Publications by authors named "William C Holland"

13 Publications

  • Page 1 of 1

Sulfo-Gambierones, Two New Analogs of Gambierone Produced by .

Mar Drugs 2021 Nov 24;19(12). Epub 2021 Nov 24.

Laboratoire Phycotoxines, DYNECO, Ifremer, F-44000 Nantes, France.

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera and . Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic species using Neuro2a cell-based assay identified as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of due to their sensitive detection by mass spectrometry.
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http://dx.doi.org/10.3390/md19120657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8703632PMC
November 2021

Algal toxins in Alaskan seabirds: Evaluating the role of saxitoxin and domoic acid in a large-scale die-off of Common Murres.

Harmful Algae 2020 02 23;92:101730. Epub 2019 Dec 23.

US Geological Survey, Alaska Science Center, Anchorage, AK, United States.

Elevated seawater temperatures are linked to the development of harmful algal blooms (HABs), which pose a growing threat to marine birds and other wildlife. During late 2015 and early 2016, a massive die-off of Common Murres (Uria aalge; hereafter, murres) was observed in the Gulf of Alaska coincident with a strong marine heat wave. Previous studies have documented illness and death among seabirds resulting from exposure to the HAB neurotoxins saxitoxin (STX) and domoic acid (DA). Given the unusual mortality event, corresponding warm water anomalies, and recent detection of STX and DA throughout coastal Alaskan waters, HABs were identified as a possible factor of concern. To evaluate whether algal toxins may have contributed to murre deaths, we tested for STX and DA in a suite of tissues obtained from beach-cast murre carcasses associated with the die-off as well as from apparently healthy murres and Black-legged Kittiwakes (Rissa tridactyla; hereafter, kittiwakes) sampled in the preceding and following summers. We also tested forage fish and marine invertebrates collected in the Gulf of Alaska in 2015-2017 to evaluate potential sources of HAB toxin exposure for seabirds. Saxitoxin was present in multiple tissue types of both die-off (36.4 %) and healthy (41.7 %) murres and healthy kittiwakes (54.2 %). Among birds, we detected the highest concentrations of STX in liver tissues (range 1.4-10.8 μg 100 g) of die-off murres. Saxitoxin was relatively common in forage fish (20.3 %) and invertebrates (53.8 %). No established toxicity limits currently exist for seabirds, but concentrations of STX in birds and forage fish in our study were lower than values reported from most other bird die-offs in which STX intoxication was causally linked. We detected low concentrations of DA in a single bird sample and in 33.3 % of invertebrates and 4.0 % of forage fish samples. Although these results do not support the hypothesis that acute exposure to STX or DA was a primary factor in the 2015-2016 mortality event, additional information about the sensitivity of murres to these toxins is needed before we can discount their potential role in the die-off. The widespread occurrence of STX in seabirds, forage fish, and invertebrates in the Gulf of Alaska indicates that algal toxins should be considered in future assessments of seabird health, especially given the potential for greater occurrence of HABs in the future.
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http://dx.doi.org/10.1016/j.hal.2019.101730DOI Listing
February 2020

HABscope: A tool for use by citizen scientists to facilitate early warning of respiratory irritation caused by toxic blooms of Karenia brevis.

PLoS One 2019 20;14(6):e0218489. Epub 2019 Jun 20.

National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.

Blooms of the toxic microalga Karenia brevis occur seasonally in Florida, Texas and other portions of the Gulf of Mexico. Brevetoxins produced during Karenia blooms can cause neurotoxic shellfish poisoning in humans, massive fish kills, and the death of marine mammals and birds. Brevetoxin-containing aerosols are an additional problem, having a severe impact on beachgoers, triggering coughing, eye and throat irritation in healthy individuals, and more serious respiratory distress in those with asthma or other breathing disorders. The blooms and associated aerosol impacts are patchy in nature, often affecting one beach but having no impact on an adjacent beach. To provide timely information to visitors about which beaches are low-risk, we developed HABscope; a low cost (~$400) microscope system that can be used in the field by citizen scientists with cell phones to enumerate K. brevis cell concentrations in the water along each beach. The HABscope system operates by capturing short videos of collected water samples and uploading them to a central server for rapid enumeration of K. brevis cells using calibrated recognition software. The HABscope has a detection threshold of about 100,000 cells, which is the point when respiratory risk becomes evident. Higher concentrations are reliably estimated up to 10 million cells L-1. When deployed by volunteer citizen scientists, the HABscope consistently distinguished low, medium, and high concentrations of cells in the water. The volunteers were able to collect data on most days during a severe bloom. This indicates that the HABscope can provide an effective capability to significantly increase the sampling coverage during Karenia brevis blooms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586399PMC
February 2020

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

PLoS One 2018 20;13(6):e0198358. Epub 2018 Jun 20.

National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198358PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010213PMC
December 2018

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

PLoS One 2017 18;12(10):e0185776. Epub 2017 Oct 18.

Ocean Tester, LLC, Beaufort, North Carolina, United States of America.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646788PMC
October 2017

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Mar Drugs 2017 Jul 11;15(7). Epub 2017 Jul 11.

Ifremer, Phycotoxins Laboratory, rue de l'Ile d'Yeu, BP 21105, F-44311 Nantes, France.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera and and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, , is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of and 37 other strains representing one species and ten species, one ribotype and one undetermined strain/species of were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain ( S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic in the Atlantic area.
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http://dx.doi.org/10.3390/md15070220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532662PMC
July 2017

Toxicity screening of 13 Gambierdiscus strains using neuro-2a and erythrocyte lysis bioassays.

Harmful Algae 2017 03 9;63:173-183. Epub 2017 Mar 9.

Ifremer, Phycotoxins Laboratory, rue de l'Ile d'Yeu, BP 21105, F-44311 Nantes, France.

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 86pg MTX equivalents (eq) cell. All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell. The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell. 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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http://dx.doi.org/10.1016/j.hal.2017.02.005DOI Listing
March 2017

New scenario for speciation in the benthic dinoflagellate genus Coolia (Dinophyceae).

Harmful Algae 2016 05 15;55:137-149. Epub 2016 Mar 15.

Bachok Marine Research Station, Institute of Ocean and Earth Sciences, University of Malaya, 16310 Bachok, Kelantan, Malaysia.

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.
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http://dx.doi.org/10.1016/j.hal.2016.02.010DOI Listing
May 2016

Rapid Extraction and Identification of Maitotoxin and Ciguatoxin-Like Toxins from Caribbean and Pacific Gambierdiscus Using a New Functional Bioassay.

PLoS One 2016 28;11(7):e0160006. Epub 2016 Jul 28.

National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Fisheries & Habitat Research, 101 Pivers Island Road, Beaufort, NC, 28516, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965106PMC
August 2017

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

PLoS One 2016 13;11(4):e0153348. Epub 2016 Apr 13.

National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153348PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830512PMC
September 2016

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay.

Toxicon 2013 Apr 10;65:15-33. Epub 2013 Jan 10.

National Oceanic and Atmospheric Administration, National Ocean Service, National Centers for Coastal Ocean Science, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, NC 28516, USA.

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC-MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.
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http://dx.doi.org/10.1016/j.toxicon.2012.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468995PMC
April 2013

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

J Phycol 2012 Aug 10;48(4):902-15. Epub 2012 May 10.

NOS/NOAA, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USAMarine Conservation Molecular Facility, Duke University Marine Laboratory, Nicholas School of the Environment, 135 Marine Lab Road, Beaufort, North Carolina 28516, USADepartment of Botany, United States National Herbarium, National Museum of Natural History, Smithsonian Institution, 4210 Silver Hill Road, Suitland, Maryland, 20746, USATropical Marine Science Institute, 14 Kent Ridge Road, National University of Singapore, Singapore City 119223, SingaporeAquatic Ecosystem Health, Department of Environmental and resource Management, GPO Box 2454, Brisbane, Quennsland 4001, AustraliaLaboratoire Des Micro-Algues Toxiques, Institut Louis Malardé, BP 30 98713 Papeete, TahitiNOS/NOAA, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA.

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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http://dx.doi.org/10.1111/j.1529-8817.2012.01146.xDOI Listing
August 2012

Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus.

Toxicon 2010 Oct 16;56(5):711-30. Epub 2010 Jun 16.

NOS/NOAA, Center for Coastal Fisheries and Habitat Research, Beaufort, NC 28516, USA.

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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http://dx.doi.org/10.1016/j.toxicon.2010.05.017DOI Listing
October 2010
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