ANALYSIS OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING SEQUENCES OF THE 5.8S RIBOSOMAL DNA AND INTERNAL TRANSCRIBED SPACER REGIONS1

The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS-1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS-1 (WKS-I type) or A. tamarense CU-15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS-1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS-1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150-177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28-bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.     

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

In 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). I. RFLP ANALYSIS OF SSU rRNA GENES1

Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni. The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.     

DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF SIX ALEXANDRIUM SPECIES (DINOPHYCEAE)1

In Japan, the bloom seasons of two toxic species, namely, Alexandrium catenella (Whedon et Kof.) Balech and Alexandrium tamiyavanichii Balech, sometimes overlap with those of three nontoxic Alexandrium species, namely, Alexandrium affine (H. Inouye et Fukuyo) Balech, Alexandrium fraterculus (Balech) Balech, and Alexandrium pseudogoniaulax (Biecheler) T. Horig. ex Y. Kita et Fukuyo. In this study, a multiplex PCR assay has been developed that enables simultaneous detection of six Alexandrium species on the basis of differences in the lengths of the PCR products. The accuracy of the multiplex PCR system was assessed using 101 DNA templates of the six target Alexandrium species and 27 DNA templates of 11 nontarget species (128 DNA templates in total). All amplicons obtained from the 101 DNA templates of the target species were appropriately identified, whereas all 27 DNA templates of the nontarget species were not amplified. Species‐specific identification by the multiplex PCR assay was certainly possible from single cells of the target species.     

FLUORESCENCE IN SITU HYBRIDIZATION USING rRNA‐TARGETED PROBES FOR SIMPLE AND RAPID IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM TAMARENSE AND ALEXANDRIUM CATENELLA1

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA‐targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5‐isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters.     

Geographic differences in paralytic shellfish poisoning toxin profiles among Japanese populations of Alexandrium tamarense and A. catenella (Dinophyceae)

To reconsider whether toxin profile could be used as a marker for populations from different geographical areas, clonal isolates of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech from Ofunato Bay (Iwate Prefecture), Atsumi Bay (Aichi Prefecture), Tanabe Bay (Wakayama Prefecture), Harima‐Nada (Kagawa Prefecture), Uranouchi Bay (Kochi Prefecture), Hiroshima Bay (Hiroshima Prefecture) and Yamakawa Bay (Kagoshima Prefecture), which were identified on the basis of morphotaxonomy, immunological and molecular biological techniques, were subjected to analysis of paralytic shellfish poisoning toxins by high performance liquid chromatography‐fluorometric method. All the isolates except A. tamarense OF152 from Ofunato Bay contained mainly N‐sulfocarbamoyl toxins (C1 +2) with various amounts of derivatives, and a typical north‐to‐south trend of decreasing toxicity was observed. In both A. tamarense and A. catenella, toxin profiles were rather constant within a geographical area and divergent among different geographical areas. The toxin profiles of A. tamarense from Harima‐Nada were well conserved among different bloom years. Toxin profile showed that isolates of A. tamarense from Ofunato Bay, A. tamarense from Harima‐Nada isolated in 1988 and A. catenella from Uranouchi Bay were heterogeneous. However, only two or three groups of isolates with different toxin profiles were observed in a geographical region, suggesting that several representative isolates express the genotype in a given region. These observations confirmed that toxin composition could be used as a marker to discriminate different geographical populations of these species.     

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes

To improve labeling-intensity of whole-cell fluorescence in situ hybridization (FISH) in the molecular identification of toxic Alexandrium tamiyavanichii, two DNA probes (TAMID2 plus TAMIS1 designed from the LSU and SSU rDNA regions, respectively) were used to test the labeling intensity of targeted cultured A. tamiyavanichii cells. The cross-reactivity of the DNA probe to natural seawater samples and six Alexandrium species: A. affine, A. catenella, A. fraterculus, A. insuetum, A. pseudogonyaulax and A. tamarense, was also tested. The labeling intensity of the DNA probe TAMID2S1, a combination of two separate probes that target different regions of the rRNA, was 1.7–2.7 times higher than that of the single DNA probe TAMID2. With cultured A. tamiyavanichii cells in the dead growth phase at 30 days, the TAMID2S1 intensity was 1.9 times higher than that of TAMID2. During a 30-day culture, the labeling intensity of A. tamiyavanichii cells hybridized with TAMID2S1 decreased to 49.4% of the original intensity. No cross-reactivity to various microorganisms in natural seawater samples was found. The two DNA probes together, designated as TAMID2S1, readily detected A. tamiyavanichii added to natural seawater samples, even aged cultured cells.     

Genus Alexandrium Halim, 1960 (Dinophyta) from the Pacific coast of Russia: Species composition, distribution, and dynamics

At present 8 species of Alexandrium genus have been found in seas and adjacent waters of Russia: A. acatenella, A. catenella, A. insuetum, A. margalefii, A. ostenfeldii, A. pseudogonyaulax, A. tamarense, and A. tamutum. The distribution and population density of Alexandrium species varied within the surveyed area of the Pacific: in the Sea of Japan and Sea of Okhotsk, 7 species were recorded; 3 species were recorded along the Pacific coast of Kamchatka; and 2 species were found in the Bering Sea. A. tamarense was the most widespread and abundant species over the area. A. insuetum was recorded only in the Sea of Japan, and A. catenella, in the Sea of Okhotsk (Terpeniya Bay). The highest concentration of Alexandrium spp. (2–7 million cells/l) was recorded along the Pacific coast of Kamchatka and in the Bering Sea; in the Sea of Okhotsk, a rather high concentration (51000 cells/l) was registered in Aniva Bay; in the Sea of Japan, the highest concentration was recorded in Peter the Great Bay (6000 cells/l). The distribution of cysts (spores) in surface sediments of the Pacific coast of Russia as a whole reflected the pattern of distribution of vegetative cells of Alexandrium. Cysts of Alexandrium cf. tamarense prevailed all over the area, with the maximum concentration along the Pacific coast of Kamchatka. Beyond that type of cysts, insignificant numbers of cysts of Alexandrium cf. minutum were recorded in Peter the Great Bay and Aniva Bay. Analysis of seasonal dynamics revealed that cells of Alexandrium spp. occurred in Peter the Great Bay from June up to September, and along the Pacific coast of Kamchatka from April to October. In the first region, the maximum density was recorded in August; it was provided by A. pseudogonyaulax (59% of the total density of Alexandrium), A. tamarense (35%), and A. insuetum (6%). In the second region, it was recorded in July, thanks only to development of A. tamarense.     

PATTERNS IN HOST RANGE FOR TWO STRAINS OF AMOEBOPHRYA (DINOPHYTA) INFECTING THECATE DINOFLAGELLATES: AMOEBOPHYRA SPP. EX ALEXANDRIUM AFFINE AND EX GONYAULAX POLYGRAMMA1

The endoparasitic dinoflagellate Amoebophrya ceratii (Koeppen) Cachon uses a number of its free‐living relatives as hosts and may represent a species complex composed of several host‐specific parasites. Two thecate host–parasite systems [Amoebophrya spp. ex Alexandrium affine (Inoue and Fukuyo) Balech and ex Gonyaulax polygramma Stein], were used to test the hypothesis that two strains of Amoebophrya have a high degree of host specificity. To test this hypothesis, a series of cross‐infection experiments were conducted, with 10 thecate and three athecate dinoflagellate species as potential hosts. Surprisingly, the two strains of Amoebophrya lacked host specificity and had wider host ranges than previously recognized. Among the host species tested, Amoebophrya sp. ex Alexandrium affine was capable of infecting only species of genus Alexandrium (Alexandrium affine, Alexandrium catenella, and Alexandrium tamarense), while the parasite from Gonyaulax polygramma infected species covering five genera (Alexandrium, Gonyaulax, Prorocentrum, Heterocapsa, and Scripsiella). In the context of previous reports, these results suggest that host specificity of Amoebophrya strains varies from extremely species‐specific to rather unspecific, with specificity being stronger for strains isolated from athecate hosts. Information on host specificity of Amoebophrya strains provided here will be helpful in assessing the possibility of using these parasites as biological control agents for harmful algal blooms, as well as in defining species of Amoebophrya in the future.     

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

A 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.     

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.     

Toxic mucus traps: A novel mechanism that mediates prey uptake in the mixotrophic dinoflagellate Alexandrium pseudogonyaulax

The functional role of harmful substances (i.e. toxins) produced by marine planktonic algae is still, in many cases, unknown. This study describes a novel mechanism by which the phototrophic dinoflagellate Alexandrium pseudogonyaulax secretes a toxic mucus trap where prey items are caught and immobilized prior to ingestion. Prey cells remain entrapped and immobile in the mucus trap, but most stay intact, readily available as whole-cell prey. It is shown that food uptake by A. pseudogonyaulax increases its growth rate considerably even in nutrient-replete, high-light conditions. The increase in growth rate was more enhanced in light-limited treatments and A. pseudogonyaulax grew significantly faster when fed Heterocapsa rotundata, than when fed Teleaulax acuta under both light conditions. For comparison, strains of Alexandrium catenella and Alexandrium minutum were studied for their mixotrophic capabilities. None of these strains were mixotrophic under the conditions provided. In addition, the toxic effects on various protistan targets of these Alexandrium strains as well as Alexandrium tamarense and Alexandrium ostenfeldii were compared to that of A. pseudogonyaulax. A. tamarense and A. catenella did immobilize and lyse target cells through substances leaked directly into the water, differing from all the strains of A. pseudogonyaulax studied. Results show that the toxic effect of A. pseudogonyaulax is non-specific causing nearly 100% immobilization of a variety of protistan targets at relatively low cell concentrations (500 cells ml⁻¹ of donor cell). A critical donor cell density was not required as only one A. pseudogonyaulax cell was able to cause immobilization of target cells. For the first time, the connection between excreted toxins and phagotrophy is evident in an Alexandrium species and this particular strategy has the potential to severely impact competing phytoplankton communities.     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

IDENTIFICATION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING PCR AND rDNA-TARGETED PROBES

A PCR (polymerase chain reaction)-based assay for the detection of Alexandrium species in cultured samples using rDNA-targeted probes was developed. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S ribosomal RNA gene (rDNA) from cultured isolates of A. tamarense (Lebour) Taylor, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech and A. lusitanicum Balech were amplified using PCR and sequenced. Sequence comparisons showed that the 5.8S and ITS1-ITS2 regions contain sequences specific for the Alexandrium genus, especially at the 3' end of the 5.8S coding region. PCR primers and a radioactive ³²P-labeled DNA probe were devised for this region. The cross-reactivity of the PCR primers and probe was tested against cultured isolates of Alexandrium and other dinoflagellates and diatoms. All the Alexandrium isolates screened reacted toward the genus-specific probe; in contrast, the other groups of microalgae (dinoflagellates and diatoms) did not react with the probe. Furthermore, the PCR amplification technique combined with the use of the rDNA-target probe allowed us to develop a method for the detection of Alexandrium cells in cultured samples. This PCR method might offer a new approach for the identification and enumeration of the HAB (harmful algal bloom) species present in natural phytoplankton populations.