Functional study of IN-2-containing isoforms of BgTai and reporter assays to assess the contribution of IN-1 of TbTai to transduce the JH signal.
(A) Effects, at transcript level, of dsTai-in-2 treatment in N4; N4 females received two 3-µg doses of dsTai-in-2, one on N4D0 and the other on N4D3, and transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects of the dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (C) Effects, at transcript level, of dsTai-in-1 plus dsTai-2 treatment in N4; specimens received an injection of dsTai-in-1 (3 µg) plus dsTai-in-2 (3 µg) in N4D0, and the treatment was repeated in N4D3; controls received an equivalent treatment with dsMock. (D) Effects of the dsTai-in-1 plus dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (E–G) Dorsal and ventral view of specimens resulting from dsTai-in-1 plus dsTai-in-2 treatment; normal N5 obtained from this or from dsMock treatments (E); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 plus dsTai-in-2 treatments (F); normal N6 obtained from dsMock treatments (G). Each point of quantitative data in histograms A to D 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 . Scale bars in E–G = 3 mm. (H) Reporter assays to study the JH-dependent interaction of TcMet and TcTai DEL-1 or TcTai IN-1 with kJHRE in Drosophila S2 cells. The cells were transfected with a kJHRE-reporter vector (−477 to +1883, pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pIZT-Rluc) and a plasmid expressing the full ORF of TcMet and/or TcTai IN-1 or TcTai DEL-1. Cells were treated with 1 µM of JH III for 24 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System. Each bar indicates the mean ± SEM (n = 6). The asterisks indicate statistically significant differences between measurements (***p<0.001; **p<0.01) using a two-tailed t-test (n = 6).