Yeong Du Yoo

Mixotrophy in the phototrophic harmful alga Cochlodinium polykrikoides (Dinophycean): Prey species, the effects of prey concentration, and grazing impact

Abstract We first reported here that the harmful alga Cochlodinium polykrikoides, which had been previously known as an autotrophic dinoflagellate, was a mixotrophic species. We investigated the kinds of prey species and the effects of the prey concentration on the growth and ingestion rates of C. polykrikoides when feeding on an unidentified cryptophyte species (Equivalent Spherical Diameter, ESD = 5.6 μm). We also calculated grazing coefficients by combining field data on abundances of C. polykrikoides and co-occurring cryptophytes with laboratory data on ingestion rates obtained in the present study. Cocholdinium polykrikoides fed on prey cells by engulfing the prey through the sulcus. Among the phytoplankton prey offered, C. polykrikoides ingested small phytoplankton species that had ESDs ≤ 11 μm (e.g. the prymnesiophyte Isochrysis galbana, an unidentified cryptophyte, the cryptophyte Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellate Amphidinium carterae). It did not feed on larger phytoplankton species that had ESDs ≥ 12 μm (e.g. the dinoflagellates Heterocapsa triquetra, Prorocentrum minimum, Scrippsiella sp., Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum). Specific growth rates of C. polykrikoides on a cryptophyte increased with increasing mean prey concentration, with saturation at a mean prey concentration of approximately 270 ng C ml−1 (i.e. 15,900 cells ml−1). The maximum specific growth rate (mixotrophic growth) of C. polykrikoides on a cryptophyte was 0.324 d−1, under a 14:10 h light-dark cycle of 50 μE m−2 s−1, while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.166 d−1. Maximum ingestion and clearance rates of C. polykrikoides on a cryptophyte were 0.16 ng C grazer−1d−1 (9.4 cells grazer−1d−1) and 0.33 μl grazer−1h−1, respectively. Calculated grazing coefficients by C. polykrikoides on cryptophytes were 0.001–0.745 h−1 (i.e. 0.1–53% of cryptophyte populations were removed by a C. polykrikoides population in 1 h). The results of the present study suggest that C. polykrikoides sometimes has a considerable grazing impact on populations of cryptophytes.

Feeding by the Heterotrophic Dinoflagellate Oxyrrhis marina on the Red-Tide Raphidophyte Heterosigma akashiwo: a Potential Biological Method to Control Red Tides Using Mass-Cultured Grazers

Abstract As part of the development of a method to control the outbreak and persistence of red tides using mass-cultured heterotrophic protist grazers, we measured the growth and ingestion rates of cultured Oxyrrhis marina (a heterotrophic dinoflagellate) on cultured Heterosigma akashiwo (a raphidophyte) in bottles in the laboratory and in mesocosms (ca. 60 liter) in nature, and those of the cultured grazer on natural populations of the red-tide organism in mesocosms set up in nature. In the bottle incubation, specific growth rates of O. marina increased rapidly with increasing concentration of cultured prey up to ca. 950 ng C ml−1 (equivalent to 9,500 cells ml−1), but were saturated at higher concentrations. Maximum specific growth rate (μmax), KGR (prey concentration sustaining 0.5 μmax) and threshold prey concentration of O. marina on H. akashiwo were 1.43 d−1, 104 ng C ml−1, and 8.0 ng C ml−1, respectively. Maximum ingestion and clearance rates of O. marina were 1.27 ng C grazer−1 d−1 and 0.3 μl grazer−1 h−1, respectively. Cultured O. marina grew well effectively reducing cultured and natural populations of H. akashiwo down to a very low concentration within 3 d in the mesocosms. The growth and ingestion rates of cultured O. marina on natural populations of H. akashiwo in the mesocosms were 39% and 40%, respectively, of those calculated based on the results from the bottle incubation in the laboratory, while growth and ingestion rates of cultured O. marina on cultured H. akashiwo in the mesocosms were 55% and 36%, respectively. Calculated grazing impact by O. marina on natural populations of H. akashiwo suggests that O. marina cultured on a large scale could be used for controlling red tides by H. akashiwo near aquaculture farms that are located in small ponds, lagoons, semi-enclosed bays, and large land-aqua tanks to which fresh seawater should be frequently supplied.

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 phototrophic red-tide dinoflagellates on the ubiquitous marine diatom Skeletonema costatum.

ABSTRACT We investigated feeding by phototrophic red‐tide dinoflagellates on the ubiquitous diatom Skeletonema costatum to explore whether dinoflagellates are able to feed on S. costatum, inside the protoplasm of target dinoflagellate cells observed under compound microscope, confocal microscope, epifluorescence microscope, and transmission electron microscope (TEM) after adding living and fluorescently labeled S. costatum (FLSc). To explore effects of dinoflagellate predator size on ingestion rates of S. costatum, we measured ingestion rates of seven dinoflagellates at a single prey concentration. In addition, we measured ingestion rates of the common phototrophic dinoflagellates Prorocentrum micans and Gonyaulax polygramma on S. costatum as a function of prey concentration. We calculated grazing coefficients by combining field data on abundances of P. micans and G. polygramma on co‐occurring S. costatum with laboratory data on ingestion rates obtained in the present study. All phototrophic dinoflagellate predators tested (i.e. Akashiwo sanguinea, Amphidinium carterae, Alexandrium catenella, Alexandrium tamarense, Cochlodinium polykrikoides, G. polygramma, Gymnodinium catenatum, Gymnodinium impudicum, Heterocapsa rotundata, Heterocapsa triquetra, Lingulodinium polyedrum, Prorocentrum donghaiense, P. micans, Prorocentrum minimum, Prorocentrum triestinum, and Scrippsiella trochoidea) were able to ingest S. costatum. When mean prey concentrations were 170–260 ng C/ml (i.e. 6,500–10,000 cells/ml), the ingestion rates of G. polygramma, H. rotundata, H. triquetra, L. polyedrum, P. donghaiense, P. micans, and P. triestinum on S. costatum (0.007–0.081 ng C/dinoflagellate/d [0.2–3.0 cells/dinoflagellate/d]) were positively correlated with predator size. With increasing mean prey concentration of ca 1–3,440 ng C/ml (40–132,200 cells/ml), the ingestion rates of P. micans and G. polygramma on S. costatum continuously increased. At the given prey concentrations, the maximum ingestion rates of P. micans and G. polygramma on S. costatum (0.344–0.345 ng C/grazer/d; 13 cells/grazer/d) were almost the same. The maximum clearance rates of P. micans and G. polygramma on S. costatum were 0.165 and 0.020 μl/grazer/h, respectively. The calculated grazing coefficients of P. micans and G. polygramma on co‐occurring S. costatum were up to 0.100 and 0.222 h, respectively (i.e. up to 10% and 20% of S. costatum populations were removed by P. micans and G. polygramma populations in 1 h, respectively). Our results suggest that P. micans and G. polygramma sometimes have a considerable grazing impact on populations of S. costatum.

Feeding and Grazing Impact by Small Marine Heterotrophic Dinoflagellates on Heterotrophic Bacteria

ABSTRACT. We investigated the feeding of the small heterotrophic dinoflagellates (HTDs) Oxyrrhis marina, Gyrodinium cf. guttula, Gyrodinium sp., Pfiesteria piscicida, and Protoperidinium bipes on marine heterotrophic bacteria. To investigate whether they are able to feed on bacteria, we observed the protoplasm of target heterotrophic dinoflagellate cells under an epifluorescence microscope and transmission electron microscope. In addition, we measured ingestion rates of the dominant heterotrophic dinoflagellate, Gyrodinium spp., on natural populations of marine bacteria (mostly heterotrophic bacteria) in Masan Bay, Korea in 2006–2007. Furthermore, we measured the ingestion rates of O. marina, G. cf. guttula, and P. piscicida on bacteria as a function of bacterial concentration under laboratory conditions. All HTDs tested were able to feed on a single bacterium. Oxyrrhis marina and Gyrodinium spp. intercepted and then ingested a single bacterial cell in feeding currents that were generated by the flagella of the predators. During the field experiments, the ingestion rates and grazing coefficients of Gyrodinium spp. on natural populations of bacteria were 14–61 bacteria/dinoflagellate/h and 0.003–0.972 day−1, respectively. With increasing prey concentration, the ingestion rates of O. marina, G. cf. guttula, and P. piscicida on bacteria increased rapidly at prey concentrations of ca 0.7–2.2 × 106 cells/ml, but increased only slowly or became saturated at higher prey concentrations. The maximum ingestion rate of O. marina on bacteria was much higher than those of G. cf. guttula and P. piscicida. Bacteria alone supported the growth of O. marina. The results of the present study suggest that some HTDs may sometimes have a considerable grazing impact on populations of marine bacteria, and that bacteria may be important prey.

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.

Mixotrophy in the Newly Described Phototrophic Dinoflagellate Woloszynskia cincta from Western Korean Waters: Feeding Mechanism, Prey Species and Effect of Prey Concentration

ABSTRACT. 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/m2/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.

Feeding by the Pfiesteria-Like Heterotrophic Dinoflagellate Luciella masanensis

ABSTRACT. To explore the feeding ecology of the Pfiesteria‐like dinoflagellate (PLD) Luciella masanensis (GenBank Accession no. AM050344, previously Lucy), we investigated the feeding behavior and the kinds of prey species that L. masanensis fed on and determined its growth and ingestion rates of L. masanensis when it fed on the dinoflagellate Amphidinium carterae and an unidentified cryptophyte species (equivalent spherical diam., ESD=5.6 μm), which were the dominant phototrophic species when L. masanensis and similar small heterotrophic dinoflagellates were abundant in Masan Bay, Korea in 2005. Additionally, these parameters were also measured for L. masanensis fed on blood cells of the perch Lateolabrax japonicus and the raphidophyte Heterosigma akashiwo in the laboratory. Luciella masanensis fed on prey cells by using a peduncle after anchoring the prey with tow filament, and was able to feed on diverse prey such as cryptophytes, raphidophytes, diatoms, mixotrophic dinoflagellates, and the blood cells of fish and humans. Among the prey species tested in the present study, perch blood cells were observed to be the optimal prey for L. masanensis. Specific growth rates of L. masanensis feeding on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte, either increased continuously or became saturated with increasing the mean prey concentration. The maximum specific growth rate of L. masanensis feeding on perch blood cells (1.46/day) was much greater than that of A. carterae (0.59/day), the cryptophyte (0.24/day), or H. akashiwo (0.20/day). The maximum ingestion rate of L. masanensis on perch blood cells (2.6 ng C/grazer/day) was also much higher than that of A. carterae (0.32 ng C/grazer/day), the cryptophyte (0.44 ng C/grazer/day), or H. akashiwo (0.16 ng C/grazer/day). The kinds of prey species which L. masanensis is able to feed on were the same as those of Pfiesteria piscicida, but very different from those of another PLD Stoeckeria algicida. However, the maximum growth and ingestion rates of L. masanensis on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte were considerably lower than those of P. piscicida. Therefore, these three dinoflagellates may occupy different ecological niches in marine planktonic communities, even though they have a similar size and shape and the same feeding mechanisms.

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

ABSTRACT. 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) ≤11 μm (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 ≥12 μm (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 °C under a 14:10 h light–dark cycle of 20 μE/m2/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 μl/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.

NaOCl produced by electrolysis of natural seawater as a potential method to control marine red-tide dinoflagellates

Abstract As part of the development of a method to control the outbreak and persistence of red tides using sodium hypochlorite (NaOCl), we investigated the effect of NaOCl on the survival of red-tide dinoflagellates, diatoms, heterotrophic protists, planktonic crustaceans, fin-fish, shellfish, and macroalgae. Because NaOCl introduced into natural waters would be subject to dilution, as well as breakdown in sunlight to NaCl, the survival of organisms was determined after 10 min exposure and 1 h exposure to NaOCl, and again after transfer to fresh seawater for 6 or 24 h. The lethal total residual chlorine (TRC) concentration that killed 50% of the test organisms (LC50) for the red-tide dinoflagellates Gymnodinium catenatum, Cochlodinium polykrikoides, Akashiwo sanguinea, Lingulodinium polyedrum, Prorocentrum micans, Alexandrium affine, and Gymnodinium impudicum ranged from 57 to 157 ppb for 10 min exposure and from 30 to 106 ppb for 1 h exposure. Complete mortality of all red-tide species occurred at TRC concentrations of ~ 500 ppb. The LC50 of the diatoms Skeletonema costatum and Thalassiosira rotula, 3083–3383 ppb for 10 min exposure and 3128–3433 ppb for 1 h exposure, were much higher than for red-tide dinoflagellates. But the LC50s of the heterotrophic dinoflagellates Polykrikos kofoidii and Oxyrrhis marina were similar to those of red-tide dinoflagellates. The ciliate Strombidinopsis sp. had LC50s of 306 ppb for 10 min exposure and 119 ppb for 1 h exposure, which are higher than those for dinoflagellates. The LC50s of the calanoid copepods Acarlia spp. and Pseudodiaptomus sp. were 1397–1493 ppb for 10 min exposure and 744–987 ppb for 1 h exposure, and those for larvae of the brine shrimp Artemia franciscana were 4905 ppb for 10 m exposure and 2814 ppb for 1 h exposure. The LC50s of juvenile gray mullet Mugil cephalus and juvenile black rockfish Sebastes schlegeli were 1234–1883 ppb for 10 min exposure and 1234–1440 ppb for 1 h exposure, whereas those of adult Manila clam Ruditapes philippinarum and spat of the abalone Nordotis discus were> 20,000 ppb. The LC50s of the macroalgae Griffithsia japonica (Rhodophyta) and Ulva pertusa (Chlorophyta) were 1519–12,365 ppb for 10 min exposure and 1085–12,558 ppb for 1 h exposure. The present study therefore suggests that, if NaOCl is introduced into waters containing red-tide organisms at TRC concentrations of 300–500 ppb for 10 min exposure and 200–400 ppb for 1 h exposure, red tides can be effectively controlled without serious harmful effects on other marine organisms, except heterotrophic dinoflagellates.