A CTL-Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

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 −20C 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).

本研究采集了从六龄期草地贪夜蛾幼虫体内抽取的100微升血淋巴样本，并在乳酸杆菌植物乳杆菌或磷酸盐缓冲盐溶液注射后24小时进行混合。该混合样本与400微升提取溶液（乙腈：甲醇=1:1）混合，其中含有0.02毫克/毫升的内标（L-2-氯苯丙氨酸）。完成30秒涡旋混合和30分钟的低温超声波提取后，样本在-20°C下保存30分钟以沉淀蛋白质，随后以13,000×g的转速离心15分钟。收集上清液并在氮气下吹干。随后，用100微升溶液（乙腈：水=1:1）重新溶解样本，并以13,000×g的转速离心。再次收集上清液用于LC-MS/MS分析。血淋巴样本中的代谢物提取物利用Thermo Fisher Scientific公司（美国马萨诸塞州）生产的UPLC-Q-Exactive质谱仪进行分析。色谱分离采用Waters公司（美国米尔福德）的ACQUITY HSS T3色谱柱。流动相由0.1%甲酸在水和乙腈中的溶液（95:5，溶剂A）以及0.1%甲酸在水和乙腈：异丙醇中的溶液（5:47.5:47.5，溶剂B）组成。