Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.     

Analysis of rRNA gene content in the Mediterranean dinoflagellate <Emphasis Type="Italic">Alexandrium catenella</Emphasis> and <Emphasis Type="Italic">Alexandrium taylori</Emphasis>: implications for the quantitative real-time PCR-based monitoring methods

A number of species belonging to the genus Alexandrium are among the main toxic microalgae responsible for Harmful Algal Blooms (HABs). The monitoring of coastal waters for the presence of these microalgae is essential to identify correlations between cell abundances and environmental factors that regulate bloom dynamics. In the attempt to improve the monitoring sensitivity and the rapidity at which a large number of field samples can be processed, several molecular methods for the detection of genetically distinct HAB species have been developed during the last years. In particular, real-time PCR has been shown to be a powerful method for quantitative detection of HAB species in environmental samples. When a plasmid is used as a standard, the knowledge of the amount of target gene per cell is essential for the determination of the cell number in the field sample. In this study, we analyzed the rRNA gene content variability in several Alexandrium catenella and Alexandrium taylori strains isolated from the Mediterranean Sea using a real-time PCR-based approach. The rRNA gene content was also analyzed in different growth phases, from early exponential to stationary conditions. The results showed a general variability in the rRNA gene content depending on the strain and, for the species A. taylori, in relation also to the growth phase. These results should be taken into account for the application of the real-time quantitative PCR-based techniques for monitoring purposes in coastal seawaters.     

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.     

Morphology,toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China

The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate)

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.     

Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR

The presence of neurotoxic species within the genus Alexandrium along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of Alexandrium catenella and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying A. catenella in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter⁻¹, 2 orders of magnitude below threshold abundances that can impact food webs. A. catenella was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter⁻¹). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter⁻¹, whereas the maximum STX concentration of 26 ng liter⁻¹ occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but A. catenella was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of A. catenella at subbloom and bloom abundances.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

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.     

Description of the new phototrophic dinoflagellate Alexandrium pohangense sp. nov. from Korean coastal waters

The planktonic phototrophic dinoflagellate Alexandrium pohangense sp. nov. isolated from the coastal waters off Korea is described from living and fixed cells by light and scanning electron microscopy (SEM). DNA sequence data were collected from the small subunit (SSU), the large subunit (LSU), internal transcribed spacer regions (ITS1 and ITS2), and 5.8S of the ribosomal DNA (rDNA). The SSU and LSU rDNA sequences of the new dinoflagellate were 4–7% and 14–17%, respectively, different from those of Alexandrium minutum, Alexandrium ostenfeldii, Alexandrium tamutum, Alexandrium margalefii, and Alexandrium pseudogonyaulax, the most closely related species. In addition, the 5.8S rDNA sequence of the new dinoflagellate was also 12% different from those of A. minutum, A. ostenfeldii, A. tamutum, and Alexandrium peruvianum. In a phylogenetic tree based on LSU rDNA sequences, A. pohangense formed a clade with A. margalefii, and this clade was clearly distinct from the clade of A. minutum, Alexandrium diversaporum, A. tamutum, Alexandrium leei, A. ostenfeldii, and Alexandirum andersoni. Moreover, in a phylogenetic tree based on SSU rDNA sequences, A. pohangense was positioned at the base of the clade containing A. leei and A. diversaporum. Morphological analysis showed that A. pohangense has a Kofoidian plate formula of Po, 4′, 6′′, 6c, 8s, 5′′′, and 2′′′′, which confirmed its assignment to the genus Alexandrium. This dinoflagellate has a wide rectangular 1′ plate, the upper left side of which is slightly bent, protruding, and touching the 2′ plate, unlike A. margalefii, which has a wide rectangular 1′ plate that does not touch the 2′ plate, or A. pseudogonyaulax and Alexandrium camurascutulum, which have a narrower elongated pentagonal 1′ plate that touches the 2′ plate. Furthermore, the 1′ plate of A. pohangense meets the 1′′ plate as a straight vertical line, whereas that of A. camurascutulum meets the 1′′ plate as an inclined line because it is lifted by the intrusion of the 1′′ plate. In addition, A. pohangense had a relatively small ventral pore whose majority was located on the 4′ plate, unlike A. margalefii or A. pseudogonyaulax, which have a relatively large ventral pore whose majority is located on the 1′ plate. Furthermore, A. pohangense had pores of two different sizes on the cell surface, unlike A. margalefii and A. pseudogonyaulax, which have similar pores of only one size. On the basis of morphological and phylogenetic criteria, it is proposed that this is a new species of the genus Alexandrium.     

A PCR IMMUNOASSAY METHOD FOR THE DETECTION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES

PCR primers targeting the internal transcribed spacer (ITS)-5.8S rDNA regions specific for the genus Alexandrium were used to develop an ELISA assay method to detect and enumerate this genus in cultured isolates. The solid-phase ELISA involves the application of a biotinylated labeled primer to target the specific ITS-5.8S rDNA region; the PCR-amplified products, generated in the presence of digoxigenin-11-deoxiuracil triphosphate nucleotide, are captured on the streptavidin-coated microplate. The captured molecules were hybridized to an anti-digoxigenin antibody conjugated with alkaline phosphatase. The presence and number of the Alexandrium cells in the samples resulted in a proportional appearance of color generated by the phosphatase activity in the presence of a chromogenic substrate and measured in a plate reader. This PCR and immunoassay solid-phase assay proved to be a useful technique to detect the presence of Alexandrium sp. in cultured isolates and seawater samples.     

Identification of unique microbiomes associated with harmful algal blooms caused by Alexandrium fundyense and Dinophysis acuminata

Biotic interactions dominate plankton communities, yet the microbial consortia associated with harmful algal blooms (HABs) have not been well-described. Here, high-throughput amplicon sequencing of ribosomal genes was used to quantify the dynamics of bacterial (16S) and phytoplankton assemblages (18S) associated with blooms and cultures of two harmful algae, Alexandrium fundyense and Dinophysis acuminata. Experiments were performed to assess changes in natural bacterial and phytoplankton communities in response to the filtrate from cultures of these two harmful algae. Analysis of prokaryotic sequences from ecosystems, experiments, and cultures revealed statistically unique bacterial associations with each HAB. The dinoflagellate, Alexandrium, was strongly associated with multiple genera of Flavobacteria including Owenweeksia spp., Maribacter spp., and individuals within the NS5 marine group. While Flavobacteria also dominated Dinophysis-associated communities, the relative abundance of Alteromonadales bacteria strongly co-varied with Dinophysis abundances during blooms and Ulvibacter spp. (Flavobacteriales) and Arenicella spp. (Gammaproteobacteria) were associated with cells in culture. Eukaryotic sequencing facilitated the discovery of the endosymbiotic, parasitic dinoflagellate, Amoebophrya spp., that had not been regionally described but represented up to 17% of sequences during Alexandrium blooms. The presence of Alexandrium in field samples and in experiments significantly altered the relative abundances of bacterial and phytoplankton by both suppressing and promoting different taxa, while this effect was weaker in Dinophysis. Experiments specifically revealed a negative feedback loop during blooms whereby Alexandrium filtrate promoted the abundance of the parasite, Amoebophrya spp. Collectively, this study demonstrates that HABs formed by Alexandrium and Dinophysis harbor unique prokaryotic and eukaryotic microbiomes that are likely to, in turn, influence the dynamics of these HABs.     

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.     

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.     

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 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.     

Environmental drivers of paralytic shellfish toxin producing Alexandrium catenella blooms in a fjord system of northern Southeast Alaska

Paralytic shellfish poisoning (PSP) is a persistent problem that threatens human health and the availability of shellfish resources in Alaska. Regular outbreaks of marine dinoflagellates in the genus Alexandrium produce paralytic shellfish toxins (PSTs) that make shellfish consumption unsafe, and impose economic hardships on Alaska’s shellfish industry. Phytoplankton and environmental monitoring spanning 2008–2016, and a pilot benthic cyst survey in 2016, were focused in the Juneau region of Southeast Alaska to investigate Alexandrium catenella distributions and conditions favorable to bloom development. Overwintering Alexandrium cysts were found in near-shore sediments throughout the study region. Alexandrium catenella cells were present in the water column across a range of sea surface temperatures (7–15 °C) and surface salinities (S = 4–30); however, an optimal temperature/salinity window (10–13 °C, 18–23) supported highest cell concentrations. Measurable levels of PSTs were associated with lower concentrations (100 cells L⁻¹) of A. catenella, indicating high cell densities may not be required for shellfish toxicity to occur. Several interacting local factors were identified to support A. catenella blooms: 1) sea surface temperatures ≥7 °C; 2) increasing air temperature; 3) low to moderate freshwater discharge; and 4) several consecutive days of dry and calm weather. In combination, these bloom favorable conditions coincide with toxic bloom events during May and June in northern Southeast Alaska. These findings highlight how integrated environmental and phytoplankton monitoring can be used to enhance early warning capacity of toxic bloom events, providing more informed guidance to shellfish harvesters and resource managers in Alaska.     

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.