Toxic marine microalgae and noxious blooms in the Mediterranean Sea: A contribution to the Global HAB Status Report.

We review the spatial distribution of toxic marine microalgal species and the impacts of all types of harmful algal events (Harmful Algal Blooms, HABs) in the Mediterranean Sea (MS), including the Black Sea, the Sea of Marmara, coastal lagoons and transitional waters, based on two databases compiled in the Ocean Biogeographic Information System (OBIS). Eighty-four potentially toxic species have been detected in the MS (2,350 records), of which 16 described from these waters between 1860 and 2014 and a few suspected to have been introduced. More than half of these species (46) produce toxins that may affect human health, the remainders ichthyotoxic substances (29) or other types of toxins (9). Nevertheless, toxicity-related events are not frequent in the MS (308 records in 31 years), and mainly consist of impacts on aquaculture, caused by the dinoflagellates Dinophysis and Alexandrium, along with a few actual shellfish poisoning cases. Pseudo-nitzschia blooms are widespread, but domoic acid in shellfish rarely exceeds regulatory levels. Fish kills are probably less sporadic than reported, representing a problem at a few places along the southern MS coasts and in the Ebro River Delta. Since the last decade of the 20th century, blooms of the benthic dinoflagellates Ostreopsis cf. ovata have regularly occurred all along rocky shores of the MS, at times with human health problems caused by toxic aerosol. New records of Gambierdiscus and Fukuyoa, until now reported for the westernmost and easternmost MS coasts, raise concerns about the risk of ciguatera, a syndrome so far known only for subtropical and tropical areas. Recent discoveries are the dinoflagellates Vulcanodinium rugosum, responsible for the presence of pinnatoxins in French lagoons' shellfish, and the azaspiracid-producers Azadinium spp. Mucilages and discolorations have a major impact on tourism in summer. Reports of toxic species and HABs have apparently increased in the MS over the last half century, which is likely related to the increased awareness and monitoring operations rather than to an actual increase of these phenomena. Indeed, while the case of Ostreopsis appears as a sudden upsurge rather than a trend, no actual increase of toxic or noxious events has so far emerged in intensively studied areas, such as the French and Spanish coasts or the Adriatic Sea. Moreover, some cases of decrease are reported, e.g., for Alexandrium minutum blooms disappearing from the Harbour of Alexandria. Overall, main HAB risks derive from cases of massive development of microalgal biomass and consequent impacts of reduced coastal water quality on tourism, which represents the largest part of the marine economy along the MS coasts.

Phytoplankton community structure changes during autumn and spring in response to environmental variables in Methana, Saronikos Gulf, Greece.

Phytoplankton community was investigated during two contrasting periods using offshore plankton samples in the volcanic area of Methana peninsula (Saronikos Gulf): the first at early autumn (warm period, September 2016) and the second one at early spring (cold period, March 2017). In order to investigate the phytoplankton community structure in the complex geo-biochemical conditions of the area, samples were collected from stations near the CO2 hydrothermal vents, at the hydrothermal sulfur and radioactive springs and at a fishery nearby Methana town. Three major phytoplankton groups, Bacillariophyceae, Dinophyceae, and Prymnesiophyceae, were studied, using inverted microscopy. In early autumn, Dinophyceae were dominant in the majority of the stations with cell concentrations of Prorocentrum spp. up to ~ 35.5 × 103 cells l-1. In early spring, the dominant class was Bacillariophyceae with dominant genus Nitzschia/Pseudo-nitzschia presenting cell concentrations up to ~ 33.9 × 103 cells l-1. Furthermore, Prymnesiophyceae appeared in both spring and autumn samples with small fluctuations. Total phytoplankton cell concentrations followed a seasonal trend, presenting slightly lower values in the hydrothermal-effected area in comparison with the broader Saronikos Gulf, confirming the prevalence of oligotrophic conditions. Seasonal variation was very strong, revealing an association with water temperature and nutrient content. Those environmental variables proved to have a strong effect that was reflected in the phytoplankton community structure.

Use of Mass Spectrometry to Determine the Diversity of Toxins Produced by Gambierdiscus and Fukuyoa Species from Balearic Islands and Crete (Mediterranean Sea) and the Canary Islands (Northeast Atlantic).

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions.

Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool.

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.

Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data.

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future.

Assessing the neurotoxic effects of palytoxin and ouabain, both Na⁺/K⁺-ATPase inhibitors, on the myelinated sciatic nerve fibres of the mouse: an ex vivo electrophysiological study.

Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na⁺/K⁺-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na⁺/K⁺-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT50) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT50 of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC50) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 µM (n = 5) and IC50 of 370.0 ± 18.00 µM (incubation 24.5 h). Finally, when the two compounds were combined--e.g. pre-incubation of the nerve fibre in 250.0 µM ouabain for 1 h and then exposure to 1.0 nM PlTX--ouabain offered minor a neuroprotection of 9.1-17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 µM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay--the standard ex vivo test for PlTX toxicity.

Occurrence of palytoxin-group toxins in seafood and future strategies to complement the present state of the art.

Palytoxin (PlTX) and palytoxin-like (PlTX-like) compounds in seafood have been raising scientific concern in the last years. The constant increase in record numbers of the causative dinoflagellates of the genus Ostreopsis together with the large spatial expansion of this genus has led to intensification of research towards optimization of methods for determination of PlTX presence and toxicity. In this context, identification of seafood species which could possibly contain PlTXs constitutes an important issue for public health protection. In the present paper, worldwide occurrence of PlTX-like compounds in seafood is reviewed, while potential future strategies are discussed. PlTX has been reported to be present in several species of fish, crustaceans, molluscs and echinoderms. In one occasion, PlTX has been identified in freshwater puffer fish whereas all other records of PlTXs refer to marine species and have been recorded in latitudes approximately between 43°N and 15°S. PlTX determination in seafood has relied on different methodologies (mainly LC-MS, mouse bioassay and hemolysis neutralization assay) that have evolved over time. Future recommendations include systematic screening of PlTX in those species and areas where PlTX has already been recorded implementing updated methodologies.

Update on methodologies available for ciguatoxin determination: perspectives to confront the onset of ciguatera fish poisoning in Europe.

Ciguatera fish poisoning (CFP) occurs mainly when humans ingest finfish contaminated with ciguatoxins (CTXs). The complexity and variability of such toxins have made it difficult to develop reliable methods to routinely monitor CFP with specificity and sensitivity. This review aims to describe the methodologies available for CTX detection, including those based on the toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting any of these methodological approaches for routine monitoring of ciguatera may be dependent upon the applicability of the method. However, identifying a reference validation method for CTXs is a critical and urgent issue, and is dependent upon the availability of certified CTX standards and the coordinated action of laboratories. Reports of CFP cases in European hospitals have been described in several countries, and are mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTX-producing tropical genus Gambierdiscus in the eastern Atlantic Ocean of the northern hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in Europe [1]. The question of the possible contribution of climate change to the distribution of toxin-producing microalgae and ciguateric fish is raised. The impact of ciguatera onset on European Union (EU) policies will be discussed with respect to EU regulations on marine toxins in seafood. Critical analysis and availability of methodologies for CTX determination is required for a rapid response to suspected CFP cases and to conduct sound CFP risk analysis.

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.