Identification and characterization of a marine bacterium extract from Mameliella sp. M20D2D8 with antiviral effects against influenza A and B viruses.

Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

Description and Characterization of the Odontella aurita OAOSH22, a Marine Diatom Rich in Eicosapentaenoic Acid and Fucoxanthin, Isolated from Osan Harbor, Korea.

Third-generation biomass production utilizing microalgae exhibits sustainable and environmentally friendly attributes, along with significant potential as a source of physiologically active compounds. However, the process of screening and localizing strains that are capable of producing high-value-added substances necessitates a significant amount of effort. In the present study, we have successfully isolated the indigenous marine diatom Odontella aurita OAOSH22 from the east coast of Korea. Afterwards, comprehensive analysis was conducted on its morphological, molecular, and biochemical characteristics. In addition, a series of experiments was conducted to analyze the effects of various environmental factors that should be considered during cultivation, such as water temperature, salinity, irradiance, and nutrients (particularly nitrate, silicate, phosphate, and iron). The morphological characteristics of the isolate were observed using optical and electron microscopes, and it exhibited features typical of O. aurita. Additionally, the molecular phylogenetic inference derived from the sequence of the small-subunit 18S rDNA confirmed the classification of the microalgal strain as O. aurita. This isolate has been confirmed to contain 7.1 mg g-1 dry cell weight (DCW) of fucoxanthin, a powerful antioxidant substance. In addition, this isolate contains 11.1 mg g-1 DCW of eicosapentaenoic acid (EPA), which is one of the nutritionally essential polyunsaturated fatty acids. Therefore, this indigenous isolate exhibits significant potential as a valuable source of bioactive substances for various bio-industrial applications.

Yihiella yeosuensis gen. et sp. nov. (suessiaceae, dinophyceae), a novel dinoflagellate isolated from the coastal waters of Korea.

A small (7-11 µm long) dinoflagellate with thin amphiesmal plates was isolated into culture from a water sample collected in coastal waters of Yeosu, southern Korea, and examined by LM, SEM, and TEM, and molecular analyses. The hemispheric episome was smaller than the hyposome. The nucleus was oval and situated from the central to the episomal region of the cell. A large yellowish-brown chloroplast was located at the end of the hyposome, and some small chloroplasts extended into the periphery of the episome. The dinoflagellate had a single elongated apical vesicle (EAV) and a type E eyespot, which are key characteristics of the family Suessiaceae. Unlike other genera in this family, it had two long furrow lines, one on the episome and the other on the hyposome, and encircling the dorsal, and lateral sides of the cell body. The pyrenoid lacked starch sheaths, but tubular invaginations into the pyrenoid matrix from the cytoplasm were observed. In the TEM, the dinoflagellate was observed to have cable-like structures (CLSs) near the eyespot but so far not observed in other dinoflagellates. The SSU rDNA sequences examined were 1.2%-5.1% different from those of other genera in the family Suessiaceae, whereas the LSU (D1-D3) rDNA sequences of this dinoflagellate were 15.1%-31.5% different. The dinoflagellate lacked a 51-bp fragment in domain D2 of the LSU rDNA, but it had an ~100-bp fragment in domain D2. This feature has been found previously only in the genera Leiocephalium and Polarella, two other genera of the Suessiaceae. The molecular phylogeny and sequence divergence based on SSU, and LSU rDNA indicate that the Korean dinoflagellate holds a taxonomically distinctive position and we consider it to be a new species in a new genus in the family Suessiaceae, named Yihiella yeosuensis gen. et sp. nov.

Most Low-Abundance "Background" Symbiodinium spp. Are Transitory and Have Minimal Functional Significance for Symbiotic Corals.

Speculation surrounds the importance of ecologically cryptic Symbiodinium spp. (dinoflagellates) that occur at low abundances in reef-building corals and in the surrounding environment. Evidence acquired from extensive sampling, long-term monitoring, and experimental manipulation can allow us to deduce the ecology and functional significance of these populations and whether they might contribute to the response of coral-dinoflagellate mutualisms to climate change. Quantitative PCR was used here to diagnose the prevalence, seasonal variation, and abundances of Symbiodinium spp. within and between colonies of the coral, Alveopora japonica. Consistent with broader geographic sampling, only one species comprised 99.9 %, or greater, the population of symbionts in every sample. However, other Symbiodinium including the non-mutualistic species, Symbiodinium voratum, were often detected, but at estimated cell densities thousands-fold less than the dominant symbiont. The temporal variation in prevalence and abundances of these "background" Symbiodinium could not be definitively related to any particular environmental factor including seasonality and water chemistry. The prevalence (proportion detected among host samples), but not abundance, of S. voratum may weakly correspond to increases in environmental inorganic silica (SiO2) and possibly nitrogen (NO3). When multiple background Symbiodinium occurred within an individual polyp, the average cell densities were positively correlated, suggesting non-specific processes of cell sorting and retention by the animal. While these findings substantiate the existence of a broader, yet uncharacterized, diversity of Symbiodinium, we conclude that only those species which can occur in high abundance and are temporally stable are ultimately important to coral-dinoflagellate mutualisms. Many transient Symbiodinium spp., which occur only at trace abundances in the coral's microbiome, belong to different functional guilds and likely have little, if any, importance to a coral's physiology. The successful integration between host and symbiont into a stable functional unit should therefore be considered when defining host-symbiont specificity.

Heterotrophic feeding as a newly identified survival strategy of the dinoflagellate Symbiodinium.

Survival of free-living and symbiotic dinoflagellates (Symbiodinium spp.) in coral reefs is critical to the maintenance of a healthy coral community. Most coral reefs exist in oligotrophic waters, and their survival strategy in such nutrient-depleted waters remains largely unknown. In this study, we found that two strains of Symbiodinium spp. cultured from the environment and acquired from the tissues of the coral Alveopora japonica had the ability to feed heterotrophically. Symbiodinium spp. fed on heterotrophic bacteria, cyanobacteria (Synechococcus spp.), and small microalgae in both nutrient-replete and nutrient-depleted conditions. Cultured free-living Symbiodinium spp. displayed no autotrophic growth under nitrogen-depleted conditions, but grew when provided with prey. Our results indicate that Symbiodinium spp.'s mixotrophic activity greatly increases their chance of survival and their population growth under nitrogen-depleted conditions, which tend to prevail in coral habitats. In particular, free-living Symbiodinium cells acquired considerable nitrogen from algal prey, comparable to or greater than the direct uptake of ammonium, nitrate, nitrite, or urea. In addition, free-living Symbiodinium spp. can be a sink for planktonic cyanobacteria (Synechococcus spp.) and remove substantial portions of Synechococcus populations from coral reef waters. Our discovery of Symbiodinium's feeding alters our conventional views of the survival strategies of photosynthetic Symbiodinium and corals.

Genetics and morphology characterize the dinoflagellate Symbiodinium voratum, n. sp., (Dinophyceae) as the sole representative of Symbiodinium Clade E.

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free-living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well-separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) µm. Unique among species in the genus, a high proportion (approximately 10-20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.

Gymnodinium smaydae n. sp., a new planktonic phototrophic dinoflagellate from the coastal waters of Western Korea: morphology and molecular characterization.

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3-10.9 µm long and 5.1-10.0 µm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4-0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1-5.8S-ITS2, and LSU rDNA region differed by 1.5-3.8%, 6.0-17.4%, and 9.1-17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.

Gyrodinium moestrupii n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence.

The heterotrophic dinoflagellate Gyrodinium moestrupii n. sp. is described based on live cells and cells prepared for light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU), internal transcribed spacers (ITS1 and ITS2), 5.8S, and the large subunit (LSU) of the rDNA were analyzed. The cells have a slender, fusiform body, taper to a sharp point at both apices, and are widest in the middle. The conical episome and hyposome are equal in size. A distinct elliptical bisected apical groove (AG) is present. Gyrodinium moestrupii has longitudinal surface striations (LSS) containing 14 and 23 lines in the episome and hyposome, respectively, whereas Gyrodinium dominans, morphologically the most similar species, has 14 and 18 lines, respectively. In addition, the episome and hyposome of G. moestrupii show distinct twists to the right and left, respectively, unlike those of Gyrodinium gutrula or G. dominans, which are not markedly twisted. The cingulum is displaced by 0.3-0.4 × cell length. Length and width of cells starved for 2 d were 23.9-38.2 and 12.0-18.6 µm, respectively, whereas those of cells satiated with Alexandrium minutum were 30.1-61.4 and 20.7-35.6 µm, respectively. The cells contain a pusule system, trichocysts, a lamellar-like structure, and a fibrous bundle, but lack chloroplasts. The SSU rDNA sequence differed by 0.2-3.9% from those of the three most closely related sequenced species for which data are currently available: G. cf. gutrula (FN669511), G. dominans (FN669510), and Gyrodinium rubrum (AB120003). The LSU rDNA was 3.2-13.9% different from G. dominans (AY571370), Gyrodinium spirale (AY571371), and G. rubrum (AY571369). The phylogenetic trees demonstrated that this novel species belongs within the Gyrodinium clade. Based on the morphological and molecular data, we propose to name it G. moestrupii n. sp.

First report of the photosynthetic dinoflagellate genus Azadinium in the Pacific Ocean: morphology and molecular characterization of Azadinium cf. poporum.

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3' apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well-defined taxonomic position.

First report of the epiphytic benthic dinoflagellates Coolia canariensis and Coolia malayensis in the waters off Jeju Island, Korea: morphology and rDNA sequences.

Coolia spp. are epiphytic and benthic dinoflagellates. Herein, we report for the first time, the occurrence of Coolia canariensis and Coolia malayensis in Korean waters. The morphology of the Korean strains of C. canariensis and C. malayensis isolated from the waters off Jeju Island, Korea was similar to that of the original Canary lslands strains and Malaysian strains, respectively. We found several pores and a line of small knobs on the pore plate, and perforations within the large pores of both C. canariensis and C. malayensis. The plates of the Korean strains of C. canariensis and C. malayensis were arranged in a Kofoidian series of Po, 3', 7'', 6c, 6s, 5''', and 2'''', and Po, 3', 7'', 7c, 6-7s, 5''', and 2'''', respectively. When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of C. canariensis was identical to that of the Biscayan strains, but it was 2-3% different from the Canary lslands strain VGO0775 and the Australian strain. In addition, the sequences of small subunit (SSU) and/or LSU rDNA from the two Korean strains of C. malayensis were < 1% different from the Malaysian strains of C. malayensis and the Florida strain CCMP1345 and New Zealand strain CAWD39 ("Coolia monotis"). In phylogenetic trees based on LSU rDNA sequences, the Korean strains of C. malayensis belonged to a clade including the Malaysian strains and these two strains. Therefore, based on genealogical analyses, we suggest that the Korean strain of C. canariensis is closely related to two Atlantic strains and the Australian strain, whereas the Korean strains of C. malayensis are related to the Malaysian strains of C. malayensis and the Florida and New Zealand strains.

First report of the epiphytic dinoflagellate Gambierdiscus caribaeus in the temperate waters off Jeju Island, Korea: morphology and molecular characterization.

Gambierdiscus spp. are epiphytic, benthic dinoflagellates. Some species have been shown to be toxic and cause ciguatera fish poisoning. We report, for the first time, the occurrence of Gambierdiscus caribaeus isolated from the waters off Jeju Island in Korea. Its morphology was similar to that of the original Belize strains of G. caribaeus. Gambierdiscus caribaeus has been reported in the tropical and subtropical waters of the Pacific, Gulf of Mexico, Caribbean Sea, and Floridian coast. Our report extends its range to the North Pacific Ocean. The plates of the Korean strain were arranged in a Kofoidian series of Po, 3', 7'', 6c, 6s, 5''', 1p, and 2'''', morphologically closer to other strains of G. caribaeus than to G. carpenteri. When properly aligned, its small subunit (SSU) rDNA was 0.5% different from those of Gambierdiscus sp. C-1, a strain that was isolated from the waters off eastern Japan, but was 2.4-4.0% different from those of the NOAA strains of G. caribaeus and 3.1-3.4% different from those of the NOAA strains of G. carpenteri. Additionally, the D1-D3 large subunit (LSU) rDNA sequence of the Korean strain of G. caribaeus was 4.7-5.3% different from those of the NOAA strains of G. caribaeus and 7.1-7.5% different from those of all reported G. carpenteri strains, including the NOAA strains. In phylogenetic trees based on SSU and LSU rDNA sequences, our Korean strain was basal to the clade consisting of the NOAA strains of G. caribaeus, which in turn was sister clade to all reported G. carpenteri strains.

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence.

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 µm long and 6.6-15.7 µm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.

Feeding by the newly described, nematocyst-bearing heterotrophic dinoflagellate Gyrodiniellum shiwhaense.

We explored the feeding ecology of the newly described, nematocyst-bearing heterotrophic dinoflagellate Gyrodiniellum shiwhaense (GenBank accession number=FR720082). Using several different types of microscopes and high-resolution video-microscopy, we investigated feeding behavior and types of prey species that G. shiwhaense feeds upon. Additionally, we measured its growth and ingestion rates on its optimal algal prey, the cryptophyte Teleaulax sp. and the dinoflagellate Amphidinium carterae, as a function of prey concentration. These rates were measured for other edible prey at single prey concentrations at which the growth and ingestion rates of G. shiwhaense were saturated. After anchoring the prey with a tow filament, G. shiwhaense fed using a peduncle, ingesting small algal species with equivalent spherical diameters (ESDs) of <13 µm. However, it did not feed on larger algal species that had ESDs≥13 µm or the small diatom Skeletonema costatum. The specific growth rates for G. shiwhaense feeding upon Teleaulax sp. and A. carterae increased rapidly with increasing mean prey concentration before saturating at concentrations of ca. 180-430 ng C/ml. The maximum specific growth rate of G. shiwhaense on Teleaulax sp. and A. carterae were 1.05 and 0.82/d, respectively. However, Heterosigma akashiwo did not support positive growth of G. shiwhaense. The maximum ingestion rates of G. shiwhaense on Teleaulax sp. and A. carterae were 0.35 and 0.54 ng C/grazer/d, respectively. The calculated grazing coefficients attributable to G. shiwhaense on co-occurring cryptophytes and Amphidinium spp. were 0.01-1.87/d and 0.08-2.60/d, respectively. Our results suggest that G. shiwhaense can have a considerable grazing impact on algal populations.

Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration.

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 µm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 µm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 µE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 µl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.

Tetraselmis jejuensis sp. nov. (Chlorodendrophyceae), a Euryhaline Microalga Found in Supralittoral Tide Pools at Jeju Island, Korea.

We found the euryhaline microalga, Tetraselmis jejuensis sp. nov., which was adapted to supralittoral tide pools with salinities varying from 0.3-3.1%. Fifteen strains of T. jejuensis were isolated from Daejeong (DJ) and Yongduam (YO), and clonal cultures were established in the laboratory. Morphological characterization revealed that the cells have a compressed shape, four flagella emerging from a depression near the apex in two opposite pairs, a cup-shaped chloroplast containing one pyrenoid surrounded by starch, and eyespot regions not located near the flagellar base. T. jejuensis cells showed distinct characteristics compared to other Tetraselmis species. First, a regular subunit pattern with honeycomb-like structures was predominantly displayed on the surface in the middle of the cell body. Second, the pyrenoid was invaded by both cytoplasmic channels comprising electron-dense material separated from the cytoplasm, and two branches of small cytoplasmic channels (canaliculi) in various directions, which characterize the subgenus Tetrathele. Eyespot regions containing a large number of osmiophilic globules, packed closely together and arranged in subcircular close packing of diverse sizes, were dispersed throughout the chloroplast. In the phylogenetic analysis of small subunit (SSU) rDNA sequences, the 15 strains isolated from DJ and YO separated a newly branched clade in the Chlorodendrophyceae at the base of a clade comprising the T. carteriiformi/subcordiformis clade, T. chuii/suecica clade, and T. striata/convolutae clade. The strains in the diverging clade were considered to belong to the same species. The SSU rDNA sequences of the DJ and YO strains showed a maximum difference of 1.53% and 1.19% compared to Tetraselmis suecica (MK541745), the closest species of the family based on the phylogenetic analysis, respectively. Based on morphological, molecular, and physiological features, we suggest a new species in the genus Tetraselmis named Tetraselmis jejuensis, with the species name "jejuensis" referring to the collection site, Jeju Island, Korea.

Characterization of the Proteolytic Activity of a Halophilic Aspergillus reticulatus Strain SK1-1 Isolated from a Solar Saltern.

Salterns are hypersaline environments that are inhabited by diverse halophilic microorganisms, including fungi. In this study, we isolated a fungal strain SK1-1 from a saltern in the Republic of Korea, which was identified as Asperillus reticulatus. This is the first reported saline-environment-derived A. reticulatus that belongs to the Aspergillus penicillioides clade and encompasses xerophilic fungi. SK1-1 was halophilic, obligately requiring NaCl for growth, with a maximum radial growth of 6%-9% (w/v) NaCl. To facilitate the biotechnological application of halophilic fungi, we screened the SK1-1 strain for proteolytic activity. Proteases have widespread applications in food processing, detergents, textiles, and waste treatment, and halophilic proteases can enable protein degradation in high salt environments. We assessed the proteolytic activity of the extracellular crude enzyme of SK1-1 using azocasein as a substrate. The crude protease exhibited maximum activity at 40-50 °C, pH 9.5-10.5, and in the absence of NaCl. It was also able to retain up to 69% of its maximum activity until 7% NaCl. Protease inhibitor assays showed complete inhibition of the proteolytic activity of crude enzymes by Pefabloc® SC. Our data suggest that the halophilic A. reticulatus strain SK1-1 produces an extracellular alkaline serine protease.

A new potentially toxic dinoflagellate Fukuyoa koreansis sp. nov. (Gonyaulacales, Dinophyceae) from Korean coastal waters: Morphology, phylogeny, and effects of temperature and salinity on growth.

To clarify an unspecified toxic Gambierdiscus-like species isolated from seawaters off Jeju Island, Korea, its morphology and molecular phylogeny based on the small subunit (SSU) and partial large subunit (LSU) rRNA gene sequences were examined. Cells were narrow in ventral view and broad in lateral view with a smooth surface. The round thecal pores were evenly distributed, with an average diameter of 0.41 µm. Cell depth, width and height were 51.7 ± 4.5 µm, 43.0 ± 4.2 µm and 55.0 ± 4.7 µm, respectively, and depth-to-width (D/W) and height-to-width (H/W) ratios were 1.1 ± 0.2 µm and 1.3 ± 0.02 µm, respectively. The nucleus was located in the hypotheca. Scanning electron microscope observations revealed that the cells displayed a plate formula of Po, 4', 6'', 6c, 6s, 5''' and 2''', and transmission electron microscope observation demonstrated that the cells contained crystal-like particles. Morphological features indicated that the unspecified Korean isolate belonged to the genus Fukuyoa, and based on the H/W and D/W ratios, the apical pore H/W ratio and thecal pore size, it could be differentiated from other Fukuyoa species. The phylogenetic analyses based on the SSU and LSU rRNA sequences revealed that the Korean isolate was nested within the genus Fukuyoa with high support, and it grouped with F. cf. yasumotoi isolated from Japan. Based on the morpho-molecular data, a new species, Fukuyoa koreansis sp. nov. is proposed. The maximum growth rate (0.254 d-1) of F. koreansis was observed at 25°C and a salinity of 25. The required levels of temperature and salinity for growth distinguished Fukuyoa koreansis from Gambierdiscus species.

Feeding by the newly described mixotrophic dinoflagellate Paragymnodinium shiwhaense: feeding mechanism, prey species, and effect of prey concentration.

To investigate the feeding by the newly described mixotrophic dinoflagellate Paragymnodinium shiwhaense (GenBank accession number=AM408889), we explored the feeding process and the kinds of prey species that P. shiwhaense is able to feed on using several different types of microscopes, including a transmission electron microscope and high-resolution video-microscopy. In addition, we measured the growth and ingestion rates of P. shiwhaense on its optimal algal prey Amphidinium carterae as a function of prey concentration. We also measured these parameters for edible prey at a single concentration at which the growth and ingestion rates of P. shiwhaense on A. carterae were saturated. Paragymnodinium shiwhaense feed on algal prey using a peduncle after anchoring the prey by a tow filament. Among the algal prey offered, P. shiwhaense ingested small algal species that had equivalent spherical diameters (ESDs) < or =11 microm (e.g. the prymnesiophyte Isochrysis galbana, the cryptophytes Teleaulax sp. and Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellates Heterocapsa rotundata and A. carterae). However, it did not feed on larger algal species that had ESDs > or =12 microm (e.g. the dinoflagellates Prorocentrum minimum, Heterocapsa triquetra, Scrippsiella trochoidea, Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum) or the small diatom Skeletonema costatum. The specific growth rates for P. shiwhaense feeding upon A. carterae increased rapidly with increasing mean prey concentration before saturating at concentrations of ca. 350 ng C/ml (5,000 cells/ml). The maximum specific growth rate (i.e. mixotrophic growth) of P. shiwhaense on A. carterae was 1.097/d at 20 degrees C under a 14:10 h light-dark cycle of 20 microE/m(2)/s, while its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was -0.224/d. The maximum ingestion and clearance rates of P. shiwhaense on A. carterae were 0.38 ng C/grazer/d (5.4 cells/grazer/d) and 0.7 microl/grazer/h, respectively. The calculated grazing coefficients for P. shiwhaense on co-occurring Amphidinium spp. was up to 0.07/h (i.e. 6.7% of the population of Amphidinium spp. was removed by P. shiwhaense populations in 1 h). The results of the present study suggest that P. shiwhaense can have a considerable grazing impact on algal populations.

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.

Complete mitochondrial genome of Micractinium singularis MM0003 (Chlorellaceae, Trebouxiophyceae).

The mitochondrial genome of Micractinium singularis MM0003 was completely sequenced. This mitogenome has 75,931 bp in length and consists of 62 genes including 32 protein-coding, 3 rRNA, and 27 tRNA genes. The overall GC content of the genome is 27.5%.