Gene expression analysis as a tool for age estimation of blowfly pupae

Forensic entomology, the use of insects in medicolegal investigations, mainly focus on the estimation of the postmortem interval (PMI) calculating the age of necrophagous specimens like blowflies, typically examined using maggot stages. While staging the age of the larvae is possible at a quite detailed scale, the age of the pupae is not easy to specify without rearing up to the adult stage so far, which is time-consuming or might be difficult. However, the pupal stage represents about 50% of the immature development time and therefore pupal age may serve as an important tool in entomological PMI estimation. Using a specific ddRT-PCR protocol we were able to monitor a differential gene expression in Calliphora vicina pupae at different ages by development dependent banding patterns on an agarose gel. Transcripts of interest were isolated from the gel and their nucleotide sequence determined. Primer/ probe sets for quantitative RT-PCR using an ABI 7300 real-time instrument were designed and applied on cDNA from pupae of different development stages. Quantitation of these transcripts leads to age specific expression patterns. So far they have been worked out for pupae at the beginning, the middle and the end of their metamorphosis. (C) 2009 Elsevier Ireland Ltd. All rights reserved.