Publications by authors named "Katherine Belov"

135 Publications

How much is enough? Sampling intensity influences estimates of reproductive variance in an introduced population.

Ecol Appl 2021 Oct 6:e02462. Epub 2021 Oct 6.

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, 2006, Australia.

Conservation introductions to islands and fenced enclosures are increasing as in situ mitigations fail to keep pace with population declines. Few studies consider the potential loss of genetic diversity and increased inbreeding if released individuals breed disproportionately. As funding is limited and post-release monitoring expensive for conservation programs, understanding how sampling effort influences estimates of reproductive variance is useful. To investigate this relationship, we used a well-studied population of Tasmanian devils (Sarcophilus harrisii) introduced to Maria Island, Tasmania, Australia. Pedigree reconstruction based on molecular data revealed high variance in number of offspring per breeder and high proportions of unsuccessful individuals. Computational subsampling of 20%, 40%, 60%, and 80% of observed offspring resulted in inaccurate estimates of reproductive variance compared to the pedigree reconstructed with all sampled individuals. With decreased sampling effort, the proportion of inferred unsuccessful individuals was overestimated and the variance in number of offspring per breeder was underestimated. To accurately estimate reproductive variance, we recommend sampling as many individuals as logistically possible during the early stages of population establishment. Further, we recommend careful selection of colonizing individuals as they may be disproportionately represented in subsequent generations. Within the conservation management context, our results highlight important considerations for sample collection and post-release monitoring during population establishment.
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http://dx.doi.org/10.1002/eap.2462DOI Listing
October 2021

Improved high-throughput MHC typing for non-model species using long-read sequencing.

Mol Ecol Resour 2021 Sep 22. Epub 2021 Sep 22.

School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia.

The major histocompatibility complex (MHC) plays a critical role in the vertebrate immune system. Accurate MHC typing is critical to understanding not only host fitness and disease susceptibility, but also the mechanisms underlying host-pathogen co-evolution. However, due to the high degree of gene duplication and diversification of MHC genes, it is often technically challenging to accurately characterise MHC genetic diversity in non-model species. Here we conducted a systematic review to identify common issues associated with current widely used MHC typing approaches. Then to overcome these challenges, we developed a long-read based MHC typing method along with a new analysis pipeline. Our approach enables the sequencing of fully phased MHC alleles spanning all key functional domains and the separation of highly similar alleles as well as the removal of technical artefacts such as PCR heteroduplexes and chimeras. Using this approach, we performed population-scale MHC typing in the Tasmanian devil (Sarcophilus harrisii), revealing previously undiscovered MHC functional diversity in this endangered species. Our new method provides a better solution for addressing research questions that require high MHC typing accuracy. Since the method is not limited by species or the number of genes analysed, it will be applicable for studying not only the MHC but also other complex gene families.
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http://dx.doi.org/10.1111/1755-0998.13511DOI Listing
September 2021

First evidence of deviation from Mendelian proportions in a conservation programme.

Mol Ecol 2021 08 19;30(15):3703-3715. Epub 2021 Jun 19.

Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia.

Classic Mendelian inheritance is the bedrock of population genetics and underpins pedigree-based management of animal populations. However, assumptions of Mendelian inheritance might not be upheld in conservation breeding programmes if early viability selection occurs, even when efforts are made to equalise genetic contributions of breeders. To test this possibility, we investigated deviations from Mendelian proportions in a captive metapopulation of the endangered Tasmanian devil. This marsupial population is ideal for addressing evolutionary questions in conservation due to its large size, range of enclosure types (varying in environmental conditions), good genomic resources (which aid interpretation), and the species' biology. Devil mothers give birth to more offspring than they can nurse in the pouch, providing the potential for intense viability selection amongst embryos. We used data from 140 known sire-dam-offspring triads to isolate within-family selection from population-level mechanisms (such as mate choice or inbreeding), and compared observed offspring genotypes at 123 targeted SNPs to neutral (i.e., Mendelian) expectations. We found lower offspring heterozygosity than expected, and subtle patterns that varied across a gradient of management intensity from zoo-like enclosures to semi-wild environments for some loci. Meiotic drive or maternal-foetal incompatibilities are consistent with our results, although we cannot statistically confirm these mechanisms. We found some evidence that maternal genotype affects annual litter size, suggesting that family-level patterns are driven by differential offspring mortality before birth or during early development. Our results show that deviations from Mendelian inheritance can occur in conservation programmes, despite best-practice management to prevent selection.
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http://dx.doi.org/10.1111/mec.16004DOI Listing
August 2021

Koala cathelicidin PhciCath5 has antimicrobial activity, including against Chlamydia pecorum.

PLoS One 2021 14;16(4):e0249658. Epub 2021 Apr 14.

School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia.

Devastating fires in Australia over 2019-20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249658PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046226PMC
September 2021

Annotation of immune genes in the extinct thylacine (Thylacinus cynocephalus).

Immunogenetics 2021 06 5;73(3):263-275. Epub 2021 Feb 5.

School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW, Australia.

Advances in genome sequencing technology have enabled genomes of extinct species to be sequenced. However, given the fragmented nature of these genome assemblies, it is not clear whether it is possible to comprehensively annotate highly variable and repetitive genes such as those involved in immunity. As such, immune genes have only been investigated in a handful of extinct genomes, mainly in human lineages. In 2018 the genome of the thylacine (Thylacinus cynocephalus), a carnivorous marsupial from Tasmania that went extinct in 1936, was sequenced. Here we attempt to characterise the immune repertoire of the thylacine and determine similarity to its closest relative with a genome available, the Tasmanian devil (Sarcophilus harrisii), as well as other marsupials. Members from all major immune gene families were identified. However, variable regions could not be characterised, and complex families such as the major histocompatibility complex (MHC) were highly fragmented and located across multiple small scaffolds. As such, at a gene level we were unable to reconstruct full-length coding sequences for the majority of thylacine immune genes. Despite this, we identified genes encoding functionally important receptors and immune effector molecules, which suggests the functional capacity of the thylacine immune system was similar to other mammals. However, the high number of partial immune gene sequences identified limits our ability to reconstruct an accurate picture of the thylacine immune repertoire.
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http://dx.doi.org/10.1007/s00251-020-01197-zDOI Listing
June 2021

Comprehensive Knowledge of Reservoir Hosts is Key to Mitigating Future Pandemics.

Innovation (N Y) 2020 Nov 3;1(3):100065. Epub 2020 Nov 3.

CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Beijing 100101, China.

Coronavirus disease 2019 (COVID-19) and other epidemics (such as severe acute respiratory syndrome [SARS], Ebola, and H1N1) are stark reminders that knowledge of animal behavior and ecosystem health are key to controlling the spread of zoonotic diseases early in their onset. However, we have very limited information about the set of behavioral and ecological factors that promote viral spillover and the effects that has on ecosystem health and disease transmission. Thus, expanding our current knowledge of reservoir hosts and pandemics represents an urgent and critical tool in ecological epidemiology. We also propose to create an integrative database that ranks animal species in terms of their likelihood as hosts for specific infectious diseases. We call for a global and cooperative effort of field and laboratory scientists to create, maintain, and update this information in order to reduce the severity of future pandemics.
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http://dx.doi.org/10.1016/j.xinn.2020.100065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833268PMC
November 2020

Platypus and echidna genomes reveal mammalian biology and evolution.

Nature 2021 Apr 6;592(7856):756-762. Epub 2021 Jan 6.

Tree of Life Programme, Wellcome Sanger Institute, Cambridge, UK.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.
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http://dx.doi.org/10.1038/s41586-020-03039-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081666PMC
April 2021

Characterization of reproductive gene diversity in the endangered Tasmanian devil.

Mol Ecol Resour 2021 Apr 26;21(3):721-732. Epub 2020 Nov 26.

School of Life and Environmental Sciences, Faculty of Science, University of Sydney, Sydney, NSW, Australia.

Interindividual variation at genes known to play a role in reproduction may impact reproductive fitness. The Tasmanian devil is an endangered Australian marsupial with low genetic diversity. Recent work has shown concerning declines in productivity in both wild and captive populations over time. Understanding whether functional diversity exists at reproductive genes in the Tasmanian devil is a key first step in identifying genes that may influence productivity. We characterized single nucleotide polymorphisms (SNPs) at 214 genes involved in reproduction in 37 Tasmanian devils. Twenty genes contained nonsynonymous substitutions, with genes involved in embryogenesis, fertilization and hormonal regulation of reproduction displaying greater numbers of nonsynonymous SNPs than synonymous SNPs. Two genes, ADAMTS9 and NANOG, showed putative signatures of balancing selection indicating that natural selection is maintaining diversity at these genes despite the species exhibiting low overall levels of genetic diversity. We will use this information in future to examine the interplay between reproductive gene variation and reproductive fitness in Tasmanian devil populations.
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http://dx.doi.org/10.1111/1755-0998.13295DOI Listing
April 2021

Deciphering genetic mate choice: Not so simple in group-housed conservation breeding programs.

Evol Appl 2020 Oct 19;13(9):2179-2189. Epub 2020 May 19.

School of Life and Environmental Sciences Faculty of Science The University of Sydney Sydney Australia.

Incorporating mate choice into conservation breeding programs can improve reproduction and the retention of natural behaviors. However, different types of genetic-based mate choice can have varied consequences for genetic diversity management. As a result, it is important to examine mechanisms of mate choice in captivity to assess its costs and benefits. Most research in this area has focused on experimental pairing trials; however, this resource-intensive approach is not always feasible in captive settings and can interfere with other management constraints. We used generalized linear mixed models and permutation approaches to investigate overall breeding success in group-housed Tasmanian devils at three nonmutually exclusive mate choice hypotheses: (a) advantage of heterozygous individuals, (b) advantage of dissimilar mates, and (c) optimum genetic distance, using both 1,948 genome-wide SNPs and 12 MHC-linked microsatellites. The managed devil insurance population is the largest such breeding program in Australia and is known to have high variance in reproductive success. We found that nongenetic factors such as age were the best predictors of breeding success in a competitive breeding scenario, with younger females and older males being more successful. We found no evidence of mate choice under the hypotheses tested. Mate choice varies among species and across environments, so we advocate for more studies in realistic captive management contexts as experimental or wild studies may not apply. Conservation managers must weigh up the need to wait for adequate sample sizes to detect mate choice with the risk that genetic changes may occur during this time in captivity. Our study shows that examining and integrating mate choice into the captive management of species housed in realistic, semi-natural group-based contexts may be more difficult than previously considered.
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http://dx.doi.org/10.1111/eva.12981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513713PMC
October 2020

Author Correction: Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

Sci Rep 2020 Jul 22;10(1):12453. Epub 2020 Jul 22.

The University of Sydney, School of Medical Sciences, Camperdown, NSW, 2006, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-68759-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376021PMC
July 2020

Inbreeding depression in one of the last DFTD-free wild populations of Tasmanian devils.

PeerJ 2020 16;8:e9220. Epub 2020 Jun 16.

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia.

Background: Vulnerable species experiencing inbreeding depression are prone to localised extinctions because of their reduced fitness. For Tasmanian devils, the rapid spread of devil facial tumour disease (DFTD) has led to population declines and fragmentation across the species' range. Here we show that one of the few remaining DFTD-free populations of Tasmanian devils is experiencing inbreeding depression. Moreover, this population has experienced a significant reduction in reproductive success over recent years.

Methods: We used 32 microsatellite loci to examine changes in genetic diversity and inbreeding in the wild population at Woolnorth, alongside field data on breeding success from females to test for inbreeding depression.

Results: Wefound that maternal internal relatedness has a negative impact on litter sizes. The results of this study imply that this population may be entering an extinction vortex and that to protect the population genetic rescue should be considered. This study provides conservation managers with useful information for managing wild devils and provides support for the "Wild Devil Recovery Program", which is currently augmenting small, isolated populations.
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http://dx.doi.org/10.7717/peerj.9220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304431PMC
June 2020

A demonstration of conservation genomics for threatened species management.

Mol Ecol Resour 2020 Nov 24;20(6):1526-1541. Epub 2020 Jul 24.

School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW, Australia.

As species extinction rates increase, genomics provides a powerful tool to support intensive management of threatened species. We use the Tasmanian devil (Sarcophilus harrisii) to demonstrate how conservation genomics can be implemented in threatened species management. We conducted whole genome sequencing (WGS) of 25 individuals from the captive breeding programme and reduced-representation sequencing (RRS) of 98 founders of the same programme. A subset of the WGS samples was also sequenced by RRS, allowing us to directly compare genome-wide heterozygosity with estimates from RRS data. We found good congruence in interindividual variation and gene-ontology classifications between the two data sets, indicating that our RRS data reflect the genome well. We also attempted genome-wide association studies with both data sets (regarding breeding success), but the genomic data suffered from small sample size, while the RRS data suffered from lack of precision, highlighting a key trade-off in the design of conservation genomic research. Nevertheless, we identified a number of candidate genes that may be associated with variation in breeding success. Individual heterozygosity, as measured by WGS or RRS, was not associated with breeding success in captivity but was negatively associated with litter sizes of breeding females in the RRS data set. Our findings enable conservation managers to have confidence in RRS data while understanding its limitations, and provide avenues for further investigation into which processes underlie variation in breeding success in captive Tasmanian devils. We caution, however, that deep functional insights using RRS may be impaired by a lack of precision, especially when marker density is low.
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http://dx.doi.org/10.1111/1755-0998.13211DOI Listing
November 2020

Marsupial Gut Microbiome.

Front Microbiol 2020 29;11:1058. Epub 2020 May 29.

School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia.

The study of the gut microbiome in threatened wildlife species has enormous potential to improve conservation efforts and gain insights into host-microbe coevolution. Threatened species are often housed in captivity, and during this process undergo considerable changes to their gut microbiome. Studying the gut microbiome of captive animals therefore allows identification of dysbiosis and opportunities for improving management practices in captivity and for subsequent translocations. Manipulation of the gut microbiome through methods such as fecal transplant may offer an innovative means of restoring dysbiotic microbiomes in threatened species to provide health benefits. Finally, characterization of the gut microbiome (including the viral components, or virome) provides important baseline health information and may lead to discovery of significant microbial pathogens. Here we summarize our current understanding of microbiomes in Australian marsupial species.
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http://dx.doi.org/10.3389/fmicb.2020.01058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272691PMC
May 2020

The Value of Reference Genomes in the Conservation of Threatened Species.

Genes (Basel) 2019 10 25;10(11). Epub 2019 Oct 25.

School of Life & Environmental Sciences, The University of Sydney, Sydney 2006, Australia.

Conservation initiatives are now more crucial than ever-over a million plant and animal species are at risk of extinction over the coming decades. The genetic management of threatened species held in insurance programs is recommended; however, few are taking advantage of the full range of genomic technologies available today. Less than 1% of the 13505 species currently listed as threated by the International Union for Conservation of Nature (IUCN) have a published genome. While there has been much discussion in the literature about the importance of genomics for conservation, there are limited examples of how having a reference genome has changed conservation management practice. The Tasmanian devil (), is an endangered Australian marsupial, threatened by an infectious clonal cancer devil facial tumor disease (DFTD). Populations have declined by 80% since the disease was first recorded in 1996. A reference genome for this species was published in 2012 and has been crucial for understanding DFTD and the management of the species in the wild. Here we use the Tasmanian devil as an example of how a reference genome has influenced management actions in the conservation of a species.
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http://dx.doi.org/10.3390/genes10110846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895880PMC
October 2019

Looking like the locals - gut microbiome changes post-release in an endangered species.

Anim Microbiome 2019 Oct 3;1(1). Epub 2019 Oct 3.

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, 2006, Australia.

Background: Captivity presents extreme lifestyle changes relative to the wild, and evidence of microbiome dysbiosis in captive animals is growing. The gut microbiome plays a crucial role in host health. Whilst captive breeding and subsequent reintroduction to the wild is important for conservation, such efforts often have limited success. Post-release monitoring is essential for assessing translocation success, but changes to the microbiome of released individuals are poorly understood. The Tasmanian devil was previously shown to exhibit loss of microbiome diversity as a result of intense captive management. This current study examines changes in the devil gut microbiome in response to translocation and aims to determine if perturbations from captivity are permanent or reversible.

Methods: Using 16S rRNA amplicon sequencing, we conducted temporal monitoring of the gut microbiome of released devils during two translocation events, captive-to-wild and wild-to-wild. To investigate whether the microbiome of the released devils changed following translocation, we characterized their microbiome at multiple time points during the translocation process over the course of 6-12 months and compared them to the microbiome of wild incumbent devils (resident wild-born devils at the respective release sites).

Results: We showed that the pre-release microbiome was significantly different to the microbiome of wild incumbent animals, but that the microbiomes of animals post-release (as early as 3 to 4 weeks post-release) were similar to wild incumbents. The gut microbiome of released animals showed significant compositional shifts toward the wild incumbent microbiome of both translocation events.

Conclusion: Our results suggest that the devil gut microbiome is dynamic and that loss of microbiome diversity in captivity can be restored following release to the wild. We recommend the broader application of microbiome monitoring in wildlife translocation programs to assess the impacts of translocation on animal microbiomes.
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http://dx.doi.org/10.1186/s42523-019-0012-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7807427PMC
October 2019

From reference genomes to population genomics: comparing three reference-aligned reduced-representation sequencing pipelines in two wildlife species.

BMC Genomics 2019 Jun 3;20(1):453. Epub 2019 Jun 3.

Faculty of Science, The University of Sydney, School of Life and Environmental Sciences, Sydney, Australia.

Background: Recent advances in genomics have greatly increased research opportunities for non-model species. For wildlife, a growing availability of reference genomes means that population genetics is no longer restricted to a small set of anonymous loci. When used in conjunction with a reference genome, reduced-representation sequencing (RRS) provides a cost-effective method for obtaining reliable diversity information for population genetics. Many software tools have been developed to process RRS data, though few studies of non-model species incorporate genome alignment in calling loci. A commonly-used RRS analysis pipeline, Stacks, has this capacity and so it is timely to compare its utility with existing software originally designed for alignment and analysis of whole genome sequencing data. Here we examine population genetic inferences from two species for which reference-aligned reduced-representation data have been collected. Our two study species are a threatened Australian marsupial (Tasmanian devil Sarcophilus harrisii; declining population) and an Arctic-circle migrant bird (pink-footed goose Anser brachyrhynchus; expanding population). Analyses of these data are compared using Stacks versus two widely-used genomics packages, SAMtools and GATK. We also introduce a custom R script to improve the reliability of single nucleotide polymorphism (SNP) calls in all pipelines and conduct population genetic inferences for non-model species with reference genomes.

Results: Although we identified orders of magnitude fewer SNPs in our devil dataset than for goose, we found remarkable symmetry between the two species in our assessment of software performance. For both datasets, all three methods were able to delineate population structure, even with varying numbers of loci. For both species, population structure inferences were influenced by the percent of missing data.

Conclusions: For studies of non-model species with a reference genome, we recommend combining Stacks output with further filtering (as included in our R pipeline) for population genetic studies, paying particular attention to potential impact of missing data thresholds. We recognise SAMtools as a viable alternative for researchers more familiar with this software. We caution against the use of GATK in studies with limited computational resources or time.
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http://dx.doi.org/10.1186/s12864-019-5806-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547446PMC
June 2019

Tasmanian devils with contagious cancer exhibit a constricted T-cell repertoire diversity.

Commun Biol 2019 13;2:99. Epub 2019 Mar 13.

School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia.

The Tasmanian devil () is threatened by a contagious cancer, known as Devil Facial Tumour Disease (DFTD). A highly diverse T-cell receptor (TCR) repertoire is crucial for successful host defence against cancers. By investigating TCR beta chain diversity in devils of different ages, we show that the T-cell repertoire in devils constricts in their second year of life, which may explain the higher DFTD prevalence in older devils. Unexpectedly, we also observed a pronounced decline in TCR diversity and T cell clonal expansion in devils after DFTD infection. These findings overturned the previous assumption that DFTD did not directly impact host immunity.
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http://dx.doi.org/10.1038/s42003-019-0342-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416256PMC
April 2020

Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics.

J Virol 2019 06 15;93(11). Epub 2019 May 15.

Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample. The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.
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http://dx.doi.org/10.1128/JVI.00205-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532096PMC
June 2019

Complex problems need detailed solutions: Harnessing multiple data types to inform genetic management in the wild.

Evol Appl 2019 Feb 26;12(2):280-291. Epub 2018 Dec 26.

Faculty of Science, School of Life and Environmental Sciences The University of Sydney Sydney New South Wales Australia.

For bottlenecked populations of threatened species, supplementation often leads to improved population metrics (genetic rescue), provided that guidelines can be followed to avoid negative outcomes. In cases where no "ideal" source populations exist, or there are other complicating factors such as prevailing disease, the benefit of supplementation becomes uncertain. Bringing multiple data and analysis types together to plan genetic management activities can help. Here, we consider three populations of Tasmanian devil, , as candidates for genetic rescue. Since 1996, devil populations have been severely impacted by devil facial tumour disease (DFTD), causing significant population decline and fragmentation. Like many threatened species, the key threatening process for devils cannot currently be fully mitigated, so species management requires a multifaceted approach. We examined diversity of 31 putatively neutral and 11 MHC-linked microsatellite loci of three remnant wild devil populations (one sampled at two time-points), alongside computational diversity projections, parameterized by field data from DFTD-present and DFTD-absent sites. Results showed that populations had low diversity, connectivity was poor, and diversity has likely decreased over the last decade. Stochastic simulations projected further diversity losses. For a given population size, the effects of DFTD on population demography (including earlier age at death and increased female productivity) did not impact diversity retention, which was largely driven by final population size. Population sizes ≥500 (depending on the number of founders) were necessary for maintaining diversity in otherwise unmanaged populations, even if DFTD is present. Models indicated that smaller populations could maintain diversity with ongoing immigration. Taken together, our results illustrate how multiple analysis types can be combined to address complex population genetic challenges.
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http://dx.doi.org/10.1111/eva.12715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346650PMC
February 2019

Too much of a good thing? Finding the most informative genetic data set to answer conservation questions.

Mol Ecol Resour 2019 May;19(3):659-671

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia.

Molecular markers are a useful tool allowing conservation and population managers to shed light on genetic processes affecting threatened populations. However, as technological advancements in molecular techniques continue to evolve, conservationists are frequently faced with new genetic markers, each with nuanced variation in their characteristics as well as advantages and disadvantages for informing various questions. We used a well-studied population of Tasmanian devils (Sarcophilus harrisii) from Maria Island, Tasmania, to illustrate the issues associated with combining multiple genetic data sets and to help answer a question posed by many population managers: which data set will provide the most precise and accurate estimates of the population processes we are trying to measure? We analysed individual heterozygosity (as internal relatedness, IR) of 96 individuals, calculated using four genetic marker types (putatively neutral microsatellites, major histocompatibility complex-linked microsatellites, reduced representation sequencing, and candidate region resequencing). We found no correlation in IR values across marker types, suggesting that various genetic markers reflect different aspects of genomic diversity. In addition, some marker types were more informative than others for conservation decision-making. Reduced representation sequencing provided the highest precision (lowest error) for estimating population-level genetic diversity, and most closely reflected genome-wide heterozygosity both theoretically and empirically. Within the conservation context, our results highlight important considerations when choosing a molecular technique for wildlife genetics.
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http://dx.doi.org/10.1111/1755-0998.12997DOI Listing
May 2019

Lessons learnt from the Tasmanian devil facial tumour regarding immune function in cancer.

Mamm Genome 2018 12 17;29(11-12):731-738. Epub 2018 Sep 17.

School of Life and Environmental Science, The University of Sydney, Sydney, NSW, Australia.

Genetic and genomic technologies have facilitated a greater understanding of the Tasmanian devil immune system and the origins, evolution and spread of devil facial tumour disease (DFTD). DFTD is a contagious cancer that has caused significant declines in devil populations across Tasmania. Immune responses to DFTD are rarely detected, allowing the cancer to pass between individuals and proliferate unimpeded. Early immunosenscence in devils appears to decrease anti-tumour immunity in older animals compared to younger animals, which may increase susceptibility to DFTD and explain high DFTD prevalence in this age group. Devils also have extremely low major histocompatibility complex (MHC) diversity, and multiple alleles are shared with the tumour, lowering histocompatibility barriers which may have contributed to DFTD evolution. DFTD actively evades immune attack by down-regulating cell-surface MHC I molecules, making it effectively invisible to the immune system. Altered MHC I profiles should activate natural killer (NK) cell anti-tumour responses, but these are absent in DFTD infection. Recent immunisation and immunotherapy using modified DFTD cells has induced an anti-DFTD immune response and regression of DFTD in some devils. Knowledge gained from immune responses to a transmissible cancer in devils will ultimately reveal useful insights into immunity to cancer in humans and other species.
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http://dx.doi.org/10.1007/s00335-018-9782-3DOI Listing
December 2018

Disentangling the mechanisms of mate choice in a captive koala population.

PeerJ 2018 21;6:e5438. Epub 2018 Aug 21.

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.

Successful captive breeding programs are crucial to the long-term survival of many threatened species. However, pair incompatibility (breeding failure) limits sustainability of many captive populations. Understanding whether the drivers of this incompatibility are behavioral, genetic, or a combination of both, is crucial to improving breeding programs. We used 28 years of pairing data from the San Diego Zoo koala colony, plus genetic analyses using both major histocompatibility complex (MHC)-linked and non-MHC-linked microsatellite markers, to show that both genetic and non-genetic factors can influence mating success. Male age was reconfirmed to be a contributing factor to the likelihood of a koala pair copulating. This trend could also be related to a pair's age difference, which was highly correlated with male age in our dataset. Familiarity was reconfirmed to increase the probability of a successful copulation. Our data provided evidence that females select mates based on MHC and genome-wide similarity. Male heterozygosity at MHC class II loci was associated with both pre- and post-copulatory female choice. Genome-wide similarity, and similarity at the MHC class II DAB locus, were also associated with female choice at the post-copulatory level. Finally, certain MHC-linked alleles were associated with either increased or decreased mating success. We predict that utilizing a variety of behavioral and MHC-dependent mate choice mechanisms improves female fitness through increased reproductive success. This study highlights the complexity of mate choice mechanisms in a species, and the importance of ascertaining mate choice mechanisms to improve the success of captive breeding programs.
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http://dx.doi.org/10.7717/peerj.5438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108315PMC
August 2018

Bioluminescent murine models of bacterial sepsis and scald wound infections for antimicrobial efficacy testing.

PLoS One 2018 16;13(7):e0200195. Epub 2018 Jul 16.

Australian Centre for Antimicrobial Resistance Ecology, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, South Australia, Australia.

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200195PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047774PMC
January 2019

Adaptation and conservation insights from the koala genome.

Nat Genet 2018 08 2;50(8):1102-1111. Epub 2018 Jul 2.

Sydney School of Veterinary Science, Faculty of Science, University of Sydney, Sydney, New South Wales, Australia.

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.
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http://dx.doi.org/10.1038/s41588-018-0153-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197426PMC
August 2018

Gomesin peptides prevent proliferation and lead to the cell death of devil facial tumour disease cells.

Cell Death Discov 2018 Dec 14;4:19. Epub 2018 Feb 14.

1QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006 Australia.

The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease.
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http://dx.doi.org/10.1038/s41420-018-0030-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841354PMC
December 2018

MHC diversity and female age underpin reproductive success in an Australian icon; the Tasmanian Devil.

Sci Rep 2018 03 8;8(1):4175. Epub 2018 Mar 8.

Centre for Integrative Ecology, School of Life and Environmental Science, Deakin University, Geelong, 3216, VIC, Australia.

Devil Facial Tumour Disease (DFTD), a highly contagious cancer, has decimated Tasmanian devil (Sarcophilus harrisii) numbers in the wild. To ensure its long-term survival, a captive breeding program was implemented but has not been as successful as envisaged at its launch in 2005. We therefore investigated the reproductive success of 65 captive devil pair combinations, of which 35 produced offspring (successful pairs) whereas the remaining 30 pairs, despite being observed mating, produced no offspring (unsuccessful pairs). The devils were screened at six MHC Class I-linked microsatellite loci. Our analyses revealed that younger females had a higher probability of being successful than older females. In the successful pairs we also observed a higher difference in total number of heterozygous loci, i.e. when one devil had a high total number of heterozygous loci, its partner had low numbers. Our results therefore suggest that devil reproductive success is subject to disruptive MHC selection, which to our knowledge has never been recorded in any vertebrate. In order to enhance the success of the captive breeding program the results from the present study show the importance of using young (2-year old) females as well as subjecting the devils to MHC genotyping.
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http://dx.doi.org/10.1038/s41598-018-20934-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843591PMC
March 2018

Immunization Strategies Producing a Humoral IgG Immune Response against Devil Facial Tumor Disease in the Majority of Tasmanian Devils Destined for Wild Release.

Front Immunol 2018 19;9:259. Epub 2018 Feb 19.

School of Medicine, University of Tasmania, Hobart, TAS, Australia.

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government's Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials ( = 19 and  = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.
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http://dx.doi.org/10.3389/fimmu.2018.00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826075PMC
April 2019

Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

Sci Rep 2018 02 5;8(1):2412. Epub 2018 Feb 5.

The University of Sydney, School of Medical Sciences, Camperdown, NSW, 2006, Australia.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.
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http://dx.doi.org/10.1038/s41598-018-20744-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799185PMC
February 2018

Koala immunology and infectious diseases: How much can the koala bear?

Dev Comp Immunol 2018 05 31;82:177-185. Epub 2018 Jan 31.

Animal Research Centre, University of the Sunshine Coast, 90 Sippy Downs Drive, Sippy Downs 4556, Australia. Electronic address:

Infectious diseases are contributing to the decline of the iconic Australian marsupial, the koala (Phascolarctos cinereus). Infections with the obligate intracellular bacteria, Chlamydia pecorum, cause debilitating ocular and urogenital-tract disease while the koala-retrovirus (KoRV) has been implicated in host immunosuppression and exacerbation of chlamydial pathogenesis. Although histological studies have provided insight into the basic architecture of koala immune tissues, our understanding of the koala immune response to infectious disease has been limited, until recently, by a lack of species-specific immune reagents. Recent advances in the characterisation of key immune genes have focused on advancing our understanding of the immune response to Chlamydia infection, revealing commonalities in disease pathologies and immunity between koalas and other hosts and paving the way for the development of a koala Chlamydia vaccine. This review summarises these recent findings and highlights key aspects of the koala immune system requiring further attention with particular regard to their most prominent infectious diseases.
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http://dx.doi.org/10.1016/j.dci.2018.01.017DOI Listing
May 2018

Long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline.

Sci Rep 2017 Nov 20;7(1):15838. Epub 2017 Nov 20.

Faculty of Science, Health, Education & Engineering, University of the Sunshine Coast, Locked Bag 4, Maroochydore DC, Qld, 4558, Australia.

The koala retrovirus (KoRV) is implicated in several diseases affecting the koala (Phascolarctos cinereus). KoRV provirus can be present in the genome of koalas as an endogenous retrovirus (present in all cells via germline integration) or as exogenous retrovirus responsible for somatic integrations of proviral KoRV (present in a limited number of cells). This ongoing invasion of the koala germline by KoRV provides a powerful opportunity to assess the viral strategies used by KoRV in an individual. Analysis of a high-quality genome sequence of a single koala revealed 133 KoRV integration sites. Most integrations contain full-length, endogenous provirus; KoRV-A subtype. The second most frequent integrations contain an endogenous recombinant element (recKoRV) in which most of the KoRV protein-coding region has been replaced with an ancient, endogenous retroelement. A third set of integrations, with very low sequence coverage, may represent somatic cell integrations of KoRV-A, KoRV-B and two recently designated additional subgroups, KoRV-D and KoRV-E. KoRV-D and KoRV-E are missing several genes required for viral processing, suggesting they have been transmitted as defective viruses. Our results represent the first comprehensive analyses of KoRV integration and variation in a single animal and provide further insights into the process of retroviral-host species interactions.
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http://dx.doi.org/10.1038/s41598-017-16171-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696478PMC
November 2017
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