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.     

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.     

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.     

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.     

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 >45 m, 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 40 m 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 10 μmol photons m⁻² s⁻¹. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. 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 50 m.     

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.     

Dynamics of ciguatoxins from Gambierdiscus polynesiensis in the benthic herbivore Mugil cephalus: Trophic transfer implications

This study investigates ciguatoxin dynamics in mullet after controlled feeding of Gambierdiscus polynesiensis cells as a model to characterize the absorption, distribution, retention and accumulation of ciguatoxins into the second trophic level of southwestern Pacific coral reef ecosystems. Mullet (Mugil cephalus) were fed once every other day over a period of 16 days for nine toxic feedings, and ciguatoxin activity was assessed over time in blood and seven tissues using the Neuro2a assay. Within 3 h of feeding on G. polynesiensis cells, ciguatoxins attained maximal blood concentrations, indicating rapid absorption of toxins into the systemic circulation. The time course for distribution of the estimated total tissue burden of ciguatoxin closely followed the time course for blood toxin levels, indicating a rapid distribution of the ciguatoxins throughout the fish body. The large majority (95%) of the ciguatoxin ingested dose was eliminated from the examined fish tissues 24 h after a single toxic meal, indicating little retention potential for ciguatoxin. We found no evidence for ciguatoxin accumulation after nine repeated feedings spaced two days apart, indicating that mullet did not accumulate ciguatoxin. These results provide the first experimental evidence supporting the central tenet of Randall's food chain hypothesis that ciguatoxins enter the food chain by transfer from unicellular algae to herbivorous and detritus-feeding fish. We propose that a time-dependent transformation of oxopene ciguatoxins may be necessary for the concentration of ciguatoxin through higher trophic levels.     

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.     

Gambierdiscus species exhibit different epiphytic behaviors toward a variety of macroalgal hosts

Ciguatera fish poisoning is a common form of seafood poisoning caused by toxins (ciguatoxins) that accumulate in demersal (reef) food webs. The precursors of ciguatoxins are produced by dinoflagellates of the genus Gambierdiscus, and enter the food web via herbivory and detritivory. The Gambierdiscus genus was recently revised and new research on the physiology and ecology of the revised species is needed. While it has been demonstrated that Gambierdiscus spp. are predominately epiphytic, the variability in epiphytic behavior among the various Gambierdiscus species is not known. Five Gambierdiscus species isolated from the Greater Caribbean Region were the focus of this study (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and G. yasumotoi). Cells of Gambierdiscus were grown in wells with algae fragments from eight different macroalgal host genera (Acanthophora, Caulerpa, Dasya, Derbesia, Dictyota, Laurencia, Polysiphonia, and Ulva) where the epiphytic behavior and growth of the different Gambierdiscus species were monitored over 29 days. The results of this experiment demonstrate that epiphytic behavior (growth and attachment) differs among the Gambierdiscus species toward the various macroalgal hosts. Results tended to be specific to Gambierdiscus – host pairings with few commonalities in the way a particular Gambierdiscus species interacted across hosts or how the various Gambierdiscus species responded to a particular host. The Gambierdiscus – host pairings that resulted in the highest growth and attachment combinations were examined in terms of known cellular toxicity and host palatability to determine which pairings could represent the most likely vectors for the transfer of ciguatoxins (or precursors) into the demersal food web. Two pairings, Gambierdiscus belizeanus – Polysiphonia and G. belizeanus – Dictyota, best met these criteria, providing a hypothetical approach to better focus sampling and monitoring efforts on such potential vectors in the benthic environment.     

TRP channel blamed for burning cold after a tropical fish meal

EMBO J (2012) 31 19, 3795–3808 doi:10.1038/emboj.2012.207; published online July312012Ciguatera is one of the most common forms of food poisoning, occurring after consumption of fish contaminated with ciguatoxins. New work by Vetter et al (2012) reveals the key molecular players that underlie the altered temperature sensation associated with ciguatera. In particular, they show that ciguatoxins act on sensory neurons that express TRPA1, an ion channel implicated in the detection of noxious cold.Imaging yourself in the following idyllic settings: a white sandy tropical beach, blue sea and sky; and a barbecue on which your catch of the day, a 4-kg red snapper, is being grilled for dinner. But then, just a few hours after savouring the delicious fish meal, you undergo another unforgettable but far less heavenly experience: it starts with severe nausea, vomiting and diarrhoea, followed by disturbing neurological symptoms including headache, numbness and burning of the skin. You contracted ciguatera, a food-borne disease that affects an estimated 500 000 persons each year, particularly in the tropical and sub-tropical coastal regions (Dickey and Plakas, 2010).So what causes ciguatera? The prime culprits are dinoflagellates of the genus Gambierdiscus, small microalgae that produce a group of fat-soluble toxins called ciguatoxins and grow on macroalgae in coral reefs (Yasumoto et al, 1977). Gambierdiscus-containing macroalgae serve as food for herbivorous fish, which results in the introduction of ciguatoxins into the food web. These smaller, herbivorous fish are on the menu of large, carnivorous reef fish, such as mackerel, red snapper, or barracuda, which can accumulate ciguatoxins in the fatty parts of their body over years, apparently without any distress or obvious sign of disease. However, when such a mouth-watering but toxin-loaded catch appears on your menu, just a few bites (or sips of fish broth) can be sufficient to induce ciguatera (Figure 1A). Disturbingly, affected fish looks, smells, and tastes normal, and ciguatoxins are resistant to grilling, drying, or cooking of the fish, so there is no straightforward method to predict whether your tropical culinary dream will be followed by a ciguatera nightmare. Yes, there are commercial kits available to test for the presence of the toxin in fish, but these are considered too cumbersome, unreliable, and/or expensive to be of general practical use. And yes, there are persistent rumours of tropical fishermen feeding part of their catch to the cat first…Open in a separate windowFigure 1Ciguatera and cold allodynia. (A) Ciguatoxins, polycyclic polyether toxins, are produced by dinoflagellates of the genus Gambierdiscus, which reside in macroalgae in tropical coral reefs. These Gambierdiscus-containing macroalgae are eaten by herbivorous fish, which in turn serve as food for larger carnivorous fish, which accumulate the toxins in their bodies. Human consumption of these toxin-loaded fish causes ciguatera. (B) Ciguatoxins cause cold allodynia by increasing the cold sensitivity of TRPA1-expressing nociceptor neurons. Left, sensory nerve ending of a normal TRPA1-expressing nociceptor. At temperatures in the warm or innocuous cold region, the membrane potential (E_m) is around −65 mV, and both TRPA1 and the voltage-gated Na⁺ (Na_V) channels are closed. Cooling <10°C causes sufficient TRPA1 activation to cause action potential (AP) firing, experienced as burning cold. Right, ciguatoxin causes modification of the voltage-gated Na⁺ channels, resulting in significant opening at rest and depolarization of E_m to around −55 mV. Increased neuronal excitability and depolarization-induced activation of TRPA1 result in action potential firing and burning pain at innocuously cold temperatures.Apart from the gastrointestinal torment, which mostly fades away after a day or so, one of the most striking and disturbing symptoms of ciguatera is a form of oversensitivity to cold, termed as cold allodynia (Isbister and Kiernan, 2005). For instance, ciguatera sufferers have reported that a refreshing dive in the ocean actually caused burning pain, or that drinking cool beer felt like too hot coffee. The mechanisms whereby ciguatoxins provoke this peculiar form of altered temperature sensitivity, which can last for weeks to years, were fully unknown.In their paper, Vetter et al (2012) provide the first insights into the molecular mechanisms underlying cold allodynia induced by P-CTX-1, the most potent ciguatoxin present in the Pacific Ocean. First, they demonstrate that ciguatoxin-induced cold allodynia can be reproduced in a mouse model: following injection of minute quantities of P-CTX-1 into the sole of the paw, these mice exhibited clear signs of pain at moderately cool ambient temperatures, which were alleviated by warming. Next, they provide evidence that P-CTX-1 acts by sensitizing a specific subset of sensory neurons, characterized by the expression of the cation channel TRPA1. Finally, they show that pharmacological inhibition or genetic disruption of TRPA1 in mice strongly reduce the severity of cold allodynia upon P-CTX-1 injection.TRPA1 is a member of the TRP superfamily of cation channels, many of which play key roles in the sensory system as molecular sensors of temperature and of a variety of chemical ligands (Talavera et al, 2008). TRPA1 was known to be expressed in a subset of pain-sensing neurons (nociceptors), where it acts as a sensor of a wide variety of pungent/irritating substances (e.g., mustard oil) and of noxious cold (Story et al, 2003; Jordt et al, 2004; Karashima et al, 2009). TRPA1, like most other temperature-sensitive TRP channels, is voltage dependent, and thermal activation reflects a temperature-induced shift of its voltage-dependent activation curve towards more negative potentials (Voets et al, 2004; Karashima et al, 2009).Although TRPA1-expressing neurons show exquisite sensitivity to P-CTX-1, Vetter et al (2012) show that TRPA1 by itself is insensitive to P-CTX-1. Instead, P-CTX-1 responsiveness requires the combined presence of TRPA1 and of voltage-gated Na⁺ channels. In nociceptor neurons of non-intoxicated individuals, TRPA1 and the voltage-gated Na⁺ channels are largely closed at the resting membrane potential of around −65 mV, both at warm and at innocuously cool temperatures. Only upon cooling into the noxious cold range (<10°C), sufficient TRPA1 activation occurs to cause depolarization beyond the threshold for action potential firing (Figure 1B). This creates the sensation of burning cold (Karashima et al, 2009), which represents an important alarm signal, warning the body for potential cold-induced tissue damage (frostbite). However, as Vetter et al (2012) demonstrate, poisoning with P-CTX-1 strongly deregulates the cold sensitivity of the TRPA1-expressing nociceptors, such that they greatly overdo their alarm function. Reconfirming earlier work (Isbister and Kiernan, 2005), they show that P-CTX-1 acts as a potent modifier of voltage-gated Na⁺ channels in these neurons. In particular, at a concentration as low as 1 nM, P-CTX-1 causes a hyperpolarizing shift of the voltage dependence of activation of Na⁺ channels. This has a dual effect on the cold sensitivity of the TRPA1-expressing nociceptors (Figure 1B): (1) modulation of the voltage-gated Na⁺ channels increases the excitability of the neurons, an effect that is further enhanced by the inhibitory effect of P-CTX-1 on voltage-gated K⁺ channels and (2) P-CTX-1-treated voltage-gated Na⁺ channels allow Na⁺ influx at the resting membrane potential of these neurons, resulting in membrane depolarization, which in turn facilitates the voltage-sensitive activation of TRPA1 (Karashima et al, 2009). These mechanisms explain why in individuals suffering from ciguatera innocuous cooling is already sufficient to provoke action potential firing of TRPA1-expressing nociceptors, and is perceived as a burning pain.In addition to provide a molecular and cellular basis for ciguatera-associated cold allodynia, the study by Vetter et al (2012) also further strengthens the theory that TRPA1 is a relevant cold sensor in vivo, which has been disputed in several studies (Jordt et al, 2004; Knowlton et al, 2010). In particular, by using non-invasive functional MRI brain imaging, Vetter et al (2012) show for the first time significant differences in brain activity between wild-type and TRPA1-deficient mice when cooling their paw, and this difference is even more pronounced after the injection of P-CTX-1.Some important open questions remain. For example, given that P-CTX-1 is also acting on voltage-gated Na⁺ channels in TRPA1-negative sensory neurons, it is surprising that the toxin did not cause increased sensitivity to other stimuli, such as heat or mechanical stimuli. Moreover, it would be interesting to know whether TRPA1, which is also highly expressed on sensory neurons that innervate the gastrointestinal tract, plays a role in the gastrointestinal symptoms of ciguatera. If so, then TRPA1 would be an even more appealing target for the development of specific drugs to create relief from ciguatera symptoms.     

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.

     

Heavy metals, lipid peroxidation, and ciguatera toxicity in the liver of the caribbean barracuda (Sphyraena barracuda)

Human consumption of over 400 species of tropical fish containing polyether toxins (e.g. ciguatoxins, maitotoxins) causes ciguatera fish poisoning. The Caribbean barracuda (Sphyraena barracuda) is one of the most potent ciguatoxic fish. The objective of this study was to determine whether toxicity of 14 barracuda livers was correlated with lipid peroxidation. A significant correlation (p = 0.015, Pearson’s correlation) between lipid peroxidation and toxicity of barracuda liver was found. Because iron and copper are well-known catalysts of hydroxyl radical production and lipid peroxidation in biological systems, the correlation between the concentrations of these metals in barracuda liver and lipid peroxidation and toxicity was also investigated. Cadmium was significantly correlated (p = 0.014) with the toxicity of barracuda livers. This study provides the first data concerning the concentration of iron, copper, and cadmium in the liver of the Caribbean barracuda. Of the three metals studied in barracuda liver, iron was the most abundant, followed by copper and cadmium. Lipid peroxidation was highly variable and detected in five (36%) of the liver samples. Lipid peroxidation was not statistically significantly correlated (p > 0.05) with concentrations of iron, copper, and cadmium in barracuda liver. Collectively, these findings provide additional evidence that lipid peroxidation can be a mechanistic component of ciguatera toxicity in the Caribbean barracuda.     

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.     

RAPID PROCEDURE FOR DETECTING CIGUATOXINS IN FISH

Flesh and viscera/gill tissues of six amberjacks (Seriola dumerilii), suspected positive for ciguatoxins, were each extracted and the toxins partially purified. Both flesh and viscera/gill of only five fish were toxic to mice exhibiting ciguatoxins (CTX) symptoms. The methanol extracts of the five fish were pooled and concentrated, the volume of flesh extract was 50.0 mL (129.4 mg toxins/mL) and viscera/gill had 25.0 mL (25.5 mg toxins/mL). Pooled extracts exhibited CTX symptoms in mice but only flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh was 198.17 g fish, equivalent to 58.3 mouse unit. An efficient fractionation and purification procedure was developed for the extracts using an HPTLC and silica gel 60 plate with a chromatographic solvent mixture of chloroform:methanol:water (60:35:8, v/v). The system yielded 10 fractions for flesh and 9 for viscera/gill. Scanned plates were subdivided into three equal zones, each scraped, methanol extracted and tested in mice. The 2nd zone (R_f fractions between 0.40 and 0.66) was very toxic to mice compared to 1st or 3rd zones and the mice had CTX symptoms. The scanner for this 2nd zone had a cluster of minor peaks on both sides of the major one with a sum total area of 62.47% indicating multiplicity of CTX in amber-jack fish. The major peak, at retention time of 1.48 s and a single area of 43.28%, is believed to be the main ciguatoxins present. The HPTLC is a rapid and sensitive procedure for ciguatoxins in fish flesh extracts with a detection limit of 40.0 ± 1.9 picogram toxins.     

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.     

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.