Cause Ciguatera Fish Poisoning Research Articles

Structural Assignment of the Product Ion Generated from a Natural Ciguatoxin-3C Congener, 51-Hydroxyciguatoxin-3C, and Discovery of Distinguishable Signals in Congeners Bearing the 51-Hydroxy Group.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C55H79O15), 439 (C24H39O7), 149 (C10H13O), 135 (C9H11O), and 115 (C6H11O2). Additionally, characteristic product ions were observed at m/z 509 (C28H45O8), 491 (C28H43O7), 481 (C26H41O8), 463 (C26H39O7), 439 (C24H39O7), 421 (C24H37O6), 171 (C9H15O3), 153 (C9H13O2), 141 (C8H13O2), and 123 (C8H11O).

Survey of epiphytic microalgae to evaluate risk of ciguatera fish poisoning across natural and artificial reefs in North Carolina

Epiphytic microalgae are important contributors to the carbon and nutrient cycles yet are often overlooked during ecological surveys. In reef habitats, epiphytes are often found living on host organisms, including seaweeds or corals, and can influence community composition of higher trophic level taxa. Hence, understanding how epiphytes respond to different reef substrate materials can help inform designs of substrates intentionally deployed to form artificial reefs which can encourage high biodiversity and ecological functioning. One such epiphyte, Gambierdiscus spp., is a harmful benthic dinoflagellate which produces toxins that bioaccumulate and cause ciguatera fish poisoning (CFP) when contaminated fish is consumed by humans. CFP is one of the most frequently reported seafood-associated illnesses around the world, occurring most often near tropical reefs. We surveyed the epiphytes amongst 13 natural and artificial reef sites located off the coast of North Carolina to determine the role of the reefs’ foundational substrate (e.g., natural marl, steel or concrete) on structuring the epiphyte community with an emphasis on Gambierdiscus spp. abundance. No Gambierdiscus spp. were detected among the sampled reefs, likely due to suboptimal water temperatures. An ex-situ substrate preference experiment for Gambierdiscus spp. was performed using marl to represent natural rocky reefs, and steel and concrete to represent artificial reefs. Experimental results indicated that Gambierdiscus spp. grew fastest in the presence of marl and density decreased significantly when exposed to steel. However, steel artificial reefs had the highest average epiphyte biomass and species richness amongst the sampled reefs. 18s rRNA gene sequence analysis revealed that natural reefs were more likely to be dominated by dinoflagellates, whereas steel and concrete reefs were dominated by diatoms. We found that epiphyte composition was related to material at a phylum level, but seaweed hosts played a more significant role at the species level. These findings suggest that CFP risk is relatively low on the reefs studied but natural reefs would likely be preferentially colonized by Gambierdiscus spp. should ambient conditions become appropriate.

Kelimpahan Dinoflagellata Bentik Berbahaya di Habitat Lamun dan Makroalga di Pulau Pari, Kepulauan Seribu, Indonesia

<p class="Papertext"><strong>The Abundance of the Harmful Benthic Dinoflagellate in the Seagrass and Macroalgae Habitats on Pari Island, Thousand Islands, Indonesia. </strong>Benthic dinoflagellates are an important part of benthic ecosystems and part of their ecological functions. However, ciguatoxin (CTX) producer dinoflagellates, which could cause Ciguatera Fish Poisoning (CFP) disease, is a known threat to marine biota and the island’s coastal communities, such as in Pari Island. Thus, this research aimed is to study the population of five harmful and toxin producer benthic dinoflagellate genera, namely <span style="text-decoration: underline;">Amphidinium</span>, <span style="text-decoration: underline;">Coolia</span>, <span style="text-decoration: underline;">Gambierdiscus</span>, <span style="text-decoration: underline;">Ostreopsis</span>, and <span style="text-decoration: underline;">Prorocentrum</span>, in macroalgae and seagrass habitats of Pari Island. Benthic dinoflagellate samples were collected in Juni 2019 using artificial substrates in the form of a 10×15 cm nylon screen. Environmental variables, such as nutrient concentration (nitrogen and phosphorus) and water temperature were also measured. The results showed that the average benthic dinoflagellate density in seagrass habitats was four times higher compared to the macroalgae habitats. <em>Gambierdiscus</em> were found as the most abundant benthic dinoflagellate in seagrass habitats, while <em>Prorocentrum</em> were more abundant in macroalgae habitats. Principal Component Analysis (PCA) showed that the N:P ratio was the most important regulating parameter for most benthic dinoflagellate genera in this study, except <em>Ostreopsis</em>, which was found to be more abundant in phosphorus-rich macroalga habitats. The data also shows an increase of benthic dinoflagellate cell density up to seven times compared to the previous research data in 2012 and 2013. That trend indicates an increase in the disturbance and pressure on the macroalgae and seagrass habitats of Pari Island, which require special attention to prevent the occurrence of toxic Benthic Harmful Algal Blooms (bHABs) and CFP cases in the island.</p>

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 μg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at −20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

Morphological and molecular characterization of Gambierdiscus caribaeus (Dinophyceae), with a confirmation of its occurrence in the Colombian Caribbean Tayrona National Natural Park

Abstract Dinoflagellates in the genus Gambierdiscus are known to produce potent neurotoxins known as ciguatoxins. These toxins vector through the food web and can reach sufficiently high levels to cause ciguatera fish poisoning (CFP). In many regions of the Caribbean, little is known about the occurrence of CFP-causing species or the incidence of CFP, despite its apparent regional increase over the last few decades. Given the intraspecific variability in content and potencies of these toxins among Gambierdiscus species, the precise identification of local species of this genus is crucial. In this study we confirm the presence of the epibenthic dinoflagellate Gambierdiscus caribaeus in the Colombian continental Caribbean. Cells were collected in seagrass beds of Thalassia testudinum in Bonito Gordo, a site located on the western side of Bahía Concha, Tayrona National Natural Park, near the city of Santa Marta, Colombia. Subsequently, a single strain of Gambierdiscus could be established for further identification. Morphological (light and scanning electron microscopy), and molecular results (partial LSU and ITS rDNA sequencing) were consistent with the original description of G. caribaeus, as well as with the morphological characteristics shown by other authors in organisms obtained near the sampling area.

Accumulation of C-CTX1 in Muscle Tissue of Goldfish (Carassius auratus) by Dietary Experience.

Simple SummarySome marine microalgae usually present in warm waters can produce ciguatoxins (CTXs); these toxins can accumulate in fish through the trophic chain, causing the food poisoning known as ciguatera in humans. It is important to understand how these compounds could be incorporated into fish muscle. For this purpose, this study was conducted using goldfish, an omnivorous freshwater species, daily fed raw fish flesh contaminated with a known toxicity concentration of CTX, seeking the accumulation profile in muscle and any signs of intoxication. Toxicity was detectable from day eight of the toxic diet and reached its maximum after two weeks. Signs of poisoning were observed after two weeks in all treated fish. However, two individuals developed strong symptoms, and one of them was separated and fed non-toxic food for 60 days; it showed recovery signs after the first week, and no toxicity was observed at the end of that non-toxic period. These results demonstrate that this toxin can accumulate in the muscle tissue of goldfish and produce associated symptomatology. Moreover, goldfish can recover and eliminate the CTX from its muscle if the toxin source is not available.Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g−1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g−1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.

The dinoflagellate causing ciguatera fish poisoning, Prorocentrum lima, in Karimunjawa island waters - Central Java

Prorocentrum lima is one of the microalgae species in Dinoflagellates group, which could cause Ciguatera Fish Poisoning (CFP) syndrome. P. lima commonly found it attached to various kind of macroalgae species and could cause human poisoning through biomagnification process in the food chain. Research on the abundance of P. lima and the environmental factors influences had been conducted in Karimunjawa Island, on 24 August 2018, and the objective of the research is to analyze the P. lima distributions in Karimunjawa waters, based on the cells abundance and environmental factors. The research was carried out by collecting macroalgae Padina and Sargassum at four stations, in the reef flat of Karimunjawa Island. Macroalgae were put inside plastic bottles and vigorously shaken by vortex (12500 rpm, 1 minute). Water samples separated from the macroalgae, filtered through a series of sieves, and then observed under a light microscope. The environmental factors are analyzed by Principal Component Analysis (PCA). Results showed a high abundance of P. lima found at Station 1 (Tanjung Gelam beach), which are 266 cells/ml on Padina and 210 cells/ml on Sargassum. Based on PCA analysis, Station 1 was characterized by water current, light intensity, phosphate, and depth. Those environmental factors should support the higher abundance of P. lima cells at the station.

Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in a Subtropical Region of North Atlantic Ocean (Canary Islands): Morphological Characterization and Biogeography.

Dinoflagellates belonging to the genus Gambierdiscus produce ciguatoxins (CTXs), which are metabolized in fish to more toxic forms and subsequently cause ciguatera fish poisoning (CFP) in humans. Five species of Gambierdiscus have been described from the Canary Islands, where CTXs in fish have been reported since 2004. Here we present new data on the distribution of Gambierdiscus species in the Canary archipelago and specifically from two islands, La Palma and La Gomera, where the genus had not been previously reported. Gambierdiscus spp. concentrations were low, with maxima of 88 and 29 cells·g−1 wet weight in samples from La Gomera and La Palma, respectively. Molecular analysis (LSUrRNA gene sequences) revealed differences in the species distribution between the two islands: only G. excentricus was detected at La Palma whereas four species, G. australes, G. caribaeus, G. carolinianus, and G. excentricus, were identified from La Gomera. Morphometric analyses of cultured cells of the five Canary Islands species and of field specimens from La Gomera included cell size and a characterization of three thecal arrangement traits: (1) the shape of the 2′ plate, (2) the position of Po in the anterior suture of the 2′ plate, and (3) the length–width relationship of the 2″″ plate. Despite the wide morphological variability within the culture and field samples, the use of two or more variables allowed the discrimination of two species in the La Gomera samples: G. cf. excentricus and G. cf. silvae. A comparison of the molecular data with the morphologically based classification demonstrated important coincidences, such as the dominance of G. excentricus, but also differences in the species composition of Gambierdiscus, as G. caribaeus was detected in the study area only by using molecular methods.

Investigation of ciguatoxins in invasive lionfish from the greater caribbean region: Implications for fishery development.

Lionfish, native to reef ecosystems of the tropical and sub-tropical Indo-Pacific, were introduced to Florida waters in the 1980s, and have spread rapidly throughout the northwestern Atlantic, Caribbean Sea and the Gulf of Mexico. These invasive, carnivorous fish significantly reduce other fish and benthic invertebrate biomass, fish recruitment, and species richness in reef ecosystems. Fisheries resource managers have proposed the establishment of a commercial fishery to reduce lionfish populations and mitigate adverse effects on reef communities. The potential for a commercial fishery for lionfish is the primary reason to identify locations where lionfish accumulate sufficient amounts of ciguatoxin (CTX) to cause ciguatera fish poisoning (CFP), the leading cause of non-bacterial seafood poisoning associated with fish consumption. To address this issue, an initial geographic assessment of CTX toxicity in lionfish from the Caribbean and Gulf of Mexico was conducted. Lionfish samples (n = 293) were collected by spearfishing from 13 locations (74 sampling sites) around the Caribbean and Gulf of Mexico between 2012 and 2015. The highest frequencies of lionfish containing measurable CTX occurred in areas known to be high-risk regions for CFP in the central to eastern Caribbean (e.g., 53% British Virgin Islands and 5% Florida Keys). Though measurable CTX was found in some locations, the majority of the samples (99.3%) contained CTX concentrations below the United States Food and Drug Administration guidance level of 0.1 ppb Caribbean ciguatoxin-1 (C-CTX-1) equivalents (eq.). Only 0.7% of lionfish tested contained more than 0.1 ppb C-CTX-1 eq. As of 2018, there has been one suspected case of CFP from eating lionfish. Given this finding, current risk reduction techniques used to manage CTX accumulating fish are discussed.

Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species have very similar morphological characteristics and are difficult or impossible to distinguish using light microscopy. DNA sequencing has been an important tool in the definition of Gambierdiscus species, but it can be time-consuming and relatively expensive. To provide an alternative approach, a PCR-RFLP protocol was developed for efficient, rapid, and cost-effective identification of Gambierdiscus strains isolated from the Gulf of Mexico and Caribbean Sea, where CFP cases and Gambierdiscus spp. have been reported. The assay targets the D1-D2 hypervariable regions of the large subunit ribosomal RNA gene and uses a single restriction enzyme, BsrI. This method produces distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea, and also distinguishes them from four Pacific endemic species. This method was successfully used to type 465 clonal isolates of Gambierdiscus from the U.S. Virgin Islands and Akumal Beach - Mexico This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.

Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1μM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.

Ciguatoxicity of Gambierdiscus and Fukuyoa species from the Caribbean and Gulf of Mexico.

Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there is 2- to 27-fold variation in toxicity within species, there was a 1740-fold difference between the least and most toxic species. Consequently, production of CTX or CTX-like compounds is more dependent on the species present than on the random occurrence of high or low toxicity strains. Seven of the eight species tested (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, Gambierdiscus ribotype 2, G. silvae and F. ruetzleri) exhibited low toxicities, ranging from 0 to 24.5 fg CTX3C equivalents cell-1, relative to G. excentricus, which had a toxicity of 469 fg CTX3C eq. cell-1. Isolates of G. excentricus from other regions have shown similarly high toxicities. If the hypothesis that G. excentricus is the primary source of ciguatoxins in the Atlantic is confirmed, it should be possible to identify areas where CFP risk is greatest by monitoring only G. excentricus abundance using species-specific molecular assays.

The Epiphytic Genus Gambierdiscus (Dinophyceae) in the Kermadec Islands and Zealandia Regions of the Southwestern Pacific and the Associated Risk of Ciguatera Fish Poisoning.

Species in the genus Gambierdiscus produce ciguatoxins (CTXs) and/or maitotoxins (MTXs), which may cause ciguatera fish poisoning (CFP) in humans if contaminated fish are consumed. Species of Gambierdiscus have previously been isolated from macroalgae at Rangitahua (Raoul Island and North Meyer Islands, northern Kermadec Islands), and the opportunity was taken to sample for Gambierdiscus at the more southerly Macauley Island during an expedition in 2016. Gambierdiscus cells were isolated, cultured, and DNA extracted and sequenced to determine the species present. Bulk cultures were tested for CTXs and MTXs by liquid chromatography-mass spectrometry (LC-MS/MS). The species isolated were G. australes, which produced MTX-1 (ranging from 3 to 36 pg/cell), and G. polynesiensis, which produced neither MTX-1 nor, unusually, any known CTXs. Isolates of both species produced putative MTX-3. The risk of fish, particularly herbivorous fish, causing CFP in the Zealandia and Kermadec Islands region is real, although in mainland New Zealand the risk is currently low. Both Gambierdiscus and Fukuyoa have been recorded in the sub-tropical northern region of New Zealand, and so the risk may increase with warming seas and shift in the distribution of Gambierdiscus species.

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales, Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia).

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food-borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8-D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography-Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G.lapillus sp. nov. (plate formula Po, 3', 0a, 7″, 6c, 7-8s, 5‴, 0p, 2″″ and distinctive by size and hatchet-shaped 2' plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal-derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.

Temperature and salinity effects and toxicity of Gambierdiscus caribaeus (Dinophyceae) from Thailand

:Species of the benthic dinoflagellate genus Gambierdiscus are the putative producer of the toxins causing ciguatera fish poisoning (CFP) in tropical and subtropical areas. Although CFP events and distribution of G. caribaeus have been reported in Thailand, little is known about the conditions that enhance the growth of Gambierdiscus in Thailand. Hence, this study was aimed at determining the effects of temperature and salinity and their interactions on the growth of Thai G. caribaeus. The strain tested showed the highest cell yield in IMK/2 medium. Based on growth rate (μ), Thai G. caribaeus grew in the temperature range of 20°C–35°C and the salinity range of 20–40. The suboptimal condition (≥ 80% of the maximal growth rate) for the strain tested was 20.5°C–30.5°C and salinity of 25.0–37.5. The optimal condition was 25°C and salinity of 30. Furthermore, the Thai G. caribaeus revealed high toxicity to mice. Our results suggest that tolerance of up to 35°C may allow these organisms to be distributed in tropical areas, including Thailand, where the water temperature often exceeds 30°C.

Characterization of Gambierdiscus and Coolia (Dinophyceae) isolates from Thailand based on morphology and phylogeny

SummaryThe benthic dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub‐tropical fish causing ciguatera fish poisoning (CFP). Other co‐occurring genera such as Coolia have also been implicated in causing CFP. Little is known about the diversity of the two genera Gambierdiscus and Coolia along the Thai coasts. The results of morphological analyses based on observation under light microscopy and scanning electron microcopy showed that strains of Gambierdiscus from Thailand displayed the typical Gambierdiscus plate formula: Po, 4′, 0a, 6″, 6c,?s, 5′′′, 0p and 2′′′′. Morphological examination of Thai Gambierdiscus enabled it to be identified as Gambierdiscus caribaeus: round and anterior‐posteriorly compressed cell shape, broad 2′′′′ plate, rectangular 2′ plate, and symmetrical 3″ plate. The phylogenetic analyses based on the large subunit (LSU) rDNA D8/D10 sequences of Gambierdiscus from Thailand confirmed the morphological identification. The thecal plate formula for all of the Coolia isolates from Thailand was Po, 4′, 0a, 6″,?c,?s, 5′′′, 0p and 2′′′′. Most, but not all, of these isolates could be identified morphologically as Coolia malayensis. An LSU rDNA D1/D2 phylogenetic analysis confirmed identity of C. malayensis isolates identified morphologically. The remaining unidentified isolates fell in the C. tropicalis clade.

High abundance of the potentially maitotoxic dinoflagellate Gambierdiscus carpenteri in temperate waters of New South Wales, Australia

Species of the genus Gambierdiscus are epiphytic dinoflagellates well known from tropical coral reef areas at water temperatures from 24 to 29°C. Gambierdiscus spp. are able to produce ciguatoxins (CTXs) known to bioaccumulate in fish, and the ingestion of tropical fish that accumulated CTXs and possibly also maitotoxins (MTXs) can cause ciguatera fish poisoning (CFP) in humans. In Australia, ciguatera poisonings have been reported in tropical parts of Queensland and the Northern Territory. Here, we report for the first time the seasonal abundance (April–May 2012/13) of Gambierdiscus spp. (up to 6565–8255cellsg−1 wet weight algae) from Merimbula and Wagonga Inlets in temperate southern New South Wales, Australia (37°S) at water temperatures of 16.5–17°C. These are popular shellfish aquaculture and recreational fisheries areas with no reports of ciguatera poisoning. Sequencing of a region of the 28S rRNA gene led to the conclusive identification of Gambierdiscus carpenteri. The cells differed however from the Belize type description, including the absence of a thecal groove, dorsal rostrum and variable hatchet- to rectangular-shaped 2′ plate, and were morphologically more similar to Gambierdiscus toxicus. To study the dinoflagellate community structure in detail, a pyrosequencing approach based on the 18S rRNA gene was applied, which confirmed the presence of a single Gambierdiscus species only. Neither CTXs nor MTXs were detected in natural bloom material by LC–MS/MS; however, the extracts were found to be toxic via mouse-bioassay, with symptoms suggestive of poisoning by MTX-like compounds. Understanding the abundance of Gambierdiscus populations in areas with no apparent human health impacts is important towards defining the alternate conditions where sparse populations can create ciguatera problems.

Genetic Diversity and Distribution of the Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in Coastal Areas of Japan

BackgroundThe marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.Methods/Principal FindingA total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.Conclusions/SignificanceThis study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.

THE DISTRIBUTION OF TOXIC DINOFLAGELLATES ON SEA GRASS Enhalus acoroides AT PARI ISLAND, SERIBU ISLANDS

Benthic dinoflagellates causing Ciguatera Fish Poisoning (CFP), could be found attached either on macroalgae or sea grasses. Research on density and distribution of benthic dinoflagellates on sea grass leaves was conducted in Pari Island waters, Seribu Islands, in April 2012. Research was carried out by collecting Enhalus acoroides leaves from each site, and put inside the plastic jars containing seawater. In order to separate the dinoflagellates species from the leaves, the plastic jars were shaken vigorously and the seawater filtered through a series of sieves (125μm and 20μm). The resulted residue was than observed using Sedgewick rafter cell under light microscope. Based on samples collected, eight benthic dinoflagellates were found, where five of them were potentially toxic. They were Prorocentrum concavum, P. lima, P. rhathymum, Ostreopsis lenticularis, and O. siamensis. The highest abundance was found in the reef flat on the southern side of the island (652 sel/cm2 seagrass leaf). Research showed that the density and distribution of toxic dinoflagellates on sea grass at Pari Island, Seribu Islands were more influenced by local water currents.Keywords: benthic dinoflagellates, Ciguatera Fish Poisoning, Pari Island, seagrass

THE DISTRIBUTION OF TOXIC DINOFLAGELLATES ON SEA GRASS Enhalus acoroides AT PARI ISLAND, SERIBU ISLANDS

<p>Benthic dinoflagellates causing Ciguatera Fish Poisoning (CFP), could be found attached either on macroalgae or sea grasses. Research on density and distribution of benthic dinoflagellates on sea grass leaves was conducted in Pari Island waters, Seribu Islands, in April 2012. Research was carried out by collecting Enhalus acoroides leaves from each site, and put inside the plastic jars containing seawater. In order to separate the dinoflagellates species from the leaves, the plastic jars were shaken vigorously and the seawater filtered through a series of sieves (125μm and 20μm). The resulted residue was than observed using Sedgewick rafter cell under light microscope. Based on samples collected, eight benthic dinoflagellates were found, where five of them were potentially toxic. They were Prorocentrum concavum, P. lima, P. rhathymum, Ostreopsis lenticularis, and O. siamensis. The highest abundance was found in the reef flat on the southern side of the island (652 sel/cm2 seagrass leaf). Research showed that the density and distribution of toxic dinoflagellates on sea grass at Pari Island, Seribu Islands were more influenced by local water currents.</p><p>Keywords: benthic dinoflagellates, Ciguatera Fish Poisoning, Pari Island, seagrass</p>