First Report of Ciguatoxins in Two Starfish Species: Ophidiaster ophidianus and Marthasterias glacialis

Toxins 2015, 7, 3740-3757; doi:10.3390/toxins7093740 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Article First Report of Ciguatoxins in Two Starfish Species: Ophidiaster ophidianus and Marthasterias glacialis Marisa Silva 1,2,†, Inés Rodriguez 3,†, Aldo Barreiro 1,2, Manfred Kaufmann 2,4,5, Ana Isabel Neto 2,6, Meryem Hassouani 7, Brahim Sabour 7, Amparo Alfonso 3, Luis M. Botana 3 and Vitor Vasconcelos 1,2,* 1 Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto 4619-007, Portugal; E-Mails: mari[email protected] (M.S.); [email protected] (A.B.) 2 Interdisciplinary Center of Marine and Environmental Research—CIMAR/CIIMAR, University of Porto, Rua dos Bragas, 289, Porto 4050-123, Portugal; E-Mails: mkaufm[email protected] (M.K.); [email protected] (A.I.N.) 3 Department of Pharmacology, Faculty of Veterinary, University of Santiago of Compostela, Lugo 27002, Spain; E-Mails: ines.rodri[email protected] (I.R.); [email protected] (A.A.); [email protected] (L.M.B.) 4 Centre of Life Sciences, University of Madeira, Marine Biology Station of Funchal, Funchal 9000-107, Portugal 5 Center of Interdisciplinary Marine and Environmental Research of Madeira—CIIMAR-Madeira, Edif. Madeira Tecnopolo, Caminho da Penteada, Funchal 9020-105, Portugal 6 Department of Marine Biology, University of Azores, Ponta Delgada 9501-801, Portugal 7 Phycology Research Unit—Biotechnology, Ecosystems Ecology and Valorization Laboratory. Faculty of Sciences El Jadida, University Chouaib Doukkali, El Jadida BP20, Morocco; E-Mails: [email protected] (M.H.); [email protected] (B.S.) † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: vm[email protected]; Tel.: +351-223-401-814; Fax: +351-223-390-608. Academic Editor: Michio Murata Received: 24 July 2015 / Accepted: 16 September 2015 / Published: 21 September 2015 Abstract: Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by OPEN ACCESS Toxins 2015, 7 3741 dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk. Keywords: ciguatera; new vectors; Madeira Island; São Miguel Island; Morocco 1. Introduction Ciguatoxins (CTXs) are lipophilic compounds that result from the biotransformation in finfish of precursor gambiertoxins produced by dinoflagellates from the genus Gambierdiscus [1–3]. Geographically, CTXs are typical for the tropical and sub-tropical regions of the Atlantic (C-CTX), Indian (I-CTX) and Pacific (P-CTX) Oceans [1,4–8]. Both the structure and potency of CTXs appear to be geographically determined, in resemblance to Maitoxin that shares the same producer, and to date more than 30 CTX analogues have been described [3–5,7,9–13]. CTXs are polyethers with rigid structure, formed by rings 13–14 fused by ether linkages (Figure 1) [2]. These potent biotoxins are odorless, tasteless, heat resistant and lipid soluble, so they are not destroyed by cooking processes [7]. Regarding action mechanism, CTXs bind to non-selective, non-voltage activated ion channels, causing their opening, leading to the increase of intracellular calcium levels to toxic concentrations [14]. In terms of symptomatology, during the acute period in the first 24 h gastrointestinal problems such as diarrhea, nausea, abdominal pain and vomiting appear. In addition, cardiovascular complications can occur. Neurologic symptoms that can emerge from within a few hours to two weeks after exposure, like paresthesias, dysesthesias, and hyperesthesias [15]. Ciguatera fish poisoning (CFP) is the most common type of intoxication syndrome, even in non-endemic areas, due to the large quantity of fish exports, and this makes it a worldwide concern [16]. The most common route of intoxication is through ingestion of contaminated fish, being estimated 10,000 to 50,000 intoxications per year [4,17]. Recent reports document the presence of CTXs as far Toxins 2015, 7 3742 north as the Mediterranean [18]. In addition, high abundance of Gambierdiscus cells are not visible as blooms, as in other harmful algal bloom species, and this hinders the process of monitoring and managing of these blooms [19]. Figure 1. Structures Caribbean (C) and Pacific (P) CTX-group toxin. The epimers, P-CTX-2 (52-epi P-CTX-3), P-CTX-4A (52-epi P-CTX-4B) and C-CTX-2 (56-epi C-CTX-1) are indicated in parenthesis. The common vectors for these phycotoxins are finfish, some mollusks (e.g., turban snail, Lunella cinerea, and in giant clams, Tridacna gigas), achieving higher concentrations in top predatory fish like groupers (Family: Serranidae), barracuda (Family: Sphyraenidae) and snapper (Family: Lutjanidae) [20]. Regarding the regulatory status in the European Union (EU), EFSA proposes the use of toxicity equivalency factors (TEFs), based on their acute intraperitoneal LD50 in mice, determining eleven values: P-CTX-1 = 1, P-CTX-2 = 0.3, P-CTX-3 = 0.3, C-CTX-2 = 0.3, P-CTX-3C = 0.2, 2,3-dihydroxy PCTX-3C = 0.1, 51-hydroxy P-CTX-3C = 1, P-CTX-4A = 0.1, C-CTX-1 = 0.1 and P-CTX-4B = 0.05. In the same scientific opinion on CTXs, EFSA claims that a concentration of 0.01 μg P-CTX-1 equivalents/kg of fish as expectable not to exert effects in sensitive individuals. In addition, due to very few reported cases for CTX occurrences European markets no limits nor ARfD (acute reference dosage) have been established until now, nor advisement regarding analytical methodologies to use [1]. Nevertheless, Commission Regulation (EC) nr 854/2004stated mandates that checks have to be made to ensure that fishery products containing CTX are forbidden to enter the market [21]. Other countries like the United States of America, have safety levels (<0.1 μg/kg C-CTX-1 equivalents and <0.01 μg/kg P-CTX-1 equivalents), other countries opted for more radical measures, like Japan that banned the importation of some fish species reported as CFP vectors (e.g., barracuda) [1,22]. Toxins 2015, 7 3743 The aim of our work was to search for new vectors, to better access the human health risk. We screened the Madeira Island (Madeira archipelago), São Miguel Island (Azores archipelago), and the Moroccan coast by scuba diving expeditions and intertidal harvesting of 22 edible and inedible species. The inedible species were collected due to their importance in the food chain. We hope that our work contributes to the development and establishment of monitoring procedures as well as legislation in the EU to better protect consumers and public health. 2. Results and Discussion In this work, a total of 94 samples were analyzed for CTX plus its analogues from 14 sampling points distributed in three different locations: Madeira Island (Figure 2), São Miguel Island (Figure 3) and along the northwestern Moroccan Coast (Figure 4). Twenty-two species of benthic organisms, including bivalves (mussels), gastropods (sea-snails, sea-slugs and limpets), echinoderms (starfishes, sea-urchins and sea-cucumbers) and crustaceans (barnacles) were collected. The decision to sample these particular species was linked to the fact we wanted to screen edible and commercially important species and inedible species for their importance in the trophic chain. The number of samples collected and average number of specimens needed to set a pooled sample are detailed (Table 1). Figure 2. Location of the sampling points: (A) São Miguel island coast, Azores archipelago: 1, Cruzeiro; 2, Mosteiros; 3, Étar; 4, São Roque; 5, Lagoa; and 6, Caloura. (B) Madeira island coast: 1, Reis Magos and 2, Caniçal. (C) Northwestern Moroccan coast: 1, Casablanca Corniche; 2, El Jadida Haras; 3, El Jadida Sâada; 4, Sidi Bouzid; 5, Mrizika; and 6, Oualidia. Toxins 2015, 7 3744 Table 1. Species sampled and their trophic level, average number of specimens comprising a pooled sample (AvNr) and number of samples collected (NrP Samples) on Madeira Island in September 2012, São Miguel Island, Azores, in June 2013 and Morocco in July 2013 and their edibility. Availability of animals is dependent on their geographical distribution and ecology. Species Trophic Level Sampling Site(s) NrP Samples AvNr Edible Ref. Aplysia depilans Grazer Morocco 3 1 No [23] Arbacia lixula Grazer Madeira/Azores/Morocco 9 4 No [24] Charonia lampas 3rd level predator Madeira/Morocco 3 1 Yes [25] Cerithium vulgatum Grazer Morocco 1 40 Yes [26] Diadema africanum Grazer Madeira 2 1 No [27] Echinaster sepositus 2nd level predator Madeira 1 3 No [28] Gibbula umbilicalis Grazer Morocco 3 100 Yes [29] Holothuria(Platyperona)sanctori Deposit feeder Morocco 4 1 Yes [30,31] Marthasterias glacialis 2nd level predator Madeira/Azores/Morocco 8 1 No [32] Monodonta lineata Grazer Morocco 5 86 Yes [29] Mytilus spp. Filter feeder Morocco 4 30 Yes [33] Onchidella celtica Grazer Morocco 1 50 No [34] Ophidiaster ophidianus Detritivorous Madeira/Azores 5 1 No [28] Pattela aspera Grazer Madeira 2 15 Yes [32] Patella spp. Grazer Morocco 4 12 Yes [32] Pattela candei Grazer Azores 3 10 Yes [32] Paracentrotus lividus Grazer Madeira/Azores/Morocco 7 1 Yes [35] Pollicipes pollicipes Filter feeder Morocco 3 35 Yes [32] Sphaerechinus granularis Grazer Azores 4 1 Yes [36] Umbraculum umbraculum Grazer Madeira 1 1 No [37] Stramonita haemostoma 2nd level predator Madeira/Azores/Morocco 5 15 No [38] After sample extraction, the CTXs profile was determined in each sample by Ultra Performance Liquid Chromatography- Mass Spectometry (UPLC-MS) method. Initially, samples were screened by UPLC-MS in Selected Ion Monitoring (SIM) mode for 20 CTXs analogues described in the literature (Table 2) and in seven samples some suspicious peak was detected, all of them from starfish. Table 2. Screened CTXs UPLC-MS in Positive Mode. Compound Name Mass Polarity I-CTX-3 and I-CTX-41157.6 Positive Unknown CTX 1143.6 Positive Caribean-CTX 1141.7 Positive C-CTX-1127 1127.6 Positive CTX-1B 1111.6 Positive 54-deoxy-CTX-1B 52-epi-54-deoxy-CTX-1B 1095.6 Positive M-CTX-4A and M-CTX-4B 1079.6 Positive CTX-4A and CTX-4B 1061.6 Positive MTX small 1060 Positive Toxins 2015, 7 3745 Table 2. Cont. Compound Name Mass Polarity 2,3-OH-CTX-3C M-CTX-3C methylacetal 1055.6 Positive 2-OH-CTX-3C and M-CTX-3C 1041.6 Positive Analogs CTX 1040.6 Positive 51-OH-CTX-3C 1039.6 Positive 49-epo-CTX-3C and CTX-3C 1023.6 Positive Unknown CTX 1159.6 Positive Afterwards, the scan of each sample was carefully analyzed, checking the characteristic mass pattern fragmentation of CTX; that is, the formation of sodium and ammonium adducts and losses of water. In this sense, Figure 3 shows the SIM and the spectrum of CTX-3C standard. The mass spectrum from Figure 3B showed the ion CTX3C [M + H] + m/z 1023.5, and the water losses m/z 1005.5 associated with [M + H − H 2 O] + and m/z 987.5 due to second water lost [M + H − 2H 2 O] + . In addition, the sodium adduct [M + Na] + m/z 1045.7 is also detected. Figure 3. Selected Ion Monitoring (SIM) chromatogram (A) and mass spectrum (B) of standard CTX-3C. SIM obtained by UPLC-MS/MS and spectrum obtained by UPLC-MS-IT-TOF. Toxins 2015, 7 3746 Through this analysis, three CTX analogues were detected. From SIM data of samples, m/z 1111.5 was found and from scan mass mode (m/z 800–1300) of samples, two molecules with m/z 1109.5 and m/z 1123.5 were found. The mass spectrum of these molecules (Figure 4) shows the typical fragmentation pattern of CTX-like compounds, with several losses of water and adducts of ammonium and sodium. The mass spectrum in Figure 4A shows two intense masses, one ([M + H] + ) at m/z 1111.5 and other ([M + Na] + ) at m/z 1133.5. Moreover, five losses of water were observed while the ammonium adduct did not appear, probably due to its low intensity. Therefore CTX-1B, m/z 1111.5, could be proposed, although not confirmed due to the absence of a standard. The mass spectrum in Figure 4B shows an intense mass ([M + NH 4 ] + ) at m/z 1116.6 associated with ammonium adduct from m/z 1109.5. The other three masses shown in the spectrum correspond to one, two and three losses of water at m/z 1099.6 and 1073.5 and 1055.5, respectively, from m/z 1109.5 molecule. The mass spectrum in Figure 4C shows an ion at [M + H] + m/z 1123.5 and two intense masses, [M + NH 4 ] + at m/z 1140.5 and [M + Na] + at m/z 1145.5. In addition, the four losses of water and the potassium adduct of m/z 1123.5 were also detected in the spectrum. Figure 4. Cont. Toxins 2015, 7 3747 Figure 4. MS2 spectrum of CTX analogue at m/z 1111.5 [M+H] + (A); CTX analogue at m/z 1109.5 [M+H] + (B); and CTX analogue m/z 1123.5 [M+H] + (C). Spectra obtained by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight UPLC-MS-IT-TOF. Once the presence of CTX analogues was identified, the toxin amount was quantified (Table 3). In order to calculate concentrations of CTX analogues, it was assumed that related analogues would give a similar response to that of CTX-3C, because of this, and since there is an absence of correspondence with EFSAs, TEFs results will be expressed in μg CTX-3C equivalents/Kg. Therefore, the calibration curve was done with CTX-3C standard. The quantification of these toxins was carried out using SIM acquisition in positive mode. For CTX-3C standard, a good seven-point calibration curve with range between 18.75–600 ng/mL was obtained (R2 = 0.999). The limit of detection (LOD) was 1.125 µg/Kg and the limit of quantification (LOQ) was 3.75 µg/Kg. Table 3. Presumed CTX detected in UPLC-MS (FW-Fresh weight). Sample Location Species 1111 (μg CTX-3C Equivalents/KgFw) 1123 (μg CTX-3C Equivalents/KgFw) 1109 (μg CTX-3C Equivalents/KgFw) 341 #1 Madeira O. ophidianus 21.55 7.24 341 #2 Madeira O. ophidianus 46.49 29.80 411 #2 Azores M. glacialis <LOQ <LOQ 412 Azores O. ophidianus 19.73 424 Azores O. ophidianus 124.04 30.90 426 #1 Azores M. glacialis 7.92 435 Azores O. ophidianus 58.78 4.40 Only 7.45% of the 94 samples analyzed were quantifiable for CTX. All of the measurable samples were one of two starfish species, O. ophidianus and M. glacialis, from Portuguese territory. This is the first report of CTX in echinoderms. Average concentrations detected ranged from 4.40 μg CTX-3C equivalents/Kg fresh weigh (fw), to 124.04 μg CTX-3C equivalents/Kg fw in O. ophidianus, if it were Toxins 2015, 7 3748 possible to apply EFSAs, TEFs both would be above the U.S. American safety level (<0.1 μg/kg C-CTX-1 equivalents and <0.01 μg/kg P-CTX-1 equivalents) [22]. Regarding statistics, the first step of the gamma hurdle model was a GLZ performed with the data of presence/absence of CTX with Binomial distribution of the error. The results of the model’s analysis of deviance are shown in Table 4, as well as the coefficients rescaled to a logistic probability (0,1). Sampling site had no significant effect. Among sampling sites, the coefficients showed the lowest probability of occurrence in Morocco, and the highest in Madeira. The second part of the model, which considers the variation in CTX concentration, analyzed with a Gamma distribution of the error among those samples showing positive results, did not show significance of sampling site as factor either, although its p value was closer to significance (Table 4). Looking at model coefficients, it is clear that the largest concentrations found occurred in Azores Islands, and lowest in Morocco (zero). This differs from the probability results of the presence/absence data. The analysis performed with starfish species as factor (Table 5) showed significant differences in both the presence/absence and the analysis of the positives (differences in putative CTX concentrations), conclusively, O. ophidianus had both higher probability of showing a positive result, and significantly higher amounts of toxin per body mass. This can be explained by to their distinct feeding habits, since O. ophidianus is a detritivorous acquiring CTX from the sediment bed, against the predatory ones from M. glacialis. In addition, Llewellyn (2010) suggested that the rise of water temperature, derived by climate change, can increase the incidence of CFP in Papua New Guinea from 35–70 per thousand people in 1990 to 160–430 per thousand people in 2050 [39]. In addition, it is known that the rise of water temperature can influence the growth rate of the producers [40], toxin production as well as the uptake rate of the vectors [41,42], and that could be the main reason of the differences between sampling sites, though Morocco and Madeira are at the same latitude. Nevertheless, the main species that we detected quantifiable amounts of CTX is absent in Morocco. Since Madeira and Azores archipelagos present oligotrophic waters, making these ecosystems poor in bivalves, the red starfish (O. ophidianus) presents a possible alternative as key species for CTX monitoring. We have to interpret the results from our models carefully, since the actual number of data is low. This may affect the non-significance of the models from sampling sites (Table 4) where the differences found might become significant by simply adding more data. 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Available online: http://www.R-project.org/ (accessed on 2 June 2015). © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).