Insect Identification with ESEM Imaging and DNA Barcoding for Forensic Use

Flies are among the first insects to arrive at a corpse to lay eggs. The developing larvae can be used to determine post-mortem interval (PMI) using species specific growth models. The larvae can be difficult to identify due to minute morphological characteristics and rearing larvae into adults is time consuming and may be unsuccessful. We investigated the potential of coupling Environmental Scanning Electron Microscopy (ESEM) and DNA barcoding to enable the acquisition of high resolution images while obtaining DNA in a minimally destructive manner. This study focused on the widespread and forensically relevant species Chrysomya rufifacies as a model. Specimens were prepared in three different drying treatments: unaltered, dried, dried and imaged. Following the imaging process, three different DNA extraction methods with different levels of sample destruction were tested. ESEM imaging showed that there were no visual differences between specimens that underwent drying treatments and ones that were not dried. No significant differences in DNA yield were observed for drying treatments and extraction methods post extraction. However, extraction methods demonstrated significant differences in yield of PCR products. Sequenced PCR products aligned with 100% identity to the C. rufifacies mitochondrial genome reference sequence. Lower nucleotide identities between the sequenced PCR products and other forensically relevant fly species demonstrated that combining ESEM and DNA barcoding is a promising approach to rapid and reliable identification of insect larvae recovered from forensic samples.