Effect of ciguatoxins on the cardiocirculatory system

The aim of the present review was to collect the main observations reported until now concerning the cardio-circulatory effects of polyether toxins, called ciguatoxins, which are involved in an endemic intoxication named ciguatera found in tropical and subtropical countries. Ciguatera is caused by the ingestion of fishes contaminated with the dinoflagellate Gamberdiscus toxicus. Due to both tropical fish exportation destined for food and tourism, the disease has now spread out to temperate areas. Several toxins have been isolated and purified from different fish species living in different geographical areas. They are classified into three main groups by the nature of certain cycles of their carbon skeleton. Clinical reports show evidence that ciguatera intoxication affect both electrocardiograms and blood pressure. In most cases, ciguateric intoxication mainly evoked bradycardia, hypotension, and the alteration of S-T segment in the electrocardiogram. Isolated and purified ciguatoxins strongly altered the morphology of cardiac tissue inducing swelling of the cells and alterations of cellular organelles. These toxins impair the conduction of cardiac nerves and increase the opening probability of Na+ channels in intracardiac ganglions. Depending on the concentration applied, the substances exerted either a fast positive inotropic effect or a negative inotropic effect on the contraction of mammalian atrial and ventricular cardiac muscle. These effects were attributed to a release of noradrenaline and acetylcholine from neural terminals of the autonomic nervous system present in cardiac tissue. They also exert a slow delayed inotropic effect on the contraction which has been attributed to a direct effect of the toxins on tetrodotoxin-sensitive voltage-dependent Na+ channels of cardiac membranes. Ciguatoxins depolarized the membrane of mammalian atrial and ventricular preparations and shifted the threshold of sodium current activation to more negative membrane potentials. In conclusion, the inotropic effects of ciguatoxins on cardiac tissues mainly depend on the toxin concentration sensitivity of autonomic nerve terminals, which released noradrenaline and/or acetylcholine, while the ciguatoxin-induced increase of the sodium influx could be involved in the cardiac cell swelling which coincides with reports in which ciguatoxins induced a mannitol-inhibited swelling of the Node of Ranvier.     

A pilot study of the cognitive and psychological correlates of chronic ciguatera poisoning

Ciguatera fish poisoning is the most frequently reported seafood-toxin illness in the world, caused by the consumption of coral reef fishes contaminated with a group of natural toxins produced by minute phytoplankton (dinoflagellates). These toxins are potent and cause both acute and chronic neurologic disease in humans.Although ciguatera fish poisoning is associated with established neurotoxins in animal models, and with known peripheral nervous system effects in humans, this pilot study was the first to explore possible central nervous system effects associated with chronic ciguatera poisoning in humans. In a matched cohort design, 12 cases and 12 their age and gender matched friend controls underwent a battery of neuropsychological tests. The results indicated that although their scores on objective neuropsychological tests did not differ significantly from matched controls, persons with ciguatera reported a higher degree of toxicity-related symptomatology and endorsed significantly more depressive symptomatology than controls. Future research should explore larger numbers of persons with both acute and chronic ciguatera, with and without mannitol treatment.     

Origin and transfer of toxins involved in ciguatera

1. Ciguatera is a disease caused by sodium channel activator toxins and results from the consumption of warm water fish contaminated by the ciguatoxin class of polyether toxins.2. Other toxins, including okadaic acid and maitotoxin, have no proven role in causing human illness associated with ciguatera.3. Ciguatera often affects only a discrete region of a reef, with flare-ups of ciguatera being both temporally and spatially unpredictable.4. The ciguatoxins likely arise through the biotransformation and acid-catalysed spiroisomerisation of gambiertoxin-4A produced by Gambierdiscus toxicus and it is unlikely that other toxic benthic dinoflagellates are involved.5. Events leading to a ciguatera outbreak are initiated by environmental and genetic factors that favour the proliferation of gambiertoxins, with an apparent role for anthropomorphic effects; however, the precise factors involved are yet to be determined.6. The gambiertoxins and/or ciguatoxins are transferred from the benthos to herbivorous species (fish, invertebrates etc) and then to carnivorous fish via marine food chains.7. Factors influencing the concentration of ciguatoxins that accumulate in fish include the rate of dietary intake, the efficiency of assimilation, the degree and nature of any toxin biotransformation, the rate of depuration, and the rate of growth of fish.     

Species of dinoflagellates of the genus Gambierdiscus (Dinophyceae) in the Mexican Caribbean Sea

Some dinoflagellates with benthic habits are related to ciguatera intoxication by fish consumption, especially in tropical areas. In the Mexican Caribbean, ciguatera is relatively common, but only one paper seems to have been published on the subject, and there are very few publicactions on phytoplankton and benthic microalgae. Material collected along the coast of the State of Quintana Roo with phytoplankton net (54 mm) and directly from sediment and epiphytes of macroscopic plants, was searched for toxic and other associated dinoflagellates. Samples were studied by light and scanning electron microscopy. Morphological characters were useful for species identification, but eventually physiological, ecological and molecular characters could also be used. Three species of Gambierdiscus, related to the production of ciguatera toxins, were identified: G. belizeanus, G. toxicus and G. yasumotoi. They are distributed in shallow coastal areas, including coastal lagoons.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Detection of ciguatoxins: advantages and drawbacks of different biological methods

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.     

A Phylogenetic Re-Analysis of Groupers with Applications for Ciguatera Fish Poisoning

Background

Ciguatera fish poisoning (CFP) is a significant public health problem due to dinoflagellates. It is responsible for one of the highest reported incidence of seafood-borne illness and Groupers are commonly reported as a source of CFP due to their position in the food chain. With the role of recent climate change on harmful algal blooms, CFP cases might become more frequent and more geographically widespread. Since there is no appropriate treatment for CFP, the most efficient solution is to regulate fish consumption. Such a strategy can only work if the fish sold are correctly identified, and it has been repeatedly shown that misidentifications and species substitutions occur in fish markets.

Methods

We provide here both a DNA-barcoding reference for groupers, and a new phylogenetic reconstruction based on five genes and a comprehensive taxonomical sampling. We analyse the correlation between geographic range of species and their susceptibility to ciguatera accumulation, and the co-occurrence of ciguatoxins in closely related species, using both character mapping and statistical methods.

Results

Misidentifications were encountered in public databases, precluding accurate species identifications. Epinephelinae now includes only twelve genera (vs. 15 previously). Comparisons with the ciguatera incidences show that in some genera most species are ciguateric, but statistical tests display only a moderate correlation with the phylogeny. Atlantic species were rarely contaminated, with ciguatera occurrences being restricted to the South Pacific.

Conclusions

The recent changes in classification based on the reanalyses of the relationships within Epinephelidae have an impact on the interpretation of the ciguatera distribution in the genera. In this context and to improve the monitoring of fish trade and safety, we need to obtain extensive data on contamination at the species level. Accurate species identifications through DNA barcoding are thus an essential tool in controlling CFP since meal remnants in CFP cases can be easily identified with molecular tools.     

Gene expression patterns in peripheral blood leukocytes in patients with recurrent ciguatera fish poisoning: Preliminary studies

Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent and 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved.     

Morphology,toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.     

Effect of ciguatoxin 3C on voltage-gated Na+ and K+ currents in mouse taste cells

The marine dinoflagellate Gambierdiscus toxicus produces highly lipophilic, polycyclic ether toxins that cause a seafood poisoning called ciguatera. Ciguatoxins (CTXs) and gambierol represent the two major causative agents of ciguatera intoxication, which include taste alterations (dysgeusiae). However, information on the mode of action of ciguatera toxins in taste cells is scarce. Here, we have studied the effect of synthetic CTX3C (a CTX congener) on mouse taste cells. By using the patch-clamp technique to monitor membrane ion currents, we found that CTX3C markedly affected the operation of voltage-gated Na(+) channels but was ineffective on voltage-gated K(+) channels. This result was the exact opposite of what we obtained earlier with gambierol, which inhibits K(+) channels but not Na(+) channels. Thus, CTXs and gambierol affect with high potency the operation of separate classes of voltage-gated ion channels in taste cells. Our data suggest that taste disturbances reported in ciguatera poisoning might be due to the ability of ciguatera toxins to interfere with ion channels in taste buds.     

Ciguatera — Parameters of a tropical health problem

Ciguatera fish poisoning is a significant health and resource problem in the tropical world, largely because of its uneven and unpredictable distribution in space and through time. Here, the problem is reviewed with evidence from the Pacific Basin. The contemporary distribution of ciguatera and the species commonly perceived to be toxic are considered and a hypothesis relating the greater prevalence of ciguatera in the eastern Pacific to reduced species diversity is presented. The problem is also considered as a public health phenomenon (the mean reported incidence for the Pacific region as a whole in 1981 was 109/100,000) and attention is given to island dwellers' adaptation to the problem, their explanations of its etiology, as well as its detection, prophylaxis, and cure.The primary research was made possible by a Fulbright-Hays Research Aboard Grant (#.441Ah70055); subsequent research was supported by a University of Hawaii Research and Training Revolving Fund Award.     

TRPV1 as a key determinant in ciguatera and neurotoxic shellfish poisoning

Ciguatera fish poisoning and neurotoxic shellfish poisoning are distinct clinical entities characterized by gastrointestinal and neurological disturbances, following the consumption of certain reef fish and shellfish containing toxic polyether compounds sporadically present in certain toxic marine dinoflagellates. The biotransformation and bioaccumulation of gambierol and brevetoxin, and their congeners, are believed to be involved in the pathogenesis of these "food-chain diseases", for which no effective treatments are available. Here, we describe for the first time the potent effect of gambierol and brevetoxin on TRPV1 channels, a key player in thermal and pain sensation. Our findings may lead to promising new therapeutic interventions.     

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.     

Macroalgal palatability and the flux of ciguatera toxins through marine food webs

The benthic dinoflagellate Gambierdiscus toxicus produces polyether toxins that cause ciguatera fish poisoning in humans. The toxins initially enter food webs when fish forage on macroalgae, or other substrates, hosting this epiphytic dinoflagellate. Population studies of G. toxicus and risk assessments in ciguatera-prone regions often rely on quantifying dinoflagellates on macroalgae. Underlying these studies is the assumption that the algae sampled represent a readily consumable resource equally available for benthic grazers. However, many algal hosts of G. toxicus possess a variety of defenses against grazing, and host–dinoflagellate associations may act as toxin sources or sinks depending on their palatability. Marine macroalgae may tolerate or avoid herbivory by exhibiting fast growth, by having poor nutritional quality, by utilizing spatial or temporal escapes or by using chemical or structural defenses. Thus, rapidly consumed algae that cope with herbivores by growing fast, such as many filamentous turfs, could be responsible for a high toxin flux even at low dinoflagellate densities. In contrast, ubiquitous unpalatable algae with much higher dinoflagellate densities might contribute little to toxin flux, and effectively act as refuges for G. toxicus. To date, G. toxicus has been reported from 56 algal genera, two cyanobacteria, one diatom, and one seagrass; 63% of these contain species that are defended from fish grazing and other grazers via chemical, morphological or structural defenses, by low nutritional quality, or by a combination of defensive strategies. High dinoflagellate densities on unpalatable macroalgae could indicate passive accumulation of cells on undisturbed hosts, rather than population explosions or active toxin sources for food webs. Understanding the flow of ciguatoxins in nature requires consideration of the ecology of both G. toxicus and its algal hosts. The complexity of marine algal–herbivore interactions also has consequences for other benthic dinoflagellates that produce toxins, which accumulate in consumers.     

Effects of temperature,salinity and composition of the dinoflagellate assemblage on the growth of Gambierdiscus carpenteri isolated from the Great Barrier Reef

Increases in reported incidence of ciguatera fish poisoning (hereafter ciguatera) have been linked to warmer sea temperatures that are known to trigger coral bleaching events. The drivers that trigger blooms of ciguatera-causing dinoflagellates on the Great Barrier Reef (GBR) are poorly understood. This study investigated the effects of increased temperatures and lowered salinities, often associated with environmental disturbance events, on the population growth of two strains of the potentially ciguatera-causing dinoflagellate, Gambierdiscus carpenteri (NQAIF116 and NQAIF380). Both strains were isolated from the central GBR with NQAIF116 being an inshore strain and NQAIF380 an isolate from a stable environment of a large coral reef aquarium exhibit in ReefHQ, Townsville, Australia. Species of Gambierdiscus are often found as part of a mixed assemblage of benthic toxic dinoflagellates on macroalgal substrates. The effect of assemblage structure of dinoflagellates on the growth of Gambierdiscus populations has, however, not been explored. The study, therefore investigated the growth of G. carpenteri within mixed assemblages of benthic dinoflagellates. Population growth was monitored over a period of 28 days under three salinities (16, 26 and 36) and three temperature (24, 28 and 34 °C) conditions in a fully crossed experimental design. Temperature and salinity had a significant effect on population growth. Strain NQAIF380 exhibited significantly higher growth at 28 °C compared to strain NQAIF116, which had highest growth at 24 °C. When strain NQAIF116 was co-cultured with the benthic dinoflagellates, Prorocentrum lima and Ostreopsis sp., inhibitory effects on population growth were observed at a salinity of 36. In contrast, growth stimulation of G. carpenteri (strain NQAIF116) was observed at a salinity of 26 and particularly at 16 when co-cultured with Ostreopsis-dominated assemblages. Range expansion of ciguatera-causing dinoflagellates could lead to higher frequency of reported ciguatera illness in populated temperate Australian regions, outside the tropical range of the GBR. Therefore, the findings on salinity and temperature tolerance of two strains of G. carpenteri indicates potential adaptability to different local environmental conditions. These are baseline data for future investigations into the potential southward range expansion of ciguatera-causing dinoflagellates originating from the GBR.     

Ciguatera fish poisoning in the Pacific Islands (1998 to 2008)

Background

Ciguatera is a type of fish poisoning that occurs throughout the tropics, particularly in vulnerable island communities such as the developing Pacific Island Countries and Territories (PICTs). After consuming ciguatoxin-contaminated fish, people report a range of acute neurologic, gastrointestinal, and cardiac symptoms, with some experiencing chronic neurologic symptoms lasting weeks to months. Unfortunately, the true extent of illness and its impact on human communities and ecosystem health are still poorly understood.

Methods

A questionnaire was emailed to the Health and Fisheries Authorities of the PICTs to quantify the extent of ciguatera. The data were analyzed using t-test, incidence rate ratios, ranked correlation, and regression analysis.

Results

There were 39,677 reported cases from 17 PICTs, with a mean annual incidence of 194 cases per 100,000 people across the region from 1998–2008 compared to the reported annual incidence of 104/100,000 from 1973–1983. There has been a 60% increase in the annual incidence of ciguatera between the two time periods based on PICTs that reported for both time periods. Taking into account under-reporting, in the last 35 years an estimated 500,000 Pacific islanders might have suffered from ciguatera.

Conclusions

This level of incidence exceeds prior ciguatera estimates locally and globally, and raises the status of ciguatera to an acute and chronic illness with major public health significance. To address this significant public health problem, which is expected to increase in parallel with environmental change, well-funded multidisciplinary research teams are needed to translate research advances into practical management solutions.     

Detection of Gambierdiscus and Fukuyoa single cells using recombinase polymerase amplification combined with a sandwich hybridization assay

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health.