Regulation of NF-κB signaling by NLRC (NLRC3-like) gene in the common carp (Cyprinus carpio).

Among teleost NLRs, NLR-C subfamily is a large group of proteins that were teleost-specific and evolution analysis showed that NLR-Cs are most likely to evolve from NLRC3 gene (thus also called as NLRC3Ls). Presently, although there have been rich studies investigating teleost NLRC3 and NLRC3L, the data on the regulatory mechanism was limited. In this study, immune regulation of inflammatory signaling pathway mediated by common carp NLRC3L gene (CcNLRC) has been investigated. Confocal microscopy analysis showed that CcNLRC was located in cytoplasm, and in HEK293T cells, dual-luciferase reporter assay showed the regulation of NF-κB signaling by CcNLRC, in which CcNLRC could alter/decrease RIPK2-induced activation of NF-κB. These results indicated that CcNLRC may function as a negative NLR in the regulation of inflammatory response in common carp. Our data will allow to gain more insights into the molecular mechanism of teleost specific NLR (NLRC3L).

Enterocytozoon hepatopenaei (EHP) Infection Alters the Metabolic Processes and Induces Oxidative Stress in Penaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the differences in histology, metabolism, oxidative stress and growth between healthy and EHP-infected Penaeus vannamei were analyzed using an EHP challenge experiment. Histology showed that EHP caused lesions in the hepatic tubules of P. vannamei, such as hepatic tubular atrophy and epithelial cell shedding, with mature spores. Meanwhile, white feces may appear when the infection is severe. Furthermore, the content of total protein, glycogen, ATP and glucose in the EHP challenge group was significantly reduced. The qPCR results showed that EHP infection changed the expression of key genes in glucose metabolism, among which hexokinase (HK), phosphofructokinase (PFK), pyruvatekinase (PK), citrate synthase (CS) and isocitric dehydrogenase (IDH) were significantly down-regulated, while phosphoenolpyruvate carboxykinase (PEPCK), fructose bisphosphatase (FBP) and glucose-6-phosphatase (G6P) were significantly up-regulated. Obviously, the expression of growth-related genes was disordered. Simultaneously, the antioxidant genes manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferases (GST) and nuclear factor E2-related factor2 (Nrf2) were up-regulated to varying degrees in the EHP challenge group, and EHP infection induced significant increases in the oxidative damage products lipid peroxide (LPO) and malondialdehyde (MDA). Ultimately, the shrimp weight of the challenge group was 6.85 ± 0.86 g, which was significantly lower than that of the control group (8.95 ± 0.75 g). Taken together, we speculate that EHP changes the substance metabolism and growth process by causing oxidative damage to the hepatopancreas, which may lead to the growth retardation of P. vannamei.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Identification of CCCH-type zinc finger antiviral protein 1 (ZAP) gene from Pacific white shrimp (Penaeus vannamei): Characterization and expression analysis in response to viral infection.

Zinc-finger proteins (ZFPs) are a huge family that exert multiple roles in the cells. ZFPs could be divided into nine types based on the numbers and positions of conserved Cys and His residues, in which CCCH-type ZFP was one of the most widely studied types. CCCH-type zinc finger antiviral protein 1 (ZAP), a CCCH-type ZFP that can inhibit the replication of certain RNA viruses and DNA viruses by mediating degradation of viral RNA and repressing mRNA translation, plays significant roles in the host innate immune defenses against viral infections. Presently, there have been numerous reports investigating the antiviral ability of ZAP, while no data is available about ZAP gene in the species of shrimps or even crustaceans. In this study, a novel protein containing CCCH-type zinc finger motifs (ZnF-CCCH), CCCH-type zinc finger antiviral protein 1 (ZAP) gene, was identified from Pacific white shrimp (Penaeus vannamei) and its role in antiviral immunity was further investigated. Similar to mammalian ZAPs, in addition to ZnF-CCCH, PvZAP also possesses central WWE domains and C-terminal PARP domain. Phylogenetic analysis showed that PvZAP was close to that of the crustacean Pacific oyster, separating from the cluster of vertebrate ZAP proteins. Upon in vivo infection by IHHNV, gene expression of PvZAP was strongly up-regulated in the hepatopancreas and gills of both adult and juvenile shrimps, where adult individuals showed higher fold changes of up-regulation than in juvenile individuals. These results suggested that PvZAP might play an important role in the innate immune defense of Pacific white shrimp against IHHNV infection. This allows us to gain new insights into the immunological function of ZAP in the innate immunity of shrimp species and even crustaceans.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.

Transcriptome analysis of IHHNV infection in Penaeus vannamei at different developmental stages.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is the smallest known virus in shrimp, which causes runt-deformity syndrome (RDS) and leads to huge economic loss every year in penaeid shrimp farming. Previous studies have shown that the juvenile Penaeus vannamei is more susceptible to IHHNV infection than the adults, but the mechanism is still unclear. In order to investigate the mechanism of pathogenic differences in IHHNV infection of P. vannamei at different developmental stages, the juvenile and adult P. vannamei were studied by transcriptome high-throughput sequencing to analyze their response to IHHNV infection. GO and KEGG enrichment were analyzed to search for differentially expressed genes (DEGs) related to immunity, growth and metabolism. The results showed that many immune-related genes of the juvenile and adult P. vannamei responded differently to IHHNV infection. For the adult P. vannamei, the expression of most immune-related genes was significantly up-regulated, which means that a cellular defense response was triggered after IHHNV infection. However, most immune-related genes in juvenile P. vannamei were inhibited, indicating that the immune system of juvenile the P. vannamei is imperfect and makes it to be more susceptible to IHHNV. Similarly, the growth-related genes of P. vannamei were changed during IHHNV infection. For the juvenile P. vannamei, the growth-related genes were significantly down-regulated, which resulted in a growth hormone disorder and prevented the juvenile P. vannamei from growth. In the adult P. vannamei, most molting-related genes were significantly up-regulated, indicating that IHHNV infection leads the adult P. vannamei to early molting to eliminate pathogen in the body. Metabolic process data showed that energy metabolism pathway was affected when P. vannamei infected with IHHNV. The adult P. vannamei infected with IHHNV can cause energetically costly and lead to the disturbance of the metabolism, activate complex immune systems to resist the invasion of pathogens. The results of this study clarified the response mechanism of P. vannamei at different developmental stages to IHHNV infection, which can provide new insights to IHHNV effective control and a reference for the study of sensitive period of different shrimp virus to host infection.

Effects of crustacean hyperglycaemic hormone RNA interference on regulation of glucose metabolism in Litopenaeus vannamei after ammonia-nitrogen exposure.

To unveil the adaptation of Litopenaeus vannamei to elevated ambient ammonia-N, crustacean hyperglycaemic hormone (CHH) was knocked down to investigate its function in glucose metabolism pathway under ammonia-N exposure. When CHH was silenced, haemolymph glucose increased significantly during 3-6 h, decreased significantly during 12-48 h and recovered to the control groups' level at 72 h. After CHH knock-down, dopamine (DA) contents reduced significantly during 3-24 h, which recovered after 48 h. Besides, the expressions of guanylyl cyclase (GC) and DA1R in the hepatopancreas decreased significantly, while DA4R increased significantly. Correspondingly, the contents of cyclic AMP (cAMP), cyclic GMP (cGMP) and diacylglycerol (DAG) and the expressions of protein kinase A (PKA), protein kinase G (PKG), AMP active protein kinase α (AMPKα) and AMPKγ were significantly down-regulated, while the levels of protein kinase C (PKC) and AMPKß were significantly up-regulated. The expressions of cyclic AMP response element-binding protein (CREB) and GLUT2 decreased significantly, while GLUT1 increased significantly. Moreover, glycogen content, glycogen synthase and glycogen phosphorylase activities in hepatopancreas and muscle were significantly increased. Furthermore, the levels of key enzymes hexokinase, pyruvate kinase and phosphofructokinase in glycolysis (GLY), rate-limiting enzymes citrate synthase in tricarboxylic acid and critical enzymes phosphoenolpyruvate carboxykinase, fructose diphosphate and glucose-6-phosphatase in gluconeogenesis (GNG) were significantly decreased in hepatopancreas. These results suggest that CHH affects DA and then they affect their receptors to transmit glucose metabolism signals into the hepatopancreas of L. vannamei under ammonia-N stress. CHH acts on the cGMP-PKG-AMPKα-CREB pathway through GC, and CHH affects DA to influence cAMP-PKA-AMPKγ-CREB and DAG-PKC-AMPKß-CREB pathways, thereby regulating GLUT, inhibiting glycogen metabolism and promoting GLY and GNG. This study contributes to further understand glucose metabolism mechanism of crustacean in response to environmental stress.

The exploration of neuroendocrine regulation of crustacean hyperglycemic hormone (CHH) on innate immunity of Litopenaeus vannamei under ammonia-N stress.

To unveil the neuroendocrine-immune (NEI) mechanism of crustaceans under high ambient ammonia-N, crustacean hyperglycemic hormone (CHH) in L. vannamei was knocked down under 20 mg/L ammonia-N exposure. The results showed that the expression of CHH in the eyestalks decreased significantly when CHH was silenced. After CHH was knocked down, the levels of CHH, ACh, DA, NE, and 5-HT in the haemolymph decreased significantly. Correspondingly, the expressions of GC, ACh7R, DM1, DA1R, and 5-HT7R in haemocytes down-regulated significantly, while DA4R and α2AR up-regulated significantly. Besides, the expression of Toll3 reduced significantly. And significantly changes occurred in the levels of G protein effectors (AC and PLC), second messengers (cAMP, cGMP, CaM, and DAG), protein kinases (PKA, PKC and PKG), and nuclear transcription factors (CREB, Dorsal, Relish and NKRF). Furthermore, immune defense proteins (BGBP and PPO3, Crustin A, ALF, LYC, TNFα, and IL-16), phagocytosis-related proteins (Cubilin, Integrin, Peroxinectin, Mas-like protein, and Dynamin-1) and exocytosis-related proteins (SNAP-25, VAMP-2 and Syntaxin) changed significantly. Eventually, a significant decrease in the levels of THC, haemocytes phagocytosis rate, plasma PO, antibacterial and bacteriolytic activities was detected. Therefore, these results indicate that under ammonia-N stress, the combination of CHH and GC mainly affects exocytosis of shrimp through the cGMP-PKG-CREB pathway. Simultaneously, CHH stimulates the release of biogenic amines, and then activate G protein effectors after binding to their specific receptors, to regulate exocytosis mainly via the cAMP-PKA-CREB pathway and influence phagocytosis primarily by the cAMP-PKA-NF-κB pathway. CHH can enhance ACh, and then activate G protein effectors after binding to the receptors, and finally regulate exocytosis mainly through the cAMP-PKA-CREB pathway and regulate phagocytosis by the cAMP-PKA-NF-κB pathway. CHH can also promote Toll3-NF-κB pathway, thereby affecting the expressions of immune defense factors. This study contributes to a further understanding of the NEI mechanism of crustacean in response to environmental stress.

Effects of Nrf2-Keap1 signaling pathway on antioxidant defense system and oxidative damage in the clams Ruditapes philippinarum exposure to PAHs.

NF-E2-related factor 2 (Nrf2) is a master regulator of antioxidant defense system which can maintain the oxidation balance in the cell. In our previous study, we first cloned the Nrf2 gene in clams and preliminarily explored the role of the Nrf2 at the transcription level. In this study, RNA interference (RNAi) technology was used to interfere with the expression of Nrf2 after being exposed to benzo(a)pyrene (BaP) for 5 days to verify the role of Nrf2 in the antioxidant defense system. Besides, we examined the mRNA expression and enzyme activities of antioxidases and the oxidative damage. The positive correlations between the Nrf2 with the mRNA expression and the enzyme activities of antioxidases indicated that Nrf2 was required for the induction of these antioxidant genes. Additionally, the mRNA expression and the enzyme activities of the glutathione peroxidase (GPx) in the Nrf2-dsRNA group were significantly higher than those in the control groups on the fifth day, indicating that the GPx is more sensitive to oxidative stress. Moreover, the oxidative damage in the RpNrf2-dsRNA group was markedly increased than control groups, indicating that Nrf2 transcriptional regulation may play an essential role in defending against oxidative damage. This study provides a foundation for further research on the mechanism of detoxification and antioxidation of polycyclic aromatic hydrocarbons (PAHs) in the clams at the transcription level and the protein level.

The molecular mechanism of AhR-ARNT-XREs signaling pathway in the detoxification response induced by polycyclic aromatic hydrocarbons (PAHs) in clam Ruditapes philippinarum.

The aryl hydrocarbon receptor (AhR) has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes to mitigate environmental stresses. In this study, we interfere the expression of AhR gene to investigate the mechanism of AhR signaling pathway in the detoxification and antioxidation defense system that induced by Polycyclic Aromatic Hydrocarbons (PAHs) exposure by RNA interference (RNAi). The gene expressions of aryl hydrocarbon receptor nuclear translocator (ARNT), heat shock protein 90 (Hsp90) were evaluated after being exposed to benzo(a)pyrene (BaP) (4 µg/L) for 5 days and the positive correlations between AhR, ARNT, HSP90 indirectly indicating that AhR may have the ability to bind to ligands such as PAHs in Ruditapes philippinarum (R. philippinarum). Besides, the activities of detoxification enzymes were determined to investigate the role of AhR signaling pathway played in the metabolic detoxification. What's more, the gene expressions of protein kinase C (PKC) signaling pathway, mitogen-activated protein kinase (MAPKs) signaling pathway, NF-E2-related factor 2 (Nrf2) signaling pathway and antioxidant defense system indicated that AhR may regulate the Nrf2-Keap1 signaling pathway through Kelch-like ECH-associated protein-1 (Keap1) and MAPKs, PKC signaling pathways. In conclusion, adoption of RNA interference technology to explore the role of RpAhR gene played in the detoxification and antioxidation defense system under the PAHs stress at different time points can informe molecular endpoints for application towards ecotoxicology monitoring of bivalves.

Transcriptome analysis of hemocytes from the white shrimp Litopenaeus vannamei with the injection of dopamine.

As a crucial neuroendocrine-immune factor, dopamine (DA) could regulate the immune system of Litopenaeus vannamei. To understand the immune mechanisms and regulatory pathways of DA in L. vannamei, the transcriptome analysis of hemocytes of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were performed in this study. Moreover, quantitative real-time PCR (qPCR) method was applied to validate the accuracy of transcriptome sequencing and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, 12, and 24 h) after DA injection. The results showed that a total of 51382 unigenes with a N50 length of 2341 bp were generated. And 1397 and 457 DEGs were obtained by comparative transcriptome at 3 and 12h respectively. Moreover, the results of functional annotation and enriched pathway showed that the DEGs were involved in phagosome (ko04145), lysosome (ko04142), Endocytosis (ko04144), and NOD-like receptor signaling pathway (ko04621). Besides, the Pearson's correlation coefficient (R) between transcriptome sequencing and qPCR was 0.845, which confirmed the reliability of the transcriptome sequencing results and the accuracy of assembly. Furthermore, the expression pattern of 15 candidate DEGs, containing 9 up-regulated and 6 down-regulated DEGs at 3 h, indicated the regulation of DA in physiological functions especially in the immune system. Therefore, these results revealed that DA induced the expressions of membrane receptors or proteins, activated intracellular signaling pathways, regulated cellular and humoral immune systems, controlled antioxidation and apoptosis, and was involved in the regulation of neuroendocrine system. These findings are helpful to promote the understanding on the effects of biogenic amines on physiological functions and regulatory networks of crustacean, and offer a substantial material and foundation for researching the immune response of crustacean.

Evidence that dopamine is involved in neuroendocrine regulation, gill intracellular signaling pathways and ion regulation in Litopenaeus vannamei.

The transport of ions and ammonia in gills may be regulated by neuroendocrine factors. In order to explore the mechanism of dopamine (DA) regulation, we investigated hemolymph neuroendocrine hormones, gill intracellular signaling pathways, ion and ammonia transporters, hemolymph osmolality and ammonia concentration in Litopenaeus vannamei after injection of 10-7 and 10-6 mol DA per shrimp. The data showed a significant increase in crustacean hyperglycemic hormone (CHH) concentration at 1-12 h and a transient significant decrease in corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol concentration under DA stimulation. The up-regulation of guanylyl cyclase (GC) mRNA, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) concentration, together with the down-regulation of DA receptor D4 mRNA and up-regulation of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), diacylglycerol (DAG) and protein kinase C (PKC) concentration suggested the activation of complicated intracellular signaling pathways. The expression of cAMP response element-binding protein (CREB), FXYD2 and 14-3-3 protein mRNA was significantly increased by PKA regulation. The increase in Na+/K+-ATPase (NKA) activity and the stabilization of V-type H+-ATPase (V-rATPase) activity were accompanied by an up-regulation of K+ channel, Na+/K+/2Cl- cotransporter (NKCC), Rh protein and vesicle associated membrane protein (VAMP) mRNA, resulting in an increase in hemolymph osmolality and a decrease in hemolymph ammonia concentration. These results suggest that DA stimulates the secretion of CHH and inhibits the release of cortisol, which activates intracellular signaling factors to facilitate ion and ammonia transport across the gills, and may not affect intracellular acidification.

The molecular mechanism of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by BaP in the scallop Chlamys farreri.

In this study, we cloned the full-length cDNA of the Kelch-like ECH-associated protein 1 (Keap1) from the scallops Chlamys farreri (C. farreri). Sequences alignment and phylogenetic analysis showed that CfKeap1 was highly specific in the scallops, and the amino acid sequence identity value is closer to that in zebrafish Keap1b and Nothobranchius furzeri Keap1b than Keap1a. The highest transcription level of CfKeap1 expression was detected in the digestive glands. The gene expressions of CfKeap1, NF-E2-related nuclear factor 2 (Nrf2), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive glands were evaluated by quantitative real-time PCR (qRT-PCR) after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 µg/L) for 15 days, which indicated that the activation of Nrf2 and Keap1 expression can be significantly induced under BaP exposure. RNA interference (RNAi) experiments were conducted to examine the expression profiles of CfKeap1, Nrf2, antioxidant genes (Cu/Zn-SOD, CAT and GPx), mitogen-activated protein kinase (MAPKs) and protein kinase C (PKC) signaling pathways key genes in digestive glands and gills when exposed to BaP. Results showed that the mRNA level of CfKeap1 was significantly decreased by 60.69% and59.485%. The changes of CfKeap1 and Nrf2 suggested that the enhancement of Keap1 expression stimulating Nrf2 degradation. Furthermore, the expression of antioxidant genes were consistent with the Nrf2 gene, which suggesting that Nrf2-Keap1 signaling pathway is required for the induction of antioxidant genes. Besides, the changes of PKC, c-Jun N-terminal kinase (JNK) and p38 genes expression suggested that PKC and MAPKs signaling pathways played a synergistic role with Nrf2-Keap1 signaling pathway in the anti-oxidative defense system of bivalve molluscs. In conclusion, these data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with Cul3-based E3 Ubiquitin Ligase (E3), and the Nrf2-Keap1 signaling pathway played an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.

Ammonia-N exposure alters neurohormone levels in the hemolymph and mRNA abundance of neurohormone receptors and associated downstream factors in the gills of Litopenaeus vannamei.

Effects of ammonia-N (0.05, 2, 10 and 20 mg l-1) on the neuroendocrine regulation of ammonia transport were investigated in Litopenaeus vannamei The results showed that corticotrophin-releasing hormone, adrenocorticotropic hormone, dopamine, noradrenaline and 5-hydroxytryptamine concentrations in all ammonia-N groups increased significantly between 3 and 12 h. Cortisol increased significantly between 3 and 24 h. All hormones except crustacean hyperglycemic hormone were reduced to control levels. mRNA abundance of guanylyl cyclase increased significantly during the experiment. Dopamine receptor D4 and α2 adrenergic receptor mRNA abundance in treatments decreased significantly at the beginning, and eventually returned to the control level, whereas mRNA abundance of the 5-HT7 receptor increased significantly only within the first 12 h. Changes in protein kinase (PKA, PKG) mRNA abundance were similar to the patterns of biogenic amines and crustacean hyperglycemic hormone, peaking at 6 and 12 h, respectively, whereas PKC mRNA abundance decreased within 24 h. 14-3-3 protein, FXYD2 and cAMP-response element binding protein mRNA abundance increased significantly and peaked at 6 h. ß-catenin and T-cell factor mRNA abundance increased significantly throughout the experiment and peaked at 12 h. The upregulation of Rh protein, K+ channel, Na+/K+-ATPase, V-type H+-ATPase and vesicle associated membrane protein (VAMP) mRNA, together with downregulation of Na+/K+/2Cl- cotransporter mRNA, indicated an adjustment of general branchial ion-/ammonia-regulatory mechanisms. Meanwhile, hemolymph ammonia concentration was significantly increased in most ammonia-N exposure groups. Histological investigation revealed the hepatopancreatic damage caused by ammonia-N. Results suggest that hormones, biogenic amines and Wnt/ß-catenin play a principal role in adapting to ammonia-N exposure and facilitating ammonia transport.

Comparative transcriptome analysis between the short-term stress and long-term adaptation of the Ruditapes philippinarum in response to benzo[a]pyrene.

In order to monitor the pollution of polycyclic aromatic hydrocarbons (PAHs) in the seawater environment, screening biomarkers capable of monitoring PAHs is the focus of many studies. The transcriptomic profiles of the digestive gland tissue from the R. philippinarum groups after the exposure to BaP (4 µg/L) at four time points (0, 0.5, 6 and 15 days) were investigated to globally screen the key genes and pathways involved in the responses to short-term stress and long-term adaptation of BaP resistance. By comparative transcriptome analysis, 233, 282 and 58 differentially expressed genes (DEGs) were identified at 0.5 day, 6 day and 15 day (vs 0 day). The differential expression genes were related to stress response, detoxification metabolic process and innate immunity. DEGs of each group at different stages were clustered in six profiles based on gene expression pattern. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis were used on all genes to determine the biological functions and processes. We selected Multidrug resistance protein 3 (MRP3), transcriptional regulator ATRX-like isoform X2 (ATRX) as biomarker indicator genes for short-term pollution monitoring and NADH dehydrogenase [ubiquinone] 1 (NQO1), Complement C1q-like protein 4 (C1q), Glutathione-S-transferase theta (GST), E3 ubiquitin-protein ligase (E3) for long-term pollution monitoring based on the different expression patterns and the function in detoxification and antioxidant defense system. Besides, the expression of seven genes was measured through Quantitative real-time PCR (qPCR) according to their gene expression patterns which was confirmed by the DGE analysis. Taken together, adoption of transcriptomic analysis to explore the bivalves' mRNA abundance changes and detoxification metabolic mechanism under the BaP stress at different time points can aid the development of sensitive and informed molecular endpoints for application towards ecotoxicogenomic monitoring of bivalves.

Identification of the role of Rh protein in ammonia excretion of the swimming crab Portunus trituberculatus.

In Portunus trituberculatus, a full-length cDNA of Rhesus-like glycoprotein (Rh protein), encoding the entire 478 amino acid protein, has been identified in gills, and plays an essential role in ammonia (NH3/NH4+) excretion. Phylogenetic analysis of Rh-like proteins from crabs was clustered, showing high conservation of the ammonium transporter domain and transmembrane segments essential to the function of Rh protein. Rh protein of P. trituberculatus (PtRh) was detected in all tested tissues, and showed the highest expression in the gills. To further characterize the role of PtRh in ammonia metabolism and excretion, double-stranded RNA-mediated RNA interference of PtRh was employed. Knockdown of PtRh upregulated mRNA expression of ammonia excretion-related genes encoding aquaporin (AQP), K+ channels and vesicle-associated membrane protein (VAMP), increased the activity of Na+/K+-ATPase (NKA) and V-type H+-ATPase (V-ATPase), and initially reduced then elevated the expression of the Na+/H+-exchanger (NHE). dsRNA-mediated reduction in PtRh significantly reduced ammonia excretion rate and increased ammonia and glutamine (Gln) levels in the hemolymph, together with an increase of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activity, indicating a central role for PtRh in ammonia excretion and detoxification mechanisms. Taken together, we conclude that Rh protein is a primary contributor to ammonia excretion of P. trituberculatus, which may be the basis of their ability to inhabit benthic water with high ammonia levels.

Independent and simultaneous effect of crustacean hyperglycemic hormone and dopamine on the hemocyte intracellular signaling pathways and immune responses in white shrimp Litopenaeus vannamei.

Immune responses and intracellular signaling pathways were examined after hemolymph of Litopenaeus vannamei being incubated in Crustacean hyperglycemic hormone (CHH), dopamine (DA) and DA antagonist (Y). The results showed that the effect CHH and CHH + DA + Y on viability of hemocytes were no significant changes compared to the control group. However, in DA, DA + Y and CHH + DA groups, the viability of hemocytes decreased significantly. The phagocytic activity and the antibacterial activity of CHH group were increased significantly within 12h. Whereas the CHH + DA, DA were significantly lower than the control. PO in haemolymph was up-regulated after CHH and DA incubation. The proPO has the opposite change in all groups. In addition, DA + Y, CHH + DA + Y has a similar trend with the DA and CHH respectively. Furthermore, a significant increase of cAMP, CaM and cGMP were found in treatment groups except for the CaM concentration of the CHH group and the cGMP concentration of DA group. There is no significant change observed in the CHH group about CaM concentration. Whereas the cGMP of DA group decreased within 12h. The results suggest that DA could depress the immune responses by cAMP-, CaM-pathways. However, the CHH is on the contrary, which transduced the signals from cAMP, cGMP to PKA, PKC and PKG to enhance the immune response parameters.

The role of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by PAHs in the calm Ruditapes philippinarum.

The NF-E2-related factor 2 (Nrf2) is a master regulator of cellular responses against environmental stresses. In this study we cloned the full-length cDNAs of the RpNrf2 encompassed 2823 bp from the clam Ruditapes philippinarum (R. philippinarum). Sequences alignment and phylogenetic analysis showed that Nrf2 was highly specific in the clams. RpNrf2 expression was detected in gill, digestive gland, mantle and adductor, which the highest transcription level was observed in gill and digestive gland. The gene expressions of RpNrf2, Kelch-like-ECH-associated Protein 1 (Keap1), Cul3-based E3 Ubiquitin Ligase (E3), Glutathione S-transferase (GST-pi), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive gland was evaluated by real-time PCR after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 µg/L) for 15 days, which showed that the expression of Nrf2 significantly increased at day 1 and day 6 after exposure (p < 0.05), and there was a negative relationship between the mRNA levels of Nrf2 and Keap1 that indicates the enhancement of Keap1 expression stimulating Nrf2 degradation. RNA interference experiments were conducted to examine the expression profiles of RpNrf2, antioxidant and detoxification genes (GST-pi, Cu/Zn-SOD, CAT and GPx) and Lipid Peroxidase (LPO) level in digestive gland exposed to BaP. The results showed that the mRNA level of Nrf2 was significantly decreased by 63.2%, and the changes of antioxidant and detoxification genes expression were consistent with the Nrf2 gene suggesting that Nrf2 is required for the induction of antioxidant and detoxification genes. Besides, the LPO levels expressed by malondialdehyde (MDA) contents were significant higher compared with the control group at 72 h post dsRNA-Nrf2 injection. In conclusion, our data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with E3 and Nrf2 signaling pathway plays an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.

Effects of ammonia-N exposure on the concentrations of neurotransmitters, hemocyte intracellular signaling pathways and immune responses in white shrimp Litopenaeus vannamei.

The effects of ammonia-N exposure (transferred from 0.07 to 2, 10 and 20 mg L-1) on the mechanism of neuroendocrine-immunoregulatory network were investigated in Litopenaeus vannamei. The results showed that biogenic amines (dopamine, noradrenaline, 5-hydroxytryptamine) concentrations in treatment groups increased significantly within 12 h. The gene expression of guanylyl cyclase increased significantly from 3 h to 24 h. And dopamine receptor D4 and α2 adrenergic receptor gene expression in treatment groups decreased significantly within 12 h, whereas the mRNA expression of 5-HT7 receptor increased significantly within 3 h and reached the peak levels at 6 h. The second messengers (cAMP, cGMP) and Calmodulin (CaM) increased significantly in treatment groups after 3 h. The concentrations of protein kinases (PKA, PKG) shared a similar trend in cAMP and cGMP which were up-regulated and reached the peak value at 6 h, while the PKC decreased within 3 h and arrived at its bottom at 6 h. The nuclear factor kappa-b and cAMP-response element binding protein mRNA expression levels of treatment shrimps increased sharply and reached maximum values at 6 h. The total hemocyte count, phagocytic activity, antibacterial activity in treatment groups decreased dramatically within 48 h. Whereas the phenoloxidase activities slightly up-regulated. Then it was decreased significantly up to 48 h. α2-macroglobulin activity decreased at the first 3 h-stress. Then they up-regulated significantly in 6 h. The results suggest that there are two crucial neuroendocrine substances (biogenic amine and CHH), which play a principal role in adapting to ammonia-N exposure and cause immune response through cAMP-, CaM- and cGMP-dependent pathways.