EPR detection of reactive oxygen species in hemolymph of Galleria mellonella and Dendrolimus superans sibiricus (Lepidoptera) larvae

The formation of reactive oxygen species (ROS) in hemolymph and hemocytes o f Galleria mellonella and Dendrolimus superans sibiricus larvae was studied by ESR spectroscopy using spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) . The background level of ROS formation was detected in the intact hemolymp h. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hem olymph increased CP-H oxidation about two times for G. mellonella and about four times for D, superans sibiricus. This increase was completely inhibit ed by a specific inhibitor of phenoloxidase, phenylthiourea. The presence o f exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph, The data obtained in hemocytes showed only a DOPA-induced incre ase in CP-H oxidation, Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxida tion 20-30% in suspension of hemocytes of D. superans sibiricus only. Our r esults are in agreement with the contribution of superoxide radical and DOP A-derived quinones/semiquinones in the immune response of insects. (C) 1999 Academic Press.