Sequestered plastids in Mesodinium rubrum are functionally active up to 80 days of phototrophic growth without cryptomonad prey Geumog Myung a , Hyung S. Kim b , Jong Woo Park c , Jong Soo Park d , Wonho Yih a, a Department of Oceanography, Kunsan National University, San 68, Miryong-dong, Gunsan 573-701, Republic of Korea b Department of Marine Biotechnology, Kunsan National University, San 68, Miryong-dong, Gunsan 573-701, Republic of Korea c Fishery and Ocean Information Division, National Fisheries Research and Development Institute, Busan 619-705, Republic of Korea d Department of Oceanography, Kyungpook National University, Sangju 742-711, Republic of Korea 1. Introduction Mesodinium rubrum Lohmann, 1908 is a mixotrophic marine photosynthetic ciliate (Crawford, 1989; Ryther, 1967; Sieburth et al., 1978), and frequently causes non-toxic red-tide in estuarine and neritic pelagic environments (Lindholm, 1985; Taylor et al., 1971; Hart, 1934). M. rubrum requires repeated ingestion of cryptomonad preys (Yih et al., 2004; Gustafson et al., 2000) through the cytopharynx (Nam et al., 2012) mainly for the renewing retention of photosynthetic apparatus (Hansen et al., 2012; Johnson et al., 2006, 2007; Johnson and Stoecker, 2005). Sequestered plastids of M. rubrum are known to become functionally less efficient within a few weeks after the initial introduction into the ciliate (Myung et al., 2011; Stoecker et al., 2009; Gustafson et al., 2000). M. rubrum MR-MAL01 grown phototrophically for 2 months without cryptomonad prey, and then quickly resumed its fast growth when re-fed with crypto- monad strain CR-MAL01 (Myung et al., 2011). Maximum duration for a newly retained plastid to stay photosynthetically functional, however, has been rarely estimated or determined. Dimension of the maximum duration might be critical for a Mesodinium rubrum population to survive long-term starvation from its cryptomonad prey (Bielewicz et al., 2011; Laybourn-Parry, 2002). Months-long functioning of the new kleptoplastids in M. rubrum should lead us to better understand its overwintering success or long-term survival (Yih et al., 2013; Martin et al., 2007; Laybourn-Parry et al., 2000) under extreme environments even though the formation of cyst or resting stage in M. rubrum has never been known (Gibson et al., 1997; Lindholm, 1985). Here, we report on the dynamics of population density, orange fluorescence (Glazer et al., 1971) of the retained plastids (Parsons and Blackbourn, 1968), and DCMU photosynthetic capacity (Furuya and William, 1992) of phototrophically growing Harmful Algae 27 (2013) 82–87 A R T I C L E I N F O Article history: Received 22 January 2013 Received in revised form 2 May 2013 Accepted 8 May 2013 Keywords: Mesodinium rubrum Phototrophic growth Photosynthetic capacity PCR product Plastid genes A B S T R A C T The red tide ciliate Mesodinium rubrum is an obligate mixotroph which requires feeding on cryptomonad prey mainly to retain its photosynthetic apparatus. Functionality of the sequestered plastids has been known to be lowered within a few weeks. The upper limit of the functionally active duration for the newly retained plastid, however, has been rarely estimated or determined. In parallel with genetic analysis, we investigated dynamics of population density, orange fluorescence of the plastids, and DCMU ((3-(3,4- dichlorophenyl)-1,1-dimethylurea) photosynthetic capacity of phototrophically growing M. rubrum (strain MR-MAL01) for 100 days. M. rubrum populations continued their phototrophic growth for the first 6 weeks, with gradually decreasing growth rates. Rapid decline of population density began from the 8th week. The photosynthetic capacity remained quite stable, ranging from 0.7 during the 1st week down to 0.5 during the 11th week. On day 87, the photosynthetic capacity steeply decreased to 0.05. The orange fluorescence of the retained plastids became very weak during the 4th week, to be almost undetectable on day 98. Only plastid 16S rRNA gene kept strong band intensity of PCR products throughout the whole period of 100 day experiment. Interestingly, the band intensities from psaA and psbA genes all become dramatically weakened after day 77. After new prey cryptomonads (strain CR-MAL03) were offered to M. rubrum starved for 80 days, ‘CR-MAL03 type’ 1192-bp PCR product of plastid 16S rRNA gene was detected in most experimental single M. rubrum cells. Here, we demonstrate that M. rubrum can grow for 6 weeks in the absence of cryptomonad prey, and photosynthetic capacity of M. rubrum can be maintained active for 11 weeks without prey. Additionally, M. rubrum starved for 80 days was shown to be physiologically healthy enough to ingest cryptomonad preys and retain new plastids. ß 2013 Elsevier B.V. All rights reserved.  Corresponding author. Tel.: +82 63 461 4601; fax: +82 63 469 4911. E-mail address: ywonho@kunsan.ac.kr (W. Yih). Contents lists available at SciVerse ScienceDirect Harmful Algae jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/hal 1568-9883/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2013.05.001