Doublesex and mab-3 related transcription factors (Dmrts) play crucial roles in sex determination/differentiation and gonad development. The information on Dmrts and their functions are still scarce in mud crab Scylla paramamosain. In this study, 12 published transcriptome data of S. paramamosain were retrieved, pooled, and assembled. From the assembly, 7 Dmrt gene family members were identified and consisted of Spdmrt-like, Spdmrt-1a, Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdoublesex (Spdsx), and Spidmrt-2. These dmrt genes were predicted to encode 224 aa, 465 aa, 435 aa, 276 aa, 520 aa, 552 aa, and 266 aa protein precursors, respectively. The expression patterns of the dmrt genes were characterized by semi-quantitative PCR. The Spdmrt-like and Spdmrt-1a were exclusively detected in gonads, of which both expression levels in the testis were higher than that in the ovary. The Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdsx, and Spidmrt-2 were observed in various tissues; all these genes were sexually dimorphic except for dmrt-11E. Specifically, the expression level of Spdmrt-3 and Spidmrt-2 were higher in the testis than that in the ovary. On the contrary, the Spdsx and Spidmrt-1 expression level were higher in ovary than that in testis. The present study's findings provided a fundamental understanding of Dmrt gene family members involving sex determination/differentiation and gonad development in the S. paramamosain.

Sox transcription factors play essential roles in a variety of critical physiological processes. Still, members of the sox gene family have not yet been genome-wide identified in shrimps. In this study, a total of five members of the sox gene family were identified from the genome of Pacific white shrimp Litopenaeus vannamei and classified into three subgroups based on the conserved HMG-box domain. Among them, three belong to the SoxB subgroup (one in B1 and two in B2), one in the SoxC subgroup, and one in the SoxE subgroup. The five sox genes had different sex-biased expression in some tissues. Sox21, soxB1, and sox14 had a higher expression in ovary than in testis. In comparison, sox4 had a male-biased specific expression in the gonad, hepatopancreas, gill, and eyestalk. There was no difference in soxE gene expression between testis and ovary. During embryonic development, the expression level of three sox genes (soxB1, sox21, and soxE) was higher in gastrulation stage compared to previous stages, declined in limb bud stage and then increased in intramembrane nauplius stage; the expression of sox4 was detected in blastula stage and continued to increase in the following two stages and then surged in intramembrane nauplius stage; the highest expression of sox14 was in the fertilized egg stage, and the expression level decreased with the development of the embryo. These results suggest that the shrimp sox gene family may be involved in gametogenesis, tridermogenesis, and neurogenesis.

Members of sox gene family play critical roles in development, and some of them have crucial functions in sexual dimorphism. To understand the role of two SoxB2 genes, Sox14b and Sox21 of mud crab Scylla paramamosain, the full-length 1939 bp SpSox14b cDNA sequence and 861 bp SpSox21 cDNA sequence were obtained from the crab's transcriptome database, which encode 397 and 259 amino acids respectively. The results of sq-PCR showed that SpSox14b was expressed in all tissues, while SpSox21 was only expressed in the testis and brain. qRT-PCR showed that the expression level of SpSox14b in ovary was significantly higher than that of testis, and during the gonad development its expression was the highest in O2 (previtellogenesis) stage. The expression level of SpSox21 in testis was much higher than in brain, and was significantly higher in T3 (the mature sperm stage) than in other stages of testis development. Meanwhile, in different stages of larval development, SpSox21 was low expressed in zoea, then increased significantly in megalopa. Therefore we speculated that SpSox14b and SpSox21 may play different roles in the gonad development of mud crab, especially SpSox21 may be involved in the development and maintenance of testis. The expression level of SpSox14b and SpSox21 during the eye-pigment formation was significantly higher than that in other embryonic development stages, the results of whole-mount in situ hybridization showed that SpSox14b and SpSox21 were mainly located near the head and the compound eyes in eye-pigment formation stage and hatching. It suggested that they may be involved in the formation of brain nerves and are related to the regulation of body segments, and play different roles in sexual dimorphism.

Sox9 gene, a crucial member of the Sox gene family, is present in various organisms and involved in many physiological processes, especially in sex determination and gonad development. In this study, we reported a sox9 gene (designated as Spsox9) from Scylla paramamosain through analyzing published gonad transcriptome data. Meanwhile, the accuracy was validated by PCR technology, and the 3' sequences were cloned with 3' RACE technology. The full-length cDNA of Spsox9 is 2843 bp, consisting of a 243 bp 5' UTR, an 1124 bp 3' UTR, and a 1476 bp ORF encoding 491 amino acids. Furthermore, to better understand its conservation among crustacean species, the sox9 gene ortholog was identified in several other crustaceans species with their published transcriptome data, respectively. All of the Sox9 proteins identified in the current study had the common feature of Sox proteins (HMG domain) and were highly conserved among analyzed crustacean species. In all examined tissues, the Spsox9 was mainly expressed in the gonad (testis and ovary), eyestalk, and cerebral ganglion. During embryo development, Spsox9 was highly expressed in 5 pairs of appendages, 7 pairs of appendages, and eye-pigment formation stage. During ovary development, the expression level of Spsox9 remained stable in the first 4 stages (O1-O4) and decreased in the tertiary vitellogenesis (O5) stage. During testis development, the expression level of Spsox9 was highest in the spermatid stage (T2) and was significantly different from that in the spermatocyte stage (T1) and mature sperm stage (T3) (p < 0.05). In addition, Spsox9 exhibited a sex-biased expression pattern in T1 and O1. These present results indicated that the Spsox9 gene might play crucial roles in the gonad and embryo development of mud crab.