Effects of Parasitism on ROS Levels in Larval Gut

Reactive oxygen species play an important role in insect gut immunity (Bae et al., 2010), and hydrogen peroxide (H₂O₂) belonging to ROS is an important indicator of oxidative stress and immune responses (Bi and Felton, 1995). To examine if parasitism could affect the ROS level in larval guts, the ferrous oxidation-xylenol orange (FOX) assay was modified and used for comparative spectrometric analysis of H₂O₂ levels in the gut of parasitized and non-parasitized FAW larvae. H₂O₂ causes the oxidation of ferrous ion to ferric ion, and the complex formation of the reduced ion with xylenol orange produces a blue chromosphere which is detectable at 560 nm (Jiang et al., 1992; Nappi and Vass, 1998). Briefly, gut tissues were weighed and homogenized in 200 μl PBS (0.1 M, pH = 7.0). For reaction, 50 μl of homogenate was collected in a sterile tube containing 450 μl of a FOX working reagent that was composed of ammonium ferrous sulfate, H₂SO₄, reagent-grade methanol with 4 mM butylated hydroxytoluene (BHT), and xylenol orange in Milli-Q H₂O. After a 30-min incubation at room temperature, 200 μl was added to duplicate wells in a 96-well plate. The absorbance of each sample was read at 560 nm. Standard curves were conducted using commercial hydrogen peroxide.