Interactions of antioxidants and phenoloxidase reactions in Manduca sexta hemolymph

Abstract

Melanization is an important component of the insect innate immune response. Melanization occurs when an insect is exposed to pathogens such as bacteria or fungi. Phenoloxidase (PO) is a key enzyme in the synthesis of melanin. Recognition of bacterial peptidoglycan initiates a cascade of serine proteases, which activates PO. PO then hydroxylates tyrosine and oxidizes DOPA and dopamine, derived from tyrosine, to ortho-quinones. These participate in redox reactions that finally result in synthesizing melanin. Ortho-quinone formation and the redox reactions produce reactive oxygen species (ROS), which may help in eliminating pathogens but may also be harmful to the host insect. Insects may regulate oxidative damage to themselves. In this study, PO in hemolymph was activated similarly when plasma was exposed to either Lys type or DAP type bacterial peptidoglycans. However, lower amounts of Lys type peptidoglycans were able to stimulate PO more when compared to DAP type peptidoglycans. Colorimetric measurements revealed that M. sexta plasma contains a high concentration of ascorbate that significantly decreases when plasma PO is stimulated with peptidoglycan. Unexpectedly, PO activation also resulted in an increase in the total antioxidant capacity in plasma, which may be caused by the increased concentrations of DOPA produced during the melanization response, since DOPA can act as a reducing agent. Mass spectrometry, performed on M. sexta whole hemolymph and plasma, was used to reproducibly determine concentrations of total thiols, total reduced thiols, reduced glutathione, oxidized glutathione, cysteine, cystine, and glutathione-ss-cysteine, revealing that antioxidants are predominantly located in plasma and not hemocytes. Mass spectrometry was used to determine changes in concentrations of the following antioxidants and metabolites in plasma after stimulation of PO activation with peptidoglycan: total reduced thiols, total oxidized thiols, percent oxidized thiols, total glutathione, total cysteine, total homocysteine, total cysteine-glutathione, free glutathione, oxidized glutathione (GSSG), free cysteine, cystine, glutathione-ss-cysteine, ascorbate, dehydroascorbate, tyrosine, DOPA, dopamine, and tyrosine glucoside. Stimulated plasma had a significant decrease in concentrations of glutathione, cysteine, ascorbate, and tyrosine. Concentrations of DOPA significantly increased in stimulated plasma samples while the concentration of dopamine did not change, demonstrating the hydroxylation of tyrosine and suggesting that DOPA is the predominant diphenol used during melanization. These results show that the redox environment in M. sexta plasma is highly reducing and suggests that shifts in redox potentials and regulation of the redox environment may not only regulate PO activity but may also prevent oxidative damage to the host.