Identification of Crustacean Female Sex Hormone Receptor Involved in Sexual Differentiation of a Hermaphroditic Shrimp

The neurohormone crustacean female sex hormone (CFSH) contains a highly conserved interleukin-17 (IL-17) domain in the mature peptide. Although CFSH has been demonstrated to stimulate female sexual differentiation in crustaceans, its receptors (CFSHR) have been poorly reported. The present study identified an IL-17 receptor (named Lvit-IL-17R), a candidate of CFSHR, from the protandric simultaneous hermaphroditic (PSH) shrimp Lysmata vittata through GST pulldown assays and RNAi experiments. Lvit-IL-17R is a transmembrane protein with an SEFIR (similar expression as the fibroblast growth factor and IL-17R) domain, as determined through sequence analysis. A GST pulldown experiment confirmed the interactions between the type I CFSHs (CFSH1a and CFSH1b) and Lvit-IL-17R. Meanwhile, the RNAi results revealed that Lvit-IL-17R displays similar functions to type I CFSHs in regulating sexual differentiation and gonad development. In brief, Lvit-IL-17R is a potential receptor for type I CFSHs aimed at regulating the sexual differentiation of the PSH species. This study helps shed new light on the mechanism of sexual differentiation among crustaceans.


Introduction
The sexual systems in decapod crustaceans are diverse [1,2].Most species exist as functional females or males throughout their lives [1].In rare cases, some crustaceans may transform into an intersex form under the effect of parasitic infections, abnormal environmental sex determination, genetic abnormity, and pollution [3].Notably, some or all Caridean species are naturally intersex [1].These intersex species first mature as males with ovotestis containing both testicular and ovarian morphologies [2,4,5] and can be classified into two types.
In species that undergo complete sex reversal (e.g., strictly sequential protandric hermaphroditism, or SPH), female germ cells are located on the inner side of the ovotestis and are surrounded by male germ cells.As the female germ cells mature, testicular morphologies completely degenerate, and the ovotestis transforms into an ovary [4,5].In species that eventually possess both male and female reproductive functions (e.g., protandric simultaneous hermaphroditism or PSH), a relatively obvious boundary divides ovotestis into the ovarian region on the anterior sides and the testicular region on the posterior sides (in the dorsal view).Additionally, the testicular region partially degrades as the ovarian regions mature [2,6,7].
IL-17 is a specific group of cytokines that are crucial in the hosts' defense against microbial organisms and the development of inflammatory diseases widespread throughout the animal kingdom [31][32][33].After IL-17 was first identified in purple sea urchins, Strongylocentrotus purpuratus [34,35], this cytokine and its signaling pathways related to specific molecules have been reported in other invertebrates [31,36].However, IL-17 was not reported in decapod crustaceans until a recent investigation identified IL-17 homologs in Pacific whiteleg shrimps, Penaeus vannamei [31].When these IL-17 proteins were compared with other identified CFSHs, they were considered as CFSH of the species [30].Recently, a candidate CFSH receptor was identified in mud crabs, Scylla paramamosain, which have a conversed SEFIR (similar expression as the fibroblast growth factor and IL-17Rs) domain [37].Thus, it is reasonable to consider that CFSH is indeed an IL-17 homolog in decapod crustaceans, and CFSH receptors may also share similar structures with the IL-17R of other species.
The peppermint shrimp L. vittata is a small caridean species from the genus Lysmata, of which most shrimps are popular ornamental species and display a unique PSH sexual system [2,38].It is also suggested as a good model organism for PSH shrimps for its impressive reproductive fecundity and short generation time [11].Previous studies have demonstrated that two type I CFSHs (Lvit-CFSH1a and Lvit-CFSH1b) act in concert in regulating the development of the female external features of the species [10].Furthermore, because of a regulatory feedback loop between type I CFSH and IAG, CFSHs also inhibit IAG expression to suppress the male sexual differentiation of the PSH species [10].In the present study, the cDNA of Lvit-IL-17R was cloned to explore expression profiles.A glutathione s-transferase (GST) pulldown assay was employed to detect the interaction between Lvit-CFSH1a/CFSH1b and Lvit-IL-17R.Short-term and long-term silencing experiments were then performed to clarify the functional relevance between Lvit-CFSH1a/CFSH1b and Lvit-IL-17R in the sexual differentiation of the species.

Animals
L. vittata were bred in captivity at the Fisheries College of Jimei University maintained at 25-27 • C, a salinity of 30-32 PSU, and a 12 h light/dark cycle.L. vittata were provided with a commercially formulated shrimp diet on a daily basis.The gonadal development stages were defined according to Chen et al. 2019 [6].

cDNA Cloning of Lvit-IL-17R
TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract the total RNA from various tissues according to the manufacturer's protocol.An Lvit-IL-17R fragment was obtained from a transcript library.The 5 -untranslated region (5 UTR) was obtained with the SMART TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol and the rapid amplification of cDNA ends (RACE).Coding sequences (CDS) were verified using a polymerase chain reaction (PCR) with LA-Taq polymerase (TaKaRa, Dalian, China) under standard PCR conditions.The PCR primers are listed in Table A1.

The qRT-PCR Assays
The primers used for quantitative real-time PCR (qRT-PCR) were either designed with the Beacon Designer 8.21 software or sourced from a previous study [11].RT-PCR products were sequenced to ensure accuracy.The amplification efficiency of the primer pairs was also tested.qRT-PCR assays were performed with TB Green Premix Ex Taq II (2X) (TaKaRa, Dalian, China) according to the manufacturer's protocol.

Expression Profiles of Lvit-IL-17R
Reverse transcription-PCR (RT-PCR) and qRT-PCR were performed to explore the spatial expression features of Lvit-IL-17R.
First, various tissues at gonadal development stage II were dissected to detect the expression profile of Lvit-IL-17R in various tissues.Following total RNA extraction and firststrand cDNA synthesis, RT-PCR was performed with Ex-Taq polymerase (TaKaRa, Dalian, China) under standard PCR conditions.Lvit-β-actin (GenBank accession no.MT114194) was used as a positive control.PCR products were imaged and photographed using the Gel Image System (Tanon 2500B).
qRT-PCR was then performed to detect the temporal expression profiles of Lvit-IL-17R during gonadal development.The androgenic gland (AG), ovarian regions and the hepatopancreas were collected at different developmental stages (n = 4-5).Total RNA extraction and qRT-PCR assays were performed as described earlier.

GST Pulldown Assays
GST pulldown assays were conducted to detect the interactions between Lvit-CFSH1a/ CFSH1b and Lvit-IL-17R.CFSH1a and CFSH1b recombinant proteins were expressed and purified with 6 × His tag as previously described [10].The recombinant protein of the Lvit-IL-17R extracellular domain (rIL-17R) with 6 × His and GST tags was also purified using the prokaryotic expression system.The Lvit-IL-17R fragment was inserted into the PET-GST vector with EcoR I and Nhe I restriction enzyme sites and was transformed into E. coli TransB (DE3) for prokaryotic expression.After being induced at 16 • C for 20 h (isopropyl-beta-D-thiogalactopyranoside, IPTG, and 0.5 mM final concentration were added), bacterial cells were harvested.The purification of rIL-17R was then conducted using Glutathione Sepharose 4B (Solarbio, Beijing, China) following the established protocol from the supernatant of crude cell extracts.GST (with 6 × His tag) was expressed and purified using the same method as that used on the negative control after transforming the pET-GST vector into DE3.
After reloading rCFSH into the Glutathione Sepharose 4B (Solarbio, Beijing, China), rCFSH1a/rCFSH1b was added and incubated at 4 • C for 1 h.Next, unbound proteins were removed by washing them with 10 mM phosphate-buffered saline (PBS; pH 7.4).One column volume of elution buffer (50 mM Tris-Cl, 10 mM reduced glutathione, and pH 8.0) was added, and the samples were incubated for 10 min.Supernatant-bound proteins were collected through centrifugation and analyzed using SDS-PAGE and Western blotting with anti-His mouse monoclonal antibody.

Short-Term Silencing Experiment
A knockdown experiment was carried out by injecting double-stranded RNA (dsRNA) into the shrimps.A specific fragment of Lvit-IL-17R was then selected and cloned into a pGEMT-Easy vector.Next, dsRNA was synthesized with T7 and SP6 RNA Polymerase according to the manufacturer's instructions.Furthermore, the dsRNA of green fluorescent protein (GFP) was synthesized as the negative control.
Synthetic dsRNA was diluted with 10 mM PBS (pH 7.4) prior to injection.Shrimps (carapace length: 3.15 ± 0.17 mm; body weight: 48.65 ± 5.94 mg) at stage I of growth were equally and randomly assigned into three treatment groups (n = 5).They were injected with ds IL-17R (2 µg/g), ds GFP (2 µg/g), or an equal volume of dilution solution (10 mM PBS, pH 7.4).Twenty-four hours post-injection, the shrimps were anesthetized on ice for 5 min.

Long-Term Silencing Experiment
For the long-term silencing experiment, shrimps (carapace length: 3.01 ± 0.11 mm; body weight: 43.47 ± 4.38 mg) (n = 13) at stage I of growth were treated with ds IL-17R (2 µg/g), ds GFP (2 µg/g), or an equal volume of dilution solution (10 mM PBS; pH 7.4).During the 36-day experimental period, the shrimps were subjected to an injection once every 4 days (10 injections in total).Twenty-four hours after the tenth injection, the shrimps were anesthetized on ice, and the carapace length and body weight of each shrimp were recorded.
Before tissue collection, the gonad and external features of the male and female external features were photographed.Samples of the AG, the ovarian region, and the hepatopancreas were collected to examine the knockdown efficiency and the effects of IL-17R silencing on the expression levels of genes related to sexual differentiation and ovarian development.The parts of gonad tissues (testicular and partial ovarian regions) were fixed in modified Bouin's Fixative Solution (Phygene, Fuzhou, China) at 4 • C for 24 h.Following the process of gradient alcohol dehydration and paraffin embedding, tissue blocks were sliced into 6 µm sections for hematoxylin and eosin (H & E) staining.

Bioinformatics and Statistical Analyses
In addition to the qRT-PCR primers, we used Primer 5.0 software to design the other primers in this study.ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/(accessed on 5 April 2023)) was employed to predict the open reading frame (ORF).SMART (http://smart.embl-heidelberg.de/(accessed on 5 April 2023)) was used to predict the signal peptides and transmembrane domains.MEGA7 software was used to generate evolutionary trees.
Statistical analyses were conducted using SPSS 18.0 software.All data display a normal distribution, as determined by the Kolmogorov-Smirnov test.The homogeneity of variances was subsequently assessed using Levene's test.Statistical significance was assessed through the utilization of one-way ANOVA, followed by Tukey's multiple range tests, with a significance level set at p < 0.05.The F values of one-way ANOVA analysis are shown.All data are represented as mean ± SD.
A phylogenetic tree was constructed with Lvit-IL-17R, the SEFIR domain-contained proteins of crustaceans, and IL-17Rs from various categories (Figure 1A).Phylogenetic analysis demonstrated that the IL-17Rs from other categories formed five major clades: IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE.Additionally, the SEFIR domain-contained proteins of crustaceans formed a unique clade, into which Lvit-IL-17R was classified.number: ON787957), with 38.4 % amino acid similarity in the SEFIR domain.
A phylogenetic tree was constructed with Lvit-IL-17R, the SEFIR domain-contained proteins of crustaceans, and IL-17Rs from various categories (Figure 1A).Phylogenetic analysis demonstrated that the IL-17Rs from other categories formed five major clades: IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE.Additionally, the SEFIR domain-contained proteins of crustaceans formed a unique clade, into which Lvit-IL-17R was classified.
A phylogenetic tree was then constructed with the IL-17 domain-contained proteins of crustaceans (CFSH and other sequences) and the IL-17 from other categories (Figure
A phylogenetic tree was then constructed with the IL-17 domain-contained proteins of crustaceans (CFSH and other sequences) and the IL-17 from other categories (Figure 1B).Phylogenetic analysis demonstrated that the IL-17 domain-contained proteins of crustaceans were aggregated into a cluster, indicating that CFSH is an IL-17 homolog of decapod crustaceans.
In the present study, the Lvit-IL-17R expression level in the AG significantly decreased by 53.74% at stage II.Subsequently, it significantly increased to 143.39% of the stage I level during stage III.Finally, the Lvit-IL-17R level expression decreased to 78.68% of the stage I level during stage IV (F 3,15 = 36.246,p < 0.05) (Figure 2B).
In the hepatopancreas, the Lvit-IL-17R expression level significantly increased to 314.84% at stage II, and then slightly reduced to 265.63% of the stage I level, and eventually, at stage IV, it downregulated to almost the same levels (111.76%) as those during stage I (F 3,15 = 12.274, p < 0.05) (Figure 2B).decapod crustaceans.

Expression Profiles of Lvit-IL-17R
Lvit-IL-17R is widely expressed in various tissues (Figure 2A).Lvit-IL-17R in A hepatopancreas, and ovarian regions has different expression profiles.
In the present study, the Lvit-IL-17R expression level in the AG significantly creased by 53.74% at stage II.Subsequently, it significantly increased to 143.39% of stage I level during stage III.Finally, the Lvit-IL-17R level expression decreased to 78.6 of the stage I level during stage IV (F3,15 = 36.246,p < 0.05) (Figure 2B).
In the hepatopancreas, the Lvit-IL-17R expression level significantly increased 314.84% at stage II, and then slightly reduced to 265.63% of the stage I level, and even ally, at stage IV, it downregulated to almost the same levels (111.76%) as those dur stage I (F3,15 = 12.274, p < 0.05) (Figure 2B).
In the ovarian regions, the Lvit-IL-17R mRNA expression levels increased gradua with gonadal development.The Lvit-IL-17R mRNA expression levels significantly creased to 338.37% of the stage I level during stage II.The increase continued, with a p expression at stage III at 916.07% of the stage I level.With the maturation of ovarian gions, at stage IV, the Lvit-IL-17R expression level significantly decreased to 413.87% the stage I level (F3,15 = 57.831,p < 0.05) (Figure 2B).In the ovarian regions, the Lvit-IL-17R mRNA expression levels increased gradually with gonadal development.The Lvit-IL-17R mRNA expression levels significantly increased to 338.37% of the stage I level during stage II.The increase continued, with a peak expression at stage III at 916.07% of the stage I level.With the maturation of ovarian regions, at stage IV, the Lvit-IL-17R expression level significantly decreased to 413.87% of the stage I level (F 3,15 = 57.831,p < 0.05) (Figure 2B).

Ligand-Receptor Interaction Analysis
The analysis of ligand-receptor interaction was performed through the utilization of GST pulldown assays.Through prokaryotic expression, recombinant proteins CFSH1a (21.0 kDa, containing 6 × His tag), CFSH1b (24.8 kDa, containing 6 × His tag), and IL-17R (62.8 kDa, containing 6 × His tag) (Figure 3) were obtained.The results of the GST pulldown assays demonstrate that rCFSH1a and rCFSH1b can bind to rIL-17R specifically rather than GST (Figure 3).
The analysis of ligand-receptor interaction was performed through the utilization of GST pulldown assays.Through prokaryotic expression, recombinant proteins CFSH1a (21.0 kDa, containing 6 × His tag), CFSH1b (24.8 kDa, containing 6 × His tag), and IL-17R (62.8 kDa, containing 6 × His tag) (Figure 3) were obtained.The results of the GST pulldown assays demonstrate that rCFSH1a and rCFSH1b can bind to rIL-17R specifically rather than GST (Figure 3).

Ligand-Receptor Interaction Analysis
The analysis of ligand-receptor interaction was performed through the utilization of GST pulldown assays.Through prokaryotic expression, recombinant proteins CFSH1a (21.0 kDa, containing 6 × His tag), CFSH1b (24.8 kDa, containing 6 × His tag), and IL-17R (62.8 kDa, containing 6 × His tag) (Figure 3) were obtained.The results of the GST pulldown assays demonstrate that rCFSH1a and rCFSH1b can bind to rIL-17R specifically rather than GST (Figure 3).

Effects of Lvit-IL-17R Silencing on Female Sexual Differentiation
The retardation of female sexual traits (female gonopores) was observed in the absence of Lvit-IL-17R.In the PBS and dsRNA GFP treatments, female gonopores were two distinct bulges located on the coxa of the third pair of pereiopods.The top edge of the female gonopores was surrounded by lush plumose setae.Following long-term Lvit-IL-17R silencing, the female gonopores became flattened and visually inapparent.The feathery setae surrounding female gonopores became sparse and were relatively short (Figure 6A).
The retardation of female sexual traits (female gonopores) was observed in the absence of Lvit-IL-17R.In the PBS and dsRNA GFP treatments, female gonopores were two distinct bulges located on the coxa of the third pair of pereiopods.The top edge of the female gonopores was surrounded by lush plumose setae.Following long-term Lvit-IL-17R silencing, the female gonopores became flattened and visually inapparent.The feathery setae surrounding female gonopores became sparse and were relatively short (Figure 6A).The results demonstrated that Lvit-IL-17R silencing impeded the process of ovarian development.In the dsRNA Lvit-IL-17R treatment, the ovarian regions became smaller and filled with less-developed germ cells (Figure 6B).Relatively smaller oocytes and fewer follicular cells were observed during the dsRNA Lvit-IL-17R treatment (Figure 6B).The results demonstrated that Lvit-IL-17R silencing impeded the process of ovarian development.In the dsRNA Lvit-IL-17R treatment, the ovarian regions became smaller and filled with less-developed germ cells (Figure 6B).Relatively smaller oocytes and fewer follicular cells were observed during the dsRNA Lvit-IL-17R treatment (Figure 6B).We then measured the average oocyte diameter of the three treatments.The average oocyte diameters in both control groups were 58.89 ± 0.47 µm (n = 5) and 59.05 ± 1.06 µm (n = 6).This metric decreased to 37.00 ± 0.95 µm after Lvit-IL-17R silencing (n = 6) (F 2,14 = 201.459,p < 0.05) (Figure 6C).Meanwhile, subsequent to the knockdown of Lvit-IL-17R, the genes associated with ovarian development were also significantly repressed.The Lvit-Vg (F 2,14 = 244.880,p < 0.05) and Lvit-VgR (F 2,14 = 17.844, p < 0.05) expression levels were significantly downregulated by 99.72% and 65.83%, respectively (Figure 6D).
Meanwhile, Lvit-IL-17R knockdown dramatically promotes testicular development.Gonadal histology analysis revealed that the injection of Lvit-IL-17R dsRNA yielded different germ cell compositions.For both control groups, the majority of the cells were poorly developed germ cells, including spermatogonia (Sg) and spermatocytes I (Sc I), indicating less active spermatogenesis (Figure 7D).On the contrary, a more mature profile of germ cell compositions was established with the dsRNA Lvit-IL-17R treatment.Abundant mature germ cells, such as the spermatid (Sd) and spermatozoa (Sz), were observed in the testicular regions (Figure 7D).
Previous studies have shown that the IL-17 domain is highly conserved in the mature peptides of all known CFSHs [24][25][26][27][28][29][30].Also, we found that the recently identified IL-17 proteins were indeed CFSHs described in previous studies (Table A3) [30,31].Thus, we suspected that CFSH was the IL-17 homolog in decapod crustaceans.To further confirm this suspicion, we constructed a phylogenetic tree with the IL-17 domain-contained proteins of crustaceans (CFSH and other sequences) and IL-17 from other categories (Figure 1B).Via phylogenetic analysis, we demonstrated that the IL-17 domain-contained proteins of crustaceans form a unique clade similar to other known IL-17.Thus, it is reasonable to suggest that CFSH is the IL-17 in decapod crustaceans.
By examining the tissue expression profile, Lvit-IL-17R was confirmed to be widely distributed in various tissues.Previous studies have also demonstrated that CFSH could suppress IAG expression in the AG and stimulate Vg expression in the hepatopancreas and VgR expression in the ovarian region, indicating that the AG, the hepatopancreas, and the ovarian region were target tissues of CFSH in these particular species [10].To elucidate this, we examined the expression profiles in these three tissues.The results demonstrated that Lvit-IL-17R exhibited similar temporal expression profiles of Lvit-CFSH1b in the hepatopancreas and the ovarian region.However, Lvit-IL-17R displayed the opposite expression trend to those of Lvit-IAG1 and Lvit-IAG2, which are more highly expressed in the male phase rather than the female phase [11].The present findings suggest that Lvit-IL-17R may play a role in CFSH's inhibition of IAG and CFSH's promotion of ovarian development.
To explore the interaction between Lvit-IL-17R and Lvit-CFSHs, we performed GST pulldown assays with rCFSH1s and the extracellular segment of IL-17R.The results revealed that the extracellular segment of Lvit-IL-17R could bind to both Lvit-CFSHs.These findings suggest that Lvit-IL-17R is likely a receptor of type I CFSH in PSH shrimps.
According to the previous studies, both type I CFSHs (CFSH1a and CFSH1b) coregulate sexual differentiation in L.vittata [10].In detail, CFSH1a and CFSH1b regulate the development of female gonopores.Furthermore, CFSH1b has been proposed to regulate ovarian development via vitellogenesis [10].Although CFSH1a and CFSH1b suppress IAG expression in the AG, only CFSH1b is closely related to male sexual differentiation in the species [10].Thus, an in vivo silencing experiment was conducted to explore whether Lvit-IL-17R had any functional relevance with CFSHs in the PSH species.
In the short-term silencing experiment, during the subsequent knockdown of Lvit-IL-17R, there was a notable increase in the expression levels of both Lvit-IAGs, whereas Lvit-Vg and Lvit-VgR were downregulated.Similar expression trends were observed for these genes in a previous study involving Lvit-CFSH1b [10].These results suggest a correlation between Lvit-CFSH1b and Lvit-IL-17R in ovarian development and male sexual differentiation in this crustacean species.
To clarify the regulation roles of Lvit-IL-17R in sexual differentiation, we performed a long-term silencing experiment utilizing shrimps at the early gonadal development stages.The results indicate that Lvit-IL-17R knockdown hindered the development of female phenotypes (female gonopores), suggesting its regulatory role in female sexual differentiation.Moreover, the development of ovarian regions was also suppressed.Following Lvit-IL-17R knockdown, the ovarian regions were less developed with significantly smaller oocytes.Simultaneously, the transcripts of genes related to ovarian development were also inhibited.Similar results have been produced from the knockdown of either CFSH1a or CFSH1b in previous studies [10].These findings suggest a relationship between Lvit-IL-17R and the two type I CFSHs (CFSH1a and CFSH1b) aimed at the female sexual differentiation of the PSH species.
Previous studies have shown that Lvit-IAG1 and Lvit-IAG2 co-regulate the development of male external phenotypes and spermatogenesis in PSH shrimps, L. vittata [11].As IAG is regulated by type I CFSH through a negative feedback loop in both dioecious and hermaphrodite crustaceans [10,23], male sexual differentiation is also negatively regulated by CFSH1b in L. vittata [10].The present results demonstrate that Lvit-IL-17R negatively regulates male sexual differentiation in this species.Lvit-IL-17R knockdown induces the promotion of the development of both testicular regions and male-related external phenotypes.These results also correspond to studies involving CFSH1b [10].The combined results suggest that Lvit-IL-17R is a receptor of CFSH1b in the sexual differentiation of the PSH species.
Moreover, Lvit-IL-17R is also related to the biological functions of CFSH1a.A previous study demonstrated that Lvit-CFSH1a could also regulate female and male sexual differentiation by inhibiting Lvit-IAG2 expression.Following Lvit-CFSH1a knockdown, the development of female gonopores was hindered, and the expression of Lvit-IAG2 was promoted in the AG [10].In the present study, Lvit-IL-17R knockdown also resulted in similar results.Moreover, Lvit-IL-17R could also bind to Lvit-CFSH1a.These findings suggest that Lvit-IL-17R is also a receptor of Lvit-CFSH1a.

Conclusions
In summary, a receptor of type I CFSH aimed at regulating sexual differentiation was identified through protein interaction and biological function experiments.To the best of the authors' knowledge, the present study provides the first report on CFSH receptors involved in sexual differentiation in PSH species.We confirmed that type I CFSHs promote the development of both female external features and ovarian regions via Lvit-IL-17R in the PSH species.Moreover, Lvit-IL-17R is involved in the inhibition of IAG through CFSH, suppressing male sexual differentiation.These findings expand our understanding of

Figure 1 .
Figure 1.Phylogenetic tree of IL-17Rs (A) and CFSHs (B).Phylogenetic analysis was conducted using the Neighbor-Joining method, which is based on the Poisson model of MEGA7.The sequences used in the phylogenetic tree analysis of IL-17Rs and CFSHs are listed in Tables A2 and A3, respectively.The numbers indicate bootstrap values based on 1000 replicates, as shown next to the branches.Lvit-IL-17R is indicated by a red solid arrow.

Figure 1 .
Figure 1.Phylogenetic tree of IL-17Rs (A) and CFSHs (B).Phylogenetic analysis was conducted using the Neighbor-Joining method, which is based on the Poisson model of MEGA7.The sequences used in the phylogenetic tree analysis of IL-17Rs and CFSHs are listed in Tables A2 and A3, respectively.The numbers indicate bootstrap values based on 1000 replicates, as shown next to the branches.Lvit-IL-17R is indicated by a red solid arrow.

Figure 2 .
Figure 2. Spatial and temporal expression profiles of Lvit-IL-17R in L. vittata.(A) The tissue dis bution profile of Lvit-IL-17R was generated using the RT-PCR assays of shrimps at the gona development of stage II.Lvit-β-actin was used as a positive control.(B)The expression profileLvit-IL-17R in the androgenic gland, hepatopancreas, and ovarian regions during gonadal de opment through qRT-PCR.The Lvit-IL-17R expression levels were standardized using Lvit-β-a expression levels ("A, B and C", "a and b", an "α, β, and γ"; p < 0.05; n = 4-5).Original WB ima can be found in File S1.

Figure 2 .
Figure 2. Spatial and temporal expression profiles of Lvit-IL-17R in L. vittata.(A) The tissue distribution profile of Lvit-IL-17R was generated using the RT-PCR assays of shrimps at the gonadal development of stage II.Lvit-β-actin was used as a positive control.(B) The expression profiles of Lvit-IL-17R in the androgenic gland, hepatopancreas, and ovarian regions during gonadal development through qRT-PCR.The Lvit-IL-17R expression levels were standardized using Lvit-β-actin expression levels ("A, B and C", "a and b", an "α, β, and γ"; p < 0.05; n = 4-5).Original WB images can be found in File S1.

Figure 4 .
Figure 4. Short-term silencing experiment in vivo.The effectiveness of gene knockdown was evaluated in the androgenic gland (A), hepatopancreas (B), and ovarian regions (C).The expression of

Figure 4 .
Figure 4. Short-term silencing experiment in vivo.The effectiveness of gene knockdown was evaluated in the androgenic gland (A), hepatopancreas (B), and ovarian regions (C).The expression of

Figure 4 .
Figure 4. Short-term silencing experiment in vivo.The effectiveness of gene knockdown was evaluated in the androgenic gland (A), hepatopancreas (B), and ovarian regions (C).The expression of genes related to sexual differentiation (Lvit-IAG1 and Lvit-IAG2) (D,E) and ovarian development (Lvit-Vg and Lvit-VgR) (F,G) were also detected.The gene expression levels were standardized by Lvit-β-actin expression levels and represented as mean ± SD ("a and b", p < 0.05; n = 5).

Figure 5 .
Figure 5.Long-term silencing experiment in vivo.The effectiveness of gene knockdown was evaluated in the androgenic gland (A), hepatopancreas (B), and ovarian regions (C).The effect of Lvit-IL-17R knockdown on L. vittata growth was also assessed.The carapace length (D) and body weight (E) were measured ("a and b", p < 0.05; n = 5-6).

Figure 5 .
Figure 5.Long-term silencing experiment in vivo.The effectiveness of gene knockdown was evaluated in the androgenic gland (A), hepatopancreas (B), and ovarian regions (C).The effect of Lvit-IL-17R knockdown on L. vittata growth was also assessed.The carapace length (D) and body weight (E) were measured ("a and b", p < 0.05; n = 5-6).

Figure 6 .
Figure 6.Effect of long-term Lvit-IL-17R silencing on female sexual differentiation.(A) Effect of long-term Lvit-IL-17R silencing on female gonopores.Female gonopores were marked with red dotted circles.(B) Effect of long-term Lvit-IL-17R silencing on ovarian development.Ovotestes were photographed to examine tissue morphology.The histological features were further analyzed via H & E staining.Ovd: oviduct; Ova: ovary; Spd: sperm duct; Tes: testis; Ooc: oocytes; Fc: follicular cell.(C) The long and short axis lengths of each oocyte were measured and averaged, yielding the diameter for each cell ("a and b", p < 0.05; n = 5-6).(D) Expression levels of Lvit-VgR in ovarian regions and Lvit-Vg in hepatopancreas were also detected ("a and b", p < 0.05; n = 5-6).

Figure 6 .
Figure 6.Effect of long-term Lvit-IL-17R silencing on female sexual differentiation.(A) Effect of long-term Lvit-IL-17R silencing on female gonopores.Female gonopores were marked with red dotted circles.(B) Effect of long-term Lvit-IL-17R silencing on ovarian development.Ovotestes were photographed to examine tissue morphology.The histological features were further analyzed via H & E staining.Ovd: oviduct; Ova: ovary; Spd: sperm duct; Tes: testis; Ooc: oocytes; Fc: follicular cell.(C) The long and short axis lengths of each oocyte were measured and averaged, yielding the diameter for each cell ("a and b", p < 0.05; n = 5-6).(D) Expression levels of Lvit-VgR in ovarian regions and Lvit-Vg in hepatopancreas were also detected ("a and b", p < 0.05; n = 5-6).

Figure 7 .
Figure 7. Effect of long-term Lvit-IL-17R silencing on male sexual differentiation.(A) Expression levels of Lvit-IAG1 and Lvit-IAG2 in androgenic gland were detected ("a and b", p < 0.05; n = 5-6).(B) Effect of long-term Lvit-IL-17R silencing on the development of male external phenotypes.AM and AI are represented by yellow and white dash lines, respectively.Cincinnuli are marked with red solid arrows.Male gonopores were indicated by blue dotted circles.AM: appendices mas-

Figure 7 .
Figure 7. Effect of long-term Lvit-IL-17R silencing on male sexual differentiation.(A) Expression levels of Lvit-IAG1 and Lvit-IAG2 in androgenic gland were detected ("a and b", p < 0.05; n = 5-6).(B) Effect of long-term Lvit-IL-17R silencing on the development of male external phenotypes.AM and AI are represented by yellow and white dash lines, respectively.Cincinnuli are marked with red solid arrows.Male gonopores were indicated by blue dotted circles.AM: appendices masculinae; AI: appendix interna.(C) The length of AM and AI were measured, and the normalized length of AM (AM/AI) was calculated ("a and b", p < 0.05; n = 5-6).(D) Effect of long-term Lvit-IL-17R silencing on testicular development.H & E staining was employed to analyze histological features of testicular regions.Sg: spermatogonia; Sc I: primary spermatocyte; Sd: spermatid; Sz: spermatozoa.