Fig 1.
Expression profiles of DopEcR by western blot analysis and 20E upregulates DopEcR expression.
A. Expression profiles of DopEcR detected by anti-DopEcR antibody as the method description. β-actin as control. B. Specificity analysis of the antibodies against DopEcR and β-actin by western blotting with epidermis protein of 6th-72 h larvae. SDS-PAGE gel in western blot is 12.5%. C. Quantification of the data in A by ImageJ. Error bars indicate the mean ± standard deviations (SD) of three times repetition. D. qRT-PCR analysis of 20E regulation on DopEcR mRNA in larval head by time and dose. Equal volume of diluted DMSO was injected as control. The relative mRNA level was counted by 2–ΔΔCT. E. and F. Western blot analysis of 20E regulation on DopEcR in larvae head as the treatment in D. SDS-PAGE gel in western blot is 12.5%. β-actin was detected as control. Statistical analyses of C and D by Student’s t test and ImageJ. Error bars showed the mean ± SD of three times repetition. Asterisks manifest significant differences by Student’s t test (*p < 0.05; **p < 0.01).
Fig 2.
The 20E titer and dopamine titer in H. armigera and 20E repressed larval feeding and promoted pupation.
A. 20E titer of the whole body from 3rd instar larvae to adult 3 day (A3). B. Hemolymphal dopamine titer from 3rd instar larvae to adult 3 day (A3). 3F: third instar feeding larvae; 3M: third instar molting larvae; 4F: fourth instar feeding larvae; 4M: fourth instar molting larvae. 5F: fifth instar feeding larvae; 5M: fifth instar molting larvae; 6–0 h, 6–24 h, 6–48 h, 6–72 h, 6–96 h, and 6–120 h: sixth instar larvae from 0 h to 120 h; P0, P2, P4, P6, P8, P10: zero to 10-day-old pupae. A1-A3: adult day 1 to day 3 from female adult. MM: metamorphic molting. C. The food consumption was quantitated as the amount of diet eaten at 0–12, 12–24, 24–36, 36–48 and 48–60 h after 500 ng 20E injection. DMSO was used as control. The amount of the food consumption was weighted for the quantity of feeding. D. The increment weight of body was quantitated at 0–12, 12–24, 24–36, 36–48 and 48–60 h after 500 ng 20E injection. DMSO was used as control. E. The food consumption was quantitated as the amount of diet eaten at 0–12, 12–24, 24–36, 36–48 and 48–60 h after flupentixol injection. PBS was used as control. The amount of the food consumption was weighted for the quantity of feeding. F. The increment weight of body was quantitated at 0–12, 12–24, 24–36, 36–48 and 48–60 h after flupentixol injection. PBS was used as control. Error bars show the mean ± SD of three biological repeats. Significant differences were calculated by Student’s t test (*p < 0.05; **p < 0.01).
Fig 3.
Silencing DopEcR by feeding dsRNA from first instar to 6th instar larvae repressed growth, feeding and pupation.
A. The mRNA expression levels of DopEcR in tissues of 6th-96 h larvae were quantified by qRT-PCR after DopEcR knockdown. B. Phenotypes after feeding dsRNA. a. Phenotypes of larvae, with larger body size after feeding dsDopEcR. b. Phenotypes of pupae. The bar represents 1 cm. C. Western blotting showing the knockdown efficiency by dsRNA feeding (12.5% SDS gel with β-actin used as a control). ImageJ software was used to transform the image data. D. Ratio of Phenotypes. The data were calculated from 30 larvae × 3 experiments. E. F. and G. The average quantity of feeding, body weight and body length of insect from first instar larvae (1-F) to pupae (P-2), analyzed individually with 30 insects. All data were performed in triplicate. The bars indicate the mean ± SD. (*p < 0.05; **p < 0.01).
Fig 4.
DopEcR silence by dsRNA injection delayed pupation and tissue remodeling.
A. The mRNA expression levels of DopEcR in tissues of 6th-72 h larvae were quantified by qRT-PCR after DopEcR knockdown (sixth instar 6 h larvae for the first dsRNA injection, thrice at a 24 h interval, 500 ng dsRNA/larva). 20E (500 ng/larva). DMSO was a solvent control. B. Phenotypes after DopEcR knockdown as the experiments in A. Phenotypes were obtained at 98 h after first dsDopEcR injection. Scale bar = 1 cm. C and D. Statistical analysis of pupation time from sixth instar 6 h larvae developing to pupae and the percentage of different phenotypes. E. Tunel and HE-stained midgut and fat body after knockdown of DopEcR, observed at 60 h after the first dsRNA injection. LM: larval midgut; IM: imaginal midgut. Tunel Red fluorescence indicates apoptotic signal. HE staining showing the morphology of the midgut and fat body. The yellow bars represent 100 μM. F. The red fluorescence intensity (tunnel signal) were counted by ImageJ and represented with mean ± SD. Tunnel signal was calculated with red area (% of the tissue). Error bars show the mean ± SD of three biological repeats. Significant differences were calculated by Student’s t test (*p < 0.05; **p < 0.01).
Fig 5.
20E antagonizes dopamine function.
A. Apoptosis signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP+20E (5 μM), dsDopEcR+20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the NucView caspase-3 activity assay kit. a. The ratio of apoptotic cells (green) to the total cells (blue) in the field view was obtained. B. Proliferation signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP+20E (5 μM), dsDopEcR+20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the 5-ethynyl-2′-deoxyuridine (EdU) kit (Ribobio, Guangzhou, China). b. The ratio of proliferation cells (green) to the total cells (blue) in the field view was obtained. DAPI stained the nucleus (blue). Statistical analysis using the data from 100 × 3 cells. The yellow bars represent 100 μM. C. Western blot analysis of 20E (5 μM) or DA (10 μM) induced proteins phosphorylation for 30 min. a. b. and c. AKT-RFP-His, AKT -RFP-His, and CDK10-RFP-His. 7.5% SDS-PAGE gel. Error bars show the mean ± SD of three times’ biological repetition. ImageJ software was used to transform the image data. Significant differences were calculated by Student’s t test (*p < 0.05; **p < 0.01) or one-way analysis of variance (ANOVA, p < 0.05).
Fig 6.
Roles of DopEcR in 20E pathway.
A. DopEcR was localized in plasma membrane. Red: The cell membrane was marked by wheat germ agglutinin (WGA). Green: DopEcR protein stained with an anti-DopEcR antibody and secondary antibody labeled with Alexa-488. Blue: nucleus stained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Observed by confocal microscope. Scale bar = 25 μm. B. and C. qRT-PCR showing mRNA levels of 20E-response genes after DopEcR knockdown in 6th-72 h larval epidermis (500 ng dsRNA/larva) and in HaEpi cells (2 μg dsRNA/mL, 48 h once, followed 1 μM 20E for 12 h). β-actin was regard as control. D and E. Ca2+ levels by Image Pro-Plus analysis after DopEcR knockdown in HaEpi cells, which representing three repeats. Cells were treated with dsGFP (2 μM) and dsDopEcR (2 μM) for 48 h and AM ester calcium crimson dye (3 μM) in DPBS for 30 min, and then by 20E (1 μM) and CaCl2 (1 mM), respectively. F: fluorescence intensity of HaEpi cells after different treatments. F0: fluorescence intensity before different treatments. Fluorescence was recorded per 6 s by confocal microscope photographs at 555 nm wavelength laser. F. and G. DopEcR knockdown repressed the 20E-triggered cAMP increase. HaEpi cells were transfected with dsDopEcR or dsGFP for 48 h followed by incubation with 2 μM 20E. Error bars showed the mean ± SD of three times repetition. Asterisks manifest significant differences by Student’s t test (*p < 0.05; **p < 0.01).
Fig 7.
20E promotes DopEcR coupling with Gαq and Gαs and protein phosphorylation via DopEcR.
A. Co-immunoprecipitation (Co-IP) to detect DopEcR coupling with Gαq and Gαs under 20E (2 μM for 30 min) induction. DMSO was solvent control. a. b. and c. Input: the levels of Gαq-RFP-His, DopEcR-His, Gαs-RFP-His and RFP-His in the cells detected by antibodies against RFP or His. β-actin was a loading control. Co-IP: Anti-RFP antibody co-immunoprecipitated Gαq-RFP-His and DopEcR-His, Gαs-RFP-His and DopEcR-His, or RFP-His and His. Nonspecific mouse IgG was a negative control. SDS-PAGE gel was 12.5%. B. Western blot analysis 20E-induced phosphorylation of proteins (2 μM 20E for 1 h). a. b. and c. USP1-His, CDK10-His and PKAC1-His. λPP: 0.5 μM λPPase incubation for 30 min at 30 °C. 7.5% SDS-PAGE gel. C. ChIP analyzing the involvement of DopEcR in 20E-induced EcRB1 binding to the EcRE. EcRB1-RFP-His was overexpressed in HaEpi cells for 48 h. The cells were incubated with dsDopEcR (2 μg/mL) or dsGFP (2 μg/mL) for 12 h, followed by inducing of 2 μM 20E or DMSO for 6 h. Input: non-immunoprecipitated chromatin. IgG: nonspecific mouse IgG. Primer EcRE: primers targeted to EcRE-containing DNA. Primer HR3: primers targeted to HR3 ORF. *p value via Student’s t-test based on three replicates in all figures (*p < 0.05; **p < 0.01).
Fig 8.
Modeling of the ligand-binding complex of the GPCRs.
A. B. and C. Prediction of the Surflex-Dock (SFXC) program from the SYBYL X2.0 software. Overall structures of DopEcR, ErGPCR-2, and ErGPCR-1 (gray) and docked 20E (Green), respectively. D. E. and F. A closer view of the pockets relative to docking models of DopEcR-, ErGPCR-2- and ErGPCR-1-20E complex and the amino acid residues mutated in this study. The dotted lines indicate the predicted hydrogen bonds between the amino acid residues of GPCRs and 20E. Orientation is the same in all models.
Fig 9.
Detection of 20E that was bound by the GPCRs-overexpressing cell membrane proteins and the isolated GPCRs.
A. Cell membrane localization of overexpressed GFP and GPCRs. Red: the cell membrane was marked by wheat germ agglutinin (WGA). Green: green fluorescence from GFP and various GPCRs fused with GFP. Blue: nucleus stained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Observed by confocal microscope. Scale bar = 20 μm. B. Quantity of 20E bound by 50 μg membrane proteins from HaEpi cells that was overexpressing GFP, DopEcR-GFP, DopEcR-M-GFP, ErGPCR-1-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP. C. SDS-PAGE shows the partially purified GPCRs with Coomassie brilliant blue staining used in the experiments in D. D. Quantity of 20E bound by DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP. Error bars represent the SD of three replicates. Asterisks indicate significant differences according to Student’s t-tests (*p < 0.05; **p < 0.01).
Fig 10.
A. Saturation binding curves of DopEcR-GFP and DopEcR-M-GFP to 20E. B. Saturation binding curves of ErGPCR-2-GFP and ErGPCR-2-M-GFP to 20E. Nonspecific binding was analyzed with GFP-His tag. All the experiments were performed using 10 μg of isolated protein in 50 μL EIA buffer. C. SDS-PAGE to show the highly purified GPCRs used in A, B, F and G. D. The cAMP levels of HaEpi cells overexpressed with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP under 20E-triggered. HaEpi cells were transfected with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP for 48 h followed by incubation with 2 μM 20E. E. Ca2+ levels after DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP overexpressed in HaEpi cells. Cells were transfected with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP for 48 h and AM ester calcium crimson dye (3 μM) in DPBS for 30 min, and then by 20E (1 μM) and CaCl2 (1 mM), respectively. F: fluorescence intensity of HaEpi cells after different treatments. F0: fluorescence intensity before different treatments. Fluorescence was recorded per 6 s by confocal microscope photographs at 555 nm wavelength laser and then analyzed using Image Pro-Plus software. F. Saturation binding curves of DopEcR-GFP to DA. Nonspecific binding was analyzed with GFP-His tag. G. The competition curves of DopEcR-GFP to 20E and Dopamine. The ELISA plate coated with DopEcR-GFP was incubated with 20 pmol of DA in the presence of increasing concentrations of the different ligands (20E and DA). Error bars represent the SD of three replicates. Asterisks indicate significant differences according to Student’s t-tests (*p < 0.05; **p < 0.01).
Fig 11.
Diagram illustrating the roles of DopEcR in larval feeding and 20E-regulated metamorphosis.
Dopamine titer in hemolymph decreased and 20E titer in whole larvae increased from 6 instar 6 h to 6 instar 120 h larvae. Dopamine binds DopEcR to promote AKT-P levels, larval feeding and cell proliferation (1). The increased 20E binds to DopEcR to initiate 20E signal, including the interaction of DopEcR and Gαs and Gαq, increase of cAMP and Ca2+ levels, and protein phosphorylation of PKAC1, CDK10 and USP1 (2). The phosphorylated-CREB (CREB-P) binds to CRE to promote 20E-regulated gene transcription [12] (3). The phosphorylated-USP1 and CDK10 forms EcRB1/USP1 transcription complex and bind to ecdysone response element (EcRE) to regulate gene expression for insect metamorphosis [10, 11] (4).