RESUMEN
Multiple species in the toxic marine diatom genus Pseudo-nitzschia have been identified in the Northwestern Atlantic region encompassing the Gulf of Maine (GOM), including the Bay of Fundy (BOF). To gain further knowledge of the taxonomic composition and toxicity of species in this region, Pseudo-nitzschia isolates (n=146) were isolated from samples collected during research cruises that provided broad spatial coverage across the GOM and the southern New England shelf, herein referred to as the GOM region, during 2007-2008. Isolates, and cells in field material collected at 38 stations, were identified using electron microscopy (EM). Eight species (P. americana, P. fraudulenta, P. subpacifica, P. heimii, P. pungens, P. seriata, P. delicatissima and P. turgidula), and a novel form, Pseudo-nitzschia sp. GOM, were identified. Species identity was confirmed by sequencing the large subunit of the ribosomal rDNA (28S) and the internal transcribed spacer 2 (ITS2) for six species (36 isolates). Phylogenetic analyses (including neighbor joining, maximum parsimony, and maximum likelihood estimates and ITS2 secondary structure analysis) and morphometric data supported the placement of P. sp. GOM in a novel clade that includes morphologically and genetically similar isolates from Australia and Spain and is genetically most similar to P. pseudodelicatissima and P. cuspidata. Seven species (46 isolates) were grown in nutrient-replete batch culture and aliquots consisting of cells and growth medium were screened by Biosense ASP ELISA to measure total domoic acid (DA) produced (intracellular + extracellular); P. americana and P. heimii were excluded from all toxin analyses as they did not persist in culture long enough for testing. All 46 isolates screened produced DA in culture and total DA varied among species (e.g., 0.04 to 320 ng ml-1 for P. pungens and P. sp. GOM isolates, respectively) and among isolates of the same species (e.g., 0.24 - 320 ng ml-1 for P. sp. GOM). The 15 most toxic isolates corresponded to P. seriata, P. sp. GOM and P. pungens, and fg DA cell-1 was determined for whole cultures (cells and medium) using ELISA and liquid chromatography (LC) with fluorescence detection (FLD); for seven isolates, toxin levels were also estimated using LC - with mass spectrometry and ultraviolet absorbance detection. Pseudo-nitzschia seriata was the most toxic species (up to 3,500 fg cell-1) and was observed in the GOM region during all cruises (i.e., during the months of April, May, June and October). Pseudo-nitzschia sp. GOM, observed only during September and October 2007, was less toxic (19 - 380 fg cell-1) than P. seriata but more toxic than P. pungens var. pungens (0. 4 fg cell-1). Quantitation of DA indicated that concentrations measured by LC and ELISA were positively and significantly correlated; the lower detection limit of the ELISA permitted quantification of toxicity in isolates that were found to be nontoxic with LC methods. The confirmation of at least seven toxic species and the broad spatial and temporal distribution of toxic Pseudo-nitzschia spp. have significant implications for the regional management of nearshore and offshore shellfisheries resources.
RESUMEN
Marine dinoflagellates of the genus Dinophysis can produce toxins of the okadaic acid (OA) and pectenotoxin (PTX) groups. These lipophilic toxins accumulate in filter-feeding shellfish and cause an illness in consumers called diarrhetic shellfish poisoning (DSP). In 2008, a bloom of Dinophysis led to the closure of shellfish harvesting areas along the Texas coast, one of the first DSP-related closures in the U.S. This event resulted in a broad study of toxin production in isolates of Dinophysis spp. from U.S. waters. In the present study, we compared toxin profiles in geographical isolates of Dinophysis collected in the U.S. (Eel Pond, Woods Hole MA; Martha's Vineyard, MA; and Port Aransas Bay, Texas), and in those from Canada (Blacks Harbour, Bay of Fundy) and Chile (Reloncavi Estuary), when cultured in the laboratory under the same conditions. For each isolate, the mitochondrial cox1 gene was sequenced to assist in species identification. Strains from the northeastern U.S. and Canada were all assigned to Dinophysis acuminata, while those from Chile and Texas were most likely within the D. acuminata complex whereas precise species designation could not be made with this marker. Toxins were detected in all Dinophysis isolates and each isolate had a different profile. Toxin profiles of isolates from Eel Pond, Martha's Vineyard, and Bay of Fundy were most similar, in that they all contained OA, DTX1, and PTX2. The Eel Pond isolate also contained OA-D8 and DTX1-D7, and low levels (unconfirmed structurally) of DTX1-D8 and DTX1-D9. D. acuminata from Martha's Vineyard produced DTX1-D7, along with OA, DTX1, and PTX2, as identified in both the cells and the culture medium. D. acuminata from the Bay of Fundy produced DTX1 and PTX2, as found in both cells and culture medium, while only trace amounts of OA were detected in the medium. The Dinophysis strain from Texas only produced OA, and the one from Chile only PTX2, as confirmed in both cells and culture medium.