Fig. 2.

Domain organization, expression, and role of TcKnk in maintenance of cuticular chitin. (A) TcKnk is composed of an N-terminal signal peptide (SP), two DM13 domains, a dopamine monooxygenase N-terminal like (DOMON) domain, and a C-terminal GPI anchor. Domains were annotated with the SMART database and graphed with the protein-domain illustrator program DOG 1.0.3. (B) Tissue-specific expression pattern of TcKnk in the last-instar larval midgut (M), hindgut (H), and carcass (C) of T. castaneum. TcChs-A and TcChs-B transcripts were used as carcass-specific and midgut-specific markers, respectively, to assess the purity of the tissue preparations, and RpS6 was an internal loading control used to normalize for the differences in the concentrations of the cDNA templates. Numbers in parentheses indicate the cycle number for each of transcripts shown. (C) RNAi was carried out by injecting ∼200 ng of TcKnk dsRNA into penultimate instar larvae, last-instar larvae, or pharate pupae. Representative images of terminal phenotypes are displayed along with controls injected with dsRNA for TcVer (n = 20 each). (D) Quantitative analysis of chitin content from whole animals at pharate pupal and pharate adult stages was carried out by using a modified Morgan–Elson assay. Data are reported as mean ± SE (n = 5). Statistical significance was computed with Student's t test. ns, not significant. (E) At 4 d after injections, the effect of TcKnk dsRNA on transcript levels was determined by collecting dsRNA-treated insects (n = 4) from each treatment at the indicated stage of development. Depletion of transcripts was detected by extracting total RNA from each treatment followed by cDNA preparation and RT-PCR. RpS6 transcript was used as an internal loading control. Numbers in parentheses indicate the cycle number for each of transcripts shown.