Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric ("per-EV") quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age-related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let-7a, miR-21, and miR-92a) on CHO cell growth and viability. Let-7a-abundant in CHO EVs from stressed cultures-reduced CHO cell viability, while miR-92a-abundant in CHO EVs from unstressed cultures-promoted cell survival. Overexpression of miR-21 had a slight detrimental impact on CHO cell growth and viability during late exponential-phase culture, an unexpected result based on the reported antiapoptotic role of miR-21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro- and antiapoptotic effects on target cells, depending on the conditions under which they were produced.

Implications of ammonia stress for the pathogenicity of Shewanella spp. in Oreochromis niloticus: effects on hematological, biochemical, immunological, and histopathological parameters.

Environmental stressors (such as ammonia) in aquaculture could increase the risk of pathogenicity, posing a more severe threat to farmed fish. The aim of this study was to investigate the effects of ammonia stress on the pathogenicity of Shewanella spp. in Oreochromis niloticus. First, a 96-hour static test was used to determine the median lethal concentration (LC50) of unionized ammonia to Nile tilapia. After 96 h of exposure, the Un-ionized ammonia (UIA) LC50 was estimated to be 4.26 mg/L. Second, an experiment was conducted to test the effect of unionized ammonia stress on the pathogenicity of Shewanella spp. in O. niloticus for 30 days. A study involved 180 fish divided into six groups, with the first group serving as a control. The second group (AMN1/10) and the third group (AMN1/20) were not challenged and were exposed to 1/10 (0.42 mg/L) and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. Then 0.2 mL (0.14 × 105) of Shewanella spp. was intraperitoneally injected into the fourth (SH), fifth (SH + AMN1/10), and sixth (SH + AMN1/20) groups, which were subjected to 0, 1/10 (0.42 mg/L), and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. The survival rate, hematological indices, immunological parameters, and antioxidant activity of the fish significantly decreased when they were exposed to ammonia and Shewanella infection separately or together. Histopathological changes were also observed in the kidney and liver. Furthermore, both individual and combined exposures significantly altered renal and hepatic function, with notable increases in glucose and cortisol levels, as well as in the expression of proinflammatory cytokine genes (TNF-α and IL-1ß). However, the detrimental effects of co-exposure to ammonia stress and Shewanella infection were greater than those of separate exposures. As a result, we may say that increased ammonia concentrations enhance the infection of Shewanella spp. These findings could contribute to a better understanding of Shewanella infection in Nile tilapia.

Comparing the mechanisms of syntrophic volatile fatty acids oxidation and methanogenesis recovery from ammonia stress in regular and biochar-assisted anaerobic digestion: Different roads lead to the same goal.

Biochar has been recognized as a promising additive to mitigate ammonia inhibition during syntrophic methanogenesis, while the key function of biochar in this process is still in debates. This study clarified the distinct mechanisms of syntrophic volatile fatty acids -oxidizing and methanogenesis recovery from ammonia inhibition in regular and biochar-assisted anaerobic digestion. Under 5 g/L ammonia stress, adding biochar shortened the methanogenic lag time by 10.9% and dramatically accelerated the maximum methane production rate from 60.3 to 94.7 mLCH4/gVSsludge/d. A photometric analysis with a nano-WO3 probe revealed that biochar enhanced the extracellular electron transfer (EET) capacity of suspended microbes (Pearson's r = -0.98), confirming that biochar facilitated methanogenesis by boosting EET between syntrophic butyrate oxidizer and methanogens. Same linear relationship between EET capacity and methanogenic rate was not observed in the control group. Microbial community integrating functional genes prediction analysis uncovered that biochar re-shaped syntrophic partners by enriching Constridium_sensu_stricto/Syntrophomonas and Methanosarcina. The functional genes encoding Co-enzyme F420 hydrogenase and formylmethanofuran dehydrogenase were upregulated by 1.4-2.3 times, consequently enhanced the CO2-reduction methanogenesis pathway. Meanwhile, the abundances of gene encoding methylene-tetrahydrofolate transformation, a series of intermediate processes involved in acetate oxidation, in the biochar-assisted group were 28.2-63.7% higher than these in control group. Comparatively, Methanosaeta played a pivotal role driving aceticlastic methanogenesis in the control group because the abundance of gene encoding acetyl-CoA decarbonylase/synthase complex increased by 1.9 times, suggesting an aceticlastic combining H2-based syntrophic methanogenesis pathway was established in control group to resist ammonia stress. A 2nd period experiment elucidated that although depending on distinct mechanisms, the volatile fatty acid oxidizers and methanogens in both groups developed sustained and stable strategies to resist ammonia stress. These findings provided new insights to understand the distinct methanogenic recovery strategy to resist toxic stress under varied environmental conditions.

The microRNomes of Chinese hamster ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress.

A new area of focus in Chinese hamster ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs or miRNA) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR-92a and miR-23a under normal culture conditions, in contrast to the high enrichment in let-7 family miRs (let-7c, let-7b, and let-7a) under both stress conditions. The latter suggests a preserved stress-responsive function of the let-7 miR family, one of the most highly preserved miR families across species, where among other functions, let-7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected-based on their profound enrichment-important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.

Essential role of the Cytochrome P450 2 (CYP2) in the mud crab Scylla paramamosain antioxidant defense and immune responses.

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Transcriptomic analysis revealed the dynamic response mechanism to acute ammonia exposure in the ivory shell, Babylonia areolata.

The ivory shell (Babylonia areolata) is an economically important shellfish in tropical and subtropical regions, but its intensive culture and biological characteristic of hiding in the sandy substrate make it highly susceptible to ammonia stress. In this study, we investigated the dynamic changes in histopathology, oxidative stress, and transcriptome of the ivory shell at different time points under high concentration (60 mg/L) ammonia exposure. With prolonged exposure to stress, vacuoles appeared in the hepatopancreas while cell volume and intercellular space increased. The activities of superoxide dismutase (SOD) and catalase (CAT) decreased significantly under high concentrations of ammonia-induced stress while malondialdehyde (MDA) levels increased significantly. Integrated analysis of differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA), and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that lipid transport primarily contributed to maintaining cellular homeostasis during the early stage of stress (6 and 12 h). Subsequently, a significant upregulation of oxidation-reduction reactions occurred at the middle stage (24 h), leading to oxidative stress. Finally, during the later stage (48 h), metabolic decomposition provided energy for survival maintenance. Additionally, lysosome and apoptosis were identified as potential key pathways in response to acute ammonia toxicity. Overall, our findings suggest that ivory shells can respond to acute ammonia toxicity via immune and antioxidant defense mechanisms but sustained high concentrations may cause irreversible damage. This study provides valuable insights into the response mechanism of mollusks towards ammonia and serves as a data reference for breeding ammonia-tolerant varieties of ivory shells.

Physiological Responses of Methanosarcina barkeri under Ammonia Stress at the Molecular Level: The Unignorable Lipid Reprogramming.

Acetotrophic methanogens' dysfunction in anaerobic digestion under ammonia pressure has been widely concerned. Lipids, the main cytomembrane structural biomolecules, normally play indispensable roles in guaranteeing cell functionality. However, no studies explored the effects of high ammonia on acetotrophic methanogens' lipids. Here, a high-throughput lipidomic interrogation deciphered lipid reprogramming in representative acetoclastic methanogen (Methanosarcina barkeri) upon high ammonia exposure. The results showed that high ammonia conspicuously reduced polyunsaturated lipids and longer-chain lipids, while accumulating lipids with shorter chains and/or more saturation. Also, the correlation network analysis visualized some sphingolipids as the most active participant in lipid-lipid communications, implying that the ammonia-induced enrichment in these sphingolipids triggered other lipid changes. In addition, we discovered the decreased integrity, elevated permeability, depolarization, and diminished fluidity of lipid-supported membranes under ammonia restraint, verifying the noxious ramifications of lipid abnormalities. Additional analysis revealed that high ammonia destabilized the structure of extracellular polymeric substances (EPSs) capable of protecting lipids, e.g., declining α-helix/(ß-sheet + random coil) and 3-turn helix ratios. Furthermore, the abiotic impairment of critical EPS bonds, including C-OH, C═O-NH-, and S-S, and the biotic downregulation of functional proteins involved in transcription, translation, and EPS building blocks' supply were unraveled under ammonia stress and implied as the crucial mechanisms for EPS reshaping.

Effects of partial replacement of dietary flour meal with seaweed polysaccharides on the resistance to ammonia stress in the intestine of hybrid snakehead (Channa maculatus ♀ × Channa argus ♂).

The purpose of this study was to evaluate the effects of partial replacement of dietary flour meal with seaweed polysaccharides on survival rate, histology, intestinal oxidative stress levels, and expression of immune-related genes in hybrid snakeheads under acute ammonia stress. Four experimental diets were set: (C) basal diet with 0% of seaweed polysaccharides as the control group, (MR) basal diet with 10% of seaweed polysaccharides, (HR) basal diet with 15% of seaweed polysaccharides, (HF) basal diet with 10% of fish oil. After 60 days of feeding, fish fed with the diet of C group were sampled as the control group, and other fish were exposed to ammonia nitrogen for 48 h. Two concentrations of total ammonia nitrogen (TAN) were used in this study: 120 mg/L TAN (low concentration exposure group), and 1200 mg/L TAN (high concentration exposure group). After exposure to ammonia nitrogen for 48 h, fish were sampled. The results indicated that adding seaweed polysaccharides to the diet could improve the survival rate of hybrid snakeheads under high concentration of ammonia stress. Histopathological analysis demonstrated multiple abnormalities in gills and intestines after exposure to two concentrations of TAN. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and lactate dehydrogenase (LDH) were all increased in the MR group under two concentrations of TAN stress. The mRNA abundance of immune-related genes in fish intestinal tissues was significantly induced or inhibited. These results suggested that partial replacement of dietary flour meal with seaweed polysaccharides improved the ability of hybrid snakeheads to resist ammonia stress.

Effects of dietary tea tree oil on the growth, physiological and non-specific immunity response in the giant freshwater prawn (Macrobrachium rosenbergii) under high ammonia stress.

This study aimed to investigate the effects of dietary tea tree oil (TTO) on the performance, intestinal antioxidant capacity, and non-specific immunity after ammonia nitrogen stress in Macrobrachium rosenbergii. Six experimental diets were formulated with 0, 25, 50, 100, 200, 400 mg/kg TTO, respectively. A total of 900 prawns (average initial weight, 0.39 ± 0.01 g) were randomly assigned to 6 groups in triplicate in 18 tanks. After an 8-week feeding trial, 20 prawns from each tank were changed with 20 mg/L ammonia stress for 24 h. The results showed that 100 mg/kg TTO significantly increased prawns performance and survival rate compared with the control group. Moreover, 100 and 200 mg/kg TTO significantly improved intestinal antioxidant capabilities by increasing SOD enzyme activities and decreasing MDA levels. In addition, the prawns fed with 100 mg/kg TTO diet showed the highest survival rate under ammonia stress. After ammonia stress, the group of 100 mg/kg TTO significantly improved antioxidant capacity by increasing hemolymph respiratory burst activity, as well as intestinal anti-superoxide anion activity and SOD. Coincidentally, 100 mg/kg TTO significantly upregulated the intestinal relative expression of antioxidant-related genes (peroxiredoxin-5). Further, it was found that 100 mg/kg TTO activated the toll-dorsal pathway in prawns, which performed the similar function as the classic NF-κB pathway by upregulating the TNF-α and IL-1. Finally, 100 mg/kg TTO increased the levels of iNOS activities and NO contents after ammonia stress and enhanced non-specific immunity. The results indicated that 100 mg/kg TTO could significantly improve the M. rosenbergii performance, antioxidant capacity and ammonia stress resistance. We suggested that the mechanisms may be attributed to that TTO enhanced the antioxidant capacity and non-specific immunity of M. rosenbergii via the NF-κB signal pathway.

The effects of ammonia stress exposure on protein degradation, immune response, degradation of nitrogen-containing compounds and energy metabolism of Chinese mitten crab.

BACKGROUND: The Chinese mitten crab is one of the most economically important crabs that are widely farmed in China. Ammonia, which is a main physiological challenge for crab culture, grows rapidly in the intensive culture system over time, but little information is available with Chinese mitten crab on the molecular mechanisms. METHODS AND RESULTS: Therefore, to understand the mechanism of response to ammonia stress in Eriocheir japonica sinensis, comparative transcriptome analysis was used to identify the key genes and pathways in hepatopancreas challenged with ammonia stress (325.07 mg/L NH4Cl). By sequencing the transcriptome hepatopancreas of E. j. sinensis treated with ammonia, 366,472 unigenes were obtained and annotated into several public libraries for later analyses. Subsequently, 1775 differentially expressed genes (DEGs) were identified according to comparative transcriptome analysis, of which 307 were up-regulated and 1468 were down-regulated. According to the DEGs of GO and KEGG enrichment analyses, we focused on four aspects of significant enrichment in this study: protein degradation, immune response, degradation of nitrogen-containing compounds and energy metabolism. The genes involved in protein degradation and energy metabolism process showed a significant decrease which was consisting of overall biological activity of E. j. sinensis decreased. In addition, five genes involved in high concentration of ammonia were discovered and validated by qRT-PCR. CONCLUSIONS: This study will help us understand the molecular mechanisms of E. j. sinensis under high ammonia exposure and provide valuable information to the future research of other crabs with ammonia exposure.

Ammonia stress affects the structure and function of hemocyanin in Penaeus vannamei.

Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.

Distinct vulnerability to oxidative stress determines the ammonia sensitivity of crayfish (Procambarus clarkii) at different developmental stages.

Red swamp crayfish (Procambarus clarkii) has increasingly become a high-value freshwater product in China. During the intensive cultivation, excessive ammonia exposure is an important lethal factor of crayfish. We investigated the toxic effects and mechanisms of ammonia on crayfish at two different developmental stages. A preliminary ammonia stress test showed a 96-h LC50 of 135.10 mg/L and 299.61 mg/L for Stage_1 crayfish (8.47 ± 1.68 g) and Stage_2 crayfish (18.33 ± 2.41 g), respectively. During a prolonged ammonia exposure (up to 96 h), serum acid phosphatase and alkaline phosphatase showed a time-dependent decrease. Histological assessment indicated the degree of hepatopancreatic injury, which was mainly characterized as tubule lumen dilatation, degenerated tubule, vacuolization and dissolved hepatic epithelial cell, increased with exposure time. Enhanced malondialdehyde level and reduced antioxidant capacity of hepatopancreas were also observed. The mRNA expression and activity of catalase and superoxide dismutase showed an initial up-regulation within 24 h, and then gradually down-regulated with the exposure time. In the post-treatment recovery period, the Stage_2 crayfish exerted a stronger antioxidant and detoxification capacity than that of the Stage_1 crayfish, and thus quickly recovered from the ammonia exposure. Our findings provide a further understanding of the adverse effects of ammonia stress and suggest guidelines for water quality management during crayfish farming.

Genotype × ammonia interaction effects on plasma physiological indexes in turbot (Scophthalmus maximus) cultured under acute ammonia stress for different periods of times.

We evaluated six plasma physiological indexes (epinephrine, cortisol, alkaline phosphatase, superoxide dismutase, reduced glutathione, and blood glucose) in turbot (Scophthalmus maximus) cultured in five ammonia concentrations (30.28, 35.90, 42.56, 50.47, and 59.82) at different exposure times (4, 8, 12, 24, and 48 h). Then, we conducted additive main effects and multiplicative interaction (AMMI) and genotype main effects and genotype × environment interaction (GGE) biplot analyses to analyze the genotype × ammonia interaction effects on the plasma physiological indexes. The AMMI analysis results revealed that the contributions of ammonia effect first decreased sharply (4-8 h), then decreased slowly (8-24 h), and then increased slowly (24-48 h). The contributions of genotype effect first decreased sharply (4-8 h), then increased slowly (8-24 h), and then decreased slowly (24-48 h). The contributions of genotype × ammonia interactions showed a sharp rise (4-8 h), a sharp decline (8-12 h), a slow decline (12-24 h), and finally a slow increase (24-48 h). The GGE biplot analysis revealed that alkaline phosphatase and reduced glutathione contents/activities are reliable biomarkers that can be used to establish an online early warning system for behavior numerical simulation.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

ß-glucan extracted from eukaryotic single-celled microorganism Saccharomyces cerevisiae: Dietary supplementation and enhanced ammonia stress tolerance on Oreochromis mossambicus.

Ammonia is a widespread pollutant that is toxic to living organisms in aquaculture. This study aimed to evaluate the effects of a diet supplemented with beta-glucan from yeast, Saccharomyces cerevisiae (Sc-ßG), on the stress response of Oreochromis mossambicus (Tilapia) to ammonia. Fish were divided into four groups, including a control fed a basal diet and three experimental groups fed diets supplemented with Sc-ßG at 2, 5 and 10 mg/g respectively. After 8 weeks, experimental groups were exposed to ammonia at 100 mg L-1 for 1 week. Growth was measured after the 8-week feeding trial and serum, mucus, and liver tissue were sampled before and after the ammonia challenge. Compared with the control diet, feed supplemented with Sc-ßG at 10 mg/g significantly (p < 0.05) improved growth performance (7.8-9.9 g increase in weight). The cellular immune responses (myeloperoxidase, reactive oxygen species, and reactive nitrogen species), humoral immune responses (alkaline phosphatase, lysozyme, and peroxidase inhibition), and antioxidant response (catalase, superoxide dismutase, and glutathione) were tested in serum, mucus and liver tissue. Compared with the control, these responses were significantly (p < 0.05) enhanced at 10 mg/g supplementation with Sc-ßG. This study demonstrates that Sc-ßG may be applied to induce stress tolerance and improve growth performance in aquaculture.

Effects of acute ammonia toxicity on oxidative stress, DNA damage and apoptosis in digestive gland and gill of Asian clam (Corbicula fluminea).

Ammonia is one of the major pollutants associated with the main river basins due to ammonification of uneaten food and animal excretion, which usually brings detrimental health effects to aquatic invertebrate. However, the mechanisms of ammonia toxicity in aquatic invertebrate have rarely been reported. In this study, C. fluminea was exposed to different levels of ammonia (control group, 10 mg/L, and 25 mg/L) for 24 h and 48 h, and digestive gland and gill were collected to explore toxic effects on oxidative stress, DNA damage and apoptosis under ammonia stress. The results showed that ammonia poisoning could increase the activity of oxidative stress enzyme (SOD and CAT), inducing differentially expressed genes (DRAM2, GADD45, P53, BAX, BCL2, CASP8, CASP9, CASP3, HSP70 and HSP90) and different cytokines (IL-1 beta, IL-8, IL-17 and TNF-alpha) of DNA damage and apoptosis. The difference of toxic effects induced by ammonia among digestive gland and gill were also observed by real-time PCR and TUNEL staining. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in C. fluminea.

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei.

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

Effects of Moringa oleifera leaf extract on growth performance, physiological and immune response, and related immune gene expression of Macrobrachium rosenbergii with Vibrio anguillarum and ammonia stress.

In order to study the effects of Moringa oleifera leaf extract on Macrobrachium rosenbergii under high ammonia exposure, freshwater prawns were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 0.25%, 0.5% and 1.0% M. oleifera leaf extract and 0.025% Enrofloxacin for 60 days, respectively. Then, freshwater prawns were exposed to high ammonia stress for 72 h and Vibro anguillarum infection. The growth, antioxidant capabilities, related immune genes as well as resistance to infection by V. anguillarum were determined. The results showed that compared with the control group, the weight gain, specific growth rate and protein efficiency rate, haemolymph catalase (CAT), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) increased while feed conversion ratio, haemolymph aspartate aminotransferase, alanine aminotransferase, nitrogen oxide (NO), hepatopancreas heat shock proteins (HSP70), immune deficiency (IMD) expression levels decreased in the group of 0.5% M. oleifera leaf extract before the stress. After ammonia stress, the group of 0.5% M. oleifera leaf extract also could improve the haemolymph SOD, glutathione peroxidase, NO, iNOS, hepatopancreas HSP70 expression levels and reduce haemolymph CAT, hepatopancreas peroxiredoxin 5 and NF kappa B inhibitor alpha expression level compared with the control group. The rate of mortality of the prawns challenged with V. anguillarum was lower in the supplemented groups in comparison with the control group with the lowest being in the group of 0.5% M. oleifera leaf extract. Antioxidant activities as well as biochemical parameters in the enrofloxacin group (0.025%E) were not significantly enhanced both pre and post challenge in comparison with the M. oleifera leaf extract groups, showing the superiority of the natural herb over the synthetic antibiotic. In summary, this study suggested that at an inclusion rate of 0.5%, M. oleifera leaf extract could increase the growth performance, even has positive effects on physiological and immune function and prevents high ammonia stress in the Freshwater prawn, M.rosenbergii.

Exposure to acute ammonia stress influences survival, immune response and antioxidant status of pacific white shrimp (Litopenaeus vannamei) pretreated with diverse levels of inositol.

The effect of acute ammonia challenge on survival, immune response and antioxidant status of Litopenaeus vannamei pretreated with diets containing different inositol levels was investigated. Shrimp (initial mean weight 0.40 ±â€¯0.00 g) were randomly allocated in 18 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet without myo-inositol (MI) supplementation (242.6 mg inositol kg-1 diet) or diets containing diverse levels of inositol (368.8, 459.7, 673.1, 993.8 and 1674.4  mg kg-1 diet) as treatment groups for 8-week. Randomly selected 10 shrimp per tank (final mean weight approximately 11.1-13.8g) were exposed to ammonia stress (total ammonia-nitrogen, 60.21  mg L-1) for 24 h after feeding trial. The results showed that after exposed to ammonia stress, survival rates of MI-supplemented groups were enhanced by 31-77% when compared with the control group. MI supplementation increased activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in plasma, and reduced its activities in hepatopancreas. It also enhanced activities of total antioxidant capacity (T-AOC), glutathione S-transferase (GST) and glutathione peroxidase (GPX) and content of reduced glutathione (GSH), and lowered malondialdehyde (MDA) and protein carbonyl (PC) content in plasma or hepatopancreas. In addition, mRNA expression levels of ferritin (FT), arginine kinase (AK), thioredoxin (Trx), heat shock protein 70 (Hsp70), catalase (CAT) and peroxiredoxin (Prx) were significantly differentially regulated in hepatopancreas owing to MI supplementation. Therefore, it suggested that L. vannamei pretreated with higher dietary inositol content may have better ammonia stress tolerance and antioxidant status after ammonia stress, and the optimum levels ranged from 459.7 to 993.8 mg inositol kg-1 when total ammonia-nitrogen concentration was 60.21  mg L-1.

Molecular characterization of p38 MAPK from blunt snout bream (Megalobrama amblycephala) and its expression after ammonia stress, and lipopolysaccharide and bacterial challenge.

p38 mitogen-activated protein kinase (MAPK) is an important protein which plays a key role in regulating the innate immunity, so exploring its molecular characterization is helpful in understanding the resistance against microbial infections in cultured fish. Here, a full-length cDNA of p38 MAPK was cloned from liver of blunt snout bream (Megalobrama amblycephala) which covered 2419 bp with an open reading frame of 1086 bp encoding 361 amino acids. p38 MAPK contained the characteristic structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW), which are conserved in MAPK family. To investigate p38 MAPK functions, two in vivo experiments were carried out to examine its expression following ammonia exposure and bacterial challenge. Also, an in vitro experiment was conducted to assess the role of p38 MAPK in inflammation of primary hepatocytes induced by lipopolysaccharide (LPS). The results showed the ubiquitous expression of p38 MAPK in all the tested tissues with varying levels. p38 MAPK mRNA expression was significantly up-regulated by ammonia stress and Aeromonas hydrophila challenge, and altered in a time-dependent manner. Moreover, the results indicated that the inflammatory response induced by LPS in hepatocytes is p38 MAPK dependent as knockdown of p38 MAPK using siRNA technology depressed the expression of IL-1ß and IL-6. The findings in this study showed that p38 MAPK has anti-stress property, and plays key role in protection against bacterial infection and inflammation in blunt snout bream.