Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx moil

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.

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.

RR-1 cuticular protein TcCPR69 is required for growth and metamorphosis in Tribolium castaneum

Cuticle is not only critical for protecting insects from noxious stimuli but is also involved in a variety of metabolic activities.Cuticular proteins(CPs)affect cuticle structure and mechanical properties during insect growth,reproduction,and environmental adaptation.Here,we describe the identification and characterization of a member of the RR-1 subfamily of CPs with an R&R consensus(CPR)in Tribolium castaneum(TcCPR69).Although it was previously reported to be highly expressed in the wings,we found that knocking down TcCPR69 by RNA interference(RNAi)did not cause obvious wing abnormalities but markedly disrupted the growth and metamorphosis of beetles with 100%cumulative mortality;additionally,the chitin content of the pharate adult was decreased and the new abdominal cuticle was significantly thinner before molting.TcCPR69 showed chitin-binding ability and the expression levels of key genes involved in chitin metabolism(trehalase[TcTRE],chitin synthase[TcCHSA and TcCHSB],and chitinase[TcCHT5 and TcCHT10])were also decreased by TcCPR69 knockdown.TcCPR69 gene expression peaked shortly after molting and was increased 2.61 fold at 12 h after 20-hydroxyecdysone(20E)injection.This was reversed by RNAi of the ecdysone-related genes ecdysone receptor(TcECR)and fushi tarazu transcription factor 1(TcFTZ-F1).These results indicate that TcCPR69 is positively regulated by 20E signaling to contribute to cuticle formation and maintain chitin accumulation during the growth and metamorphosis of beetles.