Integrated Morphological and Transcriptomic Analyses of Gene Regulatory Mechanisms in Different Intra-Puparial Developmental Stages of Phormia regina

Phormia regina (Meigen, 1826) (Diptera: Calliphoridae), a necrophagous blow fly of significant forensic value, plays a crucial role in postmortem interval (PMI) estimation. The intra-puparial period of this species constitutes approximately half of its entire immature developmental duration. Therefore, precise aging of intra-puparial specimens is essential to improve the accuracy of PMI estimation. This study was performed at a constant 25 °C, examining intra-puparial morphology, histological sections, and body weight across different developmental stages. Additionally, full-length transcriptome profiling was conducted via a hybrid sequencing strategy combining PacBio single-molecule real-time (SMRT) sequencing and Illumina short-read next-generation sequencing (NGS). Morphological studies revealed the intra-puparial morphological characteristics, the reconstruction process of tissues and organs, and the continuous body weight loss during the intra-puparial period. Transcriptomic research identified a total of 425,349 full-length non-chimeric (FLNC) sequences. After redundancy removal, 84,852 transcript sequences were obtained, of which 46,325 transcripts were annotated. Using day-0 puparia (D0) as the control, differentially expressed gene (DEG) analysis was performed on samples from subsequent developmental stages (D1–D5), identifying 4242, 7964, 9509, 10,526, and 10,011 DEGs, respectively. Results from GO enrichment and KEGG pathway analyses provided reasonable explanations for the behavioral traits of different developmental stages. Eight candidate genes for intra-puparial development were screened, most of which were highly expressed at different time points during the intra-puparial period. Quantitative real-time PCR (qRT-PCR) results showed consistent gene expression trends with the RNA-seq data, confirming the reliability of the RNA-seq results. This study provides key morphological and molecular markers for P. regina in forensic PMI estimation.