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.
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.
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.
Nitratireductor basaltis strain UMTGB225 is a Gram-negative bacterium isolated from a marine tunicate found in Bidong Island, Terengganu, Malaysia. In this study, the genome of Nitratireductor basaltis UMTGB225 was sequenced to gain insight into the role of this bacterium and its association with tunicate hosts in a coral reef habitat.
Vibrio campbellii strain UMTGB204 was isolated from a green barrel tunicate. The genome of this strain comprises 5,652,224 bp with 5,014 open reading frames, 9 rRNAs, and 116 tRNAs. It contains genes related to virulence and environmental tolerance. Gene clusters for the biosynthesis of nonribosomal peptides and bacteriocin were also identified.
This study was carried out to characterize the detection and quantitation of several paralytic shellfish poisoning (PSP) toxin congeners using a receptor binding assay (RBA). This involved competitive binding of the toxin congeners against tritium-labeled STX for receptor sites on rat brain sodium channels. Competitive binding curves were described by a four-parameter logistic equation. Half-saturation values (EC(50)) ranged from 4.38 nM for STX to 142 nM for GTX5. Receptor binding affinity was in the order STX>GTX1/4>neoSTX>GTX2/3>dcSTX>GTX5, and this was similar to the order of mouse toxicity of these congeners. Predicted toxin concentrations from observed STXeq values and EC(50) ratios relative to STX were within 20% or better of the actual concentrations used in the assay. In contrast predicted toxin concentrations using mouse toxicity ratios relative to STX did not provide a good match to actual concentrations, except for GTX1/4. This study has shown that the rat brain sodium channel RBA will provide a reliable integration of total toxicity of various PSP toxin congeners present in a sample.
Mameliella alba strain UMTAT08 was isolated from clonal culture of paralytic shellfish toxin producing dinoflagellate, Alexandrium tamiyavanichii. Genome of the strain UMTAT08 was sequenced in order to gain insights into the dinoflagellate-bacteria interactions. The draft genome sequence of strain UMTAT08 contains 5.84Mbp with an estimated G + C content of 65%, 5717 open reading frames, 5 rRNAs and 49 tRNAs. It contains genes related to nutrients uptake, quorum sensing and environmental tolerance related genes. Gene clusters for the biosynthesis of type 1 polyketide synthase, bacteriocin, microcin, terpene and ectoine were also identified. This is suggesting that the bacterium possesses diverse adaptation strategy to survive within the dinoflagellate phycosphere. The draft genome sequence and annotation have been deposited at DDBJ/EMBL/GenBank under the accession number JSUQ00000000.
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.
In tropical regions, different species of fiddler crabs coexist on the mangrove floor, which sometimes makes it difficult to define species-specific habitat by visual inspection. The aim of this study is to find key environmental parameters which affect the distribution of fiddler crabs and to determine the habitats in which each species was most abundant. Crabs were collected from 19 sites within the mudflats of Sepang-Lukut mangrove forest. Temperature, porewater salinity, organic matter, water content, carbon and nitrogen content, porosity, chlorophyll content, pH, redox potential, sediment texture and heavy metals were determined in each 1 m2 quadrate. Pearson correlation indicated that all sediment properties except pH and redox potential were correlated with sediment grain size. Canonical correspondence analysis (CCA) indicated that Uca paradussumieri was negatively correlated with salinity and redox potential. Sand dwelling species, Uca perplexa and Uca annulipes, were highly dependent on the abundance of 250 μm and 150 μm grain size particles in the sediment. Canonical Discriminative Analysis (CDA) indicated that variation in sediment grain size best explained where each crab species was most abundant. Moreover, U. paradussumieri commonly occupies muddy substrates of low shore, while U. forcipata lives under the shade of mangrove trees. U. annulipes and U. perplexa with the high number of spoon tipped setae on their second maxiliped are specialized to feed on the sandy sediments. U. rosea and U. triangularis are more common on muddy sediment with high sediment density. In conclusion, sediment grain size that influences most sediment properties acts as a main factor responsible for sediment heterogeneity. In this paper, the correlation between fiddler crab species and environmental parameters, as well as the interaction between sediment characteristics, was explained in order to define the important environmental factors in fiddler crab distributions.
Median enterococci counts of beach water samples gradually increased at statistically significant levels (χ2: 26.53, df: 4; p<0.0001) with increasing proximity to river influx. The difference in proportion of antibiotic resistant enterococci in beach water and river water samples was statistically significant (p<0.05) for the tested antibiotics with river isolates generally presenting higher resistance frequencies. Virulence genes cyl, esp, gelE and asa were detected at varying frequencies (7.32%, 21.95%, 100% and 63.41% respectively) among river isolates. On the other hand, the prevalence of these genes was lower (0%, 20%, 67.27% and 41.82% respectively) among beach water isolates. Multi-Locus-Sequence-Typing analysis of Enterococcus faecalis presented four sequence types (ST) one of which shared six out of seven tested loci with ST6, a member of the clonal complex of multi-drug resistant strains associated with hospital outbreaks.
This study was undertaken to investigate the influence of culture conditions and medium components on production of antibacterial compounds by Serratia sp. WPRA3 (JX020764) which was isolated from marine water of Port Dickson, Malaysia. Biochemical, morphological, and molecular characteristics suggested that the isolate is a new candidate of the Serratia sp. The isolate showed strong antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria. This bacterium exhibited optimum antibacterial compounds production at 28°C, pH 7 and 200 rev/min aeration during 72 h of incubation period. Highest antibacterial activity was obtained when sodium chloride (2%), yeast extract (0.5%), and glucose concentration (0.75%) were used as salt, nitrogen, and carbon sources respectively. Different active fractions were obtained by Thin-Layer Chromatography (TLC) and Flash Column Chromatography (FCC) from ethyl acetate crude extracts namely OCE and RCE in different culture conditions, OCE (pH 5, 200 rev/min) and RCE (pH 7/without aeration). In conclusion, the results suggested different culture conditions have a significant impact on the types of secondary metabolites produced by the bacterium.
Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO4 and MnSO4 were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30-70 °C. Bacteriocin activity also was not affected within a wide pH range of 3-9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.
We report the first study on the occurrence of antibiotic-resistant enterococci in coastal bathing waters in Malaysia. One hundred and sixty-five enterococci isolates recovered from two popular recreational beaches in Malaysia were speciated and screened for antibiotic resistance to a total of eight antibiotics. Prevalence of Enterococcus faecalis and Enterococcus faecium was highest in both beaches. E. faecalis/E. faecium ratio was 0.384:1 and 0.375:1, respectively, for isolates from Port Dickson (PD) and Bagan Lalang (BL). Analysis of Fisher's exact test showed that association of prevalence of E. faecalis and E. faecium with considered locations was not statistically significant (p < 0.05). Chi-square test revealed significant differences (χ(2) = 82.630, df = 20, p < 0.001) in the frequency of occurrence of enterococci isolates from the considered sites. Resistance was highest to nalidixic acid (94.84 %) and least for chloramphenicol (8.38 %). One-way ANOVA using Tukey-Kramer multiple comparison test showed that resistance to ampicillin was higher in PD beach isolates than BL isolates and the difference was extremely statistically significant (p < 0.0001). Frequency of occurrence of multiple antibiotic resistance (MAR) isolates were higher for PD beach water (64.29 %) as compared to BL beach water (13.51 %), while MAR indices ranged between 0.198 and 0.48. The results suggest that samples from Port Dickson may contain MAR bacteria and that this could be due to high-risk faecal contamination from sewage discharge pipes that drain into the sea water.
We report the first study on the occurrence of high-level aminoglycoside-resistant (HLAR) Enterococci in coastal bathing waters and beach sand in Malaysia. None of the encountered isolates were resistant to high levels of gentamicin (500 μg/mL). However, high-level resistance to kanamycin (2,000 μg/mL) was observed in 14.2 % of tested isolates, the highest proportions observed being among beach sand isolates. High-level resistance to kanamycin was higher among Enterococcus faecalis and Enterococcus faecium than Enterococcus spp. Chi-square analysis showed no significant association between responses to tested antibiotics and the species allocation or source of isolation of all tested Enterococci. The species classification of encountered Enterococci resistance to vancomycin was highest among Enterococcus spp. (5.89 %) followed by E. faecium (4.785) and least among E. faecalis. A total of 160 isolates were also tested for virulence characteristics. On the whole, caseinase production was profoundly highest (15.01 %) while the least prevalent virulence characteristic observed among tested beach Enterococci was haemolysis of rabbit blood (3.65 %). A strong association was observed between the source of isolation and responses for each of caseinase (C = 0.47, V = 0.53) and slime (C = 0.50, V = 0.58) assays. Analysis of obtained spearman's coefficient showed a strong correlation between caseinase and each of the slime production (p = 0.04), gelatinase (p = 0.0035) and haemolytic activity on horse blood (p = 0.004), respectively. Suggestively, these are the main virulent characteristics of the studied beach Enterococci. Our findings suggest that recreational beaches may contribute to the dissemination of Enterococci with HLAR and virulence characteristics.