Limited information is available on the sex-determination system of brachyuran crabs. For decades, scientists believed that the XY/XX sex-determination system is the only sex-determination system for crabs . Recently, besides the XY/XX , the WZ/ZZ sex-determination system has been found in some crab species such as Eriocheir sinensis , S. paramamosain, S. tranquebarica, and S.serrata . Sex-specific SNP marker is considered a powerful tool for understanding the sex-determination system . Detection of female-specific SNP markers provides insights into a WZ/ZZ sex determination system in S. paramamosain . In the present study, SNP1888 was mapped on the LG32 of the genetic map, which is a sex-related linkage group, and was located very close to the positions of other sex-specific SNPs (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, SNP12) [10, 11]. The presence of female-specific SNP markers and their locations on the female linkage map supports the existence of a WZ/ZZ sex determination system in S. paramamosain.
Phosphorylation plays critical roles in several aspects of cell life, including metabolism, proliferation, apoptosis, differentiation, cell division, and DNA replication. Protein phosphorylation is catalyzed by a group of enzymes called kinases, which add a phosphate group to serine, threonine, tyrosine, or, to a lesser degree, histidine residues . Twenty-eight putative phosphorylation sites (14 sites on serine and 14 sites on threonine residues) in Sp-Pol protein in the present study suggest that Sp-Pol protein may be activated by some kinases enzymes. The prediction of six potential O-glycosylation and one N-glycosylation site indicates that glycosylation may also affect the function of Sp-Pol protein. Glycosylation, like phosphorylation, is important due to the various roles in protein folding, protein trafficking and localization, cell-cell interactions, and epitope recognition . Glycosylation can be classified into four types: N-linked, O-linked, Glypiation, and C-linked. In N-linked glycosylation, the oligosaccharide chain is attached to the amide nitrogen of asparagine. In O-linked glycosylation, the glycan is attached to the hydroxyl oxygen of serine or threonine .
Sequence homology and phylogenetic analysis showed that Sp-Pol protein had the highest similarity to a Retrovirus-related Pol polyprotein gene from P. vannamei (60.47% identity) and was also slightly homologous (20–35% identity, bit score: 50–55, expectation value: 10− 3-10− 6) to a group of uncharacterized and hypothetical proteins from crustaceans, insects and echinoderms species. Although Sp-Pol protein was closely related to a Retrovirus-related Pol polyprotein from P. vannamei and a hypothetical protein from swimming crab (Portunustri tuberculatus), it was distinct from other identified proteins. The very low similarity of Sp-Pol protein to other known homologous proteins and failure to predict functional domains indicates that this gene may not belong to the Gag-Pol polyprotein family and can be reclassified as a new family. Notably, the protein encoded by the Sp-Pol gene (204 aa) was quite smaller than Retrovirus-related Pol polyprotein from P. vannamei (541 aa) and other homologous proteins.
Spatio-temporal expression analysis showed that Sp-Pol was expressed in all the examined tissues and during all life stages, suggesting that it has a wide range of functions in different tissues and developmental stages. Expression of Sp-Pol in gonads was higher than other tissues including heart, hepatopancreas, muscle, gill, thoracic ganglion, gut, and stomach. Comparing the expression of Sp-Pol between females and males, male crabs displayed a higher level of Sp-Pol expression in the testis compared to the ovary. The higher expression of Sp-Pol in testis revealed that it is likely to play more important roles in the sexual development of male compared to female S. paramamosain. The lower expression of Sp-Pol in the ovary compared to testis may due to the mono-allelically expression pattern of this gene in the ovary. In the ovary, mono-allele which owing nucleotide acid C at the SNP1888 locus was expressed, and the other allele which owing T was not expressed. The mono-allele expression could result in several different outcomes at the transcriptional level. There is a general trend for monoallelically expressing cells to have fewer transcript levels than biallelically expressing cells .
Recently, several sexual genes have been identified to be involved in the “eyestalk-AG-testis” endocrine axis . 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 [22, 23, 40–42]. For example, it has been reported that CFSH, Sox, Dsx, Slx, Dmrt, GEM, and Fem-1 are negatively regulated by the eyestalk (X-Organ).
Considering the important involvement of eyestalk (X-Organ) in the regulation of sex-related genes in male crustaceans, we hypothesized that eyestalk may participate in the regulation of Sp-Pol, either directly or indirectly upstream of Sp-Pol. Therefore, to evaluate the regulatory role of eyestalk in the regulation of Sp-Pol, we investigated the expression level of Sp-Pol after unilateral eyestalk ablation. After unilateral eyestalk ablation, the expression level of Sp-Pol was significantly up-regulated in the testis and male hepatopancreas and downregulated in female hepatopancreas. A similar expression pattern has been reported for IAG in the male blue crab, Callinectes sapidus. The expression of IAG was found to be up-regulated after eyestalk ablation in testis and hepatopancreas . The significant changes of Sp-Pol mRNA in the hepatopancreas suggest Sp-Pol may be related to nutrient metabolism in the hepatopancreas. In decapods, the hepatopancreas is the major source for providing energy to gonad development . The increase of hepatopancreas weight during the reproductive season has been documented in several studies (i.e., material and energy storage for oogenesis) . Vitellin (Vn) produced in the hepatopancreas is the main component of yolk proteins, which is stored in oocytes and is a nutritional source for animal embryo growth . Vn is processed from its precursor, vitellogenin (Vg). In S. paramamosain, both the hepatopancreas and ovary are sites of Vg synthesis, but the hepatopancreas is the primary synthesis site . Therefore, further studies are required to evaluate the role of Sp-Pol in vitellogenesis and hepatic metabolism. Eyestalk ablation revealed that Sp-Pol expression may be regulated by inhibitory factors present in the eyestalk ganglia.
To investigate the potential role of Sp-Pol in gametogenesis, we performed FISH to locate Sp-Pol mRNA in the ovary and testis. In the ovary, Sp-Pol transcripts were detected in the oogonium cells but no signal was found in the follicle cells. In the testis, strong Sp-Pol signals were detected in the epithelia of seminiferous tubules. Signals decreased toward the centre of the tubules of the testis, which means the expression level of Sp-Pol may progressively decrease from spermatogonia to spermatids. Spermatogenesis starts with the mitotic division of spermatogonia cells that locate on one side of the seminiferous tubules. Then, spermatogonia cells differentiate into primary spermatocytes. Spermatocytes divide into two equal haploid spermatids by Meiosis II. Later, spermatids differentiate into spermatozoa in the central region of the tubule . The role of the epithelium seminiferous tubules is not well studied in crustaceans. In fish, the cellular interactions between Sertoli cells and germ cells in the seminiferous epithelium play an important role during the spermatogenesis process . The structure of seminiferous epithelium changes from a Sertoli cell monolayer (containing spermatogonia cells during the non-reproductive season) to an active spermatogenic arrangement composed of spermatocytes and spermatozoa that fill the tubular lumen (reproductive season) .