A Comprehensive Larval microRNA Atlas of Hyphantria cunea Identifies Candidate miRNAs and Potential Molecular Targets for Green Pest Management

Hyphantria cunea (Drury) causes extensive ecological damage primarily during its larval stages, characterized by voracious feeding and rapid dispersal. Given that conventional dsRNA-mediated RNA interference (RNAi) is generally recalcitrant in Lepidoptera, endogenous microRNAs (miRNAs) may represent an additional class of regulatory molecules worthy of systematic investigation. In this study, we utilized high-throughput sequencing to construct nine comprehensive miRNA libraries across three critical developmental milestones (three biological replicates per instar): the 1st, 4th, and 7th instars (L1, L4, and L7). A total of 1667 miRNA entries were catalogued, including 1080 known and 587 bioinformatically predicted, as yet unvalidated novel miRNA candidates. Comparative transcriptomic analysis revealed 52 differentially expressed miRNAs with significant stage-dependent profiles, with the most pronounced divergence observed between the L1 and L7 groups. Bioinformatic prediction identified 16,784 non-redundant target genes. GO and KEGG enrichment analyses indicated that the predicted target genes of these differentially expressed miRNAs were enriched in developmental and metabolic categories, including cellular development, protein digestion, and nutrient absorption, suggesting that these miRNAs may be associated with tissue remodeling and larval developmental transitions. Collectively, our findings expand the currently available miRNA resource for H. cunea and define stage-associated miRNA expression patterns during larval development. Rather than establishing direct functional roles, this work provides a framework and candidate molecules for future design of RNAi-based biopesticides.