In forensic entomology, the patterns associated with insect colonization of remains are used to estimate the postmortem interval (PMI) in death investigations. Blow flies (Family: Calliphoridae) are often the first to colonize remains. A 'back-calculation' method exists to estimate the PMI by considering many factors, including the retrieved blow fly species identity and their known species-specific life cycles. Accurate PMI estimation hinges on precise species identification (ID), as the developmental and maturation rates that are used for its assessment vary between species as a function of temperature and environmental conditions. Typical ID methods based on morphology are time-consuming, require specialized expertise, and are susceptible to error for the juvenile life stages in particular (i.e., eggs, larvae and pupae) because of their visual similarity across species.
Reported here is an alternative novel approach employing mass spectral analysis and chemometrics for rapid blow fly species ID of juvenile life stages in the form in which they are typically collected by investigators, thereby facilitating estimation of PMI. Analysis of multiple species representing six genera by direct analysis in real time — high resolution mass spectrometry (DART-HRMS), revealed distinct mass spectral profiles for each species. Application of Support Vector Machine (SVM) to the mass spectral data acquired enabled effective genus and species-level species differentiation. The prediction models for 10 egg species resulted in accuracies of the represented genera of 90.22% (Calliphora), 95% (Chrysomya), and 92.50% (Lucilia). The model also exhibited an identification accuracy for 5 larval and 6 pupal species of 97.33% and 97.22%, respectively. The external validation samples for the Calliphora, Chrysomya, and Lucilia genera eggs had accuracies of 93.93%, 100%, and 85.27%, respectively. The external validation samples for the larvae and pupae had accuracies of 80% and 96.67%, respectively.
The data also revealed that there are unique chemical markers that can be used to identify the insect species. For example, m/z 117.08 may be indicative of the presence of C. latifrons eggs, while m/z 88.11 was indicative of the presence of C. cadaverina larvae, and m/z 132.14, P. regina pupae.
These findings have implications for determination of the species ID of juvenile blow fly evidence, without DNA analysis or the need to rear the specimens to adulthood.

Alexa Figueroa

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