Metabolomic Analysis of Aphid Feeding

50 adult aphids were feeding on plant for 0 and 48 h, five biological replicates were used, and a previously established method was employed (Qi et al., 2016 (link)). In brief, samples were extracted with 80% methanol and then analyzed on an Agilent 1200 Rapid Resolution Liquid Chromatography (RRLC) system equipped with a ZORBAX SB-Aq column (2.1 × 100 mm, 1.8 μm) followed by an Agilent 6510 Q-TOF running in positive ionization mode. The column temperature was set at 50°C, and the flow rate was 0.3 mL min⁻¹. All metabolites were identified based on pure standards, except some Bxs: DIMBOA was confirmed by comparing the Bx profiles between maize inbred line H88 and bx1 mutant (GRMZM2G085381); DIMBOA-Glc, DIM₂BOA-Glc, HDMBOA-Glc, and MBOA were confirmed with purified standards, and the other putative Bxs were identified by their molecular masses. The abundance of each metabolite was estimated from the peak area. Peak detection and matching were performed using bioconductor XCMS and CAMERA packages (http://www.bioconductor.org/.) Samples (metabolites after integration) with P < 0.05 were considered to be differentially regulated. The acquired data were introduced to the SIMCA-P software package (v11.5, Umetric, Umea, Sweden) for Principal Component Analysis (PCA). The data was filtered by orthogonal signal correction (OSC) to remove variations from non-correlated factors.