Analysis of the roles of MAD proteins in the wing dimorphism of Nilaparvata lugens

Abstract Wing dimorphism in Nilaparvata lugens is controlled by the insulin‐like growth factor 1 (IGF‐1) signaling – Forkhead transcription factors (IIS‐FoxO) pathway. However, the role of this signal in the wing development program remains largely unclear. Here, we identified 2 R‐SMAD proteins, Nl MAD1 and Nl MAD2, in the brown planthopper (BPH) transcriptome, derived from the intrinsic transforming growth factor‐ β pathway of insect wing development. Both proteins share high sequence similarity and conserved domains. Phylogenetic analysis placed them in the R‐SMAD group and revealed related insect orthologs. The expression of Nlmad1 was elevated in the late instar stages of the macropterous BPH strain. Nlmad1 knockdown in nymphs results in malformed wings and reduced wing size in adults, which affects the forewing membrane. By contrast, Nlmad2 expression was relatively consistent across BPH strains and different developmental stages. Nlmad2 knockdown had a milder effect on wing morphology and mainly affected forewing veins and cuticle thickness in the brachypterous strain. Nl MAD1 functions downstream of the IIS‐FoxO pathway by mediating the FoxO‐regulated vestigial transcription and wing morph switching. Inhibiting Nlmad1 partially reversed the long‐winged phenotype caused by NlFoxO knockdown. These findings indicate that Nl MAD1 and Nl MAD2 play distinct roles in regulating wing development and morph differentiation in BPH. Generally, Nl MAD1 is a key mediator of the IIS‐FoxO pathway in wing morph switching.