http://dx.doi.org/10.4490/algae.2013.28.4.343 Open Access
Growth and ingestion rates of heterotrophic dinoflagellates and a ciliate on the mixotrophic dinoflagellate Biecheleria cincta
Yeong Du Yoo
1,*, Eun Young Yoon
2, Kyung Ha Lee
3, Nam Seon Kang
3and Hae Jin Jeong
31Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202, USA
2Advanced Institutes of Convergence Technology, Seoul National University-Gyeonggi Province, Suwon 443-270, Korea
3School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea
To explore the interactions between the mixotrophic dinoflagellate Biecheleria cincta (previously Woloszynskia cincta) and heterotrophic protists, we investigated whether the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Gyrodinium spirale, Oxyrrhis marina, and Polykrikos kofoidii, and the ciliate Strobilidium sp.
were able to feed on B. cincta. We also measured growth and ingestion rates of O. marina and Strobilidium sp. on B. cincta as a function of prey concentration. In addition, these rates were measured for other predators at single prey concentra- tions at which the growth and ingestion rates of O. marina and Strobilidium sp. were saturated. All grazers tested in the present study were able to feed on B. cincta. B. cincta clearly supported positive growth of O. marina, G. dominans, and Strobilidium sp., but it did not support that of G. moestrupii, G. spirale, and P. kofoidii. The maximum growth rates of Strobilidium sp. and O. marina on B. cincta (0.91 and 0.49 d-1, respectively) were much higher than that of G. dominans (0.07 d-1). With increasing the mean prey concentration, the specific growth rates of O. marina and Strobilidium sp. on B.
cincta increased, but either became saturated or slowly increased. The maximum ingestion rate of Strobilidium sp. (1.60 ng C predator-1 d-1) was much higher than that of P. kofoidii and O. marina (0.55 and 0.34 ng C predator-1 d-1) on B. cincta.
The results of the present study suggest that O. marina and Strobilidium sp. are effective protistan grazers of B. cincta.
Key Words: graze; growth; harmful algal bloom; ingestion; protist; red tide
INTRODUCTION
Phototrophic dinoflagellates are ubiquitous and some- times dominate the abundance and biomass of plankton assemblages in marine environments (Porter et al. 1985, Hallegraeff 1993, Lindberg et al. 2005, Jeong et al. 2010a, 2013b). They can sometimes form dense blooms so called red tides or harmful algal blooms in marine ecosystem (Eppley and Horrison 1975, Burkholder et al. 2008, Kang et al. 2013, Park et al. 2013a). Dense blooms dominated by phototrophic dinoflagellates can upset the balance
of food webs and cause great loss to the aquaculture and tourist industries in many countries (e.g., Park et al.
2013b). Furthermore, phototrophic dinoflagellates play diverse roles in marine planktonic food webs (Ander- son et al. 2002, Yoo et al. 2009, Jeong et al. 2010b, Hansen 2011); they are primary producers (Tillmann et al. 2009), predators feeding on diverse prey items (Park et al. 2006, Berge et al. 2008, Yoo et al. 2010b, Jeong et al. 2012), and, in turn, act as prey for diverse predators (Jeong and Latz
Received July 30, 2013, Accepted November 17, 2013
*
Corresponding Author E-mail: ydyoo@ucsd.eduTel: +1-858-534-7110, Fax: +1-858-534-7313 This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://cre- ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
feed on B. cincta. 2) We also measured the growth and/
or ingestion rates of O. marina and Strobilidium sp. on B. cincta as a function of prey concentration. 3) In addi- tion, the growth and ingestion rates were measured for G. dominans, G. moestrupii, G. spirale, and P. kofoidii at single prey concentrations at which these rates of O. ma- rina and Strobilidium sp. were saturated. 4) Additionally, we compared the growth and ingestion rates of heterotro- phic protists in the present study of B. cincta to those of other prey species reported in the literature.
The results of the present study provide a basis for un- derstanding the interactions between B. cincta and het- erotrophic protists and their population dynamics in ma- rine planktonic food webs.
MATERIALS AND METHODS
Preparation of experimental organisms
For isolation and culture of Biecheleria cincta (Gen- Bank accession No. FR690459), plankton samples col- lected with water samplers were taken from Shiwha Bay, Korea during June 2009 when the water temperature and salinity were 22.0°C and 29.3, respectively (Table 1). These samples were screened gently through a 154-µm Nitex mesh and placed in 6-well tissue culture plates. A clonal culture of B. cincta was established by two serial single cell isolations as described by Kang et al. (2011).
For the isolation and culture of the heterotrophic dino- flagellate predators Gyrodinium dominans, G. moestru- pii, G. spirale, Oxyrrhis marina, and Polykrikos kofoidii, plankton samples collected with water samplers were taken from the coastal waters off Masan, Saemankeum, or Keum estuary, Korea in 2001-2009, and a clonal culture of each species was established by two serial single-cell isolations (Table 1).
For the isolation and culture of Strobilidium sp., plank- ton samples collected with water samplers were taken from a pier in Shiwha Bay, Korea, during May 2010 when the water temperature and salinity were 17.7°C and 27.8, respectively (Table 1). A clonal culture of Strobilidium sp.
(30-50 µm in cell length) was established by two serial single cell isolations as described by Jeong et al. (2008a).
The carbon contents for B. cincta (0.1 ng C per cell), the heterotrophic dinoflagellates, and the ciliate were es- timated from the cell volume according to the methods described by Menden-Deuer and Lessard (2000). The cell volume of the predators was estimated using the methods described by Jeong et al. (2008b) and Jeong et al. (2008a) 1994, Jeong 1999, Tillmann 2004, Jeong et al. 2010b, Kim
et al. 2013, Yoo et al. 2013b). Therefore, to understand the roles of phototrophic dinoflagellates in the food web, we must collate data on growth and mortality due to preda- tion.
The phototrophic dinoflagellate Biecheleria cincta was previously named Woloszynskia cincta (Siano et al. 2009, Kang et al. 2011). However, this dinoflagellate has since been reclassified and moved from the genus Woloszyn- skia to the genus Biecheleria because it has genetic and morphological characteristics that more closely resemble Biecheleria, including an apical furrow apparatus formed by a single elongated narrow vesicle extending over the apex from the ventral to the dorsal side of the cell (Moes- trup et al. 2009, Kang et al. 2011, Balzano et al. 2012, Luo et al. 2013). In addition, it has chloroplasts and eyespots that are formed by a stack of cisternae containing brick- like material (type E sensu) (Moestrup et al. 2009, Kang et al. 2011). The presence of this species has been reported in the coastal waters of Korea (Kang et al. 2011).
Recently, Kang et al. (2011) discovered that B. cincta, originally thought to be an exclusively phototrophic di- noflagellate, is a mixotrophic dinoflagellate; it feeds on diverse prey such as the haptophyte Isochrysis galbana, the cryptophytes Teleaulax sp. and Rhodomonas salina, the raphidophyte Heterosigma akashiwo, the eugleno- phyte Eutreptiella gymnastica, and the dinoflagellates Heterocapsa rotundata and Amphidinium carterae. How- ever, to date, there have been no studies on the mortal- ity of B. cincta due to predation. Grazing pressure some- times plays an important role in controlling populations of phototrophic dinoflagellates (Watras et al. 1985, Turner 2006, Kang et al. 2013, Yoo et al. 2013a). Heterotrophic di- noflagellates and ciliates are the major components of heterotrophic protist communities (Sherr and Sherr 2007, Jeong et al. 2011, Yoo et al. 2013a). They are effective graz- ers on many phototrophic dinoflagellates (Eppley and Harrison 1975, Jeong et al. 2013a, Yoo et al. 2013b). Thus, to understand the population dynamics of B. cincta, the predator-prey relationships between B. cincta and com- mon heterotrophic dinoflagellates and ciliates should be investigated.
We established a clonal culture of Biecheleria cincta isolated from Shiwha Bay, Korea in 2009. In the present study, using this culture we investigated feeding by five common heterotrophic dinoflagellates and one ciliate on this dinoflagellate; 1) we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, G.
moestrupii, G. spirale, Oxyrrhis marina, and Polykrikos kofoidii and the ciliate Strobilidium sp. were able to
ton wheel rotating at 0.9 rpm and incubated at 20°C un- der an illumination of 20 µmol photons m-2 s-1 on a 14 : 10 h light-dark cycle.
Five milliliters aliquots were removed from each bottle after 1, 2, 6, 24, and 48-h incubation periods and then transferred into 6-well plate chambers (or slide glasses).
Approximately 100 cells of predators at different stag- es of the feeding process in the plate chamber (or slide glasses) were observed under a dissecting microscope (or inverted microscope) at a magnification of ×20-90 (or
×100-630) to determine whether the predators were able to feed on B. cincta. Cells from those predators that con- tained ingested B. cincta cells were photographed using a digital camera (Zeiss AxioCam HRc5; Carl Zeiss Ltd., Göt- tingen, Germany) on the microscope at a magnification of ×400-630.
for O. marina and Strobilidium sp., respectively.
Feeding occurrence
Experiment 1 was designed to investigate whether G.
dominans, G. moestrupii, G. spirale, O. marina, P. kofoidii, and Strobilidium sp. were able to feed on B. cincta (Table 2). The concentrations of each predator species offered were similar in terms of carbon biomass.
Approximately 4.8 × 105 B. cincta cells were added to each of two 80 mL polycarbonate (PC) bottles contain- ing G. dominans, G. moestrupii, G. spirale (100-500 cells mL-1), P. kofoidii (30 cells mL-1), and Strobilidium sp. (30 cells mL-1) (final B. cincta prey concentration = ca. 6,000 cells mL-1). One control bottle (without prey) was set up for each experiment. The bottles were placed on a plank-
Table 1.
Isolation and maintenance conditions of the experimental organismsOrganism Location Date T S Prey species Concentration
Gyrodinium dominans (HTD) Masan Bay Apr 2007 15.1 33.4 Amphidinium
carterae 30,000-40,000 Gyrodinium moestrupii (HTD) Off Saemankeum Oct 2009 21.2 31.0 Alexandrium
minutum 8,000-10,000 Gyrodinium spirale (HTD) Masan Bay May 2009 19.7 31.0 Prorocentrum
minimum 20,000-30,000
Oxyrrhis marina (HTD) Keum Estuary May 2001 16.0 27.7 Amphidinium
carterae 8,000
Polykrikos kofoidii (HTD) Masan Bay Jun 2007 20.2 32.2 Lingulodinium
polyedrum 4,000
Strobilidium sp. (CIL) Shiwha Bay May 2010 17.7 27.8 Teleaulax sp. 50,000-60,000 Biecheleria cincta (MTD) Shiwha Bay Jun 2009 22.0 29.3 Heterosigma
akashiwo 10,000-15,000 Sampling location and date, water temperature (T, oC), salinity (S, practical salinity units) for isolation, and prey species and concentrations (cells mL-1) for maintenance.
HTD, heterotrophic dinoflagellate; CIL, ciliate; MTD, mixotrophic dinoflagellate.
Table 2.
Design of the experimentsExp. no. Prey Predator
Feeding
Species Density Species Density
1 Biecheleria cincta 6,000 Gyrodinium dominans 500 Y
Gyrodinium moestrupii 200 Y
Gyrodinium spirale 100 Y
Oxyrrhis marina 500 Y
Polykrikos kofoidii 30 Y
Strobilidium sp. 30 Y
2 Biecheleria cincta 20, 60, 130, 510, 1,690, 3,670, 6,420 Oxyrrhis marina 3, 4, 6, 12, 19, 39, 73, (6) - 3 Biecheleria cincta 20, 80, 240, 850, 2,640, 5,310, 6,930 Strobilidium sp. 5, 12, 14, 24, 23, 35, 35, (12) - The numbers in the prey and predator columns are the actual initial densities (cells mL-1) of the prey and predator. Values in the parentheses in the predator column are the predator densities in the control bottles. Feeding occurrence of each predator fed Biecheleria cincta is represented by Y (feeding observed).
was taken from each bottle after a 48-h incubation period and fixed with 5% Lugol’s solution, and the abundance of O. marina (or Strobilidium sp.) and B. cincta were deter- mined by counting all or >200 cells in three 1-mL SRCs.
Prior to taking the subsamples, the conditions of O. ma- rina (or Strobilidium sp.) and its prey were assessed us- ing a dissecting microscope, as described earlier in this section.
The specific growth rate of O. marina (or Strobilidium sp.), µ (d-1) was calculated as follows:
µ = [ln (Gt/G0)] / t (1) , where G0 and Gt are the concentration of O. marina (or Strobilidium sp.) at time (t) 0 and 2 d, respectively.
Data for O. marina (or Strobilidium sp.) growth rates were fitted to a modified Michaelis-Menten equation:
µ max (x – x') KGR + (x – x')
µ = (2)
, where µmax = the maximum growth rate (d-1), x = prey concentration (cells mL-1 or ng C mL-1), x' = threshold prey concentration (the prey concentration where µ = 0), and KGR = the prey concentration sustaining 1/2 µmax. Data were iteratively fitted to the model using DeltaGraph (Delta Point Inc., Monterey, CA, USA).
Ingestion and clearance rates were calculated using the equations of Frost (1972) and Heinbokel (1978). The incu- bation time for calculating ingestion and clearance rates was the same as that for estimating growth rate. Data for O. marina (or Strobilidium sp.) ingestion rates (IR, cells predator-1 d-1 or ng C predator-1 d-1) were fitted to a modi- fied Michaelis-Menten equation:
Imax (x) KIR + (x)
IR = (3)
, where Imax = the maximum ingestion rate (cells preda- tor-1 d-1 or ng C predator-1 d-1), x = prey concentration (cells mL-1 or ng C mL-1), and KIR = the prey concentration sus- taining 1/2 Imax.
Comparison of growth and ingestion rates at single prey concentrations
Experiment 4 was designed to compare the growth and ingestion rates of G. dominans, G. moestrupii, G. spirale, and P. kofoidii when B. cincta was provided at a single prey concentration (Table 3). Growth and ingestion rates of O. marina and Strobilidium sp. at single prey concen-
Effects of prey concentration on growth and ingestion rates
Experiments 2 and 3 were designed to measure the growth, ingestion, and clearance rates of O. marina and Strobilidium sp. as a function of the prey concentrations when fed on B. cincta (Table 2).
A dense culture of ~15,000 cells mL–1 of B. cincta grown mixotrophically on the raphidophyte Heterosigma akashiwo in the f/2 medium (Guillard and Ryther 1962, Kang et al. 2011) under the illumination of 20 µmol pho- tons m-2 s-1 on a 14 : 10 h light : dark cycle was transferred to a 250-mL PC bottle containing the f/2 medium where- in H. akashiwo was undetectable. This culture was main- tained in the f/2 medium for 2 d under the illumination of 20 µmol photons m-2 s-1 on a 14 : 10 h light : dark cycle and then transferred to another 250-mL PC bottle contain- ing filtered seawater. Three 1-mL aliquots from the bottle were examined using a light microscope to determine the concentration of B. cincta cells, and the cultures were then used in further experiments.
Furthermore, dense cultures of O. marina (or Stro- bilidium sp.) growing on algal prey were transferred into 250-mL PC bottles containing filtered seawater. The bot- tles were filled to capacity with freshly filtered seawater, capped, and placed on plankton wheels rotating at 0.9 rpm and incubated at 20°C under the illumination of 20
mol photons m-2 s-1 on a 14 : 10 h light : dark cycle.
For each experiment, the initial concentrations of O.
marina (or Strobilidium sp.) and B. cincta were estab- lished using an autopipette to deliver predetermined volumes of known cell concentrations to the bottles.
Triplicate 42-mL PC experimental bottles (mixtures of predator and prey) and triplicate control bottles (prey only) were set up for each predator-prey combination.
Triplicate control bottles containing only O. marina (or Strobilidium sp.) were also established for a single preda- tor concentration. All the bottles were filled to capacity with freshly filtered seawater and capped. To determine the actual predator and prey densities at the beginning of the experiment, a 5-mL aliquot was removed from each bottle, fixed with 5% Lugol’s solution, and then examined under a light microscope to determine predator and prey abundance by enumerating the cells in three 1-mL Sedg- wick-Rafter chambers (SRCs). The bottles were refilled to capacity with freshly filtered seawater, capped, and placed on rotating wheels under the conditions described earlier in this section. Dilution of cultures associated with the refilling of bottles was taken into consideration when calculating growth and ingestion rates. A 10-mL aliquot
The growth and ingestion rates were measured in the same manner as described for Experiments 2 and 3.
Gross growth efficiency (GGE)
GGE, defined as grazer biomass produced (+) or lost (–) per prey biomass ingested, was calculated from the esti- mates of carbon contents per cell based on the cell vol- ume for each mean prey concentration.
RESULTS
Feeding occurrence and growth
It was observed that G. dominans, G. moestrupii, G. spi- rale, O. marina, P. kofoidii, and Strobilidium sp. fed on B.
cincta (Table 2, Fig. 1). All predators in the present study fed on prey by engulfing the prey cells.
B. cincta clearly supported positive growth rates for O.
marina, G. dominans, and Strobilidium sp. but did not support the growth of G. moestrupii, G. spirale, or P. ko- foidii.
Effects of prey concentrations on growth and ingestion rates
The specific growth rates of O. marina on B. cincta in- creased rapidly with increasing mean prey concentration
<ca. 12 ng C mL-1 (120 cells mL-1), but became saturated or slowly increased at higher concentrations (Fig. 2).
When the data were fitted to Eq. (2), the maximum spe- cific growth rates of O. marina was 0.49 d-1. The feeding threshold prey concentration for the growth of O. marina was 1.4 ng C mL-1 (14 cells mL-1).
The specific growth rates of Strobilidium sp. on B.
cincta increased rapidly with increasing mean prey con- centration <ca. 71 ng C mL-1 (710 cells mL-1), but became slowly increased at higher concentrations (Fig. 3). When the data were fitted to Eq. (2), the maximum specific growth rates of Strobilidium sp. was 0.91 d-1. The feeding threshold prey concentration for the growth of Strobilidi- um sp. was 11.8 ng C mL-1 (118 cells mL-1).
The ingestion rates of O. marina on B. cincta increased rapidly with increasing mean prey concentration <ca. 45 ng C mL-1 (450 cells mL-1), but became saturated or slowly increased at higher concentrations (Fig. 4). When the data were fitted to Eq. (3), the maximum ingestion rates of O.
marina was 0.35 ng C predator-1 d-1 (3.5 cells predator-1 d-1). The maximum clearance rate of O. marina was 1.47 trations were obtained in Experiment 2 and 3.
The B. cincta culture was prepared as described earlier in this section. In addition, G. dominans, G. moestrupii, G.
spirale, and P. kofoidii were cultured in the same manner as described earlier in this section.
The initial concentrations of G. dominans (or another predator) and B. cincta were established using an auto- pipette to deliver predetermined volumes of known cell concentrations to the bottles. Triplicate 42-mL PC experi- mental bottles containing mixtures of G. dominans (or another predator) and B. cincta, triplicate prey control bottles containing B. cincta only, and triplicate preda- tor control bottles containing only G. dominans (or an- other predator) were set up for B. cincta. Next, 5 mL of the f/2 medium was added to all the bottles, which were then filled to capacity with freshly filtered seawater and capped. To determine the predator and prey concentra- tions at the beginning of the experiment and the prey concentrations after 2 d, a 5-mL aliquot was removed from each bottle and fixed with 5% (v/v) Lugol’s solution;
then, all or >200 predator and prey cells from three 1-mL SRCs were enumerated. Prior to taking subsamples, the conditions of G. dominans (or other predators) and B.
cincta were assessed using a dissecting microscope. The bottles were, again, filled to capacity with freshly filtered seawater, capped, and placed on a rotating wheel at 0.9 rpm at 20°C under the illumination of 20 µmol photons m-2 s-1 on a 14 : 10 h light : dark cycle. The dilution of the cultures associated with the refilling of bottles was taken into consideration when calculating the growth and in- gestion rates.
Table 3.
Comparison in growth (GR, d-1) and ingestion rates (IR, ng C predator-1 d-1) (means ± standard errors, n = 3) protistan predators on mixotrophic dinoflagellate Biecheleria cincta at single mean prey concentrations (MPC, ng C mL-1) where GR and IR of Oxyrrhis marina and Strobilidium sp. on B. cincta were saturatedPredators MPC GR IR
Oxyrrhis marina (HTD) 600 0.436 (0.023)
0.34 (0.03) Gyrodinium dominans (HTD) 562 0.069
(0.018)
0.13 (0.02) Gyrodinium moestrupii (HTD) 487 -0.086
(0.037)
0.10 (0.03) Gyrodinium spirale (HTD) 512 -0.198
(0.020)
0.04 (0.02) Polykrikos kofoidii (HTD) 481 -0.253
(0.009)
0.55 (0.05) Strobilidium sp. (CIL) 504 0.706
(0.054)
1.60 (0.17) HTD, heterotrophic dinoflagellate; CIL, ciliate.
rates increased slowly were 25-32% (Table 4).
Comparison of growth and ingestion rates at single prey concentrations
When the mean prey concentrations were 480-600 ng C mL-1, the specific growth rate of Strobilidium sp. (0.71 d-1) on B. cincta was significantly higher than that of O.
marina (0.44 d-1) or G. dominans (0.07 d-1) (p < 0.01, two- tailed t-test). However, the growth rates of G. moestrupii, G. spirale, and P. kofoidii were negative (Table 3).
The ingestion rate of Strobilidium sp. (1.60 ng C preda- µL predator-1 h-1. GGEs of O. marina on B. cincta at prey
concentrations where the ingestion rates were saturated were 45-53% (Table 4).
The ingestion rates of Strobilidium sp. on B. cincta in- creased rapidly with increasing mean prey concentration
<ca. 236 ng C mL-1 (2,360 cells mL-1), but became slowly increased at higher concentrations (Fig. 5). When the data were fitted to Eq. (3), the maximum ingestion rates of Strobilidium sp. was 2.0 ng C predator-1 d-1 (20.0 cells predator-1 d-1). The maximum clearance rate of Strobilidi- um sp. was 1.72 µl predator-1 h-1. GGEs of Strobilidium sp.
on B. cincta at prey concentrations where the ingestion
Fig. 1.
Feeding by heterotrophic dinoflagellates (A-D) and a ciliate (E) on the mixotrophic dinoflagellate Biecheleria cincta. (A) Gyrodinium dominans with an ingested B. cincta cell. (B) Gyrodinium spirale with an ingested B. cincta cell. (C) Oxyrrhis marina with several ingested B. cincta cells. (D) Polykrikos kofoidii with two ingested B. cincta cells. (E) Strobilidium sp. with two ingested B. cincta cells. Arrows indicate ingested prey cells. All photographs were taken using an inverted microscope. Scale bars represent: A-E, 10 µm.A C
D
B
E
Fig. 4.
Ingestion rates of the heterotrophic dinoflagellate Oxyrrhis marina on the mixotrophic dinoflagellate Biecheleria cincta as a function of mean prey concentration (x). Symbols represent treatment means ± 1 SE. The curves are fitted according to the Michaelis-Menten equation [Eq. (3)] using all treatments in the experiment. Ingestion rate (ng C predator-1 d-1) = 0.35[x/(9.22 + x)], r2 = 0.777.Fig. 3.
Specific growth rates of the ciliate Strobilidium sp. on the mixotrophic dinoflagellate Biecheleria cincta as a function of mean prey concentration (x). Symbols represent treatment means ± 1 SE.The curves are fitted according to the Michaelis-Menten equation [Eq.
(2)] using all treatments in the experiment. Growth rate (d-1) = 0.910 {(x - 11.8)/[34.8 + (x - 11.8)]}, r2 = 0.911.
Fig. 2.
Specific growth rates of the heterotrophic dinoflagellate Oxyrrhis marina on the mixotrophic dinoflagellate Biecheleria cincta as a function of mean prey concentration (x). Symbols represent treatment means ± 1 SE. The curves are fitted according to the Michaelis-Menten equation [Eq. (2)] using all treatments in the experiment. Growth rate (d-1) = 0.492{(x - 1.38)/[5.67 + (x - 1.38)]}, r2 = 0.843.Fig. 5.
Ingestion rates of the ciliate Strobilidium sp. on the mixotro- phic dinoflagellate Biecheleria cincta as a function of mean prey con- centration (x). Symbols represent treatment means ± 1 SE. The curves are fitted according to the Michaelis-Menten equation [Eq. (3)] using all treatments in the experiment. Ingestion rate (ng C predator-1 d-1) = 1.98 [x/(62.2 + x)], r2 = 0.884.Table 4.
Growth and grazing data for the Oxyrrhis marina and Strobilidium sp. on Biecheleria cinctaPredator PDV µmax KGR x' Imax KIR Cmax GGE
Oxyrrhis marina (HTD) 1.1 0.49 5.67 1.38 0.35 9.22 1.47 45-53
Strombilidium sp. (CIL) 11.4 0.91 34.8 11.8 1.98 62.2 1.72 25-32 Parameters are for numerical and/or functional responses from Eqs. (2) and (3), as presented in Figs 2-5.
PDV, predator’s volume (×103 µm3); µmax,maximum growth rate (d-1); KGR, prey concentration sustaining 1/2 µmax (ng C mL-1); x’, threshold prey concentration (ng C mL-1); Imax,maximum ingestion rate (ng C predator-1 d-1); KIR,prey concentration sustaining 1/2 Imax (ng C mL-1); Cmax,maximum clearance rate (µL predator-1 h-1); GGE, gross growth efficiency, %, of predators feeding on B. cincta at the prey concentrations where the ingestion rates were saturated or the 3 highest ingestion rates were achieved; HTD, heterotrophic dinoflagellate; CIL, ciliate.
DISCUSSION
Feeding occurrence and growth
To the best of our knowledge, this study is the first re- port on feeding by heterotrophic protistan predators on B. cincta. All heterotrophic protistan predators investi- gated in the present study were able to feed on B. cincta by engulfing the cells. These heterotrophic protists com- monly occur in many marine environments (Goldman et al. 1989, Yoo et al. 2010a, Jeong et al. 2011, Yoon et al.
2012). Thus, heterotrophic protists should be considered predators of B. cincta in marine food webs.
O. marina, G. dominans, and Strobilidium sp. exhibit- ed positive growth rates when feeding on B. cincta but G.
moestrupii, G. spirale, and P. kofoidii did not. Thus, during blooms dominated by B. cincta, O. marina, G. dominans, and Strobilidium sp. are likely to be abundant, while G.
moestrupii, G. spirale, and P. kofoidii may not be present.
B. cincta can be a critical prey for selecting dominant spe- cies among heterotrophic protistan communities.
Growth and ingestion rates
The growth rates for G. moestrupii, G. spirale, and P. ko- foidii feeding on B. cincta were negative, while those for O. marina or Strobilidium sp. were relatively high (Table 3). For G. moestrupii, G. spirale, and P. kofoidii feeding on B. cincta, their ingestion rates (0.10, 0.04, and 0.55 ng C predator-1 d-1, respectively) were much lower than their carbon contents (0.4, 1.3, and 4.2 ng C cell-1, respectively) (Jeong et al. 2001b, Kim and Jeong 2004, Yoo et al. 2013b).
Thus, low ingestion rates for G. moestrupii, G. spirale, and P. kofoidii on B. cincta are likely responsible for their negative growth rates. However, growth rates for G. moes- trupii, G. spirale, and P. kofoidii are high when feeding on algal prey (Jeong et al. 2001b, Kim and Jeong 2004, Yoo et al. 2013b). The maximum growth rates for G. moestrupii, G. spirale, and P. kofoidii when feeding on optimal prey (e.g., Alexandrium minutum, Prorocentrum minimum, and Gymnodinium catenatum) are as high as 1.60, 1.13, and 1.12 d-1, respectively (Jeong et al. 2001b, Kim and Jeong 2004, Yoo et al. 2013b). We assume that the ecologi- cal niches of G. moestrupii, G. spirale, and P. kofoidii may be different from those of O. marina or Strobilidium sp., and competition among these protistan grazers might re- duce when feeding on certain prey.
Among the maximum growth (µmax) and ingestion rates (Imax) of O. marina feeing on diverse prey items, the µmax of O. marina on B. cincta is similar than that on Azadini- tor-1 d-1) on B. cincta was significantly higher than that of
P. kofoidii (0.55 ng C predator -1 d-1), O. marina (0.34 ng C predator -1 d-1), G. dominans (0.13 ng C predator -1 d-1), G.
moestrupii (0.10 ng C predator -1 d-1), or G. spirale (0.04 ng C predator -1 d-1) (p < 0.01, two-tailed t-test).
Both growth and ingestion rates of the heterotrophic protists feeding on B. cincta in the present study were not significantly correlated with the predators’ equivalent spherical diameter (p > 0.1, linear regression analysis of variance [ANOVA]) (Fig. 6A & B). Moreover, the growth rates were not significantly correlated with ingestion rates (p > 0.1, ANOVA) (Fig. 6C).
Fig. 6.
Growth (GR) and ingestion rates (IR) of heterotrophic dinoflagellates and the ciliate on the mixotrophic dinoflagellate Biecheleria cincta as a single prey concentration where the growth and ingestion rates of Oxyrrhis marina and Strobilidium sp. were saturated. GR (A) and IR (B) of the predators on B. cincta as a function of predator size (equivalent spherical diameter, ESD, µm). (C) The GR of predators on B. cincta as a function of the IR (as shown in Table 3). The p-values in (A), (B), and (C) were all p > 0.1 (linear regression ANOVA). Gd, Gyrodinium dominans; Gm, Gyrodinium moestrupii;Gs, Gyrodinium spirale; Om, O. marina; Pk, Polykrikos kofoidii; St, Strobilidium sp.
A
C B
gestion rate of O. marina feeding on B. cincta than that on the heterotrophic nanoflagellate and heterotrophic dino- flagellates may be responsible for its higher growth rates.
O. marina may capture and ingest B. cincta with more difficulty than the other algal prey, except some unpalat- able ones, but more easily than heterotrophic nanoflagel- late and dinoflagellates. Both the µmax and Imax of O. ma- rina feeding on diverse prey species, including B. cincta, were not significantly correlated with the prey’s equiva- lent spherical diameter (p > 0.1, ANOVA). Moreover, the µmax of O. marina feeding on diverse prey species was not significantly correlated with the Imax (p > 0.1, ANOVA).
Therefore, for O. marina, the nutritional value of the dif- ferent prey species, including B. cincta, may differ.
The µmax of Strobilidium sp. on B. cincta is higher than that on A. cf. poporum and the euglenophyte Eutreptiella gymnastica, although the Imax of Strobilidium sp. on B.
cincta is comparable to or lower than that on A. cf. popo- rum and E. gymnastica (Table 5). Therefore, for Strobilid- ium sp., B. cincta may have higher nutritional value than A. cf. poporum or E. gymnastica.
um cf. poporum (Table 5). However, the Imax of O. marina on B. cincta is lower than that on A. cf. poporum (Potvin et al. 2013). Therefore, the nutritional value of B. cincta for growth of O. marina may be greater than that of A.
cf. poporum. The µmax of O. marina feeding on B. cincta is lower than that on the other algal prey species except a toxic strain of Karlodinium veneficum, but higher than that on the heterotrophic nanoflagellate Cafeteria sp. and the heterotrophic dinoflagellates Luciella masanensis and Stoeckeria algicida (Table 5). Therefore, B. cincta is a better prey item for O. marina than these heterotrophic nanoflagellate and heterotrophic dinoflagellates, but less favorable prey than the other algal prey species, except K. veneficum. The Imax of O. marina feeding on B. cincta is lower than that on the other algal prey species except the mixotrophic dinoflagellate Gymnodinium aureolum, but higher than that on Cafeteria sp., Pfiesteria piscicida, L.
masanensis, and S. algicida (Table 5). Therefore, the lower ingestion rate of O. marina feeding on B. cincta than that on the other algal prey species except one species may be responsible for its lower growth rates, but the higher in-
Table 5.
Comparison of maximum growth and ingestion rates of Oxyrrhis marina and Strobilidium spp. on diverse prey speciesPredator Prey species ESD µmax Imax Reference
Oxyrrhis marina (HTD) Cafeteria sp. (HNF) 3.5 0.19 0.3 Jeong et al. (2007b)
Phaeodactylum tricornutum (DIA) 4.2 1.30 1.9 Goldman et al. (1989)
Isochrysis galbana (PRY) 5.1 0.80 2.2 Goldman et al. (1989) Dunaliella tertiolecta (CHL) 5.1 0.80 1.5 Goldman et al. (1989) Karlodinium veneficum_NT (MTD) 9.1 0.90 6.4 Adolf et al. (2007) Amphidinium carterae (MTD) 9.7 1.17 2.8 Jeong et al. (2001a) Azadinium cf. poporum (PTD) 10.0 0.50 5.0 Potvin et al. (2013) Karlodinium veneficum_T (MTD) 10.5 0.25 2.2 Adolf et al. (2007) Heterosigma akashiwo (RAP) 11.5 1.43 1.3 Jeong et al. (2003) Biecheleria cincta (MTD) 12.2 0.49 0.91 This study Eutreptiella gymnastica (EUG) 12.6 0.81 2.7 Jeong et al. (2011) Pfiesteria piscicida (HTD) 13.5 0.66 0.33 Jeong et al. (2007a) Luciella masanensis (HTD) 13.5 0.04a 0.07 Jeong et al. (2007a) Stoeckeria algicida (HTD) 13.9 0.22 0.14 Jeong et al. (2007a) Gymnodinium aureolum (MTD) 19.4 0.71 0.51 Yoo et al. (2010a)
Fibrocapsa japonica (RAP) 20.4 0.72 1.2 Tillmann and Reckermann (2002) Strobilidium spp. (CIL) Azadinium cf. poporum (PTD) 10.0 0.64 179 Potvin et al. (2013)
Biecheleria cincta (MTD) 12.2 0.91 1.98 This study Eutreptiella gymnastica (EUG) 12.6 -0.94a 2.2 Jeong et al. (2011) Rates are corrected to 20°C using Q10 = 2.8 (Hansen et al. 1997).
ESD, equivalent spherical diameter (µm); µmax, maximum growth rate (d-1); Imax, maximum ingestion rate (ng C predator-1 d-1); HTD, heterotrophic dinoflagellate; HNF, heterotrophic nanoflagellate; DIA, diatom; PRY, prymnesiophyte; CHL, chlorophyte; NT, non-toxic; MTD, mixotrophic dinofla- gellate; PTD, phototrophic dinoflagellate; T, toxic; RAP, raphidophyte; EUG, euglenophyte; CIL, ciliate.
aThe maximum value among the mean growth rates measured at given prey concentrations.
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bilidium sp. on B. cincta are affected by prey concentra- tions. The threshold prey concentration for growth of O.
marina on B. cincta (1.4 ng C mL-1) was lower than that for the growth rate of Strobilidium sp. on the same prey (11.8 ng C mL-1). Therefore, O. marina is likely to survive at low B. cincta concentrations but Strobilidium sp. is not. The KGR (the prey concentration sustaining 1/2 µmax) of 5.7 ng C mL-1 for O. marina feeding on B. cincta was also lower than that of Strobilidium sp. (34.8 ng C mL-1) feeding on the same algal prey. Thus, O. marina is likely to grow rap- idly at low B. cincta concentrations but Strobilidium sp.
would not. Additionally, the KIR (the prey concentration sustaining 1/2 Imax) of 9.2 ng C mL-1 for O. marina feed- ing on B. cincta was also lower than that of Strobilidium sp. (62.2 ng C mL-1) feeding on the same algal prey. Thus, these results indicate that, at low prey concentrations, the growth and ingestion rates of O. marina would respond more readily to changes in prey concentrations than those of Strobilidium sp.
We could not estimate the grazing impact by O. marina and Strobilidium sp. on B. cincta in this study because data on the abundance of B. cincta, O. marina, and Stro- bilidium sp. are not available. Therefore, to understand the population dynamics of B. cincta and heterotrophic protists and their interactions, the abundance of B. cinc- ta and its predators in natural environments need to be quantified.
ACKNOWLEDGEMENTS
We thank Yeong Jong Hwang and Eric Potvin for techni- cal support. This paper was supported by Basic Research Program through the National Research Foundation of Korea (NRF) grant funded by Ministry of Science, ICT and Future Planning (MSICTFP), the Korean Govern- ment NRF/MEST (2012R1A6A3A03040333) award to YD Yoo and the NRF grant funded by MSICTFP (NRF- 2010-0020702) and Mid-career Researcher Program (2012-R1A2A2A01-010987) award to HJ Jeong.
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