Joint mRNA–miRNA Analysis Reveals Immune Regulatory Networks in Drosophila During Beauveria bassiana Infection

Understanding how insects coordinate immune responses with metabolic demands is key to elucidating host–pathogen interactions. Here, we performed integrated mRNA and miRNA transcriptomic profiling in Drosophila melanogaster following infection with the entomopathogenic fungal Beauveria bassiana at 12 and 24 h post‐infection (hpi). Differential expression and time‐series clustering revealed dynamic transcriptional reprogramming, including early immune activation, sustained oxidative stress responses, and delayed metabolic compensation. To investigate post‐transcriptional regulation, we constructed high‐confidence miRNA–mRNA networks by intersecting differentially expressed miRNAs and mRNAs with dual‐target predictions from miRanda and RNAhybrid, retaining only canonical repression pairs (upregulated miRNAs with downregulated targets and vice versa). At 12 hpi, the network comprised 91 interactions involving 18 miRNAs and 72 mRNAs. Key regulators such as dme‐miR‐34‐5p and dme‐miR‐285‐5p targeted genes in immune and metabolic pathways, including Toll/IMD signaling, glutathione metabolism, and lysosome function. By 24 hpi, the network contracted to 23 interactions (12 miRNAs and 22 mRNAs), with regulatory focus shifting toward amino acid metabolism, ER stress response, and redox homeostasis. Distinct sets of upregulated and downregulated miRNAs mediated temporally resolved targeting of biosynthetic and immune‐related genes. These results reveal a stage‐specific, miRNA‐mediated regulatory architecture that balances immune activation with metabolic adaptation. Our work presents a time‐resolved dual‐omics map of the Drosophila immune response to fungal infection, offering new insights into conserved regulatory mechanisms and host–pathogen interactions.