Aquariibacter albus gen. nov., sp. nov., a new member of the order Burkholderiales, isolated from a freshwater aquarium.

A novel bacterium, strain SJAQ100T, was isolated from a freshwater aquarium and was characterized taxonomically and phylogenetically. Strain SJAQ100T was a Gram-stain-negative, aerobic, rod-shaped and non-motile bacterium. The strain grew optimally with 0 % NaCl and at 25-37 °C on Reasoner's 2A agar. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strain SJAQ100T clustered with members of Burkholderiales incertae sedis in the order Burkholderiales, but sequence similarities to known species were less than 96.5 %. The genomic DNA G+C content of strain SJAQ100T was 71.2 mol%. Genomic comparisons of strain SJAQ100T with species in the order Burkholderiales were made using the Genome-to-Genome Distance Calculator, average nucleotide identity and average amino acid identity analyses (values indicated ≤22.1, ≤78.1, and ≤68.1 % respectively). Strain SJAQ100T contained C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c as major fatty acids and Q-8 as the major quinone. The major polyamines were putrescine and cadaverine. Strain SJAQ100T contained phosphatidylethanolamine and diphosphatidylglycerol as major polar lipids. Based on the genotypic, chemotaxonomic and phenotypic results, strain SJAQ100T represents a novel genus and species, Aquariibacter albus gen. nov., sp. nov., which belongs to order Burkholderiales and the class Betaproteobacteria. The type strain is SJAQ100T (=KCTC 72203T=CGMCC 1.18869T=MCC 4385T).

Effects of light and temperature on the growth of Takayama helix (Dinophyceae): mixotrophy as a survival strategy against photoinhibition.

Takayama helix is a mixotrophic dinoflagellate that can feed on diverse algal prey. We explored the effects of light intensity and water temperature, two important physical factors, on its autotrophic and mixotrophic growth rates when fed on Alexandrium minutum CCMP1888. Both the autotrophic and mixotrophic growth rates and ingestion rates of T. helix on A. minutum were significantly affected by photon flux density. Positive growth rates of T. helix at 6-58 µmol photons · m-2  · s-1 were observed in both the autotrophic (maximum rate = 0.2 · d-1 ) and mixotrophic modes (0.4 · d-1 ). Of course, it did not grow both autotrophically and mixotrophically in complete darkness. At ≥247 µmol photons · m-2  · s-1 , the autotrophic growth rates were negative (i.e., photoinhibition), but mixotrophy turned these negative rates to positive. Both autotrophic and mixotrophic growth and ingestion rates were significantly affected by water temperature. Under both autotrophic and mixotrophic conditions, it grew at 15-28°C, but not at ≤10 or 30°C. Therefore, both light intensity and temperature are critical factors affecting the survival and growth of T. helix.

Gonyaulax whaseongensis sp. nov. (Gonyaulacales, Dinophyceae), a new phototrophic species from Korean coastal waters.

The planktonic phototrophic dinoflagellate Gonyaulax whaseongensis sp. nov., isolated from coastal waters of western Korea, was described from living and fixed cells under light and scanning electron microscopy, and its rDNA was sequenced. Gonyaulax whaseongensis had a plate formula of 2pr, 4', 6'', 6c, 6''', 1p, and 1'''' with S-type ventral organization like the other species in the genus. However, this dinoflagellate had a narrow cingulum (ca. 2.6 µm), small displacement of the cingulum, slight overhang and steep angle between the ends of the cingulum, quadrangular sixth precingular plate, reticulated cell surface without longitudinal lines or ridges, and two unequal antapical spines, together which distinguish this from all other reported Gonyaulax species. In addition, the SSU and LSU rDNA sequences were 8%-12% and 11%-24%, respectively, different from those of Gonyaulax polygramma, Gonyaulax spinifera, Gonyaulax fragilis, Gonyaulax membranacea, and Gonyaulax digitale, the putatively closest related species in the phylogenetic analysis.

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.

Feeding by heterotrophic dinoflagellates and ciliates on the free-living dinoflagellate Symbiodinium sp. (Clade E).

To investigate heterotrophic protists grazing on Symbiodinium sp., we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Gyrodinium spirale, Oblea rotundata, Oxyrrhis marina, and Polykrikos kofoidii and the ciliates Balanion sp. and Parastrombidinopsis sp. preyed on the free-living dinoflagellate Symbiodinium sp. (clade E). We measured the growth and ingestion rates of O. marina and G. dominans on Symbiodinium sp. as a function of prey concentration. Furthermore, we compared the results to those obtained for other algal prey species. In addition, we measured the growth and ingestion rates of other predators at single prey concentrations at which these rates of O. marina and G. dominans were saturated. All predators tested in the present study, except Balanion sp., preyed on Symbiodinium sp. The specific growth rates of O. marina and G. dominans on Symbiodinium sp. increased rapidly with increasing mean prey concentration < ca. 740-815 ng C/ml (7,400-8,150 cells/ml), but became saturated at higher concentrations. The maximum growth rates of O. marina and G. dominans on Symbiodinium sp. (0.87 and 0.61/d) were much higher than those of G. moestrupii and P. kofoidii (0.11 and 0.04/d). Symbiodinium sp. did not support positive growth of G. spirale, O. rotundata, and Parastrombidinopsis sp. However, the maximum ingestion rates of P. kofoidii and Parastrombidinopsis sp. (6.7-10.0 ng C/predator/d) were much higher than those of O. marina and G. dominans on Symbiodinium sp. (1.9-2.1 ng C/predator/d). The results of the present study suggest that Symbiodinium sp. may increase or maintain the populations of some predators.

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.

Vegetation as a key driver of the distribution of microbial generalists that in turn shapes the overall microbial community structure in the low Arctic tundra.

Understanding the variability of microbial niches and their interaction with abiotic and biotic factors in the Arctic can provide valuable insights into microbial adaptations to extreme environments. This study investigates the structure and diversity of soil bacterial communities obtained from sites with varying vegetation coverage and soil biogeochemical properties in the low Arctic tundra and explores how bacteria interact under different environmental parameters. Our findings reveal differences in bacterial composition and abundance among three bacterial niche breadths (specialists, common taxa, and generalists). Co-occurrence network analysis revealed Rhizobiales and Ktedonobacterales as keystone taxa that connect and support other microbes in the habitat. Low-elevation indicators, such as vascular plants and moisture content, were correlated with two out of three generalist modular hubs and were linked to a large proportion of generalists' distribution (18%). Structural equation modeling revealed that generalists' distribution, which influenced the remaining microbial communities, was mainly regulated by vegetation coverage as well as other abiotic and biotic factors. These results suggest that elevation-dependent environmental factors directly influence microbial community structure and module formation through the regulation of generalists' distribution. Furthermore, the distribution of generalists was mainly affected by macroenvironment filtering, whereas the distribution of specialists was mainly affected by microenvironment filtering (species-engineered microbial niche construction). In summary, our findings highlight the strong top-down control exerted by vegetation on generalists' distribution, which in turn shapes the overall microbial community structure in the low Arctic tundra.

Food web structure for high carbon retention in marine plankton communities.

Total annual net primary productions in marine and terrestrial ecosystems are similar. However, a large portion of the newly produced marine phytoplankton biomass is converted to carbon dioxide because of predation. Which food web structure retains high carbon biomass in the plankton community in the global ocean? In 6954 individual samples or locations containing phytoplankton, unicellular protozooplankton, and multicellular metazooplankton in the global ocean, phytoplankton-dominated bottom-heavy pyramids held higher carbon biomass than protozooplankton-dominated middle-heavy diamonds or metazooplankton-dominated top-heavy inverted pyramids. Bottom-heavy pyramids predominated, but the high predation impact by protozooplankton on phytoplankton or the vertical migration of metazooplankton temporarily changed bottom-heavy pyramids to middle-heavy diamonds or top-heavy inverted pyramids but returned to bottom-heavy pyramids shortly. This finding has profound implications for carbon retention by plankton communities in the global ocean.

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.

Feeding diverse prey as an excellent strategy of mixotrophic dinoflagellates for global dominance.

Microalgae fuel food webs and biogeochemical cycles of key elements in the ocean. What determines microalgal dominance in the ocean is a long-standing question. Red tide distribution data (spanning 1990 to 2019) show that mixotrophic dinoflagellates, capable of photosynthesis and predation together, were responsible for ~40% of the species forming red tides globally. Counterintuitively, the species with low or moderate growth rates but diverse prey including diatoms caused red tides globally. The ability of these dinoflagellates to trade off growth for prey diversity is another genetic factor critical to formation of red tides across diverse ocean conditions. This finding has profound implications for explaining the global dominance of particular microalgae, their key eco-evolutionary strategy, and prediction of harmful red tide outbreaks.

Feeding by the harmful phototrophic dinoflagellate Takayama tasmanica (Family Kareniaceae).

The trophic mode of a phototrophic dinoflagellate is a critical factor in the dynamics of its harmful algal bloom. Recent discoveries of the mixotrophic capabilities of phototrophic dinoflagellates have changed the traditional view of bloom dynamics and prediction models. Here, mixotrophy in the harmful phototrophic dinoflagellate Takayama tasmanica was examined. Moreover, growth and ingestion rates of T. tasmanica on each of Alexandrium minutum CCMP1888 and Alexandrium tamarense CCMP1493, suitable prey, were determined as a function of prey concentration. This study reported for the first time that T. tasmanica is a mixotrophic species. Among the phytoplankton species offered as prey, T. tasmanica fed on all prey species whose equivalent spherical diameter (ESD) was greater than 30 µm, but also A. minutum whose ESD was 19 µm. In contrast, T. tasmanica did not feed on the phototrophic dinoflagellates Heterocapsa triquetra, Gymnodinium aureolum, Scrippsiella acuminata (previously S. trochoidea), Cochlodinium polykrikoides, Alexandrium affine, Alexandrium insuetum, and Alexandrium pacificum that its sister species Takayama helix is able to feed on. With increasing mean prey concentration, ingestion rates of T. tasmanica on A. minutum increased, but became saturated at the prey concentrations of >2130 cells mL-1 (1070 ng C mL-1). The maximum ingestion rate (MIR) of T. tasmanica on A. minutum was 0.5 ng C predator-1 d-1 (1.0 cells predator-1 d-1) which is only 64% of the body carbon of a T. tasmanica cell. Growth rates of T. tasmanica on A. minutum were not affected by prey concentrations. Thus, the low maximum ingestion rate is likely to be responsible for the small increases of its growth rate through mixotrophy. In addition, neither growth nor ingestion rates of T. tasmanica feeding on Alexandrium tamarense were affected by prey concentrations. The maximum ingestion rate of T. tasmanica on A. minutum was considerably lower than that of T. helix on the same prey species. Therefore, the mixotrophic ability of T. tasmanica is weaker than that of T. helix, and also T. tasmanica may have an ecological niche different from that of T. helix in marine ecosystems.

Feeding by the heterotrophic nanoflagellate Katablepharis remigera on algal prey and its nationwide distribution in Korea.

Heterotrophic nanoflagellates are ubiquitous in natural waters, and most heterotrophic nanoflagellates are known to grow on bacteria. Recently, the heterotrophic nanoflagellate Katablepharis japonica has been reported to be an effective predator of diverse toxic or harmful algal prey. To date, 7 Katablepharis species have been identified, and therefore important questions arise as to whether other Katablepharis species can feed on algal prey, and further whether the types of prey of other Katablepharis species differ from those of K. japonica. To answer these important questions, feeding by Katablepharis remigera on diverse algal prey was examined. Specific growth and ingestion rates of K. remigera feeding on the raphidophytes Heterosigma akashiwo and Chattonella subsalsa were determined. Furthermore, the abundance of K. remigera at 28 stations along the coastline of Korea from January 2015 to October 2017 was quantified using qPCR method and newly designed specific primer-probe sets. Among 25 potential algal prey tested, K. remigera fed on only H. akashiwo and C. subsalsa; however, it did not feed on a diatom, a prymnesiophyte, a prasinophyte, cryptophytes, dinoflagellates, Mesodinium rubrum, a mixotrophic ciliate, and another raphidophyte Fibrocapsa japonica. The number of prey types on which K. remigera could feed (2 species) was considerably smaller than that of K. japonica (14 species). With the increase in the mean prey concentration, the specific growth rates of K. remigera on H. akashiwo and C. subsalsa increased as well before becoming saturated. The maximum specific growth rates of K. remigera on H. akashiwo and C. subsalsa were 0.717 and 0.129 d-1, respectively. In addition, the maximum ingestion rates of K. remigera on H. akashiwo and C. subsalsa were 0.333 and 0.661 ng C predator-1 d-1 (3.33 and 0.23 cells predator-1 d-1), respectively. The results of this study clearly indicate that K. remigera is an effective predator of 2 red tide-causing raphidophyte species, and additionally, the feeding activity of K. remigera differs greatly from that of K. japonica. The abundance of K. remigera was ≥0.1 cells mL-1 at 24 stations located in the East, West, and South Sea of Korea. Thus, K. remigera has a nationwide distribution in Korea. The highest abundance of K. remigera in Korean waters was 24.9 cells mL-1 in March 2017, when there was no red tide caused by H. akashiwo or Chattonella spp. In most regions where red tides caused by H. akashiwo or Chattonella spp. occurred in 2000-2017, K. remigera was detected. Thus, the abundance of K. remigera may increase during red tides caused by H. akashiwo and Chattonella spp.

Interactions between the mixotrophic dinoflagellate Takayama helix and common heterotrophic protists.

The phototrophic dinoflagellate Takayama helix that is known to be harmful to abalone larvae has recently been revealed to be mixotrophic. Although mixotrophy elevates the growth rate of T. helix by 79%-185%, its absolute growth rate is still as low as 0.3d-1. Thus, if the mortality rate of T. helix due to predation is high, this dinoflagellate may not easily prevail. To investigate potential effective protistan grazers on T. helix, feeding by diverse heterotrophic dinoflagellates such as engulfment-feeding Oxyrrhis marina, Gyrodinium dominans, Gyrodinium moestrupii, Polykrikos kofoidii, and Noctiluca scintillans, peduncle-feeding Aduncodinium glandula, Gyrodiniellum shiwhaense, Luciella masanensis, and Pfiesteria piscicida, pallium-feeding Oblea rotunda and Protoperidinium pellucidum, and the naked ciliates Pelagostrobilidium sp. (ca. 40µm in cell length) and Strombidinopsis sp. (ca. 150µm in cell length) on T. helix was explored. Among the tested heterotrophic protists, O. marina, G. dominans, G. moestrupii, A. glandula, L. masanensis, P. kofoidii, P. piscicida, and Strombidinopsis sp. were able to feed on T. helix. The growth rates of all these predators except Strombidinopsis sp. with T. helix prey were lower than those without the prey. The growth rate of Strombidinopsis sp. on T. helix was almost zero although the growth rate of Strombidinopsis sp. with T. helix prey was higher than those without the prey. Moreover, T. helix fed on O. marina and P. pellucidum and lysed the cells of P. kofoidii and G. shiwhaense. With increasing the concentrations of T. helix, the growth rates of O. marina and P. kofoidii decreased, but those of G. dominans and L. masanensis largely did not change. Therefore, reciprocal predation, lysis, no feeding, and the low ingestion rates of the common protists preying on T. helix may result in a low mortality rate due to predation, thereby compensating for this species' low growth rate.

Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method.

Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml-1 eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml-1 within 24h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml-1 to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800cellsml-1A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1m3 stock culture of A. pohangense at 4000cellsml-1 is calculated to remove all C. polykrikoides cells in ca. 200m3 within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml-1 with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land.

Removal of two pathogenic scuticociliates Miamiensis avidus and Miamiensis sp. using cells or culture filtrates of the dinoflagellate Alexandrium andersonii.

Scuticociliatosis, which is caused by parasitic protistan pathogens known as scuticociliates, is one of the most serious diseases in marine aquaculture worldwide. Thus, elimination of these ciliates is a primary concern for scientists and managers in the aquaculture industry. To date, formalin and other toxic chemicals have been used as anti-scuticociliate agents, but issues regarding their secondary effects often arise. Consequently, development of safer methods is necessary. To find out a safe method of controlling scuticociliate populations in aqua-tanks or small-scale natural environments, cultures of 14 phototrophic dinoflagellates were tested to determine whether they were able to control populations of the common scuticociliates Miamiensis avidus and Miamiensis sp. isolated from Korean waters. Among the dinoflagellates tested, both cells and culture filtrates of Alexandrium andersonii effectively killed M. avidus and Miamiensis sp. The minimal concentration of cells and equivalent culture filtrates of A. andersonii to kill all M. avidus cells within 48h of incubation was ca. 2500 and 4500 cells ml-1, respectively; whereas those needed to kill all Miamiensis sp. cells were ca. 1000 and 4500 cells ml-1, respectively. It was estimated that 1m3 of the stock culture containing 20,000A. andersonii cells ml-1 could eliminate all M. avidus cells in 7m3 of waters within the aqua-tanks on land and all Miamiensis sp. cells in 19m3 of waters within 48h. None of the brine shrimp Artemia salina nauplii incubated with concentrations of 50-4500A. andersonii cells ml-1 for 24h was dead. Furthermore, none of the flounder Paralichthys olivaceus juveniles incubated with a mean concentration of ca. 2280A. andersonii cells ml-1 for 96h was dead. Therefore, A. andersonii cultures may be used as a safe biological method for controlling populations of scuticociliates and can replace toxic formalin. The results of this study provided the basis for developing the method to control scuticociliate populations and understanding interactions between scuticociliates and phototrophic dinoflagellates in marine ecosystems.

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods.

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing >500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014-2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93-1.03 times) and the second closest (1.16-1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense.

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1d-1. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10cellsml-1 of A. pohangense was 20-60%, while that at the equivalent culture filtrates was 20-70%. With increasing A. pohangense cell-concentration (up to 1000cellsml-1 or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC50) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3cellsml-1, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0cellsml-1, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml-1 and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.

Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates.

Takayama spp. are phototrophic dinoflagellates belonging to the family Kareniaceae and have caused fish kills in several countries. Understanding their trophic mode and interactions with co-occurring phytoplankton species are critical steps in comprehending their ecological roles in marine ecosystems, bloom dynamics, and dinoflagellate evolution. To investigate the trophic mode and interactions of Takayama spp., the ability of Takayama helix to feed on diverse algal species was examined, and the mechanisms of prey ingestion were determined. Furthermore, growth and ingestion rates of T. helix feeding on the dinoflagellates Alexandrium lusitanicum and Alexandrium tamarense, which are two optimal prey items, were determined as a function of prey concentration. T. helix ingested large dinoflagellates ≥15µm in size, except for the dinoflagellates Karenia mikimotoi, Akashiwo sanguinea, and Prorocentrum micans (i.e., it fed on Alexandrium minutum, A. lusitanicum, A. tamarense, A. pacificum, A. insuetum, Cochlodinium polykrikoides, Coolia canariensis, Coolia malayensis, Gambierdiscus caribaeus, Gymnodinium aureolum, Gymnodinium catenatum, Gymnodinium instriatum, Heterocapsa triquetra, Lingulodinium polyedrum, and Scrippsiella trochoidea). All these edible prey items are dinoflagellates that have diverse eco-physiology such as toxic and non-toxic, single and chain forming, and planktonic and benthic forms. However, T. helix did not feed on small flagellates and dinoflagellates <13µm in size (i.e., the prymnesiophyte Isochrysis galbana; the cryptophytes Teleaulax sp., Storeatula major, and Rhodomonas salina; the raphidophyte Heterosigma akashiwo; the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum minimum; or the small diatom Skeletonema costatum). T. helix ingested Heterocapsa triquetra by direct engulfment, but sucked materials from the rest of the edible prey species through the intercingular region of the sulcus. With increasing mean prey concentration, the specific growth rates of T. helix on A. lusitanicum and A. tamarense increased continuously before saturating at prey concentrations of 336-620ngC mL-1. The maximum specific growth rates (mixotrophic growth) of T. helix on A. lusitanicum and A. tamarense were 0.272 and 0.268d-1, respectively, at 20°C under a 14:10 h light/dark cycle of 20µE m-2 s-1 illumination, while its growth rates (phototrophic growth) under the same light conditions without added prey were 0.152 and 0.094d-1, respectively. The maximum ingestion rates of T. helix on A. lusitanicum and A. tamarense were 1.23 and 0.48ng C predator-1d-1, respectively. The results of the present study suggest that T. helix is a mixotrophic dinoflagellate that is able to feed on a diverse range of toxic species and, thus, its mixotrophic ability should be considered when studying red tide dynamics, food webs, and dinoflagellate evolution.