Cloning, expression, and function of the Spdmrt-like gene in Scylla paramamosain.

BACKGROUND: The mud crab Scylla paramamosain is an economically important species for aquaculture in China and has sexually dimorphic between females and males. Understanding sex differentiation in this species is essential for the development of monosex aquaculture. The Dmrt genes play a vital role in sex differentiation in animals. METHODS AND RESULTS: In this study, two dmrt-like transcript variants, Spdmrt-like-tv1 and Spdmrt-like-v2, were cloned. SpDmrt-like-tv1 contained a DM domain, while SpDmrt-like-tv2 contained a DM and a DMA domain. Spdmrt-like-tv1 and Spdmrt-like-tv2 were both specifically expressed in testis. During testicular development, the expression level of Spdmrt-like-tv1 increased from stage I to stage II (P > 0.05) and then decreased from stage II to stage III (P < 0.05). The expression level of Spdmrt-like-tv2 in stages I and II was significantly higher than that in stage III (P < 0.05). During embryonic development, the expression level of Spdmrt-like-tv1 was higher in the mid-embryonic stage compared with the early and late stages, but the differences were not significant. Moreover, the expression level of Spdmrt-like-tv2 was stable and remained high throughout embryonic development. Furthermore, the expression level of Spdmrt-like-tv2 was significantly higher than that of Spdmrt-like-tv1. Knockdown of Spdmrt-like variants indicated that the regulative target gene of Spdmrt-like-tv1 was Spsox21, and the regulative target genes of Spdmrt-like-tv2 were Spfoxl2 and Spsox21. Combined with the results in our previously published peer-reviewed articles that the expression of Spfoxl2 in the testis was significantly higher than that in the ovary, and Spfoxl2 negatively regulated Spvtg expression. Spsox21 played a role in the development and maintenance of testis as well as in the process of neural development and regulation of body segmentation. CONCLUSION: Therefore, we suggest that Spdmrt-like-tv1 and Spdmrt-like-tv2 might be involved in testicular development and embryonic development, and Spdmrt-like-tv2 might play more important roles in these two developmental processes by regulating the expression of Spfoxl2 and Spsox21 due to its high expression.

Comparative Transcriptome Reveals the Potential Modulation Mechanisms of Spfoxl-2 Affecting Ovarian Development of Scylla paramamosain.

Previously, we reported the identification, tissue distribution and confirmed the roles of Spfoxl-2 in regulating vitellogenin (vtg) expression in Scylla paramamosain. Here, we primally analyzed its potential target genes in the ovary with RNAi and RNA-Seq technology. By comparing the transcriptome data of two groups (ovaries that injected with EGFP and Foxl-2 siRNA, respectively), we found 645 DEGs (differentially expressed genes), including several conserved crucial genes involved in ovarian development, such as vtg, vitellogenin receptor (vtgR), adenylate cyclase (AC), cyclinB, and cell division cycle 2 (cdc2). In addition, these DEGs were also enriched in pathways related to ovary development, including relaxin signaling pathway, ovarian steroidogenesis, and progesterone-mediated oocyte maturation. Moreover, several genes were selected for qRT-PCR to validate the accuracy of the bioinformatic result. To the best of our knowledge, the current study was the first report about foxl-2 function through comparative transcriptome analysis in crustacean species, which identified not only relevant genes and pathways involved in ovarian development of S. paramamosain, but also provided new insights into the regulatory mechanisms of foxl-2 at the molecular level in crustacean.

Identification and functional analysis of the doublesex gene in the mud crab Scylla paramamosain.

Doublesex (Dsx) is a crucial member of the Dmrt gene family and plays a vital role in sex determination and differentiation among the animal kingdom. In the present study, a doublesex (designated as Spdsx) gene was identified and characterized for the first time in the mud crab, Scylla paramamosain. The Spdsx cDNA contains an 801 bp open reading frame (ORF) encoding 266 amino acids with a conserved DM domain. Meanwhile, to elucidate the conservation of Dsx, its orthologus were identified in several crustacean species as well. In addition, the expression pattern of Spdsx in various adult tissues and during embryo development was analyzed with qRT-PCR technology. Finally, the roles of Spdsx might play in the testis, androgenic gland, and ovary were analyzed by RNAi technology. The main results are as follows: (1) the Spdsx gene widely existed in analyzed crustacean species, and the multiple sequences alignment result indicated the conservation of Dsx was low except for the DM domain; (2) only one dsx gene was identified in analyzed crab and lobster, while 2 dsx genes (dsx-1 and dsx-2) were identified in shrimps; (3) the Spdsx gene was widely expressed in analyzed tissues, and the expression level in androgenic gland was obviously higher than that in other tissues. Interestingly, the expression level of Spdsx in the ovary was significantly higher than that in testis (p < 0.05); (4) The expression pattern of Spdsx during embryo development was divided into two groups: remained stable from blastula stage to 5 pairs of appendages stage; after 5 pairs of appendages stage, the expression level increased and remained stable from 7 pairs of appendages stage to hatching stage; (5) After the silencing of Spdsx, the expression level of marker genes in testis, ovary, and androgenic gland significantly changed, among which the expression level of vtg and vtgR in ovary down-regulated, the dmrt-like and dmrt-1a (exclusively expressed in testis) in testis up-regulated and the IAG in androgenic gland down-regulated. All the results above demonstrated that the Spdsx play crucial roles in regulating the reproduction system development of mud crab.

dLp/HDL-BGBP and MTP Cloning and Expression Profiles During Embryonic Development in the Mud Crab Scylla paramamosain.

Lipids are the main energy source for embryonic development in oviparous animals. Prior to the utilization and catabolism, lipids are primarily transported from the yolk sac to embryonic tissues. In the present study, cDNA encoding a circulatory large lipid transfer protein (LLTP) superfamily member, the precursor of large discoidal lipoprotein (dLp) and high-density lipoprotein/ß-1,3-glucan-binding protein (HDL-BGBP), named dLp/HDL-BGBP of 14,787 bp in length, was cloned from the mud crab Scylla paramamosain. dLp/HDL-BGBP was predicted to encode a 4,831 amino acids (aa) protein that was the precursor of dLp and HDL-BGBP, which were both detected in hemolymph by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. For the intracellular LLTP, three microsomal triglyceride transfer protein (MTP) cDNAs of 2,905, 2,897, and 3,088 bp in length were cloned from the mud crab and were predicted to encode MTP-A of 881 aa, MTP-B of 889 aa, and MTP-C of 919 aa, respectively, which were different merely in the N-terminal region and shared an identical sequence of 866 aa. During embryonic development, the expression level of dLp/HDL-BGBP consecutively increased from the early appendage formation stage to the eye pigment-formation stage, which indicated that HDL-BGBP is probably the scaffolding protein for yolk lipid. For the MTP gene, MTP-C accounted for ~70% of MTP mRNA from the blastocyst stage to the nauplius stage, as well as the pre-hatching stage; MTP-C and MTP-A expression levels were comparable from the early appendage formation stage to the late eye pigment-formation stage; MTP-A was extremely low in blastocyst and gastrula stages; MTP-B was expressed at a relatively low-level throughout embryo development. The variations in the expression profiles among MTP transcripts suggested that MTP might play roles in the lipid droplet maturation and lipoprotein assembly during embryonic development.

Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain).

Forkhead box protein L2 (Foxl2) is involved in multiple physiological processes, such as ovarian development, granulosa cell differentiation, ovarian follicle development, and oocyte growth. In this study, a Spfoxl2 gene encoded 530 amino acid protein with characteristic forkhead (FH) domain was identified from transcriptome data of mud crab Scylla paramamosain and validated the accuracy by PCR technology. Meanwhile, the orthologues of the Spfoxl2 gene in other 14 crustacean species were identified with the same method. Further multiple sequence alignment analysis revealed the Foxl2 was highly conserved, especially in the FH domain, even completely identical in several species. Besides, the semi-quantitative PCR (Sq-PCR) result showed Spfoxl2 gene was mainly expressed in the gonad (testis and ovary). Further quantitative real-time PCR (qRT-PCR) result demonstrated its expression level in the testis was significantly higher than that in the ovary (p < 0.01). In addition, the qRT-PCR result showed that in zoea V, megalopa, and larval I, the expression level of Spfoxl2 in megalopa is the highest. In addition, a putative Foxl2 binding site was identified on the promoter region of Spvtg, and knockdown of Spfoxl2 mediated by RNAi technology increased the expression of Spvtg in the ovary, suggesting Spfoxl2 might be the upstream negative regulator of Spvtg. Overall, this study provided new insights into the role of Spfoxl2 in ovary development through regulating Spvtg expression in S. paramamosain.

Discovery of the Dmrt gene family members based on transcriptome analysis in mud crab Scylla paramamosain.

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.

Large lipid transfer proteins in hepatopancreas of the mud crab Scylla paramamosain.

Large lipid transfer proteins (LLTPs) are extensively involved in various physiological processes. In the present study, five LLTP sequences encoding apolipocrustacein 1 (apoCr 1), apoCr 2, precursor of the large discoidal lipoprotein (dLp) and high density lipoprotein/ß-glucan binding protein (HDL-BGBP) (dLp-BGBP), microsomal triglyceride transfer protein (MTP) and clotting protein (CP) were identified in the hepatopancreas of Scylla paramamosain. Of these, apoCr 2, dLp-BGBP, and MTP were newly identified in this species, and the former two proteins were classified into the APO family while the later into the MTP family in phylogenetic trees. The apoCr 1 expression level was dramatically increased in the hepatopancreas towards ovarian maturation, which was extremely greater than that in the ovaries concurrently, likely to meet the considerable requirements of yolk protein and lipids for embryo development. The dLp-BGBP expression level in male crabs was comparable to that in female crabs, supporting HDL-BGBP acts as a major circulatory lipid carrier. The close phylogenetic relationship between dLp-BGBP and the scaffolding protein of lipid transfer particle implied dLp might facilitate lipid transfer between the hepatopancreas and HDL-BGBP-containing lipoproteins. The MTP expression level was positively related to ovarian development in both the hepatopancreas and ovaries, indicating MTP may be involved in lipoprotein assembly in the hepatopancreas and lipid droplet maturation in the ovaries. CP may play a crucial role in embryo development based on high expression level observed in the testes of mature crabs. Our findings provide novel insights into LLTP superfamily members and their functions in decapods.

Synergistic oxidation - filtration process analysis of catalytic CuFe2O4 - Tailored ceramic membrane filtration via peroxymonosulfate activation for humic acid treatment.

This work synthesized catalytic CuFe2O4 tailored ceramic membrane (CuFeCM), and systematically investigated the intercorrelated oxidation - filtration mechanism of peroxymonosulfate (PMS)/CuFeCM catalytic filtration for treating humic acid (HA). PMS/CuFeCM filtration exhibited enhanced HA removal efficiency while reduced the irreversible fouling resistance as compared with the conventional CM filtration. Results from HA characterizations showed that PMS/CuFeCM catalytic filtration oxidized HA into conjugated structures of smaller molecular weight. The unsaturated bonds further caused the re-agglomeration of HA, hence enhancing the size exclusion of CuFeCM. Meanwhile, oxidized HA particles with changing physicochemical properties reduced the total attractive interaction energy between CuFeCM and HA, mainly attributed to the reduced acid-base interaction energy according to the Extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) analysis. The changing of HA properties and HA-CuFeCM physicochemical interactions rendered more re-agglomerated HA particles retained above membrane with less attachment, which induced decreasing irreversible fouling resistance and facilitated easier external fouling removal by hydraulic cleaning. Overall, the PMS/CuFeCM configuration demonstrated in this study could provide a new insight into the synergistic oxidation - filtration interaction mechanism of hybrid catalytic ceramic membrane filtration process.