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The changes in the cell physiology (growth rate, cell size, and cell DNA content), photosynthetic efficiency, toxicity, and sexuality under variable light and nutrient (phosphates) conditions were evaluated in cultures of the dinoflagellate Alexandrium minutum obtained from a red tide in the Ría de Vigo (NW Spain). The cells were grown at low (40 and 150 µE m−2 s−1), moderate (400 µE m−2 s−1), and high (800 µE m−2 s−1) light intensities in a medium with phosphate (P+) and without (P−). Cultures were acclimated to the irradiance conditions for one week, and the experiment was run for ~1 month. The cell size and DNA content were monitored via flow cytometry. Two different clonal strains were employed as a monoculture (in a P− or P+ medium) or, to foster sexuality and resting cyst formation, as a mixed culture (only in a P− medium). A. minutum growth was favored by increasing light intensities until 400 µE m−2 s−1. The DNA content analyses indicated the accumulation of S-phase cells at the highest light intensities (400 and 800 µE m−2 s−1) and therefore the negative effects on cell cycle progression. Only when the cells were grown in a P− medium did higher light intensities trigger dose-dependent, significantly higher toxicities in all the A. minutum cultures. This result suggests that the toxicity level is responsive to the combined effects of (high) light and (low) P stress. The cell size was not significantly affected by the light intensity or P conditions. The optimal light intensity for resting cyst formation was 150 µE m−2 s−1, with higher irradiances reducing the total encystment yield. Encystment was not observed at the lowest light intensity tested, indicative of the key role of low-level irradiance in gamete and/or zygote formation, in contrast to the stressor effect of excessive irradiance on planozygote formation and/or encystment. [ABSTRACT FROM AUTHOR]

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]

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters. [ABSTRACT FROM AUTHOR]

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions. [ABSTRACT FROM AUTHOR]

Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency. [ABSTRACT FROM AUTHOR]

Simple Summary: Alexandrium minutum is one of the causing organisms for the occurrence of harmful algae bloom (HABs) in marine ecosystems. This species produces saxitoxin, one of the deadliest neurotoxins which can cause human mortality. However, molecular information such as genes and proteins catalog on this species is still lacking. Therefore, this study has successfully characterized several new molecular mechanisms regarding A. minutum environmental adaptation and saxitoxin biosynthesis. Ultimately, this study provides a valuable resource for facilitating future dinoflagellates' molecular response to environmental changes. The toxin-producing dinoflagellate Alexandrium minutum is responsible for the outbreaks of harmful algae bloom (HABs). It is a widely distributed species and is responsible for producing paralytic shellfish poisoning toxins. However, the information associated with the environmental adaptation pathway and toxin biosynthesis in this species is still lacking. Therefore, this study focuses on the functional characterization of A. minutum unigenes obtained from transcriptome sequencing using the Illumina Hiseq 4000 sequencing platform. A total of 58,802 (47.05%) unigenes were successfully annotated using public databases such as NCBI-Nr, UniprotKB, EggNOG, KEGG, InterPRO and Gene Ontology (GO). This study has successfully identified key features that enable A. minutum to adapt to the marine environment, including several carbon metabolic pathways, assimilation of various sources of nitrogen and phosphorus. A. minutum was found to encode homologues for several proteins involved in saxitoxin biosynthesis, including the first three proteins in the pathway of saxitoxin biosynthesis, namely sxtA, sxtG and sxtB. The comprehensive transcriptome analysis presented in this study represents a valuable resource for understanding the dinoflagellates molecular metabolic model regarding nutrient acquisition and biosynthesis of saxitoxin. [ABSTRACT FROM AUTHOR]

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment. [ABSTRACT FROM AUTHOR]

A paralytic shellfish poisoning (PSP) episode developed in summer 2018 in the Rías Baixas (Galicia, NW Spain). The outbreak was associated with an unprecedentedly intense and long-lasting harmful algal bloom (HAB) (~one month) caused by the dinoflagellate Alexandrium minutum. Paralytic shellfish toxins (PSTs) were analyzed in extracts of 45 A. minutum strains isolated from the bloom by high-performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-PCOX-FLD). PSTs were also evaluated in tissues from marine fauna (invertebrates and fish) collected during the episode and in dolphin samples. The analysis of 45 A. minutum strains revealed a toxic profile including GTX1, GTX2, GTX3 and GTX4 toxins. With regard to the marine fauna samples, the highest PSTs levels were quantified in bivalve mollusks, but the toxins were also found in mullets, mackerels, starfish, squids and ascidians. This study reveals the potential accumulation of PSTs in marine invertebrates other than shellfish that could act as vectors in the trophic chain or pose a risk for human consumption. To our knowledge, this is the first time that PSTs are reported in ascidians and starfish from Spain. Moreover, it is the first time that evidence of PSTs in squids is described in Europe. [ABSTRACT FROM AUTHOR]

The synthetic pathways responsible for the production of the polyunsaturated fatty acids 22:6n-3 and 20:5n-3 were studied in the Dinophyte Alexandrium minutum. The purpose of this work was to follow the progressive incorporation of an isotopic label (13CO2) into 11 fatty acids to better understand the fatty acid synthesis pathways in A. minutum. The Dinophyte growth was monitored for 54 h using high-frequency sampling. A. minutum presented a growth in two phases. A lag phase was observed during the first 30 h of development and had been associated with the probable temporary encystment of Dinophyte cells. An exponential growth phase was then observed after t30. A. minutum rapidly incorporated 13C into 22:6n-3, which ended up being the most 13C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher 13C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the 13C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C18 PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least 13C-enriched FAs during this experiment. They were assumed to be produced by the n-3 PUFA pathway. However, they could not be further elongated or desaturated to produce n-3 C20-C22 PUFA, because the AEs of the n-3 C18 PUFAs were lower than those of the n-3 C20-C22 PUFAs. Thus, the especially high atomic enrichment of 22:6n-3 (55.8% and 54.9% in neutral lipids (NLs) and polar lipids (PLs), respectively) led us to hypothesize that this major PUFA was synthesized by an O2-independent Polyketide Synthase (PKS) pathway. Another parallel PKS, independent of the one leading to 22:6n-3, was also supposed to produce 20:5n-3. The inverse order of the 13C atomic enrichment for n-3 PUFAs was also suspected to be related to the possible β-oxidation of long-chain n-3 PUFAs occurring during A. minutum encystment. [ABSTRACT FROM AUTHOR]

The dinoflagellate Alexandrium minutum (A. minutum) which can produce paralytic shellfish toxins (PSTs) is often used as a model to study the migration, biotransformation, accumulation, and removal of PSTs. However, the mechanism is still unclear. To provide a new tool for related studies, we tried to label PSTs metabolically with 15N stable isotope to obtain 15N-PSTs instead of original 14N, which could be treated as biomarker on PSTs metabolism. We then cultured the A. minutum AGY-H46 which produces toxins GTX1-4 in f/2 medium of different 15N/P concentrations. The 15N-PSTs' toxicity and toxin profile were detected. Meanwhile, the 15N labeling abundance and 15N atom number of 15N-PSTs were identified. The 14N of PSTs produced by A. minutum can be successfully replaced by 15N, and the f/2 medium of standard 15N/P concentration was the best choice in terms of the species' growth, PST profile, 15N labeling result and experiment cost. After many (>15) generations, the 15N abundance in PSTs extract reached 82.36%, and the 15N atom number introduced into GTX1-4 might be 4–6. This paper innovatively provided the initial evidence that 15N isotope application of labeling PSTs in A. minutum is feasible. The 15N-PSTs as biomarker can be applied and provide further information on PSTs metabolism. [ABSTRACT FROM AUTHOR]

Marine dinoflagellates are a valuable source of bioactive molecules. Many species produce cytotoxic compounds and some of these compounds have also been investigated for their anticancer potential. Here, we report the first investigation of the toxic dinoflagellate Alexandrium minutum as source of water-soluble compounds with antiproliferative activity against human lung cancer cells. A multi-step enrichment of the phenol–water extract yielded a bioactive fraction with specific antiproliferative effect (IC50 = 0.4 µg·mL−1) against the human lung adenocarcinoma cells (A549 cell line). Preliminary characterization of this material suggested the presence of glycoprotein with molecular weight above 20 kDa. Interestingly, this fraction did not exhibit any cytotoxicity against human normal lung fibroblasts (WI38). Differential gene expression analysis in A549 cancer cells suggested that the active fraction induces specific cell death, triggered by mitochondrial autophagy (mitophagy). In agreement with the cell viability results, gene expression data also showed that no mitophagic event was activated in normal cells WI38. [ABSTRACT FROM AUTHOR]