Integrated transcriptomic and RNAi approaches uncover a key role for ApApoD1 in the desiccation tolerance of Acyrthosiphon pisum

Insects rely on cuticular hydrocarbons (CHCs) to prevent desiccation. CHCs are synthesized in oenocytes and transported through the hemolymph to the epicuticle by lipid carriers (e.g., lipophorin); as a lipid-binding lipocalin, apolipoprotein D (ApoD) can shuttle small hydrophobic ligands, suggesting a mechanistic link to CHC trafficking and deposition. We characterized the gene ApApoD1, encoding a lipocalin-family apolipoprotein D, in the pea aphid (Acyrthosiphon pisum) and examined its role in drought tolerance. ApApoD1 is highly conserved among aphids, predominantly expressed in cuticle tissues, and its expression is upregulated in response to desiccation stress. Using an RNA interference (RNAi) approach delivered via a star polycation (SPc) nanocarrier, we achieved 48% silencing of ApApoD1 transcripts. Silencing ApApoD1 resulted in a 48.9% reduction in total CHC content (particularly C₂₇-C₃₁ alkanes), accompanied by a 1.8-fold increase in water loss rate. Consequently, ApApoD1-silenced aphids showed significantly reduced survival time under extreme desiccation (5% relative humidity [RH]). Transcriptome analysis (RNA-seq) identified 765 differentially expressed genes (DEGs) due to ApApoD1 silencing, including downregulation of lipid metabolic and cuticle structural genes and upregulation of immune pathway genes (Toll and immune deficiency [Imd]). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of these DEGs highlighted significant involvement of lipid metabolism, cuticle biosynthesis, and immune signaling pathways. Our findings demonstrate that ApApoD1 is a critical regulator of cuticular lipid accumulation and desiccation tolerance in aphids, and suggest that disrupting this gene via RNAi could increase pest susceptibility to drought stress, providing a potential strategy for aphid management.