Kyeong Ah Seong

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 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.

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.

The newly described heterotrophic dinoflagellate Gyrodinium moestrupii, an effective protistan grazer of toxic dinoflagellates.

Few protistan grazers feed on toxic dinoflagellates, and low grazing pressure on toxic dinoflagellates allows these dinoflagellates to form red‐tide patches. We explored the feeding ecology of the newly described heterotrophic dinoflagellate Gyrodinium moestrupii when it fed on toxic strains of Alexandrium minutum, Alexandrium tamarense, and Karenia brevis and on nontoxic strains of A. tamarense, Prorocentrum minimum, and Scrippsiella trochoidea. Specific growth rates of G. moestrupii feeding on each of these dinoflagellates either increased continuously or became saturated with increasing mean prey concentration. The maximum specific growth rate of G. moestrupii feeding on toxic A. minutum (1.60/d) was higher than that when feeding on nontoxic S. trochoidea (1.50/d) or P. minimum (1.07/d). In addition, the maximum growth rate of G. moestrupii feeding on the toxic strain of A. tamarense (0.68/d) was similar to that when feeding on the nontoxic strain of A. tamarense (0.71/d). Furthermore, the maximum ingestion rate of G. moestrupii on A. minutum (2.6 ng C/grazer/d) was comparable to that of S. trochoidea (3.0 ng C/grazer/d). Additionally, the maximum ingestion rate of G. moestrupii on the toxic strain of A. tamarense (2.1 ng C/grazer/d) was higher than that when feeding on the nontoxic strain of A. tamarense (1.3 ng C/grazer/d). Thus, feeding by G. moestrupii is not suppressed by toxic dinoflagellate prey, suggesting that it is an effective protistan grazer of toxic dinoflagellates.

Molecular Characterization and Morphology of the Photosynthetic Dinoflagellate Bysmatrum caponii from Two Solar Saltons in Western Korea

Species belonging to the genus Bysmatrum are peridinoid, thecate, photosynthetic dinoflagellates. The plate formula of Bysmatrum spp., arranged in a Kofoidian series, is almost identical to that of Scrippsiella spp. Bysmatrum spp., which were originally classified as Scrippsiella spp., but were transferred to the genus Bysmatrum spp. because of separation of the intercalary plates 2a and 3a by plate 3′. Whether this transfer from Scrippsiella spp. to Bysmatrum spp. is reasonable should be genetically confirmed. Dinoflagellates were isolated from 2 solar saltons located in western Korea in 2009–2010 and 3 clonal cultures from Sooseong solar saltons and 2 clonal cultures from Garolim solar saltons were successfully established. All of these dinoflagellates were identified as Bysmatrum caponii based on morphology analysis by light and electron microscopy. The plates of all Korean strains of B. caponii were arranged in a Kofoidian series of Po, X, 4′, 3a, 7″, 6c, 4s, 5‴, 0 (p), and 24’. When properly aligned, the ribosomal DNA (rDNA) sequences of the 3 Sooseong strains of B. caponii were identical, as were those of the 2 Garolim strains. Furthermore, the sequences of the 3 Sooseong strains were 0.01% different from those of the Garolim strains. However, the sequences of SSU rDNA of these Korean B. caponii strains were 9% different from that of Bysmatrum subsalsum and > 10% from that of any other dinoflagellate thus far reported. In the phylogenetic trees generated using SSU and LSU rDNA sequences, these Korean B. caponii strains formed a clade with B. subsalsum which was clearly divergent from the Scrippsiella clade. However, this Bysmatrum clade was phylogenetically close to the Protoperidinium and/or Peridinium clades. The results of the present study suggest that Bysmatrum spp. are markedly different genetically from Scrippsiella spp..

Differential interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and common heterotrophic protists and copepods: Killer or prey

To investigate interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and different heterotrophic protist and copepod species, feeding by common heterotrophic dinoflagellates (Oxyrrhis marina and Gyrodinium dominans), naked ciliates (Strobilidium sp. approximately 35μm in cell length and Strombidinopsis sp. approximately 100μm in cell length), and calanoid copepods Acartia spp. (A. hongi and A. omorii) on P. shiwhaense was explored. In addition, the feeding activities of P. shiwhaense on these heterotrophic protists were investigated. Furthermore, the growth and ingestion rates of O. marina, G. dominans, Strobilidium sp., Strombidinopsis sp., and Acartia spp. as a function of P. shiwhaense concentration were measured. O. marina, G. dominans, and Strombidinopsis sp. were able to feed on P. shiwhaense, but Strobilidium sp. was not. However, the growth rates of O. marina, G. dominans, Strobilidium sp., and Strombidinopsis sp. feeding on P. shiwhaense were very low or negative at almost all concentrations of P. shiwhaense. P. shiwhaense frequently fed on O. marina and Strobilidium sp., but did not feed on Strombidinopsis sp. and G. dominans. G. dominans cells swelled and became dead when incubated with filtrate from the experimental bottles (G. dominans+P. shiwhaense) that had been incubated for one day. The ingestion rates of O. marina, G. dominans, and Strobilidium sp. on P. shiwhaense were almost zero at all P. shiwhaense concentrations, while those of Strombidinopsis sp. increased with prey concentration. The maximum ingestion rate of Strombidinopsis sp. on P. shiwhaense was 5.3ngC predator-1d-1 (41 cells predator-1d-1), which was much lower than ingestion rates reported in the literature for other mixotrophic dinoflagellate prey species. With increasing prey concentrations, the ingestion rates of Acartia spp. on P. shiwhaense increased up to 930ngCml-1 (7180cellsml-1) at the highest prey concentration. The highest ingestion rate of Acartia spp. on P. shiwhaense was 4240ngC predator-1d-1 (32,610 cells predator-1d-1), which is comparable to ingestion rates from previous studies on other dinoflagellate prey species calculated at similar prey concentrations. Thus, P. shiwhaense might play diverse ecological roles in marine planktonic communities by having an advantage over competing phytoplankton in anti-predation against potential protistan grazers.

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.

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.

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica, a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea-a suitable algal prey item-heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d-1. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13ngC predator-1d-1 (0.06 cells predator-1d-1). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019ngC predator-1d-1 (266 bacteria predator-1d-1), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 107 cells ml-1 was 0.01ngC predator-1d-1 (48 Synechococcus predator-1d-1). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.