Morphogen-driven melanin pathway dynamics regulated by Wnt1 and apontic-like underlie larval spot coloration in Bombyx mori

In insects, conspicuous larval pigmentation patterns serve critical ecological roles such as warning signals and mimicry, yet their underlying genetic regulation remains poorly understood. In this study, I investigated the molecular mechanisms underlying black and yellow pigmentation patterns in three distinct larval spot types of the silkworm Bombyx mori: large, diffuse L-spots of the Multilunar (L) mutant; small, sharply defined +p-spots of the Normal strain; oval pM-hybrid spots of an interspecific hybrid with Bombyx mandarina. Each spot type comprises a yellowish center surrounded by a black periphery, forming crescent-shaped pigmentation patterns. Chemical treatments confirmed that both colors are melanin-based. Using quantitative PCR and RNA interference (RNAi), I analyzed six melanin synthesis genes (Tyrosine Hydroxylase, Dopa Decarboxylase, laccase2, yellow, tan, and ebony) and discovered that black pigmentation involves both dopa/dopamine- and NBAD-melanin synthesis, whereas yellow pigmentation primarily reflects only the latter. I further examined Wnt1 and apontic-like (apt-like) using qPCR, RNAi, and TALEN-mediated mosaic analysis. Wnt1 expression localized to presumptive spot areas, functioning dose-dependently to regulate both spot size and pigment composition: high Wnt1 levels induced larger spots with yellow centers, while reduced Wnt1 expression resulted in black pigmentation and smaller spots. Wnt1-activated transcription factor apt-like was required for pigmentation in all spot types without influencing spot size. Taken together, the results of this study reveal a morphogen-driven gene regulatory network in which Wnt1 dosage and downstream transcriptional cascades orchestrate pigment placement and patterning, offering new insights into the modular genetic control of insect pigmentation.