TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis

Results

In Drosophila melanogaster (Dm), it was reported that loss of Dm’mad, Dm’tkv, or Dm’dpp caused precocious metamorphosis, even in the early larval stages (20). Therefore, we first examined whether Dpp signaling plays a role in regulating Gryllus metamorphosis. For these studies, RNAi targeting Gb’mad, Gb’tkv, and Common mediator (Co)-Smad (Gb’medea) were individually injected into third-instar nymphs. The nymphs that received RNAi targeting Gb’mad or Gb’tkv achieved adult metamorphosis at the seventh instar rather than the eighth instar in both sexes [male n = 12/15, female n = 14/16 for Gb’mad (Fig. 1A) and male n = 10/12, female n = 12/15 for Gb’tkv (Fig. 1B)]. In addition, an overall reduction in body size and weight were observed for both RNAi-treated nymphs (Fig. 1 C and D). Following the injection of RNAi targeting Gb’mad, dysgenesis of the wing pads (Fig. 1 E and G) and ovipositor (Fig. 1 F and H) were observed during the sixth instar and the precocious adult stage. Finally, RNAi-mediated depletion of Gb’medea led to precocious adult metamorphosis that occurred at the seventh instar (n = 10/21). As a result, malformation of the wing pads (Fig. S1 D and E) and ovipositor (Fig. S1F) were observed compared with the control nymphs treated with RNAi targeting DsRed2 (n = 31) (Fig. S1 A–C).

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Fig. 1.

Phenotypes observed after depletion of Gb’mad and Gb’tkv was achieved with RNAi in the nymph stage of G. bimaculatus. (A and B) The effects of RNAi targeting Gb’mad or Gb’tkv in nymphs on day 1 of the third instar. In each box, the control nymph is on the left, and the RNAi-treated nymph is on the right. The instar and adult stages for each box are indicated at the bottom. The RNAi-treated nymphs remained small but underwent precocious adult metamorphosis at the seventh instar. (C and D) Body length (C) and weight (D) of male () and female () adults that developed following injections of RNAi targeting DsRed2 (as a control) or Gb’mad. The data presented are the mean ± SD. *P < 0.05 according to Student’s t test. (E) The wing pads (indicated by red asterisks) of the sixth-instar Gb’mad RNAi nymphs exhibited abnormal growth and displayed an extended side. (F) The morphology of the ovipositor (indicated by arrows) in the Gb’mad RNAi sixth-instar nymphs was smaller than that of the control nymphs (Fig. 2O and Fig. S4J). (G) Precocious adults were produced following the injection of RNAi targeting Gb’mad. The wings of these adults were significantly smaller than those of controls and were wrinkled. (H) The ovipositors of the adults produced following the injection of RNAi targeting Gb’mad were cleaved at the tip and became abnormally short. (Scale bars: 10 mm in A and B; 2 mm in E, G, and H; 1 mm in F.)

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Fig. S1.

Wing pad and ovipositor development following the injection of RNAi targeting DsRed2 (control) (A–C), Gb’medea (D–F), Gb’gbb (G–I), and Gb’dpp + Gb’gbb (J–L) into sixth-instar nymphs and adult crickets as indicated. Wing pads are marked with asterisks. The wing pads of the Gb’medea- (D), Gb’gbb- (G), and Gb’dpp + Gb’gbb- (J) targeted sixth-instar nymphs exhibited abnormal growth and displayed an extended side, whereas the wings of the precocious adults (E, H, and K) were significantly reduced and wrinkled. The ovipositors of the precocious adults (F, I, and L) were cleaved at the tip and became short. (M) The male () and female () adults that developed following injections of RNAi targeting DsRed2 (as a control), Gb’dpp, or Gb’dpp + Gb’dpp-like1 + Gb’dpp-like2. (Scale bars: 2 mm in A–L; 10 mm in M.)

On the other hand, we identified three different Gryllus bone morphogenetic protein (BMP) homologs, Gb’dpp (BMP2/4 homolog), Gb’dpp-like1 (BMP2-like homolog), and Gb’dpp-like2 (BMP3 homolog). Therefore, each of these homologs were targeted with RNAi [Gb’dpp, n = 33; Gb’dpp-like1, n = 29; and Gb’dpp-like2, n = 31]. Combinations of these homologs also were targeted: Gb’dpp + Gb’dpp-like1 (n = 25), Gb’dpp + Gb’dpp-like2 (n = 28), Gb’dpp-like1 + Gb’dpp-like2 (n = 27), and Gb’dpp + Gb’dpp-like1 + Gb’dpp-like2 (n = 28). However, all the nymphs that received these RNAi treatments developed normally to become adults (Fig. S1M). It is possible that the absence of an effect in these experiments may be caused by the presence of other redundant ligands.

To identify ligands that may be redundant for Gb’dpp, RNAi was used to target various Gryllus homologs of the Drosophila TGF-β family members (21), including Gb’gbb, activinß (Gb’actβ), maverick (Gb’mav), and myoglianin (Gb’myo).

First, we investigated whether depletion of Gb’gbb mRNA could be linked to the effects associated with loss of Gb’tkv, Gb’mad, or Gb’medea. Following the injection of RNAi targeting Gb’gbb into third-instar nymphs, precocious differentiation of adult features was observed, and these features were similar to those exhibited by the nymphs that underwent depletion of Gb’mad or Gb’medea by RNAi. However, substantially fewer Gb’gbb-depleted nymphs were obtained (n = 6/33) (Fig. S1 G–I). Because Gbb forms a heterodimeric complex with Dpp in Drosophila (22⇓–24), we hypothesized that the simultaneous depletion of Gb’dpp and Gb’gbb would be sufficient to impair normal adult development, especially in the wing pads and ovipositor. Therefore, we next injected RNAi targeting Gb’dpp and RNAi targeting Gb’gbb into third-instar nymphs. Of a total of 16 nymphs, 14 exhibited precocious adult metamorphosis. Furthermore, the wing pads and ovipositors of the resulting sixth-instar nymphs and precocious adults resembled those of the nymphs that received RNAi targeting Gb’mad or Gb’medea (Fig. S1 J–L). In contrast, the combined targeting of Gb’dpp-like1/2 and Gb’gbb with RNAi did not affected the ratio of appearance of precocious adult metamorphosis (Gb’dpp-like1 + Gb’gbb, n = 2/15 and Gb’dpp-like2 + Gb’gbb, n = 4/17). Overall, these results demonstrate that the Dpp signaling pathway is triggered by heterodimeric ligand complexes of Dpp and Gbb and that Dpp/Gbb signaling via Tkv and Mad/Medea is critical for ensuring the completion of adult metamorphosis.

When RNAi targeting myoglianin was injected into third-instar nymphs, a supernumerary nymphal molt was observed in 52 of 59 of the injected nymphs. In comparison, the control nymphs injected with RNAi targeting DsRed2 (n = 33) underwent normal molting between the fourth and eighth instars and then became adults (Fig. 2A; see also Fig. S4N). The molting of the myoglianin-targeted nymphs specifically involved a progression series of third–3′–3′′–fourth–4′–4′′–fifth instar or third–3′–fourth–4′–4′′–4′′′–fifth instar (instead of third–fourth–fifth instar); they then underwent a sixth-instar molt (Fig. 2 A and B). We subsequently identified the myoglianin homolog as metamorphosis-inducing factor (Gb’myo), and its predicted amino acid sequence contains hallmarks of the TGF-β family members (Fig. S2 A–C). Moreover, although the injection with RNAi targeting Gb’myo blocked the morphological transition from one nymphal instar to the next, the number of supernumerary molts at each instar was restricted to one to three molts. Moreover, when the sixth-instar nymphs became adults after these supernumerary molts, their body size and weight were significantly greater than those of the controls (Fig. 2 B, T, and U), and the developmental period for metamorphosis was approximately twice that of the controls (see also Fig. S4N). However, progressive morphogenesis of the wing pads (Fig. 2 G–K) and the ovipositor primordial (Fig. 2 P–S) remained unchanged in the supernumerary nymphs over an extended period, whereas the control nymphs developed normal wing pads (Fig. 2 C–F) and ovipositors (Fig. 2 L–O). Furthermore, when RNAi targeting the TGF-β signaling factor smox/Smad2 (Gb’smox) (Fig. S3 A, D, and E; n = 17/22) and RNAi targeting the type I receptor baboon (Gb’babo) (Fig. S3B) (n = 10/15) were injected into third-instar nymphs, phenotypes similar to those associated with the control nymphs were observed. Based on these RNAi results, targeting of Gb’myo, Gb’babo, and Gb’smox appears to preserve the status quo, and after molting wings and ovipositors are able to form normally, possibly because of the loss of the RNAi effects.

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Fig. 2.

Phenotypes observed after depletion of Gb’myo was achieved with RNAi in G. bimaculatus. (A and B) RNAi targeting DsRed2 (control) or Gb’myo were injected into third-instar nymphs on day 1. Morphological variations during supernumerary molts (3′–3′′–fourth–4′–4′′) and during metamorphosis were subsequently observed in A and B, respectively. In A, the control nymph is on the left and the RNAi-treated nymph is on the right in each box. The instar and adult stages for each box are indicated at the bottom (male: ; female: ). (C–E) Lateral views of third- (C), fourth- (D), and fifth- (E) instar nymphs injected with RNAi targeting DsRed2 on day 1 of the third instar. The red lines indicate the contours of the wing pads (indicated by asterisks). T1–3; thorax 1–3. (F) Dorsal view of the wing pads (indicated by asterisks) in a representative sixth-instar nymph injected with RNAi targeting DsRed2 on day 1 of the third instar. (G–J) Lateral views of supernumerary 3′- (G), 3′′- (H), 4′- (I), and 4′′- (J) instar nymphs injected with RNAi targeting Gb’myo on day 1 of the third instar. (K) Dorsal view of a representative supernumerary 4′′-instar nymph injected with RNAi targeting Gb’myo on day 1 of the third instar. (L–O) Ventral views of third- (L), fourth- (M), fifth- (N), and sixth- (O) instar nymphs injected with RNAi targeting DsRed2 on day 1 of the third instar. Morphological alterations in the ovipositors (indicated by arrows) at the abdomen 8 (Abd8; indicated by arrowheads) were observed. (P–S) Ventral views of supernumerary 3′- (P), 3′′- (Q), 4′- (R), and 4′′- (S) instar nymphs injected with RNAi targeting Gb’myo on day 1 of the third instar. (T and U) Body length (T) and weight (U) of nymphs and adults treated with RNAi targeting DsRed2 (black) or Gb’myo (red). Weeks postinjection (w) are indicated on the x axis. The data presented are the mean ± SD. (Scale bars: 10 mm in A and B; 0.5 mm in C and L–O; 2 mm in F and K.)

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Fig. S2.

(A) The amino acid sequence of Gb’Myo. The predicted RXXR processing site is underlined, and conserved cysteine residues are marked with asterisks. (B) The Gb’Myo TGF-β family domain amino acid sequence was aligned with orthologs in Drosophila melanogaster, Tribolium castaneum, and Bombyx mori. Residues outlined in red are common between two or more of the sequences. (C) Pairwise comparisons were made among G. bimaculatus TGF-β family members. The numbers represent the percent amino acid identity from the processing site (underlined in A) to the C terminus. Gb’Myo exhibited the highest identity with Gb’Activineβ (42%).

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Fig. S3.

Phenotypes of G. bimaculatus nymphs after depleting Gb’smox and Gb’babo with RNAi and following treatment with a JH analog (JHA). (A and B) RNAi targeting Gb’smox or Gb’babo were injected into nymphs on day 1 of the third instar. Control crickets (labeled in black) are shown on the left of each box; RNAi-treated crickets (labeled in red) are shown on the right. The RNAi-treated nymphs underwent progression series of third–3′–3′′–fourth–4′–4′′–fifth or third–3′–fourth–4′–4′′–fifth (instead of third–fourth–fifth) and subsequently developed into large-sized adults compared with the nymphs that were administered DsRed2 RNAi (control). (C) Following the injection of methoprene, a JH analog, third-instar nymphs underwent a progression series of third–3′–fourth–4′–fifth and subsequently developed into large-sized adults compared with the nymphs that were injected with ethanol (control). Control crickets (labeled in black) are shown on the left of each box. Crickets treated with a JH analog (labeled in blue) are shown on the right of each box. (D and E) Body length (D) and weight (E) of the nymphs and adults treated with ethanol (control; black), methoprene (JHA; blue), or RNAi targeting Gb’smox (red) are shown. Weeks postinjection are indicated on the x axis. The data presented are the mean ± SD. (Scale bar: 10 mm.)

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Fig. S4.

RNAi-mediated knockdown of Gb’myo in G. bimaculatus nymphs. (A) After RNAi targeting Gb’myo was injected into fourth-instar nymphs on day 1 (the crickets shown on the far right in each box and labeled in red), a fourth–4′–4′′–4′′′–fifth progression (instead of fourth–fifth) resulted in large-sized adults compared with nymphs injected with RNAi targeting DsRed2 (control; crickets shown on the left in each box and labeled with black text). (B) The phenotypes of fourth-instar nymphs injected with RNAi targeting Gb’myo. These phenotypes resemble those of the third-instar nymphs injected with the same RNAi. (C) The fifth-instar nymphs that received a second dose of RNAi targeting Gb’myo initiated supernumerary molts such as fifth–5′–5′′ or fifth–5′–sixth (instead of fifth–sixth–seventh–eighth–adult) and then developmentally arrested and died. (D) Phenotypes of the fifth- (Left) and sixth- (Center) instar control nymphs administered RNAi targeting DsRed2 on day 1 of the fifth instar and a 5′ nymph (Right) that received RNAi targeting Gb’myo on day 1 of the fifth instar. The nymphs that received RNAi targeting Gb’myo died after supernumerary molts of fifth–5′–5′′ or fifth–5′–sixth (instead of fifth–sixth–seventh–eighth–adult). (E–H) The effect of RNAi targeting DsRed2 (control) or Gb’myo on the wing pad (E and G) and ovipositor (arrows in F and H) that developed in fifth-instar nymphs. In the nymphs treated with RNAi targeting Gb’myo, the wing pad morphology in the resultant 5′-instar nymphs was abnormal (G), and the ovipositor was smaller than in the sixth-instar control nymphs (H). T1–3; thorax 1–3. (I–L) The effect of RNAi targeting DsRed2 (control) or Gb’myo on the wing pad (asterisks in I and K) and ovipositors (arrows in J and L) of sixth-instar nymphs. In the nymphs treated with RNAi targeting Gb’myo, wing pad morphology in the resultant 6′-instar nymphs was abnormal (K), and the ovipositor was smaller than in the control nymphs (L). (M) A qPCR analysis revealed transcript levels of Gb’myo, Gb’jhamt, and Gb’CYP15A1 on day 1 of 5′-instar nymphs injected with RNAi targeting Gb’myo. The transcript levels at day 5 of the fifth-instar nymphs injected with RNAi targeting DsRed2 (control) were set to 1. Asterisks represent significant differences between the control and the Gb’myo RNAi-treated nymphs (Student’s t test; *P < 0.05; **P < 0.005). The data presented are the mean ± SD. (N) A comparison of the different life stages and supernumerary molts induced by RNAi targeting Gb’myo at various stages of development. (Scale bars: 10 mm in A–D; 2 mm in E and I; 1 mm in F and J.)

To investigate further the status quo preservation that characterized the RNAi targeting of Gb’myo, a second dose of RNAi targeting Gb’myo was injected into fourth-instar nymphs (Fig. S4 A–C) (n = 15/15 for ), into fifth-instar nymphs (Fig. S4 D, G, and H) (n = 14/15), and into sixth-instar nymphs (Fig. S4 K and L) (n = 10/10) within the first 24 h after ecdysis. Changes in the wing pads and ovipositor for these stages (Fig. S4 E–L) and in the relative amounts of Gb’myo transcripts (Fig. S4M) and the temporal profile of these changes (Fig. S4N) suggest that Gb’myo may determine the molting characteristics that occur between different nymphal instars. Furthermore, loss of the functions mediated by the Gb’Myo protein resulted in developmental arrest and death at the sixth instar.

Methoprene is an analog of JH, and it also was applied to nymphs during the third instar. This treatment resulted in supernumerary molting and larger adults (Fig. S3 C–E) (n = 17/23). A similar phenotype was observed for the nymphs that received Gb’myo-targeted RNAi. Therefore, we hypothesized that Gb’myo-RNAi phenotype might be caused by a constant JH titer.

To examine a potential dependence of nymphal instars on the concentration of JH in the hemolymph, JH III production was monitored. Periodic changes in JH III production were observed (Fig. 3A), and at the final (eighth) instar, the titer of JH III declined to a low level on day 1 and then was not synthesized until day 7 to allow adult molting (Fig. 3A). To examine further whether periodic changes in the JH III titer depended on Gb’Myo function, JH III titers were quantified on day 5 for the supernumerary (3′ and fourth) instars that had received RNAi targeting Gb’myo. Loss of Gb’myo mRNA resulted in constitutively higher JH III titer levels, whereas the introduction of RNAi targeting Gb’mad lowered the JH III titer levels only on day 1 of the fourth and sixth instars (Fig. 3B). In combination, these data suggest that Gb’Mad and Gb’Myo play crucial roles in controlling JH biosynthesis.

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Fig. 3.

Expression profiles of Gb’myo, Gb’dpp, Gb’jhamt, and Gb’CYP15A1 transcripts in G. bimaculatus during development and the effect of RNAi targeting Gb’myo and Gb’mad on the hemolymph titer of JH. (A) Developmental changes in JH III titer in the hemolymph of male (dotted line) and female (solid line) nymphs that were collected from the fourth to the eighth instars. (B) JH III titer measurements in the hemolymph of nymphs treated with RNAi targeting Gb’myo (red) or Gb’mad (blue) in the third instar. Asterisks represent significant differences between control and RNAi nymphs: *P < 0.05 according to Student’s t test. (C–F) Temporal expression of Gb’myo (C), Gb’dpp (D), Gb’jhamt (E), and Gb’CYP15A1 (F) as detected in qPCR analyses of nymph heads. Relative fold changes in the mRNA levels were plotted, and the average expression level in the heads on day 1 of the third instar (D1 third) was set to 1. The mRNA levels were also normalized to Gb’β-actin mRNA levels. Developmental stages were defined as days (D) after molting. Nymphs were unsexed during the third to fifth instars and were sexed during the sixth to eighth instars and the adult (ad) stage (male data: dotted lines; female data: solid lines). The data presented are the mean ± SD. (G–M) Expression levels of Gb’myo (G), Gb’baboon (H), Gb’dpp (I), Gb’tkv (J), Gb’jhamt (K), and Gb’CYP15A1 (L) in the corpus allatum–corpus cardiacum (CA–CC) complex on day 3 of the seventh instar were examined by whole-mount in situ hybridization. A control experiment using the Gb’myo sense probe is shown in M. (N and O) Expression levels of Gb’myo, Gb’dpp, Gb’jhamt, and Gb’CYP15A1 as detected in qPCR analyses of RNA samples collected from the CA (N) and CC (O) of seventh-instar nymphs. The expression level of Gb’jhamt was set to 1. The data presented are the mean ± SD.

To investigate the spatial and temporal expression patterns of Gb’myo mRNA, quantitative RT-PCR (qPCR) was performed. Gb’myo mRNA was found to be highly expressed in the head and thorax 1 (Fig. S5A), but the levels of Gb’myo mRNA exhibited periodic changes in each of the instars, with a peak in Gb’myo mRNA detected on day 3 (Fig. 3C). Although stepwise increases in the levels of Gb’myo mRNA were observed throughout the developmental stages, they were not observed in adulthood. Moreover, the levels of Gb’myo mRNA exhibited no obvious differences between males and females during all nymphal and adult stages. When the levels of Gb’jhamt mRNA were detected, peaks in expression were initially observed on day 1 in each instar, decreased by day 3, and then disappeared completely on the day before molting (Fig. 3E). This pattern may be associated with the ecdysis process, which is closely tied to the JH cycle. In contrast, Gb’dpp mRNA was found to be constitutively expressed in the head throughout the nymphal stages (Fig. 3D). A slight change was observed in the transcript levels of Gb’CYP15A1, a cytochrome P450 gene that is essential for JH biosynthesis (25); the highest transcript levels were detected in the eighth-instar females (Fig. 3F). These results suggest that although Gb’dpp may play a role in regulating Gb’jhamt expression, Gb’myo appears to act as a rate-limiting factor in the Gb’jhamt expression pathway.

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Fig. S5.

(A) Relative levels of Gb’myo expression in the head, thorax 1, thorax 2 and 3, leg, and abdomen of third-, sixth-, and eighth-instar G. bimaculatus nymphs after normalization to Gb’β-actin mRNA. The average expression level in the heads of third-instar nymphs was set to 1. The data are presented as the mean ± SD. (B) The effect of Gb’smox depletion by RNAi on the transcript levels of Gb’smox, Gb’brk, and Gb’jhamt at day 5 of 3′ and fourth instars. The transcript levels of Gb’smox, Gb’brk, or Gb’jhamt on day 5 (D5 fourth) or day 1 (D1 fourth) of the control fourth-instar nymphs, respectively, were set to 1. The data presented are the mean ± SD. (C) A qPCR analysis revealed the transcript levels of Gb’medea, Gb’brk, and Gb’jhamt on day 1 following the injection of RNAi targeting Gb’medea into third-instar nymphs. The transcript levels at day 1 for the fourth instar (D1 fourth) nymphs that were injected with RNAi targeting DsRed2 (control) were set to 1. The data presented are the mean ± SD. (D) A qPCR analysis revealed the transcript levels of Gb’gbb, Gb’brk, and Gb’jhamt on day 1 following the injection of RNAi targeting Gb’gbb into third-instar nymphs. The transcript levels at day 1 for the fourth-instar (D1 fourth) nymphs injected with RNAi targeting DsRed2 (control) were set to 1. The data presented are the mean ± SD. (E) Relative transcript levels of Gb’dad detected in the heads of 3′- and fourth-instar nymphs on day 5 or fourth- and sixth-instar nymphs on day 1 following treatment with RNAi targeting Gb’smox (maroon) or Gb’mad (blue). The transcript levels on day 5 (D5 fourth for Gb’smox RNAi) or day 1 (D1 fourth for Gb’mad RNAi) of the fourth-instar nymphs that received RNAi targeting DsRed2 (control) were set to 1. The data presented are the mean ± SD. Asterisks in B–E represent significant differences between control and RNAi nymphs (Student’s t test; *P < 0.05; **P < 0.005; n.s., not significant). (F) Relative levels of Gb’jhamt, Gb’CYP15A1, Gb’myo, and Gb’dpp detected in brain extracts collected from seventh-instar nymphs. The expression level of Gb’jhamt was set to 1. The data presented are the mean ± SD.

The spatial expression patterns of Gb’myo, Gb’babo, Gb’dpp, Gb’tkv, Gb’jhamt, and Gb’CYP15A1 also were detected in the head with whole-mount in situ hybridization. All these genes were found to be predominantly expressed in the CA on day 3 of the seventh instar (Fig. 3 G–M). Similar results were obtained when the transcripts of these genes were detected in the CA by qPCR (Fig. 3 N and O). Thus, it appears that expression of Gb’myo in the CA correlates with the regulation of Gb’jhamt expression and JH biosynthesis.

Because both Gb’myo and Gb’dpp were found to be expressed in the CA, we investigated whether these genes are involved in the regulation of Gb’jhamt transcription. First, we confirmed that RNAi targeting of Gb’myo was effective in the heads of supernumerary nymphs (Fig. 4A). An increase in Gb’jhamt expression also was detected in the supernumerary instars on day 1 (3′–fourth–4′), and these levels were significantly higher in these supernumerary nymphs on day 5 as compared with the undetectable levels of Gb’jhamt that characterized the controls (Fig. 4B). Consistent with the supernumerary molting of the nymphs that had received RNAi targeting Gb’smox, Gb’jhamt mRNA levels were up-regulated in 3′- and fourth-instar nymphs on day 5 (Fig. S5B). In contrast, no significant changes were observed in each of the supernumerary instars that expressed the Gb’CYP15A1 transcript (Fig. 4C). When RNAi targeting Gb’mad (Fig. 4D), Gb’medea (Fig. S5C), or Gb’gbb (Fig. S5D) was injected into third-instar nymphs, Gb’jhamt transcript levels were lower in both the fourth- and sixth-instar nymphs on day 1, whereas no apparent effect on Gb’CYP15A1 mRNA levels were observed in the Gb’mad-depleted nymphs (Fig. 4D). Taken together, these results demonstrate that the precocious metamorphosis in nymphs that received RNAi targeting Gb’mad, Gb’medea, or Gb’gbb derives from repression of Gb’jhamt expression, and they also suggest that the up-regulation of Gb’jhamt in nymphs that received RNAi targeting Gb’myo or Gb’smox may depend on timely regulation of the Gb’Dpp/Gbb signaling pathway.

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Fig. 4.

The effects of RNAi-mediated depletion of Gb’myo and Gb’mad on the expression of Gb’jhamt, Gb’CYP15A1, and Gb’brk. (A–C) RNAi targeting DsRed2 control or Gb’myo were injected on day 1 of the third instar. Transcript levels of Gb’myo (A), Gb’jhamt (B), and Gb’CYP15A1 (C) were subsequently determined on days 1 and 5 in the heads of the supernumerary third-, 3′-, fourth-, and 4′-instar nymphs. The transcript levels determined on day 1 of the third-instar control nymphs (D1 third) for A–C were set to 1. The data presented are the mean ± SD. (D) Transcript levels of Gb’mad, Gb’jhamt, and Gb’CYP15A1 also were determined on day 1 of the fourth and sixth instars following the injection of RNAi targeting Gb’mad. The transcript levels of these genes in control nymphs on day 1 of the fourth instar (D1 fourth) were set to 1. The data presented are the mean ± SD. (E) Gb’brk mRNA levels in the heads of fourth- (3′) and sixth- (fourth-) instar nymphs on days 1 and 5 after the injection of RNAi targeting Gb’myo (red) and on day 1 for the fourth- and sixth-instar nymphs that received RNAi targeting Gb’mad (blue). The transcript levels of both sets of control nymphs on day 1 of the fourth instar were set to 1. The data presented are the mean ± SD. (F) After RNAi-mediated depletion of Gb’brk (green) or Gb’mad + Gb’brk (yellow) in the third instar, transcript levels of Gb’brk or Gb’jhamt were measured on days 1 and 5 of the fourth and sixth instars as indicated. The transcript levels measured on day 5 (D5 fourth) or day 1 (D1 fourth) of the control fourth-instar nymphs, respectively, were set to 1. The data presented are the mean ± SD. Asterisks in A, B, and D–F represent significant differences between the control and RNAi nymphs. n.s., not significant; *P < 0.05; **P < 0.005; ***P < 0.001 according to Student’s t test.

Following the injection of RNAi targeting Gb’jhamt into third-instar nymphs, precocious metamorphosis was observed, and these features were similar to those of RNAi depletion targeting Gb’mad (Fig. S6A). To examine whether the increase in Gb’jhamt expression caused by Gb’myo-targeted RNAi can be prevented by the knockdown of Gb’mad or Gb’jhamt, Gb’myo RNAi + Gb’mad RNAi and Gb’myo RNAi + Gb’jhamt RNAi were injected into third-instar nymphs (n = 9/12 and n = 15/16, respectively; Fig. S6 B and C). Changes in overall body size (Fig. S6 D and E) and relative transcript levels (Fig. S6F) were observed. Moreover, the supernumerary molting phenotype was rescued when Gb’jhamt was targeted for depletion. Thus, it appears that supernumerary molts are caused by alterations in Gb’jhamt expression. However, the mechanisms underlying regulation of Gb’jhamt expression by Gb’Myo signaling are unknown.

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Fig. S6.

Rescue of the Gb’myo-RNAi phenotype by dual targeting of Gb’mad and Gb’jhamt with RNAi in the nymphal stage of G. bimaculatus. (A) The effects of RNAi targeting Gb’jhamt in nymphs on day 1 of the third instar. (Left) Adult control males and males treated with Gb’jhamt-targeted RNAi (). (Right) Control females and females treated with Gb’jhamt-targeted RNAi (). The RNAi-treated nymphs underwent precocious adult metamorphosis at the seventh instar. (B and C) The supernumerary molts caused by targeting of Gb’myo by RNAi (the middle cricket in the first three boxes of B and C) were inhibited by simultaneously injecting RNAi targeting Gb’mad and RNAi targeting Gb’jhamt (the crickets at the far right in all boxes in B and C), and adults of approximately normal body size eventually formed (far right boxes in B and C). As a control, RNAi targeting DsRed2 was injected into nymphs; the resulting crickets are shown at the left of each box in B and C. (D and E) Body length (D) and weight (E) of the nymphs and adults treated with RNAi targeting Gb’myo and RNAi targeting Gb’jhamt were compared with nymphs and adults treated with RNAi targeting DsRed2. Weeks postinjection are indicated on the x axis. The data presented are the mean ± SD. (F) A qPCR analysis of the transcript levels of Gb’myo, Gb’jhamt, and Gb’CYP15A1 detected on day 5 for the fifth instar (D5 fifth) and day 1 for the sixth instar (D1 sixth) after third-instar nymphs were injected with RNAi targeting Gb’myo and RNAi targeting Gb’jhamt. The transcript levels for the fifth-instar nymphs injected with DsRed (control) on day 5 (D5 fifth) were set to 1. The asterisks represent significant differences between the control nymphs and nymphs treated with RNAi targeting Gb’myo + Gb’jhamt (Student’s t test; *P < 0.05; **P < 0.005). The data presented are the mean ± SD. (Scale bars: 10 mm.)

In recent studies, the transcriptional repressor Brinker (Brk) has been found to be a Dpp target that negatively regulates Dpp signaling in Drosophila (26, 27). Therefore, we examined the levels of Gb’brk mRNA in nymphs that received RNAi targeting genes related to Gb’Dpp/Gbb signaling (Gb’mad, Gb’medea, and Gb’gbb) or Gb’Myo signaling (Gb’myo and Gb’smox). In the former experiments, depletion of Gb’mad (Fig. 4E), Gb’medea (Fig. S5C), and Gb’gbb (Fig. S5D) resulted in an increase in Gb’brk mRNA levels in fourth- and sixth-instar nymphs on day 1. These results suggest that Gb’brk expression is negatively regulated by Gb’Dpp/Gbb signaling (Fig. 5A). Thus, we speculated that the transcriptional repressor Gb’Brk plays a role in negatively regulating Gb’Dpp/Gbb signaling, and it may regulate the repression of Gb’jhamt. To examine the latter possibility, RNAi targeting Gb’brk was injected into third-instar nymphs. Although the control animals exhibited normal molting, the majority (25 of 27) of the Gb’brk RNAi-treated nymphs arrested in the early developmental stages. In addition, increased expression of Gb’jhamt mRNA was detected in the Gb’brk RNAi-treated nymphs during the fourth and sixth instars on day 1, but no effect was observed on day 5 (Fig. 4F). These results suggest that Gb’Brk may be associated with the negative regulation of Gb’jhamt (Fig. 5B). To investigate whether the reduction in Gb’jhamt expression in the Gb’mad-depleted nymphs was caused by concomitant up-regulation of Gb’brk (Fig. 5A), dual RNAi targeting Gb’mad and Gb’brk were injected into third-instar nymphs. Subsequently, Gb’mad RNAi-dependent repression of Gb’jhamt that previously was observed in the fourth and sixth instars on day 1 was not rescued by depletion of Gb’brk (Fig. 4F). Thus, repression of Gb’jhamt in the nymphs that received RNAi targeting Gb’mad appeared to be independent of increased Gb’brk expression (Fig. 5C). Consequently, our results suggest that both an up-regulation of Gb’jhamt and a down-regulation of Gb’brk are controlled by the Gb’Dpp/Gbb/Mad signaling pathway (Fig. 5 D and F). Gb’brk expression was markedly decreased on days 1 or 5 in the supernumerary nymphs (3′ and fourth instars) with depletion of Gb’myo (Fig. 4E) and Gb’smox (Fig. 5D and Fig. S5B). Therefore, we propose that induction of Gb’jhamt and repression of Gb’brk that are dependent on the function of Gb’Mad may be blocked by Gb’Myo/Smox signaling (Fig. 5 E and F).

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Fig. 5.

Regulation of Gb’jhamt expression. (A–E) Schematic diagrams of Gb’brk and Gb’jhamt transcriptional regulation based on the results obtained from experiments targeting Gb’mad (A and E), Gb’brk (B), Gb’mad + Gb’brk (C), and Gb’smox (D) genes by RNAi. Gray denotes gene depletion and transcriptional regulatory effects by RNAi. Red arrows indicate the down- and up-regulation of target gene expression. (F) A diagram depicting the function of Dpp/Gbb (blue) and Myo (pink) signaling pathways in the regulation of jhamt expression and JH action. P indicates the phosphorylation of Mad and Smox.

Previous studies have showed that Daughters against dpp (dad) is an inhibitory Smad that is able to antagonize Dpp signaling genetically in Drosophila (28). Regulation of dad also has been reported to be affected by the function of Mad and Smox (29). To understand how Gb’Myo signaling prevents Gb’Dpp/Gbb signaling, we investigated whether the Gb’Dpp/Gbb and Gb’Myo signaling pathways are associated with expression of Gb’dad. When RNAi targeting Gb’mad was injected into third-instar nymphs, lower levels of Gb’dad mRNA were detected (Fig. S5E). In contrast, depletion of Gb’smox by RNAi had no effect on Gb’dad expression (Fig. S5E). These results suggest that Gb’dad may represent a target gene downstream of Gb’Dpp/Gbb signaling and that Gb’Myo signaling may regulate the expression of Gb’brk and Gb’jhamt through the control of the Gb’Dpp/Gbb signaling pathway (Fig. 5 E and F).

Overall, the results of the experiments performed suggest that Gb’Myo signaling suppresses Gb’jhamt expression that is induced by Gb’Dpp/Gbb signaling and that this suppression leads to an inhibition of JH biosynthesis and an induction of metamorphosis.