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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/β-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. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) is one of the most economically-important marine crabs in China. However, research on mechanisms of reproductive regulation is not sufficient. Vitellogenesis-inhibiting hormone (VIH) is a member of the crustacean hyperglycemia hormones (CHH) family, which plays an essential role in the regulation of gonadal development and maturation in crustaceans, and current studies on the regulation of Vih transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab Vih (SpVih) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of SpVih. In this study, the electrophoretic mobility shift assay (EMSA) experiment confirmed that the nuclear protein extracted from the eyestalk could bind to the key region of SpVih promoter, and these specific bindings were dependent on the presence of Oct4/Sox9 binding sites. Two specific binding complex bands were detected in the supershift group of EMSA supershift experiments by Oct4 and Sox9 antibodies, further confirming the specific recognition of these two transcription factors on the key regulatory region of SpVih. In vitro, Oct4 and Sox9 gene overexpression vectors and SpVih core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result, SpVih activity increased with the concentration of transcription factors. In vivo , when Oct4 and Sox9 dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of SpVih decreased significantly after interference with Oct4 or Sox9 , and the expression level of SpVtg in the ovary and hepatopancreatic increased. Both in vitro and in vivo experiments showed that Oct4 and Sox9 had a positive regulatory effect on SpVih. The GST pull-down experiment was carried out by purified Oct4 and Sox9 proteins, and the results showed that there was an interaction between them. It was speculated that they regulated the expression of SpVih through the interaction. [ABSTRACT FROM AUTHOR]

Ubiquitin C-terminal hydrolases (UCHLs) are a subset of deubiquitinating enzymes, and are involved in numerous physiological processes. However, the role of UCHLs during gonad development has not been studied in crustaceans. In this study, we have first cloned and analyzed expression profiling of Sp-uchl3 and Sp-uchl5 genes frommud crab Scylla paramamosain. The full-length cDNA of Sp-uchl3 is of 1804 bp. Its expression level in the ovary was significantly higher than in other tissues (p < 0.01), and during gonadal development, its expression in both O1 and O5 stages was significantly higher than in the other three stages of ovaries (p < 0.05), while in T3 it was higher than in the former two stages of testes (p < 0.05). Meanwhile, the full-length cDNA of Sp-UCHL5 is 1217 bp. The expression level in the ovarywas significantly higher than in other tissues (p < 0.01). Its expression in ovaries was higher than in testes during gonadal development (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage in ovarian development, and with no significant difference in the testis development (p > 0.05). These results provide basic data showing the role of Sp-UCHL3 and Sp-UCHL5 in the gonad development of the crab. [ABSTRACT FROM AUTHOR]

Vitellogenesis in crustaceans is controlled by several steroid hormones. In humans, the expression of SF-1 , a gene that regulates gonadal development and the synthesis of steroid hormones, is affected by DDX20. However, how the homologous gene FTZ-F1 is regulated by DDX20 and its association with vitellogenesis remains unknown in the mud crab Scylla paramamosain. In this study, SpDDX20 and SpFTZ-F1 were identified in the transcriptome of mature ovarian tissue from the mud crab. qRT-PCR results revealed that the expression levels of SpFTZ-F1 and SpVTG in the ovaries of crab in the experimental group injected with dsDDX20 (EO) were significantly higher (P < 0.05) than those in the negative control group injected with dsEGFP (NO) and the blank control group injected with SPSS (BO). The differentially expressed genes (DEGs) identified by comparative transcriptome analysis of the EO group and NO group were enriched into five pathways related to ovarian steroidogenesis. The expression of CYP17 , CYP3A4 , CYP1A1 and 3β-HSD were up-regulated in pathways related to steroid hormone production and biosynthesis. The expression of the INSR , IRS and PI3K genes in the insulin signaling pathway were significantly increased (P < 0.05). The expression level of the TGF-β gene was up-regulated (P < 0.05) in the transforming growth factor pathway, whereas the expression level of the Smad2 gene was down-regulated (P < 0.05). The expression of GnRHR , GS , AC and PKA genes in the gonadotropin-releasing hormone signaling pathway were up-regulated. Our data provide a foundation for investigating the relationship between DDX20 and FTZ-F1 in the regulation of vitellogenin expression in S. paramamosain. [Display omitted] • Full-length cDNA of SpDDX20 and SpFTZ-F1 were cloned and characterized from Scylla paramamosain. • The expression of SpDDX20 and SpFTZ-F1 had tissue and gender specificity. • The expression level of SpFTZ-F1 was up-regulated after SpDDX20 interference. • Five ovarian steroidogenesis pathways were differentially expressed after SpDDX20 knockdown. • The expression level of SpVTG was up-regulated after knockdown of SpDDX20. [ABSTRACT FROM AUTHOR]

We identified extracellular signal-regulated kinase 2 ( erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5′ Rapid amplification of cDNA end (RACE) technique was used to obtain the 5′ untranslated region (UTR). The full-length cDNA of Sp- erk2 is 1516 bp, including a 5′-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3′-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp- erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp- erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp- erk2 is important for ovarian development. [ABSTRACT FROM AUTHOR]

Abstract: A total of 5160 high quality ESTs (expressed sequence tags) averaging 357bp were collected from normalized cDNA libraries created from testis, ovary and mixed organs of mud crab Scylla paramamosain. Clustering and assembly of these ESTs resulted in a total of 3837 unique sequences with 576 overlapping contigs and 3261 singletons. Comparisons with the GenBank non-redundant (Nr) protein database (BLASTx, e-values <10−5) revealed putative functions or matched homologs from other organisms for 847 (22%) of the ESTs. Several gonad development related genes such as cathepsin C, thioredoxin peroxidase, vitellogenin receptor precursor, 50S ribosomal protein L24 and ubiquitin-conjugating enzyme E2 isoform 2 were identified from this EST project and demonstrated as gonad differential expression genes by rqRT-PCR. Sixty five different types of SSRs (simple sequence repeats) were identified from the total 411 EST-SSR motifs. A home-made cDNA microarray containing 5664 spots was developed and the hybridization results indicated that 39 unique transcripts were differentially expressed in testis and ovaries (P<0.05). The expression levels of eleven unique transcripts examined by rqRT-PCR were matched with microarray fairly. These results will provide a useful resource for functional genomic studies on the biology of reproduction of mud crab. [Copyright &y& Elsevier]

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/β-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. [Display omitted] • Five LLTPs, apoCr 1, apoCr 2, dLp-BGBP, MTP and CP, in mud crabs • apoCr 1 was the major egg yolk protein and specific lipid carrier to ovaries. • dLp might mediate lipid transfer between HDL-BGBP and hepatopancreas. • MTP likely involved in lipoprotein assembly and lipid droplet maturation. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) has become an important mariculture crab along the southeast coast of China due to its strong adaptability, delicious taste, and rich nutrition. Several vertebrate steroid hormones and their synthesis-related genes and receptors have been found in crustaceans, but there are few reports on their synthesis process and mechanism. 3-beta-hydroxysteroid dehydrogenase (HSD3B) is a member of the Short-chain Dehydrogenase/Reductase (SDR) family, and an indispensable protein in vertebrates' steroid hormone synthesis pathway. In this study, the SpHsd3b gene sequence was obtained from the transcriptome data of S. paramamosain , and its full-length open reading frame (ORF) was cloned. The spatial and temporal expression pattern of SpHsd3b was performed by quantitative real-time PCR (qRT-PCR). SpHsd3b dsRNA interference (RNAi) and HSD3B inhibitor (trilostane) were used to analyze the function of Sp HSD3B. The results showed that the SpHsd3b gene has an 1113 bp ORF encoding 370 amino acids with a 3β-HSD domain. Sp HSD3B has lower homology with HSD3B of vertebrates and higher homology with HSD3B of crustaceans. SpHsd3b was expressed in all examined tissues in mature crabs, and its expression was significantly higher in the testes than in the ovaries. SpHsd3b expression level was highest in the middle stage of testicular development, while its expression was higher in the early and middle stages of ovarian development. RNAi experiment and trilostane injection results showed that Sp HSD3B had regulatory effects on several genes related to gonadal development and steroid hormone synthesis. 15-day trilostane suppression could also inhibit ovarian development and progesterone level of hemolymph. According to the above results, crustaceans may have steroid hormone synthesis pathways like vertebrates, and the Hsd3b gene may be involved in the gonadal development of crabs. This study provides further insight into the function of genes involved in steroid hormone synthesis in crustaceans. • Steroid hormone synthesis pathway-related gene— Hsd3b was identified from Scylla paramamosain. • SpHsd3b knockdown can decrease the expression of gonadal development and steroid hormone synthesis-related genes. • Trilostane can inhibit ovarian development and lower progesterone level of hemolymph. • crustaceans may have steroid hormone synthesis pathways like vertebrate. [ABSTRACT FROM AUTHOR]

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different Sp TLRs were analyzed. It was found that different Sp TLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish , myd88 , dorsal , anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain. • Nine TLRs were identified from the transcriptome data of mud crab Scylla paramamosain. • The novel Sptlr-6 had a high expression level in the hepatopancreas of S. paramamosain. • PGN, Poly I: C and Vibrio injection induced the expression of Sptlr-6 in the hepatopancreas. • Sptlr-6 played a crucial role via the Dorsal/Relish-AMPs pathway in response to Vibrio challenge. [ABSTRACT FROM AUTHOR]

• Sp-IAG promoter has a dsx and a foxl-2 binding sites that can regulate its expression. • Cell experiment and RNAi experiment proved that dsx can enhance Sp-IAG expression. • Cell experiment and RNAi experiment showed that foxl-2 can inhibit Sp-IAG expression. Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG , we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG , respectively. The results showed that dsx could significantly promote the expression of Sp-IAG , while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx , while significantly increased after interfering with foxl-2 , which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG. [ABSTRACT FROM AUTHOR]

Antimicrobial peptide (AMP) is a crucial component of the innate immune system in crustaceans. In mud crab, Scylla paramamosain , a commercially important species, a glycine-rich antimicrobial peptide (Sp gly-AMP) gene was newly identified and putatively encoded a 26aa signal peptide and 37aa mature peptide. To understand the function of Sp gly-AMP, the expression profile of Spgly-amp gene was characterized, which showed Spgly-amp was expressed widely in most tissues of adult crabs with the highest expression level in hemocytes. After Vibrio parahaemolyticus , PGN, or Poly I:C stimulations, the expression level of Spgly - amp was significantly up-regulated in the hemocytes. In antimicrobial assays, chemically synthesized Sp gly-AMP peptides exhibited strong antibacterial activities against both Gram-positive and Gram-negative bacteria and high thermal stability after high-temperature heating. These findings in the present study verified the importance of the Sp gly-AMP in defense of pathogenic bacteria infection in the mud crab and provided a promising candidate of antimicrobial agents in the crab aquaculture. • A glycine-rich antimicrobial peptide (Sp gly-AMP) gene was newly identified from mud crab. • The orthologs of gly-AMP in several crabs are highly conserved. • After bacterial stimulations, the Spgly - amp mRNA was significantly induced in the hemocytes. • Sp gly-AMP peptides exhibit strong antibacterial activities and high thermal stability. [ABSTRACT FROM AUTHOR]

A member of the immunoglobulin superfamily designated leucine-rich repeats and immunoglobulin-like domains protein-1 (lrig-1) encoding a protein with 1109 amino acids with a characteristic IGc2 domain was identified from the transcriptome data of mud crab Scylla paramamosain. Lrig-1 contained: one signaling peptide; one LRR_NT domain; nine LRR domains; three LRR_TYP domains; one LRR_CT domain; three IGc2 regions; one transmembrane region; C-terminal cytoplasmic tail. lrig-1 was widely expressed in all tissues of mud crab and was responsive in hemocytes to first and second Vibrio parahaemolyticus infections. lrig-1 knockdown mediated by RNAi repressed expression of several antimicrobial peptides significantly. Its orthologs in 19 crustacean species were identified and showed high conservation. These results suggest that lrig-1 have a vital role in mud crabs against V. parahaemolyticus infection through expression of multiple antimicrobial peptides. The results obtained in the present study imply the potential roles the lrig-1 played in immune priming in crabs. • lrig-1 regulates mud crab's immune response to Vibrio parahaemolyticus during two infections. • RNAi-mediated downregulation of lrig-1 inhibits multiple antimicrobial peptides expression. • Lrig-1 orthologues in the other 19 crustacean species showed high conservation with mud crab. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the innate immune responses of invertebrates have been less studied. In this study, Sp TNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. Sp TNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of Sp TNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of Sp TNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that Sp TNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (Sp LITAF) can regulate the expression of Sp TNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of Sp LITAF were found to be regulated by WSSV infection. Knocking down of Sp LITAF increased the WSSV copy number and expression of VP28 gene. Taken together, these results proved the protective function of Sp TNF, which is regulated by Sp LITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis. • Sp TNF is initially down-regulated but subsequently up-regulated in hemocytes of mud crabs during WSSV infection. • Sp TNF enhances mud crab immunity against WSSV infection by activating the apoptosis, NF-κB pathway, and AMP synthesis. • Sp LITAF can regulate the expression of Sp TNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. • Expression and nuclear translocation of Sp LITAF are regulated by WSSV infection. • Sp LITAF can regulate the replication of WSSV in mud crabs. [ABSTRACT FROM AUTHOR]

In our previous study, we found that the Spfoxl-2 transcript was highly expressed in gonads and explored its potential target genes in the ovary of Scylla paramamosain. In the current study, we primally analyzed its potential target genes in the testis through RNAi and RNA-Seq technology and compared with that in the ovary. The results showed that a total of 7892 unigenes were differentially expressed after Spfoxl-2 silencing in the testis, including plenty of conserved genes involved in testicular development, such as Dmrt family genes, Sox family genes, Caspase family genes, Cdk family genes, Kinesin family genes, Fox family genes and other genes. Further analysis revealed that these differentially expressed genes (DEGs) were enriched in crucial pathways involved in spermatogenesis, such as DNA replication, Cell cycle, Spliceosome, Homologous recombination, Meiosis and Apoptosis. The comparison results of potential target genes in the ovary and testis reveal 135 common potential target genes, including some genes involved in the immune response. According to our knowledge, the present work was the first to disclose the functions of foxl-2 in the testis of crustacean species using transcriptome analysis. It not only identifies key genes and pathways involved in the testicular development of S. paramamosain , but also reveals a new molecular-level understanding of the function of foxl-2 in testicular development. [Display omitted] • The foxl-2 was highly expressed in the testis of mud crab. • The knockdown of foxl-2 highly decreased the expression of genes involved in testis development. • The functions of foxl-2 in the testis and ovary are significantly different. • The knockdown of foxl-2 inhibits the expression of antimicrobial peptide genes in the testis and ovary. [ABSTRACT FROM AUTHOR]

In this study, the 5′-flanking region of molt-inhibiting hormone (MIH) gene was cloned by Tail-PCR. It is 2024 bp starting from the translation initiation site, and 1818 bp starting from the predicted transcription start site. Forecast analysis results by the bioinformatics software showed that the transcription start site is located at 207 bp upstream of the start codon ATG, and TATA box is located at 240 bp upstream of the start codon ATG. Potential transcription factor binding sites include Sp1, NF-1, Oct-1, Sox-2, RAP1, and so on. There are two CpG islands, located at −25— +183 bp and −1451— −1316 bp respectively. The transfection results of luciferase reporter constructs showed that the core promoter region was located in the fragment −308 bp to −26 bp. NF-kappaB and RAP1 were essential for mih basal transcriptional activity. There are three kinds of polymorphism CA in the 5′-flanking sequence, and they can influence mih promoter activity. These findings provide a genetic foundation of the further research of mih transcription regulation. [ABSTRACT FROM AUTHOR]

Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5′-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain . The full-length cDNA of SpVIH is 634 bp including 105 bp 5′UTR, 151 bp 3′UTR and 378 bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790 bp containing two exons and one intron. The 5′-flanking promoter regions of SpVIH we isolated are 3070 bp from the translation initiation (ATG) and 2398 bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between p Sp VIH-4 and p Sp VIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of p Sp VIH-4 was created. The results demonstrated that the transcriptional activity of p Sp VIH-4△ decreased significantly (p < 0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription factors which regulate the expression of SpVIH . [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The expression level of Spdsx in the ovary was significantly higher than that in testis. • The Spdsx positively regulate the expression of vtg and vtgR in ovary. • The Spdsx negatively regulate the expression of dmrt-like and dmrt-1a in testis. • The Spdsx positively regulate the expression of IAG in androgenic gland. [ABSTRACT FROM AUTHOR]

Abstract: The full-length (7816bp) cDNA of Vitellogenin (Vtg) encoding 2560 aa with an estimated molecular mass of 287.743kDa was cloned from the green mud crab Scylla paramamosain. Semi-quantitative PCR (sq-PCR) revealed a specific expression pattern of Sp-vtg gene in ovaries and hepatopancreas. With the development of ovaries, the expression level of Sp-vtg gene showed an increasing trend both in ovaries and hepatopancreas, and the expression level of Sp-vtg gene in hepatopancreas and ovary was stable after stage IV. By in situ hybridization, the positive signals of Sp-vtg gene were detected in the cytoplasm of oocytes in stage I, in the follicle cell and the surrounding of the nucleus in stage III, and in the nucleus in stage V. Furthermore, the signals become stronger with the later development stages of ovary. Moreover, in situ hybridization analysis revealed that positive signals of Sp-vtg gene are present in the hepatopancreatic tubule, and the signals increase during the development, becoming the strongest in stage V. Our results indicate that both ovaries and hepatopancreas are sites of Vitellogenin gene synthesis in S. paramamosain. [Copyright &y& Elsevier]

Abstract: The maturation promoting factor (MPF) is a key regulator of controlling G2/M phase transition in the meiotic maturation of oocyte and spermatocyte in animals, which is a complex of CDC2 (CDK1) and cyclin B. To better understand the molecular mechanism of oocyte and spermatocyte maturation in mud crab (Scylla paramamosain), the full length cDNA of cdc2 (Sp-cdc2) and cyclin B (Sp-cyclin B) were cloned and characterized. The full length cDNA of Sp-cdc2 gene is of 1593bp encoding a protein of 299 amino acids. Real-time quantitative PCR analysis revealed that the expression level of Sp-cdc2 in the ovary was higher than in other tissues (P<0.01); and its expression level was not significantly different in different stages of ovary development (P>0.05), meanwhile there was higher expression in T3 stage than in T1 and T2 stages (P<0.05). The full length cDNA of Sp-cyclin B is 1492bp encoding a protein of 391 amino acids. The real-time PCR results showed that its expression level in the ovary was the highest in all examined tissues (P<0.01), and the gonad expression level in O5 stage was significantly higher than in previous 4 stages and the testis (P<0.05), and was also significantly higher in T2 stage than in T1 stage (P<0.05). In situ hybridization analysis showed that the expressions of Sp-cdc2 and Sp-cyclin B transcripts were presented in similar distribution patterns in different developing stages of ovary and testis. The positive signals of Sp-cdc2 and Sp-cyclin B mRNA were detected in the oocytoplasm of oogonia and pre-vitellogenic and primary vitellogenic oocytes, while these two genes had higher expression level in the spermatid and secondary spermatocyte following primary spermatocyte. These results suggested that Sp-cdc2 and Sp-cyclin B may play essential roles in the oogenesis and spermatogenesis of the crab. [Copyright &y& Elsevier]

Abstract: The small ubiquitin-related modifier-1 (SUMO-1) is a member of a family of ubiquitin-related proteins. SUMO pathway, which is involved in gene expression in eukaryotic posttranslational processing, plays important roles in gene expression, genomic stability and the occurrence of cells, development and other biological processes. Scylla paramamosain is one of the important economic breeding crabs in the southeast coast of China. To date, little is known about the distinct roles of SUMO in crustacean, especially in crabs. In the present study, we report the identification and characterization of mud crab, S. paramamosain SUMO-1 (SpSUMO-1) gene using an approach which combines expressed sequence tag (EST) and rapid amplification cDNA end (RACE). The full length cDNA of SpSUMO-1 gene (GenBank: HM581660) is of 732bp, including a 282bp open reading frame which encodes a protein of 93 amino acids. Tissue distribution analysis showed that SpSUMO-1 was expressed more abundantly in the ovary than in other tissues (P<0.01). And the expression profiles of SpSUMO-1 in the different gonad developing stages revealed that the highest expression of SpSUMO-1 occurred at proliferation stage, and then decreased gradually as the ovarian development progressed, while in the testis, the expression level of SpSUMO-1 was relatively stable at different stages of testis development. The distribution of SpSUMO-1 mRNA and its protein was observed in the crab gametogenesis by in situ hybridization and immunocytochemical method respectively. In oogenesis, SpSUMO-1 transcripts presented at the cytoplasm and nucleus of oocytes from proliferation stage to primary vitellogenesis stage, but only appeared in the nucleus of oocytes in secondary and tertiary vitellogenesis stages. Meanwhile, SpSUMO-1 protein was localized in the cytoplasm of oogonia and different developing oocytes. On the other hand, the SpSUMO-1 transcript was detected throughout the spermatogenesis, with the strong positive signals of SpSUMO-1 presented at the nuclei of primary and secondary spermatocytes, spermatids and spermatozoa. Interestingly, the positive signals of acrosomal tubules of spermatozoa were also detected. SpSUMO-1 protein was localized in spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, but the positive signal was only detected in the nucleus of spermatozoa. All these results suggested that SUMO-1 may play essential roles in the gametogenesis of the crustacea. [Copyright &y& Elsevier]

• The Spfoxl2 was exclusively expressed in gonad. • The expression level of Spfoxl2 in the testis was significantly higher than that in the ovary. • The Foxl2 was highly conserved among crabs, while less conserved among analyzed other decapods. • The knockdown of Spfoxl2 mediated by RNAi technology repressed the expression of Spvtg in the ovary. 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. [ABSTRACT FROM AUTHOR]

• 7 members of Dmrt gene family of Scylla paramamosain were identified based on transcriptome data. • Majority of dmrt genes were differently expressed between testis and ovary. • The dmrt_like gene was exclusively expressed in testis. • The dmrt-1a gene was exclusively expressed in gonad. • Different from many organisms, the expression of dsx gene in ovary was higher than that in testis. 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. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is an economically important cultured crab species in China. Cyclins and cyclin-dependent kinases (CDKs) play important roles in regulations of cell cycle and ovarian development. MiRNAs can negatively regulate gene expression at the post-transcriptional level through base-complementary pairing with the 3'-untranslated region (3-UTR) of the target gene. In this study, bioinformatics prediction showed that miR-9c and miR-263a identified from our group's gonad miRNAome of S. paramamosain may bind to the 3' UTR region of cyclin A , cyclin B , cyclin E , cyclin H , CDK1, and CDK2. Furthermore, the results of double luciferase reporter gene assay showed that the luciferase activities of HEK293T cells co-transfected with miR-9c mimics/miR-9c inhibitor and the 3'-UTR plasmid vectors of the five genes (cyclin A , cyclin B , cyclin H , CDK1, and CDK2) were significantly decreased/increased compared with those in the NC (negative control) and BC (blank control) groups. The results in miR-263a were similar to miR-9c, but all of the six genes could be regulated by miR-263a. In in vivo experiments, agomiR-9c (miR-9c enhancer) injection resulted in decreases of cyclin A and CDK1 expression level, and reverse effects were observed by injecting antagomiR-9c. AgomiR-263a decreased the expression of cyclin A , cyclin B , cyclin H , CDK1, and CDK2, but antagomiR-263a increased their expression. Both the in vitro and in vivo experiments confirmed functions of miR-9c and miR-263a in cell cycle progress of ovarian development by expression regulation of cyclin A , cyclin B , cyclin E , cyclin H , CDK1, and CDK2. The findings provide new insights into the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates. [Display omitted] • The regulation of miR-9c and miR-263a on cyclins and CDKs was confirmed in Scylla paramamosain. • MiR-263a can inhibit the expression of cyclin A , cyclin B , cyclin E , cyclin H , CDK1 , and CDK2. • MiR-9c only inhibit the expression of cyclin A and CDK1. • MiR-263a may play a more essential role than miR-9c in regulating cell cycle genes in S. paramamosain. • The change of vtg expression level manifests that cyclins and CDKs can regulate ovarian development. [ABSTRACT FROM AUTHOR]

• Full-length cDNA of SpSox9 was cloned and characterized from Scylla paramamosain. • The ortholog of SpSox9 identified from 16 decapoda crustacean species was highly conserved. • The expression of the Spsox9 was sexually dimorphic expressed in early gonad development. • The Spsox9 was mainly expressed in gonad (testis and ovary), eyestalk and cerebral ganglion. • The Spsox9 was highly expressed in 5 and 7 pairs of appendages, and eye-pigment formation stage. 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. [ABSTRACT FROM AUTHOR]

The regulatory role of microRNAs in gonad development and reproduction is attracting more and more attention in recent years.Vitellogenesis−/Gonadal-inhibiting hormone (VIH/GIH) is a member of the crustacean hyperglycemic hormone (CHH) family. It plays a key role in regulating ovarian development and gonadal maturation in crustaceans. In this study, based on our preliminary studies of Spvih (Scylla paramamosain vih, Spvih), the regulation of expression of Spvih gene was further investigated. Predicted by bioinformatics software, we found that miR-34/miR-277 may play a regulatory role in Spvih expression. Analysis of luciferase activity of HEK293T cells transfected with the synthetic miR-34/miR-277 mimics and miR-34/miR-277 inhibitor revealed that miR-277 mimics significantly reduced the activity and miR-277 inhibitor significantly increased the activity compared to the control group. However, there was no significant difference in activity between miR-34 mimics and miR-34 inhibitor. The relative expression of Spvih and miR-277 was detected in the eyestalk, which had been injected with stroke-physiological saline solution(SPSS)and chemically synthesized miR-277 agomir and miR-277 antagomir, respectively. The results showed that compared with the control group SPSS, the expression of miR-277 in the eyestalk was increased significantly after miR-277 agomir injection, while the expression of Spvih was decreased significantly. After the injection of miR-277 antagomir, the expression of miR-277 in the eyestalk decreased significantly, while the expression of Spvih increased significantly. The inhibitory effect of miR-277 on Spvih expression was further verified. Based on miR-277 agomir and miR-277 antagomir injected into the base of the crab, the relative expression of the Spvtg gene in the ovarian and hepatopancreas was detected. The results showed that compared with the control group SPSS, the expression of Spvtg in the ovary and hepatopancreas were significantly increased after injection of miR-277 agomir, while the expression of Spvtg was significantly decreased after injection of miR-277 antagomir. Our study may provide an evidence of how miRNA works on the regulation of Spvih , which indirectly affects the ovarian development of mud crab, and suggest a new strategy for controlling crab reproduction. • It is the first time to report the relationship of miRNA and vih gene in crab. • By targeting the 3′UTR of the Spvih gene miR-277 could inhibit its expression. • miR-277 might be an efficient indirect regulator of vtg expression. • miRNA injection is a new strategy for induction the ovarian development in crab. [ABSTRACT FROM AUTHOR]

• Full-length cDNA of SpSox14b and SpSox21 were cloned and characterized from Scylla paramamosain. • The expression of SpSox14b and SpSox21 had tissue and gender specificity. • SpSox14b was expressed in all tissues, while SpSox21 was only expressed in testis and brain. • SpSox14b and SpSox21 were high-expressed in the eye-pigment formation during embryogenesis. • WISH showed the SpSox14b and SpSox21 were mainly located in brain and eye areas in embryogenesis. 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. [ABSTRACT FROM AUTHOR]