RTKs were screened to determine their involvement in the JH signal pathway in HaEpi cells and larvae.

(A) The roles of RTKs in JH III-induced Kr-h1, Vg, Jhi-1, and Jhi-26 expression were determined by RNAi of Rtk genes (1 μg/mL dsRNA, 48 h, 1 μM JH III for 12 h). DMSO as solvent control. The relative mRNA levels were calculated via the 2-ΔΔCT method and the bars indicate the mean ± SD. n = 3. Multiple sets of data were compared by analysis of variance (ANOVA). The different lowercase letters show significant differences. (B) The examples of phenotype after Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck knockdown in larvae. Scale = 1 cm. (C) Phenotype percentage and pupation time after Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck knockdown in larvae. The time was recorded from the bursting of the head shell of the 5th instar to pupal development. Images were collected after more than 80% of the larvae had pupated in the DMSO control group. Two-group significant differences were calculated using Student’s t test (*p<0.05, **p<0.01 indicate the significant difference between the percentages of the delayed pupation in dsGFP + JH III control group and gene knockdown) based on three replicates, n = 30 × 3 larvae.

RTKs involved in JH III-regulated Ca2+ increase and protein phosphorylation.

(A) The level of Ca2+ after Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck knockdown in HaEpi cells. The cells were incubated with dsRNA (the final concentration was 1 μg/mL for 48 h) and AM ester calcium crimson dye (3 μM, 30 min). F0: the fluorescence intensity of HaEpi cells without treatment. F: the fluorescence intensity of HaEpi cells after different treatments. DMSO as solvent control. (B) The interference efficiency of dsRNA in HaEpi cells. (C) Western blotting was performed to analyze TAI-His and MET1-His phosphorylation after treatment with dsRNA and JH III (1 μM, 3 h). Phos-tag: phosphate affinity SDS‒PAGE gel, Normal: normal SDS‒PAGE gel, which was a 7.5 or 10% SDS‒PAGE gel. The results of three independent repeated western blots were statistically analyzed by ImageJ software. The p value was calculated by Student’s t test based on three independent replicate experiments. The error bar indicates the mean ± SD.

CAD96CA and FGFR1 could bind JH III.

(A) Cell membrane localization of the overexpressed CAD96CA-CopGFP-His, FGFR1-CopGFP-His, NRK-CopGFP-His and OTK-CopGFP-His. GFP: green fluorescence of RTKs fused with a green fluorescent protein. WGA: red fluorescence, the cell membrane was labeled with wheat germ agglutinin. DAPI: nuclear staining. Merge: the pictures of different fluorescent-labeled cells were combined. The cells were observed with a fluorescence microscope. Scale bar = 20 μm. (B) Coomassie brilliant blue staining of the SDS‒PAGE gel showed the purity of the separated CAD96CA-CopGFP-His, FGFR1-CopGFP-His, NRK-CopGFP-His, and OTK-CopGFP-His proteins. (C) Saturation binding curves of CAD96CA-CopGFP-His, FGFR1-CopGFP-His, NRK-CopGFP-His and OTK-CopGFP-His. (D) Saturation binding curves of CAD96CA-CopGFP-His were incubated with the indicated compounds. (E) The binding and competition curves of CAD96CA and methoprene. (F) Saturation binding curves of FGFR1-CopGFP-His were incubated with the indicated compounds. (G) The binding and competition curves of FGFR1 and methoprene. (H) The binding curves of CAD96CA mutants and JH III. (I) The binding curves of FGFR1 mutants with JH III. Data are mean ± SE of three replicates.

The roles of CAD96CA and FGFR1 in larval development were determined by CRISPR/Cas9 system-mediated mutants.

(A and B) Schematic showing the injection mixture of the CRISPR/Cas9 system. The black line refers to the genome of H. armigera; the yellow blocks correspond to exons. The Cas9 nuclease (in grey) was targeted to genomic DNA by Cad96ca-gRNA or Fgfr1-gRNA with an ∼20-nt guide sequence (orange) and a scaffold (blue). The guide sequence pairs with the DNA target (orange sequence on the top strand), which requires the upstream sequence of the 5’-CGG-3’ adjacent motif (PAM; green). Cas9 induces a double-strand break (DSB) ∼3 bp upstream of the PAM (black triangle). (C) Summary of G0 mutations. (D) Images showing WT and mutant H. armigera phenotypes. The scale represents 1 cm. (E) Morphology and statistical analysis of WT and mutant H. armigera. Both Cad96ca and Fgfr1 mutant larvae showed earlier pupation than WT controls. (F and G) qRT‒PCR showing the mRNA levels of the JH/20E response genes in WT and mutant H. armigera. (H) Schematic showing the CRISPR/Cas9 editing in HaEpi cells by pIEx-4-BmU6-Cad96ca-gRNA-Cas9-GFP-P2A-Puro and pIEx-4-BmU6-Fgfr1-gRNA-Cas9-GFP-P2A-Puro recombination vectors. (I) qRT‒PCR showing the mRNA levels of Kr-h1 in WT and mutant HaEpi cells. (J) pIEx-GCaMP5G was overexpressed in WT and mutant HaEpi cells, and calcium mobilization was detected. Green fluorescence shows the calcium signal. The concentration of JH III was 1 μM, and that of CaCl2 was 1 mM. The scale bar represents 100 μm.

CAD96CA and FGFR1 participated in JH-induced calcium ion mobilization.

(A) The level of Ca2+ after Cad96ca and Fgfr knockdown in Sf9 cells. The cells were incubated with dsRNA (the final concentration was 1 μg/mL for 48 h). F0: the fluorescence intensity of Sf9 cells without treatment. F: the fluorescence intensity of Sf9 cells after different treatments. DMSO as solvent control. (B) Effect of JH III on calcium ion levels in S2 cells after Cad96ca and Htl knockdown. (C) The response of calcium ion levels to JH III in HEK-293T cells. (D) The analysis of calcium ion flow after HEK-293T cells overexpressed RTK. DMSO as solvent control. His as tag control. (E and F) The calcium was quantitated after HEK-293T cells overexpressed CAD96CA-His, FGFR1-His, and mutants. The error bar indicates the mean ± SD.

A diagram illustrating CAD96CA and FGFR1 transmitting juvenile hormone signal for gene expression.

(1) JH binds to cell membrane receptors CAD96CA and FGFR1 to increase intracellular calcium, phosphorylation of MET1 and TAI to enhance their function in gene expression to maintain larval status. (2) On the other hand, JH enters cells freely via diffusion to bind its intracellular receptor MET. MET interacts with TAI and then binds to the JH response element (JHRE, containing the E-box core sequence, in the Kr-h1 promoter region) to promote gene expression to keep larval status. Therefore, JH III transmits signal by either cell membrane receptor and intracellular receptor at different stages in the signaling.