Elsevier

Harmful Algae

Volume 99, November 2020, 101913
Harmful Algae

Gambierdiscus and Fukuyoa as potential indicators of ciguatera risk in the Balearic Islands

https://doi.org/10.1016/j.hal.2020.101913Get rights and content

Highlights

  • G. australes and F. paulensis are well distributed and established in the balearic Islands, a region free of ciguatera poisoning.

  • Overall, low CTX-like toxicity was detected in G. australes and F. paulensis strains.

  • Presence of MTX-like activity was detected in G. australes strains.

Abstract

Gambierdiscus and Fukuyoa are genera of toxic dinoflagellates which were mainly considered as endemic to marine intertropical areas, and that are well known as producers of ciguatoxins (CTXs) and maitotoxins (MTXs). Ciguatera poisoning (CP) is a human poisoning occurring after the consumption of fish or more rarely, shellfish containing CTXs. The presence of these microalgae in a coastal area is an indication of potential risk of CP. This study assesses the risk of CP in the Balearic Islands (Western Mediterranean Sea) according to the distribution of both microalgae genera, and the presence of CTX-like and MTX-like toxicity in microalgal cultures as determined by neuro-2a cell based-assay (neuro-2a CBA). Genetic identification of forty-three cultured microalgal strains isolated from 2016 to 2019 revealed that all of them belong to the species G. australes and F. paulensis. Both species were widely distributed in Formentera, Majorca and Minorca. Additionally, all strains of G. australes and two of F. paulensis exhibited signals of CTX-like toxicity ranging respectively between 1 and 380 and 8–16 fg CTX1B equivalents (equiv.) • cell−1. Four extracts of F. paulensis exhibited a novel toxicity response in neuro-2a cells consisting of the recovery of the cell viability in the presence of ouabain and veratridine. In addition, G. australes showed MTX-like toxicity while F. paulensis strains did not. Overall, the low CTX-like toxicities detected indicate that the potential risk of CP in the Balearic Islands is low, although, the presence of CTX-like and MTX-like toxicity in those strains reveal the necessity to monitor these genera in the Mediterranean Sea.

Introduction

Gambierdiscus (Adachi and Fukuyo, 1979) and Fukuyoa (Gómez et al., 2015) (Dinophyceae) are marine benthic dinoflagellates that live attached to different substrates such as macroalgae, corals, rocks and sands in well-illuminated habitats but also at very low light levels (>45 m depth) (Tester et al., 2013). Historically, the genera Gambierdiscus and Fukuyoa were known to be distributed primarily in tropical and subtropical areas of the Caribbean Sea, the Pacific and Indian Ocean. However, in recent decades, both genera have been reported in warm-temperate areas. The genus Gambierdiscus was recently recorded in the North East Atlantic Ocean (Fernández-Zabala et al., 2019; Fraga et al., 2011; Rodríguez et al., 2017), North West Atlantic Ocean (Litaker et al., 2009), South West Atlantic (Nascimento et al., 2015), the Mediterranean Sea (Aligizaki and Nikolaidis, 2008; Tudó et al., 2018), the Red Sea (Catania et al., 2017), Sea of Japan (Jang et al., 2018) and the South Pacific Ocean (Kohli et al., 2014a; Larsson et al., 2018). In contrast species of the genus Fukuyoa (formerly within the genus Gambierdiscus), have been reported in the Atlantic Ocean (Gómez et al., 2015), the Mediterranean Sea (Laza-Martínez et al., 2016; Aligizaki et al., 2018), the South Pacific Ocean (Rhodes et al., 2017), the China Sea and the Asia Pacific region (Larsson et al., 2019, 2018; Leung et al., 2018).

Gambierdiscus and Fukuyoa produce multiple secondary metabolites, among which are included ciguatoxins (CTXs) and maitotoxins (MTXs) (Chinain et al., 2010; Holmes et al., 1990; Lewis and Holmes, 1993; Munday et al., 2017; Satake et al., 1996). CTXs are lipophilic polyethers, that bind to voltage-gated sodium channels (VGSCs), thereby inhibiting the inactivation process of VGSCs resulting in intracellular sodium increase (Hidalgo et al., 2002; Molgó et al., 1993; Nicholson and Lewis, 2006; Strachan et al., 1999). Moreover, CTXs are potassium channel inhibitors (Inserra et al., 2017). MTXs are amphiphilic polyethers that bind to Ca2+ independent voltage gated channels and non-selective ion channels causing an increase of intracellular Ca2+ (Reyes et al., 2014).

CTXs in fish or invertebrates are responsible for the human intoxication known as Ciguatera Poisoning (CP) (Bagnis, 1993; Bagnis et al., 1980). CTXs enter marine food webs through invertebrates and herbivorous fish, where they may be biotransformed along the food webs and bioaccumulated at different trophic levels, eventually reaching humans (Bagnis et al., 1980; Yasumoto et al., 1977). Regarding MTXs, their implication in CP is unlikely. Although its intraperitoneal administration in mice is more toxic than CTXs, their oral potency is almost non-detectable (Munday et al., 2017). In addition, their bioaccumulation along the food webs is low (Litaker et al., 2010; Munday, 2014; Yasumoto et al., 1971) and they have not been found in the tissue of fish involved in CP cases. However, snapper (Chrysophrys auratus) (previously Pagrus auratus), that had been experimentally fed with G. australes contained MTXs in their viscera, liver and muscle (Kohli et al., 2014b).

Although, epidemiological records of CP are not available at a global level, it is estimated that CP affects between 25,000 – 500,000 people per year (Fleming et al., 1998; Friedman et al., 2017; Skinner et al., 2011). CP effects include gastrointestinal, neurological, and cardiovascular symptoms, and the latter two can last for months or years (Friedman et al., 2017). Fatal cases of CP are rare (Chan, 2016; Diogène et al., 2017). CP occurs mainly in tropical and subtropical areas (35 °N - 35 °S), but in more recent decades, CP cases have been reported in temperate areas, previously free of CP (Bravo et al., 2015; Chinain et al., 2019; Gouveia et al., 2010).

In the Mediterranean Sea, the presence of CTXs in fish, or confirmed CP cases have not been demonstrated. Follow-up investigations of previous descriptions of CP cases in the eastern Mediterranean did not find CTXs in fish tissue (Bentur and Spanier, 2007; Herzberg, 1973; Raikhlin-Eisenkraft and Bentur, 2002; Raiklin-Eisenkraft et al., 1988; Spanier et al., 1989). The detection of possible CTX-compounds in Siganus sp. by Bentur and Spanier (2007) was performed using a Cigua-Check strip test, which was later considered unreliable (Bienfang et al., 2011). In addition, the clinical symptoms described, including hallucinations, are rare in CP cases (Chinain et al., 2019) and they are indicative of ichthyoallyeinotoxism, which is often mistaken for cases of CP (De Haro and Pommier, 2006).

At present, five confirmed species of the genus Gambierdiscus and Fukuyoa live in the Mediterranean Sea (Aligizaki et al., 2018; Laza-Martínez et al., 2016; Litaker et al., 2009; Tudó et al., 2018). The presence of certain CTX-producing species in the area can be indicative of a higher risk of CP in comparison to areas where they are absent (Chinain et al., 2019, 2010; Friedman et al., 2017). Nonetheless, evaluating CTX-production by these species is important to estimate the risk, since CTX production varies accordingto species, and high and low CTX-producers species have been characterized (Litaker et al., 2017; Pisapia et al., 2017). For the estimation of CTX production in Gambierdiscus, growth phases and strain variability among isolates of the same species have to be taken into account (Reverté et al., 2018; Rossignoli et al., 2020).

The goal of this study was to assess the potential risk of CP based on the presence in the Balearic Islands of the genera Gambierdiscus and Fukuyoa (Western Mediterranean Sea), and their potential production of compounds with CTX-like and MTX-like activity. This is the first study that provides information about the risk of CP in the Balearic Islands, according to the presence of the genera Gambierdiscus and Fukuyoa in several sampling locations, and their evaluation of toxin production of several strains.

Section snippets

Reagents and equipment

CTX1B was provided by Dr. Lewis, University of Queensland (Lewis et al., 1991). Neuroblastoma murine cells (neuro-2a) were purchased from ATCC LGC standards (USA). Poly-l-lysine, foetal bovine serum (FBS), l-glutamine solution, ouabain, veratridine, phosphate buffered saline (PBS), penicillin, streptomycin, RPMI-1640 medium, sodium pyruvate, thiazolyl blue tetrazolium bromide (MTT) and SKF96365 were purchased from Merck KGaA (Germany). Dimethyl sulfoxide (DMSO) and absolute methanol were

Presence of Gambierdiscus and Fukuyoa genera in the Western Mediterranean Sea

Presence of Gambierdiscus and Fukuyoa genera was assessed using samples from live and Lugol's iodine preserved samples collected during 2016 to 2019. A total of 110 isolates from the genera Gambierdiscus and Fukuyoa were obtained from the epiphytic samples and 26 isolates from the epilithic samples. Epiphytic samples were obtained from macrophytes of the genera Lobophora, Cystoceira, Jania, Padina, and Dictyota. Furthermore, other dinoflagellates co-occurred with the Gambierdiscus and Fukuyoa

Discussion

The presence of the genus Gambierdiscus in the Eastern Mediterranean Sea was reported in 2003 (Aligizaki & Nikolaidis, 2008). Reported species include G. carolinianus (Holland et al., 2013), Gambierdiscus sp., G. cf. belizeanus and G. silvae (Aligizaki et al., 2018). G. australes was detected later in the Balearic Islands, as presented in a brief communication (Tudó et al., 2018). The first detection of the genus Fukuyoa was in 2016 in the Western Mediterranean Sea (Laza-Martínez et al., 2016)

Conclusions

Fukuyoa and Gambierdiscus cells found in samples from the Balearic Islands from 2016 to 2019 have been identified as F. paulensis and G. australes. These two species seem to be well-established in the area. Considering the other studies, CTX-toxicity exhibited by most of the G. australes and F. paulensis strains was low. However, one strain of G. australes (IRTA-SMM-17–168) was classified as a very high producer in comparison to previous studies. In addition, it is not possible to discard that

Authors contribution

Conceptualization A.T. (Àngels Tudó), M.C. (Mònica Campàs), M.F. (Margarita fernández-Tejedor) and J.D. (Jorge Diogène); methodology A.T., A.T.F. (Anna Toldrà), M.R. (María Rey) and I.T. (Irene Todolí); data curation A.T., K.A. (Karl B. Andree), M.F., J.D. formal analysis A.T., K.A., M.F., M.C., F.S. (Francesc X. Sureda) and J.D.; writing original draft preparation A.T., M.C., A.T.F and J.D.; writing, review and editing A.T., A.T.F., M.R., M.C., K.A., M.F., F.S. and J.D. All authors have read

Declaration of Competing Interest

The authors declare that there is no conflict of interest.

Acknowledgements

The authors acknowledge José Luis Costa and Vanessa Castan for their assistance in the samplings and to Ferran Pellisé for sampling in Menorca. We would like to thank to José Manuel Fortuño from SEM facilities from Institut Ciències del Mar (CSIC). The authors acknowledge the financial support from the European Food Safety Authority (EFSA) through the EUROCIGUA project (GP/EFSA/AFSCO/2015/03) and the Ministerio de Ciencia, Innovación y Universidades (MICINN), the Agencia Estatal de

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