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A Simple and Rapid Cryopreservation Technique for Ciliates: a Long-Term Storage Procedure Used for Marine Scuticociliates

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A Simple and Rapid Cryopreservation Technique for Ciliates: a Long-Term Storage Procedure Used for Marine Scuticociliates Journal of Eukaryotic Microbiology ISSN 1066-5234 ORIGINAL ARTICLE A Simple and Rapid Cryopreservation Technique for Ciliates: A Long-Term Storage Procedure Used for Marine Scuticociliates Yongqiang Liua,b, Bei Nana,b, Lili Duana,b, Ting Chenga,b, William A. Bourlandc, Mingjian Liua & Yan Zhaod a Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China b Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China c Department of Biological Sciences, Boise State University, Boise, Idaho 83725-1515, USA d Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Keywords ABSTRACT Freezing; liquid nitrogen; methodology; protozoa; recovery efficiency. Pseudocohnilembus persalinus is a free-living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, Correspondence long-term laboratory maintenance for this species is mainly achieved by sub- M. Liu, Institute of Evolution & Marine culture that requires rigorous culture environments and, too often, cultures of Biodiversity, Ocean University of China, the organism die out for a variety of reasons. Successful transport of viable Qingdao 266003, China cultures also poses problems for researchers. This study describes a simple Telephone number: +86 532 82031676; and rapid protocol for long-term cryopreservation of P. persalinus. The effects FAX number: +86 532 82031676; of physiological states of individuals before freezing, the type and concentra- e-mail: [email protected] tion of cryoprotectant, and optimal temperatures for freezing and thawing and were assessed. A cryopreservation protocol, using a mixture of 30% glycerol Y. Zhao, Research Center for Eco-Environ- and 70% concentrated P. persalinus cell culture, incorporating rate-controlled mental Sciences, Chinese Academy of freezing at À80 °C before liquid nitrogen storage, maintained a high recovery Sciences, Beijing 100085, China efficiency after 8 wk of storage. These results suggest that broader application Telephone number: +86 10 62841639; of this protocol to build a cryopreserved marine protozoa culture bank for bio- FAX number: +86 10 62841639; logical studies may be possible. e-mail: [email protected] Received: 6 January 2019; revised 22 February 2019; accepted March 15, 2019. Early View publication 12 April, 2019 doi:10.1111/jeu.12730 CILIATES are ideal model organisms for biological research isolations of trophic cells into proteose peptone-based or and have already been used in various fields, such as genet- bacterized media such as wheatgrass medium, and incu- ics, ecology, epigenetics, developmental cellular biology, bation under controlled environments (Anderson et al. and ageing studies (Gao et al. 2016; Li et al. 2018; Wang 2009; Cassidy-Hanley 2012; Zheng et al. 2015). However, et al. 2017a,b; Yan et al. 2016; Zhao et al. 2018; Zheng the above-mentioned preservation methods are inconsis- et al. 2018). In recent years, the study of ciliate biodiversity, tently successful, often due to unwanted encystment of molecular phylogeny, and cell development has steadily many species at low food concentrations or under subopti- increased, highlighting the need for the improvement of cili- mal environmental conditions (Benca tova and Tirjakova ate preservation methods, especially cryopreservation 2018; Muller€ 2007; Tsutsumi et al. 2004). For instance, (Chen et al. 2017, 2018; Pan et al. 2017; Qu et al. 2018; Pseudocohnilembus persalinus can form cysts under con- Sheng et al. 2018; Zhang et al. 2018; Zhao et al. 2017). ditions of food deficiency, pH changes, low oxygen con- For most cultivatable ciliate species, to maintain use- centration, the accumulation of excretory products, able, stable populations generally involves repeated overcrowding, low temperatures, and dessication (Fenchel © 2019 International Society of Protistologists 836 Journal of Eukaryotic Microbiology 2019, 66, 836–848 Liu et al. Cryopreservation for a Marine Ciliate 1990; Tsutsumi et al. 2004). Many ciliate species are cap- micropipettes from raw cultures, washed 3 or 4 times, able of excystment, growth, and then re-encystment after and then transferred to Petri dishes with filtered, auto- being transferred to a fresh culture medium (Day et al. claved seawater at 25 °C. Rice grains were added to pro- 2007). However, for some species, problems with the mote the growth of bacteria, the food of the ciliates. long-term stability of the cysts held at “normal” environ- Living cells were observed, using bright field and differ- mental temperatures (5–20 °C) may decrease the percent- ential interference contrast microscopy at 100–1,0009 age of excystment over time (Muller€ et al. 2008). Thus, magnification. The ciliature and argyrome structures were there are still many practical difficulties in the preservation revealed, using the protargol, Chatton-Lwoff silver nitrate of cyst-forming ciliates due to low recovery efficiency. and the silver carbonate staining methods (Corliss 1953; Cryopreservation in liquid nitrogen seems to be an opti- Ma et al. 2003; Wilbert 1975). Measurements were made mal method for preservation of cyst-forming protozoa. It under 1,0009 magnification with a calibrated ocular has the potential to ensure long-term stability of these micrometer. organisms ex situ, and to maintain characteristic genetic traits since biological activities will be completely stopped Culture medium, mass cultivation, and induction of at the storage temperature (À196 °C). Thus, cryopreserva- encystment tion could be a useful alternative or complement to long- term culture maintenance for ciliate research (Ashwood- Escherichia coli (HST04 damÀ/dcmÀ Competent Cells, Smith and Farrant 1980; Mazur 1984). Previous studies of Code No. 9129; TaKaRa Co., Ltd., Beijing, China) cells protist cryopreservation have mainly focused on a broad were used as food for Pseudocohnilembus persalinus. diversity of rumen-parasitic and freshwater ciliates (Muller€ The E. coli were cultivated at 37 °C in a flask with Luria- et al. 2008). The low-temperature storage of Tetrahymena Bertani (LB) broth on a shaker at 200 rpm for 24 h and species is probably the most time-tested approach, and grown to an optical density of 2.0 at 600 nm (OD600). successful long-term storage of T. thermophila has been Then 25 ml of culture solution (OD600 = 2.0) was col- accomplished by this method (Cassidy-Hanley et al. 2010; lected and washed 2 or 3 times, using sterile filtered sea- Simon 1982). However, large-scale cultivation methods for water by centrifugation at 6,500 g for 5 min. Finally, 10 ml marine ciliate species are less dependable than for fresh- sterile filtered seawater was added to the bacterial sus- water species due to less well-defined living conditions pension which was stored at 4 °C for use as food for the (such as salinity), and the cryoprotectants that have been ciliates. used for freshwater species are sometimes harmful to Pseudocohnilembus persalinus cells were cultured at marine ciliates (Fuller 2004). 25 °C in a stationary incubator, and 10 ll of prepared bac- A practical method of cryopreservation and recovery for terial suspension was added every 6–8 h to 5 ml of the the marine ciliate Uronema marinum was reported by ciliate culture before freezing or after thawing. Encyst- Anderson et al. (2009), using a 2-ml system containing ment was induced by starving the P. persalinus cells 20% glycerol, 10% fetal bovine serum, and 70% U. mar- (2 days after initiation of culture, abundance about inum cells for cryopreservation. It allowed successful 4 9 104 cells/ml) in sterilized seawater for about 5 days. recovery of U. marinum. However, the recovery was time- consuming, required a high concentration of cells for cry- Immunofluorescence staining and imaging opreservation, and yielded a low survival abundance after recovery (Anderson et al. 2009; Cassidy-Hanley et al. Immuofluoresence (IF) staining was performed according 2010). to previous studies (Gao et al. 2013; Wang et al. 2017a,b). Based on previous studies, we developed a repeatable, The individuals of Pseudocohnilembus persalinus were effective, and user-friendly cryopreservation method for immunostained with an anti-acetylated a-tubulin (Lys40) the marine scuticociliate Pseudocohnilembus persalinus monoclonal antibody (Catalog: 32-2700, 1:500; Thermo that allowed recovery of viable cells after storage in liquid Scientific, Invitrogen, Shanghai, China) to label the cilia. nitrogen for up to 8 wk. The cells could be successfully Cells were incubated with the primary antibodies at 4 °C recovered with high efficiency using this protocol. The overnight and then were incubated in the secondary anti- results have enabled us to begin establishing cryobanks of body (Goat anti-Mouse Ig G1, Alexa Fluor 555, Catalog: A- free-living marine ciliates. 21127, 1:500; Thermo Scientific, Invitrogen, Maryland) at 25 °C for 1 h. Digital images were obtained by using a Motion Blitz EoSens Mini1 camera (Mikrotron, Unterschlei MATERIALS AND METHODS b heim, Germany) mounted on a Leica Sp8 confocal microscope with a 1009 oil immersion objective (numeri- Sampling, identification, and preliminary cultivation cal aperture is 1.40). Pseudocohnilembus persalinus was collected from surface seawater in Qingdao, northern China (36°03043″N; Swimming paths and relative swimming velocity 120°19012″E), on 3rd September 2016 at a water tempera- ture of 24 °C and a salinity
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