University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 2011 Sequence and analysis of the mitochondrial DNA control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae) Terina Bruhn University of Wollongong Recommended Citation Bruhn, Terina, Sequence and analysis of the mitochondrial DNA control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae), Master of Science thesis, University of Wollongong. School of Biological Sciences, University of Wollongong, 2011. http://ro.uow.edu.au/theses/3236 Research Online is the open access institutional repository for the University of Wollongong. For further information contact Manager Repository Services: [email protected]. University of Wollongong Sequence and analysis of the mitochondrial DNA control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae) Terina Bruhn 2011 Master of Science - Research (Biological Science) School of Biological Sciences, University of Wollongong, NSW Declaration I, Terina Bruhn declare that this thesis “Sequence and analysis of the mitochondrial DNA control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae)” is wholly my own work unless otherwise referenced or acknowledged below . The document has not been submitted for qualifications at any other academic institution. ii Acknowledgements I would like to thank my supervisors Dr Mark Dowton, Dr James Wallman and Dr Peter Gunn for lending their considerable expertise to assist me to compile this degree. I would like to thank the University of Wollongong and the staff of laboratory 108, in particular Tracey Gibson who gave generously of her time. I would like to acknowledge the considerable amount of time that the NSW Police, Forensic Services Group has invested in this project, and thank management for their support and for allowing me the opportunity to further my studies. On a personal note, I would like to thank my husband Glenn, and my sons Joshua and Denzel for their support and patience. iii Abstract Blowflies of the genus Chrysomya are forensically important, myiasis-causing flies. Their larvae are frequently recovered from decomposing human corpses, and may assist in criminal investigations by helping to determine the postmortem interval. Larvae of some Chrysomya species can be difficult to identify because of their morphological similarity. This is especially so along the east coast of Australia, where this genus is much more speciose than in other parts of the continent. Molecular techniques, especially those based around DNA sequencing, can improve the reliability of insect identification. The hypervariable non-coding control region of the mitochondrial genome is one region of DNA that can be particularly informative, because of its high nucleotide divergence and size variation. This project examined the potential of the mitochondrial control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae): Ch. saffrenea, Ch. megacephala, Ch. semimetallica, Ch. varipes, Ch. incisuralis, Ch. nigripes, Ch. flavifrons, Ch. rufifacies and Ch. latifrons latifrons to determine its usefulness as a potential identification tool and as a molecular marker for evolutionary studies. The mitochondrial control region was successfully amplified in two overlapping pieces, but direct sequencing of both of these products was unsuccessful. Cloning of amplification products from single individuals confirmed the heteroplasmic nature of the control region in Australian Chrysomy a species, with intraindividual heteroplasmy due to indels, variation in the number of nucleotides in homopolymer runs (poly- adenine and poly-thymidine), variation in the number of short tandem repeats (AT), and the presence of duplicated genes. Length heteroplasmy reported in this study was mainly surrounding long monoadenosine/monothymidine and dinucleotide repeats, PCR polymerase artefact due to slipped strand mispairing may have contributed to the length variation observed in the present study. Length variation was also observed between species, due primarily to the presence or absence of a duplicated tRNA Ile gene and/or partially duplicated tRNA Gln pseudogene. Duplicate tRNA Ile genes were present in Ch. saffranea , Ch. megacephala , Ch. incisuralis , Ch. flavifrons , Ch. rufifacies and Ch. nigripes . The partially duplicated iv tRNA Gln pseudogene was identified in many of the same flies as those with the duplicate tRNA Ile gene. The data suggest the duplication of the tRNA Ile and tRNA Gln in a common ancestor of the Australian Chrysomya and may be used to infer evolutionary relationships among the Australian Chrysomya blowflies. The IQM gene cluster as described by Lessinger et al (2004) was identified in all nine species of Chrysomya . However, intraindividual variation, due to point mutations, insertions and deletions, were common in the IQM gene cluster across most species sequenced. Phlogenetic analysis reports that the parsimony tree resolved all Chrysomya species with bootstrap proportions over 99%, supporting the grouping of sister species which was also supported in the neighbour joining approach, and the separation of Ch. incisuralis and Ch. rufifacies from the rest of the Chrysomya species. Strong posterior support 100% validates the relationships between sister species Ch. semimetallica and Ch. latifrons as well as between Ch. megacephala and Ch. saffranea. Phylogenetic analysis of the mtDNA control region Target 3 amplicon resulted in Ch. nigripes being placed in a clade consisting of Ch. nigripes , Ch. varipes and Ch. flavifrons , with strong support at 98%, (see Appendix 19 Neighbour Joining Approach – All at Once Alignment). Together, these findings indicate that there may be enough variation between the blowfly species to separate and identify the Chrysomya blowflies at a species level. However, the presence of heteroplasmy makes the routine use of this region impractical, due to the need to clone amplification products prior to sequencing. Other regions, such as COI or ITS2 appear less problematic. v Table of Contents Acknowledgements iii Abstract iv Abbreviations viii CHAPTER 1- INTRODUCTION 1 1.1 Literature Review 1 1.2 Successional Patterns of Insects and Post Mortem Interval 1 1.3 Chrysomya Blowflies 3 1.4 Morphological Identification of Forensically Important Blowflies 4 1.5 Molecular Identification of Diptera 6 1.5.1 Nuclear Markers 6 - Nuclear ribosomal DNA (rDNA) genes 6 - Microsatellites 10 - Single Nucleotide Polymorphism (SNPs) 13 - Nuclear Protein Coding genes 13 1.5.2 Mitochondrial Markers 15 - Animal Mitochondrial DNA 15 - Insect Mitochondrial DNA 16 - mtDNA tRNA genes 17 - mtDNA Protein Coding genes 19 - Mitochondrial ribosomal RNA genes (12S and 16S) 23 1.6 Heteroplasmy in Mitochondrial DNA 26 1.7 Mitochondrial DNA Control Region of Insects 27 1.8 Mitochondrial DNA Control Region of Diptera: Calliporidae 29 1.9 Primer Design and Selection 31 1.10 Conclusions 34 1.11 Aims of the project 35 CHAPTER 2 – MATERIALS AND METHODS 36 2.1 Specimens 36 2.1.1 Australian Chrysomya Specimens 36 2.1.2 DNA extraction 36 2.2 PCR Amplification 38 2.2.1 Amplification of the Control Region and Surrounding Genes 38 2.2.2 Nested Amplification of the Control Region using internal 40 primers from “Long Target” 2.2.3 Amplification of the Control Region using internal primers 41 from genomic DNA 2.3 Agarose Gel Electrophoresis 43 2.4 Cloning 43 2.4.1 Colony PCR Screen 45 2.4.2 Plasmid DNA Purification 45 2.4.3 Restriction Enzyme Analysis 45 2.5 Sequencing 46 2.6 Sequence Analysis 47 2.7 Phylogenetic Analysis 47 vi CHAPTER 3 – RESULTS 49 3.1 PCR & Sequencing 49 3.1.1 Amplification of the entire Control Region 49 3.1.2 Nested Amplification using Internal Primers 49 3.1.3 Amplification of the Control Region using internal primers 52 3.2 Cloning Optimisation 54 3.3 Target 3 57 3.3.1 Transformations of Target 3 57 3.3.2 Colony PCR Screening of Target 3 57 3.3.3 Plasmid DNA Purification of Target 3 58 3.3.4 Restriction Enzyme Analysis of Target 3 59 3.4 Target 4 61 3.4.1 Transformations of Target 4 61 3.4.2 Colony PCR Screening of Target 4 61 3.4.3 Plasmid DNA Purification of Target 4 62 3.4.4 Restriction Enzyme Analysis of Target 4 63 3.5 Sequencing and Analysis 64 3.5.1 Introduction 64 3.5.2 Target 3 65 3.5.3 tRNA Genes of Target 3 66 3.5.4 Conserved Sequence Blocks (CSBs) of Target 3 70 3.5.5 Length variation of Target 3 72 3.5.6 Target 4 74 3.5.7 tRNA Genes of Target 4 75 3.5.8 Conserved Sequence Blocks of Target 4 79 3.5.9 Length variation of Target 4 81 3.6 Phylogenetic Analysis 83 3.6.1 Neighbour-Joining Analysis 83 3.6.2 Maximum Parsimony Analysis 84 CHAPTER 4 – DISCUSSION & CONCLUSIONS 85 4.1 Introduction 85 4.2 Amplification of the mtDNA Control Region of Australian Chrysomya 85 Blowflies 4.2.1 Cloning confirms the existence of heteroplasmy in Australian 85 Chrysomya 4.2.2 Heteroplasmy in the Control Region of Australian Chrysomya 86 4.3 Organisation of the Australian Chrysomya mtDNA Control Region and 88 surrounding genes 4.3.1 Presence of the IQM gene cluster 88 4.3.2 Duplicated tRNA genes and tRNA pseudogenes 90 4.3.3 Tracing the history of gene rearrangement in Chrysomya 91 4.3.4 Conserved Sequence Blocks in the Control Region of Australian 94 Chrysomya 4.4 Phylogenetic Analysis and the Identification of the Australian Chrysomya 95 Blowflies 4.5 Conclusions 99 REFRENCES 100 APPENDICES 112 vii Abbreviations