A Review of Sexual Determination and Differentiation in Crustacean

Jianwen Li

College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, China.

DOI: 10.4236/jbm.2022.107002                

Abstract

The prerequisite for monosex culture is to elucidate the molecular mechanism of sex determination and differentiation in crustaceans, as well as to explore the key genes that play a role in this process. Monosex culture technology based on crustacean economic species is of great importance in terms of genetic breeding and economic benefits of aquaculture. As a result, study into the mechanisms of sex determination and differentiation in crustaceans not only contributes to the current absence of basic theories of crustacean sexual mechanism, but also encourages technical innovation in aquaculture to increase overall economic efficiency. This study synthesizes and evaluates available research on sex determination and differentiation in crustaceans, and then provides recommendations for future research objectives and priorities in the field.

Keywords

Crustacean, Sex Determination, Sex Differentiation

1. Introduction

Crustaceans are members of the arthropoda phylum, which includes a diverse range of species, many of which, such as shrimp and crabs, are vital for farming around the world and provide significant economic benefits [1] [2]. Individuals of both sexes undergo mating and reproduction processes in the common culture process, consuming large amounts of energy and affecting the growth rate [3]. Monosex culture based on crustacean sex regulation technology is therefore of great importance to improve the benefits of farming economic species in crustaceans. However, because elucidating the molecular mechanisms of sex determination and sex differentiation in crustaceans, as well as identifying the key genes involved in this process, is a precondition for monosex culture, sex-related studies in crustaceans have attracted a lot of attention. As a result, this study summarizes available research on sex determination and differentiation in crustaceans with the objectives of providing a reference and theoretical basis for monosex culture in each crustacean species.

2. Sexual Determination and Differentiation in Crustacean

Animals form their sexes mainly through two processes: sex determination and sex differentiation [4]. There are two types of sex determination: genetical determination, in which the organism’s genomic chromosomes determine sex, and environmental determination, in which the organism’s sex is determined by environmental circumstances [5]. After sex determination, a process is termed sex differentiation occurs in which the sex traits of an embryo or larva, such as the reproductive system, move from absent to present, resulting in the acquisition of female or male sexual characteristics [4]. As described in this study, there are two types of sex determination in crustaceans: genetical determination and environmental determination.

2.1. Genetical Determination

In comparison to highly advanced vertebrates, crustaceans’ sex determination system is rudimentary and diversified. By counting the male to female ratio of the offspring of Penaeus monodon after hybridization as well as triploid induction culture, Benzie et al. presumed that the sex determination type might be ZW/ZZ type [6]. The sex-determining type of Cargon cataphrastus was also presumed by the above method to be ZW/ZZ type [7]. In addition, Malecha et al. inferred the ZW/ZZ type of Macrobrachium rosenbergii from the female to male ratio of the crossed offspring of sex-reversed individuals [8].

As bioinformatics and molecular biology have developed, bioinformatics tools such as amplified fragment length polymorphism (AFLP) and genetic linkage map construction have become incredibly common in the identification of sex determination processes in crustaceans. Li et al. proposed that the Marsupenaeus japonicus chromosomal genotype would be ZW/ZZ type by using AFLP markers in a two-way pseudo-testcross strategy to construct genetic maps of this specie [9]. Zhang et al. Constructed genetic linkage map using AFLP and microsatellite markers, and the results suggest that the chromosomal genome of Litopenaeus vannamei may be of ZW/ZZ type [10]. Yu et al. then used genetic linkage mapping based on high-density linkage mapping to further localize sex-determining regions on chromosomes of L. vannamei, identifying one marker associated with females that showed female heterozygozity, bolstering the hypothesis of its ZW/ZZ sex-determining type [11]. Robinson et al. used the same method to identify three SNP loci significantly associated with sex in P. monodon, and the feminization gene Fem-1 was also found at this location, implying that these three SNP loci are the female sex-linked loci transmitted from maternal to offspring females, further supporting its ZW/ZZ-type sex determination mechanism [12]. In 2015, Cui et al. localized sex-determining loci in the genetic map through the construction of genetic map of Eriocheir sinensis and consequently identified SNP loci closely linked to females and genetic linkage clusters corresponding to W and Z chromosomes, indicating that its chromosomal genome is of ZW/ZZ type of sex determination [7]. The summary of genetical determination typies mentioned in this section are shown in Table 1.

2.2. Environmental Determination

Crustaceans have a primitive sex determination system compared to vertebrates. Environmental factors like as temperature, season, light, and salinity, for example, have been demonstrated to affect the sex determination and differentiation process of several crustaceans [13].

After clutching eggs, the embryos of female Fenneropenaeus chinensis can produce different sex ratios of offspring males and females, depending on temperature [14]. The parent shrimp of Gammarus duebeni produced significantly different sex ratios of offspring groups when living in different water temperatures [15]. When the quality of the surrounding environment deteriorates in Daphnia magna, the response to the environment switches from sexual reproduction to parthenogenesis and the production of male individuals, followed by the regulation of male gonadal development by the sex-regulatory key gene Doublesex to ensure population survival [16].

Light and salinity factors in the crustacean culture environment can affect the sex determination and differentiation process. The ration of G. duebeni male-to- female can be affected by light [17], while variations in salinity can impact the gonadal development cycle of E. sinensis [18]. The summary of environmental determination typies mentioned in this section are shown in Table 2.

3. Sex Regulation in Crustacean

3.1. Sex-Regulated Organs

The androgenic gland (AG) is a male crustacean endocrine organ that regulates male sexual development and secondary sexual characteristics by the secretion of insulin-like androgenic hormone (IAG) [19]. In 1947, Crosin et al. first utilized

Table 1. Genetical determination typies in crustacean.

Table 2. Effects of environmental factors on sex differentiation of crustaceans.

histological sections to identify the androgenic gland as a paragland wrapped around the lateral side of the vas deferens in the reproductive system of Callinectes sapidus [20]. Subsequently, Taketomi Y et al. injected male androgenic gland extracts into male Procambarus clarkii in 1990, resulting in rapid development of external male sexual features [21]. Thereafter, Sagi et al. removed the male androgenic gonads of M. rosenbergii at the stage of sex differentiation to obtain sex-reversed individuals with male to female reversal whose external sexual characteristics showed female, and then mated the sex-reversed individuals with normal males to successfully obtain all-male offspring [22]. Meanwhile, by transplanting male androgenic gonads into sexually differentiated females of M. rosenbergii, Rungsin et al. obtained reproductively competent neomales [23]. Studies in other crustaceans have shown that androgenic gland adenotomy and transplantation will lead to feminization of males and androgenesis of females, respectively, and that male reproductive pathways, such as spermatogenesis, were observed to be significantly inhibited after androgenic adenotomy in some species [19] [24] [25] [26] [27] [28]. In conclusion, the removal or transplantation of androgenic gland during the early development of isopods and decapods may result in the production of sex-reversed individuals, and androgenic gland removal may causes spermatogenic pathway blockage in males, implying that androgenic gland play an important role in sex differentiation and reproductive function in crustaceans.

In decapod crustaceans, the X-organ complex of the eyestalk secretes a number of neurohormones that regulate various physiological processes, including reproduction, such as molt-inhibiting hormone (MIH), gonadal-inhibiting hormone (GIH), and crustacean hyperglycemic hormone (CHH) [29]. The endocrine pathway “eyestalk-androgenic gland-testis” [30] regulates sexually mature individuals in M. rosenbergii, in which the X-organ complex of the eyestalk negatively regulates the development and maturation of the gonad, and its released CHH interacts with the IAG secreted by the gonad. Therefore, the removal of the eyestalk in crustaceans promotes the growth and development of androgenic gonad cells, which in turn promotes the expression of IAG to further promote the development and maturation of the individual reproductive system [31] [32]. Up to now, the method of accelerating crustacean sex maturation by eyestalk removal and thus cultivating parent shrimp for efficient offspring reproduction has been widely used in the industrial culture of some crustaceans and has further improved the overall economic efficiency of aquaculture.

3.2. Crustacean Sex Determination and Differentiation Genes

The elucidation of the molecular mechanisms of sex determination and differentiation in crustaceans, as well as the discovery of key genes that play a role in this process, are prerequisites for achieving monosex culture, and the androgenic gland hormones secreted by the crustacean androgenic gland are currently the most important key regulators of sex differentiation in crustaceans [28]; in addition to this, with the development of molecular biology techniques and high- throughput sequencing technologies nowadays, a series of homologous genes based on model organism sex determination pathways such as Dmrt, Sxl, Tra-2, Fem-1, etc. have been identified in crustaceans, and this section will review the information on the studies that have been conducted on these genes. The summaries of sex-related genes mentioned in this section are shown in Table 3.

Table 3. Sex-related genes and their roles in crustacean.

3.2.1. Insulin-Like Adrogenic Gland Hormone (IAG)

The androgenic gland, a unique endocrine organ identified only in male crustaceans, secretes the insulin-like adrogenic gland hormone (IAG), which is involved in the regulation of sexual differentiation and secondary sexual characteristics [19]. In 2007, Manor et al. identified the first IAG gene, Cq-IAG, in a cDNA library of the androgenic gland of Cherax quadricarinatus, whose tissue expression and in situ hybridization (ISH) results indicated that the gene was specifically expressed in the terminal ampulla [33]. In 2009, Tomer Ventura et al. identified the Mro-IAG in a M. rosenbergii cDNA library and further RNA interfered with the Mro-IAG in mature male individuals, identifying that the regeneration of male external sexual characteristics was inhibited in interfered individuals; silenced individuals showed an atypical male growth pattern and a reproductive phenotype with stagnant spermatogenesis and spermatophore development, and an overall hypertrophic and hyperplastic gonad [34]. In 2012, Tomer Ventura et al. obtained M. rosenbergii neofemales with male reversal to females by RNA interfering with the Mro-IAG of male individuals in incomplete sexual differentiation, in which the appearance of secondary sexual characteristics was delayed and reduced in the interfered individuals, and the neofemales were then mated with normal males to successfully acquire all-male offspring [35]. In conclusion, IAG is not only involved in sex regulation, sex and gonadal development, but also in spermatogenesis in reproduction. Up to now, IAGs have been identified in decapod Scylla paramamosain [36], C. sapidus [37], Portunus pelagicus [38], F. chinensis [39], L. vannamei [40], M. japonicus [41], P. monodon [42], M. rosenbergii [34], Macrobrachium nipponense [43], and C. quadricarinatus [33], Procambarus virginalis [44], and the isopod Armadillidium vulgare [45].

3.2.2. Sex-Lethel (Sxl)

Sex-lethel (Sxl) is a key component of the sex determination cascade in Drosophila, which produces various spliceosomes in response to sex chromosomal signals and regulates sex-specific shearing of downstream Tra, Dsx [46]. EsSxl was found to be expressed in both male and female tissues of E. sinensis, and its embryonic expression was identified to be insignificant from the blastula stage to the egg-nauplius stage, but it increased significantly from the nauplius stage to the original zoea stage, suggesting that EsSxl may play a role in embryonic development [47]. Sxl tissue expression was similar to EsSxl in L. vannamei. All six splice variants of PvanSxl were expressed in fertilized eggs during embryonic development, but PvanSxl2 and PvanSxl4 were expressed at the highest levels up to blastocoele; PvanSxl3 expression increased in the gastrula stage, and PvanSxl1, PvanSxl3, PvanSxl6 expression decreased in the mysis stage. Furthermore, PvanSxl ISH results revealed that it was involved in both male and female gametogenesis and gonadal development [48]. Among the four splice variants of Sxl identified and obtained in C. quadricarinatus, CqSxl3 is tissue-specific, with higher expression in the testis than in the ovaries; CqSxl1 and CqSxl4 are widely expressed in a variety of tissues, and CqSxl2 is less expressed in the shrimp, while transcript levels of CqSxl1/3/4 slowly increase with embryonic development [49]. Up to now, in addition to the Sxl homologs identified in E. sinensis [47], L. vannamei [48], and C. quadricarinatus [49], Sxl homologs have been identified in P. clarkii [50], Sagmariasus verreauxi [51]. In decapods, it has been reported that Sxl is not sex-specific and that it is involved in sex differentiation, embryonic development, and gonadal development.

3.2.3. Transform-2 (Tra-2)

In Drosophila, Transform-2 (Tra-2) is involved in the sex determination cascade, where it controls sex-specific splicing of Dsx pre-mRNA production, as well as sex differentiation and development [52]. Leelatanawit et al. identified the PmTra-2 sequence in crustaceans by constructing a cDNA library of P. monodon testis, but tissue expression revealed that PmTra-2 was insignificant expressed in ovaries and testis and was a nonsex-specific gene [53]. In F. chinensis, Tra-2c has three spliced mRNA transcripts, FcTra-2a, FcTra-2b and FcTra-2c, and the sequence analysis revealed that the three splice variants are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA [54]. Among which, FcTra-2a and FcTra-2b were not significantly differentially expressed in the male and female gonads of the shrimp while the expression of FcTra-2c was significantly higher in the ovaries than in the testis [54]. In addition, the expression of FcTra-2c was detected to be significantly increased at the mysis stage during the early stages of shrimp embryonic development, thus FcTra-2 may be involved in the sex determination of females [54]. Luo et al. identified four Tra-2 splice variants from E. sinensis, termed Estra-2a, Estra-2b, Estra-2 and Estra-2d, and the sequence analysis revealed that the four splice variants are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA [55]. In addition, four splice variants of Estra-2 were all highly expressed in the fertilized egg, and in the 2 - 4 cell and blastula stages compared with larval stages, suggesting their maternal origin in early embryonic developmental stages, and tissue expression showed that Estra-2a was expressed at higher levels in male somatic tissues than in other shrimp tissues; Estra-2b and Estra-2d were both showed small sex differences in gonads; Estra-2c was expressed at higher levels in the ovary, suggesting that Estra-2c is involved in sexual differentiation of the crab [55]. In addition, Tra-2 plays a role in the sex differentiation and embryonic development of M. nipponense [56] [57]. In summary, in crustaceans, Tra-2 is associated with sex determination and differentiation, and embryonic development processes. Up to now, Tra-2 genes have been identified and obtained in P. monodon [53], F. chinensis [54], M. nipponense [56], E. sinensis [55], S. verreauxi [51], and Palaemon serratus [58].

3.2.4. Doublesex and Mab-3 Related Transcription (Dmrt)

The Dmrt family is involved in sex determination and sexual differentiation in insects, nematodes, and vertebrates [59], and Doublesex (Dsx), a member of the Dmrt family, is also a member of the bottom of the sex determination cascade in Drosophila, where sex-specific splicing produced by regulation of Tra and Tra-2 determines female and male differentiation, respectively [60]. It has been reported that the Dmrt family has a highly conserved structure and function in sex determination and differentiation [59] [61].

Up to now, Dmrt have been identified in M. nipponense [62], S. verreauxi [51], P. serratus [58], M. rosenbergii [59] [63] [64], E. sinensis [65], S. paramamosain [66]. In E. sinensis, EsDmrt-like was detected only in the testis and was highly expressed in the immature stage, which is similar to the vertebrate DMRT1 gene expression pattern, and EsDmrt-like mRNA was localized in the supporting cells around the germinal tubules and developing germ cells such as spermatogonia, spermatocytes, and spermatocytes and not expressed in spermatozoa, the above results suggest an important role of EsDmrt-like in the development/differentiation of the male crab testis [65], as evidenced by further EsDmrt-like RNA interference results [67]. The MniDmrt11E identified in M. nipponense was highly expressed in both ovaries and spermatocytes of mature males and females, and was mainly localized in ovarian oocytes and spermatocytes of spermatocytes; during embryonic development, the expression level of MniDMRT11E was higher at the oogenesis stage than at other stages; at different stages of ovarian development, the expression of MniDMRT11E gradually increased from stage I to III and decreased to the lowest at the end of stage IV; after further RNA interference, the expression of VG significantly decreased and the expression of IAG significantly increased in mature individuals, the above results indicated that MniDMRT11E is associated with embryonic development, sexual maturation and gonadal development [62]. Furthermore, RNA silencing MroDmrt11E in M. rosenbergii resulted in a signficant decrease in IAG expression, suggesting that IAG is positively regulated by MroDmrt11E; additional research has shown putative Dmrt binding sites in the promoter region of the male sex differentiation effector gene IAG [64].

Doublesex (Dsx) have been identified in M. rosenbergii [59], F. chinensis [68], L. vannamei [69], and Daphnia magna [70]. Sex-biased expression of Dsx was associated to male sex development in Daphnia magna, indicating that Dsx may have a conservation role in sex determination in crustacean species [70]. LvDsx expression was reported to be higher in the testis than in the ovaries in L. vannamei [69]. The ablation of the eyestalk in M. rosenbergii significantly increased Dsx expression levels in the testis and adrogenic gland [59]. In F. chinensis, FcDsx showed a sex-biased expression pattern in various tissues, and its expression level increased with developmental stage. One possible Dsx binding site was identified in the FcIAG promoter region, and knocking down FcDsx reduced FcIAG expression, suggesting that FcDsx may be an upstream regulator of FcIAG [68].

In summary, Dmrt is an important gene in crustacean sex differentiation, as well as gonad and embryonic development. Second, gene structure and functional studies have shown that the crustacean Dmrt interacts with IAG, which is an essential gene for sex regulation in crustaceans. However, there are yet insufficiently defined mechanisms of action to investigate the regulatory pathways of this cascade, necessitating further experimental investigation.

3.2.5. Feminization-1 (Fem-1)

The feminization-1 (Fem-1) gene is characterized by one of the most common protein-protein interaction motifs, ankyrin repeat motifs, displays many expression patterns in vertebrates and invertebrates, and plays an essential role in the sex-determination/differentiation pathway in Caenorhabditis elegans [71]. In the model species C. elegans, a system analogous to the Drosophila sex regulatory cascade occurs, in which Fem-1, together with Fem-2 and Fem-3, plays a crucial part in the sex determination cascade by downregulating Tra-1 expression in nematodes [71] [72]. Up to now, Fem-1 has been identified in P. serratus [58], E. sinensis [73], L. vannamei [74], M. nipponense [75], S. paramamosain [66], P. monodon [12]. In M. nipponense, Mnfem-1 is highly expressed in both unfertilized eggs and cleavage stage and thereafter dropped to a low level from blastula to zoea during embryogenesis, indicating that the Mnfem-1 in early embryos is maternally inherited [75]. In L. vannamei, natural antisense transcript regulation leads to the expression of Fem-1 in the female gonads, resulting in the expression of Fem-1 in the female gonads, thereby allowing sexual differentiation in female individuals, and in addition, LvFem-1 expression in spermatogonia suggests that it is also required for male gonad differentiation [74]. Tissue expression pattern of Fem-1 in E. sinensis revealed persistently high levels of Fem-1 expression in the spermatophore, ovary, hepatopancreas and muscle suggesting its potential role in the final stages of gonad development [73]. Based on the high expression of Fem-1 in early embryonic development of crustaceans, Song et al. and Ma et al. suggested that Fem-1 may be maternally inherited in decapods [73] [75]. In summary, the Fem-1 gene may be involved in the process of sex and gonadal differentiation in crustaceans, but the exact process and mechanism of its role need further experimental investigation.

3.2.6. Other Sex Determination and Differentiation-Related Genes

In recent years with the high throughput sequencing technology and the construction of cDNA libraries in crustaceans, many other sex-related genes have been identified in addition to the homologous genes found in crustaceans based on the inclusion of model organism sex regulatory pathways. In the crustacean Artemia, silencing of ArMasc by RNA interference resulted in a significant upregulation of its male-to-female ratio, suggesting that ArMasc is involved in the sex determination process [76]; the Sox family is involved in male gonad development and reproduction in crustaceans [58] [69] [77] [78] [79]; BMP7 is involved in the gonad development process [80] [81]; CFSH regulates part of decapod sex development [82] [83] [84] [85] [86]; ubiquitin C-terminal hydrolases (UCHLs) regulate ovarian development as well as EGFR regulates the formation of male secondary sexual characteristics [87]. In addition, with the development of sequencing technology and molecular biology, more sex-related genes have been successfully mined, such as Vasa, Foxl2, Vitellogenin (Vg), Ecdysone Receptor (EcR), Sry, Ftz-f1, etc. [88] - [95], however, there is still a lack of research on the functional mechanisms of genes in sex determination, differentiation and gonadal development.

4. Conclusions

Many species of crustaceans have a promising future in aquaculture, and with the development of science and economy, academic problems in the field of aquaculture have attracted much attention with the rapid development of aquaculture industry in China, among which monosex culture has attracted much attention because of its advantages in immunization and farming practice. The prerequisite for monosex culture is to elucidate the molecular mechanisms of sex determination and differentiation in crustaceans and to identify the key genes that play a role in this process. However, up to now, only Sagi et al. have successfully achieved monosex reproduction in M. rosenbergii and disclosed the technique of preparing all-male shrimp, and no research has been done to achieve sex reversal in other economic species such as L. vannamei, therefore needs to refine the existing sex theory and to elucidate the patterns of sex determination and differentiation in crustacean species as a whole through more researches [19] [28].

To date, the mechanisms of sex determination and differentiation have been initially elucidated in model organisms such as Bombyx mori and C. elegans, while studies on sex determination in crustaceans are relatively backward, most notably: The genome sequences of most crustacean species have not yet been elucidated, and therefore the bioinformatic function prediction around gene sequences is difficult and the prediction accuracy is low. On the other hand, it needs to elucidate the genome sequence of each crustacean species through bioinformatics to facilitate the mining of key genes for sex determination and differentiation.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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