Marine Dinoflagellate Alexandrium Tamarense Research Articles

Promotion of cyst formation in the toxic dinoflagellate Alexandrium (Dinophyceae) by natural bacterial assemblages from Hiroshima Bay, Japan

The relationship between the abundance of the toxic marine dinoflagellate Alexandrium tamarense (Lebour) Balech and Alexandrium-cyst-formation-promoting bacteria (Alex-CFPB) was investigated in the water column of Hiroshima Bay (Japan) from 1997 to 1998. Cell density of A. tamarense increased gradually from February to the middle of April, then peaked at the end of April and blooms declined rapidly in the beginning of May in both years. All seawater fractions collected from 5 m depth, where the density of A. tamarense cells was highest and which also contained the bulk of planktonic bacteria, promoted cyst formation of A. catenella (Whedon and Kofoid) Balech. This promotion was not caused by effects from nutrient limitation. The number of Alex-CFPB in seawater samples, analyzed by means of the most probable number (MPN) method, increased from the beginning of the Alexandrium bloom and reached 3.60 x 10 3 and 1.00 x 10 3 cells ml -1 at the peak bloom period at the end of April in 1997 and 1998, respectively. As the blooms declined, the number of Alex-CFPB decreased rapidly to less than 10 cells ml-1. Alexandrium-cyst-formation-inhibiting bacteria (Alex-CFIB) were not detected. These results show a clear positive correlation between the abundance of A. tamarense and Alex-CFPB during blooms, which suggests that Alex-CFPB play a significant role in the process of encystment and bloom dynamics of Alexandrium in the field.

Phylogenetic analysis of selected toxic and non-toxic bacterial strains isolated from the toxic dinoflagellate Alexandrium tamarense

Recent evidence has implicated bacterial involvement in the production of paralytic shellfish poison toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. Preliminary reports of the identification of toxin-producing bacteria isolated from the toxic marine dinoflagellate Alexandrium tamarense suggested that they belonged to the gamma sub-division of the Proteobacteria, specifically related to the bacterium Moraxella. Digoxigenin-labelled alpha, beta and gamma ribosomal rRNA probes, hybridized to both toxigenic and non-toxigenic bacteria isolated from several strains of Alexandrium tamarense, indicated that the bacteria belonged to the alpha sub-division of the Proteobacteria. Phylogenetic analysis of small subunit ribosomal RNA coding regions confirmed this and provided strong evidence that these bacteria are likely to represent a new genus in that group.

LSU rDNA‐BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

ABSTRACTIn a previous study large‐subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter‐ and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment‐length polymorphism (RFLP) analysis and to devise RFLP‐based assays for discriminating among representatives of that group. Computer‐assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR‐amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.

IDENTIFICATION OF GROUP‐ AND STRAIN‐SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). II. SEQUENCE ANALYSIS OF A FRAGMENT OF THE LSU rRNA GENE1

ABSTRACTA fragment of the large‐subunit (LSU) ribosomal RNA gene (rDNA) from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech was cloned and sequenced to assess inter‐ and intraspecific relationships. Cultures examined were from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo vessels and included both toxic and nontoxic isolates. Parsimony analyses revealed eight major classes of sequences, or “ribotypes,” indicative of both species‐ and strain‐specific genetic markers. Five ribotypes subdivided members of the A. tamarense/catenella/ fundyense species cluster (the “tamarensis complex”) but did not correlate with morphospecies designations. The three remaining ribotypes were associated with cultures that clearly differ morphologically from the tamarensis complex. These distinct sequences were typified by 1) A. affine, 2) A. minutum and A. lusitanicum, and 3) A. andersoni. LSU rDNA from A. minutum and A. lusitanicum was indistinguishable. An isolate's ability to produce toxin, or lack thereof, was consistent within phylogenetic terminal taxa. Results of this study are in complete agreement with conclusions from previous work using restriction fragment‐length polymorphism analysis of small subunit rRNA genes, but the LSU rDNA sequences provided finer‐scale species and population resolution.The five divergent lineages of the tamarensis complex appeared indicative of regional populations; representatives collected from the same geographic region were the most similar, regardless ofmorphotype, whereas those from geographically separated populations were more divergent even when the same morphospecies were compared. Contrary to this general pattern, A. tamarense and A. catenella from Japan were exceptionally heterogeneous, displaying sequences associated with Australian, North American, and western European isolates. This diversity may stem from introductions of A., tamarense to Japan from genetically divergent sources in North America and western Europe. Alexandrium catenella from Japan and Australia appeared identical, suggesting that these two regional populations share a recent, common ancestry. One explanation for this genetic continuity was suggested by A. catenella cysts transported from Japan to Australia via ships' ballast water: the cysts contained LSU rDNA sequences that were indistinguishable from those of known populations of A. catenella in both Japan and Australia. Ships ballasted in South Korea and Japan have also fostered a dispersal of viable A. tamarense cysts to Australia, but their LSU rDNA sequences indicated they are genetically distinct from A. tamarense/catenella previously found in Australia and genetically distinct from each other, as well. Human‐assisted dispersal is a plausible mechanism for inoculating a region with diverse representatives of the tamarensis complex from geographically and genetically distinct source populations. The D1‐D2 region of Alexandrium LSU rDNA is a valuable taxo‐nomic and biogeographic marker and a useful genetic reference for addressing dispersal hypotheses.