Cryopreserving Coral Sperm for Reef Conservation & Restoration (Part 1)
Stony corals are ecosystem engineers; they build reefs that support one quarter of all marine organisms and contribute billions of dollars to global economies each year through tourism, coastline protection, and fisheries (Spurgeon et al. 1992; Jones et al. 1994; Wells et al. 2006). However, coral reefs are declining globally at an alarming rate, which threatens the critical ecosystem services they provide (Pandolfi et al. 2003; Wild et al. 2010). The combined and accelerating effects of multiple local and global stressors – including ocean warming, ocean acidification, nutrient pollution, coastal development, overfishing, and predator and disease outbreaks – may outpace corals’ natural capacity to adapt and evolve (Hughes et al. 2003; Palumbi et al. 2014).
If coral reefs are to persist into the “Anthropocene” - the current geological age during which human activity has been the dominant influence on climate and the environment - interventions are urgently needed.
A healthy coral reef off the coast of Eleuthera, the Bahamas. Photo credit: Liv Williamson
Cryopreservation is the long-term storage of biological materials such as proteins, cells, organs, and tissues at extremely low temperatures - usually -196°C, the temperature of liquid nitrogen. Under such cold conditions, all enzymatic and biochemical activity essentially ceases, including processes leading to DNA degradation and cell death. Free from deterioration, this material can (at least in theory) be thawed at some point in the future and remain alive and viable.
As such, cryopreservation is a powerful tool for maintaining diverse genetic repositories of existing species that are endangered in the wild and preventing further loss of genetic variation as populations decline. Further, thawed gametes (the collective term for eggs and sperm) can be used to rescue dwindling or even extinct populations in the future when conditions are more favorable for their survival.
Photo credit: San Diego Zoo
“It is urgent that scientists and stakeholders test a variety of interventions to halt the rapid decline of coral reefs worldwide. Without these immensely diverse ecosystems, we could lose over 25% of all species in the oceans.”
Leona Chemnick, Marlys Houck, and Dr. Oliver Ryder in The Frozen Zoo - a collection of cell and tissue samples at San Diego Zoo's Institute for Conservation Research. Photo credit: San Diego Zoo
These techniques have already been utilized successfully for the conservation of endangered species. Since 1976, the San Diego Zoo has maintained a genetic repository with frozen cells and tissues from more than 10,000 individual animals representing over 1,000 species of birds, reptiles, amphibians, and mammals (www.institute.sandiegozoo.org/resources/frozen-zoo). Called the “Frozen Zoo”, this gene bank not only stores genetic material from these species, but has also been thawed and used to introduce new genetic material into endangered populations of pheasants, rhinos, and snakes.
Similarly, the University of Nottingham’s “Frozen Ark” (www.frozenark.org) stores thousands of tissue, cells, and DNA samples from endangered animals including the scimitar horned oryx, which is now extinct in the wild. A particular focus of cryopreservation has been freezing gametes (eggs and sperm), due to their ability to generate offspring and pass preserved genetic material directly to a new generation when thawed and fertilized. The ability to store high-quality biological samples long-term has facilitated genetic exchanges between captive areas, zoos, and research centers for the purposes of artificial insemination, gamete micromanipulation, in vitro culture, and grafting.
Researchers at the San Diego Zoo are using cryopreservation and other cutting-edge reproductive technologies to attempt to boost populations of critically-endangered northern white rhinos. Photo credit: AP Photo/Chris O’Meara
To date, relatively limited work has been done on the cryobiology of gametes from aquatic species, perhaps because of the very different physical and chemical conditions that they face compared with terrestrial animals. However, the conservation applications of cryopreservation are increasingly relevant and urgently needed for threatened aquatic species, particularly tropical reef-building corals.
Why is cryopreservation necessary for corals?
Cryopreservation of coral genetic material, particularly sperm, may be extremely beneficial to prevent further loss of coral cover and diversity and boost recovery of damaged populations for the following reasons:
First, this can serve as a valuable gene bank, allowing scientists to store genetic material from a range of individuals that are threatened by rising sea temperatures, increasing ocean acidity, disease outbreaks, pollution, and other stressors driving coral mortality. It is estimated that coral cover has already been reduced by 80% in parts of the Caribbean (Gardner et al. 2003), meaning the genetic material from 80% of corals has likely also been lost. To prevent further loss of genetic variation that may prove important in the face of current or future conditions, efforts should be made to freeze gametes from as many species and as many locations as possible.
An overview of the life cycle of broadcast-spawning coral species. Image credit: Liv Williamson
Second, cryopreserving coral sperm can help restoration practitioners selectively breed gametes of parent colonies that may not be able to mate in nature because they are too far from one another – a process termed “assisted gene flow”. Recently, Dr. Mary Hagedorn and colleagues succeeded in fertilizing fresh Acropora palmata (A. palmata) coral eggs from Curaçao with cryopreserved coral sperm from Curaçao, Florida, and Puerto Rico (2018). Each treatment resulted in viable, healthy larvae that grew into a total of 4,700 juveniles from cryopreserved sperm.
This study proved that 1) gametes from distant A. palmata populations that cannot breed in nature are reproductively compatible and 2) assisted gene flow is possible throughout the Caribbean. This may become a promising approach to help boost genetic diversity by injecting genes from more distant populations while local ones lose diversity. This could also become a useful tool to selectively breed for specific traits by crossing gametes from colonies deemed to have some fitness advantage (such as resistance to bleaching or disease) in order to create offspring with these desirable traits.
Figure: Hagedorn et al., 2018
Gamete bundles fill the ocean during an Orbicella faveolata (mountainous star coral) spawning event off the coast of Key Largo, FL. Photo credit: Liv Williamson
Third, nature’s best recovery strategy – sexual reproduction, or the introduction of new genetic diversity by mixing male and female gametes to generate new individuals – is breaking down on reefs due to dwindling coral populations and environmental changes (Kaniewska et al. 2015, Teo & Todd 2018, Shlesinger & Loya 2019). Unlike humans, corals cannot choose to reproduce at any time during the year. For most species, their spawning is restricted to just a few nights each year, when all the colonies on a reef carefully synchronize the release of their gametes in hopes they will meet and fertilize one another.
Unfortunately, as coral populations decline worldwide, a negative feedback loop forms: the fewer corals in a given area, the lower their collective reproductive success becomes. This is due to the “Allee effect”, a decline in individual fitness at low population density. As coral cover decreases and the distance between colonies increases, it will become increasingly unlikely that their gametes will meet in high enough concentrations to achieve adequate fertilization (Levitan & McGovern 2005, Teo & Todd 2018).
Furthermore, even for colonies that remain close enough together for their gametes to mix, environmental changes such as increasing ocean temperature may be throwing off their reproductive timing (Baird et al. 2009, Shlesinger & Loya 2019). Corals have evolved to spawn with careful precision, utilizing cues from the lunar cycle, water temperature, day length, and water motion to release their gametes with incredible precision (Babcock et al. 1986, Oliver & Babcock 1992, Levy et al. 2007). However, as conditions change due to human impacts, corals may not be able to “read” environmental cues with enough precision. Peak fertilization occurs when spawning is well-synchronized (Levitan et al. 2007), so any shift in timing poses a major threat to the successful formation of offspring.
A Lobactis scutaria female releases her eggs during a spawning event. Photo credit: Liv Williamson
A bleached Pseudodiploria strigosa (symmetrical brain coral) colony in Eleuthera, the Bahamas. Photo credit: Liv Williamson
Finally, heat waves have become increasingly frequent and severe on reefs around the world, triggering what is known as coral “bleaching” (Hughes et al. 2017). Stony corals depend on tiny, single-celled algae called Symbiodiniaceae that live within their tissues and use sunlight to make energy for their coral hosts. When corals experience stressful conditions such as high seawater temperatures, they eject the algae from their tissues, becoming white in appearance (hence the term “bleaching”). Without Symbiodiniaceae, corals lose the primary source of nutrition they need to survive and grow.
Although corals can recover by re-acquiring Symbiodiniaceae if the stressful conditions subside, these events damage the reproductive potential of reefs. With depleted energetic resources, colonies that have recently experienced severe bleaching often fail to spawn (Fisch et al. 2019), and their reduction in reproductive output may last years after a disturbance (Levitan et al. 2014). Few reefs on Earth have avoided bleaching, and the time between severe bleaching events is diminishing (Hughes et al. 2018). If corals do not have sufficient time to recover after disturbances, reproduction and recruitment on impacted reefs may totally collapse and prevent the ecosystem from bouncing back.
Orbicella faveolata (mountainous star coral) releasing gamete bundles during August 2019 spawning event. Photo credit: Liv Williamson
For all these reasons, it is urgent that scientists and stakeholders test a variety of interventions to halt the rapid decline of coral reefs worldwide. Without these immensely diverse ecosystems, we could lose over 25% of all species in the oceans, many of which human beings rely on for food, fisheries, and pharmaceuticals.
Cryopreservation is a low-risk strategy that can prevent further loss of genetic diversity and help mitigate the intensifying problems of scarce parent colonies and gametes (Hagedorn & Carter 2016). If sperm is collected and frozen from male colonies whenever they spawn, it can be thawed and used to fertilize fresh eggs any time females spawn, even if that is hours, days, months or years apart from the males in question. On shorter time scales, this can help minimize the negative impacts of desynchronized spawning and still enable managers to generate offspring (and new genetic diversity).
Tune in to Part 2 of this article series to learn how cryopreservation science is being used in corals, and what we can expect to see in the near future. Until then stay happy, healthy, and hopefully warmer than -196 C.
-Liv
A coral reef off the coast of Abaco, the Bahamas. Photo credit: Liv Williamson