Intersex(ix), a gene involved in the sex-determining cascade of Drosophila melanogaster, works in concert with the female-specific product of doublesex (dsx) at the end of the hierarchy to implement the sex-specific differentiation of sexually dimorphic characters in female individuals. In this study, the ix homolog was identified in the brown planthopper (BPH), Nilaparvata lugens, which contained two splice variants expressed in both female and male insects. We found that Nlix played a vital role in the early nymphal development of BPH, showing an accumulated effect. RNAi-mediated knockdown of Nlix at 4th instar led to the external genital defects in both sexes, consequently resulting in the loss of reproductive ability in female and male individuals. After dsRNA injection, the males were normal on testes, while the females had defective ovarian development. Nlix was also required for early embryogenesis. Notably, when the dsNlix microinjection was performed in newly emerged females, the copulatory bursas were abnormally enlarged while the other tissues of the reproductive system developed normally. Our results demonstrated the pleiotropic roles of Nlix in embryogenesis and development of the reproductive system in a hemimetabolous insect species.

The sex determination mechanism for hemipteran species remains poorly understood. During the sex determination of the brown planthopper (BPH), Nilaparvata lugens , one species of Hemiptera, the functions of doublesex ( Nldsx ) and NlTra-2 ( NlTra-2 ) genes were identified in our previous studies. Here, we identify an upstream gene for Nldsx in the sex determination cascade, NlFmd , which acts as female determinant gene for N. lugens . The sex-specific transcript of NlFmd ( NlFmd-F ) encodes an arginine/serine-, and proline-rich protein that is essential for female development. The knockdown of NlFmd resulted in the development of pseudomales, with sex-specific alternative Nldsx processing, and maternal RNA interference (RNAi) against NlFmd generates male-only progeny. Moreover, homologous genes for NlFmd have also been identified in two rice planthopper species, the white-backed planthopper (WBPH, Sogotalla furcifera ) and the small brown planthopper (SBPH, Laodelphax striatellus ), and these genes appear to be involved in the sex determination cascades for these species. Our data suggest that the sex determination cascade in Delphacidae is conserved.