Hydrogen Peroxide Regulates Butterfly Wing Eyespot Color Patterns

Butterfly wing color patterns are determined during the early pupal stage. It has been proposed that the interactions between wing epidermal cells and the facing inner cuticle surface may initiate signal transduction pathways that determine cell fate regarding presumptive scale colors. Here, we hypothesized that hydrogen peroxide plays a key role in color pattern determination. To examine this hypothesis, we pharmacologically treated fresh pupae of the Blue Pansy butterfly Junonia orithya using two application methods to experimentally manipulate the levels of hydrogen peroxide in pupal wing tissues. When systemically injected into pupae, hydrogen peroxide reduced eyespot size, and two scavengers of hydrogen peroxide, catalase and dimethylthiourea (DMTU), increased eyespot size. When topically applied to pupal wing tissues, hydrogen peroxide induced severe color pattern modifications, catalase increased eyespot size, and a cuticle inhibitor, fluorescent brightener 28 (FB28), reduced eyespot size. Mechanical load tests revealed that the level of hydrogen peroxide influences the cuticle hardness of the pupal body. These results suggest that hydrogen peroxide regulates eyespot color patterns in butterfly wings and that this regulation may be mediated by modulating the chemical and mechanical properties of the facing cuticle during the early pupal stage.