Carolyn J. Hogg

The Tasmanian devil microbiome—implications for conservation and management

BackgroundThe Tasmanian devil, the world’s largest carnivorous marsupial, is at risk of extinction due to devil facial tumour disease (DFTD), a fatal contagious cancer. The Save the Tasmanian Devil Program has established an insurance population, which currently holds over 600 devils in captive facilities across Australia. Microbes are known to play a crucial role in the health and well-being of humans and other animals, and increasing evidence suggests that changes in the microbiota can influence various aspects of host physiology and development. To improve our understanding of devils and facilitate management and conservation of the species, we characterised the microbiome of wild devils and investigated differences in the composition of microbial community between captive and wild individuals.ResultsA total of 1,223,550 bacterial 16S ribosomal RNA (rRNA) sequences were generated via Roche 454 sequencing from 56 samples, including 17 gut, 15 skin, 18 pouch and 6 oral samples. The devil’s gut microbiome was dominated by Firmicutes and showed a high Firmicutes-to-Bacteroidetes ratio, which appears to be a common feature of many carnivorous mammals. Metabolisms of carbohydrates, amino acids, energy, cofactors and vitamins, nucleotides and lipids were predicted as the most prominent metabolic pathways that the devils gut flora contributed to. The microbiota inside the female’s pouch outside lactation was highly similar to that of the skin, both co-dominated by Firmicutes and Proteobacteria. The oral microbiome had similar proportions of Proteobacteria, Bacteroidetes, Firmicutes and Fusobacteria.ConclusionsCompositional differences were observed in all four types of microbiota between devils from captive and wild populations. Certain captive devils had significantly lower levels of gut bacterial diversity than wild individuals, and the two groups differed in the proportion of gut bacteria accounting for the metabolism of glycan, amino acids and cofactors and vitamins. Further studies are underway to investigate whether alterations in the microbiome of captive devils can have impacts on their ability to adapt and survive following re-introduction to the wild.

Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (Sarcophilus harrisii).

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population.

BackgroundThe Tasmanian devil (Sarcophilus harrisii) has undergone a recent, drastic population decline due to the highly contagious devil facial tumor disease. The tumor is one of only two naturally occurring transmissible cancers and is almost inevitably fatal. In 2006 a disease-free insurance population was established to ensure that the Tasmanian devil is protected from extinction. The insurance program is dependent upon preserving as much wild genetic diversity as possible to maximize the success of subsequent reintroductions to the wild. Accurate genotypic data is vital to the success of the program to ensure that loss of genetic diversity does not occur in captivity. Until recently, microsatellite markers have been used to study devil population genetics, however as genetic diversity is low in the devil and potentially decreasing in the captive population, a more sensitive genotyping assay is required.MethodsUtilising the devil reference genome and whole genome re-sequencing data, we have identified polymorphic regions for use in a custom genotyping assay. These regions were amplified using PCR and sequenced on the Illumina MiSeq platform to refine a set a markers to genotype the Tasmanian devil insurance population.ResultsWe have developed a set of single nucleotide polymorphic (SNP) markers, assayed by amplicon sequencing, that provide a high-throughput method for monitoring genetic diversity and assessing familial relationships among devils. To date we have used a total of 267 unique SNPs within both putatively neutral and functional loci to genotype 305 individuals in the Tasmanian devil insurance population. We have used these data to assess genetic diversity in the population as well as resolve the parentage of 21 offspring.ConclusionsOur molecular data has been incorporated with studbook management practices to provide more accurate pedigree information and to inform breeding recommendations. The assay will continue to be used to monitor the genetic diversity of the insurance population of Tasmanian devils with the aim of reducing inbreeding and maximizing success of reintroductions to the wild.

Impacts of early viability selection on management of inbreeding and genetic diversity in conservation

Maintaining genetic diversity is a crucial goal of intensive management of threatened species, particularly for those populations that act as sources for translocation or re‐introduction programmes. Most captive genetic management is based on pedigrees and a neutral theory of inheritance, an assumption that may be violated by selective forces operating in captivity. Here, we explore the conservation consequences of early viability selection: differential offspring survival that occurs prior to management or research observations, such as embryo deaths in utero. If early viability selection produces genotypic deviations from Mendelian predictions, it may undermine management strategies intended to minimize inbreeding and maintain genetic diversity. We use empirical examples to demonstrate that straightforward approaches, such as comparing litter sizes of inbred vs. noninbred breeding pairs, can be used to test whether early viability selection likely impacts estimates of inbreeding depression. We also show that comparing multilocus genotype data to pedigree predictions can reveal whether early viability selection drives systematic biases in genetic diversity, patterns that would not be detected using pedigree‐based statistics alone. More sophisticated analysis combining genomewide molecular data with pedigree information will enable conservation scientists to test whether early viability selection drives deviations from neutrality across wide stretches of the genome, revealing whether this form of selection biases the pedigree‐based statistics and inference upon which intensive management is based.

No evidence of inbreeding depression in a Tasmanian devil insurance population despite significant variation in inbreeding

Inbreeding depression occurs when inbred individuals experience reduced fitness as a result of reduced genome-wide heterozygosity. The Tasmanian devil faces extinction due to a contagious cancer, devil facial tumour disease (DFTD). An insurance metapopulation was established in 2006 to ensure the survival of the species and to be used as a source population for re-wilding and genetic rescue. The emergence of DFTD and the rapid decline of wild devil populations have rendered the species at risk of inbreeding depression. We used 33 microsatellite loci to (1) reconstruct a pedigree for the insurance population and (2) estimate genome-wide heterozygosity for 200 individuals. Using heterozygosity-fitness correlations, we investigated the effect of heterozygosity on six diverse fitness measures (ulna length, asymmetry, weight-at-weaning, testes volume, reproductive success and survival). Despite statistically significant evidence of variation in individual inbreeding in this population, we found no associations between inbreeding and any of our six fitness measurements. We propose that the benign environment in captivity may decrease the intensity of inbreeding depression, relative to the stressful conditions in the wild. Future work will need to measure fitness of released animals to facilitate translation of this data to the broader conservation management of the species in its native range.

Adaptation and conservation insights from the koala genome

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.The assembly of the genome of the koala provides insights into its adaptive biology and identifies gene expansions that contribute to its ability to detoxify eucalyptus-derived compounds and perceive plant secondary metabolites.

Significant decline in anticancer immune capacity during puberty in the Tasmanian devil

Tasmanian devils (Sarcophilus harrisii) are at risk of extinction in the wild due to Devil Facial Tumour Disease (DFTD), a rare contagious cancer. The prevalence of DFTD differs by age class: higher disease prevalence is seen in adults (2–3 years) versus younger devils (<2 years). Here we propose that immunological changes during puberty may play a role in susceptibility to DFTD. We show that the second year of life is a key developmental period for Tasmanian devils, during which they undergo puberty and pronounced changes in the immune system. Puberty coincides with a significant decrease in lymphocyte abundance resulting in a much higher neutrophil:lymphocyte ratio in adults than subadults. Quantitative PCR analysis of gene expression of transcription factors T-bet and GATA-3 and cytokines interferon gamma (IFN-γ) and interleukin 4 (IL-4) revealed a drastic increase in GATA-3 and IL-4 expression during puberty. These changes led to a significantly lower IFN-γ:IL-4 ratio in 2-year-olds than <1 year olds (on average 1.3-fold difference in males and 4.0-fold in females), which reflects a major shift of the immune system towards Th2 responses. These results all indicate that adult devils are expected to have a lower anticancer immune capacity than subadults, which may explain the observed pattern of disease prevalence of DFTD in the wild.

Increasing generations in captivity is associated with increased vulnerability of Tasmanian devils to vehicle strike following release to the wild

Captive breeding of threatened species, for release to the wild, is critical for conservation. This strategy, however, risks producing captive-raised animals with traits poorly suited to the wild. We describe the first study to characterise accumulated consequences of long-term captive breeding on behaviour, by following the release of Tasmanian devils to the wild. We test the impact of prolonged captive breeding on the probability that captive-raised animals are fatally struck by vehicles. Multiple generations of captive breeding increased the probability that individuals were fatally struck, a pattern that could not be explained by other confounding factors (e.g. age or release site). Our results imply that long-term captive breeding programs may produce animals that are naïve to the risks of the post-release environment. Our analyses have already induced changes in management policy of this endangered species, and serve as model of productive synergy between ecological monitoring and conservation strategy.

Pedigree analysis reveals a generational decline in reproductive success of captive Tasmanian devil (Sarcophilus harrisii): implications for captive management of threatened species

Captive breeding programs are an increasingly popular tool to augment the conservation of threatened wild populations. Many programs keep detailed pedigrees, which are used to prescribe breeding targets to meet demographic and genetic goals. Annual breeding targets are based on previous productivity, but do not account for changes in reproductive success that may occur over generations in captivity and which may impair the ability of a program to meet its goals. We utilize a large studbook from the Tasmanian devil (Sarcophilus harrisii) captive breeding program to investigate biological, genetic, and environmental factors that affect variation in reproductive success among individuals and over generations of captive breeding. Reproductive success declined with increasing generations in captivity: wild-born females had a 56.5% chance of producing a litter compared to a 2.8% chance for generation 5 captive-born females (N = 182) and when they did, wild-born females produced more offspring (3.1 joeys, 95% CI: 2.76-3.38, compared to 2.7 joeys, 95% CI: 2.55-2.90, in captive-born females [N = 105]). Reproductive success also declined as dam age at first breeding increased. Our results reveal a conflict with the widely cited conservation strategy to limit opportunity for selection by extending generation length through delaying reproduction, as captive breeding programs that delay female breeding with this goal in mind risk reduced productivity. Our data demonstrate the benefit of pedigree analysis to identify biological processes that reveal crucial trade-offs with conservation best-practice.

Are any populations ‘safe’? Unexpected reproductive decline in a population of Tasmanian devils free of devil facial tumour disease

Abstract Context. Conservation management relies on baseline demographic data of natural populations. For Tasmanian devils (Sarcophilus harrisii), threatened in the wild by two fatal and transmissible cancers (devil facial tumour disease DFTD: DFT1 and DFT2), understanding the characteristics of healthy populations is crucial for developing adaptive management strategies to bolster populations in the wild. Aims. Our analysis aims to evaluate contemporary reproductive rates for wild, DFTD-free Tasmanian devil populations, and to provide a baseline with which to compare the outcome of current translocation activities. Methods. We analysed 8 years of field-trapping data, including demographics and reproductive rates, across 2004–16, from the largest known DFTD-free remnant population at Woolnorth, Tasmania. Key results. Surprisingly, we found a dramatic and statistically significant decline in female breeding rate when comparing data collected from 2004–2009 with data from 2014–2016. Unfortunately we do not have any data from the intermediate years. This decline in breeding rate was accompanied by a subtle but statistically significant decline in litter sizes. These changes were not associated with a change in body condition over the same period. Furthermore, we could not attribute the decline in breeding to a change in population size or sex ratio. Preliminary analysis suggested a possible association between annual breeding rate and coarse measures of environmental variation (Southern Oscillation Index), but any mechanistic associations are yet to be determined. Conclusions. The decline in breeding rates was unexpected, so further monitoring and investigation into potential environmental and/or biological reasons for the decline in breeding rate are recommended before the arrival of DFTD at Woolnorth. Implications. Our results provide valuable data to support the conservation management of Tasmanian devils in their native range. They also highlight the importance of continued monitoring of ‘safe’ populations, in the face of significant threats elsewhere.