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.
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.
Pseudo-nitzschia nanaoensis sp. nov. is described from waters around Nan'ao Island (South China Sea), using morphological data and molecular evidence. This species is morphologically most similar to P. brasiliana, but differs by a denser arrangement of fibulae, interstriae, and poroids, as well as by the structure of the valvocopula and the narrow second band. Pseudo-nitzschia nanaoensis constitutes a monophyletic lineage and is well differentiated from other species on the LSU and ITS2 sequence-structure trees. Pseudo-nitzschia nanaoensis makes up the basal node on the LSU tree, and forms a sister clade with a group of P. pungens and P. multiseries on the ITS2 tree. The ability of cultured strains to produce domoic acid was assessed, including its possible induction by the presence of a copepod and brine shrimp, by liquid chromatography-tandem mass spectrometry. However, no strains showed detectable domoic acid.
Some diatoms of the genera Pseudo-nitzschia and Nitzschia produce the neurotoxin domoic acid (DA), a compound that caused amnesic shellfish poisoning (ASP) in humans just over 30 years ago (December 1987) in eastern Canada. This review covers new information since two previous reviews in 2012. Nitzschia bizertensis was subsequently discovered to be toxigenic in Tunisian waters. The known distribution of N. navis-varingica has expanded from Vietnam to Malaysia, Indonesia, the Philippines and Australia. Furthermore, 15 new species (and one new variety) of Pseudo-nitzschia have been discovered, bringing the total to 52. Seven new species were found to produce DA, bringing the total of toxigenic species to 26. We list all Pseudo-nitzschia species, their ability to produce DA, and show their global distribution. A consequence of the extended distribution and increased number of toxigenic species worldwide is that DA is now found more pervasively in the food web, contaminating new marine organisms (especially marine mammals), affecting their physiology and disrupting ecosystems. Recent findings highlight how zooplankton grazers can induce DA production in Pseudo-nitzschia and how bacteria interact with Pseudo-nitzschia. Since 2012, new discoveries have been reported on physiological controls of Pseudo-nitzschia growth and DA production, its sexual reproduction, and infection by an oomycete parasitoid. Many advances are the result of applying molecular approaches to discovering new species, and to understanding the population genetic structure of Pseudo-nitzschia and mechanisms used to cope with iron limitation. The availability of genomes from three Pseudo-nitzschia species, coupled with a comparative transcriptomic approach, has allowed advances in our understanding of the sexual reproduction of Pseudo-nitzschia, its signaling pathways, its interactions with bacteria, and genes involved in iron and vitamin B12 and B7 metabolism. Although there have been no new confirmed cases of ASP since 1987 because of monitoring efforts, new blooms have occurred. A massive toxic Pseudo-nitzschia bloom affected the entire west coast of North America during 2015-2016, and was linked to a 'warm blob' of ocean water. Other smaller toxic blooms occurred in the Gulf of Mexico and east coast of North America. Knowledge gaps remain, including how and why DA and its isomers are produced, the world distribution of potentially toxigenic Nitzschia species, the prevalence of DA isomers, and molecular markers to discriminate between toxigenic and non-toxigenic species and to discover sexually reproducing populations in the field.
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.
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.