Melanization is a common phenomenon in insects, and melanin synthesis is a conserved physiological process that occurs in epidermal cells. Moreover, a comprehensive understanding of the mechanisms of melanin synthesis influencing insect pigmentation are well-suited for investigating phenotype variation. The Asian multi-colored (Harlequin) ladybird beetle, Harmonia axyridis, exhibits intraspecific polymorphism based on relative levels of melanization. However, the specific characteristics of melanin synthesis in H. axyridis remains elusive. In this study, we performed gene-silencing analysis of the pivotal inverting enzyme, tyrosine hydroxylase (TH), and DOPA decarboxylase (DDC) in the tyrosine metabolism pathway to investigate the molecular and regulatory mechanism of melanin synthesis in H. axyridis. Using RNAi of TH and DDC genes in fourth instar larvae, we demonstrated that dopamine melanin was the primary contributor to the overall body melanization of H. axyridis. Furthermore, our study provides the first conclusive evidence that dopamine serves as a melanin precursor for synthesis in the early pupal stage. According to transcription factor Pannier, which is essential for the formation of melanic color on the elytra in H. axyridis, we further demonstrated that suppression of HaPnr can significantly decrease expression levels of HaTH and HaDDC. These results in their entirety lead to the conclusion that transcription factor Pannier can regulate dopamine melanin synthesis in the dorsal elytral epidermis of H. axyridis.that different phenotypic individuals exhibited varying behavioral traits. Thus, elucidating the molecular mechanism of melanin synthesis in H. axyridis would provide crucial information on the sole ontogenies and evolutionary histories of the attendant melanization.Melanin is the final product in the melanization process and has a prominent role in wound healing, cuticle sclerotization, innate immunity, defensive reactions, cuticular coloration, and camouflage in insects [16][17][18]. Melanin synthesis is a conserved physiological process in insects that occurs in epidermal cells [19,20], and in the melanin synthesis pathway, the precursor DOPA (3, 4-dihydroxyphenylalanine) and dopamine are synthesized from tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) enzymes, respectively [21]. Tyrosine hydroxylase and DOPA decarboxylase enzymes are abound in central nervous system and epidermal cells of insects. Dopamine is an important catecholamine neurotransmitter in invertebrates and vertebrates [18]. In insects, dopamine is the major contributor for melanin biosynthesis, while in mammals DOPA is dominated [17,18]. The biochemistry of tyrosine metabolism is abounding in insects because tyrosine is the initiatory material of melanin formation. Tyrosine hydroxylase is the first key enzyme in this pathway that can convert tyrosine to DOPA. Then, DOPA decarboxylase catalyzes DOPA to dopamine. DOPA and dopamine are crucial precursors of sclerotization and melanization in insects. There was research re...

The GATA transcription factor Pannier is identified as the major regulatory gene in color pattern formation in the Asian multi-colored ladybird beetle (Harmonia axyridis). however, the mechanisms of Pannier in regulating melanin synthesis and development in H. axyridis remain elusive. In this study, we identified and characterized Pannier in H. axyridis (HaPnr) and showed it to have two alternative spliced variants named HaPnr-α and HaPnr-β. Analyses of developmental stage expression patterns revealed that HaPnr, HaPnr-α and HaPnr-β were constitutively expressed throughout all developmental stages. To examine the role of HaPnr in H. axyridis development, RNA interference was performed in late larvae (the fourth instar) and early pupae (the first day of pupa stage). The transcript levels of HaPnr were effectively suppressed after the injection of double-stranded RNA of HaPnr (dsHaPnr). The fourth instar larvae injected with dsHaPnr reduced the pupation rates to only 61.50%, compared with 88.5% in the dsGFP-injected group. The un-pupated larvae gradually died after 1 week, and visually unaffected pupae emerged into abnormal adults with malformed hind wings and melanin absent from the cuticle. These abnormal adults gradually died 10 days after eclosion. However, when early pupae were injected with dsHaPnr, the normal eclosion rate was achieved at 88.41% on day 6 after the injection. In addition, these successful eclosion adults also showed an absence of melanin in the cuticle, but they could mate normally and have normal fecundity as compared with the control. We further demonstrated that the suppression of HaPnr-α or HaPnr-β individually did not affect the pupation and eclosion process. The suppression of HaPnr-α expression resulted in elytra melanin decreasing in both the conspicua and the succinea subgroup in H. axyridis. Even though the suppression of HaPnr-β expression only affected the melanin synthesis in the succinea subgroup, it significantly prolonged the time taken for melanin synthesis to occur in the conspicua subgroup in H. axyridis. These results indicate that HaPnr plays an essential role in insect development, especially during their metamorphosis, and also support our hypothesis that HaPnr could regulate melanin synthesis in H. axyridis under the combined action with its two splicing variants, HaPnr-α and HaPnr-β.