Transcriptomic analysis of gills provides insights into the molecular basis of molting in Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) is an economically important freshwater aquaculture species and is a model species for research on the mechanism of molting. This study aimed to identify important candidate genes associated with the molting process and to determine the role of gills in the regulation of molting with the help of transcriptomic analysis. The transcriptomes of crabs at different molting stages-postmolt (PoM), intermolt (InM), premolt (PrM) and ecdysis (E)-were de novo assembled to generate 246,232 unigenes with a mean length of 851 bp. A total of 86,634 unigenes (35.18% of the total unigenes) were annotated against reference databases. Significantly upregulated genes were identified in postmolt compared to intermolt (1,475), intermolt compared to premolt (65), premolt compared to ecdysis (1,352), and ecdysis compared to postmolt (153), and the corresponding numbers of downregulated genes were 1,276, 32, 1,573 and 171, respectively. Chitin synthase, endochitinase, chitinase A, chitinase 3, chitinase 6 and chitin deacetylase 1 were upregulated during the postmolt and ecdysis stages, while phosphoglucomutase 3 (PGM3), glucosamine 6-phosphate deaminase (GNPDA) and glucosamine glycoside hydrolase (nagZ) were upregulated during the intermolt and premolt stages compared to the other stages. The upregulated genes were enriched in several lipid-related metabolic pathways, such as "fatty acid elongation", "glycerophospholipid metabolism" and "sulfur metabolism". Meanwhile, three signaling pathways, including the "phosphatidylinositol signaling system", the "calcium signaling pathway" and the "GnRH signaling pathway" were also enriched. Tetraspanin-18, an important effector gene in the lysosomal pathway involved in cell apoptosis, up-regulate with the beginning of molting (in premolt stage) and reach the top in the ecdysis stage, and barely expressed in the intermolt stage. The expression variations in the tetraspanin-18 gene indicated that it may play an important role in the beginning of molting cycle, which might be regulated by the stress of salinity. This study revealed that the gills could participate in chitin degradation, in reestablishment of the exoskeleton and the signaling process. Based on transcriptomic analysis of the gills, we not only explored novel molecular mechanisms of molting in E. sinensis but also acquired foundational genetic data for E. sinensis.

Functional characterization of purinergic receptor P2Y14 in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Extracellular nucleotides and nucleotide sugars are important danger-associated signaling molecules that play critical roles in regulation of immune responses in mammals through activation of purinergic receptors located on the cell surface. However, the immunological role of extracellular UDP-glucose-activated P2Y14 receptor (P2Y14R) in fish still remains unknown. In this study, we identified and characterized a P2Y14R paralog in the Japanese flounder (Paralichthys olivaceus). The mRNA transcripts of P2Y14R are detected in all examined Japanese flounder tissues. Compared with the UDP-activated P2Y6 receptor, however, P2Y14R gene is highly expressed in Japanese flounder head kidney macrophages (HKMs). In addition, P2Y14R is significantly upregulated following inflammatory stimulation with LPS and poly (I:C) in the HKMs, suggesting a role of P2Y14R in response to inflammation in fish. Furthermore, activation of P2Y14 receptor with its potent and selective agonist MRS 2905 resulted in a decreased expression of LPS-induced pro-inflammatory cytokine IL-1beta gene in the HKMs. In contrast, inhibition of P2Y14 receptor activity or down-regulation of the endogenous expression of P2Y14R by small interfering RNA significantly upregulates the LPS-induced pro-inflammatory cytokine IL-1beta gene expression in the HKMs, demonstrating that P2Y14R is involved in inflammation regulation in fish. Moreover, stimulation of the Japanese flounder HKMs with UDP-glucose evoked a rapid increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent manner, indicating the involvement of P2Y14R in activation of ERK1/2 signaling in fish immune cells. Taken together, we demonstrated that the inducible P2Y14R plays an important role in regulation of fish innate immunity.

A novel complement C3 like gene (Lv-C3L) from Litopenaeus vannamei with bacteriolytic and hemolytic activities and its role in antiviral immune response.

As a core component of the complement system, complement component 3 (C3) plays a central role in the opsonization of pathogens, immune defense and immune regulation in the mammalian for its activation is required to trigger classical as well as alternative complement pathways. However, the molecular mechanism underlying C3 activation in invertebrates remains unknown. Several C3 genes have been characterized in invertebrates but very few in crustacean. To understand the molecular characterization and immunological functions of shrimp C3, we characterized a novel complement C3 like gene (designated Lv-C3L) with full-length cDNA sequence identified from pacific white shrimp Litopenaeus vannamei in the present study. The full length cDNA of Lv-C3L sequence was 4769 bp (GenBank accession number: MH638255) containing a 4077 bp open reading frame (ORF), which encodes 1358 amino acids contained a putative signal peptide of 17 amino acids. Six model motifs of C3 were found in Lv-C3L including typical A2M domain, a highly conserved thioester region (GCGEQ) and proteolytic cleavage site of ANATO. In addition to typical conservative domains, Lv-C3L also contains a particular GLN-rich region which might be involved in the protein interaction and transcriptional activation. The transcripts of Lv-C3L were mainly detected in hemocytes and gill which might be involved in defense response. At 36 h post V.parahaemolyticus and B.thuringensis infection, the expression level of Lv-C3L gene in hemocytes were significantly upregulated. At 48 h and 72 h post WSSV infection, the expression level of Lv-C3L gene in hemocytes and gill were significantly upregulated. These results indicated that Lv-C3L gene play a pivotal role in innate immune responses to the WSSV and G+/G- bacterial infection. The obvious immune function of Lv-C3L was described as an effective membrane rupture in bacteriolytic and hemolytic activities on V.parahaemolyticus, V.anguillarum and rabbit erythrocytes. Combining with WSSV copy number, WSSV-VP28 gene expression profile and shrimp cumulative mortality analysis, RNAi knockdown of Lv-C3L gene could obviously promote the in vivo propagation of WSSV in shrimp. This is the first report in crustaceans that Lv-C3L, as a key complement like components, is involved in shrimp antiviral immune response. It is speculated that complicated complement response cascade may exist in shrimp. These results collectively indicated that the complement pathway in shrimp might play an important protective role against pathogenic infection and activation of complement pathway including C3 could restrict the propagation of WSSV.

Identification of Litopenaeus vannamei BiP as a novel cellular attachment protein for white spot syndrome virus by using a biotinylation based affinity chromatography method.

White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target.

Functional characterization of purinergic P2Y2 and P2Y12 receptors involved in Japanese flounder (Paralichthys olivaceus) innate immune responses.

G-protein-coupled P2Y receptors activated by extracellular nucleotides play important roles under different physiological and pathophysiological conditions in mammals. To investigate the immunological relevance of P2Y receptors in fish, we identified and characterized the P2Y2 and P2Y12 receptors in Japanese flounder Paralichthys olivaceus. The P. olivaceus P2Y2 and P2Y12 receptors harbor seven transmembrane domains but share only 24% sequence identity. Real-time PCR analysis revealed the constitutive but unequal mRNA expression pattern of P2Y2R and P2Y12R in normal Japanese flounder tissues with the dominant expression of P2Y2R in head kidney and blood and P2Y12R in hepatopancreas. In addition, the expression of P2Y2 and P2Y12 receptors was markedly modulated by PAMPs stimulation and Edwardsiella tarda infection. Furthermore, blockage of P2Y12R potently increased ADP-activated pro-inflammatory cytokine IL-1beta gene expression in the head kidney macrophages (HKMs). Moreover, inhibition of P2Y2 and P2Y12 receptor activity with their respective potent antagonists significantly altered some of the LPS-induced pro-inflammatory cytokine gene expression in the HKMs. However, blockade of P2Y12R did not affect the poly(I:C)-induced pro-inflammatory cytokine gene expression examined in the HKMs. Collectively, we have for the first time reported the role of purinergic P2Y2 and P2Y12 receptors in fish innate immunity. Our findings have also addressed the importance of extracellular ATP and its metabolites in fish innate immune responses.

Comparative analysis of dual specificity protein phosphatase genes 1, 2 and 5 in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder.

Characterization of Japanese flounder (Paralichthys olivaceus) Caspase1 involved in extracellular ATP-mediated immune signaling in fish.

Caspase1 is a member of inflammatory Caspases that play important roles in the innate immune system. Although several teleost caspase1 genes have been identified, their partner proteins and implication in extracellular ATP-mediated immune signaling in fish are still very limited. Here we identified and characterized a caspase1 gene, named JfCaspase1, from Japanese flounder Paralichthys olivaceus. JfCaspase1 mRNA was constitutively expressed in all examined normal tissues with high expression in skin and gills and moderate expression in the enriched Japanese flounder head kidney macrophages (HKMs) and peripheral blood leukocytes (PBLs). JfCaspase1 was initially down-regulated but significantly up-regulated at the later stage upon LPS and poly(I:C) challenges in the HKMs. JfCaspase1 was also up-regulated in the Japanese flounder immune-related tissues including head kidney, gill and spleen by bacterial challenge with Edwardsiella tarda. JfCaspase1 protein is comprised of 384 amino acid residues with a calculated molecular mass of 43.75 kDa and is phylogenetically close to fish Caspase1 proteins. JfCaspase1 was co-immunopercipitated with Japanese flounder apoptosis-associated speck-like protein (ASC) when co-expressed in HeLa cells, suggesting that there is a potential interaction between the two proteins. In addition, we showed that extracellular ATP, a potent signaling molecule in activating innate immune response, rapidly up-regulates JfCaspase1 expression and enhances its enzymatic activity both in the HKMs and PBLs. Our findings indicated that the inflammatory JfCaspase1 interacted with ASC protein is implicated in the extracellular ATP-mediated immune signaling in fish.

Expression and functional characterization of vitronectin gene from Japanese flounder (Paralichthys olivaceus).

Vitronectin (Vtn) is a multifunctional protein that plays significant roles in cell adhesion, migration, spreading and survival, and in the regulation of membrane attack complex formation and the terminal pathway of complement activation in innate immune response. However, the expression and immune significance of Vtn in fish remains largely unknown. In order to understand the involvement of Vtn in fish innate immune response, here we cloned and characterized a full-length Vtn ortholog cDNA, termed PoVtn, from Japanese flounder Paralichthys olivaceus. The deduced PoVtn protein is comprised of 438 amino acids with a 19-amino-acid signal peptide sequence (1Met-19Ala) at the N-terminus. Protein domain analysis revealed that PoVtn possesses a conserved N-terminal somatomedin B domain followed by a conserved RGD motif and four haemopexin-like domains. Sequence analysis revealed that PoVtn has two potential glycosylation sites and shares 44-74% sequence identity with other teleost Vtn proteins. PoVtn mRNA was ubiquitously distributed in all examined normal tissues and showed the highest expression in Japanese flounder hepatopancreas tissue. PoVtn expression was induced by LPS and poly(I:C) challenges in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) and shows a pathogen-associated molecular pattern- and cell type-dependent manner. The expression of PoVtn was also modulated by bacterial challenge with Edwardsiella tarda in Japanese flounder immune-related tissues including head kidney, gill and spleen. Furthermore, overexpression of PoVtn in Japanese flounder FG-9307 cells significantly attenuated the LPS- and poly(I:C)-induced proinflammatory cytokines IL-1beta and TNF-alpha gene expression. Taken our findings together, we for the first time characterized Vtn gene expression in response to inflammatory stimuli in fish. Our results suggested a potential role of PoVtn in regulating fish innate immunity.

Identification and functional analysis of dual-specificity MAP kinase phosphatase 6 gene (dusp6) in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Dual-specificity phosphatase 6 (Dusp6) is a member of mitogen-activated protein kinase (MAPK) phosphatases that play crucial roles in regulating MAPK signaling and immune response. The immunological relevance of Dusp6 in fish, however, remains largely uncharacterized. In the present study, a full-length Japanese flounder dusp6 cDNA ortholog, termed PoDusp6, was identified and characterized from Paralichthys olivaceus. The deduced PoDusp6 protein is comprised of 383 amino acids with a conserved N-terminal regulatory rhodanese homology domain and a C-terminal catalytic domain. Immunofluorescence microscopy revealed that PoDusp6 protein is mainly localized in cytoplasm. Sequence analysis indicates that PoDusp6 is highly conserved (>70% identity) throughout the evolution from teleost to mammals. In unstimulated conditions, PoDusp6 mRNA was present in all examined tissues and showed the highest expression in Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments revealed that the expression of PoDusp6 was down-regulated at the early stage after LPS and poly(I:C) stimulations but significantly up-regulated at the later stage in the HKMs. The similar expression pattern was also observed in the Japanese flounder immune-related tissues including head kidney, gill and spleen upon bacterial challenge with Edwardsiella tarda. Overexpression of PoDusp6 in Japanese flounder FG-9307 cells led to a significant down-regulation of proinflammatory cytokine genes IL-1beta, TNF-alpha and IFN-gamma, and antiviral gene Mx. Interestingly, inhibition of Dusp6 activity also down-regulated the LPS-induced IL-beta gene expression but did not affected on the LPS-induced IFN-gamma and TNF-alpha expression in the HKMs. Our findings suggest that the expression of PoDusp6 is modulated by immune stimuli and PoDusp6 may act as an essential modulator in fish inflammatory response.

Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP release in fish.

Cloning and characterization of apoptosis-associated speck-like protein containing a CARD domain (ASC) gene from Japanese flounder Paralichthys olivaceus.

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. However, few studies have been performed in lower vertebrates such as in teleost. Here we identified and characterized a novel ASC gene (namely PoASC) from Japanese flounder Paralichthys olivaceus. The complete cDNA sequence of PoASC contains a 22 bp 5'-untranslated sequence, a 612 bp open reading frame, and a 438 bp 3'-untranslated sequence. The deduced PoASC protein is comprised of 203 amino acids with a conserved N-terminal PYD domain and a C-terminal CARD domain and shows 35-62% sequence identity with other vertebrate ASC proteins. PoASC mRNA transcripts was detected in various Japanese flounder tissues and is dominantly expressed in hepatopancreas. Oligomeric speck-like structures were observed when PoASC was exogenously expressed in Japanese flounder FG-9307 cells. Immune challenge experiments revealed that PoASC gene expression was significantly induced in the Japanese flounder head kidney macrophages and peripheral blood leukocytes by the canonical TLR ligands LPS, Poly(I:C) and zymosan stimulations. In addition, the induction of PoASC was also observed in Edwardsiella tarda challenged head kidney and gill tissues. Furthermore, we for the first time showed that extracellular ATP, an important signaling molecule in triggering innate immune response and activation of NLR inflammasome, significantly up-regulates PoASC expression in the Japanese flounder head kidney macrophages in a dose-dependent manner. Together, these findings addressed the involvement of PoASC in TLR and extracellular ATP-mediated innate immune signaling in the Japanese flounders.

Identification and characterization of ATP-gated P2X2 receptor gene dominantly expressed in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

P2X2 receptor (P2X2R) belongs to the family of purinergic receptors that have been shown to play important roles in regulating host innate immune response. Although the immunologic significance of P2X2R has been studied in mammals, the presence and immune relevance of P2X2R in fish remains unclear. In this study we extended our previous observations by identifying and characterizing a P2X2R ortholog (termed PoP2X2R) from Japanese flounder (Paralichthys olivaceus). Quantitative real-time PCR analysis revealed that PoP2X2R mRNA transcripts are widely distributed in all examined normal tissues and are dominantly expressed in hepatopancreas tissue. In addition, we for the first time showed that multiple P2XR subtypes, including P2X2R, P2X4R and P2X7R are co-expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood lymphocytes (PBLs), indicating that they may assemble into hetero-receptor complex or interact in the form of homotrimers to trigger diverse purinergic signaling in the Japanese flounder immune cells. Compared with the known Japanese flounder P2X4 and P2X7 receptors, however, PoP2X2R is much more abundantly expressed in the Japanese flounder HKM cells, suggesting that PoP2X2R may play an important role in this type of immune cells. Glycosylation and immunohistochemistry analyses revealed that PoP2X2R is a glycoprotein expressed on the plasma membrane. Immune challenges experiments showed that PoP2X2R was significantly induced by LPS, poly(I:C) and zymosan stimulations in the HKM and PBL cells, and by Edwardsiella tarda infections in spleen and gill tissues as well. Taken together, we have identified and characterized a new P2X2R member that is involved in fish innate immune response.

Identification and characterization of thirty novel microsatellite DNA markers from the Chinese mitten crab Eriocheir sinensis expressed sequence tags.

BACKGROUND: The Chinese mitten crab Eriocheir sinensis is an economically important decapod crustacean in China. Despite a widespread distribution and production in China, the resources of E. sinensis have experienced a dramatic decline in the past decades. Here we describe a new set of novel polymorphic microsatellite loci to facilitate the investigation of genetic structure and artificial breeding. RESULTS: In this study, a set of 30 novel polymorphic microsatellite markers for E. sinensis was developed from EST databases. The number of alleles per locus ranged from three to twenty. The observed and expected heterozygosities ranged from 0.047 to 0.932 and from 0.047 to 0.935, respectively. CONCLUSIONS: These informative microsatellite markers will be useful in studies of genetics, genomics and marker-assisted selection breeding in E. sinensis.

CHHBP: a newly identified receptor of crustacean hyperglycemic hormone.

Crustacean hyperglycemic hormone (CHH) is a neurohormone found only in arthropods that plays a pivotal role in the regulation of hemolymph glucose levels, molting and stress responses. Although it was determined that a membrane guanylyl cyclase (GC) acts as the CHH receptor in the Y-organ during ecdysteroidogenesis, the identity of the CHH receptor in the hepatopancreas has not been established. In this study, we identified CHH binding protein (CHHBP), as a potential receptor by screening the annotated unigenes from the transcriptome of ITALIC! Eriocheir sinensis, after removal of the eyestalk. Analysis of the binding affinity between CHH and CHHBP provided direct evidence that CHH interacts with CHHBP in a specific binding mode. Subsequent analysis showed that CHHBP is expressed primarily in the hepatopancreas where it localizes to the cell membrane. In addition, real-time PCR analysis showed that ITALIC! CHHBPtranscript levels gradually increase in the hepatopancreas following eyestalk ablation. RNAi-mediated suppression of ITALIC! CHHBPexpression resulted in decreased glucose levels. Furthermore, the reduction of blood glucose induced by ITALIC! CHHBPRNAi reached the same level as that observed in the eyestalk ablation group, suggesting that CHHBP is involved in glucose metabolism regulated by CHH. In addition, compared with the control group, injection of CHH was unable to rescue the decreased glucose levels in ITALIC! CHHBPRNAi crabs. CHH induced transport of 2-NBDG to the outside of cells, with indispensable assistance from CHHBP. Taken together, these findings suggest that CHHBP acts as one type of the primary signal processor of CHH-mediated regulation of cellular glucose metabolism.

Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Nucleotide oligomerization domain (NOD)-like receptor (NLR) family with a caspase activation and recruitment domain (CARD) containing 3 (NLRC3) protein is an important cytosolic pattern recognition receptor that negatively regulates innate immune response in mammals. Hitherto, the immunological significance of NLRC3 protein in fish remains largely uncharacterized. Here we identified and characterized a novel NLRC3 gene (named poNLRC3) implicated in regulation of fish innate immunity from Japanese flounder Paralichthys olivaceus. The poNLRC3 protein is a cytoplasmic protein with an undefined N-terminal domain, a NACHT domain, a fish-specific NACHT associated domain, six LRR motifs, and a C-terminal fish-specific PYR/SPYR (B30.2) domain but only shares less than 40% sequence identities with the known Japanese flounder NLRC proteins. poNLRC3 gene is ubiquitously expressed in all tested tissues and is dominantly expressed in the Japanese flounder head kidney macrophages (HKMs). We for the first time showed that poNLRC3 expression was significantly modulated by the stimulation of extracellular ATP, an important danger/damage-associated molecular pattern in activating innate immunity in P. olivaceus. Importantly, we revealed that poNLRC3 plays an important role in positively regulating ATP-induced IL-1beta and IL-6 gene expression, suggesting the involvement of poNLRC3 in extracellular ATP-mediated immune signaling. In addition, we showed that poNLRC3 mRNA expression was up-regulated in response to LPS and Edwardsiella tarda immune challenges. Finally, we showed that down-regulating the endogenous poNLRC3 expression with small interfering RNA significantly reduced LPS-induced proinflammatory cytokine gene expression in the Japanese flounder HKM cells. Altogether, we have identified a novel inducible fish NLR member, poNLRC3, which is involved in extracellular ATP-mediated immune signaling and may positively regulate the LPS-induced innate immune response in the Japanese flounder HKM cells.

Identification and characterization of nascent polypeptide-associated complex alpha from Chinese mitten crab (Eriocheir sinensis): A novel stress and immune response gene in crustaceans.

Disease in aquatic animals is tightly linked to environmental challenges and their immune responses are greatly modified by their external environment. The chaperone protein nascent polypeptide-associated complex alpha (NACA) has been suggested to play important roles in the cellular response to stress and immune challenges, while the related biological functions remain largely unknown in invertebrates. In the present study we identified a NACA gene (termed EsNACA) from Chinese mitten crab Eriocheir sinensis and analyzed its expression changes in response to ambient (salinity and pH) stresses and immune challenges. The EsNACA protein is comprised of 209 amino acid residues with a conserved DNA binding domain, a C-Jun binding domain, a NAC domain and an ubiquitin-associated domain and shows the highest sequence identity (87%) with its counterpart in shrimp Penaeus monodon. EsNACA mRNA transcripts are presented in all tested normal tissues with predominant expression in hepatopancreas and lower expression in hemocytes. In addition, EsNACA expression was significantly altered in response to the ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, muscle, hemocytes and intestine tissues. Furthermore, EsNACA gene expression was substantially induced upon LPS and Poly(I:C) immune stimulations in E. sinensis hemocytes in vitro. Finally, EsNACA expression was up-regulated in E. sinensis hemocytes, gill, hepatopancreas, intestine and muscle tissues in response to Vibrio anguillarum challenges in vivo. Taken together, our findings for the first time show that EsNACA is an inducible gene involved in stress and immune response in crustaceans.

Molecular characterization of purinergic receptor P2X4 involved in Japanese flounder (Paralichthys olivaceus) innate immune response and its interaction with ATP release channel Pannexin1.

P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity.

Identification and expression analysis of nascent polypeptide-associated complex alpha gene in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Nascent polypeptide-associated complex (NAC) is a conserved heterodimeric protein consisting of alpha and beta subunits. In addition to acting as a protein translation chaperone by forming a heterodimer with the beta subunit, NAC alpha (NACA) also shows important immune significance independent of NAC beta in mammalian cells. In lower vertebrates, however, the immunological relevance of NACA has not been revealed yet. In the present study, we identified and characterized a NACA gene (termed poNACA) involved in innate immune response in Japanese flounder Paralichthys olivaceus. poNACA encodes a 215-amino-acid protein, with an apparent molecular weight of 23.5 kDa and an isoelectric point of 4.51. Tissue distribution analysis revealed that poNACA gene was constitutively expressed in all examined tissues and showed dominant expression in hepatopancreas and gonad tissues. In enriched Japanese flounder head kidney macrophages and peripheral blood leucocytes, the expression of poNACA mRNA transcript was significantly induced by LPS, Poly(I:C) and zymosan stimulations. In vivo experiments further revealed that poNACA gene expression was up-regulated in head kidney, gill and spleen tissues in response to Edwardsiella tarda challenges. Furthermore, overexpression of poNACA in Japanese flounder FG-9307 cells resulted in increased gene expression of IL-1beta, IL-11 and TNF-alpha, and myxovirus resistance (Mx). Taken together, our findings indicate that an immune response gene, poNACA, involved in innate immune regulation in P. olivaceus has been identified.

Identification and characterization of the cDNAs encoding the two subunits of Chinese mitten crab (Eriocheir sinensis) calcineurin: their implications in stress and immune response.

Calcineurin (CN), the only Ca(2+)/calmodulin-activated serine/threonine protein phosphatase, is a key effector participating in Ca(2+)-dependent signal transduction pathways in a number of cellular processes under normal, stress and pathological conditions. However, the expression and the relevance of CN in stress and immune response have not been characterized in crustaceans. Here, we identified the cDNAs that encode the two subunits of CN (termed EsCN-A and EsCN-B, respectively) in Chinese mitten crab Eriocheir sinensis and analysed their expression patterns in response to stress and immune challenges. The catalytic subunit EsCN-A is comprised of 511 amino acids with a theoretical molecular mass of 57.5 kDa and shows 80% sequence identity with human beings CN-A alpha isoform, while the regulatory subunit EsCN-B protein is composed of 170 amino acids with an estimated molecular mass of 19.3 kDa and shares 88% sequence identity with human beings CN-B type 1. Tissue distribution analysis reveals that both EsCN-A and EsCN-B mRNA transcripts are expressed in all tested tissues with the greatest expression in hepatopancreas and the lowest expression in haemocytes. In addition, both EsCN-A and EsCN-B genes could be significantly up-regulated but with different expression patterns by ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, haemocytes, intestine and muscle. Furthermore, EsCN-A and EsCN-B were up-regulated by LPS and Poly(I:C) immune stimulations in E. sinensis haemocytes in vitro. Moreover, EsCN-A and EsCN-B mRNA were significantly up-regulated in haemocytes, gill, hepatopancreas, intestine and muscle in response to Edwardsiella tarda challenge in vivo. Finally, we revealed the importance of EsCN in LPS-induced nitric oxide production in E. sinensis haemocytes. Together our observations suggest that EsCN, the important downstream effector of CaM-mediated signalling pathway(s), may possess vital roles in stress and immune response in the Chinese mitten crab.

Calmodulin is a stress and immune response gene in Chinese mitten crab Eriocheir sinensis.

Calmodulin (CaM) is a multifunctional calcium sensor protein that participates in various cellular processes under normal, stress and pathological conditions. In crabs, however, the involvement of CaM in response to environmental stress and immune challenges has not been revealed yet. In the present study, a CaM cDNA (EsCaM) was identified from Chinese mitten crab Eriocheir sinensis and its mRNA expression patterns in response to ambient (salinity and pH) stress and immune challenges was examined. EsCaM encodes a 149-amino-acid protein with a calculated molecular mass of 16.8 kDa and an isoelectric point of 4.09. In unstimulated healthy E. sinensis, EsCaM mRNA transcript was detected in all tested tissues with predominant expression in hepatopancreas and the lowest expression in haemocytes. Ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stress significantly altered EsCaM mRNA expression in gill, hepatopancreas, haemocytes, intestine and muscle in Chinese mitten crab. In addition, EsCaM gene expression was significantly and rapidly induced as early as 2 h after LPS and Poly(I:C) immune stimulations in haemocytes in vitro. Furthermore, EsCaM expression was significantly up-regulated in E. sinensis haemocytes, gill, hepatopancreas, intestine and muscle in response to Edwardsiella tarda and Vibrio anguillarum challenges. Collectively, our findings suggest that EsCaM is an important stress and immune response gene in Chinese mitten crab.