Identification of a Putative CFSH Receptor Inhibiting IAG Expression in Crabs

The crustacean female sex hormone (CFSH) is a neurohormone peculiar to crustaceans that plays a vital role in sexual differentiation. This includes the preservation and establishment of secondary female sexual traits, as well as the inhibition of insulin-like androgenic gland factor (IAG) expression in the androgenic gland (AG). There have been no reports of CFSH receptors in crustaceans up to this point. In this study, we identified a candidate CFSH receptor from the mud crab Scylla paramamosain (named Sp-SEFIR) via protein interaction experiments and biological function experiments. Results of GST pull-down assays indicated that Sp-SEFIR could combine with Sp-CFSH. Findings of in vitro and in vivo interference investigations exhibited that knockdown of Sp-SEFIR could significantly induce Sp-IAG and Sp-STAT expression in the AG. In brief, Sp-SEFIR is a potential CFSH receptor in S. paramamosain, and Sp-CFSH controls Sp-IAG production through the CFSH-SEFIR-STAT-IAG axis.


Introduction
In dioecious crustaceans, there are apparent differences between males and females, and this sexual dimorphism is caused by sex determination and differentiation [1,2]. At present, studies on the regulatory mechanisms of sexual differentiation mainly focus on endocrine regulation and related transcription factors [1,[3][4][5]. Crustaceans have unique endocrine systems. Insulin-like androgenic gland (IAG) hormone generated from the androgenic gland (AG) and crustacean female sex hormone (CFSH) secreted by the eyestalk ganglion are considered vital hormones that regulate sexual differentiation [6,7]. It is widely recognized that IAG acts principally in male sexual differentiation [6]. Although IAG receptors have been found in a variety of species [8][9][10][11][12][13], no CFSH receptor (CFSHR) has been identified.
In 2014, the identification of CFSH was initially reported from the eyestalk ganglion of the Atlantic blue crab Callinectes sapidus [7]. Subsequently, its central functions in female sexual differentiation have been well-established in a number of species [7,[14][15][16]. Aside from the eyestalk ganglion, CFSH transcripts have been detected in various tissues of many species, suggesting that CFSH has multiple biological functions [17][18][19][20][21][22][23][24][25][26]. The potential relationship between CFSH and female reproduction was speculated in the kuruma prawn Marsupenaeus japonicus and the giant freshwater prawn Macrobrachium rosenbergii [22,24]. Of note, results of recent investigations estimated that CFSH could also regulate differentiating the sex of males through inhibiting IAG expression in AG [15,16,27]. In the mud crab Scylla paramamosain as well as the Chinese mitten crab Eriocheir sinensis, transcripts of CFSH were detected in both males and females. Moreover, CFSH may suppress IAG expression in AG of these two species [16,27]. Moreover, identical outcomes were reported

Molecular Cloning and Phylogenetics of Sp-SEFIR
The full length of Sp-SEFIR (GenBank accession number: ON787957) cDNA is 2515 base pairs (bp), including a 70 bp 5 untranslated region (UTR), a 1959 bp ORF and a 486 bp 3 UTR. The polyadenylation signal (AATAAA) is located at 149 bp upstream of the polyA sequence ( Figure A1). The ORF segment was responsible for the synthesis of a polypeptide consisting of 652 amino acids (aa), with a molecular weight of 73801.91 Da and a theoretical isoelectric point (pI) of 5.27. The precursor polypeptide was inferred to possess a 23 aa signal peptide, a 23 aa transmembrane domain, a 123 aa SEFIR domain and two low-compositional complexity regions (11 and 30 aa, respectively) ( Figure 1A).
Phylogenetic analysis demonstrated that IL-17R was clustered into five clusters: IL-17RA, IL-17RB, IL-17RC, IL-17RD, as well as IL-17RE. Sp-SEFIR was classified as IL-17RD ( Figure 1B). (B) The phylogenetic tree was created with conserved SEFIR domain of IL-17R utilizing the NJ approach. The bootstrap test (1000 replicates) was utilized to display the proportion of replicate trees wherein the related taxa clustered together, and this information was presented adjacent to the branches. Sp-SEFIR was marked in red.

Tissue Distribution of Sp-SEFIR
RT-PCR demonstrated that Sp-SEFIR had a broad distribution across different tissues in males. The Sp-SEFIR mRNA expression level was relatively higher in the Y organ, testis, androgenic gland, stomach, hepatopancreas and muscle (Figure 2A).

Tissue Distribution of Sp-SEFIR
RT-PCR demonstrated that Sp-SEFIR had a broad distribution across different tissues in males. The Sp-SEFIR mRNA expression level was relatively higher in the Y organ, testis, androgenic gland, stomach, hepatopancreas and muscle (Figure 2A). The standardized expression levels of Sp-SEFIR, normalized by β-actin expression patterns, were expressed as the mean ± SEM. Statistical analysis was performed utilizing one-way analysis of variance (ANOVA) and then using Duncan's multiple range tests; "a and b", p < 0.05; n = 5). The standardized expression levels of Sp-SEFIR, normalized by β-actin expression patterns, were expressed as the mean ± SEM. Statistical analysis was performed utilizing one-way analysis of variance (ANOVA) and then using Duncan's multiple range tests; "a and b", p < 0.05; n = 5).

Expression Profile of Sp-SEFIR during AG Development
The expression profile of Sp-SEFIR in AG throughout AG advancement (stage I-III) was determined using qRT-PCR. The findings revealed that the expression patterns of Sp-SEFIR mRNA were elevated along with the development of AG to reach a peak at stage II before significantly being reduced at stage III ( Figure 2B).

Immunofluorescence Localization of Sp-SEFIR in AG
Consistent with the previous study, two forms of glandular cells (A and B) were observed in AG [41]. Type A glandular cells have a round, less heterochromatic nucleus, with a lightly stained cytoplasm and indistinct borders ( Figure 3A). In type B glandular cells, the cytoplasmic staining is dark and uniform, with hyperchromatic nuclei and well-defined borders ( Figure 3A). Sp-SEFIR is mainly located on the membrane of Type B glandular cells ( Figure 3B).

Expression Profile of Sp-SEFIR during AG Development
The expression profile of Sp-SEFIR in AG throughout AG advancement (stage I-III) was determined using qRT-PCR. The findings revealed that the expression patterns of Sp-SEFIR mRNA were elevated along with the development of AG to reach a peak at stage II before significantly being reduced at stage III ( Figure 2B).

Immunofluorescence Localization of Sp-SEFIR in AG
Consistent with the previous study, two forms of glandular cells (A and B) were observed in AG [41]. Type A glandular cells have a round, less heterochromatic nucleus, with a lightly stained cytoplasm and indistinct borders ( Figure 3A). In type B glandular cells, the cytoplasmic staining is dark and uniform, with hyperchromatic nuclei and well-defined borders ( Figure 3A). Sp-SEFIR is mainly located on the membrane of Type B glandular cells ( Figure 3B). Two types of glandular cells (type A and type B) were observed. Type A glandular cells have a round nucleus with one or two nucleoli, with a lightly stained cytoplasm and indistinct borders. In type B glandular cells, the cytoplasmic staining is dark and uniform, with hyperchromatic nuclei and well-defined borders. Immunofluorescence localization of Sp-SEFIR was performed with mouse anti-Sp-SEFIR serum (B) or mouse preimmune serum (negative control) (C). Sp-SEFIR was mainly located on the membrane of Type B glandular cells (B). Solid yellow arrows indicated type A glandular cells; solid red arrows indicated type B glandular cells.

Ligand-Receptor Interaction Analysis
Ligand-receptor interaction analysis was conducted using GST pull-down assays. Through prokaryotic expression, we obtained rHisCFSH (20 kDa) ( Figure A2), rGSTCFSH (47 kDa) ( Figure 4) and rSEFIR (65 kDa) ( Figure A3). Results of GST pull-down assay with rGSTCFSH and total protein of AG demonstrated that Sp-SEFIR was a CFSH-binding component of AG ( Figure 4). The further GST pull-down assay with rHisCFSH and rSEFIR confirmed that Sp-CFSH could specifically bind to extracellular regions of Sp-SEFIR ( Figure 5). Two types of glandular cells (type A and type B) were observed. Type A glandular cells have a round nucleus with one or two nucleoli, with a lightly stained cytoplasm and indistinct borders. In type B glandular cells, the cytoplasmic staining is dark and uniform, with hyperchromatic nuclei and well-defined borders. Immunofluorescence localization of Sp-SEFIR was performed with mouse anti-Sp-SEFIR serum (B) or mouse preimmune serum (negative control) (C). Sp-SEFIR was mainly located on the membrane of Type B glandular cells (B). Solid yellow arrows indicated type A glandular cells; solid red arrows indicated type B glandular cells.

Ligand-Receptor Interaction Analysis
Ligand-receptor interaction analysis was conducted using GST pull-down assays. Through prokaryotic expression, we obtained rHisCFSH (20 kDa) ( Figure A2), rGSTCFSH (47 kDa) ( Figure 4) and rSEFIR (65 kDa) ( Figure A3). Results of GST pull-down assay with rGSTCFSH and total protein of AG demonstrated that Sp-SEFIR was a CFSHbinding component of AG ( Figure 4). The further GST pull-down assay with rHisCFSH and rSEFIR confirmed that Sp-CFSH could specifically bind to extracellular regions of Sp-SEFIR ( Figure 5).

Analysis of Gene Expression in AG following Sp-SEFIR Silencing In Vivo
Previous studies have shown that CFSH could suppress IAG and STAT expression in S. paramamosain [27,28]. To study the involvement of Sp-SEFIR in this inhibition, we first carried out in vivo RNA interference experiments. Based on current results, compared to CPS-injected therapy, Sp-SEFIR expression was 42% inhibited ( Figure 6A). Meanwhile, the knockdown of Sp-SEFIR significantly induced Sp-IAG and Sp-STAT expression in AG ( Figure 6B,C).

Analysis of Gene Expression in AG following Sp-SEFIR Silencing In Vivo
Previous studies have shown that CFSH could suppress IAG and STAT expression in S. paramamosain [27,28]. To study the involvement of Sp-SEFIR in this inhibition, we first carried out in vivo RNA interference experiments. Based on current results, compared to CPS-injected therapy, Sp-SEFIR expression was 42% inhibited ( Figure 6A). Meanwhile, the knockdown of Sp-SEFIR significantly induced Sp-IAG and Sp-STAT expression in AG ( Figure 6B,C). , and Sp-STAT (C) were determined after in vivo injection with CPS, GFP dsRNA or SEFIR dsRNA. The gene expression levels were standardized by β-actin expression levels and expressed as mean ± SEM ("a and b", p < 0.05; one-way ANOVA followed by Duncan's multiple range tests; n = 9).

Analysis of Gene Expression in AG after Medication with rCFSH following Sp-SEFIR Silencing In Vitro
To further explore the involvement of Sp-SEFIR in IAG regulation via CFSH, we conducted interference experiments of Sp-SEFIR in vitro. According to the results, the addition of SEFIR-dsRNA inhibited Sp-SEFIR expression by 48% in vitro compared to the PBS therapy ( Figure A4A). Furthermore, we confirmed that the addition of rHisCFSH (10 −6 M) could suppress Sp-IAG expression in AG, as previously reported ( Figure A4B) [27,28].
Following Sp-SEFIR silencing in the in vitro AG explant culture system, we added rHisCFSH (   , and Sp-STAT (C) were determined after in vivo injection with CPS, GFP dsRNA or SEFIR dsRNA. The gene expression levels were standardized by β-actin expression levels and expressed as mean ± SEM ("a and b", p < 0.05; one-way ANOVA followed by Duncan's multiple range tests; n = 9).

Analysis of Gene Expression in AG after Medication with rCFSH following Sp-SEFIR Silencing In Vitro
To further explore the involvement of Sp-SEFIR in IAG regulation via CFSH, we conducted interference experiments of Sp-SEFIR in vitro. According to the results, the addition of SEFIR-dsRNA inhibited Sp-SEFIR expression by 48% in vitro compared to the PBS therapy ( Figure A4A). Furthermore, we confirmed that the addition of rHisCFSH (10 −6 M) could suppress Sp-IAG expression in AG, as previously reported ( Figure A4B) [27,28].
Following Sp-SEFIR silencing in the in vitro AG explant culture system, we added rHisCFSH (10 −6 M) and detected the mRNA expression pattern of Sp-IAG and Sp-STAT. The findings demonstrated that the knockdown of Sp-SEFIR could also significantly induce Sp-IAG and Sp-STAT expression in vitro ( Figure 7).

Analysis of Gene Expression in AG following Sp-SEFIR Silencing In Vivo
Previous studies have shown that CFSH could suppress IAG and STAT expression in S. paramamosain [27,28]. To study the involvement of Sp-SEFIR in this inhibition, we first carried out in vivo RNA interference experiments. Based on current results, compared to CPS-injected therapy, Sp-SEFIR expression was 42% inhibited ( Figure 6A). Meanwhile, the knockdown of Sp-SEFIR significantly induced Sp-IAG and Sp-STAT expression in AG ( Figure 6B,C).

Analysis of Gene Expression in AG after Medication with rCFSH following Sp-SEFIR Silencing In Vitro
To further explore the involvement of Sp-SEFIR in IAG regulation via CFSH, we conducted interference experiments of Sp-SEFIR in vitro. According to the results, the addition of SEFIR-dsRNA inhibited Sp-SEFIR expression by 48% in vitro compared to the PBS therapy ( Figure A4A). Furthermore, we confirmed that the addition of rHisCFSH (10 −6 M) could suppress Sp-IAG expression in AG, as previously reported ( Figure A4B) [27,28].
Following Sp-SEFIR silencing in the in vitro AG explant culture system, we added rHisCFSH (
Although a significant number of nucleotide sequences of CFSH have been documented in decapod crustaceans, the IL-17 domain exhibits a high degree of conservation in all identified sequences [29,30]. Interestingly, IL-17 has been identified in various invertebrates, but not in crustaceans [48]. In both vertebrates and invertebrates, IL-17 binds to different IL-17Rs through a functional dimer. Consequently, a signaling system is established between the ligand and the receptor, resulting in the initiation of downstream signals [49][50][51][52]. CFSH might be evolved from an IL-17-like original protein [24], and possibly bound to IL-17R-like polypeptides.
Here, we obtained a transcript encoding an IL-17R-like polypeptide and named it Sp-SEFIR. Sp-SEFIR shared structures similar to those of other invertebrate IL-17Rs, including a signal peptide, a transmembrane domain, a SEFIR domain and two regions with low compositional complexity. In sea vase Ciona intestinalis, the predicted IL-17R protein includes a signal peptide domain at the N-terminal, a transmembrane domain at the central part and a SEFIR domain at the cytoplasmic tail [49]. The classification of Sp-SEFIR into IL-17RD was additionally supported by the phylogenetic analysis. The results suggest that Sp-SEFIR might be the homologous protein of IL-17RD, and it belongs to the single transmembrane interleukin-17 receptor.
Our results demonstrated that Sp-SEFIR had high expression in AG. In a previous study, temporal expression profiles of CFSH and IAG during the development of androgenic glands have been identified in male S. paramamosain [27]. According to the previous study, CFSH expression showed a significant elevation during stages I and II but a marked decline during stage III. On the contrary, IAG expression levels were relatively lower at stages I-II compared to stage III [27]. In the present study, we noticed that Sp-SEFIR and CFSH exhibited similar temporal expression profiles and displayed the opposite expression trend to IAG. The current findings suggested that Sp-SEFIR may play a role in CFSH's inhibition of IAG.
On the basis of the SMART software prediction (http://smart.embl-heidelberg.de/ (accessed on 8 December 2022)), Sp-SEFIR was a single transmembrane protein. Immunofluorescence analysis further confirmed the membrane localization of Sp-SEFIR. To explore the interaction between Sp-CFSH and Sp-SEFIR, we carried out GST pull-down assays with rHisCFSH and the extracellular segment of Sp-SEFIR. The results revealed that the extracellular segment of Sp-SEFIR could bind to Sp-CFSH. The findings mentioned above indicate that Sp-SEFIR is a putative receptor for CFSH. Still, further investigations in appropriate cell lines are needed to verify the putative ligand-receptor interactions between Sp-CFSH and Sp-SEFIR in the future research.
According to the research, it was proposed that CFSH has the ability to inhibit STAT and thereby block IAG expression in the mud crab S. paramamosain [28]. Both in vivo and in vitro silencing experiments were performed to investigate the probable involvement of Sp-SEFIR in this process. We noticed that following Sp-SEFIR silencing, both Sp-IAG and Sp-STAT expression levels significantly increased. Thus, it is reasonable to speculate that Sp-CFSH binds to Sp-SEFIR, inhibits Sp-STAT activity and eventually suppresses IAG expression. To verify this hypothesis, in vitro experiments were conducted. The results showed that the addition of rCFSH could suppress Sp-IAG expression in AG explants, which was consistent with the previous results [27]. Similar to the result of in vivo experiment, Sp-SEFIR silencing could induce Sp-IAG and Sp-STAT expression in 9 of 19 AG explants, indicating that knockdown of Sp-SEFIR could relieve inhibition of rCFSH on Sp-IAG and Sp-STAT expression. Collectively, these results suggest that Sp-SEFIR works as a CFSH receptor aimed at regulating IAG expression by suppressing Sp-STAT expression in S. paramamosain.
Furthermore, we noticed that Sp-SEFIR exhibited a broad distribution in multiple tissue types, indicating that the biological functions of CFSH are not limited to regulating sexual differentiation. According to the previous study, CFSH probably evolved from an original protein that resembles IL-17 for the conserved IL-17 domain in the mature peptide [14,29,30]. IL-17 is widely involved in the immune response by activating different downstream signal pathways (MAPK, NF-κB and JAK/STAT pathways) in vertebrates and invertebrates [34,37,39,[50][51][52][53][54][55]. Based on sequence similarity, we speculate that CFSH likely has an involvement in the immune response of various organs. Further comprehensive investigations are required to elucidate the functions of CFCH in various physiological actions.

Animals
According to previous studies, the development stages of AG were classified as follows: Stage I, AG was small and contained fewer secretory cells, which were attached to the spermaduct; Stage II, AG was clearly linear, cells were clustered or cross-linked into cords, and glands expanded into the surrounding connective tissues; Stage III, AG was largest, and tissue hyperplasia occurred in some areas, which contained the largest number of secretory cells; Stage IV, development of AG stopped, AG degenerated rapidly, and its size was smaller than that in stage II and III [41,56].
In this study, S. paramamosain at AG development stage I (body weight: 48.4 ± 4.5 g, carapace width: 6.6 ± 0.5 cm), stage II (body weight: 146.7 ± 9.8 g, carapace width: 11.7 ± 1.0 cm) and stage III (body weight: 249.1 ± 13.2 g, carapace width: 14.7 ± 0.5 cm) were selected as experimental materials. Upon arrival at the laboratory, individuals were subjected to a period of acclimatization, during which they were exposed to controlled conditions consisting of 27 ± 1 • C in addition to a salinity level of 26 ± 0.5 ppm.

cDNA Cloning of Sp-SEFIR
The transcriptome of S. paramamosain was utilized to obtain the Sp-SEFIR transcript. Moreover, the AG's total RNA was retrieved by utilizing TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the guidelines provided by the supplier. The 3 and 5 untranslated regions (UTR) of Sp-CFSHR were acquired through the utilization of rapid amplification of cDNA ends (RACE) technique, employing the SMART TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) in accordance with the producer's guidelines. The validation of open reading frame (ORF) was performed via synthesizing the first-strand cDNA from 1 µg of total RNA utilizing the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The ORF of Sp-SEFIR was confirmed by specific primers Sp-SEFIR-OF/OR (Table A1). Utilizing LA Taq polymerase (TaKaRa, Dalian, China), polymerase chain reaction (PCR) was conducted as per these criteria: 94 • C for 5 min; 35 cycles of 94 • C for 30 s, 56 • C for 30 s and 72 • C for 100 s, and then through 72 • C for 10 min final extension. PCR outcomes were observed with 1.5% agarose (Biowest, Kansas City, MO, USA) gel electrophoresis before being connected to the pMD19-T vector (Takara, Dalian, China) for sequencing. Table A1 lists the primer sequences.

Quantitative Real-Time PCR (qRT-PCR) Assays
The primers utilized for qRT-PCR were obtained from previous investigations [27,28]. The determination of the amplification effect of all primer pairs was conducted prior to their utilization in qRT-PCR tests. The cDNA underwent a dilution of a four-fold magnitude utilizing water that was free of RNases. Components in a 20 µL qRT-PCR reaction system were: 10 µL of 2 × PCR main mixture containing SYBR Green, 2 µL of diluted cDNA, 0.5 µL of every primer and 7 µL of water. The experiment was conducted using a 7500 rapid RT-PCR (Applied Biosystems, CA, USA), and the experimental parameters utilized for the reaction were: 95 • C for 2 min, then 40 cycles of 95 • C for 15 s, 58 • C for 30 s, and 72 • C for 30 s. The 2 −∆∆Ct approach was employed to measure the outcome, with the reference gene being β-actin (GenBank accession number: GU992421).

Tissue Distribution of Sp-SEFIR
The reverse transcription-PCR (RT-PCR) was utilized to detect the distribution profile of Sp-SEFIR in various tissues (eyestalk ganglion, cerebral ganglion, thoracic ganglion, Y organ, heart, testis, androgenic gland, stomach, hepatopancreas, muscle and epidermis) of S. paramamosain (n = 3). The procedures for extracting total RNA and first-strand cDNAs synthesized in accordance with the guidelines outlined in the relevant Section 4.2. The Sp-SEFIR-F/R was used as a primer, β-actin (GenBank accession no: GU992421) was amplified as a positive control, which was achieved utilizing Ex Taq polymerase (TaKaRa, Dalian, China), and the circumstances were: 94 • C for 5 min; 35 cycles of 94 • C for 30 s, 55 • C for 30 s and 72 • C for 30 s before an extension at 72 • C for 5 min. The PCR outcomes underwent analysis through the utilization of 1.5% agarose gel electrophoresis and were subsequently imaged with a UV detector (Geldoc, Thermo Fisher Scientific, Madrid, Spain).

Sp-SEFIR Expression Profile throughout AG Development
qRT-PCR determined the Sp-SEFIR mRNA expression patterns in AG at stage I-III (n = 5). Total RNA was extracted, first-strand cDNAs were synthesized, and qRT-PCR was conducted as mentioned before (Sections 4.2 and 4.3).

Immunofluorescence Assays
Immunofluorescence assays for Sp-SEFIR were performed with AG attached to the subterminal portion of ejaculatory ducts. We entrusted Shanghai GL Biochem Co., Ltd. (Shanghai, China) to produce the Sp-SEFIR antibody.
The tissues were first preserved in modified Bouin's fixative (25 mL 37-40% formaldehyde, 75 mL saturation picric acid in addition to 5 mL glacial acetic acid) for one night at 4 • C. Following gradient alcohol drying, tissues were immersed in paraffin and prepared for 5 µm slices. After being dewaxed and rehydrated, parts of tissue sections were subjected to Hematoxylin-Eosin (HE) staining for histological observation. Meanwhile, other tissue sections were repaired in antigen repair solution (EDTA Antigen Retrieval Solution, pH 8.0; Sangon Biotech, Shanghai, China) at 99 • C for 20 min and cooled down to room temperature naturally. After antigen retrieval, slides were blocked with 5% bovine serum antigen (BSA) in 1 × PBS for 30 min at room temperature. After that, the tissue samples were incubated with Sp-SEFIR antibodies (1:500 dilution) or preimmune serum (negative control) at 37 • C for 2 h. Upon using PBS for washing, tissue samples were incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor ® 488) preadsorbed (1:100, Abcam, Cambridge, MA, USA) diluted with 1% BSA in 1 × PBS for 45 min at room temperature. Subsequently, tissue samples were incubated with DAPI (1 µg/mL, Beyotime, Shanghai, China) for nuclear staining. Following washing one time in PBS, the slides were locked up using mounting medium, antifading (Solarbio, Beijing, China) and photographed with the BGIMAGING Cellview 4.11 system.

Expression of Sp-CFSH Recombinant Proteins and Sp-SEFIR Extracellular Domain Recombinant Protein
In this study, we expressed two recombinant Sp-CFSH proteins: Sp-CFSH recombinant protein with only 6 × His tag (rHisCFSH) and Sp-CFSH recombinant protein containing both 6 × His tag and GST (rGSTCFSH). rHisCFSH was expressed and purified according to the previous study [27,28]. rGSTCFSH was also expressed utilizing a prokaryotic expression system. The fragment encoding mature peptide of Sp-CFSH was cloned into the pET-GST vector, utilizing BamH I and Nhe I restriction enzyme sites. The generated constructs (pET-GST-CFSH-His), which included GST and 6 × His tags, were transformed into E. coli TransB (DE3) and induced for 20 h at 16 • C, following the application of isopropyl-beta-D-thiogalactopyranoside (IPTG) at a level of 1 mM. Upon harvest using centrifugation (8000× g, 10 min, 4 • C), the bacteria were disrupted by ultrasonic waves. We selected the supernatant for further purification with glutathione sepharose 4B (Solarbio, Beijing, China) according to the guidelines.
The recombinant protein of Sp-SEFIR extracellular fragment containing GST and 6 × His tag (rSEFIR) was also expressed with the same prokaryotic expression system as rGSTCFSH. The fragment encoding the extracellular segment of Sp-SEFIR was cloned into the pET-GST vector with restriction enzyme sites (EcoR I and Nhe I). The generated constructs (pET-GST-SEFIR-His) were transformed into E. coli TransB (DE3) and induced for 6 h at 25 • C after adding IPTG (0.2 mM final concentration). rSEFIR was purified from the supernatant of crude cell extracts with glutathione sepharose 4B (Solarbio, Beijing, China).

GST Pull-Down Assays
Herein, we conducted two GST pull-down assays to detect the protein-protein interaction between Sp-CFSH and Sp-SEFIR. First, we performed GST pull-down assay with rGSTCFSH and the total protein of AG to detect whether Sp-SEFIR was a CFSH-binding component of AG. After that, a GST pull-down assay with rHisCFSH and rSEFIR was performed, aimed at exploring binding regions of Sp-SEFIR.
The total protein of AG was extracted with PP11-Universal Protein Extraction Reagent (Aidlab, Beijing, China) according to the instructions. rGSTCFSH was obtained as previously described (Section 4.7). Upon being incubated at 4 • C for 30 min, rGSTCFSH was immobilized in the Glutathione Sepharose 4B (Solarbio, Beijing, China). Then, the total protein of AG was added and incubated at 4 • C for 2 h, and the unbound protein was washed away using PBS. A column volume of glutathione elution buffer was added and incubated for 10 min to elute bound proteins, and the supernatant was collected by centrifugation. The proteins that were eluted underwent analysis through the utilization of SDS-PAGE and Western blot techniques, with the aid of the Sp-SEFIR antibody and the anti-His mouse monoclonal antibody. GST protein (with 6 × His tag) was utilized as the negative control.
rHisCFSH and rSEFIR were obtained as described above (Section 4.7). Upon being incubated at 4 • C for 30 min, rSEFIR was immobilized on the Glutathione Sepharose 4B (Solarbio, Beijing, China). Subsequently, rCFSH was introduced and subjected to incubation at a temperature of 4 • C for 1 h. The protein that did not bind was eliminated through PBS washing. A column volume of glutathione elution buffer was added and incubated for 10 min to elute the bound proteins, and the supernatant was collected by centrifugation (500× g, 5 min, 4 • C). The eluted proteins were analyzed by SDS-PAGE and Western blot using anti-His mouse monoclonal antibody. GST protein (with 6 × His tag) was used as the negative control.

Silencing Experiment In Vivo
Fragments of Sp-SEFIR and green fluorescent protein gene (GFP) were cloned into pGEMT-Easy Vector to prepare the linearized DNA templates. The dsRNA synthesis was performed using T7 and SP6 polymerase. GFP dsRNA was synthesized as the negative control. Individuals (body weight: 48.4 ± 4.5 g, carapace width: 6.6 ± 0.5 cm, n = 27) in stage I of AG development were equally divided at random into three groups: GFP-dsRNAinjected, SEFIR-dsRNA-injected and crustacean physiological saline (CPS)-injected [57]. Crabs were injected with either 1 µg/g of dsRNA or an equivalent amount of CPS. The injection was repeated 24 h after the first injection. Then, 72 h after first injection, crabs were anesthetized on ice for 5 min and then AGs were dissected. The interference efficiency of Sp-SEFIR and expression levels of Sp-IAG as well as Sp-STAT, were detected using qRT-PCR. Furthermore, the RNAs extraction, cDNA synthesis and qRT-PCR analysis of samples were conducted in accordance with the methods previously outlined.

In Vitro Experiment: Sp-SEFIR Interference
The in vitro explant culture system was modified and used to investigate the regulatory role of Sp-SEFIR in the inhibitory process of IAG expression mediated by CFSH [27].
Individuals (body weight: 146.7 ± 9.8 g, carapace width: 11.7 ± 1.0 cm, n = 7) at AG stage II were selected for in vitro experiments. The AG explants of the same individual were split into three equal groups and treated with 200 µL L-15 medium containing GFP-dsRNA (1 µg/mL final), SEFIR-dsRNA (1 µg/mL final), or CPS with the identical quantity, respectively. Upon incubating at 26 • C for 6 h, the culture media was changed with 200 µL of L-15 media that contained a concentration of 10 −6 M rCFSH. After a 12 h cultivation period, AG explants were collected to extract total RNA. Expression levels of Sp-IAG, Sp-STAT were detected using qRT-PCR. RNA extraction, cDNA synthesis, as well as qRT-PCR analysis of samples, were performed according to the criteria mentioned above (Sections 4.2 and 4.3).

Statistical Analysis
Statistical analysis was conducted utilizing SPSS 18.0 program. The provided data exhibited a normal distribution in accordance with the Kolmogorov-Smirnov test. Levene's test was employed to assess the homogeneity of variance, and significant variations were determined employing one-way analysis of variance (ANOVA), subsequently employing the Duncan test. p < 0.05 was judged significant, while p < 0.01 was judged extremely significant. All findings are expressed as mean ± SEM.

Conclusions
To summarize, we detected a CFSH receptor aimed at regulating IAG expression via the protein interactions experiment and the biological function experiment. As far as we know, this study represents the initial documentation of CFSH receptors. Furthermore, we confirm that CFSH regulates IAG expression in AG through the CFSH-SEFIR-STAT-IAG axis in the mud crab S. paramamosain. Our findings, as mentioned earlier, offer novel perspectives on molecular pathways that have roles in differentiating sex in crustaceans, in addition to substantiating the pleiotropic effects of CFSH.  Acknowledgments: We express our gratitude to all laboratory members for their valuable input and productive deliberations.

Conflicts of Interest:
The authors declare no conflict of interest.
Appendix A Table A1. Primers used in this study.