Benzoxazinoids profiling in insects and plants

To assess benzoxazinoid sequestration by the insect larvae, we measured the content of benzoxazinoids in larval tissues of third instar WCR and BCB larvae fed on hybrid DFI 45321 maize seedlings, as well as WCR larvae fed either on the wild type B73 or on bx1 mutant maize. As previously reported, the sequestration occurs when the chemical compounds are transferred from the gut to other body tissues, such as muscles and haemolymph^15,16,18. Therefore, the benzoxazinoids were measured from larvae from which the gut was removed (n = 5). Eight to ten larvae were pooled per biological replicate. We also quantified the amount of benzoxazinoids in the maize seedlings of the genotypes that were used in the experiments (n = 5, five days old). Maize seedlings or larvae (guts removed) were flash frozen in liquid nitrogen and ground into a fine powder. To 100 mg of plant or 20 mg of larval tissue per sample, 1 ml or 400 µl of the extraction buffer (MeOH: H₂O: formic acid (FA); 50: 50: 0.5%) were added, respectively. Extracts were vortexed for 1 min and centrifuged at 20,000 g for 20 min at 4 °C. Supernatants were used for further analyses. Plant samples of the hybrid DFI 45321 and the wild type B73 were diluted 50 times, and samples of the the maize mutant bx1 were diluted 20 times. Larval extracts were directly used without dilution for the analyses. We measured the main benzoxazinoids that had previously been found in maize plants and in WCR larvae: 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-β-d-glucopyranose (HDMBOA-Glc), 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-β-d-glucopyranose (DIMBOA-Glc), 2‐β‐d‐glucopyranosyloxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (HMBOA-Glc), 6-methoxy-2-benzoxazolinone N-glucoside (MBOA-Glc) and 6-methoxy-2-benzoxazolinone (MBOA)^{21,28,73–75}. The quantification was performed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The equipment was composed of an Ultimate 3000 RSLC (Dionex, Thermo Fisher Scientific) interfaced to a 4000 QTRAP (AB Sciex) through an electrospray probe. Benzoxazinoids were separated on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters) in gradient mode using H₂O + formic acid 0.05% and acetonitrile + formic acid 0.05% as phases A and B, respectively. The flow rate was set at 0.4 ml/min and the column temperature at 30 °C. An injection of 2.5 μl was made. The gradient started at 2% B, linearly increased to 40% B in 3 min, then to 100% B in 2 min, and the column was finally washed at 100% B for 2 min and reconditioned at 2% B for 4 min. The mass spectrometer was operated in MRM mode using specific transitions for each benzoxazinoid compound. The instrument was run in negative ionization from 0.0–4.28 min for the detection of DIMBOA-Glc HMBOA-Glc and MBOA-Glc, and in positive ionization from 4.29–5.0 min for the detection of HDMBOA-Glc and MBOA. The gas temperature in the MS source (TEM) was set to 550 °C and the nebulizing (GS1), drying (GS2) and curtain (CUR) gas flows set to 55, 50 and 15 psi, respectively. Quantification was done by external calibration using standards purified from plants at 1, 5, 20, 100, 500 and 2000 ng/ml. The lowest limits of quantification on pure standards were 0.5–1.0 ng/ml for benzoxazinoid glucosides and 2.0 ng/ml for benzoxazinoid aglucones.