Health Assessments of Koalas after Wildfire: A Temporal Comparison of Rehabilitated and Non-Rescued Resident Individuals.

Many koalas (Phascolarctos cinereus) required rehabilitation after the 2019/20 Australian megafires. Little is known about how the post-release health of rehabilitated koalas compares to non-rescued resident koalas. We evaluated health parameters in rehabilitated koalas and resident koalas in burnt and unburnt habitat in southern New South Wales, Australia. Health checks were undertaken within six weeks of fire (rehabilitated group), 5-9 months post-fire and 12-16 months post-fire. Body condition improved significantly over time in rehabilitated koalas, with similar condition between all groups at 12-16 months. Rehabilitated koalas therefore gained body condition at similar rates to koalas who remained and survived in the wild. The prevalence of Chlamydia pecorum was also similar between groups and timepoints, suggesting wildfire and rehabilitation did not exacerbate disease in this population. While there was some variation in measured serum biochemistry and haematology parameters between groups and timepoints, most were within normal reference ranges. Our findings show that koalas were generally healthy at the time of release and when recaptured nine months later. Landscapes in the Monaro region exhibiting a mosaic of burn severity can support koalas, and rehabilitated koala health is not compromised by returning them to burnt habitats 4-6 months post-fire.

Koala Genome Survey: An Open Data Resource to Improve Conservation Planning.

Genome sequencing is a powerful tool that can inform the management of threatened species. Koalas (Phascolarctos cinereus) are a globally recognized species that captured the hearts and minds of the world during the 2019/2020 Australian megafires. In 2022, koalas were listed as 'Endangered' in Queensland, New South Wales, and the Australian Capital Territory. Populations have declined because of various threats such as land clearing, habitat fragmentation, and disease, all of which are exacerbated by climate change. Here, we present the Koala Genome Survey, an open data resource that was developed after the Australian megafires. A systematic review conducted in 2020 demonstrated that our understanding of genomic diversity within koala populations was scant, with only a handful of SNP studies conducted. Interrogating data showed that only 6 of 49 New South Wales areas of regional koala significance had meaningful genome-wide data, with only 7 locations in Queensland with SNP data and 4 locations in Victoria. In 2021, we launched the Koala Genome Survey to generate resequenced genomes across the Australian east coast. We have publicly released 430 koala genomes (average coverage: 32.25X, range: 11.3-66.8X) on the Amazon Web Services Open Data platform to accelerate research that can inform current and future conservation planning.

Best genome sequencing strategies for annotation of complex immune gene families in wildlife.

BACKGROUND: The biodiversity crisis and increasing impact of wildlife disease on animal and human health provides impetus for studying immune genes in wildlife. Despite the recent boom in genomes for wildlife species, immune genes are poorly annotated in nonmodel species owing to their high level of polymorphism and complex genomic organisation. Our research over the past decade and a half on Tasmanian devils and koalas highlights the importance of genomics and accurate immune annotations to investigate disease in wildlife. Given this, we have increasingly been asked the minimum levels of genome quality required to effectively annotate immune genes in order to study immunogenetic diversity. Here we set out to answer this question by manually annotating immune genes in 5 marsupial genomes and 1 monotreme genome to determine the impact of sequencing data type, assembly quality, and automated annotation on accurate immune annotation. RESULTS: Genome quality is directly linked to our ability to annotate complex immune gene families, with long reads and scaffolding technologies required to reassemble immune gene clusters and elucidate evolution, organisation, and true gene content of the immune repertoire. Draft-quality genomes generated from short reads with HiC or 10× Chromium linked reads were unable to achieve this. Despite mammalian BUSCOv5 scores of up to 94.1% amongst the 6 genomes, automated annotation pipelines incorrectly annotated up to 59% of manually annotated immune genes regardless of assembly quality or method of automated annotation. CONCLUSIONS: Our results demonstrate that long reads and scaffolding technologies, alongside manual annotation, are required to accurately study the immune gene repertoire of wildlife species.

A targeted approach to investigating immune genes of an iconic Australian marsupial.

Disease is a contributing factor to the decline of wildlife populations across the globe. Koalas, iconic yet declining Australian marsupials, are predominantly impacted by two pathogens, Chlamydia and koala retrovirus. Chlamydia is an obligate intracellular bacterium and one of the most widespread sexually transmitted infections in humans worldwide. In koalas, Chlamydia infections can present as asymptomatic or can cause a range of ocular and urogenital disease signs, such as conjunctivitis, cystitis and infertility. In this study, we looked at differences in response to Chlamydia in two northern populations of koalas using a targeted gene sequencing of 1209 immune genes in addition to genome-wide reduced representation data. We identified two MHC Class I genes associated with Chlamydia disease progression as well as 25 single nucleotide polymorphisms across 17 genes that were associated with resolution of Chlamydia infection. These genes are involved in the innate immune response (TLR5) and defence (TLR5, IFNγ, SERPINE1, STAT2 and STX4). This study deepens our understanding of the role that genetics plays in disease progression in koalas and leads into future work that will use whole genome resequencing of a larger sample set to investigate in greater detail regions identified in this study. Elucidation of the role of host genetics in disease progression and resolution in koalas will directly contribute to better design of Chlamydia vaccines and management of koala populations which have recently been listed as "endangered."

Genome assembly of the numbat (Myrmecobius fasciatus), the only termitivorous marsupial.

The numbat (Myrmecobius fasciatus) is an endangered Australian marsupial, and the last surviving member of the Myrmecobiidae family. The numbat regularly undergoes torpor and is unique amongst marsupials as it is the only diurnal and termitivorous species. Here we sequenced the first draft genome of the numbat using 10× Genomics Chromium linked-read technology, resulting in a 3.42 Gbp genome with a scaffold N50 of 223 kbp. A global transcriptome from liver, lung and tongue was also generated to aid genome annotation, identifying 21,465 protein-coding genes. To investigate adaptation to the numbat's termitivorous diet and arid/semi-arid range, we interrogated the most highly expressed transcripts within the tongue and manually annotated taste, vomeronasal and aquaporin gene families. Antimicrobial proteins and proteins involved in digestion were highly expressed in the tongue, alongside umami taste receptors. However, sweet taste receptors were not expressed in this tissue, which combined with the putative contraction of the bitter taste receptor gene repertoire in the numbat genome, may indicate a potential evolutionary adaptation to their specialised termitivorous diet. Vomeronasal and aquaporin gene repertoires were similar to other marsupials. The draft numbat genome is a valuable tool for conservation and can be applied to population genetics/genomics studies and to investigate the unique biology of this interesting species.

A reference genome for the critically endangered woylie, Bettongia penicillata ogilbyi.

Biodiversity is declining globally, and Australia has one of the worst extinction records for mammals. The development of sequencing technologies means that genomic approaches are now available as important tools for wildlife conservation and management. Despite this, genome sequences are available for only 5% of threatened Australian species. Here we report the first reference genome for the woylie (Bettongia penicillata ogilbyi), a critically endangered marsupial from Western Australia, and the first genome within the Potoroidae family. The woylie reference genome was generated using Pacific Biosciences HiFi long-reads, resulting in a 3.39 Gbp assembly with a scaffold N50 of 6.49 Mbp and 86.5% complete mammalian BUSCOs. Assembly of a global transcriptome from pouch skin, tongue, heart and blood RNA-seq reads was used to guide annotation with Fgenesh++, resulting in the annotation of 24,655 genes. The woylie reference genome is a valuable resource for conservation, management and investigations into disease-induced decline of this critically endangered marsupial.