Age estimation of forensically important blowfly, Chrysomya megacephala (Diptera: Calliphoridae) pupae using micro-computed tomography imaging

Accurate estimation of the minimum post-mortem interval (minPMI) is important in the investigation of forensic cases. Various thanatological methods are being used to estimate this interval. However, entomology approach is the most reliable method for this minPMI estimation especially when death has occurred over 72 hours and involved insects or other arthropods evidence at the death scene. The current methods of age estimation are daunting and destructive especially when dealing with pupal stage. The aims of this study were to characterize the morphological changes during intra-puparial period of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and their relation with minPMI estimation by using a high resolution micro-Computed Tomography (micro-CT). Gravid C. megacephala were collected from a rural area in Sungai Buloh, Selangor and cultured in the laboratory at 23.83±0.25°C with light: dark hour of 12:12 to initiate oviposition. The resulting larvae were reared until pupal stage. A pupa was collected at first (24 hours), second (48 hours), third (72 hours), and fourth quarter (96 hours) of the intra-puparial period. The pupal samples were placed directly into 70% ethanol for preservation. Micro-CT scanning was employed to acquire microstructural information following pupal sample staining for contrast enhancement. Eight age-informative internal morphological landmarks were mapped from the micro-CT scanning. The present study enhanced the potential value of micro-CT for the estimation of minPMI based on the internal morphological changes of C. megacephala pupae. This novel method is a promising tool for improving medico-legal investigations in forensic entomology.