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Fulltext Effects of substratum and depth on benthic harmful dinoflagellate assemblages

Lee LK, Lim ZF, Gu H, Chan LL, Litaker RW, Tester PA, et al.

Sci Rep, 2020 07 09;10(1):11251.
PMID: 32647125 DOI: 10.1038/s41598-020-68136-6

Microhabitats influence the distribution and abundance of benthic harmful dinoflagellate (BHAB) species. Currently, much of the information on the relationships between BHABs and microhabitat preferences is based on non-quantitative anecdotal observations, many of which are contradictory. The goal of this study was to better quantify BHAB and microhabitat relationships using a statistically rigorous approach. Between April 2016 to May 2017, a total of 243 artificial substrate samplers were deployed at five locations in the Perhentian Islands, Malaysia while simultaneous photo-quadrat surveys were performed to characterize the benthic substrates present at each sampling site. The screen samplers were retrieved 24 h later and the abundances of five BHAB genera, Gambierdiscus, Ostreopsis, Coolia, Amphidinium, and Prorocentrum were determined. Substrate data were then analyzed using a Bray-Curtis dissimilarity matrix to statistically identify distinct microhabitat types. Although BHABs were associated with a variety of biotic and abiotic substrates, the results of this study demonstrated differing degrees of microhabitat preference. Analysis of the survey results using canonical correspondence analysis explained 70.5% (horizontal first axis) and 21.6% (vertical second axis) of the constrained variation in the distribution of various genera among microhabitat types. Prorocentrum and Coolia appear to have the greatest range being broadly distributed among a wide variety of microhabitats. Amphidinium was always found in low abundances and was widely distributed among microhabitats dominated by hard coral, turf algae, sand and silt, and fleshy algae and reached the highest abundances there. Gambierdiscus and Ostreopsis had more restricted distributions. Gambierdiscus were found preferentially associated with turf algae, hard coral and, to a lesser extent, fleshy macroalgae microhabitats. Ostreopsis, almost always more abundant than Gambierdiscus, preferred the same microhabitats as Gambierdiscus and were found in microbial mats as well. With similar habitat preferences Ostreopsis may serve as an indicator organism for the presence of Gambierdiscus. This study provides insight into how BHAB-specific microhabitat preferences can affect toxicity risks.
Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays

Shang L, Xu Y, Leaw CP, Lim PT, Wang J, Chen J, et al.

Sci Total Environ, 2021 Aug 01;780:146484.
PMID: 33774286 DOI: 10.1016/j.scitotenv.2021.146484

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24 h exposed to the live culture (2000-6710 cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48 h when exposed to 500-6000 cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100 cells·mL-1 within 24 h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.
Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum

Luo Z, Hu Z, Tang Y, Mertens KN, Leaw CP, Lim PT, et al.

J Phycol, 2018 10;54(5):744-761.
PMID: 30144373 DOI: 10.1111/jpy.12780

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.
Toxic bloom of Pseudo-nitzschia cuspidata (Bacillariophyceae) and domoic acid contamination of bivalve molluscs in Malaysia Borneo

Teng ST, Abdullah N, Hanifah AH, Tan SN, Gao C, Law IK, et al.

Toxicon, 2021 Sep 30;202:132-141.
PMID: 34600910 DOI: 10.1016/j.toxicon.2021.09.018

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.
Corrigendum to "Toxic bloom of Pseudo-nitzschia cuspidata (Bacillariophyceae) and domoic acid contamination of bivalve molluscs in Malaysia Borneo" [Toxicon 202 (2021) 132-141]

Teng ST, Abdullah N, Hanifah AH, Tan SN, Gao C, Law IK, et al.

Toxicon, 2022 Jan 15;205:99.
PMID: 34872755 DOI: 10.1016/j.toxicon.2021.12.002
Six marine thecate Heterocapsa (Dinophyceae) from Malaysia, including the description of three novel species and their cytotoxicity potential

Hanifah AH, Teng ST, Law IK, Abdullah N, Chiba SUA, Lum WM, et al.

Harmful Algae, 2022 Dec;120:102338.
PMID: 36470602 DOI: 10.1016/j.hal.2022.102338

Thirty-four strains of Heterocapsa were established from Malaysian waters and their morphologies were examined by light, scanning, and transmission electron microscopy. Three species, H. bohaiensis, H. huensis, and H. rotundata, and three new species, H. borneoensis sp. nov., H. limii sp. nov., and H. iwatakii sp. nov. were described in this study. The three species were differentiated morphologically by unique characteristics of cell size, shape, displacement of the cingulum, shape and position of nucleus, the number and position of pyrenoids, and body scale ultrastructure. The species delimitations were robustly supported by the molecular data. A light-microscopy-based key to species of Heterocapsa is established, with two major groups, i.e., species with a single pyrenoid, and species with multiple pyrenoids. Bioassays were conducted by exposing Artemia nauplii to Heterocapsa densities of 1-5 × 105 cells mL-1, and treatments exposed to H. borneoensis showed naupliar mortality, while no naupliar death was observed in the treatments exposed to cells of H. bohaiensis, H. huensis, H. limii, and H. iwatakii. Naupliar death was observed during the initial 24 h for both tested H. borneoensis strains, and mortality rates increased up to 50% after 72-h exposure. This study documented for the first time the diversity and cytotoxic potency of Heterocapsa species from Malaysian waters.
Regional comparison on ciguatoxicity, hemolytic activity, and toxin profile of the dinoflagellate Gambierdiscus from Kiribati and Malaysia

Zhu J, Lee WH, Yip KC, Wu Z, Wu J, Leaw CP, et al.

Sci Total Environ, 2023 May 10;872:162236.
PMID: 36791857 DOI: 10.1016/j.scitotenv.2023.162236

The dinoflagellates Gambierdiscus and Fukuyoa can produce Ciguatoxins (CTXs) and Maitotoxins (MTXs) that lead to ciguatera poisoning (CP). The CP hotspots, however, do not directly relate to the occurrence of the ciguatoxic Gambierdiscus and Fukuyoa. Species-wide investigations often showed no association between CTX level and the molecular identity of the dinoflagellates. In the Pacific region, Kiribati is known as a CP hotspot, while Malaysia has only three CP outbreaks reported thus far. Although ciguatoxic strains of Gambierdiscus were isolated from both Kiribati and Malaysia, no solid evidence on the contribution of ciguatoxic strains to the incidence of CP outbreak was recorded. The present study aims to investigate the regional differences in CP risks through region-specific toxicological assessment of Gambierdiscus and Fukuyoa. A total of 19 strains of Gambierdiscus and a strain of Fukuyoa were analyzed by cytotoxicity assay of the neuro-2a cell line, hemolytic assay of fish erythrocytes, and high-resolution mass spectrometry. Gambierdiscus from both Kiribati and Malaysia showed detectable ciguatoxicity; however, the Kiribati strains were more hemolytic. Putative 44-methylgambierone was identified as part of the contributors to the hemolytic activity, and other unknown hydrophilic toxins produced can be potentially linked to higher CP incidence in Kiribati.
Fulltext Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay

Lau WLS, Teng ST, Lim HC, Hii KS, Leong SCY, Leaw CP, et al.

Trop Life Sci Res, 2023 Mar;34(1):99-120.
PMID: 37065805 DOI: 10.21315/tlsr2023.34.1.7

Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species' identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.
Fulltext A new record of the spiny lobster, Panulirusfemoristriga (von Martens, 1872) from the coastal waters of Malaysia, with revision of global distribution

Ng WL, Chen CA, Mustafa S, Leaw CP, Teng ST, Zakaria SNFB, et al.

Biodivers Data J, 2022;10:e77973.
PMID: 35237095 DOI: 10.3897/BDJ.10.e77973

BACKGROUND: Spiny lobsters of the family Palinuridae Latreille, 1802 are known to be industrial crustaceans in the global fishing market amongst other crustacean marine species. Panulirusfemoristriga has been reported in the Maldives, Japan, Taiwan, Vietnam, the Philippines, Indonesia (Ambon, Irian Jaya, Celebes Island, Seram Island), the Polynesian Islands, Solomon Islands, New Hebrides, Wallis and Futuna and off the coast of northern Australia, but there is uncertainty about their distributions due to the morphological similarity with Panulirusfemoristriga, Panuliruslongipesbispinosus and Panulirusbrunneiflagellum. However, the identification on P.femoristiga can only be confirmed if the morphological descriptions are mentioned in literature.
NEW INFORMATION: A specimen of the spiny lobster Panulirusfemoristriga Von Martens, 1872 was discovered in Semporna, located on the west coast of Sabah State, Malaysia Borneo. While the status of P.femoristriga has been classified as "least concern" on the International Union for Conservation of Nature Red List, studies on the species' population size, habitat and distribution are still inadequate. This study adopted both morphological and molecular approaches for species delimitation.The phylogenetic position of the Sabah P.femoristriga was revealed by the mitochondrial cytochrome c oxidase gene (COI) marker. This represents the first record of the species in the coastal waters of Sabah, despite its wide geographical distribution in the Indo-West Pacific. A revision on the species global distribution was also conducted by harvesting all literature with species named Panuliruslongipesfemoristriga and Panulirusfemoristriga which were available online including those prior to year 2001 before the presence of P.femoristriga is confirmed. Due to the uncertainties on the morphological distribution in previous literature, further studies are required to fill in the missing data for confirmation.
Intraspecific genetic diversity with unrestricted gene flow in the domoic acid-producing diatom Nitzschia navis-varingica (Bacillariophyceae) from the Western Pacific

Tan SN, Kotaki Y, Teng ST, Lim HC, Gao C, Lundholm N, et al.

Harmful Algae, 2025 Jan;141:102769.
PMID: 39645396 DOI: 10.1016/j.hal.2024.102769

The benthic pennate diatom Nitzschia navis-varingica, known for producing domoic acid (DA) and its isomers, is widely distributed in the Western Pacific (WP) region. To investigate the genetic differentiation and gene flow patterns among the populations in the WP, the genetic diversity of 354 strains of N. navis-varingica was analysed using two nuclear-encoded rDNA loci: the large subunit rDNA (LSU rDNA) and the internal transcribed spacer 2 (ITS2). Frustule morphology of each strain was examined by TEM. The LSU rDNA phylogeny revealed a monophyletic lineage encompassing all strains, with sequence divergences of <0.9 %. Phylogenetic and population genetic analyses of ITS2 identified eight distinct clades (designated as Groups A to H) with moderate to high genetic heterogeneity (0.5-19.7 %). The low genetic differentiations between the geographically separated populations (pairwise FST of <0.03) suggested high gene flow and lack of spatial genetic structuring. Molecular clock analysis of the ITS2 phylogeny traced the evolutionary history of N. navis-varingica to the Eocene Epoch, and the split between clades likely occurred from the mid-Miocene to Pleistocene Epochs (10.8-1.2 Ma). The population dispersal in the WP were likely influenced by historical events like the Quarternary glacial cycles during the period, contributing to its homogenous distributions in the region.
A non-toxigenic but morphologically and phylogenetically distinct new species of Pseudo-nitzschia, P. sabit sp. nov. (Bacillariophyceae)

Teng ST, Lim PT, Lim HC, Rivera-Vilarelle M, Quijano-Scheggia S, Takata Y, et al.

J Phycol, 2015 Aug;51(4):706-25.
PMID: 26986792 DOI: 10.1111/jpy.12313

A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.
Diverse harmful microalgal community assemblages in the Johor Strait and the environmental effects on its community dynamics

Hii KS, Mohd-Din M, Luo Z, Tan SN, Lim ZF, Lee LK, et al.

Harmful Algae, 2021 07;107:102077.
PMID: 34456026 DOI: 10.1016/j.hal.2021.102077

Coastal ecosystems are often subjected to anthropogenic disturbances that lead to water quality deterioration and an increase in harmful algal bloom (HAB) events. Using the next-generation molecular tool of 18S rDNA metabarcoding, we examined the community assemblages of HAB species in the Johor Strait, Malaysia between May 2018 and September 2019, covering 19 stations across the strait. The molecular operational taxonomic units (OTUs) of HAB taxa retrieved from the dataset (n = 194) revealed a much higher number of HAB taxa (26 OTUs) than before, with 12 taxa belong to new records in the strait. As revealed in the findings of this study, the diversity and community structure of HAB taxa varied significantly over time and space. The most common and abundant HAB taxa in the strait (frequency of occurrence >70%) comprised Heterosigma akashiwo, Fibrocapsa japonica, Pseudo-nitzschia pungens, Dinophysis spp., Gymnodinium catenatum, Alexandrium leei, and A. tamiyavanichii. Also, our results demonstrated that the HAB community assemblages in the strait were dependent on the interplay of environmental variables that influence by the monsoonal effects. Different HAB taxa, constitute various functional types, occupied and prevailed in different environmental niches across space and time, leading to diverse community assemblages and population density. This study adds to the current understandings of HAB dynamics and provides a robust overview of temporal-spatial changes in HAB community assemblages along the environmental gradients in a tropical eutrophic coastal ecosystem.
Habitat complexity affects benthic harmful dinoflagellate assemblages in the fringing reef of Rawa Island, Malaysia

Yong HL, Mustapa NI, Lee LK, Lim ZF, Tan TH, Usup G, et al.

Harmful Algae, 2018 09;78:56-68.
PMID: 30196925 DOI: 10.1016/j.hal.2018.07.009

Few studies have investigated the effect of fine-scale habitat differences on the dynamics of benthic harmful dinoflagellate assemblages. To determine how these microhabitat differences affect the distribution and abundance of the major benthic harmful dinoflagellate genera in a tropical coral reef ecosystem, a field study was undertaken between April-September 2015 and January 2016 on the shallow reef flat of the fringing reef of Rawa Island, Terengganu, Malaysia. Sampling of benthic dinoflagellates was carried out using an artificial substrate sampling method (fiberglass screens). Benthic microhabitats surrounding the sampling screens were characterized simultaneously from photographs of a 0.25-m2 quadrat based on categories of bottom substrate types. Five taxonomic groups of benthic dinoflagellates, Ostreopsis, Gambierdiscus, Prorocentrum, Amphidinium, and Coolia were identified, and cells were enumerated using a light microscope. The results showed Gambierdiscus was less abundant than other genera throughout the study period, with maximum abundance of 1.2 × 103 cells 100 cm-2. While most taxa were present on reefs with high coral cover, higher cell abundances were observed in reefs with high turf algal cover and coral rubble, with the exception of Ostreopsis, where the abundance reached a maximum of 3.4 × 104 cells 100 cm-2 in habitats with high coral cover. Microhabitat heterogeneity was identified as a key factor governing the benthic harmful dinoflagellate assemblages and may account for much of the observed variability in dominant taxa. This finding has significant implications for the role of variability in the benthic harmful algal bloom (BHAB) outbreaks and the potential in identifying BHAB-related toxin transfer pathways and the key vectors in the food webs.
Growth and epiphytic behavior of three Gambierdiscus species (Dinophyceae) associated with various macroalgal substrates

Mustapa NI, Yong HL, Lee LK, Lim ZF, Lim HC, Teng ST, et al.

Harmful Algae, 2019 Nov;89:101671.
PMID: 31672230 DOI: 10.1016/j.hal.2019.101671

Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.
Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae)

Lim ZF, Luo Z, Lee LK, Hii KS, Teng ST, Chan LL, et al.

Harmful Algae, 2019 Mar;83:95-108.
PMID: 31097256 DOI: 10.1016/j.hal.2019.01.007

Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0-1.6 pg cell-1) and P. caipirignum (3.1 pg cell-1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.
Fulltext Identification of Fish Species and Toxins Implicated in a Snapper Food Poisoning Event in Sabah, Malaysia, 2017

Dao HV, Uesugi A, Uchida H, Watanabe R, Matsushima R, Lim ZF, et al.

Toxins (Basel), 2021 09 15;13(9).
PMID: 34564661 DOI: 10.3390/toxins13090657

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian countries, few have been confirmed by ciguatoxins identification, resulting in limited information for the correct diagnosis of this food-borne disease. In the present study, ciguatoxin-1B (CTX-1B) in red snapper (Lutjanus bohar) implicated in a CFP case in Sabah, Malaysia, in December 2017 was determined by single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Continuous consumption of the toxic fish likely resulted in CFP, even when the toxin concentration in the fish consumed was low. The identification of the fish species was performed using the molecular characterization of the mitochondrial cytochrome c oxidase subunit I gene marker, with a phylogenetic analysis of the genus Lutjanus. This is the first report identifying the causative toxin in fish-implicated CFP in Malaysia.
Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution

Lim HC, Tan SN, Teng ST, Lundholm N, Orive E, David H, et al.

J Phycol, 2018 04;54(2):234-248.
PMID: 29377161 DOI: 10.1111/jpy.12620

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.
Prolonged high biomass diatom blooms induced formation of hypoxic-anoxic zones in the inner part of Johor Strait

Mohd-Din M, Abdul-Wahab MF, Mohamad SE, Jamaluddin H, Shahir S, Ibrahim Z, et al.

Environ Sci Pollut Res Int, 2020 Dec;27(34):42948-42959.
PMID: 32725555 DOI: 10.1007/s11356-020-10184-6

The Johor Strait has experienced rapid development of various human activities and served as the main marine aquaculture area for the two countries that bordered the strait. Several fish kill incidents in 2014 and 2015 have been confirmed, attributed to the algal blooms of ichthyotoxic dinoflagellates; however, the cause of fish kill events after 2016 was not clarified and the causative organisms remained unknown. To clarify the potential cause of fish kills along the Johor Strait, a 1-year field investigation was conducted with monthly sampling between May 2018 and April 2019. Monthly vertical profiles of physical water parameters (temperature, salinity, and dissolved oxygen levels) were measured in situ at different depths (subsurface, 1 m, 5 m, and 8 m) depending on the ambient depth of the water column at the sampling stations. The spatial-temporal variability of macronutrients and chlorophyll a content was analyzed. Our results showed that high chlorophyll a concentration (up to 48.8 μg/L) and high biomass blooms of Skeletonema, Chaetoceros, Rhizosolenia, and Thalassiosira were observed seasonally at the inner part of the strait. A hypoxic to anoxic dead zone, with the dissolved oxygen levels ranging from 0.19 to 1.7 mg/L, was identified in the inner Johor Strait, covering an estimated area of 10.3 km2. The occurrence of high biomass diatom blooms and formation of the hypoxic-anoxic zone along the inner part Johor Strait were likely the culprits of some fish kill incidents after 2016.
Mapping harmful microalgal species by eDNA monitoring: A large-scale survey across the southwestern South China Sea

Abdul Manaff AHN, Hii KS, Luo Z, Liu M, Law IK, Teng ST, et al.

Harmful Algae, 2023 Nov;129:102515.
PMID: 37951609 DOI: 10.1016/j.hal.2023.102515

A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.
Phylogeny and ultrastructure of a non-toxigenic dinoflagellate Amphidoma fulgens sp. nov. (Amphidomataceae, Dinophyceae), with a wide distribution across Asian Pacific

Kuwata K, Lum WM, Takahashi K, Benico G, Takahashi K, Lim PT, et al.

Harmful Algae, 2024 Sep;138:102701.
PMID: 39244236 DOI: 10.1016/j.hal.2024.102701

Amphidoma languida, a marine thecate dinoflagellate that produces the lipophilic toxin azaspiracids (AZAs), is primarily found in the Atlantic. Although this species has not been recorded in the Asian Pacific, environmental DNAs related to Am. languida have been widely detected in the region by metabarcoding analysis. Their morphology and AZA production remain unclear. In this study, the morphology, ultrastructure, phylogeny, and AZA production of nine Amphidoma strains isolated from Japan, Malaysia, and Philippines were investigated. Phylogenetic trees inferred from rDNAs (SSU, ITS, and LSU rDNA) showed monophyly of the nine Pacific strains and were sister to the Am. languida clade, including the toxigenic strains from the Atlantic. Cells were ellipsoid, 8.7-16.7 µm in length and 7.4-14.0 µm in width, with a conspicuous apical pore complex. A large nucleus in the hyposome, parietal chloroplast with a spherical pyrenoid in the episome, and refractile bodies were observed. Thecal tabulation was typical of Amphidoma, Po, cp, X, 6', 6'', 6C, 5S, 6''', 2''''. A ventral pore was located on the anterior of 1' plate, beside the suture to 6' plate. The presence of a ventral depression, on the anterior of anterior sulcal plate, was different from Am. languida. A large antapical pore, containing approximately 10 small pores, was observed. Cells were apparently smaller than Am. trioculata, a species possessing three pores (ventral pore, ventral depression, and antapical pore). TEM showed the presence of crystalline structures, resembling guanine crystals, and cytoplasmic invaginations into the pyrenoid matrix. Flagellar apparatus lacking the striated root connective is similar to peridinioids and related dinoflagellates. AZAs were not detected from the Pacific strains by LC-MS/MS. This non-toxigenic Amphidoma species, here we propose as Amphidoma fulgens sp. nov., is widely distributed in the Asian Pacific. Moreover, molecular comparison also suggested that most of the environmental DNA sequences previously reported as Am. languida or related sequences from the Asian Pacific were attributable to Am. fulgens.

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