Invasion and Amplification of Endogenous Retroviruses in Dasyuridae Marsupial Genomes.

Retroviruses are an ancient viral family that have globally coevolved with vertebrates and impacted their evolution. In Australia, a continent that has been geographically isolated for millions of years, little is known about retroviruses in wildlife, despite the devastating impacts of a retrovirus on endangered koala populations. We therefore sought to identify and characterize Australian retroviruses through reconstruction of endogenous retroviruses from marsupial genomes, in particular the Tasmanian devil due to its high cancer incidence. We screened 19 marsupial genomes and identified over 80,000 endogenous retrovirus fragments which we classified into eight retrovirus clades. The retroviruses were similar to either Betaretrovirus (5/8) or Gammaretrovirus (3/8) retroviruses, but formed distinct phylogenetic clades compared to extant retroviruses. One of the clades (MEBrv 3) lost an envelope but retained retrotranspositional activity, subsequently amplifying throughout all Dasyuridae genomes. Overall, we provide insights into Australian retrovirus evolution and identify a highly active endogenous retrovirus within Dasyuridae genomes.

Imprinted X chromosome inactivation in marsupials: The paternal X arrives at the egg with a silent DNA methylation profile.

X chromosome inactivation (XCI) is an epigenetic process that results in the transcriptional silencing of one X chromosome in the somatic cells of females. This phenomenon is common to both eutherian and marsupial mammals, but there are fundamental differences. In eutherians, the X chosen for silencing is random. DNA methylation on the eutherian inactive X is high at transcription start sites (TSSs) and their flanking regions, resulting in universally high DNA methylation. This contrasts XCI in marsupials where the paternally derived X is always silenced, and in which DNA methylation is low at TSSs and flanking regions. Here, we examined the DNA methylation status of the tammar wallaby X chromosome during spermatogenesis to determine the DNA methylation profile of the paternal X prior to and at fertilization. Whole genome enzymatic methylation sequencing was carried out on enriched flow-sorted populations of premeiotic, meiotic, and postmeiotic cells. We observed that the X displayed a pattern of DNA methylation from spermatogonia to mature sperm that reflected the inactive X in female somatic tissue. Therefore, the paternal X chromosome arrives at the egg with a DNA methylation profile that reflects the transcriptionally silent X in adult female somatic tissue. We present this epigenetic signature as a candidate for the long sought-after imprint for paternal XCI in marsupials.

Hemoplasmas in wild rodents and marsupials from the Caatinga Biome, Brazil.

A total of 231 blood samples from wild mammals belonging to the orders Rodentia (n = 142) and Didelphimorphia (n = 89) were screened by real-time PCR assay (qPCR), being six Rhipidomys sp., 118 Thrichomys laurentius, nine Rattus rattus, four Kerodon rupestris, five Necromys lasiurus, 42 Didelphis albiventris and 47 Monodelphis domestica. Results using qPCR showed that 32 of the total 231 (13.85 %) samples were positive for hemoplasma sequences of the 16S rRNA gene. Sequences from two D. albiventris showed 99.77-99.89 % identity with 'Candidatus Mycoplasma haemoalbiventris' and 99.09 % with 'Candidatus Mycoplasma haemodidelphidis', respectively. Furthermore, one M. domestica and five T. laurentius showed 99.72-99.77 % identity with Mycoplasma sp., and one K. rupestris showed 98.13 % identity with 'Candidatus Mycoplasma haematohydrochaerus'; and from two Rattus rattus showed 99.65-99.89 % identity with Mycoplasma sp. and 'Candidatus Mycoplasma haemomuris'. The 23S rRNA gene sequences obtained from the two D. albiventris showed 100 % identity with 'Ca. M. haemoalbiventris' whereas the sequences from the R. rattus showed only 85.31 % identity with 'Candidatus Mycoplasma haematohydrochaerus'. Two T. laurentius and one K. rupestris showed 84.66-92.97 % identity with 'Candidatus Mycoplasma haemosphiggurus'. Based on phylogenetic and Neighbor-Net network analyses of the 16S and 23S rRNA genes, potential novel species are described. In addition, 'Ca. M. haemoalbiventris' was detected in Didelphis albiventris, and Mycoplasma sp. was detected in Rattus sp. rodents from the Caatinga biome, Brazil.

Adaptive interventions for advancing in situ wildlife disease management.

There is a critical need for advancements in disease management strategies for wildlife, but free-living animals pose numerous challenges that can hinder progress. Most disease management attempts involve fixed interventions accompanied by post hoc outcome assessments focused on success or failure. Though these approaches have led to valuable management advances, there are limitations to both the rate of advancement and amount of information that can be gained. As such, strategies that support more rapid progress are required. Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei mites, is a globally emerging and re-emerging panzootic that exemplifies this problem. The bare-nosed wombat (Vombatus ursinus), a marsupial endemic to southeastern Australia, is impacted by sarcoptic mange throughout its geographic range and enhanced disease management capabilities are needed to improve upon existing in situ methods. We sought to advance in situ wildlife disease management for sarcoptic mange in free-living bare-nosed wombats, implementing an adaptive approach using fluralaner (Bravecto, MSD Animal Health) and a structured process of learning and method-optimisation. By using surveillance of treated wombats to inform real-time management changes, we have demonstrated the efficacy of topically administered fluralaner at 45 and 85 mg/kg against sarcoptic mange. Importantly, we observed variation in the effects of 45 mg/kg doses, but through our adaptive approach found that 85 mg/kg doses consistently reduced mange severity. Through modifying our surveillance program, we also identified individual-level variation in wombat observability and used this to quantify the level of surveillance needed to assess long-term management success. Our adaptive intervention represents the first report of sarcoptic mange management with fluralaner in free-living wildlife and evaluation of its efficacy in situ. This study illustrates how adapting interventions in real time can advance wildlife disease management and may be applicable to accelerating in situ improvements for other host-pathogen systems.

Characterising the Tasmanian devil (Sarcophilus harrisii) pouch microbiome in lactating and non-lactating females.

Wildlife harbour a diverse range of microorganisms that affect their health and development. Marsupials are born immunologically naïve and physiologically underdeveloped, with primary development occurring inside a pouch. Secretion of immunological compounds and antimicrobial peptides in the epithelial lining of the female's pouch, pouch young skin, and through the milk, are thought to boost the neonate's immune system and potentially alter the pouch skin microbiome. Here, using 16S rRNA amplicon sequencing, we characterised the Tasmanian devil pouch skin microbiome from 25 lactating and 30 non-lactating wild females to describe and compare across these reproductive stages. We found that the lactating pouch skin microbiome had significantly lower amplicon sequence variant richness and diversity than non-lactating pouches, however there was no overall dissimilarity in community structure between lactating and non-lactating pouches. The top five phyla were found to be consistent between both reproductive stages, with over 85% of the microbiome being comprised of Firmicutes, Proteobacteria, Fusobacteriota, Actinobacteriota, and Bacteroidota. The most abundant taxa remained consistent across all taxonomic ranks between lactating and non-lactating pouch types. This suggests that any potential immunological compounds or antimicrobial peptide secretions did not significantly influence the main community members. Of the more than 16,000 total identified amplicon sequence variants, 25 were recognised as differentially abundant between lactating and non-lactating pouches. It is proposed that the secretion of antimicrobial peptides in the pouch act to modulate these microbial communities. This study identifies candidate bacterial clades on which to test the activity of Tasmanian devil antimicrobial peptides and their role in pouch young protection, which in turn may lead to future therapeutic development for human diseases.

Conversion of Mouse-Derived Hybridomas to Tasmanian Devil Recombinant IgG Antibodies.

The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.

Pharmacokinetics of a topical application of moxidectin in bare-nosed wombats (Vombatus ursinus).

Sarcoptic mange is a debilitating disease that affects bare-nosed wombats (Vombatus ursinus). One of the drugs currently used for treatment is moxidectin, as it has a relatively high efficacy against endo and ectoparasites and side effects are uncommon in domestic species, thus it is considered a relatively safe drug to use at the recommended doses. Developing further understanding of the pharmacokinetics of moxidectin will aid in developing treatment regimens for sarcoptic mange in wombats. Here we analyzed the pharmacokinetic parameters of using 100 ml of moxidectin (5 g/l) applied topically. We found that mean peak plasma concentration was 0.50 ng/ml and half-life was 8 days. Moxidectin was excreted in scats with the mean peak concentration of 2461.43 ng/g (on a dry matter basis). Our study has provided the pharmacokinetic parameters of a commonly used treatment for sarcoptic mange in wombats. There were no adverse side effects recorded in the wombats after applying moxidectin topically. This study replicated real-world conditions using topical application on free-living wombats. The relatively low plasma concentration suggests the drug is not accumulating in the blood stream and is excreted via scats.

Do Tasmanian devil declines impact ecosystem function?

Tasmanian eucalypt forests are among the most carbon-dense in the world, but projected climate change could destabilize this critical carbon sink. While the impact of abiotic factors on forest ecosystem carbon dynamics have received considerable attention, biotic factors such as the input of animal scat are less understood. Tasmanian devils (Sarcophilus harrisii)-an osteophageous scavenger that can ingest and solubilize nutrients locked in bone material-may subsidize plant and microbial productivity by concentrating bioavailable nutrients (e.g., nitrogen and phosphorus) in scat latrines. However, dramatic declines in devil population densities, driven by the spread of a transmissible cancer, may have underappreciated consequences for soil organic carbon (SOC) storage and forest productivity by altering nutrient cycling. Here, we fuse experimental data and modeling to quantify and predict future changes to forest productivity and SOC under various climate and scat-quality futures. We find that devil scat significantly increases concentrations of nitrogen, ammonium, phosphorus, and phosphate in the soil and shifts soil microbial communities toward those dominated by r-selected (e.g., fast-growing) phyla. Further, under expected increases in temperature and changes in precipitation, devil scat inputs are projected to increase above- and below-ground net primary productivity and microbial biomass carbon through 2100. In contrast, when devil scat is replaced by lower-quality scat (e.g., from non-osteophageous scavengers and herbivores), forest carbon pools are likely to increase more slowly, or in some cases, decline. Together, our results suggest often overlooked biotic factors will interact with climate change to drive current and future carbon pool dynamics in Tasmanian forests.

Plethora of New Marsupial Genomes Informs Our Knowledge of Marsupial MHC Class II.

The major histocompatibility complex (MHC) plays a vital role in the vertebrate immune system due to its role in infection, disease and autoimmunity, or recognition of "self". The marsupial MHC class II genes show divergence from eutherian MHC class II genes and are a unique taxon of therian mammals that give birth to altricial and immunologically naive young providing an opportune study system for investigating evolution of the immune system. Additionally, the MHC in marsupials has been implicated in disease associations, including susceptibility to Chlamydia pecorum infection in koalas. Due to the complexity of the gene family, automated annotation is not possible so here we manually annotate 384 class II MHC genes in 29 marsupial species. We find losses of key components of the marsupial MHC repertoire in the Dasyuromorphia order and the Pseudochiridae family. We perform PGLS analysis to show the gene losses we find are true gene losses and not artifacts of unresolved genome assembly. We investigate the associations between the number of loci and life history traits, including lifespan and reproductive output in lineages of marsupials and hypothesize that gene loss may be linked to the energetic cost and tradeoffs associated with pregnancy and reproduction. We found support for litter size being a significant predictor of the number of DBA and DBB loci, indicating a tradeoff between the energetic requirements of immunity and reproduction. Additionally, we highlight the increased susceptibility of Dasyuridae species to neoplasia and a potential link to MHC gene loss. Finally, these annotations provide a valuable resource to the immunogenetics research community to move forward and further investigate diversity in MHC genes in marsupials.

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.

Regional Intraspecific Differences of Thermal Biology in a Marsupial Hibernator.

AbstractDuring periods of torpor, hibernators can reduce metabolic rate (MR) and body temperature (Tb) substantially. However, to avoid physiological dysfunction at low temperatures, they defend Tb at a critical minimum, often between ~0°C and 10°C via an increase in MR. Because thermoregulation during torpor requires extra energy, individuals with lower Tb's and thus minimal MR during torpor should be selected in colder climates. Such inter- and intraspecific variations occur in some placental mammals, but for the evolutionary separate marsupials, available information is scarce. Marsupial eastern pygmy possums (Cercartetus nanus; ~22 g body mass), widely distributed along the Australian southeastern coast including subtropical to alpine areas, were used to test the hypothesis that the defended Tb of torpid individuals is related to the climate of their habitat. Possums were captured from five regions, 1,515 km apart, with midwinter (July) minimum environmental temperatures (min Tenv's) ranging from -3.9°C to 6.6°C. Captive possums in deep torpor were slowly cooled with ambient temperature (Ta), while their MR was measured to determine the minimum torpor metabolic rate (TMR), the Ta at which their MR increased for thermoregulation (min Ta), and the corresponding minimum Tb (min Tb). Partial least squares regression analysis revealed that Ta and Tenv were the strongest explanatory variables for the min Tb. The min Tb and Ta were also correlated with latitude but not elevation of the capture sites. However, the best correlations were observed between the min Tenv and the min Tb and Ta for individuals experiencing min Tenv>0°C; these individuals thermoconformed to min Ta's between -0.8°C and 3.7°C, and their min Tb ranged from 0.5°C to 6.0°C and was 0.5°C-2.6°C below the min Tenv at the capture site. In contrast, individuals experiencing a min Tenv of -3.9°C regulated Tb at 0.6°C±0.2°C or 4.5°C above the Tenv. The minimum TMR of all possums did not differ with Ta and thus did not differ among populations and was 2.6% of the basal MR. These data provide new evidence that thermal variables of marsupials are subject to regional intraspecific variation. It suggests that min Tb is a function of the min Tenv but only above 0°C, perhaps because the Tb-Ta differential for torpid possums in the wild, at a min Tenv of -3.9°C, remains small enough to be compensated by a small increase in MR and does not require the physiological capability for a reduction of Tb below 0°C.

Two-stage evolution of mammalian adipose tissue thermogenesis.

Brown adipose tissue (BAT) is a heater organ that expresses thermogenic uncoupling protein 1 (UCP1) to maintain high body temperatures during cold stress. BAT thermogenesis is considered an overarching mammalian trait, but its evolutionary origin is unknown. We show that adipose tissue of marsupials, which diverged from eutherian mammals ~150 million years ago, expresses a nonthermogenic UCP1 variant governed by a partial transcriptomic BAT signature similar to that found in eutherian beige adipose tissue. We found that the reconstructed UCP1 sequence of the common eutherian ancestor displayed typical thermogenic activity, whereas therian ancestor UCP1 is nonthermogenic. Thus, mammalian adipose tissue thermogenesis may have evolved in two distinct stages, with a prethermogenic stage in the common therian ancestor linking UCP1 expression to adipose tissue and thermal stress. We propose that in a second stage, UCP1 acquired its thermogenic function specifically in eutherians, such that the onset of mammalian BAT thermogenesis occurred only after the divergence from marsupials.

Evergrowing incisors of diprotodont marsupials record age and life history.

OBJECTIVE: Tooth growth and wear are commonly used tools for determining the age of mammals. The most speciose order of marsupials, Diprotodontia, is characterised by a pair of procumbent incisors within the lower jaw. This study examines the growth and wear of these incisors to understand their relationship with age and sex. DESIGN: Measurements of mandibular incisor crown and root length were made for two sister species of macropodid (kangaroos and wallabies); Macropus giganteus and Macropus fuliginosus. Histological analysis examined patterns of dentine and cementum deposition within these teeth. Broader generalisability within Diprotodontia was tested using dentally reduced Tarsipes rostratus - a species disparate in body size and incisor function to the studied macropodids. RESULTS: In the macropodid sample it is demonstrated that the hypsodont nature of these incisors makes measurements of their growth (root length) and wear (crown length) accurate indicators of age and sex. Model fitting finds that root growth proceeds according to a logarithmic function across the lifespan, while crown wear follows a pattern of exponential reduction for both macropodid species. Histological results find that secondary dentine deposition and cementum layering are further indicators of age. Incisor measurements are shown to correlate with age in the sample of T. rostratus. CONCLUSIONS: The diprotodontian incisor is a useful tool for examining chronological age and sex, both morphologically and microstructurally. This finding has implications for population ecology, palaeontology and marsupial evolution.

Extant and extinct bilby genomes combined with Indigenous knowledge improve conservation of a unique Australian marsupial.

Ninu (greater bilby, Macrotis lagotis) are desert-dwelling, culturally and ecologically important marsupials. In collaboration with Indigenous rangers and conservation managers, we generated the Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura). We developed and tested a scat single-nucleotide polymorphism panel to inform current and future conservation actions, undertake ecological assessments and improve our understanding of Ninu genetic diversity in managed and wild populations. We also assessed the beneficial impact of translocations in the metapopulation (N = 363 Ninu). Resequenced genomes (temperate Ninu, 6; semi-arid Ninu, 6; and Yallara, 4) revealed two major population crashes during global cooling events for both species and differences in Ninu genes involved in anatomical and metabolic pathways. Despite their 45-year captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life history. Here we investigated the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus, an XY1Y2 sex chromosome system and olfactory receptor gene expansions. Together, we demonstrate the holistic value of genomics in improving key conservation actions, understanding unique biological traits and developing tools for Indigenous rangers to monitor remote wild populations.

Climate change and population persistence in a hibernating marsupial.

Climate change has physiological consequences on organisms, ecosystems and human societies, surpassing the pace of organismal adaptation. Hibernating mammals are particularly vulnerable as winter survival is determined by short-term physiological changes triggered by temperature. In these animals, winter temperatures cannot surpass a certain threshold, above which hibernators arouse from torpor, increasing several fold their energy needs when food is unavailable. Here, we parameterized a numerical model predicting energy consumption in heterothermic species and modelled winter survival at different climate change scenarios. As a model species, we used the arboreal marsupial monito del monte (genus Dromiciops), which is recognized as one of the few South American hibernators. We modelled four climate change scenarios (from optimistic to pessimistic) based on IPCC projections, predicting that northern and coastal populations (Dromiciops bozinovici) will decline because the minimum number of cold days needed to survive the winter will not be attained. These populations are also the most affected by habitat fragmentation and changes in land use. Conversely, Andean and other highland populations, in cooler environments, are predicted to persist and thrive. Given the widespread presence of hibernating mammals around the world, models based on simple physiological parameters, such as this one, are becoming essential for predicting species responses to warming in the short term.

Changes in the Skin Microbiota in Two Bare-nosed Wombats (Vombatus ursinus) with Differing Recovery Trajectories following Treatment for Sarcoptic Mange.

We report tracking of bacterial skin microbiota for two bare-nosed wombats (Vombatus ursinus) following in situ treatment for sarcoptic mange. Sarcoptes scabiei, the etiologic agent, has dramatic effects on skin microbiota. Our case reports show differing disease trajectory and bacterial beta diversity between the two treated individuals.

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).

The missing toxic link: Exposure of non-target native marsupials to second-generation anticoagulant rodenticides (SGARs) suggest a potential route of transfer into apex predators.

Anticoagulant rodenticides (ARs) are used globally to control rodent pests. Second-generation anticoagulant rodenticides (SGARs) persist in the liver and pose a significant risk of bioaccumulation and secondary poisoning in predators, including species that do not generally consume rodents. As such, there is a clear need to understand the consumption of ARs, particularly SGARs, by non-target consumers to determine the movement of these anticoagulants through ecosystems. We collected and analysed the livers from deceased common brushtail possums (Trichosurus vulpecula) and common ringtail possums (Pseudocheirus peregrinus), native Australian marsupials that constitute the main diet of the powerful owl (Ninox strenua), an Australian apex predator significantly exposed to SGAR poisoning. ARs were detected in 91 % of brushtail possums and 40 % of ringtail possums. Most of the detections were attributed to SGARs, while first-generation anticoagulant rodenticides (FGARs) were rarely detected. SGAR concentrations were likely lethal or toxic in 42 % of brushtail possums and 4 % of ringtail possums with no effect of age, sex, or weight detected in either species. There was also no effect of the landscape type possums were from, suggesting SGAR exposure is ubiquitous across landscapes. The rate of exposure detected in these possums provides insight into the pathway through which ARs are transferred to one of their key predators, the powerful owl. With SGARs entering food-webs through non-target species, the potential for bioaccumulation and broader secondary poisoning of predators is significantly greater and highlights an urgent need for routine rodenticide testing in non-target consumers that present as ill or found deceased. To limit their impact on ecosystem stability the use of SGARs should be significantly regulated by governing agencies.

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.