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Shauna A Murray - One of the best experts on this subject based on the ideXlab platform.
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development of a quantitative pcr assay for the detection and enumeration of a potentially ciguatoxin producing dinoflagellate Gambierdiscus lapillus gonyaulacales dinophyceae
PLOS ONE, 2019Co-Authors: Anna Liza Kretzschmar, Arjun Verma, Gurjeet S Kohli, Shauna A MurrayAbstract:Ciguatera fish poisoning (CFP) is an illness contracted through the ingestion of seafood containing ciguatoxins. It is prevalent in tropical regions worldwide, including in Australia. Ciguatoxins are produced by some species of Gambierdiscus. Therefore, screening of Gambierdiscus species identification through quantitative PCR (qPCR), along with the determination of species toxicity, can be useful in monitoring potential ciguatera risk in these regions. In Australia, CFP is prevalent in tropical Queensland and increasingly in sub-tropical regions of Australia, but has a report rate of approximately 10%. Yet the identity, distribution and abundance of ciguatoxin producing Gambierdiscus spp. is largely unknown. In this study, we developed a rapid qPCR assay to quantify the presence and abundance of Gambierdiscus lapillus, a likely ciguatoxic species first described from Australia. We assessed the specificity and efficiency of the qPCR assay. The assay was tested on 25 environmental samples from the Heron Island reef in the southern Great Barrier Reef, a ciguatera endemic region, to determine the presence and patchiness of this species across samples from Chnoospora sp., Padina sp. and Sargassum sp. macroalgal hosts.
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Characterisation of Two Toxic Gambierdiscus spp. (Gonyaulacales, Dinophyceae) from the Great Barrier Reef (Australia): G. lewisii sp. nov. and G. holmesii sp. nov.
Protist, 2019Co-Authors: Anna Liza Kretzschmar, Michaela E. Larsson, Mona Hoppenrath, Martina A. Doblin, Shauna A MurrayAbstract:Ciguatera fish poisoning (CFP) is a human illness caused via consumption of seafood contaminated with neurotoxins produced by some species from the epiphytic dinoflagellate genus Gambierdiscus. In this study, we describe two new species of Gambierdiscus isolated from Heron Island in the Southern Great Barrier Reef, Queensland, Australia. These new species were analysed using light microscopy, scanning electron microscopy, and phylogenetic analyses of nuclear encoded ribosomal ITS, SSU as well as D1-D3 and D8-D10 of the LSU gene regions. Gambierdiscus lewisii sp. nov. (Po, 3′, 0a, 7″, 6c,? s, 5‴, 0p, 2′‴) is distinguished by its strong reticulate-foveate ornamentation and is genetically distinct from its sister species, G. pacificus. Gambierdiscus holmesii sp. nov. (Po, 3′, 0a, 7″, 6c, 6s?, 5‴, 0p, 2′‴) is morphologically distinct from the genetically similar species G. silvae because of a strongly ventrally displaced apical pore complex and a characteristic fold at the anterior edge of the sulcus. Both G. lewisii and G. holmesii produce putative Maitotoxin-(44-Methylgambierone) and compounds which show ciguatoxin and maitotoxin-like activities. Identification of two new Gambierdiscus species will enable us to more accurately assess the risk of CFP in Australia and internationally.
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Morphological and molecular phylogenetic identification and record verification of Gambierdiscus excentricus (Dinophyceae) from Madeira Island (NE Atlantic Ocean)
Marine Biodiversity Records, 2019Co-Authors: Mona Hoppenrath, A. Liza Kretzschmar, Manfred J. Kaufmann, Shauna A MurrayAbstract:The marine benthic dinoflagellate genus Gambierdiscus currently contains ~ 16 species that can be highly morphologically similar to one another, and therefore molecular genetic characterization is necessary to complement the morphological species determination. Gambierdiscus species can produce ciguatoxins, which can accumulate through the food chain and cause ciguatera fish poisoning. Recent studies have suggested that Gambierdiscus excentricus may be one of the main species responsible for ciguatoxin production in the temperate and tropical regions of the eastern Atlantic. The present study definitively identifies the species, G. excentricus , from Madeira Island, Northeast-Atlantic Ocean (32° 38′ N 16° 56′ W) by examining the morphology of a strain using light and scanning electron microscopy and sequencing regions of the ribosomal DNA (D8-D10 LSU, SSU rDNA). Variability in the shape of the apical pore and the microarchitecture of the apical pore plate were documented for the first time, as well as variability in the width of the second antapical plate. The first SSU rDNA sequence for the species is reported. Because G. excentricus is known to produce high levels of CTX-like compounds, its presence and toxicity should be regularly monitored to establish whether it is the primary cause of the ciguatera poisoning events on Madeira Island.
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development of a quantitative pcr assay for the detection and enumeration of a potentially ciguatoxin producing dinoflagellate Gambierdiscus lapillus gonyaulacales dinophyceae
bioRxiv, 2019Co-Authors: Anna Liza Kretzschmar, Arjun Verma, Gurjeet S Kohli, Shauna A MurrayAbstract:Abstract Ciguatera fish poisoning is an illness contracted through the ingestion of seafood containing ciguatoxins. It is prevalent in tropical regions worldwide, including in Australia. Ciguatoxins are produced by some species of Gambierdiscus. Therefore, screening of Gambierdiscus species identification through quantitative PCR (qPCR), along with the determination of species toxicity, can be useful in monitoring potential ciguatera risk in these regions. In Australia, the identity, distribution and abundance of ciguatoxin producing Gambierdiscus spp. is largely unknown. In this study we developed a rapid qPCR assay to quantify the presence and abundance of Gambierdiscus lapillus, a likely ciguatoxic species. We assessed the specificity and efficiency of the qPCR assay. The assay was tested on 25 environmental samples from the Heron Island reef in the southern Great Barrier Reef, a ciguatera endemic region, in triplicate to determine the presence and patchiness of these species across samples from Chnoospora sp., Padina sp. and Sargassum macroalgal hosts. Author’s summary Ciguatera fish poisoning is a human disease contracted by ingesting seafood contaminated with a group of neurotoxins. The group of neurotoxins, named ciguatoxins, are synthesised by species of single celled marine algae from the genus Gambierdiscus. Ciguatera fish poisoning occurs worldwide, particularly in tropical nations. Pacific Island nations are disproportionately impacted, and this impact is predicted to increase as the effects of climate change unfold. Few effective monitoring and mitigation strategies exist for ciguatera fish poisoning, and reporting rates of the disease are estimated to be approximately 20% at best. A global ciguatera strategy was developed by a group of researchers coordinated by UNESCO’s Intergovernmental Oceanographic Commission to characterise the cause and mode of action of ciguatera fish poisoning, as a matter of urgency. In this study, we designed a qPCR assay to detect a species of microalgae, Gambierdiscus lapillus. Gambierdiscus lapillus produces compounds with ciguatoxin-like properties, which may lead to ciguatoxin uptake in fish in the Australian region. This assay was sensitive and able to detect the presence of Gambierdiscus lapillus in a range of environmental samples from the Great Barrier Reef region, Australia.
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toxicology of Gambierdiscus spp dinophyceae from tropical and temperate australian waters
Marine Drugs, 2018Co-Authors: Michaela E. Larsson, Richard J. Lewis, Shauna A Murray, Tim D Harwood, Olivier Laczka, S W A Himaya, Martina A. DoblinAbstract:Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.
Santiago Fraga - One of the best experts on this subject based on the ideXlab platform.
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Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in a Subtropical Region of North Atlantic Ocean (Canary Islands): Morphological Characterization and Biogeography.
Toxins, 2019Co-Authors: Isabel Bravo, Francisco Rodríguez, Isabel Ramilo, Pilar Rial, Santiago FragaAbstract: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.
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Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.
Harmful algae, 2019Co-Authors: Lisbet Díaz-asencio, R. Wayne Litaker, Mark W. Vandersea, Santiago Fraga, Nicolas Chomérat, Rachel J. Clausing, Donaida Chamero-lago, Miguel Gómez-batista, Angel R. Moreira-gonzález, Patricia A. TesterAbstract:Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.
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role of modular polyketide synthases in the production of polyether ladder compounds in ciguatoxin producing Gambierdiscus polynesiensis and g excentricus dinophyceae
Journal of Eukaryotic Microbiology, 2017Co-Authors: Santiago Fraga, Lesley Rhodes, Kirsty F Smith, Gurjeet S Kohli, Katrina Campbell, Uwe John, Shauna A MurrayAbstract:Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates.
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“Canary Islands (NE Atlantic) as a biodiversity ‘hotspot’ of Gambierdiscus : Implications for future trends of ciguatera in the area”
Harmful algae, 2017Co-Authors: Francisco Rodríguez, Santiago Fraga, Isabel Ramilo, Pilar Rial, Rosa Isabel Figueroa, Pilar Riobó, Isabel BravoAbstract:Abstract In the present study the geographical distribution, abundance and composition of Gambierdiscus was described over a 600 km longitudinal scale in the Canary Islands. Samples for cell counts, isolation and identification of Gambierdiscus were obtained from five islands (El Hierro, Tenerife, Gran Canaria, Fuerteventura and Lanzarote). Average densities of Gambierdiscus spp. between 0 and 2200 cells g −1 blot dry weight of macrophyte were recorded. Morphological (light microscopy and SEM techniques) and molecular analyses (LSU and SSU rDNA sequencing of cultures and single cells from the field) of Gambierdiscus was performed. Five Gambierdiscus species ( G. australes, G. caribaeus, G. carolinianus, G. excentricus and G. silvae ), together with a new putative species ( Gambierdiscus ribotype 3) were identified. These results suggest that some cases of CFP in the region could be associated with the accumulation of ciguatoxins in the marine food web acquired from local populations of Gambierdiscus . This unexpected high diversity of Gambierdiscus species in an area which a priori is not under risk of ciguatera, hints at an ancient settlement of Gambierdiscus populations, likely favored by warmer climate conditions in the Miocene Epoch (when oldest current Canary Islands were created), in contrast with cooler present ones. Currently, warming trends associated with climate change could contribute to extend favorable environmental conditions in the area for Gambierdiscus growth especially during winter months.
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Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin‐Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae)
The Journal of eukaryotic microbiology, 2017Co-Authors: Gurjeet S Kohli, Santiago Fraga, Lesley Rhodes, Kirsty F Smith, Katrina Campbell, Uwe John, Shauna A MurrayAbstract:Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates.
Patricia A. Tester - One of the best experts on this subject based on the ideXlab platform.
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Species-specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae).
Journal of phycology, 2019Co-Authors: R. Wayne Litaker, Patricia A. Tester, Mark W. VanderseaAbstract:The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species-specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species-specific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species-specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes.
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Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.
Harmful algae, 2019Co-Authors: Lisbet Díaz-asencio, R. Wayne Litaker, Mark W. Vandersea, Santiago Fraga, Nicolas Chomérat, Rachel J. Clausing, Donaida Chamero-lago, Miguel Gómez-batista, Angel R. Moreira-gonzález, Patricia A. TesterAbstract:Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.
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Ciguatoxicity of Gambierdiscus and Fukuyoa species from the Caribbean and Gulf of Mexico.
PloS one, 2017Co-Authors: R. Wayne Litaker, William C Holland, Steven R. Kibler, Francesco Pisapia, D. Ransom Hardison, Philipp Hess, Patricia A. TesterAbstract: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.
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Gambierdiscus and Fukuyoa species in the greater Caribbean: Regional growth projections for ciguatera-associated dinoflagellates
Ecological Modelling, 2017Co-Authors: Steven R. Kibler, William C Holland, Patricia A. Tester, D. Ransom Hardison, Eric D. Davenport, R. Wayne LitakerAbstract:Abstract Satellite-derived sea surface temperature data from the MODIS Aqua instrument were used to project rates of growth for four Gambierdiscus and one Fukuyoa species, dinoflagellates associated with ciguatera fish poisoning. Dinoflagellate growth rates were modelled using experimental temperature vs. growth equations and projected bottom temperatures with light penetration and bathymetry masks. Daily projected growth rates for each species were used to calculate monthly, yearly and multiyear averages between 2003 and 2013. The resulting projections were then used to characterize patterns of regional Gambierdiscus/Fukuyoa abundance and compare these to ciguatera fish poisoning incidences in the Greater Caribbean Region. Model output indicated the highest growth potential was in the shelf waters of the Caribbean Sea, with moderate growth in the Bahamas, southern Florida and Gulf of Mexico; the lowest growth potential was in the northern Gulf of Mexico and along the U.S. south Atlantic coast. Mean projected growth rates generally coincided with distribution of ciguatera fish poisoning incidences in the region, with some exceptions in the southwestern Caribbean. The results of this study indicate spatial differences in Gambierdiscus and Fukuyoa growth likely play a prominent role in governing the occurrence of ciguatera fish poisoning in the greater Caribbean. Growth and distribution models may be an effective tool for ciguatera risk assessment.
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rapid extraction and identification of maitotoxin and ciguatoxin like toxins from caribbean and pacific Gambierdiscus using a new functional bioassay
PLOS ONE, 2016Co-Authors: Richard J. Lewis, Marco Inserra, Irina Vetter, William C Holland, Ransom D Hardison, Patricia A. Tester, Wayne R LitakerAbstract:Background Ciguatera is a circumtropical disease produced by polyether sodium channel toxins (ciguatoxins) that enter the marine food chain and accumulate in otherwise edible fish. Ciguatoxins, as well as potent water-soluble polyethers known as maitotoxins, are produced by certain dinoflagellate species in the genus Gambierdiscus and Fukuyoa spp. in the Pacific but little is known of the potential of related Caribbean species to produce these toxins. Methods We established a simplified procedure for extracting polyether toxins from Gambierdiscus and Fukuyoa spp. based on the ciguatoxin rapid extraction method (CREM). Fractionated extracts from identified Pacific and Caribbean isolates were analysed using a functional bioassay that recorded intracellular calcium changes (Ca2+) in response to sample addition in SH-SY5Y cells. Maitotoxin directly elevated Ca2+i, while low levels of ciguatoxin-like toxins were detected using veratridine to enhance responses. Results We identified significant maitotoxin production in 11 of 12 isolates analysed, with 6 of 12 producing at least two forms of maitotoxin. In contrast, only 2 Caribbean isolates produced detectable levels of ciguatoxin-like activity despite a detection limit of >30 pM. Significant strain-dependent differences in the levels and types of ciguatoxins and maitotoxins produced by the same Gambierdiscus spp. were also identified. Conclusions The ability to rapidly identify polyether toxins produced by Gambierdiscus spp. in culture has the potential to distinguish ciguatoxin-producing species prior to large-scale culture and in naturally occurring blooms of Gambierdiscus and Fukuyoa spp. Our results have implications for the evaluation of ciguatera risk associated with Gambierdiscus and related species.
Mireille Chinain - One of the best experts on this subject based on the ideXlab platform.
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Application of solid phase adsorption toxin tracking (SPATT) devices for the field detection of Gambierdiscus toxins.
Harmful algae, 2017Co-Authors: Mélanie Roué, D. Tim Harwood, Hélène Taiana Darius, Jérôme Viallon, André Ung, Clémence Mahana Iti Gatti, Mireille ChinainAbstract:Ciguatera fish poisoning is a food-borne illness caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the Gambierdiscus genus. Since most surveillance programs currently rely on the survey of Gambierdiscus cell densities and species composition, supplementary toxin-based methods allowing the time- and spatially integrated sampling of toxins in ciguateric environments are needed for a more reliable assessment and management of the risks associated with Gambierdiscus proliferation. Solid Phase Adsorption Toxin Tracking (SPATT) filters use porous synthetic resins capable of adsorbing toxins directly from the water column. To assess the ability of these passive monitoring devices to retain Gambierdiscus toxins, SPATT bags filled with 10g of HP20 resin were deployed for 48h in two French Polynesian locations at high (Nuku Hiva Island) vs. low to moderate (Kaukura Atoll) risk of ciguatera. CTXs could be detected in SPATT bags extracts from Nuku Hiva Island, as assessed by the mouse neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography - tandem mass spectrometry (LC-MS/MS) analyses. Results of in vitro experiments suggest that the saturation limit of CTXs on HP20 resin, for a deployment time of 48h, is ≃ 55ng P-CTX-3C equiv. g-1 resin. Despite the non detection of maitotoxin (MTX), LC-MS/MS analyses showed that two other compounds also produced by Gambierdiscus species were retained on SPATT bags, i.e. iso-P-CTX-3B/C and a putative MTX analogue, known as MTX-3. This study, the first to demonstrate the suitability of SPATT technology for the in situ monitoring of Gambierdiscus toxins, highlights the potential application of this tool for routine ciguatera risk assessment and management programs.
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LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species.
Harmful algae, 2017Co-Authors: Yihua Lyu, Mireille Chinain, Michael L. Parsons, Mindy L. Richlen, Tomohiro Nishimura, Masao Adachi, Tyler B. Smith, Taylor R. Sehein, Tianling ZhengAbstract:The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.
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development of semi quantitative pcr assays for the detection and enumeration of Gambierdiscus species gonyaulacales dinophyceae 1
Journal of Phycology, 2012Co-Authors: Mark W. Vandersea, Michael J. Holmes, William C Holland, Patricia A. Tester, Mireille Chinain, Maria A. Faust, Steven R. Kibler, Thomas F Schultz, Wayne R LitakerAbstract:Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.
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Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus.
Toxicon : official journal of the International Society on Toxinology, 2010Co-Authors: R. Wayne Litaker, Michael J. Holmes, William C Holland, Mireille Chinain, Maria A. Faust, Mark W. Vandersea, Steven R. Kibler, Amy W. Nau, Patricia A. TesterAbstract:Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed.
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Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae)
Phycologia, 2009Co-Authors: R. Wayne Litaker, Michael J. Holmes, William C Holland, Mireille Chinain, Maria A. Faust, Mark W. Vandersea, Steven R. Kibler, Patricia A. TesterAbstract:Abstract Litaker R.W., Vandersea M.W., Faust M.A., Kibler S.R., Chinain M., Holmes M.J., Holland W.C. and Tester P.A. 2009. Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae). Phycologia 48: 344–390. DOI: 10.2216/07-15.1 Gambierdiscus species produce toxins that cause ciguatera fish poisoning (CFP), the most common nonbacterial illness associated with fish consumption worldwide. Understanding the role that individual Gambierdiscus species play in causing CFP is hampered because the morphologically similar species in the genus are difficult to distinguish. Ambiguities in the description of the type species Gambierdiscus toxicus also exist. This paper presents detailed line drawings along with additional scanning electron microscopy (SEM) and light micrographs to more fully characterize the six known Gambierdiscus species in addition to describing four new species, Gambie...
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Species-specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae).
Journal of phycology, 2019Co-Authors: R. Wayne Litaker, Patricia A. Tester, Mark W. VanderseaAbstract:The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species-specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species-specific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species-specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes.
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Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.
Harmful algae, 2019Co-Authors: Lisbet Díaz-asencio, R. Wayne Litaker, Mark W. Vandersea, Santiago Fraga, Nicolas Chomérat, Rachel J. Clausing, Donaida Chamero-lago, Miguel Gómez-batista, Angel R. Moreira-gonzález, Patricia A. TesterAbstract:Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.
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Ciguatoxicity of Gambierdiscus and Fukuyoa species from the Caribbean and Gulf of Mexico.
PloS one, 2017Co-Authors: R. Wayne Litaker, William C Holland, Steven R. Kibler, Francesco Pisapia, D. Ransom Hardison, Philipp Hess, Patricia A. TesterAbstract: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.
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Toxicity screening of 13 Gambierdiscus strains using neuro-2a and erythrocyte lysis bioassays.
Harmful algae, 2017Co-Authors: Francesco Pisapia, William C Holland, R. Wayne Litaker, Tomohiro Nishimura, Masao Adachi, Santiago Fraga, Lam Nguyen-ngoc, D. Ransom Hardison, Véronique Séchet, Zouher AmzilAbstract:Abstract Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species. This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes , G. balechii , G. caribaeus , G. carpenteri , G. pacificus , G. scabrosus and one strain of an undetermined species ( Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes , a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay. Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86 pg MTX equivalents (eq) cell −1 . All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell −1 . The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell −1 . As was true for CTX, the highest MTX-toxicity was also found in G. excentricus . Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean.
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Gambierdiscus and Fukuyoa species in the greater Caribbean: Regional growth projections for ciguatera-associated dinoflagellates
Ecological Modelling, 2017Co-Authors: Steven R. Kibler, William C Holland, Patricia A. Tester, D. Ransom Hardison, Eric D. Davenport, R. Wayne LitakerAbstract:Abstract Satellite-derived sea surface temperature data from the MODIS Aqua instrument were used to project rates of growth for four Gambierdiscus and one Fukuyoa species, dinoflagellates associated with ciguatera fish poisoning. Dinoflagellate growth rates were modelled using experimental temperature vs. growth equations and projected bottom temperatures with light penetration and bathymetry masks. Daily projected growth rates for each species were used to calculate monthly, yearly and multiyear averages between 2003 and 2013. The resulting projections were then used to characterize patterns of regional Gambierdiscus/Fukuyoa abundance and compare these to ciguatera fish poisoning incidences in the Greater Caribbean Region. Model output indicated the highest growth potential was in the shelf waters of the Caribbean Sea, with moderate growth in the Bahamas, southern Florida and Gulf of Mexico; the lowest growth potential was in the northern Gulf of Mexico and along the U.S. south Atlantic coast. Mean projected growth rates generally coincided with distribution of ciguatera fish poisoning incidences in the region, with some exceptions in the southwestern Caribbean. The results of this study indicate spatial differences in Gambierdiscus and Fukuyoa growth likely play a prominent role in governing the occurrence of ciguatera fish poisoning in the greater Caribbean. Growth and distribution models may be an effective tool for ciguatera risk assessment.
