In recent years, pea aphids have become major pests of alfalfa. Our previous study found that “Gannong 5” is a highly aphid-resistant alfalfa variety and that “Lie Renhe” is a susceptible one. The average field susceptibility index of “Gannong 5” was 31.31, and the average field susceptibility index of “Lie Renhe” was 80.34. The uptake and balance of amino acids in insects are usually dependent on amino acid transporters. RT-qPCR was used to detect the relative expression levels of seven amino acid transporter differential genes in the different instar pea aphids fed on resistant and susceptible alfalfa varieties after 24 h, and two key genes were selected. When pea aphids fed on “Gannong 5”, the expression of ACYPI004320 was significantly higher than that in pea aphids fed on “Lie Renhe”; however, the expression of ACYPI000536 was significantly lower than that in pea aphids fed on “Lie Renhe”. Afterward, the RNA interference with pea aphid ACYPI000536 and ACYPI004320 genes was performed using a plant-mediated method, and gene function was verified via liquid chromatography–mass spectrometry and pea aphid sensitivity to aphid-resistant and susceptible alfalfa varieties. The results showed that the down-regulation of the ACYPI000536 gene expression led to an increase in the histidine and lysine contents in pea aphids, which, in turn, led to an increase in mortality when pea aphids fed on the susceptible variety “Lie Renhe”. The down-regulation of the ACYPI004320 gene expression led to an increase in phenylalanine content in pea aphids, which, in turn, led to a decrease in mortality when pea aphids fed on the resistant variety “Gannong 5”.

The pea aphid (Acyrthosiphon pisum) is a destructive phloem-feeding pest of alfalfa. Its overreliance on chemical insecticides necessitates the development of sustainable, plant-based resistance strategies. Plant flavonoids are important in induced defense, but their dynamic responses to aphid feeding and subsequent insecticidal effects remain unclear. Using the highly resistant alfalfa variety ‘Gannong No. 5’ (GN5), this study integrated behavioral assays, performance bioassays, untargeted and targeted metabolomics, and exogenous flavonoid feeding assays to investigate how pea aphid infestation alters flavonoid metabolism and thereby affects aphid performance. Bioassays confirmed strong antibiosis of GN5 against A. pisum. Behavioral choice assays showed that, at 2 h post-release, aphids significantly avoided plants pre-infested for 12 h and 48 h, but not those pre-infested for 24 h; by 8 h post-release, they significantly avoided all pre-infested plants regardless of infestation duration. Prolonged pre-infestation (48 h) also reduced average fecundity per female. Non-targeted metabolomics revealed substantial metabolic reprogramming after 48 h of aphid feeding, with most flavonoids and isoflavonoids significantly upregulated. Targeted metabolomics identified 28 flavonoids, among which only sakuranetin and chrysin were significantly upregulated after 48 h, indicating their specific induction. Finally, performance bioassays confirmed insecticidal effects in a concentration-dependent manner: sakuranetin at 0.1 μg/μL reduced reproduction, and at 10.0 μg/μL reduced survival; chrysin at 0.1 μg/μL reduced both survival and reproduction. Collectively, these results demonstrate that pea aphid feeding triggers the induction of specific defensive flavonoids in GN5, which may contribute to antibiosis. This study provides a theoretical basis for exploring flavonoid-based approaches in sustainable aphid management.