Identification of Sex-Related Genes from the Three-Spot Swimming Crab Portunus sanguinolentus and Comparative Analysis with the Crucifix Crab Charybdis feriatus

Simple Summary Crabs within the family Portunidae are important marine species in both aquaculture and fishery sectors. The current aquaculture status of most portunids still relies on wild-caught fisheries due to the lack of essential knowledge regarding their reproductive biology and underlying governing mechanism. In the present study, we compared the differentially expressed genes (DEGs) between the different sexes of Portunus sanguinolentus based on their gonadal transcriptome profiles and subsequently contrasted them with the gonadal DEGs of Charybdis feriatus, the other member of the family Portunidae. In total, 40,964 DEGs between the ovaries and testes of P. sanguinolentus were uncovered, with 27,578 up-regulated and 13,386 down-regulated in females. After comparison, C. feriatus has approximately 63.5% of genes in common with P. sanguinolentus, with 62.6% showing similar expression patterns. Interestingly, the DMRT gene was specifically expressed in male P. sanguinolentus, while its homologous gene—doublesex (DSX)—was specifically expressed in male C. feriatus. The DEGs obtained from the gonadal transcriptome of P. sanguinolentus are a beneficial resource for future genetic and genomic research in P. sanguinolentus and its close species. The transcriptomic comparison analysis might provide references for better understanding the sex determination and differentiation mechanisms among portunids. Abstract Crabs within the family Portunidae are important marine species in both aquaculture and fishery sectors. The current aquaculture status of most portunids, however, still relies on wild-caught fisheries due to the lack of essential knowledge regarding their reproductive biology and underlying governing mechanism. With the advancement of sequencing technology, transcriptome sequencing has been progressively used to understand various physiological processes, especially on non-model organisms. In the present study, we compared the differentially expressed genes (DEGs) between sexes of Portunus sanguinolentus based on their gonadal transcriptome profiles and subsequently contrasted them with the gonadal DEGs of Charybdis feriatus, the other member of Family Portunidae. In total, 40,964 DEGs between ovaries and testes were uncovered, with 27,578 up- and 13,386 down-regulated in females. Among those, some sex-related DEGs were identified, including a dmrt-like (DMRT) gene which was specifically expressed in males. C. feriatus has approximately 63.5% of genes common with P. sanguinolentus, with 62.6% showing similar expression patterns. Interestingly, the DMRT gene was specifically expressed in male P. sanguinolentus while its homologous gene—doublesex (DSX)—was specifically expressed in male C. feriatus. The DEGs obtained from the gonadal transcriptome of P. sanguinolentus are a beneficial resource for future genetic and genomic research in P. sanguinolentus and its close species. The transcriptomic comparison analysis might provide references for better understanding the sex determination and differentiation mechanisms among portunids.


Introduction
Portunidae are important marine crab species with a diversity and speciation of around 300 members [1], among which many are considered important marine aquaculture and captured-fishery species [2]. The culture of crabs has been gaining attention since the last decade because of the high demand for live crabs and crab products in the global market. In particular, Portunus sanguinolentus and Charybdis feriatus are potential marine aquaculture species in China due to their high meat quality and fast growth rate [3]. However, the current crab culture is still largely dependent on wild capture fishery [4]. The unregulated fisheries practice, combined with increasing overfishing and environmental deterioration, resulted in the quick depletion of many crab resources [5]. P. sanguinolentus is generally named the three-spot swimming crab for its three distinct red to chestnut spots on the back of its carapace. It can be found in Indo-Pacific waters from the east coast of South Africa to Hawaii [6]. So far, genetic studies on P. sanguinolentus are limited. Thus, the availability of P. sanguinolentus gonadal transcriptomic profiles will benefit greatly to the revealing of the regulatory roles of ovary and testis in portunids and serve as a useful comparison for other marine crabs.
Charybdis feriatus, naturally distributed in the coastal areas of Guangdong, Zhejiang, Guangxi, Hainan, and Fujian provinces of China and the waters of Indo-Pacific regions, is a large, highly prized marine portunid crab species. Juvenile crabs inhabit sandy shores while adults preferentially move to the muddy offshore areas [7]. They are easy to distinguish according to their prominent red and white carapace and an obvious cross on the median surface of their carapace [8]. Despite belonging to different genera, P. sanguinolentus and C. feriatus share not just morphological similarities, but also life cycles and regeneration ability. However, the information of a genome, transcriptome, and associated molecular markers of these two species are rare at the moment.
RNA-seq is a practical tool for uncovering differential gene expression under certain conditions of non-model organisms. It has been successfully performed in many aquaculture invertebrates for various purposes, including identification of differentially expressed genes (DEGs) in different tissues or under specific conditions in crabs like Scylla paramamosain [9,10], Scylla olivacea [4], Portunus trituberculatus [11,12], Eriocheir sinensis [13,14] and Sinopotamon henanensis [15]. RNAseq analysis is also used in resolving comparative genomic-level issues, especially for non-model organisms [16]. In principle, comparative RNA-seq studies between closely related species could offer excess genomic resources and simultaneously supply information about the processes of differentiation between species [17].
The present study aimed at screening the DEGs between sexes of P. sanguinolentus based on its gonadal transcriptome data. In addition to contributing to the genetic resources available for portunid species, we compared the gonadal DEGs of P. sanguinolentus with that of C. feriatus obtained from previous research [18] in an attempt to understand the similarities and differences in sex-biased genes of both portunid species. These data would be helpful for future studies on the gonad molecular regulatory mechanism and sex differentiation mechanism of marine crabs.

Transcriptome Analysis and Validation
The present experiment was approved by the Institutional Animal Care and Use Ethics Committee of Shantou University. The detailed transcriptome analysis method was described in [19]. The ovaries in stage III-IV and testes in stage II-III without vas deferens were extracted from four females and four males P. sanguinolentus, respectively. Females P. sanguinolentus were named as PS-F and males as PS-M. In brief, total RNA was extracted from the gonads of PS-F and PS-M, converted into cDNA libraries, and sequenced on Illumina HiSeq 3000 platform. Clean reads were subjected to de novo assembly and annotated to public databases.

Analysis of Differentially Expressed Genes of P. sanguinolentus
StringTie was used to calculate FPKMs (Fragment per kilobase of exon model per million mapped reads) [20] of transcripts of the testes (PS-M) and ovaries (PS-F) samples of P. sanguinolentus. FPKMs were calculated based on the length of the fragments and reads count mapped to this fragment. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. DEGs were detected by the EBSeq package with raw counts as inputs. Unigene with false discovery rate (FDR) ≤ 0.001 and the absolute value of log2 Ratio ≥ 1 was considered significantly different expressed gene. GOseq with the Wallenius noncentral hyper-geometric distribution model was used for Gene Ontology (GO) enrichment analysis (p-value ≤ 0.05), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using KOBAS with the hyper-geometric distribution model.

Comparison of the DEGs between P. sanguinolentus and C. feriatus
Using the previously published data of C. feriatus (CF) [18], the whole gonadal transcriptome profiles between the two crab species were compared using BLAST. The assembled sequences from the two species were considered as the same transcriptome and given a new ID considering as "new unigene" when the identity reached 98%. At least one transcript was mapped to one "new unigene" in both libraries. Raw fragments detected in each sample of the two profiles and FPKM values were accordingly considered as the FPKM value of the "new unigene". If the new unigene is composed of more than one transcript, the FPKM values of more than one transcript are added together as the FPKM of the "new unigene". Subsequently, the expression values (FPKM) of the "new unigenes" among females (PS-F vs. CF-F) and males (PS-M vs. CF-M) of the two portunid species were compared, respectively. The threshold of the DEGs was set as FDR < 0.001 and the absolute value of log2 Ratio > 1. The GO and KEGG annotations of the "new unigenes" were performed following Section 2.2.

DEGs of P. sanguinolentus
To identify the DEGs between males and females of P. sanguinolentus, the mapped reads were normalized to calculate the unigenes expressions between the PS-M and PS-F using FPKM value. The details of unigene expressions can be found in Supplementary  Table S2. Among all unigenes, 40,964 DEGs were obtained with 27,578 up-and 13,386 down-regulated in female P. sanguinolentus ( Figure 1, Supplementary Table S3). Some genes directly or indirectly related to sex determination and differentiation, like AIP, CYCB, DmX-like (DMX), ER, FEM1, GPR, HSP90, PG, wnt4 precursor (WNT4P), VASA, VTG, VTGR, crustacean hyperglycemic hormone (CHH) and ovary development-related protein (ODR) were up-regulated in females, while male-reproductive-related genes such as Dmc1-like (DMC1), MRLIM, MSD, sex peptide receptor (SPR), insulin receptor (IR) and spermatogenesis-associated protein 2 (SPATA2) were up-regulated in males. Besides, SRY-like (SRY) and dmrt-like (DMRT) gene were specifically expressed in males. These DEGs were further analyzed using KEGG pathway enrichment to determine their metabolic pathways. The significant enrichment KEGG pathway (p value < 0.0001) were Ribosome biogenesis in eukaryotes, Aminoacyl-tRNA biosynthesis, Parkinson's disease, Proteasome as well as Glyoxylate and dicarboxylate metabolism ( Figure 2).

Comparison of Unigenes between P. sanguinolentus and C. feriatus
The expression levels (FPKM values) of the unigenes in both libraries were mostly (~99%) between 0-100 (Table 1). 9,436 "new unigenes" with more than 98% similar identities on both libraries ( Table 2, Supplementary Table S4) were identified. Among the 9,436 unigenes, approximately 63.5% of genes were shared between the libraries of P. sanguinolentus and C. feriatus, with 62.6% showed similar expression patterns. More DEGs were shared among females (n = 5085) of the two species compared to males (n = 906). The number of upregulated (n = 7080) and specifically expressed (n = 256) DEGs were higher in P. sanguinolentus females when compared to C. feriatus females. In contrast, the males of C. feriatus had higher upregulated and specifically expressed DEGs compared to those of P. sanguinolentus ( Figure 5). Some genes related to sex determination and differentiation in P. sanguinolentus and C. feriatus were screened out from the shared up-and down-regulated DEGs between the two portunid species (Figure 6), such as AIP, ODR, CYCB, DMX, GPR, PG, WNT4P, VASA, and VTGR were up-regulated in females, and some were up-regulated in males including SPR and SPATA2. We also verified those genes with the same expression tendency in both P. sanguinolentus and C. feriatus, revealing the same expression patterns with RNA-seq ( Figure 7). Additionally, DMRT was specifically expressed in PS-M while no DMRT gene was found to be differentially expressed in C. feriatus, but its homologous gene DSX was specifically expressed in CF-M.

DEG Profiles of P. sanguinolentus
High-throughput transcriptome sequencing allows analysis of DEGs under different physiological conditions and treatments [21,22]. Our previous study identified a total of 119,718 unigenes with an average length of 904 nt in P. sanguinolentus [19]. As a continuation, in the present study, an analysis of DEGs in male and female P. sanguinolentus was performed, aiming to explore potentially valuable genes to better comprehend the basics of sex determination and differentiation biological mechanisms in P. sanguinolentus and possibly serve as references for other crustacean species. A comprehensive grasp of the molecular mechanisms of sex preference and the regulatory pathways that occur in the gonads may lead to future manipulation of desired traits and will impart data resources for future gene expression, functional and reproductive investigation for P. sanguinolentus.
Comparing between sexes, the expression of upregulated unigenes in P. sanguinolentus ovary was almost twice that of the testis. This expression pattern of more transcripts upregulated in females was also found in the comparative gonadal transcriptome of other invertebrates, such as in the gonadal transcriptome of the S. paramamosain [23,24] and C. feriatus [18], as well as crustacean Caligus rogercresseyi [25]. This pattern of expression might be due to the complex role of the ovary in the female reproduction process. After DEG annotation, most DEGs were involved in ribosome biogenesis in eukaryotes (p value: 2.81 × 10 −5 ). Ribosomes are the workplace for protein biosynthesis and are directly associated with translation, localization, and cell growth, cycle, and proliferation [26]. The high enrichment of DEGs in ribosome biogenesis could be due to the different reproduction-related processes occurring within the testis and ovary of P. sanguinolentus, including gametogenesis and hormonal regulation. Additionally, growth-related GO items such as cellular process, cell, binding in the classify of biological process, cellular component, and molecular function were found to be enriched based on the DEGs of P. sanguinolentus. A similar pattern was detected in the gonadal DEGs of S. paramamosain [10] and E. sinensis [27], highlighting the potential involvement of a high number of sex-biased genes in the gonadal growth and development of portunid species.

Genes Differentially Expressed in Females and Males of P. sanguinolentus
The FPKMs of the unigenes obtained from gonads of P. sanguinolentus were mainly in the range of 0-100, which is accordant with C. feriatus. However, several unigenes exhibited FPKM values beyond ten thousand, among which was the ODR gene, specifically expressed in females of P. sanguinolentus and C. feriatus, suggesting that the ODR were vital for female reproductive development. Additionally, as expected, PG and VTGR were upregulated in both females of P. sanguinolentus and C. feriatus. The former is involved in oogenesis and ovarian development and maturation of animals including crustaceans [28,29] while the latter existed in the oocyte membranes [30]. PGs participate in regulating the oocyte maturation of animals including crustaceans [28,31,32]. And it has been reported to stimulate ovarian maturation in Parapanaeopsis hardwickii [33]. Furthermore, PG can also promote the synthesis of VTG mRNA, as reported in freshwater crayfish Cherax albidus [34]. The female up-regulated VTG gene is crucial to the development of oocyte and embryo [35]. VTGs were up-regulated in the mature female gonad of P. trituberculatus [36], suggesting that the up-regulation of VTG might be a preparation for oocyte development. Additionally, estrogen hormones may also increase the synthesis of VTG in oviparous vertebrates by the increase in VTG gene transcription, and the estrogen hormone is regulated by HSP90 [37,38]. Paolucci et al. [39] showed that the presence and up-regulation of PG and ER resulted in the accumulation of VTG in crayfish. A previous study also showed that HSP90 was involved in the regulation of VTG synthesis [38] and was upregulated in the copulated ovary of D. melanogaster [40]. In the present study, PG, VTG, HSP90, and ER genes were differentially expressed in our transcriptomic sequencing data, with a higher level of expression in ovaries than in testes. This indicates the involvement of PG, VTG, HSP90, and ER genes in the oocyte development of crabs and highlights the conserved oogenesis maturation pathway in animals.
Specifically expressed in the gonads, the VASA gene is actively expressed during early gametogenesis [41,42]. VASA exists in both invertebrates and vertebrates [41] and plays a vital role in germ cell development, proliferation, maintenance, and gametogenesis [41], including the gonadal development and gametogenesis of crustaceans [42][43][44]. Specifically, the VASA gene is expressed in germ cells encoding for RNA-dependent helicase during the whole developmental stages of pacific white shrimp Litopenaeus vannamei [43]. The present study found that the VASA gene was up-regulated in ovaries, which is in accordance with that in S. paramamosain [23] and C. feriatus [18], verifying its reproduction-related function. And it was found that Vasa mainly function in auxiliary cells of oyster ovaries [45].
The upregulation of CYCB in the females of P. sanguinolentus and C. feriatus was expected as cyclin B was commonly known to be related to the meiotic maturation of oocytes [46] and active during crab's ovarian maturation [46]. In line with the results of this study, a high expression level of CYCB in the ovary was also observed in S. paramamosain [47], E. sinensis [48], Macrobrachium rosembergii [49], and P. monodon [46], suggesting that cyclin B is crucial for the ovarian development and maturation of crustaceans. Some female up-regulated genes in P. sanguinolentus that were potentially involved in the regulation of gametogenesis, reproduction, gonadal differentiation, and development, such as FEM-1 [11], CHH [50], and GPR [23] were identified in this study. It is reported that FEM-1 might be involved in the early sex determination and late gonad development of crabs [51]. CHH may be involved in the inhibition of molting and ovarian development of crabs [52,53]. GPR is involved in the reproductive system's maturation [23]. WNT4P was also found to be upregulated in females. WNT4, a member of the WNT family, plays a vital role in female reproductive development in mammals, controlling steroidogenesis in the gonad and supporting oocyte development [54,55]. Lack of WNT4 in mice led to a striking reduction in the number of developing oocytes and resulted in gonadal masculinization of the female [56]. Besides, DMX, which contains a DNA-binding motif called DM domain, was up-regulated in both males of P. sanguinolentus and C. feriatus, which might be potentially related to the sex determination mechanisms of the two species. The up-regulated gene AIP was a useful probe for studies on specific gene expression and prostatic secretory mechanisms regulated by androgen [57].
Additionally, some sex determination and differentiation-related genes were found to be upregulated in males, including DMC1, MRLIM1, MSD, SPR, and SPATA2. The upregulated expression of DMC1 in male P. sanguinolentus was in accordance with that in the male giant tiger shrimp Penaeus monodon [58]. DMC1 was specifically expressed in embryonic ovaries and testicular germ cells during meiosis of Chinese mitten crab [59]. A similar up-regulation pattern of MRLIM1 in males of both P. sanguinolentus and C. feriatus was also observed in the oriental river prawn Macrobrachium nipponense [60]. The MRLIM1 was reported to be involved in the endocrinal function of the eyestalk [61]. MSD might be related to male sterility. SPR, upregulated in both males of P. sanguinolentus and C. feriatus, was also expressed in embryonic and larval stages of Drosophila as well as in the adult male nervous system, while the expression of SP was confined to the male reproductive system [62]. Maurizio et al. [63] reported that SPATA2 was expressed in testis of rats and originated from Sertoli cells in the period of infantile-juvenile and increases gradually during growing up. Some genes like IR, SRY, and DMRT were specifically expressed in males. IR was found to be the receptor of insulin-like androgenic gland hormone (IAG) [64]. IAG is regarded as the key regulator in crustacean male sexual differentiation [65]. SRY was discovered in the human Y chromosome for the first time as a sex-determining factor and contains a conserved high-mobility group (HMG) box [66], SOX14 was similar to the HMG box with SRY on sequences [67].
In short, some genes showed similar upregulation patterns in both females and males of P. sanguinolentus and C. feriatus. This might signify the close relationship between the two species and indicate that the genes related to sex determination and differentiation mechanisms were relatively conservative between portunids. The GO annotation of the "new unigenes" indicated that metabolic process, protein binding, and membrane were respective enriched most amount of unigenes, which was accordance with the GO annotation of total unigenes in P. sanguinolentus [19], C. feriatus [18], E. sinensis [26], S. paramamosain [23] and P. trituberculatus [11].

Differences between the DEGs of P. sanguinolentus and C. feriatus
When comparing the DEGs of P. sanguinolentus and C. feriatus, the DMRT gene was specifically expressed in male P. sanguinolentus, whereas its homologous gene DSX gene was specifically expressed in male C. feriatus instead of the DMRT gene. DMRT and DSX belong to the same DM DNA-binding domain superfamily [68]. DMRT, which has a function in sex differentiation and reproductive development, was also found to be differentially expressed in other crabs [11,69] but not in E. sinensis [26] and S. paramamosain [10]. Whereas, DSX gene functions in the sexual differentiation of crustaceans and is indispensable for male trait development [70]. The species-specific expression pattern here might be related to the different sex determination regulation mechanisms and the DM domain gene evolutions in different species, which warrants further research. DMRT1 is one of the well-conserved genes associated with sex differentiation from invertebrate to vertebrate, despite the great diversity of animal sex-determination mechanisms [71]. The difference in the expression of DMRT and DSX between the two species in our study was speculated on the account of the divergence of sex determination mechanisms between P. sanguinolentus and C. feriatus, although future validation is urgently needed to support this hypothesis.

Conclusions
The present study provides a comprehensive excavation of DEGs of P. sanguinolentus through the comparison of its gonadal transcriptome profiles. As a whole, functional analyses of the present dataset identified many gonadal DEGs (such as female up-regulated genes including AIP, ODR, CYCB, DMX, GPR, PG, WNT4P, SOX21b, VASA, CHH, and VTGR, male up-regulated genes including DMC1, MRLIM, MSD, SPR, SPATA2, IR, and SRY) that are potentially involved in reproduction, specifically sex determination and differentiation. In addition, we found that the DMRT gene was specifically expressed in male P. sanguinolentus and the DSX gene specifically in male C. feriatus. We proposed to focus on sex determination mechanisms of P. sanguinolentus and C. feriatus in future work to verify the difference revealed in the present study. Our results illustrate the application of high throughput transcriptome sequencing as a basis for mining tissue specific functional genes in a non-model species. The mine of sex-related genes from gonads of P. sanguinolentus in the present study will be helpful for the study of the mechanism of sex determination and differentiation as a reference for crabs. These transcriptome data will contribute to future gene functional and genomic analysis of portunid species.   Institutional Review Board Statement: The present experiment was approved by the Institutional Animal Care and Use Ethics Committee of Shantou University (approval code SUMC2021-211).

Conflicts of Interest:
The authors confirm that this article content has no conflict of interest.