This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.
Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.
Screening of the occurrence of potentially toxic diatoms was carried out at two sites of cage cultures in Tebrau Straits, Johor. Phytoplankton samples from Sungai Pendas and Teluk Sengat were collected using a 20 μm mesh plankton net and salinity was recorded in-situ. Nitzschia and Pseudo-nitzschia cells were isolated and established into clonal cultures. All cultures were tested for domoic acid using HPLC-UV analysis and verified by LC-MS analysis. Three Nitzschia spp. and one Pseudo-nitzschia sp. were identified from these locations. Toxic and non-toxic strains of Nitzschia navis-varingica are found at the cage culture areas. Cellular toxin content in the toxic strain of N. navis-varingica is 1.8 pg cell(-1). This is a new record from Malaysia and this species was isolated from estuarine water with salinity 28 PSU. The discovery of toxic Nitzschia species in Tebrau Straits indicates the potential for domoic acid accumulation in seafood.
Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions.
Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.
Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.
Coolia is a widespread and ecologically important genus of benthic marine dinoflagellates found in tropical regions. Historically, there has been taxonomic confusion about the taxonomy and toxicity of this group. The goal of this study was to resolve morphological questions concerning Coolia tropicalis and determine the taxonomic identity of the Australian Coolia isolate which has been reported to produce cooliatoxins. To accomplish this, the morphology of tropical strains from Belize (the type locality of C. tropicalis), Malaysia, Indonesia, and Australia were examined and compared to published reports. The morphological analysis showed that C. tropicalis differs from the original description in that it has a slightly larger size (35-47 μm long by 30-45 μm wide versus 23-40 μm long by 25-39 μm wide), and the shape of fourth apical plate, and the length of Po plate (7.4-12 μm versus 7 μm). Based on both morphology and phylogenetic analysis using LSU D1- D3 rDNA sequences, the clones of C. tropicalis from Malaysia, Indonesia, and Belize were found to form a monophyletic clade within the genus. The strain producing cooliatoxin was found to be C. tropicalis, not Coolia monotis as originally assumed. To explore the factors influencing the growth of Coolia species, the growth rates of C. tropicalis and Coolia malayensis were determined at different temperatures and salinities. Both species tolerated a wide range of temperatures, but cannot survive at temperatures <20°C or >35°C. C. monotis, the dominant species reported in the literature, probably does not produce toxins.
Microcystins (MCs) are hepatotoxic cyanotoxins implicated in several incidents of human and animal toxicity. Microcystin-(Lysine, Arginine) or MC-LR is the most toxic and encountered variant of MCs where oxidative stress plays a key role in its toxicity. This study investigated the oxidative damages induced in the liver and heart of Balb/C mice by an intraperitoneal injected acute dose of MC-LR. Thereafter, the potential protective effect of garlic (Allium sativum) extract supplementation against such damages was assessed through the evaluation of oxidative stress and cytotoxicity markers. Lipid peroxidation (LPO), carbonyl content (CC), glutathione content (GSH), alkaline phosphatase activity (ALP), lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH) activities were measured. Results showed important oxidative damages in hepatic and cardiac cells of mice injected with the toxin. However, these damages have been significantly reduced in mice supplemented with garlic extract. Thus, this study demonstrated for the first time the effective use of garlic as an antioxidant agent against oxidative damages induced by MC-LR. As well, this study supports the use of garlic as a potential remedy against pathologies related to toxic agents.
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.
Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.
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.
The genus Amphidinium is an important group of athecated dinoflagellates because of its high abundance in marine habitats, its member's ability to live in a variety of environmental conditions and ability to produce toxins. Furthermore, the genus is of particular interest in the biotechnology field for its potential in the pharmaceutical arena. Taxonomically the there is a history of complication and confusion over the proper identities and placements of Amphidinium species due to high genetic variability coupled with high morphological conservation. Thirteen years has passed since the most recent review of the group, and while many issues were resolved, some remain. The present study used microscopy, phylogenetics of the 28S region of rDNA, secondary structure of the ITS2 region of rDNA, compensatory base change data, and cytotoxicity data from Amphidinium strains collected world-wide to elucidate remaining confusion. This holistic approach using multiple lines of evidence resulted in a more comprehensive understanding of the morphological, ecological, and genetic characteristics that are attributed to organisms belonging to Amphidinium, including six novel species: A. fijiensis, A. magnum, A. paucianulatum, A. pseudomassartii, A. theodori, and A. tomasii.