GENETIC RESCUE TOOLKIT

A growing number of organizations are growing collections of cryopreserved tissue and cell line samples, including the San Diego Frozen Zoo with over 1,000 taxa preserved, the American Museum of Natural History, the Frozen Ark, Northeastern University’s Ocean Genomics Legacy, and the for-profit company Cryogenetics (which has a special focus on commercial fisheries). Bio-banking should be a more common practice and coupled with any comprehensive and targeted sequencing initiatives.

There are many levels of genomic sequencing techniques that can be used for conservation purposes.

The field of genomics forms the foundation for genetic insight and intervention applications; however, any use of genomics research for conservation begins with one building block: a reference genome. While there are many genome initiatives, they have accumulated primarily the genomes of non-endangered species, hence there is a lack of available reference genomes for conservation purposes. Without high quality reference genomes, genetic studies for conservation are limited to older, much less informative sequencing techniques, and therefore have fewer conservation applications. A number of challenges need to be overcome to generate reference genomes for managed and threatened species, but with more sequencing, the full potential of genetic rescue innovations can be developed.

Metabarcoding approaches enable the characterization of entire ecological communities without prior knowledge of species likely to be present. This application of eDNA monitoring contributes a powerful genomic tool for conservation that will continue to improve as the technologies and bioinformatic abilities are developed. A large-scale program centered around cataloguing biodiversity in different habitats and locations can provide critical baseline information and identify critically imperiled habitats and may eventually allow for population level inferences of target taxa without the need for invasive or labor-intensive sampling. Notably, as invasive species expand into new marine environments, early detection can be difficult. eDNA enables a rapid assessment of the expanding presence of an invasive species which can greatly aid in controlling its spread  

The level of resolution provided by eDNA data is contingent upon the quality of genomic data used to sort and allocate sequence identities. The application of eDNA monitoring to specific conservation goals will require genomic references for target species. Narrowing the focus of questions to be addressed with eDNA can decrease the references needed for an effective database and reduce the processing burden.  

Localized populations or groups can be compared across the genome, transcriptome, and proteome to determine genetic basis of adaptation to local environments and responses to environmental stimuli. Such analyses can guide the formulation, execution, and monitoring of species management planning through enhanced understanding of adaptive and demographic processes related to landscapes- or “Landscape Genomics”. 

These insights have been useful in guiding connectivity planning for fragmented populations, and identifying instances where populations have extremely low genetic diversity and require genetic rescue. These insights are also extremely important in conservation planning in response to climate change and for understanding organismal response to disease and stress. Collectively, genomics tools applied appropriately can uncover insights on populations with various levels of suitable adaptations providing an opportunity to manage populations with greater precision and intent. Such information can ultimately transform conservation practice from a “one size fits all” to more targeted restoration and management.  

More conventional technologies include in vitro fertilization (IVF) and even somatic cell nuclear transfer (aka cloning), which require gametes (spermatozoa and oocytes) from the target species. Two newer methods exist in the absence of living oocyte donors for mammals: interspecies somatic cell nuclear transfer (iSCNT) and stem cell embryogenesis (SCE). iSCNT has been demonstrated to successfully produce surviving offspring of several wild felids (Gómez et al., 2009) and endangered gray wolves (Kim et al., 2007). There must be cellular and reproductive compatibility between the donor and surrogate species to generate viable embryos and pregnancies. Comparative genomics and epigenomics should be used to determine the best surrogate. The black-footed ferret is the subject of a restoration strategy centered around this technology (Wisely et al., 2015). 

The revolutionary potential of SCE technology is that it can reduce the reproductive resources needed to yield offspring. A skin cell is first transformed into a stem cell (called an induced pluripotent stem cell, or iPSC). Once a stem cell, it can be reprogrammed to develop into sperm or egg cells. Once sperm and egg cells are made scientists can apply IVF to generate an embryo, implant the embryo into a surrogate mother, and give birth to a new unique individual. This process is currently being pioneered to save the Northern White Rhinoceros (Ceratotherium simum cottoni) from extinction. Currently, SCE has only been achieved in mice and stem cell technologies overall are most advanced in mammals. 

iSCNT and SCE are not yet viable options for reproducing endangered birds. The only technology that allows the cryopreservation and reproduction of cells in birds is germline transfer/transmission (GTT) using primordial germ cells (PGCs), which was developed in 2006 (van de Lavoir et al. 2006).  GTT requires the collection of cells from the target species. Like iSCNT, once donor cells are collected they can be transferred and transmitted through a surrogate species reproductive system. Research teams in 2012 transmitted domestic chicken PGCs through a male guinea fowl using this technique (van de Lavoir et al. 2012). While the technique of GTT could be universal for all birds, PGCs have only been successfully cultured for the domestic chicken, which currently prevents the technique from being applied for conservation of wild species. 

The major complication for reproductive technologies is that every species’ reproductive biology is unique, even among very closely related species. Substantial investment in genomic resource generation, cell culture conditions, and gamete/embryonic transfer techniques is required as these must be adapted for surrogate and donor species. Additionally, such endeavors require extensive planning to specify short- and long-term goals, and ways by which success will be measured. 

Gene drive methods are in advanced development in mosquitos as a mechanism to combat malaria (Hammond et al. 2016) and testing is ongoing in rodents (Leitschuh et al. 2018). However, the technique is controversial due to the concerns over uncontrollable negative effects on ecosystems or from a release of gene drive to unintended areas. Other research outlines large technical hurdles facing gene drives, such as inbreeding, poor homing, and development of resistance mutations (Unckless et al. 2018). Overcoming these challenges will need to be coupled with development of gene-drive systems with population percentage thresholds and/or mechanisms for spatial/temporal control.