Silencing of Chitin-Binding Protein with PYPV-Rich Domain Impairs Cuticle and Wing Development in the Asian Citrus Psyllid, Diaphorina citri

SIMPLE SUMMARY: Molting is extremely important for insect growth and development, which is accompanied the degradation of old cuticle and synthesis of new cuticle. Chitin and proteins, as major components of insect cuticle, maintain the rigidity of the exoskeleton. The functions of chitin-binding proteins have not, to date, been characterized in Diaphorina citri. In the current study, we identified a cuticle protein (DcCP64) according to chitin column purification and LC-MS/MS analysis. Silencing of DcCP64 induced an abnormal phenotype and increased the permeability of the abdomen and wings. Additionally, the mortality and malformation rate significantly increased, and the molting rate decreased after inhibition of DcCP64. Transcriptome sequencing analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in MAPK and FoxO signaling pathways. Our results provide a basis for further functional research on DcCP64 in D. citri. ABSTRACT: Chitin is a major component of the arthropod exoskeleton, always working together with chitin-binding proteins to maintain the functions of extracellular structures. In the present study, we identified a cuticle protein 64 from Diaphorina citri using a chitin-binding assay. Bioinformatics analysis revealed that DcCP64 contained eight conserved PYPV motifs but lacked a Rebers–Riddiford (R–R) consensus and other chitin-binding domains. RT-qPCR analysis suggested that DcCP64 had the highest expression level in the wing and fifth-instar nymph stage. Knockdown of DcCP64 by RNA interference (RNAi) resulted in a malformed-wing phenotype, higher mortality and decreased molting rate. Furthermore, transcriptomics analysis revealed that 1244 differentially expressed genes (DEGs) were up-regulated and 580 DEGs were down-regulated, compared with dsDcCP64 groups and dsGFP groups. KEGG enrichment analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in the MAPK and FoxO signaling pathways. Moreover, inhibition of DcCP64 significantly affected the cuticle surface, and increased the permeability of the abdomen and wings. Further chitin- and cellulose-binding assay confirmed the chitin-binding properties of recombinant DcCP64 in vitro. These results indicate that DcCP64 might play an important role in the cuticle and wing development of D. citri.