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MicroRNA (miRNA) is a posttranscriptional downregulator that plays a vital role in a wide variety of biological processes. In this study, we constructed five ovarian and testicular small RNA libraries using two somatic libraries as reference controls for the identification of sex-biased miRNAs and gonadal differentially expressed miRNAs (DEMs) of the Chinese mitten crab, Eriocheir sinensis. A total of 535 known and 243 novel miRNAs were identified, including 312 sex-biased miRNAs and 402 gonadal DEMs. KEGG pathway analysis showed that DEM target genes were statistically enriched in MAPK, Wnt, and GnRH signaling pathway, and so on. A number of the sex-biased miRNAs target genes associated with sex determination/differentiation, such as IAG, Dsx, Dmrt1, and Fem1, while others target the genes related to gonadal development, such as P450s, Wnt, Ef1, and Tra-2c. Dual-luciferase reporter assay in vitro further confirmed that miR-34 and let-7b can downregulate IAG expression, miR-9-5p, let-7d, let-7b, and miR-8915 can downregulate Dsx. Taken together, these data strongly suggest a potential role for the sex-biased miRNAs in sex determination/differentiation and gonadal development in the crab.

Germ cell-specific genes play an important role in establishing the reproductive system in sexual organisms and have been used as valuable markers for studying gametogenesis and sex differentiation. Previously, we isolated a vasa transcript as a germ cell marker to trace the origin and migration of germ cells in the oriental river prawn Macrobrachium nipponense. Here, we identified a new germ cell-specific marker MnTdrd RNA and assessed its temporal and spatial expression during oogenesis and embryogenesis. MnTdrd transcripts were expressed in high abundance in unfertilized eggs and embryos at cleavage stage and then dropped significantly during late embryogenesis, suggesting that MnTdrd mRNA is maternally inherited. In situ hybridization of ovarian tissue showed that MnTdrd mRNA was initially present in the cytoplasm of previtellogenic oocyte and localized to the perinuclear region as the accumulation of yolk in vitellogenic oocyte. Whole-mount in situ hybridization of embryos showed that MnTdrd-positive signals were only localized in one blastomere until 16-cell stage. In the blastula, there were approximately 16 MnTdrd-positive blastomeres. During embryonized-zoea stage, the MnTdrd-positive cells aggregated as a cluster and migrated to the genital rudiment which would develop into primordial germ cells (PGCs). The localized expression pattern of MnTdrd transcripts resembled that of the previously identified germ cell marker vasa, supporting the preformation mode of germ cell specification. Therefore, we concluded that MnTdrd, together with vasa, is a component of the germ plasm and might have critical roles in germ cell formation and differentiation in the prawn. Thus, MnTdrd can be used as a novel germ cell marker to trace the origin and migration of germ cells.

Largemouth bass (Micropterus salmoides) is an economically important fish. It can spawn many times during a breeding season, and there are no obvious morphological characteristics to distinguish male and female juvenile fish. So far, little is known about the genes regulating their sexual development in this species. Here, we performed RNA sequencing (RNA-Seq) analysis of the testis, ovary, and somatic tissue to identify sex-related genes in the largemouth bass. A total of 51,672 unigenes were obtained via the transcriptome analysis, and 5900 differential expression genes (DEGs), including 3028 up-regulated and 2872 down-regulated DEGs, were obtained in the somatic tissue, testis, and ovary. DEGs were retrieved by making comparisons: somatic tissue vs testis (1733-up and 1382-down), testis vs ovary (841-up and 807-down), and ovary vs somatic tissue (454-up and 683-down). Finally, functional annotation identified 22 key sex-related DEGs, including 13 testis-biased DEGs (dmrt1, cyp11b1, sox9, spata4, spata22, spata17, fshr, fem-1a, wt1, daz1, amh, vasa, and piwi1) and 9 ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, bmp15, fem-1b, fig. la, and piwi2). This result was further confirmed by the tissue expression detection via RT-PCR and RT-qPCR. Protein-protein interacting (PPI) network analysis revealed that the testis-specific dmrt1 interacts directly with the testis-biased DEGs (cyp11b1 and spata4) and the ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, and bmp15), suggesting that the dmrt1 as a sex-determining gene can play a dual role through inducing the testis-biased DEGs and inhibiting the ovary-biased DEGs during the testicular development. Our present results provide useful molecular data for a better understanding of sexual development in the largemouth bass.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4 kb, corresponding to ∼ 4× coverage of the estimated 5.38 Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

Germ plasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germ plasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germ plasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germ plasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germ plasm that functions in early germ cell specification.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M−phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial–temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Characterized by ankyrin repeat motifs, the feminization-1 (fem-1) gene plays an essential role in sex determination/differentiation in Caenorhabditis elegans. However, there are only a few reports on fem-1 in crustaceans. In this study, a fem-1 gene (Mrfem-1) was first isolated from the giant freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of Mrfem-1 was 2607 bp long, containing an open reading frame encoding 615 amino acids, and presenting eight ankyrin repeats. The full-length cDNA has been submitted to GenBank with the accession no. MT160093. According to the RT-PCR results, Mrfem-1 was exclusively expressed in the ovary. The expression level of Mrfem-1 had increased with ovarian maturation and reached the highest peak at vitellogenic stage. In situ hybridization results showed that positive signals were concentrated in the cytoplasm of previtellogenic stage, and scattered in the cytoplasm and follicular cells at vitellogenic stage, suggesting that Mrfem-1 might be associated with ovarian maturation. Moreover, two effective siRNAs targeting Mrfem-1 were found and their effectiveness verified in vitro. These results on Mrfem-1 will help us to better understand the fem family and provide a new resource for subsequent investigations of siRNA-mediated regulation on ovarian development in M. rosenbergii.

During recent years, China has become a hotspot for the domestication of mandarin fish, and this is of great commercial value. Although the food preference of domesticated mandarin fish has been studied, little is known about genes regulating their growth. We raised hybrid mandarin fish on artificial feed for 3 months, the results showed that the survival rate of hybrid mandarin fish was 60.00%. Their total length and body weight were 18.34 ±0.43 cm and 100.44 ±4.87 g. The absolute length and weight gain rates were 0.14 cm/d and 1.08 g/d, respectively. Finally, RNA sequencing (RNA-Seq) was performed to identify potential genes and pathways activated in response to growth performance. The transcriptome analysis generated 68, 197 transcripts and 45,871 unigenes. Among them, 1025 genes were up-regulated and 593 genes were down-regulated between the fast- and slow-growth fish. Finally, we obtained 32 differentially expressed genes, which were mainly related to fatty acid biosynthesis (e.g. FASN and ACACB), collecting duct acid secretion (e.g. ATP6E and KCC4), cell cycle (e.g. CDC20 and CCNB), and the insulin-like growth factor (IGF) system (IGFBP1). These pathways might be related to the growth of hybrid mandarin fish. In addition, more potential single nucleotide polymorphisms (SNPs) were detected in the fast-growth fish than in the slow-growth fish. The results suggest that the interaction of metabolism and abundant alleles might determine the growth of hybrid mandarin fish after food conversion.

Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.

As post-transcriptional regulators, microRNAs (miRNAs) play an important role in growth and reproductive processes. So far, there is limited information regarding crustacean miRNAs. To explore the potential role of miRNAs in the gonadal development of the prawn Macrobrachium rosenbergii, we constructed seven small RNA libraries from ovarian and testicular tissues at various stages using somatic tissue as the control. A total of 1,954 known and 129 novel miRNAs were retrieved. By comparing differentially expressed miRNAs (DEMs) between testes and ovaries, forty-one miRNAs were identified with sex-biased expression patterns, including 17 ovary-biased and 24 testis-biased patterns. Furthermore, the putative target genes of the sex-biased miRNAs, such as cyclin L1, mitogen-activated protein kinase 7 (MAPK 7), heat shock protein (HSP), and zinc finger protein, were significantly enriched in many reproduction-related pathways including the Gonadotropin-releasing hormone (GnRH) pathway, glycolysis, gluconeogenesis pathway, ovarian steroidogenesis, estrogen signaling pathway, MAPK pathway, Wnt pathway, and insulin signaling pathway, implicating potential regulatory roles of miRNAs in reproduction. These data aid in the further investigation of the mechanism of gonadal development and reproductive regulation mediated by miRNA in M. rosenbergii.

EsDmrt-like is a novel testis-specific Dmrt gene identified from the Chinese mitten crab Eriocheir sinensis . To explore its biological functions, we performed RNA interference (RNAi) by injection of long double-stranded RNA (dsRNA) targeting the coding region of the EsDmrt-like gene. At 24 h post-injection with EsDmrt-like -dsRNA, the relative expression level of EsDmrt-like mRNA significantly decreased by 80% ( P EsDmrt-like -dsRNA group disappeared in four of the five tested individuals. Taken together, the expressions of EsDmrt-like mRNA and its protein were successfully inhibited by RNAi. After extending RNAi treatment for one month, the crab testicular development exhibited inhibitory effects. The average testis size was reduced and the gonadosomatic index (GSI) was significantly decreased by 75% compared with the controls. The histological and immunohistochemical analysis showed that the spermatogenesis was blocked, with rare spermatozoa in the seminiferous tubules, and the immunohistochemical signal in the spermatogonia and spermatocytes became extremely weak in the EsDmrt-like -dsRNA group. These data demonstrated a critical role for EsDmrt-like in testicular development and spermatogenesis in E. sinensis , and in vivo repetitive injection of EsDmrt-like -dsRNA could work efficiently to inhibit male testicular development. Statement of relevance The Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture species and has high commercial value as a food source. EsDmrt-like is a novel testis-specific Dmrt gene identified from the E. sinensis . We performed RNAi by repetitive injection of long double-stranded RNA (dsRNA) targeting the coding region of the EsDmrt-like gene, and certified that EsDmrt-like plays a critical role during spermatogenesis and testicular development.

Insulin-like androgenic gland hormone gene (IAG) is a sex regulator specifically expressed in male crustaceans, controlling the male sexual differentiation, spermatogenesis and reproductive strategy. Our previous study reported the cloning and characterization of the prawn Macrobrachium nipponense IAG (MnIAG). In this study, we further identified a 2214-bp MnIAG 5'-flanking region, and analyzed its transcription factor binding sites and transcriptional activity. The results showed that there were two potential promoter core sequences, three TATA boxes and one CAAT box existing in the MnIAG 5'-flanking region as well as many potential transcription factor binding sites, such as SRY, Sox-5, GATA-1, etc. Notably, the transcriptional activity was weak in this region, and a negative regulatory region was found in -604 to -231bp. In addition, we constructed M. nipponense yeast libraries and identified proteins interacting with the MnIAG protein by yeast two hybridization assay. The yeast two-hybrid screening yielded ten positive clones, of which five were annotated by NCBI database, namely heat shock protein 21, NADH dehydrogenase, zinc finger protein, beta-N-acetylglucosaminidase and a hypothetical protein. The identification of MnIAG putative regulatory region and proteins that interact with IAG will facilitate our understanding of the regulatory role of MnIAG and provide a foundation for deep insight into the prawn sex differentiation mechanism and signaling transduction pathways.

Genetic linkage maps provide excellent tools for detecting quantitative trait loci (QTL) and understanding genome organization and evolution. By taking advantage of a large number of microsatellite markers isolated from the genome of the Chinese mitten crab Eriocheir sinensis in our previous study, here we constructed a first generation genetic linkage map for this species using 1300 microsatellite markers that genotyped a F1 full-sib family from an intercross between northern and southern populations in China. A total of 674 markers (52%) were informative and grouped on the two parental maps. The number of linkage groups is quite close to the actual number of the crab haploid chromosomes ( n = 73). The female linkage map contained 466 markers which mapped to 71 linkage groups, spanning a total genetic distance of 2430.9 cM with an average marker space of 6.2 cmd The male map contained 457 markers which mapped to 70 linkage groups, spanning 2658.4 cM with an average marker space of 6.9 cmd The crab genome length was estimated to be 3390.0 cM, covering 71.7% of genome for the female map, and 3678.8 cM covering 72.3% of genome for the male map. Furthermore, the linkage maps were used for preliminary detection of QTL. Nine growth-related QTL for body length, width and weight were mapped to seven linkage groups. Additionally, two QTL were identified to be associated with sexual precocity. The linkage maps and the identified QTL will be valuable for marker-assisted selection breeding programs of the mitten crab. Statement of relevance A first generation genetic linkage map of the Chinese mitten crab was created using 1300 microsatellite markers. Nine growth-related QTL and two sexual precocity-related QTL were identified, which will be valuable for further studies of the genetic mechanisms of development and sexual precocity, and marker-assisted selection breeding programs for this species.

As an essential mediator in the Gonadotropin-releasing hormone (GnRH) signaling pathway, GnRH receptor (GnRHR) coupled to GnRH, plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction. To detect the existence of GnRHR and potential GnRH signaling pathway, we cloned and characterized GnRHR in the Chinese mitten crab, Eriocheir sinensis (named EsGnRHR). The full-length EsGnRHR cDNA is 2038 bp in length, including an open reading frame (ORF) of 1566 bp, a 57 bp 5′-untranslated region (5′-UTR) and a 415 bp 3′-UTR. Prediction of transmembrane domains in protein sequence revealed that the EsGnRHR protein contained seven hydrophobic transmembrane regions (TMs). Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary, and weakly distributed in the testis and brain. In situ hybridization further demonstrated that EsGnRHR mRNA was localized at the protocerebrum and deutocerebrum. In the ovary and testis, the hybridization signal was dominantly at the earlier developmental stages. The signal was mainly localized in the cytoplasm cell in the ovary, and in the epithelium cell in the testis. During the different stages of gonadal development, EsGnRHR displayed increasing trends in both female and male when analyzed by quantitative real-time PCR, suggesting that EsGnRHR was involved in controlling gonadal development. Our study provides important information for further research on the molecular mechanisms underlying crab development.

Vertebrate gonadotropin-releasing hormone (GnRH) plays a central role in the regulation of gonadial development by binding with its specific membrane receptor, GnRH receptor (GnRHR), which is an incipient key node in the GnRH signaling pathway. To explore the potential GnRH pathway in invertebrates, in this study, we reanalyze the transcriptomic library data of the oriental prawn Macrobrachium nipponense achieved previously in our laboratory. A total of 26 transcripts representing the genes assigned to the vertebrate GnRH signaling cascade were identified, including a transcript encoding a GnRH-like receptor ( Mn-GnRHR ). The Mn-GnRHR transcript contains an open reading frame (ORF) of 1083 bp, encoding a protein of 360 amino acid residues. The putative protein is characterized by seven typical transmembrane (TM) domains of vertebrates GnRHR and is most closely related to the invertebrate Octopus vulgaris GnRHR as demonstrated by phylogenetic analysis. Reverse-transcription PCR revealed that the Mn-GnRHR mRNA was distributed predominantly in the brain, gonad, eyestalk, muscle and weakly or not in other tissues examined. In situ hybridization further demonstrated that the Mn-GnRHR mRNA was mainly distributed in the small- and medium-sized neurons located in the protocerebrum and deutocerebrum. In the ovary,the Mn-GnRHR mRNA was localized in the ooplasm of previtellogenic and vitellogenic oocytes and meiotically-mature oocytes as well as the follicular cells around oocytes. Quantitative real time PCR showed that the Mn-GnRHR transcript was expressed stably in ovaries at various stages. The above data provided the first evidence of the presence of GnRH-like receptors in the crustacean, which may function through GnRH signaling pathway to regulate ovarian development.

The Chinese mitten crab Eriocheir sinensis is the most economically important cultivated crab species in China, and its genome has a high number of chromosomes (2n = 146). To obtain sufficient markers for construction of a dense genetic map for this species, we employed the recently developed specific-locus amplified fragment sequencing (SLAF-seq) method for large-scale SNPs screening and genotyping in a F1 full-sib family of 149 individuals. SLAF-seq generated 127,677 polymorphic SNP markers, of which 20,803 valid markers were assigned into five segregation types and were used together with previous SSR markers for linkage map construction. The final integrated genetic map included 17,680 SNP and 629 SSR markers on the 73 linkage groups (LG), and spanned 14,894.9 cM with an average marker interval of 0.81 cM. QTL mapping localized three significant growth-related QTL to a 1.2 cM region in LG53 as well as 146 sex-linked markers in LG48. Genome-wide QTL-association analysis further identified four growth-related QTL genes named LNX2, PAK2, FMRFamide and octopamine receptors. These genes are involved in a variety of different signaling pathways including cell proliferation and growth. The map and SNP markers described here will be a valuable resource for the E. sinensis genome project and selective breeding programs.

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P &lt, 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P &lt, 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH2-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex-reversed females (neomales). To provide molecular evidence for the proposed system, novel sex-linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male-specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female-specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female-specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female-linked SCAR markers can be applied for rapid detection of prawn gender. These sex-specific SCAR markers and sex-associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).

The androgenic gland (AG), a male-specific endocrine organ in crustacean, is responsible for the maintenance of male characteristics and gender differentiation. In this study, an AG-specific gene, the Macrobrachium nipponesne insulin-like androgenic gland factor (MnIAG) was isolated from a transcriptome library of M. nipponesne and its full-length cDNA sequences were obtained by RACE method. The cDNA was 1,547 bp in length and encoded a precursor protein of 175 amino acids. The deduced precursor protein consisted of a signal peptide, B chain, C peptide and an A chain, which exhibited the same structural organization as that of previously identified insulin-like androgenic gland in crustacean. The mature peptide of the MnIAG owned two additional conserved cysteine residues, which were also found in the Palaemonidae species reported. Results of the tissue distribution and in situ hybridization showed the MnIAG expressed exclusively in androgenic gland. The quantitative RT-PCR results demonstrated that the MnIAG transcript was present at blastula stage and later developmental stages with low levels, which suggested that the primordial cells of the AG might form at these stages.

Germ cells are specified by the inheritance of maternal germline determinants (preformation mode) or inductive signals from somatic cells (epigenesis mode) during embryogenesis. However, the germline specification in decapod crustaceans is unclear so far. Using vasa homolog (MnVasa) as a germ cell marker, here we probed the early events of germline specification in the oriental river prawn Macrobrachium nipponense. Quantitative RT-PCR analysis of unfertilized eggs and embryos demonstrated that the prawn MnVasa mRNA is a maternal factor. Whole-mount in situ hybridization further indicated that MnVasa transcripts are maternally supplied to only one blastomere at the very early cleavage stages. As cleavage proceeds, the MnVasa-positive blastomere undergoes proliferation and increases in number. During gastrulation, the MnVasa-positive cells are found to be around a blastopore and could migrate into an embryo through the blastopore. At the zoea stage, clusters of the MnVasa-positive cells distribute not only in the gonad rudiment in the cephalothorax but also at an extragonadic site, dorsal to the posterior hindgut in the abdomen, suggesting that MnVasa-positive cells could migrate anteriorly to the genital rudiment through the hindgut. Based on the dynamic localization and number of MnVasa-positive cells during embryogenesis, we concluded that the MnVasa-positive cells are primordial germ cells (PGC) or founder cells of PGC that are separated from soma at the early cleavage stage. MnVasa mRNA might have a key function in the specification of the prawn germline cells as a maternal determinant. These results provide the first evidence that the germline specification in decapod crustaceans follows a preformation mode.

SRY-related HMG box (Sox) genes are characterized by the presence of a DNA-binding HMG domain and involved in a diverse range of developmental processes. In this study, we identified a novel Sox gene, designated as EsSoxB2-1, from the Chinese mitten crab Eriocheir sinensis. The EsSoxB2-1 encodes a protein of 259 amino acids, sharing the highest identity with the beetle Tribolium castaneum SOX21b. Unlike insect Sox21b, however, EsSoxB2-1 is intronless and exhibits a gonad-specific expression pattern at both mRNA and protein level. Two core promoters in 5′ flanking region were demonstrated to be essential for inducing transcriptional regulatory activity. The transcription of EsSoxB2-1 mRNA begins in spermatogonia stage, while the translation of EsSOXB2-1 protein initiates at spermiogenesis stage. Interestingly, EsSOXB2-1 protein was exclusively localized in the nucleus of spermatid and spermatozoa even at the end of acrosome reaction, and was bound to the uncondensed chromatin in nucleoplasm of mature spermatozoa. Knockdown of EsSoxB2-1 by RNAi leads to abnormal transformation of the nucleus during spermiogenesis. Together, these findings demonstrated the requirement of EsSoxB2-1 for the spermatozoa nucleus maturation and also suggested that EsSoxB2-1 would be delivered into fertilized eggs along with chromatins as a paternal transcription factor for regulating early embryonic development.

Cdc2 kinase is a catalytic subunit of maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocyte. To understand the role of Cdc2 kinase on the oocyte maturation in crustacean, a complete cDNA sequence of Cdc2 kinase was cloned from Chinese mitten crab Eriocheir sinensis and its spatial-temporal expression profiles were analyzed during oogenesis at RNA and protein levels. The crab Cdc2 cDNA (1364 bp) encodes for a 299 amino acids protein with calculated molecular weight of 34.7 kDa. The Cdc2 mRNAs level showed no significant change in the ovary during oogenesis, whereas higher protein level was found at previtellogenesis, late vitellogenesis and germinal vesicle breakdown (GVBD) stages. Two forms (35 kDa and 34 kDa) of Cdc2 proteins were simultaneously identified in ovary at all stages. Immunocytochemistry analysis revealed that Cdc2 proteins locate exclusively in ooplasm of previtellogenic oocyte, and then relocate into germinal vesicle at vitellogenesis stage and accumulate on meiotic spindle at oocyte maturation. These findings suggest that Cdc2 kinase has essential roles in inducing GVBD and generating meiotic apparatus during the crab oocyte maturation.

The meiotic maturation of oocyte in animals is regulated by maturation promotion factor (MPF), a complex of Cdc2 and cyclin B. Although the role of MPF during oocyte maturation has been well studied in a wide variety of eukaryotic organisms, little is known for crustacean species. In this study, a full-length cDNA of cyclin B was cloned from the Chinese mitten crab using degenerate RT-PCR and RACE methods. The crab cyclin B cDNA was 3,794 bp containing an unusually long 3' untranslation region (UTR) of 2,403 bp and an open-reading frame encoding for a protein of 410 amino acids, with a calculated molecular mass of 45 kDa. The long 3'UTR harbors many cytoplasmic polyadenylation elements (CPE), and the GY-box, Brd-box, K-box that are perfectly complementary to the 5'-ends of various Drosophila microRNAs. The crab cyclin B transcript was predominantly expressed in ovary and testis. Semi-quantitative RT-PCR analysis revealed that the amount of cyclin B mRNA was high at previtellogenesis and late vitellogenesis stages, while low at early and middle vitellogenesis, suggesting that differential expression of cyclin B is closely related to oogonial proliferation (mitosis) and oocyte meiotic maturation.

The feminization-1 (fem-1) gene is characterized by one of the most common protein-protein interaction motifs, ankyrin repeat motifs, displays many expression patterns in vertebrates and invertebrates, and plays an essential role in the sex-determination/differentiation pathway in Caenorhabditis elegans. In this study, a fem-1 homolog, designated as Mnfem-1, was first cloned from the oriental river prawn Macrobrachium nipponense. The prawn Mnfem-1 gene consists of six exons and five introns. The full-length cDNA (2603bp) of Mnfem-1 contains an open reading frame (ORF) encoding a protein of 622 amino acids. The Mnfem-1 RNA and protein are exclusively expressed in the ovary in adult prawns as revealed by RT-PCR and immunofluorescence analysis, respectively. In situ hybridization results showed that strong positive signals were concentrated at the edge of the previtellogenic and vitellogenic oocyte. During embryogenesis, Mnfem-1 is highly expressed in both unfertilized eggs and embryos at cleavage stage and thereafter dropped to a low level from blastula to zoea, indicating that the Mnfem-1 in early embryos is maternal. After hatching, the Mnfem-1 expression significantly increased in the larvae at length of 2cm, an important stage of sex differentiation. Yeast two hybridization results showed that the Mnfem-1 protein can be potentially interactive with cathepsin L and proteins containing the domains of insulinase, ankyrin or ubiquitin. Our results suggested that Mnfem-1 could have roles in prawn ovarian development and sex determination/differentiation.

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn (Marsupenaeus japonicus), subtractive suppression hybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed by evaluation of the differential expression profile by macroarray and quantitative real-time RT-PCR analyses. The cathepsin C (dipeptidyl peptidase I) gene was thus found to exhibit a significantly higher expression around the onset of cortical rod (CR) formation (early CR stage, appearance of round CRs), progress to a higher mRNA level until the middle CR stage (elongation of CRs), then rapidly revert to a low expression level at the late CR stage (occurrence of germinal vesicle breakdown, GVBD), as also observed at the non-CR stage (previtellogenesis and vitellogenesis). In situ hybridization analyses revealed that the sites of the expression of cathepsin C transcripts in the ovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specific gene was subsequently determined by rapid amplification of the cDNA 3' and 5' ends (3' and 5' RACE). The deduced amino acid sequence of the 230-residue mature peptide shared 67-70% identity to the known cathepsin C in mammals. Western blot analysis showed that expression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs as revealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration of its involvement in the final stages of oocyte maturation in crustacean species.

In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.

The penaeid shrimp develops cortical rods (CRs) in the oocyte after completing yolk accumulation during ovarian maturation. In this study, CRs from the mature ovary of the kuruma prawn (Marsupenaeus japonicus) were isolated and their proteins were characterized. Under reducing conditions of SDS-PAGE, the CR demonstrated five major bands of approximately 210, 150, 140, 130 and 30 kDa. Of 32 clones of monoclonal antibodies raised against the CR proteins, 21 were found to be immunoreactive by Western blotting, demonstrating 150, 140 and 130 kDa proteins simultaneously. This result suggested that the proteins were post-translational or post-transcriptional products from the same gene, or that they originated from the same gene family. CRs were identified by immunohistochemical analysis by 31 clones of monoclonal antibodies in paraffin sections and by 26 clones in cryostatic ones. Most of the antibodies also stained the yolk in both developing and mature oocytes, suggesting that CR proteins accumulated in developing oocytes, then assembled to form the CR structure.

Cyclin-dependent kinases (cdks) are key regulators of the cell cycle. In mammals, cdk2 plays an essential role in the meiosis of spermatocytes and oocytes. To investigate the role of cdk2 kinase during gametogenesis in crustaceans, we cloned a complete cDNA sequence of cdk2 from the freshwater giant prawn, Macrobrachium rosenbergii, and examined its localization and expression in the developing gonads. The prawn cdk2 cDNA is 1,745 bp in length and encodes a putative protein of 305 amino acids. The deduced protein contains a conserved cyclin binding motif PSTAIRE and shares high homology with reported cdk2 kinases of other species. RT-PCR analysis showed a wide distribution of the cdk2 mRNA in all tested organs including the testis, ovary, heart, muscles, hepatopancreas and gills, and the highest level of expression in the ovary and testis. Localization by in situ hybridization of cdk2 mRNA in the ovary showed high expression in the ooplasm of previtellogenic and the nuclei of late vitellogenic oocytes. In testicular sections, cdk2 transcript is low in spermatogonia, high in spermatocytes, but reduced in spermatids and sperm. The high expression of the cdk2 transcripts in meiotic spermatocytes and oocytes indicated that the cdk2 gene has the conservative function in the germ cells meiosis during gametogenesis.

Background The oriental river prawn, Macrobrachium nipponense, is an economically and nutritionally important species of the Palaemonidae family of decapod crustaceans. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for M. nipponense using high-throughput sequencing technologies. Methodology and Principal Findings Total RNA was isolated from eyestalk, gill, heart, ovary, testis, hepatopancreas, muscle, and embryos at the cleavage, gastrula, nauplius and zoea stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, we generated a total of 984,204 high quality reads (338.59Mb) with an average length of 344 bp. Clustering and assembly of these reads produced a non-redundant set of 81,411 unique sequences, comprising 42,551 contigs and 38,860 singletons. All of the unique sequences were involved in the molecular function (30,425), cellular component (44,112) and biological process (67,679) categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literature, many putative genes involved in sex determination, including DMRT1, FTZ-F1, FOXL2, FEM1 and other potentially important candidate genes, were identified for the first time in this prawn. Furthermore, 6,689 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset. Conclusions The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in M. nipponense. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will be essential for accelerating aquaculture breeding programs with this species.

Dmrt is a family of genes related to the sexual regulators Doublesex of Drosophila melanogaster and Mab-3 of Caenorhabditis elegans. Dmrt genes are widely conserved and known for their involvement in sex determination and differentiation across phyla. In this study, we report here the identification of a novel Dmrt gene, named EsDmrt-like, from Chinese mitten crab Eriocheir sinensis. EsDmrt-like encodes a protein of 236 amino acids without intron. The protein contains a conserved DNA-binding DM domain that is characteristic of Dmrt genes. The DM domain shares 98% identity with that of Drosophila Dmrt99B and vertebrate Dmrt5, but outside the DM domain, there is little homology in sequence and no other conserved domain such as DMA specific to the Dmrt99B and Dmrt3-5. Interestingly, the expression pattern of EsDmrt-like is quite similar with that of vertebrate Dmrt1. Reverse transcription-polymerase chain reaction showed that EsDmrt-like transcripts were detectable only in testis with much higher expression at immature stage. In situ hybridization to gonad sections indicated that the EsDmrt-like mRNA was exclusively localized in Sertoli cells around the periphery of seminiferous tubules and developing germ cells including spermatogonium, spermatocyte, and spermatid, but absent in spermatozoa. This finding strongly suggests an essential role for EsDmrt-like in the male testicular development/differentiation of the crab.

Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawnMarsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and37∘Cfor 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

Ferritin, a major iron storage protein of most living organisms，plays a crucial role in iron metabolism. A cDNA encoding a heavy-chain homologue of ferritin was isolated and sequenced in the freshwater giant prawn, Macrobrachium rosenbergii . The cloned cDNA was 1012 bp long containing an iron-responsive element (IRE) sequence in the 5′-untranslated region and a complete open-reading frame encoding for a 172 amino acid residues protein with a conserved domain for the ferroxidase center characteristic for heavy chains of vertebrate ferritin. Prawn ferritin transcripts are expressed in muscle, heart, hepatopancreas, stomach, hemocytes, ovary and testis. Quantitative real-time PCR revealed that the abundance of ferritin transcript was highest in the hepatopancreas, followed by the testis. The expression of the ferritin transcript in the muscle significantly increased 6-fold at 3 h after injection of iron. In the ovary, a high expression of ferritin transcript was first detected with nearly a 4-fold increase after 3 h post-injection that remained elevated for 48 h. Heart ferritin mRNA expression increased up to 8-fold at 24 h. No significant difference was found in the hepatopancreas, hemocytes and testis. These results strongly suggested that the expression of prawn ferritin is regulated by iron at the transcriptional level as found in insects, and iron increased prawn ferritin transcripts in a tissue-specific manner.

Noncoding mRNA-like transcripts play important roles in a wide range of biological events such as cell differentiation and organogenesis. We report here the identification of a novel germ cell-specific mRNA-like transcript (RtGST-1) from a rainbow trout oocyte cDNA library. The novel transcript of 1,165 bp was confirmed to be full-length based on Northern blot and 5' RACE analyses. The transcript is polyadenylated but does not contain a significant open reading frame (ORF). The presumable ORF (encodes a peptide of 71 amino acids) has poor codon usage for rainbow trout and the AUG codon is in a poor context for translation initiation. RT-PCR showed that the novel gene is specifically expressed in ovaries of various stages and immature testis, whereas no transcripts of this gene were detected in mature testis and somatic tissues. Quantitative real-time PCR analysis revealed that the mRNA level of this novel gene is extremely high in early previtellogenesis stage ovaries relative to vitellogenesis stage ovaries. In situ hybridization analysis further demonstrated that the novel transcript is localized exclusively in early previtellogenic oocytes and spermatocytes. To our knowledge, this study represents the first report of a germline-specific mRNA-like transcript in fish.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.