Exploring unified methods of killing and storing insect samples for forensic entomotoxicology using diazepam in Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae

Forensic entomologists use the maturity of necrophagous larvae to estimate the minimum post-mortem interval (PMImin), ideally taking account of effects that xenobiotics in the corpse may have on insect maturation. Forensic toxicologists may employ larvae to detect drugs in drug-related deaths when human samples are unavailable. Yet current pre-analytical practices of these two professions differ significantly, impeding the successful use of the same samples. Potential benefits of shared pre-analytical practices and opportunities for enhanced collaboration have yet to be fully explored. We employed Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae, raised in the presence of diazepam, to investigate the effects of two standard investigative practices on larvae for drug detection and for quantifying mass and length as proxies of age. Specimens were killed by either blanching or freezing and stored at -20℃ for either intermediate or long periods. Blanched larvae showed smaller changes in size and body integrity during storage, thereby producing the most reproducible estimates of PMImin. Consequently, data obtained from blanched larvae were used to evaluate the impact of diazepam on larval development. Diazepam exerted no significant effect on larval mass, and a weak effect on length. Diazepam recovery was significantly higher from blanched larvae, suggesting that freeze-killing causes drug loss. This model system demonstrates the value to forensic entomologists of the standard technique of blanching larvae, followed by storage at -20℃ for toxicological analysis. We recommend that forensic toxicologists consider blanching to kill larvae before storage at low temperatures, at least for certain drugs. This approach offers the dual benefit of high-quality specimens for both PMI estimation and drug detection.