Susceptibility to a sexually transmitted disease in a wild koala population shows heritable genetic variance but no inbreeding depression.

The koala, one of the most iconic Australian wildlife species, is facing several concomitant threats that are driving population declines. Some threats are well known and have clear methods of prevention (e.g., habitat loss can be reduced with stronger land-clearing control), whereas others are less easily addressed. One of the major current threats to koalas is chlamydial disease, which can have major impacts on individual survival and reproduction rates and can translate into population declines. Effective management strategies for the disease in the wild are currently lacking, and, to date, we know little about the determinants of individual susceptibility to disease. Here, we investigated the genetic basis of variation in susceptibility to chlamydia using one of the most intensively studied wild koala populations. We combined data from veterinary examinations, chlamydia testing, genetic sampling and movement monitoring. Out of our sample of 342 wild koalas, 60 were found to have chlamydia. Using genotype information on 5007 SNPs to investigate the role of genetic variation in determining disease status, we found no evidence of inbreeding depression, but a heritability of 0.11 (95% CI: 0.06-0.23) for the probability that koalas had chlamydia. Heritability of susceptibility to chlamydia could be relevant for future disease management, as it suggests adaptive potential for the population.

Inbreeding and disease avoidance in a free-ranging koala population.

Habitat destruction and fragmentation are increasing globally, forcing surviving species into small, isolated populations. Isolated populations typically experience heightened inbreeding risk and associated inbreeding depression and population decline; although individuals in these populations may mitigate these risks through inbreeding avoidance strategies. For koalas, as dietary specialists already under threat in the northern parts of their range, increased habitat fragmentation and associated inbreeding costs are of great conservation concern. Koalas are known to display passive inbreeding avoidance through sex-biased dispersal, although population isolation will reduce dispersal pathways. We tested whether free-ranging koalas display active inbreeding avoidance behaviours. We used VHF tracking data, parentage reconstruction, and veterinary examination results to test whether free-ranging female koalas avoid mating with (a) more closely related males; and (b) males infected with sexually transmitted Chlamydia pecorum. We found no evidence that female koalas avoid mating with relatively more related available mates. In fact, as the relatedness of potential mates increases, so did inbreeding events. We also found no evidence that female koalas can avoid mating with males infected with C. pecorum. The absence of active inbreeding avoidance mechanisms in koalas is concerning from a conservation perspective, as small, isolated populations may be at even higher risk of inbreeding depression than expected. At risk koala populations may require urgent conservation interventions to augment gene flow and reduce inbreeding risks. Similarly, if koalas are not avoiding mating with individuals with chlamydial disease, populations may be at higher risk from disease than anticipated, further impacting population viability.

Developing noninvasive methodologies to assess koala population health through detecting Chlamydia from scats.

Wildlife diseases are a recognized driver of global biodiversity loss, have substantial economic impacts, and are increasingly becoming a threat to human health. Disease surveillance is critical but remains difficult in the wild due to the substantial costs and potential biases associated with most disease detection methods. Noninvasive scat surveys have been proposed as a health monitoring methodology to overcome some of these limitations. Here, we use the known threat of Chlamydia disease to the iconic, yet vulnerable, koala Phascolarctos cinereus to compare three methods for Chlamydia detection in scats: multiplex quantitative PCR, next generation sequencing, and a detection dog specifically trained on scats from Chlamydia-infected koalas. All three methods demonstrated 100% specificity, while sensitivity was variable. Of particular interest is the variable sensitivity of these diagnostic tests to detect sick individuals (i.e., not only infection as confirmed by Chlamydia-positive swabs, but with observable clinical signs of the disease); for koalas with urogenital tract disease signs, sensitivity was 78% with quantitative PCR, 50% with next generation genotyping and 100% with the detection dog method. This may be due to molecular methods having to rely on high-quality DNA whereas the dog most likely detects volatile organic compounds. The most appropriate diagnostic test will vary with disease prevalence and the specific aims of disease surveillance. Acknowledging that detection dogs might not be easily accessible to all, the future development of affordable and portable "artificial noses" to detect diseases from scats in the field might enable cost-effective, rapid and large-scale disease surveillance.

Fresh is best: Accurate SNP genotyping from koala scats.

Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next-generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq™ protocol. We compare genotype results from two-day-old scat DNA and 14-day-old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14-day-old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq™ process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.