Investigation of alternative molecular approaches for enhanced traceability of illegally traded Temminck's Ground Pangolins (Smutsia temminckii)

Abstract

Conservation regulatory bodies created to limit the overexploitation of wildlife are reliant on regulatory adherence. This over-reliance on regulation means that complementary approaches to assist with enforcement are often required. Wildlife forensics is one such approach that can be used to address the illegal trade in wildlife and pangolins in particular. With estimates from the legal trade being that over one million pangolins were trafficked in a ten-year period, it is clear that alternative approaches to curb illegal trade, which represents >98% of pangolin trade, are needed. Geographic traceability uses a range of markers and georeferenced samples for tracking the origins of trafficked pangolins and their derivatives. In this study, the applicability of two molecular profiling approaches (stable isotopes and host microbes) were evaluated for their traceability potential, for three pangolin populations sampled from two sites in the Kalahari (xeric savanna) and one site in the Lowveld (mesic savanna). The feasibility of carbon and nitrogen stable isotope traceability using pangolin scales was evaluated through mass spectrometry (Chapter 2). Results revealed that intra-scale and inter-individual variation are important considerations for traceability, whilst inter-scale variation and scale harvesting methods (boiling or direct flaming of carcasses) are not. Given the high levels of intra-scale variation, development of a time and cost-effective sampling protocol suitable for forensic investigations was attempted. It was found that the use of duplicate samples of homogenised scales provides an averaged effect on scale variability. However, scale tips are comparatively isotopically enriched and should be taken into consideration during forensic analyses. Although preliminary results indicate that geographic separation based on isotopic ratios between Lowveld and Kalahari regions may be possible, geographic partitioning through use of stable isotopes could not be conclusively demonstrated due to the small sample size. Baseline estimates of microbial diversity were established using two metagenomics approaches (cloning versus next generation sequencing (NGS)) targeting the 16S rRNA gene of bacteria and two taxonomic assignment methods (phylogeny testing versus a heuristic search tool; Chapter 3). These differed significantly with respect to microbial community composition with results suggesting that phylogeny testing in combination with NGS holds potential for pangolin traceability. Although whole microbial communities differed by 70.3% between the two Kalahari sites, constraints with respect to the number of localities sampled and the number of cloning colonies characterised are duly noted. The results also revealed that Clostridium sordellii and genospecies C. novyi sensu lato appear to be key site-specific taxa for presence/absence and topotype traceability methods, respectively.Using a primer-set developed to target the phylogenetically-informative phospholipase C gene, the potential health risks and geographic traceability efficacy of the C. novyi sensu lato pathogenic genospecies group was investigated (Chapter 4). Although the prevalence was moderate at 17.65% and thus not feasible for traceability purposes, the potential public and animal health risks that the genospecies group poses, particularly for the illicit trade of pangolins, is of concern. Expanded studies using high throughput metagenomics and additional stable isotopes hold promise for enhanced traceability of illegally traded pangolins, particularly when used together.