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

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

Bloom of a freshwater green alga Botryococcus braunii (Botryococcaceae, Trebouxiophyceae) and the associated mass fish mortality in a man-made lake, Sarawak, Malaysia

Mass mortality of fish (∼8,500 fishes), mainly Oreochromis placidus, was noted in a man-made lake located at Kuching, Sarawak (Malaysia). A field investigation was conducted to collect water samples and fishes. Patches of discoloration in brick red were observed in the lake and clear oil layer was found on the surface of the water. Microscopic observation and enumeration of the water samples showed that the plankton composition was dominated by a green algal species Botryococcus sp., with the colony densities ranging 1.2×103–7.4×106 colonies L−1. Detailed morphological assessment by light microscopy revealed the dominant species as Botryococcus braunii Kützing. Molecular characterization using an rDNA marker further supported the species identity as B. braunii in the L race. Fish gill observation showed that cells of B. braunii and the oily substances were found in the dead fish gills. The race-L B. braunii bloom was reported, for the first time, to be associated with a fish kill event in a freshwater lake in Malaysia and confirmed the species as one of the algal types causing harmful effects to the environment

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

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2ʹ/4ʹ suture length, narrower plates 1ʹ and 6ʹʹ, with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1ʹ having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6–14 pg cell− 1 ) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4–7.3 pg cell− 1 ). None of the strains contained detectable levels of PSTs and cycloimines

Saxitoxin biosythesis genes (SXT) in the paralytic shellfish toxins-producing dinoflagellates, alexandrium species (Dinophyceae)

Saxitoxin (STX) is a neurotoxin that causes Paralytic Shellfish Poisoning (PSP). The toxin is produced by a group of marine eukaryotic dinoflagellates and prokaryotic cyanaobacteria. The toxin is accumulated in the edible shellfish and responsible for human intoxication after consumption. The biosynthetic gene,sxt/ is one of the genes involved in the PSP toxins biosynthetic pathway. The gene encodes O-carbomoyltransferase (OCASE) which was proposed to carbamoylate the hydroxyl-methyl side chain of the saxitoxin precursor. The present study was conducted to characterize the sxt genes in the marine dinoflagellates, Alexandrium species from Malaysian waters. Clonal cultures of Alexandrium species obtained from Harmful Algae Culture Collection, UNIMAS were used in this study. Toxicity of toxic and non-toxic Alexandrium species was confirmed by ELISA kit and HPLC method. Alexandriumminutum and A. tamiyavanichii were confirmed toxic, while A. leei, A. tamarense and A. tamutum are not toxic. A primer pair was insilico designed to screen for the presence of sxtI in both toxic and non-toxic Alexandrium species, the primer pair successfully amplified the gene from two toxic species, A. minutum and A. tamiyavancihii, with the amplicon size of around 200 bp, but was not detected in non-toxic .....Alexandrium species. This study revealed the presence of sxtI in toxic dinoflagellates

Toxic bloom of Pseudo-nitzschia cuspidata (Bacillariophyceae) and domoic acid contamination of bivalve molluscs in Malaysia Borneo

In March 2018, an algal bloom of Pseudo-nitzschia was detected, for the first time, in a semi-enclosed lagoon in Miri, Sarawak, Malaysia Borneo. The plankton samples were collected for cell enumeration and species identification by electron microscopy and molecular characterization. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to detect and quantify the neurotoxin domoic acid (DA) in both the plankton and shellfish samples. The abundance of Pseudo-nitzschia cells ranged from 5.6 � 105 to 3.5 � 106 cell L-1 during the bloom event. Morphological observation of the cells by transmission electron microscopy showed that the plankton samples comprised a single Pseudo-nitzschia morphotype resembling P. cuspidata. The ITS2 sequence-structure phylogenetic inference further supported the species identity as Pseudo-nitzschia cuspidata. Low levels of DA were detected in the plankton samples, with cellular DA, particulate DA, and dissolved DA of 257�504 fg DA cell-1, 676 ng L-1, and 15 ng L-1, respectively. The amount of DA, 8 μg g-1 tissue, was found present in the shellfish sample (Magallana sp.) which is below the regulatory limit of 20 μg DA g-1 tissue. The study documented, for the first time, DA contamination in shellfish that associated with bloom of P. cuspidata in the Western Pacific region

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2ʹ/4ʹ suture length, narrower plates 1ʹ and 6ʹʹ, with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1ʹ having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6–14 pg cell−1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4–7.3 pg cell−1). None of the strains contained detectable levels of PSTs and cycloimines