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.

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.

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.

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.

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.

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.

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.

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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.

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.