Are cyanobacteria involved in Ciguatera Fish Poisoning-like outbreaks in New Caledonia?

Harmful Algae 7 (2008) 827–838

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Are cyanobacteria involved in Ciguatera Fish Poisoning-like outbreaks in New Caledonia? Dominique Laurent a,*, Anne-Sophie Kerbrat a, H. Taiana Darius b, Emmanuelle Girard c, Stjepko Golubic d, Evelyne Benoit c, Martin-Pierre Sauviat e, Mireille Chinain b, Jordi Molgo c, Serge Pauillac f a Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox, UMR152 IRD – Universite´ Paul Sabatier Toulouse III, centre IRD de Noume´a, BPA5, 98848 Noume´a, New Caledonia b Laboratoire des Microalgues Toxiques, Institut Louis Malarde´, BP30, 98713 Papeete, Tahiti, French Polynesia c CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Mole´culaire - UPR9040, Gif sur Yvette F-91198, France d Biological Science Center, Boston University, 5 Cummington Street, Boston, MA 02215, USA e Laboratoire d’Optique et Biosciences, INSERM U 696-CNRS UMR 7645-X ENSTA, Ecole Polytechnique, 91128 Palaiseau, France f Laboratoire des Biotoxines, Institut Pasteur de Nouvelle-Cale´donie, BP61, 98845 Noume´a, New Caledonia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 April 2008 Accepted 18 April 2008

From 2001 to 2005, numerous cases of seafood poisonings were reported in a tribe from Lifou (Loyalty Islands Province, New Caledonia) of which 35 were thoroughly examined. Observations outlined by the epidemiological and clinical data (including severity and rapid onset of certain symptoms following consumption of either giant clams (Tridacna spp.) or grazing and molluscivorous fish together with the apparent inefficacy of traditional remedies, were not in favour of a classical Ciguatera Fish Poisoning (CFP) outbreak. From 2005 onwards, an environmental offshore survey of the affected area was conducted. Screening of the damaged coral area revealed the presence of large populations of cyanobacteria identified as Hydrocoleum Ku¨tzing, but the absence of Gambierdiscus spp., the well-known dinoflagellate causative agent of CFP. In vivo and in vitro toxicological studies of extracts obtained from cyanobacteria and giant clams, strongly suggested the co-occurrence of ciguatoxin-like, anatoxin-like and paralytic shellfish toxins in these samples. These new findings shed new light on the complexity of the CFP symptomatology and treatment and also on the diversity and origin of the CFP toxins. Furthermore they provide new evidence of the overall variability of seafood poisonings following the ingestion of different sea products living in a marine environment where significant harmful populations of microalgae and cyanobacteria coexist. This is the first report on the involvement of cyanobacteria in CFP-like outbreaks following the consumption of giant clams or fish specimens. Consequently, it is recommended that CFP risk assessment programs now include monitoring of cyanobacteria besides the obvious screening of CFP-promoting dinoflagellates. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Ciguatera Fish Poisoning Cyanobacteria Epidemiology Hydrocoleum Toxicology

1. Introduction Ciguatera Fish Poisoning (CFP) is a form of human poisoning resulting from the ingestion of some species of tropical marine fish, which have accumulated naturally occurring toxins through their diet. CFP is most prevalent in circumtropical regions with coral reef environments (Bagnis et al., 1979; Lewis, 1992; Pottier et al., 2001; Hamilton et al., 2002a). Besides the obvious public health issues that arises from CFP – more than 50,000 people suffering annually

* Corresponding author. Tel.: +687 26 07 61; fax: +687 26 43 26. E-mail address: [email protected] (D. Laurent). 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.04.005

from severe gastrointestinal, neurological and cardiovascular disorders (Ruff and Lewis, 1994; Marquais and Sauviat, 1999; Lewis et al., 2000) – lies its often underestimated negative socioeconomic impact resulting from the loss of work days and from the strain on the development of the fisheries (Lewis, 1992; Dalzell, 1994). Typically, in south Pacific regions, CFP is characterized by a number of clinical symptoms beginning with gastrointestinal disorders 3–6 h after the meal and followed by neurological disorders in the next 6 h. In some rare cases (usually the most severe ones), the symptoms manifest in as little as a few minutes just after the meal (Bagnis et al., 1979; Legrand and Bagnis, 1991). Until now, occidental therapy for CFP remains primarily supportive and limited to symptom treatment (Lehane

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and Lewis, 2000). Likewise, the use of traditional herbal medicine is practiced with some efficiency to reduce some symptoms of CFP in the South Pacific area (Bourdy et al., 1992; Laurent et al., 1993; Boydron-Le Garrec et al., 2005). The primary causative agents of CFP are unicellular algae classified as benthic dinoflagellates belonging to the genus Gambierdiscus (Yasumoto et al., 1977a; Bagnis et al., 1980; Chinain et al., 1999a). So far, five potentially toxinogenic species have been described (Chinain et al., 1999b). These dinoflagellates are the source of two classes of potent polyether marine neurotoxins, the lipid-soluble ciguatoxins (CTXs, mainly responsible for CFP) and the water-soluble maitotoxins (MTXs). These toxic compounds enter the food chain via herbivorous fish that graze and browse on the filamentous or calcareous macro-algae that colonize dead corals and provide substrate for toxic Gambierdiscus spp. Subsequently, the toxins are concentrated and biotransformed along the food chain when carnivorous fish prey on smaller herbivorous fish. Although humans can be exposed to the CTXs at any level of the food chain, they are particularly at risk when a large predatory fish is consumed (Lehane, 1999). Deterioration of environmental conditions is thought to be primarily responsible for CFP outbreaks. In a healthy reef environment with live and growing corals, the density of Gambierdiscus spp., which are benthic dinoflagellates with limited motility, is generally low (Faust, 1995). Accordingly, biomass of microalgae ingested by grazing and herbivorous fish is also low and contributes little to the toxicity of carnivorous fish. However, following coral reef disturbances, both natural (storms, hurricanes, tsunamis, submarine volcano eruptions, coral bleaching, etc.) and

man-made (shipwreck, construction of docks and piers, dredging and landfill, chemical pollution, eutrophication, etc.) there is an increase in dead coral surfaces, which serve as substrate for macroalgae, which in turn, support microalgae including toxic dinoflagellates (Banner, 1976). Nevertheless, the abundance of Gambierdiscus spp. does not always lead to CFP outbreaks, as there are no consistent correlation between the toxicity of a given bloom and its biomass (Bagnis et al., 1988; Chinain et al., 1999a; Turquet et al., 2001). These observations also apply to the toxicity of Gambierdiscus clones (Holmes et al., 1991; Holmes and Lewis, 1994). The reasons for such differences in toxin production remain unknown. Although the biosynthesis of CTXs appears restricted to certain genetically defined strains (Bomber et al., 1989; Holmes et al., 1991; Chinain et al., 1999b; Lehane and Lewis, 2000), physiological or environmental factors in triggering toxin production under natural conditions cannot be totally excluded. In addition, other sources of toxic substances that can be transmitted through trophic chains must be considered. During the year 2005, following a public health alert by the health authorities on the island of Lifou (Loyalty Islands Province, New Caledonia) where cases of CFP were reported following consumption of fish and molluscs, a panel of experts from the ‘‘Institut de Recherche pour le De´veloppement’’ (IRD) was commissioned to assist the affected tribal population. Based on their previous experience in seafood harvesting and resulting intoxication episodes, the inhabitants designated a ‘‘high-risk ciguatoxic area’’ (h-rc area), located offshore the Hune¨te¨ village (Fig. 1). First observations by snorkelling and subsequent microscopic analyses established an absence of Gambierdiscus spp.

Fig. 1. Area of study. (A) Map of New Caledonia with Lifou Island, Loyalty Islands Province, (B) location of Hune¨te¨ village on Lifou Island above the high-risk ciguatoxic area of the coast. *Sampling sites.

D. Laurent et al. / Harmful Algae 7 (2008) 827–838

around the zone of damaged corals, but the presence of large populations of filamentous cyanobacteria identified as Hydrocoleum Ku¨tzing ex Gomont (Blennothrix Ku¨tzing ex Anagnostidis et Koma´rek 1988). These observations were inconsistent with the classical CFP phenomena and prompted a thorough survey of the entire affected area. Although phylogenetically unrelated to dinoflagellates, cyanobacteria have already been considered as a likely source of CTX-like compounds. As soon as the 1950s, Randall assumed that a benthic organism, most likely a blue-green alga, was the source of the toxin (Randall, 1958). Halstead, following the presence of benthic cyanobacteria Lyngbia majuscula in the gut of a large number of poisonous fishes, suggested the possibility that these cyanobacteria might serve as a primary source of CTXs or its phytochemical precursor (Halstead, 1967). Indeed, typical CFP signs of intoxication (quiescence, piloerection, diarrhoea, lachrymation, cyanosis, dyspnoea, convulsive spasms and death within 24 h stemming from respiratory failure) have been demonstrated in mice injected intraperitoneally with extracts of both Trichodesmium (Oscillatoria) erythraeum and molluscs (Hahn and Capra, 1992; Endean et al., 1993). Benthic marine cyanobacteria, such as Lyngbya majuscula, produce a great diversity of biologically active components (over 70), many of which (e.g. aplysiatoxin, debromoaplysiatoxin, lyngbyatoxins, antillatoxin) have been shown to be highly toxic (Osborne et al., 2001). Likewise, freshwater cyanobacteria produce a wide range of neurotoxins such as: (i) anatoxin-a and anatoxina(s) (genera Anabaena, Aphanizomenon, Nostoc and Oscillatoria), (ii) paralytic shellfish toxins (PSTs) including saxitoxin and its 21 closely related tetrahydropurine analogues (Anabaena, Aphanizomenon and Cylindrospermopsis), (iii) hepatotoxins such as microcystins (Microcystis, Planktothrix, Anabaena, Nostoc, Hapalosiphon and Anabaenopsis), (iv) nodularins (Nodularia), (v) cylindrospermopsins (Cylindrospermopsis, Aphanizomenon, Raphidiopsis and Umezakia) and (vi) dermatotoxins (Oscillatoria, Lyngbya and Schizothrix) (Mankiewicz et al., 2003). These toxins, when they accumulate in inland, drinking or recreational waters are considered hazardous to human and animal health (Carmichael, 2001; Codd et al., 2005). The present paper reports for the first time the possible involvement of toxic marine benthic cyanobacteria (Hydrocoleum spp.) affecting human subjects with symptoms closely resembling those of CFP. These cases of intoxication were caused by the consumption of either giant clams (Tridacna sp.) or fish (mainly grazing and molluscivorous species) caught inshore, in the area of Hune¨te¨ on Lifou Island, designated as high-risk for CFP. 2. Materials and methods 2.1. Epidemiological survey The inhabitants of Lifou numbering 10,000 live in three tribal districts: Wetr, Lossi and Gaitcha (Fig. 1). The tribe of Hune¨te¨, belonging to the district of Wetr, is populated by 300 inhabitants and is located at the northern part of the island. Since 2001, most of the tribal residents have experienced, at least once, mild or severe signs of seafood poisoning. With the help of the sanitary services of the district, 35 patients intoxicated during the peak of the CFP outbreak (from 2001 to 2005) were identified and volunteered to fill out a consent form and an epidemiological questionnaire. Epidemiological and clinical forms were designed according to a field reference guidebook on CFP, co-edited by the Secretariat of the Pacific Community (SPC) and IRD (Laurent et al., 2005). This questionnaire was divided into four parts: (i) information about the person in care of the patient and filling out the form (doctor,

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health worker, etc.); (ii) information about the patient (name, age, sex, etc.); (iii) information about the seafood product(s) that caused the poisoning (type, size, and origin), and (iv) patient’s medical history (clinical picture and parameters). 2.2. Environmental survey Surveys of potential causative toxic agents were carried out on six transects across the intertidal and subtidal (infralittoral) zones, from the coast to the offshore blue water, three placed across the areas designated as high-risk (h-rc) for CFP, and three across the eastern area, which is considered safe. Three collecting sites were located along each transect (Fig. 1, map 1B). Samples of 100–150 g of benthic algal biomass (brown turf or Phaeophyceae as Turbinaria spp.) were collected since March 2005, on a semi-monthly basis in each of the 18 sites, and their microalgal and cyanobacterial contents were determined. The used methodology entails shaking seaweeds in bag with filtered seawater to dislodge epiphytic microorganisms, filtering the water on sieves (mesh sizes of 500, 250 and 35 mm) to separate microorganisms from detritus and observing the presence of dinoflagellates or cyanobacteria with a microscope in the fraction collected in the 35 mm sieve (Chinain et al., 1999a). For taxonomic determination, an aliquot of each sample of dinoflagellates and of cyanobacteria was preserved in 5% formalin/seawater solution. 2.3. Microscopic observations The cell densities of Gambierdiscus spp. from all sampling locations were assessed microscopically (200) using a Malassez counting chamber (n = 10) and the values are expressed in cells g-1 algal wet weight. Due to the sticky texture and the cell variability, both in size and shape, of the resultant cyanobacteria biomass, neither accurate weighing nor microscopic assessments of the cyanobacteria cell densities were possible. Photomicrographs were made by using Zeiss Universal microscope with plain transmitted light and digital camera or by Nomarski Interference Contrast (DIC) and film camera. 2.4. Sample collections and extraction In the field, cyanobacteria appeared as bundles or tufts of several millimetres long filaments, agglutinated to form a dark coloured mat on dead coral limestone (consisting of mainly broken branch corals). When a large amount of cyanobacteria was observed, samples were collected and shaken immediately in a plastic bag filled with filtered seawater, and the resulting suspension was successively sieved (mesh sizes of 500, 250 and 35 mm). The retentate was rinsed with filtered seawater, stored in plastic ziploc bags and brought back to the laboratory. Upon standing at room temperature, stored cyanobacteria produced reddish-brown exudates, which coloured the ambient seawater. The purity (discrimination from other microorganisms) of each collection was checked by microscopy and samples were saved in 5% formalin–seawater solution for a thorough examination of the cyanobacterial bloom composition and subsequent morphological identification. Then cyanobacteria and the exudates were separated by centrifugation (2000  g for 20 min). In order to identify the water-soluble toxin(s) present in the exudates, two desalting methods were used. In accordance with the first one, exudates were freeze-dried and the powder was dissolved in absolute ethanol. According to the second one, exudates were filtered on a XAD4 Amberlite resin column and then rinsed with methanol (Fig. 2A). Toxicity of each fraction was analyzed using the mouse bioassay (see Section 2.5). The ethanol

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sample was partitioned between water-soluble and lipid-soluble extracts, as described above. Finally, all extracts were stored frozen at 20 8C until use. 2.5. Mouse bioassay All extracts were emulsified in a 0.4 mL solution of 0.1% Tween 60 (Sigma) in 0.9% NaCl and injected intraperitoneally to 19–21 g mice (OF1 female). Three extract doses were injected to duplicate mice and one control mouse was given the emulsifying solution alone under similar conditions, so that only 7 animals were used per extract. Symptoms and behavioural changes were observed for a period of approximately 48 h with food and water provided ad libitum. Mice were sacrificed if they survived more than 2 days post-extract injection. 2.6. Mechanical and electrophysiological experiments Mechanical and electrophysiological recordings were performed on phrenic nerve–hemidiaphragm muscle and cutaneous pectoris nerve–muscle preparations isolated from Swiss-Webster mice and Rana temporaria frogs, respectively. The isolated preparations were mounted in a Plexiglas chamber, and bathed in a standard physiological solution gassed with pure O2. Twitch tension or resting membrane potential and miniature endplate potentials (MEPPs) were recorded using conventional techniques, as previously described (Molgo´ et al., 1990; Llanos et al., 2006). Twitches were evoked by stimulating either the motor nerve associated to the muscle, or directly the muscle. Resting membrane potential and MEPPs recordings were made continuously from the same endplate before and during treatment with a given extract. Stock solutions of water-soluble extracts were diluted with physiological saline just before experiments (performed at room temperature). 2.7. Receptor binding assay (RBA) Fig. 2. Fractionating method for water-soluble (A) and lipid-soluble (B) extracts of cyanobacteria and giant clams.

fraction of the first desalting method and the aqueous fraction of the second one were analyzed by mechanical and electrophysiological experiments performed on vertebrate neuromuscular preparations. In order to characterize the lipid-soluble toxin(s), cyanobacterial pellets were extracted with 2 L methanol per kg of crude material and further partitioned between 1 L of dichloromethane and 2  500 mL of 60% aqueous methanol per kg. The dichloromethane phase was then defatted by partition between 200 mL of cyclohexane and 100 mL of 80% aqueous methanol (Fig. 2B). This latter phase was finally concentrated in a rotary evaporator and tested for the presence of CTX-like compounds. Giant clams (Tridacna sp.) are sessile filter-feeders, which can bioaccumulate natural toxins or microorganisms floating in the ambient seawater. Some of these bivalves were harvested within the h-rc and ‘‘safe’’ areas (5 and 4 specimens, respectively). Whole clams were pooled according to their respective harvesting area (850 g for the h-rc area and 250 g for the safe area). The methanol extract of each pool was partitioned between water-soluble and lipid-soluble extracts, as described above. Five specimens of parrotfish (Scarus sp.) were caught within the h-rc area and 3 within the safe area. Their flesh and liver were pooled (800 and 28 g, respectively, for the h-rc area and 400 and 11 g, respectively, for the safe area). The methanol extract of each

Lipid-soluble extracts, likely to contain CTX-like compounds, were assayed in binding experiments involving competition with tritiated brevetoxins [3H]PbTx-3 for the site-receptor 5 of voltagegated sodium channels on rat brain synaptosomes. Brevetoxins (PbTx-2 and PbTx-3) were obtained from Latoxan (Rosans, France). [3H]PbTx-3 (15 Ci mmol 1) was prepared by Amersham PerkinElmer Life Science by the reduction of PbTx-2 with [3H] sodium borohydride according to the method reported by Poli et al. (1986). Purity was near 99% as determined by HPLC analysis. A stock solution of 1 mCi in 1 mL ethanol was kept at 80 8C and dilutions were made immediately prior to use. The final assay concentration of [3H]PbTx-3 was 0.85 nM. P-CTX-3C was used as internal standard for sample calibration and was obtained from a clonal culture of Gambierdiscus polynesiensis (Chinain et al., 1999a). Extraction and purification of P-CTX-3B, P-CTX-3C and P-CTX-4A was done following the procedure previously described by Legrand et al. (1989, 1992) and Pauillac et al. (1995). Rat brain synaptosomes were prepared following the protocol described by Dechraoui et al. (1999). Protein concentration of each preparation was determined by assaying aliquots in duplicate using the Bradford protein assay with serum albumin (BSA) as a standard (Sigma). Rat brain synaptosomes were stored at 80 8C for several weeks and were used at final concentration of 60– 80 mg mL 1. The RBA was performed using the test tube format (Dechraoui et al., 1999). Radioactivity was determined using a PerkinElmer Microbeta Trilux 1450 liquid scintillation counter in 2 mL PerkinElmer Betaplate scintillation cocktail. Non-specific binding

D. Laurent et al. / Harmful Algae 7 (2008) 827–838

was measured in the presence of a saturating concentration of PbTx-3 (0.67 mM) and subtracted from the total binding to yield specific binding. For all samples, 2 aliquots were tested and measured in parallel. P-CTX-3C was used as internal standard for sample calibration. Unknown sample concentrations were calculated from the IC50 values, i.e. concentration of sample (mg mL 1 for cyanobacteria and giant clam samples) which causes 50% inhibition, determined using Graphpad Prism v 4.1. RBA toxicity results of samples were expressed as equivalents P-CTX-3C mg 1 of extract for cyanobacteria and giant clam samples. Graphpad Prism v 4.1 was also used to analyze binding competition curves. 2.8. Chromatographic purification and receptor binding assay Lipid-soluble extract of giant clam was purified as described by Hamilton et al. (2002b) and the fractionation was bioguided using RBA. Briefly, 1539 mg of lipophilic extract was dissolved into hexane/acetone (3:1) and applied to a glass column (23.5 cm  0.85 cm; bed volume, Vb = 53 mL) packed with Florisil (60–100 mesh), pre-washed with three Vb of acetone/methanol (9:1) and five Vb of hexane/acetone (3:1). Nine fractions corresponding to one Vb were eluted with hexane–acetone– methanol (h:a:m) mixtures of increasing polarity. The RBA active fractions 5 and 6 (eluted with 3:1:0 and 0:9:1, respectively) were combined to yield 128 mg of which a 108 mg portion was applied onto a Sephadex LH-20 (Pharmacia) column (0.6 cm  17 cm; Vb = 19.2 mL) eluted with methylene chloride/methanol (1:1). After running about 6 mL of eluant through the column, the active fraction was collected in 4 mL and dried under nitrogen atmosphere, yielding 58 mg that was further analyzed by high pressure liquid chromatography (HPLC). HPLC was performed on a KONTRON analyzing system, using a Asahipack, Shodex column (ODP 50, 6E; 6 mm  250 mm), isocratic elution (MeCN/H2O, 75:25), at a flow rate of 1.5 mL/ min and a pressure of 104 bars. Each fraction was characterized by UV monitoring at 210 nm, R = 0.1. Six fractions were collected (RT = 0–10; 10–20; 20–26; 26–34; 34–48; and 48–60 min). Standards of CTX-4A, CTX-3B and CTX-3C were injected using identical chromatographic conditions. Finally, each fraction was tested on RBA. 2.9. Experimental animals All efforts were made to minimise the suffering of animals (i.e. the frogs were killed by double pithing and the mice by dislocation of the cervical vertebrae followed by immediate exsanguination), and a minimum number of animals was used. All animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC). 3. Results 3.1. Epidemiological survey Cases of poisonings have been recorded for the past 5 years, beginning from the year 2001 to this date, without any annual major peak; however the intoxication rates tend to increase during the hot season (from November to April). The epidemiological survey began with interviews of 35 native residents of the Hune¨te¨ tribe, who were affected by seafood poisoning over the last 5 years. This number accounts for an incidence rate of 12%. This value is assumed to be underestimated as all the previously intoxicated patients were not included in this voluntary-based retrospective study.

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In addition, some inhabitants, notably the chief of the fishermen clan, were also questioned about their fishing habits, the existence of the high-risk (h-rc) areas and the natural and anthropogenic environmental changes recorded during the last 10 years. Intuitively, inhabitants have delimited an area where they do not catch fish anymore to avoid intoxications. This h-rc area lies 500 m along the coast and extends to about 50 m offshore, up to the limits of the reef platform (Fig. 1). This area was affected by the construction of an access road and an embarkation ramp in 1990, making a large break into the coral reef calcareous ridge. The bitumen-surfaced coral road was subsequently destroyed by heavy rains during the passage of a hurricane in 2003, and part of this material was deposited over the reef flat. The results of our epidemiological survey confirmed the association of ciguateratype occurrences with this area. From the 35 reported cases of seafood poisoning (Table 1), two were induced by a giant clam (Tridacna sp.) collected in the h-rc area and 33 were associated with fish consumption (28 harvested from the h-rc area and 5 from the safe ones). The implicated fishes were herbivorous grazers (18 cases with parrotfish, Scarus sp., 2 with long-nosed unicorn fish, Naso sp., 2 with hump headed maori wrass, and 1 with rabbitfish), molluscivorous (6 cases by longnosed emperor fish, Lethrinus sp.) and carnivorous (2 cases by goatfish and 2 by blue-spotted grouper). The implication of grazing and molluscivorous fish in these poisoning events is particularly disturbing, indeed in New Caledonia, parrotfish and long-nosed emperor fish are widely consumed because of their safety reputation. Medical data (Table 1) showed that fish or giant clam intoxications were accompanied with the specific clinical pattern of signs and symptoms of CFP such as gastrointestinal disorders, general fatigue, pain in the limbs and joints, reversal of hot and cold sensations as well as tingling sensation upon contact with water. Sometimes, cardiovascular symptoms like hypotension were also noted. However, the severity of these symptoms (which caused one third of the victims to be hospitalized) was far greater than usually encountered in New Caledonia. For about one third of the cases poisoned by the fish and for the two cases by the giant clam, symptoms developed immediately after the meal with a strong alteration to taste and a burning sensation on the tongue and the throat, which is not a common symptom of CFP. Besides these rapidly expressed symptoms, the two patients intoxicated by giant clams, developed dizziness, cold sensations, diarrhoea, nausea and vomiting. The most severe case experienced muscular pain, headaches, weakness in the legs leading to inability to walk, slow heart rate (46 beats min 1) and a reduced blood pressure (95/50 mmHg), which necessitated hospitalization. However, both of these patients also exhibited specific symptoms of CFP such as reversal of hot and cold sensations and itching involving interactions between CTXs and specific Na+ channels isoforms located in the membrane of sensory neurons (Benoit et al., 2005). These rapid poisonings were caused by eating parrotfish (5 cases), long-nosed emperor fish (3 cases), long-nosed unicorn fish (1 case), goatfish (1 case) and giant clam (2 cases). In Hune¨te¨, the inhabitants have experienced numerous CFP episodes in their life, which they claimed to have successfully treated with indigenous herbal medicine. However, during the recent episodes, all the victims reported the use of folk remedies, but the majority of them pointed out the inefficacy of many commonly accepted traditional treatments. 3.2. Environmental survey The initial inspection of the h-rc area, by diving in March 2005, revealed an outward expanding degradation of the coral reef

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Table 1 Clinical and epidemiological data collected through the survey conducted in Lifou from 2001 to 2004 Area

High-risk area

Contaminated seafood

Parrotfish (G)

Long-nosed emperor fish (M)

Giant clam Safe area

Parrotfish (G)

Long-nosed emperor fish (M) Blue-spotted grouper (C)

Sex

Age

Intoxication date (year/month)

Onset of symptoms

01/12

Symptoms Neurolog.

Digest.

Others

11 h

(Y)

(Y)

? 2h 2h 6h Immediately Immediately Immediately Immediately 8h 4h Some h 0h 3h 2h 7h 10 h 8h 1.5 h Immediately Some h Immediately ? 2h Immediately 1.5 h 2h

(N) (N) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (N) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y)

(Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y) (Y)

Dizziness, hypotension, bradycardia Unusual taste (N) (N) Dizziness; paresis Asthenia Asthenia Asthenia Asthenia Hungry and cold feeling Joint pain; asthenia (N) Asthenia; dizziness (N) (N) Severe joint pain Asthenia Cold feeling Dizziness Unusual taste Unusual taste Unusual taste

Recurrence

Treatment

(N)

Ho; Re

(Y) (N) (N) (Y) (Y) (Y) (Y) (Y) (N) (Y) (N) (Y) (Y) (N) (N) (N) (Y) (Y) (Y) (N) (Y) (N) (N) (N) (Y)

Re Re Re Re; Co Re Re Re Ho; Re Ho; Re Ho; Re Re Co; Re Re Ho; Re Ho; Re Ho Re Ho Re Ho; Re Re Re Ho; Re Re Co; Re Co; Re

(Y) (Y) (Y) (Y)

? (N) Ho; Re Ho; Re

(Y)

Co

Reminiscence

Christophe H.

m

35

Kyle H. Vaha H. Re´my H. Rock H.

f f m m

4 26 41 51

Marie I. Denis U. Jeanette U. Ambroise W. Eugene E. Marie de Rosaire I. Christophe I. He´loise A. Joseph A. Marie-Anne H. Anne-Marie H. Basie´ I. Anne-Marie H. Euge`ne E. Marianna I. Benjamin Q. Lizie´ Q. Damien W.

f m f m m f m f m f f m f m f m f m

39 63 44 41 43 41 40 39 47 31 36 46 36 43 39 31 26 36

? 04/07 04/07 04/11 02/10 03/03 05/03 05/04 04/01 03/10 03/10 02/09 02/05 02/12 02/12 01/12 01/12 02/06 03/03 01/12 04/12 05/03 01/12 01/04 01/04 04/11

Eliane Q. Claudio I. Johannes I. Marguerite W.

f m m f

67 20 57 48

02/03 02/04 02/04 04/09

Immediately Immediately 1h 3h

(Y) (Y) (Y) (Y)

(Y) (Y) (Y) (Y)

Wanadrio W.

m

58

04/09

3h

(Y)

(N)

Franc¸ois W.

m

55

05/02

Immediately

(Y)

(Y)

Asthenia Burning feeling Dizziness Immediate burning of the mouth Burning in the mouth Sore throat; cold feeling Sore throat; cold feeling Asthenia; burning in the mouth Painful sensation on contact with water (N)

Ciane Q.

f

49

03/02

12 h

(Y)

(Y)

(N)

(N)

Co; Re

3 months

Thomas Q.

m

57

03/02

4h

(Y)

(Y)

(N)

(Y)

Co; Re

3 months

(C): carnivorous; (G): grazor; (H): herbivorous; (M): molluscivorous; (Y): yes; (N): no; Ho: hospitalisation; Re: remedies; Co: consultation.

(N) 1 month

(N) 6 months (N) 3 months

(N)

(Y) A long time (Y) (Y) (N)

1 month ? Some days A very long time (N)

(N)

D. Laurent et al. / Harmful Algae 7 (2008) 827–838

Hump-headed Maori wrass (H) Long-nosed unicorn fish (H) Rabbitfish (H) Goatfish (C)

Name

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833

Fig. 3. Marine benthic cyanobacteria associated with ciguatera-like fish poisoning on Lifou Island. (A) Massive growth of Hydrocoleum spp. over the rubble of dead corals. (B) Trichomes of H. lyngbyaceum (photomicrograph made by using Zeiss Universal microscope with plain transmitted light and digital camera). Note the hourglass-shaped end cell with thickened outer wall (calyptra). (C) Trichomes of the larger species H. glutinosum, which produces dark mats (photomicrograph made by using Nomarski Interference Contrast (DIC) and film camera). Scale bar in (B) is 10 mm long for (B) and (C) and 5 mm long for (A).

environment beginning from the embarkation ramp (see Fig. 1). Within the first 50 m around the ramp, the reef was severely damaged. The area was covered by broken branched corals and polluted by rusting steel drums, discarded around the blocks of fringing reef flat. The whole area was overgrown with turf of various algae, and coated by bright orange and dark, almost black cyanobacterial mats. Approximately 100 m away from this damaged zone, the seabed became gradually lighter between the blocks of fringing reef flat, while the bed of turf disappeared progressively. The most striking feature of this area was the absence of echinoderms and Phaeophyceae of the genus Turbinaria, although some fishes were present. In contrast, the regions outside of the high-risk area, described as safe by the tribesmen, were covered with live corals and supported different Turbinaria species, and large number of the holothurian Stichopus chloronotus. Patches of algal turf were observed scattered on the blocks of fringing reef flat. During the environmental survey, the presence of Gambierdiscus spp., or other dinoflagellates, was never detected. Instead, extensive mats (Fig. 3, A) dominated by species of Hydrocoleum Ku¨tzing ex Gomont (=Blennothrix Ku¨tzing ex Anagnostidis et Koma´rek 1988) were observed on dead branched corals during the warm period from November to April. The most common organism H. lyngbyaceum Ku¨tzing (Fig. 3B) was characterized by trichomes of fairly constant diameter with mean and standard deviation of 11.52  0.79 mm. Masses of dark filamentous cyanobacteria were also dominated by the larger H. glutinosum Gomont (Fig. 3C) with trichomes 18.88  0.94 mm wide. Both species are characterized by short, 2–5 mm long cells, by straight and shortly attenuated trichome ends with capitated, hourglass-shaped end cells, covered by a thickened cell wall (calyptra). Other cyanobacteria of the Oscillatoriales order (Oscillatoria subuliformis, O. bonnemaisonii, Phormidium laysanense and Spirulina cf. weissii) were also observed in the h-rc area, with Phormidium laysanense appearing only in the cold season. Massive growth of cyanobacteria, including trichome fragments and motile hormogonal stages, apparently contributed to the diet of filterfeeding molluscs as well as of the fish foraging in this area. Observations outlined by the epidemiological data (severity and rapid onset of certain symptoms, implication of giant clams in the intoxication, apparent inefficacy of traditional remedies) and the absence of Gambierdiscus cells in the h-rc area were not in favour of a classical CFP, but rather suggested the co-occurrence of other toxins, likely produced by the cyanobacteria observed in these regions.

3.3. Toxicological and electrophysiological studies: water-soluble extracts Symptoms exhibited by mice injected intraperitoneally with water-soluble extracts of cyanobacteria or giant clams included ataxia, laboured breathing, frequently convulsive spasms, and paralysis followed by death occurring within the first minutes with doses up to 4–5 mg g 1 body weight, or by complete spontaneous recovery. No toxic effects were observed following intraperitoneally injection of water-soluble parrotfish extracts. Tested on muscle contraction on the isolated mouse hemidiaphragm, the water-soluble extract of the cyanobacterial exudate desalted by Amberlite resin, produced a decrease of muscle contraction elicited by nerve stimulation (Fig. 4). The

Fig. 4. Time-dependent effect of different doses of the water-soluble extract of cyanobacterial exudates on the nerve-evoked muscle contraction of the isolated mouse hemidiaphragm. (A) The amplitude of the contraction was measured before and during the addition of various doses of extract, normalized to its control value (i.e. in the absence of extract), and plotted as a function of time. (B) Tracings of muscle twitches recorded under the conditions specified by the letters in (A). Note the dose-dependent blockade of the twitch response in the presence of extract, and the quasi-reversibility of the effect.

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Fig. 6. Competition-displacement curves on rat brain membrane preparation using RBA of lipid-soluble extracts from cyanobacteria and giant clams samples. Each point represents the mean  S.E.M. of 2 experiments. The Hill slope of both sigmoidal curves were 1.9 and 1.6 for cyanobacteria and giant clam samples, respectively, given by Graphpad Prism (v 4.1).

Fig. 5. Effect of the absolute ethanol extract of cyanobacteria (25 mg mL 1) on nerve-evoked miniature end-plate potentials (MEPPs) (A and B) and on the resting membrane potential of skeletal muscle fibres (C), recorded from isolated mouse hemidiaphragms. (A) Tracings of MEPPs recorded before (a) and after 90 min (b) exposure to the extract. (B) Mean frequency of MEPPS (S.E.M.) recorded before (n = 5) and after (n = 4) the addition of the extract to the physiological solution. (C) Mean resting action potential (S.E.M.) before (n = 6) and after (n = 4) the addition of the extract to the physiological solution. Note the increase in MEPPs frequency induced by the extract (in A and B), and the absence of extract’s significant effect on the resting membrane potential (in C).

kinetics and steady-state values of this decrease depended on the dose of the extract (23.75 mg mL 1 inhibited more than 90% of the contraction in less than 2 min), and was completely reversible (in about 30 min). The dose required to block 50% of the contraction was estimated to be about 5 mg mL 1. It is worth noting that contraction elicited by direct muscle stimulation was also reversibly decreased by the extract (result not shown). This indicates that the inhibition of muscle contraction was not due to an action of the extract on the elements involved in neuromuscular transmission. These results, in addition to the rapid onset of symptoms in humans (like burning sensation in mouth and throat), the symptoms observed in mice and the known ability of cyanobacteria to produce paralytic shellfish toxins, strongly suggest that the water-soluble extract may contain saxitoxin (STX) and/or neoSTX that would act by blocking voltage-dependent sodium channels in nerve and muscle through interactions with their receptor-site 1 (for a review, see Catterall, 1992). The absolute ethanol extract of cyanobacteria was tested on the membrane potential of isolated mouse hemidiaphragm and frog cutaneous pectoris nerve–muscle preparations (Fig. 5). At doses lower than 10 mg mL 1, no apparent effect was detected. At doses higher than 10 mg mL 1, the ethanol extract produced an about 10fold increase in the mean frequency of MEPPs (i.e. enhancement of spontaneous quantal acetylcholine release), in both preparations, without significantly affecting the resting potential of muscles fibres. Signs observed in mice were hyperexcitability and, at doses up to 5 mg mL 1, convulsive spasms, diarrhoea, paralysis and death.

All these results strongly suggest that the extract contained anatoxin-a (or homoanatoxin-a), an agonist of acetylcholine receptors, and/or anatoxin-a (s), an inhibitor of acetylcholinesterase. However, the anatoxin-a hypothesis is prominent since excessive salivation, a symptom characterizing anatoxin-a (s) poisoning (Codd et al., 1999), was not observed in mice. The presence of CTX-like compounds could also be suspected despite the fact that the excitatory effects on vertebrate neuromuscular junctions were different from those recorded with CTXs (i.e. an about 5–10 times higher increase in the mean frequency of MEPPs and depolarization of muscle fibre membrane) (Molgo´ et al., 1990). 3.4. Toxicological and RBA: lipid-soluble extracts Signs exhibited by mice injected with lethal amounts of lipidsoluble extracts of cyanobacteria or giant clams included quiescence, trembling, perspiration, vasodilatation of caudal artery, diarrhoea, reduced reflexes and hind-limb paralysis. Death occurred with doses up to 1 mg g 1 body weight. It is noteworthy that lipidsoluble liver extracts taken from parrotfish harvested from the h-rc area exhibited a slightly stronger toxicity in mice (e.g. severe hindlimb paralysis) compared to those caught within the safe area. The presence of CTX-like compounds in the lipid-soluble extracts of cyanobacteria and giant clams was studied by RBA, using site-receptor 5 of voltage-gated Na+ channels on rat brain synaptosomes (Fig. 6). Dose–response curves show that cyanobacterial extract (IC50 = 257.5  11.8 mg mL 1, n = 2) seemed to contain compounds 3-fold less efficient in binding to sodium channel than giant clam extract (IC50 = 78.7  10.5 mg mL 1, n = 2). CTX concentration in both extracts was estimated using purified P-CTX3C as an internal standard and expressed as P-CTX-3C equivalents for comparison purposes. Therefore, toxicity results were 2.41  0.11 and 7.95  1.06 pg P-CTX-3C equivalents/mg of extract for cyanobacteria and giant clam, respectively. The IC50 values (mg/mL of extract) in RBA for the HPLC fractions (Section 2.8) were the following: 415 (0–10 min), 733 (10–20 min), 619 (20–26 min), 69 (26–34 min), 146 (34–48 min) and 243 (48– 60 min). Under identical chromatographic conditions, the retention time of the standard CTXs was CTX-3B (31.18 min), CTX-3C (35.07 min), and CTX-4A (39.2 min). 4. Discussion The analysis of the data collected via the epidemiological questionnaire allowed us on the one hand, to identify a high-risk

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seafood catching area and the involved toxic species and on the second hand, to look for potential differences in clinical signs and symptoms observed in humans with regard to the seafood species consumption and catching area. The designation of an area as ciguatoxic by the natives of Hune¨te¨ is based on previous experiences of intoxication. It turned out to be accurate, as it correlates well with our results, starting with the environmental disturbances we observed during the snorkelling survey of the area. Although one cannot exclude that certain fish may move between the two areas, it has long been established that the distribution of Gambierdiscus is notoriously patchy, even on spatial scales of a few meters (Yasumoto et al., 1979). Although the home range of coral reef fish is poorly documented, it is generally assumed in fish behaviour studies that parrotfish have a restricted area of distribution even limited to a unique coral block, whereas Lethrinidae (e.g. emperor fish) are considered as mobile fishes capable of moving over relatively long distance. Few studies were carried out on cyanobacteria palatability for grazing or herbivorous fish; however more recent studies conducted in tropical oceans suggest that some benthic large mat-forming cyanobacteria occupying a significant portion of the available substrate might constitute an available resource and play ecological roles as food and shelter for marine mesograzers (CruzRivera and Paul, 2006). Despite the fact that cyanobacteria are well known to produce feeding deterrent compounds, some herbivorous fish can even so eat cyanobacteria; e.g. the rabbitfish (Siganus fuscescens) is known to feed upon L. majuscula (Capper et al., 2006). In addition, parrotfish have been claimed to feed on films of microscopic and filamentous algae which growth on hard substrate, consuming a great part of detritus and probably cyanobacteria (Randall, 2005). They are generally recognised as safe in New Caledonia, except for the big specimens that can potentially carry substantial level of toxins. The weak toxicity observed with parrotfish extracts could be explained by the small size of these specimens (around 500 g per fish), which have probably not accumulated sufficient toxin amount during their lifetime. A crucial issue that remains to address is the estimation of the half-life of marine biotoxins. The detoxification rate of CTXs in fish (through excretion and/or metabolism) is assumed to be very slow. Indeed, an estimation of the excretion of CTXs in a family of moray eels indicated a half-life of 264 days (Lewis and Holmes, 1993). Nevertheless, the depuration kinetics and persistence of PSTs in marine fish is unknown yet. Regarding molluscivorous fish such as emperor fish, their potential intoxication via giant clams or other filter-feeding molluscs remained to be assessed as they are generally considered to be safe and CTX-free (Laboute and Grandperrin, 2000). The co-occurrence of water-soluble and lipid-soluble toxins in extracts of marine pelagic cyanobacteria has already been reported (Endean et al., 1993). These authors established an apparent relationship between the toxins present in Trichodesmium erythraeum and those found in the flesh of a narrow-barred Spanish mackerel Scomberomorus commersoni implicated in a poisoning resembling CFP. It is noteworthy that the pelagic genus Trichodesmium, is not only morphologically similar, but also closely genetically related to the benthic genus Hydrocoleum (Abed et al., 2006), which was the subject of our study.

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can contaminate a great variety of marine animals such as oysters, scallops, clams and crustaceans and were responsible for numerous human intoxications (Llewellyn et al., 2006). For example, in Palau Islands, STX and other derivatives have been found in extracts of the giant clam (Tridacna crocea) caught in an area containing numerous Pyrodinium bahamense var compressa (Hwang, 2003). In this study, the presence of PSTs was detected by mouse bioassay in both giant clam and cyanobacteria extracts and by electrophysiological experiments with cyanobacteria extracts. These results agree with the burning sensation of mouth and throat, soon after ingestion of giant clams or some fishes. Indeed, PSTs are known to induce burning and tingling sensations on the lips and face within minutes of ingesting toxic bivalves. Sensations of numbness and paresthesia can spread to the arms and legs. Victims may experience incoherent speech, giddiness, motor difficulties, paralysis and respiratory failure (Llewellyn et al., 2006). As already pointed out, in south Pacific regions, the clinical symptoms of CFP are diverse, but typically involve short-term gastrointestinal disorders (3–6 h post-meal) followed by longer term sensory disturbances affecting the peripheral nervous system. However, more severe cases but fortunately rare (Bagnis et al., 1979; Legrand and Bagnis, 1991) are associated with an uncommon rapid onset of symptoms (just a few minutes postmeal). Nonetheless, it must be pointed out that such early symptoms have never been proven to be exclusively caused by CTX-producing Gambierdiscus spp. HPLC-fluorometric analysis according to the method of Lawrence et al. (2005) based upon a pre-column derivatization, failed to reveal STX and its main derivatives (dc-STX, neo-STX, GTX-1&4, GTX-2&3, GTX-5 or dc-GTX-2&3), therefore both in vivo and in vitro toxic effects must be attributed to the presence of one or more new toxins. Although the availability of meal remnants involved in human poisonings would probably confirm the presence of paralytic toxins. 4.2. Neurotoxins hypothesis Anatoxin-a, isolated from fresh-water cyanobacteria Anabaena flos-aquae and Oscillatoria sp. (Araoz et al., 2005), is an alkaloid with a low MW of 165 Da (Carmichael et al., 1979). A neurotoxic alkaloid with similar action in mice injected i.p. was partially purified from pelagic cyanobacteria belonging to the genus Trichodesmium (Hawser et al., 1991) and from water-soluble extracts of T. erythraeum and finally from the flesh of a pelagic fish Scomberomorus commersoni (Endean et al., 1993). Such a paralytic activity was exhibited by a small lipid-soluble toxin co-isolated with CTX from the gut content of parrotfish (Yasumoto et al., 1977b). The algae found in the gut contents being very low, it was concluded that algae were not the toxin producers. It is noteworthy, however, that freshwater cyanobacteria Oscillatoria spp., were shown to produce anatoxin-a, and were conclusively associated with illness and death of dogs (Edwards et al., 1992), and that consumption of giant clams in two Polynesian atolls (Fangatau and Pukarua) likewise caused death of dogs (personal communication). 4.3. Ciguatoxins hypothesis

4.1. Paralytic shellfish toxins hypothesis Freshwater cyanobacteria (genera Anabaena, Aphanizomenon and Cylindrospermopsis) and marine dinoflagellates (in the Tropics, Alexandrium tamarense, A. minutum, A. tamiyavanichi, Gymnodinium catenatum and Pyrodinium bahamense) are the only proven producers of PSTs. These toxins when produced by dinoflagellates

In our experiments with mice injected i.p., one of the observed signs was hind-limb paralysis which is characteristic of scaritoxin (Endean et al., 1993). This toxin was previously isolated from the flesh of the parrotfish Scarus gibbus (Bagnis et al., 1974; Chungue et al., 1977) and from viscera of a turban shell collected in Marcus Island, Japan (Yasumoto and Kanno, 1976). Its structure was later

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shown to be identical to CTX-4A isolated from cultures of the marine dinoflagellate Gambierdiscus toxicus (Fig. 7) (Satake et al., 1996). Similarly, a CTX-like compound was also isolated from Trichodesmium (Oscillatoria) erythraeum and from four species of molluscs (3 oysters and 1 snail) and one species of molluscivorous fish collected subsequently to blooms of this cyanobacterium (Hahn and Capra, 1992). Our results were in favour of toxins with a polarity closer to that of CTX-3C than CTX-4A (scaritoxin). Moreover, as they were first co-purified by molecular weight sieve effect on a Sephadex LH-20 resin column (according to the protocol used for the CTXs), we can reasonably assume that these giant clam lipid-soluble toxins must possess a MW of about 1000 Da. Subsequent reverse phase LC/MS analysis would fully characterize these compounds, or at least confirm their CTX-like nature by demonstrating compatible mass range and the characteristic pattern of ions formation of this class of ladder-like polyether compounds (multiple losses of water and formation of sodium and ammonium adducts). There are a limited number of toxin studies dealing with giant clams, Bagnis (1967) reported such intoxication cases with a triple vasomotor, digestive and nervous syndrome in the atoll of Bora Bora (Society Islands, French Polynesia) following the consumption of specimens caught in a limited area of the lagoon containing ciguateric fishes. A survey of the endemicity of CFP conducted in 1974 in the Gambier Islands (French Polynesia), established that 4% of the total cases of poisonings were due to giant clams consumption (Bagnis, 1974). Furthermore, Kanno et al. (1976) determined the occurrence of lipid-soluble toxins in the hepatopancreas of Tridacna maxima. Recently, our epidemiological survey in Raivavae (Australes archipelago, French Polynesia) brought to light human intoxications caused by giant clams harvested from an area designated to yield ciguatoxic fish (personal communication). Giant clams or other molluscs poisoning incidence rate would appear low at first sight, but it is probably underestimated as Pacific islanders have a strong tradition of eating a great variety of crustaceans, molluscs and fish so these latter could be wrongly accused of causing the illness.

These results suggested the simultaneous production of paralytic water-soluble toxins (likely PSP toxins or anatoxin-a) and lipid-soluble toxins (CTX-like toxins) (Fig. 7) by the benthic marine cyanobacteria Hydrocoleum spp., as earlier supposed by Endean et al. (1993) in pelagic marine cyanobacteria Trichodesmium erythraeum. These results were obtained with taxonomically impure field samples, so we cannot rule out the possible role of noncyanobacterial microorganisms in toxin production, but our overall observations (absence of dinoflagellates in any of our samples during these last 2 years) and tests support the production of toxin by these cyanobacteria. The common hypothesis on the occurrence of ciguateric areas through the succession of events such as ecological disturbances promoting the bleaching and the death of coral reefs, thus offering ‘‘new surfaces’’ for colonization of macro-algae that are finally used as supports by toxic dinoflagellates, is not confirmed by the facts observed in this study. In Hune¨te¨, broken branched corals amassed between the blocks of fringing reef flat were a substrate favourable to cyanobacterial blooms constituting new likely progenitors of ciguatoxins. Our results were in favour of a second trophic pathway of CTXs through the marine food chain via molluscs such as giant clams (Fig. 8). These toxins may also enter the food chain directly by grazer fish or via the molluscs by molluscivorous fish, this pathway would account for the strong involvement of grazer and herbivorous fish as well as for the burning sensation on the tongue and the throat, symptoms more characteristic of STX-like toxin poisonings. In order to confirm this hypothesis, toxins content of grazer and herbivorous fish specimens caught in area where exist large populations of Hydrocoleum spp. will be further investigated. Of course, in the case of Hune¨te¨, we cannot rule out the hypothesis of a double intoxication by microalgae and cyanobacteria despite the fact that during the environmental survey in the two last years, no Gambierdiscus cell was observed in any of the samples examined. But is it the same syndrome? Perhaps, in this precise case, we should rather talk about ‘‘Ciguatera Shellfish Poisoning’’ instead of Ciguatera Fish Poisoning, for the consumption of giant clams has induced the specific symptoms of CFP. Nevertheless, the links between cyanobacteria and fish or molluscs and fish are not well

Fig. 7. Chemical structures of involved toxins. (A) Anatoxin-a. (B) Anatoxin-a (s). (C) Saxitoxins. Saxitoxin (STX) and derivatives belong to a 20 member family with a tetrahydropurin structure and differ by the presence of H, OH, or S in position R1, R2 or R3 and by chemical groups carbamoyl, N-sulfocarbamoyl or decarbamoyl in position R4. (D) Ciguatoxins. P-CTX-1 is the major Pacific ciguatoxin. P-CTX-4A (=scaritoxin) is similar to GT-4B, a gambiertoxin, precursor of ciguatoxin, isolated from Gambierdiscus toxicus.

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experiments on PSP toxins. We thank Vincent Arnold and Morgane Lacombe (IRD) for their help in the environmental survey, and Dr. Isabelle de Fremicourt for the epidemiological data collection. Philippe Cruchet and Taina Revel from ILM are gratefully acknowledged for their skillful assistance in HPLC analysis and RBA experiments, respectively. We are much indebt to the tribe of Hune¨te¨, especially to Basie Ijezie (the small chief), Martial Ehnyimane (the chief of the fisher clan) and Antoine Holue for technical assistance.[SS]

References

Fig. 8. Schematic presentation of concentration and biotransformation passages of CFP substances along food chain in disturbed coral reef environments. The new hypothetic proposed pathway leading to human intoxication by ciguatoxins, anatoxin-a and Paralytic Shellfish Poisoning toxins is in bold.

established yet. After all, it is noteworthy that the term ‘‘cigua’’ is the Cuban name of a small turban shell Livona pica which produces a similar illness upon ingestion (Poey, 1866). 5. Conclusion To our knowledge, this is the first report of the biogenesis of PSTs, neurotoxins and CTX-like compounds by marine benthic cyanobacteria of the genus Hydrocoleum, and their possible implication in human poisonings. These mat-forming cyanobacteria are common in intertidal and shallow subtidal zones of New Caledonian and Polynesian lagoons (Abed et al., 2006), and this study strongly suggests that toxic benthic cyanobacteria may enter the food chain of bivalves such as giant clams, and molluscivorous and grazing fish (Lethrinidae and Scaridae, respectively), which are otherwise considered a low-risk species in New Caledonia, but may actually represent a potential threat to human health in areas harboring high population densities of cyanobacteria. Cyanobacteria are gaining considerable importance in coral reef ecosystems, worldwide. Like dinoflagellates, their proliferation could be related to anthropogenic factors such as deforestation, discharge of wastewaters and industrial activities. Incidences of Ciguatera appear to be on increase in the context of global warming and climate change (Hales et al., 1999; Chateau-Degat et al., 2005), and so are occurrences of massive blooms of cyanobacteria (e.g. Paul et al., 2005), potentially leading to an increasing number of severe seafood poisoning outbreaks. Our results call attention to the complexity of ciguatera-like fish poisoning and bring new insights on the overall variability as well as distinctions in symptoms and efficacy of medical treatment depending whether the toxin complex is produced by harmful algae or cyanobacteria. Although preliminary and requiring complementary studies on identification, characterization and distribution of the toxins as well as on the incidence of the cyanobacteria in the intertropical regions, our study stresses the need for forthcoming CFP risk assessment programs to include monitoring of cyanobacteria, in addition to the compulsory screening of well-known CFP-promoting dinoflagellates. Finally, this study will help providing some thinking in the ciguatera field with regards to ecology and biology to give some explanations on otherwise unknown phenomena and poisonings. Acknowledgements We are grateful to Fre´de´ric Grosso and Sophie Krys from AFSSA (Agence Franc¸aise de Se´curite´ Sanitaire des Aliments) for the HPLC

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