Descriptive analysis of protein expression variations during pupal development of <i>Chrysomya megacephala</i> (Diptera: Calliphoridae) using label-free proteomic techniques

Age estimation is a critical aspect of forensic entomology, especially in the examination of pupae. The use of proteins as a means for age identification shows great promise. In this study, proteomic techniques were employed to investigate differentially expressed proteins (DEPs) during the intrapuparial stage of <i>Chrysomya megacephala</i>. Specimens were sampled at four distinct time points: 0 h (Group A), 24 h (Group B), 48 h (Group C), and 72 h (Group D). Our analysis uncovered 56 DEPs between Groups B and A, 116 DEPs between Groups C and A, and a total of 152 DEPs between Groups D and A. These DEPs were categorized into nine clusters based on their expression patterns. Cluster 1 exhibited an increasing trend in protein expression, while Cluster 4 displayed the opposite pattern. Clusters 2, 6, and 9 showed an initial rise followed by a decline, whereas Cluster 3 demonstrated the reverse trend. Cluster 8 indicated an initial rise, a subsequent drop, and another rise, while Cluster 7 showed an initial decrease, followed by an increase and a minor decrease. Notably, the C-type lectin domain-containing (CTLD) protein and Failed axon connections (Fax) protein consistently displayed an upward trend. These two DEPs were selected for validation using the parallel reaction monitoring technique-targeted proteomics, confirming the trends observed in the initial analysis. In summary, this study highlights the potential of using proteins as reliable biomarkers for estimating pupal age.