Pathogen-targeted hydroxyl radical generation during melanization in insect hemolymph: EPR study of a probable cytotoxicity mechanism

The main component of the insect immune system is melanotic encapsulation of pathogenic organisms. Molecular mechanisms of destruction of an encapsulated pathogen are poorly understood. Reactive oxygen species (ROS) are considered as probable cytotoxic agents responsible for destruction of pathogenic organisms in insect hemolymph. In the present work the formation of H₂O₂ during melanization in Galleria mellonella hemolymph in the presence of catalase inhibitor NaN₃ was detected. Enhanced rates of H₂O₂ generation were observed in the hemolymph of insects activated by injection of bacterial cells. Using spin trapping technique in combination with electron paramagnetic resonance spectroscopy we demonstrated that production of H₂O₂ in the hemolymph causes the formation of highly toxic reactive oxygen species, hydroxyl radical. However, neither H₂O₂ nor hydroxyl radical were detected in the absence of NaN₃ in agreement with the high catalase activity in the hemolymph. These observations allow us to propose a unique mechanism of pathogen-targeted cytotoxicity based on localized hydroxyl radical generation within a melanotic capsule.