A CTL-Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections
Description
Hemolymph samples (100 L) from Helicoverpa armigera sixth-instar larvae at 24 h post-Lactobacillus plantarum or -PBS injection were mixed with 400 L extraction solution (acetonitrile:methanol = 1:1) containing 0.02 mg/mL internal standard (L-2-chlorophenylalanine). After vortexing for 30 s and low-temperature ultrasonic extraction for 30 min was complete, the samples were kept at −20C for 30 min to precipitate the proteins and centrifuged for 15 min at 13,000 × g. The supernatants were collected and blow-dried under nitrogen. The samples were resolubilized with 100 L solution (acetonitrile:water = 1:1) and centrifuged at 13,000 × g. The supernatants were obtained for LC-MS/MS analysis. The metabolite extracts from hemolymph samples were analyzed using a UPLC-Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). Chromatographic separation was conducted using an ACQUITY HSS T3 column (Waters, Milford, USA). The mobile phases consisted of 0.1% formic acid in water:acetonitrile (95:5; solvent A) and 0.1% formic acid in water:acetonitrile:isopropanol (5:47.5:47.5; solvent B).