Big Idea: Advanced Coral Toolkit

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CONTEXT

Recent estimates show that 50 percent of the world’s corals have already been lost and project that by 2050 as much as 90 percent will be gone. Consensus is building that even the strongest corals will need to withstand more frequent and more severe stressors in the future. While many laboratories and academic institutions are beginning to use modern genomics to identify the genetic and epigenetic variations that convey resilience to specific stressors, there is no global plan to deal with the widespread failure of reefs or to maintain coral reefs beyond 2050. The most comprehensive approach is being taken in Australia with a coordinated approach from across government and non-governmental organizations seeking to better understand and abate the threats to coral.

The success of managed breeding programs in agriculture and terrestrial conservation provide a potential model for the development of a coordinated effort to breed corals capable of surviving future climate-related stressors. This, with the scientific innovations described here, will enable a scalable response to ensure coral reefs and the species they support have a chance of being conserved and restored.

Because of the severity, scope, and immediacy of the problem, the scientists interviewed for this Ocean Genomic Horizon Scan all felt that a multi-track approach to contending with the coral crisis is needed. These scientists tend to be more open-minded about the development of genomic solutions for coral bleaching than are those focused on other threats. Despite this willingness to innovate, even having sufficient coral samples for experimentation is currently a limiting factor in research (S. Palumbi, pers. comm.).

Before resilient or novel strains of coral can be developed, advances must be made in several fundamental facets of coral biology. Key technological hurdles need to be overcome so that corals and their eggs can be cryopreserved, and coral stem cells can be isolated and grown. These capabilities would lay the foundation for coral breeding programs and enable future efforts for foundational coral science and applied coral restoration.

One thing that is clear: we need more time. Even with the global urgency, openness to innovation, and funding dedicated to coral restoration, scientists need more time to discover the underlying biology for coral responses to warming oceans. They will then need to develop tools, like genomic assays and stem cells, and interventions, like genome edits, and rigorously test those interventions in lab trials, then larger scale field trials. Finally, methods of intervening at global scale will need to be invented and tested. The timescales of all of this research are not well aligned with the predicted time frame for the devastating effects of climate change. We will need to employ both short term and long term thinking to solve the coral crisis.

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BIG IDEA

The program below outlines complementary strategies for near- and long-term solutions for corals: First, a near-term plan to refine and scale techniques for cryopreserving samples of wild corals from reefs today, in order to ensure a continual source of genetic and cellular resources for research and eventual re-population of reefs around the world. If successful, this project will enable more labs to have corals to work with in the future, which will give scientists time to do necessary research and field trials. The long-term strategy is to advance, refine and scale needed technologies in coral biology and genomics to generate new strains of corals that are better able to survive in the warmer climate of the future. This work will involve fundamental biology of coral stem cells to enable genetic engineering of temperature resilience. Taken together, these two parallel projects could achieve what’s been assumed to be impossible: the restoration of the world’s coral reefs in a ruthlessly warming climate.

Near-Term Plan: The Frozen Ark of Coral

Cryopreservation is a vital preventative measure for preserving threatened coral species before they fully succumb to the effects of climate change. Unlike the engineering of resilient coral strains, cryopreservation is something we can do today to give corals more time for facilitated adaptation in the future.


Coral Eggs, Sperm, and Larvae

Current methods involve the freezing of sperm and larvae harvested from reefs. Despite several attempts, no one has been able to freeze and thaw corals eggs in a viable technique. Sperms and larval samples could serve as a source of materials for research and captive breeding efforts. However, the problem of not yet being able to freeze eggs currently limits the options in research and restoration. This is compounded by the fact that most corals only spawn on two days per year. Therefore, collection events must be orchestrated on a large scale with little room for error or missteps. Read More

Inducible Spawning

As described above, coral eggs are necessary for coral breeding but cannot yet be cryopreserved. However, corals can be induced to spawn eggs if kept under ideal conditions with appropriate environmental simulation and husbandry. Scalable inducible spawning techniques would provide coral breeders a year-round source of eggs, allowing banked coral sperm to be efficiently used whenever necessary. The Project Coral laboratory at the Horniman Museum is a pioneer in this area, but is unable to provide more than one-on-one training at this time.  By scaling up the team’s laboratory and training capabilities, other practitioners could be more easily trained to perform the inducible spawning technique.

Microfragments

The microfragmentation method potentially offers an immediate solution to the scalability of coral cryopreservation. Researchers have found that when large fragments of living coral are cut into microfragments of ~1 cm2, these small pieces will grow at exceptional rates to reconstitute larger corals. Because microfragments are derived from fragments of adult corals, they can be collected year-round. Further, the microfragments contain almost all cells necessary for forming adult corals, including stem cells and symbiotic microbes. Therefore, they represent a more complete sample of coral material in each individual frozen sample than the germline or larvae samples. They do not, however, contain gametes. Therefore, microfragment banking will be complementary to gamete banking.  Indeed, using this method, it may be entirely feasible to collect, freeze, and store samples of every species of coral in the world before critical ocean temperatures are reached.

Multiple groups have developed husbandry methods for making and growing microfragments in lab environments. The Hagedorn lab has also identified a suite of cyroprotectants that are non-toxic to the fragments and allow them to survive the freezing process. The remaining hurdle is to optimize the thawing process so that preserved microfragments derived from many species of coral can grow in open water after years in the freezer. Once the thawing technique is optimized, the entire procedure from collection, to freezing and thawing will need to be standardized and potentially automated. Read More

Long-Term Plan: Technology for Genetic Engineering of Corals

To rationally select parental corals for use in breeding programs or genetically engineered traits, it will be essential to determine the genetic basis of traits and inheritance. To do so, a reliable technique is needed to easily assess novel strains for their potential breeding values.


Coral Stem Cells

Ongoing developments in coral stem cell technology and the clonal nature of coral will make it possible to isolate coral stems cells and propagate them in culture. Paired with insight from ongoing population genomics experiments, coral stem cells can then be subjected to genome engineering, experimentation, and assessment to determine genotype-phenotype interactions. This work will inform understanding about the relative breeding value of a particular coral strain, and enable appropriate decisions to be made about parental lineages.

Stem cells derived from novel coral breeds could be propagated in a continuous manner in bioproduction facilities to support industrial-scale cellular micropropagation of coral larvae for direct out planting of high-performance coral breeds. Moreover, stem cells could be cleaved from juvenile corals, cloned, and then sub-pooled for genetic experimentation, such that a single coral can be independently tested for many different traits, even with a destructive assay. Finally, stem cells could themselves be transplanted directly into wild corals, in order to recolonize dying coral colonies with adaptive genes, as a coral stem cell therapy approach.

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PARTNERS

  • Hagedorn at the Smithsonian Institute Marine GEO facility is a leading figure in the coral cryopreservation space and has recruited a global team of specialists inside and outside of coral biology. Her recent work with Dr. Kristen Marhaver at Caribbean Marine Biological Institute (CARMABI) demonstrated the successful use of cryopreserved sperm to fertilize and develop 4700 juvenile corals, the largest living wildlife population ever created with cryopreserved material. Dr. Hagedorn is collaborating with Dr. Jon Daley, soon to be a professor at U. of New South Wales, on microfragment cryopreservation.
  • Craggs at the Horniman Museum and Gardens Aquarium runs Project Coral, the leading group in the inducible spawning sub-field, and has pioneered the inducible spawning technique over the last decade. The group has contributed to work demonstrating successful IVF fertilization of captive-spawned eggs with thawed sperm
  • Traylor-Knowles at the University of Miami and Dr. Rosental at Ben Gurion University recently isolated putative stem cells from a single coral species are currently proposing research to isolate and characterize stem cells from additional species and demonstrate transplantation from donor corals to recipient corals. Beyond the work in corals, the team has succeeded in stem cell isolation and transplantation in golden star tunicate (Botryllus schlosseri), a marine colonial invertebrate.
  • Great Barrier Reef Foundation: the lead charity dedicated to protecting the Great Barrier Reef through funding solutions grounded in science, technology, engineering and on-ground action to ensure its long-term conservation.

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BUDGET

Cryopreservation Component

ITEMTimelineUSD
Total:$620,000
1) Determine the cryobiology of microfragments and eggs from priority coral species.12 months$240,000
2) Investigate optimal sample form and handling for cryopreservation.6 months$90,000
3) Compare and assess slow cooling and methods for cryopreservation of microfragments and eggs from priority coral species.6 months$90,000
4) Identify factors affecting post-thaw recovery and growth of coral microfragments and eggs.4 months$67,000
5) Optimize post-warming recovery conditions and develop grow-out methods for coral microfragments and eggs.4 months$67,000
6) Investigate alternative sources of adult coral tissues such as cell culture, polyp bail-out, and tissue balls.4 months$66,600

Inducible Spawning Component

ITEMTimelineUSD
Total:$295,000
1) Horniman Project Coral facility extended to accommodate additional training lab.12 months$150,000
2) Complete Visiting Spawners program: 3 fellows in residential training at Horniman.18 months$120,000
3) Publish open source curriculum.12 months$25,000

Stem Cell Component

ITEMTimelineUSD
Total:$780,000
1) Isolation and characterization of coral stem cells, genomes, and gene expression.12 months$430,000
2) Transplantation and localization of coral stem cells in live coral.12 months$150,000
3) Propagation of coral stem cells in a cell culture/ cryopreservation of culture.12 months$200,000

PROJECT TOTAL: 3 Years and $1,695,000

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RISKS & CHALLENGES

Enabling thawing of microfragments: It is anticipated that cryopreservation of coral fragments will be challenging and may require modification of protocols to suit individual species.

  • Mitigation: The cryobiology of adult tissues and cells is relatively unexplored and could provide an alternative biobanking source if cryopreservation methods can be developed.

Stem cell isolation and transplantation: Identifying totipotent cells for multiple species with the same approach may prove challenging. Stem cell transplantation efficacy and long-term survival is poorly known.

  • Mitigation: In prior studies the team has been able to keep progenitor cells from one coral (Pocillopora damicornis) and one anemone (Nematostella vectensis) alive in culture for nine months. These early results show that stem cell culture is possible, reducing the technical risk for the proposed research.

Regulatory Concerns: High-volume bioproduction may be subject to numerous technical issues. CITES/CBD compliance for stem cells is still unknown but the uncertainty may cause time consuming legal complications. Also, still uncertain are any regulatory issues related to the release of stem cells for restoration or experimentation. Keeping the regulators and concerned stakeholder informed and engaged should foster collaboration. Ultimately, beyond the scope of this Big Idea, is the potential to release engineered corals into the wild.  Such releases will require a deliberate and authentic stakeholder engagement process to ensure that the development of these tools prioritized the development of comprehensive environmental safeguards.

Toward long-term precision coral breeding: With this plan is the assumption that various nations will cooperate to allow the collection of the world’s coral species into the frozen ark, which will require a substantial amount to diplomacy by the conservation community. Eventually, international coral breeding and reef restoration programs will need to emerge to take advantage of these proposed technologies, which may not prove to be the case or happen within our expected timelines. In the initial phases, successful coral crossing will also be subject to other bottlenecks in the coral field including: poor contemporary knowledge of adaptive traits at both genotype and pedigree and limited genetic sequence coverage, as well as challenges with larval settlement rates. In the longer term, restoration efforts could see inbreeding and outbreeding which would need to be accounted for with genetic analysis of offspring.

  • Mitigation: The proposed Frozen Ark of Coral will enable time for policy and diplomacy, just as it will provide time for necessary research. Hopefully, this will not create an excuse for delaying action, but rather will encourage careful planning and testing prior to major restoration efforts. The economic benefits of reefs to the countries that are near them should inspire governments to move as quickly as possible to safely restore such valuable natural resources.

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TIMELINE

1. Cryopreservation of Coral Eggs & Microfragments


While some of the cryobiology of coral fragments and eggs is known (Hagedorn et al. 2013), successful cryopreservation has not yet been fully achieved. The Hagedorn lab has had limited success in whereby fragments lived for up to 48 hours post-thaw, and work has begun to modify these methods to extend post-thaw survival. In addition, while coral eggs have not been successfully frozen and thawed, the eggs of many other animal species are routinely cryopreserved for veterinary science and captive breeding efforts. The methods currently under development will need to be tested and adapted for corals from the genus Acroporidae and other reef-building coral species, beginning with coral from regions that have had minimal impact from warming.

MILESTONES:

a) Establish microfragment production and husbandry methods for priority coral species.
b) Develop cryopreservation methods for microfragments and eggs from priority coral species.
c) Develop methods for recovery and grow-out of cryopreserved microfragments.

2.  Inducible Spawning Training Program:


Today, inducible spawning is the only way to get eggs on-site at coral facilities; however, only one lab has mastered this technique. Due to the potential impact of inducible spawning for coral breeding efforts, it is essential that this technology be quickly disseminated to coral labs around the world.

MILESTONES:

a) Accommodate new tanks and equipment, develop standardized protocols, and expand training capabilities in the Project Coral Lab, Horniman Museum.
b) Establish a fellowship program to support training and refining Horniman technology development.
c) Assemble an open-source body of software, hardware, and wetware materials, as well as codebases and protocols to support other inducible spawning efforts.

Successful project outcomes will be presented as invited keynotes at coral conferences (ICRS, ECRS, and Reef Futures, for example) and as lectures at aquarium supply conferences in order to reach coral traders and expand coral breeding platform involvement.

NEXT STEPS:

Once the methods for collecting and freezing eggs ,and thawing microfragments have been optimized, the protocols can be standardized and automated in collaboration with process engineers. The automated protocols will speed the building of the frozen ark of coral and enable the participation of citizen scientists from around the world

3.  Stem Cell Isolation and Transplantation


The ability to isolate and grow coral stem cells has been identified by leading coral biologists as a major priority to advance the understanding of genetic resilience in corals. In theory, coral stem cells will enable genetic manipulation at a single cell stage (crucial for genome editing) and subsequent growth into adult corals. However, no basic stem cell capability exists in coral biology, and no stem cell lineages (or cell lines) are available at the present time.

MILESTONES:

a) Deliver isolated stem cell populations from three coral species
b) Grow isolated stem cells in a cell culture environment
c) Demonstrate stem cell transplantation into living coral colonies
d) Demonstrate stem cell propagation and cryopreservation in a cell culture environment.

REFERENCES

  • NAS Ocean Studies Board (2018), Interventions to Increase the Resilience of Coral Reefs, US National Academies of Sciences
  • Flint & Woolliams (2008), Precision Animal Breeding, Philosophical Trans. Royal Society of London
  • For additional references, please see “Coral Horizon Scan” in Chapter 3.