The analysis of temporal gene expression to estimate the age of forensically important blow fly pupae: results from three blind studies.

The estimation of the minimum postmortem interval can be implemented by age estimation of corpse-associated primary colonizers such as the blow flies (Diptera: Calliphoridae). In cases where pupae represent the oldest stages found on a body, their age must be added to the duration of prepupal development to estimate the minimum postmortem interval. Although methods to age blow fly larvae have been well established using morphology, length or weight and age estimation of pupae has proved challenging. In a previous work, we quantified the changes in mRNA levels of four differentially expressed genes during the metamorphosis of Calliphora vicina pupae, hence representing molecular markers for pupal age (i.e., time elapsed since pupariation). Here, we demonstrate how these data can be used to estimate pupal age with inverse prediction. We present three blind studies conducted under various conditions and show that age of C. vicina pupae can be well estimated based on gene expression data. As these data are quantitative and can be processed automatically, gene expression has the potential to outperform morphological analysis in age estimation of forensically relevant blow fly pupae.