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Development of new molecular techniques for wildlife forensic applications

Coghlan, Megan Louise (2014) Development of new molecular techniques for wildlife forensic applications. PhD thesis, Murdoch University.

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Wildlife forensics is an emerging scientific field, borrowing much of the underpinning knowledge and methodologies from the conservation biology and human forensic sectors. Research in this field has only recently begun to gain momentum, prompted by the recognition that illegal wildlife trade is a major causative factor in the decline of a large number of animal and plant species in the wild. With significant advancements in scientific capabilities witnessed over the last few decades, new techniques are now accessible for application in the wildlife forensic arena. Wildlife forensics encompasses key areas of scientific analysis including morphology and microscopy, chemistry, pathology, and genetics. This thesis focuses solely on the development of a molecular toolkit with the aim to build capacity across a number of applications within wildlife forensic science.

One of the most important functions of wildlife forensic genetics is to accurately identify the species of seized wildlife material. A central theme of this thesis is the investigation of how analysis of regions of mitochondrial DNA can be used to identify the species of wildlife specimens from single-source samples through to complex mixtures. In Chapter Two, 99 illegally trafficked avian eggs were DNA sequenced using two gene regions for species identification, with 54% being identified to species level. The remaining extracted eggs were identified to either genus or family taxonomic levels only. This result highlighted the deficiencies of current reference sequence databases, needed to make robust species level identifications. In Chapter Four, feather and tissue remains from aircraft collisions with birds (birdstrikes) were also identified using multiple mitochondrial gene regions with species level identifications being successfully made in 86% of cases. This information provided valuable insight for aerodrome safety officials, and formed the basis of birdstrike mitigation strategy planning.

For the first time, the most advanced DNA sequencing technology currently available, next generation sequencing (NGS), was applied to wildlife forensic samples. The species composition within birdstrike gastrointestinal tracts were analysed to determine diet, and within traditional Chinese medicines (TCM) for determining the legality of seized and legally purchased products (Chapters Four and Five). This technology was shown to give powerful insights into the composition of species-rich samples, providing over 790,000 DNA sequences across all extracts from both chapters, detecting over 20 animal genera and 105 plant families. The sequencing depth afforded by NGS techniques allowed for the identification of plant, vertebrate, and invertebrate DNA content in parallel and at deep sequencing depths, an outcome that is not previously possible using Sanger sequencing.

Moving beyond species identifications, in some cases it is important from a law enforcement perspective, to be able to determine if there is relatedness between seized samples. The current DNA markers of choice in the forensic sector are short tandem repeats (STRs). Well-developed markers exist for human forensic applications, however there is currently a shortage of available and characterised STRs for most of the highly targeted species in the illegal wildlife trade. In this thesis, a new approach to developing STR loci for use across multiple species was investigated (Chapter Three). For the first time in wildlife forensics, NGS methods were applied to the analysis of five STR loci, with direct comparison of this new method to that of traditional capillary electrophoresis across the same five loci. NGS-based genotyping facilitated the detailed characterisations of: repeat unit structure, flanking region base composition, and identified possible species-specific single nucleotide polymorphisms. The NGS method was consistent with capillary electrophoresis methods in delivering the same DNA profile result, but was far more informative.

Taken together, this thesis presents a number of new molecular methodologies for species identification and identity testing in wildlife forensic applications. The dramatic advancement in molecular biological data generation afforded by NGS techniques is applied to wildlife forensics for the first time, and the result is extremely encouraging. Through incorporation of NGS techniques, wildlife forensic genetics research and application can continue to progress in size and scale towards successful prosecution of illegal wildlife poaching and trade.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Bunce, Michael and Haile, James
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