Can Lucilia sericata Larvae Produce Ethyl Glucuronide? An Experimental Study for Forensic Implications

Can Lucilia sericata Larvae Produce Ethyl Glucuronide? An Experimental Study for Forensic Implications Learning overview This study investigates the ability of Lucilia sericata larvae to absorb ethanol and produce ethyl glucuronide (EtG) after feeding on ethanol-containing substrates. The results indicate that the metabolic and excretory systems of Lucilia sericata (Meigen, 1826) (Diptera, Calliphoridae) larvae are not responsible for EtG production. These findings emphasize critical interpretive considerations for toxicological analysis and underscore the need to better understand the metabolic capacities of necrophagous insects in forensic investigations. Impact statement This discovery is essential for understanding the biochemical interactions between larvae and their environment. Moreover, the absence of EtG synthesis in larvae despite ethanol exposure reaffirms the value of EtG as a reliable external biomarker of antemortem alcohol consumption, as it is not subject to insect-driven metabolic interference. This approach has the potential to enhance the accuracy of postmortem investigations by offering deeper insights into the biological and environmental conditions preceding death. By incorporating larval analysis into forensic protocols, practitioners may improve their ability to reconstruct the circumstances surrounding death. Abstract This study examines whether Lucilia sericata larvae are capable of producing ethyl glucuronide (EtG) after feeding on ethanol-containing substrates. The goal is to determine whether EtG detected in larvae collected from a corpse may be attributed to postmortem synthesis or reflects actual antemortem ethanol exposure. The findings aim to refine the accuracy of postmortem toxicological investigations by providing critical insights into the biological and environmental contexts preceding death. Forensic entomotoxicology explores the role of necrophagous insects in toxicological analysis, particularly in cases where conventional biological samples are unavailable or compromised [1]. Ethyl glucuronide (EtG) is a direct biomarker of alcohol consumption, formed as a result of repeated alcohol intake [2]. Lucilia sericata (Meigen, 1826) (Diptera, Calliphoridae) is a widely distributed necrophagous species in Italy, often among the first to colonize remains, and is extensively studied in forensic entomotoxicology [3,4]. Understanding whether these insects can absorb ethanol from their feeding substrate and metabolize it into EtG has important implications in forensic toxicology and pathology. To date, no studies have definitively addressed this question. To test this hypothesis, Lucilia sericata larvae were reared in a laboratory setting and divided into three groups: one fed meat spiked with ethanol (G_EtOH), one fed meat spiked with EtG (G_EtG), and a control group fed unaltered meat without any additives (G_Neg). 40 g of minced meat were used for each of the 3 substrates. For the G_EtOH group, 12.5 ml of ethanol were added to the meat; for the G_Etg group, 200 μg of EtG in aqueous solution were added. G_EtOH, G_EtG and G_Neg larvae were transferred to their specific substrate immediately after hatching. Each tested group consisted of between 20 and 25 larvae. EtG analysis was performed using Ultra-High-Performance Liquid Chromatography analysis (UHPLC), while ethanol detection was carried out using headspace Gas Chromatography-Flame Ionization Detector (GC-FID), employing both validated and adapted methods for larval matrices [5,6]. The larvae were left free to feed on their substrate and were subsequently sample at 24 and 72 hours. The pupae, formed at the end of the larval cycle, were sampled at 120 hours. This allowed us to observe whether different times corresponded to different results in terms of EtG formation. The analyses for EtG by UHPLC and ethanol by GC-FID were conducted on groups of three larvae or pupae each (300 mg total sample), to allow a better resolution of the analysis results. At the conclusion of the study, EtG was not detected in either the larvae or pupae from the G_EtOH group (ethanol fed group), although ethanol was present, confirming its absorption. The G_EtG and G_Neg groups yielded expected results, testing positive and negative for EtG, respectively. This is the result of an initial exploratory study with possible applications in the forensic field. These results reinforce the interpretation of EtG as an indicator of antemortem alcohol use when found in larvae, as its presence reflects preexisting levels in the corpse rather than de novo synthesis by the insect. This study highlights critical interpretative issues in toxicological analysis and supports the growing importance of understanding insect metabolism in forensic science. With ongoing advances in analytical techniques and experimental practice, the use of carrion-feeding larvae as forensic tools may increasingly become standard practice.