Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Description of a new planktonic mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the coastal waters off Western Korea: morphology, pigments, and ribosomal DNA gene sequence.

The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate-like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension-like furrow. The cingulum is as wide as 0.2-0.3 x cell length and displaced by 0.2-0.3 x cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4-19.3 and 6.1-16.0 microm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17-18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst-nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.

Alexandrium diversaporum sp. nov., a new non-saxitoxin producing species: Phylogeny, morphology and sxtA genes.

Species of the PST producing planktonic marine dinoflagellate genus Alexandrium have been intensively scrutinised, and it is therefore surprising that new taxa can still be found. Here we report a new species, Alexandrium diversaporum nov. sp., isolated from spherical cysts found at two sites in Tasmania, Australia. This species differs in its morphology from all previously reported Alexandrium species, possessing a unique combination of morphological features: the presence of 2 size classes of thecal pores on the cell surface, a medium cell size, the size and shape of the 6″, 1', 2⁗ and Sp plates, the lack of a ventral pore, a lack of anterior and posterior connecting pores, and a lack of chain formation. We determined the relationship of the two strains to other species of Alexandrium based on an alignment of concatenated SSU-ITS1, 5.8S, ITS2 and partial LSU ribosomal RNA sequences, and found A. diversaporum to be a sister group to Alexandrium leei with high support. A. leei shares several morphological features, including the relative size and shapes of the 6″, 1', 2⁗ and Sp plates and the fact that some strains of A. leei have two size classes of thecal pores. We examined A. diversaporum strains for saxitoxin production and found them to be non-toxic. The species lacked sequences for the domain A4 of sxtA, as has been previously found for non-saxitoxin producing species of Alexandrium.

A new clade, based on partial LSU rDNA sequences, of unarmoured dinoflagellates.

The order Gymnodiniales comprises unarmoured dinoflagellates. However, the lack of sequences hindered determining the phylogenetic positions and systematic relationships of several gymnodinioid taxa. In this study, a monophyletic clade was defined for the species Ceratoperidinium margalefii Loeblich III, Gyrodinium falcatum Kofoid & Swezy, three Cochlodinium species, and two Gymnodinium-like dinoflagellates. Despite their substantial morphotypic differentiation, Cochlodinium cf. helix, G. falcatum and 'Gymnodinium' sp. 1 share a common shape of the acrobase. The phylogenetic data led to the following conclusions: (1) C. margalefii is closely related to several unarmoured dinoflagellates. Its sulcus shape has been observed for the first time. (2) G. falcatum was erroneously assigned to the genus Gyrodinium and is transferred to Ceratoperidinium (C. falcatum (Kofoid & Swezy) Reñé & de Salas comb. nov.). (3) The genus Cochlodinium is polyphyletic and thus artificial; our data support its separation into three different genera. (4) The two Gymnodinium-like species could not be morphologically or phylogenetically related to any other gymnodinioid species sequenced to date. While not all studied species have been definitively transferred to the correct genus, our study is a step forward in the classification of inconspicuous unarmoured dinoflagellates. The family Ceratoperidiniaeceae and the genus Ceratoperidinium are emended.

SURVEY FOR KARLOTOXIN PRODUCTION IN 15 SPECIES OF GYMNODINIOID DINOFLAGELLATES (KARENIACEAE, DINOPHYTA)(1).

Toxin analysis of 15 species of Kareniaceae revealed the presence of karlotoxin, KmTx 2, in only a single species (Karlodinium veneficum) but with variable activity in strains from the Swan (Km(Swan) Tx 2-1, 2.1 pg · cell(-1) ; and Km(Swan) Tx 2-2, 0.53 pg · cell(-1) ), Huon (Km(Huon) Tx 2, 0.86 pg · cell(-1) ), and Derwent rivers (<0.001 pg · cell(-1) ) in Australia. A newly isolated Southern Ocean species, Karlodinium conicum, contained a novel poorly hemolytic karlotoxin analogue (Kmconicum Tx, 2.8 pg · cell(-1) ). The hemolytic potency (HD50%) of the Australian karlotoxins were as follows: Km(Swan) Tx 2-1 (65.9 ± 4.8 ng) and Km(Swan) Tx 2-2 (63.4 ± 3.7 ng), Km(Huon) Tx 2 (343 ± 4.9 ng), and Kmconicum Tx (>4,000 ng). Species from the closely related genera Takayama (T. helix, T. tasmanica, T. tuberculata), Karenia (K. asterichroma, K. brevis, K. mikimotoi, K. papilionacea, K. umbella), and Karlodinium (Ka. australe, Ka. antarcticum, Ka. ballantinum, Ka. corrugatum, Ka. decipiens) were all consistently negative for karlotoxin production. Brevetoxin (PbTx) was only detected in K. brevis, and hemolytic activity was only observed in Ka. veneficum strains.

NOVEL UNARMORED DINOFLAGELLATES FROM THE TOXIGENIC FAMILY KARENIACEAE (GYMNODINIALES): FIVE NEW SPECIES OF KARLODINIUM AND ONE NEW TAKAYAMA FROM THE AUSTRALIAN SECTOR OF THE SOUTHERN OCEAN(1).

Six new species of unarmored dinoflagellates in the family Kareniaceae were isolated from the Australian sector of the Southern Ocean in March 2006: Takayama tuberculata de Salas sp. nov, Karlodinium antarcticum de Salas sp. nov., Karl. ballantinum de Salas sp. nov., Karl. conicum de Salas sp. nov., Karl. corrugatum de Salas sp. nov., and Karl. decipiens de Salas et Laza-Martínez sp. nov. These new taxa were characterized using light and electron microscopy and sequencing of the LSU rDNA and are well supported based either on their morphology or molecular phylogeny. Takayama tuberculata, isolated just north of the polar front (55°-57° S), is genetically close to T. tasmanica, but smaller, with a significantly reduced number of amphiesmal vesicles. Medium-sized Karl. antarcticum, also isolated from near the polar front, is characterized by its long ovoid cell outline and very long apical groove. The small Karl. ballantinum has a very short apical groove. The large Karl. conicum has a distinct conical epicone and spherical posterior nucleus. The small Karl. corrugatum, from just south of the polar front, has distinctive parallel striations on the epicone surface and a distinctively shaped and placed ventral pore. The large and widespread Karl. decipiens, distributed through Southern Ocean waters from the polar front to Tasmanian coastal waters, and coastal Spain, has a helicoidal chloroplast arrangement and a large central nucleus. This study represents the first description of species in the potentially ichthyotoxic family Kareniaceae recorded from the Southern Ocean.

Development of a real-time PCR probe for quantification of the heterotrophic dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in environmental samples.

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter(-1), with higher abundance (maximum, 112 cells liter(-1)) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter(-1). P. shumwayae was not detected during the survey.