# Pharmacological Effects of NADPH Oxidase Inhibitors on Butterfly Wing Morphogenesis and Color Pattern Formation in Junonia orithya

## TL;DR

This study shows that NADPH oxidase inhibitors disrupt wing shape and color patterns in a butterfly species, suggesting a role for this enzyme in wing development.

## Contribution

The novel finding is that NADPH oxidase (NOX) and hydrogen peroxide are involved in butterfly wing morphogenesis and color pattern formation.

## Key findings

- NOX inhibitors caused severe deformities in wing morphology and color patterns when applied to pupal tissue.
- Systemic injection of VAS2870 increased eyespot size in male butterflies but decreased it in females.
- DMSO alone mildly enlarged eyespots without affecting other wing features.

## Abstract

Butterfly wing morphology is determined during the early pupal stage, when the peripheral portion of the pupal wing tissue undergoes apoptosis. We hypothesized that the development of wing morphology and color patterns may involve NADPH oxidase (NOX). To test this hypothesis, we treated pupae of the blue pansy butterfly Junonia orithya with NOX inhibitors. When VAS2870, isuzinaxib, or diphenyleneiodonium chloride (DPI) was topically applied to pupal wing tissue, wing morphology and color pattern elements were severely deformed. When systemically injected into pupae, VAS2870 increased eyespot size in males but decreased eyespot size in females, likely due to the sexual dimorphism of this species. These results suggest that NOX and probably hydrogen peroxide play important roles in wing morphogenesis and color pattern fate determination in butterfly wings.

During the early pupal stage in butterflies, the peripheral portion of wing tissue undergoes apoptosis to finalize adult wing morphology, and wing color patterns are determined coordinately. We hypothesized that the development of wing morphology and color patterns may involve NADPH oxidase (NOX). To test this hypothesis, we treated pupae of the blue pansy butterfly Junonia orithya with NOX inhibitors. When VAS2870, isuzinaxib, or diphenyleneiodonium chloride (DPI) in dimethyl sulfoxide (DMSO) was topically applied to the pupal wing tissue via the sandwich method, wing morphology and color pattern elements, including eyespots, parafocal elements, submarginal bands, and marginal bands, were severely deformed as if the marginal area were surgically removed. The topical application of DMSO alone mildly deformed and enlarged eyespots without affecting other color patterns and wing morphology. When systemically injected into pupae, VAS2870 increased eyespots in males but decreased eyespots in females, likely due to the sexual dimorphism of this species. These results suggest that NOX and probably hydrogen peroxide play important roles in wing morphogenesis and color pattern fate determination in butterfly wings. Sexually dimorphic eyespot size in this species may also be explained by the sexually differential activities of NOX.