Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages.

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Acylated Inulin as a Potential Shale Hydration Inhibitor in Water Based Drilling Fluids for Wellbore Stabilization.

Shale hydration dispersion and swelling are primary causes of wellbore instability in oil and gas reservoir exploration. In this study, inulin, a fructo-oligosaccharide extracted from Jerusalem artichoke roots, was modified by acylation with three acyl chlorides, and the products (C10-, C12-, and C14-inulin) were investigated for their use as novel shale hydration inhibitors. The inhibition properties were evaluated through the shale cuttings hot-rolling dispersion test, the sodium-based bentonite hydration test, and capillary suction. The three acylated inulins exhibited superb hydration-inhibiting performance at low concentrations, compared to the commonly used inhibitors of KCl and poly (ester amine). An inhibition mechanism was proposed based on surface tension measurements, contact angle measurements, Fourier-transform infrared analysis, and scanning electron microscopy. The acylated inulin reduced the water surface tension significantly, thus, retarding the invasion of water into the shale formation. Then, the acylated inulin was adsorbed onto the shale surface by hydrogen bonding to form a compact, sealed, hydrophobic membrane. Furthermore, the acylated inulins are non-toxic and biodegradable, which meet the increasingly stringent environmental regulations in this field. This method might provide a new avenue for developing high-performance and ecofriendly shale hydration inhibitors.

Effect of Chemical Composition of Metal-Organic Crosslinker on the Properties of Fracturing Fluid in High-Temperature Reservoir.

To investigate the effect of the chemical composition of a metal-organic crosslinker on the performances of fracturing fluid in high-temperature conditions, four zirconium (Zr) crosslinkers and one aluminum-zirconium (Al-Zr) crosslinker with a polyacrylamide were used. The crosslinkers possessed the same Zr concentration, but they differed in component amounts and the order of the addition of the crosslinker components, leading to different chemical compositions in the crosslinkers. The fracturing fluids prepared by different tested crosslinkers were compared in terms of properties of rheological behavior, sand-carrying ability, microstructure, and gel breaking characteristics. The results showed that the fracturing fluids prepared by zirconium lactic acid, ethanediamine, and sorbitol crosslinkers offered the slowest viscosity development and highest final viscosity compared to the zirconium lactic acid crosslinker and the zirconium lactic acid and ethanediamine crosslinker. The zirconium sorbitol, lactic acid, and ethanediamine crosslinker exhibited a faster crosslinking rate and a higher final viscosity than the zirconium lactic acid, ethanediamine, and sorbitol crosslinker; the crosslinker showed crosslinking density and crosslinking reactivity, resulting in more crosslinking sites and a higher strength in the fracturing fluid. The Al-Zr-based crosslinker possessed better properties in temperature and shear resistance, viscoelasticity, shear recovery, and sand-carrying ability than the Zr-based crosslinker due to the synergistic crosslinking effect of aluminum and zirconium ions. The tertiary release gelation mechanism of the Al-Zr-based fracturing fluid achieved a temperature resistance performance in the form of continuous crosslinking, avoiding the excessive crosslinking dehydration and reducing viscosity loss caused by early shear damage. These results indicated that the chemical compositions of metal-organic crosslinkers were important factors in determining the properties of fracturing fluids. Therefore, the appropriate type of crosslinker could save costs without adding the additional components required for high-temperature reservoirs.

A novel C1qDC (PoC1qDC) with a collagen domain in Paralichthys olivaceus mediates complement activation and against bacterial infection.

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection.

Hedgehog signaling is essential in the regulation of limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds-HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells.

Identification and evaluation of potential probiotics against skin-ulceration disease in the Chinese tongue sole (Cynoglossus semilaevis).

In this study, three highly pathogenic bacterial strains (Vibrio harveyi TB6, Vibrio alginolyticus TN1, and Vibrio parahaemolyticus TN3) were isolated from skin ulcers and intestines of diseased Chinese tongue sole (Cynoglossus semilaevis). The bacteria were investigated using hemolytic activity tests, in vitro co-culture with intestinal epithelial cells, and artificial infection of C. semilaevis. A further 126 strains were isolated from the intestines of healthy C. semilaevis. The three pathogens were used as indicator bacteria, and the antagonistic strains were identified from the 126 strains. The activities of exocrine digestive enzymes in the strains were also tested. Four strains with antibacterial and digestive enzyme activities were obtained and the best strains, Bacillus subtilis Y2 and Bacillus amyloliquefaciens Y9, were selected according to their ability to protect epithelial cells from infection. In addition, the effects of strains Y2 and Y9 at the individual level were investigated, finding that the activities of the immune-related enzymes superoxide dismutase, catalase, acid phosphatase, and peroxidase were significantly increased in the sera of the treatment group compared with the control group (p < 0.05). The specific growth rate (SGR, %) was also increased, especially in the Y2 group, and was significantly higher compared with the controls (p < 0.05). The result of the artificial infection test showed that the cumulative mortality within 72 h in the Y2 group was the lowest (50.5%), and in the Y9 group (68.5%) it was significantly lower than that in the control group (100%) (p < 0.05). Analysis of the intestinal microbial communities indicated that Y2 and Y9 could alter the composition of the intestinal flora, increasing both species richness and evenness, and inhibiting the growth of Vibrio in the intestine. These results suggested food supplemented with Y2 and Y9 could improve both immune function and disease resistance, as well as have a positive effect on the growth performance and the intestinal morphology of C. semilaevis.

Molecular characterization of TRPA1 and its function in temperature preference in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species, and its growth and development are regulated by temperature, but the molecular mechanisms of the responses to temperature remain unclear. Herein, we identified TRPA1 from E. sinensis, a member of the TRP family of heat receptor potential channels, performed RACE cloning and bioinformatics analysis, and investigated the effect of TRPA1 on temperature responses and molting by real-time PCR and RNA interference (RNAi). The open reading frame of Es-TRPA1 is 3660 bp, and the encoded protein has a molecular weight of 136.91 kDa, and is expressed in embryos and juveniles. RNAi-mediated silencing decreased Es-TRPA1 expression in juvenile crabs, molting rate was decreased, mortality was increased, and crabs avoided cold areas (4 °C) much less than control juvenile crabs. The results suggest that Es-TRPA1 is involved in regulating temperature adaptation and molting processes in E. sinensis. The findings lay a foundation for further exploration of temperature regulation mechanisms in E. sinensis and other crustaceans.

Transcriptome and gut microbiota analyses reveal a possible mechanism underlying rifampin-mediated interruption of the larval development of chironomid Propsilocerus akamusi (Diptera: Chironomidae).

Chironomids, the most abundant insect group found in freshwater habitats, are known to be pollution tolerate and serve as important bioindicators of contaminant stress. Gut microbiota has recently been shown to potentially provide a number of beneficial services to insect hosts. However, the antibiotic-mediated interruption of chironomid gut microbial community and its subsequent influence on host body are still unclear. In the present study, the effects of rifampin on chironomid larvae were investigated at both transcriptome and microbiome level to assess the relationship between gut bacteria and associated genes. Our data indicated that the rifampin-induced imbalance of gut ecosystem could inhibit the development of chironomid larvae via decreasing the body weight, body length and larval eclosion rate during 96-h treatment. Both the community structure and taxonomic composition were significantly altered due to the invasion of rifampin in digestive tracts. The relative abundance of phylum Deferribacterota and Bacteroidota were dramatically increased with rifampin exposure. A set of genes involved in amino acid synthesis as well as xenobiotic metabolism pathways were greatly changed and proved to have tight correlation with certain genus. Bacterial genus Tyzzerella was positively correlated with detoxifying PaCYP6GF1 and PaCYP9HL1 genes. This study provides a reference for understanding the environmental risks of antibiotic and aims to accelerate new biological insights into the effects of antibiotic on the fitness of chironomids and into the microbe mediated-regulatory mechanism of aquatic insects.

Fracturing Fluid Polymer Thickener with Superior Temperature, Salt and Shear Resistance Properties from the Synergistic Effect of Double-Tail Hydrophobic Monomer and Nonionic Polymerizable Surfactant.

To develop high-salinity, high-temperature reservoirs, two hydrophobically associating polymers as fracturing fluid thickener were respectively synthesized through aqueous solution polymerization with acrylamide (AM), acrylic acid (AA), 2-acrylamido-2-methylpropanesulfonic acid (AMPS), nonionic polymerizable surfactant (NPS) and double-tail hydrophobic monomer (DHM). The thickener ASDM (AM/AA/AMPS/NPS/DHM) and thickener ASD (AM/AA/AMPS/DHM) were compared in terms of properties of water dissolution, thickening ability, rheological behavior and sand-carrying. The results showed that ASDM could be quickly diluted in water within 6 min, 66.7% less than that of ASD. ASDM exhibited salt-thickening performance, and the apparent viscosity of 0.5 wt% ASDM reached 175.9 mPa·s in 100,000 mg/L brine, 100.6% higher than that of ASD. The viscosity of 0.5 wt% ASDM was 85.9 mPa·s after shearing for 120 min at 120 °C and at 170 s-1, 46.6% higher than that of ASD. ASDM exhibited better performance in thickening ability, viscoelasticity, shear recovery, thixotropy and sand-carrying than ASD. The synergistic effect of hydrophobic association and linear entanglement greatly enhancing the performance of ASDM and the compactness of the spatial network structure of the ASDM was enhanced. In general, ASDM exhibited great potential for application in extreme environmental conditions with high salt and high temperatures.

Genome-scale metabolic network model of Eriocheir sinensis icrab4665 and nutritional requirement analysis.

BACKGROUND: Genome-scale metabolic network models (GEMs) provide an efficient platform for the comprehensive analysis the physical and biochemical functions of organisms due to their systematic perspective on the study of metabolic processes. Eriocheir sinensis is an important economic species cultivated on a large scale because it is delicious and nutritious and has a high economic value. Feed improvement is one of the important methods to improve the yield of E. sinensis and control water pollution caused by the inadequate absorption of feed. RESULTS: In this study, a GEM of E. sinensis, icrab4665, was reconstructed based on the transcriptome sequencing, combined with KEGG database, literature and experimental data. The icrab4665 comprised 4665 unigenes, 2060 reactions and 1891 metabolites, which were distributed in 12 metabolic subsystems and 113 metabolic pathways. The model was used to predict the optimal nutrient requirements of E. sinensis in feed, and suggestions for feed improvement were put forward based on the simulation results. The simulation results showed that arginine, methionine, isoleucine and phenylalanine had more active metabolism in E. sinensis. It was suggested that the amount of these essential amino acids should be proportionally higher than that of other amino acids in the feed to ensure the amino acid metabolism of E. sinensis. On the basis of the simulation results, we further suggested increasing the amount of linoleic acid, EPA and DHA in the feed to ensure the intake of essential fatty acids for the growth of E. sinensis and promote the accumulation of cell substances. In addition, the amounts of zinc and selenium in the feed were also suggested to be properly increased to ensure the basic metabolism and growth demand of E. sinensis. CONCLUSION: The largest GEM of E. sinensis was reconstructed and suggestions were provide for the improvement of feed contents based on the model simulation. This study promoted the exploration of feed optimization for aquatic crustaceans from in vivo and in silico. The results provided guidance for improving the feed proportion for E. sinensis, which is of great significance to improve its yield and economic value.

Microinjection-based CRISPR/Cas9 mutagenesis in the decapoda crustaceans Neocaridina heteropoda and Eriocheir sinensis.

CRISPR/Cas9 technology has been applied to many arthropods. However, application of this technology to crustaceans remains limited because of the unique characteristics of embryos. Our group has developed a microinjection system to introduce the CRISPR/Cas9 system into Neocaridina heteropoda embryos (one-cell stage). Using the developed method, we mutated the target gene Nh-scarlet (N. heteropoda scarlet), which functions in eye development and pigmentation. The results showed that both eye color and shape were altered in individuals in which Nh-scarlet was knocked out. Furthermore, this system was also successfully applied to another decapod crustacean, Eriocheir sinensis. DNA sequencing revealed that the zoeae with red eyes had an edited version of Es-scarlet. This study provides a stable microinjection method for freshwater crustaceans, and will contribute to functional genomics studies in various decapods.

Insulin-like signaling promotes limb regeneration in the Chinese mitten crab (Eriocheir sinensis).

In the pond culture of Chinese mitten crabs, limb autotomy seriously affects the feeding efficiency, immunity and survival. Therefore, it is crucial to understand the mechanism of limb regeneration of mitten crabs, so that culture strategies could be developed to reduce the limb impairment rate. The insulin-like signaling (ILS) pathway is evolutionarily conserved, and plays key roles in the growth and immunity of various species. In this study, a full-length cDNA of insulin-like receptor (EsInR) was identified from Eriocheir sinensis, and its mRNA expression patterns during limb regeneration was evaluated. The cDNA of EsInR includes a 4326 bp ORF encoding a protein of 1441 amino acids, with conserved α-and ß-subunits. The EsInR and genes related to ILS were found to be upregulated during limb regeneration, which indicated that ILS plays a key role in limb regeneration of E. sinensis. Our experiment revealed that inhibition of ILS through injection of the InR inhibitor GSK1838705A at the blastema formation stage significantly reduced the limb regeneration rate compared to control group. In addition, injection of GSK1838705A also reduced the size of newly formed limbs after the molting cycle. Furthermore, we found that genes related to myogenesis were downregulated following injection of InR inhibitor both before and after molting. The results also indicated that cyclins and CDK1 were downregulated, while CKIs were upregulated following treatment with the InR inhibitor. These results suggest that ILS regulates limb regeneration in E. sinensis by promoting muscle growth and regeneration in response to autotomy stress. Thus, we identified a conserved insulin-like receptor in E. sinensis, and provide new evidence for the involvement of ILS in the regulation of limb autotomy and regeneration in crustaceans.

New insight into the effect of riluzole on cadmium tolerance and accumulation in duckweed (Lemna turionifera).

Cadmium (Cd) damages plant photosynthesis, affects roots and leaves growth, and triggers molecular responses. Riluzole (RIL), which protected neuronal damage via inhibiting excess Glu release in animals, has been found to improve Cd tolerance in duckweed in this study. Firstly, RIL treatment alleviated leaf chlorosis by protecting chlorophyll and decreased root abscission under Cd stress. Secondly, RIL declines Cd accumulation by alleviating excess Glu release during Cd shock. RIL mitigate Glu outburst in duckweed during Cd stress by a decline in Glu in roots. The Cd2+ influx was repressed by RIL addition with Cd shock. Finally, differentially expressed genes (DEGs) of duckweed under Cd stress with RIL have been investigated. 2141 genes were substantially up-regulated and 3282 genes were substantially down-regulated with RIL addition. RIL down-regulates the genes related to the Glu synthesis, and genes related to DNA repair have been up-regulated with RIL treatment under Cd stress. These results provide new insights into the possibility of RIL to reduce Cd accumulation and increase Cd tolerance in duckweed, and lay the foundation for decreasing Cd accumulation in crops.

Identification and characterization of Trpm4 gene involved in regulating Japanese flounder (Paralichthys olivaceus) inflammatory response.

The transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed Ca2+ -impermeable cation channel involved in modulating inflammatory and immune responses in mammals. However, the role of TRPM4 channel in fish immunity remains unclear. In this report, from a comparative immunological point of view, we identified and characterized a Trpm4 gene from Japanese flounder (Paralichthys olivaceus) and analysed its potential role in regulating the fish inflammatory response. The Japanese flounder Trpm4 gene is expressed in a wide range of tissues and encodes a 1264-amino acid protein which expresses on the cell surface and shares several conserved domains with its mammalian counterparts. In vitro inflammatory challenge and in vivo bacterial infection experiments revealed that Japanese flounder Trpm4 expression was significantly modulated following different immune challenges, indicating the implication of Trpm4 in the fish immune response. Overexpression of TRPM4 significantly attenuated LPS- and poly(I:C)-induced pro-inflammatory cytokine expression in Japanese flounder FG-9307 cells. In contrast, pharmacological inhibition of the endogenous TRPM4 channel activity in Japanese flounder head kidney macrophages resulted in increased pro-inflammatory cytokine expression following LPS and poly(I:C) stimulations. Taken together, these findings indicate that TRPM4 channels may play a conserved role in regulating inflammatory response(s) in fish.

A novel microRNA and its pfk target control growth length in the freshwater shrimp Neocaridina heteropoda.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression and play roles in a wide range of physiological processes, including ontogenesis. Herein, we discovered a novel miRNA, novel miR-26, which inhibits translation of the phosphofructokinase (PFK) gene by targeting the 3' untranslated region (UTR) of pfk directly, thereby inhibiting molting and body length growth of the freshwater shrimp Neocaridina heteropoda Lowering expression of pfk by RNA interference (RNAi) led to a longer ecdysis cycle and smaller individuals. This phenotype was mirrored in shrimps injected with novel miR-26 agomirs, but the opposite phenotype occurred in shrimps injected with novel miR-26 antagomirs (i.e. the ecdysis cycle was shortened and body length was increased). After injection of 20-hydroxyecdysone (ecdysone 20E), expression of the novel miR-26 was decreased, while expression of pfk was up-regulated, and the fructose-1,6-diphosphate metabolite of PFK accumulated correspondingly. Furthermore, expression of eIF2 (eukaryotic initiation factor 2) increased under stimulation with fructose-1,6-diphosphate, suggesting that protein synthesis was stimulated during this period. Taken together, our results suggest that the novel miR-26 regulates expression of pfk and thereby mediates the molting and growth of N. heteropoda.

Characterization of three connexin32 genes and their role in inflammation-induced ATP release in the Japanese flounder Paralichthys olivaceus.

Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish.

MMP-14 regulates innate immune responses to Eriocheir sinensis via tissue degradation.

Matrix metalloproteinases (MMPs) are a cluster of enzymes that degrade the extracellular matrix (ECM) and some intracellular proteins; as such, they play an important role in tissue regeneration, infant growth, animal reproduction, and immunity. Most research into MMPs focuses mainly on their effects on the mammalian immune system. However, it is not clear how MMPs affect immune processes in crustaceans. Here, we cloned the open reading frame (ORF) of Eriocheir sinensis (Chinese mitten crab) MMP-14 (EsMMP-14) to explore the role of MMPs in crustacean innate immune responses. RT-PCR results showed that stimulation of crab with LPS and poly I:C upregulated expression of EsMMP-14 markedly. Besides, following the stimulation of 20-Hydroxyecdysone, the expression level of EsMMP-14 increased robustly, suggesting that EsMMP-14 involved in the molt process of E. sinensis. Hematoxylin and eosin staining of hepatopancreas and intestine revealed that knocking down EsMMP-14 maintained morphology following infection by Bacillus thuringiensis. Moreover, downregulated expression of EsMMP-14 increased the survival rate of infected E. sinensis. These results show that EsMMP-14 plays a role in innate immune responses of E. sinensis and fills a gap in our knowledge about the function of MMPs in crustaceans.

Functional characterization of two ecto-nucleoside triphosphate diphosphohydrolase 2 genes in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5'-triphosphates and nucleoside 5'-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five apyrase conserved regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced proinflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.

Identification and characterization of apoptosis-related gene serine/threonine kinase 17A (STK17A) from Japanese flounder Paralichthys olivaceus.

Apoptosis plays important roles in regulation of the immune response and has a direct impact on disease resistance in teleost. Death associated protein kinase (DAPK)-related Serine/Threonine kinase 17A (STK17A) is a positive apoptosis regulator. However, the expression and function of STK17A in fish still remains uninvestigated. In this study, we identified and characterized a STK17A gene (termed PoSTK17A) from Japanese flounder Paralichthys olivaceus. We also investigated the pro-apoptotic role of PoSTK17A in fish. Real-time quantitative PCR analysis revealed that PoSTK17A is widely present in various Japanese flounder tissues, and dominantly expressed in liver. Immune challenge experiments showed that PoSTK17A expression was upregulated by inflammatory challenge, Edwardsiella tarda infection and DNA-damaging agent cisplatin treatment as well. Immunofluorescence microscopy revealed that the recombinant PoSTK17A proteins are mainly located in the nucleus of Japanese flounder FG-9307 cells, and human Hela and MCF7 cells. However, PoSTK17A was translocated from the nucleus to cytoplasm following cisplatin treatment. Overexpression of PoSTK17A significantly increased the apoptosis in human MCF7 cells through both cisplatin-dependent and independent manners. Importantly, PoSTK17A also promotes the ATP-gated P2X7 receptor-mediated apoptosis in Japanese flounder FG-9307 cells. Collectively, we characterized an inducible STK17A gene (PoSTK17A) that may play a conserved pro-apoptotic role in fish.

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.