Santiago Fraga

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin.

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY, TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES†

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.

Influence of upwelling relaxation on dinoflagellates and shellfish toxicity in Ria de Vigo, Spain

Abstract Outbreaks of paralytic shellfish poisoning (PSP) along the north-west coast of Spain have become a serious threat to the extensive mussel farming industry in that region over the last decade. During the summer, high phytoplankton productivity is supported by the sustained upwelling of nutrient-rich deep water into the rias. An episode of PSP in the autumn of 1985 in Ria de Vigo coincided with the sudden appearance, rapid numerical increase, and dominance of two chain-forming dinoflagellates, Gymnodinium catenatum and Protogonyaulax affinis . Field data suggest that warm offshore surface water was transported into the ria as the summer upwelling ceased. This occurred when winds changed from northerly (upwelling favourable) to southerly or westerly (upwelling unfavourable); the injected water contained established populations of oceanic and neritic dinoflagellates. The simultaneous appearance and dominance of two dinoflagellates that form long chains leads us to speculate that the small-scale downwelling of water within the ria favoured efficient swimmers among the phytoplankton. These data not only implicate these two species as possible sources of the PSP toxins in local mussels, but they also suggest the feasibility of developing a bloom prediction capability for some dinoflagellate species based in part on an upwelling index that can indicate when offshore surface waters might be forced into the rias.

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.

Phylogenetic relationships among the Mediterranean Alexandrium (Dinophyceae) species based on sequences of 5.8S gene and Internal Transcript Spacers of the rRNA operon

A phylogenetic analysis of the genus Alexandrium, including both the most common and rare species from coastal areas of the Mediterranean Sea was carried out. Nucleotide sequences of 5.8 S gene and Internal Transcribed Spacer regions of the rRNA operon were examined and analysed together with isolates of Alexandrium spp. from elsewhere in the world. These rDNA ribosomal markers were useful in delineating the phylogenetic position of species in the genus, as well as in determining relationships among isolates within each species collected from different localities. Results of phylogeographical analyses within the ‘Alexandrium tamarense’ species complex identified three lineages in the Mediterranean Sea: the Mediterranean (ME), Western European (WE) and Temperate Asian (TA) clades. The phylogenetic grouping of the isolates is consistent with the ribotype clades, but not with the morpho-species that constitute the complex. Additional non-toxic isolates were included in the ME clade. The NA (North Atlantic) clade is the fourth group within the ‘Alexandrium tamarense’ species complex identified by phylogenetic analyses. Based on its higher genetic diversity and phylogeographical relationships, it can be hypothesized that the NA clade represents the ancestral group of the ‘Alexandrium tamarense’ species complex. Alexandrium minutum isolates of the NW Mediterranean clustered with strains from Brittany and Australia. Alexandrium minutum constituted a sister clade of A. tamutum, which is another species strongly associated with the Mediterranean area. Another typical Mediterranean species, A. taylori, was placed as a sister clade of A. pseudogoniaulax by the phylogenetic analysis. Finally, the phylogenetic relationships of some Alexandrium morpho-species that were infrequently observed in the Mediterranean Sea have been resolved.

Resting cysts of the toxigenic dinoflagellate genus Alexandrium in recent sediments from the Western Mediterranean coast, including the first description of cysts of A. kutnerae and A. peruvianum

Cyst studies carried out in 2002–2003 on Mediterranean Sea sediment from seven different sites along the Catalan and Balearic coasts (Western Mediterranean) revealed a higher diversity of Alexandrium species in the region than was previously known. The cysts of eight species of the toxigenic, marine dinoflagellate genus Alexandrium are described, and some, such as A. kutnerae, A. margalefi, A. peruvianum and A. pseudogoniaulax, are reported from the area for the first time. This is also the first record of resting cysts of A. taylori in Mediterranean sediment, and the first description known to date of resting cysts for A. kutnerae and A. peruvianum. All the cysts were characterized by a smooth wall except for the paratabulated cyst of A. pseudogoniaulax, and most of them had a prominent yellow/orange accumulation body. Nevertheless, we have also detected an unparatabulated cyst of A. pseudogoniaulax, both from the sediment and in cultures. The cyst of A. kutnerae had a roughly cylindrical shape with rounded ends which makes it impossible to distinguish from resting cysts of A. tamarense and A. catenella, while the flattened round cysts of A. peruvianum were very similar to those of A. taylori. The cyst concentration data revealed A. catenella and A. minutum to be the most abundant cysts in the region, and they were detected in semi-enclosed waters, such as harbours. This highlights the importance of water exchange in the accumulation of cyst beds of these species, which has already been reported in the region by other authors for A. minutum. This paper contributes to the biogeographic distribution of some Alexandrium species, such as A. kutneare, A. margalefi, A. peruvianum and A. pseudogoniaulax, which have been reported only infrequently in a global context.

COOLIA CANARIENSIS SP. NOV. (DINOPHYCEAE), A NEW NONTOXIC EPIPHYTIC BENTHIC DINOFLAGELLATE FROM THE CANARY ISLANDS

A new photosynthetic dinoflagellate species, Coolia canariensis S. Fraga sp. nov., is described based on samples taken from tidal ponds on the rocky shore of the Canary Islands, northeast Atlantic Ocean. Its morphology was studied by LM and SEM. It is almost spherical and has a thick smooth theca with many scattered pores. Plate 1′ is the biggest of the epithecal plates, and 7″ is twice as wide as it is long. Phylogeny inferred from the D1/D2 regions of the LSU nuclear rDNA of three strains of C. canariensis and several strains of other Coolia species, C. monotis, C. sp., showed that C. canariensis strains clustered in a well‐supported clade distinct from the other species. No toxins were detected using mouse bioassay, liquid chromatography with Fluorescence detection (LC‐FLD) or liquid chromatography‐mass spectrometry (LC‐MS). Its pigment composition is of the peridinin type of dinoflagellates. Together with this new species, many other strains of C. monotis from the Atlantic Ocean and Mediterranean Sea have been analyzed for toxin presence, and no evidence of toxin production related to yessotoxins (YTXs) was found, as was previously suggested for C. monotis from Australia.

Evidence of saxitoxin derivatives as causative agents in the 1997 mass mortality of monk seals in the Cape Blanc Peninsula

Monk seals in Cape Blanc (Western Sahara coast) suffered a mass mortality during May-July 1997 which was attributed to a morbillivirus. High performance liquid chromatography (HPLC) analysis on tissues of seals killed during the outbreak and on related fauna showed peaks with retention times coincident with those of some saxitoxin derivatives but their identity was not proved. Here we present results of further HPLC analyses that unambiguously prove the identity of these toxins by mass spectrometry (MS), supporting the hypothesis that this mortality of monk seals was caused by biotoxins rather than by a morbillivirus.

The Life History and Cell Cycle of Kryptoperidinium foliaceum, A Dinoflagellate with Two Eukaryotic Nuclei

Kryptoperidinium foliaceum is a binucleate dinoflagellate that contains an endosymbiont nucleus of diatom origin. However, it is unknown whether the binucleate condition is permanent or not and how the diatom nucleus behaves during the life history processes. In this sense, it is also unknown if there is a sexual cycle or a resting stage during the life history of this species, two key aspects necessary to understand the life history strategy of this dinoflagellate. To answer these questions, life history and cell cycle studies were performed with the following results: (i) Kryptoperidinium foliaceum has a sexual cycle and in the dinoflagellate strains studied, the binucleate condition is permanent. Sexuality in the host was confirmed by the presence of fusing gamete pairs and planozygotes in clonal cultures (revealing homothallism), but signs of meiosis in the endosymbiont were not observed. The endosymbiont nucleus likely fuses first, because fusing gamete pairs were found to have two dinoflagellate nuclei but only one endosymbiont nucleus. After complete gamete fusion, the planozygotes had apparently normal endosymbiont and dinoflagellate nuclei. (ii) Asexual division studies using flow cytometry showed that the S phase in the endosymbiont (diatom) nucleus starts 6-8h later than in the host nucleus, but there was no evidence of mitosis in the former. (iii) Sexual and asexual cysts were formed in culture. Neither cysts from natural samples nor those formed in culture exhibited a dormancy period before germination.

Review of the main ecological features affecting benthic dinoflagellate blooms

Abstract Both benthic and planktic dinoflagellates can produce harmful algal blooms. However most of the studies conducted so far emphasized on planktic species. In the present review, we assessed the main ecological factors affecting the population dynamics of bloomforming benthic dinoflagellates, with particular emphasis on Ostreopsis and Gambierdiscus. Based on the basic equation of population dynamics, we mainly focused on growth, predation, mortality, immigration and dispersion. Factors determining the dynamics of benthic dinoflagellate populations are very different from the well-studied case of planktic dinoflagellates. The relative movement of cells and water is the main difference as benthic dinoflagellates depend on a fixed substratum while planktic dinoflagellates depend on a water body. Any alteration in the substratum will affect benthic dinoflagellate populations, as for example the changes in seaweeds concentrations due to predation by sea urchins. We also evaluated the impact of global changes on dinoflagellates bloom occurrence.