Two novel glutathione S-transferase (GST) genes in the toxic marine dinoflagellate Alexandrium pacificum and their transcriptional responses to environmental contaminants.

The present study identified two novel glutathione S-transferase (GST) genes from the toxic dinoflagellate Alexandrium pacificum and examined their molecular characteristics and transcriptional responses to algicides and environmental contaminants. Bioinformatic analysis revealed that both ApGSTs are cytosolic, belonging to the chi-like class (ApGST1) and an undefined class (ApGST2). The overall expression of ApGSTs showed similar patterns depending on the exposed contaminants, while they were differently regulated by polychlorinated biphenyl (PCB). Copper treatments (CuCl2 and CuSO4) did not significantly induce the expression of ApGSTs. The highest up-regulations of ApGST1 and ApGST2 were under 6-h treatments of 0.10 and 0.50 mg L-1 NaOCl. Interestingly, only ApGST1 increased significantly after 0.10, 0.50, and 1.00 mg L-1 of PCB exposure (6 h). Intracellular reactive oxygen species (ROS) increased considerably under NaOCl; however, it was not significantly higher in the PCB-treated cells. GST activity was increased by NaOCl and PCB treatments, but only PCB caused apoptosis. These results suggest that GSTs are involved in the first line of phase II detoxification, protecting dinoflagellate cells against oxidative damage.

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

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

Toxic Effects and Tumor Promotion Activity of Marine Phytoplankton Toxins: A Review.

Phytoplankton are photosynthetic microorganisms in aquatic environments that produce many bioactive substances. However, some of them are toxic to aquatic organisms via filter-feeding and are even poisonous to humans through the food chain. Human poisoning from these substances and their serious long-term consequences have resulted in several health threats, including cancer, skin disorders, and other diseases, which have been frequently documented. Seafood poisoning disorders triggered by phytoplankton toxins include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP), and azaspiracid shellfish poisoning (AZP). Accordingly, identifying harmful shellfish poisoning and toxin-producing species and their detrimental effects is urgently required. Although the harmful effects of these toxins are well documented, their possible modes of action are insufficiently understood in terms of clinical symptoms. In this review, we summarize the current state of knowledge regarding phytoplankton toxins and their detrimental consequences, including tumor-promoting activity. The structure, source, and clinical symptoms caused by these toxins, as well as their molecular mechanisms of action on voltage-gated ion channels, are briefly discussed. Moreover, the possible stress-associated reactive oxygen species (ROS)-related modes of action are summarized. Finally, we describe the toxic effects of phytoplankton toxins and discuss future research in the field of stress-associated ROS-related toxicity. Moreover, these toxins can also be used in different pharmacological prospects and can be established as a potent pharmacophore in the near future.

Increased nitrate concentration differentially affects cell growth and expression of nitrate transporter and other nitrogen-related genes in the harmful dinoflagellate Prorocentrum minimum.

The molecular mechanisms through which dinoflagellates adapt to nitrate fluctuations in aquatic environments remain poorly understood. Here, we sequenced the full-length cDNA of a nitrate transporter (NRT) gene from the harmful marine dinoflagellate Prorocentrum minimum Schiller. The cDNA length was 2431 bp. It encoded a 529-amino acid protein, which was phylogenetically clustered with proteins from other dinoflagellates. Nitrate supply promoted cell growth up to a certain concentration (∼1.76 mM) but inhibited it at higher concentrations. Interestingly, at the inhibitory concentrations, nitrite levels in the medium were considerably increased. Nitrate concentration affected the expression of PmNRT, nitrite transporter (PmNiRT), nitrate reductase (PmNR), and nitrite reductase (PmNiR). Specifically, PmNRT was upregulated after 24 h, with ∼6-fold change compared with the control level, in both nitrate-depleted and nitrate-repleted cultures. In addition, PmNR transcript levels increased to the maximum of 4-fold at 48 h but decreased thereafter. In contrast, PmNiR levels remained unchanged in both nitrate-repleted and nitrate-depleted cultures. Therefore, P. minimum likely copes with nitrate fluctuations in its environment by regulating a set of genes responsible for nitrate uptake.

Molecular cloning and oxidative-stress responses of a novel Phi class glutathione S-transferase (GSTF) gene in the freshwater algae Closterium ehrenbergii.

Glutathione S-transferases (GSTs) belong to a family of enzymes involved in diverse biological processes, including detoxification and protection against oxidative damage. Here, we determined the full-length sequence (915 bp) of a novel Phi class cytosolic glutathione S-transferase (GSTF) gene from the green algae Closterium ehrenbergii. We examined the gene structure and expression patterns in response to metals and endocrine disrupting chemicals (EDCs). It was significantly upregulated by metals, but responded differently to EDCs. The highest up-regulation of CeGSTF was registered under 0.1 mg/L CuCl2 and 0.01 mg/L CuSO4 treatments. In a 72-h course experiment with treatment of 0.1 mg/L CuCl2 , CeGSTF was dramatically induced at 6 h, and then gradually decreased with increasing exposure time. This was consistent with the increase in both GST activity and ROS production in copper-treated cells. These results suggest that CeGSTF may be involved in detoxification mechanisms associated with oxidative stress in green algae.

Characterization and Ca2+-induced expression of calmodulin (CaM) in marine dinoflagellates.

Calmodulin (CaM) is one of the major Ca2+-binding proteins in the cells, and it plays multiple roles in several Ca2+ signaling pathways and regulating the activities of other proteins. In the present study, we characterized CaM genes from the marine dinoflagellates Amphidinium carterae, Cochlodinium polykrikoides, Prorocentrum micans, and P. minimum, and examined their expression patterns upon the addition and chelation of calcium. Their cDNAs had same ORF length (450 bp) and encoded the same protein, but with few nucleotide differences in the ORF and different 3'- and 5' untranslated regions (UTRs). The four CaM proteins consist of four EF-hand Ca2+-binding motifs, two N-terminal domains and two C-terminal domains, and they were highly conserved within eukaryotes. The CaM gene expressions in the tested species increased by calcium treatments; however, they were significantly down-regulated by the calcium-chelator EGTA. The CaM genes of the test species were inducible and regulated by different calcium doses, suggesting their major role in calcium regulation in dinoflagellates.

Molecular cloning of heat shock protein 70 and HOP from the freshwater green algae Closterium ehrenbergii and their responses to stress.

Heat shock proteins (HSPs) and HSP70-HSP90 organizing proteins (HOPs) are related, and they function together to maintain cellular homeostasis and respond to stress. In the present study, we reported the first molecular characteristics of HSP70 (designated as CeHSP70) and HOP (designated as CeHOP) genes from the freshwater green algae Closterium ehrenbergii and examined the changes in their expression profiles under heat stress and toxic chemicals treatment. CeHSP70 presented the conserved motif patterns and EEVD domain specific to cytosolic HSP70; CeHOP contained a typical domain of TPR repeats. Real-time PCR analysis showed that thermal stress considerably up-regulated both CeHOP and CeHSP70. In addition, the genes were significantly induced by CuCl2, CuSO4, and NiSO4, but not by K2Cr2O7, herbicide, and endocrine disrupting chemicals. These results suggest that CeHOP and CeHSP70 function together and play a role in responses to specific stressors and indicate their possible use as sensitive specific biomarkers in risk assessments.

The herbicide alachlor severely affects photosystem function and photosynthetic gene expression in the marine dinoflagellate Prorocentrum minimum.

Alachlor is one of the most widely used herbicides and can remain in agricultural soils and wastewater. The toxicity of alachlor to marine life has been rarely studied; therefore, we evaluated the physiological and transcriptional responses in the marine dinoflagellate Prorocentrum minimum. The herbicide led to considerable decreases in P. minimum cell numbers and pigment contents. The EC50 was determined to be 0.373 mg/L. Photosynthesis efficiency and chlorophyll autofluorescence dramatically decreased with increasing alachlor dose and exposure time. Real-time PCR analysis showed that the photosynthesis-related genes PmpsbA, PmatpB, and PmrbcL were induced the most by alachlor; the transcriptional level of each gene varied with time. PmrbcL expression increased after 30 min of alachlor treatment, whereas PmatpB and PmpsbA increased after 24 h. The PmpsbA expression level was highest (5.0 times compared to control) after 6 h of alachlor treatment. There was no significant change in PmpsaA expression with varying treatment time or concentration. Additionally, there was no notable change in the expression of antioxidant genes PmGST and PmKatG, or in ROS accumulation. These suggest that alachlor may affect microalgal photosystem function, with little oxidative stress, causing severe physiological damage to the cells, and even cell death.

Small heat shock protein genes of the green algae Closterium ehrenbergii: Cloning and differential expression under heat and heavy metal stresses.

The freshwater green algae Closterium ehrenbergii has been considered as a model for eco-toxicological assessment in aquatic systems. Heat shock proteins (HSPs) are a class of highly conserved proteins produced in all living organisms, which participate in environmental stress responses. In the present study, we determined the cDNA sequences of small heat shock protein 10 (sHSP10) and sHSP17.1 from C. ehrenbergii, and examined the physiological changes and transcriptional responses of the genes after exposure to thermal shock and toxicants treatments. The open reading frame (ORF) of CeHSP10 was 300 bp long, encoding 99 amino acid (aa) residues (10.53 kDa) with a GroES chaperonin conserved site of 22 aa. The CeHSP17.1 had a 468 bp ORF, encoding 155 aa with a conserved C-terminal α-crystallin domain. For heat stress, cells presented pigment loss and possible chloroplast damage, with an up-regulation in the expression of both sHSP10 and sHSP17.1 genes. As for the heavy metal stressors, an increase in the production of reactive oxygen species was registered in a dose dependent manner, with a significant up-regulation of both sHSP10 and sHSP17.1 genes. These results suggest that sHSP genes in C. ehrenbergii may play a role in responses to stress environments, and they could be used as an early detection parameter as biomarker genes in molecular toxicity assessments.

Origin and roles of a novel copper-zinc superoxide dismutase (CuZnSOD) gene from the harmful dinoflagellate Prorocentrum minimum.

Superoxide dismutase (SOD) acts as the first line of defence against reactive oxygen species (ROS) within cells. In this study, we characterized a novel SOD gene (PmCuZnSOD) from the dinoflagellate Prorocentrum minimum, and examined its structural features, putative origin and gene expression. The SOD cDNA is 895 bp in length, containing dinoflagellate splice-leader (dinoSL) sequence, 714-bp ORF (237 aa), and poly (A) tail. In addition, PmCuZnSOD is coded on the dinoflagellate nuclear genome without introns and in a non-tandem repeat manner; however, the encoded protein is probably localized in chloroplasts. Phylogenetic analysis indicated that it might be acquired from cyanobacteria via horizontal gene transfer (HGT) and then the gene possibly relocated from the chloroplast to the nuclear genome. Excess copper dramatically increased the PmCuZnSOD transcripts and SOD activity in cells, caused by ROS generation and decrease of photosynthetic efficiency in the treated cells. These suggest that CuZnSOD may function to defend against oxidative stress for the survival of the dinoflagellate.

A Novel Cyclophilin B Gene in the Red Tide Dinoflagellate Cochlodinium polykrikoides: Molecular Characterizations and Transcriptional Responses to Environmental Stresses.

The marine dinoflagellate Cochlodinium polykrikoides is one of the most common ichthyotoxic species that causes harmful algal blooms (HABs), which leads to ecological damage and huge economic loss in aquaculture industries. Cyclophilins (CYPs) belong to the immunophilin superfamily, and they may play a role in the survival mechanisms of the dinoflagellate in stress environments. In the present study, we identified a novel cyclophilin gene from C. polykrikoides and examined physiological and gene transcriptional responses to biocides copper sulphate (CuSO4) and sodium hypochlorite (NaOCl). The full length of CpCYP was 903 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the polyA tail, comprising a 639 bp ORF, a 117 bp 5'-UTR, and a 147 bp 3'-UTR. Motif and phylogenetic comparisons showed that CpCYP was affiliated to group B of CYP. In biocide stressors, cell counts, chlorophyll a, and photosynthetic efficiency (Fv/Fm) of C. polykrikoides were considerably decreased in both exposure time- and dose-dependent manners. In addition, CpCYP gene expression was significantly induced after 24 h exposure to the biocide-treated stress conditions. These results indicate an effect of the biocides on the cell physiology and expression profile of CpCYP, suggesting that the gene may play a role in environmental stress responses.