Genus Gambierdiscus Research Articles

Cellular and nuclear morphological variability within a single species of the toxigenic dinoflagellate genus Gambierdiscus: Relationship to life-cycle processes

Dinoflagellates belonging to the genus Gambierdiscus are the causative agent of ciguatera fish poisoning (CFP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in temperate latitudes. Taxonomic studies of Gambierdiscus have yet to completely couple the morphological features of member species with their genetics. In this study, the cellular and nuclear morphology of a single strain of one species of Gambierdiscus was determined in cells grown under different culture conditions. The results showed a wide-ranging variability of cell sizes, together with a clear relationship between cell size and nuclear morphology. Thus, small cells were associated with round to oval or slightly U-shaped nuclei and large cells with obviously U-shaped nuclei. Most cells exhibited the typical anterio-posteriorly compressed lenticular, shape of Gambierdiscus, with the exception of a few small globular-shaped specimens. In all cells, regardless of their size, the arrangement of the thecal plates was typical of lenticular Gambierdiscus. Dividing cells were consistently the largest. In these cells, nuclear morphology, karyokinesis, and cytokinesis were characterized. Cells underwent division only during the dark period, thus demonstrating their spontaneous synchronized division. Cellular forms related to the sexual cycle were also present in the cultures and included gamete pairs and putative meiotic planozygotes. The effect of the culture medium was studied by means of principal component analyses, which showed a positive correlation between the medium used and nuclear size and shape but not cell size.

Genus Gambierdiscus in the Canary Islands (NE Atlantic Ocean) with Description of Gambierdiscus silvae sp. nov., a New Potentially Toxic Epiphytic Benthic Dinoflagellate

Species of the dinoflagellate genus Gambierdiscus are the cause of Ciguatera Fish Poisoning, common in tropical areas. Nevertheless, until recently this syndrome was not reported in the NE Atlantic Ocean. A new photosynthetic dinoflagellate species, Gambierdiscus silvae sp. nov. is described based on samples taken from tide pools on rocky shores of the Canary Islands (NE Atlantic Ocean). Its morphology was studied by light and scanning electron microscopy. The new species is anterioposteriorly compressed, lenticular in shape with an epitheca slightly higher than the hypotheca. It is round in apical view and has a thick smooth theca with many scattered pores. Plate formula is Po, 4', 0a, 6″, 6c, 6s?, 5'″, 0p, 2″″. Plate 2' is hatchet-shaped and Plate 2″″ is very wide and the largest of the hypotheca. Phylogenies inferred from the large subunit nuclear rRNA showed that three G. silvae strains clustered in a well supported sister clade to G. polynesiensis, distinct from the other species. G. australes was observed for the first time in the Atlantic, together with G. excentricus already reported from these islands. This work increases the number of Gambierdiscus species described and shows their unexpected diversity in the Canary Islands.

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.

Sampling harmful benthic dinoflagellates: Comparison of artificial and natural substrate methods

This study compared two collection methods for Gambierdiscus and other benthic harmful algal bloom (BHAB) dinoflagellates, an artificial substrate method and the traditional macrophyte substrate method. Specifically, we report the results of a series of field experiments in tropical environments designed to address the correlation of benthic dinoflagellate abundance on artificial substrate and those on adjacent macrophytes. The data indicated abundance of BHAB dinoflagellates associated with new, artificial substrate was directly related to the overall abundance of BHAB cells on macrophytes in the surrounding environment. There was no difference in sample variability among the natural and artificial substrates. BHAB dinoflagellate abundance on artificial substrates reached equilibrium with the surrounding population within 24h. Calculating cell abundance normalized to surface area of artificial substrate, rather than to the wet weight of macrophytes, eliminates complications related to the mass of different macrophyte species, problems of macrophyte preference by BHAB dinoflagellates and allows data to be compared across studies. The protocols outlined in this study are the first steps to a standardized sampling method for BHAB dinoflagellates that can support a cell-based monitoring program for ciguatera fish poisoning. While this study is primarily concerned with the ciguatera-associated genus Gambierdiscus, we also include data on the abundance of benthic Prorocentrum and Ostreopsis cells.

Transcriptome sequencing reveals single domain Type I-like polyketide synthases in the toxic dinoflagellate Gambierdiscus polynesiensis

Ciguatoxins (CTXs) are potent neurotoxins responsible for the food-borne illness known as ciguatera that occurs after consumption of contaminated fish. Benthic dinoflagellates of the genus Gambierdiscus spp. are known as the main producers of CTXs. CTXs are polycyclic polyethers, presumed to be synthesized by polyketide synthase (PKS) complexes; however, the mechanisms of CTX biosynthesis remain unresolved. Here, we investigated a de novo transcriptome assembly of Gambierdiscus polynesiensis TB-92 clone, a highly toxic producer of Pacific ciguatoxins, and focused on the identification of PKS transcripts. A cDNA library generated using a spliced leader (SL) priming approach, which specifically targets the dinoflagellate nuclear transcriptome, was sequenced by Roche 454. This strategy produced 1,221,335 raw reads, assembled into 16,336 unique contigs. Contigs were subjected to BLAST search, annotated with Gene Ontology (GO) terms and enriched with enzyme codes (EC) from Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Thirty-three PKS-related sequences were thus identified. Twenty-two contigs encoded single domain β-ketoacyl synthases (KS) with sequence similarity to Type I PKSs, as reported in other dinoflagellates. A conserved motif previously observed near the 5′ end of dinoflagellate KS domain transcripts was present in G. polynesiensis, and distinguished two groups of KS domain sequences. Ketoreductase (KR), acyltransferase (AT), and acyl carrier protein (ACP) domains were also found on single domain containing transcripts. KEGG pathway mapping placed three of the KS sequences containing the PKS conserved domain (cd00833) in the fatty acid biosynthesis pathway. No contigs were found encoding the conserved domains typically found in elongating ketosynthase domains of fatty acid synthases (cd00832, Type I or cd00834, Type II). Contigs mapping to other parts of the fatty acid biosynthesis pathway similarly encoded individual domains, suggesting that fatty acid synthesis takes place in multiprotein complexes. Other than the three KS domains, none of the sequences mapping to the fatty acid biosynthesis pathway overlapped with those annotated as PKSs. These data lend support to the idea that PKSs may contribute to both polyketide and fatty acid synthesis in dinoflagellates. This dataset provides important background to future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

Effects of temperature, salinity and their interaction on growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a seafood poisoning that is widespread in tropical, subtropical and temperate regions of the world. In the main island of Japan, distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3, have been reported. To discuss the bloom dynamics of these Japanese species/phylotypes of Gambierdiscus, first we tested six culture media to optimize growth conditions and then clarified the effects of temperature and salinity and temperature–salinity interactions on growth. All strains of the species/phylotypes tested showed the highest cell yields when they were cultivated in IMK/2 medium. G. australes, G. scabrosus and Gambierdiscus sp. type 2 grew in the range 17.5–30°C, whereas Gambierdiscus sp. type 3 grew in 15–25°C. The semi-optimal temperature ranges (≥80% of the maximal growth rate) of the former three species/phylotypes were 19–28°C, 24–31°C and 21–28°C, respectively, whereas that of the latter phylotype was 22–25°C. Hence, Gambierdiscus sp. type 3 may be adapted to relatively lower water temperatures of ≤25°C. In contrast, G. australes, G. scabrosus and Gambierdiscus sp. type 2 presumably possess adaptability to relatively high water temperatures. The optimal temperature for G. scabrosus was 30°C, whereas the optimal temperature for the others was 25°C. G. australes and Gambierdiscus sp. type 3 grew in a salinity range of 25–40 whereas G. scabrosus and Gambierdiscus sp. type 2 grew in salinity 20–40. Furthermore, the semi-optimal salinity range of G. australes, G. scabrosus, Gambierdiscus spp. type 2 and type 3 were salinity 27–38, 24–36, 22–36 and 29–37, respectively. Among the species/phylotypes, G. scabrosus and Gambierdiscus sp. type 2 grew even at salinity 20 where the others did not grow, thus possessing adaptability to low salinity waters. Our results clearly demonstrate that the optimal and tolerable temperature–salinity conditions differ among Japanese Gambierdiscus species/phylotypes. Considering these results, temperature–salinity interactions may play an important role in bloom dynamics and the distribution of the Gambierdiscus species/phylotypes in Japanese coastal waters.

Distribution, abundance and diversity of Gambierdiscus spp. from a ciguatera-endemic area in Marakei, Republic of Kiribati

Ciguatera is a serious seafood poisoning syndrome caused by the consumption of ciguatoxin-contaminated finfish from tropical and subtropical regions. This study examined the community structure of ciguatera-associated dinoflagellates and the distribution pattern, taxonomy and toxicity of Gambierdiscus spp. from a high-risk area of Marakei, Republic of Kiribati. The genera Gambierdiscus, Prorocentrum, Ostreopsis, Amphidinium and Coolia were present, and generally the former three dominated the dinoflagellate assemblage. Among these three, Gambierdiscus was the most abundant dinoflagellate genus observed at three of the four sites sampled, two of which (Sites 1 and 2) were on the northern half of the island and two (Sites 3 and 4) on the southern half. The following patterns of abundance were observed among sites: (1) Average Gambierdiscus spp. abundance at the northern sites exceeded the southern sites by a factor of 19–54; and (2) Gambierdiscus spp. abundance at shallow sites (2–3m) exceeded deeper sites (10–15m). The distribution of Gambierdiscus spp. at Marakei corresponded with previously observed patterns of fish toxicity, with fish from southern locations being much less toxic than fish sampled north of the central channel. DNA sequencing identified three Gambierdiscus species (Gambierdiscus carpenteri, Gambierdiscus belizeanus, Gambierdiscus pacificus) and three previously unreported ribotypes (Gambierdiscus sp. type 4, Gambierdiscus sp. type 5, Gambierdiscus sp. type 6) in the samples; Gambierdiscus sp. type 4 may represent a Pacific clade of Gambierdiscus sp. ribotype 1. Toxicity analyses determined that Gambierdiscus sp. type 4 isolates were more toxic than the Gambierdiscus sp. type 5 and G. pacificus isolates, with toxin contents of 2.6–6.0 (mean: 4.3±1.4), 0.010 and 0.011fg P-CTX-1 eq cell−1, respectively. Despite low densities of Gambierdiscus spp. observed at Marakei relative to other studies in other parts of the world, the presence of low and moderately toxic populations may be sufficient to render the western coast of Marakei a high-risk area for ciguatera. The long history of toxicity along the western side of Marakei suggests that large-scale oceanographic forcings that regulate the distribution of Gambierdiscus spp. along the western side of Marakei may have remained relatively stable over that time. Chronic as well as acute exposure to ciguatoxins may therefore pose an important human health impact to the residents of Marakei.

Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

Ecology of the ciguatera causing dinoflagellates from the Northern Great Barrier Reef: Changes in community distribution and coastal eutrophication

Ciguatera fish poisoning (CFP) is known to be caused by the ciguatoxins from the dinoflagellate genus Gambierdiscus, however, there is the potential for other toxins such as okadaic acid and dinophysistoxins from the genus Prorocentrum, and palytoxin from the genus Ostreopsis, to contaminate seafood. These genera may also be indicators of ecosystem health and potentially impact on coral reef ecosystems and the role they may play in the succession of coral to macroalgae dominated reefs has not been researched. Sixteen GBR field sites spanning inshore, mid-lagoon and outer lagoon (offshore) regions were studied. Samples were collected from September 2006 to December 2007 and abundance of benthic dinoflagellates on different host macroalgae and concentration of nutrients present in the water column were determined. The maximum abundance of Prorocentrum, Ostreopsis and Gambierdiscus found was 112, 793 and 50 cells per gram wet weight of host macroalgae, respectively. The average level of Dissolved Inorganic Nitrogen (DIN) in the water column across all sites (0.03mg/L) was found to be more than double the threshold critical value (0.013mg/L) for healthy coral reefs. Compared to a previous study 1984, there is evidence of a major shift in the distribution and abundance of these dinoflagellates. Inshore reefs have either of Prorocentrum (as at Green Island) or Ostreopsis (as at Magnetic Island) dominating the macroalgal surface niche which was once dominated by Gambierdiscus, whilst at offshore regions Gambierdiscus is still dominant. This succession may be linked to the ongoing eutrophication of the GBR lagoon and have consequences for the sources of toxins for ongoing cases of ciguatera.

Giant clams: New vectors of ciguatera?

s / Toxicon 75 (2013) 205–223 206 immediate evaluation by a physician. In these cases, the risk of drowning is great because of pain and muscular malfunction. On our beaches envenomation by Physalia physalis is an emerging phenomenon whose magnitude remains to be evaluated in the coming years, and whose causes are unknown. http://dx.doi.org/10.1016/j.toxicon.2013.08.004 Giant clams: New vectors of ciguatera? M. Roue , P. Cruchet , A. Ung , T. Darius , M. Chinain , D. Laurent a,b Universite de Toulouse (UPS), UMR-152 (Pharma-Dev), Toulouse cedex 9, France b Institut de Recherche pour le Developpement (IRD), UMR-152 (PharmaDev), Papeete, Tahiti, French Polynesia c Institut Louis Malarde, Laboratoire des Microalgues Toxiques, Papeete, Tahiti, French Polynesia Ciguatera Fish Poisoning is a form of ichtyosarcotoxism caused by the consumption of tropical coral reef fishes that have accumulated ciguatoxins, neurotoxins produced by dinoflagellates of the genus Gambierdiscus. Ciguatera is prevalent worldwide in subtropical and tropical regions and is regarded as the most prevalent form of intoxication in French Polynesia. Giant clams, although frequently consumed in the South Pacific, are rarely incriminated in human intoxications. However, they are sometimes involved in strong and atypical Ciguatera incidents, as observed in French Polynesia, New Caledonia and Vanuatu. The symptoms of this particular poisoning include the characteristics of Ciguatera Fish Poisoning (itching, paresthesia and reversal of hot/cold sensations for example) associated with additional symptoms like the burning of the mouth and the throat that appear very quickly and may be sometimes followed by severe paralysis. Giant clams could thus constitute another link in the ciguatera food chain and the name “Ciguatera Shellfish Poisoning” was proposed to designate this newly ecotoxicological phenomenon. In order to decipher the origin of these uncommon human intoxications, giant clams were collected in Raivavae (French Polynesia) and in Lifou (New Caledonia) where Ciguatera Shellfish Poisoning incidents were reported. Here, we present the results relative to the bioguided (via the neuroblastoma cytotoxicity assay) fractionation by lowand high-pressure chromatography of extracts prepared from ciguatera-suspected samples. Concurrently, cyanobacterial mats have been frequently observed near giant clams in the toxic areas of Raivavae and Lifou. Although phylogenetically unrelated to dinoflagellates, cyanobacteria are also suspected to be a potential source of ciguatoxin-like compounds. Indeed, during cyanobacterial blooms, filter-feeding bivalve molluscs such as giant clams are likely to become contaminated, providing a new link for the transfer of cyanotoxins to upper trophic levels including humans. This possible relationship between the proliferation of toxic marine cyanobacteria, giant clams toxicity and CSP intoxications will be discussed. This study provides a better understanding of the eco-toxicological phenomenon of ciguatera and should contribute to the improvement of seafood poisoning risk assessment and management programs in ciguatera-prone coral reef regions worldwide. http://dx.doi.org/10.1016/j.toxicon.2013.08.005 Coupling a Torpedo microplate-receptor binding assay with mass spectrometry to detect freshwater and marine toxins acting on nicotinic acetylcholine receptor

Gambierdiscus (Dinophyceae) species diversity in the Flower Garden Banks National Marine Sanctuary, Northern Gulf of Mexico, USA

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10μmolphotonsm−2s−1. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50m.

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.

New records of the genus Gambierdiscus in marginal seas of the Indian Ocean

The occurrence of the dinoflagellates belonging to the ciguatera-related genus Gambierdiscus is reported for the first time from the north-western Arabian Gulf and northern Red Sea in the Indian Ocean basin. Gambierdiscus yasumotoi was recorded from two sampling sites on the southern Kuwait coast, and co-occurrence of G. cf. belizeanus and G. yasumotoi was found also in the Gulf of Aqaba in Jordan. The morphology of cells is described on the basis of light microscopy and scanning electron microscopy examination.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P-CTX-1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin-resistant, TRPA1-expressing peptidergic C-fibres and tetrodotoxin-sensitive A-fibres. P-CTX-1 does not directly open heterologously expressed TRPA1, but when co-expressed with Na(v) channels, sodium channel activation by P-CTX-1 is sufficient to drive TRPA1-dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P-CTX-1 on TRPA1-deficient nociceptive C-fibres and of ciguatoxin-induced cold allodynia in TRPA1-null mutant mice. Functional MRI studies revealed that ciguatoxin-induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1-deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia.

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

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.

Development of a monoclonal antibody against the left wing of ciguatoxin CTX1B: Thiol strategy and detection using a sandwich ELISA

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL.

Harmful Algae in Benthic Systems: A GEOHAB Core Research Program

Harmful events caused by benthic microalgae have recently garnered a high level of attention in temperate areas, upon reports of Ostreopsis species causing fauna damage, food intoxication and respiratory illness. A resurgence of interest has also occurred in ciguatera fish poisoning (CFP), a syndrome associated with the consumption of reef fish contaminated by ciguatoxins produced by the dinoflagellate genus Gambierdiscus in tropical and subtropical areas. To foster research on events caused by benthic microalgae, the SCOR-IOC Global Ecology and Oceanography on Harmful Algal Blooms (GEOHAB) program started a Core Research Project on Benthic Harmful Algal Blooms (BHABs). During an Open Science Meeting (OSM) in 2010, scientists studying benthic and planktonic microalgae identified the main gaps of knowledge and the research, technology and infrastructure needs in the field of the ecology and oceanography of BHABs. The issues addressed during the OSM covered the state of taxonomy, sampling methods, biogeography, genetic diversity, ecology and ecophysiology of BHABs, along with the identification of research priorities and approaches to be taken in order to improve understanding and prediction of BHABs.

The occurrence of the ciguatera fish poisoning producing dinoflagellate genus Gambierdiscus in Pakistan waters

Five benthic species of the genus Gambierdiscus (Dinophyceae) were observed for the first time in the coastal waters of Pakistan, Northern Indian Ocean. The morphology of the epiphytic, ciguatera-related toxic species G. toxicus, G. belizeanus, G. polynesiensis, G. australes and G. cf. yasumotoi are presented here, described by the Kofoid system of thecal plates Po, 3`, 7, 6c, 8s, 5`, 1p, 2 with differences in cell shape, cell size, plates, pores around the apical pore plate by using light and scanning electron microscopy. The occurrence of these potentially toxic dinoflagellate species in Pakistani coastal areas of Manora Channel and Balochistan during high temperatures of 28- is cause of concern for human health impacts from ciguatera fish poisoning.

Preliminary study for rapid determination of phycotoxins in microalgae whole cells using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Rapid and sensitive methods for identification of several phycotoxins produced by microalgae species such as yessotoxins (YTXs) for Protoceratium reticulatum, okadaic acid (OA) and pectenotoxins (PTXs) for Prorocentrum spp. and Dinophysis spp., Palytoxins (PLTXs) for Ostreopsis spp., ciguatoxins (CTXs) for Gambierdiscus spp. or domoic acid (DA) for Pseudo-nitzschia spp. are of great importance to the shellfish and fish industry. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to detect several phycotoxins in whole cells of some microalgae which are known as toxin producers. To achieve an appropriate MALDI matrix and a sample preparation method, several matrices and solvent mixtures were tested. The most appropriate matrix system for toxin detection was obtained with 10 µg μL(-1) of DHB in 0.1% TFA/ACN (3:7, v/v) by mixing the intact cells with the matrix solution directly on the MALDI target (dried-droplet technique). Toxin detection by this procedure is much faster than current procedures based on solvent extraction and chromatographic separation. This method allowed the rapid detection of main phycotoxins in some dinoflagellate cells of genus Ostreopsis, Prorocentrum, Protoceratium, Gambierdiscus, Dinophysis and diatoms from Pseudo-nitzschia genus.

Gambierdiscus excentricus sp. nov. (Dinophyceae), a benthic toxic dinoflagellate from the Canary Islands (NE Atlantic Ocean)

A new benthic toxic dinoflagellate is described from the Canary Islands, Spain. Gambierdiscus excentricus sp. nov. was isolated from seaweeds growing in tidal ponds and was observed in winter and summer. Its morphology was studied by means of light microscopy (LM) and scanning electron microscopy (SEM); G. excentricus is a lenticular species having a Po plate ventrally displaced in relation to other species of the genus Gambierdiscus. Phylogenetic trees from large subunit (LSU) of ribosomal RNA gene sequences displayed a topology confirming that G. excentricus clustered in its’ own group, separated from the rest of Gambierdiscus species and with Gambierdiscus australes as its closest relative. Pigment composition studied from G. excentricus cultures, included peridinin, as the major carotenoid, chlorophyll a and the accessory chlorophylls c 1 and c 2. The Neuroblastoma cell-based assays for ciguatoxins (CTX) and maitotoxin (MTX) confirmed G. excentricus as a CTX- and MTX-like compounds producer. The finding of a toxic species of Gambierdiscus in the Canary Islands may explain the recent reported cases of ciguatera in the area.