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Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a 'master signal' that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS–FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS–FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements. Author summary: Many insects are capable of developing into either long-winged or short-winged adults, but the underlying molecular basis remains largely unknown. Pioneer studies showed that the insulin/insulin-like growth factor signaling pathway acts as a 'master signal' that directs wing buds to develop into long or short wings in the wing-dimorphic planthopper, Nilaparvata lugens. However, downstream effectors mediating the IIS pathway effects are unknown. Our findings highlight that vestigial, a key wing-patterning gene, is a main downstream effector that mediates the IIS activity on the development of alternative wing morphs during the wing-morph decision stage. The molecular mechanism of wing formation, including the function of vestigial, has been studied in great depth in the model insect Drosophila melanogaster. Our data provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network in regulating developmental plasticity of wings in insects. [ABSTRACT FROM AUTHOR]

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with the ability to bind to a CCAAT box in nearly all eukaryotes. However, the function of NF-Y in the life-history traits of insects is unclear. Here, we identified three NF-Y subunits, Nl NF-YA, Nl NF-YB, and Nl NF-YC, in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens. Spatio-temporal analysis indicated that NlNF-YA , NlNF-YB , and NlNF-YC distributed extensively in various body parts of fourth-instar nymphs, and were highly expressed at the egg stage. RNA interference (RNAi)-mediated silencing showed that knockdown of NlNF-YA , NlNF-YB , or NlNF-YC in third-instar nymphs significantly extended the fifth-instar duration, and decreased nymph-adult molting rate. The addition of 20-hydroxyecdysone could specifically rescue the defect in adult molting caused by NlNF-YA RNAi, indicating that Nl NF-Y might modulate the ecdysone signaling pathway in the BPH. In addition, NlNF-YA RNAi, NlNF-YB RNAi, or NlNF-YC RNAi led to small and moderately malformed forewings and hindwings, and impaired the normal assembly of indirect flight muscles. Adult BPHs treated with NlNF-YA RNAi, NlNF-YB RNAi, or NlNF-YC RNAi produced fewer eggs, and eggs laid by these BPHs had arrested embryogenesis. These findings deepen our understanding of NF-Y function in hemipteran insects. [ABSTRACT FROM AUTHOR]

The brown planthopper Nilaparvata lugens is one of the most destructive insect pests in Asia, demonstrating high fertility and causing huge crop losses by sucking sap of rice as well as transmitting viruses. However, functional genomic studies on N. lugens are seriously constrained by lack of genetic tools. Here, we employed two eye pigmentation genes to generate germ-line mutations in N. lugens using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system. We showed that injection of single guide RNA of the cinnabar gene of N. lugens ( Nl-cn ) into pre-blastoderm eggs induced insertion and deletion (indels) in the founder generation (G 0 ), which were heritably transmitted to the following G 1 generation, leading to bright red compound eyes and ocelli. Mutations of N. lugens white ( Nl-w ) generated a high mutant rate of up to 27.3%, resulting in mosaic eyes consisting of white and lightly pigmented ommatidia in both G 0 and G 1 individuals. The specificity of CRISPR/Cas9-mediated mutagenesis was further bolstered by PCR and RNA interference-based knockdown analysis. These results show that CRISPR/Cas9-mediated gene editing is achievable in a hemipteran insect, offering a valuable tool for the study of functional genomics and pest management in this planthopper species. [ABSTRACT FROM AUTHOR]

• Two Ubx isoforms (NlUbx) were identified in the planthopper (BPH) Nilaparvata lugens. • Knockdown of NlUbx in short-winged BPH nymphs induced the development of T3 wingbuds. • Knockdown of NlUbx in long-winged BPH nymphs transformed hindwings to forewings. • Parental RNAi of NlUbx induced leg-like appendages in the A1 of offspring nymphs. The homeotic complex (Hox) gene Ultrabithorax (Ubx) plays pivotal roles in modifying specific morphological differences among the second (T2), the third thoracic (T3), and the first abdomen (A1) segment in several insects. Whether Ubx regulates wing dimorphism and other morphological traits in the delphacid family (order Hemiptera) remains elusive. In this study, we cloned a full-length Ubx ortholog (NlUbx) from the wing-dimorphic planthopper Nilaparvata lugens , and identified two NlUbx isoforms. RNA-interference (RNAi)-mediated silencing of NlUbx in short-winged BPH nymphs significantly induced the development of wing-like appendages from T3 wingbuds, and this effect is likely mediated by the insulin/insulin-like signaling pathway. RNAi knockdown of NlUbx in long-winged BPH nymphs led to a transformation from hindwings to forewings. Additionally, silencing of NlUbx not only dramatically changed the T3 morphology, but also led to jumping defect of T3 legs. First-instar nymphs derived from parental RNAi had an additional leg-like appendages on A1. These results suggest that Ubx plays a role in determining some morphological traits in delphacid planthoppers, and thus help in understanding evolution of morphological characteristics in arthropods. [ABSTRACT FROM AUTHOR]