Preservation of Calliphora vicina (Diptera: Calliphoridae) pupae for use in post-mortem interval estimation

Post-mortem interval (PMI) is frequently calculated using immature stages of carrion frequenting Calliphoridae (Diptera). This is based on identification to species level, followed by age estimation of the samples. These two processes depend on suitable preservation of insects for subsequent analyses, yet preservation methods for the pupal stage are poorly defined and inappropriate methods may result in discolouration or nucleic acid degradation. This study examined the effects of 21 common preservation methods on Calliphora vicina pupae of 4 and 7d old, assessing consequences of the various methods for DNA-based species identification, age estimation using morphological analyses, and differential gene expression (DGE) studies. Pupae were examined within two weeks of preservation and again after 6-8 months. Of the methods tested, hot-water-killing (HWK) followed by storage in 80% ethanol at -20°C or 4°C was the best treatment for external morphology and histological analyses respectively. DNA based species identification was possible following all methods. RNA integrity and amplification were best when pupae were stored at -80°C or in RNAlater (-20°C), however HWK and storage in 80% ethanol at -20°C was also acceptable, and thus the latter is proposed as a universal preservative method for pupae. This study proposes a preservation method for pupae that enables DNA-based species identification, internal and external morphological analysis for age estimation, and DGE study to be carried out on a single specimen, enabling a multidisciplinary approach to age estimation from a single pupa.