Molecular differentiation of Central European blowfly species (Diptera, Calliphoridae) using mitochondrial and nuclear genetic markers.

A challenging step in medical, veterinary and forensic entomology casework is the rapid and accurate identification of insects to estimate the period of insect activity (PIA), which usually approximates the post-mortem interval (PMI). The morphological identification of insect evidence is hampered by species similarities, especially at the early larval stages. However, DNA-based species identification is more accurate and reliable. In this study, we improved the suitability and efficacy of the standard mitochondrial cytochrome c oxidase subunit I (COI) barcode region of 658 bp combined with an additional region of 616 bp of the same gene. We also tested the usefulness of other mitochondrial and nuclear loci, such as the non-coding region included in mitochondrial Cyt-b-tRNA(ser)-ND1 (495-496 bp) and the second internal transcribed spacer (ITS2) region of nuclear ribosomal DNA (rDNA) (310-337 bp). We classified a total of 54 specimens from five blowfly species belonging to three Calliphoridae genera commonly found in Central Europe: Phormia (P. regina), Calliphora (C. vicina) and Lucilia (L. sericata, L. ampullacea and L. caesar). Additionally included were the Cyt-b (307 bp) sequences for P. regina species and GenBank recorded information about the studied loci for select species. The results revealed the robustness of COI (616 bp) and ITS2 (310-337 bp) as diagnostic tools to be added to the widely established COI barcode (658 bp). Their higher discriminatory power allows for more precise and reliable identifications, even within more complex genera (Lucilia). This work also contributes new nucleotide sequences that are useful for accurate species diagnosis and new sequence data of Calliphoridae interspecific variability in the European Westphalia region (Germany).