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 dsNlCHS1-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.