Genome-wide nascent transcription profiling uncovers gene regulatory elements and dynamic immune responses in the major arbovirus vector Aedes aegypti

Aedes aegypti mosquitoes are the principal vectors for epidemic arboviruses, including dengue, Zika, and chikungunya viruses. In these insect vectors, the concerted action of immune pathways shapes virus replication and transmission; yet, mechanistic and dynamic understanding of the transcriptional immune responses in mosquitoes remain limited. To address this knowledge gap, we profiled nascent transcription in mosquito cells and midgut tissue using Precision Run-On sequencing (PRO-seq). We identified exact transcription start nucleotides (TSNs), characterized promoter architectures, and generated the first genome-wide list of putative enhancers in Aedes aegypti, substantially expanding the regulatory annotation of this non-model organism. To investigate dynamic immune responses, we stimulated Aag2 cells with heat-inactivated E. coli and measured RNA synthesis and mRNA levels from matched samples. Bacterial stimulation induced rapid and temporally organized transcriptional responses associated with distinct biological functions and transcription factor motifs. Integration with mRNA-seq revealed that temporal patterns of mRNA accumulation were shaped by RNA synthesis, gene length, and stability, indicating multilayered regulation of mosquito immune responses. Implementing PRO-seq for mosquito midgut enabled characterization of tissue-specific transcription and TSN usage. Importantly, we demonstrate for the first time that PRO-seq captures in vivo microbial transcription within an organism, co-profiling host and microbiome nascent RNA synthesis. Altogether, this study substantially expands the regulatory annotation and functional genomic landscape of Aedes aegypti, reveals multilayered regulation of mosquito immune responses, and extends transcriptional profiling to host and microbiome activity in vivo. These insights advance understanding of gene regulation and immunity in this important arbovirus vector.