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1. Asynchrony of Gambierdiscus spp. Abundance and Toxicity in the U.S. Virgin Islands: Implications for Monitoring and Management of Ciguatera

   https://doi.org/10.3390/toxins13060413  

   Liefer, Justin D.; Richlen, Mindy L.; Smith, Tyler B.; DeBose, Jennifer L.; Xu, Yixiao; Anderson, Donald M.; Robertson, Alison (June 2021, Toxins)

   Ciguatera poisoning (CP) poses a significant threat to ecosystem services and fishery resources in coastal communities. The CP-causative ciguatoxins (CTXs) are produced by benthic dinoflagellates including Gambierdiscus and Fukuyoa spp., and enter reef food webs via grazing on macroalgal substrates. In this study, we report on a 3-year monthly time series in St. Thomas, US Virgin Islands where Gambierdiscus spp. abundance and Caribbean-CTX toxicity in benthic samples were compared to key environmental factors, including temperature, salinity, nutrients, benthic cover, and physical data. We found that peak Gambierdiscus abundance occurred in summer while CTX-specific toxicity peaked in cooler months (February–May) when the mean water temperatures were approximately 26–28 °C. These trends were most evident at deeper offshore sites where macroalgal cover was highest year-round. Other environmental parameters were not correlated with the CTX variability observed over time. The asynchrony between Gambierdiscus spp. abundance and toxicity reflects potential differences in toxin cell quotas among Gambierdiscus species with concomitant variability in their abundances throughout the year. These results have significant implications for monitoring and management of benthic harmful algal blooms and highlights potential seasonal and highly-localized pulses in reef toxin loads that may be transferred to higher trophic levels. 

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2. LC–HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4

   https://doi.org/10.3390/md18040182  

   Kryuchkov, Fedor; Robertson, Alison; Miles, Christopher O.; Mudge, Elizabeth M.; Uhlig, Silvio (April 2020, Marine Drugs)

   Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC–HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC–HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications. 

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3. Reductive Amination for LC–MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish

   https://doi.org/10.3390/toxins14060399  

   Kryuchkov, Fedor; Robertson, Alison; Mudge, Elizabeth M.; Miles, Christopher O.; Van Gothem, Soetkien; Uhlig, Silvio (June 2022, Toxins)

   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. 

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4. Depuration Kinetics and Growth Dilution of Caribbean Ciguatoxin in the Omnivore Lagodon rhomboides: Implications for Trophic Transfer and Ciguatera Risk

   https://doi.org/10.3390/toxins13110774  

   Bennett, Clayton T.; Robertson, Alison (November 2021, Toxins)

   Modeling ciguatoxin (CTX) trophic transfer in marine food webs has significant implications for the management of ciguatera poisoning, a circumtropical disease caused by human consumption of CTX-contaminated seafood. Current models associated with CP risk rely on modeling abundance/presence of CTX-producing epi-benthic dinoflagellates, e.g., Gambierdiscus spp., and are based on studies showing that toxin production is site specific and occurs in pulses driven by environmental factors. However, food web models are not yet developed and require parameterizing the CTX exposure cascade in fish which has been traditionally approached through top-down assessment of CTX loads in wild-caught fish. The primary goal of this study was to provide critical knowledge on the kinetics of C-CTX-1 bioaccumulation and depuration in the marine omnivore Lagodon rhomboides. We performed a two-phase, 17 week CTX feeding trial in L. rhomboides where fish were given either a formulated C-CTX-1 (n = 40) or control feed (n = 37) for 20 days, and then switched to a non-toxic diet for up to 14 weeks. Fish were randomly sampled through time with whole muscle, liver, and other pooled viscera dissected for toxin analysis by a sodium channel-dependent MTT-based mouse neuroblastoma (N2a) assay. The CTX levels measured in all tissues increased with time during the exposure period (days 1 to 20), but a decrease in CTX-specific toxicity with depuration time only occurred in viscera extracts. By the end of the depuration, muscle, liver, and viscera samples had mean toxin concentrations of 189%, 128%, and 42%, respectively, compared to fish sampled at the start of the depuration phase. However, a one-compartment model analysis of combined tissues showed total concentration declined to 56%, resulting in an approximate half-life of 97 d (R2 = 0.43). Further, applying growth dilution correction models to the overall concentration found that growth was a major factor reducing C-CTX concentrations, and that the body burden was largely unchanged, causing pseudo-elimination and a half-life of 143–148 days (R2 = 0.36). These data have important implications for food web CTX models and management of ciguatera poisoning in endemic regions where the frequency of environmental algal toxin pulses may be greater than the growth-corrected half-life of C-CTX in intermediate-trophic-level fish with high site fidelity. 

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5. Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis

   https://doi.org/10.3390/md21040244  

   Leynse, Alexander K.; Mudge, Elizabeth M.; Turner, Andrew D.; Maskrey, Benjamin H.; Robertson, Alison (April 2023, Marine Drugs)

   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. 

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