The Molecular Properties and Roles of Pannier in Harmonia axyridis’s Metamorphosis and Melanin Synthesis

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.