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

On-site rapid detection of toxic Alexandrium tamiyavanichii: integrating the species-specific hydrolysis probe in insulated isothermal polymerase chain reaction (iiPCR)

On-site investigation of phytoplankton samples is important for rapid detection of harmful algal species and for early warning of harmful algal bloom. Molecular detection method by DNA amplification in a portable insulated isothermal PCR (iiPCR) device provides a simple and rapid detection based on fluorescent probe within an hour of reaction time. The assay was developed for a paralytic shellfish toxinproducing dinoflagellate Alexandrium tamiyavanichii. The assay presents the data as positive or negative on the presence or absence of A. tamiyavanichii cells, with a limit of detection (LOD) at five target cells per reaction. While the assay is incapable to accurately quantify cell density, it exhibits high detection accuracy and strongly correlated with quantitative PCR (qPCR) data. The user repeatability of iiPCR assay was evaluated; the results showed that no significant differences in the assay run by different operators. Field applicability of the assay was further validated by environmental samples. Despite the shortcoming of the assay, the overall performance of the assay to detect cells, its low-cost effectiveness, and portability for on-site detection, iiPCR has proven its potential as an early screening tool for harmful algae monitoring. Nyuk Fong Kon1 & Winnie Lik Sing Lau2 & Ing Kuo Law2 & Po Teen Lim2 & Chui Pin Leaw

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

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

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

Morphology and molecular characterization of marine dinoflagellates (Dinophyceae)

There is a wide diversity of dinoflagellates inhabiting the complex nature of the marine environment. Marine dinoflagellates are potentially responsible for toxic red tides which can cause harmful effects to the surrounding ecosystem, marine life as well as human health. In this study, marine dinoflagellates were characterized based on morphology and molecular information. Field samplings were carried out at Samariang, Sarawak and clonal cultures were established. Morphological observations of cultured samples were carried out using epiflourescence microscope. The cultures established were subjected to genomic DNA isolation for molecular characterization. Gene amplification was performed to amplify the nuclear-encoded ribosomal RNA gene (rDNA). The amplicons obtained were subjected to further purification prior to DNA sequencing. In further, the nucleotide sequences obtained were analysed and used in phylogenetic reconstruction. Sequence signatures of species found were assigned for DNA barcoding. A species inventory of benthic dinoflagellates in Samariang with molecular signatures as potential DNA barcode was obtained in this study. The results of the present study showed that the Kudat isolate resembled C. malayensis with identical morphological features. The phylogenetic tree constructed for Coolia species showed that the Kudat isolate was claded together with other C. malayensis from Langkawi Island, Port Dickson and Sabah. In addition, three Prorocentrum species from Samariang and one from Kudat were identified. Their phylogenetic tree showed that they are claded into respective species clades which further supported the morphology evidence. Furthermore, one Alexandrium species was identified from Kuala Abai, Sabah. The cells had rhomboidal first apical plate (1´), wide sixth precingular plate (6´´) and posterior sulcal plate (Sp) that was wider than longer. The phylogenetic tree constructed for Alexandrium species further proved that the isolate was Alexandrium tamutum. Therefore, the morphologies and molecular data obtained were well support each other

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