Impact of flunitrazepam on Calliphora vicina (Diptera: Calliphoridae) microbiome dynamics

This study explored the effects of flunitrazepam, a benzodiazepine, on the microbiome diversity of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera: Calliphoridae). By examining the microbial shifts throughout developmental stages, the research contributes valuable data to the field of forensic entomotoxicology. Two colonies of 500 adults were fed minced beef liver, spiked and non-spiked with 25 mg of flunitrazepam, and reared under controlled conditions (24°C, relative humidity of 45%, 12:12 light-dark cycle). Following oviposition, egg clusters were transferred, and the experiment was carried out in triplicate under the same experimental conditions. A total of 54 specimens, including all developmental stages, were collected for microbiome investigation via Illumina MiSeq. Both colonies had a 19-day development cycle from eggs to teneral. However, flunitrazepam-fed specimens were heavier, particularly during the pupa and teneral stages. Microbiome analysis revealed significant differences in diversity and composition between the colonies and across developmental stages. Pseudomonadota (Proteobacteria) dominated the control adults, while Bacteroidota and Bacillota (Firmicutes) were more prevalent in flunitrazepam-fed adults. Additionally, Enterobacterales, Lactobacillales, and Morganellaceae showed notable variations across different stages. This study highlights the significant impact of flunitrazepam on the microbiome dynamics of C. vicina, revealing notable morphological changes related to the specimens' weight toward the end of the development cycle and alterations in microbiome composition. These findings have important implications for forensic entomotoxicology, particularly in the accurate estimation of the minimum postmortem interval (mPMI).