A rapid and simple micro-assay to assess catalase activity in individual mosquito tissues.

Abstract

Oxidative stress generated as a normal byproduct of aerobic metabolism is minimized by the enzyme catalase (CAT; EC 1.11.1.6), which reduces hydrogen peroxide to molecular oxygen and water. In various mosquitoes, hydrogen peroxide and/or CAT activity have been implicated in oxidative responses to viral and protozoal pathogens as well as in ovarian maturation and insecticide resistance. We combined features of various CAT assays to develop a simple micro-assay that enables comparison of enzyme activities in individual mosquito tissues on a microscope slide. Activity recovered in the supernatant of mosquito whole body homogenates was inhibited by the CAT-specific inhibitor 3-amino-1,2,4-triazole. Activity was higher in blood-fed mosquitoes, consistent with exogenous enzyme in vertebrate blood. Triton X-100 improved evaluation of dissected organs, and accurate comparisons required careful removal of extraneous tissues. In unfed mosquitoes baseline CAT activity was lower in ovaries than in midgut or fatbody, but increased as oocytes matured after a blood meal, and was detectable in a single mature egg. CAT has unusual kinetics and can be difficult to assay directly. Our observations provide a simple approach for direct evaluation of CAT activity independent of changes in transcript levels and results of RNAi-based interference.