Developmental and gene expression changes during intra-puparial development in Chrysomya albiceps (Diptera: Calliphoridae)
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Date
2021-11
Authors
Van der Westhuizen, Lucinda
Journal Title
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Publisher
University of the Free State
Abstract
Criminal investigations are dependent on postmortem interval estimations (PMI) to solve crimes by determining when a homicide occurred. Time of death is estimated using several components, including pathology; however, a pathologist can only determine time of death up to 72 hours postmortem. Therefore, during and beyond this period, entomological evidence is very valuable as the age of the eldest immature fly stage available is used to determine the time of death. In forensic investigations, it is important to have validated growth information at hand for entomological samples, involving all developmental stages, to determine the age of the samples collected. In forensic investigations, blowflies (Diptera: Calliphoridae) are important as primary colonizers of cadavers, especially in exposed areas, and current age estimation methods of entomological samples are based on morphological changes of the immature stages. Larvae, often encountered at crime scenes, already have established age estimation methods and preservation protocols. However, pupal developmental and preservation studies are in the minority. The main reason for the lack of data is based on the difficulty of working with pupal samples due to the puparium which conceals morphological changes. Considering the importance of the pupal stage, additional techniques such as molecular analysis are required to augment techniques to improve age estimation of the pupal stage. This dissertation reports on a study that followed a multidisciplinary approach to age estimation of Chrysomya albiceps (blowfly) pupae, which are abundant at crime scenes due to their predatory nature. Investigating the morphological changes occurring within the puparium and the possibility to link this to differential gene expressions changes. Two methods for age estimation were therefore developed: external morphology changes and temporal gene expression changes. In addition, different preservation protocols were tested to determine the most effective preservative dependant on the type of analysis. Validated collection and preservation methods are not readily available for pupal samples and the use of unvalidated collection and preservation methods ultimately hinders age estimation. Laboratory colonies of C. albiceps, reared from wild samples, collected at the University of the Free State, were utilized for pupal sample collection during three trials to obtain optimum
number of samples. Three preservation protocols (pierced before hot water kill (HWK) or not pierced before HWK and placing in 70% ethanol or 10% buffered formalin) for morphological examination were tested on 580 – 590 pupae aiming to retain the native morphology. For nucleic acid integrity, to conduct age estimation by gene expression, 78 pupae were subjected to liquid nitrogen as a killing method and samples were preserved at -80˚C. After morphological and molecular examination of pupae the following preservation method is proposed: for morphological examination pupae are pierced prior to HWK and then stored in 70% ethanol, for long- and short-term preservation as ethanol is the best for retaining native morphology; and for gene expression analysis, pupae are immediately subjected to liquid nitrogen and stored at -80˚C. In addition to previously identified markers, we proposed four new landmarks and based on these results, age estimation can be refined up to 96 hours at 26˚C. The temporal gene expression levels based on 78 pupal samples were quantified using qRT-PCR. It emerged that actin, which was previously used as a reference and target gene, cannot be used as a reference gene due to the changes in expression levels during pupal development and that RpS17, 18S and Rp49 are the best reference genes for normalization. This study made a valuable contribution to the forensic science field in criminal investigations by validating current suggested reference genes, analysing previously identified target genes and identifying new morphological markers which can be used as a baseline for future C. albiceps studies.
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Keywords
Dissertation (M.Sc. (Genetics))--University of the Free State, 2021, Criminal investigation, Postmortem interval estimations (PMI), Pathologist, Genetics (Crime investigation)