A Simple Method for Estimating the Spatiotemporal Distribution of Phenoloxidase Proteins in Insect Tissues.

Laccase2 (Lac2), a member of the phenoloxidase (PO) family, is an essential oxidase for melanin pigmentation in insects. The identification of the in vivo spatial distribution of Lac2 is crucial for understanding the molecular mechanisms underlying color pattern formation. However, it is technically difficult to determine the distribution because Lac2 expression peaks at late pupal stages, when adult cuticle sclerotization has already begun. Here, we report a simple and rapid protocol for estimating the distribution of endogenous PO proteins, prophenoloxidases (proPOs) and phenoloxidases (POs), in insect tissues. In this method, the spatial distribution of endogenous PO proteins is estimated based on staining patterns formed by dopamine melanin synthesis in tissues incubated in a solution containing isopropanol and dopamine. We validated that tissues collected at approximately 80% of the total pupal duration yielded staining patterns corresponding to adult melanin-forming regions in three insect species. By comparing staining patterns across developmental stages, this protocol enables estimation of the timing of color pattern formation. Furthermore, the contrast between stained and unstained regions within the same tissue allows region-specific sampling, thereby facilitating an investigation of the underlying molecular mechanisms regulating spatial PO distribution. Taken together, this method facilitates the study of melanin biosynthesis and enables the identification of the genes involved in regulating color pattern formation. This protocol does not require antibodies, transgenic lines, or specialized equipment and can be completed within a short time frame. Its effectiveness has been validated in multiple coleopteran and lepidopteran species, demonstrating its broad applicability as a versatile tool for studying insect pigmentation and color pattern formation. Key features • A simple tissue staining protocol to estimate the spatial distribution of endogenous PO proteins without the use of antibodies or transgenic lines. • Comparing staining patterns across different developmental stages to estimate both the spatial distribution of PO proteins within tissues and the timing of color pattern formation. • RNAs can be extracted from the tissue after staining, enabling gene expression analyses between stained and unstained regions. • This protocol has been validated in two coleopteran species and one lepidopteran species, demonstrating broad applicability across diverse insect taxa.