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- Toxin profile of Ostreopsis cf. ovata from Portuguese continental coast and Selvagens Islands (Madeira, Portugal)Publication . SoliƱo, Lucia; Garcia-Altares, Maria; Godinho, Lia; Reis Costa, PedroThe toxigenic dinoflagellate Ostreopsis cf. ovata is known to produce a range of palytoxin (PLTX) - related compounds named ovatoxins (OVTX). O. cf. ovata presents a wide variability in toxin production and its toxic profile is strain-specific. Several OVTXs, denominated from -a to -h and -I have been reported from different strains of this benthic microalgae up to now, mainly in Mediterranean isolates. However, less is known about the toxin profile of the strains present in the Atlantic coasts of Europe. In this work, strains of O. cf. ovata isolated from the South coast of Portugal mainland (Algarve) and Selvagens Island (Madeira, Portugal) were cultured and tested for toxicity by hemolytic assay. Toxin profiles were qualitatively elucidated by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). The strain from Algarve presented lower toxic potency than the strain from Selvagens island (12.3 against 54.8 pg of PLTX equivalents per cell) showing in both cases the characteristic toxin profile of Mediterranean strains. The major component, OVTX-a, was concomitant with OVTX from -b to -g and isobaric PLTX. Regarding the morphological characteristics of these strains, as well as their toxin fingerprint, it is likely they are closely related to strains from Mediterranean coasts. The present study reports for the first time the occurrence of several OVTX congeners and iso-PLTX in O. cf. ovata from Portuguese waters. This study provides valuable information to characterize the risk of OVTXs-related outbreaks in Portugal.
- New insights into the occurrence of paralytic shellfish toxins in the oceanic pufferfish Lagocephalus lagocephalus (Linnaeus, 1758) from Madeira Island, PortugalPublication . SoliƱo, Lucia; Gouveia, Neide; TimĆ³teo, Viriato; Reis Costa, PedroPufferfish are some of the most poisonous marine organisms due to their capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). Several species are consumed, mainly in Asiatic countries, for the quality of their flesh, which may cause severe human poisoning every year due to errors in preparation. Lagocephalus lagocephalus the oceanic pufferfish, is a cosmopolitan species present in European Atlantic and Mediterranean coasts. Although considered toxic, only a few studies have previously addressed the occurrence of toxic compounds in this species. In the present study, 29 individuals of L. lagocephalus caught in Madeira Island (NE Atlantic) were dissected and analysed by HPLC-FLD for PSTs detection. Saxitoxin (STX) and decarbamoyl STX (dcSTX) were detected in liver, intestine and gonads but not in the muscle. Neosaxitoxin (NEO) could also be detected in a liver sample. The total STX equivalent (eq) concentrations in liver ranged from < LOQ to 973.4 Ī¼g STX eq kgā1. The presence of toxins in the intestines and gonads points to the fish capability to acquire toxins through feeding or by association with endosymbiotic, toxin-producing microbes as well as the ability to accumulate them in specific organs. Although pufferfish trade in Europe is forbidden by European Union regulations, the increasing occurrence of this species in new areas and the expected raise of blooms of PSTs-producer organisms claims for a better knowledge on oceanic pufferfish toxicity and biology. This will be needed to provide accurate information to stakeholders and population to prevent illegal trade of these species and poisoning incidents.
- Distribution, identification and cytotoxicity of Gambierdiscus (Dinophyceae) in the Atlantic Selvagens Islands (Madeira, Portugal): a ciguatera gateway to EuropePublication . Godinho, Lia; SoliƱo, Lucia; Churro, Catarina; Timoteo, Viriato; Santos, Carolina; Gouveia, Neide; DiogĆØne, Jorge; Reis Costa, PedroThe emerging threat of ciguatera poisoning (CP) in Europe has been associated with fish captured in the Canary Islands (Spain) and Selvagens Islands (Portugal). The first are heavily populated islands where numerous scientific studies have been carried out. Conversely, the Selvagens Islands are a nature reserve with low human pressure that have been rarely surveyed in terms of the marine benthic microalgae, including the epiphytic ciguatera-causing dinoflagellate species. To investigate the harmful microalgal diversity of the Selvagens Islands, a scientific cruise to these remote islands took place in September, 2018. The Gambierdiscus species composition and distribution, and the associated epiphytic dinoflagellate community, were assessed using artificial substrate devices. Gambierdiscus cells were found in all samples, reaching concentrations of up to 725 cells 100 cm(-2). G. australes was the only species identified after morphological and molecular analysis of the retrieved cultures. Species identification was confirmed by molecular characterization based on the LSU D8-D10 region. Nevertheless, phylogenetic studies indicated that some strains diverged from the G. australes clade suggesting genetic differentiation. Toxicity was estimated by neuro-2a cell-based assay in four strains, ranging from 2.46-83 fg of CTX1B eq. cell(-1). The epiphytic dinoflagellate community that co-occurred with Gambierdiscus comprised other toxic or potentially toxic dinoflagellates, such as Ostreopsis, Prorocentrum, Amphidinium and Coolia species. Oceanographic and meteorological data were also obtained to characterize the occurrence of Gambierdiscus. This study is the first stage in understanding the role of the Selvagens Islands in the incubation and proliferation of the ciguatera-causing dinoflagellates Gambierdiscus in the NE Atlantic.
- Cytotoxic and hemolytic activities of extracts of the fish parasite dinoflagellate amyloodinium ocellatumPublication . Moreira, MĆ”rcio; SoliƱo, Lucia; Marques, CĆ”tia L.; LaizĆ©, Vincent; PousĆ£o-Ferreira, Pedro; Fidalgo E Costa, Pedro; Soares, FlorbelaThe dinoflagellate Amyloodinium ocellatum is the etiological agent of a parasitic disease named amyloodiniosis. Mortalities of diseased fish are usually attributed to anoxia, osmoregulatory impairment, or opportunistic bacterial infections. Nevertheless, the phylogenetic proximity of A. ocellatum to a group of toxin-producing dinoflagellates from Pfiesteria, Parvodinium and Paulsenella genera suggests that it may produce toxin-like compounds, adding a new dimension to the possible cause of mortalities in A. ocellatum outbreaks. To address this question, extracts prepared from different life stages of the parasite were tested in vitro for cytotoxic effects using two cell lines derived from branchial arches (ABSa15) and the caudal fin (CFSa1) of the gilthead seabream (Sparus aurata), and for hemolytic effects using erythrocytes purified from the blood of gilthead seabream juveniles. Cytotoxicity and a strong hemolytic effect, similar to those observed for Karlodinium toxins, were observed for the less polar extracts of the parasitic stage (trophont). A similar trend was observed for the less polar extracts of the infective stage (dinospores), although cell viability was only affected in the ABSa15 line. These results suggest that A. ocellatum produces tissue-specific toxic compounds that may have a role in the attachment of the dinosporesā and trophontsā feeding process.
- Global impact of ciguatoxins and ciguatera fish poisoning on fish, fisheries and consumersPublication . SoliƱo, Lucia; Reis Costa, PedroCiguatera fish poisoning (CFP) is one of the most devastating food-borne illnesses caused by fish consumption. Ciguatoxins (CTXs) are potent neurotoxins synthesized by the benthic microalgae Gambierdiscus spp. and Fukuyoa spp. that are transmitted to fish by grazing and predation. Despite the high incidence of CFP, affecting an estimated number of 50,000 persons per year in tropical and subtropical latitudes, the factors underlying CTXs occurrence are still not well understood. Toxin transfer and dynamics in fish and food-webs are complex. Feeding habits and metabolic pathways determine the toxin profile and toxicity of fish, and migratory species may transport and spread the hazard. Furthermore, CTX effect on fish may be a limiting factor for fish recruitment and toxin prevalence. Recently, new occurrences of Gambierdiscus spp. in temperate areas have been concomitant with the detection of toxic fish and CFP incidents in non-endemic areas. CFP cases in Europe have led to implementation of monitoring programs and fisheries restrictions with considerable impact on local economies. More than 400 species of fish can be vectors of CTXs, and most of them are high-valued commercial species. Thus, the risk uncertainty and the spread of Gambierdiscus have serious consequences for fisheries and food safety. Here, we present a critical review of CTXs impacts on fish, fisheries, and humans, based on the current knowledge on CFP incidence and CTXs prevalence in microalgae and fish.
- High Levels of Tetrodotoxin (TTX) in Trumpet Shell Charonia lampas from the Portuguese CoastPublication . Reis Costa, Pedro; GirĆ”ldez, Jorge; Rodrigues, Susana Margarida; LeĆ£o, JosĆ© Manuel; Pinto, EstefanĆa; SoliƱo, Lucia; Gago-MartĆnez, AnaTetrodotoxin (TTX) is a potent neurotoxin, considered an emerging toxin in Europe where recently a safety limit of 44 Āµg TTX kg-1 was recommended by authorities. In this study, three specimens of the large gastropod trumpet shell Charonia lampas bought in a market in south Portugal were analyzed using a neuroblastoma cell (N2a) based assay and by LC-MS/MS. N2a toxicity was observed in the viscera of two individuals analyzed and LC-MS/MS showed very high concentrations of TTX (42.1 mg kg-1) and 4,9-anhydroTTX (56.3 mg kg-1). A third compound with m/z 318 and structurally related with TTX was observed. In the edible portion, i.e., the muscle, toxin levels were below the EFSA recommended limit. This study shows that trumpet shell marine snails are seafood species that may reach the markets containing low TTX levels in the edible portion but containing very high levels of TTX in non-edible portion raising concerns regarding food safety if a proper evisceration is not carried out by consumers. These results highlight the need for better understanding TTX variability in this gastropod species, which is critical to developing a proper legal framework for resources management ensuring seafood safety, and the introduction of these gastropods in the markets.
- Effects of the marine biotoxins Okadaic acid and Dinophysistoxins on fishPublication . Corriere, Mauro; SoliƱo, Lucia; Reis Costa, PedroNatural high proliferations of toxin-producing microorganisms in marine and freshwater environments result in dreadful consequences at the socioeconomically and environmental level due to water and seafood contamination. Monitoring programs and scientific evidence point to harmful algal blooms (HABs) increasing in frequency and intensity as a result of global climate alterations. Among marine toxins, the okadaic acid (OA) and the related dinophysistoxins (DTX) are the most frequently reported in EU waters, mainly in shellfish species. These toxins are responsible for human syndrome diarrhetic shellfish poisoning (DSP). Fish, like other marine species, are also exposed to HABs and their toxins. However, reduced attention has been given to exposure, accumulation, and effects on fish of DSP toxins, such as OA. The present review intends to summarize the current knowledge of the impact of DSP toxins and to identify the main issues needing further research. From data reviewed in this work, it is clear that exposure of fish to DSP toxins causes a range of negative effects, from behavioral and morphological alterations to death. However, there is still much to be investigated about the ecological and food safety risks related to contamination of fish with DSP toxins.
- No Ī²-N-Methylamino-L-alanine (BMAA) was detected in stranded cetaceans from Galicia (North-West Spain)Publication . SoliƱo, Lucia; Kim, Sea-Yong; LĆ³pez, Alfredo; Covelo, Pablo; Rydberg, Sara; Reis Costa, Pedro; Lage, SandraThe neurotoxin Ī²-N-methylamino-L-alanine (BMAA), a non-proteinogenic amino acid produced by several species of both prokaryotic (cyanobacteria) and eukaryotic (diatoms) microorganisms, has been proposed to be associated with the development of neurodegenerative diseases. At first, BMAA appeared to be ubiquitously present worldwide in various organisms, from aquatic and terrestrial food webs. However, recent studies, using detection methods based on mass spectrometry, instead of fluorescence detection, suggest that the trophic transfer of BMAA is debatable. This study evaluated BMAA in 22 cetaceans of three different species (Phocoena phocoena, n = 8, Delphinus delphis, n = 8, and Tursiops truncatus, n = 6), found stranded in North-West Spain. BMAA analysis of the liver, kidney, or muscle tissues via sensitive liquid chromatography with tandem mass spectrometry did not reveal the presence of this compound or its isomers. The absence recorded in this study highlights the need to better understand the trophic transfer of BMAA and its anatomical distribution in marine mammals.
- Are pelagic seabirds exposed to amnesic shellfish poisoning toxins?Publication . SoliƱo, Lucia; Ferrer-Obiol, Joan; Navarro-Herrero, Leia; Gonzalez-Solis, Jacob; Reis Costa, PedroMarine birds have been hypothesized to be underreported victims of harmful algal blooms (HABs). Toxic blooms of Pseudo-nitzschia spp., the primary amnesic toxin producer microalgae, domoic acid (DA) are known to cause massive mortalities of coastal seabirds and marine mammals around the world. However, these fatalities are only detected when birds die nearby the coastline and little is known about possible outbreaks of pelagic seabirds in oceanic areas. Here we aim to understand whether pelagic seabirds are exposed to amnesic shellfish poisoning (ASP) toxins. For this purpose, we tracked pelagic seabirds feeding on small epipelagic fish and squid, reported to be vectors of DA, which are obtained in high productivity zones where intense Pseudo-nitzschia blooms regularly occur. In particular, we tracked Cory's (Calonectris borealis) and Scopoli's (C. diomedea) shearwaters breeding in Gran Canaria (Canary Is.) and in Menorca (Balearic Is.) and feeding on the Canary Current region and the Catalonian coast, respectively. We sampled birds for blood at the recovery of the GPS (Global Positioning System) and analyzed it for DA determination by Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). Among the 61 samples analyzed from Gran Canaria, and 87 from Menorca, 31 (50.8%) and 28 (32.2%) from each location presented detectable levels of DA ranging 1.0-10.6 ng mL(-1). This work reveals that DA can be detected at variable levels in the blood of ASP-asymptomatic shearwaters and suggests a chronic exposure of shearwaters to DA, highlighting the need for further studies on DA effects. These results are of high relevance due to the vulnerability of these marine birds, which populations are in continuous decline. Since global warming is expected to alter and increase the occurrence of HABs, marine toxins might become an additional stressor for seabirds and exacerbate the already precarious conservation status of many species.
- Impaired fish swimming performance following dietary exposure to the marine phycotoxin okadaic acidPublication . Corriere, Mauro; Baptista, Miguel; Paula, Jose R.; Repolho, Tiago; Rosa, Rui; Reis Costa, Pedro; SoliƱo, LuciaFish are frequently exposed to harmful algal blooms (HAB) and to related toxins. However, the biological effects of okadaic acid (OA), the most abundant and frequent HAB-toxin in Europe, South America and Asia, have been poorly investigated. In this study, fish swimming performance and metabolic rates were investigated in juveniles of Zebra seabream (Diplodus cervinus) exposed to OA-group toxins via dietary route, during three days. Fish fed on contaminated food accumulated up to 455.5 mu g OA equiv. Kg(-1). Significant lower mean critical swimming speed (Ucrit) were observed in fish orally exposed to OA (and its related isomer dinophysistoxin-1, DTX-1) than fish feeding on non-toxic diet. A tendency to higher demands of oxygen consumption was also recorded in OA-exposed fish at higher current velocities. This study indicates that fish may not be affected by OA-group toxins under basal conditions, but suggests a decrease in fitness linked to a reduction in swimming performance of fish exposed to OA under increased stimulus. OA and related toxins are suggested to have a cryptic effect on swimming performance that may be enhanced when fish deals with multiple stressors. Considering that a reduction in swimming performance may have impact on critical activities, such as foraging and escaping from predators, this study highlights the ecological risk associated with dinoflagellate toxic blooms, biotoxins food web transfer and fish contamination.