NlAgo3 and NlBiwi are essential for piRNAs biogenesis and female reproduction in Nilaparvata lugens.

P-element-induced wimpy testis (PIWI) -interacting RNAs (piRNAs) are short non-coding RNAs crucial for silencing transposable elements (TEs) and maintaining genome stability, particularly in germ cells. While most research on piRNAs and PIWI proteins has traditionally focused on model organisms such as Drosophila, emerging studies are now extending to other insect orders. However, little is known about piRNAs in insects of the Hemiptera order. Here, we investigated the role of piRNAs in the brown planthopper (BPH), Nilaparvata lugens, a significant pest of rice that belongs to the Hemiptera order. We identified and characterized two PIWI subfamily protein genes in N. lugens: NlAgo3 (a homolog of Ago3) and NlBiwi (the BPH Piwi). Both proteins contain Piwi/Argonaute/Zwille (PAZ) and PIWI domains. Sequence alignment and phylogenetic analysis demonstrated that NlAgo3 and NlBiwi are conserved across other insect orders. NlAgo3 and NlBiwi are highly expressed in female adults and their ovaries in N. lugens. Importantly, knockdown of NlAgo3 and NlBiwi significantly impaired eggs laying, leading to female sterility, suggesting that these proteins play a crucial role in female reproduction in N. lugens. Subsequently, through small RNA sequencing, we characterized piRNAs in N. lugens and identified a length peak in the range of 26-28 nucleotides, with differences in piRNA abundance between females and males. Additionally, we observed that piRNA clusters were distributed across each chromosome, with a higher density on the sex chromosomes. Moreover, RNAi-mediated knockdown of NlAgo3 and NlBiwi severely impaired piRNA production in BPH, highlighting that these proteins are essential for piRNA biogenesis in N. lugens, consistent with findings in other species. Overall, this study provides valuable insights into the role of piRNAs in the reproductive biology of N. lugens, a major pest of rice. By clucidating the characteristics of piRNAs in this insect species, this research enhances our understanding of piRNA-mediated gene regulation in Hemiptera insects. These findings have potential implications for the development of novel strategies for pest control strategies.