Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMI_min ) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post-feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9-33 carbons, alkenes (18%), and methyl-branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post-feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMI_min estimations. However, future research is needed to validate these results under natural conditions in the field.