Hydrogen peroxide production in immune-reactive Drosophila melanogaster

Upon infection with the wasp parasitoid Leptopilina boulardi, the blood cells or hemocytes of Drosophila melanogaster larvae become activated and manifest a type of communal phagocytosis wherein eggs of the parasitoid are enveloped by multicellular, melanotic capsules. Hemocytes engaged in this collaborative response generate reactive oxygen intermediates (ROI). These molecules, together with melanogenic intermediates, are believed to destroy intrahemocoelic parasites. Cellular uptake of 2',7'-dichlorofluorescin diacetate (DCF-DA) and the oxidation of its deacetylated form (DCF) to yield the fluorescent product dichlorofluorescein (DC) was used as an intracellular probe for oxidant generation. The selective uptake of the fluorescent probe only by activated plasmatocytes from immune-reactive larvae identified these hemocytes as the source of ROI. Inhibition of DCF oxidation by catalase established hydrogen peroxide (H2O2) as 1 of the principal oxidants generated during melanotic encapsulation. A sensitive spectrometric assay for assessing iron oxidation and complex formation with xylenol orange (FOX assay) also was used to document in vitro-enhanced H2O2-mediated oxidations by hemolymph from immune-competent larvae. Cumulative evidence now establishes both superoxide anion (O2-*) and its dismutation product H2O2 in the cellular encapsulation response of D. melanogaster.