The Regulatory Mechanism of Sexual Development in Decapod Crustaceans

Crustacean culture has been developing rapidly in various parts of the world. Therefore, it is important to understand their reproductive biology. Insulin-like androgenic gland hormone (IAG) secreted from the androgenic gland (AG) is widely accepted as a key regulator of sexual differentiation in male crustaceans. However, recently several sex-related genes (i.e., CFSH, DEAD-box family, Tra-2, Sxl, Dsx, Fem-1, Sox gene family, Foxl2, and Dmrt gene family) have been identified via transcriptomic analysis in crustaceans, indicating that sexual differentiation in crustaceans is more complicated than previously expected. It has been found that several non-coding RNAs (i.e., miRNAs, lncRNAs, and piRNAs) and IAG receptors may be involved in the sexual development of decapods. Identification and study of the regulation mechanism of sex-related genes, non-coding RNAs, and IAG receptors will provide valuable information regarding sexual development in decapods. In this review, the roles of hormonal and genetic factors in both males and females are discussed. In males, crustacean female sex hormone (CFSH), Sxl, Dmrt gene family, Dsx, Sox gene family, GEM, Fem-1, l-GnRH-III, and corazonin play important roles in IAG regulation in the “eyestalk-IAG-testis” endocrine axis. Unlike males, the regulation mechanism and interaction of sexual genes are relatively unknown in females. However, CFSH, IAG, Fem-1, FAMeT, Slo, UCHLs, Erk2, Cdc2, EGFR, Vg, VgR, and VIH seem to play crucial roles during ovarian development. This study summarizes the available information in the field, highlights gaps, and lays the foundations for further studies and a better understanding of the regulatory mechanism of sexual development in decapods.


INTRODUCTION
The order Decapoda includes approximately 17,000 species of crayfish, shrimps, crabs, and lobsters (De Grave et al., 2009). Some decapods are economically cultivated and some maintained in domestic aquariums, but some are invasive and are considered as pathogenic vectors. Crustacean culture is a rapidly developing industry, due to the high economic value as a source of animal protein, employment, and foreign exchange gains. Crustaceans production reached 9.4 million tons in 2018 (6.4 and 3 million tons were provided from capture fisheries and aquaculture, respectively), with an estimated sale value of US$ 69.3 billion (FAO, 2020).
Understanding the reproductive biology and mechanism of sexual differentiation are key issues for sexual manipulation and improving the reproductive efficiency of decapods. Male and female decapods have different growth performances at different growth stages, which is important in artificial selective breeding and farming . For example, in crayfish and prawns (freshwater Palaemonidae), males grow larger and faster, and as such, all-male individual rearing is desired. In contrast, females grow larger and faster than males in shrimps (marine Penaeidae) and crabs . In hatcheries, females are considered more valuable than males for increasing the size of the population since males can copulate with more females with no negative effect on the percentage of berried females (Harlıoǧlu and Farhadi, 2017b). Studies showed that male and female decapods have different biochemical composition, nutritional value, and flesh quality . Another benefit of sexual manipulation is the ecological application of non-reproductive and monosex crustaceans in controlling invasive species. For example, all-male specimens of some Macrobrachium genus have been successfully used as biocontrol vectors on snails in some African countries (Sokolow et al., 2014). The advantages of sexual manipulation in decapod crustaceans are summarized in Table 1.

Purpose
Advantage/Description/ Example References

Monosex culture
Selective harvesting in M. Rosenbergii Sagi and Aflalo, 2005 Increasing stability of mating systems (e.g., sex change in groupers) Harlıoǧlu and Farhadi, 2017a Avoiding energy diversion (e.g., lipids) toward reproductive processes. Rodgers et al., 2006 All-female monosex culture Females grow larger and faster than males in shrimp (marine Penaeidae) and crab  In hatcheries, females are considered more valuable Harlıoǧlu and Farhadi, 2017a All-male monosex culture In crayfish and prawns (freshwater Palaemonidae), males grow larger and faster  Ecological application Mono-sex culture of non-indigenous crustaceans Policar and Kozák, 2015 Mono-sex crustaceans in controlling invasive species, for example, all-male specimens of some Macrobrachium genus have been successfully used as biocontrol vectors on snails in some African countries Sokolow et al., 2014 Flesh quality Male and female decapods have different biochemical composition, nutritional value, and flesh quality Wu et al., 2019

Sex Determination
The sexual development in crustaceans begins with a sex-specific genetic cascade mediated by a chromosomal mechanism of sex determination. In Penaeidae shrimp and prawns a general ZZ/ZW (homogametic male) mechanism seems to exist (Aflalo et al., 2006;Coman et al., 2008;Staelens et al., 2008). In other decapods such as freshwater crayfish (Astacidea) and true crabs (Brachyura), both ZZ/ZW and XY/XX mechanisms occur (Parnes et al., 2003;Cui et al., 2015;Mlinarec et al., 2016). Recently, various genetic techniques have been successfully applied to identify the sex-specific DNA sequences and markers in decapods. Restriction-site associated DNA sequencing (RADseq) is based on NGS technology and can discover massive single nucleotide polymorphisms (SNPs) in various species by sequencing parts of the genome at high depth, without a reference genome. RAD-seq has been successfully applied to develop sex-specific markers in several decapod species . For example, sex-specific SNP markers and high-density genetic linkage maps showed male homogamety and female heterogamety, presenting reliable genetic evidence for a WZ/ZZ sex-determination system in mud crabs, Scylla tranquebarica, and Scylla serrata (Shi et al., 2018;Waiho et al., 2019b). The discovery of five male-specific SNP markers in crucifix crab (Charybdis feriatus) provided reliable genetic evidence for a XX/XY sexdetermination system in C. feriatus . The sexual determination process in Drosophila species is controlled through alternative splicing of pre-mRNA of a series of genes involved in the sex determination pathway, including Tra-2, Sxl, and Dsx. A homologue gene of Tra-2 has been identified in Penaeus monodon, however, no sex dimorphism on the expression and alternative splicing patterns was observed (Leelatanawit et al., 2009). Up to now, the sex determination mechanisms in crustacean species are still largely unknown. Kato et al. (2011) identified two Dsx homologues (DapmaDsx1 and DapmaDsx2), which act as a key regulator of the male phenotype in Daphnia magna. However, in contrast to Dsx for Tra no significant difference was detected in the splicing or expression patterns (Kato et al., 2010). In ornate spiny lobster (Panulirus ornatus), Sxl and Tra-2 isoform-encoding transcripts showed broad tissue expression, with no clear bias between males and females, suggesting that Sxl-Tra-Dsx may be unique to Drosophilidae and not conserved among decapods. However, the male-specific iDMY was conserved between the eastern (Sagmariasus verreauxi) and ornate spiny lobsters (P. ornatus), indicating a conserved central function in sex determination (Ventura et al., 2020).

Sexual Differentiation and Maturation
The complex process of sexual differentiation results in sexspecific phenotypic development. Our understanding of sexual differentiation in the decapods far exceeds what is known about sex determination. Neuropeptides from the eyestalk and mandibular organ regulate the sexual development in decapods (Khalaila et al., 2002;Alfaro-Montoya, 2010). Eyestalk is the major neuroendocrine control center and eyestalk ablation influences gonadal development in crustaceans (Fingerman, 1987). The CHHs (crustacean hyperglycemic hormones) secreted from the X-organ have been classified as subfamily I peptides containing a CHH precursor related peptide (CPRP) and the subfamily II peptides lack the CPRP (Jia et al., 2012). The subfamily II peptides including moult inhibiting hormone (MIH), gonad/vitellogenesis-inhibiting hormone (GIH or VIH), and mandibular organ-inhibiting hormone (MOIH) control the moulting and gonadal maturation (Lacombe et al., 1999;Böcking et al., 2002). Serotonin (5-HT), dopamine, and gonadotropinreleasing hormones (l-GnRH-III) are other important factors affecting the sexual development pathway of decapods (Tinikul et al., 2008(Tinikul et al., , 2011Siangcham et al., 2013).
The insulin-like androgenic gland hormone (IAG) secreted from androgenic gland (AG) is a key regulator in sexual differentiation. Recent studies have shown that IAG itself is controlled by several genes and hormonal factors (Shen et al., 2014;Shi L. et al., 2019;Zhong et al., 2019). The detailed mechanisms and the role of sex-related genes and miRNAs in the IAG regulation are discussed in detail in other sections of the present review. In the present review, we focus on the downstream mechanisms such as novel sex-related genes and miRNAs that play important role in the sexual development process. The present review aims to highlight gaps in knowledge that must be filled to allow further development of sexual manipulation techniques.

SEXUAL MANIPULATION IN DECAPOD CRUSTACEANS
Androgenic gland is the key regulator of primary and secondary sexual characters in male decapods. It controls behavior, spermatogenesis, and sexual differentiation by secretion of IAG. AG is associated with the subterminal part of the sperm duct and attached to the posterior vas deferens in male decapods. IAG silencing had adverse effects on sexual development (i.e., spermatogenesis) in male Macrobrachium rosenbergii (Ventura et al., 2009). Silencing accomplished via double-stranded RNA (dsRNA) injection caused considerable sex-related changes such as ovarian activation and testicular degeneration in male Cherax quadricarinatus (Rosen et al., 2010). Similar results were observed in M. rosenbergii and C. quadricarinatus after AG ablation (Barki et al., 2006).
At present, sexual manipulation in decapods only applies through the manipulation of AG. Such as AG ablation and IAG suppression in males, which produce neofemales (phenotypically female but genetically male) in M. rosenbergii (Aflalo et al., 2006;Ventura et al., 2009). Neofemale M. rosenbergii (complete and functional sex reversal) was obtained by long-term IAG silencing at PL30 stage . AG implantation into females induced sex reversal in females, called neomales (phenotypically male but genetically female) (Aflalo et al., 2006;Ventura et al., 2009).
Until now, the only successful AG manipulation that led to complete and functional sex reversal has been recorded in M. rosenbergii (Aflalo et al., 2006;. In Macrobrachium sp., IAG is expressed exclusively and abundantly in the AG rather than in the testis or other tissues Ma et al., 2013). However, studies showed that in other decapod groups such as shrimps (Mareddy et al., 2011;Li et al., 2012b), crayfish , and crabs Jiang et al., 2020a) the IAG is expressed not only in the AG, but also in other tissues. The AG-specific expression pattern of IAG could explain why AG manipulation led to complete sex reversal only in M. rosenbergii. The broad tissue expression pattern of IAGs in other decapods indicates that IAGs may have some additional functions.
Recently, several sex-related genes and non-coding RNAs have been identified in decapods, suggesting that sexual differentiation mechanisms in decapods are more complicated than previously expected (Chandler et al., 2018). Further studies are required to identify more sex-related factors such as genes, non-coding RNAs and IAG hormones, which could be involved in the sexual development of decapods. Understating the function of these genetic factors may provide novel insights into the development of new sexual manipulation techniques. Sex-related genes, hormones, and their potential roles in decapods are summarized in Table 2.

Sox Gene Family
The Sox (Sry-related high-mobility group (HMG) box) family of transcription factors is extensively distributed in different animal groups, and plays crucial roles in development processes including cell pluripotency, neurogenesis, and sex determination. To date, the Sox gene has been isolated from several decapods including Macrobrachium nipponense (Hu et al., 2020), Penaeus Play a crucial role in female phenotypes and acts as a negative regulator inhibiting the development of AG Ventura et al., 2014;Zmora and Chung, 2014;Veenstra, 2015;Nguyen et al., 2016;Toullec et al., 2017;Kotaka and Ohira, 2018;Liu C. et al., 2018;Jiang et al., 2020b Vitellogenin (  vannamei (Peng et al., 2015), Palaemon serratus (González-Castellano et al., 2019), E. sinensis (Liu et al., 2016), and S. paramamosain (Liao et al., 2020;Lin et al., 2020). SoxE1 is involved in testis development in M. nipponense (Hu et al., 2020). In a recent study, Lin et al. (2020) reported that the SoxB2 gene plays a key role during gonad development in the mud crab. Sox gene plays critical roles in sexual dimorphism and participates in the formation of brain nerves and is involved in the regulation of body segments (Liao et al., 2020). In E. sinensis, Sox protein was exclusively localized in the nucleus of spermatid and spermatozoa even at the end of acrosome reaction and was bound to the uncondensed chromatin in the nucleoplasm of mature spermatozoa. SoxB2-1 knockdown leads to an abnormal transformation of the nucleus during spermiogenesis. These results revealed the importance of the Sox gene family in testis development and spermatogenesis in males (Liu et al., 2016;Lin et al., 2020).
Targeting the coding region of the Dmrt gene by repetitive injection of dsRNA revealed that Dmrt plays important roles in the spermatogenesis and testicular development of E. sinensis (Ma et al., 2016a). RNAi-targeting of MroDmrt99B in male M. rosenbergii did not affect the expression level of IAG, while MroDmrt11E silencing resulted in a significant reduction in the expression level of IAG (Yu et al., 2014), indicating that IAG is positively regulated by MroDmrt11E. In M. rosenbergii, eyestalk removal significantly increased the expression level of Dsx in the testis and AG. Putative Dsx/Mab-3 binding site was identified on the promoter region of male sexual differentiation effector gene IAG (Yu et al., 2014).

Vasa Gene
Vasa is an important regulatory gene for reproductive system development (Lasko and Ashburner, 1988). In decapod crustaceans, the Vasa gene has been identified in F. chinensis (Zhou et al., 2010) and P. vannamei , M. rosenbergii (Nakkrasae and Damrongphol, 2007), and mud crab S. paramamosain . Vasa gene can be used as a specific germ cell marker to track the origin and precise route of germ cell migration toward the gonad, and elevates our knowledge on the reproductive and developmental processes of the germline in decapods . In F. chinensis, the strongest hybridization signal was detected in the spermatogonia and the intensity gradually declined toward the centre of testis tubules indicating that the transcript level of Vasa progressively decreased from spermatogonia to spermatids (Zhou et al., 2010). Vasa RNA was detected in the nucleus and the cytoplasm of the spermatozoa in M. rosenbergii (Nakkrasae and Damrongphol, 2007). It seems that Vasa gene plays various roles in the regulation of spermatogenesis. Further studies are required to investigate the exact role of the Vasa gene in the germ cells of decapods.

Sex-lethal (Sxl)
It has been documented that sex-lethal (Sxl) is involved in the sex determination process of insects. In Drosophila, female embryos expressed a fully functional Sxl protein while males produced a shorter peptide lacking an RNA binding domain.
In males, the truncated Sxlm has no regulatory effect, leading to the production of an equally inactive default splice variant of Tra (Mullon et al., 2012). In decapods, Sxl has been isolated from Chinese mitten crab (Shen et al., 2014), redclaw crayfish C. quadricarinatus (Zheng et al., 2019), red swamp crayfish P. clarkii (Shi L. et al., 2019), S. verreauxi (Chandler et al., 2015), M. nipponense , and P. vannamei (López-Cuadros et al., 2018). Sxl plays crucial roles in sexual differentiation, embryonic development, and gonad development but not in the sex determination of decapods (Shen et al., 2014;López-Cuadros et al., 2018;Zheng et al., 2019). In E. sinensis, Sxl was expressed in different stages of embryos development. The expression of Sxl had a gradual increase from the cleavage stage to the egg-nauplius stage, however, Sxl rapidly increased in the original zoea stage. The increase of Sxl at the last stages of embryos indicates that Sxl may play a role in embryonic development (Shen et al., 2014). Among the four isoforms of Sxl in P. clarkii, CqSxl3 demonstrated tissue specificity with a higher expression level in the testis than in the ovary. CqSxl4 and CqSxl1 were extensively expressed in various tissues while CqSxl2 was almost undetectable. The transcript levels of CqSxl1/3/4 slowly increased along with embryonic development (Zheng et al., 2019). The transcript pattern of Sxl in females and males show that Sxl may not be sex-specific in decapods (Shen et al., 2014;López-Cuadros et al., 2018;Zheng et al., 2019).

Bone morphogenetic proteins (BMPs)
It has been demonstrated that bone morphogenetic proteins (BMPs) such as BMPRIB and BMPRII play an important role in the development of ovary in S. paramamosain (Shu et al., 2016). BMP7 may also play a role in the testis development of E. sinensis, probably by promoting the germ cell differentiation/meiotic mitosis and maintaining the self-renewal of spermatogonia or facilitating the successful fertilization (Xu et al., 2018).
Gem-associated Protein 2-like Isoform X1 (GEM) In M. nipponense, gem-associated protein 2-like isoform X1 (GEM) was highly expressed in the testis and ovary and during larval development indicating that GEM plays important roles in gonad and larval development. In situ hybridization showed that GEM may promote oogenesis and spermatogenesis. RNAi experiment revealed that GEM negatively controls the expression of IAG in M. nipponense .

REGULATORY MECHANISM OF SEX IN MALE DECAPODS
The "eyestalk-AG-testis" endocrine axis is the main regulatory mechanism of sex in male decapods. The X-Organ-Sinus-gland complex (XO-SG) of the eyestalk regulates the activity of AG through the XO-SG-AG-Gonad axis (Rodríguez et al., 2007). Eyestalk ablation causes hyperplasia and hypertrophy in AG cells (Khalaila et al., 2002), elevates IAG expression, and accelerates the testicular development in males (Nagaraju, 2011). XO-SG secretes neurohormones such as GIH, CHH, MIH, and VIH which are suspected to directly regulate the expression of IAG (Li et al., 2015b) and have inhibitory effects on reproduction and sexual development (Suwansa-ard et al., 2015). Recently, several sexual genes have been identified to be involved in the "eyestalk-IAG-testis" endocrine axis (Guo et al., 2019). It has been revealed that some of these genes are regulated by the eyestalk neurohormones and are located at the upstream of IAG and the sexual differentiation cascade (Shen et al., 2014;Ma et al., 2016a;Shi X. et al., 2019;Zheng et al., 2019;Zhong et al., 2019).

Upstream Mechanisms of Sex in Male Decapods
Crustacean female sex hormone (CFSH) , Sxl (Shi L. et al., 2019;Zheng et al., 2019), Dmrt gene family (Abayed et al., 2019), Dsx (Yu et al., 2014), Sox gene family (Li et al., 2012a;Zhang et al., 2014), GEM , Fem-1 (Ma et al., 2016b), l-GnRH-III, and Crz (Siangcham et al., 2013) may play key roles in IAG regulation at the upstream of IAG in the "eyestalk-IAG-testis" endocrine axis. Liu C. et al. (2018) suggested that CFSH is not a femalespecific hormone and the expression pattern of CFSH is opposite to the expression pattern of IAG during the development of AG in males. Recombinant CFSH (rCFSH) reduced the expression of IAG in AG by approximately 60% . CFSH from the eyestalk is a negative regulator and inhibits AG development in decapods. Jiang et al. (2020b) found no difference in the expression level of Sox-5 and AP-1 in AG exposed to rCFSH, however, STAT significantly declined, indicating that CFSH controls the expression of STAT, inhibiting its function to control IAG in the mud crab. Further experiment showed that RNAi mediated STAT gene knockdown lowered IAG expression indicating that CFSH regulates IAG by inhibiting STAT. In a study by Jiang et al. (2020b), promoter activity was significantly increased in pGL3-pSp-IAG1 (Sox-5binding site) and pGL3-pSp-IAG6 (Sox-5-binding site) than in previous plasmids (pGL3-basic and pGL3-pSp-IAG5), indicating that the Sox-5-binding site is probably involved in enhancing this activity, consistent with site-directed mutation (SDM) results of decreased promoter activity in pGL3-pSp-IAG6-Mut .
The Sox gene plays a crucial role in male sexual differentiation and sex determination of vertebrates (Ma K. et al., 2012). In zebrafish, Sox-5 binds to the Dmrt1 promoter (double sex and mab-3-related transcription factor 1) and inhibits Dmrt1 expression that may affect sexual differentiation (Gao et al., 2005). In medaka, Sox-5 knockout reverses female to male by controlling dmrt1bY expression (Schartl et al., 2018). In decapods, 5 flanking regions for IAG have only been studied in F. chinensis, M. nipponense, and S. paramamosain, which contains a cis-acting element of Sox-5-binding site, indicating that the transcriptional regulation mechanism of IAG may be conserved in decapods (Li et al., 2012a;Zhang et al., 2014). The finding of transcription factor binding sites of Sox in the 5 flanking region of IAG indicates a regulatory role of Sox at the upstream of IAG.
Sxl is regulated by neuropeptide hormones from the eyestalk (Shen et al., 2014) and acts as the upstream regulator of IAG (Shi L. et al., 2019;Zheng et al., 2019). Sxl transcription in the testis was downregulated 3 days after eyestalk removal while in the ovary it was upregulated 7 days after eyestalk removal (Shen et al., 2014). Sxl silencing significantly decreased the IAG level in C. quadricarinatus and P. clarkii (Shi L. et al., 2019;Zheng et al., 2019). In P. clarkii, no direct relation was observed between the Sxl and IAG and the expression level of Sxl was not affected by IAG-dsRNA injection (Shi L. et al., 2019). In contrast, after IAG silencing, the expression level Sxl1, and Sxl2 decreased significantly in M. rosenbergii (Tan et al., 2020b).
Similarly, Dsx is likely to be negatively regulated by the neuropeptide hormones from the eyestalk and Dsx may be involved in the regulation of IAG in M. rosenbergii (Zhong et al., 2019). Dsx knockdown induced a significant decrease in the expression of IAG (Yu et al., 2014;Li et al., 2018). IAG silencing significantly reduced the expression of two genes, MroiDmrt1b and MroiDmrt1c in M. rosenbergii, thereby indicating their possible roles in the IAG-switch (Abayed et al., 2019;Tan et al., 2020a). Nevertheless, the up-regulation of Dsx at the nauplius stage before the first up-regulation of IAG  indicate that Dsx may act as an upstream regulator of IAG (Yu et al., 2014;Li et al., 2018). Abayed et al. (2019) suggested that Mr-IAG is correlated with MroiDmrt1b and MroiDmrt1c, however, it is difficult to conclude whether the relationship is downstream or upstream.
The Fem-1 gene might be at the upstream of the sex determination cascade when compared with IAG in M. nipponense (Ma et al., 2016b), since IAG was first expressed at the blastula stage (Ma et al., 2013). In contrast to IAG, the Fem-1 showed a high expression level from the unfertilized egg to cleavage, and then the expression declined to a low level from blastula to zoea. Fem-1 probably influences sexual development by interacting with cathepsin L in embryogenesis and vitellogenesis (Ma et al., 2016b). Silencing of IAG results in testicular degeneration and sexual shifts in decapods (Rosen et al., 2010). The insulinase domain protein in Drosophila willistoni may hydrolyze insulin protein in individual development (Clark et al., 2007), indicating that Fem-1 probably controls IAG indirectly and feminizes male-related phenotypes similar to IAG silencing (Ma et al., 2016b).
Crz and l-GnRH-III are other regulatory factors at the upstream of IAG, In M. rosenbergii, Crz has inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and IAG suppression. In contrast, it seems that IAG is positively regulated by l-GnRH-III (Siangcham et al., 2013).

Downstream Mechanisms of Sex in Male Decapods
To better understand the multiple functions of IAG, it is necessary to clarify the mechanisms of how IAG is transported. An insulin family-based signaling system has been proposed based on the structure similarity of IAG to the insulin/IGF family (Sharabi et al., 2016). Several peptide receptors including tyrosine kinase insulin receptor (TKIR), G-protein-coupled receptor (GPCR), insulin receptor (IR), and insulin-like growth factorbinding peptide (IGFBP) have been identified to be the receptor for IAG (Rosen et al., 2010;Aizen et al., 2016;Chandler et al., 2017;Jiang et al., 2020c;Ventura et al., 2020).
IGFBPs act not only as carriers of IGFs to release at the target site, but also function as modulators of IGF availability and activity (Govoni et al., 2005). In vitro study of IGFBP in the red claw crayfish C. quadricarinatus revealed that recombinant Cq-IGFBP protein interacts with the IAG hormone (Rosen et al., 2013). In M. rosenbergii, the highest expression level of IAGBP was detected in AG (Yang et al., 2020). IAGBP-dsRNA significantly reduced the transcript level of IAG in M. rosenbergii and M. nipponense (Li et al., 2015a;Yang et al., 2020). These results indicate that IAGBP probably participates in IAG signaling. Aizen et al. (2016) suggested that IAG hormone in S. verreauxi is regulating phosphorylation through a tyrosine kinase insulin receptor (Sv-TKIR). The Sv-TKIR receptor signaling pathway is mediated through the MAPK/ERK pathway and IAG activates this signaling pathway in a dose-dependent manner (Aizen et al., 2016). This activation confirms the existence of receptors in the testis which can bind to AG ligands, coinciding with the testis regression in response to IAG silencing (Rosen et al., 2010;Aizen et al., 2016).
Insulin receptors (IRs) belong to the large class of tyrosine kinase receptors. IRs play important roles in signaling pathways, which mediate insulin and insulin-like peptides (ILP) signaling (Jin Chan and Steiner, 2000). The expression of IR in M. rosenbergii (Mr-IR) was detected in most tissues in both males and females, including the AG and gonads (Sharabi et al., 2016). Mr-IR silencing resulted in hypertrophy of AG and increased the expression level of Mr-IAG, with an unusual mass of immature spermatozoa cells in the distal vas deferens. RNAi experiments revealed that MrIR functions as a receptor for MrIAG; and the knockdown of MrIR, using both siRNA and dsRNA approaches, induced sex reversal (neofemales) in M. rosenbergii (Tan et al., 2020a). Mr-IR could also regulate Mr-IAG expression, possibly through cross-talk with eyestalk-borne neuropeptide (s) or potentially via auto/paracrine feedback with the AG itself (Sharabi et al., 2016). The regulatory mechanism of the eyestalk-IAG-testis endocrine axis, sex-related genes, hormones, and miRNAs are presented in Figure 1.

Fem-1 Gene Family
Fem-1 gene family includes three members, Fem-1a, Fem-1b, and Fem-1c (Ventura-Holman et al., 2003). It has been demonstrated that the sex determination pathway in the roundworm Caenorhabditis elegans is controlled by Fem-1, together with Fem-2 and Fem-3 (Doniach and Hodgkin, 1984). Fem-1 is known to be required for sexual development, involving both somatic and germline sex determination together with other Fem genes (Fem-2 and Fem-3) in C. elegans. Fem-1 negatively regulates Tra-1, which is a key gene for sexual development in females (Doniach and Hodgkin, 1984;Mehra et al., 1999). To date, Fem-1 has been isolated from several decapod species including P. serratus (González-Castellano et al., 2019), E. sinensis , P. vannamei (Galindo-Torres et al., 2019), M. nipponense (Ma et al., 2016b,a), S. paramamosain (Gao et al., 2014), and P. monodon (Robinson et al., 2014). Based on the high expression of Fem-1 in early embryonic development it might be maternal genes in decapods Ma et al., 2016b). The high expression level of Fem-1 in unfertilized eggs and embryos at the cleavage stage and the low expression level from blastula to zoea in M. nipponense, indicate that Fem-1 in early embryos is maternal. The expression of Fem-1 significantly increased in the larvae, an important stage of sexual differentiation. Fem-1 protein can be potentially interactive with cathepsin L and proteins containing the domains of insulinase, ankyrin or ubiquitin (Ma et al., 2016b). Galindo-Torres et al. (2019) suggested that a negative post-transcriptional regulation controls the expression of Fem-1 in female gonads, which allows the occurrence of sexual differentiation in female P. vannamei. The expression of Fem-1 in spermatogonia indicates it's also requirment for differentiation of the male gonad (Galindo-Torres et al., 2019). In E. sinensis, the expression pattern of Fem-1 shows a certain degree of sexually dimorphic expression in some tissues. The higher expression level of Fem-1 consistently detected in the testis, ovary, hepatopancreas, and muscles shows its potential role in the last stages of gonadal development .

Ubiquitin C-Terminal Hydrolases (UCHLs)
Ubiquitin C-terminal hydrolases (UCHLs) are involved in several physiological processes. However, little information is available regarding the role of UCHLs in gonadal development of crustaceans. UCHLs have been identified only in S. paramamosain (Han et al., 2018). Han et al. (2018) reported that the expression level of UCHL3 and UCHL5 in the ovary was significantly higher than in other tissues which provides basic data indicating that UCHL3 and UCHL5 may play important roles in the ovarian development of S. paramamosain.

Epidermal Growth Factor Receptor (EGFR)
Epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor superfamily found in many species. EGFR is a pleiotropic glycoprotein that plays a role in regulating cell proliferation, migration, and differentiation. In mud crab, EGFR is mainly localized in the oocyte perinuclear region with notably obvious signals. EGFR might promote ovarian development by stimulating the expression of VgR and Cyclin B . The role of EGFR in M. rosenbergii seems to be mostly related to growth control. EGFR-dsRNA injection resulted in delay in the appearance of a male secondary sexual characteristic, growth reduction, and affected eye formation. It seems that Mr-IAG is probably not a ligand of Mr-EGFR (Sharabi et al., 2013).

Upstream Mechanisms of Sex in Female Decapods
The upstream regulation mechanisms in female decapods include the role of several neurohormones and neuropeptides such as methyl farnesoate (MF), ecdysone, ecdysone receptor (EcR), CHH, GIH, MIH, 5-HT, DA, VIH, octopamine, and red pigment concentrating hormone (RPCH). The major source of these neuropeptides is the X-organ located in the eyestalk ganglia. These neuropeptides involved in carbohydrate and lipid metabolism, moulting, and ovarian development (Böcking et al., 2002;Zhou et al., 2019). The RPCH is not only involved in the regulation of pigmentation but also the regulation of ovarian maturation in crustaceans (Fingerman, 1997). EcR and farnesoic acid O-methyltransferase (FAMeT) are important genes and have a high expression in the eyestalk (Gong et al., 2015). FAMeT is an important rate-limiting enzyme for the conversion from Farnesoic acid (FA) to methyl farnesoate (MF) in the final stage of MF synthesis (Holford et al., 2004). MF is synthesized from the mandibular organ. MF plays important roles in ovarian development (Rodríguez et al., 2002). Based on the high structural similarity, MF is considered to be the homologue of juvenile hormones (JH) from insects . Localization studies indicated that FAMeT is co-localized in the neurosecretory cells that produce CHH and MIH. Higher levels of FAMeT mRNA transcript and protein levels were reported along with higher levels of CHH and GIH . MF biosynthesis is regulated by secreted neuropeptides from the X-organ which regulates the activity of FAMeT and thereby vitellogenesis (Gunawardene et al., 2002).

Downstream Mechanisms of Sex in Female Decapods
CFSH, IAG, Vg, UCHLs, Erk2, SUMO, 14-3-3 zeta, Slo, CKS1, Cdc2, MAPK1, FAMeT, Wnt4, and EGFR probably play important roles in the sexual development of females. Unlike males, the downstream mechanisms in females are relatively unknown. Except for CFSH and IAG, the regulation mechanism of other genes is unknown and information is limited to the molecular characterization and expression profile.
The bilateral eyestalk ablation and silencing of CFSH altered reproductive structures (i.e., sharper abdomens, absent or misplaced gonopores, fewer and shorter setae on pleopods) and impaired mating in female C. sapidus (Zmora and Chung, 2014). It seems that CFSH plays an important role in sexual differentiation rather than sex determination because the absence of CFSH neither resulted in sex reversal nor ovarian degeneration (Zmora and Chung, 2014). The expression level of CFSH changed at different moulting stages and the highest expression level was detected in the pre-moult stage. Moreover, the expression of CFSH increased at the beginning of gonopore development in females and decreased in males, indicating that the expression of CFSH affects the formation of gonopores in S. paramamosain (Jiang et al., 2020a). CFSH may also participate in the regulation of sexual differentiation of early juveniles (Jiang et al., 2020a).
The role of IAG is not limited to males, IAG may play an inhibitory role in ovarian development in female decapods. The expression level of IAG is maintained at low levels during the undeveloped stage (stage I) to late vitellogenic stage (stage IV) and then increased at the mature stage (stage V), indicating that IAG participates in inhibiting oocyte growth and vitellogenesis (Huang et al., 2014). In C. sapidus, ovarian development and season affected the expression level of both IAG and insulinlike peptide binding protein (ILPBP) in the ovary. These results indicated that ovarina IAG and ILPBP may be involved in the ovarian development (Huang et al., 2017).
The interaction of sex-related genes in females is poorly understood. Therefore, further studies are required to study the function and interaction of these genes. The regulatory mechanism of sexual development in female decapods and the roles of sex-related genes, hormones, and miRNAs are presented in Figure 2.

MicroRNAs (miRNAs)
MicroRNAs (miRNAs) are small non-coding RNA with a length of about 22 bp. miRNAs inhibit gene expression through translational repression by binding to the 3 -untranslated regions (UTRs) of the target mRNAs (Christensen et al., 2009). Moreover, one miRNA can control numerous genes, and one gene can be regulated by multiple miRNAs (Jingsheng et al., 2015). There are two main stages for miRNAs generation. The first step is the synthesis of the 70-100 bp precursor-miRNA which has a characteristic hairpin structure generated from a primary miRNA transcript by the Drosha and its RNA-binding cofactor DiGeorge syndrome critical region gene 8 (DGCR8) in the nucleus. In the second step, Exprotin5-RAN-GTP carries the pre-miRNA to the cytoplasm for further processing by Dicer (a RNAse III endonuclease) to form the mature miRNA (Zeng, 2006;Winter et al., 2009). In mammals, miRNAs control gonad development (ovary and testis), induce differentiation and maturation of oocytes and spermatozoa, and influence zygote development (Scherr et al., 2007). In the rat, miR-141-3p inhibits ovarian granulosa by targeting DAPK1 gene and participates in the etiology of polycystic ovary syndrome . It has been reported that miR-143 plays a key role in follicle stimulating hormone (FSH) which induces estradiol production and granulosa cell proliferation via binding to the 3 -UTR of Kirsten rat sarcoma (KRAS) mRNA (Zhang et al., 2017). miRNAs are also essential for ovulation and oocyte maturation in invertebrates (Kadener et al., 2009). In decapods, several sexrelated miRNAs have been identified in S. paramamosain Waiho et al., 2019a;Zhou et al., 2019Zhou et al., , 2020, Portunus trituberculatus (Meng et al., 2018(Meng et al., , 2020, E. sinensis (Song et al., 2014;He et al., 2015), M. rosenbergii (Liu X. et al., 2019;Qiao et al., 2019) M. nipponense (Sb et al., 2015;Jin et al., 2017), P. monodon , and Fenneropenaeus merguiensis (Saetan and Chotigeat, 2019). The roles of sex-related miRNAs in decapods are summarized in Table 3.
In males, miRNAs such as miR-17-3p, let-7c, and miR-21-5p demonstrated different expression levels during different stages of testicular development indicating their probable involvement in spermatogenesis (He et al., 2015). MiR-34 probably regulates the reproductive function via binding to the 3 -UTRs of MIH, EcR, FAMeT, and CHH genes .
In females, miR-34 and miR-277 may play a key role in the ovarian development of S. paramamosain. AgomiR-34 reduced the expression level of MIH, CHH, VIH, EcR, RPCH, and FAMeT genes, whereas antagomiR-34 elevates the expression level of these genes . Injection of agomiR-277 in female mud crab increased the expression of the Spvtg gene in the ovary and hepatopancreas of mud crab. On the contrary, the expression of Spvih was significantly decreased after injection with agomiR-277, indicating that miR-277 could positively regulate the expression of the Spvtg gene in mud crab indirectly (by negative regulation on VIH) . It has been demonstrated that miR-133 and miR-2 downregulated cyclin B gene expression during meiotic oocyte maturation in E. sinensis (Song et al., 2014). In addition, the expression level of miR-87 and miR-7 was significantly increased and they were predicted to target cyclin-dependent kinase 2 (Cdk2)7 and cell division cycle4 (Cdc4), which are important for regulation of the G1-to-S-phase transition during the cell cycle (Schnackenberg et al., 2007). Meng et al. (2020) showed that MF synthesis and FAMeT expression are directly regulated by miR-4171 and miR-263a. Also, miR-317, miR-2b, and miR-466f-3p repress the expression of calcium signal transduction (CaM). The eyestalk ablation showed a negative association between miR-263a, miR-4171, and FAMeT and between miR-2b, miR-317, and CaM. Taken together, these results indicate that multiple miRNAs operate cooperatively to regulate the expression of FAMeT and CaM, and thereby participate in inducing ovarian maturation after eyestalk ablation.

PIWI-Interacting RNAs (piRNAs)
PIWI-interacting RNAs (piRNAs) are widely presented in germ cells and are involved in gonad development and gametogenesis. piRNAs play a key role in the gametogenesis processes by silencing transposable elements (Rastetter et al., 2015). piRNAs are longer (26-31 nt) than miRNAs (21-23 nt). However, piRNAs are seldom expressed in somatic cells and show a higher composition present compared to miRNAs during gametogenesis (Aravin et al., 2006). Small RNAs function by guiding a crucial co-factor-an Argonaute/PIWI family of proteins to target RNAs (Hilz et al., 2016). Two major pathways are known for piRNA synthesis: (A) primary biogenesis: piRNAs synthesis from several portions of long single-stranded precursor RNAs transcribed from both uni-directional and bi-directional piRNA clusters in the genomic loci (Mohn et al., 2014), and (B) secondary biogenesis, also known as the ping-pong cycle, selectively amplifies specific piRNAs targeting active transposon (Brennecke et al., 2007). Scarce information is available regarding the role of piRNAs in the sexual development of crustaceans. Waiho et al. (2020) identified gonadal piRNAs of S. paramamosain and out of 115,491 novel piRNAs, 596 were differentially expressed. Furthermore, 389,887 potential piRNA-target genes were predicted. The expression level of four piRNAs and nine genes with high piRNA interactions were validated with the inclusion of additional immature specimens including lowdensity lipoprotein receptor-related protein 2 (LRP2) that is involved in reproduction and growth, midasin (MDN1) in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonad development and differentiation.
Long Non-coding RNAs (lncRNAs) Long non-coding RNAs (lncRNAs) are non-protein-coding RNAs longer than 200 nt (Rinn and Chang, 2012). lncRNAs are divided into genic and intergenic based on their proximity to the protein-coding genes in a genome (Gibb et al., 2011). Unlike small non-coding RNAs, the annotation and identification of lncRNA are difficult due to their low expression level (Derrien et al., 2012). Similar to protein-coding genes, they are mostly transcribed by RNA polymerase II and can be post-transcriptionally modified by splicing, capping, and polyadenylation (Guttman and Rinn, 2012). lncRNAs usually have a small number of exons and a shorter transcript size (average of 800 nt) compared to protein-coding genes (Derrien et al., 2012). lncRNAs often demonstrate tissue-specific expression patterns and even sometimes are localized in a specific cellular compartment (Ginger et al., 2006). Although several lncRNAs have been identified, the molecular function of only a small number of lncRNAs has been experimentally validated. lncRNAs play an important role in gene regulation in three ways including serving as decoys, scaffolds, and guides. Many lncRNAs serve as decoys and affect the transcription of genes by preventing access to DNA by regulatory proteins (Hung et al., 2011). Some lncRNAs act as scaffolds and bring proteins into a discrete complex. Other lncRNAs regulate different developmental and cellular processes by guiding a specific protein complex to a specific promoter in response to certain molecular signals (Tsai et al., 2010). In addition, lncRNAs regulate several molecular processes such as alternative splicing and mRNA transport (Tripathi et al., 2010). In decapods, little information is available regarding the sex-related lncRNAs. Yang et al. (2018) provided fundamental resources for further studies on the roles of lncRNAs in decapod sexual development. Several lncRNAs negatively regulated the expression of some genes, indicating their potential role in the sexual differentiation of mud crab. lncRNAs including lncRNA-ncr2, lncRNA-ncr10, and lncRNA-ncr14 suppressed the expression of their target genes .

CONCLUSION AND FUTURE PERSPECTIVE
Although relatively adequate information is available regarding molecular mechanisms of sexual development in males, limited information is available in females. Several sex-related genes have been identified to be involved in the "eyestalk-IAG-testis" endocrine axis in males. Most of these genes are negatively regulated by the eyestalk neurohormones and have a regulatory effect on the AG and IAG expression. For example, it has been documented that CFSH, Sox, Dsx, Slx, Dmrt, GEM, and Fem-1 regulate the expression of IAG. IAG expression is negatively regulated by GEM, Fem-1, and CFSH. In contrast, Slx, Dsx, and Dmrt silencing significantly decrease the expression of IAG indicating the existence of positive regulation. IAG, Sox, Dmrt, Dsx, Vasa, BMPs, and Slx exhibit male-biased expression patterns and play crucial roles in testis development and spermatogenesis. In contrast, CFSH, Fem-1, FAMeT, Slo, Tra-2, UCHLs, Erk2, Cdc2, EGFR, Vg, VgR, and VIH have female-biased expression patterns and play important roles during ovarian development.
Also, it has been revealed that several non-coding RNAs (i.e., miRNAs, lncRNAs, and piRNAs) may affect the sexual development of decapods. Studies on non-coding RNAs are limited to some miRNAs and further studies are needed to clarify the roles of other non-coding RNAs (i.e., piRNA and lncRNAs) in the regulation mechanism of sex in decapods. For example, miR-34 indirectly regulates the reproductive function via binding to the 3 -UTRs of MIH, CHH, EcR, and FAMeT genes and suppresses their expression.
Further research is required to (1) identify more sex-related genes and non-coding RNAs; (2) investigate the role and function of sex-related genes and non-coding RNAs; (3) understand the interaction of these sex-related genes together and with noncoding RNAs, (4) understand the role and regulation mechanism of IAG in female decapods, and (5) identify more IAG receptors.

AUTHOR CONTRIBUTIONS
AF designed the framework of the draft and collected the literature. AF and HM drafted the manuscript. Other authors revised and finalized the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was supported by the National Key Research