AQUATIC MICROBIAL ECOLOGY Vol. 36: 165–170, 2004 Published July 21 Aquat Microb Ecol Ingestion of cryptophyte cells by the marine photosynthetic ciliate Mesodinium rubrum Wonho Yih1,*, Hyung Seop Kim1, Hae Jin Jeong2, Geumog Myung1, Young Geel Kim1 1Department of Oceanography, Kunsan National University, San 68, Miryong-dong, Kunsan 573-701, RO Korea 2School of Earth and Environmental Science, College of Natural Sciences, Seoul National University, Seoul 151-747, RO Korea ABSTRACT: We investigated the mechanism of capturing and ingesting cryptophyte cells by a labo- ratory strain of the marine photosynthetic ciliate Mesodinium rubrum Lohmann 1908 (= Myrionecta rubra Jankowski 1976), a cosmopolitan red tide species. When offered cryptophytes as food, M. rubrum, originally grown photosynthetically for 2 wk, used its bifurcated oral tentacles to instantly seize prey cells when encountered. Immediately after capturing a prey cell, M. rubrum swam in a zigzag pattern (30 to 60 µm long linear paths) for >4 s, without showing the large jumps (with ca. 2000 µm long linear paths) that were usually observed when the predator was not feeding. M. rubrum with a cryptophyte attached to its tentacles became motionless while the prey cell was moved to the oral surface of the predator, a process that took <10 s. Engulfment of a captured prey cell by M. rubrum occurred through a cytostome-like structure and took ca. 15 s. Once engulfed, the prey was slowly delivered to the posterior end of the ciliate over a period of ca. 63 s. The whole feeding process lasted approximately 92 s. With increasing mean prey concentration, specific growth rates of M. rubrum feeding on the cryptophyte increased, with saturation at a mean prey concentration of 44 cells ml–1. The maximum specific growth rate (mixotrophic growth) of M. rubrum feeding on the cryptophyte was 0.521 d–1, under continuous illumination of 60 µE m–2 s–1, while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.357 d–1. The inges- tion rate of M. rubrum feeding on cryptophytes increased continuously with increasing prey concen- tration. The maximum ingestion rate was 8.9 cryptophytes ciliate–l d–1. M. rubrum may sometimes exert considerable impact on prey populations. KEY WORDS: Feeding · Growth rate · Grazing · Life cycle · Phagotrophy · Red tide Resale or republication not permitted without written consent of the publisher INTRODUCTION M. rubrum was first suggested by Hargraves (1991) based on photographic evidence, and the inclusion of Mesodinium rubrum Lohmann 1908 (= Myrionecta cryptophyte cells in the protoplasm of M. rubrum was rubra Jankowski 1976) (Lynn & Small 2002) is a plank- later documented by Gustafson et al. (2000) using data tonic ciliate that sometimes forms red tides (Powers from cytological, epifluorescence microscopy and flow 1932, Taylor et al. 1971, Lindholm 1985). The nutri- cytometry. The pre-capture behavior and feeding pro- tional mode of M. rubrum has been under debate for a cess of M. rubrum on cryptophytes, however, have not long time, and it is listed as an obligate phototroph yet been documented. (Sieburth et al. 1978) and/or a species with the poten- To investigate the feeding process of Mesodinium tial for heterotrophy (Smith & Barber 1979). The inclu- rubrum grazing on cryptophytes, we established a sion of functional cryptophyte plastids (Barber et culture of M. rubrum from coastal waters of Korea and al. 1969, Hibberd 1977) as ‘incomplete symbionts’ followed the capture and ingestion of prey using a (Oakley & Taylor 1978) has also led to the speculation video system. We also measured ingestion rates of M. that M. rubrum is a potential mixotrophic ciliate (Lind- rubrum feeding on cryptophytes as a function of prey holm & Mörk 1989). Ingestion of cryptophyte cells by concentration. *Email: [email protected] © Inter-Research 2004 · www.int-res.com 166 Aquat Microb Ecol 36: 165–170, 2004 The results of the present study provide a basis for Feeding processes. To record the feeding process of understanding the feeding mechanism of Mesodinium Mesodinium rubrum on cryptophytes, actively swim- rubrum grazing on cryptophytes and the impact of ming M. rubrum cells that had been starved (i.e. grown such grazing on prey populations. photosynthetically) for 2 wk were added to 6-well tissue culture plates containing prey. Images of feed- ing M. rubrum were taken continuously over the fol- MATERIALS AND METHODS lowing 10 h using a Nikon camera and a video camera system (Watec 202B video camera) mounted on an Clonal culture of a cryptophyte. The clonal culture Olympus compound microscope at magnifications of of an unidentified cryptophyte (CR-MAL01) was estab- 400× (for the Nikon camera) and 200× (for the video lished by isolating single cells from water samples col- camera). lected at Gomso Bay, Korea (35° 40’ N, 126° 40’ E) in Ingestion and growth rates. This experiment was February 2002. The cryptophyte culture was grown designed to measure growth and ingestion rates of at 15°C in enriched f/2 seawater media (Guillard & Mesodinium rubrum, as a function of the prey concen- Ryther 1962) minus silicate under continuous illumina- tration, when feeding on cryptophytes of 5 different tion of 25 µE m–2 s–1 provided by cool-white fluorescent concentrations (0, 75, 1500, 7500, 15 000 cells ml–1). lamps. For feeding experiments, we used cultures in Photosynthetically grown, 2 wk old cultures of M. exponential growth phase. rubrum were diluted with f/2 media in 500 ml PC bot- Isolation and culturing of Mesodinium rubrum. tles to obtain experimental cultures at 1500 cells ml–1. Individual M. rubrum cells were isolated from natural The abundances of M. rubrum and prey were deter- assemblages of Gomso Bay during May 2001, when mined by enumerating cells in three 1 ml Sedgwick- water temperature and salinity were approximately Rafter counting chambers (SRCs). 18°C and 31.5 psu, respectively. Each of the isolated M. Initial concentrations of cryptophytes were estab- rubrum cells was washed by serially transferring into 3 lished using an autopipette by delivering predeter- droplets of f/2 medium and then put into 2 ml of f/2 mined volumes of cryptophyte culture (CR-MAL01) to medium with 5 prey cells held in a 3 ml well of a the bottles. Duplicate 500 ml PC experimental bottles 24-well tissue culture plate. The culture plates were (mixtures of predator and prey) and duplicate control incubated at 15°C under continuous illumination of bottles (prey only) were set up at each predator-prey 60 µE m–2 s–1 provided by cool white fluorescent light. combination. Duplicate control bottles containing only Every 5 to 6 d, cryptophyte prey (CR-MAL01) were Mesodinium rubrum were also established at 1 preda- added to each 3 ml well containing M. rubrum, with tor concentration. All the experimental bottles were the density of cryptophytes adjusted to be 5 times that kept under continuous illumination of 60 µE m–2 s–1 of of the M. rubrum cells. Once dense cultures of M. cool white fluorescent and culture-room temperature rubrum were obtained, they were transferred to 500 ml of 15°C. To determine predator and prey densities at polycarbonate (PC) bottles containing f/2 medium. the same time of day (i.e. 16:00 h) from the beginning Subsequent transfers were made at intervals of 5 to 6 d to the end of the 10 d long experiment, a 5 ml aliquot by placing one-third of a culture in a 500 ml PC bottle was removed from each bottle and fixed with 5% into a new PC bottle containing f/2 medium and cryp- Lugol’s solution, and all or >200 M. rubrum and prey tophytes adjusted to be 5 times that of the M. rubrum cells in three 1 ml SRCs were enumerated. cells. Experiments were conducted when a large The specific growth rate of Mesodinium rubrum (µ,d–1) volume of M. rubrum culture (MR-MAL01) became was calculated by averaging the instantaneous growth available. rates (IGR) for each sampling interval, calculated as: Cell dimension and volume. Mean size, surface area ln(SS͞ ) and volume of the prey cryptophyte and Mesodinium IGR = tt21× 24 (1) tt− rubrum cells (Table 1) were obtained with an elec- 21 tronic particle counter (Coulter Multisizer II, Coulter where t2 – t1 = 24 h and St1 and St2 = the concentration Corporation). of M. rubrum at consecutive samplings. The final t2 for Table 1. Prey cryptophyte species and ciliate Mesodinium rubrum used in the present study. Mean and standard error (SE) of equivalent spherical diameter (ESD) and cell volume were measured by the Coulter Multisizer II electronic particle counter Species Strain no. ESD (µm) Cell volume (µm3) Sampling area (and date) Unidentified cryptophyte CR-MAL01 5.3 ± 0.0047 76.1 ± 0.22 Gomso Bay (Feb 8, 2002) Mesodinium rubrum MR-MAL01 22.0 ± 0.040 5996 ± 30.0 Gomso Bay (May 31, 2001) Yih et al.: Ingestion of cryptophyte cells by Mesodinium rubrum 167 calculation was 72 h, which provided the highest specific growth rate. AB Data for Mesodinium rubrum growth rate (GR) were fitted to a Michaelis-Menten equation: µ (’)xx− µ = max (2) Kxx+−(’) GR –1 where µmax = the maximum GR (d ); x = prey concen- tration (cells ml–1); x’ = threshold prey concentration (the prey concentration where µ = 0); and KGR = the 1 µ CD prey concentration sustaining ⁄2 max. Data were itera- tively fitted to the model using DeltaGraph® (Delta Point). Ingestion rate (IR) was calculated using the equa- tions of Frost (1972) and Heinbokel (1978). Incubation time for calculating ingestion rate was the same as for estimating growth rate. Ingestion rate data were fitted to a Michaelis-Menten equation: EF Ix() IR = max (3) Kx+ () IR –1 –1 where Imax = the maximum IR (cells Mesodinium d ); –1 x = prey concentration (cells ml ), and KIR = the prey 1 concentration sustaining ⁄2 Imax.