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Bioactive venoms and toxins are emerging as a promising source of drug leads. Optimized through evolution, these compounds display remarkable selectivity and ligand affinity toward a range of relevant pharmacological targets. The successful development of new drugs from toxins is hampered in some areas by the chemical complexity of the active compounds, which limits the possibility of using chemical synthesis or recombinant strategies for drug lead generation. Marine paralytic shellfish toxins produced by marine microalgae is one such family of compounds. These compounds are highly potent blockers of voltage-gated ion channels, involved in regulating a range of physiological processes and thus versatile targets for drug development. To overcome the supply issue, the current paper describes the development of a scalable production method to generate gram amounts of gonyautoxin-1,4 by mass cultivation of the dinoflagellate Alexandrium pacificum in artificial seawater. By selecting a high-producing strain and running a series of growth optimization experiments, we have scaled up production from 100 mL to 1150 L, with cellular yields of toxin 30 times higher than in a natural bloom. This allows commercial production of gram amounts of these promising compounds, thereby enabling their use in a range of applications beyond the analytical scale.

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.

Alexandrium pacificum, a globally distributed dinoflagellate, is well-known for causing harmful algal blooms and producing Paralytic Shellfish Toxins (PSTs), a threat to marine life and human health. The frequency and intensity of Alexandrium blooms have increased in recent decades, driven, in some cases, by increasing temperatures. Here, we investigated the temperature-dependent (15 °C, 20 °C, 25 °C, and 30 °C) growth rates and paralytic shellfish toxin profiles of eight A. pacificum strains while concurrently examining differences in sequences of the saxitoxin biosynthetic gene, sxtA4 . While maximum cell densities were lowest at 30 °C, toxin production per cell was highest at higher temperatures that inhibited growth, with greater diversity of toxin analogs peaking at 30 °C, as confirmed by the higher Shannon’s diversity index obtained for the toxin profiles with the increasing temperatures. Furthermore, genetic analysis of the sxtA4 gene showed that greater genetic diversity—quantified by nucleotide diversity ( π ) ranging from 9.91 to 30.21 across strains—was positively correlated with this wider array of toxin analogs (Shannon’s diversity index; p  < 0.0001). Conserved regions within the gene were identified, suggesting that these regions may play important structural or functional roles in the saxitoxin biosynthetic pathway. These findings highlight the role of temperature, genetic diversity, and sxtA4 conserved regions in influencing toxin production and profiles in Alexandrium . Further research into the genetic mechanisms underlying saxitoxin biosynthesis will improve our understanding of Alexandrium ’s adaptability to changing temperatures. Such insights are essential for effective ecosystem management and safeguarding public health.

The marine dinoflagellate Alexandrium is known to produce saxitoxins (STXs), causing paralytic shellfish poisoning (PSP). STX biosynthesis is catalyzed by eight enzymes encoded in their corresponding sxt core genes. SxtU encodes a short-chain dehydrogenase that participates in the last step of the STX synthesis; however, its characteristics are insufficiently elucidated in toxic dinoflagellates. Herein, we characterized the full-length sxtU from the toxic Alexandrium pacificum and evaluated its transcriptional responses under varying salinity and temperature. The ApsxtU cDNA was 936 bp in length, comprising an 819-bp open reading frame (68.9% GC). The 3-dimensional structure contained the Rossmann fold with seven β-sheets and eight α-helices in each monomer. Phylogenetic analysis showed that ApsxtU formed a clade with that of Alexandrium tamarense and toxic cyanobacteria. In the tested conditions, STX eq of A. pacificum was the highest in the exponential phase at 16 °C (96.27 fmol cell-1), at 33 psu (59.67 fmol cell-1), and decreased at other conditions. ApsxtU expression levels increased in time and reached the highest in the stationary phase at 40 psu but were not statistically correlated with toxin contents. These suggest that sxtU may be involved in STXs synthesis and also in other metabolic functions in dinoflagellates.

In this study we characterized the genome scale of the transcriptome of the marine dinoflagellate Alexandrium pacificum under various stresses, focusing on the identification of stress-responsive genes. RNA sequencing generated 18.1 Gb nucleotides that were assembled to 297,808 transcripts. Of them, 207 and 134 transcripts were assigned to the antioxidant enzyme system and heat shock protein (HSP) families, respectively. In addition, the carotenoid biosynthesis pathway and other typical stress-responsive genes (e.g., cold shock proteins and mitogen-activated protein kinases) were widely expressed under stresses. Phylogenetic analysis revealed that these genes might be acquired from different origins via separate evolutionary events. The A. pacificum CuZnSOD and HSP70 were evidently responded to algicides and metals. These findings clearly suggest that A. pacificum possesses many different stress-resistant genes from different sources, which are used for defense and/or adaptation in diverse environments. These may allow the organism to survive and develop when introduced to new environments.

Alexandrium pacificum, a dinoflagellate known for causing harmful algal blooms (HABs), has garnered significant attention due to its potential toxicity to marine ecosystems, fisheries, and human health. However, the effects of this toxin-producing alga on shrimp are not yet comprehensively understood. This study aimed to assess the hepatopancreas damage induced by A. pacificum in the economically important shrimp species E. carinicauda and to elucidate the underlying molecular mechanisms through histology, antioxidant enzyme activity, and transcriptome analysis. The shrimp were assigned to either a control group or an exposed group, with the latter involving exposure to A. pacificum at a concentration of 1.0 × 104 cells/mL for 7 days. A histological analysis subsequently revealed pathological changes in the hepatopancreas tissue of the exposed group, including lumen expansion and the separation of the basement membrane from epithelial cells, while antioxidant enzyme activity assays demonstrated that exposure to A. pacificum weakened the antioxidant defense system, as evidenced by the reduced activities of catalase, superoxide dismutase, and glutathione, along with increased malondialdehyde levels. Transcriptome analysis further identified 663 significantly upregulated genes and 1735 significantly downregulated ones in the exposed group, with these differentially expressed genes being primarily associated with pathways such as protein processing in the endoplasmic reticulum, mitophagy, glycolysis/gluconeogenesis, sphingolipid metabolism, and glycerophospholipid metabolism. This study provides novel insights into the toxicological effects of A. pacificum on aquatic organisms and enhances the current understanding of the ecotoxicological risks posed by HABs.

Microplastics (MP) widely distributed in aquatic environments have adverse effects on aquatic organisms. Currently, the impact of MP on toxigenic red tide microalgae is poorly understood. In this study, the strain of Alexandrium pacificum ATHK, typically producing paralytic shellfish toxins (PST), was selected as the target. Effects of 1 and 0.1 μm polystyrene MP with three concentration gradients (5 mg L−1, 25 mg L−1 and 100 mg L−1) on the growth, chlorophyll a (Chl a), photosynthetic activity (Fv/Fm) and PST production of ATHK were explored. Results showed that the high concentration (100 mg L−1) of 1 μm and 0.1 μm MP significantly inhibited the growth of ATHK, and the inhibition depended on the size and concentration of MP. Contents of Chl a showed an increase with various degrees after MP exposure in all cases. The photosynthesis indicator Fv/Fm of ATHK was significantly inhibited in the first 11 days, then gradually returned to the level of control group at day 13, and finally was gradually inhibited in the 1 μm MP treatments, and promotion or inhibition to some degree also occurred at different periods after exposure to 0.1 μm MP. Overall, both particle sizes of MP at 5 and 25 mg L−1 had no significant effect on cell toxin quota, and the high concentration 100 mg L−1 significantly promoted the PST biosynthesis on the day 7, 11 and 15. No significant difference occurred in the cell toxin quota and the total toxin content in all treatments at the end of the experiment (day 21). All MP treatments did not change the toxin profiles of ATHK, nor did the relative molar percentage of main PST components. The growth of ATHK, Chl a content, Fv/Fm and toxin production were not affected by MP shading. This is the first report on the effects of MP on the PST-producing microalgae, which will improve the understanding of the adverse impact of MP on the growth and toxin production of A. pacificum.

The presence of zooplankton in marine ecosystems is an important factor affecting toxin production in dinoflagellates. However, whether the production of paralytic shellfish toxins (PSTs) by Gymnodinium catenatum and Alexandrium pacificum is affected by substances produced by zooplankton is not yet fully understood. This study assessed the effects of zooplankton extracts on cell abundance, pigment concentration and composition, chlorophyll-a-specific primary production (Chl-a SP), and PST production in G. catenatum and A. pacificum, isolated from Korean coastal waters. In addition, a novel group of hydroxybenzoate PSTs known as GC toxins, which lack commercial reference standards, was evaluated. No significant differences were observed in cell abundance, pigment composition, or Chl-a SP in the experimental group of G. catenatum strains exposed to zooplankton extracts compared to the control group. However, the production of PSTs, including GC toxins, was significantly enhanced when the strain was exposed to extracts of Calanus finmarchicus. Meanwhile, in the A. pacificum strain, some experimental groups showed significant differences in cell abundance and pigment composition, while Chl-a SP significantly decreased in all experimental groups. In addition, PST production was stimulated by the addition of C. finmarchicus extracts. No GC toxins were detected in the A. pacificum strain. This study demonstrated that substances derived from C. finmarchicus enhance the production of PSTs in G. catenatum and A. pacificum. Our findings will improve the current understanding of the occurrence and development mechanisms of PSTs in marine ecosystems and contribute to developing strategies to efficiently secure standard reference toxin products using toxic dinoflagellates. KCI Citation Count: 0

Salinity is an important factor in the physiological regulation of algae; however, its influence on the genomic responses in toxic dinoflagellates is insufficiently understood. In the present study, we evaluated the effect of salinity stress on the physiology, photosynthesis, and molecular responses of the toxic dinoflagellate Alexandrium pacificum (group IV). When exposed cells to different salinities of 20–40 psu, we detected the lowest cell density (3.25 × 103 cells mL−1) and highest cell size (30.6 µm) at 20 psu. Photosynthesis efficiency considerably decreased at 20 and 40 psu compared to the control (33 psu). Quantitative real-time polymerase chain reaction revealed that psbA, psbD, and atpC expression levels were significantly downregulated under conditions of salinity stress for 72 h. In contrast, the expression levels of antioxidant genes MnSOD and GPx were greatly upregulated at 20 psu (13.2- and 15.2-fold changes at 6 h; 8.8- and 8.3-fold changes at 24 h, respectively). The expression levels of other antioxidant genes, CuZnSOD, GST, and APx, increased steadily over time under salinity stress. Such conditions increased the relative levels of reactive oxygen species by 2.2-fold in 6 h and 2.4-fold in 24 h at 20 psu. These results suggest that low salinity may cause cellular oxidative stress, leading to a decrease in photosynthesis and affecting specific antioxidant systems in toxic dinoflagellates.

Salinity is an important factor for regulating metabolic processes in aquatic organisms; however, its effects on toxicity and STX biosynthesis gene responses in dinoflagellates require further elucidation. Herein, we evaluated the physiological responses, toxin production, and expression levels of two STX synthesis core genes, sxtA4 and sxtG, in the dinoflagellate Alexandrium pacificum Alex05 under different salinities (20, 25, 30, 35, and 40 psu). Optimal growth was observed at 30 psu (0.12 cell division/d), but cell growth significantly decreased at 20 psu and was irregular at 25 and 40 psu. The cell size increased at lower salinities, with the highest size of 31.5 µm at 20 psu. STXs eq was highest (35.8 fmol/cell) in the exponential phase at 30 psu. GTX4 and C2 were predominant at that time but were replaced by GTX1 and NeoSTX in the stationary phase. However, sxtA4 and sxtG mRNAs were induced, and their patterns were similar in all tested conditions. PCA showed that gene transcriptional levels were not correlated with toxin contents and salinity. These results suggest that A. pacificum may produce the highest amount of toxins at optimal salinity, but sxtA4 and sxtG may be only minimally affected by salinity, even under high salinity stress.