Chitin synthase 1 and five cuticle protein genes are involved in serosal cuticle formation during early embryogenesis to enhance eggshells in Nilaparvata lugens

Many holo- and hemimetabolous insects enhance their eggshells during embryogenesis by forming a serosal cuticle (SC). To date, scholarly understanding of the SC composition and SC-related gene functions has been limited, especially for hemimetabolous insects. In this study, we initially performed transmission electron microscopic (TEM) observation and chitin staining of the SC in Nilaparvata lugens , a hemimetabolous rice pest known as the brown planthopper (BPH). We confirmed that the SC was a chitin-rich lamellar structure deposited gradually during the early embryogenesis. Parental RNA interference (RNAi) against Nilaparvata lugens chitin synthase 1 ( NlCHS1 ) in newly emerged and matured females resulted in decreases of egg hatchability by 100% and 76%, respectively. Ultrastructural analyses revealed loss of the lamellar structure of the SC in ds NlCHS1- treated eggs. According to temporal expression profiles, five cuticle protein coding genes, NlugCpr1/2/3/8/90 , were specifically or highly expressed during the SC formation period, and NlugCpr1/2/3/90 were further detected in 72 h eggshells extract by ultra-performance liquid chromatography-tandem mass spectrometry/mass spectrometry. NlugCpr2/3/90 were likely three SC-specific cuticle proteins. TEM observations of the SC following parental RNAi against NlugCpr1 / 2 / 3 / 8 / 90 demonstrated that NlugCpr3 / 8 / 90 were essential for SC formation. The study provided an understanding of the SC formation process and SC-related cuticle proteins in BPHs, which offer potential targets for pest control in the egg stage as well.