The Frozen Ark Project – Biobanking endangered animal samples for conservation and research

In the midst of the current ‘Anthropocene’ period and its resultant species decline, The Frozen Ark Project has a vital role to play in safeguarding the world’s ‘at risk’ species. Dr. Mafalda Costa and Prof. Michael W Bruford explain more about this important Project…

Global biodiversity is declining at an overwhelming speed, with many species on the path to extinction as a direct consequence of human activities. This not only compromises the survival of the other species that share the planet with us, but also reduces our quality of life and endangers our own future.

According to the latest edition of the WWF’s Living Planet Report (WWF 2016), worldwide populations of vertebrates have already declined by 58% in the period spanning 42 years leading up to 2012. It is predicted that if no action is taken this will amount to a loss of two-thirds of the world’s vertebrates by 2020. In another study on vertebrates, a detailed analysis focused on 177 mammal species found that all of them have lost 30% or more of their geographic ranges and more than 40% have experienced severe population declines (Ceballos et al 2017). Invertebrate species are not faring better, with countless having already disappeared due to habitat destruction. As an example, a recent study reports that during the last 27 years the total flying insect biomass in Germany’s protected areas have declined by more than 75% (Hallmann et al 2017).

With each species that disappears, vast amounts of information about their biology, ecology and evolutionary history is irreplaceably lost. The Frozen Ark Project (www.frozenark.org), a charity with headquarters at the University of Nottingham, is one of the many organisations that are working hard to halt the loss of biodiversity by ensuring that all biological material that has been collected from threatened animal species is properly curated and used responsibly and sustainably in conservation, management and research. We aim to achieve this goal by: i) coordinating global efforts in animal biobanking; (ii) sharing expertise; (iii) offering help to organisations and governments that wish to set up biobanks in their own countries; (iv) providing the physical and informatics infrastructure that will allow conservationists and researchers to search for, locate and use this material wherever possible without having to resample from wild populations.

The origins of the Ark
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 Dame Anne McLaren, a leading figure in developmental biology.

Starting in 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 almost all Partula species within just 15 years, because of a governmental biological control plan that went horribly wrong (Clarke et al 1984). 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 introduced to the islands in the ’70s, 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. Currently, Partula species (including some that later became extinct in the wild) are held in 15 American and European zoos and a few promising reintroductions have since been carried out (e.g. Coote et al 2004).

The extinction story of the Partula snails resonated with Bryan and Ann, who realised that systematic collection and preservation of tissue, DNA and viable cells of endangered species should become standard practice, ultimately inspiring the birth of Frozen Ark.

Partnerships: connecting the World’s animal biobanks
The Frozen Ark Project operates as a federated model, building partnerships with organisations worldwide that share the same vision and goals. The founding partners of the Frozen Ark consortium include the (i) University of Nottingham, which houses the office and laboratory space designed specifically for the project, (ii) the Natural History Museum (NHM) in London, which provides dedicated space for the preparation and storage of Frozen Ark samples and stores duplicate collections from other Frozen Ark partners, and (iii) ZSL, which oversees the Partula snail conservation programme and initially facilitated the relationship with the zoo community.

The Frozen Ark consortium has grown steadily since the project’s launch, with new national and international organisations joining every year. The 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. The activities of the charity are driven by the Board of Trustees, with the help of an Advisory Board. The research work is now being carried out at Cardiff University under the supervision of the Frozen Ark’s Interim Director, Professor Mike Bruford.

Biobanking: preserving genetic material for the present and future
When the Convention on Biological Diversity (CBD) was signed in Rio in 1992, biodiversity conservation, and its sustainable use, was meant to become a priority for the international community. In 2010, the CBD 196 signatory countries agreed on 20 ambitious biodiversity targets to reverse the current declining trend by 2020 and address its failure to meet targets set for 2010. Aichi Target 13 addresses the loss of genetic diversity of domesticated species, their wild relatives, and, importantly, socio-economically and culturally valuable species, within which category many endangered species fall. The overall purpose of this target is “to minimize the erosion of genetic diversity, which occurs when genes are lost from a gene pool”. Species with small or isolated populations (gene pools) are particularly at risk of genetic erosion. The UK has committed to “maintain and safeguard genetic diversity through the development and application of strategies, which allow for the different genetic characteristics of a species to continue to exist over time” and, to this effect, both in-situ and ex-situ conservation of selected species should be enhanced inside and outside protected areas. Zoos and aquariums are critical to help the UK reach this target. To support conservation breeding programmes, viable biological material is needed to ensure that genetic diversity can be maintained through assisted reproductive technologies, DNA profiling and by understanding the genetic origin and diversity of captive and closely-managed wild populations.

Biological material (e.g. tissue or blood) from animals held in zoos and aquariums can be easily obtained post-mortem or from living animals during routine veterinary work. Given the high number of threatened species, biobanks can provide a safe storage for many types of biological material, particularly the highly valuable germ cells (sperm and eggs), which are key for the successful management of small populations. The example of the black-footed ferret (Mustela nigripes) illustrates how cryobanked material, together with an effective management and captive breeding programme, can make all the difference. The species was listed as “extinct in the wild” in 1996, but has since been reintroduced back to its habitat and is now gradually recovering. More recently, researchers were able to return lost genetic diversity to the extant population by using 10 to 20-year-old cryopreserved sperm and artificial insemination. The resulting offspring were integrated in the ex-situ breeding programme, enhancing the gene diversity of the species (Howard et al 2016).

Since the 1970s, the role of cryopreserved biomaterial in UK conservation programmes has been rapidly growing. While preserved genetic material from endangered species is already available at several UK institutions, and potentially accessible on a one-off basis from individual collections, we are still lacking a central reference point to link these often isolated biobanking efforts. Additionally, even when tissue or DNA is available, its associated metadata is not, rendering these samples of little value in a conservation or research context. The Frozen Ark and its partners continually receive requests for samples, yet few institutions possess the means or the infrastructure to actively handle these requests, emphasising the need for a coordinated effort to improve the general state of many UK animal frozen collections. The importance of frozen samples is becoming clearer every day and we expect that demand for high quality DNA will increase exponentially in the upcoming years, to match the increase in the number of studies dedicated to answer questions arising in conservation, genomics, proteomics, gene regulation and transcriptome processes. To address the challenge, we have joined forces with the NHM, the National Museums Scotland, the University of Edinburgh and the recently launched European Association of Zoos and Aquaria (EAZA) biobank, which includes a biobanking partner at the RZSS, to enhance the UK’s frozen collections for non-model and endangered animal taxa. This initiative will enable us to create the infrastructure that will link the many diverse frozen collections of animal genetic material found in zoos, aquariums, museums, research institutes and universities across the UK. The aim is to harness existing samples, create common quality standards among partners and collaborators, develop protocols for sample collection, transport and storage and then make these samples available for conservation and research purposes. While it is anticipated that some institutions will wish to retain their own frozen collection, key specimens (high priority taxonomic groups, unique specimens, extinct populations and endangered species) will be duplicated within one of our core facilities.

The development of this overarching infrastructure in the UK is particularly timely due to the 2014 implementation of the Nagoya Protocol on access and benefit sharing of genetic resources. The Nagoya Protocol is a supplementary agreement to the CBD, which was designed to oversee the movement of genetic material across borders and to create incentives to ensure equitable sharing of benefits arising from genetic resources found in developing countries. Although important, the Nagoya Protocol has unintentionally hindered the circulation of samples between countries for non-commercial research purposes, especially for much needed research on threatened taxa. Harnessing and managing existing genetic material in the UK is more likely to be Nagoya-compliant, efficient, and sustainable for the future.

The database: making samples available to the conservation and research communities
Since its inception, it has been an ambition of Frozen Ark to establish a global database to identify where genetic material from endangered species is already stored in institutions around the world, to identify which gene pools need to be prioritised for collection and preservation, and to make existing stored samples available for conservation and research. We are now closer than ever to achieve this aim. With our UK partners, we are currently developing a comprehensive web-enabled sample database using the open-source software Specify (The University of Kansas Biodiversity Institute). This software is already routinely used in international museum collections and animal biobanks. Specify allows the rapid and efficient aggregation of diverse datasets into a single repository that can be queried, updated and accessed freely. There is also the potential for links to other databases used in museums and zoos. This bioinformatics resource will provide a user-friendly web environment for sample requests, sample-use monitoring, reporting, updating, and gathering of statistical information on how genetic material is being used. In the upcoming months, we will be collating the databases of UK organisations that wish to increase the visibility of their frozen collections and make them available for requests. It is expected that, on the long-term, the resource will expand to accommodate international collections, including the ones of Frozen Ark partners.

Research: DNA degradation in the field and in the Ark
The main research interest of Frozen Ark 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 (e.g. Nagy 2010; Brunstein 2012). To address this research need, we will be running a series of experiments on DNA degradation. Andrew Beazer is investigating how the interaction of temperature and relative humidity contributes to DNA degradation over time, and comparing methods for short-term sample storage under the tested conditions. The aim is to replicate a range of real field conditions in the laboratory to provide researchers with information on how best to store and transport tissue samples, without compromising the overall DNA quality, before these are safely deposited in a long-term storage biobank. Laurie Fabian is focused on what happens with samples that are already securely stored in the biobank. Specifically, he is looking at the effects of increasing freeze-thaw cycles on DNA quality. His research aims to provide animal biobanks with a reference point on how many times samples can be frozen and thawed before the DNA is deemed “too damaged” for further use.

There is still a long way to go…
Despite all that has been achieved by the project so far, we are still a long way from fully realising Bryan and Ann’s dream for the Frozen Ark, but the current team is committed to deliver the ambitious programme that we set out for ourselves at the end of 2016. We are grateful for all the support the project received so far from the research, museum, zoo and aquarium communities and from our funders, individual donors, students and Friends of the Ark. We predict that once the online database is established and made available to end-users it will become an invaluable resource for UK researchers and conservationists. We hope other institutions at home and abroad feel encouraged to establish their own animal biobanks and join the Frozen Ark consortium, as a concerted worldwide effort has a much better chance of succeeding.

References:

• Brunstein J (2012) Freeze-thaw cycles and nucleic acid stability: what’s safe for your samples. Medical laboratory observer 47(9): 44-47.
• Ceballos G, Ehrlich PR & R Dirzo (2017) Biological annihilation via the ongoing sixth mass extinction signaled by vertebrate population losses and declines. Proceedings of the National Academy of Sciences 114(30): E6089–E6096.
• Clarke B, Murray J & MS Johnson (1984) The extinction of endemic species by a program of biological control. Pacific Science 38(2): 97-104.
• Coote T, Clarke D, Hickman CS, Murray J & P Pearce-Kelly (2004) Experimental release of endemic Partula species, extinct in the wild, into a protected area of natural habitat on Moorea. Pacific Science 58(3): 429-434.
• Hallmann CA, Sorg M, Jongejans E, Siepel H, Hofland N, Schwan H, Stenmans W, Müller A, Sumser H, Hörren T, Goulson D & H de Kroon (2017) More than 75 percent decline over 27 years in total flying insect biomass in protected areas. PLoS ONE 12(10): e0185809.
• Howard JG, Lynch C, Santymire RM, Marinari PE & DE Wildt (2016) Recovery of gene diversity using long-term cryopreserved spermatozoa and artificial insemination in the endangered black-footed ferret. Animal Conservation 19: 102-111.
• Nagy ZT (2010) A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity and Evolution 10(1): 91-105.
• WWF (2016) Living Planet Report 2016. Risk and resilience in a new era. WWF International, Gland, Switzerland.

Images supplied by Mafalda Costa

About the Authors: Mafalda Costa is a research officer at the Frozen Ark responsible for the day-to-day management of the project, including partner liaison, developing grants and publications, research and dissemination. Mafalda did her PhD on the conservation genetics of European polecats at the University of Lisbon and Cardiff University.

Michael W Bruford is Professor of Biodiversity at the School of Biosciences, Cardiff University and the Interim Director of Frozen Ark and is a conservation geneticist. He is co-Chair of the IUCN SSC’s Conservation Genetics Specialist Group. If you have an interest or want to find out more about Frozen Ark, please do not hesitate to get in touch with us: info@frozenark.org.