Abstract
Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs), which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R) consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, Tribolium castaneum. Depletion of TcCPR27 by RNA interference (RNAi) causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s) of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the TcCPR4 gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult T. castaneum. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for TcCPR4 resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs) in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.
Highlights
Insects have a protective exoskeleton made up of a multi-layered cuticle that helps to withstand various environmental and pathogenic challenges
We have identified a cuticular structural protein, TcCPR4, which is predominantly localized in the pore canals of rigid cuticles of the red flour beetle
Loss of function of TcCPR4 by RNA interference causes abnormal and amorphous pore canal fibers resulting in less organized pore canals that do not traverse the procuticle vertically
Summary
Insects have a protective exoskeleton made up of a multi-layered cuticle that helps to withstand various environmental and pathogenic challenges. It consists of three morphologically and functionally distinct layers, the outermost waterproof envelope, the protein-rich epicuticle and the innermost chitin/protein-rich procuticle [1,2,3]. During cuticle maturation and tanning (sclerotization and pigmentation), some of the CPs are cross-linked by quinones or quinone methides produced by laccase 2-mediated oxidation of N-acylcatechols [4,5,6]. This study examines the role of a CP in Tribolium castaneum (red flour beetle), TcCPR4, which is present in rigid adult cuticles
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