Tribolium castaneum RR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle

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.

Fig 1. Identification of TcCPR4 protein in elytral cuticle of TcCPR27-deficient adult T. castaneum.

(A) dsRNAs for TcCPR27 (dsCPR27) and TcVer (dsVer) (100 ng per insect) were injected into a mixture of penultimate instar and last instar larvae (n = 20). Proteins from the elytra of each dsRNA-treated newly emerged adults were extracted, and then the extracts were analyzed by SDS-PAGE. Two proteins with apparent molecular masses of 16 and 22 kDa (red arrows) were more extractable in TcCPR27-deficient elytra. (B) Nucleotide and deduced amino acid sequences of TcCPR4. The two proteins were cut out from the gel, digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. Results were compared with conceptual trypsinization products of the computed proteome of T. castaneum. Both proteins exhibited the same six matched peptides that are highlighted in gray. The broken line between R⁹¹ and K⁹² indicates that there are two matched peptides, one is N⁸⁰-R⁹¹ and the other is N⁸⁰-K⁹². Predicted signal peptide is underlined. The box indicates the RR-1 motif. The potential polyadenylation signal (AATAAA) is indicated in bold.

Fig 2. Localization of TcCPR4 protein in adult cuticles of T. castaneum.

(A) Immunohistochemical analysis was performed to determine the locations of TcCPR4 in adult cuticles. Cryosections of pharate adults (5 d-old pupae) were incubated with the anti-TcCPR4 antibody, which was then detected by Alexa Fluor 488 goat anti-rabbit IgG (green). E: elytron, H: hindwing, VA: ventral abdomen, DA: dorsal abdomen, L: leg, PC: pupal cuticle. Scale bar = 200 μm. (B) Immunolocalization of TcCPR4 and TcCPR27 in elytral cuticle. Cryosections of 3 d-, 4 d- and 5 d-old pupae were incubated with the anti-TcCPR4 or anti-TcCPR27 antibody [21]. Anti-TcCPR4 and anti-TcCPR27 antibodies were detected by Alexa Fluor 488 goat anti-rabbit IgG (green) and Alexa Fluor 546 rabbit anti-chicken IgG (red), respectively. Nuclei were stained with To-Pro-3 (blue). D: elytral dorsal cuticle, V: ventral elytral cuticle, EC: epithelial cell. Scale bar = 20 μm.

Fig 3. Immunogold labeling followed by TEM analysis for TcCPR4.

Ultra-thin sections (~90 nm) of pharate adults (5 d-old pupae) were incubated with anti-TcCPR4 antibody. Anti-TcCPR4 antibody was detected using goat anti-rabbit IgG conjugated to 10 nm gold particles. The pore canal with pore canal fibers (PCF) in their core (2) and apical plasma membrane protrusions (APMP) (3) were enlarged. Scale bar in panel 1 = 1 μm and in panels 2 and 3 = 200 nm.

Fig 4. Injection of dsTcCPR4 prevents severe elytral morphological defects produced by RNAi for TcCPR27.

dsRNAs for TcCPR4, TcCPR27, TcCPR4/27 and TcVer (100 ng per insect) were injected into late instar larvae (n = 40). (A) The expression of TcCPR4 was analyzed by real-time PCR. cDNAs were prepared from total RNA isolated from 5 d-old pupae (n = 3). Expression levels of TcCPR4 are presented relative to the levels in dsVer-injected control insects. The transcript levels of T. castaneum ribosomal protein S6 (TcRpS6) were measured to normalize for differences in the concentrations of cDNA templates between samples. An asterisk indicates a significant difference in transcript levels of TcCPR4 between control and test insects (p < 0.05, t-test). Data are shown as mean ± SE (n = 3). (B) Insects treated with those dsRNAs developed and grew normally. The dsTcCPR4-treated adults exhibited elytra indistinguishable from those of dsTcVer-treated control insects. Elytra from the resulting dsTcCPR27-treated adults were malformed, wrinkled, warped and fenestrated as reported previously [8], while those from the resulting dsTcCPR4/27-treated adults were less severely affected and expanded well enough to yield a decrease in desiccation-induced mortality produced by TcCPR27 RNAi (see S7 Fig.).

Fig 5. Localization of TcCPR4 protein in elytral cuticle from TcCPR27-deficient insects.

Ultra-thin sections (~90 nm) of pharate adults (5 d-old pupae) that had been injected with dsRNA for TcCPR27, TcCPR4/27 (co-injection) or TcVer (100 ng per insect) in late instar larvae were incubated with anti-TcCPR4 antibody. Anti-TcCPR4 antibody was detected by goat anti-rabbit IgG conjugated to 10 nm gold particles. TcCPR4 protein is mainly present in vertical PCFs of elytral cuticle from dsTcVer-treated insects (A-C), while it is distributed in the entire procuticle of the elytra from TcCPR27-deficient insects (D-F). The number of gold particles is drastically decreased in TcCPR4/27-deficient insects (G-I). EN: envelope, EP: epicuticle, PCF: pore canal fibers, APMP: apical plasma membrane protrusion. Scale bar = 500 nm.

Fig 6. Wheat germ agglutinin (WGA)-gold labeling TEM of dorsal elytral cuticle of T. castaneum.

Ultra-thin sections (~90 nm) of pharate adults (5 d-old pupae) that had been injected dsTcVer (200 ng per insect) in late instar larvae were incubated with gold-labeled (10 nm) WGA (EY Laboratories) to detect chitin in the dorsal side of elytral cuticle. dsRNA for chitin synthase-A (dsTcChs-A) which is required for cuticular chitin synthesis [30] was injected as a negative control. Chitin was detected in both horizontal laminae and vertical PCFs of elytral cuticle from dsTcVer-treated insects (A and C). Few or no gold particles were observed in TcChs-A-deficient insects (B and D). EN: envelope, EP: epicuticle, PRO: procuticle, PCF: pore canal fibers, APMP: apical plasma membrane protrusion. Scale bar in A and B = 1 μm and C and D = 500 nm.

Fig 7. Ultrastructure of elytral cuticle of TcCPR4-, TcCPR27- and TcCPR4/27-deficient pharate adults.

Elytra from pharate adults (5 d-old pupae) that had been injected with dsTcCPR4, dsTcCPR27, dsTcCPR4/27 and dsTcVer into late instar larvae were collected for analysis of ultrastructure by TEM. Panels E-H show enlarged images of the horizontal laminae and vertical pore canals in the procuticle of the dorsal elytral cuticles from each set of dsRNA treated-insects. EN, envelope; EP, epicuticle; PRO, procuticle; PCF, pore canal fiber; APMP, apical plasma membrane protrusion. Scale bar in A-D = 2 μm and E-H = 500 nm.

Fig 8. Schematic diagram of structure and localization of TcCPR4 and TcCPR27 proteins in rigid cuticle of adult T. castaneum.

The body regions with highly sclerotized and pigmented cuticle such as the dorsal elytron, thoracic body wall and legs of T. castaneum adults are composed of the envelope (EN), epicuticle (EP) and procuticle (PRO). In the procuticle, there are numerous chitinous horizontal laminae and vertical pore canals with pore canal fibers (PCFs) in their core that directly extend from the apical plasma membrane protrusions (APMP) to the epicuticle. TcCPR27 protein is present in both the horizontal laminae and vertical PCFs, whereas TcCPR4 is predominantly localized in the latter structure. PC, pore canal; AP, apical plasma membrane; EC, epithelial cells underlying the rigid cuticle. A. Wild type, B. TcCPR4 RNAi. C. TcCPR27 RNAi.