To what extent do we understand DFTD, and what are the conservation options for Tasmanian Devils? ABIGAIL SHEPPARD CANDIDATE NUMBER: 8972, CENTRE NUMBER: 58625 Table of Contents
Abstract ...... 3 Glossary ...... 3 Introduction ...... 5 Literature Review ...... 6 History of the Tasmanian Devil ...... 6 What is DFTD? ...... 9 Allograft Theories of Transmission ...... 14 Immune Response to DFTD ...... 16 Genetic Diversity and the MHC ...... 18 Non-Allograft Theory of Transmission ...... 22 DFTD Evolution ...... 23 Tasmanian devil conservation ...... 25 Discussion ...... 25 Which is the stronger theory: The Allograft or Non-Allograft Theory? ...... 26 What are the conservation options for Tasmanian devils? ...... 27 Captive breeding ...... 27 Vaccine development ...... 28 Disease suppression through culling ...... 29 Depopulation, Translocation and Population Reinforcement ...... 30 Fencing ...... 31 Issues with management options: ...... 32 Conclusion ...... 32 Evaluation ...... 34
2 Abstract
Devil Facial Tumour Disease (DFTD) is an emergent transmissible cancer exclusive to Tasmanian devils (Sarcophilus harrisii) which is threatening the species with extinction in the wild. Research began ten years ago (Pye, 2016)7, when nothing was known about the tumour and little was known about Tasmanian devils. The research since then has covered the cause and pathogenesis of DFTD, the immune response of the devils, the immune evasion mechanisms of the tumour, the transmission patterns of DFTD, and the impacts of DFTD on the ecosystem. I have collated this information and put it into the context of conservation strategies designed to mitigate the impacts of DFTD on Tasmanian devils and the Tasmanian ecosystem, and I have also evaluated how much is really known about DFTD.
Glossary
2-microglobulin (B2ml): a protein that stabilises the heavy chain of MHC I molecules.
Allograft: transplant of biological tissue from one individual to another of the same species.
Chromosome painting: a technique in which chromosome-specific probes, labelled with colour fluorescent dyes, are hybridised with a cellular karyotype to identify individual chromosomes of a cell and the rearrangement of nom-homologous fragments that occurs by end-joining DNA repair mechanisms.
Demethylation: a decrease in methylation patterns, resulting in a genome that may be hypo-methylated.
Hayflick limit: the number of times a cell population can divide before telomeres shorten to a critical length and cell division ceases.
Immunohistochemical testing: the process of selectively imaging antigens (e.g. proteins) in cells of a tissue by exploiting the principle of antibodies binding specifically to antigens.
Karyotype: the arrangement of chromosomal material in a cell. Rearrangements, deletions, duplications and other chromosomal mutations may be observed by examining the karyotype.
Kinship: the genetic relationship, either based on a known pedigree or molecular data, between one animal and another.
3 Major histocompatibility complex (MHC): a set of molecules primarily involved in the presentation of antigenic peptides to the immune system; first discovered for their role in graft rejection.
Methyl-CpG binding domain: enzymes that cause active demethylation.
Methylation: an epigenetic modification of DNA that may lead to changes in gene expression.
Microsatellite: (short tandem repeats) regions of highly repetitive DNA (usually two to five base pairs in length), widely used for many ecological genetics applications, such as diversity surveys, quantifying population differentiation or percentage analysis.
miRNA: small non-coding RNA molecules that regulate gene expression.
Myelin basic protein (MBP): a protein encoding a gene that is expressed in Schwann cells and an indicator that DFTD originally arose in this tissue.
Natural Killer (NK) cells: cytotoxic lymphocytes of the innate immune system that can kills cells lacking cell surface MHC.
Reverse transcriptase-polymerase chain reaction (RT-PCR): laboratory technique for studying gene expression through generating complementary DNA transcripts from RNA.
Schwann cell: cell of the peripheral nervous system; progenitor of DFTD.
Sex-determining region (SRY): a gene found on the Y chromosome that is involved in mammalian sex determination.
Single nucleotide polymorphism (SNP): a sequence variant that occurs as a substitution in the alignment of two or more sequences of bases.
4 Introduction
In July 2017, I shadowed a Biological Sciences PhD student at Southampton University. She was in the process of finding peptide candidates for a vaccine against a “transmissible cancer” called DFTD, or Devil Facial Tumour Disease. I was allowed to help her run PCRs and gel electrophoreses on Tasmanian devil genes, which I really enjoyed. At the time, I had only vaguely heard of such a thing as a cancer that can be passed between individuals, so the concept was incredibly thought-provoking to me, and I decided to research it further. This resulted in me completing an EPQ on the topic, as I believe the implications of understanding such a devastating disease can help further cancer research and our methods of saving endangered species.
The Tasmanian devil (Sarcophilus harrisii) is the largest living marsupial, belonging to the Dasyuridae family. Listed as Endangered on the International Union for Conservation of Nature and Natural Resources (IUCN) Red List, the global Tasmanian devil population has declined by more than 60% in the last ten years (IUCN, 2017)1. Research indicates that an invariably fatal infectious cancer called Devil Facial Tumour Disease (DFTD)
1 The International Union for Conservation of Nature is the global authority on the status of the natural world and the measures needed to safeguard it. It is an extremely reputable source and it is classed as an official source of information regarding species and their conservation status. It gives a comprehensive review of the conservation levels and threats to thousands of species and it often referenced by top researchers and governments. It is also used to make official decisions about the conservation efforts for endangered species by governmental bodies and non-
5 is responsible for the decline. DFTD currently occurs across the majority of the geographic range of the Tasmanian devil and continues to spread at variable rates, within the range of 7 to 50km per year, depending on the location (McCallum, 2007)2. Mark-recapture data from the most intensively studied population at the Freycinet National Park in Tasmania, estimated a decline in total population size of 30% in the 3 years after the disease first arose, with an annual decline in the adult (two years and above) population of 50% (Lachlish, 2007)3. Research on the nature of DFTD only began fairly recently, and there is still much to understand about it. The priority now should be to conserve the wild population numbers with the aim to eradicate DFTD in the future, before Tasmanian devils face extinction.
Literature Review
History of the Tasmanian Devil
The Tasmanian devil line has little known ancestry, but it is assumed that it arose in the mid-Miocene Epoch, 16 to 5 million years ago. Few carnivores could survive the switch from warm, moist conditions to cold, dry, arid conditions and so major extinctions resulted. Demise of Australian and Tasmanian megafauna (species with an average body mass higher than 45kg) was swift and extensive; the majority of species with individuals weighing over 40kg became extinct within a very short space of time, leaving only the Tasmanian devil, the quoll and a single species of thylacine extant. Devils were once found across mainland Australia but became
profit organisations. Therefore, I believe it is an incredibly reliable source to attain information about Tasmanian devils from. 2 Distribution and Impacts of Tasmanian Devil Facial Tumor Disease (McCallum, 2007) has been cited 143 times and it a well refenced piece of literature. It was written by several experts in the field of DFTD, such as Shelly Lachish and Clare Hawkins. It has been cited in multiple research papers that I have used in my project, such as Greuber, 2015 and Lachish, 2010. Both of these are also well-cited and extensively peer reviewed papers, leading me to believe that McCallum’s paper is reputable and reliable to cite. Furthermore, it was published in the journal EcoHealth, an international, peer reviewed journal focused on the integration of knowledge between ecological and health sciences. It is the official journal of the EcoHealth Alliance, a world-renowned non-profit organisation, thus I believe that it is a reliable source that can be trusted. 3 The Impact of Disease on the Survival and Population Growth Rate of the Tasmanian Devil (Lachish, 2007) was published in the Journal of Animal Ecology, as highly reputable journal for the most up to date scientific research. As a result, it has been cited 121 times, again by experts in the field including David Pemberton, Hamish McCallum and Ruth Pye, whose comprehensive literature formed much of a basis for my EPQ. It has also been extensively peer reviewed, meaning that it is a highly reliable source.
6 extinct on the mainland 3000 to 4000 years ago (Owen & Pemberton, 2011)4. Since the devils became isolated on the island of Tasmania 10,000 years ago, the population has suffered at least three population crashes. However, the lack of genetic diversity actually dates back much further than these recent genetic bottlenecks: it appears that devil population declines have coincided with changes in the abundance of prey, associated with El Niño oscillation activity about 2000 to 4000 years ago and the Last Glacial Maximum, 22,000-48,000 years ago (Owen & Pemberton, 2011)5. Devils are now renowned for their lack of genetic diversity which has had a critical role in the spread of DFTD, and has led it to be listed as endangered on the IUCN Red List of Threatened Species1.
Since the extinction of the Tasmanian tiger (Thylacinus cynocephalus) during the 1930s, the Tasmanian devil inherited the title of largest living carnivorous marsupial. Specialist scavenging has become an important niche; Tasmanian devils come from a line of polyphagous (they can eat a variety of foods), opportunistic feeders and solitary hunters. Predator-scavenger niches are apparent with evidence from the extinct thylacine (often thought to be the devil’s closest relative, the devil-thylacine relationship is close enough to infer similar evolutionary traits in the Australian and Tasmanian environments). There are seven extinct genera of thylacine, ranging from 4 to 18 kilograms and varying from smaller, foraging species to larger, hunting carnivores. Alternatively, devils could have descended from a dwarfed version of the S. larinus from the Pleistocene epoch, or possibly a different species that co-existed with the S. larinus (Owen & Pemberton, 2011). 5 Tasmanian devils are described as having “considerable evolutionary fine-tuning that has allowed them to cope with the dramatically altered climates and escalating environmental stress over the last five million years” (Morrisin, 1988)6. However, the unique niche they inhabit is at risk of being filled by other apex predators, such
4 Tasmanian Devil: A Unique and Threatened Species (David Owen and David Pemberton) was the first book I read on Tasmanian devils and DFTD. It is one of the only published pieces of work about the Tasmanian Devil that is not a scientific article, so my choices were fairly limited. It primarily contains anecdotal evidence for the history of Tasmanian devils, that is not scientifically reliable enough to reference, but it does discuss the potential evolutionary background of the devil (mostly unknown), and their wild behaviour such as latrine and mating behaviour which was important background reading. 5 David Owen is the author of nine natural history novels. Tasmanian Devils: A Unique and Threatened Animal is one of the only pieces of printed literature on the species and DFTD. However, it is a little out of date as it was published in 2005. This was before, for example, the allograft theory of transmission was being researched as extensively as it is now. This means some of the comments about the spread of DFTD and the effect on the population are a little out-dated. Having said this, it is a very good resource for the evolutionary and natural history of Tasmanian devils and closely related species such as the thylacine. 6 Morrison 1988 The Voyage of the Great Southern Ark is a documentation by Maggie Morrison of the development of Australian and Tasmanian geography and fauna following the multi-billion-year-old evolution of the continent and discussing topics of importance, such as the Tasmanian devil. However, it is not a scientific journal and was
7 as feral cats or foxes. The consequences of this would prove disastrous for other native species, such as birds and rodents who are not primarily targeted by Tasmanian devils, but whose population numbers would drop rapidly if cats or foxes became the apex predator. It is likely that the European red fox would fill the Tasmanian devil’s niche; with an abundance of food such as small mammals, reptiles and ground nesting birds and minimal competition as a result of the devil’s absence, allowing for quick establishment. Although devils eat these organisms, their diet also consists of large amounts of scavenged meat. As a result of the extinction of Tasmanian devils, more live animals would be eaten, so the replacement of the devil by the European fox would be have a large impact. The establishment of the fox on mainland Australia has led to irreversible ecosystem changes, as more small mammals and birds are eaten and less carrion is removed by devil scavenging behaviour. Extinction in the wild of the Tasmanian devil would have profound impacts; they are an internationally recognised species with an iconic status and inhabits a unique ecological niche as the largest extant carnivorous marsupial, exclusive to the island of Tasmania (Owen & Pemberton, 2011)5. Many of Tasmania’s species are endemic and the red fox particularly would be a large threat to these unique species, as they will not have faced a similar threat in their evolutionary history, and would therefore not be adapted to cope.
There are currently many management options proposed for the conservation of the Tasmanian devil. After collating information about DFTD, this paper will weigh up the most effective management schemes in the discussion. These include captive breeding programs, vaccine development, culling, depopulation, translocation, population reinforcement and fencing (Pye, 2016)7, with the aim of finding the most effective method for preventing the Tasmanian devil from going extinct.
therefore not peer reviewed for scientific accuracy and a certain ‘artistic licence’ may have been used. It was also published in 1988, long before DFTD had been discovered, and so may be out of date and therefore not as reliable. 7 R. Pye is a renowned researcher in the field of DFTD and has produced many papers on its pathology, and impact, such as A Second Transmissible Cancer in Tasmanian devils (Pye, 2015), Demonstration of immune responses against devils facial tumour disease in wild Tasmanian devils (Pye, 2016) and Regression of devil facial tumour disease following immunotherapy in immunised Tasmanian devils (Pye, 2017), including new research at the forefront of the field on a second type of DFTD (DFTD2) commented on later. She researches at the University of Tasmania, a highly regarded university and her work is published in The Journal of Veterinary Pathology; a well peer reviewed, and therefore prominent journal in the veterinary industry that can be trusted. This article was also co-written by A. Kreiss, who has also produced various well-reviewed pieces of literature on DFTD and is a researcher at the University of Tasmania. Therefore, I believe this source to be reliable.
8 FIGURE 1: DFTD DISTRIBUTION FROM 1996 TO 2015. DOTS REPRESENT EACH 1 CASE OF DFTD CONFIRMED BY HISTOLOGY, STARS REPRESENT MAIN LOCATIONS OF RESEARCH. TAKEN FROM (PYE, 2016).
What is DFTD?
Devil Facial Tumour Disease (DFTD) is an emergent, transmissible cancer exclusive to Tasmanian devils. It is passed between devils by biting, usually whilst fighting over mates, and is threatening the entire species with extinction (Pye, 2016)7. Cancer is the result of uncontrolled cell division that evades the host’s immune system surveillance function and DFTD is especially unique in that it is a clonally transmissible cancer. This means that it spreads from one individual to the next and outlives its host in the process. DFTD in the wild is aggressive and invariably fatal, exacerbated by Tasmanian devil’s limited habitat; being confined solely to the island of Tasmania. Fatality normally occurs in less than 6 months of contracting the disease, with death resulting from starvation, metastases or organ failure (Lachlish, 2007)3.
9 It is highly debated how DFTD first arose. Genomic analysis (Murchison, 2012)8 indicates that the primary tumour appeared in a female devil less than 20 years ago. First observed in north-eastern Tasmania in 1996, DFTD has spread rapidly to affect most of the devil’s geographical range, up to 90% of individuals in certain locations, and this is certainly increasing (as shown in Fig 1). Carcinogens, infectious agents and genetic predisposition are the usual inciting causes of cancer. A review was carried out by the Tasmanian conservation program Save the Tasmanian Devil in 2008 of chemical residues such as metals, herbicides and pesticides to see if they were a causing factor of DFTD found in both healthy devils and those affected by DFTD, (Ross, 2008)9. Residues of dioxins, dibenzofurans, polychlorinated biphenyls, brominated diphenyl ethers, arsenic, cadmium and lead were detected in the fat or liver of most devils tested, but at a similar level to those found in other species at the top of the food chain. There was no significant difference in residue levels between healthy and diseased devils, suggesting there is no link between chemicals and DFTD (Ross, 2008)9.
When the transmission pattern of DFTD first became obvious, a viral aetiology was suspected. However, it is now apparent for reasons discussed later, that the tumour cells are the sole aetiological agent. This description of event in nature is incredibly rare, as the tumour must not only infect the host, but also evade the host’s immune mechanisms to colonise the tissues (see “Immune Response to DFTD” section). This is only recorded
8 E. Murchison is a Reader in Comparative Oncology and Genetics at the University of Cambridge, Department of Veterinary Medicine. Her research group works on transmissible cancers in dogs and Tasmanian devils. In 2009, (cont.) she was awarded an NHMRC Overseas Biomedical Fellowship to the Wellcome Trust Sanger Institute, UK, where she was involved in the sequencing the Tasmanian devil genome as well as genetic analysis of DFTD and CTVT. (cont.) She has received many honours and awards including the Philip Leverhulme Prize 2014 and the Cancer Research UK Future Leaders in Cancer Research Prize 2014, and her TED talk “Fighting a contagious cancer”, has been watched more than 400,000 times. Given the prestige of the universities attended by E Murchison, and her reputation and success in the field, I believe her papers and research are very reliable. Furthermore, the paper I have referenced here The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer, has been cited more than 150 times. 9 Persistent Chemicals in Tasmanian Devils (Ross, 2008) is not a paper published in a scientific journal, so it is hard to assess the reliability of it because it has not been peer reviewed or checked for accuracy. However, the research was funded by Save the Tasmanian Devil, a large program backed by the Tasmanian governments aimed at conserving the species. Since this is the main conservation movement for Tasmanian devils, it is likely that the research they have carried out is scientifically accurate, and it has a comprehensive list of references of very reputable sources, and so is really a compilation of the results of these using data from studies ranging from 1983 to 2008. For this reason, although it is not strictly a scientific journal, I assume it provides reliable information.
10 in a few other cases; such as canine transmissible venereal tumour (CTVT) and more recently in soft-shelled clams (Weiss, 2015)10.
The pathology of DFTD has been extensively researched (Pye, 2016)7. The most prevalent characteristic is the presence of a locally aggressive tumour on the facial area, primarily inside the mouth on the gingival mucosa hard palate and lip (shown in photos 1 to 3 in Fig 2), on the head (cheek, lips and muzzle) and on the neck. This is due to the method of transmission of DFTD via biting when males fight aggressively over female mates. Masses within the oral cavity can prevent feeding as well as occluding (blocking) eyes and damaging whisker beds. Often, tumours show disruption of the epithelium, necrosis (death of most or all cells in an organ or tissue due to disease, injury or failure of the blood supply, exudation and bacterial contamination as well as a very common finding of metastases: 65% of cases in lungs, lymph nodes and kidneys (Loh, 2006)11 (shown in photo 4 in Fig 2).
10 The Clammy Grip Of Parasitic Tumors (Weiss, 2015) was published in Cell Press, one of the most well known and internationally recognised scientific journals. It has been cross referenced in Pye, 2016 and references several papers I have cited, such as Pearse and Swift, showing its relevence to DFTD. It was a valuable source of extra information regarding transmissible tumours and the similarities and differences. I believe it is a reliable source of information. 11 Richmond Loh’s The Pathology of Devil Facial Tumour Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii) is one of the most important papers in the area of DFTD research. It gives comprehensive information and a detailed summary of the pathology of DFTD tumours, compiling the results of many cytological and histological (cont.) tests and has been referenced over 100 times by other key researchers in the field, such as (Siddle, 2007), (Murchison, 2012) and (Lachlish, 2007). For this reason I believe this source to be very reliable.
11
FIGURE 2: DEVIL FACIAL TUMOUR DISEASE. PHOTOS 1 AND 2: RIGHT UPPER LIP AND RIGHT CHEEK,
SOLITARY ULCERATED MASS. PHOTO 3: RIGHT UPPER GUM, MULTIPLE ULCERATED MASSES. PHOTO 4:
RIGHT AND LEFT KIDNEYS, DFTD METASTASES IN CORTEX OF BOTH KIDNEYS. IMAGES FROM (PYE, 2016).
The neoplastic cells (the formation of atypical cell growth) are arranged in nodules or bundles and are enclosed by a thin pseudo-capsule. The anaplasia (poor cellular differentiation) exhibited by DFTD cells is consistent with the highly malignant nature of the tumour. Based on the immunohistochemical findings (Loh, 2006)11, DFTD has been classified as a sarcoma, which is a malignant tumour of connective or other non-epithelial tissue. This is because it is negative for epithelial markers such as epithelial membrane antigens and cytokeratins, which are keratins specific to particular tissues and so are commonly used to identify the cell of origin of various tumours.
Deep sequencing of the DFTD transcriptome (Murchison, 2012)12, shows the tumour originates from Schwann cells. Schwann cells are the principle glia (connective tissue of the nervous system) of the peripheral nervous
12 E. Murchison is a Reader in Comparative Oncology and Genetics at the University of Cambridge, Department of Veterinary Medicine. Her research group works on transmissible cancers in dogs and Tasmanian devils. In 2009, she was awarded an NHMRC Overseas Biomedical Fellowship to the Wellcome Trust Sanger Institute, UK, where she
12 system, keeping peripheral nervous system nerve fibres alive. There are numerous subtypes, two of which are myelinating and non-myelinating Schwann cells. Myelinating Schwann cells coat the axons of neurons to form the myelin sheath and repair nerves and modulate local immune reactions. The original functional roles of these cells may have provided DFTs (devil facial tumours) with the capacity to modulate immune reactions and evade immune responses (Greuber, 2015)13. Protein expression determined by immunohistochemical analyses found 100% of primary tumours, metastases, DFTD cultured cells and mouse xenografts to be strongly positive for periaxin, a Schwann cell marker. Murchison et al. also found a high expression of MBP, a gene that encodes myelin basic protein, and that nine out of ten investigated highly expressed genes were involved in the myelination process. Further evidence for DFTD being linked to the peripheral nervous system comes from research showing a lack of chromogranin A and identified expression of proteins associated with the peripheral nervous system in DFTD cells (Loh, 2006)11. Thus, convincing evidence for Schwann cell origin for DFTD is presented; the ability of Schwann cells to instigate and modulate an immune response and the plasticity retained by mature Schwann cells may have contributed to DFTD’s evolution as a transmissible cancer (Murchison, 2012)12.
was involved in the sequencing the Tasmanian devil genome as well as genetic analysis of DFTD and CTVT. She has received many honours and awards including the Philip Leverhulme Prize 2014 and the Cancer Research UK Future Leaders in Cancer Research Prize 2014, and her TED talk “Fighting a contagious cancer”, has been watched more than 400,000 times. Given the prestige of the universities attended by E Murchison, and her (cont.) reputation and success in the field, I believe her papers and research are very reliable. Furthermore, the paper I have referenced here The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer, has been cited more than 150 times, such as in Obendorf, 2010. 13 C. Greuber’s Genomic insights into a contagious cancer in Tasmanian Devils proved to be a very useful source for this EPQ. It covers the majority of information available about the genetics of Tasmanian devils, including the proposed origins of DFTD, how genomics can save the Tasmanian devils and how emerging genetic tools can be used to facilitate greater understanding of DFTD. Furthermore, this paper provides a comprehensive glossary for the more technical terminology, many of which I have used in my EPQ glossary, as the definitions are more specific to DFTD rather than the general scientific meaning. This paper was published in Cell Press, a leading publisher of cutting edge biomedical research and review journals including Cell, Neuron, Immunity and Current Biology. Due to the publisher’s reputation, it is likely that this paper is well peer reviewed, accurate and therefore reliable.
13 FIGURE 3: DFTD NEOPLASM; TASMANIAN DEVIL. CHARACTERIZED BY LARGE ROUND CELLS WITH A UNIFORM ROUND NUCLEUS, HIGH NUCLEAR TO CYTOPLASMIC RATIO, AND BLUISH CYTOPLASM. A TENDENCY FOR THE CELLS TO AGGREGATE IS ALSO SHOWN. IMAGES TAKEN FROM (LOH, 2016)
Allograft Theories of Transmission
An allograft is the transplant of an organ or tissue from one individual to another individual of the same species with a different genotype (MedicineNet, accessed 14/11/17)14. In the case of Tasmanian devils, the tissue transplanted is cancerous, hence DFTD is infamous for its unique and disastrous transmission; as one of the few known cases of transmissible cancers.
Transmissible cancers occur extremely rarely in nature, but there is evidence for the clonality of DFTD is from both kayrotypic and genetic perspectives. Firstly, each karyotype for 11 forms of DFTD were shown to be identical, this would not be possible unless they all originated from the same animal. Giemsa banding (G- banding), used to produce visible karyotypes, showed the complexity and several abnormalities of DFTD chromosome arrangement. Tasmanian devils have fourteen chromosomes including two sex chromosomes. These are similar to humans’; females and XX and males have XY chromosomes (Figure 4). The DFTD karyotype was shown to be missing both chromosomes two, chromosome six, the long arm of chromosome one,
14 https://www.medicinenet.com/ is a medical website that provides detailed information about diseases, conditions, medications and general health. It was very useful in providing general definitions for scientific or medical terminology. It is a network of U.S. Board-Certified Physicians and Allied Health Professionals working together to provide the public with current, comprehensive medical information, and therefore I believe it is a reputable and reliable source to use.
14 both sex chromosomes and had an extra four additional unidentified markers. These abnormalities are shown in Figure 4: a) is a normal karyotype for a male Tasmanian devil with fourteen chromosomes, including XY (the example is male) and b) is a karyotype of cancer cells found in each of the facial tumours studied. It has thirteen chromosomes, no sex chromosomes, the afore mentioned abnormalities and additional marker proteins M1-M4.15 It is impossible that each DFTD of the eleven strains developed the same complex arrangement by chance; it is concluded that they are all clones derived from the same original tumour, and so the allograft theory of transmission was put forward. Further proof for this is shown in (Pearse, 2006)16; one host devil had a pericentric inversion in its bodily chromosomes, when a section of chromosome is completely reversed, but its DFT had no such inversion. This is a complicated and unusual abnormality, meaning that it is unlikely that the DFT could have arisen from the host it infected, but is instead the same tumour passed between individuals.
FIGURE 4: ALLOGRAFT THEORY: TRANSMISSION OF DEVIL FACIAL-TUMOUR DISEASE. A) HEALTHY
DEVIL KARYOTYPE, B) DFTD KARYOTYPE. IMAGE TAKEN FROM (PEARSE, 2006)16.
15 More recent research based on gene mapping and chromosome painting suggests that some chromosomes were mislabelled in this original research. Accordingly, the karyotype of DFT cells is shown to have the deletion of both chromosomes 1, 1 pair of chromosome 5, and an addition to the short arm of chromosome 2 (which originated from the X chromosome and chromosome 1). The origin of the 4 markers was elucidated as being derived from chromosomes 1, 5 and X (Deakin, 2014). 16 Anne-Maree Pearse has written many journals and academic papers on DFTD and Tasmanian devil genetics. This research paper, co-written with K. Swift, was published by Nature, a very reputable science journal, and has been cited over 253 times, showing that this research is very important to the DFTD research industry. Therefore, I believe this is a reliable source.
15 There are two explanations for how a tumour allograft might establish itself in the population: The devils have a poor immune response, The devils are either genetically identical or have low genetic diversity at the major histocompatibility complex (MHC).
Immune Response to DFTD
It is logical to assume Tasmanian devils have a functional immune system. They are carnivores, specialised scavengers and opportunistic predators and have therefore been exposed to a wide range and high level of bacteria and parasites due to their diet and biting behaviour. As a result, they have primary protection against bacterial and pathogenic parasites and there is little evidence that wild Tasmanian devils succumb to disease from such pathogens (Obendorf, 2010)17. Evidence for the proficiency of their innate immune response (Kreiss, 2008)18 shows that neutrophils, a key component of the innate immune system, demonstrated efficient phagocytic uptake of Escherichia coli, showing that their phagocytes are fully functional and should, in theory, protect the devil from DFTD cells.
The devil is also shown to have a competent humoural immune response. Devils immunised with horse red blood cells had elevated titres of immunoglobulin antibody against horse red blood cells, 1 week after the first injection, indicating a rapid immune response. Rapid secondary responses were shown 8 months later after boosters, showing an excellent memory response in addition to a humoural immune response (Kreiss, 2009)19.
17 This paper Trichinella pseudospiralis infection in Tasmanian wildlife (2010) has been cross referenced 33 times and was published in The Journal of the Australian Veterinary Association, indicating that it has been peer reviewed and is therefore reliable. However, it was published in 1990, long before anything to do with the immune response of DFTD was known, it is possible that they could have developed a different or weaker immune response more recently than the research that Obendorf carried out. However, other sources appear to agree to this day, such as Siddle et al.’s research and papers by Pye et al., published in 2016. Therefore, I feel that this paper is reliable enough to reference. 18 A. Kreiss is again an expert in the field of DFTD. His name has appeared on over 30 leading research papers and all have been extensively peer reviewed and cross referenced. This paper, Assessment of cellular immune responses of healthy and diseased Tasmanian devils (Sarcophilus harrisii) was published in Developmental & Comparative Immunology, a rigourously reviewed and reputable journal. Therefore, I believe this paper to be reliable enough to reference. 19 A. Kreiss is again an expert in the field of DFTD. His name has appeared on over 30 leading research papers and all have been extensively peer reviewed and cross referenced with other researchers in the field, such as Pye, Brown
16 This shows that the devil’s immune response is certainly not lacking in any speed or effectiveness of response to foreign cells. Again, this doesn’t shed any light onto how DFTD cells spread.
As discussed, Tasmanian devils resist most bacterial and parasitic attacks using their fully functioning immune system. However, devils succumb to DFTD without developing a protective immune response. Natural Killer (NK) cells identify abnormal cells and can kill the organism’s own cells if they are infected with a virus or have become cancerous. A normal, healthy cell has a protein on its surface called MHC1 (major histocompatibility complex 1), but if the cell is infected, it stops making MHC1. If a NK cell detects this it triggers cell apoptosis (programmed cell death). In a study to determine if Tasmanian devils are capable of forming cytotoxic antitumour responses (i.e. able to kill the cancerous cells) and develop antibodies against DFTD cells and foreign tumour cells, none of the devils developed cytotoxic or humoural responses to DFTD when immunised with irradiated DFTD cells (Brown, 2011).20 However, they did form a cytotoxic response and antibodies after immunisation to xenogeneic K562 cells. Fig 5 shows the responses of different devils’ cytotoxic and antibody responses against K562 tumour cells. K562 cells are leukaemia cells and should stimulate a similar immune response in Tasmanian devils as the DFTD should. Panel A shows a devil who developed a weak response after one dose. After two doses, three of the four Tasmanian devils formed cytotoxic responses against K562 cells. The levels were statistically significant compared to pre-immune data. It appeared to occur through the activity of NK cells in FIGURE 5: PALE GREY LINES REPRESENT PRE-IMMUNE an antibody-dependent manner, although classical NK cell responses (such CYTOTOXICITY LEVELS. RESPONSES as innate killing of DFTD and foreign cancer cells), were not observed. AFTER A SINGLE DOSE ARE SHOWN AS DARK GREY LINES. BLACK LINES This suggested that Tasmanian devils do have NK cells with functional REPRESENT THE RESPONSES AFTER TWO cytotoxic pathways but they do not directly recognise DFTD cancer cells. DOSES. FIGURE FROM BROWN, 2011.20
and Siddle. This paper, The humoral immune response of the Tasmanian devil (Sarcophilus harrisii) against horse red blood cells, has been cited 51 times, indicating that it is a reliable source of information. 20 Gabriella K. Brown is a researcher at the Menzies Research Institute Tasmania, specialising in oncology. This article Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil has been cited in 21 other articles, mostly published in renowned and well peer-reviewed journals such as PLoS and Veterinary Pathology, including being cross-referenced in another article I have used Devil Facial Tumour Disease (Pye, 2016), showing that it is a reliable and up-to-date source.
17 However, it also shows that the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease which will thus have positive implications for future DFTD vaccine research.
Although there is evidence for a competent immune system in Tasmanian devils, responses against DFTD cells are absent or limited. It is apparent that Tasmanian devils are capable of producing functional cytotoxic responses in the presence of antibodies, but it is unknown why NK cells do not directly recognise DFTD cells. The fact that DFTD transmission occurs in the presence of a functional immune system suggests a capacity to evade the devil’s natural anti-tumour response. Potential mechanisms have been proposed such as limited genetic diversity among Tasmanian devils, both in nuclear satellite markers and at the major histocompatibility complex (MHC), which is discussed in greater detail subsequently.
Genetic Diversity and the MHC
Low genetic diversity is to be expected of Tasmanian devils, as they are an island species. They have been shown to exhibit low heterozygosity and allelic diversity at microsatellite loci (Pye, 2016)7. However, these results give information on population history such as relatedness and previous population bottlenecks, whereas analysis of major histocompatibility complex (MHC) diversity better represents a population’s “fitness” and ability to counter disease challenges (Pye, 2016)7.
The MHC is a cluster of genes occurring in all vertebrates and is the most polymorphic portion of the mammalian genome. They code for a set of cell surface proteins that are essential for the immune system to recognise foreign molecules. Their main function is to bind antigens from invading pathogens and display them on the cell surface so that the organism’s T-cells can recognise and subsequently destroy them. They also have highly polymorphic sections in the peptide binding region (PBR), which are subject to plenty of selection, meaning they change regularly and constantly evolve (McDowall, 2017).21
The genes for the MHC were first associated with allograft transplantation but are now known to be essential in the immune recognition of pathogens and tumour cells and play a key role in immune response to both tumours and grafts. The cell-mediated adaptive immune response is regulated by the major histocompatibility complex (MHC), named that because it is responsible for graft rejection, or tissue compatibility. For individuals to exchange grafts successfully, they have to have near-identical MHC combinations; which is normally very rare. The diversity of MHC combinations is usually a 10% difference between unrelated individuals, meaning
21 This website was comprehensive and thus a very useful source of general information about the MHC. https://www.ebi.ac.uk/interpro/potm/2005_2/Page1.htm [Accessed 12/12/17.]
18 tissue acceptance is highly unlikely. This forms the base for a hypothesis for the lack of tumour recognition in Tasmanian Devils; they are supposedly lacking in MHC diversity and thus it is far easier for them to exchange tissue grafts without immune rejection.
Alternatively, instead of the MHC having low diversity in Tasmanian devils, it is theorised that the tumour is able to up- and down-regulate the MHC response, so it can enter the devil unnoticed by their immune system.
The most common mechanism of immune evasion by tumours is down-regulation of classical cell surface MHC molecules. MHC molecules invoke vigorous T-cell responses against incompatible cells, and regulate the immunological mechanisms of tissue graft rejection (Siddle, 2007)24. The diversity of MHC genes (i.e. polymorphism) provides the foundation for immune responses against infectious agents, such as viruses and bacteria. There are two types of MHC molecules, class I and class II. Class I molecules present endogenous peptide antigens to cytotoxic T cells. Class II molecules bind to exogenously derived peptides for antigen presentation. Both have a highly polymorphic peptide binding region (PBR) that enables the recognition of a range of antigenic peptides within a population.
In populations with high class I polymorphism, grafts between unrelated individuals are rapidly rejected due to differences between class I alleles expressed on the surface of the donor cells and host cells. In a functioning immune system, the failure to recognise and target DFTD could be a consequence of two genetic possibilities; that the tumour may ‘escape’ the immune response by modulating expression of MHC genes during tumour growth, or that devils lack genetic diversity at MHC loci, resulting in an immune system failure to recognise the tumour as foreign (Siddle, 2007)24.
Support for the former hypothesis comes from studies into other types of transmissible cancer such as canine transmissible venereal tumour (CTVT) which is passed between individuals though coitus. CTVT originated from a single neoplastic clone more than 200 years ago. It passes across MHC barriers by up- and down- regulating MHC molecules, thus avoiding immunological response. Class I MHC genes can be divided into two major categories: classical and non-classical. The classical class I genes encode the major transplantation antigens. Non-classical class I genes encode highly specialised cell surface molecules that do not function as major transplantation antigens, the use of these are mostly unknown (Bordallo, 1990)22. CTVT down-regulates
22 Although this research paper Expression of a Non-Classical Class I Gene in Transgenic Mice is not directly about Tasmanian devils, there has not been any research specific to Tasmanian devils that show the same results. To give extra information about non-classical class I genes, this was an incredibly useful source to cite, and has been cross referenced with Pye, 2016, indicating that it is a reliable source to use. This was a conference paper, presented at the Experimental Immunology Branch, Bethesda, USA, a world-renowned research centre that uses state-of-the-art
19 MHC class I and class II expression and up-regulates non-classical class I expression to avoid natural killer (NK) cells recognition and response (Siddle, 2007)24. However, it is important to note that the CTVT line is very old (more than 200 years old) whereas the DFTD cell line emerged far more recently (about 10 years ago). DFTD may, in time, evolve into a less lethal form, or Tasmanian devils may evolve to be resistant to DFTD, allowing the population numbers to increase, but maintaining numbers in the meantime will be difficult.
However, research by Siddle et al. (Siddle, 2007)24 suggests that altered MHC expression, a common cause of immune evasion by tumours, is not responsible for a lack of immune response to DFTD. They suggest instead, that low MHC diversity in the devil has enables natural transmission of tumour cells between individuals. This research provided conclusive multi-locus evidence for the allograft theory of DFTD transmission, confirming that it is of a clonal rogue cell line. 90% of the sampled devils were genetically unique, whereas all examined devil facial tumours had an identical genotype at multiple microsatellite and MHC loci, demonstrating the clonal nature of the tumour. Furthermore, the tumour genotype was different from that of all examined host devils, verifying that tumour cells are not “self” but came from an external source.
Researchers at the Faculty of Veterinary Science, Sydney (Greuber, 2015)23, showed that DFTD tumours express at least three, possibly all five, classical MHC class I loci that are found in Tasmanian devils, but do not express any non-classical class I loci. Class II molecules are usually only expressed on hematopoietic (blood) and thymic cells (such as monocytes, macrophages and B cells). Despite expression of MHC class I and MHC class II molecules on the tumour cells, analysis of DFTD has shown that T lymphocytes do not infiltrate the tumour or the metastases. Consequently, T cells are not activated by the tumour itself or by tumour cells within the lymph nodes, leading to a suppressed immune response. This supports the idea that altered regulation of MHC molecules could be the cause of the rapid spread of DFTD, which would not encounter a fully functional immune response (Figure 6). Figure 6 shows MHC expression in a normal (A) versus DFTD (B) cells. The immune system should prevent the transmission of cancer cells between individuals through T cell recognition by the antigens presented on the MHC class I molecules. DFTD cells are not recognised by the immune system
microscopy research tools and techniques for cellular and molecular immunology. Thus, it is a reputable source to use. 23 C. Greuber’s Genomic insights into a contagious cancer in Tasmanian Devils proved to be a very useful source for this EPQ. It covers the majority of information available about the genetics of Tasmanian devils, including the proposed origins of DFTD, how genomics can save the Tasmanian devils and how emerging genetic tools can be used to facilitate greater understanding of DFTD. Furthermore, this paper provides a comprehensive glossary for the more technical terminology, many of which I have used in my EPQ glossary, as the definitions are more specific to DFTD rather than the general scientific meaning. This paper was published in Cell Press, a leading publisher of cutting edge biomedical research and review journals including Cell, Neuron, Immunity and Current Biology. Due to the publisher’s reputation, it is likely that this paper is well peer reviewed, accurate and therefore reliable.
20 because they lack functional MHC class I molecules on their surface. In healthy tissue (A), the role of MHC class I molecules is to present the antigens to CD8 T cells, which requires the expression of 2-microglubulin (B2m) (a structural component of the MHC molecule) and Transporters 1 and 2 (TAP1 and 2). Antigens are pumped into the endoplasmic reticulum, where they are moved onto the MHC I molecule. It can then be recognised by the CD8 cell, which activates an immune response which kills the invading cell. In DFTD, both MHC I and B2m are at very low levels. This means that tumour antigens will not be presented to the immune system/CD8 cells and the DFTD evades rejection and attack (Greuber, 2015)13.
Furthermore, genetic evidence supports clonal origins of DFTD through analysis of microsatellites, MHC genotyping and the whole genome. It was found, through analyses of 15 tumour, host and healthy blood samples, that 90% of samples devils had unique genotypes and all the tumours were identical at multiple microsatellite and MHC loci. This supports the tumours clonal/allograft nature (Siddle, 2007)24. Further analyses of more samples showed a comparable tumour genotype across all loci, even with variants of sex, age or location. In all
FIGURE 6: TAKEN FROM (GREUBER 2015)13 SHOWING DIFFERENTIAL MAJOR HISTOCOMPATIBILITY (MHC) EXPRESSION IN (A) A NORMAL DEVIL AND (B) DFTD.
studies, the tumour was shown to be distinct from that of the host; this shows that it is impossible for DFTD to have arisen from the host’s own tissues and consequently supports the allograft theory.
24Hannah Siddle is one of the leading researchers and an expert in the DFTD field. Her name appears on more than 10 papers to do with DFTD and MHC diversity, including many I have used already for my EPQ, such as (Pye, 2015) A Second Transmissible Cancer in Tasmanian Devils. However, this could show a lack of different opinions because the same researchers have input into most papers. Having said this, it is a very niche topic so there are not many researchers to have different opinions. Due to the number of reputable researchers from prestigious institutes such as the University of Sidney and the University of Southampton contributing to this paper, I believe it to be reliable.
21
Telomeres are structures at the end of chromosomes in most organisms. They consist of several thousand DNA repeats of TTAGGG associated with protein complexes. They maintain chromosomes integrity and genomic stability. Telomere length shortens during each cell replication and in general, a critically short telomere length can trigger the cell to replicate uncontrollably, which ultimately results in cells death. However, if this length is kept consistent and cell death does not occur, cell proliferation can continue, and a tumour is formed (Zhu, 2016)25. One of the most important features of neoplastic DFTD cells is that they are immortal; they don’t die when passed between devils and proliferate uncontrollably once inside a host. This requires telomere length maintenance, so that the cells don’t die. It was found that all DFTD cell telomeres are very short. DFTD cells appear to monitor and regulate the length of individual telomeres; shorter telomeres are elongated by up- regulation of telomerase-related genes, and longer telomeres are protected from further elongation. (Ujvari, 2012)26.
These independent lines of substantiation are convincing evidence for DFTD being a transmissible tumour that acts as an allograft and evades the host’s immune system. Further evidence that DFTD is self-sustaining due to self-regulation of telomere length, provides an explanation as to how it can establish itself successfully in a new host.
Non-Allograft Theory of Transmission
Although it is generally agreed that the transmission of DFTD is of an allograft nature, Xianlan Cui et al.27 have proposed an alternative, non-allograft theory (Cui, 2016)27. Before this theory arose, the hypothesis that DFTD
25This paper, The association between telomere length and cancer risk in population studies (2016), was useful for up-to-date research (2016) regarding telomeres and cancer, as there has been very limited research into DFTD cells and telomere length. It provided a relevant definition and background information. It was published in Scientific Reports, and reputable journal, and has been cross referenced in fourteen other scientific papers 26 This paper, Telomere dynamics and homeostasis in a transmissible cancer, was written by B. Ujvari, Senior Honorary Fellow at the Faculty of Veterinary Science, University of Sydney, and was published by the Royal Society of Publishing in Biological Sciences. Due to the authors credentials, such as being Chair of Comparative Oncology Special Interest Group, Cancer Research Network, University of Sydney, 2012 – 2014, and the reputation of Royal Society Publishing, I believe this source is reliable and accurate. 27 X. Cui’s research includes the development of a novel vaccine against devil facial tumours, mapping biomarkers in DFTD serum by phage display, isolation and identification of devil retrovirus and herpesvirus associated with DFTD. He researches with the Department of Primary Industries, Parks, Water and Environment, Tasmania and was educated at the University of Melbourne, a reputable University with very high standards. This paper, Sex determination by SRY PCR and sequencing of Tasmanian devil facial tumour cell lines reveals non-allograft
22 was transmitted by allograft during biting was based on two cytogenetic findings of DFTD tumours in 2006 (Pearse, 2006)16, and it was believed that the DFTD tumours were originally from a female devil. Pearse et al.’s primary findings, as discussed earlier, were that a pericentric inversion of chromosome five in the karyotype of one Tasmanian devil was found in all cultures of that devil’s normal tissue, but was not present in the two copies of chromosome five in the facial-tumour cells.
In Cui et al.’s experiments, the devil sex-determining region Y (SRY) gene was PCR amplified and sequenced, and six pairs of devil SRY PCR primers were used for the detection of devil SRY gene fragments in purified DFTD tumour cell lines. The devil SRY gene sequence was detected by PCR and sequencing of genomic DNA of DFTD tumour cells lines from six male devils, but not from six female devils. Four out of six DFTD tumour cell lines from male devils contained nucleotides 288-482 of the devil SRY gene, and the other two DFTD tumour cells contained nucleotides 381-577 and 493-708 of the SRY gene respectively. If DFTD is transmitted by tumour cells (allograft), and all of the tumour cells originated from a female, none of the DFTD tumour cells should contain a devil SRY gene sequence. Cui et al. proposes that DFTD could be induced by a tumour virus; it would do this by insertional mutagenesis (the virus inserts its own genome into the devil’s, creating mutations by adding base pairs into DNA). Assuming that the SRY gene is not completely deleted by the tumour viruses, the tumour cells from male, but not female, devils, should contain SRY gene fragments. These results therefore indicate that the different portions of the SRY gene in the DFTD tumours were originally from male hosts, rejecting the currently believed DFTD allograft transmission theory, which postulates that the tumour cell lines are all identical from the same original host.
DFTD Evolution
Genome-sequencing projects such as research carried out by Greuber et al. (2015), have helped to reveal the mutational and selective processes involved in tumour development and metastasis. It is clear that DFTD is evolving over time; four closely related but karyotypically distinct DFT strains have recently be described, showing how it is changing constantly (Greuber, 2015)13.
transmission is one of the only ones of its kind. This paper provided convincing evidence against the allograft theory, and thus I thought it vital to be in my literature review as (cont.) a potential other mode of DFTD transmission. However, I have not found another paper than suggests the same idea; the rest of the research in my literature review assumes the allograft theory of transmission to be correct. Being the only paper I can find with this view, it is hard to check its reliability, but it was also very recent (2016) and there will not therefore, be much more research on the topic yet.
23 Epigenetic processes are mitotically and meiotically heritable changes in gene expression that are not caused by changes in the primary DNA sequences and are often important in the development and evolution of human cancer. The role of epigenetics in DFT evolution had not yet been explored, but Ujvari et al. attempted to look into the effects of DNA methylation of DFTs. Cancer is an evolutionary process with a “mosaic of cells competing for space and resources” (Ujvari, 2012, p. 6)26. Cancers best able to survive can evade detection by the immune system by adapting and therefore survive. This study proposed that DFTD is evolving via epigenetic modifications and that plasticity in methylation patterns could lead to changes in gene expression over time. It was found that on a genetic level, DFT cells are very stable, except the four strains mentioned before (Pearse, 2006)16 (Deakin, 2014)15. Overall methylation patterns in tumours and nerves were remarkably similar, which again supports the hypothesis that DFTD originated from a Schwann cell and is a clonal.
However, the methylation levels were significantly higher than the normal tissue tests. Hemimethylation, where only one of the two DNA strands is methylated, was very high compared to the nerves analysed which is a feature of early cancer development and can also occur in late cancer progression and a cancer with poor prognosis. It appears that DFT underwent early hypomethylation (the loss of a methyl group in a nucleotide) when it first arose, and that DFTD is still undergoing hypomethylation 16 years later, suggesting ongoing epigenetic evolution.
Over time, DFTs have exhibited an increase in hypo-methylation, supported by an observed increase in the expression of the genes encoding methyl-CpG binding domain proteins 2 and 4 (MBD2 and MBD4), which are involved in active demethylation. Variation in epigenetic patters between tumour cells leads to differences in gene expression patters, thereby providing the tumour with evolutionary potential. An increase in ploidy (number of sets of chromosomes in a cell, and hence number of possible alleles for autosomal genes) has also been observed over time. Polyploidisation allowed cancer cells to mask the effects of deleterious alleles, sustain higher mutation rates, and allow growth rates as a result of increased genome size. Occasionally, humans born with tetraploidy (a condition in which cells contain four complete sets of chromosomes) can develop cancerous or precancerous cells, and these are connected to more aggressive, metastatic, and drug-resistant cancers (Greuber, 2015)13. Since the pathology of devil and human cancers are fairly similar, an uncontrolled proliferation of cells in a particular area, it is conceivable that Tasmanian devils could develop DFTD due to abnormally high chromosome numbers.
Understanding the role of epigenetic mutations in the evolution of DFTD as a parasitic cancer will provide important insights into the role of epigenetic plasticity in cancer evolution and progression into traditional cancers that arise and die within their hosts. This knowledge could allow researchers to target cancer more effectively, for example focusing more on DNA methylation homeostasis rather than the cancer’s static genome could allow researchers to study more closely the subtle changes that allows the cancer to inhabit the organism.
24
Tasmanian devil conservation
Having collated extensive information on the transmissible nature and malignant pathology of DFTD tumours, it is clear that Tasmanian devils are in a very precarious position. It is a very realistic concern that they may become extinct in the near future, due to the rapid and devastating effects of DFTD to date, with no apparent emergence of reduced tumour aggression or host resistance. Furthermore, it is a gamble as to whether evolution would give rise to a more benign form of DFTD, by which time the population numbers may have fallen to dangerous numbers that threaten extinction. Therefore, it would be irresponsible to neglect intervention and it is imperative that conservation and management options are discussed and put into place as soon as realistically possible. Viable options are captive breeding, disease suppression through culling, depopulation, translocation, population reinforcement and fencing. These are all discussed as proposed management solutions by Pye et al., and they are the steps the Tasmanian Government is recommended to take to reduce the impacts of DFTD on Tasmanian devil populations (Pye, 2016)7. Some management methods, such as captive breeding and translocation, have already been effectively implemented, whereas others have proved unsuccessful (such as disease suppression). Others are not yet fully developed; current vaccine research is alluded to in the discussion.
In conclusion, extensive research has been carried out on the DFTD tumour since it first arose in 1996. It has provided unique opportunities to study a transmissible cancer, and our understanding has developed significantly over the past 10 years. However, there is still much to know. It is still not agreed how this transmissible cancer is actually passed between organisms, and how it is so resilient. It is decreasing the population numbers of Tasmanian devils at an alarming rate, so more must be done and known about DFTD to save them from extinction.
Discussion
It is clear that, despite rapid acceleration in the research surrounding DFTD, researchers still do not fully understand the nature of devil facial tumour disease. Researchers cannot yet agree on the mode of transmission of DFTD, allograft or non-allograft. Until the true causal nature of DFTD is understood it, will be very hard to determine much more about it, and even harder to implement management options, whether that be conservation or vaccine development.
A major factor in our understanding of DFTD is undoubtedly its mode of transmission; how the tumour cells are passed from one devil to another. Based on current research, I have attempted to answer this question.
25 Which is the stronger theory: The Allograft or Non-Allograft Theory?
Since the first pathological and aetiological tests were carried out on devil facial tumours, it was agreed that DFTD is of an allograft nature; the cancer is passed between devils. Once this theory arose many studies were produced in quick succession confirming the theory. Many of these theories are discussed in the literature review. Significantly more research has been produced in favour of the allograft theory of transmission of cancerous cells than non-allograft theories. Research was dedicated to finding the method by which the tumour evaded the immune system’s responses; was it a lack of genetic diversity, a low MHC diversity, the modification of the MHC by the tumour itself (downregulation) or simply that Tasmanian devils have an inadequate immune system to reject the cancerous cells? However, more recently another theory of transmission has arisen, throwing the field of research into a state of disarray.
The non-allograft research, carried out by Cui et al. (Cui, 2016)27, claimed that all previous research was inherently flawed. Cui et al. rejected findings of pericentric inversion of devil chromosomes (Pearse, 2006)16, but not DFT chromosomes, ‘confirming’ the clonal nature of DFTD28 on the grounds that it is possible that the two copies of chromosome five in the DFTD tumours detected by karyotyping were duplicated from the normal chromosome five in the host. This is an example of uniparental disomy (offspring receive two copies of a chromosome, or part of a chromosome from one parent and no copy from another), which is common in human cancers (because it increases the chance of mutation leading to uncontrolled cell proliferation), and therefore more likely to occur in the tumour. Furthermore, chromosome painting and gene mapping results indicate that there is only one chromosome five in DFTD tumours, which rejects the original karyotyping of two copies of chromosome five in DFTD tumours. Therefore, as discussed, Cui suggested a viral transmission of DFTD.
There is, however, little reputable support for Cui et al.’s theory in the DFTD field of research, and no scientific literature to support it. An explanation for their findings however, can be found in Pye et al.’s research into a “second DFTD”, called DFTD2 (Pye, 2015)7. Cui et al. assumed that the only type of DFTD present in devils was DFTD1, which has the traits such as an X chromosome (hence it is assumed to come from a female devil), and a lack of chromosomal pericentric inversion. DFTD2 tumours, however, may look similar to DFTD1, but are entirely histologically distinct from tumours caused by DFTD1. DFTD2 bears no cytogenetic similarity to DFTD1 and carries a Y chromosome, which possibly explains the Y chromosome found by Cui et al. that lead them to believe that the tumour had the same genetic make-up as its current host.
To show that DFTD2 is not a variant of DFTD1 in a single devil, researchers found a distinct karyotype in five Tasmanian devils’ tumours, showing that it is an entirely separate form of DFTD. Each tumour showed identical
28 This is explained in more detail the Literature Review. See “Allograft Theories of Transmission” section.
26 complex structural abnormalities; the presence of additional material on chromosomes 1, 2 and 4, deletion of chromosome 5, and monosomy (only one chromosome present) of chromosome 6. Crucially, both chromosome X and Y were present, linking it to Cui et al.’s possible misunderstanding.
To conclude, I would argue that despite Cui et al. being very confident in the findings and conclusion, it is an example of DFTD2 being misunderstood. The overwhelming evidence for an allograft theory of transmission leads me to believe that the tumours are transferred via the clonal cells of one devil evading the immune system of another devil; and this has enabled its extremely rapid spread through the Tasmanian devil population. Furthermore, it can be argued that whilst research of this nature will be important in the future for the understanding and combatting of cancer (increased focus on chromosomal abnormalities and MHC up- and down-regulation, which would lead to more personalised treatment of cancer in humans), Tasmanian devils are facing a very imminent and real threat of extinction. It is paramount, therefore, that we instil effective conservation techniques as soon as practically possible, rather than waiting for conclusive research on the transmission of DFTD, before a vaccine or specific gene therapy is developed, which may take longer than Tasmanian devils have left in the wild before extinction.
What are the conservation options for Tasmanian devils?
Captive breeding
Captive breeding provides an insurance population in case a species becomes extinct in the wild. One of the first initiatives to protect the Tasmanian devil from extinction was the establishment of a captive insurance population which currently holds approximatively 500 individuals (Pye, 2016)7. Ideally, 95% of the genetic diversity present in the founder population should be represented within these captive devils. Different management breeding options utilised include:
Intensive Breeding: devils are kept in isolation or small groups, and mate selection is strictly controlled during breeding season to prevent inbreeding depression Extensive Breeding: devils are kept in groups in large double-fenced areas and breeding is loosely controlled; mate selection is mostly defined by each individual.
Extensive Breeding is a viable option because it eliminates the risk of DFTD with appropriate quarantine regulations, and these non-DFTD populations may be eventually self-sustaining enough to release into the wild when the dangers of DFTD have passed to repopulate Tasmania (Pye, 2016)7.
27 The target breeding carried out in some conservation efforts is invaluable for Tasmanian devils if, as Siddle discusses in Transmission of a Fatal Clonal Tumour, devils are lacking genetic diversity; whether it be generally or within their MHCs (Siddle, 2007)24. Maintaining the genetic diversity and avoiding inbreeding depression may reduce the chances of a strain of DFTD breaking out in the future, especially if Tasmanian devils are a species prone to transmissible tumours. Furthermore, captive breeding programs allow easy access for individuals and research teams to carry out investigations on devils, furthering the understanding of devil and DFTD biology. This may also have far reaching implications, such as to an understanding of human cancer and to the global effort to find a cure.
Captive breeding programs are very expensive, however, and require a lot of land, particularly if natural behaviour includes roaming and scavenging as does the devil’s. This becomes an issue when the costs do not cover suitable care for each individual. Therefore, captive breeding programs can often only support a small number of individuals, which cannot sustain the genetic diversity required for a suitable breeding population. There is also a serious concern that devils might lose their wild, or natural, behaviour. Although captive breeding programs attempt to make the enclosures as natural and stimulating as possible, they will frequently, if not always, fall short of the natural environment. This change in environment can lead to changes in the animal’s behaviour. In the Tasmanian devil, this can include decrease in foraging or scavenging abilities, increase in sleep patterns, decrease in overall activity and problems with social behaviours, such as using social latrines for communication (Owen & Pemberton, 2011)5. These changes in behaviour are a major factor in whether these animals can be reintroduced into the wild, and if it would benefit their population.
Furthermore, there have also been issues with reproductive behaviour in some captive breeding sites, with some suggesting that fecundity decreases with each generation held in captivity, which calls into question the effectiveness of the captive breeding program at all (Snyder, 1996). Finally, issues have been raised such as the possibilities that genetically important animals might not breed, which creates yet another problem with lack of genetic diversity, and also that post-breeding animals require ongoing care, which drains resources; they may not get the care required for them to be a functioning population to reintroduce to the wild.
Vaccine development
An effective vaccine against DFTD would be complimentary to other management options, such as captive breeding. It would allow the repopulation of DFTD-affected areas with captive devils from a current captive insurance population. It is also conceivable that disease-free wild devils could be vaccinated in significant numbers given that many populations have proven amenable to trapping and handling. An encouraging aspect regarding the likelihood of vaccine development is the high conservation of tumours morphology and genotype, implying that the tumour antigens are conserved (Pye, 2016)7. While this may be a major factor in the
28 transmission of DFTD (if it is indeed an allograft transmission), it can have positive implications for a vaccine, as it gives the immune system a stable target. A working vaccine would be invaluable, as it creates an opportunity to prevent the disease, rather than waiting for it to run its evolutionary course. DFTD may take hundreds, if not thousands of years to evolve to become benign and this is time the Tasmanian devil simply does not have. To put this into perspective, David Llewellyn, Tasmania’s Primary Industries Minister, predicts that Tasmanian devils could face extinction in the wild within 20 years, as of 2008 (Geographic, 2008).
The potential for herd immunity is also very encouraging. If a devil with DFTD is centred within a population that is vaccinated, it has no opportunity to pass on cancerous tumour cells, and thus the cancer will die with the host. Furthermore, the process of vaccine research improves the understanding of devil immunology and DFTD, which could be applied to other marsupials and other transmissible cancers (Pye, 2016)7.
However, similar to captive breeding, vaccine development is extortionately expensive and time consuming to produce and deliver. Sophisticated technology and techniques are required to accurately create and test a working vaccine, and the trials alone could take years. Furthermore, thousands of devils would need to be trapped for injection, and teams of conservationists would be needed to deliver the vaccine into the population. Current research also suggests that the effect of the vaccine is short-lived, there may be a need for several booster vaccines after the initial vaccination (Tovar, 2017). Finally, a vaccine would almost certainly change the dynamics of DFTD evolution. This could become an issue if a DFTD strain evolved that was resistant to vaccines, or more malignant than the current DFTD1 and DFTD2. It could therefore spread faster and be more lethal than the current strain of DFTD, and a well-meaning vaccine could accelerate the evolution of the tumour and cause more harm than good.
Disease suppression through culling
A disease suppression trial took place in a semi-isolated peninsula in the south-east of Tasmania between 2004 and 2010 (Lachish, 2010)3. It was hoped that there was a possibility of keeping a functional population in its original area, and it is ethically acceptable because devils can be euthanised before the onset of severe clinical illness. However, in all trials DFTD prevalence remained the same as other unmanaged sites (Lachish, 2010)3. This management tool is ineffective because of the long incubation period and frequency-dependent nature of DFTD, coupled with failure to capture trap-wary animals. It is essentially unfeasible unless a very high proportion is trapped and does not allow for natural resistance to DFTD to develop (and thus a possible change in tumour biology that may make it more benign). It is generally assumed that culling Tasmanian devils will only exacerbate the onset of DFTD, and make population numbers dangerously low, leading to an early extinction if not carried out with precautions (Beeton, 2011). In a computer model created at Griffith University, the effects of culling were stimulated. The removal rate to suppress the disease was higher than feasible in the
29 field. In the wild, 20% of the population is never captured, which would act as a reservoir for the disease. This means that DFTD would never be able to be eradicated by such human intervention (McCallum, 2007)2. It is also incredibly expensive, costing conservation programs around $200,00 per year (BBC, 2011).
Depopulation, Translocation and Population Reinforcement
Since culling is predicted to be unsuccessful in eliminating DFTD, conservationists have turned to other options involving moving healthy and cancerous devils around and off the island of Tasmania, allowing healthy populations to develop without DFTD.
The depopulation of a peninsula in the southeast of Tasmania has occurred, with a view to repopulating the area with healthy animals once the boundaries are secure. It is seen as an opportunity to ‘clear up’ an area ahead of future reintroduction (and is commonly used in conjunction with captive management programs). However, there is the possibility that the incursion of Tasmanian devils with DFTD will occur, and this is a particular worry on a small, isolated island such as Tasmania. This would negate any depopulation and captive breeding efforts and so be an issue both ethically and financially.
Translocation is moving an endangered species to another location. In the case of Tasmanian devils, this is away from other devils with DFTD with the aim to create a steady, healthy population elsewhere. The first translocation took place in 2012, to Maria Island (shown in Fig 7), a mountainous island off the east coast of Tasmania in the Tasman Sea. This was an ideal location for the translocation of Tasmanian devils, because it is contained within the Maria Island National Park. This means that the Tasmanian devils in the insurance population are subject to strict quarantine; visitors are not able to see or interact with them. Furthermore, the island’s natural boundaries prevent the incursion of devils with DFTD and the escaping of healthy devils to diseased populations. Unlike captive breeding programmes, the devils can mostly maintain their wild behaviour, as Maria island is very ecologically similar to Tasmania, and this also means that once established, the population needs very little maintaining. Having said this, the population will still need monitoring for interbreeding and overpopulation, and to make sure that a strain of DFTD does not spontaneously arise. Also, there is evidence that the presence of devils on Maria Island is having a negative impact on the bird life there, distrupting the natual ecosystem balance.
30 FIGURE 7: MAP TO SHOW THE LOCATION OF MARIA ISLAND (RED) IN RELATION TO TASMANIA.
Population reinforcement is the introduction of a species to its indigenous habitat with the aim of enhancing the local population. Several areas of Tasmania have had drastic declines of the devil population following the onset of DFTD, and these areas are suitable for reinforcement. This would artificially augment the local population, for example on the Freycinet Peninsula, and hopefully re-establish the ecosystem balance that existed prior to DFTD arrival. It would also improve the genetic diversity of the population. However, a major drawback to this approach is that there is a high possibility that the healthy introduced devils will be at risk of contracting DFTD and if not, devils may disperse elsewhere, essentially making the attempt to re-establish that particular population futile. This is also ethically dubious; interfering with the natural dynamics of the ecosystem and population could result in the faster onset of DFTD in the Tasmanian devils’ population, leading to a quicker extinction. Introducing otherwise healthy devils into an area where they could contract the disease is generally considered unwise and unethical.
Fencing
Fencing has been used all over the world in different contexts with varying degrees of success. For example, Asiatic wild ass in China and Mongolia are fenced out of inner Mongolia to protect them from poachers in areas where there are few restrictions and unregulated poaching, which has been very beneficial for the population and welfare of Asiatic wild ass. Botswana’s “Buffalo Fence”, which runs north to south across the Okavango Delta, prevents Cape buffalo from transmitting foot-and-mouth disease to other populations (Hart, 2017). However, it has also entrapped migrating mammals such as elephants, roan antelope and giraffe. As well as interfering with the ecosystem and the natural flow of native animals in and out of the area, it is also expensive
31 and labour-intensive to create and maintain. Again, the population would have to be monitored for inbreeding and overpopulation (Pye, 2016)7. As part of the ‘Save the Tasmanian Devil’ conservation effort (Tasmanian Government, 2015), a devil-proof barrier has been set up at Dunalley in south-eastern Tasmania, to prevent the spread of DFTD into the disease-free Tasmanian Peninsula region. However, this has not been in action long enough for any meaningful data to have been collected yet, so its’ usefulness is still unknown.
Issues with management options:
These management options ignore the possibility of DFTD evolution, the development of a less aggressive disease would favour survival of the host and, by consequence, reduce the impact of the disease. It is possible that a more benign form of DFTD might evolve given enough time and devil hosts, and it is conceivable that implementation of certain management options would interfere with this evolutionary process. However, given the rapid and devastating effects of DFTD to date, with no apparent emergence of reduced tumour aggression or host resistance, it would be irresponsible to neglect intervention and let the disease take its course knowing the implications of devil extinction as a top-order land predator, as discussed earlier.
Furthermore, breeding programmes and other conservation efforts are incredibly expensive. Captive breeding in general costs approximately $788,000 to establish successfully, translocation costs $198,000 and the surveying and monitoring of the population alone costs $422,000 per year. Additionally, there have been concerns raised over human intervention in the case of DFTD. It is argued that Tasmanian devils are not evolutionary fit enough to survive, and that DFTD is proliferating at too fast a rate to save them. Thus, humans should not intervene and let the devils go extinct naturally, rather than interfering with their ecosystem and habitat. However, I believe that much can be learnt about cancer from DFTD in Tasmanian devils, and that they hold such a key role in the Tasmanian ecosystem and that it is vital they are kept extant.
Therefore, I would conclude that the best way to manage the declining and endangered Tasmanian devil population would be a combination of multiple management ideas. Maintaining a breeding insurance population is vital, and this has been particularly successful on Maria Island, which provides a similar habitat to Tasmania.
Conclusion
In conclusion, to what extent do we understand DFTD, and what are the conservation options for saving them from extinction?
32 DFTD is a unique cancer that has developed strategies to avoid the host’s immune response and has capitalised on the biting behaviour of devils to allow transmission between individuals. This unfortunate situation has provided unique opportunities to study transmissible cancers, including mechanisms of cancer cell transfer and immune evasion. It has also highlighted the possibility that transmissible cancers in wild animals may be more common than originally thought. Accelerated research within the last 20 years, due to a high incidence of devils with tumours found on the Freycinet Peninsula, Tasmania, in 1996, has shown much about DFTD. Most studies that carried out genomic and pathological analysis of the tumour have concluded that it arose from a female devil, and that carcinogens and other chemical factors were most likely not to blame. Further research ascertained that devil facial tumours are bundles of neoplastic cells with severe anaplasia exhibited of epithelial tissues. Furthermore, we now know that DFTD arose from Schwann cells and that a probable immune evasion technique of the tumour is the downregulation of the MHC. That all of this information has been produced in such a short amount of time is undoubtedly noteworthy. It also has positive implications for human cancer research, with increased focus on the immune evasion techniques of the cancer, which will lead to more personalised and focused treatment.
However, research has also highlighted how little is currently known about the nature of DFTD, particularly its transmission. As concluded in my discussion, the allograft theory of transmission appears to be the most plausible, but this assumption is based on very little research. For example, only one paper is currently available that discusses non-allograft theories, so it is clear that our understanding in this area has much to improve upon and further research is necessary to form a more complete argument. This should include more analysis of the tumours found by Cui et al. (Cui, 2016)27, and more research into how DFTD arose in Tasmanian devils.
In terms of conservation, the preservation of the Tasmanian devil population has, and will continue, to require a coordinated approach from various governmental and non-governmental bodies. Despite the less effective attempts to remove the prevalence of DFTD via culling infected individuals and the ecological concerns of fencing, there have been recent efforts to safeguard the Tasmanian devil population. The successful establishment of an insurance population of healthy captive devils and research towards a vaccine will facilitate the reintroduction of these animals into the wild. Currently, researchers at Southampton University are trying to find peptide candidates that can prevent the onset of DFTD in Tasmanian devils. However, this research is ongoing and there are no results as of yet. Currently, this is definitely the most feasible option to save the Tasmanian devil. However, research is still ongoing and there is much to learn about DFTD. There are high hopes for an effective vaccination, and this will be the most efficient way to keep the Tasmanian devil from extinction.
33 Evaluation
This project has been a huge aid in developing my skills in researching, reading and writing about science. Throughout the course of investigating and writing my dissertation, I have become significantly better at using scientific research papers to find relevant information and evidence for arguments, as well as understanding the technical terminology. Furthermore, I have become much more efficient in analysing information critically and deciding whether or not it is appropriate for the topic I am writing about. I have also developed my scientific writing style, having read enough research papers to understand the jargon and manner in which they are written. However, inevitably, I encountered some problems whilst writing my dissertation, and I have discussed these below.
The first problem I encountered when starting this project was finding some basic information about Tasmanian devils. I quickly found that there is not much general literature about them as they are not a particularly well- known species outside of Tasmania. I managed to find and read Tasmanian Devils: A Unique and Threatened Animal5, which is, I found, the only general book about them and is already out of date having been published in 2005. This means I had to quickly move onto scientific research and journals, as everything else is very anecdotal.
The next problem I met was a lack of biological knowledge. I started this project before I had covered any topics on immunity or pathology in lessons, and trying to read scientific papers that were investigating a very small niche of this was extremely challenging. I slowly starting to learn the terms for different immune cells and responses, and the research papers started to make more sense. What I found invaluable was choosing papers that were related to my question and making detailed notes of them, making sure I understood each section and its significance to my project before continuing. I also found that this project required a glossary, as it uses many technical biological terms that needed defining, it was incredibly useful and both the reader of this project and myself, as I was writing it, could refer back to it.
Thirdly, time management was an anticipated problem before I began the project. I run a very busy schedule with many activities both in and out of school, as well as applying to universities and heading off for interviews during the main writing periods of the project. However, the weekly double periods on Tuesday afternoons proved very useful over these phases and I remained more or less on track throughout the entire project. The most effective method I found was to set myself deadlines within the general project deadlines our assessor gave us, such as finishing a subsection by a certain day or reading a certain amount of papers I had chosen in a set amount of time. This allowed me to consistently move forward with my project, and although there were the inevitable times when I had to shift my timetable around to fit in time for my EPQ, it worked fairly well.
34 The final issue I was not expecting to encounter was the number of times I changed my project title. Although they were all fairly similar, they would each require a different set of research papers and lead to a different conclusion. Therefore, I ended up reading a lot of information that turned out redundant for the purposes of my project. This led to a significant amount of wasted research time, reading papers that are not entirely relevant to my EPQ title. However, I do feel that changing the title was a very useful process. It allowed me to really think though what I wanted to explore in my EPQ, and what might have the most meaningful conclusion with regards to the future of Tasmanian devils. Researching the extra information was actually very useful, as it allowed me to evaluate sources and opinions more effectively.
Overall, I am very pleased I completed an EPQ. the process of writing an extended piece of scientific literature has been an invaluable tool in developing skills that will be vital for reading Biological Sciences at university. I have become more efficient at analysing scientific documents, as well as better at prioritising both my time and the information I need. If I were to repeat my EPQ, I would set aside more time for the initial reading phase, and complete my activity log more regularly. I would also look ahead to see which commitments may prevent me from continuing with my EPQ (such as university interviews and mock exams), and plan my time around those accordingly. However, I have mostly kept to the key deadlines, so I feel I was fairly successful in this.
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