Solving the phylogeny of toxic dinoflagellates, alexandrium halim (Dinophyceae)

The genus Alexandrium is a widely distributed dinoflagellate and has the ability to produce potent neurotoxins, saxitoxin (STX) and its deriavatives. In this study, the molecular phylogenetic approach was used to infer the relationships of Alexandrium species distributed worldwide. The clonal cultures of Alexandrium minulllm (AmKBOI, AmKB02, AmKB03, AmKB04, AmKB05 and AmKB06) was used in this study and maintained in SW II medium at 26°C under 12:12 hour light: dark cycle. The total genomic DNA of late exponential phase cultures was extracted and the genomic DNA were used for internal transcribed spacer (ITS) regions amplification and sequencing. Structural analysis of the ITS2 transcript was carried out using the sequences obtained together with related sequences from eighteen species of Alexadrium, Pyrodinium bahamense var. compressum and two outgroup taxa, Coolia malayensis and OSlreopsis lenliclilaris. The results showed conserved four helices secondary structure of ITS2 transcript for all sequences analyzed. Structural comparison between species found a number of compensatory base changes (CBCs). Structural information was then used to perform multiple sequence-structure alignment for phylogenetic anaJy ·s. The phylogenetic inference of Alexandrium species from this study showed nearly similar framework as in the LSU rDNA phylogeny

Spatial distribution of toxic Alexandrium tamiyavanichii (Dinophyceae) in the southeastern South China Sea-Sulu Sea: A molecular-based assessment using real-time quantitative PCR (qPCR) assay

In this study, a quantitative real-time PCR (qPCR) assay targeting the second internal transcribed spacer (ITS2) of the nuclear-encoded ribosomal RNA gene (rDNA) was developed for Alexandrium tamiyavanichii, a harmful tropical marine dinoflagellate. This species is of concern because it produces toxins that cause paralytic shellfish poisoning (PSP). The qPCR assay employed hydrolysis probe technology and showed high specificity, with a detection limit of 102 gene copies (less than one cell equivalent). Using this assay, the spatial distribution of A. tamiyavanichii was assessed, for the first time, in the southeastern South China Sea and the Sulu Sea. Plankton samples were collected from 71 stations during a scientific cruise from the Research Vessel Sonne as part of the joint EU project on Stratosphere ozone: Halogens in a Varying Atmosphere (SHIVA), conducted in November 2011. The highest cell densities were detected offshore of Kuching, southern Borneo (150 cells l−1) and exceeded the threshold level of 20–40 cells l−1 where the bioaccumulation of PSP toxins by shellfish is of concern. The distribution of A. tamiyavanichii was patchy horizontally with the highest cell concentrations found mainly offshore of southern Borneo, and a heterogeneous vertical distribution was observed above the pycnocline. The A. tamiyavanichii qPCR assay proved its applicability, specificity and sensitivity, and provides an alternative implementation tool for harmful microalgae monitoring programs

First Record of Marine Dinoflagellate, Alexandrium Tamutum (Dinophyceae) from Malaysia

Several species of dinoflagellates in the genus Alexandrium are known to be toxic, and have been associated with paralytic shellfish poisoning (PSP) in Malaysia. These Alexandrium species showed high morphological similarity among the toxic and non-toxic species, and detailed observation of the thecal plate’s arrangement is required for precise species identification. Co-occurrence of the toxic and non-toxic species has complicated the plankton monitoring of PSP. In this study, a clone of Alexandrium species was established from plankton samples collected from Kota Belud, Sabah. The specimen was observed under epi-fluorescence microscope, and nucleotide sequences of the nuclear-encoded ribosomal RNA gene obtained. Morphologically, the clone showed relatively wide and large sixth precingular plate (6´´) compared to that of A. minutum. The sulcal posterior plate (Sp) is similar to that of A. minutum, which is wider than long. The first apical plate (1´) is irregularly rhomboidal with a small ventral pore (vp) present on its right margin. The morphological characters resembled to the species description of A. tamutum. Phylogenetic analysis of the ITS rDNA region also revealed a monophyly of this clone with other strains of A. tamutum, and separating them from the A. minutum clade. Species-specific sequence signatures of A. tamutum were obtained in silico, which could be as potential oligonucleotide probe regions for species detection by using molecular tool. This represents the first report of A. tamutum found in Malaysian waters

Phytoplankton community changes in Kuantan Port (Malaysia), with emphasis on the paralytic-shellfish toxin-producing dinoflagellate Alexandrium tamiyavanichii

The Kuantan Port (Pahang, Malaysia, South China Sea) is a multi-cargo port located on the east coast of Peninsular Malaysia. The port has served as an important seaway to major ports in Asia-Pacific regions. In November 2013 and August 2014, two incidents of paralytic shellfish poisoning (PSP) have been consecutively reported in the Port. In this study, a field investigation was undertaken in the Port from April 2015 to May 2016 as an effort to continuously monitor the occurrence of HAB species following the PSP episodes in the year 2013–2014. Phytoplankton and hydrographic samples were collected for quantitative and qualitative assessments in a monthly interval. To precisely quantify the PSP-toxins producing species Alexandrium tamiyavanichii, a real-time quantitative PCR (qPCR) assay was applied to detect the motile cells and cysts. The results revealed the presence of A. tamiyavanichii but with extremely low cell abundances (<0.1% of the total abundances). The species was found co-existed with other Alexandrium species. Alexandrium abundance was associated with salinity and nitrogen to phosphorus ratios but negatively correlated with PO4-P and NH4-N as revealed in the canonical correspondence analysis. Low cell abundances of diarrhetic-shellfish toxins producing dinoflagellates (Dinophysis spp.) and fish-killing species (Prorocentrum sigmoides, Akashiwo sanguinea, Noctiluca scintillans, Chattonella spp.) were also encountered in the port. The results of this study would provide useful baseline information for the assessment and management of ballast water in Malaysian ports and its territorial waters

A bloom of Karlodinium australe (Gymnodiniales, Dinophyceae) associated with mass mortality of cage-cultured fishes in West Johor Strait, Malaysia

A recent (February 2014) mass mortality of fishes was observed in the cage-farming region of the West Johor Strait of Malaysia, involving over four different species of cultured fishes, numbering ∼50,000 fish. A field investigation at six stations along the West Johor Strait collected water samples and examined for the presence of harmful species. Dead fishes were collected for necropsy. The phytoplankton composition was dominated by a species of Karlodinium, at a considerably high cell concentration (0.31–2.34 × 106 cells l−1), and constituting 68.8–98.6% of the phytoplankton relative abundance at all stations. Detailed morphological assessment by light and scanning electron microscopy revealed that the species was Karlodinium australe de Salas, Bolch and Hallegraeff. This was supported by molecular evidence of the nuclear encoded large subunit ribosomal gene (LSU rDNA) and the second internal transcribed spacer (ITS2) via single-cell PCR. The sequences of LSU rDNA yielded 3.6–4.0% divergence when compared to the sister taxon, K. armiger; and >6.5% when compared to other Karlodinium species. Fish necropsy showed symptoms similar to those affected by karlotoxin ichthyotoxins. This is the first report of a mass mortality of cage-cultured and wild fishes attributed to the unarmored dinoflagellate K. australe

Quantitative real-time PCR detection of a harmful unarmoured dinoflagellate, Karlodinium australe (Dinophyceae)

We investigated a harmful algal bloom (HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green-based realtime PCR (qPCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species-specific qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species specificity of the primers designed was evaluated by screening on the non-target species (Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross-detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 � 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R2 = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single-cell PCR and rDNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose

Transcriptional and Physiological Responses of Genes Related to Nutrient Uptakes and Toxin Production of Alexandrium minutum (Dinophyceae) in Malaysia Waters

Saxitoxins (STXs) is a family of potent sodium channel blocking toxins, also causative agents of paralytic shellfish poisoning (PSP) that is produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes (sxt), sxtA and sxtG have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remained unclear. The nutrient fluxes and the bloom of Alexandrium have also been intensively studied but the genetically nutrient regulation mechanisms in nutrient uptakes on this genus are still unknown. This study aims to investigate the physiological responses of the tropical Pacific strains of A. minutum in nitrate- or ammonium-grown culture conditions, and with various nitrogen to phosphorus supply ratios (N:P). The transcriptional responses of the sxt, nutrient transporters and assimilation genes were observed. Likewise, a putative sxtI encoding Ocarbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and the relative expression was investigated. The results have revealed that the cellular toxin quota (Qt) was higher in P-deficient nitrate-grown cultures. Between cultures grown at similar N: P (<16), cells grown in excess ammonium showed higher Qt than that of the nitrate-grown cultures. sxtA1 was not expressed in the culture conditions suggesting that the gene might not be involved in STX biosynthesis of this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and up-regulated at P-deficient nitrategrown cultures and excess ammonium environment. On the other hand, relative expression of AmsxtI was induced only at P-deficient environment suggesting that it may play an important role in the P-recycling metabolism and simultaneously enhances the toxin production. Conversely, the A. minutum novel high affinity nitrate, ammonium and phosphate transporter genes (AmNrt2, AmAmt1 and AmPiPT1); as well as assimilation genes, cytosol nitrate reductase (AmNas), mitochondria glutamine syntheses (AmGSIII) and pyrimidine syntheses carbamoyl phosphate syntheses (AmCPSII) were assembled by the Sequence Read Archive (SRA) dataset. AmAmt1 was suppressed in excess ammoniumgrown, but not for AmNrt2 and AmNas, suggesting that the nitrate uptake was preferred in this species. AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII and AmPiPT1 were highly expressed in P-deficient environment showing that A. minutum is likely to be able to take up nutrients for growth under P-stress condition. Relative expression of AmCPSII was incongruent with the cells growth, but it was well correlated with Qt, postulating that the gene might involve in arginine metabolism and concurrently related to toxin production. Besides that, the AmGSIII expression in this study has found that the SXT production of the cells also is a manner to detoxify or release excess ammonium environment stress. The gene regulation of this study has provided better insights into the eco-physiology of A. minutum in relation to the toxin production and its adaptative strategies in the unfavourable environment. This will be an advantageous for the future harmful algae blooms (HABs) monitoring

Molecular characterization of the its2 transcript and saxitoxin biosynthetic genes in two toxic dinoflagellates, alexandrium (dinophyceae) from Malaysia

The genus Alexandrium is a widely distributed dinoflagellate, and has the ability to produce potent neurotoxins, saxitoxin (STX). Taxonomy of this genus still remains uncertain and species identification is yet confusing. In this study, the second internal transcribed spacer (ITS2) transcript was used to infer the phylogenetic relationships of Alexandrium species distributed worldwide. A total of 33 ITS2 transcript of Alexandrium spp. were successfully modeled in silico, with one each from Pyrodinium bahamense var. compressum, Coolia malayensis and C. monotis as outgroup) The models showed conserved four universal helices of ITS2 transcript. The phylogenetic inference based on sequence-structural information revealed nearly similar phylogenetic framework as inferred in the Large Subunit (LSU) rDNA phylogeny. However, the results showed possible phylogeographic break in the A. minutum Glade where the Asia Pacific and New Zealand A. minutum formed a distant group from the Australian and European group. Genetics of the STX biosynthesis pathway has recently become one of the major focuses in Paralytic Shellfish Poisoning (PSP) toxin-related studies after the discovery of STX biosynethetic genes in toxic cyanobacteria, and later in the toxic dinoflagellates. In the present study, two domains of a saxitoxin biosynthetic gene sxtA, S-adenosyl-L-methionine (SAM)-dependent methyltransferase coding gene (sxtA1) and the class 11 aminotransferase coding gene (sxtA4) were characterized from a toxic A. tamiyavanichii from Samariang, Sarawak. A saxitoxin biosynthetic gene encoding the O-carbamoyltransferase (sxtl) was also characterized in the toxic A. minutum from Tumpat, Kelantan. The partial coding sequences of saxitoxin starting gene, sxtAl and sxtA4 of A. tamiyavanichii were 432 bp and 639 bp, respectively. While the deduced amino acid sequences of sxtAl and sxtA4 were 144 and 213 amino acid residues, respectively. Sequences comparison revealed high similarity and identity to other PSP toxins-producing dinoflagellates (82-98% and 84-99%). In contrast, protein phylogenetic analyses revealed close relationship of both A. tamiyavanichii sxtA1 and sxtA4 to others PSP toxins-producing dinoflagellates, with sxtA of PSP toxins-producing cyanobacteria and putative toxin-related genes forming the sister Glade. On the other hand, the coding sequence of O-carbamoyltransferase (sxtl) of A. minutum was 1,920 bp long, and the deduced amino acid sequence revealed a polypeptide of 639 amino acids. Structural sequence alignment revealed high similarity and identity (50-52% and 87- 89%) to sxd from the toxic cyanobacteria. Sequence comparison of A. minutum sxtl revealed highly conserved pattern, with five phosphorylation motifs, two catalytic regions, and a zinc finger detected. Even though homology between A. minutum sxtl and other cyanobacterial sxtl was observed, protein phylogenetic analysis inferred a distant relationship with the cyanobacterial Sxtl, suggesting a paralog of SxtI in dinoflagellates

Characterization of the Saxitoxin Biosynthetic Starting Gene, sxta in the Toxic Dinoflagellate Alexandrium tamiyavanichii

Recently, molecular genetics of the Saxitoxin (STX) biosynthesis pathway has become one of the major focuses in paralytic shellfish poisoning (PSP) toxin-related studies after the recent discovery of STX biosynthetic genes in toxic cyanobacteria and later in the toxic dinoflagellates. Here we described the two domains of sxtA, SAM-dependent methyltransferase coding gene, sxtA1 and the class II aminotransferase coding gene, sxtA4 from a toxic strain of Alexandrium tamiyavanichii isolated from Samariang, Sarawak

Physiological and transcriptional responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for the saxitoxin genes and toxin production

Saxitoxins (STXs) constitute a family of potent sodium channel blocking toxins, causative agents of paralytic shellfish poisoning (PSP), and are produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes, sxtA and sxtG, have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remains unclear. This study investigates the physiological responses of a tropical Pacific strain of Alexandrium minutum growing with nitrate or ammonium, and with various nitrogen to phosphorus (N:P) supply ratios. The transcriptional responses of the sxt genes were observed. Likewise, a putative sxtI encoding O-carbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and its expression was investigated. The results revealed that the cellular toxin quota (Qt) was higher in P-depleted, nitrate- grown cultures. With cultures at similar N:P (<16), cells grown with excess ammonium showed a higher Qt than those grown with nitrate. sxtA1 was not expressed under any culture conditions, suggesting that this gene might not be involved in STX biosynthesis by this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and were up-regulated in P-depleted, nitrate-grown cultures and with excess ambient ammonium. On the other hand, AmsxtI was expressed only when induced by P-depletion, suggesting that this gene may play an important role in P-recycling metabolism, while simultaneously enhancing toxin production