Dissecting roles of <i>pannier</i> splice variants during pupal and adult morphogenesis in <i>Henosepilachna vigintioctopunctata</i>

The GATA transcription factor gene, pannier (pnr), has been extensively studied in Drosophila, revealing its crucial role in dorsal closure, heart development and the regulation of cuticular bristle patterns in adults. However, studies on the functions of pnr in the development of coleopteran insects are still scarce. Herein, we identified the pnr gene in Henosepilachna vigintioctopunctata and discovered two splicing variants named Hvpnr-α and Hvpnr-β respectively. Temporal expression analysis revealed that Hvpnr, Hvpnr-α and Hvpnr-β were expressed at various stages including egg, larval, pupal and adult stages. To investigate the developmental role of Hvpnr in H. vigintioctopunctata, RNA interference (RNAi) assays were conducted on third-instar larvae. Injection of dsHvpnr, dsHvpnr-α and dsHvpnr-β and co-injection of dsHvpnr-α and dsHvpnr-β (dsRNAs mix) all resulted in significant downregulation of the target transcripts. In pupae developed from dsHvpnr-treated larvae, the symmetric black spots on both sides of the mesothorax, metathorax and tergites approached and connected. Pupal morphometric analysis revealed that dsHvpnr, dsHvpnr-α and dsRNAs mix injections significantly narrowed the spacing of dorsal symmetric spots, contracted spiracle distances on tergite sides, diminished pronotum width and markedly reduced inter-compound eye spacing compared to controls. In addition, injections of dsHvpnr and dsRNAs mix significantly reduced the oviposition in female adults. Silencing of Hvpnr led to the disappearance of the scutellum in adults, preventing the elytra from closing and properly attaching to the dorsal side of the abdomen. It is noteworthy that dsHvpnr-α or dsRNA mix induced scutellum formation defects in adults, while knockdown of Hvpnr-β had no impact. Furthermore, in stark contrast to previous studies on ladybird species such as Harmonia axyridis and Coccinella septempunctata, silencing Hvpnr did not affect melanin synthesis in pupae and adults in H. vigintioctopunctata. These findings demonstrate that among the splice variants of Hvpnr, Hvpnr-α plays a dominant regulatory role in the post-embryonic morphogenesis of H. vigintioctopunctata. This study also shows that Hvpnr is not involved in melanin synthesis, indicating significant functional differentiation of pnr during the evolution of ladybirds.