A methodological approach to age estimation of the intra-puparial period of the forensically relevant blow fly Calliphora vicina via Fourier transform infrared spectroscopy.

Estimating the age of immature blow flies is of great importance for forensic entomology. However, no gold-standard technique for an accurate determination of the intra-puparial age has yet been established. Fourier transform infrared (FTIR) spectroscopy is a method to (bio-)chemically characterise material based on the absorbance of electromagnetic energy by functional groups of molecules. In recent years, it also has become a powerful tool in forensic and life sciences, as it is a fast and cost-effective way to characterise all kinds of material and biological traces. This study is the first to collect developmental reference data on the changes in absorption spectra during the intra-puparial period of the forensically important blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Calliphora vicina was reared at constant 20°C and 25°C and specimens were killed every other day throughout their intra-puparial development. In order to investigate which part yields the highest detectable differences in absorption spectra throughout the intra-puparial development, each specimen was divided into two different subsamples: the pupal body and the former cuticle of the third instar, that is, the puparium. Absorption spectra were collected with a FTIR spectrometer coupled to an attenuated total reflection (ATR) unit. Classification accuracies of different wavenumber regions with two machine learning models, i.e., random forests (RF) and support vector machines (SVMs), were tested. The best age predictions for both temperature settings and machine learning models were obtained by using the full spectral range from 3700 to 600 cm^-1. While SVMs resulted in better accuracies for C. vicina reared at 20°C, RFs performed almost as good as SVMs for data obtained from 25°C. In terms of sample type, the pupal body gave smoother spectra and usually better classification accuracies than the puparia. This study shows that FTIR spectroscopy is a promising technique in forensic entomology to support the estimation of the minimum post-mortem interval (PMI_min), by estimating the age of a given insect specimen.