Dual defense strategies in alfalfa: A multi-omics investigation into differential activation of defense pathways against adult and larval weevil feeding

The alfalfa weevil ( Hypera postica ) is a significant pest in alfalfa cultivation, with both adults and larvae causing significant damage by feeding on stems, leaves and leaf mesophyll, respectively. This study utilized a multi-omics approach to systematically investigate the differential defense mechanisms activated in alfalfa in response to weevil infestation. The findings revealed that larval feeding primarily activates the jasmonic acid (JA) signaling pathway which upregulates the LOX-AOS-AOC gene cluster to facilitate the synthesis of direct defense compounds. In contrast, adult feeding preferentially stimulate the phenylpropanoid pathway leading to the emission of specific volatile. For the first time, we revealed the formation of a synergistic defense barrier involving flavonoid and lignin biosynthesis, with larval-induced γ-aminobutyric acid (GABA) metabolism and adult-specific glutamate signaling contributing to distinct defense responses. Volatile analysis showed an 8.9-fold increase in phenylethanol (adult-induced) and a 2.57-fold increase in green leaf volatiles (larval-induced). These findings provide a comprehensive understanding of the stage-specific defense strategy employed by alfalfa, mediated through precise regulation of JA signaling, secondary metabolism, and volatile synthesis. This study provides a theoretical basis for breeding insect-resistant alfalfa varieties and developing environmentally sustainable pest control strategies. • This study pioneers multi-omics discovery of differential alfalfa defenses: larval infestation activates JA pathway, while adults trigger phenylpropanoid volatiles. • A flavonoid-lignin synergy orchestrates defense: MAPK barriers against adults and phosphatase-driven chemistry against larvae in perennial legumes. • Stage-specific markers (GABA/glutamate) and volatiles (phenylethanol 8.9×) enable precise breeding and eco-friendly pest control, paving the way for field applications.