Descriptive analysis of protein expression variations during pupal development of Chrysomya megacephala (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 Chrysomya megacephala. 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.