Master of Science - Research
University of Wollongong. School of Biological Sciences
Bruhn, Terina, Sequence and analysis of the mitochondrial DNA control region of nine Australian species of the genus Chrysomya (Diptera: Calliphoridae), Master of Science - Research thesis, University of Wollongong. School of Biological Sciences, University of Wollongong, 2011. http://ro.uow.edu.au/theses/3236
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 over lapping 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 Chrysomya species, with intraindividual heteroplasmy due to indels, variation in the number of nucleotides in homopolymer runs (polyadenineand 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 thelength variation observed in the present study.
Length variation was also observed between species, due primarily to the presence orabsence of a duplicated tRNAIle gene and/or partially duplicated tRNAGln pseudogene. Duplicate tRNAIle genes were present in Ch. saffranea, Ch. megacephala, Ch.incisuralis, Ch. flavifrons, Ch. rufifacies and Ch. nigripes. The partially duplicated tRNAGln pseudogene was identified in many of the same flies as those with theduplicate tRNAIle gene. The data suggest the duplication of the tRNAIle and tRNAGlnin a common ancestor of the Australian Chrysomya and may be used to inferevolutionary 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 posteriorsupport 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.