Internal morphological analysis for age estimation of blow fly pupae (Diptera: Calliphoridae) in postmortem interval estimation

Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau-Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot-water-killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot-water-killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.