*3.3. The EsFOXO-like mRNA and Protein Expression Characteristic in Hepatopancreas at Three Molting Stages*

The mRNA and protein expression of *Es*FOXO-like in hepatopancreas at pre-molt, post-molt and inter-molt were measured. It was shown that *Es*FOXO-like was highly expressed at the pre-molt stage and lowly expressed at the post-molt stage (Figure 5a). The expression level at pre-molt was 22.01-fold (*p* < 0.01) and 3.58-fold (*p* < 0.05) of that at post-molt stage and inter-molt stage, respectively.

The results showed that there was a specific band of about 70 kDa (Figure 5b) detected with FOXO1 antibody in the hepatopancreas by western blotting (Figure 5b), indicating that the FOXO1 antibody was relatively specific. Additionally, the *Es*FOXO-like protein in the hepatopancreas was increased at the pre-molt stage and decreased at the post-molt stage. Specifically, the protein expression levels of *Es*FOXO-like at the pre-molt stage were 12.62-fold (*p* < 0.01) and 3.03-fold (*p* < 0.01) of that at the post-molt stage and the inter-molt stage, respectively (Figure 5c,d).

**Figure 5.** The mRNA and protein expression patterns of *Es*FOXO-like in hepatopancreas at different molt stages. (**a**) The relative mRNA expression level of *Es*FOXO-like in hepatopancreas at different molt stages by qRT-PCR. (**b**) The specific antibody detection of FOXO1 antibody in hepatopancreas. (**c**) The protein expression levels of *Es*FOXO-like in hepatopancreas at different molt stages using western blotting. (**d**) Statistical analysis of western blot performed in (**c**). Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). The results were analyzed using one-way ANOVA. Asterisks indicate significant differences (\* *p* < 0.05 and \*\* *p* < 0.01).

#### *3.4. The EsFOXO-like mRNA and Protein Expression Levels after Inhibition of EsFOXO-like*

The inhibitory effect of the FOXO inhibitor (AS1842856) on *Es*FOXO-like expression in hepatopancreas was evaluated by qRT-PCR and western blotting, respectively. After AS1842856 injection, the *Es*FOXO-like mRNA transcripts in the AS1842856 group were significantly decreased, representing 0.11-fold (*p* < 0.05) of that in the DMSO group (Figure 6a). The western blotting assay showed that the band against the *Es*FOXO-like antibody in AS1842856 injected crabs was thinner compared to that in the PBS group (Figure 6b). The protein expression level of *Es*FOXO-like in the hepatopancreas was also decreased (Figure 6c) in the AS1842856 group (0.14-fold of that in the PBS group, *p* < 0.05).

**Figure 6.** The mRNA and protein expression levels of *Es*FOXO-like gene in hepatopancreas after being injected with AS1842856 and *Es*FOXO-like dsRNA. (**a**) The *Es*FOXO-like mRNA expression level in hepatopancreas in AS1842856 injected crabs. (**b**,**c**) The protein expression levels of *Es*FOXOlike in hepatopancreas in AS1842856 and DMSO group. (**c**) Statistical analysis of blot performed in (**b**). (**d**) The mRNA expression level of *Es*FOXO-like in hepatopancreas after knockdown of *Es*FOXO-like by RNAi. (**e**,**f**) The protein expression levels of *Es*FOXO-like in hepatopancreas in *Es*FOXO-like-RNAi group and EFPG-RNAi group. (**f**) Statistical analysis of blot performed in (**e**). Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Asterisks indicate significant differences (\* *p* < 0.05), "ns" = not significant. The results were analyzed using Student's *t* test.

Furthermore, the RNAi assay was employed to reduce the expression of *Es*FOXOlike mRNA, and the silencing efficiency of *Es*FOXO-like was detected by qRT-PCR and western blotting, respectively. The mRNA expression of *Es*FOXO-like in the hepatopancreas was significantly decreased 24 h after the injection of *Es*FOXO-like dsRNA, which was 0.46-fold compared with that of the EGFP-RNAi group (*p* < 0.05) (Figure 6d). No significant difference in the *Es*FOXO-like expression level was observed between the EGPF-RNAi group and the PBS group (Figure 6d). Similarly, the western blotting assay demonstrated that the intensity values of the *Es*FOXO-like band in *Es*FOXO-like-RNAi crabs decreased significantly (0.51-fold of that in the EGFP-RNAi group; *p* < 0.05) (Figure 6e,f).
