Cryopreservation: "Setting the stage"
Is cryopreservation the technique that the restoration community has been waiting for? We asked the expert.
photo: Smithonian researcher Dr Mary Hagedorn has created the first genome repository for endangered coral species in the Caribbean, Hawaii, and The Great Barrier Reef and has distributed this germplasm to frozen banks around the world. (Marco Garcia)
In your latest study, you successfully fertilized fresh elkhorn coral eggs from Curaçao with cryopreserved elkhorn sperm from other Caribbean locations – a breakthrough, as many colleagues in the field say. Why is this proof of concept so important for the conservation and restoration of coral reefs?
Mary Hagedorn: As you can see in most of the world’s reefs, global change is causing reductions in coral’s growth, survival, and reproductive health due to rapidly-warming and acidifying conditions. Since corals are sessile organisms and cannot easily migrate to more favorable locations, our basic idea is to help them adapt to rising temperatures by crossing individuals from different areas using cryopreserved sperm and assisted gene flow as a promising option for accelerating population adaptation and buffering species against extinction.
Which is a concept that has been applied to mammals and other animals very successfully before.
Cryopreservation has been around since the 1950’s and it has been applied to agriculture and wildlife for a very long time. Nowadays, we do not move animals around anymore, we move their germplasm, whether it is a frozen embryo or frozen sperm. Like if you want to diversify your farm, your cattle, you would call a company and decide on what breed you wanted or what diversification you wanted. You would order that, and they would send it to you frozen. That has been a very hard thing for us to conceptualize for ecosystems such as coral. But we did it. Our group has helped to create the science and apply the science for cryopreservation, because it is the only thing right now where we can say: If we do this, we will secure biodiversity and genetic diversity of the reef. And we can do it in a way that institutions like aquaria cannot. We can store much more material in a much smaller space, and it does not change over time.
photo: Elkhorn corals (Acropora palmata) once surrounded all Caribbean islands and Florida’s coast line as a natural shelterbelt against massive waves and storms. Nowadays this species is rare, more than 95 percent of its population has disappeared in the past three decades. (Paul Selvaggio)
And it gives you the option to cross corals from different locations without actually moving colonies, which is essential especially for endangered coral species such as elkhorn corals.
Yes, our paper proves that we could take cryopreserved sperm and use it to make these transregional crosses, these colonies lived hundreds of kilometers apart. We also demonstrated that we could mature the cryopreserved settlers. We have thousands of transregional elkhorn coral settlers now that are growing really well in Mote Marine Lab and Florida Aquarium. So, one can say that we set the stage for assisted gene flow throughout the ocean for coral reefs. Now we hope that this will open up the field. Hopefully, conservationists will start saying: Let’s find hotspots of biodiversity and let’s start cryopreserving and making these kinds of crosses. So far, we are one of the few groups in the world that are doing this. We have trained a few others, but they are not actually going out and applying it all that much. Our goal really is to train more people to do this.
photos: Between 2006 and 2010 Mary Hagedorn and SECORE joined forces in Puerto Rico to collect elkhorn coral spawn. Some of the sperm gathered in 2008 was cryopreserved and stored in a repository. 10 years later, Mary’s team thawed the sperm and used it to fertilize fresh coral eggs from Curaçao successfully. These photos were made during the coral spawning workshop in 2006 (Brenna Hernandez, Shedd Aquarium)
Can cryopreservation be done easily?
It takes some training, but it is not like it is rocket science. If we are working with an endangered species, we have to go out shortly before spawning, net the colonies and collect their egg-sperm bundles. SECORE divers did that for us in 2007 to 2010 in Puerto Rico. All tubes filled with gametes are brought into the laboratory, where we start to gently break apart the eggs and the sperm. The eggs float to the surface, because they are filled with fat. The sperm forms a milky solution in the tube. After about ten to 20 minutes we very gently pull off the sperm. We filter it, put it in a clean tube and set-up a coral fertility clinic, meaning, we look at the sperm’s concentration and at its motility, and we decide whether it is appropriate to cryopreserve. In a human fertility clinic, they generally will not freeze a volunteer donor’s sperm if it has less than 50 percent motility. This is because cryopreservation can cause some damage, so you want to start with the highest quality material possible in order to get good fertilization success with the thawed sperm. For corals, we often choose the same cut-off. After assessing the sperm, we start adding a cryoprotectant. It helps protect the sperm cells from the damage as it is freezing. Then we load the sperm and the cryoprotectant into cryotubes. These are special tubes that can go down to liquid nitrogen. We place a label on them and start the freezing process.
photos: For sperm collection of endangered coral species, colonies are netted shortly before spawning. When the colony starts releasing its egg-sperm bundles, they float into small collections tubes (left), which are brought into the lab afterwards. Here scientists gently break apart eggs and sperm (middle). The eggs float to the surface while the sperm forms a milky solution. (Paul Selvaggio)
Do you need any special devices for freezing coral sperm?
Yes, in addition to methodology we also had to create our own freezing apparatus, because there was nothing on the market that would work properly. We freeze the sperm by about 20 degrees per minute. After about five to six minutes it is frozen down to minus 80 degrees Celsius and we plunge it into liquid nitrogen where it freezes to -196 degrees Celsius. To transport it, we put the frozen samples into dry-shipper, which is like a big thermos that has foam that absorbs liquid nitrogen and keeps everything at liquid nitrogen temperature. We can hold the tubes for a couple of weeks in those dry-shippers and we can send them to one of the two existing coral sperm banks in the world. Theoretically, they could stay there for a thousand years.
Without the cells actually dying?
The sperm cells are still alive, but they are so slowed down that no metabolism is really happening. Once we thaw them, which we do in just filtered sea water in about 30 degrees, we assess their post-thaw motility and then we can use them on fresh eggs to create new offspring.
photo: Mary Hagedorn (right) and her technician are freezing sperm in the laboratory. (Steve Clarke / AIMS)
Your study proves that we have the methods figured out. What needs to be done to apply cryopreservation and assisted gene flow for large-scale reef restoration?
It is a step-by-step process. Let’s talk about the coral reefs in Australia. They form a U-shape across the top of Australia. You have the Western Reefs and then you have the Great Barrier Reef coming down the top of Australia and going into the South. There are obviously some very critical reefs within these areas that are important. Those reefs need to be identified and then within those reefs you need to have the species that we want to cryopreserve. So there needs to be prioritization done on species. And individuals need to be tagged and genetically analyzed, so that you know you do not have clones when you start collecting spawn.
We know from Iliana Baums and her colleagues that you would need approximately 10 individuals of a species from a reef to get 90 percent of the genetic diversity – remember the Great Barrier Reef is made up of many reefs! That is not a lot and it is really quite doable. So, if you have set-up all those processes and gained some competence in cryopreservation and genetics, you are ready to do this in a large scale. In our elkhorn study, we could have done this in a much larger scale, but we faced the problem that we only had two nights of spawning and some of our individuals were self-fertilizing, which never happened, if I recall, or does very rarely. But it really is not a huge effort to get hundreds of thousand offspring from just one trial. However, it is a lot to take care of those – so be careful what you wish for.
Selecting a site and species, analyzing the corals genetically, and doing all the rest of the work sounds very time consuming and labor-intensive. How could all this important work be funded?
We need a global program that is funded at a very high level, so that we can train teams and people to go out. My post-doc, John Daly, who will return to the Great Barrier Reef to apply cryopreservation and assisted gene flow there, will hopefully have sufficient money to start this kind of program on the Great Barrier Reef because of Australia’s Reef Restoration and Adaptation Program. But it is a problem for the rest of the world. Indonesia, for instance, does not have the money to do that kind of work nor do they have large biorepositories for holding the material. My team and I work in Hawaii and in French Polynesia – but our fieldwork is conducted on a shoestring with our small team. However, if we want this type of conservation and restoration to move forward, we will need trained teams in a number of field sites collecting material. Because the problem that I see – and I’m not trying to be a downer – is that we are going to have more and more bleaching events that affect reproduction considerably. In fact, we have an El Nino starting in the Pacific this year – you can see the warming on the maps. I do not know what that will mean for warming and the subsequent effects on reproduction of corals in the Pacific this winter and next summer. But it is important that we think about getting this work done as quickly as we can because these warming events will be getting more and more frequent. We have already seen substantial changes.
What kind of changes are you talking about?
For one of our studies we started to cryopreserve coral symbionts almost every month from 2013 to 2014 because we wanted to look into seasonal changes in the algae over the year. During this period, our freezing protocols worked well. However, after the 2015 bleaching event in Hawaii, we can no longer use the same protocol. I have a post-doc now who is looking into this and retesting their physiology comparing it to their pre-bleaching values. Things are changing, the algae are adapting, which is good, but it is changing their physiology, which means we have to go back and recalibrate our symbiont cryopreservation method. It is this constant challenge of staying current with the changes and adaptations on the reef. The genetic diversity is shrinking, which makes it even harder.
So how do we save the reefs’ diversity for future restoration approaches? Cryopreserved sperm only helps for sexual reproduction.
Yes, that’s why we are developing other tools as well. I’m working on ways to freeze tiny micro-fragments of corals. Imagine a one-millimeter by one-millimeter cube of a piece of coral. We mount them on silver pins, which are about two centimeters long, and freeze them. I’m hoping that within the next six months we will be fully successful. If we are, that will be a major breakthrough for coral conservation and restoration, because we can freeze these microfragments many days throughout the year. You do not have to wait for a few nights of coral spawning per year.
You want to freeze coral polyps, store them, and then thaw and grow them whenever they are needed?
We did this already and got them to live for two days. We are now trying to extend this. I’m really trying to make this method very robust and field-friendly. Ideally, we would like to freeze and thaw them and then have them grow in good shape for at least a month to prove that it will work. Then we could put several micro-fragments together to form a single colony, so that it can grow really quickly.
But you would not get the genetic changes, which happen within the coral population on the reef, while the cubes are frozen.
Yes, that is exactly right. But what we need to do is to quickly save genetic and biodiversity. The microfragments take up a small amount of space, and if done properly might allow us to freeze many individuals within hundreds of species worldwide. Scientists at Mote Marine Lab have shown that coral colonies produced from many microfragments can reach maturity after about three years. Scientists at the Horniman Museum have also proven that we can reproduce coral in captivity. If necessary, we can bring these thawed microfragments into captivity, mature them, and then produce a wide variety of sexual recruits. We need to produce and use as many tools as possible to help save our reefs.
photo: Successful fertilization experiment: Tiny elkhorn coral settlers, made from Florida sperm and Curacao eggs, are growing in tanks at the Mote Marine Lab. (Chris Page)
Would you say that sexual coral reproduction is heading south?
I’m still a huge fan of corals’ sexual reproduction, it is critical for adaption of our reefs. I worked on it for many years, but it is getting harder and harder for us to use it for cryopreservation. Even if you have really impacted sperm with 5 to 10 % motility, if it is fresh, you could always get embryos. But you cannot use this sperm for cryopreservation. It is not healthy enough to produce good fertilization success. Therein lies the main problem. The other thing that we are doing well now is that we can freeze larvae. After thawing, we get about 50 percent of those larvae back. And they swim and they settle, and they take up their symbionts. Nowadays, we have the capacity to freeze hundreds of thousands of larvae in a short period of time. We are trying to produce many tools for professionals so that we can try to maintain various aspects of the genetic and biodiversity of reefs around the world. Some of it will be sperm, embryos, symbionts or microfragments. Additionally, we are also freezing the reef fish. More precisely, we are freezing testicular stem cells of fish. With these frozen cells, you can resurrect that species, even it if goes extinct, by putting these thawed stem cells into a sibling species or even a sister genera. Over the next 10 years we will have so many tools that we can provide professionals with to maintain reefs, that I think we stand a fighting chance of helping corals with adaptations.
About the expert
Dr Mary Hagedorn has created the first genome repository for endangered coral species in the Caribbean, Hawaii, and The Great Barrier Reef and has distributed this germplasm to frozen banks around the world. She received her Ph.D. in Marine Biology from Scripps Institution of Oceanography and is a Senior Research Scientist at the Smithsonian Institution. In 2000, she received the prestigious George E. Burch Fellowship in Theoretic Medicine and Affiliated Theoretic Sciences, in 2005 she was nominated for the Pew Fellowship in Marine Conservation and was a 2012 finalist for the Rolex Award for Enterprise. From 2007 to 2010 Mary also worked as a scientific advisor for SECORE International and has been one of our closest supporters from the beginning.
The new study, which motivated us to talk with Mary, was conducted at CARMABI Marine Research Station on Curaçao. The related field and laboratory work, led by Mary Hagedorn, Kristen Marhaver, and Mark Vermeij, was supported by colleagues at Florida Aquarium, Mote Marine Laboratory, CRF, and by SECORE’s field team on Curaçao, Valérie Chamberland and Kelly Latijnhouwers. The paper was published under the following title:
Mary Hagedorn, Christopher A. Page, Keri ONeill, Daisy M. Flores, Lucas Tichy, Valerie F. Chamberland, Claire Lager, Nikolas Zuchowicz, Kathryn Lohr, Harvey Blackburn, Tali Vardi, Jennifer Moore, Tom Moore, Mark J. A. Vermeij, Kristen L. Marhaver (2018): Successful demonstration of assisted gene flow in the threatened coral Acropora palmata across genetically-isolated Caribbean populations using cryopreserved Sperm, bioRxiv, DOI: 10.1101/492447
Interview: Sina Löschke