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Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

Twenty-seven clones were established from elongate Alexandrium sp. cysts collected in six regions along the Russian Pacific coast. All isolates were identified as Alexandrium tamarense via detailed epifluoresence microscopy of thecal plates. Morphological differences of both cultured and wild cells from the study regions mainly occurred in the shape of the cell (length/width ratio), degree of development of the sulcal list, and the shape of the posterior sulcal (S.p.) and second antapical (2″″) plates. Cells were divided into two cell types: ‘short’ (isodiametrical or wide) and ‘tall’. Each cell type exhibits specific features of tabulation, mainly the shape of the S.p. and 2″″ plates and was dominant in each particular region of the study. The short type, with a wide S.p. and reduced length in the dorsoventral 2″″ plates, was characteristic of A. tamarense from Primorye and southern Sakhalin Island. The tall cells, i.e., with cell length exceeding width, and having and elongate S.p. and dorsoven...

Current knowledge on global phylogeographic patterns and dispersal routes of dinoflagellates in the Alexandrium tamarense species complex is largely based on isolates from the northern hemisphere. The paucity of molecular data available has limited the development of evolutionary hypotheses for southern hemisphere Alexandrium populations. To address this shortcoming, we present morphological, toxicity and molecular data from three Alexandrium isolates from South African coastal waters. Morphological descriptions indicate that these isolates correspond to the species A. catenella and A. tamiyavanichi, making this the first recorded presence of A. tamiyavanichi in African waters. Both species produce paralytic shellfish poisoning toxins, with the total average cellular toxicity of the A. catenella isolates [9.77 fmol saxitoxin (STX) eq cell−1] exceeding that of A. tamiyavanichi by a factor of 35. Complex toxin profiles showed high proportions of B1 and C1+C2 congeners (> 20% molar percentage each) ...

Light, scanning and transmission electron microscopy were carried out on Alexandrium tamarense and A. fundyense hypnozygotes (cysts) from cultures and marine sediments. Transmission electron microscopy protocols were adapted to improve the quality of ultrathin sections. Cell contents of hypnozygotes were reduced compared to vegetative stages and were largely made up of storage vesicles in a dense, granular matrix. Chloroplasts and other organelles (Golgi bodies, endoplasmic reticulum and mitochondria) were observed as whorls of undifferentiated membranes and the nucleus was compressed with strongly condensed, granular chromosomes. Two types of accumulation bodies were found, some composed of dense amorphous material and others containing polygonal crystalline inclusions. Both types contained numerous membrane profiles. In cross section, the hypnozygote wall was made up of three layers divided by membranes: an outer layer with a thin electron-dense distal surface and membrane that formed the interface with the environment; a wide middle layer of striated material and membrane (possibly involved in deposition of cyst wall material); and a narrow unstructured inner layer and membrane lying close to the cytoplasmic membrane of the cell. Comparative analysis of cyst wall structure with other dinoflagellate species showed this three layered structure is common among refractive cysts. Energy dispersive X-ray analysis of the cyst wall surface demonstrated that the principal components of the cyst wall were sulphur and silica. Copyright © (2004) International Phycological Society. Reprinted by permission of Alliance Communications Group, a division of Allen Press, Inc.

Five clonal cultures of Alexandrium tamarense (AT-2, 6, 10, A and B) were isolated during toxic shellfish events in Jinhae Bay, Korea, in 1997. Thecal plate morphology, toxin composition and content, and growth characteristics were determined for each clone under different environmental conditions in order to distinguish potentially toxic A. tamarense and to improve the monitoring and prediction system in Korean coastal waters. Examination using epifluorescence microscopy after calcofluor staining showed that the thecal morphology of the apical pore complex, the first apical plate, and the posterior sulcal plates in the five clones of A. tamarense were similar. However, paralytic shellfish toxins analysed using high-performance liquid chromatography showed a range of 0.04–5.71 pg STXeq cell−1. Two clones in particular (AT-2 and AT-6) produced higher levels of toxin, at 3.2–5.7 pg STXeq cell−1. Toxin composition profiles of AT-6 showed neosaxitoxin and C-toxins (C1, C2) as the principal toxins, wi...

In order to examine temporal changes in the germination ability, time to germination and autofluorescence properties of the resting cysts of the toxic dinoflagellate Alexandrium tamarense, a long-t...

Polymerase chain reaction was used to amplify and sequence the LSU rDNA gene of 21 isolates of Alexandrium tamarense collected from the United Kingdom. Analysis of a 621 bp sequence covering the D1/D2 region of LSU rDNA revealed two distinct genetic lineages. In all cases, the strains shown to be nontoxic by ELISA or high-performance liquid chromatography were assigned to a `Western European' lineage, whereas those producing the paralytic shellfish poisoning (PSP) toxin were assigned to the `North American' lineage, which also included toxic isolates from the Orkneys. Our data are consistent with previous findings that an isolate's ability to produce PSP toxin, or lack thereof, correlates with its phylogenetic position and supports the hypothesis that at least two distinct populations of Alexandrium exist in UK waters. Copyright © (2001) International Phycological Society. Reprinted by permission of Alliance Communications Group, a division of Allen Press, Inc.

Hypotheses concerning the molecular evolution, population structure and dispersal of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech and A. f...

A new method is described that uses the fluorochrome primuline and epifluorescence microscopy for precise enumeration of dinoflagellate cysts in natural sediments. Alexandrium tamarense (Lebour) Balech resting cysts obtained in laboratory culture were fixed with glutaraldehyde and treated with methanol. The cysts were stained using nine fluorochromes under identical procedures to find those suitable for enumerating cysts. Four fiuorochromes, acriflavine, calcofluor white M2R, nile red and primuline, were found to provide satisfactory results in terms of high stainability and fluorescence intensity. Methanol treatment after fixation was necessary for high stainability. The four fluorochromes were then examined for their applicability to enumerate naturally occurring cysts in Hiroshima Bay sediments. Primuline proved to be superior to all other dyes examined, providing higher counts of cysts. Primuline-stained cysts exhibited an intense yellow-green fluorescence under blue-light excitation which hi...

Thin sections of Prorocentrum lima (Ehrenberg) Dodge and Prorocentrum maculosum Faust, two dinoflagellates known to produce diarrhetic shellfish poisoning (DSP), were probed with a monoclonal antibody targeted against okadaic acid (OA), the primary toxin in DSP outbreaks. The antibody localizes primarily to chloroplasts and pyrenoids and, to a lesser degree, cellular Iysosomes. Controls on the primary antibody lack labelling, as do controls done on a non-producer of toxin, Prorocentrum micans Ehrenberg, and a paralytic shellfish poison (PSP) producer, Alexandrium tamarense (Lebour) Balech. OA is likely to be produced by the chloroplasts of the cell and its compartmentalization in plastids may explain why producer cells are unaffected by the toxicity of OA to eukaryotic cytoplasmic phosphatases.

The effects of different growth factors and mitogens (platelet-derived growth factor, phorbol 12 monoacetate and fetal bovine serum) on dinoflagellate proliferation were analysed in axenic clonal cultures of the bloom-forming dinoflagellates Prorocentrum triestinum Schiller and Alexandrium tamarense (Lebour) Balech. Cell yield and acclimatized maximal growth rates of all clones tested increased significantly due to the specific mitogenic effect of growth factors. This is thought to be due to the existence of a universal mechanism controlling the cell division cycle in all eukaryotic cells. Apparently dinoflagellates respond to growth factors in a similar way as mammalian and yeast cells. Because growth factors can increase growth rates, they could play a significant role in the occurrence and proliferation of dinoflagellate blooms.