Figure 1.
Structure, expression and function of BgMet in Blattella germanica metamorphosis.
(A) Organization of BgMet protein in different domains; also indicated are the regions were the two dsMet used in RNAi studies were designed. (B) Expression of BgMet mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar. (C) Effect of JH III treatment (20 µg) on BgMet expression; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (D) Effects, at transcript level, of dsMet-a treatment in N4; N4 females received two 3-µg doses of dsMet-a, one on N4D0 and the other on N4D3, and transcript levels (of Met, Kr-h1 and BR-C) were measured on N4D5; controls received an equivalent treatment with dsMock. (E) Effects, at transcript level, of dsMet-a treatment in N5; the experimental design was equivalent to that used in N4, with a double treatment, one on N5D0 and the other on N5D3; transcript levels (of Met, Kr-h1, BR-C and Tai) were measured on N5D6. (F–I) Dorsal and ventral view of specimens resulting from dsMet-a treatment in N4 and N5; normal last nymphal instar and adult obtained from dsMock treatments, either in N4 or N5 (F); nymphoid (instead of N6) with adult features, and precocious adult obtained from dsMet-a treatments in N4 (G), detail of the enlarged lateral expansions in T2 and T3 of a nymphoid with adult features compared with a control N6 (H), nymphoid with adult features, and precocious adult obtained from dsMet-a treatments in N5 (I). Each point of quantitative data in histograms represents 4 biological replicates and results are expressed as the mean ± SEM; data in A represent copies of BgMet mRNA per 1000 copies of BgActin-5c mRNA; data in C, D and E are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [29]. Photomicrographs in F–I were taken with a Zeiss DiscoveryV8 Stereo microscope with an AxioCam MRc digital camera; scale bars in F, G, I = 3 mm and in H = 2 mm.
Figure 2.
Effects of BgMet depletion in last nymphal stage of Blattella germanica.
(A) Effects at transcript level; N6 females received two 3-µg doses of dsMet-a, one on N6D0 and the other on N6D3, and Met mRNA levels were measured on N6D6; controls received an equivalent treatment with dsMock. (B) Dorsal and ventral view of specimens resulting from dsMet-a treatment in N6; normal adult obtained from dsMock treatments; mild and severe adult phenotypes obtained after Adult N6 with adultoid features, mild and severe, obtained from dsMet-a treatments. (C) Tegmina and membranous wings corresponding to normal adult obtained after dsMock treatments, and to adults treated with dsMet-a showing the mild and the severe phenotype. (D) Effects of Met depletion on N6 on the expression of EcR, RXR, E75A and ILP-1 measured on N6D6. In A and D, each point represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [29]. Photomicrographs in B–C were with a Zeiss DiscoveryV8 Stereo microscope with an AxioCam MRc digital camera; scale bars = 3 mm.