AbstractBlowflies (Diptera: Calliphoridae) are commonly the first colonisers of carrion. Forensic entomologists are able to estimate a minimum post-mortem interval by examining the eldest immature stage collected from the scene. For a minimum post-mortem interval estimation to be calculated, developmental data of the species is commonly subtracted from total developmental time. Information regarding events prior to death such as post-mortem relocation, mortuary behaviour and origin can also be determined from collected samples, in particular those from archaeological sites. The information, however, relies on accurate species identification. Currently, several methods are employed on insect samples. These involve both morphological and molecular techniques. With the constant development of molecular genetics, new methods are being developed which show potential for species identification. This thesis examines the use of such techniques on both contemporary and museum-stored insect samples.
Deep sequencing technology, nested-PCR and light microscopy were used to examine the utility of a combined approach for the identification of insect samples collected from two sets of mummified remains. In both cases, species identification was successful, and origin was determined in one case. Results showed the importance of a multi-technique approach, with emphasis on utilising both morphological and molecular techniques to assign identity.
Multi-gene analysis was used to examine the utility of several genes from both mitochondrial and nuclear DNA to assign species status of the South African blowfly Calliphora croceipalpis (Diptera: Calliphroidae). Using the Cytochrome Oxidase I gene, Internal Transcribed Spacer 2 and the Carbamoylphosphate Synthetase gene, species identity was determined. It was found that identification of C. croceipalpis must be under taken with care due to possible morphological similarities due to founder effects with Calliphora vicina and it is recommended to use a multi-gene approach for identification of calliphorids.
Inter-simple sequence repeat-polymerase chain reaction was investigated for its applicability as a rapid identification tool for forensically important calliphorids. Examining 26 calliphorid species and several populations within many of the species collected globally, the existence of species-specific bands was examined. Results showed that large amounts of interspecific and intraspecific variation were seen, with no species-specific bands detected. It is recommended that inter-simple sequence repeat not to be used as a rapid tool for calliphorid identification, but it may have a use as a population-based tool.
The analysis of molecular techniques showed that with new recent techniques, such as next generation sequencing, the information that is gained from museum-stored samples, could aid in historical findings. Significant information such as geographic origin and historical events has been determined from molecular work. The use of a multi-gene approach is recommended when analysing closely related species, due to recent divergence. Analysing both nuclear and mitochondrial genes increase the accuracy of species identification. The use of a rapid molecular technique for identifying entomological samples would be a fundamental and valuable tool. Although cytochrome oxidase I amplification and sequencing are relatively time-consuming, they are more reliable indicator of species than inter simple sequence repeat analysis. It can therefore be concluded from this study that the application of molecular techniques for the identification of both contemporary and museum samples can provide a wealth of information to help both forensic and archaeological case studies.
|Date of Award||Oct 2013|
|Supervisor||Colin Sharpe (Supervisor), Alan Thorne (Supervisor) & Michelle Harvey (Supervisor)|