Preserving and Banking Coral Figure 7. Embryonic F. scutaria cells after cryopreservation (all cells exposed to freezing in these treatments). Mean post-thaw viability of F. scutaria cells was ,50% for all cryoprotectants tested. Ten thousand events were measured for each sample. Controls (the three left bars) were live-stained and unstained cells and 100% dead cells that produced control data for the flow cytometer (i.e., 100% intact versus 100% dead). Bars with the same letters were not different, whereas bars with different letters were different (P,0.05; Kruskal-Wallis test). doi:10.1371/journal.pone.0033354.g007 difference also had practical consequences, specifically limiting the volume of supplemented cryoprotectant (and sperm) to the fertilization vials (to 50 ml). Although this reduced the chance of toxicity, it also meant that the initial sperm concentration had to be very high (108 to 109 cells/ml) to eventually achieve our fertilization target of 106 sperm/ml per vial. While never becoming a significant factor for coral that produce sperm/egg bundles (e.g., A. palmata), this could limit success for species that release sperm into the water (e.g., F. scutaria). For example, we experienced nights when insufficient amounts of concentrated sperm could be recovered to initiate IVF. One of our most interesting observations was the night-to-night variability in fertilization success using either fresh or frozen- thawed spermatozoa. Additionally, embryonic cells from F. scutaria clearly tolerated cryopreservation better when collected on certain nights than others. However, this variation observed in sperm occurred randomly and was unlinked to any specific night of gamete release and collection. Rather, we suspect that this night- to-night variability indicated that spawned coral spermatozoa (and perhaps eggs) develop fitness over time with the most viable cells being produced nearer to the middle of the spawning period. Alternatively, it may have been possible that the kinetics of spawning were influenced by variations in genotype of our sampled donors. The night-of-sperm collection had a significant impact on cryosensitivity. From a practical perspective and until more data are generated, it would appear prudent to collect and preserve biomaterials on every night of availability and then test sub- aliquots for viability before culling frozen samples producing poor fertilization results. While the night-of-collection must be consid- ered in the large-scale banking of coral spermatozoa, clearly there is subpopulation of these gametes that is resilient to low temperature exposure and able to survive freezing as demonstrat- ed by viable embryo production. Night-of -collection also influenced sperm motility, which was an issue for A. palmata. Ideally, coral sperm samples used for IVF have motility ratings .90%, but some individual colonies of this species had ,25% motility values on certain recovery nights. For example, it was our experience that not all A. palmata colonies within a given population spawned on the same night or even all in an individual colony. However, over time there was one or two nights when the majority of the colonies spawned, which coincided with the production of those sperm best able to survive the rigors of cryopreservation. By contrast, F. scutaria, which spawned over multiple nights throughout the 4 mo summer, consistently produced sperm with both high motility (.90%) and IVF success (.75%). However, after these same sperm were cryopreserved, the incidence of fertilization in culture was highest during July and August and negligible in June or September (data not shown). Therefore, based on observations of dynamic differences in IVF success after imposing a freeze-thaw stress, our findings demon- strated that coral sperm underwent some sort of functional metamorphosis during the overall spawning interval that appeared to enhance fitness and perhaps resilience to stressors. Here we also reported the first cryopreservation and banking of dissociated coral (F. scutaria) embryonic cells. These cells have relevance for ensuring or restoring reef health, for example, testing for and then remediating coral diseases, most of which have gone largely unstudied [13]. To-date, a major obstacle to investigating infectious pathogens within the stony coral has been the inability to maintain primary cultures of differentiated coral tissue for more than a few months [59–63]. Frozen embryonic cell lines would offer an inexhaustible resource for large-scale research opportu- nities, including allowing long-distance transport of specimens to facilitate basic and applied studies. In the long-term future, banked embryonic cells could serve as a resource for stem cells to grow new corals that could be reintroduced into native ecosystems to help sustain or even increase gene diversity. We envision banked coral sperm eventually being used to help diversify shrinking populations. For example, analyses of A. palmata have revealed two genetically-isolated regions within the Carib- bean, one in the eastern and one in the western Caribbean with Puerto Rico being a mixed, ‘transition’ zone [64]. Some reef tracts in the west, especially coral stands in the Florida Keys, have little genotypic diversity, with most reefs harboring only one genet (clone) [20]. These genets produce sperm-egg bundles annually, but with no mechanism for self-fertilization and because the distance is too great to neighboring reefs with unrelated genets, reproduction fails to occur [21]. Therefore, these genets living in isolated reef ecosystems likely contribute little to species evolu- PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33354