A role for Taiman in insect metamorphosis

Abstract

Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

Figure 1. Structure and expression of BgTai and their isoforms in Blattella germanica.

(A) Organization of BgTai protein in different domains; in addition to the bHLH region and the two PAS domains, it contains seven LxxLL motifs indicates as black bars, five regions rich in Glutamine, indicated with “Q”, and two Insertions/Deletions towards the 3′ region one (IN-1) of 276 bp and the other (IN-2) of 74 bp; also indicated is the region were the dsTai-core used in RNAi studies was designed. (B) Scheme representing the C-terminal region of the four isoforms of BgTai. (C) Expression of BgTai mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar; from top to down it is showed the expression of the ensemble of isoforms (amplified with primers designed to regions common to all them), and that of each isoform A, B, C and D (amplified with respective specific primers). (D) Effect of JH III treatment (20 µg) on BgTai expression, on the ensemble of isoforms, and specifically on each one; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (E) Expression of each BgTai isoform in different tissues of females in N6D0: corpora allata (CA), epidermis of the thoracic discs (EP), ovaries (O), muscle (M), brain (B), fat body (FB), and in testicles (TST) from males of the same age. Each point in C and D represents 4 biological replicates and results are expressed as the mean ± SEM, whereas those in E represent a pool of 5 specimens; data in C and E are expressed as copies of BgTai mRNA per 1000 copies of BgActin-5c mRNA; data in D 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 .

Figure 2. Function of BgTai in Blattella germanica metamorphosis.

(A) Effects, at transcript level, of dsTai-core treatment in N5; N5 females received two 3-µg doses of dsTai-core, one on N5D0 and the other on N5D3, and transcript levels (of Tai, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects, at transcript level, of dsTai-in-1 treatment in N4; the experimental design was equivalent to that used in N5, with a double treatment, one on N4D0 and the other on N4D3; transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6. (C–E) Dorsal and ventral view of specimens resulting from dsTai-in-1 treatment in N4; normal last nymphal instar and adult obtained from dsMock treatments (C); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 treatments (D); precocious adult obtained (instead of N6) from dsTai-in-1 treatments (E). (F–H) SEM images of T2 and T3 (general and detail of the lateral parts) from specimens resulting from dsTai-in-1 treatment in N4; normal last nymphal instar and adult obtained from dsMock treatments (F); nymphoid of the figure D, right (G); precocious adult from the figure E (H). (I) Dissected T2 and T3 of: N6 control (dsMock-treated), a nymphoid with adult features obtained after dsTai-in-1 treatment, a nymphoid with adult features obtained after dsKr-h1 treatment. (J) Effects of dsTai-in-1 treatment on N4 on the expression of EcR, RXR, E75A and ILP-1 measured on N5D6. Each point of quantitative data in histograms A, B and J 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 C, D, E = 3 mm, in F, G, H = upper and bottom respectively = 1 and = 0.5 mm, and in I = 1 mm.

Figure 3. 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).