by a paralytic shellfish poisoning (PSP) toxin producing strain of Alexandrium minutum, a widespread bloomforming dinoflagellate species. With respect to still conditions, the populations exposed to turbulence grew at a slower growth rate and yielded low cell numbers turbulence. Concurrently, the cellular DMSP concentration increased by ca. 20% (from 0.22±0.01 to 0.27±0.03fmolμm−3 on a cell volume basis) in the shaken cultures. DMSP was preferentially synthesized during the light period in both treatments. During the night, a slowdown of the division process caused DMSP accumulation in the cells exposed to shaking. The study suggests the existence of a tight link between the dynamics of DMSP concentration and other cell processes entrained by circadian rhythms in dinoflagellates. The observed effects of small-scale turbulence on the DMSP dynamics supports the suggested role of this compound as an overflow mechanism in metabolically unbalanced cells. Furthermore, considering all the effects on the physiology of A. minutum exposed to the same experimental setup, we propose a possible link between the DMSP and the PSP metabolisms. [Copyright &y& Elsevier]

Abstract: This paper constitutes the first cyst record of a eutrophic tropical mariculture area with harmful algal blooms since 2002 and provides contribution to the global record on dinoflagellate cysts, specifically from Northern part of Southeast Asia. Surface sediment samples from 54 stations in October 2006 and 5 stations from 2004 to 2006 Bolinao, Pangasinan were processed palynologically and by fluorescent techniques. Exploratory data analysis using Two-Way Indicator Species Analysis (TWINSPAN) to identify major patterns of distribution and Canonical Correspondence Analysis (CCA) ordinated stations and species along sediment and environmental variables have been done to determine spatial variations in community structure. Thirty-six cyst types have been identified which include the toxic species, Alexandrium minutum. Species richness and diversity correlated with silt, mud, carbonates and depth but have been observed to be generally low in the area (p <0.05). Multivariate analysis shows that sediment grain size, nutrients and water depth are important variables related to cyst distribution. Two accumulation points that can be source and sinks of blooms have been identified. The decrease in species richness and diversity of dinoflagellate cysts from 2005 to 2006 has been attributed to intensive mussel farming. Heterotrophs have dominated during the northeast and southwest monsoon seasons while autotrophs which included A. minutum, have dominated during tradewinds. Results indicate that the cysts accumulation points could possibly seed blooms particularly during the tradewinds season and thus should be monitored effectively to prevent negative public health impacts. [Copyright &y& Elsevier]

A clonal culture of a Vietnamese strain of Alexandrium minutum, AlexSp17, was subjected to different salinity treatments to determine the growth and toxin production of this strain that produces a novel toxin analogue, deoxy GTX4-12ol. The experiment was carried out in batch cultures without pre-acclimatization at seven salinity treatments from 5 to 35 psu, under constant temperature of 25°C, illumination of 140 μmol photon m s, and 12:12 light/dark photoperiod. The strain grew in all salinity treatments, with optimum growth at 10-15 psu. However, the specific growth rate (0.2 day) was lower than those reported in Malaysian strains and other strains from different geographical areas. The optimum range of salinity for the growth of this species agreed with field observations of the locality of origin. No significant change in toxin profiles was observed at different salinities. The cellular toxin quota, Qt, was not affected by the salinity-dependent growth rate. The toxin GTX4-12ol is presumed to be a transformation product of GTX4 from specific cellular reductase enzymes. Further investigation at the molecular level of toxin biosynthesis and subcellular enzyme activities is needed to provide insight in the production of this unique toxin analogue. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of the present study was to provide a flow-cytometric (FCM) approach evaluating Alexandrium minutum cellular responses to mechanical, thermal and chemical stresses. Coupling SYBR-Green I and SYTOX-Green staining, FCM analysis and sorting, and microscopic observations permitted identification and characterization of five cellular states/forms of A. minutum; (1) vegetative cells, (2) pellicle cysts, (3) degraded cells, (4) empty theca, and (5) dead cells. Storage on ice resulted in the transformation of a portion of vegetative cells into dead cells, pellicle cysts, and “degraded” cells; however, centrifugation resulted mostly in pellicle cysts and a few degraded cells. After these thermal and mechanical treatments, control and stressed cultures were monitored for 14 days. Stressed A. minutum cultures appeared to grow at the same rates as control cultures during the first seven days. During the last week of monitoring, however, cell densities of stressed cultures reached their stationary phase earlier than control cultures, suggesting incomplete recovery. Additional experiment assessing kinetics of excystment indicated that it can occur less than 9h following mechanical stress (centrifugation) and that 75% of the culture can excyst within 24h. Upon 30min of exposure to chemical stressors (saponine and H2O2), only vegetative cells, pellicle cysts, and dead cells were detected. For both chemicals, encystment was dose-dependent. Counts of pellicle cysts increased with increasing saponine or H2O2 concentration. Pellicle cysts were the most-predominant cell type at chemical treatments above 0.05gL−1 saponine and above 0.015% H2O2. Occurrence of dead cells appeared to follow an all-or-none response as dead-cell percentage increased from 3% at 0.015% H2O2 to 81% at 0.03% H2O2 without pellicle cyst formation. Overall, encystment–excystment of A. minutum upon changes of environmental conditions can occur very rapidly but can be monitored using FCM and SYBR-Green I staining. [Copyright &y& Elsevier]

Abstract: To determine the species composition, abundance and horizontal distribution of dinoflagellate cysts in recent marine sediments, samples were collected at 13 stations in Izmir Bay, Aegean Sea. At least thirty-six dinoflagellate cyst types were identified, with the assemblages mainly dominated by Lingulodinium machaerophorum, Polykrikos kofoidii, Operculodinium centrocarpum, Gymnodinium cf. nolleri and Quinquecuspis concreta. Total cyst concentrations ranged from 41 to 3292cystsg−1 dry weight sediment. The majority of the cysts occurred in the inner and middle parts of the bay, where higher cyst concentrations were observed. According to the One Way Anova test, the difference between stations was significant statistically (p<0.05). Two of the dinoflagellate cyst species have not been recorded previously as cysts or motile stages in Aegean marine waters; Gymnodinium cf. nolleri and Oblea acanthocysta. Alexandrium minutum and Alexandrium affine type cyst were observed at almost all stations although the cyst type of the Alexandrium catenella/tamarense complex was only found in the outer bay. These findings indicate potential seedbeds for initiation of future blooms and outbreaks of potentially toxic species in Izmir Bay. [Copyright &y& Elsevier]

Abstract: A parasite threat stimulates adaptive shifts in the life-history strategy (sexual recombination rate) of the toxic bloom-forming dinoflagellate Alexandrium minutum Halim. This microalgae divides asexually when clonal but can also form mobile zygotes (planozygotes) when compatible clones are crossed. Planozygotes usually form resistant dormant stages (resting cysts) although they can also divide. In this study, asexual and sexual cultures were infected with the parasite Parvilucifera sinerae (Perkinsozoa) and the resulting clones classified as susceptible (S), low susceptible (LS), or resistant (R) to the infection. R and LS clones were never of Mediterranean origin, pointing to local adaptation of the parasite. (S×S) crosses were infected faster than either of the parental clones growing asexually. By contrast, (S×R) crosses were resistant to the parasite and produced no resting cysts, even when planozygotes were formed. Therefore, in infected cultures, the planozygotes mainly divided instead of encysting, thus increasing the rate at which recombinant progeny formed. This strategy against infection seems to combine the benefits of quickly producing asexual offspring and increasing recombination. As the susceptibility of the crosses was dependent on parental sexual compatibility, and cultures established by the division of (R×S) planozygotes (F1 offspring) also formed R or LS cultures, resistance may be regulated by several genes or through maternal effects. [ABSTRACT FROM AUTHOR]

Abstract: This study assessed the effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon nutrition related processes and reproductive output of the Pacific oyster, Crassostrea gigas. Oysters were exposed to A. minutum, Paralytic Shellfish Toxins (PST) producer and compared to a control batch of oysters fed Isochrysis galbana clone Tahitian (T.Iso). The experiment was performed in June 2008, when oysters were found ripe. Several physiological variables of oysters, such as PSTs accumulation, digestive gland and histological observations as well as spermatozoa quality, were measured at the end of the exposure. Results indicate that the digestive gland was greatly impacted upon A. minutum exposure. Monoacylglycerol and diacylglycerol contents as well as phospholipids (mainly phosphatidylcholine) drastically decreased in the digestive gland of oysters exposed to A. minutum compared to control oysters. At the same time, many oysters exposed to the harmful microalga presented a strong inflammatory response in different tissues of the digestive gland: in intestine as well as in digestive ducts and tubules. Spermatozoa in oysters exposed to A. minutum were morphologically and functionally modified compared to spermatozoa of control oysters. Indeed, spermatozoa were less motile and had lower ATP content in oysters exposed to A. minutum. Meanwhile, spermatozoa produced by control oysters showed higher percentage of mortality and relative DNA content than those produced by A. minutum exposed oysters. Finally, the characteristics of the mitochondria of spermatozoa also appeared to be modified upon A. minutum exposure. The results of this study suggests that an exposure of oysters to A. minutum, reducing energy status and motility of spermatozoa associated to morphological changes at the cellular and sub-cellular levels, can have consequences on spermatozoa fertility and reproduction success. [Copyright &y& Elsevier]

While there is increasing evidence that marine bacteria are involved in the production of paralytic shellfish toxins in algal blooms, the exact roles of the bacteria and microalgae have proved elusive. A novel experimental approach to this problem involved incubating parallel cultures of toxin producing Alexandrium minutum Anokoha A in the dark and in a natural daylight cycle. High-performance liquid chromatography was used to measure paralytic shellfish toxins while bacterial growth was monitored by culture on high- and low-nutrient agar media. After a 22-day incubation period in the dark, A. minutum Anokoha A failed to produce saxitoxin while parallel light-grown cultures produced 1.17 μg per 10,000 algal cells. However, both dark- and light-grown cultures showed comparable gonyautoxin production. Copiotrophic and oligotrophic algal-associated bacteria showed similar growth patterns over the incubation period except that a dip in bacterial numbers corresponded to a peak in algal numbers in the light-grown cultures. It appears that inhibition of photosynthesis resulted in changes in the toxin profile of A. minutum Anokoha A. When used with other methods, this approach may help to elucidate the algal–bacterial-toxin connection. [ABSTRACT FROM AUTHOR]

Abstract: The effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon physiological parameters of the Pacific oyster, Crassostrea gigas, were assessed. Diploid and triploid oysters were exposed to cultured A. minutum and compared to control diploid and triploid oysters fed T. Isochrysis. Experiments were repeated twice, in April and mid-May 2007, to investigate effects of maturation stage on oyster responses to A. minutum exposure. Oyster maturation stage, Paralytic Shellfish Toxin (PST) accumulation, as well as several digestive gland and hematological variables, were assessed at the ends of the exposures. In both experiments, triploid oysters accumulated more PSTs (approximately twice) than diploid oysters. Significant differences, in terms of phenoloxidase activity (PO) and reactive oxygen species (ROS) production of hemocytes, were observed between A. minutum-exposed and non-exposed oysters. PO in hemocytes was lower in oysters exposed to A. minutum than in control oysters in an early maturation stage (diploids and triploids in April experiment and triploids in May experiment), but this contrast was reversed in ripe oysters (diploids in May experiment). In the April experiment, granulocytes of oysters exposed to A. minutum produced more ROS than those of control oysters; however, in the May experiment, ROS production of granulocytes was lower in A. minutum-exposed oysters. Moreover, significant decreases in free fatty acid, monoacylglycerol, and diacylglycerol contents in digestive glands of oysters exposed to A. minutum were observed. Concurrently, the ratio of reserve lipids (triacylglycerol, ether glycerides and sterol esters) to structural lipids (sterols) decreased upon A. minutum exposure in both experiments. Also, several physiological responses to A. minutum exposure appeared to be modulated by maturation stage as well as ploidy of the oysters. [Copyright &y& Elsevier]