RESUMO
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.
RESUMO
In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing industry. Over the last decade, PSP in the NMS has grown significantly worse; however, the origins and dynamics of the toxic Alexandrium fundyense (Balech) populations that bloom within the NMS are not well known. This study examined a collection of 412 strains isolated from the NMS and the Gulf of Maine (GOM) in 2006-2007 to investigate the genetic characteristics of localized blooms and assess connectivity with coastal populations. Comparisons of genetic differentiation showed that A. fundyense blooms in the NMS exhibited extensive clonal diversity and were genetically distinct from populations in the GOM. In both project years, genetic differentiation was observed among temporal samples collected from the NMS, sometimes occurring on the order of approximately 7 days. The underlying reasons for temporal differentiation are unknown, but may be due, in part, to life-cycle characteristics unique to the populations in shallow embayments, or possibly driven by selection from parasitism and zooplankton grazing; these results highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale and limited connectivity of NMS salt ponds provide a novel system for investigating regulators of blooms, as well as the influence of selective forces on population structure, all of which are otherwise difficult or impossible to study in the adjacent open-coastal waters or within larger estuaries.
