Cryopreservation of the dinoflagellate symbiont of the octocoral Pseudopterogorgia elisabethae
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In this paper we describe a cryopreservation protocol followed by the culture of Symbiodinium sp. isolated from the Caribbean gorgonian Pseudopterogorgia elisabethae as a potential renewable source of the dinoflagellate symbiont. Four different freezing protocols were designed: a controlled cooling device designed to cool at 1°C/min, a three-step protocol (−20°C for 2 h, −70°C for 2 h, liquid nitrogen-LN2), a two-step protocol (−70°C for 2 h, LN2), and a one-step protocol (LN2). All cells were stored in LN2 after cryopreservation. The cryoprotective agents (CPA) used were ethanol (EtOH) and methanol (MeOH) at 10 and 20%, and seawater (FSW) was used as a control. Viability measurements using cell counts showed that all cryopreservation protocols were relatively successful, and no trends were observed regarding freezing protocol or CPA used. After 19 weeks in culture the viability of samples which had high biomass was determined by the fluorescent assay CellTiter Blue™. The most viable cultures were those cryopreserved by a two-step protocol using 20% MeOH or 20% EtOH as a CPA. A genetic examination of the DNA of these samples using Symbiodinium-specific PCR primers confirmed that the composition of the culture had not changed. For the first time, we report that Symbiodinium sp. isolated from a gorgonian can be cryopreserved and subsequently cultured successfully.
KeywordsMeOH Microalgae EtOH Dinoflagellate Restriction Fragment Length Polymorphism Pattern
We are grateful to Dr. L. K. Ranzer for her assistance with cell isolation and the cryopreservation of Symbiodinium cells aboard the R/V Suncoaster. We would like to acknowledge funding from the Center of Excellence in Biomedical and Marine Biotechnology, the National Science Foundation grant MCB-0119011, the Florida Sea grant number R/LP-MB-14, the Florida Institute of Oceanography for time on the R/V Suncoaster and the Canada Research Chair program. This material is based upon work supported by the National Science Foundation under a grant awarded in 2003 to Lory Z. Santiago-Vázquez (award #0310283). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation. We acknowledge the government of the Bahamas for issuing a scientific collecting permit and allowing us to work in their territorial waters. The experiments comply with the current laws of the countries where the experiments were performed.
- Anderson RA (1999) National Center for Culture of Marine Phytoplankton’s (CCMP) role in isolating and maintaining cultures of Pfiesteria and Pfiesteria-like organisms and the procedure for cryopreserving PLO species. In: Litaker W, Scholin C, Vasta GR (eds) Molecular approaches for the identification and environmental detection of Pfiesteria piscicida and Pfiesteria-like dinoflagellates. http://www.whoi.edu/redtide/pfiesteria/molecular/Molecular_Workshop_Report.pdf:, pp 14–16
- Bodas K, Brennig C, Diller KR, Brand J (1995) Cryopreservation of blue-green and eukaryotic algae in the culture collection at the University of Texas at Austin. Cryo Lett 16:267–274Google Scholar
- Kono S, Kuwano K, Saga N (1998) Cryopreservation of Eisenia bicyclis (Laminaria, Phaeophyta) in liquid nitrogen. J Mar Biotecnol 6:220–223Google Scholar
- Morris GJ (1981) Cryopreservation. An introduction to cryopreservation in culture collections. Institute of Terrestrial Ecology. Culture Center for Algae and Protozoa, CambridgeGoogle Scholar
- Moya CE (2004) Tetrahymena thermophila used as a pharmacological model to study the cellular mechanism of action of Pseudopterosin A. Ph.D. thesis, University of California, Santa BarbaraGoogle Scholar
- Newberger NC (2006) Bioactive terpene production associated with Caribbean gorgonians from the genera Pseudopterogorgia and Eunicea: discovery of a sustainable production method. Ph.D. thesis, Florida Atlantic University, Boca RatonGoogle Scholar
- Rhodes L, Smith J, Tervit R, Roberts R, Adamson J, Adams S, Decker M (2006) Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52:152–156CrossRefGoogle Scholar