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Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south of St. Thomas, US Virgin Islands. The highest CTXproducing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 μg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions. 

Ciguatera poisoning (CP) is the most common form of phycotoxin-borne seafood poisoning globally, affecting thousands of people on an annual basis. It most commonly occurs in residential fish of coral reefs, which consume toxin-laden algae, detritus, and reef animals. The class of toxins that cause CP, ciguatoxins (CTXs), originate in benthic, epiphytic dinoflagellates of the genera, Gambierdiscus and Fukuyoa, which are consumed by herbivores and detritivores that facilitate food web transfer. A number of factors have hindered adequate environmental monitoring and seafood surveillance for ciguatera including the low concentrations in which the toxins are found in seafood causing illness (sub-ppb), a lack of knowledge on the toxicity equivalence of other CTXs and contribution of other benthic algal toxins to the disease, and the limited availability of quantified toxin standards and reference materials. While progress has been made on the identification of the dinoflagellate taxa and toxins responsible for CP, more effort is needed to better understand the dynamics of toxin transfer into reef food webs in order to implement a practical monitoring program for CP. Here, we present a conceptual model that utilizes empirical field data (temperature, Gambierdiscus cell densities, macrophyte cover) in concert with other published studies (grazing rates and preference) to produce modeling outputs that suggest approaches that may be beneficial to developing monitoring programs: 1) targeting specific macrophytes for Gambierdiscus and toxin measurements to monitor toxin levels at the base of the food web (i.e., toxin loading); and 2) adjusting these targets across sites and over seasons. Coupling this approach with other methodologies being incorporated into monitoring programs (artificial substrates; FISH probes; toxin screening) may provide an “early warning” system to develop strategic responses to potential CP flare ups in the future. 

Ciguatera Poisoning (CP) is a widespread and complex poisoning syndrome caused by the consumption of fish or invertebrates contaminated with a suite of potent neurotoxins collectively known as ciguatoxins (CTXs), which are produced by certain benthic dinoflagellates species in the genera Gambierdiscus and Fukuyoa. Due to the complex nature of this HAB problem, along with a poor understanding of toxin production and entry in the coral reef food web, the development of monitoring, management, and forecasting approaches for CP has lagged behind those available for other HAB syndromes. Over the past two decades, renewed research on the taxonomy, physiology, and toxicology of CP-causing dinoflagellates has advanced our understanding of the species diversity that exists within these genera, including identification of several highly toxic species (so called “superbugs”) that likely contribute disproportionately to ciguatoxins entering coral reef food webs. The recent development of approaches for molecular analysis of field samples now provide the means to investigate in situ community composition, enabling characterization of spatio-temporal species dynamics, linkages between toxic species abundance and toxin flux, and the risk of ciguatoxin prevalence in fish. In this study we used species-specific fluorescent in situ hybridization (FISH) probes to investigate Gambierdiscus species composition and dynamics in St. Thomas (USVI) and the Florida Keys (USA) over multiple years of sampling (2018-2020). Within each location, samples were collected seasonally from several sites comprising varying depths, habitats, and algal substrates to characterize community structure over small spatial scales and across different host macrophytes. This approach enabled the quantitative determination of communities over spatiotemporal gradients, as well as the selective enumeration of species known to exhibit high toxicity, such as Gambierdiscus silvae. The investigation found differing community structure between St. Thomas and Florida Keys sites, driven in part by differences in the distribution of toxin producing species G. silvae and G. belizeanus, which were present throughout sampling sites in St. Thomas but scarce or absent in the Florida Keys. This finding is significant given the high toxicity of G. silvae, and may help explain differences in fish toxicity and CP incidence between St. Thomas and Florida. 

Mudge, E. M.; Lewis, N.; Miles, C. O.; McCarron, P.; Uhlig, S.; Kryuchkov, F.; Gwinn, J. K.; Robertson, A.(November, 2023). Identification of Caribbean ciguatoxins from benthic dinoflagellates: Advances in knowledge on toxin chemistry and analysis. Harmful Algae UNESCO IOC, 73, 10. 

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways. 

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.