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.     

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.     

Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus, Alexandrium (Dinophyceae)

The genus Alexandrium includes organisms of interest, both for the study of dinoflagellate evolution and for their impacts as toxic algae affecting human health and fisheries. Only partial large subunit (LSU) rDNA sequences of Alexandrium and other dinoflagellates are available, although they contain much genetic information. Here, we report complete LSU rDNA sequences from 11 strains of Alexandrium, including Alexandrium affine, Alexandrium catenella, Alexandrium fundyense, Alexandrium minutum, and Alexandrium tamarense, and discuss their segmented domains and structure. Putative LSU rRNA coding regions were recorded to be around 3,400 bp long. Their GC content (about 43.7%) is among the lowest when compared with other organisms. Furthermore, no AT-rich regions were found in Alexandrium LSU rDNA, although a low GC content was recorded within the LSU rDNA. No intron-like sequences were found. The secondary structure of the LSU rDNA and parsimony analyses showed that most variation in LSU rDNA is found in the divergent (D) domains with the D2 region being the most informative. This high D domain variability can allow members of the diverse Alexandrium genus to be categorized at the species level. In addition, phylogenetic analysis of the alveolate group using the complete LSU sequences strongly supported previous findings that the dinoflagellates and apicomplexans form a clade.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

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.     

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.     

Three estuarine Australian dinoflagellates that can produce paralytic shellfish toxins

Three toxic dinoflagellates that can cause paralytic shellfishpoisoning (PSP) in humans are reported for the first time fromestuarine Australian waters. Blooms of the chain-forming, unarmouredGymnodinium catenatum Graham resulted in closures of shellfishfarms in summer-autumn 1986 and 1987 in southern Tasmanian estuaries.The chain-forming, armoured Alexandrium catenella (Whedon etKofoid) Balech occurred in April 1986 in Port Phillip Bay, Melbourne.Alexandrium minutum Halim produced red water in October 1986and 1987 in Port River, Adelaide. For the first time in thisspecies PSP toxin production was demonstrated by mouse bioassaysand HPLC analyses. Biogeographic aspects of these dinoflagellatesand the apparent global spreading of toxic plankton blooms arediscussed.     

Loop-mediated isothermal amplification method for rapid detection of the toxic dinoflagellate Alexandrium, which causes algal blooms and poisoning of shellfish

The marine dinoflagellate genus Alexandrium includes a number of species that produce potent neurotoxins responsible for paralytic shellfish poisoning, which in humans may cause muscular paralysis, neurological symptoms and, in extreme cases, death. Because of the genetic diversity of different genera and species, molecular tools may help to detect the presence of target microorganisms in marine field samples. Here we employed a loop-mediated isothermal amplification (LAMP) method for the rapid and simple detection of toxic Alexandrium species. A set of four primers were designed based upon the conserved region of the 5.8S rRNA gene of members of the genus Alexandrium . Using this detection system, toxic Alexandrium genes were amplified and visualized as a ladder-like pattern of bands on agarose gels under isothermal condition within 60 min. The LAMP amplicons were also directly visualized by eye in the reaction tube by the addition of SYBR Green I. This LAMP assay was 10-fold more sensitive than a conventional PCR method with a detection limit of 5 cells per tube when targeting DNA from Alexandrium minutum . The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of target toxic Alexandrium species during coastal water monitoring.     

MORPHOSPECIES VS. GENOSPECIES IN TOXIC MARINE DINOFLAGELLATES: AN ANALYSIS OF GYMNODZNIUM CATENATUM/GYRODINIUM IMPUDICUM AND ALEXANDRIUM MINUTUM/A. LUSITANICUM USING ANTIBODIES,LECTINS, AND GENE SEQUENCES1

Morphological features are the predominant criteria used to define species of marine dinoflagellates. Taxonomic problems with some toxic groups has led to the implementation of molecular taxonomy techniques and development of a genospecies concept. As a result, the relationships between “morphospecies” and “genospecies” has been questioned. In this study, the genetic differentiation between two sets of closely related morphospecies, Gymnodinium catenatum Graham/Gyrodinium impudicum Fraga and Alexandrium minutum Halim/Alexandrium lusitanicum Balech, were analyzed. The extent of morphological differentiation existing within these two groups is of the same order of magnitude. Analysis of cell surface antigens detected by preadsorbed serum, cell surface glycan moieties detected by lectins and sequencing of the D9 and D10 domains of the Large-subunit ribosomal RNA gene, showed that the extent of genetic differentiation existing between the dinofagellates Gymnodinium catenatum/Gyrodinium impudicum is substantial. Therefore, both morphological and genetic criteria resolve these organisms as two distinct entities. In contrast, Alexandrium minutum/Alexandrium lusitanicum were indistinguishable using the some suite of molecular markers. The findings demonstrated that classifications based on morphological criteria may be incongruous. On a practical level, molecular taxonomy provides useful tools to distinguish between morphologically similar microalgal species and furthermore can prevent misidentification of species such as Gymnodinium catenatum/Gyrodinium impudicum, a frequent occurrence when samples are fixed with Lugol's or formaldehyde solution.     

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.     

The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distributions within the Alexandrium tamarense "species complex" (Dinophyceae)

The cosmopolitan dinoflagellate genus Alexandrium, and especially the A. tamarense species complex, contain both toxic and nontoxic strains. An understanding of their evolution and paleogeography is a necessary precursor to unraveling the development and spread of toxic forms. The inclusion of more strains into the existing phylogenetic trees of the Alexandrium tamarense species complex from large subunit rDNA sequences has confirmed that geographic distribution is consistent with the molecular clades but not with the three morphologically defined species that constitute the complex. In addition, a new clade has been discovered, representing Mediterranean nontoxic strains. The dinoflagellates fossil record was used to calibrate a molecular clock: key dates used in this calibration are the origins of the Peridiniales (estimated at 190 MYA), Gonyaulacaceae (180 MYA), and Ceratiaceae (145 MYA). Based on the data set analyzed, the origin of the genus Alexandrium was estimated to be around late Cretaceous (77 MYA), with its earliest possible origination in the mid Cretaceous (119 MYA). The A. tamarense species complex potentially diverged around the early Neogene (23 MYA), with a possible first appearance in the late Paleogene (45 MYA). A paleobiogeographic scenario for Alexandrium is based on (1) the calculated possible ages of origination for the genus and its constituent groups; (2) paleogeographic events determined by plate movements, changing ocean configurations and currents, as well as climatic fluctuations; and (3) the present geographic distribution of the various clades of the Alexandrium tamarense species complex.     

Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species

AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA. METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized. We tested 11 type cultures of Saccharomyces, 27 different cultures of S. cerevisiae and four other ascomycetic genera. CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S. cerevisiae identification. HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.     

Molecular cloning and nucleotide sequence analysis of psbA from the dinoflagellates: Origin of the dinoflagellate plastid

Cloning and sequencing of psbA, the gene encoding D1 protein of photosystem II, from six species of dinoflagellates harboring a peridinin type plastid [Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra Stein, Lingulodinium polyedra (Dodge) Stein, Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech] is reported. Using the polymerase chain reaction technique, the psbA gene was detected in a satellite DNA band isolated from total DNA of A. catenella by CsCl-Hoechst 33258 gradient ultracentrifugation. This finding suggests that in dinoflagellates psbA is encoded in the plastid genome. The deduced amino acid sequences of D1 from the dinoflagellates did not reveal a typical ‘C-terminus extension’, which should be removed by proteolytic cleavage from the D1 precursor. Molecular phylogenetic analysis based on the deduced amino acid sequences of D1 revealed that the six species of dinoflagellates are monophyletic and also showed that dinoflagellates cluster with rhodophytes, a cryptophyte and heterokonts. These results support the hypothesis that the peridinin type plastid in dinoflagellates originated from an engulfed red alga.     

DINOFLAGELLATE HOST‐PARASITE STEROL PROFILES DICTATE KARLOTOXIN SENSITIVITY1

We examined the sterol profile of Karlodinium veneficum (D. Ballant.) J. Larsen, Akashiwo sanguinea (Hiraska) Ge. Hansen et Moestrup, Alexandrium tamarense (M. Lebour) Balech, Alexandrium affine (H. Inoue et Fukuyo) Balech, Gonyaulax polygramma F. Stein, and Gymnodinium instriatum (Freud. et J. J. Lee) Coats, along with their Amoebophyra parasites. There were no consistent sterol profiles that characterized the genus Amoebophyra. Instead, in five out of six comparisons, the host and parasite sterol profiles where highly correlated. The one exception, Amoebophyra sp. ex Alex. tamarense, was least like its host in sterol profile and also possessed the widest host range for infection. There was little correlation between host and parasite in fatty acid profiles, with the parasite being deficient in fatty acids characteristic of the plastid [e.g., 18:5(n‐3) associated with galactolipids of the thylakoids, as previously published by Adolf et al. (2007)]. Those hosts and parasites with sterol profiles dominated by desmethyl sterols were most sensitive to karlotoxin toxicity. In the host‐parasite pairs most sensitive to karlotoxin addition, recovery of the intact karlotoxin molecule was poorest. Given the sensitivity to karlotoxin, some species of Amoebophyra may avoid infection of K. veneficum.     

OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE1

Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A “universal” oligonucleotide primer set, along with species and genus‐specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low‐light‐emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples.     

PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.