Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium) q M. Hagedorn a,b, * , V.L. Carter a,b , J.C. Leong b , F.W. Kleinhans c a Department of Reproductive Sciences, Smithsonian National Zoological Park, Washington, DC 20008, USA b Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744, USA c Department of Physics, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA article info Article history: Received 6 August 2009 Accepted 14 October 2009 Available online 24 October 2009 Keywords: Coral Symbiodinium Algae Zooxanthellae Symbionts Water and cryoprotectant permeability Deuterium Pulse Amplitude Fluorometer abstract Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbi- odinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endo- symbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryo- preservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers world- wide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stres- ses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotec- tants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D 2 O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similari- ties in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, sug- gesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appen- dix describes our H 2 O:D 2 O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability. Published by Elsevier Inc. Introduction Coral reefs are some of the oldest and most diverse ecosystems on our planet. They are one of the ocean’s main nurseries and feed- ing grounds for fish and invertebrates, provide natural storm barriers for coastlines, and are a potential source for novel pharma- ceuticals. Throughout their range, coral reefs are dying due to hu- man influences. Even in the most remote marine bioreserves, such as the northwestern Hawaiian Islands [33], human activities are damaging fragile coral ecosystems [4]. As greenhouse gasses in- crease, atmospheric and sea-surface temperatures are also expected to increase [15,14] coupled with anthropogenic stresses, reefs will remain in crisis, threatening their existence worldwide [22,16,23]. In situ conservation practices, such as habitat preservation, are an important way to conserve coral reefs. However, reefs now face global rather than just local threats. Therefore it is critical that ex situ conservation practices are incorporated into conservation solu- tions for coral reefs. Novel ex situ conservation techniques, such as genetic banks using frozen samples, hold strong promise for rapid improvements in preserving species and genetic diversity within ecosystems. These frozen banks reflect a new and major type of preservation that can be added to conventional archives, but in this case, the living biomaterials go beyond dried materials to include gametes, embryos, somatic and stem cells, blood, and DNA. The cryo-physiology of coral larvae is fairly well-known and coral sperm has been successfully cryopreserved [18,19]. Three genome repositories worldwide now hold endangered cryopre- served coral sperm from the endangered coral, Acropora palmata (Hagedorn et al., unpublished data). Our long-term goal is to create a genetic bank for all types of coral cells and their endosymbiotic cells, such as zooxanthellae. Genome repositories have important passive and active functions. First, genetic material can remain 0011-2240/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.cryobiol.2009.10.005 q Statement of funding: We would like to thank Dr. Ruth Gates, and her entire laboratory, especially Jacqueline L. Padilla-Gamino, for their kind assistance and loan of the PAM during these experiments. Dr. Michael Stat kindly offered invaluable comments on this paper. This work was supported by the financial assistance of Mrs. Nancy Upp Potter and the Morris Animal Foundation to M.H. This paper has an HIMB contribution #1369. * Corresponding author. Address: Smithsonian Institution and Hawaii Institute of Marine Biology, P.O. Box 1346, Kaneohe, HI 96744, USA. Fax: +1 808 236 7444. E-mail address: hagedornm@si.edu (M. Hagedorn). Cryobiology 60 (2010) 147–158 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo