Generation of Superoxide Radical and Hydrogen Peroxide in Insect Hemolymph in the Course of Immune Response

Prophenoloxidase system, which is represented by awhole cascade of enzymatic reactions, one of the finalproducts of which is melanin, plays a major role ininsect immunity [1]. Reactive intermediates producedduring melanogenesis may participate in cytotoxicreactions in the course of granule formation and incap-sulation of a foreign agent in an insect organism. Thisgroup primarily involves o-semiquinones formed dur-ing enzymatic oxidation of o-hydroquinones (in partic-ular, 3,4-dihydroxyphenylalanine, DOPA) [1, 2]. Suchcompounds can interact with oxygen to form superox-ide anion (), which, after a number of transforma-tions (dismutation and Fenton reaction), is eventuallyconverted to other reactive particles, such as hydrogenperoxide and hydroxyl radical [1]. Earlier, we appliedEPR to demonstrate the formation of DOPA o-semi-quinones in the hemolymph of Galleria mellonella lar-vae during melanogenesis [3, 4]. Studies of the genera-tion of superoxide and hydrogen peroxide in insecthemolymph are represented by a few papers. It shouldbe noted that the formation of has not yet beenrecorded directly. Some authors tested generation of in insect hemolymph using the method based onreduction of nitro blue tetrazolium [1, 5, 6]. However,the nonspecificity of this method [7] does not allow thedemonstration of formation in insects' organism tobe regarded as reliable data. Today, the most informa-tive method of recording short-lived oxygen radicals inbiological systems is the spin trap method in combina-tion with EPR spectroscopy. The goal of this study wasO