Clip domain serine proteases play vital roles in various innate immune functions and in embryonic development. Nilaparvata lugens proclotting enzymes (NlPCEs) belong to this protease family. NlPCE1 was reported to be involved in innate immunity, whereas the role of other NlPCEs is unclear. In the present study, N. lugens proclotting enzyme-3 (NlPCE3) was cloned and characterized. NlPCE3 contains a signal peptide, a clip domain, and a trypsin-like serine protease domain. NlPCE3 was expressed in all tissues examined (gut, fat body, and ovary), and at all developmental stages. Immunofluorescence staining showed that NlPCE3 was mainly expressed in the cytoplasm and cytomembrane of follicular cells. Double stranded NlPCE3 RNA interference clearly inhibited the expression of NlPCE3, resulting in abnormal egg formation and obstruction of ovulation. These results indicate that NlPCE3 plays an important role in egg production in N. lugens.

The brown planthopper, Nilaparvata lugens, is a difficult-to-control insect pest affecting rice yields in Asia. As a structural component of the inter-alpha-trypsin inhibitor (ITI), the inter-alpha-trypsin inhibitor heavy chain (ITIH) has been reported to be involved in various inflammatory or malignant disorders, ovarian development, and ovulation. To reveal the function of ITIH4 in N. lugens, the gene encoding N. lugens ITIH4 (NlITIH4) was cloned and characterized. NlITIH4 contains a signal peptide, a vault protein inter-alpha-trypsin domain, and a von Willebrand factor type A domain. qPCR analysis showed that NlITIH4 was expressed at all developmental stages and in all tissues (fat body, ovary, and gut), with the highest expression in the fat body. Double stranded NlITIH4 (dsNlITIH4) injection clearly led to an RNAi-mediated inhibition of the expression of NlITIH4 and resulted in reduced survival, delayed ovarian development, and reduced egg production and egg hatching. These results indicate that NlITIH4 plays an important role in the development and reproduction of N. lugens.

Nilaparvata lugens proclotting enzymes (NlPCEs) belong to the clip domain serine protease (clip-SP) family, which is a characteristic protease family in arthropods. NlPCE3 was previously reported to regulate egg production and development in female N. lugens, but its role in male N. lugens is unclear. In the present study, qPCR analysis showed that NlPCE3 was expressed in three different tissues (gut, testis and fat body). RNAi revealed that dsNlPCE3 injection made the male vas deferens thinner and reduced the oviposition level of the females that mated with dsNlPCE3-treated males, causing eggs not to hatch. Furthermore, immunofluorescence staining showed that NlPCE3 was widely expressed in the male internal genitalia. However, after dsNlPCE3 injection, expression of NlPCE3 was diffuse in the male internal genitalia, whose peripheral cells seemed degraded. Overall, these results indicate that NlPCE3 is important for reproduction in male N. lugens.