Untargeted metabolomics for chemotaxonomy of blow fly pupae (Diptera: Calliphoridae) supported by DNA barcoding

In forensics, accurate species identification of necrophagous insects retrieved from decomposing remains is critical for estimating the postmortem interval (PMI). Blow flies (Diptera: Calliphoridae) are typically the earliest colonizers to arrive on carrion. Because juvenile life stages are morphologically similar across species, forensic entomologists traditionally rear the eggs, larvae, and pupae to adulthood to ensure a definitive identification based on features of the adults, in a process that is time-consuming and relies heavily on specimen viability. This study presents a rapid, high-throughput identification method for forensically significant blow fly pupae utilizing direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) coupled with chemometrics. A prediction model was developed for 11 species of necrophagous blow fly pupae across seven genera of Calliphoridae: Calliphora, Chrysomya, Cochliomyia, Cynomya, Lucilia, Phormia, and Protophormia. It was built using 220 species-validated test samples, and tested with 115 external validation samples. Leave-one-out cross validation accuracies of 97.27% and 98.26%, respectively, were observed for the test and validation datasets. Species identities were independently verified via DNA barcoding with cytochrome c oxidase I. These results demonstrate that DART-HRMS can successfully identify pupae-a historically underutilized developmental stage in forensic casework-based upon their unique metabolome signatures. This work provides critical forensic resources for the utilization of pupae that may facilitate PMI estimations, even for non-viable specimens.