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.
Bacillus sp. strain UMTAT18 was isolated from the harmful dinoflagellate Alexandrium tamiyavanichii Its genome consists of 5,479,367 bp with 5,546 open reading frames, 102 tRNAs, and 29 rRNAs. Gene clusters for biosynthesis of nonribosomal peptides, bacteriocin, and lantipeptide were identified. It also contains siderophore and genes related to stress tolerance.
The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3- or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.
Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene-environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.
In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments.
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.
The toxin-producing dinoflagellate Alexandrium minutum is responsible for the outbreaks of harmful algae bloom (HABs). It is a widely distributed species and is responsible for producing paralytic shellfish poisoning toxins. However, the information associated with the environmental adaptation pathway and toxin biosynthesis in this species is still lacking. Therefore, this study focuses on the functional characterization of A. minutum unigenes obtained from transcriptome sequencing using the Illumina Hiseq 4000 sequencing platform. A total of 58,802 (47.05%) unigenes were successfully annotated using public databases such as NCBI-Nr, UniprotKB, EggNOG, KEGG, InterPRO and Gene Ontology (GO). This study has successfully identified key features that enable A. minutum to adapt to the marine environment, including several carbon metabolic pathways, assimilation of various sources of nitrogen and phosphorus. A. minutum was found to encode homologues for several proteins involved in saxitoxin biosynthesis, including the first three proteins in the pathway of saxitoxin biosynthesis, namely sxtA, sxtG and sxtB. The comprehensive transcriptome analysis presented in this study represents a valuable resource for understanding the dinoflagellates molecular metabolic model regarding nutrient acquisition and biosynthesis of saxitoxin.
This study investigates bacterial diversity and potential pathogens in the international ships' ballast water at Tanjung Pelepas Port, Malaysia, using 16S rRNA amplicon sequencing. Thirty-four bacterial phylum, 305 families, 577 genera, and 941 species were detected in eight ballast water samples of different origins. The similarity of the bacterial composition between samples was found to be random and not tied to geographical locations. The bacterial abundance did not seem to be affected by related physicochemical except for temperature. Ballast water samples with a temperature lower than 25 °C showed a relatively lower bacterial abundance. A total of 33 potential pathogens were detected from all ballast water samples. Pseudomonas spp., Tenacibaculum spp., Flavobacteriaceae spp., Halomonas spp., and Acinetobacter junii are the potential pathogens with more than 10% OTU prevalence. This study would provide beneficial information for further enhancing ballast water microorganism guidelines in Malaysia.
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.
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.