*3.5. The 20E Concentration and Molting-Related Genes mRNA Expression Levels after EsFOXO-like Inhibition*

To investigate the regulation of *Es*FOXO-like gene in the 20E pathway, the 20E concentration and mRNA transcripts of *Es*EcR, *Es*RXR and *Es*MIH were detected after AS1842856 and *Es*FOXO-like dsRNA injection, respectively. The 20E concentration was significantly increased in the AS1842856 group, which was 1.65-fold (*p* < 0.05) of that in the DMSO group (Figure 7a). Similarly, the *Es*EcR and *Es*RXR mRNA expression levels in the hepatopancreas were both significantly increased in the AS1842856 group (5.03- and 5.18-fold compared to that in the DMSO group; *p* < 0.05) (Figure 7b,c). Conversely, the *Es*MIH mRNA expression

level in eyestalk decreased significantly, which was 0.12-fold compared to that in the DMSO group (*p* < 0.01) (Figure 7d).

**Figure 7.** The 20E concentration and mRNA expression levels of *Es*EcR, *Es*RXR and *Es*MIH in the AS1842856 group. (**a**) The 20E concentration in serum in AS1842856 injected crabs. The data were analyzed using Mann–Whitney U test. (**b**–**d**) Relative expression levels of *Es*EcR and *Es*RXR in the hepatopancreas and *Es*MIH expression level in the eyestalk of crabs injected with AS1842856. The DMSO group was the control group. The data were analyzed by Student's *t* test. Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Asterisks indicate significant differences (\* *p* < 0.05, \*\* *p* < 0.01).

After *Es*FOXO-like was knocked down by RNAi, the 20E concentration and the mRNA expression levels of *Es*EcR and *Es*RXR in the hepatopancreas were significantly up-regulated, which were 1.49-fold (*p* < 0.01), 5.59-fold (*p* < 0.01) and 3.88-fold (*p* < 0.01) of that in the EGFP-RNAi group, respectively (Figure 8a–c). On the contrary, the *Es*MIH mRNA expression in eyestalk was significantly lower than that (0.39-fold, *p* < 0.01) in the EGFP-RNAi group (Figure 8d).

**Figure 8.** The 20E concentration and mRNA expression levels of *Es*EcR, *Es*RXR and *Es*MIH after *Es*FOXO-like was knocked down by RNAi. (**a**) The 20E concentration in serum in *Es*FOXO-like-RNAi group. (**b**–**d**) Relative expression levels of *Es*EcR and *Es*RXR in the hepatopancreas and *Es*MIH expression level in the eyestalk of *Es*FOXO-like-RNAi crabs group. The EFPG-RNAi group was the control group. Each bar represents the mean ± standard deviation of three independent biological replicates. Asterisks indicate significant differences (\*\* *p* < 0.01), "ns" = not significant. The data were analyzed by one-way ANOVA.

#### *3.6. The mRNA Expression Level of EsmTOR after EsFOXO-like Inhibition*

In order to study whether or whether not *Es*FOXO-like regulates the 20E pathway through *Es*mTOR, the expression level of *Es*mTOR was first detected after *Es*FOXO-like inhibition by AS1842856 treatment or dsRNA-*Es*FOXO-like injection. The *Es*mTOR mRNA expression levels in the hepatopancreas were up-regulated after injection of both AS1842856 and dsRNA-*Es*FOXO-like. The mRNA transcript of *Es*mTOR in the AS1842856 group was 4.49-fold (*p* < 0.01) that of the DMSO group (Figure 9a). Similarly, the relative expression level of *Es*mTOR in the hepatopancreas was significantly (*p* < 0.05) increased in the *Es*FOXO-RNAi group, which was 2.91-fold compared to that in the EGFP-RNAi group (Figure 9b).

**Figure 9.** The expression level of *Es*mTOR gene in hepatopancreas after *Es*FOXO-like inhibition. (**a**) The *Es*mTOR expression level in AS1842856-injected crabs. The data were analyzed by Student's *t* test. (**b**) The *Es*mTOR expression level in *Es*FOXO-like-RNAi group. The data were analyzed by one-way ANOVA. Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Asterisks indicate significant differences (\* *p* < 0.05 and \*\* *p* < 0.01), "ns" = not significant.

#### *3.7. The EsmTOR mRNA Transcripts after AS1842856 and Rapamycin Injection*

To further investigate whether *Es*FOXO-like regulates the 20E pathway through *Es*mTOR or not, we evaluated the expression level of *Es*mTOR in the hepatopancreas after inhibiting *Es*FOXO-like by AS1842856 injection, followed by administering Rapamycin. The mRNA expression level of *Es*mTOR in the hepatopancreas was decreased after injection of AS1842856 + Rapamycin, and the *Es*mTOR transcript in the AS1842856 + Rapamycin group was 0.21-fold of that in the AS1842856 + DMSO group (*p* < 0.05) (Figure 10).

**Figure 10.** The expression level of *Es*mTOR gene in hepatopancreas after injection with AS1842856 and Rapamycin. AS1842856 + DMSO were injected and treated as the control group. Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Asterisks indicate significant differences (\* *p* < 0.05). The data were analyzed by Student's *t* test.
