Application of the nitroblue tetrazolium-reduction method for studies on the production of reactive oxygen species in insect haemocytes.

The in vitro generation of reactive oxygen species (ROS) in haemocytes of Galleria mellonella, Aporia crataegi, Dendrolimus sibiricus, Aglais urticae (Lepidoptera) and Gryllus bimaculatus (Orthoptera), was studied by the method of nitroblue tetrazolium (NBT) reduction. Formazan formation (product of NBT reduction) was observed in haemocytes of all the insects examined, except A. urticae. Lypopolysaccharide and zymosan reduced the number of NBT-positive cells after 1 h incubation and an increase was registered after 4 h incubation. The inhibitors of the respiratory chain enzyme (sodium azide) and melanogenesis (phenylthiourea) reduced formazan formation in nonactivated insect blood cells. No influence of sodium azide and phenylthiourea was found on the activated haemocytes. The results suggest that the generation of ROS in insect haemocytes occured as a result of processes such as respiration and melanization during phagocytosis and encapsulation.