Ectopic <i>Wnt6</i> expression induces twin—spot markings in the epidermis of the silkworm <i>Bombyx mori</i> – Potential molecular target to discover evolutionary mechanisms of adaptive body coloration in insects

Animal body color patterning, as a naturally selected adaptive trait, plays critical ecological roles (e.g., warning coloration and mimicry). These patterns develop through spatiotemporally precise effector gene expression (e.g., pigment synthetases), regulated by pattern genes. However, the key pattern genes and the underlying mechanisms that govern the formation of marking patterns remain poorly defined. This study investigated the silkworm mutant, multilunar (L), which exhibited twin-spot markings on sequential segments. We found that Wnt6 mRNA expression was significantly upregulated in the twin-spot epidermal region. The cuticular protein RR-1 motif 4 (CPR4) and cuticular protein RR-1 motif 38 (CPR38) promoters were identified as core cis-acting elements for epidermal specificity throughout larval development. CPR4-driven Wnt6 overexpression induced twin-spot markings on the epidermis of silkworm larvae, resembling the mutant L pattern, while exhibiting growth advantages compared with the CPR38-driven system. Ultrastructural analysis indicated that there was a positive correlation between epidermal bulge structures and the formation of twin spots. Mechanistically, quantitative real-time PCR (qRT-PCR) analysis confirmed that epidermis-specific overexpression of Wnt6 did not induce co-activation of coclustered Wnt members. Furthermore, the analysis results of RNA-seq and qRT-PCR suggest that Wnt6 may promote the formation of the twin-spot patterns by participating in hormone regulation, pigment production, and cell proliferation signaling pathways. This study, for the first time, establishes a heritable Wnt6 ectopic-expression system that induces a twin-spot phenotype resembling that of the mutant L, thereby providing a potential molecular target for elucidating the evolutionary mechanisms of adaptive body coloration in insects.