Biobanking: A Brave New World for Corals

by Rebecca Campbell Gibbel, MS, DVM

Saving coral biodiversity requires the contributions of many people with a variety of approaches. The political work of advocating for legislative protections and reducing the carbon emissions that lead to ocean heating is difficult but absolutely essential. Other strategies are used by teams of scientists who are racing to outplant young corals in a global effort to repopulate degraded reefs.  For hundreds of years, coral skeletons have been maintained in natural history museum collections to help researchers better understand coral species relationships and to document what exists in the present and in the past. DNA samples are now being used to aid phylogenetic studies, and just like any DNA testing, they sometimes reveal surprising relationships that can contradict assumptions. Biobanking- or methodically saving biological materials of organisms – is an old idea, but it is being modified for corals in exciting new ways. 

CORAL ARKS

Biologists are able to maintain large collections of live corals in aquaria that serve as “arks” to maintain species diversity. These land-based facilities control numerous environmental parameters in an attempt to replicate healthy sea conditions, though they may be far from any ocean. 

This concept has been adopted in both the Caribbean and the Pacific in response to different threats.  The Florida Reef Tract Rescue Project was initiated in 2016 to save coral colonies in advance of a novel and highly lethal coral disease that began spreading through Florida reefs in 2014. This disease, known as stony coral tissue loss disease (SCTLD) soon enveloped the entire Caribbean basin, driving at least one species to functional extinction within just one decade. The rescue project has collected 2283 apparently unaffected corals and disseminated them to certified public aquarium facilities throughout the US.  The hope is that these corals are a representative sample of the region’s stony corals that will persist and grow in captivity, protected from the myriad stressors that are degrading Caribbean reefs. Continuous observation data can be seen at: the Coral Rescue Monitoring Dashboard. 

Figure 1. An aquarium safeguarding samples of different species of stony corals

In the southern hemisphere, the Great Barrier Reef Legacy organization was developed in response to the many pressures impacting earth’s largest coral reef. These threats include the highly destructive crown-of-thorns starfish, Acanthaster planci, and increasing ocean temperatures which have been causing repeated mass coral bleaching and subsequent mortality. This group operates the “Forever Reef Project” which seeks to preserve as many coral species as possible, starting with Great Barrier Reef corals. The project maintains a repository of live fragments and genetic material that have been “rescued” from wild coral sources and kept in indoor holding facilities. 

The ultimate aim for both of these large projects is to create a gene bank of coral diversity. These corals are not intended to be restored to the ocean, but they will be carefully tended in hopes that selective breeding can be done to develop new genetic strains of young corals. By promoting reproduction, new coral larvae may be produced with a variety of traits that may be advantageous in a changing world.

Figure 2. The critically endangered Acropora cervicornis growing in the Florida Aquarium 

FLASH FREEZE!

And now we get to the radical-sounding  preservation approach of cryopreservation!  It seems futuristic, but it’s already here, having been steadily utilized in farm animal production for years.  The process of freezing genetic material in the form of cryopreserved sperm was developed in the early 1900s and is now in common usage for species like dairy cattle, swine, turkeys, and horses (9). Producers find it cheaper and more efficient to order tubes of frozen sperm when needed rather than moving male animals around and encouraging them to feel amorous on cue. Now coral scientists have modified the long-established techniques of freezing vertebrate gametes to cryopreserve coral sperm and even coral larvae. The goal for this work is to preserve biodiversity, not just to produce many new corals. 

Dr. Mary Hagedorn is a researcher whose laboratory at the Smithsonian Institute has cryopreserved sperm from over 30 species of coral.  It’s much harder to preserve eggs, with their high lipid content and poorly permeable membranes which resist absorption of cryoprotectant (aka antifreeze). These solutions must be added to the cells to prevent the formation of damaging ice crystals.  However, eggs of gorgonian soft corals have been successfully frozen using the method of vitrification which supercools cells to -127 °C in less than one second, preventing intracellular ice (11). Vitrified coral sperm and larvae can be preserved in this glass-like state indefinitely, which really does sound like science fiction! Freezing at day 2 of embryonic life results in more successful preservation, when the coral’s internal anatomy has started to develop, but is not yet overly complex. 

Figure 3. Days 1 to 5 of coral larval development, from Daly et al., 2018.

COMING BACK TO LIFE…

Successfully freezing samples is only the first step. It is particularly challenging to successfully reanimate frozen samples, again due to risks of damaging ice crystal formation. Recently a new technique of laser warming has been developed, which achieves an extraordinarily rapid thawing rate of 117,000°C/min, avoiding ice recrystallization (7).

Another useful technique involves evaluating coral sperm before cryopreservation. Scientists exposed spawned sperm samples to caffeine solutions to rapidly activate them and test their viability. The plot below shows the effects of caffeine on the motility of sperm post spawning. How did they think to try this? Maybe the discovery was made when a sleepy researcher spilled coffee in a sample? 

Figure 4. Caffeine best improves coral sperm motility, from Zuchowicz et al., 2021

It’s not enough to simply freeze and thaw coral larvae and sperm but they must be brought back to a functional state. Sperm need to successfully fertilize eggs, and larvae need to swim, recognize a substrate and attach or “settle” to begin growing.  

Recent work has been addressing these challenges. Jonathan Daly and colleagues at  the Taronga Conservation Society used reanimated frozen sperm to fertilize over 300,000 coral eggs spawned in the Australian National Sea Simulator. They achieved a fertilization rate of over 90% , which matches the rate of fresh sperm (5). 

In 2019, Cirino et al. successfully reanimated embryos from the scleractinian coral Seriatopora caliendrum. This is particularly notable because these larvae went on to settle successfully, along with their symbiotic microalgae that were also brought back from suspended animation!

DON’T FORGET THE SYMBIONTS!

Coral colonies are “holobionts” consisting of communities of interdependent organisms. In the future, if cryopreserved corals are brought back to life but their essential microalgae are no longer present in the future ocean, the corals may not survive- especially since the species of Symbiodiniaceae algae are usually paired with particular types of coral hosts. Bouwmeester et al., 2022 presented work showing that they were able to modify previous cryopreservation techniques to vitrify algal endosymbionts. After reanimation, these symbiotic algae were successfully incorporated by coral larvae, although at a low rate. It’s well known that Symbiodiniaceae are required by most corals for nutrition provisioning, but there are multitudes of additional microorganisms in the coral holobiont whose identities and roles are just being explored. They may be more important than we recognize, and it’s unknown which of them would survive the freezing and warming cycles intact. 

Figure 5. A happy scientist freezing coral sperm, and deploying personal protective equipment 

CORAL IVF:

The initial steps of cryopreservation of coral species using frozen sperm and embryos relies on a weak link, which is wild coral reproduction. For fertilization with frozen or fresh coral sperm, an external source of eggs is needed, since techniques for cryopreserving stony coral eggs have not yet been successful.  Except for limited laboratory settings that manipulate reproduction, coral eggs must be obtained through ocean spawning which occurs only a few nights a year. Unfortunately, corals that have withstood cycles of thermal bleaching and high UV irradiation in previous years may not reproduce at all during a spawning season, or they may have long-term loss of sperm motility or abnormal larval development (6). To address this gap, Powell-Palm et al., (2023) performed the extraordinary feat of freezing fragments of adult corals and reanimating them after thawing, which seems to echo Dr. Frankenstein’s work! 

A MOONSHOT? 

Cryobiologists who preserve corals might be viewed as the ultimate optimists who are saving imperiled species while waiting for humankind to solve climate change and restore the oceans to health.  Or their efforts might be viewed as just rearranging deck chairs on the Titanic and not focusing on the reality of ongoing ecological degradation.  Dr. Mary Hagedorn is one of the optimists, and she recognizes that biorepositories can be vulnerable, whether they are used to safeguard seeds, corals or research samples of human disease viruses.  If a biobank houses the only example of a species, one power outage or government budget cut can lead to extinction.  In an interview with Space.com writers, Dr. Hagedorn mused about where the safest place for her cryogenically suspended corals might be. With comments that may or may not have been tongue-in-cheek, she discussed saving frozen corals in the permanently shadowed craters of the moon. The frigid temperatures there would be suitable to preserve a biobank facility for hundreds of years, or until humans start colonizing the moon and wreaking havoc there too.  The plan would be that the coral samples could be returned to Earth on-demand and reseeded in the oceans to restore living reefs.  That at least, is the dream.

Figure 6. The cold lunar crater named Moltke, which is an unlikely place for corals to live

REFERENCES:

1. Bouwmeester, J., Daly, J., Quinn, M., Henley, E. M., Lager, C., Perry, R., Page, C. A., & Hagedorn, M. (2022). Securing algal endosymbiont communities for reef-building corals. https://doi.org/10.1101/2022.06.14.495714

2. Cirino, L., Wen, Z. H., Hsieh, K., Huang, C. L., Leong, Q. L., Wang, L. H., … & Lin, C. (2019). First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates. Scientific Reports, 9(1), 18851.

3. Daly, J., Zuchowicz, N., Nuñez Lendo, C.I. et al. Successful cryopreservation of coral larvae using vitrification and laser warming. Sci Rep 8, 15714 (2018). https://doi.org/10.1038/s41598-018-34035-0  

4. Drury, C., Caruso, C., & Quigley, K. (2022). Selective breeding to enhance the adaptive potential of corals. In Coral reef conservation and restoration in the omics age (pp. 71-84). Cham: Springer International Publishing.
5. Frozen coral sperm successfully used in coral breeding trials. Great Barrier Reef Foundation. (2022). https://www.barrierreef.org/news/news/Frozen%20coral%20sperm%20successfully%20used%20in%20coral%20breeding%20trials#:~:text=How%20is%20coral%20sperm%20cryopreserved,a%20chilly%20%2D196%20degrees%20Celsius 
6. Hagedorn, M., Carter, V. L., Lager, C., Ciani, J. F. C., Dygert, A. N., Schleiger, R. D., & Henley, E. M. (2016). Potential bleaching effects on coral reproduction. Reproduction, Fertility and Development, 28(8), 1061-1071.

7. Jin, B., & Mazur, P. (2015). High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse. Scientific reports, 5(1), 9271.  
8. Kuthunur, S. (2024, May 7). Should we seal DNA samples of Earth’s endangered species in a moon crater? Space.com. https://www.space.com/coral-reefs-moon-climate-change.
9. Moore, S. G., & Hasler, J. F. (2017). A 100-Year Review: Reproductive technologies in dairy science. Journal of dairy science, 100(12), 10314-10331.

10. Powell-Palm, M. J., Henley, E. M., Consiglio, A. N., Lager, C., Chang, B., Perry, R., … & Hagedorn, M. (2023). Cryopreservation and revival of Hawaiian stony corals using isochoric vitrification. Nature Communications, 14(1), 4859.

11. Tsai, S., Yen, W., Chavanich, S., Viyakarn, V., & Lin, C. (2015). Development of cryopreservation techniques for gorgonian (Junceella juncea) oocytes through vitrification. PloS one, 10(5), e0123409.

12. Zuchowicz, N., Daly, J., Bouwmeester, J., Lager, C., Henley, E. M., Nuñez Lendo, C. I., & Hagedorn, M. (2021). Assessing coral sperm motility. Scientific reports, 11(1), 61.