toxins Article An Update on Ciguatoxins and CTX-like Toxicity in Fish from Different Trophic Levels of the Selvagens Islands (NE Atlantic, Madeira, Portugal) Pedro Reis Costa 1,2,* , Pablo Estévez 3 , Lucía Soliño 1,2 , David Castro 3, Susana Margarida Rodrigues 1, Viriato Timoteo 4, José Manuel Leao-Martins 3 , Carolina Santos 5, Neide Gouveia 4,†, Jorge Diogène 6,* and Ana Gago-Martínez 3,* 1 IPMA—Portuguese Institute of the Sea and Atmosphere, Av. Brasília, 1449-006 Lisbon, Portugal; [email protected] (L.S.); [email protected] (S.M.R.) 2 CCMAR—Centre of Marine Sciences, Campus of Gambelas, University of Algarve, 8005-139 Faro, Portugal 3 Biomedical Research Center (CINBIO), Department of Analytical and Food Chemistry, Campus Universitario de Vigo, University of Vigo, 36310 Vigo, Spain; [email protected] (P.E.); [email protected] (D.C.); [email protected] (J.M.L.-M.) 4 Regional Fisheries Management—Madeira Government, DSI-DRP, Estrada da Pontinha, 9004-562 Funchal, Madeira, Portugal; [email protected] (V.T.); [email protected] (N.G.) 5 Instituto das Florestas e Conservação da Natureza, IP-RAM, Secretaria Regional do Ambiente, e Recursos Naturais e Alterações Climáticas, Regional Government of Madeira, Rua João de Deus, n.º 12 E/F, R/C-C, 9050-027 Funchal, Madeira, Portugal; [email protected] 6 IRTA, Ctra Poble Nou km 5.5, 43540 Sant Carles de la Ràpita, Spain * Correspondence: [email protected] (P.R.C.); [email protected] (J.D.); [email protected] (A.G.-M.) † Present address: Direção Regional de Agricultura e Desenvolvimento Rural, Citation: Costa, P.R.; Estévez, P.; 9000-254 Funchal, Madeira, Portugal. Soliño, L.; Castro, D.; Rodrigues, S.M.; Timoteo, V.; Leao-Martins, J.M.; Abstract: The Selvagens Islands, which are a marine protected area located at the southernmost point Santos, C.; Gouveia, N.; Diogène, J.; of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and et al. An Update on Ciguatoxins and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried CTX-like Toxicity in Fish from Different Trophic Levels of the out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from Selvagens Islands (NE Atlantic, different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) Madeira, Portugal). Toxins 2021, 13, and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). 580. https://doi.org/10.3390/ Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as toxins13080580 zebra seabream (Diplodus cervinus) and barred hogfish (Bodianus scrofa), reaching levels up to 0.75 µg CTX1B equivalent kg−1. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but Received: 10 July 2021 discrepancies between CBA and LC-MS/MS in D. cervinus and top predator species, such as the Accepted: 17 August 2021 yellowmouth barracuda (Sphyraena viridis) and amberjacks (Seriola spp.), suggest the presence of fish Published: 20 August 2021 metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for Publisher’s Note: MDPI stays neutral fisheries and environmental management of the Selvagens Islands. with regard to jurisdictional claims in published maps and institutional affil- Keywords: ciguatoxins; Selvagens Islands; seafood safety; Gambierdiscus; ciguatera iations. Key Contribution: This article reports the first comprehensive evaluation of CTX in fish (n = 56) from the Selvagens Islands (Madeira, Portugal). Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Ciguatera poisoning (CP) is one of the most common nonbacterial illnesses associated Attribution (CC BY) license (https:// with fish and other seafood consumption worldwide. The illness has been known since the creativecommons.org/licenses/by/ 16th century when ciguateric-like poisonings affected classic sea explorers, such as captains 4.0/). of the Spanish navy in the Gulf of Guinea in 1521 or the crew of captain James Cook in his Toxins 2021, 13, 580. https://doi.org/10.3390/toxins13080580 https://www.mdpi.com/journal/toxins Toxins 2021, 13, 580 2 of 12 second voyage to the South Pacific in 1772–1775 [1,2]. CP is widespread in the tropical and subtropical regions and particularly associated with the consumption of reef fish species that have bioaccumulated ciguatoxins (CTXs). These compounds are potent neurotoxins that act at the voltage-gated sodium channels (VGSC) increasing ion permeability and cell disruption leading to persistent neurological impairment [3]. The neurological symptoms include paraesthesia, dysesthesia, vertigo, and sensory abnormalities such as metallic taste, pruritus, arthralgia, myalgia, dental pain, and cold allodynia, a pathognomonic CP symptom that is characterized by burning pain in response to a cold stimulus [4–6]. Additionally, a variety of gastrointestinal symptoms including abdominal pain, nausea and vomiting, and cardiovascular symptoms, such as heart rhythm disturbances, may also affect poisoned patients. Some symptoms may become chronic and in extreme cases of severity CP may cause the death of patients [7]. The source of CTXs was linked to benthic dinoflagellates of the genus Gambierdiscus in the late 1970s [8,9]. Gambierdiscus toxicus was the first species described in the genus and, for a certain period, the only species known of this genus. Although the Gambierdiscus genus remains largely understudied, so far, at least 21 species have been described. Gam- bierdiscus and Fukuyoa are epibenthic dinoflagellates, which can be ingested by herbivorous fish. Some CTXs are produced by these dinoflagellates, but toxin congeners are products of fish metabolism. Gambierdiscus and Fukuyoa dinoflagellates are endemic to tropical and subtropical regions which are coincident with the world’s highest incidence areas of ciguat- era, namely the South Pacific, the Indian Ocean, and the Caribbean Sea [10]. Nevertheless, increasing occurrence and spread of Gambierdiscus to temperate regions has been reported and seems to be favored by the climate warming trends [11,12]. Coincident with CP out- breaks, Gambierdiscus have been detected in NE Atlantic subtropical-temperate regions, such as the Canary and Madeira Islands, and even in the Mediterranean Sea [13–17]. The first human poisonings in Europe due to the consumption of autochthonous fish occurred in 2004, in the Canary Islands, Spain [18]. Since then, several outbreaks have been reported in the Canary Islands and also in the nearby Portuguese islands, i.e., Madeira and Selvagens [19]. The first reported outbreak in Portugal dates from 2008 when 11 persons reported CP symptoms after consuming a 30 kg amberjack (Seriola sp) caught around the Selvagens Islands [20,21]. The symptoms reported by the crew members matched with symptoms previously reported by nature wardens of the natural park of the Selvagens Islands. CP was retrospectively diagnosed in the six wardens who had consumed fish caught locally [20]. In the following year, CP symptoms were observed in 20–30 people that ingested amberjack purchased in the Canary Islands but caught in the Selvagens Islands [22]. The Selvagens Islands are at the southernmost point of the Portuguese maritime area, located in the temperate-subtropical northeast Atlantic, closer to the Spanish Tenerife Island than to the Madeira Island, and 600 km from the African continental coast of Morocco (Figure1). These small islands have a high fish species diversity and abundance, including important commercial species [23,24]. The total fish biomass has been estimated to be 3.2 times higher in the Selvagens Islands than in the Madeira habitat, and 10 times higher, when only top predators’ biomass were considered [24]. The Selvagens Islands are a unique ecosystem, considered to be one of the last remaining intact ecosystems of the eastern Atlantic Ocean [25]. Despite several human poisonings associated with fish caught in waters surrounding the Selvagens Islands, only a limited number of studies have tried to assess and investigate the presence of CTX in fish from this potential ciguateric hotspot. Following the first human poisoning, in 2008, Otero and colleagues [21] analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) two amberjacks (S. dumerili and S. fasciata) caught in the Selvagens Islands and described a suite of toxins composed of several CTX, including CTX1B, CTX3C, and C-CTX1. At the same time, Boada et al. [22] reported the presence of C-CTX1 in amberjack specimens associated with human poisonings in Tenerife but that had been caught in the Selvagens Islands. A retrospective analysis carried out by Toxins 2021, 13, x FOR PEER REVIEW 3 of 12 Toxins 2021, 13, 580 3 of 12 Boada et al. [22] reported the presence of C-CTX1 in amberjack specimens associated with human poisonings in Tenerife but that had been caught in the Selvagens Islands. A ret- state-of-the-artrospective analysis LC-MS/MS carried instruments out by state-of-t to thesehe-art specimens LC-MS/MS confirmed instruments that C-CTX1 to these was spec- theimens main confirmed toxin analogue that C-CTX1 but also was suggested the main the to presencexin analogue of several