TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål)

Abstract

The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

Keywords: Nilaparvata lugens; RNA interference; juvenile hormone; target of rapamycin; vitellogenesis.

Figure 1

Phylogenetic analysis of Rheb (A), target of rapamycin (TOR) (B) and S6K (S6 kinase) (C) in insects. The tree was constructed by MEGA6 with the Maximum Likelihood method based on the amino acid sequences alignment [30]. Bootstrap test with 1000 replicates was used to analyze the evolutionary relationship and bootstrap values were shown in the cladogram. The scale bar indicates genetic distance.

Figure 1

Phylogenetic analysis of Rheb (A), target of rapamycin (TOR) (B) and S6K (S6 kinase) (C) in insects. The tree was constructed by MEGA6 with the Maximum Likelihood method based on the amino acid sequences alignment [30]. Bootstrap test with 1000 replicates was used to analyze the evolutionary relationship and bootstrap values were shown in the cladogram. The scale bar indicates genetic distance.

Figure 2

Relative mRNA levels of Rheb (A), TOR (B) and S6K (C) in the fat body of adult females upon different amino acid (AA) treatments. AD3d, newly emerged females were reared on artificial diets without AAs for three days continuously; AF3d, females were supplied with AAs for three days; AD2d, females were reared without AAs for two days; AR3d, females lacking AAs on the first and second day were supplied with AAs on the third day. Relative expression levels of Rheb, TOR and S6K mRNA in the fat body were detected by qRT-PCR using β-actin as a reference. Values were shown as mean ± SE of three independent experiments and asterisk denoted significant differences from controls (Student’s t-test, * denotes p < 0.05 and “ns” denotes p ≥ 0.05).

Figure 3

TOR pathway transduces AAs signaling that regulates vitellogenin (Vg) synthesis. (A) Knockdown efficiency of target genes in the fat body of the dsRNA of gfp, Rheb, TOR and S6K. The relative mRNA levels of these respective genes in control (gfp dsRNA) were set to 1 and the numbers above each bar indicated the percentage knockdown efficiency of corresponding gene; (B) Effect of the TOR pathway genes knockdown on the mRNA and protein levels of Vg (Student’s t-test, * denotes p < 0.05); (C) Effect of AAs on S6K phosphorylation. Western blot analysis was performed using antibody against the phospho-S6K (Thr-389); (D) RNAi-mediated knockdown of Rheb or TOR decreased S6K phosphorylation levels; (E) TOR inhibitor rapamycin effectively decreased S6K phosphorylation and Vg expression levels.

Figure 4

TOR pathway works through juvenile hormone (JH) biosynthesis to regulate Vg synthesis. (A) Relative mRNA levels of jmtN in the CA and met in the fat body of adult females after knockdown of TOR pathway genes (TOR or Rheb) and rapamycin treatment; (B) Relative mRNA and protein levels of Vg in the fat body of the dsRNA of gfp, Rheb and TOR further treated with JH III. Newly emerged females were injected with dsRNA of gfp, TOR or Rheb and reared for two days. After that, the insects were topically applied with JH III or acetone and provided with artificial diets for another day (Student’s t-test, * denotes p < 0.05); (C) Protein levels of Vg in the fat body of rapamycin-treated adult females were further topically applied with JH III.

Figure 5

JH regulates Vg synthesis independently of TOR pathway. (A) RNAi-mediated knockdown of JH pathway genes (jmtN or met) and JH III application had no effect on the expression of genes related to TOR pathway (TOR, Rheb and S6K) in the fat body; (B) Total protein was extracted and phosphorylation status of S6K in the fat body was detected by Western blot.

Figure 6

AAs, TOR, Rheb and Vg are required for ovary development and fecundity. (A) Newly emerged females were injected with dsRNA of TOR, Rheb, Vg or a control gfp and reared on artificial diets lacking AAs (−AAs) or normal diets (+AAs) for 6 days. Ovaries were dissected and photographed with a stereo microscopy SMZ18 (Nikon, Tokyo, Japan). Scale bar, 500 μm; (B) AAs deprivation and RNAi-mediated depletion of TOR or Rheb significantly reduced the numbers of laid eggs by females. Ten females were analyzed per group and three independent groups were evaluated. Since females lacking AAs laid no eggs, only egg numbers derived from the females feeding AAs (except for Vg dsRNA females) were used for statistical analysis. Student’s t-test was performed to determine significant differences in mean eggs numbers for knockdown females as compared with the gfp dsRNA-injected control group (* denotes p < 0.05).

Figure 6

AAs, TOR, Rheb and Vg are required for ovary development and fecundity. (A) Newly emerged females were injected with dsRNA of TOR, Rheb, Vg or a control gfp and reared on artificial diets lacking AAs (−AAs) or normal diets (+AAs) for 6 days. Ovaries were dissected and photographed with a stereo microscopy SMZ18 (Nikon, Tokyo, Japan). Scale bar, 500 μm; (B) AAs deprivation and RNAi-mediated depletion of TOR or Rheb significantly reduced the numbers of laid eggs by females. Ten females were analyzed per group and three independent groups were evaluated. Since females lacking AAs laid no eggs, only egg numbers derived from the females feeding AAs (except for Vg dsRNA females) were used for statistical analysis. Student’s t-test was performed to determine significant differences in mean eggs numbers for knockdown females as compared with the gfp dsRNA-injected control group (* denotes p < 0.05).

Figure 7

A proposed model for the TOR pathway and JH in regulating Vg synthesis responding to AAs stimulation. After feeding with normal diet, free AAs concentrations in the hemolymph are increased, the expression levels of the TOR pathway genes are elevated and S6K phosphorylation in the fat body is up-regulated. The activated TOR pathway works through some brain factors, like allatotropin, to induce JH biosynthesis in the corpora allata, and JH, in turn, activates expression of Vg gene in the fat body. Thus, the TOR pathway induces JH biosynthesis that, in turn, regulates AA-mediated vitellogenesis. JHAMT: juvenile hormone acid methyltransferase.