Molecular characterization of adenosine monophosphate deaminase 1 and the correlation analysis between its mRNA expression levels and inosine monophosphate content in large yellow croaker (Larimichthys crocea).

Declining flesh quality has drawn considerable attention in the farmed large yellow croaker (LYC; Larimichthys crocea) industry. Inosine monophosphate (IMP) is the primary flavor substance in aquatic animals. Adenosine monophosphate deaminase 1 (AMPD1) plays a critical role in IMP formation by catalyzing the deamination of AMP to IMP in the purine nucleotide cycle. To further evaluate the correlation between ampd1 mRNA expression levels and IMP content in the LYC muscle tissue, the relevant open reading frame (ORF) of L. crocea (Lcampd1) was cloned, and the IMP content and Lcampd1 mRNA expression in the muscles of LYCs of different sizes were examined. The ORF cDNA of Lcampd1 was 2211 bp in length and encoded a polypeptide of 736 amino acids (AAs). The deduced protein, LcAMPD1, possesses conserved AMPD active regions (SLSTDDP) and shows high homology with AMPD proteins of other teleost fishes. The genomic DNA sequence of Lcampd1 exhibits a high degree of evolutionary conservation in terms of structural organization among species. Phylogenetic analysis of the deduced AA sequence revealed that teleost fish and mammalian AMPD1 were separate from each other and formed a cluster with AMPD3, suggesting that AMPD1 and AMPD3 arose by duplication of a common primordial gene. In healthy LYC, Lcampd1 mRNA was expressed only in the muscle tissue. The IMP content in the muscle of LYCs with different average body weights was measured by high-performance liquid chromatography; the results showed that the IMP content in the muscle of LYCs with greater body weight was significantly higher than that in LYC with lower body weight. Moreover, a similar trend in Lcampd1 expression was observed in these muscle tissues. The Pearson correlation analysis further showed that the Lcampd1 mRNA expression was positively correlated with IMP content in the muscles of different-sized LYCs. These results suggest the potential function of Lcampd1 in determining the IMP content in LYC and provide a theoretical basis for flesh quality improvement, as well as a scientific basis for the development of the molecular breeding of LYC.

Cloning and expression characterization of elongation of very long-chain fatty acids protein 6 (elovl6) with dietary fatty acids, ambient salinity and starvation stress in Scylla paramamosain.

Introduction: Elongation of very long-chain fatty acids protein 6 (ELOVL6) played crucial roles in regulating energy expenditure and fatty acid metabolism. Many studies have performed to investigate the physiological roles and regulatory mechanisms of elovl6 in fish and animals, while few studies were reported in crustaceans. Methods: Here we reported on the molecular cloning, tissue distribution and expression profiles in response to dietary fatty acids, ambient salinity and starvation stress in Scylla paramamosain by using rapid amplification of cDNA ends (RACE) and quantitative real-time PCR. Results: Three elovl6 isoforms (named elovl6a, elovl6b and elovl6c) were isolated from S. paramamosain in the present study. The complete sequence of elovl6a was 1345 bp, the full-length sequence of elovl6b was 1419 bp, and the obtained elovl6c sequence was 1375 bp in full length. The elovl6a, elovl6b and elovl6c encoded 287, 329 and 301 amino acids respectively, and exhibited the typical structural features of ELOVL protein family members. Phylogenetic analysis showed that the ELOVL6a from S. paramamosain clustered most closely to ELOVL6 from Portunus trituberculatus and Eriocheir sinensis, while the ELOVL6b and ELOVL6c from S. paramamosain gathered alone into a single branch. Quantitative real-time PCR exhibited that the relatively abundant expression of elovl6b was observed in intestine and stomach, and the elovl6a and elovl6c were highly expressed in hepatopancreas. In addition, studies found that replacing fish oil with soybean oil could significantly increase the transcriptional levels of three elovl6 in hepatopancreas of S. paramamosain, and the expression of elovl6a and elovl6c in hepatopancreas were more sensitive to dietary fatty acids than the elovl6b. Compared with the normal sea water group (27‰), the expression of sterol-regulatory element binding protein1c (srebp-1), elovl6a, elovl6b and elovl6c were upregulated in the low salinity groups, particularly in 7‰. On the contrary, the starvation stress suppressed the expression of srebp-1, elovl6a, elovl6b and elovl6c. Discussion: These results may contribute to understand the functions of elovl6 in fatty acid synthesis and regulatory mechanisms in crustaceans.

Characterization of the glucocorticoid receptor of large yellow croaker (Larimichthys crocea) and its expression in response to salinity and immune stressors.

Glucocorticoids are steroidal hormones critical to stress responses in vertebrates. To gain further insight into the role of the glucocorticoid receptor (GR) in acute stress responses in teleost fish, the relevant cDNA of large yellow croaker (Larimichthys crocea; LcGR) was cloned using the rapid amplification of cDNA ends (RACE) technique. Multiple alignment of the amino acids (aa) of LcGR and the GR of other teleosts indicated LcGR contained four commonly conserved domains and lacked the 9-aa insert seen in GR1. Phylogenetic analysis of the amino acid sequence revealed that LcGR grouped most closely with the GR2 of other teleosts and can therefore be considered a GR2 subtype. In healthy L. crocea, Lcgr mRNA was found to be expressed at high levels in the gill, brain, and muscle tissue, expressed at intermediate levels in heart and stomach tissue, and expressed at low levels in the kidney, intestine, head kidney, liver, and spleen tissue. The response of L. crocea to acute low-salinity stress was tested, with a significant increase in plasma cortisol concentration after 3 h, peaking after 6 h, and gradually returning to base levels. Regarding changes of Lcgr expression in different body tissues under the stress, there was up-regulation of the Lcgr transcript in the brain, liver, and gill tissues, but not in muscle tissue. Responses to pathogen mimics were also tested. Injection with lipopolysaccharide resulted in Lcgr expression, with an increase-decrease-increase trend in the head kidney. In contrast, a down-regulation of Lcgr expression in the head kidney was observed throughout the experimental period upon injection of polyinosinic:polycytidylic acid, revealing different roles of Lcgr for different types of pathogens. The results offer novel insights about the effects of different stressors on GR gene expression in L. crocea, and can facilitate further investigations into stress responses in other mariculture fish species.

The expression regulation of Cyclins and CDKs in ovary via miR-9c and miR-263a of Scylla paramamosain.

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.

Genome-Wide Identification and Expression Profile of the Sox Gene Family During Embryo Development in Large Yellow Croaker, Larimichthys crocea.

Members of the Sox gene family play crucial roles during reproduction and development, but their genome-wide identification has not yet been performed in large yellow croaker, Larimichthys crocea. In this study, a total of 26 members of the Sox gene family were identified from the genome of large yellow croaker and classified into seven subgroups based on the conserved HMG-box domain they contain. Among the identified Sox gene family members, eight belonged to the SoxB subgroup (five in B1 and three in B2), four belonged to the SoxC subgroup, four belonged to the SoxD subgroup, six belonged to the SoxE subgroup, three belonged to the SoxF subgroup, and one belonged to the SoxK subgroup. During evolution, members of the SoxE subgroup (Sox8, Sox9, Sox10), Sox1, Sox4, Sox6, and Sox11 evolved into two copies, which may be a result of teleost-specific whole-genome duplication. Sox genes were distributed unevenly across 15 chromosomes. The number of introns in large yellow croaker Sox genes varied from 0 to 14. Results of the expression profile during embryogenesis revealed that most of the members of the Sox gene family had lower expression, except several Sox genes, and expression patterns also differed among each Sox gene group and duplicated gene. This study systematically characterized and analyzed the Sox gene family in large yellow croaker and provided new insights into its function during embryogenesis.

Genome-Wide Association Study of Growth and Body-Shape-Related Traits in Large Yellow Croaker (Larimichthys crocea) Using ddRAD Sequencing.

Large yellow croaker (Larimichthys crocea) is an economically important marine fish species of China. Due to overfishing and marine pollution, the wild stocks of this croaker have collapsed in the past decades. Meanwhile, the cultured croaker is facing the difficulties of reduced genetic diversity and low growth rate. To explore the molecular markers related to the growth traits of croaker and providing the related SNPs for the marker-assisted selection, we used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic bases of growth traits in a cultured population and identify the SNPs that associated with important growth traits by GWAS. A total of 220 individuals were genotyped by ddRAD sequencing. After quality control, 27,227 SNPs were identified in 220 samples and used for GWAS analysis. We identified 13 genome-wide significant associated SNPs of growth traits on 8 chromosomes, and the beta P of these SNPs ranged from 0.01 to 0.86. Through the definition of candidate regions and gene annotation, candidate genes related to growth were identified, including important regulators such as fgf18, fgf1, nr3c1, cyp8b1, fabp2, cyp2r1, ppara, and ccm2l. We also identified SNPs and candidate genes that significantly associated with body shape, including bmp7, col1a1, col11a2, and col18a1, which are also economically important traits for large yellow croaker aquaculture. The results provided insights into the genetic basis of growth and body shape in large yellow croaker population and would provide reliable genetic markers for molecular marker-assisted selection in the future. Meanwhile, the result established a basis for our subsequent fine mapping and related gene study.

Identification and expression of transcription factor sox2 in large yellow croaker Larimichthys crocea.

As an important transcription and pluripotency factor, Sox2 plays its functions essentially in the regulation of self-renewal and pluripotency of embryonic and neural stem cells, as well as embryogenesis, organogenesis, neurogenesis and regeneration. The data is lacking on Sox2 in large yellow croaker (Larimichthys crocea) (Lc-Sox2) which is a limitation on the generation of induced pluripotent stem cells (iPSCs). In this study, Lc-sox2 was cloned by RACE (rapid amplification of cDNA ends) and analyzed by Bioinformatics. The quantitative real-time PCR (qRT-PCR) and whole mount in situ hybridization (WISH) were used to detect the expression of Lc-sox2. The full-length cDNA sequence of Lc-sox2 is 2135 bp and encodes a 322-aa (amino acids). Lc-Sox2 possesses a highly conserved HMG-box as DNA-binding domain, maintains highly conserved with vertebrates, particularly with teleosts. In tissues, Lc-sox2 was expressed with gender difference in brain and eye (female > male), in embryos, Lc-sox2 was expressed with a zygotic type that the high level expression began to appear in the gastrula stage. The spatio-temporal expression patterns of Lc-sox2 suggested the potential involvement in embryogenesis, neurogenesis, gametogenesis and adult physiological processes of large yellow croaker. Our results contributed to better understanding of Sox2 from large yellow croaker.

Nanos3 not nanos1 and nanos2 is a germ cell marker gene in large yellow croaker during embryogenesis.

In this study, three nanos gene subtypes (Lcnanos1, Lcnanos2 and Lcnanos3) from Larimichthys crocea, were cloned and characterized. We determined the spatio-temporal expression patterns of each subtype in tissues as well as the cellular localization of mRNA in embryos. Results showed that deduced Nanos proteins have two main homology domains: N-terminal CCR4/NOT1 deadenylase interaction domain and highly conserved carboxy-terminal region bearing two conserved CCHC zinc-finger motifs. The expression levels of Lcnanos1 in testis were significantly higher than other tissues, followed by heart, brain, eye, and ovary. Nevertheless, both Lcnanos2 and Lcnanos3 were restrictedly expressed in testis and ovary, respectively. No signals of Lcnanos1 and Lcnanos2 expression were detected at any developmental stages during embryogenesis. On the contrary, the signals of Lcnanos3 were detected in all stages examined. Lcnanos3 transcripts were firstly localized to the distal end of cleavage furrow at the 2-cell stage. Subsequently, mounting positive signals started to appear in a small number of cells as the embryo developed to blastula stage and early-gastrula stage. As development proceeded, positive signals were found in the primitive gonadal ridge. These cells of Lcnanos3 positive signals implied the specification of the future PGCs at this stage. It also suggested that PGCs of croaker originate from four clusters of cells which inherit maternal germ plasm at blastula stage. Furthermore, we preliminarily analyzed the migration route of PGCs in embryos of L. crocea. In short, this study laid the foundation for studies on specification and development of germ cell from L. crocea during embryogenesis.

Molecular Characterization and Expression Profiles of Sp-uchl3 and Sp-uchl5 during Gonad Development of Scylla paramamosain.

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 from mud 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 ovary was 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.

Molecular cloning and expression of Octamer-binding transcription factor (Oct4) in the large yellow croaker, Larimichthys crocea.

Octamer-binding transcription factor 4 (Oct4) is a crucial pluripotent transcription factor in controlling pluripotency of embryonic stem cells (ESCs), formation of primordial germ cells (PGCs), early embryonic development, and gonadal germ cells. To understand the roles of Oct4 in the large yellow croaker (Larimichthys crocea) (Lc-Oct4), the full-length cDNA of Oct4 was cloned and analyzed. Lc-Oct4 includes a 104-bp 5' untranslated region (UTR), a 567-bp 3' UTR and a 1431-bp coding region encoding a protein of 476 amino acids (aa) with a predicted molecular mass (Mm) of 52.40 kDa and an isoelectric point (PI) of 6.34. Quantitative real time PCR (qRT-PCR) in tissues showed that Lc-Oct4 was only expressed in ovary. During ovarian development, the expression level in 635 days post hatching (635-dph) ovary was about 6.3-fold higher than in 270-dph and 1000-dph ovary. The in situ hybridization analysis showed that Lc-Oct4 had a high expression in the different developmental stages of oocytes with the highest level in stages II (later) and III oocytes. Lc-Oct4 was also expressed in spermatogonia and primary spermatocytes. Furthermore, the qRT-PCR analysis of Lc-Oct4 in different developmental embryos revealed that the expression from high to low was multiple-cell stage > mid-blastula stage = mid-gastrula stage, and the expression in multiple-cell stage was at less 1.6-fold higher than in other stages. Accordingly, the whole mount in situ hybridization (WISH) in embryos demonstrated that Lc-Oct4 was expressed only in early embryonic development from 2-cell stage to early-gastrula stage with the peak at 16-cell stage, but not detected in PGCs area. In conclusion, Lc-Oct4 participated in the oogenesis and early embryonic development in large yellow croaker. Overall, this study provided better understanding of Lc-Oct4 gene and lay the foundation for its function research in the large yellow croaker.

Predict the Relationship between Gene and Large Yellow Croaker's Economic Traits.

The importance of a gene's impact on traits is well appreciated. Gene expression will affect the growth, immunity, reproduction and environmental resistance of some fish, and then affect the economic performance of fish-related business. Studying the connection between gene and character can help elucidate the growth of fishes. Thus far, a collected database containing large yellow croaker (Larimichthys crocea) genes does not exist. The gene having to do with the growth efficiency of fish will have a huge impact on research. For example, the protein encoded by the IFIH1 gene is associated with the function of viral infection in the immune system, which affects the survival rate of large yellow croakers. Thus, we collected data through the published literature and combined them with a biological genetic database related to the large yellow croaker. Based on the data, we can predict new gene-trait associations which have not yet been discovered. This work will contribute to research on the growth of large yellow croakers.

Characterization and Spatiotemporal Expression of Klf4 in Large Yellow Croaker Larimichthys crocea.

As one of transcription factors and pluripotency factors, Klf4 plays a crucial role in regulation of cellular processes. In this article, we characterized Klf4 of large yellow croaker (Lc-Klf4), which encodes a 452-amino acid protein (Lc-Klf4) with three highly conserved C2H2 zinc fingers. Lc-Klf4 shares high conservative functions in teleosts with the closest relationship with Stegastes partitus. The spatiotemporal expression showed that Lc-Klf4 was expressed widely in adult tissues with gender difference as follows: brain>gill>eye>heart in female; heart>testis>gill>brain in male; male>female in heart, gill, and testis; and female>male in eye. During growth, the highest expression level of Lc-Klf4 was at 635 dph (days posthatching) in testis and at 270 dph and 635 dph in brain. Besides, Lc-Klf4 was widely and highly distributed in different developmental spermatids especially in spermatocytes. The expression of Lc-Klf4 in embryos was exhibited zygotically beginning from late gastrula stage with high level in closure of blastopore stage and appearance of optic vesicle stage. This expression pattern was supported by whole-mount in situ hybridization with high expression in back and head of late embryos. In conclusion, the spatiotemporal expression patterns of Lc-Klf4 illustrated that Klf4 involves in spermatogenesis, embryogenesis, and adult physiological processes.

Isolation, characterization, and expression of proto-oncogene cMyc in large yellow croaker Larimichthys crocea.

cMyc is a vital transcription factor that involves in the regulation of cell proliferation, growth, differentiation, and apoptosis. In the present study, cMyc in Larimichthys crocea (Lc-cMyc) was cloned and analyzed for investigating its function. The full-length cDNA of Lc-cMyc was 2089 bp encoding a 440-amino-acid protein (Lc-cMyc). Lc-cMyc had the characteristic helix-loop-helix-leucine-zipper (HLH-LZ) DNA-binding domain and highly conservative in evolution. The expression of Lc-cMyc was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization, respectively. In tissues, the gender differences of Lc-cMyc expression existed only in gonad and Lc-cMyc was extremely significantly expressed in ovary with the highest level in 635-dph ovary, especially in stages II (late) and III (early) oocytes. A certain degree of expression was examined in head kidney of both sexes and testis with high expression in spermatocyte. In embryos, Lc-cMyc was expressed at all embryonic stages. In early embryogenesis (from two-cell stage to mutiple-cell stage), Lc-cMyc was expressed very highly with a peak at two-cell stage. In late embryogenesis (from blastula stage to 1-day-post-hatching stage), the high expression of Lc-cMyc was detected as the following order: 1-day-post-hatching stage > pre-hatching stage > the appearance-of-optic-vesicles stage = mutiple-cell stage > beginning-of-heart-pulsation stage. The distribution of Lc-cMyc concentrated gradually in the back of embryos until in the head. In conclusion, the spatio-temporal expression patterns of Lc-cMyc indicated an essential role in oogenesis and embryogenesis and contributed to insight into the molecular mechanisms of regulating pluripotency in large yellow croaker.

Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion.

Characterization and expression analysis of Lc-Sox4 in large yellow croaker Larimichthys crocea.

The characterization and expression of Sox4 in large yellow croaker (Lc-Sox4) were studied in this paper. Lc-Sox4 contains a protein of 371 amino acids with a conserved high mobility group box. Quantitative real-time PCR displayed that the expression of Lc-Sox4 had tissue and gender specificity existing in brain, gonad, heart, intestine, and head kidney with male>female, in eye with female>male. During embryogenesis, Lc-Sox4 was expressed highest in one-day-post-hatching stage, next in formation-of-eye-lens stage. The expression pattern of Lc-Sox4 was different from that of Lc-Sox11a. The expression of Lc-Sox4 was significantly lower than that of Lc-Sox11a in the all tested tissues and embryonic stages except in heart, spleen, mutiple-cell, formation-of-eye-lens, and one-day-post-hatching stages (with Lc-Sox4 higher than Lc-Sox11a). There was overlapping expression between Lc-Sox4 and Lc-Sox11a in brain, gill, female eye, testis, formation-of-eye-lens stage and one-day post hatching stage. The whole mount in situ hybridization results indicated that Lc-Sox4 was expressed at all embryonic stages except 2-cell stage. The positive signals were mainly distributed in the central nervous system and notochord at one-day-post-hatching stage. In short, we first identified and analyzed the temporal and spatial expression patterns of Lc-Sox4 to elucidate its important influence on the development of nervous system, visual system and heart. We also detected the overlapping expression between Lc-Sox4 and Lc-Sox11a which may reveal the functional redundancy of them. These data would shed light on the molecular mechanism of development in large yellow croaker.

Transcriptome and analysis on the complement and coagulation cascades pathway of large yellow croaker (Larimichthys crocea) to ciliate ectoparasite Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics.

Molecular cloning, characterization and expression of Lc-Sox11a in large yellow croaker Larimichthys crocea.

Sox genes play important roles in various developmental processes such as sex determination, embryogenesis, oogenesis, neurogenesis, and larval development. In order to clarify the roles of Sox genes in the developmental process of large yellow croaker, the full-length cDNA of the Sox11a gene (Lc-Sox11a) was cloned for the first time. Bioinformatics analysis indicated that Lc-Sox11a contains a protein of 366 amino acids with a Ser-rich region, a C-terminal conserved region, and a high mobility group box. The expression of Lc-Sox11a in different tissues of both sexes and in different developmental embryonic stages revealed that Lc-Sox11a were expressed with tissue and gender specificity, of which the expression level in female was ovary>brain>eye>gill; in male was brain>testis>gill. The gender differences occurred in the brain and eye with the male brain>female brain, female eye>male eye. Moreover, the expression of Lc-Sox11a in the gonad and brain at different growth stages was detected. Significant up-regulated expression of Lc-Sox11a was found in the ovary and the male brain at 1000dph (days post hatching) compared with 270dph and 635dph. However, significant down-regulated expression of Lc-Sox11a occurred in the testis with growth. Besides, the expression of Lc-Sox11a in the female brain showed a trend of first rising then falling, with the highest peak in 635dph. The results of in situ hybridization displayed that Lc-Sox11a was widely distributed only in cytoplasm of oocytes at each stage in oogenesis. In early stage of oocytes, Lc-Sox11a was expressed weakly and evenly. As the appearance of vacuoles and synthesis of yolks, positive signals of Lc-Sox11a distributed intensively in the residual cytoplasm. In spermatogenesis, Lc-Sox11a was distributed in cytoplasm of all male germ cells except spermatozoon with spermatogonium>spermatocyte>spermatid. During embryogenesis, Lc-Sox11a was expressed in most embryonic stages, the highest expression occurred in the formation-of-eye-lens stage, closely followed by the closure-of-blastopore stage, then the beginning-of-heart-pulsation stage. The results of whole mount in situ hybridization showed that the expression of Lc-Sox11a began to increase beginning with the multiple-cell stage, with the major distribution of Lc-Sox11a in the brain and eye areas in the pre-hatching stage.

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.

Differential expression of three estrogen receptors mRNAs in tissues, growth development, embryogenesis and gametogenesis from large yellow croaker, Larimichthys crocea.

The biological activity of estrogens in target organs is mainly mediated by estrogen receptors (ERs). Herein, we addressed the isolation and expression analysis of three nuclear estrogen receptors, namely LcERα, LcERß1, and LcERß2 from Larimichthys crocea by means of SMART-RACE, qRT-PCR, and in situ hybridization. Results in different tissues showed that both LcERα and LcERß2 had the highest expression levels in female liver, followed by testis, but LcERß1 expression level was significantly higher in testis and ovary than in other tissues. Expression of LcERα and LcERß2 was significantly higher than LcERß1 in female liver, and LcERß2 was significantly higher than LcERα and LcERß1 in male liver. Moreover, we analyzed the expression of LcERs in gonad and liver at three different growth stages during the same breeding season. Significant up-regulated expression of LcERα and LcERß2 were found in female liver at 1000dph compared with at 270dph. The expression of LcERß2 was prominently higher in male liver than LcERα, LcERß1 and LcAR, while LcERß1 was lower than other receptors in male and female liver at all the three stages. In ovary, LcERα at 270dph was lower than at 635dph and 1000dph, but had no significant change in testis. The two LcERß subtypes and LcAR highly expressed in the early testis, and gradually decrease with the development of testis. In embryogenesis, a significant increase in the expression of LcERα and LcERß2 were observed after appearance of optic vesicles phase (11.8hpf). LcERß1 gradually decrease with the embryogenesis but increased dramatically at 1dph. Results of in situ hybridization showed that signals of LcERα and LcERß1 mRNA were mainly detected in Stage I-Stage IV oocytes, as well as in follicle cells around the Stage II-Stage IV and degenerated oocytes. Signals of LcERß2 were detected in the cytoplasm of Stage I and Stage II oocytes but not in the follicle cells of all oocytes stages. In parallel, LcERα and LcERß1 were detected in all cell types of spermatogenesis, but in terms of LcERß2, little or no signals were detected during spermatogenesis. Based on these results, we deduced that both LcERα and LcERß2 play a major role in mediating the physiological effects of estrogen in female liver, and LcERß2 maybe also play an important role in regulation of vitellogenesis in male liver. Differential expression of LcERs and LcAR imply their physiological functions during development and differentiation of gonad. The signals for LcERα and LcERß1 in follicle cells suggested that the follicle cell maybe an important site of estrogen action, by which estrogens exert influences on the maturation oocytes and ovulation. Furthermore, the steroid hormones produced by follicle cells may be related to the differential distributions among ER subtypes. Besides, we deduced that LcERα and LcERß1 rather than LcERß2 may play a major role in spermatogenesis of croaker. However, the differential expression of LcERß2 during gametogenesis also implicates its certain functions in mediating physiological process of estrogen action.