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Exploring mixotrophy of dinoflagellate species is critical to understanding red-tide dynamics and dinoflagellate evolution. Some species in the dinoflagellate genus Karenia have caused harmful algal blooms. Among 10 Karenia species, the mixotrophic ability of only two species, Karenia mikimotoi and Karenia brevis, has been investigated. These species have been revealed to be mixotrophic; however, the mixotrophy of the other species should be explored. Moreover, although K. mikimotoi was previously known to be mixotrophic, only a few potential prey species have been tested. We explored the mixotrophic ability of Karenia bicuneiformis, Karenia papilionacea, and Karenia selliformis and the prey spectrum of K. mikimotoi by incubating them with 16 potential prey species, including a cyanobacterium, diatom, prymnesiophyte, prasinophyte, raphidophyte, cryptophytes, and dinoflagellates. Cells of K. bicuneiformis, K. papilionacea, and K. selliformis did not feed on any tested potential prey species, indicating a lack of mixotrophy. The present study newly discovered that K. mikimotoi was able to feed on the common cryptophyte Teleaulax amphioxeia. The phylogenetic tree based on the large subunit ribosomal DNA showed that the mixotrophic species K. mikimotoi and K. brevis belonged to the same clade, but K. bicuneiformis, K. papilionacea, and K. selliformis were divided into different clades. Therefore, the presence or lack of a mixotrophic ability in this genus may be partially related to genetic characterizations. The results of this study suggest that Karenia species are not all mixotrophic, varying from the results of previous studies.

The mortality rate of red-tide dinoflagellates owing to predation is a major parameter that affects their population dynamics. The dinoflagellates Ansanella granifera and Ansanella sp. occasionally cause red tides. To understand the interactions between common heterotrophic protists and A. granifera, we explored the feeding occurrence of nine heterotrophic protists on A. granifera and the growth and ingestion rates of the heterotrophic dinoflagellate Gyrodinium dominans on A. granifera as a function of prey concentration and those of Oxyrrhis marina at a single high prey concentration. The heterotrophic dinoflagellates Aduncodinium glandula, G. dominans, Gyrodinium moestrupii, Luciella masanensis, Oblea rotunda, O. marina, Polykrikos kofoidii, and Pfiesteria piscicida and the naked ciliate Strombidium sp. were able to feed on A. granifera. With increasing mean prey concentrations, the growth and ingestion rates of G. dominans feeding on A. granifera rapidly increased and became saturated or slowly increased. The maximum growth and ingestion rates of G. dominans on A. granifera were 0.305 d-1 and 0.42 ng C predator-1 d-1 (3.8 cells predator-1 d-1), respectively. Furthermore, the growth and ingestion rates of O. marina on A. granifera at 1,700 ng C mL-1 (15,454 cells mL-1) were 0.037 d-1 and 0.19 ng C predator-1 d-1 (1.7 cells predator-1 d-1), respectively. The growth and ingestion rates of G. dominans and O. marina feeding on A. granifera were almost the lowest among those on the dinoflagellate prey species. Therefore, G. dominans and O. marina may prefer A. granifera less than other dinoflagellate prey species. The low mortality rate of A. granifera may positively affect its bloom formation.

Noctiluca scintillans is a heterotrophic dinoflagellate that causes red-colored oceans during the day (red tides) and glowing oceans at night (bioluminescence). This species feeds on diverse prey, including phytoplankton, heterotrophic protists, and eggs of metazoans. Thus, many scientists have conducted studies on the ecophysiology of this species. It is easy to cultivate N. scintillans at a scale of 100 L because N. scintillans cells usually stay near the surface, while prey cells stay below the surface in large water tanks. To obtain mass-cultured N. scintillans cells, we developed an automatic system for cultivating N. scintillans on a scale of 100 L. The system consisted of four tanks containing fresh nutrients, the chlorophyte Dunaliella salina as prey, N. scintillans for growth, and N. scintillans for storage, respectively. The light intensities supporting the high growth rates of D. salina and N. scintillans were 300 and 20 μmol photons m-2 s-1, respectively. Twenty liters of D. salina culture from the prey culture tank were transferred to the predator culture tank, and subsequently 20 L of nutrients from the nutrient tank were transferred to the prey culture tank every 2 d. When the volume of N. scintillans in the predator culture tank reached 90 L 6 d later, 70 L of the culture were transferred to the predator storage tank. To prevent N. scintillans cells from being separated from D. salina cells in the predator culture tank, the culture was mixed using an air pump, a sparger, and a stirrer. The highest abundance of N. scintillans in the predator culture tank was 45 cells mL-1, which was more than twice the highest abundance when this dinoflagellate was cultivated manually. This automatic system supplies 100 L of N. scintillans pure culture with a high density every 10 d for diverse experiments on N. scintillans.

Water temperature affects plankton survival and growth. The dinoflagellate Shimiella gracilenta survives using theplastids of ingested prey, indicating kleptoplastidy. However, studies on the effects of water temperature on kleptoplastidicdinoflagellates are lacking. We explored the growth and ingestion rates of S. gracilenta as a function of water temperature.Furthermore, using data on its spatiotemporal distribution in Korean coastal waters during 2015–2018, we predictedits distribution under elevated temperature conditions of +2, +4, and +6°C. Growth rates of S. gracilenta with andwithout Teleaulax amphioxeia prey as well as ingestion rates were significantly affected by water temperature. Growthrates of S. gracilenta with and without prey were positive or zero at 5–25°C but were negative at ≥30°C. The maximumgrowth rate of S. gracilenta with T. amphioxeia was 0.85 d-1, achieved at 25°C, and 0.21 d-1 at 20°C without prey. The ingestionrate of S. gracilenta on T. amphioxeia at 25°C (0.05 ng C predator-1 d-1) was greater than that at 20°C (0.04 ng Cpredator-1 d-1). Thus, feeding may shift the optimal temperature for the maximum growth rate of S. gracilenta from 20 to25°C. In spring and winter, the distributions of S. gracilenta under elevated temperature conditions were predicted notto differ from those during 2015–2018. However, S. gracilenta was predicted not to survive at some additional stationsunder elevated temperature conditions of +2, +4, and +6°C in summer or under elevated temperature conditions of +6°Cin autumn. Therefore, global warming may affect the distribution of S. gracilenta.

Some species in the dinoflagellate genus Alexandrium are bioluminescent. Of the 33 formally described Alexandrium species, the bioluminescence capability of only nine species have been tested, and eight have been reported to be bioluminescent. The present study investigated the bioluminescence capability of seven Alexandrium species that had not been tested. Alexandrium mediterraneum, A. pohangense, and A. tamutum were bioluminescent, but A. andersonii, A. hiranoi, A. insuetum, and A. pseudogonyaulax were not. We also measured the bioluminescent intensity of A. affine, A. fraterculus, A. mediterraneum, A. ostenfeldii, A. pacificum, A. pohangense, A. tamarense, and A. tamutum. The mean 200-second-integrated bioluminescence intensity per cell ranged from 0.02 to 32.2 × 104 relative luminescence unit per cell (RLU cell-1), and the mean maximum bioluminescence intensity per cell per second (BLMax) ranged from 0.01 to 10.3 × 104 RLU cell-1 s-1-1. BLMax was significantly correlated with the maximum growth rates of Alexandrium species, except for A. tamarense. A phylogenetic tree based on large subunit ribosomal DNA (LSU rDNA) showed that the bioluminescent species A. affine, A. catenella, A. fraterculus, A. mediterraneum, A. pacificum, and A. tamarense formed a large clade. However, the toxicity or mixotrophic capability of these species was split. Thus, their bioluminescence capability in this clade was more consistent than their toxicity or mixotrophic capability. Phylogenetic trees based on LSU rDNA and the luciferase gene of Alexandrium were consistent except for A. pohangense. The results of the present study can provide a basis for understanding the interspecific diversity in bioluminescence of Alexandrium.

To explore the ecophysiological characteristics of the kleptoplastidic dinoflagellate Shimiella gracilenta, we determined its spatiotemporal distribution in Korean coastal waters and growth and ingestion rates as a function of prey concentration. The abundance of S. gracilenta at 28 stations from 2015 to 2018 was measured using quantitative realtime polymerase chain reaction. Cells of S. gracilenta were detected at least once at all the stations and in each season, when temperature and salinity were 1.7–26.4°C and 9.9–35.6, respectively. Moreover, among the 28 potential prey species tested, S. gracilenta SGJH1904 fed on diverse prey taxa. However, the highest abundance of S. gracilenta was only 3 cells mL-1 during the study period. The threshold Teleaulax amphioxeia concentration for S. gracilenta growth was 5,618 cells mL-1, which was much higher than the highest abundance of T. amphioxeia (667 cells mL-1). Thus, T. amphioxeia was not likely to support the growth of S. gracilenta in the field during the study period. However, the maximum specific growth and ingestion rates of S. gracilenta on T. amphioxeia, the optimal prey species, were 1.36 d-1 and 0.04 ng C predator- 1 d-1, respectively. Thus, if the abundance of T. amphioxeia was much higher than 5,618 cells mL-1, the abundance of S. gracilenta could be much higher than the highest abundance observed in this study. Eurythermal and euryhaline characteristics of S. gracilenta and its ability to feed on diverse prey species and conduct kleptoplastidy are likely to be responsible for its common spatiotemporal distribution.

A small dinoflagellate, ~13 μm in cell length, was isolated from Jinhae Bay, Korea. Light microscopy showed that it was similar to the kleptoplastidic dinoflagellate Gymnodinium gracilentum nom. inval. rDNA sequences were obtained and its anatomy and morphology described using light and scanning and transmission electron microscopy. Phylogenetic analyses indicated that it belonged to the family Kareniaceae. However, its large subunit (LSU) rDNA sequences were 5.2-9.5% different from those of the other five genera in the family, and its clade was clearly divergent from that of each genus. Its overall morphology was different from those of the other five genera in the family and from Gymnodinium. Unlike Gymnodinium, this dinoflagellate did not have a horseshoe-shaped apical groove, nuclear envelope chambers, or a nuclear fibrous connective (NFC). It had an apical line of narrow amphiesmal vesicles and an elongated apical furrow crossing the apex. Cells were covered with polygonal amphiesmal vesicles arranged in 16 rows. Starved cells did not contain their own plastids, eyespots, pyrenoids, peridinin, or fucoxanthin. However, they could survive without added prey for approximately one month using chloroplasts from the cryptophyte prey Teleaulax amphioxeia, indicating kleptoplastidy. Because this taxon is genetically distinct at the generic rank from the other genera in Kareniaceae, it is placed in Shimiella gen. nov., and because G. gracilentum was invalid, the new bionomial S. gracilenta sp. nov. is proposed.

The genus Alexandrium often forms harmful algal blooms causing human illness and large-scale mortality of fish and shellfish. Thus, Alexandrium bloom dynamics are primary concerns for scientists, government officials, aquaculture farmers, and the public. To understand bloom dynamics, mortality due to predation needs to be assessed; however, interactions between many Alexandrium species and their potential predators have not previously been reported. Thus, feeding by five common heterotrophic dinoflagellates (Oxyrrhis marina, Gyrodinium dominans, Polykrikos kofoidii, Pfiesteria piscicida, and Oblea rotunda) and a naked ciliate (Strombidinopsis sp.) on 12 Alexandrium species was examined. Furthermore, the growth and ingestion rates of P. kofoidii on A. minutum CCMP 1888 (previously A. lusitanicum), A. minutum CCMP 113, and A. tamarense were measured as a function of prey concentration. The growth rates of P. kofoidii on the other Alexandrium species at single high prey concentrations were measured, at which the growth rates on A. minutum CCMP 1888 and A. tamarense were saturated. Feeding occurrence by these predators on 12 Alexandrium species could be categorized into 6 different prey groups. Each Alexandrium species was consumed by at least one predator; however, there was no Alexandrium species that was eaten by all six predators. Cells of A. minutum CCMP 1888, A. minutum CCMP 113, and A. tamarense were fed upon by four predators, but A. affine and A. pacificum by only one predator species, P. kofoidii or Strombidinopsis sp. Furthermore, A. minutum CCMP 1888 and A. tamarense supported high growth rates of P. kofoidii, but the other Alexandrium species did not support, but rather inhibited P. kofoidii growth. With increasing prey concentrations, the growth and ingestion rates of P. kofoidii on A. minutum CCMP 1888 and A. tamarense increased and became saturated, whereas those on A. minutum CCMP 113 continuously decreased. The maximum growth rates of P. kofoidii on A. tamarense and A. minutum CCMP 1888 were 1.010 and 0.765 d−1, respectively, and P. kofoidii maximum ingestion rates were 26.2 and 11.1 ng C predator−1d−1, respectively. In contrast, the growth rates of P. kofoidii on the other Alexandrium species at single high prey concentrations were almost zero (A. pacificum) or negative. Based on the feeding occurrence and growth and ingestion rates of predators on 12 Alexandrium species, it is suggested that common heterotrophic protistan predators respond differently to different Alexandrium species, and thus ecological niches of the Alexandrium species may be different from each other. These results may provide an insight into the roles of protistan predators in bloom dynamics of Alexandrium species.

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.

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.

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 0.3 d−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. 40 μm in cell length) and Strombidinopsis sp. (ca. 150 μm 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.

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

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.

The dinoflagellate genus Scrippsiella is known to cause red tides. Mortality due to predation should be assessed in order to understand the population dynamics of Scrippsiella species. However, predation has been explored only in a few species. In this study, we examined feeding by common heterotrophic dinoflagellates Oxyrrhis marina, Gyrodinium dominans, Polykrikos kofoidii, Oblea rotunda, and Pfiesteria piscicida, and a ciliate Strombidinopsis sp., on four Scrippsiella species, of similar size, namely Scrippsiella acuminata, Scrippsiella donghaiensis, Scrippsiella lachrymosa, and Scrippsiella masanensis. All the heterotrophic protists tested could feed on all the four Scrippsiella species. However, the numerical and functional responses of P. kofoidii to the mean prey concentration were apparently different between the Scrippsiella species. With increasing prey concentration, the growth and ingestion rates of P. kofoidii on S. lachrymosa increased rapidly, and then saturated similar to those on S. acuminata, as previously reported, but those on S. donghaiensis continuously decreased. The cells of S. donghaiensis lysed P. kofoidii cells. In contrast, the growth and ingestion rates of P. kofoidii on S. masanensis were not significantly related to the prey concentration. At similarly high mean prey concentration, the growth and ingestion rates of G. dominans were significantly different between the four Scrippsiella species Therefore, differences in the growth and/or ingestion rates of G. dominans and P. kofoidii on the four Scrippsiella species might result in different ecological niches of both the predator and prey species.