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.     

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.     

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.     

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.     

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.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

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.     

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.     

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.     

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.     

Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae)1

Alexandrium catenella (Whedon et Kof.) Balech, A. tamarense (M. Lebour) Balech, and A. fundyense Balech comprise the A. tamarense complex, dinoflagellates responsible for paralytic shellfish poisoning worldwide. The relationships among these morphologically defined species are poorly understood, as are the reasons for increases in range and bloom occurrence observed over several decades. This study combines existing data with new ribosomal DNA sequences from strains originating from the six temperate continents to reconstruct the biogeography of the complex and explore the origins of new populations. The morphospecies are examined under the criteria of phylogenetic, biological, and morphological species concepts and do not to satisfy the requirements of any definition. It is recommended that use of the morphospecies appellations within this complex be discontinued as they imply erroneous relationships among morphological variants. Instead, five groups (probably cryptic species) are identified within the complex that are supported on the basis of large genetic distances, 100% bootstrap values, toxicity, and mating compatibility. Every isolate of three of the groups that has been tested is nontoxic, whereas every isolate of the remaining two groups is toxic. These phylogenetic groups were previously identified within the A. tamarense complex and given geographic designations that reflected the origins of known isolates. For at least two groups, the geographically based names are not indicative of the range occupied by members of each group. Therefore, we recommend a simple group‐numbering scheme for use until the taxonomy of this group is reevaluated and new species are proposed.     

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.     

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.     

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.     

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.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Quantification methods for Alexandrium catenella, a toxic dinoflagellate: Comparison of competitive enzyme-linked immunosorbent assay and sandwich hybridization integrated with a nuclease protection assay

Alexandrium catenella (Whedon et Kofoid) Balech, a toxic dinoflagellate, is a bloom-forming planktonic species in cold water coastal regions. It produces strong paralytic shellfish poisoning (PSP) toxins which are transmitted via tainted shellfish. These toxins can affect humans, other mammals, fish and birds. In this study, polyclonal antiserum against A. catenella was produced, and a competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect A. catenella. The antiserum against A. catenella showed good specificity, the linear detection range was relatively large, between 38 and 600,000 cells. In addition, specific probes were designed to target the small subunit ribosomal RNA (SSU rRNA) of A. catenella, and quantitative sandwich hybridization integrated with a nuclease protection assay (NPA-SH) was established in order to identify and quantify A. catenella. The NPA-SH assay did not show good specificity as well as cELISA, by which A. catenella and A. tamarense could not be distinguished. Samples in different cell growth phases were analyzed with cELISA and NPA-SH. The results showed that the cell concentration calculated by cELISA was very similar with microscopy, while that of NPA-SH was sometimes higher than that of microscopy, especially in log phase. Comparing the two methods, both assays allow rapid identification of A. catenella without time-consuming microscopy when multiple sites need to be considered in routine monitoring. Meanwhile, cELISA was more specific and accurate in detection of A. catenella than NPA-SH.     

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.