Production of safe cyanobacterial biomass for animal feed using wastewater and drinking water treatment residuals.

The growing interest in microalgae and cyanobacteria biomass as an alternative to traditional animal feed is hindered by high production costs. Using wastewater (WW) as a cultivation medium could offer a solution, but this approach risks introducing harmful substances into the biomass, leading to significant safety concerns. In this study, we addressed these challenges by selectively extracting nitrates and phosphates from WW using drinking water treatment residuals (DWTR) and chitosan. This method achieved peak adsorption capacities of 4.4 mg/g for nitrate and 6.1 mg/g for phosphate with a 2.5 wt% chitosan blend combined with DWTR-nitrogen. Subsequently, these extracted nutrients were employed to cultivate Spirulina platensis, yielding a biomass productivity rate of 0.15 g/L/d, which is comparable to rates achieved with commercial nutrients. By substituting commercial nutrients with nitrate and phosphate from WW, we can achieve a 18 % reduction in the culture medium cost. While the cultivated biomass was initially nitrogen-deficient due to low nitrate levels, it proved to be protein-rich, accounting for 50 % of its dry weight, and contained a high concentration of free amino acids (1260 mg/g), encompassing all essential amino acids. Both in vitro and in vivo toxicity tests affirmed the biomass's safety for use as an animal feed component. Future research should aim to enhance the economic feasibility of this alternative feed source by developing efficient adsorbents, utilizing cost-effective reagents, and implementing nutrient reuse strategies in spent mediums.

The Halophilic Bacterium Paracoccus haeundaensis for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) from Single Carbon Sources.

The study objective was to evaluate the potential production of polyhydroxyalkanoates (PHAs), a biodegradable plastic material, by Paracoccus haeundaensis for which PHA production has never been reported. To identify the most effective nitrogen-limited culture conditions for PHAs production from this bacterium, batch fermentation using glucose concentrations ranging from 4 g l-1 to 20 g l-1 with a fixed ammonium concentration of 0.5 g l-1 was carried out at 30°C and pH 8.0. A glucose supplement of 12 g l-1 produced the highest PHA concentration (1.6 g l-1) and PHA content (0.63 g g-1) thereby identifying the optimal condition for PHA production from this bacterium. Gas chromatography-mass spectrometry analysis suggests that P. haeundaensis mostly produced copolymer types of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from glucose concentrations at 12 g l-1 or higher under the nitrogen-limited conditions. When several other single carbon sources were evaluated for the most efficient PHA production, fructose provided the highest biomass (2.8 g l-1), and PHAs (1.29 g l-1) concentrations. Results indicated that this bacterium mostly produced the copolymers P(3HB-co-3HV) from single carbon sources composing a range of 93-98% of 3-hydroxybutyrate and 2-7% of 3-hydroxyvalerate, whereas mannose-supplemented conditions produced the only homopolymer type of P(3HB). However, when propionic acid as a secondary carbon source were supplemented into the media, P. haeundaensis produced the copolymer P(3HB-co-3HV), composed of a 50% maximum monomeric unit of 3-hydroxyvaleric acid (3HV). However, as the concentration of propionic acid increased, cell biomass and PHAs concentrations substantially decreased due to cell toxicity.

Fibrivirga algicola gen. nov., sp. nov., an algicidal bacterium isolated from a freshwater river.

An aerobic, gram-stain-negative, pink-colored, non-motile and rod-shaped algicidal bacterium, designated as JA-25T was isolated from freshwater in Geumgang River, Republic of Korea. Strain JA-25T grew at 15-30 °C and pH 6-9, and did not require NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JA-25T belongs to the family 'Spirosomaceae' and is most closely related to Fibrella aestuarina BUZ 2T (93.6%). Strain JA-25T showed < 90% sequence similarity to other members of the family 'Spirosomaceae'. The average nucleotide identity(ANI), in silico DNA-DNA hybridization and average amino acid identity(AAI) values based on the genomic sequences of JA-25T and F. aestuarina BUZ 2T were 74.4, 20.5, and 73.6%, respectively. Strain JA-25T showed an algicidal effect on the marine flagellate alga Heterocapsa triquetra, but no effect on fresh water cyanobacterium (Nostoc). In genome analysis, RIPP-like peptides were detected and predicted to resemble the indolmycin biosynthetic gene cluster, which possibly influence its algicidal effect. Furthermore, a bacteriorhodopsin gene with photoheterotrophic characteristics was detected. The genomic DNA G + C content was 52.5 mol%. The major cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:1 ω5c, C16:0 (> 10%). The major respiratory quinone was menaquinone 7 and major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids. Considering the phylogenetic inference, phenotypic, and chemotaxonomic data, strain JA-25T should be classified as a novel species in the novel genus Fibrivirga, with the proposed name Fibrivirga algicola sp. nov. The type strain is JA-25T (= KCCM 43334T = NBRC 114259T).

Molecular Phylogeny and Taxonomy of the Genus Spumella (Chrysophyceae) Based on Morphological and Molecular Evidence.

The genus Spumella, established by Cienkowsky in 1870, is characterized by omnivory, two (rarely three) flagella, a short stick-like structure beneath the flagella, a threadlike stalk, cell division via constriction and cyst formation. Since the first phylogenetic study of Spumella-like flagellates, their paraphyly has consistently been shown, with separation into several genera. More recently, Spumella was carefully investigated using molecular and morphological data to propose seven new species. Classification of this genus and knowledge of its species diversity remain limited because Spumella-like flagellates are extremely difficult to identify based on limited morphological characters. To understand the phylogeny and taxonomy of Spumella, we analyzed molecular and morphological data from 47 strains, including 18 strains isolated from Korean ponds or swamps. Nuclear SSU, ITS and LSU rDNA data were used for maximum likelihood and Bayesian analyses. The molecular data divided the strains into 15 clades, including seven new lineages, each with unique molecular signatures for nuclear SSU rRNA from the E23-2 to E23-5 domains, the spacer between the E23-8 and E23-9 domains of the V4 region and domain 29 of the V5 region. Our results revealed increased species diversity in Spumella. In contrast to the molecular phylogeny results, the taxa showed very similar cell morphologies, suggesting morphological convergence into simple nanoflagellates to enable heterotrophy. Three new species produced stomatocysts in culture. Aspects of stomatocyst morphology, including collar structure, surface ornamentation, and cyst shape, were very useful in differentiating the three species. The general ultrastructure of Spumella bureschii strain Baekdongje012018B8 and S. benthica strain Hwarim032418A5 showed the typical chrysophyte form for the leucoplast, a vestigial chloroplast surrounded by four envelope membranes, supporting the hypothesis that Spumella evolved from a phototroph to a heterotroph via the loss of its photosynthetic ability. Seven new species are proposed: S. benthica, S. communis, S. longicolla, S. oblata, S. rotundata, S. similis, and S. sinechrysos.

Tunicothrix halophila n. sp., a Secondarily Oligomerized Parabirojimid Hypotrich (Ciliophora, Spirotrichea) From Hypersaline Costal Water in Korea.

Tunicothrix halophila n. sp. was discovered in a hypersaline marine sample from Jeju Island, Korea. It is characterized by the highly reduced number of dorsal bristles. In addition, the main character of the genus Tunicothrix (e.g., alveolar layer) is absent/indistinct. To figure out its identity and phylogenetic relationship, we examined the new species based on modern morphological methods and molecular phylogenetic analyses. Since the parabirojimids are of basal position to core hypotrichs and a smaller data set could show incorrect phylogenetic relationships among the hypotrichs, we used a huge data set composed of 1,460 DNA sequences to infer the phylogenetic tree. The reduction of dorsal bristles is very likely a secondarily evolved character in hypotrichs, resulting in the independent phenotypic adaptation in the hypersaline ecosystems as shown in other hypersaline hypotrichs. Furthermore, the so-called right marginal row 1 in other congeners is found to produce a pretransverse and transverse cirrus and thus we recommend using the term frontoventral row. Based on our data, we can justify Tunicothrix halophila n. sp. as a new species; however, despite the phenotypic distinctiveness, we refrain to establish a new genus because of the missing data and the non-monophyly of Tunicothrix.

Genetic Impairment of Cellulose Biosynthesis Increases Cell Wall Fragility and Improves Lipid Extractability from Oleaginous Alga Nannochloropsis salina.

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga Nannochloropsis salina does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene (CesA) expression would lead to protein accumulation without the accumulation of storage C polymers in N. salina. Three cesA mutants were generated by the CRISPR/Cas9 approach. Cell wall thickness and cellulose content were reduced in the cesA1 mutant, but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Further, N. salina cells with a thinned cell wall were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability. Taken together, the previous and current studies strongly suggest the presence of a controlling mechanism that regulates photoassimilate partitioning toward C and N metabolic pathways as well as the cellulose metabolism as a potential target for cost-effective microalgal cell disruption and as a useful protein production platform.

Enhanced removal of cesium by potassium-starved microalga, Desmodesmus armatus SCK, under photoheterotrophic condition with magnetic separation.

This study investigated the feasibility of using photoheterotrophic microalga, Desmodesmus armatus SCK, for removal of cesium (Cs+) followed by recovery process using magnetic nanoparticles. The comparison of three microalgae results indicated that D. armatus SCK removed the most Cs+ at both 25 °C and 10 °C. The results also revealed that the use of microalga grown in potassium (K+)-starved condition improves the accumulation of Cs+. Heterotrophic mode with addition of volatile fatty acids (VFAs), especially acetic acids (HAc), also enhanced removal of Cs+ by K+-starved D. armatus SCK; maximum removal efficiency of Cs+ was almost 2-fold higher than that of cells grown without organic carbon source. The Cs+ taken up by this microalga was efficiently harvested using magnetic nanoparticles, polydiallyldimethylammonium (PDDA)-FeO3. Finally, this strain eliminated more than 99% of radioactive 137Cs from solutions of 10, 100, and 1000 Bq mL-1. Therefore, use of K+-starved microalga, D. armatus SCK, with VFAs could be promising means to remove the Cs from the liquid wastes.

Mixotrophy in the marine red-tide cryptophyte Teleaulax amphioxeia and ingestion and grazing impact of cryptophytes on natural populations of bacteria in Korean coastal waters.

Cryptophytes are ubiquitous and one of the major phototrophic components in marine plankton communities. They often cause red tides in the waters of many countries. Understanding the bloom dynamics of cryptophytes is, therefore, of great importance. A critical step in this understanding is unveiling their trophic modes. Prior to this study, several freshwater cryptophyte species and marine Cryptomonas sp. and Geminifera cryophila were revealed to be mixotrophic. The trophic mode of the common marine cryptophyte species, Teleaulax amphioxeia has not been investigated yet. Thus, to explore the mixotrophic ability of T. amphioxeia by assessing the types of prey species that this species is able to feed on, the protoplasms of T. amphioxeia cells were carefully examined under an epifluorescence microscope and a transmission electron microscope after adding each of the diverse prey species. Furthermore, T. amphioxeia ingestion rates heterotrophic bacteria and the cyanobacterium Synechococcus sp. were measured as a function of prey concentration. Moreover, the feeding of natural populations of cryptophytes on natural populations of heterotrophic bacteria was assessed in Masan Bay in April 2006. This study reported for the first time, to our knowledge, that T. amphioxeia is a mixotrophic species. Among the prey organisms offered, T. amphioxeia fed only on heterotrophic bacteria and Synechococcus sp. The ingestion rates of T. amphioxeia on heterotrophic bacteria or Synechococcus sp. rapidly increased with increasing prey concentrations up to 8.6×106 cells ml-1, but slowly at higher prey concentrations. The maximum ingestion rates of T. amphioxeia on heterotrophic bacteria and Synechococcus sp. reached 0.7 and 0.3 cells predator-1 h-1, respectively. During the field experiments, the ingestion rates and grazing coefficients of cryptophytes on natural populations of heterotrophic bacteria were 0.3-8.3 cells predator-1h-1 and 0.012-0.033d-1, respectively. Marine cryptophytes, including T. amphioxeia, are known to be favorite prey species for many mixotrophic and heterotrophic dinoflagellates and ciliates. Cryptophytes, therefore, likely play important roles in marine food webs and may exert a considerable potential grazing impact on the populations of marine bacteria.

Haloplanus salinarum sp. nov., an extremely halophilic archaeon isolated from a solar saltern.

An extremely halophilic archaeal strain SP28T was isolated from the Gomso solar saltern, Republic of Korea. Cells of the new strain SP28T were pleomorphic and Gram stain negative, and produced red-pigmented colonies. These grew in medium with 2.5-4.5 M NaCl (optimum 3.1 M) and 0.05-0.5 M MgCl2 (optimum 0.1 M), at 25-50 °C (optimum 37 °C) and at a pH of 6.5-8.5 (optimum pH 8.0). Mg2+ was required for growth. A concentration of at least 2 M NaCl was required to prevent cell lysis. Polar lipids included phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one glycolipid chromatographically identical to sulfated mannosyl glucosyl diether. 16S rRNA and rpoB' gene sequence analyses showed that strain SP28T is closely related to Haloplanus ruber R35T (97.3 and 94.1 %, 16S rRNA and rpoB' gene sequence similarity, respectively), Haloplanus litoreus GX21T (97.0 and 92.1 %), Haloplanus salinus YGH66T (96.0 and 91.9 %), Haloplanus vescus RO5-8T (95.9 and 90.9 %), Haloplanus aerogenes TBN37T (95.6 and 90.3 %) and Haloplanus natans RE-101T (95.3 and 89.8 %). The DNA G+C content of the novel strain SP28T was 66.2 mol%, which is slightly higher than that of Hpn.litoreus GX21T (65.8 mol%) and Hpn.ruber R35T (66.0 mol%). DNA-DNA hybridization values betweenHpn.ruber R35T and strain SP28T and between Hpn.litoreus GX21T and strain SP28T were about 24.8 and 20.7 %, respectively. We conclude that strain SP28T represents a novel species of the genus Haloplanus and propose the name Haloplanus salinarum sp. nov. The type strain is SP28T (=JCM 31424T=KCCM 43210T).

Transcriptional Regulation of Cellulose Biosynthesis during the Early Phase of Nitrogen Deprivation in Nannochloropsis salina.

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N-), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N- stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis. However, few studies have characterised microalgal cell wall metabolism, including oleaginous Nannochloropsis sp. microalgae subjected to nitrogen deficiency. Here, we investigated N-induced changes in cellulose biosynthesis in N. salina. We observed that N- induced cell wall thickening, concurrently increased the transcript levels of genes coding for UDPG pyrophosphorylase and cellulose synthases, and increased cellulose content. Nannochloropsis salina cells with thickened cell wall were more susceptible to mechanical stress such as bead-beating and sonication, implicating cellulose metabolism as a potential target for cost-effective microalgal cell disruption.

Halomonas aestuarii sp. nov., a moderately halophilic bacterium isolated from a tidal flat.

Strain Hb3T was isolated from a tidal flat in Jeollabuk-do Gunsan, Republic of Korea. Cells were Gram-stain-negative, oxidase- and catalase-positive, rod-shaped and motile. The strain grew optimally at 25-35 °C, at pH 6.0-6.5 and with 3.0-10.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Hb3T belonged to the genus Halomonas. Strain Hb3T was related most closely to Halomonas ventosae Al12T (98.6 % 16S rRNA gene sequence similarity), Halomonas denitrificans M29T (98.6 %) and Halomonas saccharevitans AJ275T (98.4 %). Moreover, multilocus sequence analysis using the gyrB, rpoD and secA genes supported the phylogenetic position of strain Hb3T. The genomic G+C content of strain Hb3T was 67.9 mol%. DNA-DNA hybridization values for strain Hb3T versus H. ventosae Al12T, H. denitrificans M29T and H. saccharevitans AJ275T were 38.0, 54.5 and 47.4 %, respectively. The major quinone was ubiquinone Q-9 and the major fatty acids were C18 : 1ω7c, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0 and C19 : 0 cyclo ω8c. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, amino lipid, six unidentified phospholipids and an unidentified lipid comprised the polar lipid profile. On the basis of the data presented in this report, strain Hb3T represents a novel species of the genus Halomonas. The name Halomonas aestuarii sp. nov. is proposed for this novel species. The type strain is Hb3T (=KCTC 52253T=JCM 31415T).

Cryptophyte gene regulation in the kleptoplastidic, karyokleptic ciliate Mesodinium rubrum.

Photosynthesis in the ciliate Mesodinium rubrum is achieved using a consortium of cryptophyte algal organelles enclosed in its specialized vacuole. A time-series microarray analysis was conducted on the photosynthetic ciliate using an oligochip containing 15,654 primers designed from EST data of the cryptophyte prey, Teleaulax amphioxeia. The cryptophycean nuclei were transcriptionally active over 13 weeks and approximately 13.5% of transcripts in the ciliate came from the sequestered nuclei. The cryptophyte nuclei and chloroplasts could divide in the ciliate, which were loosely synchronized with host cell division. A large epigenetic modification occurred after the cryptophyte nuclei were sequestered into the ciliate. Most cryptophyte genes involved in the light and dark reactions of photosynthesis, chlorophyll assimilation, as well as in DNA methylation, were consistently up-regulated in the ciliate. The imbalance of division rate between the sequestered cryptophyte nuclei and host nuclei may be the reason for the eventual cessation of the kleptoplastidy.

The fate of cryptophyte cell organelles in the ciliate Mesodinium cf. rubrum subjected to starvation.

Mesodinium rubrum Lohmann is a mixotrophic ciliate and one of the best studied species exhibiting acquired phototrophy. To investigate the fate of cryptophyte organelles in the ciliate subjected to starvation, we conducted ultrastructural studies of a Korean strain of M. cf. rubrum during a 10 week starvation experiments. Ingested cells of the cryptophyte Teleaulax amphioxeia were first enveloped by ciliate membrane, and then prey organelles, including ejectisomes, flagella, basal bodies and flagellar roots, were digested. Over time, prey nuclei protruded into the cytoplasm of the ciliate, their size and volume increased, and their number decreased, suggesting that the cryptophyte nuclei likely fused with each other in the ciliate cytoplasm. At 4 weeks of starvation, M. cf. rubrum cells without cryptophyte nuclei started to appear. At 10 weeks of starvation, only two M. cf. rubrum cells still possessing a cryptophyte nucleus had relatively intact chloroplast-mitochondria complexes (CMCs), while M. cf. rubrum cells without cryptophyte nuclei had a few damaged CMCs. This is the first ultrastructural study demonstrating that cryptophyte nuclei undergo a dramatic change inside M. cf. rubrum in terms of size, shape, and number following their acquisition.

Do All Dinoflagellates have an Extranuclear Spindle?

The syndinean dinoflagellates are a diverse assemblage of alveolate endoparasites that branch basal to the core dinoflagellates. Because of their phylogenetic position, the syndineans are considered key model microorganisms in understanding early evolution in the dinoflagellates. Closed mitosis with an extranuclear spindle that traverses the nucleus in cytoplasmic grooves or tunnels is viewed as one of the morphological features shared by syndinean and core dinoflagellates. Here we describe nuclear morphology and mitosis in the syndinean dinoflagellate Amoebophrya sp. from Akashiwo sanguinea, a member of the A. ceratii complex, as revealed by protargol silver impregnation, DNA specific fluorochromes, and transmission electron microscopy. Our observations show that not all species classified as dinoflagellates have an extranuclear spindle. In Amoebophrya sp. from A. sanguinea, an extranuclear microtubule cylinder located in a depression in the nuclear surface during interphase moves into the nucleoplasm via sequential membrane fusion events and develops into an entirely intranuclear spindle. Results suggest that the intranuclear spindle of Amoebophrya spp. may have evolved from an ancestral extranuclear spindle and indicate the need for taxonomic revision of the Amoebophryidae.

Ultrastructure and molecular phylogeny of Mesodinium coatsi sp. nov., a benthic marine ciliate.

Mesodinium is a globally distributed ciliate genus forming frequent and recurrent blooms in diverse marine habitats. Here, we describe a new marine species, Mesodinium coatsi n. sp., originally isolated from interstitial water of surface sand samples collected at Mohang Beach, Korea. The species was maintained under a mixotrophic growth condition for longer than 1 yr by providing a cryptomonad, Chroomonas sp., as the sole prey. Cell morphology and subcellular structure were examined by light microscopy, scanning, and transmission electron microscopy, and molecular phylogeny was inferred from nuclear-encoded 18S rDNA sequence data. Like other Mesodinium species, M. coatsi consisted of two hemispheres separated by two types of kinetids, and had tentacles located at the oral end of the cell. Several food vacuoles were observed in the cytoplasm, and partially digested prey cells sometimes existed in food vacuoles. Kinetids and the associated accessory structures were quite similar to those previously reported, but M. coatsi was differentiated from other marine Mesodinium species by ultrastructural characters of the dikinetids, polykinetids, and tentacles. We also provided a detailed illustration of infraciliature. Molecular phylogeny revealed that M. coatsi and Mesodinium chamaeleon were closely related to each other.

The effect of starvation on plastid number and photosynthetic performance in the kleptoplastidic dinoflagellate Amylax triacantha.

The dinoflagellate Amylax triacantha is known to retain plastids of cryptophyte origin by engulfing the mixotrophic ciliate Mesodinium rubrum, itself a consumer of cryptophytes. However, there is no information on the fate of the prey's organelles and the photosynthetic performance of the newly retained plastids in A. triacantha. In this study, we conducted a starvation experiment to observe the intracellular organization of the prey's organelles and temporal changes in the photosynthetic efficiency of acquired plastids in A. triacantha. The ultrastructural observations revealed that while the chloroplast-mitochondria complexes and nucleus of cryptophyte were retained by A. triacantha, other ciliate organelles were digested in food vacuoles. Acquired plastids were retained in A. triacantha for about 1 mo and showed photosynthetic activities for about 18 d when measured by a pulse-amplitude modulation fluorometer.

Ultrastructure of the oral apparatus of Mesodinium rubrum from Korea.

Mesodinium rubrum Lohmann is a photosynthetic marine ciliate that has functional chloroplasts of cryptophyte origin. Little is known about the oral ultrastructure of M. rubrum compared with several reports on the sequestration of nuclei and plastids from prey organisms, such as Geminigera cryophila and Teleaulax species. Here, we describe the fine structure of the oral apparatus of a M. rubrum strain from Gomso Bay, Korea. The cytopharynx was cone-shaped and supported by 20-22 ribbons of triplet microtubules. At the anterior end of the cytopharynx, an annulus anchored small cylinders composed of 11 microtubules. The small cylinders were spaced at regular intervals, each reinforced by one set of the triplet microtubules. At the opening of the cytostome, larger 14-membered microtubular cylinders were set adjacent to the small, 11-membered microtubular cylinders, each pair surrounded by separate membranes, however, only the large cylinders extended into the oral tentacles. There were 20-22 oral tentacles each having one to five extrusomes at its tip. At the anterior end of the oral apparatus, microtubular bands supporting the cytostome curved posteriad, extending beneath the cell cortex to the kinetosomes of the somatic cirri. The microtubular bands were connected by striated fibers and originated from kinetosomes anchored by fibers. Each cirrus consisted of eight cilia associated with 16 kinetosomes. The ultrastructure of M. rubrum from Korea provides information useful for taxonomic characterization of the genus Mesodinium and relevant to developing a better understanding of the acquisition of foreign organelles through phagocytosis by M. rubrum.

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.

DINOPHYSIS CAUDATA (DINOPHYCEAE) SEQUESTERS AND RETAINS PLASTIDS FROM THE MIXOTROPHIC CILIATE PREY MESODINIUM RUBRUM(1).

"Phototrophic"Dinophysis Ehrenberg species are well known to have chloroplasts of a cryptophyte origin, more specifically of the cryptophyte genus complex Teleaulax/Geminigera. Nonetheless, whether chloroplasts of "phototrophic"Dinophysis are permanent plastids or periodically derived kleptoplastids (stolen chloroplasts) has not been confirmed. Indeed, molecular sequence data and ultrastructural data lead to contradictory interpretations about the status of Dinophysis plastids. Here, we used established cultures of D. caudata strain DC-LOHABE01 and M. rubrum strain MR-MAL01 to address the status of Dinophysis plastids. Our approach was to experimentally generate D. caudata with "green" plastids and then follow the ingestion and fate of "reddish-brown" prey plastids using light microscopy, time-lapse videography, and single-cell TEM. Our results for D. caudata resolve the apparent discrepancy between morphological and molecular data by showing that plastids acquired when feeding on M. rubrum are structurally modified and retained as stellate compound chloroplasts characteristic of Dinophysis species.

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.