Genome-wide identification, phylogeny, expression and eyestalk neuroendocrine regulation of vitellogenin gene family in the freshwater giant prawn Macrobrachium rosenbergii.

Vitellogenin (Vg) is the precursor of vitellin, which is an important female-specific protein stored in oocytes as the major nutrient and energy sources for embryogenesis in oviparous animals. In this study, we performed comprehensive genome-wide analysis of Vg gene family in the prawn Macrobrachium rosenbergii, and eight Vg genes designated as MrVg1a, MrVg1b and MrVg2-7 were identified. MrVg1a clusters with the previously described MrVg1b near the end of chromosome 46 and MrVg2 is on the chromosome 42 while MrVg3-7 cluster on the chromosome 23. All the putative MrVg proteins are characterized by the presence of three conserved functional domains: LPD-N, DUF1943 and vWD. Phylogenetic analysis revealed that MrVg1a shares 93% identity with MrVg1b and groups together into a branch while MrVg2-7 group into another branch, suggesting that MrVg1a, 1b and MrVg2-7 might diversify from a common ancestral gene. All the corresponding MrVg transcripts especially for MrVg1 exhibit high expression in the female hepatopancreas at late vitellogensis stage but extremely low in the ovaries except MrVg5, indicating that hepatopancreas is the major site of MrVgs synthesis. In the male, interestingly, MrVg5 and MrVg6 are also highly expressed in the testis, suggesting their potential involvement in testicular development. Bilateral ablation of eyestalk significantly upregulate all the MrVgs mRNA in the female hepatopancreas and the MrVg1 in ovary, but have no effect on the expression of MrVg2-7 in the ovary, demonstrating that eyestalk hormones could promote the ovarian development mostly by inducing the synthesis of MrVgs in the hepatopancreas but rarely in the ovary. Our results provide new insights into the prawn MrVgs family and improve our understanding of the potential role for each member of the family in the gonadal development of M. rosenbergii.

Transcriptome analysis of the post-larvae of giant freshwater prawn (Macrobrachium rosenbergii) after IAG gene knockdown with microRNA interference.

The insulin-like androgenic gland hormone gene (IAG) of crustaceans plays pivotal roles in the regulation of sex differentiation. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that function as post-transcriptional gene regulators. However, little information about the regulatory relationship between miRNA and Macrobrachium rosenbergii IAG (MrIAG) were exposed. In this study, we used the 3' untranslated region (UTR) of MrIAG to predict potential target sites of miRNAs. The results showed that miR-184 has one target site in the 3'UTR of MrIAG. Dual-luciferase report assay in vitro confirmed that miR-184 can significantly down-regulate MrIAG expression. Besides, we constructed mutant plasmids of 3'UTR of MrIAG. The result displayed that after co-transfection of mutant plasmids and miR-184 agomir, the activity of luciferase was not affected compared to the control. These results indicated that miR-184 could directly regulate MrIAG. In addition, we found that overexpression of miR-184 in M. rosenbergii can lead to significant changes in the transcription level of genes. Compared with control group, we identified 1510 differentially expressed genes (DEGs) in the miR-184 injection group. Some DEGs were involved in sex differentiation, gonad development, growth and molting were found. qRT-PCR verification was performed on eight DEGs randomly, and the results showed that the expression level of sex-, growth-, and metabolism-related genes changed significantly after MrIAG gene knockdown. Collectively, findings from this study suggest that miR-184, by mediating IAG expression, may be involved in many physiological processes in M. rosenbergii. The current study lays a basic understanding for short-term silencing of MrIAG with miR-184, and facilitates miRNA function analysis in M. rosenbergii in future.

Identification of a novel germ cell marker MnTdrd from the oriental river prawn Macrobrachium nipponense.

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.

Germ plasm and the origin of the primordial germ cells in the oriental river prawn Macrobrachium nipponense.

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.

Identification and characterization of sex-biased and differentially expressed miRNAs in gonadal developments of the Chinese mitten crab, Eriocheir sinensis.

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.

Lidar system based on lens assisted integrated beam steering.

We present a demonstration of solid-state light detection and ranging (Lidar) at 1550 nm by applying integrated two-dimensional (2D) lens assisted beam-steering (LABS) technology. LABS has O(logN) power consumption for N antennas and allows a simple control complexity with digital signal input. A time-of-flight coaxial Lidar is demonstrated with this beam-steering technology. The integrated beam-steering chip and lens both transmit and receive the light. The Lidar has 16 scanning angles, 19.5 m ranging distance, and a 3 cm ranging error. This Letter proves the potential application of 2D LABS in Lidar and paves the way for a fully integrated Lidar system.

Identification and Characterization of a Luteinizing Hormone Receptor (LHR) Homolog from the Chinese Mitten Crab Eriocheir sinensis.

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 < 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 < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

Identification of putative regulatory region of insulin-like androgenic gland hormone gene (IAG) in the prawn Macrobrachium nipponense and proteins that interact with IAG by using yeast two-hybrid system.

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.

Global analysis of the ovarian microRNA transcriptome: implication for miR-2 and miR-133 regulation of oocyte meiosis in the Chinese mitten crab, Eriocheir sinensis (Crustacea:Decapoda).

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules that downregulate gene expression by base pairing to the 3'-untranslated region (UTR) of target messenger RNAs (mRNAs). Up to now, rare information for the miRNAs is available in decapod crustaceans. Our previous studies showed that many miRNA-binding sites are present in the 3'-UTR of the cyclin B in the Chinese mitten crab Eriocheir sinensis, suggesting that the translation or post-transcription of the crab cyclin B might be regulated by miRNAs during meiosis of oocyte. RESULTS: To identify ovarian miRNAs in the mitten crab, ovarian small RNAs were subjected to high-throughput sequencing using an Illumina Genome Analyzer. Of 14,631,328 reads, 55 known miRNAs representing 44 miRNA families were identified and 136 novel miRNA candidates were predicted. The 5' seed sequences of four miRNAs, miR-2, miR-7, miR-79 and miR-133, were revealed to complementary to miRNA binding sites in 3'-UTR of the cyclin B. Quantitative real time PCR analysis showed that miR-2 and miR-133 are much more abundant in the first metaphase (MI) of meiosis than in germinal vesicle (GV) stage. But their increasing expressions are independent of induction of gonadotropin-releasing hormone (GnRH). Further expression analysis using double-luciferase reporter genes assay showed that miR-2 and miR-133 can downregulate the 3'-UTRs of the crab cyclin B gene, indicating that they could inhibit the translation of the cyclin B. Western blot analysis confirmed that cyclin B protein is completely disappeared in fertilized egg at the metaphase-anaphase transition of meiosis I, suggesting that miR-2 and miR-133 could function in destruction of cyclin B near the end of MI. CONCLUSIONS: A high number of miRNAs have been identified from the crab ovarian small RNA transcriptom for the first time. miR-2 and miR-133 exhibit differential expression during the meiotic maturation of the oocytes and have activity in regulating the 3'-UTR of the crab cyclin B gene. This result is inconsistent with recent finding that miRNA activity is globally suppressed in mouse oocytes.

The cloning of the cdk2 transcript and the localization of its expression during gametogenesis in the freshwater giant prawn, Macrobrachium rosenbergii.

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.

Molecular characterization and expression analysis of an insulin-like gene from the androgenic gland of the oriental river prawn, Macrobrachium nipponense.

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.

Female-only sex-linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii.

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).

Potential role for the germ cell-specific Rad21 in early meiosis of oocyte and spermatocyte in the Chinese mitten crab Eriocheir sinensis.

Rad21/Rec8 family proteins are vital for sister chromatid segregation in mitosis and homologous recombination in meiosis, but no molecular data are available in crustacean species. In this study, a germ cell-specific Rad21 named EsRad21 was identified in the crab Eriocheir sinensis. EsRad21 mRNA has an open reading frame of 2310 base pairs (bp) encoding a 769 amino acids (aa) protein. RT-PCR showed that EsRad21 mRNA was particularly expressed in testis and ovary. The RT-qPCR results further revealed that the EsRad21 mRNA exhibited similar expression pattern in gonads at various developmental stages. EsRad21 mRNA expression level was the highest in testis at early spermatogenesis stage and ovaries at previtellogenesis stage, thereafter decreased significantly at middle spermatogenesis and vitellogenesis, and finally reach the lowest level at late spermatogenesis and vitellogenesis. In situ hybridization (ISH) analysis showed that EsRad21 mRNA was exclusively expressed in germline cells, but not in gonadal somatic cells. Notably, hybridized signal was detected on chromosomes of metaphase spermatocytes. EsRad21 is thus an underlying helpful indicator of the early phases of germ cell development. RNAi knockdown of EsRad21 downregulated the expression of other meiosis-related genes like Smc5-Smc6 and SPO11 and resulted in high mortality of individuals after 24 h post injection of EsRad21 dsRNA. Taken together, our results showed a potential role for EsRad21 in early meiosis of oocytes and spermatocytes in E. sinensis. This is the first report on the molecular characterization of the Rad21 transcript in a crustacean species.

Effects of titanium dioxide (TiO2)/activated carbon (AC) nanoparticle on the growth and immunity of the giant freshwater prawn, Macrobrachium rosenbergii: potential toxicological risks to the aquatic crustaceans.

Due to their unique physicochemical characteristics, nanomaterials exhibit many excellent properties and functions, leading to their applications in numerous fields. The large-scale production and widespread application of nanomaterials have inevitably resulted in their release into the environment, especially the water environment. Several studies have confirmed that exposure to nanomaterials can be toxic to aquatic organisms. However, few studies have focused on the effects of nanomaterial exposure on growth and immunity in crustaceans. In the present study, juvenile Macrobrachium rosenbergii were exposed to different concentrations of titanium dioxide (TiO2)/activated carbon (AC) composite nanomaterial (0.1 and 0.5 mg/L) for 45 days. The effects of nanoparticle exposure on digestion and antioxidant-related enzyme activities, as well as the expression of growth and immunity-related genes and signaling pathway, were evaluated. Our results show that in response to low concentration of TiO2/AC nanoparticle (0.1 mg/L), most of the enzyme activities related to digestion and antioxidation (TPS, LPS, AMS, SOD, and CAT) were diminished. On the contrary, the GSH-Px activity increased under the 0.1 mg/L group of TiO2/AC nanoparticle concentration. Additionally, the level of digestive and antioxidant enzyme activities we detected was increased when exposed to 0.5 mg/L TiO2/AC nanoparticle. By comparison to the expression level of growth-related genes in the control group, MSTN, CaBP, E75, Raptor, EcR, and EGF were significantly inhibited at 0.1 and 0.5 mg/L concentrations of TiO2/AC nanoparticle, whereas the expression level of genes (TLR, JAK, STAT, PPAF, ACP, and AKP) related to immunity was increased when exposed to different concentrations of TiO2/AC nanoparticle. Compared with the control group (0 mg/L concentration), 5166 DEGs were identified in the TiO2/AC nanoparticle group, and a large number of DEGs were involved in molting, energy metabolism, stress tolerance, and germ cell development. Moreover, KEGG analysis revealed that many DEGs were assigned into signaling pathways related to metabolic growth and immune stress. These results showed that exposure to TiO2/AC nanoparticle will result in the changes of enzyme activity and routine mRNA expression, suggesting that TiO2/AC nanoparticle which existed in aquatic environment might affect the physiology of M. rosenbergii. This study will provide significant information for the evaluation of nanomaterial toxicity on aquatic crustaceans.

Identification of GnRH-like peptide and its potential signaling pathway involved in the oocyte meiotic maturation in the Chinese mitten crab, Eriocheir sinensis.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.

Large-scale isolation of microsatellites from Chinese Mitten Crab Eriocheir sinensis via a Solexa Genomic Survey.

Microsatellites are simple sequence repeats with a high degree of polymorphism in the genome; they are used as DNA markers in many molecular genetic studies. Using traditional methods such as the magnetic beads enrichment method, only a few microsatellite markers have been isolated from the Chinese mitten crab Eriocheir sinensis, as the crab genome sequence information is unavailable. Here, we have identified a large number of microsatellites from the Chinese mitten crab by taking advantage of Solexa genomic surveying. A total of 141,737 SSR (simple sequence repeats) motifs were identified via analysis of 883 Mb of the crab genomic DNA information, including mono-, di-, tri-, tetra-, penta- and hexa-nucleotide repeat motifs. The number of di-nucleotide repeat motifs was 82,979, making this the most abundant type of repeat motif (58.54%); the second most abundant were the tri-nucleotide repeats (42,657, 30.11%). Among di-nucleotide repeats, the most frequent repeats were AC motifs, accounting for 67.55% of the total number. AGG motifs were the most frequent (59.32%) of the tri-nucleotide motifs. A total of 15,125 microsatellite loci had a flanking sequence suitable for setting the primer of a polymerase chain reaction (PCR). To verify the identified SSRs, a subset of 100 primer pairs was randomly selected for PCR. Eighty two primer sets (82%) produced strong PCR products matching expected sizes, and 78% were polymorphic. In an analysis of 30 wild individuals from the Yangtze River with 20 primer sets, the number of alleles per locus ranged from 2--14 and the mean allelic richness was 7.4. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in four of the 20 microsatellite loci after sequential Bonferroni corrections. This method is cost- and time-effective in comparison to traditional approaches for the isolation of microsatellites.

Identification and characterization of a new germline-specific marker vasa gene and its promoter in the giant freshwater prawn Macrobrachium rosenbergii.

Vasa gene encodes a protein member of DEAD-box superfamily of ATP-dependent RNA helicases, which plays a key role in germline development in metazoans. In present study, we identified a new germline-specific marker Mrvasa in the prawn Macrobrachium rosenbergii, whose genomic DNA sequence consists of 14 exons and 13 introns. A 2516 bp of full-length Mrvasa cDNA encodes a protein of 603 amino acids. It contains nine conserved motifs, a zinc-finger motif, and RGG repeats. RT-PCR indicated that Mrvasa mRNA was specifically expressed in gonads. QPCR analysis further revealed that the expression of Mrvasa mRNA is much higher in testis than in ovary. In testis, the relative expression level of Mrvasa mRNA in late developing stage is significantly higher than that in early-middle developing stage. During ovarian development, no significant difference in expression was found. In situ hybridization demonstrated that Mrvasa mRNA was localized in germline cells including spermatogonia, spermatocytes, and spermatozoa in testes, and previtellogenic and vitellogenic oocytes in ovary. We then isolated the Mrvasa promoter and determined the transcription core region of this promoter. This is the first report on identification of vasa core promoter in crustaceans. Our results will provide a useful germline-specific marker Mrvasa for tracing germline cell formation and development in M. rosenbergii.

Comprehensive Transcriptome Analysis of Gonadal and Somatic Tissues for Identification of Sex-Related Genes in the Largemouth Bass Micropterus salmoides.

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.

Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (ß-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis.

Red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH) are crustacean neuropeptides involved in broad physiological processes including body color changes, circadian rhythm, and ovarian growth. In this study, the full-length cDNA of RPCH and PDH were identified from the brain of the Chinese mitten crab Eriocheir sinensis. The deduced RPCH and PDH mature peptides shared identical sequence to the adipokinetic hormone/RPCH peptides family and the ß-PDH isoforms and were designated as Es-RPCH and Es-ß-PDH, respectively. Es-RPCH and Es-ß-PDH transcripts were distributed in the brain and eyestalks. The positive signals of Es-RPCH and Es-ß-PDH were localized in the neuronal clusters 6, 8, 9, 10, and 17 of the brain as revealed by in situ hybridization. The expression level of Es-RPCH and Es-ß-PDH mRNA in nervous tissues were all significantly increased at vitellogenic stage, and then decreased at the final meiotic maturation stage. The administrated with synthesized Es-RPCH peptide results in germinal vesicles shift toward the plasma membrane in vitellogenic oocyte, and significant decrease of the gonad-somatic index (GSI) and mean oocyte diameter as well as the expression of vitellogenin mRNA at 30 days post injection in vivo. Similar results were also found when injection of the Es-ß-PDH peptide. In vitro culture demonstrated that Es-RPCH and Es-ß-PDH induced germinal vesicle breakdown of the late vitellogenic oocytes. Comparative ovarian transcriptome analysis indicated that some reproduction/meiosis-related genes such as cdc2 kinase, cyclin B, 5-HT-R and retinoid-X receptor were significantly upregulated in response to Es-RPCH and Es-ß-PDH treatments. Taken together, these results provided the evidence for the inductive effect of Es-RPCH and Es-ß-PDH on the oocyte meiotic maturation in E. sinensis.

A novel Dmrt gene is specifically expressed in the testis of Chinese mitten crab, Eriocheir sinensis.

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.