Lead exposure and source attribution for a mammalian scavenger before and after a culling program.

Lead-based ammunition is a significant source of environmental lead and threatens species that scavenge lead-shot carcasses, particularly in areas with intensive shooting. With the impacts of lead on avian scavengers well established, there is increasing focus on the effects of lead on mammalian scavengers. We investigated lead exposure in a morphologically specialized mammalian scavenger, the Tasmanian devil (Sarcophilus harrisii), by analyzing their blood lead levels (BLLs) before and after a marsupial culling program using linear mixed effects models. We compared lead isotope signatures in devil blood to those in the culling ammunition to inform potential source attributions. We sampled 23 devils before culling and 15 after culling, finding no significant difference in mean BLLs pre and post-culling. However, devils captured closer to forestry coupes where culling had occurred had higher BLLs, and a greater proportion of devils displayed elevated BLLs post-culling (33 % compared to 18 % pre-culling). The highest BLL (7.93 µg/dL) was found in a devil post-culling and this individual had lead isotope signatures that matched the ammunition samples analyzed, suggesting the individual was exposed to lead from scavenging on culled carcasses. While 18 % of the devil blood lead samples had isotope signatures consistent with the ammunition samples, most were measurably different, indicating other sources of lead in the landscape. BLLs in our study landscape were similar to published BLLs for wild devils across Tasmania. That said, lead isotope signatures in the blood of individual devils sampled both before and after culling shifted closer to those of ammunition samples post-culling. Our results indicate that while some individual devils may have been exposed to lead from culling, most devils in the landscape did not show evidence of recent exposure. However, even low lead levels can adversely impact wildlife health and immunity, a particular concern for devils, a species endangered by disease.

Genome of the endangered eastern quoll (Dasyurus viverrinus) reveals signatures of historical decline and pelage color evolution.

The eastern quoll (Dasyurus viverrinus) is an endangered marsupial native to Australia. Since the extirpation of its mainland populations in the 20th century, wild eastern quolls have been restricted to two islands at the southern end of their historical range. Eastern quolls are the subject of captive breeding programs and attempts have been made to re-establish a population in mainland Australia. However, few resources currently exist to guide the genetic management of this species. Here, we generated a reference genome for the eastern quoll with gene annotations supported by multi-tissue transcriptomes. Our assembly is among the most complete marsupial genomes currently available. Using this assembly, we infer the species' demographic history, identifying potential evidence of a long-term decline beginning in the late Pleistocene. Finally, we identify a deletion at the ASIP locus that likely underpins pelage color differences between the eastern quoll and the closely related Tasmanian devil (Sarcophilus harrisii).

Human habitat modification, not apex scavenger decline, drives isotopic niche variation in a carnivore community.

Top carnivores can influence the structure of ecological communities, primarily through competition and predation; however, communities are also influenced by bottom-up forces such as anthropogenic habitat disturbance. Top carnivore declines will likely alter competitive dynamics within and amongst sympatric carnivore species. Increasing intraspecific competition is generally predicted to drive niche expansion and/or individual specialisation, while interspecific competition tends to constrain niches. Using stable isotope analysis of whiskers, we studied the effects of Tasmanian devil Sarcophilus harrisii declines upon the population- and individual-level isotopic niches of Tasmanian devils and sympatric spotted-tailed quolls Dasyurus maculatus subsp. maculatus. We investigated whether time since the onset of devil decline (a proxy for severity of decline) and landscape characteristics affected the isotopic niche breadth and overlap of devil and quoll populations. We quantified individual isotopic niche breadth for a subset of Tasmanian devils and spotted-tailed quolls and assessed whether between-site population niche variation was driven by individual-level specialisation. Tasmanian devils and spotted-tailed quolls demonstrated smaller population-level isotopic niche breadths with increasing human-modified habitat, while time since the onset of devil decline had no effect on population-level niche breadth or interspecific niche overlap. Individual isotopic niche breadths of Tasmanian devils and spotted-tailed quolls were narrower in human-modified landscapes, likely driving population isotopic niche contraction, however, the degree of individuals' specialisation relative to one another remained constant. Our results suggest that across varied landscapes, mammalian carnivore niches can be more sensitive to the bottom-up forces of anthropogenic habitat disturbance than to the top-down effects of top carnivore decline.

Intergenomic signatures of coevolution between Tasmanian devils and an infectious cancer.

Coevolution is common and frequently governs host-pathogen interaction outcomes. Phenotypes underlying these interactions often manifest as the combined products of the genomes of interacting species, yet traditional quantitative trait mapping approaches ignore these intergenomic interactions. Devil facial tumor disease (DFTD), an infectious cancer afflicting Tasmanian devils (Sarcophilus harrisii), has decimated devil populations due to universal host susceptibility and a fatality rate approaching 100%. Here, we used a recently developed joint genome-wide association study (i.e., co-GWAS) approach, 15 y of mark-recapture data, and 960 genomes to identify intergenomic signatures of coevolution between devils and DFTD. Using a traditional GWA approach, we found that both devil and DFTD genomes explained a substantial proportion of variance in how quickly susceptible devils became infected, although genomic architectures differed across devils and DFTD; the devil genome had fewer loci of large effect whereas the DFTD genome had a more polygenic architecture. Using a co-GWA approach, devil-DFTD intergenomic interactions explained ~3× more variation in how quickly susceptible devils became infected than either genome alone, and the top genotype-by-genotype interactions were significantly enriched for cancer genes and signatures of selection. A devil regulatory mutation was associated with differential expression of a candidate cancer gene and showed putative allele matching effects with two DFTD coding sequence variants. Our results highlight the need to account for intergenomic interactions when investigating host-pathogen (co)evolution and emphasize the importance of such interactions when considering devil management strategies.

Disease-driven top predator decline affects mesopredator population genomic structure.

Top predator declines are pervasive and often have dramatic effects on ecological communities via changes in food web dynamics, but their evolutionary consequences are virtually unknown. Tasmania's top terrestrial predator, the Tasmanian devil, is declining due to a lethal transmissible cancer. Spotted-tailed quolls benefit via mesopredator release, and they alter their behaviour and resource use concomitant with devil declines and increased disease duration. Here, using a landscape community genomics framework to identify environmental drivers of population genomic structure and signatures of selection, we show that these biotic factors are consistently among the top variables explaining genomic structure of the quoll. Landscape resistance negatively correlates with devil density, suggesting that devil declines will increase quoll genetic subdivision over time, despite no change in quoll densities detected by camera trap studies. Devil density also contributes to signatures of selection in the quoll genome, including genes associated with muscle development and locomotion. Our results provide some of the first evidence of the evolutionary impacts of competition between a top predator and a mesopredator species in the context of a trophic cascade. As top predator declines are increasing globally, our framework can serve as a model for future studies of evolutionary impacts of altered ecological interactions.

Global change influences scavenging and carrion decomposition.

Carrion decomposition is fundamental to nutrient cycling in terrestrial ecosystems because it provides a high-quality resource to diverse organisms. A conceptual framework incorporating all phases of carrion decomposition with the full community of scavengers is needed to predict the effects of global change on core ecosystem processes. Because global change can differentially impact scavenger guilds and rates of carrion decomposition, our framework explicitly incorporates complex interactions among microbial, invertebrate, and vertebrate scavenger communities across three distinct phases of carcass decomposition. We hypothesize that carrion decomposition rates will be the most impacted when global change affects carcass discovery rates and the foraging behavior of competing scavenger guilds.

Early onset developmental and epileptic encephalopathy and Rett-like phenotype in a 15-year-old girl affected by Cornelia de Lange syndrome type 2 due to a SMC1A gene mutation.

Developmental and epileptic encephalopathies (DEE) are conditions in which a mutated gene may cause abnormal functioning of the central nervous system, resulting in both encephalopathy and epileptogenesis. We present a case of a girl with a DEE characterized by a Rett-like phenotype in association with febrile and afebrile clusters of focal seizures. The girl presented typical development until the age of 18 months, followed by regression. The first febrile bilateral tonic-clonic seizure was observed at 30 months of age, and the following month seizures recurred in clusters of several episodes per day every 10 days. These seizures were characterized by behavioural arrest, emotional symptoms, head turning, and followed by bilateral tonic-clonic seizures. The administration of valproic acid and levetiracetam led to prolonged seizure control. However, from the age of 7 years, she had monthly recurrent clusters of focal seizures and non-convulsive status epilepticus which occurred at different ages. Brain and spinal cord MRI showed mild non-progressive hemispheric cerebellar atrophy. A next generation sequencing panel for epilepsy identified the de novo splicing mutation c.2973+1G>A of the SMC1A gene.

Over-eruption in marsupial carnivore teeth: compensation for a constraint.

Pronounced over-eruption of the canine teeth, causing the cervical enamel margin to extend beyond the alveolar bone and exposing the root, occurs with age and growth in Australian marsupial carnivores, much more than in eco-morphologically equivalent placental carnivores. Suppression of functional tooth replacement is characteristic of marsupials, where most placentals have the primitive diphyodont pattern of two generations of incisor, canine and premolar teeth. Canine and molar tooth dimensions of four species of marsupial carnivores (thylacine Thylacinus cynocephalus, Tasmanian devil Sarcophilus harrisii and two quolls Dasyurus spp.) and canine dimensions of seven eco-morphologically equivalent placental carnivore species were measured from museum specimens. Canine dimensions were measured in a time series on live wild-living individual devils and quolls. The canine teeth and to a lesser extent the molar teeth of marsupial carnivores continue to erupt through life, resulting in a net increase in tooth height and diameter, a phenomenon not evident in placental carnivores. Potential mechanisms causing over-eruption include tooth wear and gradual release of occlusal pressure as the individual grows. Over-eruption in marsupial carnivores may be a compensatory response for tooth size limits imposed by monophyodont tooth replacement, ensuring that animal's teeth are scaled to jaw size from juvenile to adulthood.

Top predator restricts the niche breadth of prey: effects of assisted colonization of Tasmanian devils on a widespread omnivorous prey.

Few landscape-scale experiments test the effects of predators on the abundance and distribution of prey across habitat gradients. We use the assisted colonization of a top predator, the Tasmanian devil (Sarcophilus harrisii), to test the impacts of predation on the abundance, habitat use and temporal activity of a widespread prey species, the omnivorous common brushtail possum (Trichosurus vulpecula). Before introduction of devils to Maria Island, Tasmania, Australia, in 2012, possums were abundant in open grasslands as well as forests. Predation by devils caused high mortality of possums in grasslands, but individuals with access to trees had a higher survival probability. Possum abundance declined across the whole island from 2012-2016, as possums disappeared almost completely from grasslands and declined in drier forests with more open understorey. Abundance remained stable in wet forests, which are not preferred habitat for possums but provide better refuge from devils. Abundance and habitat use of possums remained unchanged at a control site on the adjacent Tasmanian mainland, where the devil population was low and stable. This study demonstrates how spatial variation in predator-caused mortality can limit both abundance and habitat breadth in generalist prey species, excluding them entirely from certain habitats.

Dominant carnivore loss benefits native avian and invasive mammalian scavengers.

Scavenging by large carnivores is integral for ecosystem functioning by limiting the build-up of carrion and facilitating widespread energy flows. However, top carnivores have declined across the world, triggering trophic shifts within ecosystems. Here, we compare findings from previous work on predator decline against areas with recent native mammalian carnivore loss. Specifically, we investigate top-down control on utilization of experimentally placed carcasses by two mesoscavengers-the invasive feral cat and native forest raven. Ravens profited most from carnivore loss, scavenging for five times longer in the absence of native mammalian carnivores. Cats scavenged on half of all carcasses in the region without dominant native carnivores. This was eight times more than in areas where other carnivores were at high densities. All carcasses persisted longer than the three-week monitoring period in the absence of native mammalian carnivores, while in areas with high carnivore abundance, all carcasses were fully consumed. Our results reveal that top-carnivore loss amplifies impacts associated with carnivore decline-increased carcass persistence and carrion access for smaller scavengers. This suggests that even at low densities, native mammalian carnivores can fulfil their ecological functions, demonstrating the significance of global carnivore conservation and supporting management approaches, such as trophic rewilding.

Cathelicidin-3 Associated With Serum Extracellular Vesicles Enables Early Diagnosis of a Transmissible Cancer.

The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (Sarcophilus harrisii). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis; 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population.

Long-Distance Movements of Feral Cats in Semi-Arid South Australia and Implications for Conservation Management.

Movements that extend beyond the usual space use of an animal have been documented in a range of species and are particularly prevalent in arid areas. We present long-distance movement data on five feral cats (Felis catus) GPS/VHF-collared during two different research projects in arid and semi-arid Australia. We compare these movements with data from other feral cat studies. Over a study period of three months in the Ikara-Flinders Ranges National Park, 4 out of 19 collared cats moved to sites that were 31, 41, 53 and 86 km away. Three of the cats were males, one female; their weight was between 2.1 and 4.1 kg. Two of the cats returned to the area of capture after three and six weeks. During the other study at Arid Recovery, one collared male cat (2.5 kg) was relocated after two years at a distance of 369 km from the area of collar deployment to the relocation area. The movements occurred following three years of record low rainfall. Our results build on the knowledge base of long-distance movements of feral cats reported at arid study sites and support the assertion that landscape-scale cat control programs in arid and semi-arid areas need to be of a sufficiently large scale to avoid rapid reinvasion and to effectively reduce cat density. Locally, cat control strategies need to be adjusted to improve coverage of areas highly used by cats to increase the efficiency of control operations.

Extracellular vesicle proteomes of two transmissible cancers of Tasmanian devils reveal tenascin-C as a serum-based differential diagnostic biomarker.

The iconic Tasmanian devil (Sarcophilus harrisii) is endangered due to the transmissible cancer Devil Facial Tumour Disease (DFTD), of which there are two genetically independent subtypes (DFT1 and DFT2). While DFT1 and DFT2 can be differentially diagnosed using tumour biopsies, there is an urgent need to develop less-invasive biomarkers that can detect DFTD and distinguish between subtypes. Extracellular vesicles (EVs), the nano-sized membrane-enclosed vesicles present in most biofluids, represent a valuable resource for biomarker discovery. Here, we characterized the proteome of EVs from cultured DFTD cells using data-independent acquisition-mass spectrometry and an in-house spectral library of > 1500 proteins. EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions. Furthermore, hallmark proteins of epithelial-mesenchymal transition were enriched in DFT2 EVs relative to DFT1 EVs. These findings were validated in EVs derived from serum samples, revealing that the mesenchymal marker tenascin-C was also enriched in EVs derived from the serum of devils infected with DFT2 relative to those infected with DFT1 and healthy controls. This first EV-based investigation of DFTD increases our understanding of the cancers' EVs and their possible involvement in DFTD progression, such as metastasis. Finally, we demonstrated the potential of EVs to differentiate between DFT1 and DFT2, highlighting their potential use as less-invasive liquid biopsies for the Tasmanian devil.

Spatial variation in gene expression of Tasmanian devil facial tumors despite minimal host transcriptomic response to infection.

BACKGROUND: Transmissible cancers lie at the intersection of oncology and infectious disease, two traditionally divergent fields for which gene expression studies are particularly useful for identifying the molecular basis of phenotypic variation. In oncology, transcriptomics studies, which characterize the expression of thousands of genes, have identified processes leading to heterogeneity in cancer phenotypes and individual prognoses. More generally, transcriptomics studies of infectious diseases characterize interactions between host, pathogen, and environment to better predict population-level outcomes. Tasmanian devils have been impacted dramatically by a transmissible cancer (devil facial tumor disease; DFTD) that has led to widespread population declines. Despite initial predictions of extinction, populations have persisted at low levels, due in part to heterogeneity in host responses, particularly between sexes. However, the processes underlying this variation remain unknown. RESULTS: We sequenced transcriptomes from healthy and DFTD-infected devils, as well as DFTD tumors, to characterize host responses to DFTD infection, identify differing host-tumor molecular interactions between sexes, and investigate the extent to which tumor gene expression varies among host populations. We found minimal variation in gene expression of devil lip tissues, either with respect to DFTD infection status or sex. However, 4088 genes were differentially expressed in tumors among our sampling localities. Pathways that were up- or downregulated in DFTD tumors relative to normal tissues exhibited the same patterns of expression with greater intensity in tumors from localities that experienced DFTD for longer. No mRNA sequence variants were associated with expression variation. CONCLUSIONS: Expression variation among localities may reflect morphological differences in tumors that alter ratios of normal-to-tumor cells within biopsies. Phenotypic variation in tumors may arise from environmental variation or differences in host immune response that were undetectable in lip biopsies, potentially reflecting variation in host-tumor coevolutionary relationships among sites that differ in the time since DFTD arrival.

Isotopic niche variation in Tasmanian devils Sarcophilus harrisii with progression of devil facial tumor disease.

Devil facial tumor disease (DFTD) is a transmissible cancer affecting Tasmanian devils Sarcophilus harrisii. The disease has caused severe population declines and is associated with demographic and behavioral changes, including earlier breeding, younger age structures, and reduced dispersal and social interactions. Devils are generally solitary, but social encounters are commonplace when feeding upon large carcasses. DFTD tumors can disfigure the jaw and mouth and so diseased individuals might alter their diets to enable ingestion of alternative foods, to avoid conspecific interactions, or to reduce competition. Using stable isotope analysis (δ13C and δ15N) of whiskers, we tested whether DFTD progression, measured as tumor volume, affected the isotope ratios and isotopic niches of 94 infected Tasmanian devils from six sites in Tasmania, comprising four eucalypt plantations, an area of smallholdings and a national park. Then, using tissue from 10 devils sampled before and after detection of tumors and 8 devils where no tumors were detected, we examined whether mean and standard deviation of δ13C and δ15N of the same individuals changed between healthy and diseased states. δ13C and δ15N values were generally not related to tumor volume in infected devils, though at one site, Freycinet National Park, δ15N values increased significantly as tumor volume increased. Infection with DFTD was not associated with significant changes in the mean or standard deviation of δ13C and δ15N values in individual devils sampled before and after detection of tumors. Our analysis suggests that devils tend to maintain their isotopic niche in the face of DFTD infection and progression, except where ecological conditions facilitate a shift in diets and feeding behaviors, demonstrating that ecological context, alongside disease severity, can modulate the behavioral responses of Tasmanian devils to DFTD.

Contemporary and historical selection in Tasmanian devils (Sarcophilus harrisii) support novel, polygenic response to transmissible cancer.

Tasmanian devils (Sarcophilus harrisii) are evolving in response to a unique transmissible cancer, devil facial tumour disease (DFTD), first described in 1996. Persistence of wild populations and the recent emergence of a second independently evolved transmissible cancer suggest that transmissible cancers may be a recurrent feature in devils. Here, we compared signatures of selection across temporal scales to determine whether genes or gene pathways under contemporary selection (six to eight generations) have also been subject to historical selection (65-85 Myr). First, we used targeted sequencing, RAD-capture, in approximately 2500 devils in six populations to identify genomic regions subject to rapid evolution. We documented genome-wide contemporary evolution, including 186 candidate genes related to cell cycling and immune response. Then we used a molecular evolution approach to identify historical positive selection in devils compared to other marsupials and found evidence of selection in 1773 genes. However, we found limited overlap across time scales, with only 16 shared candidate genes, and no overlap in enriched functional gene sets. Our results are consistent with a novel, multi-locus evolutionary response of devils to DFTD. Our results can inform conservation by identifying high priority targets for genetic monitoring and guiding maintenance of adaptive potential in managed populations.

The effects of weather variability on patterns of genetic diversity in Tasmanian bettongs.

While the effects of climate (long-term, prevailing weather) on species abundance, range and genetic diversity have been widely studied, short-term, localized variations in atmospheric conditions (i.e., weather) can also rapidly alter species' geographical ranges and population sizes, but little is known about how they affect genetic diversity. We investigated the relationship between weather and range-wide genetic diversity in a marsupial, Bettongia gaimardi, using dynamic species distribution models (SDMs). Genetic diversity was lower in parts of the range where the weather-based SDM predicted high variability in probability of B. gaimardi occurrence during 1950-2009. This is probably an effect of lower population sizes and extinction-recolonization cycles in places with highly variable weather. Spatial variation in genetic diversity was also better predicted by mean probabilities of B. gaimardi occurrence from weather- than climate-based SDMs. Our results illustrate the importance of weather in driving population dynamics and species distributions on decadal timescales and thereby in affecting genetic diversity. Modelling the links between changing weather patterns, species distributions and genetic diversity will allow researchers to better forecast biological impacts of climate change.

A triple threat: high population density, high foraging intensity and flexible habitat preferences explain high impact of feral cats on prey.

Alien mammalian carnivores have contributed disproportionately to global loss of biodiversity. In Australia, predation by the feral cat and red fox is one of the most significant causes of the decline of native vertebrates. To discover why cats have greater impacts on prey than native predators, we compared the ecology of the feral cat to a marsupial counterpart, the spotted-tailed quoll. Individual prey are 20-200 times more likely to encounter feral cats, because of the combined effects of cats' higher population densities, greater intensity of home-range use and broader habitat preferences. These characteristics also mean that the costs to the prey of adopting anti-predator behaviours against feral cats are likely to be much higher than adopting such behaviours in response to spotted-tailed quolls, due to the reliability and ubiquity of feral cat cues. These results help explain the devastating impacts of cats on wildlife in Australia and other parts of the world.

A transmissible cancer shifts from emergence to endemism in Tasmanian devils.

Emerging infectious diseases pose one of the greatest threats to human health and biodiversity. Phylodynamics is often used to infer epidemiological parameters essential for guiding intervention strategies for human viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Here, we applied phylodynamics to elucidate the epidemiological dynamics of Tasmanian devil facial tumor disease (DFTD), a fatal, transmissible cancer with a genome thousands of times larger than that of any virus. Despite prior predictions of devil extinction, transmission rates have declined precipitously from ~3.5 secondary infections per infected individual to ~1 at present. Thus, DFTD appears to be transitioning from emergence to endemism, lending hope for the continued survival of the endangered Tasmanian devil. More generally, our study demonstrates a new phylodynamic analytical framework that can be applied to virtually any pathogen.