Gambierdiscus species diversity and community structure in St. Thomas, USVI and the Florida Keys, USA.

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 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 (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. Intrasite comparisons along a depth gradient found higher concentrations of Gambierdiscus spp. at deeper locations. Among the macrophytes sampled, Dictyota may be a likely vector for toxin transfer based on their widespread distribution, apparent colonization by G. silvae, and palatability to at least some herbivore grazers. Given its ubiquity throughout both study regions and sites, this taxa may also serve as a refuge, accumulating high concentrations of Gambierdiscus and other benthic dinoflagellates, which in turn can serve as source populations for highly palatable and ephemeral habitats nearby, such as turf algae. These studies further demonstrate the successful application of FISH probes in examining biogeographic structuring of Gambierdiscus communities, targeting individual toxin-producing species, and characterizing species-level dynamics that are needed to describe and model ecological drivers of species abundance and toxicity.

Benthic harmful microalgae and their impacts in South America.

Public awareness about Benthic Harmful Algal Blooms (BHABs) and their negative impacts has increased substantially over the past few decades. Even so, reports of BHABs remain relatively scarce in South America (SA). This paper provides a comprehensive overview of the current state of knowledge on BHABs in the continent, by integrating data from published articles, books, and technical reports. We recorded ∼300 different occurrences of potentially toxic BHAB species over the Caribbean, Atlantic and Pacific coasts, mostly in marine (>95%) but also in estuarine areas located from 12°36' N to 54°53' S. Over 70% of the data was published/released within the past 10 years, and ∼85% were concentrated in Brazil, Venezuela, Ecuador and Colombia. Benthic species were mainly associated with macroalgae, seagrass and sediment. Incidental detection in the plankton was also relevant, mainly in places where studies targeting BHAB species are still rare, like Argentina, Uruguay, Chile and Peru. The study listed 31 infrageneric taxa of potentially toxic benthic dinoflagellates and eight of estuarine cyanobacteria occurring in SA, with the greatest species diversity recorded in the equatorial-tropical zone, mainly in northeastern Brazil (Atlantic), Venezuela and Colombia (Caribbean), and the Galapagos Islands, Ecuador (Pacific). Local strains of Amphidinium, Gambierdiscus, Coolia and Prorocentrum spp. produced toxic compounds of emerging concern. Prorocentrum lima species complex was the most common and widely distributed taxon, followed by Ostreopsis cf. ovata. In fact, these two dinoflagellates were associated with most BHAB events in SA. Whereas the former has caused the contamination of multiple marine organisms and cases of Diarrhetic Shellfish Poisoning in subtropical and temperate areas, the latter has been associated with faunal mortalities and is suspected of causing respiratory illness to beach users in tropical places. Ciguatera Poisoning has been reported in Colombia (∼240 cases; no deaths) and Venezuela (60 cases; two deaths), and may be also a risk in other places where Gambierdiscus spp. and Fukuyoa paulensis have been reported, such as the Galapagos Islands and the tropical Brazilian coast. Despite the recent advances, negative impacts from BHABs in SA are intensified by limited research/training funding, as well as the lack of official HAB monitoring and poor analytical capability for species identification and toxin detection in parts of the continent.

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

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.

Ciguatera in Mexico (1984⁻2013).

Historical records of ciguatera in Mexico date back to 1862. This review, including references and epidemiological reports, documents 464 cases during 25 events from 1984 to 2013: 240 (51.72%) in Baja California Sur, 163 (35.12%) in Quintana Roo, 45 (9.69%) in Yucatan, and 16 (3.44%) cases of Mexican tourists intoxicated in Cuba. Carnivorous fish, such as snapper (Lutjanus) and grouper (Epinephelus and Mycteroperca) in the Pacific Ocean, and great barracuda (Sphyraena barracuda) and snapper (Lutjanus) in the Atlantic (Gulf of Mexico and Caribbean Sea), were involved in all cases. In the Mexican Caribbean, a sub-record of ciguatera cases that occurred before 1984 exists. However, the number of intoxications has increased in recent years, and this food poisoning is poorly studied in the region. Current records suggest that ciguatera fish poisoning in humans is the second most prevalent form of seafood poisoning in Mexico, only exceeded by paralytic shellfish poisoning (505 cases, 21 fatalities in the same 34-year period). In this study, the status of ciguatera in Mexico (epidemiological and treatment), and the fish vectors are reviewed. Dinoflagellate species Gambierdiscus, Ostreopsis, and Prorocentrum are related with the reported outbreaks, marine toxins, ecological risk, and the potential toxicological impact.

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

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa 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, G. excentricus, 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 G. excentricus 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 Ca2+ 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 G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus 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 (G. australes 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 G. excentricus 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 G. excentricus in the Atlantic area.

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species.

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.

Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea.

A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS-rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54-deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

Primer informe del género Gambierdiscus (Dinophyceae) y otros dinoflagelados bentónicos en el Parque Nacional Isla del Coco, Costa Rica, Pacífico Tropical Oriental / First report of the genus Gambierdiscus (Dinophyceae) and other benthic dinoflagellates from Isla del Coco National Park, Costa Rica, Eastern Tropical Pacific

The Eastern Tropical Pacific is a region reported as free of ciguatera poisoning that causes serious gastrointestinal, neural and cardiovascular problems, even death. But with this study we found a high incidence of benthic microalgae involved in ciguatera poisoning in Isla del Coco National Park (PNIC, from its name in Spanish), Costa Rica. Between 2006 and 2011, during expeditions to PNIC, 420 phytoplankton samples with the interest of finding benthic dinoflagellates involved in the ciguatera poisoning were collected and analyzed. Samples were taken with phytoplankton nets, towed vertically and horizontally or carried by diving, between 5 to 30 m depth, over reef areas, and by direct extraction from benthic macroalgae. We found the dinoflagellates Gambierdiscus spp., Coolia tropicalis, Coolia cf. areolota, Prorocentrum concavum, Prorocentrum compressum, Amphidinium carterae and Ostreopsis siamensis. The quantity of dinoflagellates by macroalgae weight was high, mainly for Gambierdiscus. Ostreopsis and Prorocentrum, the most widely distributed genera throughout the collection sites. Gambierdiscus is a ciguatera producing genus. Two different sizes of Gambierdiscus were found, and comparing our samples with other studies, we conclude that they are different to any previously reported. They possibly represent two new species. Coco Island is an oceanic island and because of its protection status, it is an ideal site for studying the evolution of marine phytoplankton. Also, long-term monitoring is important due to the variety of potentially toxic dinoflagellates living in this marine ecosystem. This is the first study to report benthic dinoflagellates implicated in ciguatera poisoning in other areas of the Eastern Tropical Pacific.

El Pacífico Tropical Oriental se ha indicado como una región de los océanos libre de ciguatera, una intoxicación que causa serios trastornos gastrointestinales, neurológicos y cardiovasculares, en casos extremos, la muerte. Pero con el presente estudio hemos encontrado una incidencia alta de microalgas bénticas involucradas en la producción de ciguatera en el Parque Nacional Isla del Coco (PNIC), Costa Rica. En campañas oceanográficas realizadas entre el 2006 y el 2011 al PNIC se recolectaron 420 muestras de fitoplancton con el interés de encontrar especies de dinoflagelados bentónicos implicados en la producción de ciguatera. Las muestras se tomaron con red de fitoplancton o mediante buceos, entre los 5 y 30 m de profundidad en zonas de arrecife y por extracción directa de macroalgas. Se encontró a los dinoflagelados Gambierdiscus spp., Coolia tropicales, Coolia cf. areolota, Prorocentrum concavum, Prorocentrum compressum, Amphidinium carterae y Ostreopsis siamensis. Se determinó que la cantidad de dinoflagelados por peso de macroalga era elevada, principalmente para el género Gambierdiscus. Los géneros Ostreopsis y Prorocentrum mostraron tener la mayor distribución en los sitios de colecta. Gambierdiscus es un género productor de ciguatera. Se encontraron dos tamaños diferentes de individuos de Gambierdiscus, Comparando los especímenes de Gambierdiscus encontradas con otros estudios, se concluye que son diferentes a las descritas anteriormente, por lo que podría tratarse de especies nuevas, lo que se confirmará con cultivos y análisis moleculares. Al ser la Isla del Coco un sitio oceánico aislado y en estado de conservación, es un sitio ideal para el estudio de la evolución de especies de fitoplancton marino. Además, el monitoreo a largo plazo es muy importante debido a la variedad de dinoflagelados bentónicos potencialmente tóxicos que habitan en este ecosistema marino. Este es el primer estudio donde se da a conocer la incidencia de dinoflagelados bentónicos implicados en ciguatera para el Pacífico Tropical Oriental.