Katerina Aligizaki

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.

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.

Genetic diversity of the genus Ostreopsis Schmidt: Phylogeographical considerations and molecular methodology applications for field detection in the Mediterranean Sea

Abstract This study reports some recent phylogeographical considerations on the genus Ostreopsis distribution worldwide, with particular attention to the Mediterranean Sea, and new recent advances on the quali-quantitiative detection of Ostreopsis species along coastal areas of the Mediterranean Sea based on the PCR and quantitative real time PCR (qrt-PCR) assays. It was found that O. cf. ovata is widely dispersed throughout tropical and warm temperate coastal areas. In the Atlantic/Mediterranean region it represents a panmictic population that is highly divergent from Indo-Pacific populations. Furthermore, we demonstrated that the developed qrt-PCR assay is specific, robust and high sample throughput for the quantification of the toxic O. cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional methods of microscopy and applied for the survey of benthic toxic microalgal species in marine ecosystems.

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.