Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay.

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.

Spesies genus Chattonella (Raphidophyceae) ialah sekumpulan protista marin yang biasa ditemui di perairan laut pantai. Sesetengahnya dikenali sebagai mikroalga berbahaya yang membentuk ledakan alga berbahaya dan menyebabkan kematian ikan secara besar-besaran dalam akuakultur ikan sirip. Di Malaysia, ledakan alga Chattonella telah direkodkan sejak tahun 1980-an di Selat Johor. Dalam kajian ini, dua strain Chattonella telah didirikan dari selat, dan pemeriksaan morfologi mendedahkan ciri-ciri yang menyerupai Chattonella subsalsa. Pencirian molekul seterusnya mengesahkan identiti spesies sebagai C. subsalsa. Untuk mengesan dengan tepat sel-sel C. subsalsa di dalam persekitaran, ujian penghibridan in-situ berpendarfluor (FISH) ke atas sel keseluruhan telah dibangunkan. Prob oligonukleotida spesies telah direka secara spesifik secara siliko berdasarkan jujukan nukleotida subunit besar (LSU) dan spacer transkripsi dalaman 2 (ITS2) gen DNA ribosom (rDNA). Calon terbaik kawasan tanda dalam LSU-rRNA dan ITS2-rDNA telah dipilih berdasarkan kecekapan penghibridan dan parameter prob. Prob telah disintesis sebagai prob biotinilasi dan diuji dengan penguatan isyarat tyramide dengan FISH (FISH-TSA). Keputusan menunjukkan kekhususan prob ke atas sel sasaran. FISH-TSA telah terbukti sebagai alat yang berpotensi dalam pengesanan alga berbahaya di alam sekitar dan boleh digunakan untuk program pemantauan alga berbahaya.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae).

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL-1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL-1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

Description of two new species Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov. (Raphidophyceae) from South China Sea, with a report of wild fish mortality.

Fisheries damage caused by Chattonella red tide has been recorded in Southeast Asia. Molecular studies have clarified the presence of two species, Chattonella marina complex and Chattonella subsalsa in the region, unlike East Asia that had only C. marina complex. To elucidate the phylogeography of Chattonella in Asia, further phylogenetic and morphological examinations were carried out with 33 additional culture strains, including the strains isolated during a bloom of Chattonella sp. (up to 142 cells mL-1) that was associated with a wild fish mortality along the northeastern coast of Peninsular Malaysia in 2016, and those from Yellow Sea, where the Chattonella genotypes have not been determined. LSU rDNA and ITS2 trees showed five intrageneric clades in the genus Chattonella, which were clades I and II (C. subsalsa), clade III (C. marina complex) and two new clades, namely clade IV from Thailand and Malaysia, and clade V from Peninsular Malaysia. The positions of the two new clades were different in LSU rDNA and ITS2 trees. LSU rDNA divergences of clades IV and V from the other clades were ≥ 4.01% and ≥ 5.70%, while their ITS2 divergences were ≥ 7.44% and ≥ 16.43%, respectively. Three and five compensatory base changes (CBCs) were observed in the clades IV and V, respectively, when compared to each of their closest clade. Cells from clades IV and V showed similar morphology to C. marina complex and C. subsalsa clade II, including the presence of button-like granules on cell surface and oboe-shaped mucocysts. However, cell size, the number and shape of chloroplasts in Chattonella clades IV and V, and the non-stacked thylakoids penetrated the pyrenoid in C. subsalsa clade II, were distinctive. Based on the diagnostic chloroplast shape, we proposed the designation of clades IV and V to two new species, Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov.

Sexual reproduction and genetic polymorphism within the cosmopolitan marine diatom Pseudo-nitzschia pungens.

Different clades belonging to the cosmopolitan marine diatom Pseudo-nitzschia pungens appear to be present in different oceanic environments, however, a 'hybrid zone', where populations of different clades interbreed, has also been reported. Many studies have investigated the sexual reproduction of P. pungens, focused on morphology and life cycle, rather than the role of sexual reproduction in mixing the genomes of their parents. We carried out crossing experiments to determine the sexual compatibility/incompatibility between different clades of P. pungens, and examined the genetic polymorphism in the ITS2 region. Sexual reproduction did not occur only between clades II and III under any of experimental temperature conditions. Four offspring strains were established between clade I and III successfully. Strains established from offspring were found interbreed with other offspring strains as well as viable with their parental strains. We confirmed the hybrid sequence patterns between clades I and III and found novel sequence types including polymorphic single nucleotide polymorphisms (SNPs) in the offspring strains. Our results implicate that gene exchange and mixing between different clades are still possible, and that sexual reproduction is a significant ecological strategy to maintain the genetic diversity within this diatom species.

Phylogenetic relationships of Pseudo-nitzschia subpacifica (Bacillariophyceae) from the Mexican Pacific, and its production of domoic acid in culture.

Pseudo-nitzschia is a cosmopolitan genus, some species of which can produce domoic acid (DA), a neurotoxin responsible for the Amnesic Shellfish Poisoning (ASP). In this study, we identified P. subpacifica for the first time in Todos Santos Bay and Manzanillo Bay, in the Mexican Pacific using SEM and molecular methods. Isolates from Todos Santos Bay were cultivated under conditions of phosphate sufficiency and deficiency at 16°C and 22°C to evaluate the production of DA. This toxin was detected in the particulate (DAp) and dissolved (DAd) fractions of the cultures during the exponential and stationary phases of growth of the cultures. The highest DA concentration was detected during the exponential phase grown in cells maintained in P-deficient medium at 16°C (1.14 ± 0.08 ng mL-1 DAd and 4.71 ± 1.11 × 10-5 ng cell-1 of DAp). In P-sufficient cultures DA was higher in cells maintained at 16°C (0.25 ± 0.05 ng mL-1 DAd and 9.41 ± 1.23 × 10-7 ng cell-1 of DAp) than in cells cultured at 22°C. Therefore, we confirm that P. subpacifica can produce DA, especially under P-limited conditions that could be associated with extraordinary oceanographic events such as the 2013-2016 "Blob" in the northeastern Pacific Ocean. This event altered local oceanographic conditions and possibly generated the presence of potential harmful species in areas with economic importance on the Mexican Pacific coast.

Growth and epiphytic behavior of three Gambierdiscus species (Dinophyceae) associated with various macroalgal substrates.

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.

Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution.

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.

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity.

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2', and G. belizeanus by the asymmetrical Plate 3'. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell-1, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.

High diversity of Pseudo-nitzschia along the northern coast of Sarawak (Malaysian Borneo), with descriptions of P. bipertita sp. nov. and P. limii sp. nov. (Bacillariophyceae).

Forty-eight isolates of Pseudo-nitzschia species were established from the Miri coast of Sarawak (Malaysian Borneo) and underwent TEM observation and molecular characterization. Ten species were found: P. abrensis, P. batesiana, P. fukuyoi, P. kodamae, P. lundholmiae, P. multistriata, P. pungens, P. subfraudulenta, as well as two additional new morphotypes, herein designated as P. bipertita sp. nov. and P. limii sp. nov. This is the first report of P. abrensis, P. batesiana, P. kodamae, P. fukuyoi, and P. lundholmiae in coastal waters of Malaysian Borneo. Pseudo-nitzschia bipertita differs from its congeners by the number of sectors that divide the poroids, densities of band striae, and its cingular band structure. Pseudo-nitzschia limii, a pseudo-cryptic species in the P. pseudodelicatissima complex sensu lato, is distinct by having wider proximal and distal mantles, a higher number of striae, and greater poroid height in the striae of the valvocopula. The species were further supported by the phylogenetic reconstructions of the nuclear-encoded large subunit ribosomal gene and the second internal transcribed spacer. Phylogenetically, P. bipertita clustered with its sister taxa (P. subpacifica + P. heimii); P. limii appears as a sister taxon to P. kodamae and P. hasleana in the ITS2 tree. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of both hemi- and compensatory base changes. Toxicity analysis showed detectable levels of domoic acid in P. abrensis, P. batesiana, P. lundholmiae, and P. subfraudulenta, but both new species tested below the detection limit.

New scenario for speciation in the benthic dinoflagellate genus Coolia (Dinophyceae).

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.

Diatom Nitzschia navis-varingica (Bacillariophyceae) and its domoic acid production from the mangrove environments of Malaysia.

The distribution of the toxic pennate diatom Nitzschia was investigated at four mangrove areas along the coastal brackish waters of Peninsular Malaysia. Eighty-two strains of N. navis-varingica were isolated and established, and their identity confirmed morphologically and molecularly. Frustule morphological characteristics of the strains examined are identical to previously identified N. navis-varingica, but with a sightly higher density of the number of areolae per 1µm (4-7 areolae). Both LSU and ITS rDNAs phylogenetic trees clustered all strains in the N. navis-varingica clade, with high sequence homogeneity in the LSU rDNA (0-0.3%), while the intraspecific divergences in the ITS2 data set reached up to 7.4%. Domoic acid (DA) and its geometrical isomers, isodomoic A (IA) and isodomoic B (IB), were detected in cultures of N. navis-varingica by FMOC-LC-FLD, and subsequently confirmed by LC-MS/MS, with selected ion monitoring (SIM) and multiple reaction monitoring (MRM) runs. DA contents ranged between 0.37 and 11.06pgcell-1. This study demonstrated that the toxigenic euryhaline diatom N. navis-varingica is widely distributed in Malaysian mangrove swamps, suggesting the risk of amnesic shellfish poisoning and the possibility of DA contamination in the mangrove-related fisheries products.

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus.

The increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins.

A non-toxigenic but morphologically and phylogenetically distinct new species of Pseudo-nitzschia, P. sabit sp. nov. (Bacillariophyceae).

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.

Three novel species in the Pseudo-nitzschia pseudodelicatissima complex: P. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov. (Bacillariophyceae) from the Strait of Malacca, Malaysia.

A study on the morphology and phylogeny of 18 strains of Pseudo-nitzschia established from the Strait of Malacca, Peninsular Malaysia, was undertaken. Morphological data combined with molecular evidence show that they constitute three new species, for which the names, P. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov., are proposed. The three new species closely resemble species in the P. pseudodelicatissima complex sensu lato. Morphologically, P. batesiana differs from other species in the complex by having a smaller part of cell overlapping in the chain, whereas P. lundholmiae differs by having fewer poroid sectors and P. fukuyoi by having a distinct type of poroid sectors. Nucleotide sequences of the LSU rDNA (D1-D3) of the three new species reveal significant nucleotide sequence divergence (0.1%-9.3%) from each other and from other species in the P. pseudodelicatissima complex s.l. The three species are phylogenetically closely related to species in the P. pseudodelicatissima complex, with P. batesiana appearing as a sister taxon to P. circumpora, P. caciantha, and P. subpacifica; whereas P. lundholmiae and P. fukuyoi are more closely related to P. pseudodelicatissima and P. cuspidata. The three species show 2-3 compensatory base changes (CBCs) in their ITS2 transcripts when compared to the closely related species. The ITS2 with its structural information has proven its robustness in constructing a better resolved phylogenetic framework for Pseudo-nitzschia.

MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) FROM MALAYSIAN BORNEO, INCLUDING THE NEW SPECIES PSEUDO-NITZSCHIA CIRCUMPORA SP. NOV.

Field sampling was undertaken to investigate the occurrence of Pseudo-nitzschia Peragallo species in eight locations along the coast of Malaysian Borneo. A total of 108 strains of Pseudo-nitzschia species were isolated, and their morphology examined with SEM and TEM. Additionally, molecular data from nuclear-encoded partial LSU rDNA, and ITS regions, were characterized. A total of five species were confidently identified based on a combination of distinct morphological characteristics and supporting molecular evidence: P. brasiliana Lundholm, Hasle & Fryxell, P. cuspidata (Hasle) Hasle, P. dolorosa Lundholm & Moestrup, P. micropora Priisholm, Moestrup & Lundholm, and P. pungens (Grunow) Hasle var. pungens. However, one morphotype from Sarawak, while somewhat similar to P. caciantha, showed significant morphological distinction from this and any other of the currently described species. Most notably this morphotype possessed a characteristic pore arrangement in the poroids, with the fine pores in each perforation sector arranged in circles. Pair-wise sequence comparison of the LSU rDNA between this unidentified morphotype and P. caciantha Lundholm, Moestrup & Hasle, revealed 2.7% genetic divergence. Phylogenetic analyses strongly supported the monophyly of the morphotype. Based upon these supporting data it is here described as a new species, Pseudo-nitzschia circumpora sp. nov. A key to the six species of Pseudo-nitzschia from Malaysian Borneo is presented. Molecular signatures for all species were established based on structural comparisons of ITS2 rRNA transcripts.