2.6. Lipid Peroxidation (MDA) Analysis

Lipid peroxidation was measured using the malondialdehyde (MDA) assay kit (Sigma-Aldrich) by reacting MDA with thiobarbituric acid substance (TBARS). Approximately 25–30 mg of larval sample was homogenized on 20% trichloroacetic acid (w/v) (1.5 mL) with 0.05 mL of 1% BHT in methanol. To the primary solution, 2.95 mL of 50 mM thiobarbituric acid was added, then mixed and heated for 10 min at 100 °C. The protein precipitates were extracted by centrifugation at 2000× g, and the absorbance was measured using the Evolution 300 spectrophotometer (Thermo Scientific, Loughborough, UK) at 532 nm. The MDA standard curve was plotted, and the absorbance of samples was compared against the standard. TBA-MDA concentration was expressed as nmol MDA/mg of tissue [49].