β2-tubulin and its promoter in the brown planthopper: A versatile tool for genetic control strategies

At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.