Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Nucleophosmin 1a translocated from nucleus to cytoplasm and facilitate GCRV replication.

To decipher the functional characterization of Nucleophosmin 1a (NPM1a) from grass carp (Ctenopharyngodon idellus) (CiNPM1a), its cDNA was cloned and bioinformatic analysis were conducted. The full-length cDNA sequence of CiNPM1a is 1732 bp, which encodes 307 amino acids. CiNPM1a contains conserved domains of Nucleoplasmin domain, NPM1-C terminal domain, as well as nuclear localization signals, nuclear export signal (NES) and acid patches. There are 52 and 20 consensus amino acids exist in the Nucleoplasmin domain and the NPM1-C terminal domain of all blasted species. In addition, the immune function of CiNPM1a were analyzed. The Ciirf7, Ciifn1 and Ciifn2 transcription was inhibited, whereas the vp2 and vp7 expressions were enhanced in CiNPM1a overexpressing cells after GCRV infection (P < 0.05). Moreover, the Ciirf7, Ciifn1 and Ciifn2 mRNA levels were significantly up-regulated, but the vp2 and vp7 expressions were significantly down-regulated in CiNPM1a knockdown cells after infection. This indicated that CiNPM1a played negative roles in the induction of Type I IFN reaction and thus the GCRV replication. Finally, the NES domain that affect the nucleous-cytoplasm shuttle and the replication of GCRV were investigated. The deletion of NES1 and NES(1 + 2+3) absolutely limited the transloacation of CiNPM1a△NES1 protein and CiNPM1a △NES(1 + 2+3) protein to cytoplasm after infection, and the deletion of NES2 resulted in partially limitation of protein shuttle. In general, Ciirf3, Ciirf7, Ciifn1 and Ciifn2 expressions were enhanced in the CiNPM1a△NES1, CiNPM1a△NES2 and CiNPM1a△NES3 overexpression groups, and the deletion of functional domains in CiNPM1a led to significantly reduction of the vp2 and vp7 replication. The results indicated that CiNPM1a may be a target molecular for GCRV infection curation, and a candidate molecular for resistance strain breeding of grass carp.

Astragalus polysaccharide improves the growth, meat quality, antioxidant capacity and bacterial resistance of Furong crucian carp (Furong carp♀ × red crucian carp♂).

To evaluate the functional effects of APS (Astragalus polysaccharide) on Furong crucian carp, APS-supplemented diets (0.00 %, 0.05 %, 0.10 % and 0.15 %) were prepared and utilized in feeding experiment. The results showed that the 0.05 % APS group has the highest weight gain rate and specific growth rate, and the lowest feed coefficient rate. In addition, 0.05 % APS supplement could improve muscle elasticity, adhesiveness and chewiness. Moreover, the 0.15 % APS group had the highest spleen-somatic index and the 0.05 % group had the maximum intestinal villus length. 0.05 % and 0.10 % APS addition significantly increased T-AOC and CAT activities while MDA contents decreased in all APS groups. The plasma TNF-α levels in all APS groups significantly increased (P<0.05), and the 0.05 % group showed the highest TNF-α level in spleen. In APS addition groups, the tlr8, lgp2 and mda5 gene expressions were significantly elevated, while xbp1, caspase-2 and caspase-9 expressions decreased in uninfected and A. hydrophila-infected fish. Finally, higher survival rate and slower disease outbreak rate were observed in APS-supplemented groups after being infected by A. hydrophila. In conclusion, Furong crucian carp fed by APS-supplemented diets possesses elevated weight gain rate and specific growth rate, and improved meat quality, immunity and disease resistance.

Enhancing Electromechanical Properties of PZT-Based Piezoelectric Ceramics by High-Temperature Poling for High-Power Applications.

Defect engineering is a proven method to tune the properties of perovskite oxides. In demanding high-power piezoelectric ceramic applications, acceptor doping is the most effective method to harden ceramics, but it inevitably degrades the ceramics' electromechanical properties. Herein, a poling method based on acceptor doping, namely, high-temperature poling, is implemented by applying an electric field above the Curie temperature for poling to achieve a balance of the properties of piezoelectric coefficient d33 and mechanical quality factor Qm. After high-temperature poling, the piezoelectric property of 0.6 mol % Mn-doped Pb0.92Sr0.08(Zr0.533Ti0.443Nb0.024)O3 is d33 = 483 pC/N and Qm = 448. Compared with the traditional poling, the piezoelectric coefficient d33 of the high-temperature poling ceramics increased by approximately 40%, and Qm also increased by nearly 18%. Therefore, high d33 and Qm were exhibited by our PZT piezoelectric ceramics. Rayleigh's law analysis, XRD, and transmission electron microscopy analysis show that, after high-temperature poling, the considerably increased d33 is related to the large increase in the reversible domain wall motion in the intrinsic effect, while the slightly increased Qm is related to the inhibited irreversible domain wall motion in the extrinsic effect. This study reports a method for high-temperature poling and provides insights into the design of high-power piezoelectric ceramics with high d33 and Qm.

Grass carp IL-20 binds to IL-20R2 but induces STAT3 phosphorylation via IL-20R1.

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and has a variety of biological functions in tissue homeostasis and regulation of host immune defenses. It signals through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptor) and a subunit with a short intracellular domain (R2 type receptor). In this study, the R1 type receptor (CiIL-20R1/CRFB8) and the R2 type receptor (CiIL-20R2/CRFB16) were identified in grass carp Ctenopharyngodon idella. Expression analysis revealed that IL-20R2 was highly expressed in the gills and skin in healthy fish. Infection with Flavobacterium columnare resulted in the downregulation of both receptors in the gill at 48 and 72 h, whilst infection with grass carp reovirus induced their expression in the head kidney and spleen at 72 h. In the primary head kidney leucocytes, the expression levels of IL-20R1 and IL-20R2 were decreased after stimulation with 250 ng/mL IL-1ß but not affected by IFN-γ. Co-immunoprecipitation analysis showed that CiIL-20R2/CRFB16 but not CiIL-20R1/CRFB8 bound to CiIL-20L. Furthermore, it was shown that CiIL-20R1/CRFB8 was responsible for activating the phosphorylation of STAT3, whilst CiIL-20R2/CRFB16 was not involved. Structural modeling analysis showed that key residues involved in the interaction between IL-20 and receptors were highly conserved between grass carp and humans, suggesting that the signal transduction and functions of IL-20/IL-20R axis are evolutionarily conserved.

Functional and expression analysis reveals the involvement of integrin αvß3 in antiviral immunity of grass carp (Ctenopharyngodon idella).

Integrins are α-ß heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvß3 (GCαvß3) originally assembled by αv (GCαv) and ß3 (GCß3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCß3 shared high similarity (75.2-95.1%) and identity (58.6-90.7%) with their homologs from other vertebrates. Both GCαv and GCß3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCß3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCß3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvß3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvß3 did. In addition, both GCαv and GCß3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvß3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.

Sequence, Expression, and Anti-GCRV Function of the Ferritin from the Grass Carp, Ctenopharyngodon idellus.

Ferritin possesses an immune function to defend against pathogen infection. To elucidate the immunity-protecting roles of ferritin from Ctenopharyngodon idellus (Ciferritin) against virus infection, the cDNA and promoter sequences of Ciferritin were determined, and the correlations between Ciferrtin expressions and promoter methylation levels were analyzed. In addition, the functional role of Ciferrtin on GCRV (grass carp reovirus) infection was assessed. The full-length cDNA of Ciferritin is 1053 bp, consists of a 531 bp open-reading frame, and encodes 176 amino acids. Ciferritin showed the highest sequence identity with the ferritin middle subunit of Mylopharyngodon piceus (93.56%), followed by the subunits of Megalobrama amblycephala and Sinocyclocheilus rhinocerous. Ciferritin contains a conserved ferritin domain (interval: 10−94 aa), and the caspase recruitment domain (CARD) and Rubrerythrin domain were also predicted. In the spleen and kidney, significantly higher Ciferritin expressions were observed at 6, 12, 24, or 168 h post GCRV infection than those in the PBS injection group (p < 0.05). The Ciferrtin expression level in the progeny of maternal-immunized grass carp was significantly higher than that in the progeny of common grass carp (p < 0.05). Ciferritin promoter methylation level in the progeny from common grass carp was 1.27 ± 0.15, and in the progeny of the maternal-immunized group was 1.00 ± 0.14. In addition, methylation levels of "CpG9" and "CpG10" loci were significantly lower in the progeny of maternal-immunized fish than those in the common group. Except for the "CpG5", methylation levels of all other detected "CpG" loci negatively correlated with Ciferritin expression levels. Furthermore, the total methylation level of "CpG1−10" negatively correlated with the Ciferritin expressions. The Ciferritin expression level was significantly up-regulated, and the VP7 protein levels were significantly reduced, at 24 h post GCRV infection in the Ciferritin over-expression cells (p < 0.05). The results from the present study provide sequence, epigenetic modification and expression, and anti-GCRV functional information of Ciferritin, which provide a basis for achieving resistance to GCRV in grass carp breeding.

Fish TNF and TNF receptors.

Tumor necrosis factors (TNFs) are a group of cytokines that play critical roles in regulating a diverse range of physiological processes in vertebrates. TNFs function by activating a large number of structurally related receptors, leading to TNF mediated biological processes which are evolutionarily conserved. Fish have a much diversified TNF family, partly due to the whole genome duplication events which have occurred in this lineage, providing an excellent model to investigate the neo- and sub-functionalised properties of TNF superfamily. Fish possess most of the TNFs and receptors found in mammals and also some homologues exclusively present in fish. It seems that TNFSF4 (OX40), TNFSF7 (CD27) and TNFSF8 (CD30) and their cognate receptors are absent in teleosts. It has been shown that fish viruses are able to produce TNFR homologues to establish infection by manipulating the host immune system. Understanding the roles of TNFSFs in fish immune defence and the pathogenesis of fish diseases will provide insights into the functions of TNFSFs from an evolutionary perspective and better strategies for improving fish health and welfare in aquaculture. This review summarises recent advances in the study offish TNF biology and focuses on the molecular properties and immunological functions of the TNF and TNFR superfamily.

GH and GHR gene cloning, expression and their associations with growth-related traits of the barbel chub (Squaliobarbus curriculus).

Growth hormone (ScGH) and growth hormone receptor (ScGHR) genes from the barbel chub (Squaliobarbus curriculus), in addition to their cDNAs, were cloned. The associations between their mRNA expression levels and growth-related traits were analysed, and the differences in the levels of expression of growth regulation-related genes between the largest and smallest individuals were compared. The full-length 1182-bp cDNA of ScGH contained a 633-bp open reading frame (ORF), and the length of the gene had 2492 bp. The full-length 2825-bp cDNA of ScGHRa contained a 1818-bp ORF, and the gene had 6970 bp. The full-length 2822-bp cDNA of ScGHRb contained a 1737-bp ORF, and the gene had 8149 bp. Quantitative real-time PCR revealed that ScGH was only expressed in the pituitary. ScGHRa was expressed predominantly in muscle, and the expression level of ScGHRb was the highest in the liver. The ScGHRa mRNA levels in the muscle were significantly negatively correlated with the caudal peduncle length. However, no correlation between growth-related traits and ScGH and ScGHRb expression levels were found. Pituitary ScGH, liver GHRb and liver insulin-like growth factor I (igf-1) expression levels were significantly higher in the largest individuals than those in the smallest S. curriculus individuals. Contrarily, the largest individuals had significantly lower expression levels of muscle igf-1 and liver myog than the smallest individuals. Overall, our results provide novel molecular information for growth-regulation study of S. curriculus.

Characterization and immune function of the interferon-ß promoter stimulator-1 in the barbel chub, Squaliobarbus curriculus.

To elucidate the immunity-protecting role of the interferon-ß promoter stimulator-1 (ScIPS-1) in barbel chub Squaliobarbus curriculus, the full-length cDNA of ScIPS-1 was cloned and expression levels in response to stimulation were investigated. In addition, the function of ScIPS-1 and its domains were analyzed. The full-length cDNA of ScIPS-1 is 2524 bp and encodes 601 aa. The N-terminal caspase activation and recruitment domain, central proline-rich domain, C-terminal transmembrane domain, C2HC-zinc finger, and Cwf21 domains were identified. The mRNA level of ScIPS-1 was the highest in the kidney, whereas the highest protein level was observed in the liver. The ScIPS-1 expressions were significantly up-regulated after lipopolysaccharide and poly I:C treatment. The ScIPS-1 protein level was up-regulated at 12 h in the head kidney and was up-regulated at 12 h and then down-regulated from 12 to 48 h in the liver after grass carp reovirus (GCRV) infection. The CiIFN and CiMx transcription levels were significantly enhanced in pEGFP-C1-IPS-1 and pcDNA3.1-ΔCwf21 overexpressing cells after GCRV infection. The results indicate that ScIPS-1 may function in the immune response against pathogens and provide a basis for achieving resistance to diseases in fish breeding.

Sequence and expression analysis of the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 from the barbel chub Squaliobarbus curriculus.

MDA5 is a cytoplasmic viral double-stranded RNA recognition receptor that plays a pivotal role in the aquatic animal innate immune system. To decipher the role of MDA5 of Squaliobarbus curriculus (ScMDA5) in the immune response, full-length cDNA of ScMDA5 was cloned using the RACE technology, mRNA and protein expression levels of ScMDA5 signalling pathway members in response to stimulation were detected and effects of overexpression of ScMDA5 on the immune response were investigated. ScMDA5 comprises 3597 bp and is composed of an open reading frame (2958 nucleotides long) that translates into a putative peptide of 985 amino acid residues. ScMDA5 possesses two N-terminal caspase-recruiting domains, DEAD-like helicases superfamily, helicase superfamily C-terminal and RIG-I_C-RD domains, and differences in these domains among species were mainly observed with respect to their length and location. ScMDA5 was closely clustered with those of Carassius auratus, Ctenopharyngodon idellus and Mylopharyngodon piceus. ScMDA5 transcripts were most abundant in the spleen and the lowest in the liver. Expression levels of ScMDA5 in healthy tissues were significantly correlated with those of ScIRF3, ScIRF7 and ScIFN. Besides, mRNA expression levels of ScIRF3 were significantly correlated with those of ScIRF7 (0.956, P < 0.01). Expression level changes, including downregulation, upregulation and initial upregulation followed by downregulation, were found in ScMDA5 signalling pathway molecules in tissues after grass carp reovirus infection. Protein levels of ScMDA5 were the highest in the liver and the lowest in the spleen in detected healthy tissues. Overexpression of ScMDA5 led to significantly enhanced CiIRF7 and CiMx transcription in grass carp ovary cells (P < 0.05). The results of this study helped to clarify the role of ScMDA5 in the immune reaction against grass carp reovirus and provided fundamental information for fish breeding to achieve strong resistance to infection.

Molecular cloning and expression analysis of coagulation factor VIII and plasminogen involved in immune response to GCRV, and immunity activity comparison of grass carp Ctenopharyngodon idella with different viral resistance.

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella.

Molecular characterization and expression of TLR7 and TLR8 in barbel chub (Squaliobarbus curriculus): Responses to stimulation of grass carp reovirus and lipopolysaccharide.

The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus, designated as ScTLR7 and ScTLR8, were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8, MyD88, and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P: 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P: 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways.

Involvement of interferon regulatory factor 3 from the barbel chub Squaliobarbus curriculus in the immune response against grass carp reovirus.

The barbel chub Squaliobarbus curriculus is an important commercial fish species in China, and has shown significant resistance to grass carp reovirus (GCRV). In this study, the cDNA sequence of interferon regulatory factors 3 (IRF3) from Squaliobarbus curriculus, designated as ScIRF3, was cloned, and its effect against GCRV was investigated. The full-length 1837 base pair (bp) cDNA of ScIRF3 contained a complete open reading frame of 1374 bp and encoded a putative polypeptide of 457 amino acid residues. The ScIRF3 protein contained conserved domains, including an N-terminal DNA-binding domain, a C-terminal IRF association domain, and a serine-rich domain. Phylogenetic analysis showed that ScIRF3 was closely clustered with IRF3s from Carassius auratus and Ctenopharyngodon idellus. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScIRF3 in Squaliobarbus curriculus were the highest in the spleen and lowest in the muscle. After GCRV infection, expression levels of both ScIRF3 and type I interferon (IFN) were initially up-regulated and subsequently down-regulated in the spleen and intestine. Correlation analysis showed that the expression level of type I IFN is significantly positively correlated with that of ScIRF3 (Pearson correlation coefficient: 0.883, P: 0.004) in the intestine. The expression level of type I IFN was also significantly up-regulated and the GCRV titer was significantly decreased (P < .05) in GCRV-infected ScIRF3-overexpressing Ctenopharyngodon idellus kidney cells. These results indicate that ScIRF3 may play a role in the type I IFN immune response against GCRV in Squaliobarbus curriculus and can also inhibit GCRV replication in Ctenopharyngodon idellus kidney cells.

Molecular cloning and functional characterization of interferon regulatory factor 7 of the barbel chub, Squaliobarbus curriculus.

The interferon regulatory factor 7 (IRF7) is a critical regulator of type-I interferon-dependent immune reaction that defense against virus. To investigate the antiviral function of IRF7 of barbel chub Squaliobarbus curriculus (ScIRF7), the cDNA of ScIRF7 was cloned and characterized. The full length cDNA of ScIRF7 was 1870 bp, consisted of 41 bp 5'-UTR, 560 bp 3'-UTR and a 1269 bp open reading frame (ORF). The ORF encoded 423 amino acids with a molecular weight of 49.426 KDa and a theoretical isoelectric point of 5.71. The putative ScIRF7 protein possesses typical domains of IRF family including a conserved N-terminal DBD-binding domain (DBD), a C-terminal IRF association domain and a serine-rich domain. In the DBD, four tryptophans were found to be highly conserved among all species, whilst in another conserved tryptophan site of mammals, the corresponding amino acids were methionine for fishes. The expression level of ScIRF7 was highest in the spleen and lowest in the liver. The expression level of IFN-ß was highest in the gill and lowest in the liver. After GCRV infection, expression levels changes of ScIRF7 showed an overall tendency of firstly up-regulation and then down-regulation in the spleen and the gill; and expression levels of ScIRF7 in peripheral blood lymphocyte at 24 h post-infection was highest among all time points. In pEGFP-ScIRF7 overexpressing cells, the mRNA level of ScIRF7 was firstly up-regulation and then down-regulation; and the expression of IFN-ß was significantly up-regulated at 12 h post-infection than that of control group (P < 0.05), which was significantly higher than those in pEGFP-N1 overexpressing cells. The results indicated that ScIRF7 may play a key role in immune responses of barbel chub Squaliobarbus curriculus against GCRV and may also functions in the Ctenopharyngodon idellus kidney cells.

Characterization of the distal-less homologue gene, PfDlx, involved in regulating the expression of Pif in the pearl oyster, Pinctada fucata.

Distal-less (Dlx) homeobox transcription factors play an important role in regulating various aspects of vertebrate biology. In vertebrates and invertebrates, distal-less is a highly conserved and well-studied transcription factor. In pearl oyster, we have identified a homologue of this gene, Dlx, and cloned the full-length cDNA. Genomic structure analysis revealed that PfDlx genomic DNA contained three exons and two introns. Their deduced amino acid sequences all showed the highest identity with homologues in Crassostrea gigas. Analyses of PfDlx mRNA in tissues and developmental stages showed high expressions in gonad, polar body stage, 2-4 cells and 32 cells. After shell notching, the changes in expression of Dlx shows that it reached a maximum at 24h. In co-transfection experiments, PfDlx significantly activates reporter constructs containing a Pif promoter. Through using RNAi techniques, we demonstrated that down-regulation of Dlx in P. fucata did not significantly disrupt the development of the nacreous layer in scanning electron microscopy, but it significantly down-regulated the expression of Pif gene. Thus, our work suggests that PfDlx might participate in regulating the expression of the Pif gene in the pearl oyster.

DNA methylation is associated with expression level changes of galectin gene in mantle wound healing process of pearl oyster, Pinctada fucata.

Galectin is one important member of pattern recognition proteins that plays a pivotal role in regulating innate immune response of invertebrates. In this study, we cloned the promoter sequence of a tandem-repeat galectin from the pearl oyster Pinctada fucata (P. fucata). The quantitative real-time PCR analysis revealed that galectin mRNA expression in mantle tissues were firstly up-regulated from time points of 2 h-24 h, and then down-regulated from time points of 24 h-168 h after mantle injury. The genome methylation level of mantle tissue was inversely related to galectin mRNA expression (Pearson correlation: -0.554, P: 0.154). The "CpG4-6" methylation level in promoter region of galectin was significant positive correlated with the mRNA expression (Pearson correlation: 0.313, P: 0.049). The results indicated that galectin gene may be involved in immune response in mantle wound healing process of P. fucata, and DNA methylation may be a regulation factor of gene expression.

Genetic mapping and QTL analysis of growth-related traits in Pinctada fucata using restriction-site associated DNA sequencing.

The pearl oyster, Pinctada fucata (P. fucata), is one of the marine bivalves that is predominantly cultured for pearl production. To obtain more genetic information for breeding purposes, we constructed a high-density linkage map of P. fucata and identified quantitative trait loci (QTL) for growth-related traits. One F1 family, which included the two parents, 48 largest progeny and 50 smallest progeny, was sampled to construct a linkage map using restriction site-associated DNA sequencing (RAD-Seq). With low coverage data, 1956.53 million clean reads and 86,342 candidate RAD loci were generated. A total of 1373 segregating SNPs were used to construct a sex-average linkage map. This spanned 1091.81 centimorgans (cM), with 14 linkage groups and an average marker interval of 1.41 cM. The genetic linkage map coverage, Coa, was 97.24%. Thirty-nine QTL-peak loci, for seven growth-related traits, were identified using the single-marker analysis, nonparametric mapping Kruskal-Wallis (KW) test. Parameters included three for shell height, six for shell length, five for shell width, four for hinge length, 11 for total weight, eight for soft tissue weight and two for shell weight. The QTL peak loci for shell height, shell length and shell weight were all located in linkage group 6. The genotype frequencies of most QTL peak loci showed significant differences between the large subpopulation and the small subpopulation (P<0.05). These results highlight the effectiveness of RAD-Seq as a tool for generation of QTL-targeted and genome-wide marker data in the non-model animal, P. fucata, and its possible utility in marker-assisted selection (MAS).

Molecular cloning, characterization and expression analysis of trypsin-like serine protease from triangle-shell pearl mussel (Hyriopsis cumingii).

Trypsin-like serine protease (TLS) is ubiquitous in animals and plays a number of diverse roles, including dietary protein digestion, hemolymph coagulation, antimicrobial activity and immune responses, among others. This study reports the isolation of a 1048 bp full-length cDNA sequence of TLS from triangle-shell pearl mussel (Hyriopsis cumingii), including a 12 bp 5' UTR (untranslated region), a 172 bp 3' UTR, and an open reading frame (ORF) of 864 bp by rapid amplification of cDNA ends (RACE). Bioinformatic analysis shows that the gene belongs to the trypsin-like serine protease superfamily, and contains a 15 residues N-terminal signal peptide and a conserved C-terminal domain. In comparison to other serine proteases, the catalytic triad were identified as His-98, Asp-149, and Ser-240. Quantitative real-time PCR (qPCR) showed a broad expression of the TLS gene in ten tested tissues. Time-course expression analysis demonstrated that the expression level of the TLS mRNA was significantly up-regulated in eight tested tissues (liver, intestine, gill, heart, axe foot, adductor muscle, kidney and gonad), but down-regulated in mantle and stomach after Aeromonas hydrophila injection. This is one of the results indicate that TLS may be involved in innate defense reactions against A. hydrophila in triangle-shell pearl mussel.