Measurement of Hydrogen Peroxide Production.

The production of H₂O₂ was determined using the Hydrogen Peroxide Assay Kit (Beyotime Biotech) according to the manufacturer’s instructions. In this assay, H₂O₂ converts Fe²⁺ to Fe³⁺, which then complexes with xylenol orange dye to yield a purple product having an absorbance maximum at 560 nm, which could be detected by a spectrometer.

Mosquito midgut epithelia were dissected in ice-cold PBS and opened longitudinally in PBS to remove the gut contents. Briefly, 10 midguts were homogenized in 200 μL lysis buffer and centrifuged at 12,000 × g at 4 °C for 3 to 5 min, and the supernatant was collected. Aliquots of 50 μL of supernatants and 100 μL of test solutions from the Hydrogen Peroxide Assay Kit were incubated at room temperature for 20 min and measured immediately with a spectrometer at a wavelength of 560 nm. The concentration of H₂O₂ released was calculated according to a hydrogen peroxide standard curve. The measurement was repeated three times.