Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral Mary Hagedorn1,2*, Virginia Carter1,2, Kelly Martorana1,2, Malia K. Paresa1,2, Jason Acker3, Iliana B. Baums4, Eric Borneman5, Michael Brittsan6, Michael Byers7, Michael Henley8, Michael Laterveer9, Jo-Ann Leong 2 , Megan McCarthy10, Stuart Meyers10, Brian D. Nelson11, Dirk Petersen9, Terrence Tiersch12, Rafael Cuevas Uribe12, Erik Woods7, David Wildt1 1 Department of Reproductive Sciences, Smithsonian Conservation Biology Institute, Front Royal, Virginia, United States of America, 2 Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, Hawaii, United States of America, 3 Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta Canada, 4 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 5 Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America, 6 Columbus Zoo and Aquarium, Powell, Ohio, United States of America, 7 General BioTechnology, Indianapolis, Indiana, United States of America, 8 Invertebrate Exhibit, Smithsonian National Zoological Park, Washington, District of Colombia, United States of America, 9 Rotterdam Zoo, Rotterdam, The Netherlands, 10 Department of Anatomy, Physiology and Cell Biology, University of California Davis, Davis, California, United States of America, 11 Fishes Department, New England Aquarium, Central Wharf, Boston, Massachusetts, United States of America, 12 Aquaculture Research Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, United States of America Abstract Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (.90% in A. palmata; .84% in F. scutaria; P.0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30uC/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (,5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had,50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (2196uC) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems. Citation: Hagedorn M, Carter V, Martorana K, Paresa MK, Acker J, et al. (2012) Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral. PLoS ONE 7(3): e33354. doi:10.1371/journal.pone.0033354 Editor: Sebastian C. A. Ferse, Leibniz Center for Tropical Marine Ecology, Germany Received October 5, 2011; Accepted February 10, 2012; Published March 8, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by: National Oceanic and Atmospheric Administration (grant # NA07NMF4630109 to MH; grant # NA08NMF4630462 to IB); National Science Foundation (OCE – 0825979 to IB); Morris Animal Foundation (grant # D07ZO-053 to MH); Columbus Zoo and Aquarium Conservation Fund (to MH); Rotterdam Zoo, Green Foundation, Clyde and Connie Woodburn Foundation (to SECORE); Smithsonian Institution, Louisiana State University Agricultural Center, University of California Davis and Hawaii Institute of Marine Biology and the Canadian Natural Sciences and Engineering Research Council (NSERC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Mike Brittsan is employed by Columbus Zoo and Aquarium as a Curator, Erik Woods and Michael Byers are scientists employed by General BioTechnology, Micheal Laterveer and Dirk Petersen are Senior Aquarists employed by the Rotterdam Zoo, Brian Nelson is a Senior Aquarist employed by the Fishes Department, New England Aquarium are commercial institutions, however the Mary Hagedorn and David Wildt are Senior Scientists, Michael Henley is an animal keeper employed by the Smithsonian National Zoological Park part of the Smithsonian Institution. The Smithsonian is a Trust Instrumentality which is part federal, part Trust and a 5013C. This work was funded through support from: National Oceanic and Atmospheric Administration (grant # NA07NMF4630109 to MH; grant # NA08NMF4630462 to IB); National Science Foundation (OCE – 0825979 to IB); Morris Animal Foundation (grant # D07ZO-053 to MH); Columbus Zoo and Aquarium Conservation Fund (to MH); Rotterdam Zoo, Green Foundation, Clyde and Connie Woodburn Foundation (to SECORE); Smithsonian Institution, Louisiana State University Agricultural Center, University of California Davis and Hawaii Institute of Marine Biology and the Canadian Natural Sciences and Engineering Research Council (NSERC). Nevertheless, this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: hagedornm@si.edu Introduction Coral reefs are living, complex ecosystems. While occupying a global spatial footprint about the size of Bolivia, coral reefs still have high diversity (.800 species recognized in the stony corals) [1] and provide invaluable ecosystem services to the planet – as nurseries for marine fish and invertebrates, natural storm barriers for coastlines and for food and pharmaceuticals used by humans. As a group, corals are evolutionarily ancient [2], but recently coral reefs have been experiencing unprecedented degradations. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33354