The mechanism of reactive oxygen species generation, DNA damage and apoptosis in hemocytes of Litopenaeus vannamei under ammonia nitrogen exposure.

Ammonia-N poses a significant threat to aquatic animals. However, the mechanism of ROS production leading to DNA damage in hemocytes of crustaceans is still unclear. Additionally, the mechanism that cells respond to DNA damage by activating complex signaling networks has not been well studied. Therefore, we exposed shrimp to 0, 2, 10, and 20 mg/L NH4Cl for 0, 3, 6, 12, 24, 48, and 72 h, and explored the alterations in endoplasmic reticulum stress and mitochondrial fission, DNA damage, repair, autophagy and apoptosis. The findings revealed that ammonia exposure led to an increase in plasma ammonia content and neurotransmitter content (DA, 5-HT, ACh), and significant changes in gene expression of PLC and Ca2+ levels. The expression of disulfide bond formation-related genes (PDI, ERO1) and mitochondrial fission-related genes (Drp1, FIS1) were significantly increased, and the unfolded protein response was initiated. Simultaneously, ammonia-N exposure leads to an increase in ROS levels in hemocytes, resulting in DNA damage. DNA repair and autophagy were considerably influenced by ammonia-N exposure, as evidenced by changes in DNA repair and autophagy-related genes in hemocytes. Subsequently, apoptosis was induced by ammonia-N exposure, and this activation was associated with a caspase-dependent pathway and caspase-independent pathway, ultimately leading to a decrease in total hemocytes count. Overall, we hypothesized that neurotransmitters in the plasma of shrimp after ammonia-N exposure bind to receptors on hemocytes membrane, causing endoplasmic reticulum stress through the PLC-IP3R-Ca2+ signaling pathway and leading to mitochondrial fission. Consequently, this process resulted in increased ROS levels, hindered DNA repair, suppressed autophagy, and activated apoptosis. These cascading effects ultimately led to a reduction in total hemocytes count. The present study provides a molecular support for the understanding of the detrimental toxicity of ammonia-N exposure to crustaceans.

Mechanisms of neurocentral-eyestalk-intestinal immunotoxicity in whiteleg shrimp Litopenaeus vannamei under ammonia nitrogen exposure.

Ammonia-N, as the most toxic nitrogenous waste, has high toxicity to marine animals. However, the interplay between ammonia-induced neuroendocrine toxicity and intestinal immune homeostasis has been largely overlooked. Here, a significant concordance of metabolome and transcriptome-based "cholinergic synapse" supports that plasma metabolites acetylcholine (ACh) plays an important role during NH4Cl exposure. After blocking the ACh signal transduction, the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the cerebral ganglia increased, while the release of NPF in the thoracic ganglia and NE in the abdominal ganglia, and crustacean hyperglycemic hormone (CHH) and neuropeptide F (NPF) in the eyestalk decreased, finally the intestinal immunity was enhanced. After bilateral eyestalk ablation, the neuroendocrine system of shrimp was disturbed, more neuroendocrine factors, such as corticotropin releasing hormone (CRH), adrenocorticotropic-hormone (ACTH), ACh, DA, 5-HT, and norepinephrine (NE) were released into the plasma, and further decreased intestinal immunity. Subsequently, these neuroendocrine factors reach the intestine through endocrine or neural pathways and bind to their receptors to affect downstream signaling pathway factors to regulate intestinal immune homeostasis. Combined with different doses of ammonia-N exposure experiment, these findings suggest that NH4Cl may exert intestinal toxicity on shrimp by disrupting the cerebral ganglion-eyestalk axis and the cerebral ganglion-thoracic ganglion-abdominal ganglion axis, thereby damaging intestinal barrier function and inducing inflammatory response.

Mechanisms of ammonotelism, epithelium damage, cellular apoptosis, and proliferation in gill of Litopenaeus vannamei under NH4Cl exposure.

Excessive ammonia-N in coastal environment and aquaculture threatens the health of marine organisms. To explore the mechanism of gill damage induced by ammonia-N, transcriptome of Litopenaeus vannamei 's gill was carried out under 20 mg/L NH4Cl for 0, 6, and 48 h. K-means clustering analysis suggested that ammonia excretion and metabolism-related genes were elevated. GO and KEGG enrichment analysis suggested that glycosyltransferase activity and amino acid metabolism were affected by ammonia. Moreover, histological observation via three staining methods gave clues on the changes of gill after ammonia-N exposure. Increased mucus, hemocyte infiltration, and lifting of the lamellar epithelium suggested that gill epithelium was suffering damage under ammonia-N stress. Meanwhile, the composition of extracellular matrix (ECM) in connective tissue changed. Based on the findings of transcriptomic and histological analysis, we further investigated the molecular mechanism of gill damage under multiple concentrations of NH4Cl (0, 2, 10, 20 mg/L) for multiple timepoints (0, 3, 6, 12, 24, 48, 72 h). First, ammonia excretion was elevated via ion channel, transporter, and exocytosis pathways, but hemolymph ammonia still kept at a high level under 20 mg/L NH4Cl exposure. Second, we focused on glycosaminoglycan metabolism which was related to the dynamics of ECM. It turned out that the degradation and biosynthesis of chondroitin sulfate (CS) were elevated, suggesting that the structure of CS might be destructed under ammonia-N stress and CS played an important role in maintaining gill structure. It was enlightening that the destructions occurred in extracellular regions were vital to gill damage. Third, ammonia-N stress induced a series of cellular responses including enhanced apoptosis, active inflammation, and inhibited proliferation which were closely linked and jointly led to the impairment of gill. Our results provided some insights into the physiological changes induced by ammonia-N and enriched the understandings of gill damage under environmental stress.

The immunotoxicity mechanism of hemocytes in Chlamys farreri incubated with noradrenaline and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide alone or in combination.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is the active intermediate metabolite of benzo[a]pyrene (B[a]P) and is considered the ultimate immunotoxicant. The neuroendocrine immunoregulatory network of bivalves is affected under pollutant stress. Besides, bivalves are frequently affected by pollutants in marine environments, yet the combined effects of neuroendocrine factors and detoxification metabolites on bivalves under pollutant stress and the signal pathways that mediate this immunoregulation are not well understood. Therefore, we incubated the hemocytes of Chlamys farreri with the neuroendocrine factor noradrenaline (NA) and the B[a]P detoxification metabolite BPDE, alone or in combination, to examine the immunotoxic effects of NA and BPDE on the hemocytes in C. farreri. Furthermore, the effects of NA and BPDE on the hemocyte signal transduction pathway were investigated by assessing potential downstream targets. The results revealed that NA and BPDE, alone or in combination, resulted in a significant decrease in phagocytic activity, bacteriolytic activity and the total hemocyte count. In addition, the immunotoxicity induced by BPDE was further exacerbated by co-treatment with NA, and the two showed synergistic effects. Analysis of signaling pathway factors showed that NA activated G proteins by binding to α-AR, which transmitted information to the Ca2+-NF-κB signaling pathway to regulate the expression of phagocytosis-associated proteins and regulated cytokinesis through the cAMP signaling pathway. BPDE could activate PTK and affect phagocytosis and cytotoxicity proteins through Ca2+-NF-κB signal pathway, also affect the regulation of phagocytosis and cytotoxicity by inhibiting the AC-cAMP-PKA pathway to down-regulate the expression of NF-κB and CREB. In addition, BPDE and NA may affect the immunity of hemocytes by down-regulating phagocytosis-related proteins through inhibition of the lectin pathway, while regulating the expression of cytotoxicity-related proteins through the C-type lectin. In summary, immune parameters were suppressed through Ca2+ and cAMP dependent pathways exposed to BPDE and the immunosuppressive effects were enhanced by the neuroendocrine factor NA.

Highly Active Pyrochlore-Type Praseodymium Ruthenate Electrocatalyst for Efficient Acid-Water Oxidation.

To produce directly combustible hydrogen from water, highly active, acid-resistant, and economical catalysts for oxygen evolution reaction (OER) are needed. An electrocatalyst based on praseodymium ruthenate (Pr2Ru2O7) is presented here that greatly outperforms RuO2 for acid-water oxidation. Specifically, at 10 mA cm-2, this electrocatalyst presents a low overpotential (η) of 213 mV and markedly superior stability. Moreover, Pr2Ru2O7 presents a significant rise in turnover frequency (TOF) and a highly intrinsic mass activity of 1618.8 A gRu-1 (η = 300 mV), exceeding the most commonly reported acid OER catalysts. Density functional theory calculations and electronic structure study demonstrate that the Ru 4d-band center related to the longer Ru-O bond with a large radius of Pr ion in this pyrochlore is lower than that in RuO2, which would optimize the binding between the adsorbed oxygen species and catalytic metal sites and enhance the catalytic intrinsic activity.

The immunotoxicity mechanism of NH4Cl exposure to Litopenaeus vannamei based on the cerebral ganglion-eyestalk-haemocytes axis.

Ammonia nitrogen, as a water environmental toxin, poses a potential threat to aquatic animals. Although NH4Cl stress is known to cause immunotoxicity, mechanistic pathways linking stress networks in the neuroendocrine system to immunotoxicity remain poorly understood. In this study, firstly, using transcriptome analysis of cerebral ganglion and eyestalk in shrimp, we identified significant changes in genes related to biogenic amines, acetylcholine, crustacean hyperglycemic hormones, and neuropeptide F. Additionally, expression patterns of neuroendocrine factors in different tissues of shrimp were evaluated to explore the sources of these factors. Here, we showed that NH4Cl exposure activates acetylcholine (ACh) neurons in cerebral ganglion of shrimp and dramatically upregulates high affinity choline transporter 1 (ChT1) gene expression. The knockdown of ChT1 gene enhanced the immunity of haemocytes in shrimp compared with saline and GFP dsRNA groups. And after eyestalk ablation, the levels of neuroendocrine factors in the cerebral ganglion and thoracic ganglion were disturbed, and haemocytes parameters induced by NH4Cl were significantly decreased. Combined with different doses of NH4Cl exposure experiments, we demonstrated that: (1) In a short period of NH4Cl exposure, the neuroendocrine factors CRH-ACTH-cortisol and 5-HT-DA in the cerebral ganglion-eyestalk axis of shrimp play a major role in regulating haemocytes immunity; (2) With the prolongation of exposure, the immunotoxicity induced by NH4Cl was mainly due to the release of more ACh in the cerebral ganglion, which promoted the release of NPF in the thoracic ganglion, and CHH and NPF in the eyestalk, as well as weakened the effect of biogenic amines. Subsequently, these neuroendocrine factors regulate immunity through intracellular signaling pathways. Collectively, these results established a new mechanism that NH4Cl might directly regulate haemocytes immunotoxicity through the cerebral ganglion and thoracic ganglion; or through the cerebral ganglion-eyestalk axis or cerebral ganglion-thoracic ganglion axis cause haemocytes immunotoxicity.

Potential molecular mechanism underlying the harmed haemopoiesis upon Benzo[a]pyrene exposure in Chlamys farreri.

Benzo[a]pyrene (B[a]P), a ubiquitous contamination in the marine environments, has the potential to impact the immune response of bivalves by affecting the hemocyte parameters, especially total hemocyte count (THC). THC is mainly determined by haematopoietic mechanisms and apoptosis of hemocytes. Many studies have found that B[a]P can influence the proliferation and differentiation of hemocytes. However, the link between the toxic mechanisms of haematopoietic and environmental pollutants is not explicitly stated. This study is to investigate the toxic effects of B[a]P on haematopoietic mechanisms in C. farreri. Through the tissue expression distribution experiment and EDU assay, gill is identified as a potential haematopoietic tissue in C. farreri. Subsequently, the scallops were exposed to B[a]P (0.05, 0.5, 5 µg/L) for 1d, 3d, 6d, 10d and 15d. Then BPDE content, DNA damage, gene expression of haematopoietic factors and haematopoietic related pathways were determined in gill and hemocytes. The results showed that the expression of CDK2 was significantly decreased under B[a]P exposure through three pathways: RYR/IP3-calcium, BPDE-CHK1 and Notch pathway, resulting in cell cycle arrest. In addition, B[a]P also significantly reduced the number of proliferating hemocytes by affecting the Wnt pathway. Meanwhile, B[a]P can significantly increase the content of ROS, causing a downregulation of FOXO gene expression. The gene expression of Notch pathway and ERK pathway was also detected. The present study suggested that B[a]P disturbed differentiation by multiple pathways. Furthermore, the expression of SOX11 and CD9 were significantly decreased, which directly indicated that differentiation of hemocytes was disturbed. In addition, phagocytosis, phenoloxidase activity and THC were also significant decreased. In summary, the impairment of haematopoietic activity in C. farreri further causes immunotoxicity under B[a]P exposure. This study will improve our understanding of the immunotoxicity mechanism of bivalve under B[a]P exposure.

Potential pathway and mechanisms underlining the immunotoxicity of benzo[a]pyrene to Chlamys farreri.

The long-distance migration of polycyclic aromatic hydrocarbons (PAHs) promotes their release into the marine environment, posing a serious threat to marine life. Studies have shown that PAHs have significant immunotoxicity effects on bivalves, but the exact mechanism of immunotoxicity remains unclear. This paper aims to investigate the effects of exposure to 0.4, 2, and 10 µg/L of benzo(a)pyrene (B[a]P) on the immunity of Chlamys farreri under environmental conditions, as well as the potential molecular mechanism. Multiple biomarkers, including phagocytosis rate, metabolites, neurotoxicity, oxidative stress, DNA damage, and apoptosis, were adopted to assess these effects. After exposure to 0.4, 2, and 10 µg/L B[a]P, obvious concentration-dependent immunotoxicity was observed, indicated by a decrease in the hemocyte index (total hemocyte count, phagocytosis rate, antibacterial and bacteriolytic activity). Analysis of the detoxification metabolic system in C. farreri revealed that B[a]P produced B[a]P-7,8-diol-9,10-epoxide (BPDE) through metabolism, which led to an increase in the expression of protein tyrosine kinase (PTK). In addition, the increased content of neurotransmitters (including acetylcholine, γ -aminobutyric acid, enkephalin, norepinephrine, dopamine, and serotonin) and related receptors implied that B[a]P might affect immunity through neuroendocrine system. The changes in signal pathway factors involved in immune regulation indicated that B[a]P interfered with Ca2+ and cAMP signal transduction via the BPDE-PTK pathway or neuroendocrine pathway, resulting in immunosuppression. Additionally, B[a]P induced the increase in reactive oxygen species (ROS) content and DNA damage, as well as an upregulation of key genes in the mitochondrial pathway and death receptor pathway, leading to the increase of apoptosis rate. Taken together, this study comprehensively investigated the detoxification metabolic system, neuroendocrine system, and cell apoptosis to explore the toxic mechanism of bivalves under B[a]P stress.

Toxicological mechanism of ammonia-N on haematopoiesis and apoptosis of haemocytes in Litopenaeus vannamei.

Ammonia, as an important pollutant, contributed to the reduction of immunity, disruption of physiology in animals. RNA interference (RNAi) was performed to understand the function of astakine (AST) in haematopoiesis and apoptosis in Litopenaeus vannamei under ammonia-N exposure. Shrimps were exposed to 20 mg/L ammonia-N from 0 to 48 h with injection of 20 µg AST dsRNA. Further, shrimps were exposed to 0, 2, 10 and 20 mg/L ammonia-N also from 0 to 48 h. The results showed that the total haemocytes count (THC) decreased under ammonia-N stress and the knockdown of AST resulted in a further decrease of THC, suggesting that 1) the proliferation was decreased through the reduction of AST and Hedgehog, the differentiation was interfered by Wnt4, Wnt5 and Notch, and the migration was inhibited by the decrease of VEGF; 2) oxidative stress was induced under ammonia-N stress, leading to the increase of DNA damage with the up-regulated gene expression of death receptor, mitochondrial and endoplasmic reticulum stress pathways; 3) the changes of THC resulted from the decrease of proliferation, differentiation and migration of haematopoiesis cells and the increase of apoptosis of haemocytes. This study helps to deepen our understanding of risk management in shrimp aquaculture.

Effects of dietary trans-cinnamaldehyde on growth performance, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei.

The purpose of this study was to evaluate the effects of dietary trans-cinnamaldehyde (TC) on growth performance, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei. Shrimp were randomly divided into 4 groups, with 3 replicants in each group and 70 shrimp in each replicant. The contents of TC in the four groups were 0, 0.4, 0.8 and 1.2 g kg-1, respectively. Samples were taken after 56 days, followed by a 7-day vibrio harveyi challenge experiment. The results showed that TC significantly improved the growth performance by enhancing the activity of digestive enzymes in shrimp (P < 0.05). TC also reduced the content of crude fat (P < 0.05). The addition of TC to the diet attenuated lipid deposition, as evidenced by a reduction in the content of crude fat and a decrease in plasma levels of cholesterol and triglycerides (P < 0.05). The expression of key genes for fatty acid and triglycerides synthesis were significantly down-regulated and key genes for fatty acid ß-oxidation were significantly up-regulated (P < 0.05). In addition, the immune response and antioxidant capability of shrimp were significantly enhanced by the addition of TC to the diet (P < 0.05). Meanwhile, TC could improve intestinal health by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria, but had no significant effect on alpha diversity and beta diversity (P > 0.05). In addition, the results of histopathological sections and plasma transaminase studies showed that TC could improve the health status of hepatopancreas and was a safe nutritional supplement. After the 7-day Vibrio harveyi challenge, the cumulative mortality of shrimp decreased with increasing levels of dietary TC compared with control group (P < 0.05). These results suggested that TC could be used as a nutritional supplement for shrimp to enhance disease resistance and reduce lipid accumulation.

Effects of ammonia-N stress on molecular mechanisms associated with immune behavior changes in the haemocytes of Litopenaeus vannamei.

High concentration of ammonia-N will inhibit the immune defense of aquatic animals. The neuroendocrine-immune (NEI) regulatory mechanism under ammonia-N stress has been systematically studied, but the final response mechanism of ammonia-N affecting the immune system remains unclear. To investigate the relationship among immune factors of Litopenaeus vannamei (L. vannamei) exposed to 0, 2, 10 and 20 mg/L ammonia-N, the determination of complement components, C-type lectins, proPO system, signal transduction pathway and phagocytosis as well as exocytosis were performed. The results showed that the expressions of complement components including C1q, MBL, ficolin and alpha-2 macroglobulin (A2M) and the complement receptor integrin were decreased significantly in ammonia-N treatment groups at 6,12 and 24 h. C-type lectins and signal transduction factors changed significantly. The decrease of phagocytosis-related genes and phagocytic activity were similar to the changes of complement components, C-type lectins and the signal pathway. The mRNA abundance of exocytosis-related genes was significantly down-regulated under ammonia-N exposure. Correspondingly, significantly changes occurred in the expressions of PPAE and PPO3, immune factors-related genes (Pen3, crustin, stylicins, ALFs and LYC) and inflammatory factors (HSP90, TNFα, IL-16) in haemocytes. Eventually, the serine proteinase activity, PO activity, antibacterial activity and bacteriolytic activity in plasma were decreased significantly. In addition, we speculated that under ammonia-N stress, phagocytosis and exocytosis were affected by complement components, and C-type lectins through intracellular signal transduction pathway. Complement components may involve in the regulation of proPO-activating system to response to ammonia-N stress. This study helped to further understanding the relationship among immune factors of crustacean in response to environmental stress, which implied that when it comes to the decrease of immunity affected by environmental stress, we should not only focus on the mechanism of upstream neuroendocrine response, but also pay attention to the role of immune factors.

Immunotoxicity pathway and mechanism of benzo[a]pyrene on hemocytes of Chlamys farreri in vitro.

Benzo[a]pyrene (B[a]P), a typical PAHs widely existing in the marine environment, has been extensively studied for its immunotoxicity due to its persistence and high toxicity. Nevertheless, the immunotoxicity mechanism remain incompletely understood. In this study, isolated hemocytes of Chlamys farreri were exposed at three concentrations of B[a]P (5, 10 and 15 µg/mL), and the effects of B[a]P on detoxification metabolism, signal transduction, humoral immune factors, exocytosis and phagocytosis relevant proteins and immune function at 0, 6, 12, 24 h were studied. Results illustrated the AhR, ARNT and CYP1A1 were significantly induced by B[a]P at 12 h. Additionally, the content of B[a]P metabolite BPDE increased in a dose-dependent manner with pollutants. Under B[a]P stimulation, the expressions of PTK (Src, Fyn) and PLC-Ca2+-PKC pathway gene increased significantly, while the transcription level of AC-cAMP-PKA pathway gene decreased remarkably. Additionally, the expressions of nuclear transcription factors (CREB, NF-κB), complement system genes and C-type lectin genes up-regulated obviously. The gene expressions of phagocytosis and exocytosis related proteins were also notably affected. 5 µg/mL B[a]P could promote phagocytosis in a transitory time, but with the increase of exposure time and concentration of B[a]P, the phagocytosis, antibacterial and bacteriolytic activities gradually decreased. These results indicated that similar to vertebrates, BPDE, the metabolite of B[a]P, mediated downstream signal transduction via PTK in bivalves. The declined of the immune defense ability of hemocytes might be closely related to the inhibition of AC-cAMP-PKA pathway and the imbalance of intracellular Ca2+ pathway. In addition, the results manifested that complement and lectin systems play a significant role in regulating immune response. In this study, the direct relationship between detoxification metabolism and immune signal transduction in bivalves under B[a]P stress was demonstrated for the first time, which provided important information for the potential molecular mechanism of B[a]P-induced immune system disorder in bivalves.

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 biogenic amines on the immune response and immunoregulation mechanism in hemocytes of Litopenaeus vannamei in vitro.

The effects of three biogenic amines (DA, NE and 5-HT) on the immune signaling pathway and immune response of hemocytes in shrimp were investigated through in vitro experiments. The results showed that the G protein effectors (AC, PLC), the second messengers (cAMP, DAG), Calmodulin (CaM) and protein kinases (PKA, PKC) of DA and NE groups shared a similar trend in which all intracellular signaling factors increased significantly and reached the maximum at 3 h. The concentrations of AC, cAMP and PKA in 5-HT groups decreased significantly compared with the control group, while the concentrations of PLC, CaM, DAG and PKC in 5-HT groups increased markedly. The immune parameters such as total hemocyte count (THC), cell viability, antibacterial activity and bacteriolytic activity, as well as prophenoloxidase (proPO) activity in three biogenic amines groups decreased significantly, while the phenoloxidase (PO) activity increased significantly. The phagocytic activity in DA and NE groups decreased significantly, while that in 5-HT groups increased markedly and reached the highest level at 1 h. Among these three biogenic amines, DA showed the strongest effect on the immune activity of the hemocytes, whereas 5-HT had the least effect. In addition, we speculated that DA and NE might regulate phagocytosis by activating intracellular AC-cAMP-PKA pathway while 5-HT might inhibit intracellular AC-cAMP-PKA pathway. Moreover, the activation of proPO system might be related to PLC-DAG-PKC and PLC-CaM pathway.

Comparative transcriptome analysis of eyestalk from the white shrimp Litopenaeus vannamei after the injection of dopamine.

Dopamine (DA) is a crucial neuroendocrine-immune factor regulating the stress response of Litopenaeus vannamei. To understand the regulatory mechanisms of DA in L. vannamei, the eyestalks of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were chosen to perform transcriptome analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of transcriptome data and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, and 12 h) after DA injection. The transcriptome data showed that 79,434 unigenes were generated. Therein 204 and 434 DEGs were obtained at 3 and 12 h respectively. Besides, the results of enriched pathways showed that the DEGs were involved in GnRH signaling pathway (ko04912) dopaminergic synapse (ko04728), glutamatergic synapse (ko04724), synapse (GO:0045202), synaptic vesicle transport (GO:0048489). Moreover, the Pearson's correlation coefficient (R) of 13 candidate DEGs between transcriptome sequencing and RT-PCR was 0.948, which confirmed the reliability and the accuracy of the transcriptome sequencing results. Furthermore, the results of interaction analysis uncovered 4 pairs of DEGs between eyestalks and hemocytes. Therefore, these results revealed that DA promoted the sensitivity of eyestalk to gonadal related hormones, induced the expression of neuroendocrine factor, enhanced the synaptic behavior and neural signal transduction, regulated immune systems and antioxidation, inhibited the visual function, and promoted the molting. These findings will benefit to foster the understanding on the effects of biogenic amines on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response.

Effects of feeding level and C/N ratio on water quality, growth performance, immune and antioxidant status of Litopenaeus vannamei in zero -water exchange bioflocs-based outdoor soil culture ponds.

Two trials were conducted as follows: the first trial was a 90-day experiment to determine the effects of reducing feeding level on shrimp status; the second trial (90 days) is based on the first trial to explore the suitable C/N ratio of biological flocs for shrimp culture in outdoor soil ponds. Results showed that the BFV levels increased gradually and then tended to be stable in the treatment groups. Concentrations of TAN and NO2-N were maintained low level in each treatment pond during experimental period. The final body weight, biology body length and yield of the shrimp in each trial with no significantly different (P > 0.05) while food coefficient and THC of the shrimp in 70% feeding level and C/N12 treatment were slightly lower than in the 100% feeding level and C/N16 treatment respectively (p < 0.05). The antibacterial activity and bacteriolytic activity in C/N16 treatment group were higher than in C/N12 (p < 0.05), while there were no significant difference between the two feeding levels (70%,100%) (P > 0.05). The shrimp in 70% feeding level and C/N12 treatment had the higher T-AOC in both the plasma and the hepatopancreas when compared with 100% feeding level and C/N16 treatment group (p < 0.05). The SOD activity of plasma in 70% treatment group was higher than in 100% (p < 0.05), while it was no significant difference between the two C/N ratios (12,16) in both the plasma and the hepatopancreas (P > 0.05). The effects of two feeding levels and C/N ratios on the GSH level and the ratio of GSH/GSSG in the plasma and the hepatopancreas of shrimp showed no significant difference (P > 0.05). The results showed that 70% feeding level and C/N12 ratio could provide adequate nutrition for shrimp to maintain a normal physical health status with the presence of bioflocs.

Crustacean hyperglycemic hormone (CHH) regulates the ammonia excretion and metabolism in white shrimp, Litopenaeus vannamei under ammonia-N stress.

To understand the adaptation of Litopenaeus vannamei to high environmental ammonia-N, RNA interference was used to investigate the function of crustacean hyperglycemic hormone (CHH) in the physiological process of neuroendocrine signaling transduction, and ammonia excretion and metabolism. The shrimp were exposed to 25 mg/L NH4Cl and injected with 20 µg/shrimp CHH dsRNA for 72 h. The results showed that hemolymph ammonia content increased under ammonia-N stress and further increased after CHH knockdown, suggesting that CHH can promote ammonia excretion. Moreover, after CHH knockdown, the levels of CHH, DA, and Wnts decreased significantly, the expression of receptor GC, DA1R, Frizzled and LRP 5/6 also decreased, while DA4R increased remarkably. Moreover, PKA and PKG decreased, while PKC markedly increased, and nuclear transcription factors (CREB and TCF) as well as effector proteins (ß-catenin, FXYD2, and 14-3-3) were significantly downregulated. Furthermore, ammonia transporters Na+/K+-ATPase (NKA), K+channel, Rh protein, AQP, V-ATPase, and VAMP decreased significantly, while Na+/H+ exchangers (NHE) and Na+/K+/2Cl- cotransporter (NKCC) increased significantly. These results suggest that CHH regulates ammonia excretion in three ways: 1) by mainly regulating ion channels via PKA, PKC, and PKG signaling pathways; 2) by activating related proteins primarily through Wnt signaling pathway; and 3) by exocytosis, mostly induced by the PKA signaling pathway. In addition, the levels of Gln, uric acid, and urea increased in accordance with the activities of GDH/GS, XDH, and arginase, respectively, suggesting that ammonia excretion was inhibited but ammonia metabolism was slightly enhanced. This study deepens our understanding of the mechanism by which crustaceans respond to high environmental ammonia-N.

Effects of dopamine on immune signaling pathway factors, phagocytosis and exocytosis in hemocytes of Litopenaeus vannamei.

Dopamine (DA) is an important neuroendocrine factor, which can act as neurotransmitter and neurohormone. In this study, we explored the immune defense mechanism in Litopenaeus vannamei with injection of dopamine at 10-7 and 10-6 mol shrimp-1, respectively. The genes expressions of dopamine receptor (DAR), G proteins (Gs, Gi, Gq), phagocytosis and exocytosis-related proteins, as well as intracellular signaling pathway factors, and immune defense parameters were measured. Results showed that mRNA expression levels of dopamine receptor D4 (D4), Gi, nuclear transcription factors and exocytosis-related proteins decreased significantly and reached the minimum at 3 h, while the genes expressions of Gs, Gq and phagocytosis-related proteins reached the highest and lowest levels at 3 h and 6 h, respectively. The second messenger synthetases increased significantly in treatment groups within 3 h. Simultaneously, the second messengers and protein kinases shared a similar trend, which were significantly elevated and reached the peak value at 3 h. Ultimately lead to the total hemocyte count (THC), proPO activity and phagocytic activity decreased significantly, reaching minimum values at 3 h, 3 h and 6 h, respectively. While PO activity showed obvious peak changes, which maximum value reached at 3 h. These results suggested that DA receptor could couple with G protein after DA injection and might regulate immunity through cAMP-PKA, DAG-PKC or CaM pathway.

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.