Vol. 66: 63–75, 2012 AQUATIC MICROBIAL ECOLOGY Published online March 14 doi: 10.3354/ame01559 Aquat Microb Ecol Direct evidence for symbiont sequestration in the marine red tide ciliate Mesodinium rubrum P. J. Hansen1,*, M. Moldrup1, W. Tarangkoon1,2, L. Garcia-Cuetos3, Ø. Moestrup3 1Marine Biological Laboratory, Department of Biology, University of Copenhagen, Strandpromenaden 5, Helsingør 3000, Denmark 2Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya, Trang 92150, Thailand 3Phycology Laboratory, Department of Biology, University of Copenhagen, Øster Farimagsgade 2D, Copenhagen 1353, Denmark ABSTRACT: The red tide ciliate Mesodinium rubrum (= Myrionecta rubra) is known to contain symbionts of cryptophyte origin. Molecular data have shown that the symbiont is closely related or similar to free-living species of the Teleaulax/Plagioselmis/Geminigera clade. This suggests that the symbiont of M. rubrum is either a temporary symbiont or a quite recently established symbiont. Here, we present data from a number of experiments in which we offered M. rubrum a phototrophic dinoflagellate and 8 different cryptophyte species belonging to 5 different clades. Mesodinium rubrum was only able to grow when fed the 2 cryptophyte species belonging to the genus Teleaulax, T. acuta and T. amphioxeia. Using the nucleomorph large subunit rDNA gene as marker, we were able to discriminate the 2 Teleaulax species, allowing monitoring of the exchange of the symbionts in M. rubrum. Over a period of 35 d, M. rubrum was able to exchange its symbionts from T. amphioxeia symbionts to T. acuta symbionts. This research suggests that M. rubrum can only utilize prey within the Teleaulax/Plagioselmis/Geminigera clade for sustained high growth rates and provides the first time-frame of endosymbiont replacement by M. rubrum. KEY WORDS: Mesodinium rubrum · Symbionts · Ingestion · Cryptophytes Resale or republication not permitted without written consent of the publisher INTRODUCTION tained in culture have been fed cryptophytes belong- ing to the genus Geminigera for the cold-water strain Mesodinium rubrum is a phototrophic ciliate that and Teleaulax for the all the temperate strains. Both hosts cryptophyte symbionts consisting of chloro- genera are closely related and belong to the same plasts, mitochondria, nucleomorphs, cytoplasm and a clade, the Teleaulax/Plagioselmis/Geminigera (TPG) so-called ‘symbiont nucleus’ (Taylor et al. 1971, Hib- clade (Hoef-Emden 2008). Very little is known about berd 1977, Hansen & Fenchel 2006). The ciliate is well the growth of M. rubrum on cryptophytes outside of known for its ability to form dense red tides worldwide the TPG clade. Recently, 2 Rhodomonas strains, CR- (e.g. Taylor et al. 1971, Lindholm 1985), and it has MAL03 and CR-MAL06, have been tested as food for recently been identified as prey for the diarrhetic the Korean strain of M. rubrum, resulting in a very shellfish poisoning (DSP)-causing dinoflagellates low M. rubrum growth rate only on CR-MAL03 (Park Dino physis spp. (e.g. Park et al. 2006, Nishitani et al. et al. 2007, Myung et al. 2011). However, M. rubrum 2008a,b, Riisgaard & Hansen 2009), creating a re- obtains most of its carbon requirements through pho- newal of interest within the scientific community. tosynthesis (Smith & Hansen 2007). It only needs to Mesodinium rubrum is an obligate mixotroph ingest 1 cryptophyte cell (Teleaulax sp.) per day to requiring both food uptake and light for sustained grow at its maximum growth rate (~0.5 d−1; Smith & growth. Currently, all isolates of M. rubrum main- Hansen 2007). In terms of ingested carbon, the food *Email: [email protected] © Inter-Research 2012 · www.int-res.com 64 Aquat Microb Ecol 66: 63–75, 2012 uptake only accounts for 1 to 2% of the carbon strains belonging to different clades as well as the requirements per day. Furthermore, M. rubrum is possible occurrence of symbiont turnover. The aim of able to divide its chloroplasts when starved, and its the present investigation was to study the growth symbionts can photoacclimate (Hansen & Fenchel responses of the Danish isolate of M. rubrum when 2006, Moeller et al. 2011), indicating some genetic offered 8 different species of cryptophytes belonging control of the symbionts. Because M. rubrum meets to 5 different clades, in addition to one dinoflagellate most of its carbon requirements by photosynthesis, species. The possible exchange of symbionts was the question arises why M. rubrum has to eat to sus- examined using transmission electron microscopy tain growth. One explanation could be that M. (TEM) and molecular techniques. rubrum harbors permanent symbionts but needs to ingest prey to obtain some essential micronutrients. Another explanation could be a need for symbiont MATERIALS AND METHODS renewal when the symbionts have undergone a cer- tain number of divisions within the host. This hypo - Algal cultures thesis implies that the chloroplasts have to be acquired at regular intervals for sustained growth. A total of 8 species of cryptophytes and 1 species of Previous molecular analyses of the nucleomorph and dinoflagellate were used as food (Table 1). The selec- chloroplast genes of the chloroplasts of Mesodinium tion represents a broad range of possible prey found rubrum and its prey have revealed identical se- in Mesodinium rubrum’s natural environment. All quences for these 2 organisms in the 3 cultured cultures were grown on a glass table in h/20 medium strains of M. rubrum (Johnson et al. 2007, Park et al. (Guillard 1975) at a salinity of 30 and a temperature 2007, Garcia-Cuetos et al. 2010), tending to agree of 20 ± 1°C. Cool white light (100 µmol photons with the latter hypothesis. m−2 s−1) was provided from beneath in a light:dark In 2010, Park et al. (2010) studied possible renewal cycle of 14:10 h. of symbionts using 2 strains belonging to the TPG clade (CR-MAL01=Teleaulax amphioxeia and CR- MAL11 =Teleaulax acuta; Park et al. 2010). A Meso- Cultures of the ciliate Mesodinium rubrum dinium rubrum culture fed CR-MAL11 for 5 mo still contained chloroplast markers from CR-MAL01 (see A culture of Mesodinium rubrum was established Fig. 4 in Park et al. 2010). The aim of their study was from single cells isolated from surface seawater sam- the replacement of the chloroplasts in Dinophysis ples collected in Frederikssund, Denmark, during a caudata, and the results obtained for M. rubrum were bloom event on April 17 2007, as described in Riis- not discussed in detail. Some ambiguity, such as the gaard & Hansen (2009). Prior to the experiment, the extent of plastid renewal, was left unanswered. In a cultures, normally grown in f/2 medium (Guillard recent study, Myung et al. (2011) tried to study possi- 1975), were transferred to h/20 medium and accli- ble renewal of the plastids by feeding M. rubrum with mated for a couple of months. The change of medium a strain belonging to the Rhodomonas clade. They fed was carried out to respond to the needs of certain cryptophyte CR-MAL03 to a 2 mo starved M. rubrum cryptophytes species, such as Hanusia phi and Guil- culture for 2 d and found chloroplast markers of both lardia theta. the original prey item (CR-MAL01) and the subsequent prey item (CR- Table 1. Protist strains used as prey in the experiments. nmSSU (nSSU) rDNA: MAL03) over a period of 37 d after the nucleomorph (nuclear) small subunit ribosomal DNA. na: not available beginning of the experiment. The presence of both genetic markers Species Culture collection GenBank accession no. makes it difficult to conclude whether ID no. nmSSU rDNA nSSU rDNA there was full symbiont replacement Heterocapsa rotundata K-0483 (SCCAP) na na and to what extent the chloroplasts in- Chroomonas vectensis CCMP-432 na HM126534 herited from CR-MAL03 were photo- Guillardia theta CCMP-2712 AF083031 X57162 Hanusia phi CCMP-325 GQ396278 U53126 synthetically active. Hemiselmis tepida CCMP-442 GQ396279 HM126533 To answer these questions, more Proteomonas sulcata CCMP-321 na HM126536 information is needed concerning the Rhodomonas salina K-1487 (SCCAP) na HM126532 growth performances of Mesodinium Teleaulax amphioxeia K-0434 (SCCAP) GQ396273 AJ421146 Teleaulax acuta K-1486 (SCCAP) HQ651177 HM126531 rubrum on a variety of cryptophyte Hansen et al.: Symbionts in Mesodinium rubrum 65 Growth and survival responses of unfed M. rubrum and monocultures of the given prey Mesodinium rubrum species. All experiments were carried out in triplicate. In a second set of experiments, the cryptophyte The 8 cryptophyte species and 1 dinoflagellate spe- Proteomonas sulcata and the dinoflagellate Hetero- cies were tested as prey for Mesodinium rubrum capsa rotundata were used as prey. In this set of (Table 1). Initial attempts to culture Mesodinium experiments, Teleaulax amphioxeia was used a ‘con- rubrum on all 9 prey species, offered separately in trol food’ to demonstrate the potential performance multi-well dishes, revealed that long-term growth of the Mesodinium rubrum isolate. was only sustained with cryptophytes belonging to Finally, in a third set of experiments, the growth the genus Teleaulax, specifically T. acuta and T. responses of Mesodinium rubrum were studied when amphioxeia. offered 5 additional species of cryptophytes: Chroo - To explore the relationship between Mesodinium monas vectensis, Rhodomonas salina, Hanusia phi, rubrum and its prey, growth experiments were per- Guillardia theta and Hemiselmis tepida. The setup of formed using all prey types. The experiments were the experiment was the same as in the 2 previous sets carried out using 65 ml tissue culture bottles filled to of experiments, except that all the mixed cultures capacity. Samples (2 ml) were drawn at Days 4, 8, 12 were diluted on Day 4 to diminish effects of elevated (or 10) and in one case 16, and bottles were refilled to pH on the outcome of the experiments. capacity with fresh h/20 medium.