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 of 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 hemolymph. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hemolymph increased CP-H oxidation about two times for G. mellonella and about four times for D. superans sibiricus. This increase was completely inhibited by a specific inhibitor of phenoloxidase, phenylthiourea. The presence of exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph. The data obtained in hemocytes showed only a DOPA-induced increase in CP-H oxidation. Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxidation 20-30% in suspension of hemocytes of D. superans sibiricus only. Our results are in agreement with the contribution of superoxide radical and DOPA-derived quinones/semiquinones in the immune response of insects. Copyright 1999 Academic Press.