Harmful Algae 49 (2015) 10–18

Mixotrophy in the newly described dinoflagellate Alexandriumpohangense: A specialist for feeding on the fast-swimmingichthyotoxic dinoflagellate Cochlodinium polykrikoides

An Suk Lim a, Hae Jin Jeong a,b,*, Ji Hye Kim a, Se Hyeon Jang a, Moo Joon Lee a, Kitack Lee c

a School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Koreab Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do 443-270, Republic of Koreac School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea

A R T I C L E I N F O

Article history:

Received 4 June 2015

Received in revised form 31 July 2015

Accepted 31 July 2015

Keywords:

Grazing

Growth

HAB

Ingestion

Protist

Red tide

A B S T R A C T

Newly described Alexandrium pohangense is a phototrophic dinoflagellate, and its trophic mode should

be explored to understand its roles in marine ecosystems. To investigate feeding of A. pohangense and its

ecological roles, its trophic mode, prey type, feeding mechanism, functional and numerical responses,

and grazing impact were analyzed. Among diverse algal prey tested, it fed only on the fast swimming

ichthyotoxic dinoflagellate Cochlodinium polykrikoides. A. pohangense ingested C. polykrikoides cells by

engulfment, after immobilizing the prey cell using excreted materials. Thus, A. pohangense has an

effective mechanism for feeding on fast-swimming prey through prey immobilization. With increasing

mean prey concentrations, the specific growth and ingestion rates of A. pohangense increased rapidly

before saturating at C. polykrikoides concentrations of 138 ng C ml�1 (197 cells ml�1) and 99 ng C ml�1

(141 cells ml�1), respectively. The maximum growth rate of A. pohangense fed with C. polykrikoides was

0.487 d�1, while the growth rate of A. pohangense without added C. polykrikoides was 0.091 d�1. The

maximum ingestion rate of A. pohangense on C. polykrikoides was 4.99 ng C predator�1 d�1

(7.1 cells predator�1 d�1). The grazing coefficients attributable to A. pohangense on co-occurring C.

polykrikoides, calculated by combining field data on abundance with the ingestion rates obtained in the

present study, were 0.09–1.57 d�1, which indicates that A. pohangense could have a considerable grazing

impact on C. polykrikoides populations.

� 2015 Elsevier B.V. All rights reserved.

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1. Introduction

Trophic modes (i.e., exclusively autotrophic, mixotrophic, andheterotrophic) in dinoflagellates are important to understand theirecological roles in marine ecosystems (e.g., Jeong et al., 2010a). Inthe last two decades, many phototrophic dinoflagellates have beenrevealed as mixotrophic (Bockstahler and Coats, 1993; Jacobsonand Anderson, 1996; Stoecker, 1999; Li et al., 2000; Skovgaardet al., 2000; Park et al., 2006; Berge et al., 2008; Burkholder et al.,2008; Jeong et al., 2010a, 2012; Kang et al., 2011). Mixotrophicdinoflagellates play diverse roles in food webs (Jeong et al., 1999a,2010a; Glibert et al., 2009; Hansen, 2011; Sanders, 2011; Seongand Jeong, 2011); they are able to feed on diverse prey items such

* Corresponding author at: School of Earth and Environmental Sciences, College

of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea.

Tel.: +82 2 880 6746; fax: +82 2 874 9695.

E-mail address: hjjeong@snu.ac.kr (H.J. Jeong).

http://dx.doi.org/10.1016/j.hal.2015.07.010

1568-9883/� 2015 Elsevier B.V. All rights reserved.

as bacteria, algae, heterotrophic protists, and metazoans (forexamples, see Jeong et al., 2010a). In turn, they serve as prey forvarious predators (Jeong et al., 1999b, 2008, 2015). The mixo-trophic abilities of some dinoflagellates contribute considerably totheir growth, and to the formation of red-tide patches (Jeong et al.,2010a, 2015; Lee et al., 2014a). Therefore, the mixotrophic abilitiesof newly described phototrophic dinoflagellate species should beexplored.

Several Alexandrium species are toxic (Cembella et al., 2002;Anderson et al., 2012; Yoo et al., 2013), producing diversephycotoxins such as saxitoxins, goniodomins, and spirolides(Anderson et al., 2012). Saxitoxins cause paralytic shellfishpoisoning and are related to the formation of harmful algal bloomscausing large-scale mortality of finfish and shellfish; they also causediseases in humans (Hallegraeff, 1993; Cembella et al., 2002;Anderson et al., 2012). Moreover, some Alexandrium species secretelytic compounds that kill other protists (Tillmann et al., 2007;Blossom et al., 2012). Therefore, the growth and distribution ofAlexandrium species are critical factors for scientists, government

Table 1Taxa, size, and concentration of algal prey species offered to Alexandrium

pohangense. Mean equivalent spherical diameter (ESD, mm) + standard deviation

(SD) of the mean for algae were measured by an electronic particle counter (Coulter

Multisizer II, Coulter Corporation, Miami, FL, USA). The numbers of counted cells

were >2000 cells for each algal species. Y: Feeding by A. pohangense, N: Not fed on

by A. pohangense, L: the prey cell was lysed with added A. pohangense cultures, I: the

prey cells were immobilized with added A. pohangense cultures, T: Toxic, NT: non-

toxic dinoflagellate. The initial concentration of A. pohangense was ca. 300 cell ml�1.

Prey species ESD (SD) Initial prey

concentration

cell mL�1

Effects Feeding

by

A. pohangense

DiatomSkeletonema costatum 5.9 (1.1) 150,000 N N

PrymnesiophytesIsochrysis galbana 4.8 (0.2) 150,000 I N

CryptophytesTeleaulax sp. 5.6 (1.5) 100,000 L N

Storeatula major 6.0 (1.7) 50,000 L N

Rhodomonas salina 8.8 (1.5) 50,000 L N

RhaphidophytesHeterosigma akashiwo 11.5 (1.9) 30,000 I N

Mixotrophic dinoflagellatesHeterocapsa rotundata (T) 5.8 (0.4) 100,000 I N

Amphidinium carterae (NT) 9.7 (1.6) 30,000 I N

Prorocentrum minimum (T) 12.1 (2.5) 20,000 I N

Hetereocapsa triquetra (T) 15.0 (1.3) 30,000 I N

Gymnodinium aureolum

(NT)

19.5 (4.9) 3000 I N

Scrippsiella trochoidea (T) 22.8 (2.7) 15,000 I N

Cochlodinium polykrikoides

(NT)

25.9 (2.9) 2000 I Y

Prorocentrum micans (T) 26.6 (2.8) 3000 I N

Akashiwo sanguinea (NT) 30.8 (3.5) 1000 I N

Gymnodinium catenatum

(T)

33.9 (1.6) 1000–3000 I N

Naked ciliateMesodinium rubrum 22 (0.04) 2000 L N

A.S. Lim et al. / Harmful Algae 49 (2015) 10–18 11

officials, and the aquaculture industry. Approximately 30 Alexan-

drium species have been described so far, of which only five species(i.e., A. minutum, A. tamarense, A. catenella, A. ostenfeldii, and A.

pseudogonyaulax) are known to be mixotrophic (Nygaard andTobiesen, 1993; Jacobson and Anderson, 1996; Gribble et al., 2005;Jeong et al., 2005a,b; Yoo et al., 2009; Blossom et al., 2012).Therefore, to understand the ecological roles and the bloomdynamics of newly described Alexandrium species, it is necessary toinvestigate their mixotrophic ability.

Cochlodinium polykrikoides is a bloom-forming dinoflagellatefound in the coastal waters of many countries (Jeong et al., 2008;Mulhollannd et al., 2009; Tang and Gobler, 2010; Park et al.,2013a). This dinoflagellate is known to kill fish in aqua-cages byclogging their gills using mucus, thus sometimes causing greatlosses in the aquaculture industry (Park et al., 2013b). Therefore,many countries spend considerable budgets to minimize the lossdue to C. polykrikoides. Owing to these efforts, the mixotrophicability, optimal temperature, salinity, light intensity, and interac-tions with competing species of C. polykrikoides have been welldocumented (Park et al., 2001; Jeong et al., 2004; Kim et al., 2004;Tang and Gobler, 2010; Gobler et al., 2012; Lim et al., 2014a).However, information on effective predators of this dinoflagellateis still lacking. The chain-forming C. polykrikoides is one of thefastest swimming phototrophic dinoflagellates and may not beeasy for potential predators to capture (maximum swimmingspeed = �1450 mm s�1; Jeong et al., 1999a). Large ciliates, Strom-

bidinopsis spp., are effective protistan predators of C. polykrikoides

(Jeong et al., 1999b, 2008). To better understand C. polykrikoides

bloom dynamics and harmful effects, its mortality rate due topredation should be explored.

Recently, Alexandrium sp. was isolated from the waters offPohang, Korea, during a Cochlodinium polykrikoides red tide, and aclonal culture was established. Based on morphological andgenetic analyses, it was revealed as a new species and describedas Alexandrium pohangense (Lim et al., 2015a). In the present studyusing this culture, mixotrophic ability, prey type, and feedingmechanisms of this dinoflagellate were investigated. Additionally,the growth and ingestion rates of A. pohangense on C. polykrikoides,the only prey, as a function of prey concentration, weredetermined. Furthermore, grazing impact of A. pohangense on C.

polykrikoides populations was estimated using data on theabundance of these two species in the field, and ingestion ratesobtained in this study. The results of the present study provided abasis for understanding the interactions between A. pohangense

and C. polykrikoides and their ecological roles in marine ecosys-tems.

2. Materials and methods

2.1. Preparation of experimental organisms

Phytoplankton species used in this study were grown at 20 8Cin enriched f/2 seawater media (Guillard and Ryther, 1962)under continuous illumination of 100 mE m�2 s�1 provided bycool white fluorescent lights (Table 1). The mean equivalentspherical diameter (ESD) � standard deviation was measuredusing an electronic particle counter (Coulter Multisizer II, CoulterCorporation, Miami, FL, USA). The carbon content of phytoplanktonwas estimated from the cell volume according to Strathmann(1967).

Alexandrium pohangense was isolated from plankton samplescollected from waters off Pohang, Korea, in September 2014, whenthe water temperature and salinity were 23.3 8C and 31.1,respectively (Lim et al., 2015a). Only cultures in the exponentialgrowth phase were used.

2.2. Prey type

Experiment 1 was designed to investigate whether Alexandrium

pohangense was able to feed on potential prey when unialgal dietsof diverse microalgal species were provided (Table 1). The initialconcentrations of each algal species were similar in terms ofcarbon biomass.

A dense culture of Alexandrium pohangense growing photosyn-thetically in f/2 media at 20 8C and under a 14:10 h light-dark (LD)cycle at 100 mE m�2 s�1 was transferred to a 2-L PC bottlecontaining f/2 medium. The culture was maintained in f/2 mediafor 2 d under the same conditions described above. Three 1-mlaliquots were then removed from the bottle and A. pohangense

densities were determined using a compound light microscope.To observe the ingestion of eukaryotic algal prey under a light

microscope and/or an epifluorescence microscope, the targetinitial concentrations of Alexandrium pohangense and each algalprey species were established as described below (Table 1).Triplicate 42-ml PC experimental bottles and predator controlbottles were set up for each target algal species. The bottles werefilled to capacity with freshly filtered seawater, capped, and thenplaced on a shelf and incubated at 20 8C under a 14:10 h LD cycle ofcool white fluorescent light at 100 mE m�2 s�1. After 6, 12, 24, and48 h of incubation, a 5-ml aliquot was subsampled from eachbottle. Two tenths to one ml aliquots of the subsample were placedon slides with cover glasses. The protoplasm of >200 A. pohangense

cells was carefully examined using a light microscope and/orepifluorescence microscope at a magnification of 100–400� to

A.S. Lim et al. / Harmful Algae 49 (2015) 10–1812

determine whether A. pohangense had fed on the target algal preyspecies. A. pohangense cells containing the ingested cells werephotographed at a magnification of 400–1000� using digitalcameras mounted on the microscopes. During these feedingexperiments, the cells of all prey species were immobilized, andthe cells of four species were lysed after A. pohangense cells wereadded. To test whether the filtrates of A. pohangense cultureaffected the behavior of prey species, an A. pohangense culture ofapproximately 300 cells ml�1 was filtered through 5-mm pores(Millipore co.) and added to the wells of a 6-well plate chambercontaining the algal prey species with the same concentrations asin Table 1. In addition, seawater filtered through 5-mm pores wasadded to the wells of another 6-well plate chamber containing thetarget algal species as a control. Five min after the filtrates of A.

pohangense were added, >200 prey cells were examined using alight microscope to determine whether the target prey cells wereimmobilized. In addition, 6 h after the filtrates of A. pohangense

were added, the water in the wells was examined using a lightmicroscope to determine whether target prey cells were lysed.

2.3. Feeding mechanisms

Experiment 2 was designed to investigate the feeding mecha-nisms of Alexandrium pohangense on Cochlodinium polykrikoides

dinoflagellates, the only prey species. The initial concentrations ofthe predator and prey were the same as in Experiment 1.

The initial concentrations of Alexandrium pohangense and thetarget algal species were established using an autopipette todeliver a predetermined volume of culture with a known celldensity to the experimental bottles. One 42-ml PC bottle (mixturesof A. pohangense and Cochlodinium polykrikoides) was set up. Thebottle was filled to capacity with freshly filtered seawater, capped,and then mixed well. After 6, 24, and 48 h of incubation on theshelf, the feeding behavior of >60 A. pohangense cells wasmonitored using a light microscope and/or epifluorescencemicroscope at a magnification of 100–630 �. The feeding processeswere observed, from the time a prey cell was captured to the timethat it was engulfed by the predator. A series of photographsshowing the process of A. pohangense feeding on C. polykrikoides

were taken using a video recording system (Sony DXC-C33; SonyCo., Tokyo, Japan) mounted on a light microscope at a magnifica-tion of 100–630�.

2.4. Prey concentration effects

Experiment 3 was designed to determine the specific growthand ingestion rates of Alexandrium pohangense on Cochlodinium

polykrikoides as a function of prey concentration. A dense culture ofA. pohangense maintained in an f/2 medium and growingphotosynthetically was transferred to a 1-l PC bottle. Three 1-mlaliquots from the bottle were examined using a compoundmicroscope to determine A. pohangense cell concentrations.Triplicate 42-ml PC experimental bottles (containing mixtures ofpredators and prey), triplicate prey control bottles (containingprey only), and triplicate predator control bottles (containingpredators only) were established. To ensure similar waterconditions, we filtered the water of a predator culture through a0.7-mm GF/F filter, and added this to the prey control bottles in thesame amount as the volume of the predator culture added into theexperimental bottles for each predator-prey combination. Weadded 5 ml f/2 medium to all the bottles, which were then filled tocapacity with freshly filtered seawater and capped. To determinethe actual predator and algal prey concentrations at the start of theexperiment and after 2 d, a 5-ml aliquot was removed from eachbottle and fixed with 5% Lugol’s solution; all or >200 predator andprey cells in triplicate 1-ml Sedgwick-Rafter chambers were then

enumerated. The bottles were refilled to capacity with filteredseawater, capped, and placed on the shelf because C. polykrikoides

has negative growth on the rotating wheel. The dilution of thecultures associated with refilling the bottles was considered whencalculating the growth and ingestion rates.

The specific growth rate of Alexandrium pohangense wascalculated as follows:

m ¼ lnðAt1 � At0Þt1 � t0

(1)

where At0 is the initial concentration of A. pohangense and At1 is thefinal concentration after time t (in this instance, t = 2 d).

Data for Alexandrium pohangense growth rate were fitted to thefollowing equation:

m ¼ mmaxðx � x0ÞKGR þ ðx � x0Þ (2)

where mmax = the maximum growth rate (d�1), x = prey concen-tration (cells ml�1 or ng C ml�1), x0 = threshold prey concentration(i.e. the prey concentration where m = 0), and KGR = the preyconcentration sustaining ½mmax. Data were iteratively fitted to themodel using DeltaGraph1 software (SPSS Inc., Chicago, IL, USA).

Ingestion and clearance rates were calculated using theequations of Frost (1972) and Heinbokel (1978). The incubationtimes for calculating the ingestion and clearance rates were thesame as for estimating the growth rate.

Ingestion rate data were fitted to a Michaelis-Menten equation:

IR ¼ ImaxðxÞK IR þ ðxÞ (3)

where Imax = the maximum ingestion rate (cells predator�1 d�1 orng C predator�1 d�1), x = prey concentration (cells ml�1 orng C ml�1), and KIR = the prey concentration sustaining ½Imax.

2.5. Potential grazing impact

By combining field data on the abundance of Alexandrium spp.and Cochlodinium polykrikoides with the ingestion rates ofAlexandrium pohangense on C. polykrikoides obtained in the presentstudy, we estimated the grazing coefficients attributable to A.

pohangense and the co-occurring C. polykrikoides. Data on theabundance of Alexandrium spp. and the co-occurring C. poly-

krikoides used in this estimate were obtained by counting thewater samples taken from the waters off Pohang, Korea when C.

polykrikoides red tide patches were observed in 2014. Themorphological features of A. pohangense under the light micro-scope were difficult to distinguish from those of other Alexandrium

spp., the abundance of Alexandrium spp. having size and shapesimilar to A. pohangense was used for the calculation.

The grazing coefficients (g, d�1) were calculated as:

g ¼ CR � A � 24 (4)

where CR (ml predator�1 h�1) is the clearance rate of Alexandrium

pohangense feeding on Cochlodinium polykrikoides at a preyconcentration, and A is Alexandrium spp. concentration(cells ml�1). The CR values were calculated as:

CR ¼ IR

x(5)

where IR (cells eaten predator�1 h�1) is the ingestion rate of A.

pohangense on C. polykrikoides, and x (cells ml�1) is C. polykrikoides

concentration. Since the laboratory experiments were performedat 20 8C, these CR values were corrected using Q10 = 2.8 (Hansenet al., 1997).

A.S. Lim et al. / Harmful Algae 49 (2015) 10–18 13

2.6. Swimming speed

To measure the swimming speed of Alexandrium pohangense, aculture of A. pohangense (ca. 500 cells ml�1) growing autotrophi-cally under a 14:10 h LD cycle of 100 mE m�2 s�1 in f/2 media wastransferred into a 50-ml cell culture flask and allowed to acclimatefor 30 min. The video camera focused on one field seen as a circle ina cell culture flask under a dissecting microscope at 20 8C andswimming of A. pohangense cells was then recorded at amagnification of 20� using a video recording system (Samsung,SV-C660, Seoul, Korea) and taken using a CCD camera (Hitachi, HP-D20BU, Tokyo, Japan). Swimming of all cells seen for the first

Fig. 1. The process by which Alexandrium pohangense cells feed on Cochlodinium polykrikoi

chain of Cp after A. pohangense cells (final concentration = ca. 300 cells ml�1) were added

ingested Cp cell. (I) An Ap cell engulfing one of two Cp cells in a chain. (J) The Ap cell engu

numbers in (C–H) indicate elapsed time (min:sec). Arrows indicate Cp cells. Scale bars = 2

and I–K.

10 min (n = 20) was analyzed and the swimming speed of each cellwas calculated based on the linear displacement of the cell duringsingle-frame playback.

3. Results

3.1. Prey type and feeding mechanisms

Among the phytoplankton species offered, Alexandrium pohan-

gense fed exclusively on the mixotrophic dinoflagellate Cochlodi-

nium polykrikoides (Fig. 1; Table 1), and did not even try to attemptto feed on other phytoplankton species except for C. polykrikoides.

des cells. (A) An intact 4-celled Cochlodinium polykrikoides (Cp) chain. (B) A disrupted

. (C–H) The process of an Ap cell engulfing a Cp cell. (H) The Ap cell with completely

lfing the other Cp cell of the chain. (K) The Ap cell with two ingested Cp cells. Given

0 mm for (A and B) and 10 mm for (C–K). Predators and prey are the same cells in C–H

A.S. Lim et al. / Harmful Algae 49 (2015) 10–1814

Chains consisting of four Cochlodinium polykrikoides cells werebroken into one or two cells and became immobilized within30 min after Alexandrium pohangense cells (final con-centration = �300 cells ml�1) were added to water containing C.

polykrikoides chains (Fig. 1A and B). An A. pohangense cell was ableto engulf either one (Fig. 1C–H) or two cells in a row through thesulcus (Fig. 1I–K). The time (mean � standard error, n = 6) for a preycell to be completely engulfed and fed on by A. pohangense aftercontact was made was 118 � 17 s (Supplementary Video 1). Thebreakage of the C. polykrikoides chains and immobilization of the cellswas also observed within 30 min when prey cells were exposed to thefiltrates of a culture containing approximately 300 A. pohangense

cells ml�1.Alexandrium pohangense immobilized cells of most of the tested

phytoplankton species within 5 min of being added, even thoughthey did not feed on them (Table 1). Additionally, the cryptophytesTeleaulax sp., Rhodomonas salina, Storeatula major, and the nakedciliate Mesodinium rubrum were completely lysed within 6 h afterA. pohangense cells were added (Fig. 2). The immobilization of thecells of most phytoplankton species within 5 min and lysis of thefour species within 6 h were also observed after target prey cellswere exposed to the filtrates from a culture containing approxi-mately 300 A. pohangense cells ml�1.

3.2. Growth and ingestion rates of Alexandrium pohangense on

Cochlodinium polykrikoides

Alexandrium pohangense grew well feeding on Cochlodinium

polykrikoides. With increasing mean prey concentration, the specificgrowth rates of A. pohangense increased rapidly before saturating at

Fig. 2. (A) An intact Rhodomonas salina (Rs) cell without Ap and (B) a lysed Rs cell with a

added culture of Ap. Scale bars = 10 mm for A–D.

a C. polykrikoides concentration of 138 ng C ml�1 (197 cells ml�1)(Fig. 3). When the data were fitted to Eq. (2), the maximum specificgrowth rate (i.e., mixotrophic growth) of A. pohangense fed on C.

polykrikoides at 20 8C under a 14:10 h LD cycle of 100 mE m�2 s�1

was 0.487 d�1, while that without C. polykrikoides (i.e., autotrophicgrowth) was 0.091 d�1. The KGR (i.e. the prey concentrationsustaining ½mmax) was 11.3 ng C ml�1 (16 cells ml�1).

With increasing mean prey concentration, the ingestion rates ofAlexandrium pohangense increased rapidly at a Cochlodinium

polykrikoides concentration of 99 ng C ml�1 (141 cells ml�1), butslowly at higher prey concentrations (Fig. 4). When the data werefitted to Eq. (3), the maximum ingestion rate of A. pohangense on C.

polykrikoides was 4.99 ng C predator�1 d�1 (7.1 cells pre-dator�1 d�1) and KIR (the prey concentration sustaining ½Imax)was 30.0 ng C predator�1 d�1 (42.8 cells predator�1 d�1). Themaximum clearance rate of A. pohangense on C. polykrikoides was3.2 ml predator�1 h�1.

3.3. Potential grazing impact

The calculated grazing coefficients attributable to Alexandrium

pohangense on co-occurring Cochlodinium polykrikoides in thewaters of Yongil Bay, Pohang, Korea in 2014 were 0.089–1.567 d�1

when the abundances of A. pohangense and C. polykrikoides were 1–13 cells ml�1 and 3–259 cells ml�1, respectively (Fig. 5).

3.4. Swimming speed

The average (�SE, n = 20) and maximum swimming speeds of A.

pohangense were 218 (�12) and 340 mm s�1, respectively.

dded culture of Ap. (C) An intact Teleaulax sp. cell (Tel) and (D) lysed Tel cells with

Fig. 3. Specific growth rates of Alexandrium pohangense on Cochlodinium

polykrikoides as a function of the mean prey concentration (x, ng C ml�1).

Symbols represent treatment means � 1 SE. The curve was fitted by a Michaelis-

Menten equation [Eq. (2)] using all treatments in the experiment. Growth rate (GR,

d�1) = 0.487 [(x + 2.6792)/(11.34 + (x � 2.679))], r2 = 0.564.

Fig. 5. Calculated grazing coefficients (g, d�1) attributable to Alexandrium

pohangense on natural populations of Cochlodinium polykrikoides (see text for

calculation). n = 13.

Table 2Comparison of the prey species of 6 Alexandrium species whose mixotrophy has

been reported. ESD, equivalent spherical diameter (mm). Ap: Alexandrium

pohangense, Am: Alexandrium minutum, At: Alexandrium tamarense, Ac: Alexandrium

catenella, Ao: Alexandrium ostenfeldii Aps: Alexandrium pseudogonyaulax. T: toxic,

NT: non-toxic, Y: feeding on the prey species, N: not able to feed on the species.

Prey species ESD Ap Am At Ac Ao Aps

DiatomSkeletonema costatum 5.9 N Y Y

PrymnesiophytesIsochrysis galbana 4.8 N Y

A.S. Lim et al. / Harmful Algae 49 (2015) 10–18 15

4. Discussion

4.1. Prey species and feeding mechanisms

This study clearly shows that Alexandrium pohangense is amixotrophic dinoflagellate. A. pohangense was able to feed only onCochlodinium polykrikoides among the diverse phytoplanktonspecies tested in this study. A. pohangense is the first reportedmixotrophic dinoflagellate predator for C. polykrikoides. Interest-ingly, the prey type that A. pohangense was able to feed on wasdifferent from that of A. minutum, A. tamarense, A. catenella, A.

ostenfeldii, and A. pseudogonyaulax, whose mixotrophic abilitieswere revealed (Table 2); A. tamarense was not able to feed on C.

polykrikoides (Jeong et al., 2005a). However, A. minutum, A.

tamarense, A. catenella, A. ostenfeldii, and A. pseudogonyaulax wereable to feed on cryptophytes, dinoflagellates, ciliates, and even

Fig. 4. Ingestion rates of Alexandrium pohangense on Cochlodinium polykrikoides as a

function of the mean prey concentration (x). Symbols represent treatment

means � 1 SE. The curve was fitted by a Michaelis-Menten equation [Eq. (3)] using

all treatments in the experiment. (A) Ingestion rate (IR, ng C predator�1 d�1) = 4.99 [x/

(30.0 + x)], r2 = 0.665.

diatoms, which A. pohangense was unable to feed on (Nygaard andTobiesen, 1993; Jacobson and Anderson, 1996; Jeong et al.,2005a,b; Yoo et al., 2009; Blossom et al., 2012). Therefore, theecological niche of A. pohangense is likely to be different from thatof other Alexandrium spp. Several studies suggested that protistanpredators and prey have specific recognition relationships such aschemosensory mechanisms (Hansen and Calado, 1999; Woottonet al., 2007; Roberts et al., 2011; Sieg et al., 2011). Therefore, A.

CryptophytesTeleaulax acuta Y Y Y

Teleaulax sp. 5.6 N Y

Storeatula major 6.0 N

Rhodomonas salina 8.8 N Y

RhaphidophytesHeterosigma akashiwo 11.5 N Y

Mixotrophic dinoflagellatesHeterocapsa rotundata (T) 5.8 N Y Y Y

Amphidinium carterae (NT) 9.7 N Y

Prorocentrum minimum (T) 12.1 N Y

Hetereocapsa triquetra (T) 15.0 N N Y

Gymnodinium aureolum

(NT)

19.5 N N

Scrippsiella trochoidea (T) 22.8 N N

Cochlodinium polykrikoides

(NT)

25.9 Y N

Prorocentrum micans (T) 26.6 N N

Gymnodinium catenatum

(T)

33.9 N N

Dinophysis sp. (T) Y

Naked ciliateMesodinium rubrum 22.0 N Y

Unidentified ciliate Y

Reference (1) (2, 3, 4) (4, 5) (6) (5)

(1) This study, (2) Nygaard and Tobiesen (1993), (3) Jeong et al. (2005a), (4) Yoo

et al. (2009), (5) Blossom et al. (2012), (6) Jacobson and Anderson (1996).

A.S. Lim et al. / Harmful Algae 49 (2015) 10–1816

pohangense may have receptors and ligands involved in recogniz-ing prey species that are different from those of the otherAlexandrium species, and it would be beneficial to analyze thesecell surface components and mechanisms related to prey speciesrecognition at a molecular level.

The maximum swimming speed of Alexandrium pohangense

(340 mm s�1) measured in the present study was much lowerthan that of Cochlodinium polykrikoides, one of the fastestswimming phototrophic dinoflagellates (�1450 mm s�1; Jeonget al., 1999a). Theoretically, a slow-swimming A. pohangense cellmay not be able to capture a fast-swimming C. polykrikoides cell.Furthermore, A. pohangense does not have tow filaments ornematocysts used to anchor swimming prey cells as doGymnodinium aureolum and Paragymnodinium shiwhaense (Jeonget al., 2010b; Yoo et al., 2010). Interestingly, A. pohangense splits achain of C. polykrikoides into one or two cells, and thenimmobilizes and engulfs the C. polykrikoides cells through thesulcus. Therefore, A. pohangense has an effective mechanism tocapture and ingest very fast-swimming cells. Some otherAlexandrium species cause immobilization and/or lysis (Tillmannand John, 2002; Tillmann et al., 2007; Blossom et al., 2012); A.

pseudogonyaulax produces a mucus trap to immobilize some algalspecies (Blossom et al., 2012); A. ostenfeldii lyses someheterotrophic and autotrophic protists (Tillmann et al., 2007).However, these studies suggested that known toxins such assaxitoxins and spirolides were not involved in this immobiliza-tion activity (for examples, see Tillmann and John, 2002). A.

pohangense does not have putative sxtA saxitoxin genes (Lim et al.,2015a). Therefore, the lytic substrates of A. pohangense may not besaxitoxins and thus it is necessary to identify them. The filtrates ofA. pohangense cultures containing approximately 300 cells ml�1

immobilized the cells of all tested target prey species within5 min and lysed the cells of four species within 6 h. Thus, A.

pohangense cells may excrete substances into the ambient watersthat cause immobilization or lysis of prey cells.

Ma et al. (2011) reported that the lytic compounds ofAlexandrium tamarense showed a high affinity toward brassicas-terol, a sterol component of cell membranes. Interestingly,Rhodomonas salina and Storeatula major, which were lysed by A.

pohangense, have brassicasterol as a major sterol (>90% of the cellmembrane) (Adolf et al., 2006; Ma et al., 2011). Therefore, lytic

Table 3Optimal prey, maximum mixotrophic growth rate (MMGR), autotrophic growth rate (AG

dinoflagellate predator species.

Predator ESD Optimal prey ESD

Alexandrium pohangense 32.0 Cochlodinium polykrikoides 25.8

Ansanella granifera 10.5 Pyramimonas sp. 5.6

Prorocentrum donghaiense 13.2 Teleaulax sp. 5.6

Heterocapsa triquetra 15.0 Teleaulax sp. 5.6

Alexandrium minutum 16.7 Teleaulax acuta

Karlodinium armigera 16.7 Rhodomonas baltica 10.7

Cochlodinium polykrikoides 25.8 Teleaulax sp. 5.6

Prorocentrum micans 26.6 Teleaulax sp. 5.6

Amylax triacantha 30.0 Mesodinium rubrum 22.0

Gonyaulax polygramma 32.5 Teleaulax sp. 5.6

Alexandrium catenella 32.6 Teleaulax acuta

Alexandrium pseudogonyaulax 35.4 Heterocapsa rotundata 5.8

Lingulodinium polyedrum 36.6 Scrippsiella trochoidea 25.1

Fragilidium subglobosum 45.0 Ceratium tripos 59.5

Fragilidium cf. mexicanum 54.5 Lingulodinium polyedrum 37.9

ESD, equivalent spherical diameter (mm); T, temperature (8C); LI, light intensity (mE m�

rate (d�1); RMAG, ratio of mixotrophic to autotrophic growth rate; MIR, maximum inga Indicates the capability of feeding by both peduncle and engulfment. Berge et al. (

engulfment.

(1) This study, (2) Lee et al. (2014b), (3) Jeong et al. (2005a), (4) Blossom et al. (2012), (

(2005c), (9) Hansen and Nielsen (1997), (10) Jeong et al. (2010a), (11) Jeong et al. (199

compounds of A. pohangense may be related to brassicasterol. Inaddition, karlotoxins produced by Karlodinium veneficum (previ-ously K. micrum) were suggested to cause lysis of Oxyrrhis marina,which possesses 5,22-cholestadien-24b-methyl-3b-ol (brassicas-terol) and 5-cholesten-3b-ol (cholesterol) are the major mem-brane sterols of the cell membranes (Deed and Place, 2006).Therefore, the relationships between lytic compounds and sterolsamong protists should be explored.

4.2. Growth and ingestion rates and contribution of mixotrophy

The maximum growth rate (i.e., mixotrophic growth) ofAlexandrium pohangense on Cochlodinium polykrikoides (0.49 d�1)is higher than that of Alexandrium catenella on the optimal prey,cryptophyte Teleaulax acuta or Alexandrium pseudogonyaulax onHeterocapsa rotundata (0.06–0.32 d�1) even though the sizes ofthese predators are similar (Table 3). Additionally, the ratio ofmixotrophic to autotrophic growth rates (RMAG) of A. pohangense

on C. polykrikoides (4.9) is greater than that of other Alexandrium

predators on the optimal prey species (0.8–1.5). Furthermore, theRMAG of A. pohangense engulfment feeding is the greatest amongthe reported engulfment-feeding mixotrophic dinoflagellatesexcept Karlodinium armiger (Table 3; Fig. 6). Therefore, mixotrophymay provide A. pohangense an advantage in forming red tidepatches over these competitors.

The maximum ingestion rate of Alexandrium pohangense onCochlodinium polykrikoides (4.99 ng C predator�1 d�1) is highestamong engulfment-feeding mixotrophic dinoflagellates exceptFragilidium spp. (Table 3). A. pohangense is smaller than Fragilidium

spp.; thus, the smaller size of A. pohangense may be partiallyresponsible for the lower ingestion rate compared to Fragilidium

spp. However, the maximum ingestion rate of A. pohangense ismuch greater than that of Gonyaulax polygramma or Amylax

triacantha, which are similar in size to A. pohangense. Therefore,size may not be a critical factor affecting maximum ingestion ratesof the engulfment-feeding mixotrophic dinoflagellates. Fragilidium

spp. are able to feed voraciously on diverse dinoflagellates,including the heterotrophic species Protoperidinium cf. divergens

(Jeong et al., 1997, 1999a; Skovgaard et al., 2000). It is possible thatthe ingestion rates of engulfment-feeding mixotrophic dinofla-gellates are intrinsic characteristics of the genus or species.

R), and maximum ingestion rates (MIR) of each engulfment-feeding mixotrophic-

T LI MMGR AGR RMAG MIR Ref

20 100 0.49 0.10 4.9 4.99 (1)

20 20 1.43 0.39 3.7 0.97 (2)

20 20 0.51 0.38 1.4 0.03 (3)

20 20 0.28 0.18 1.5 0.04 (3)

15 17 0.11 0.13 0.8 (4)

15 180 0.65 0.06 10.8 0.97 (5)

20 50 0.32 0.17 2.0 0.16 (6)

20 20 0.20 0.11 1.9 0.04 (3)

15 20 0.68 �0.08 2.54 (7)

20 50 0.28 0.19 1.5 0.18 (8)

15 17 0.06 0.06 1.0 (4)

15 120 0.32 0.22 1.5 (4)

20 50 0.30 0.18 1.7 0.36 (3)

15 45 0.50 0.16 3.1 6.27 (9, 10)

22 20 0.36 �0.05 7.00 (10, 11)

2 s�1); MMGR, maximum mixotrophic growth rate (d�1); AGR, autotrophic growth

estion rate (ng C predator�1 d�1).

2008) suggested that Karlodinium armiger feeds on Rhodomonas baltica mainly by

5) Berge et al. (2008), (6) Jeong et al. (2004), (7) Park et al. (2013c), (8) Jeong et al.

9a).

Fig. 6. Ratio of maximum mixotrophic growth rate (MGR) of predators relative to

autotrophic growth rate (AGR) as a function of the cell size (equivalent spherical

diameters, ESD, mm) when each predator fed on the optimal prey species (see

Table 3). The temperature effect was not considered because the optimal

temperature for each dinoflagellate species is different from that for the others.

Ac: Alexandrium catenella. Ag: Ansanella granifera. Am: Alexandrium minumtum. Ap:

Alexandrium pohangense. Aps: Alexandrium pseudogonyaulax. Cp: Cochlodinium

polykrikoides. Fs: Fragilidium subglobosum. Gp: Gonyaulax polygramma. Ht:

Heterocapsa triquetra. Ka: Karlodinium armiger. Lp: Lingulodinium polyedrum. Pd:

Prorocentrum donghaiense. Pm: Prorocentrum micans.

A.S. Lim et al. / Harmful Algae 49 (2015) 10–18 17

4.3. Grazing impact

Prior to this study, only a few large ciliates and heterotrophicdinoflagellates, among protists, have been reported to feed onCochlodinium polykrikoides (Jeong et al., 1999b, 2005d, 2006, 2007,2008; Cho, 2006; Lim et al., 2014b). For field samples, the grazingimpact of these heterotrophic protistans on co-occurring C.

polykrikoides has not yet been analyzed. The grazing coefficients(g) attributable to A. pohangense on co-occurring C. polykrikoides

calculated in the present study can be up to 1.57 d�1 (i.e., up to 79%of the C. polykrikoides populations were removed by A. pohangense

in a day). Therefore, A. pohangense could have a considerablegrazing impact on populations of co-occurring C. polykrikoides. C.

polykrikoides has often caused huge red tides in the coastal watersof many countries and caused massive fish mortality and economicloss (Gobler et al., 2008; Mulhollannd et al., 2009; Park et al.,2013b; Lim et al., 2014a, 2015b). Therefore, A. pohangense may helpto decrease fish mortality due to C. polykrikoides red tides.

Acknowledgements

We thank Kyung Ha Lee, Sung Yeon Lee, and Ji Eun Kwon fortechnical supports. This work was supported by the NationalResearch Foundation of Korea Grant funded by the KoreaGovernment/MICTFP (NRF - 2015-M1A5A1041806), the KoreaMeteorological Administration Research and Development pro-gram under grant (CATER2014-6010), and Management of marineorganisms causing ecological disturbance and harmful effectProgram of Korea Institute of Marine Science and TechnologyPromotion (KIMST) award to HJJ.[SS]

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.hal.2015.07.010.

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