THE FROZEN ARK PROJECT

Saving the DNA and viable cells of the worldʼs endangered species

 

WHY IS THIS PROJECT IMPORTANT?

It is clear that, despite the best efforts of conservation, thousands of extinctions have occurred before animals could be rescued. There has not been enough knowledge or money to stem the tide. This pattern is being repeated across all animal groups and emphasises the importance of collecting the DNA and cells of endangered animals before they go extinct. The loss of a species destroys the results of millions of years of evolution. If the cells and DNA are preserved, a very great deal of information about the species is saved. The Frozen Ark Project is not a substitute for conservation, but a practical and timely backup of the genetic material.

WHEN DID YOU START UP?

The Frozen Ark Project was founded in 2004 by Professor Bryan Clarke FRS, a geneticist at the University of Nottingham, his wife Dr Ann Clarke, an immunologist with experience in reproductive biology, and their friend Anne McLaren FRS, a leading figure in developmental biology. But the idea for the Project came to Bryan and Ann much earlier than that after Bryan’s work in the French Polynesia.

WAS THE ORIGINAL MOTIVATION SCIENTIFIC OR CONSERVATION?

It was mainly for conservation inspired by the extinction story of Partula snails. From the 1960’s, Bryan carried out comprehensive studies on the evolution and speciation of land snails of the genus Partula, which are endemic to the volcanic islands of French Polynesia. It was during this work that he, Ann and colleagues witnessed the disappearance of most Partula species in a span of 15 years, because of a governmental biological control plan that went horribly wrong. In the late ’60s, the giant African land snail (Lissachatina fulica) was introduced to the islands as a delicacy, but soon turned into a serious agricultural pest, thriving in the absence of natural predators. To control the African land snails, the carnivorous Florida rosy wolfsnail (Euglandina rosea) was then introduced to the islands, but instead of eating its intended target species it annihilated the endemic land snails and by the early ’80s most of the islands were Partula-free. As a last resort, Bryan and his team managed to collect and bring back to Britain live specimens of the remnant 12 Partula species. Tissue samples were taken and frozen to preserve the DNA, enabling the continuation of the speciation study, and an international captive breeding programme was established at the Zoological Society of London (ZSL) with the aim of eventually returning the small land snails to their natural habitat.

HOW MANY SPECIES DO YOU HAVE STORED AND HOW MANY OF EACH SPECIES?

The Frozen Ark Consortium as a whole currently stores approximately 48,000 samples belonging to 5,500 species.

WHAT IS ACTUALLY STORED – TISSUE, BLOOD, EGGS AND SPERM?

As the Frozen Ark works as a federated model, this means that the type of samples stored by each partner highly depends on their own interests, in-house expertise, infrastructure and individual budgets. For instance, university laboratories and museums mainly store tissue (including blood, skins and bones, and even whole animals), DNA and non-invasive samples such as hairs, feathers and faecal samples, while it is much more common to find, additionally to tissue and DNA, storage of viable cells and gametes in biobanks associated with big zoos or at independent research laboratories. At the Frozen Ark headquarters, we mainly store tissue, DNA and blood samples. While storing viable cells and gametes in liquid nitrogen is the current gold standard, this is still a very expensive and demanding method, not accessible to all Frozen Ark partners. If we were aiming just for that gold standard, we would lose out on the wealth of samples that exist already in institutions across the globe and that can still be of use. It is for this reason that the Frozen Ark is currently working to harness existing samples, create (among partners) common quality standards and procedures for sample collection, transport and storage and then make these samples available for research and conservation management. Different types of samples are useful in different contexts. A conservation (population) genetics or a pedigree study will probably only need access to tissue/DNA or non-invasive samples, a comparative genomics study requires high quality tissue/DNA samples, whilst a breeding program of an endangered species will rely on the use of cryopreserved gametes, especially if the individuals are no longer able to reproduce without assisted reproductive technology or have died since.

DO YOU NEED A CERTAIN NUMBER OF SAMPLES FOR EACH SPECIES TO ENSURE DIVERSITY?

Short answer: yes! We are currently focusing in increasing the representation by taxa, but ideally the next step will be to collect and manage genetic material at the population level.

WHAT CAN YOU ACTUALLY DO WITH THE SAMPLES YOU HAVE COLLECTED?

Samples stored in Frozen Ark collections (and indeed in other similar animal biobanks) can be used in varied ways, but ultimately, they are intended to facilitate a greater range of genetic (e.g. population genetics) and genomic (e.g. comparative genomics, adaptation) research or to be used actively in conservation programmes (e.g. captive breeding, genetic rescue).

IS THE IDEA TO STORE AS MANY SAMPLES AS POSSIBLE SO THAT AT LEAST WE HAVE IT WHEN TECHNOLOGY HAS IMPROVED TO THE POINT WHERE WE CAN USE IT?

At its inception, the focus of the Frozen Ark was indeed to collect samples from as many endangered species as possible to avoid the complete loss of valuable genetic information and to safeguard this material for future generations. However, in more recent years, our focus has shifted to some extent and our current work is much more guided by the need of making existing samples available to present-day research and conservation. We are currently directing efforts to link and coordinate a variety of existing UK’s animal frozen collections and are also offering additional infrastructure and developing an online tool that will allow conservationists and researchers to search for, locate, and use genetic material wherever possible without having to resample from wild populations. We also want to ensure that genetic material is used responsibly and sustainably in research.

HOW DO YOU STORE EVERYTHING AND HOW DO YOU KNOW THIS IS THE BEST WAY OF STORING IT? COULD YOU BE DAMAGING IT AT THE LOW TEMPERATURES FOR EXAMPLE?

The lower the temperature the better. Liquid nitrogen is considered the gold standard of sample preservation and it is used for long-term storage of DNA, RNA, protein samples, and also for living cells (e.g. reproductive cells). Keeping samples at -196°C reduces the likelihood of damage from ice crystal formation and virtually all biological and chemical processes are halted at this temperature. On the down side, this storage method requires specific handling training and is fairly expensive due to the type of equipment needed, the associated running costs and the need for a regular supply of liquid nitrogen, so in smaller biobanks is normally used for the most important specimens and/or certain types of biological material (e.g. sperm/eggs). In university laboratories, DNA is most commonly stored at 4°C, -20°C or -80°C, either dry or in a buffer fluid or ethanol (this avoids chemical and enzymatic degradation). Ethanol is commonly used for preservation of animal samples and DNA stored in ethanol at -80°C is very stable for long periods. However, ethanol needs to be replaced when initially used to store tissue as the water present in tissue mixes with the ethanol reducing the concentration and making it less effective for preservation. Ethanol is easy to use, but it is relatively expensive, is flammable and evaporates quickly. There are not many long-term studies looking at DNA degradation over time under different temperatures, but it is acknowledged that samples maintained in pure ethanol at -80°C remain suitable for use for years, whilst samples stored in liquid nitrogen are useable for decades. Ultimately, the final intended use for the sample determines whether that sample is still valuable or not.

DO YOU RESEARCH WAYS OF COLLECTING, STORING AND THEN USING SAMPLES?

Our main research focus is to understand the best methods for collection, transport, storage and curation of different types of biological samples from a large variety of animal species, each one carrying out their own specific technical challenges. While it is acknowledged in many fields of biology that appropriate preservation of tissue samples is vital to isolate good-quality DNA, there is a surprising lack of comprehensive and systematic studies testing available methods for specific tissue storage of wild animal species and how continued re-use of frozen samples impacts DNA stability. To address some of these questions, we are currently running a series of experiments on DNA degradation. We are specifically testing how temperature and humidity and increased freeze-thaw cycles impact DNA quality.

HOW DO YOU GUARANTEE THE INTEGRITY OF THE PROCESS AND SAMPLES, E.G. CAN THEY BE THAWED AND REFROZEN, WHAT HAPPEN IF ELECTRICITY FAILS?

We are really interested in understanding the impact of freeze-thaw cycles in stored samples and DNA and how many of these cycles can a sample go through before it is rendered too “damaged” for further use. In a context of an active collection, processes need to be in place to minimise effects of freeze-thaw cycles, whether that be aliquoting samples on their arrival to the biobank or having back-up power systems (e.g. generators) and/or alarms in place to alert staff of any power failure. The Frozen Ark is also working to ensure that the most valuable samples are backed up at more than one location.

DO YOU HAVE A SINGLE STORAGE FACILITY FOR THIS OR DO YOU WORK WITH OTHER ORGANISATIONS AND INSTITUTIONS TO FORM A GLOBAL RESOURCE?

The Frozen Ark Project operates a federated model, building partnerships with organisations worldwide that share the same vision and goals. The Frozen Ark Consortium currently comprises 27 partners, distributed across five continents with representation in the UK through universities, zoos, ZSL, the Royal Zoological Society of Scotland (RZSS), the National Museums Scotland (NMS) and the NHM. The consortium also includes partners from Ireland, Germany, Denmark, Norway, the USA, Colombia, South Africa, India, Vietnam, Malaysia, South Korea, Australia and New Zealand and has a direct link to the IUCN (International Union for Conservation of Nature) through its Conservation Genetics Specialist Group.

ARE THERE SIMILAR INITIATIVES ELSEWHERE IN THE WORLD?

There are other projects similar to the Frozen Ark. There are two main biobanks for plants, the Global Seed Vault in Svalbard (opened in 2008) and the Millennium Seed Bank at the Kew Gardens that opened in 2010. The Global Seed Vault holds the world’s largest seed collection of crop plants and it is designed to challenge natural and man-made disasters, whereas the Millennium Seed Bank partnership is the largest ex-situ plant conservation programme for wild plants. There are several generic biobanking facilities for terrestrial animals, such as (for example) the San Diego and Australian Frozen Zoos, the AMNH – Ambrose Monell CryoCollection of the American Museum of Natural History, and the Cryo-Initiative at the Smithsonian Institution. There is also a global bank for marine species (Ocean Genome Legacy) and a few more focused in particular taxa, such as the Amphibian Ark. Where Frozen Ark differs in relation to these and many more other small animal biobanks across the world is its federated model, as opposed to a single physical facility, and its focus on endangered animals, including invertebrates.

IS THERE A PLAN OF USING A PERMANENTLY FROZEN SITE FOR FROZEN ARK COLLECTIONS, LIKE THE SEED BANK AT SVALBARD?

The Svalbard Vault is intended to be a safe storage for crop seeds, but we have recently seen how this facility was in risk of flooding due to unusually warm conditions. While having a location in the coldest regions of the planet is probably a safe bet, helping to maintain the low temperatures needed to preserve samples, we think that a model based in several locations/duplicate collections suits the Frozen Ark better. Also, due to international legislation, especially the Nagoya Protocol on access and benefit sharing of genetic resources, the movement of genetic material across borders has become increasingly difficult, so we encourage institutions such as museums, zoos, aquaria, universities or even governments to establish such animal biobanks in their own countries and whenever possible duplicate collections in different locations, ensuring that samples requests to Frozen Ark collections are Nagoya-compliant.

IS THE GOAL TO COLLECT ALL SPECIES OR ALL ENDANGERED SPECIES?

In theory, we would like to have a representation of all the endangered species within our Consortium collection, but this in itself is an ambitious goal to achieve. Firstly, the list of endangered species is continuously changing, secondly many species that are already potentially endangered will go extinct without being discovered. To put it into perspective, from the ~1.4 M animal species formally described in the Catalogue of Life only a very small fraction (~61 000) had their conservation status assessed in the latest IUCN red list (2016), and from these, 20% were included in a threatened category. One of our present aims is to identify existing sampling gaps and develop a method for sample prioritisation.

CAN YOU ONLY USE SAMPLES TAKEN FROM LIVE ANIMALS OR COULD YOU USE DEAD, OR EVEN EXTINCT ANIMALS SUCH AS THE WOOLLY MAMMOTH?

It depends. For Frozen Ark purposes, our samples are mainly collected from live animals or recently deceased animals (taken from road-killed, or from post-mortem processing of zoo animals). However, for other genetic applications we can still extract short fragments of DNA from specimens that died decades, hundreds or sometimes thousands of years ago depending on the way they were conserved (usually referred to as ancient DNA or aDNA). Ancient DNA can be recovered from archaeological or palaeontological finds, museum specimens, fossil remains, etc.

HOW OLD CAN A SAMPLE BE AND STILL BE USEFUL?

Provided that environmental conditions are suitable, DNA can be preserved for a long time. For instance, in 2013, frozen remains of a woolly mammoth were found in the Siberian permafrost. It was estimated that it had been there for 28,000 years, at an average temperature of -10◦C, but still contained DNA of high quality. If DNA is stored in liquid nitrogen at -196◦C, it should survive intact for many hundreds, and possibly thousands of years. The utility of a sample highly depends on the intended use for that sample. For example, cryopreserved sperm for 20 years in liquid nitrogen has been used successfully in the conservation programme of the black-footed ferret, but we just do not know (as nobody tested it) for how long these cryopreserved cells would still be successfully used to produce new offspring. We are currently testing (with colleagues at the Global Genome Biodiversity Network) how DNA degrades at -80 degrees, this isn’t known accurately yet – both in terms of the timescale or level of degradation.

WHAT SPECIES DO YOU HAVE SAMPLES FROM THAT ARE EITHER EXTREMELY RARE OR EXTINCT?

A few examples of the samples we held at Nottingham are: the Scimitar-horned oryx (Extinct in the Wild), Amur leopard (critically endangered), Colombian spider monkey (critically endangered), Siberian tiger (endangered), Flores scops owl (endangered), and dhole (Asiatic red dog) (endangered).