**2. Materials and Methods**

#### *2.1. Crabs and Sample Preparation*

Healthy Chinese mitten crabs (*E. sinensis*) weighing about 10 g were provided by a crab farm in Lianyungang, Jiangsu province. The crabs were cultivated in aerated water at 25–26 ◦C for at least one week to acclimate to the test conditions.

Nine crabs were selected for measuring the tissue distribution of *Es*FOXO mRNA expression levels. Hemocytes, heart, hepatopancreas, stomach, muscles, gills, and eyestalks were collected. Meanwhile, the hepatopancreas was collected from three crabs at each molting stage, including the post-molt stage, the inter-molt stage and the pre-molt stage [21,22]. Additionally, hemolymph (about 750 μL) was collected from the walking legs of the Chinese mitten crabs with a pre-cooling anticoagulant solution [41]. The hemolymph from three crabs was pooled into one sample and then centrifuged at 800× *g*, 4 ◦C for 10 min to collect the hemocytes and serum. In total, three replicate samples were processed for the analyses of tissue distribution of the *Es*FOXO-like mRNA, and three replicate samples were processed for each molting stage (made from 9 individual crab samples for each molting stage). The tissues were stored at −80 ◦C in TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Three crabs were pooled together as one parallel, and there were three parallels (including 9 individuals) for sampling at each molting stage and in various tissues [42,43].

#### *2.2. Identification and Sequence Analysis of EsFOXO-like*

A gene encoding FOXO (designed as *Es*FOXO-like) was identified by screening and downloading the genome of *E. sinensis* from NCBI (NCBI accession No. CL100111224\_L02). Briefly, all the gene sequences annotated as FOXO were searched and obtained based on the *E. sinensis* genome annotation results. Then, the sequence alignment and domain analysis was performed, and, finally, the *Es*FOXO-like gene was screened. The primers *Es*FOXO-F and *Es*FOXO-R were designed to clone the ORF of *Es*FOXO-like according to the sequence of *Es*FOXO-like (Table 1). The Takara Ex Taq® DNA Polymerase (RR001Q, Takara, Otsu, Shiga, Japan) was used as the polymerase in the PCR reaction system. PCR amplification of hepatopancreas cDNA was performed as follows: one cycle at 95 ◦C for 5 min and 35 cycles at 95 ◦C for 30 s, 57 ◦C for 30 s, 72 ◦C for 1 min and 72 ◦C for 10 min. The PCR product was gel-purified and inserted into a pMD19-T simple vector (Takara) and verified by DNA sequencing. The recombinant plasmid (pMD19-T-*Es*FOXO-like) was transformed into competent cells of *Escherichia coli* Trans5α (TransGen Biotech, Beijing, China), as follows: The recombinant plasmid (5 μL) and *Escherichia coli* Trans5α (100 μL) were mixed and then placed on ice for 45 min, at 42 ◦C in a water bath for 90 s and on ice again for 2 min immediately. The transformants were incubated in Luria–Bertani (LB) medium, and three positive clones were selected and sequenced. The validated sequence of *Es*FOXO-like has been submitted with the GenBank accession number OR115551.


**Table 1.** Sequences of the primers used in the study.

Tm: Annealing temperature; Size: Amplicon size.

The cDNA sequence of *Es*FOXO-like was blasted against the GenBank database (www.ncbi.nlm.nih.gov/blast) (accessed on 8 November 2022). The amino acid sequence and protein domain of *Es*FOXO-like were analyzed by the Expert Protein Analysis System (https://web.expasy.org/translate/) (accessed on 15 November 2022) and the online SMART tool (http://smart.embl-heidelberg.de/) (accessed on 20 November 2022), respectively. Multiple sequence alignment was performed with the Clustal X multiple alignment program. The phosphorylation sites were predicted by NetPhos-3.1 tool (https://services.healthtech.dtu.dk/services/NetPhos-3.1/) (accessed on 1 March 2023). The phylogenetic tree of the FOXO gene was constructed by using the maximum likelihood method and the MEGA 11.0 software. The reliability of the branching was tested using 1000 bootstrap samples.

#### *2.3. FOXO Inhibitor and Rapamycin Treatment*

In order to investigate the influence of *Es*FOXO-like on both the concentration of 20E and the expression levels of genes associated with molting, the expression level of *Es*FOXO-like was experimentally suppressed. For this purpose, the FOXO inhibitor AS1842856 (S8222, Selleck, UT, USA) was used, for which the inhibitory effect time was set to 24 h [44,45]. AS1842856 was dissolved in dimethyl sulfoxide (DMSO) (1% diluted in PBS) (Beyotime). Eighteen crabs at the inter-molt stage were divided into AS1842856 and DMSO groups. The crabs from each group were injected with 100 μL AS1842856 (1 μg/μL) and DMSO, respectively. The inhibitor and DMSO were injected into the hemolymph from the membrane of the third posterior walking leg on the right with slight modifications to the previously described methods [46]. Then, at 24 h post-injection of AS1842856 and DMSO, the hepatopancreas was collected and used for the detection of the mRNA expression of *Es*FOXO, *Es*EcR, *Es*RXR and *Es*mTOR. The eyestalks were also collected for detecting the mRNA expression of *Es*MIH. Finally, the upper liquid of hemolymph after centrifugation was obtained as serum, and the serum was isolated for the detection of ecdysone concentration.

Rapamycin (HY-10219, MCE, Dallas, TX, USA) was used to inhibit mTOR activity [47]. According to a previous study, it has been confirmed that mTOR could stimulate 20E synthesis [30]. Moreover, the mTOR inhibitor Rapamycin can cause impaired secretion of

20E [29]. Therefore, to explore whether *Es*FOXO-like regulates the 20E pathway through *Es*mTOR or not, rapamycin was administrated to *Es*FOXO-like inhibited crabs. Rapamycin was also dissolved in DMSO [48], and the effect time for Rapamycin was set to 12 h [49]. Eighteen inter-molt crabs were divided into the AS1842856 + Rapamycin group and the AS1842856 + DMSO group. Overall, 100 μL AS1842856 (1 μg/μL) was administered to *Es*FOXO-like inhibited crabs 24 h after the AS1842856 injection. Then, the crabs in the AS1842856 + Rapamycin group and the AS1842856 + DMSO group received 100 μL Rapamycin (2 μg/μL) and DMSO injection, respectively. At 12 h after injection, the mRNA expression levels of *Es*EcR and *Es*RXR in the hepatopancreas, *Es*MIH expression levels in the eyestalk and the 20E concentration in serum were collectively detected.

#### *2.4. RNA Isolation, cDNA Synthesis and Quantitative Real-Time PCR Analysis*

Total RNA was extracted from different tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized with 1 μg of total RNA using the Prime Script™ RT reagent Kit with gDNA Eraser (Takara, Otsu, Shiga, Japan) according to the protocol of the manufacturer. The cDNA was synthesized by a primer mix containing Oligo dT and random hexamers, and the synthesis was conducted at 37 ◦C for 15 min, 85 ◦C for 5 s. The cDNA was stored at −80 ◦C for quantitative real-time PCR (qRT-PCR) analysis.

The qRT-PCR reactions were performed using the SYBR® Premix Ex Tap™ (Takara, Otsu, Shiga, Japan) with a PCR amplification procedure of 95 ◦C for 30 s, 40 cycles at 95 ◦C for 5 s and 60 ◦C for 30 s on the ABI PRISM 7500 Sequence Detection System. An incremental increase of 0.5 ◦C/5 s was conducted for melting curve analyses. The standard curve was performed using ten-fold dilutions of the cDNA templates from the hepatopancreas for each primer pair to determine the efficiency of each primer. In addition, non-template controls were tested to check for primer-dimers and contaminated samples. The gene-specific primers of *Es*EcR (GenBank accession No. KF732874.1) and *Es*β-actin (No. HM053699) were designed according to our previous studies [42,50] (Table 1). The gene-specific primers of *Es*FOXO-like, *Es*mTOR (No. MT920347.1), *Es*MIH (No. DQ341280.1) and *Es*RXR (No. MK604180.1) were designed using the qRT-PCR primer design website (https://www. genscript.com/tools/real-time-pcr-taqman-primer-design-tool) (accessed on 13 December 2022). Additionally, in the study, *Es*β-actin acted as an internal control [46,51–53]. The specificity of primers was evaluated by 1% agarose gel electrophoresis and melting curve analysis. The relative expression levels were normalized to the control samples using the comparative threshold cycle (2−Ct) method [54].

#### *2.5. Detection of 20-Hydroxyecdysone (20E) Concentration*

The separated serum was used to detect ecdysone concentration, and it was measured using the crab ecdysone ELISA Kit according to the manufacturer's protocol [42]. The plates were first precoated with purified ecdysone antibodies. After that, 10 μL of serum (diluted 1:5) and standard samples were incubated in the thermostat bath at 37 ◦C for 30 min, the plate was washed five times and then the chromogenic agent and stop buffer were added. The absorbance (450 nm) was measured using a microtiter plate reader (BioTek, Winooski, VT, USA). Lastly, the 20E concentration of the samples was calculated by the standard curve that was constructed based on absorbance and standard concentration.

#### *2.6. RNA Interference Assay*

The double-stranded RNA (dsRNA) of *Es*FOXO-like and EGFP were synthesized according to the method described in a previous report [42]. T7 promoter-linked primers, including *Es*FOXO-RNAi-F and *Es*FOXO-RNAi-R and EGFP-RNAi-F and EGFP-RNAi-R (Table 1), were used to amplify the DNA fragment of *Es*FOXO-like and EGFP, respectively. The fragments were used as templates to synthesize dsRNA. The dsRNA was synthesized by using the in vitro Transcription T7 Kit (for siRNA synthesis) (6140, Takara) according to the instructions. Additionally, the RNA integrity was examined by electrophoresis, and the concentration was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fisher

Scientific, Wilmington, DE, USA). The dsRNAs of *Es*FOXO-like and EGFP were dissolved in PBS at a final concentration of 1 μg μL<sup>−</sup>1.

Twenty-seven crabs were randomly divided into three groups (PBS group, EGFP-RNAi group and *Es*FOXO-like-RNAi group) with nine individuals in each group to investigate the RNAi efficacy. The EGFP-RNAi group was employed as the control group according to previous studies [42]. The crabs in the PBS group, the EGFP-RNAi group and the *Es*FOXOlike-RNAi group received an injection of 100 μL PBS, EGFP dsRNA and *Es*FOXO-like dsRNA, respectively. The hepatopancreas, eyestalk and serum were collected from three crabs in each group at 24 h after the dsRNA or PBS injection. There were three replicates for each group, and the tissues from three crabs were taken as one replicate. To evaluate the RNAi efficacy, the mRNA and protein expression levels the in hepatopancreas were detected by qRT-PCR and western blotting, respectively. In addition, the mRNA expression levels of *Es*EcR, *Es*RXR and *Es*mTOR in the hepatopancreas and the *Es*MIH expression level in the eyestalk together with 20E concentration in serum were also analyzed after knocking down *Es*FOXO-like mRNA expression.
