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.

Responses and correlation among ER stress, Ca2+ homeostasis, and fatty acid metabolism in Penaeus vannamei under ammonia stress.

The role of endoplasmic reticulum (ER) stress, Ca2+ homeostasis, and fatty acid metabolism in the environmental adaptation of aquatic animals is significant, but further confirmation of the relationship between these factors is needed. This study aimed to investigate the responses and correlations among ER stress, Ca2+ homeostasis, and fatty acid metabolism in Penaeus vannamei under ammonia stress. A total of 640 P. vannamei weighing 3.0 ± 0.4 g were selected and exposed to different total ammonia concentrations (0 mg/L for the control group and 3.80, 7.60, and 11.40 mg/L for the stress groups). The experiment involved a 96 h ammonia stress period to assess indicators related to ER stress, Ca2+ homeostasis, and fatty acid metabolism. The experimental results revealed that after 12 h, exposure to ammonia induced the ER stress response in the hepatopancreas of the shrimp. The groups exposed to concentrations of 3.8 mg/L and 7.6 mg/L exhibited an increase in ER Ca2+ efflux, a decrease in influx, an elevation in mitochondrial Ca2+ influx, an enhanced energy demand within the organism, and substantial consumption of triglycerides. The 11.3 mg/L group exhibited a significant enhancement in fatty acid metabolism. At 24 h, the ER stress response induced by ammonia in the shrimp exhibited a gradual recovery. In the 7.6 mg/L and 11.3 mg/L groups, the ER Ca2+ influx and efflux exhibited significant enhancements, while the mitochondrial Ca2+ influx decreased and the organism's energy demand increased. Moreover, there was a substantial enhancement in fatty acid metabolism. At 48 h, the ER stress response disappeared in each stress group, ER Ca2+ efflux was reduced, triglycerides were consumed, and the body's energy homeostasis was basically restored. At 96 h, a stress response reoccurred in the ER in each stress group, resulting in increased influx of Ca2+ into the ER, augmented energy demand within the organism, and notable enhancement in fatty acid metabolism. Pearson correlation analysis revealed a significant positive correlation between the NH3-N content in the hepatopancreas and the expression of ER stress-related genes, as well as between ER Ca2+ influx/efflux and energy homeostasis/fatty acid metabolism. The findings indicate that the stress induced by ammonia triggers an ER stress response in P. vannamei, resulting in ER Ca2+ efflux and mitochondrial Ca2+ influx, which, in turn, enhances fatty acid metabolism to generate additional energy for adaptation in stressful environments. This study contributes to a deeper understanding of the environmental adaptability of P. vannamei in the context of Ca2+ homeostasis.

Transcriptome analysis of Chinese mitten crabs (Eriocheir sinensis) gills in response to ammonia stress.

The Chinese mitten crab (Eriocheir sinensis) is an important commercial species in China. E. sinensis is typically farmed in rice-crab symbiosis, as an important ecological farming model. However, E. sinensis is often exposed to a high ammonia environment due to the application of nitrogen fertilizers essential for rice growth. We investigated the molecular mechanisms in the gills of E. sinensis exposed to high ammonia at transcriptional and histological levels. We randomly assigned E. sinensis to two groups (control group, CG; ammonia stress group, AG), and gill samples were excised from the CG and AG groups for histopathological and transcriptome analyses. The histopathological evaluation revealed that ammonia stress damaged the gills of E. sinensis. The transcriptome analysis showed that some essential genes, including Xanthine dehydrogenase (XDH), Ubiquitin C-terminal hydrolase-L3 (UCHL3), O-linked N-acetylglucosamine transferase (OGT), Cathepsin B (CTSB), and Ubiquitin-conjugating enzyme E2 W (UBE2W) changed significantly during ammonia exposure. These genes are related to ammonia detoxification, the immune response, and apoptosis. This study demonstrated the molecular response mechanism of E. sinensis gills to ammonia stress at the transcriptional and histological levels. This study provides insight for further study on the molecular mechanism of ammonia stress in crustaceans and supplies technical support for rice crab symbiosis.

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.

The SLC38A9-mTOR axis is involved in autophagy in the juvenile yellow catfish (Pelteobagrus fulvidraco) under ammonia stress.

The primary objective of this study was to examine the effect of acute ammonia stress on hepatic physiological alterations in yellow catfish by performing a comprehensive analysis of the metabolome and transcriptome. The present study showed that ammonia stress led to liver metabolic disruption, functional incapacitation, and oxidative damage. Transcriptomic and metabolomic analyses revealed transcriptional and metabolic differences in the liver of yellow catfish under control and high ammonia stress conditions. After 96 h of acute exposure to ammonia, the mRNA levels of 596 liver genes were upregulated, whereas those of 603 genes were downregulated. Enrichment analysis of the differentially expressed genes identified multiple signalling pathways associated with autophagy, including the endocytosis, autophagy-animal, and mammalian target of rapamycin signalling pathways. A total of 186 upregulated and 117 downregulated metabolites, primarily associated with amino acid biosynthesis pathways, were identified. Multi-omics integration revealed the solute carrier family 38 member 9 (SLC38A9)-mammalian target of rapamycin axis as a signalling nexus for amino acid-mediated modulation of autophagy flux, and q-PCR was used to assess the expression of autophagy-related genes (LC3a and sqstm1), revealing an initial inhibition followed by the restoration of autophagic flux during ammonia stress. Subsequent utilisation of arginine as a specific SLC38A9 activator during ammonia stress demonstrated that augmented SLC38A9 expression hindered autophagy, exacerbated ammonia toxicity, and caused a physiological decline (total cholesterol, total triglyceride, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase levels were significantly increased), oxidative stress, and apoptosis. Autophagy activation may be an adaptive mechanism to resist ammonia stress.

Enhanced anaerobic digestion of human feces by ferrous hydroxyl complex (FHC): Stress factors alleviation and microbial resistance improvement.

Anaerobic digestion (AD) offers a reliable strategy for resource recovery from source-separated human feces (HF), but is limited by a disproportionate carbon/nitrogen (C/N) ratio. Ferrous hydroxyl complex (FHC) was first introduced into the HF-AD system to mediate methanogenesis. Mono-digestion of undiluted HF was inhibited by high levels of volatile fatty acids (VFAs), ammonia, and hydrogen sulfide (H2S). FHC addition at optimum dosage (500-1000 mg/L) increased the cumulative methane (CH4) yield by 22.7%, enhanced the peak value of daily CH4 production by 60.5%, and shortened the lag phase by 24.7%. H2S concentration in biogas was also greatly decreased by FHC via precipitation. FHC mainly facilitated the hydrolysis, acidification, and methanogenesis processes. The production and transformation of VFAs were optimized in the presence of FHC, thus relieving acid stress. FHC elevated the activities of alkaline protease, cellulase, and acetate kinase by 32.3%, 18.2%, and 30.3%, respectively. Microbial analysis revealed that hydrogenotrophic methanogens prevailed in mono-digestion at high HF loading but were weakened after FHC addition. FHC also enriched Methanosarcina, thereby expanding the methanogenesis pathway and improving the resistance to ammonia stress. This work would contribute to improving the methanogenic performance and resource utilization for HF anaerobic digestion.

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.

IR-based device to acquire real-time online heart ECG signals of fish (Cyprinus carpio) to evaluate the water quality.

Water quality monitoring is a challenging task due to continuous pollution. The rapid development of engineering technologies has paved the way for the development of efficient and convenient computer-based online continuous water-quality assessment techniques. Techniques based on biological-responses are gaining attention, worldwide. Different biosensors have been developed in recent years to monitor real-time biological responses to evaluate water-quality. The survival and function of various organs of the organism depends on the cardiac system. Alterations in the cardiac system could signify the occurrence/initiation of stress in the organism. We developed a real-time online cardiac function assessment system-OCFAS to acquire fish ECG-signals. We obtained P-wave, R-wave, T-wave, PR-intervals, QT-intervals and QRS-complex continuously, which did not affect the normal activities of carp. We exposed Cyprinus carpio to different concentrations (National Environmental Quality Standards) of ammonia for 48 h. Our OCFAS has precisely acquired the required ECG-signals. A real-time dataset reveals sensitivity to ammonia in carp ECG-indexes. Compared with the control group the P-wave, R-wave and T-wave were weaker in ammonia-treated groups. In contrast, the PR-intervals, QT-intervals and QRS-complex were prolonged in the ammonia-treatment groups. The self-organizing map signifies that the PR-intervals, the QRS-complex and the QT-intervals are consistent with environmental stress. Linear regression analysis also quantitatively signifies that the PR interval has the highest R2 value and the lowest SSE-value, followed by the QRS complex and the QT interval. A concentration-related effect was observed in the ammonia treated groups. The integrated biomarker response (IBRv2) index was used to determine the overall stress of ammonia on carp heart ECG-indexes. IBRv2 also supports the real-time response of carp to ammonia stress. Ammonia levels in the aquaculture and water environment require special attention to avoid its adverse effects on the health of aquatic biota. Our study emphasizes the importance of online real-time fish ECG for water-quality assessment.

The Effects of Acute Exposure to Ammonia on Oxidative Stress, Hematological Parameters, Flesh Quality, and Gill Morphological Changes of the Large Yellow Croaker (Larimichthys crocea).

Ammonia is considered to be the major chemical pollutant causing fish poisoning in aquaculture. This research aimed to evaluate the impact of acute ammonia exposure on the large yellow croaker's meat quality, gill morphology, liver oxidative stress, and hematological parameters. The fish were exposed to total ammonia nitrogen concentrations of 0, 2.96, 5.92, and 8.87 mg/L for 48 h, respectively. The findings demonstrated that all ammonia-exposed fish had higher liver lactate dehydrogenase and glutamic oxalate transaminase activities. The glucose, blood urea nitrogen, and creatinine levels in 8.87 mg/L total ammonia nitrogen (TAN) were higher than other samples. The total protein, albumin, and triglyceride levels in serum decreased significantly in ammonia-exposed samples. After 48 h of ammonia exposure, superoxide dismutase activities showed a 76.1%, 118.0%, and 156.8% increase when fish were exposed to 2.96, 5.92, and 8.87 mg/L TAN, respectively. Catalase activities and glutathione contents were considerably higher (p < 0.05) in all ammonia-treated samples compared to 0 mg/L TAN. The ammonia-treated gill lamellae become thicker, shorter, and curved. Additionally, the ammonia exposure resulted in the accumulation of free amino acids and the loss of nucleotides. The inosine monophosphate and adenosine monophosphate contents in the flesh were decreased after 12 h of exposure to 2.96, 5.92, and 8.87 mg/L ammonia compared to the control group. Overall, large yellow croakers exposed to ammonia for 6 h presented not only changes in serum composition but also oxidative stress, liver and gill tissue damage and flesh quality deterioration.

Combined effects of dietary carbohydrate levels and ammonia stress on growth, antioxidant capacity and glucose metabolism in juvenile oriental river prawn (Macrobrachium nipponense).

Ammonia is a common environmental stress factor that constrains aquaculture industry development. This study evaluated the effect of carbohydrate levels and ammonia stress in oriental river prawn (Macrobrachium nipponense). The experiment had six treatments containing two water ammonia levels (0 and 5 mg/L) and three dietary carbohydrate levels (low carbohydrate diet (LCD, 10%), medium carbohydrate diet [MCD, 20%], and high carbohydrate diet [HCD, 30%]), and lasted six weeks. The results showed that the prawns fed on MCD had higher weight gain than those fed on LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower acid phosphatase and alkaline phosphatase activities during ammonia stress. Feeding the prawns on the MCD increased B cells in the hepatopancreas during ammonia stress. Interestingly, the prawns fed on MCD had significantly lower superoxide dismutase activity compared to LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower pyruvate kinase activity and pyruvate and lactic acid contents, while those fed on LCD had significantly higher succinic dehydrogenase, 6-phosphogluconic dehydrogenase, and phosphoenol pyruvate carboxykinase activities during ammonia stress. The prawns fed on the MCD increased significantly glutaminase activity and decreased the ammonia content in the serum during ammonia exposure. In addition, feeding the prawns on MCD decreased significantly the expression of apoptosis and inflammation-related genes. Taken together, the MCD supplied energy required to counteract ammonia stress, which increased growth, improved antioxidant capacity, facilitated ammonia excretion, and alleviated inflammation and apoptosis of the oriental river prawn.

Heritability and preadult survivorship costs of ectoparasite resistance in the naturally occurring Drosophila-Gamasodes mite system.

Our understanding of the evolutionary significance of ectoparasites in natural communities is limited by a paucity of information concerning the mechanisms and heritability of resistance to this ubiquitous group of organisms. Here, we report the results of artificial selection for increasing ectoparasite resistance in replicate lines of Drosophila melanogaster derived from a field-fresh population. Resistance, as ability to avoid infestation by naturally co-occurring Gamasodes queenslandicus mites, increased significantly in response to selection and realized heritability (SE) was estimated to be 0.11 (0.0090). Deployment of energetically expensive bursts of flight from the substrate was a main mechanism of host resistance that responded to selection, aligning with previously documented metabolic costs of fly behavioral defenses. Host body size, which affects parasitism rate in some fly-mite systems, was not shifted by selection. In contrast, resistant lines expressed significant reductions in larva-to-adult survivorship with increasing toxic (ammonia) stress, identifying an environmentally modulated preadult cost of resistance. Flies selected for resistance to G. queenslandicus were also more resistant to a different mite, Macrocheles subbadius, suggesting that we documented genetic variation and a pleiotropic cost of broad-spectrum behavioral immunity against ectoparasites. The results demonstrate significant evolutionary potential of resistance to an ecologically important class of parasites.

Impacts of ammonia stress on different Pacific whiteleg shrimp Litopenaeus vannamei families and the underlying adaptive mechanisms.

Ammonia stress in aquaculture systems poses a great threat to the growth and survival of the Pacific whiteleg shrimp Litopenaeus vannamei. Although the ammonia stress tolerance capacity of L. vannamei has been found to vary significantly among different breeding families, the underneath mechanisms are still largely unknown. In this study, the ammonia tolerance capacity of different L. vannamei breeding families was compared. Results confirmed the significant differences in the ammonia adaptability among different families. To ascertain the underlying adaptive strategies, ATP status, ATP synthase activity, expression and activities of ammonia excretion and metabolism-related enzymes, and apoptosis in shrimp gills were analyzed. Furthermore, transcriptomic analyses were also performed to elucidate the molecular mechanisms. Our results indicated that ammonia-tolerant L. vannamei may possess (1) enhanced ability to excrete ammonia, (2) better capacity to convert ammonia into less toxic products, and (3) sufficient energy reserves for ammonia-compensating processes.

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.

Ammonia Toxicity in the Bighead Carp (Aristichthys nobilis): Hematology, Antioxidation, Immunity, Inflammation and Stress.

Ammonia is one of the main environmental pollutants that affect the survival and growth of fish. The toxic effects on blood biochemistry, oxidative stress, immunity, and stress response of bighead carp (Aristichthys nobilis) under ammonia exposure were studied. Bighead carp were exposed to total ammonia nitrogen (TAN) concentrations of 0 mg/L, 3.955 mg/L, 7.91 mg/L, 11.865 mg/L, and 15.82 mg/L for 96 h. The results showed that ammonia exposure significantly reduced hemoglobin, hematocrit, red blood cell, white blood cell count, and platelet count and significantly increased the plasma calcium level of carp. Serum total protein, albumin, glucose, aspartate aminotransferase, and alanine aminotransferase changed significantly after ammonia exposure. Ammonia exposure can induce intracellular reactive oxygen species (ROS), and the gene expression of antioxidant enzymes (Mn-SOD, CAT, and GPx) increases at the initial stage of ammonia exposure, while MDA accumulates and antioxidant enzyme activity decreases after ammonia stress. Ammonia poisoning changes the gene expression of inflammatory cytokines; promotes the gene expression of inflammatory cytokines TNF-α, IL-6, IL-12, and IL-1ß; and inhibits IL-10. Furthermore, ammonia exposure led to increases in stress indexes such as cortisol, blood glucose, adrenaline, and T3, and increases in heat shock protein 70 and heat shock protein 90 content and gene expression. Ammonia exposure caused oxidative stress, immunosuppression, inflammation, and a stress reaction in bighead carp.

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.

Responses of Micropterus salmoides under Ammonia Stress and the Effects of a Potential Ammonia Antidote.

Ammonia is a common environmental limiting factor in aquaculture. To investigate the effects of ammonia stress and explore the protective effect of N-carbamylglutamate (NCG) on Micropterus salmoides (M. salmoides), tissue sections and parameters related to oxidative stress and the inflammatory response in M. salmoides were carried out during the ammonia stress test and feeding test. The results demonstrated that the LC50 for 24 h, 48 h, 72 h, and 96 h under ammonia stress in M. salmoides were 25.78 mg/L, 24.40 mg/L, 21.90 mg/L, and 19.61 mg/L, respectively. Under ammonia stress, the structures of the tissues were damaged, and the GSH content decreased, while the MDA content increased with the increase in stress time and ammonia concentration. The NO content fluctuated significantly after the ammonia nitrogen stress. In the 15-day feeding test, with the increased NCG addition amount and feeding time, the GSH content increased while the MDA and NO contents decreased gradually in the NCG addition groups (NL group: 150 mg/kg; NM group: 450 mg/kg; NH group: 750 mg/kg) when compared with their control group (CK group: 0 mg/kg). In the ammonia toxicology test after feeding, the damage to each tissue was alleviated in the NL, NM, and NH groups, and the contents of GSH, MDA, and NO in most tissues of the NH group were significantly different from those in the CK group. The results suggested that ammonia stress caused tissue damage in M. salmoides, provoking oxidative stress and inflammatory response. The addition of NCG to the feed enhances the anti-ammonia ability of M. salmoides. Moreover, the gill and liver might be the target organs of ammonia toxicity, and the brain and kidney might be the primary sites where NCG exerts its effects. Our findings could help us to find feasible ways to solve the existing problem of environmental stress in M. salmoides culture.

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.

High dissolved oxygen exacerbates ammonia toxicity with sex-dependent manner in zebrafish.

Ammonia nitrogen is one of the important environmental factors, and causes negative effects for fish health in ecosystem and aquaculture. The toxic effects and mechanisms of ammonia in fish deserve further investigation. In the present study, we exposed female and male zebrafish (Danio rerio) to ammonia (50 mg/L NH4Cl) with oxygenated (7.5-7.8 mg/L) or non­oxygenated (3.8-4.5 mg/L) water, to identify the combined effects of dissolved oxygen and ammonia on fish with gender difference. The results showed that oxygenated ammonia exposure increased fish mortality, gill secondary lamellas damage and gill tissue spaces, gene expressions of proinflammatory interleukin 1 beta (il-1ß) and apoptotic caspase8 as compared with non­oxygenated ammonia. Besides, oxygenated ammonia elevated plasma ammonia contents, and decreased the discharge of body ammonia through gills by depressing the enzyme activity of Na+/K+-ATPase. Notably, when zebrafish were subjected to ammonia stress, more severe mortality, gill damage and tissue inflammatory response were observed in males than females. This is the first study to clarify the gender-dependent impacts of ammonia toxicity, and the adverse effects of oxygenation on ammonia resistance in zebrafish.

Evaluation of digestate-derived biochar to alleviate ammonia inhibition during long-term anaerobic digestion of food waste.

The feasibility of using food waste anaerobic digestate-derived biochar (FWDB) to mitigate ammonia toxicity in an anaerobic digester was evaluated. The optimal conditions for preparing and adding the activated FWDB were explored using response surface experiments, and the long-term effects of adding activated FWDB on digester performance under optimum conditions were verified in semi-continuous experiments. The results showed that the optimal preparation and addition conditions for activated FWDB were pyrolysis temperature of 565 °C, particle size of 0-0.30 mm, and dosage of 15.52 g·L-1. During the long-term operation of the digesters, when the total ammonia nitrogen (TAN) concentration was higher than 2000 mg·L-1, the control and experimental digesters showed deteriorated reactor performance. Volatile fatty acids in the control digester accumulated to 20,306 mg·L-1 after the TAN concentration increased to 3391 mg·L-1, the methane yield decreased to 31 mL·g VS-1, and the digester experienced process failure. In contrast, the experimental digester with added activated FWDB only suffered a slight short-term accumulation of acetate and a slight decline in methane yield. This may be attributed to the adsorption of NH4+/NH3 by activated FWDB, which reduced the TAN concentration in the anaerobic digestion (AD) system and mitigated ammonia toxicity. Microbial analysis and metagenome predictions demonstrated that the community richness, diversity, and evenness, as well as the abundance of acetogens and related key genes (ACSM1, paaF, and acdA) were higher in the experimental digester than in the control digester. This study provides a closed-loop AD enhancement strategy by pyrolysis of digestate and in-situ supplementation into the digester.

Effects of ammonia exposure on anxiety behavior, oxidative stress and inflammation in guppy (Poecilia reticulate).

Ammonia is one of the most important aquatic environmental factors, which is of great concern. In order to evaluate the effect of ammonia on guppy (Poecilia reticulate), fish were exposed to increased concentrations (0, 12.50, 25.00, 41.67, 62.50 mg/L) of ammonia for 48 h. After exposure, we measured the anxiety behavior, antioxidant enzymes and pro-inflammation genes (TNF-α, IL-1ß and IL-6) of guppy. The results showed that ammonia stress induced fish anxiety, which was manifested by the increased latency to enter the upper half and decreased time spent in upper half compared with control fish. The guppy showed oxidative stress after 48 h of ammonia stress as evidenced by decreases in the activities of antioxidant enzymes and an increase in lipid hydroperoxide content. With prolonged ammonia stress, the expressions of HSP70, HSP90, TNF-α, IL-1ß and IL-6 mRNA at first had an increasing trend, and then decreased, all of which were significantly higher than the control levels at 12 h and 24 h after ammonia stress (P < 0.05). Ammonia significantly upregulated these genes mRNA levels after 48 h exposure, suggesting that heat shock proteins and innate immune system may try to protect cells from oxidative stress induced by ammonia stress. Our study showed that higher ammonia exposure induced oxidative stress in exposed fish, since inhibition of antioxidant enzymes activity and increases in lipid peroxidation, and inflammation occurred. Furthermore, the results will be helpful to understand the mechanism of ammonia toxicity in guppys.