Diversity and toxicity of Pacific strains of the benthic dinoflagellate Coolia (Dinophyceae), with a look at the Coolia canariensis species complex.

Coolia Meunier 1919 from benthic assemblages of Hawai'i and Guam were isolated and clonal cultures were established from single cells. Cultures were identified to species-level based on 28S rRNA and ITS-5.8S rRNA genes and tested for toxicity. In Hawai'i, two strains of C. malayensis were isolated. In Guam, a high biodiversity was identified: four strains of C. malayensis, one strain of C. palmyrensis, one strain of C. tropicalis, one strain of C. canariensis phylogroup III, and two strains forming a new phylogroup (phylogroup IV) of nontoxic C. canariensis. Morphology of the new C. canariensis phylogroup was described using light microscopy and scanning electron microscopy. Mass cultures and methanol extracts of representative cultures (C. malayensis, C. palmyrensis, C. canariensis, C. tropicalis) from Guam were prepared for liquid chromatography-mass spectrometry analysis. Chemical analyses revealed yessotoxin analogue C56H78O18S2 is produced by C. malayensis, C. canariensis phylogroup IV and C. palmyrensis, but other analogues, C57H80O18S2 and C58H86O18S2, were only found in C. malayensis (Okinawa) and C. canariensis phylogroup IV. Individual toxin profiles were also different over time for an Okinawa strain of C. malayensis (NIES-3637), highlighting intra and inter-species variation in Yessotoxin-analogue expression. Biological activity was tested using Artemia bioassay and toxicity was observed in Guam and Okinawa strains of C. malayensis. Strong support of four distinct clades within the C. canariensis species complex was recovered in phylogenetic analyses, despite morphological similarities.

Diversity and Toxicity of the Genus Coolia Meunier in Brazil, and Detection of 44-Methyl Gambierone in Coolia tropicalis.

Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina, while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia. 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis, using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations.

COOLIA CANARIENSIS SP. NOV. (DINOPHYCEAE), A NEW NONTOXIC EPIPHYTIC BENTHIC DINOFLAGELLATE FROM THE CANARY ISLANDS(1).

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.