Identification of circular RNAs as biomarkers for pupal age estimation and postmortem interval in forensically important Sarcophaga peregrina (Diptera: Sarcophagidae).

Accurate estimation of the pupal age in necrophagous flies is vital for determining the postmortem interval (PMI) in forensic entomology. Differential expression of genes (DEGs) exhibits temporal fluctuations across the developmental stages of these flies. This study evaluates circular RNAs (circRNAs) as novel molecular markers and develops a model for predicting pupal development time based on circRNA expression. Transcriptomic analysis of Sarcophaga peregrina pupae at various stages identified four circRNAs (circRNA_0037, circRNA_0531, circRNA_3373, circRNA_2847) showing significant expression differences. Using real-time quantitative PCR and regression analysis, we constructed a model to estimate development time, which accurately predicts intra-puparial periods. Additionally, we examined circRNA degradation patterns in pupae under lethal conditions and identified a clear degradation trend in circRNA_2847, suggesting its potential use for estimating the PMI. This study introduces new molecular markers and insights for estimating necrophagous fly pupal age.