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University of Cape Town DNA Barcoding of Forensically Important Flies in the Western Cape By TENIELLE MONIQUE COOKE CKXTEN001 SUBMITTED TO THE UNIVERSITY OF CAPE TOWN In partial fulfilment of the requirements for the degree MPhil (Biomedical Forensic Science) Faculty of Health Sciences UNVERSITY OF CAPE TOWN February 2016 Supervisor: Dr. Marise Heyns Co-Supervisor: Ms Laura Heathfield University of Cape Town i The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town . ii ACKNOWLEDGMENTS I would like to thank my supervisor Dr. Marise Heyns for all the support and guidance throughout my Masters studies. I would also like to acknowledge my co-supervisor Miss Laura Heathfield whose constant guidance kept me striving for excellence I would like to thank my colleagues, your constant support and friendship was much appreciated I would like to thank my parents Romal and Pauline Cooke for enduring this far with me I would like to acknowledge the support I received from my sisters Kesha and Angelique, my cousins Bronwyn and Teagan Reddy and a special thank you to Rosemary Reddy for opening her heart and doors for me I hereby acknowledge the financial assistance of the National Research Foundation (NRF) towards this research. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF Lastly, I would like to acknowledge God, without whom none of this would be possible. iii 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. iv Table of Contents Title Page………………………………………………………………………………………………………………………………………..i ACKNOWLEDGMENTS ........................................................................................................................ iii ABSTRACT ........................................................................................................................................... iv Table of Contents .................................................................................................................................... v List of Abbreviations ......................................................................................................................... vii List of Tables .................................................................................................................................... viii List of Figures ...................................................................................................................................... 1 Chapter 1: Research Proposal ................................................................................................................. 2 1.1 Introduction .................................................................................................................................. 2 1.1.1 Research problem .................................................................................................................. 3 1.1.2 Rationale and Justification ..................................................................................................... 3 1.1.3 Aims and Objectives ............................................................................................................... 4 1.2 Background ................................................................................................................................... 4 1.3 Proposed methodology ................................................................................................................. 8 Part I – Morphological species identification .................................................................................. 8 Part II – Molecular Species Identification ........................................................................................ 8 Part III – Blind Experiment ............................................................................................................... 9 1.4 Ethical considerations ................................................................................................................. 10 1.5 Social value and importance of this research ............................................................................. 10 1.6 Work plan and budget ................................................................................................................ 10 1.7 References .................................................................................................................................. 13 1.8 Appendices .................................................................................................................................. 16 Chapter 2 Literature Review ................................................................................................................. 18 2.1 Introduction ................................................................................................................................ 18 2.2 Developments in forensic entomology ....................................................................................... 19 2.2.1 Brief history .......................................................................................................................... 19 2.2.2 Early French cases ................................................................................................................ 19 2.3 Insects and the corpse ................................................................................................................ 20 2.3.1 Post mortem changes .......................................................................................................... 20 2.3.2 Insect colonisation ............................................................................................................... 20 2.3.3 Calliphoridae ........................................................................................................................ 21 2.3.4 Post mortem interval ........................................................................................................... 22 2.3.5 Estimating the post mortem interval using forensic entomology ........................................... 22 2.4 Species identification .................................................................................................................. 23 v 2.4.1 Importance and rationale .................................................................................................... 23 2.4.2 Morphological species identification ................................................................................... 24 2.4.3 Molecular species
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