DNA barcoding of forensically important flies in the Western Cape

Cooke, Tenielle Monique

DNA barcoding of forensically important flies in the Western Cape

Master Thesis

2016

Publisher

University of Cape Town

Department

Division of Forensic Medicine and Toxicology

Faculty

Faculty of Health Sciences

Abstract

One of the central applications of forensic entomology is the determination of the post mortem interval (PMI) from arthropod evidence associated with a corpse. Estimations of the PMI are based on succession and developmental patterns of specific species that visit the body. As first colonisers, Calliphoridae (blow flies) are often used by forensic entomologists to determine the PMI however, developmental rates of visiting fauna differ substantially which makes correct species identification vital. Traditional methods of identification which assign species based on keys that capitalise on morphological differences are insufficient for closely related species, especially during immature stages of the lifecycle or when the specimen is damaged. Molecular identification such as DNA barcoding has therefore become a popular method of identifying species. DNA barcoding characterises species by sequencing and analysing specific regions in the genome. This technique has been used to characterise species in various countries including parts of South Africa. Its application has also been demonstrated in a forensic setting but data for the Western Cape is minimal. This study therefore aimed to assess the utility of DNA barcoding for species level determination of four blow fly species common to the Western Cape of South Africa (Chrysomya chloropyga, Chrysomya albiceps, Chrysomya marginalis, and Lucilia sericata) as well as its ability to identify immature specimens. Ten adult specimens from each species were morphologically and molecularly identified using microscopy and DNA barcoding respectively. The standard DNA barcode, cytochrome c oxidase subunit I (COI) and a secondary marker, the second internal transcribed spacer (ITS2) were analysed. Phylogenetic analyses for both barcodes showed high interspecific divergence values which are desirable for species level differentiation by DNA barcoding. COI sequences from adult flies were also submitted and searched against BOLD for identification and only genus level identity could be achieved, indicating that, COI alone may be insufficient to discriminate between closely related species. DNA sequences from the adult specimens were then used as reference sequences for identification of seven unknown immature specimen using DNA barcoding of both COI and ITS2. Sequence similarity was assessed and identity was assigned based on >98% similarity scores, and all immatures were successfully identified. The use of more than one DNA marker to complement morphological data ensures higher confidence of species level identification. This method provides a reliable and consistent tool for entomologists to use for species identification which results in higher levels of accuracy in PMI estimations.

Keywords

Biomedical Forensic Science

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