Unarmored Dinoflagellate Research Articles

Morphology, Phylogeny and Discovery of the Antioxidant Potential of a Marine Dinoflagellate Karlodinium ballantinum (Kareniaceae, Dinophyceae) Isolated from Subic Bay, Zambales, Central Luzon, Philippines

Morphological characteristics, phylogenetic analysis, and antioxidant activity of a marine unarmored dinoflagellate Karlodinium ballantinum were examined using a culture established from Subic Bay, Zambales in November 2022. Detailed examinations were based on light microscopy and molecular phylogeny inferred from internal transcribed spacer (ITS) and LSU rDNA sequences. Cells of K. ballantinum were small and ellipsoid, possessing a straight ASC, large central nucleus, accumulation bodies, and unequally distributed chloroplasts with internal pyrenoids. Cell size ranged from 7.2–13.7 μm in length and 5.4–9.0 μm in width. Phylogenetic analyses suggested that K. ballantinum has a close affinity to K. gentienii and K. zhouanum. Upscaling the culture into 20 L, the final cell density of the culture reached approximately 177.5×106 cells/mL, which produced 0.1 g of algal biomass. The antioxidant activity of K. ballantinum, as screened using the DPPH assay, showed a 23.39% antioxidative property. This result displayed the potential of K. ballantinum to be beneficial microalgae despite being known as a causative agent of HABs. The present study is the first record of K. ballantinum in Subic Bay, Zambales, and serves as the first report of the antioxidant activity of a species under the genus Karlodinium.

Record of the unarmored dinoflagellate Gynogonadinium aequatoriale (Ceratoperidiniaceae, Dinophyceae) in the Mediterranean Sea

Abstract The records of the unarmored dinoflagellate Gynogonadinium aequatoriale have been restricted to the western equatorial and the eastern tropical Pacific Ocean. The genus is reported for the first time in the Mediterranean Sea from the coast of Taranto, Ionian Sea. This is a distinctive taxon that can only be confused with the cosmopolitan Pseliodinium fusus (=Ceratoperidinium falcatum). A comparison of these two species is provided.

Establish axenic cultures of armored and unarmored marine dinoflagellate species using density separation, antibacterial treatments and stepwise dilution selection

Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.

Phylogeny and Synonymy of Gyrodinium heterostriatum comb. nov. (Dinophyceae), a Common Unarmored Dinoflagellate in the World Oceans [plus supplementary materials

The North Sea and the English Channel are regions with a long tradition of plankton studies, where the colony-forming haptophyte Phaeocystis globosa dominates the spring phytoplankton blooms. Among its predators, we investigated an abundant unarmored dinoflagellate (~3000 cells per liter) in the North Sea in May 2019. It has been reported in the literature as Gymnodinium heterostriatum or G. striatissimum, and often identified as Gyrodinium spirale. Phylogenetic analyses using the small-, large subunit- and Internal Transcriber Spacers of the ribosomal RNA (SSU-, LSU-, ITS rRNA) gene sequences indicate that our isolates clustered within the Gyrodinium clade. The new sequences formed a sister group with sequences of the freshwater taxon Gyrodinium helveticum, being one of the infrequent marine-freshwater transitions in the microbial world. This isolate is the first characterized member of a clade of numerous environmental sequences widely distributed from cold to tropical seas. This common and abundant taxon has received several names due to its morphological plasticity (changes of size and shape, often deformed after engulfing prey) and the difficulty in discerning surface striation. We conclude that the priority is for the species name Gymnodinium heterostriatum Kofoid & Swezy 1921, a new name that was proposed for Gymnodinium spirale var. obtusum sensu Dogiel 1906. The species Gyrodinium striatissimum (Hulburt 1957) Gert Hansen & Moestrup 2000 and Gymnodinium lucidum D. Ballantine in Parke & Dixon 1964 (=G. hyalinum M. Lebour 1925) are posterior synonyms. We propose Gyrodinium heterostriatum comb. nov. for Gymnodinium heterostriatum.

Toxicity comparison among four strains of Margalefidinium polykrikoides from China, Malaysia, and USA (belonging to two ribotypes) and possible implications

The unarmored dinoflagellate Margalefidinium polykrikoides is a well-known cosmopolitan harmful alga that forms intensive blooms causing massive fish kills and mortality of other marine animals. Globally distributed populations of M. polykrikoides have been categorized into four ribotypes: East Asian, American/Malaysian, Philippines, and Mediterranean, according to their characteristic sequences in LSU rRNA gene and primary geographic distributions, while no discernible morphological feature has been observed to distinguish. Here we examined the toxicity of four strains of M. polykrikoides including MPJZB-C3 and MPJZB-D6 (East Asian ribotype, from China), CP1 (American/Malaysian ribotype, from USA), and MPCoKK23 (American/Malaysian ribotype, from Malaysia), to three aquatic animals including the marine medaka (Oryzias melastigma), rotifer (Brachionus plicatilis, L-type), and brine shrimp (Artemia salina). With equivalent initial cell densities ranged from 1000 to 7000 cells mL−1, CP1 and MPCoKK23 displayed acute toxicity in a dose-response manner to the three target animals (100% mortality at higher cell densities) within 24 h, MPJZB-C3 also displayed acute toxicity to the fish and rotifer in 24 h (up to 50% mortality) when the cell densities were above 3000 cells mL−1. The strain MPJZB-D6 did not exhibit significant toxicity to all three animals in 24 h, but, in 120 h, caused 50–100% mortalities in fish when the cell density was >3000 cells mL−1 and 17% mortality in brine shrimp when the cell density was >5000 cells mL−1. Statistical analysis of the data indicated that the strains CP1 and MPCoKK23 exhibited significantly higher toxicity (50–100% mortalities) to all three animals than the strains MPJZB-C3 and MPJZB-D6 (0–100% mortality) did within 24 h exposure, while, however, the difference in toxicity between the American and Malaysian strains and that between the two Chinese strains were both not significant in terms of the mortality of test animals. While the cell-free culture media (by filtration) and sonicated cultures were less toxic to test animals than the live cultures for all M. polykrikoides strains, sonicated cultures were even less toxic than the cell-free culture media. Among the three target animals, the marine medaka exhibited most sensitive to the toxicity, followed by rotifer and then brine shrimp. Collectively, while our results demonstrated that all strains, or both ribotypes, of M. polykrikoides are toxic, the results may also suggest the differences in toxicity to three test animals among the four cultures of M. polykrikoides reflect the inherent difference in toxicity between East Asian and American/Malaysian ribotypes, which stimulated us to propose a further investigation on whether or not the four different ribotypes also differ inherently in their toxicity in the future, via comparing more strains of all four ribotypes and relevant genetic characterizations.

Morphological variation, ultrastructure, pigment composition and phylogeny of the star-shaped dinoflagellate Asterodinium gracile (Kareniaceae, Dinophyceae)

ABSTRACTMorphological variation, ultrastructure, pigment composition and phylogeny of an oceanic, star-shaped, unarmored dinoflagellate Asterodinium gracile were examined using a culture established from Nagasaki, Japan, in 2017. Our examinations were based on LM, SEM, TEM, high-performance liquid chromatography (HPLC) and molecular phylogeny inferred from nuclear-encoded internal transcribed spacer (ITS) and 28S rDNA and chloroplast-encoded 16S rDNA sequences. Typical cells were stellate, possessing an apical, two lateral, and two antapical extensions. Cells ranged in size from 62.5 to 105.6 µm long and 21.2 to 39.5 µm in cingular width. The length and orientation of cellular extensions were variable; that is, two lateral extensions were usually symmetrically bent or retracted. Ellipsoidal cells without extensions were also observed. The apical structure complex was straight or slightly sigmoid. A spherical nucleus was located at the left side in the hyposome. The numerous discoid, yellowish-brown chloroplasts were variable in size and distributed also in the cell extensions. Among Kareniaceae, the lack of a pyrenoid in Asterodinium is unique. Nuclear-encoded ITS and 28S phylogenies suggested that A. gracile is closely related to Karenia papilionacea in the Kareniaceae, but its affinity to Brachidinium capitatum, another species with extensions, was unclear. A phylogeny of 16S rDNA showed that chloroplasts of A. gracile are related to other kareniaceans derived from a haptophyte. These chloroplasts were of the fucoxanthin type, composed of pigments similar to those in other Kareniaceae; however, the detection of 19′-hexanoyloxyfucoxanthin as a major carotenoid, the low amount of fucoxanthin, and lack of 19′-butanoyloxyfucoxanthin in A. gracile were unusual for the family.

Aspectos morfológicos y primer registro del dinoflagelado <i>Pronoctiluca spinifera</i> en el Caribe colombiano

Se registró por primera vez la presencia del dinoflagelado atecado Pronoctiluca spinifera (Lohman) (Schiller, 1933) en el Parque Nacional Natural Corales de Profundidad-PNNCPR del Caribe colombiano. Fueron hallados a uno y 40 m de profundidad, tres ejemplares con tamaño promedio de 54,84 µm de largo y 10,29 µm de ancho; de contorno fusiforme con dos proyecciones, una en forma de tentáculo (13,56 µm) y otra puntiaguda. Los sitios de muestreo se caracterizaron por temperaturas entre 27,7 y 30,3 ºC; salinidad entre 35,6 y 36,4 y clorofila a entre 0,0801 y 0,1741 mg/m3; valores típicos de ambientes oceánicos. Con el presente trabajo se amplía la distribución de P. spinifera hallándose por primera vez en el mar Caribe Colombiano en el PNNCPR.

Resting Cyst Distribution and Molecular Identification of the Harmful Dinoflagellate Margalefidinium polykrikoides (Gymnodiniales, Dinophyceae) in Lampung Bay, Sumatra, Indonesia.

Margalefidinium polykrikoides, an unarmored dinoflagellate, was suspected to be the causative agent of the harmful algal blooms – associated with massive fish mortalities – that have occurred continually in Lampung Bay, Indonesia, since the first bloom event in October 2012. In this study, after examination of the morphology of putative M. polykrikoides-like cysts sampled in bottom sediments, cyst bed distribution of this harmful species was explored in the inner bay. Sediment samples showed that resting cysts, including several morphotypes previously reported as M. polykrikoides, were most abundant on the northern coast of Lampung Bay, ranging from 20.6 to 645.6 cysts g-1 dry sediment. Molecular phylogeny inferred from LSU rDNA revealed that the so-called Mediterranean ribotype was detected in the sediment while M. polykrikoides motile cells, four-cell chain forming in bloom conditions, belonged to the American-Malaysian ribotype. Moreover, hyaline cysts, exclusively in the form of four-cell chains, were also recorded. Overall, these results unequivocally show that the species M. polykrikoides is abundantly present, in the form of vegetative cells, hyaline and resting cysts in an Indonesian area.

Morphology and phylogeny of an unarmored dinoflagellate, Karlodinium jejuense sp. nov. (Gymnodiniales), isolated from the northern East China Sea

SUMMARYAn unarmored dinoflagellate, Karlodinium jejuense sp. nov., was isolated from the northern East China Sea. The species was characterized by light, fluorescence, and scanning electron microscopy of laboratory cultures, as well as SSU rDNA, ITS region (ITS1‐5.8S‐ITS2) and LSU rDNA gene sequences. Cells were ellipsoid to oval, small‐sized, 9.9–16.0 μm in length and 6.2–12.7 μm in width. The chloroplasts were 7–10 in number and yellow‐green in color. The central nucleus was large, located in the center of the cell. The linear apical groove with outward thick rolled margins was very short, and there was no ventral pore. Small pores were irregularly distributed on the cell surface. The amphiesmal vesicles were hexagonal and pentagonal. Karlodinium jejuense was morphologically similar to K. ballantinum but differed in cell surface characteristics and swimming pattern. The partial LSU sequence of K. jejuense differed by 3.7–4.1% from that of K. ballantinum. Molecular phylogenetic analyses of multi‐gene sequences revealed that K. jejuense was an independent species nested within the Karlodinium clade. Phylogenetic analysis based on partial LSU rDNA gene sequences indicated that K. jejuense was most closely related to K. gentienii and K. ballantinum.

Acute toxicity of the cosmopolitan bloom-forming dinoflagellate Akashiwo sanguinea to finfish, shellfish, and zooplankton

The unarmored dinoflagellate Akashiwo sanguinea is a well known cosmopolitan harmful alga; however, the toxic nature of this alga has yet to be examined using multiple clonal isolates. Here we examined the toxicity of 3 clonal cultures of A. sanguinea, including JX13 and JX14, isolated from Daya Bay, South China Sea, and AS2, isolated from Chesapeake Bay, USA, to multiple aquatic animals including species of finfish, shellfish, shrimp, and zooplankton. The whole-cell cultures of A. sanguinea exhibited acute lethal effects on the shrimp Litopenaeus vannamei, the bivalve Meretrix meretrix, and 2 species of fish (Mugil cephalus and Mugilidae sp.) with 72 h mortalities ranging from 20 to 100%. The sonicated and filtrated cultures were lethal to brine shrimp Artemia salina, while the filtrates of whole-cell cultures were not, suggesting that the toxins are intracellular. Boiling and freezing led to significant reductions in toxicity. A. sanguinea toxicity differed among the Chinese strains, and the hemolytic activity of 1 Chinese strain was 3-fold greater than that of the US strain. Cultures in exponential phase displayed stronger toxicity, and the greatest toxicity of A. sanguinea was observed at 20 degrees C and a salinity of 35, conditions optimal for growth of the alga. Toxicity was enhanced by increased nutrient supply, suggesting that this species could both directly (via increased growth) and indirectly (e.g. via enhanced toxin production) become more toxic in response to eutrophication. Collectively, our findings suggest that the ability to produce and release toxin(s) may promote A. sanguinea blooms by suppressing predators and competitors.

A bloom of Karlodinium australe (Gymnodiniales, Dinophyceae) associated with mass mortality of cage-cultured fishes in West Johor Strait, Malaysia

A recent (February 2014) mass mortality of fishes was observed in the cage-farming region of the West Johor Strait of Malaysia, involving over four different species of cultured fishes, numbering ∼50,000 fish. A field investigation at six stations along the West Johor Strait collected water samples and examined for the presence of harmful species. Dead fishes were collected for necropsy. The phytoplankton composition was dominated by a species of Karlodinium, at a considerably high cell concentration (0.31–2.34×106cellsl−1), and constituting 68.8–98.6% of the phytoplankton relative abundance at all stations. Detailed morphological assessment by light and scanning electron microscopy revealed that the species was Karlodinium australe de Salas, Bolch and Hallegraeff. This was supported by molecular evidence of the nuclear encoded large subunit ribosomal gene (LSU rDNA) and the second internal transcribed spacer (ITS2) via single-cell PCR. The sequences of LSU rDNA yielded 3.6–4.0% divergence when compared to the sister taxon, K. armiger; and >6.5% when compared to other Karlodinium species. Fish necropsy showed symptoms similar to those affected by karlotoxin ichthyotoxins. This is the first report of a mass mortality of cage-cultured and wild fishes attributed to the unarmored dinoflagellate K. australe.

Algicidal activity of the thiazolidinedione derivative TD49 against the harmful dinoflagellate Heterocapsa circularisquama in a mesocosm enclosure

To assess the algicidal effects of the thiazolidinedione derivative TD49 on the unarmored dinoflagellate Heterocapsa circularisquama and to evaluate the response of the planktonic community and the environment to this chemical, we undertook mesocosm (1,300 L) and small-scale experiments. The reduction ratio for H. circularisquama in each experiment was dependent on the concentration of TD49. At a TD49 concentration >0.4 μM, the abundance of H. circularisquama decreased by 99 % in the small-scale experiment and by 84 % in the mesocosm during the initial 2 days. At 0.2 μM TD49, the abundance of H. circularisquama decreased by up to 85 % in the small-scale experiment, whereas the abundance in the mesocosm increased, implying the absence of an algicidal effect. The decrease in planktonic organisms, including H. circularisquama, following TD49 treatment was correlated with abrupt declines in culture pH and dissolved oxygen concentration. Following addition of TD49, there was a significant increase in the abundance of diatoms and cryptophyta species, and after 8 days, the dominant species in the TD49 treatments shifted to small pennate diatoms including Cylindrotheca and Entomoneis species. The growth of some species among the zooplankton community was promoted at low TD49 concentrations (≤0.4 μM), whereas high concentrations (≥1.0 μM) had a negative effect. This study demonstrates that TD49 is an effective agent for the control for H. circularisquama blooms and that large-scale mesocosms play a crucial role in assessing the application of algicides such as TD49 in natural environments.

Optimization of total RNA isolation from Cochlodinium polykrikoides

Dinoflagellates are eukaryotic microorganisms that represent a group of single-celled algae composed of a highly diversified phylum that is well known for displaying an amazing range of ecological adaptation. Cochlodinium polykrikoides is an unarmored dinoflagellate species belonging to the Family Gymnodiniaceae. It is one of the causative agents for harmful algal blooms occurring in the west coast of Sabah, which result in massive economic damages to the aquaculture and mariculture industries. The limited availability of information pertaining to the genetics of C. polykrikoides raises the need for an in-depth study into the expression mechanisms and regulation of the genes of this microorganism. However, isolation of good quality and intact RNA samples from C. polykrikoides seems to be quite a challenge for molecular biologists. Rapid degradation of freshly isolated RNA samples is a bottleneck for researchers to conduct studies on the gene expression and regulation patterns, which requires intact and high-quality RNA samples. In this study, an attempt to establish the best protocol for isolation of good quality and intact RNA from C. polykrikoides was carried out by testing four different total RNA isolation protocols. We conclude that the RNA isolation protocol using the TRIzol reagent produced the best results among the methods tried. The information from this study will allow further research pertaining to the molecular aspects of C. polykrikoides to be conducted with much ease in the future.

REPORT OF A BLOOM-FORMING DINOFLAGELLATE TAKAYAMAACROTROCHA FROM TROPICAL COASTAL WATERS OF SINGAPORE(1).

Four clonal cultures of the unarmored dinoflagellate Takayama acrotrocha (J. Larsen) de Salas, Bolch et Hallegraeff were established from Singapore coastal water on October 20, 2004, and January 1, 2007, for a HAB monitoring project. LM and SEM observations demonstrated that the isolates were not consistent with the other five species within this genus in position of nucleus, shape of the apical groove, and number and shape of chloroplasts. New morphological observations of the Singapore isolates that were not in the type description of T. acrotrocha include a narrow and shallow slit located above the entire anterior edge of the cingulum, a tube-like structure in the sulcus, numerous multilateral plate-like surface vesicles, a sulcal intrusion into the epicone, and possibly a peduncle in between the two emerging points of flagella. The presence of sulcal intrusion into the epicone was not consistent with the type description but is prominent in SEM micrographs. Phylogenetic analysis of the partial LSU rDNA sequences indicated Singapore strains of T. acrotrocha are conspecific with two isolates from Italy, but less homologous to T. helix, T. tasmanica, and T. tuberculata. Laboratory fish bioassays using Asian sea bass (Lates calcarifer) and sheepshead minnows (Cyprinodon variegates) did not indicate fish-killing activity by this species, and to our knowledge, there were no reports of fish-kills occurring during blooms of this species in Singapore and Italy. This is the first report of T. acrotrocha from tropical waters and indicates a likely cosmopolitan distribution of the species.

Morphology and bloom dynamics of Cochlodinium geminatum (Schütt) Schütt in the Pearl River Estuary, South China Sea

An unarmored dinoflagellate bloom of Cochlodinium g eminatum (Schütt) Schütt has been identified in the Pearl River Estuary, South China Sea during the severe dry season, from late October to early November, 2009, when temperature and salinity ranged between 20.0–27.2 °C and 10.6–33.4, respectively. Light and scanning electron microscopy were used to identify the characteristics of C. g eminatum and provided the clear morphological structure for this species. The organism was primarily found in chains of two cells or single cell, and no longer chains were observed. Cells were irregularly spherical or slightly dorso-ventrally, with size ranged between 28 and 36 μm and longer than wide. A large nucleus in the center with numerous golden chloroplasts was present, and the cingulum made 1.5 turns around the cell. The concentration of C. g eminatum ranged from 10 2 to greater than 10 7 cells l −1 during the bloom period. Nutrient concentration ranges during the bloom were 1.29–81.00 μM NO 3, 0.14–12.14 μM NO 2, 0.21–6.29 μM NH 4, 0.23–6.26 μM PO 4 and 3.29–171.43 μM SiO 3, respectively. Total biomass expressed in terms of chlorophyll a ranged from 2.44 to 135.45 μg l −1, with an average 19.9 μg l −1 in surface water throughout the PRE. Two main clusters corresponding to the water sectors were defined with multivariate analysis (cluster and nMDS). Based on the composition and abundance of phytoplankton, spatial variations were observed at a significant level (ANOSIM, R = 0.44, P < 0.01). Although the pairwise correlation analysis detected no significant effect of any single environmental variable on the abundance of C. g eminatum, the multivariate analysis (BIO-ENV) between biotic and abiotic variables resulted in the best variables combination with all measured factors involved (temperature, salinity, turbidity, NO 3, NO 2, NH 4, PO 4 and SiO 3) which showed a combined effect during the bloom of C. g eminatum in the Pearl River Estuary ( ρ w = 0.477).

Ultrastructure of the Harmful Unarmored Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with Reference to the Apical Groove and Flagellar Apparatus

The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three-dimensional structure of the flagellar apparatus. The apical groove is U-shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5-1.0 microm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.

Seed populations of a harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef in the East China Sea

An unarmored dinoflagellate Cochlodinium polykrikoides has formed red tides responsible for fish mass mortalities especially in coastal areas of western Japan and southern Korea almost every summer to autumn. In laboratory culture, the optimum temperature for growth of the species is ca. 27 °C. Since the species cannot survive in water of temperatures of less than 10 °C, it was considered to over-winter in some certain regions as a motile form or resting cyst, and expand its distribution after the temperature increases to a level tolerable for growth. To determine the over-wintering regions and migration pattern of C. polykrikoides, occurrences of the motile cells were surveyed in the coastal and offshore areas of western Kyushu, Japan and south coast of the Korean Peninsula from April 2006 to August 2008. Cells of C. polykrikoides were found at 14 sites during the investigated period. Motile cells occurred throughout the year in Usuka Bay, Hirado of West Japan. From offshore regions of the Goto Islands and off Shin-Nagasaki Fishing Port, motile cells of C. polykrikoides were first detected from late May, and continuously occurred until February in Nama Bay of the Kami-Goto Islands. This first appearance was before red tides of C. polykrikoides reported at coastal areas in western Kyushu. In Korea, this species was first observed in May and disappeared after October in 2007. These occurrence patterns imply that Usuka Bay in Hirado is one of the over-wintering regions in western Kyushu, and also this species is possibly transported into the northern part of the East China Sea by the Tsushima Warm Current every year.

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

Abstract The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides , a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.

Complete sequence and secondary structure of the large subunit ribosomal RNA from the harmful unarmored dinoflagellateAkashiwo sanguinea

This is the first report of the complete DNA sequence of the gene encoding the ribosomal large subunit (LSU rDNA, 3336 bp) from the naked gymnodinioid dinoflagellate Akashiwo sanguinea. No introns were found in the LSU rDNA coding region and secondary structures were predicted for both the LSU and 5.8S rRNAs. The predicted LSU structure showed most of the features seen in the consensus secondary structure model proposed for the eukaryotic nuclear LSU rRNAs. However, six helices (C1_1, C1_2, C1_3, D10, D20_1 and H1_2) are not present in the A. sanguinea LSU structure. Particularly, the C branch area (or D2 domain), was extremely reduced compared to the eukaryotic consensus sequence due to nucleotide deletion. Phylogenetic resolution against 12 divergent (D) domains and cores in LSU rDNA showed that the D1, D2 and D12 domains were highly variable and could be used as genetic markers within low taxonomic levels, particularly in the gymnodinioid complex.

ULTRASTRUCTURE OF GYMNODINIUM AUREOLUM (DINOPHYCEAE): TOWARD A FURTHER REDEFINITION OF GYMNODINIUM SENSU STRICTO

Examination of the ultrastucture of the unarmored dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen (syn: Gyrodinium aureolum Hulburt) revealed the presence of nuclear chambers, which are specialized differentiations of the nuclear envelope, similar to those described in the type species of Gymnodinium, G. fuscum (Ehrenberg) Stein and certain other Gymnodinium species. The nuclear pores were restricted to these chambers. In the flagellar apparatus a nuclear fibrous connective linked the longitudinal microtubular root and the nucleus. This structure had so far been observed only in Gymnodinium spp. and in the heterotrophic species Actiniscus pentasterias (Ehrenberg) Ehrenberg, Nematodinium armatum (Dogiel) Kofoid et Swezy and Polykrikos kofoidii Chatton. Another unusual feature of G. aureolum was the presence of a striated fiber in the longitudinal flagellum, a feature previously only found in Ceratium furca (Ehrenberg) Claparède et Lachmann and C. tripos (O.F. Müller) Nitzsch. Gymnodinium aureolum also possessed a prominent ventral protrusion associated with the peduncle and containing electron opaque material. It is concluded that G. aureolum belongs to the Gymnodinium sensu stricto group. This may be a temporary classification, however, because G. aureolum and its allies differ from the type species G. fuscum by the presence of a transverse striated root, striated collars, trichocysts, and a peduncle.