AbstractBlowflies (Diptera: Calliphoridae) are primary colonisers of cadavers; the ages of the eldest immature stages can be used to estimate minimum post-mortem interval (PMI).These estimates are obtained using calliphorid larvae, for which there are established and reliable preservation protocols and age estimation methods. The opposite is true for pupae; non-standardised crime-scene collection and preservation methods are employed, resulting in poorly preserved specimens for age estimation, which is often conducted using limited and inadequate research data. This has hindered the use of this sedentary, long-lasting stage of the blowfly lifecycle in PMI estimation. A multidisciplinary approach to age estimation of Calliphora vicina pupae was explored, including development of standard preservation protocols, with the aim of improving PMI estimation.
Initial work involved the development of standardised egg collection protocols for the purpose of minimising variation in lifecycle length and precocious egg occurrence. This enabled quantification of pupal age error, which was subsequently applied to developmental timelines. Multiple preservation protocols were then trialled on pupae with the aim of retaining native morphological form and nucleic acid integrity for species identification and proposed age estimation methods. Optimal preservation methods for each analysis were suggested and the following universal preservative protocol proposed: pupae are pierced, hot-water-killed, and stored in 80% ethanol at -20°C.
Three methods of pupal age estimation were developed using changes in external morphology, histology and temporal gene expression. The external morphological development of 23 features was recorded from 1494 pupae. These data was used to create a Pupal Age Estimator tool, comprising a manual age-range correlation method and a regression equation for age estimation. Blind sample analysis indicated that age could be estimated to within 23 hours at 22°C, approaching the observed natural variation range. Internal morphological development of 42 pupae sampled at 24-hourly intervals was examined using histology and optical coherence tomography(OCT). Six additional features were identified as suitable age markers, however full analysis was limited by the inherent tissue loss due to sectioning and the low resolution of OCT. Finally, temporal gene expression levels of 42 pupae (selected at24-hourly intervals) were quantified using qRT-PCR. Expression ratios we recalculated between three developmentally expressed genes (Ecr, LSP-2 and Trp) and two housekeeping genes (EF1α and RP49). Regression analysis of these data indicated age estimation was possible to approximately 23 hours at 22°C.
It can therefore be considered that the reliability and precision of PMI estimation using the C. vicina pupal stage is much improved from that possible at present. Pupal age estimation is critically dependent on appropriate preservation, now facilitated by the proposed standard protocols and by combining all age estimation methods presented here, a multidisciplinary approach can estimate C. vicina pupal age to within 23 hours at 22°C.
|Date of Award||Aug 2012|
|Supervisor||Alan Thorne (Supervisor), Michelle Harvey (Supervisor) & Colin Sharpe (Supervisor)|