Yeast-like symbionts (YLSs) are transovarially transmitted between brown planthopper generations, but the transmission mechanism is poorly understood. In the present study, we found that YLSs do not enter the brown planthopper oocyte until it develops to late vitellogenesis. During oocyte vitellogenesis, the diversification and morphological changes of the follicular cells surrounding the oocyte are associated with the accumulation and polarization of vitellogenin (Vg) and lipids in the oocyte, indirectly affecting the entry of YLSs into the oocyte. Additionally, the posterior follicular cells, one of the diversified follicular cell populations, form the epithelial plug that is the only structure for YLSs to enter the oocyte. In addition, actin is rearranged within the follicular cells to build an actin bridge for YLSs to move from the epithelial plug to the oocyte, indicating that actin mediates the entry of YLSs into the brown planthopper oocyte during oocyte vitellogenesis. Taken together, our results demonstrate that the entry of YLSs into the brown planthopper oocyte is triggered by oocyte vitellogenesis.

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.

SLC26A10 is a member of the SLC26 gene family, but its role in insects is still unclear. We cloned the SLC26A10 gene of Nilaparvata lugens (NlSLC26A10) and found NlSLC26A10 contained 11 transmembrane regions and a STAS domain. Expression pattern analysis showed NlSLC26A10 expression was more upregulated in adults than in nymphs, highest in the ovary. After injection of double-stranded RNA (dsRNA) of NlSLC26A10, the mRNA level of NlSLC26A10 significantly decreased and, consequently, the ovarian development of adult females was hindered; the amount and the hatchability of eggs and yeast-like symbionts in mature oocytes decreased. Further study showed that NlSLC26A10 might result in decreased juvenile hormone level and vitellogenin expression. These results indicate that NlSLC26A10 plays an essential role in the reproduction of N. lugens.