Carbohydrate Metabolism Assay in Lasioderma serricorne

To explore the effects of LsAkt RNAi on carbohydrate metabolism, the glucose, glycogen, and trehalose content assay was performed using a previously reported method (Xu et al., 2020 (link)). The contents of glucose, glycogen, and trehalose in whole insect bodies were measured by the SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, United States) at 5 d after dsRNA treatment. Each sample contained 50 individuals, and three biological replications were prepared. The insect samples were homogenized in 0.25 M Na₂CO₃ and then incubated at 70°C for 10 min. By adding 0.2 M Na-acetate and 1 M acetic acid, the mixture was adjusted to pH 5.2. To measure the glycogen content, one half of the mixture was incubated with amyloglucosidase (Sigma-Aldrich). For trehalose measurement, the other half of the mixture was incubated with trehalase (Sigma-Aldrich) at 37°C, and the treated insects were homogenized in PBS (pH 7.4, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, and 2 mM KH₂PO₄₎. The glucose level was determined using a glucose (GO) assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. After LsAkt was knocked down in L. serricorne, the transcript levels of eleven carbohydrate metabolic genes (Supplementary Table 1) were detected by qPCR at 5 d after injection.