Nitrite Stress Research Articles

Integrated miRNA-mRNA transcriptomic analysis of hepatopancreas reveals molecular strategies in Chinese mitten crab (Eriocheir sinensis) under acute nitrite stress

Nitrite is one of the most common pollutants in high-density aquaculture or in recirculated aquaculture systems. In this study, gene and miRNA expression dynamics were investigated for unveiling the genetic regulation of Eriocheir sinensis in response to acute nitrite stress. Two groups of E. sinensis with different vitality of appendages (i.e., NG and DG) were treated with nitrite-N in 6 and 24 h, respectively. A number of differentially expressed (DE) genes and miRNAs were detected in the pairwise comparisons of the two groups. We found that the DE genes and the target genes of DE miRNAs were significantly enriched into some key pathways, such as metabolic pathways, MAPK signaling pathway, and nitrogen metabolism. Notably, the number of DE genes identified from the pairwise comparison of MNG vs. FNG (NG groups at 6 h and 12 h, respectively) was much larger than that identified from MDG vs. FDG (FG groups at 6 h and 12 h, respectively), indicating that the individuals from strong vitality group had genetic superiority for tolerance of nitrite stress. The similar trend was also observed from the identification of DE miRNAs. Some key genes (e.g., hsp70, mtnd2, act7, hspg2, cam-1) involved in signaling transduction, oxidative phosphorylation, and energy metabolism were detected in this study. Besides, several miRNAs (miR-31, miR-215, miR-142-x) that mediate the calmodulin related genes were also identified, inferring that calcium signaling pathway participates in the responses to nitrite stress in crabs. This study revealed the mRNA and miRNA expression patterns of Chinese mitten crab under sustained nitrite stress and provided the genetic basis for the molecular selective breeding.

Dietary hydroxyl methionine selenium supplementation enhances growth performance, antioxidant ability and nitrite tolerance of Litopenaeus vannamei

An 8-week feeding trial and then 12 h nitrite stress was conducted to estimate the effects of dietary hydroxyl methionine selenium (HMSe) on growth performances, antioxidant response and nitrite resistance of Litopenaeus vannamei (initial weight 0.90 ± 0.05 g). The diet was supplemented with 0, 0.15, 0.30, 0.60 and 0.90 mg Se/kg as HMSe to formulate six diets, labeled as HMSe0, HMSe15, HMSe30, HMSe60 and HMSe90, respectively. The results showed that significantly higher finial body weight and weight gain were observed in HMSe30 group than those in HMSe0 and HMSe90 groups (P < 0.05). Feed efficiency and protein efficiency ratio of HMSe30 were significantly higher than those of HMSe90 (P < 0.05). The selenium contents in whole body and muscle were both significantly increased with the dietary HMSe levels increasing (P < 0.05). The hepatopancreas MDA levels of groups supplemented HMSe were significantly lower than that of HMSe0 (P < 0.05). And they decreased firstly and then increased with HMSe supplementation levels increasing, the lowest concentration was observed in HMSe15 group. The antioxidant enzymes of GSH-Px, SOD, CAT and GST activities in hepatopancreas were firstly increased and then decreased with the increase of dietary HMSe levels. GSH-Px, SOD and CAT genes expression in hepatopancreas were significantly up-regulated in HMSe15, HMSe15 and HMSe60 groups. After nitrite stress, there were higher mortality and hepatopancreas MDA concentration and lower hepatopancreas GST, CAT and SOD activities in HMSe0 group. While, these negative effects could be relieved with HMSe supplementation. In conclusion, HMSe could improve the growth performance, antioxidant capacity and nitrite tolerance of L. vannamei. The optimal supplementation for L. vannamei is 0.431 to 0.454 mg Se/kg as HMSe based on weight gain and hepatopancreas GSH-Px activities using the second-order polynomial regression analysis.

Alleviating the nitrite stress on anaerobic ammonium oxidation by pyrolytic biochar

The nitrite (NO2−) inhibition in anaerobic ammonium oxidation (anammox) process is widely reported. Here, the effects of three pyrolytic biochars (CS300, CS550 and CS800) were investigated to alleviate NO2− stress on anammox process under exposure of varied NO2−-N concentrations (70, 200, 400 and 600 mg L−1). No nitrite inhibition was observed at 70 mg N L−1. However, the total nitrogen removal efficiency (TNREs) decreased with NO2−-N concentration increased, while the biochar-amended groups achieved higher TNREs than the control (CK). At 200 mg N L−1, the TNREs were 60.2%, 99.0%, 98.5% and 86.6% for CK, CS300, CS550 and CS800, respectively. At 400 mg N L−1, the TNREs were 23.3%, 56.0%, 37.1% and 29.7% for CK, CS300, CS550 and CS800, respectively. At 600 mg N L−1 in which severe inhibition was observed, the TNREs were increased by 231% (p = 0.002), 149% (p = 0.014), and 51.0% (p = 0.166) for CS300, CS550 and CS800, respectively, as compared to CK, with the corresponding specific anammox activity increased by 3.1-, 2,0- and 1.1-folds, respectively. CS300 enriched the relative abundance of Candidatus Kuenenia and increased the gene copies of functional genes (hzsA, hdh, nirS and nirK). Besides, CS300 effectively alleviated the suppression of three membrane-associated enzyme complexes for anammox electron transport chain, indicating the possible contribution of redox-active moieties of CS300 to energy conversion metabolism for mitigating the NO2−-N inhibition. This study provided an effective strategy for alleviating NO2−-N stress by applying an environmentally compatible material (biochar) on anammox process.

Molecular characterization and expression dynamics of three key genes in the PI3K-AKT pathway reveal its involvement in the immunotoxicological responses of the giant river prawn Macrobrachium rosenbergii to acute ammonia and nitrite stress

Ammonia nitrogen and nitrite are two common forms of environmental toxicants for aquatic organisms including crustaceans. The PI3K-AKT pathway is an important intracellular signaling pathway related to cellular stress response, but involvement of this pathway in the immunotoxicological response of decapod crustaceans to aquatic toxicants such as ammonia nitrogen and nitrite still remains enigmatic. In this study, based on transcriptome mining and molecular cloning techniques, three key genes (named as MrPI3K, MrAKT and MrFoxO) in the PI3K-AKT signaling pathway were identified from the giant river prawn Macrobrachium rosenbergii. Sequence homology and phylogenetic analysis revealed that all the three genes harbored signature sequences of corresponding protein families, and shared high levels of similarities with their respective homologs from other species. MrPI3K, MrAKT and MrFoxO all displayed ubiquitous tissue distribution profiles, but their expression levels varied to a great extend among different tissues and between sexes. Following exposure to nitrite (20 mg/L nitrite-N) or ammonia (25 mg/L total ammonia-N) stresses for 24 h and 48 h, the three genes all responded by altering their expression levels at different time points, but they didn’t show uniform expression patterns following these stresses, indicating the diversified roles of these genes in different tissues and the complexity of this signaling pathway. Remarkably, MrPI3K and MrAKT were induced only in the hemocytes and intestine, respectively, indicating their specific roles in these organs. Our study demonstrated the potential utility of these genes as biomarkers of acute ammonia or nitrite toxicity in prawns, and also provided evidence that the PI3K-AKT pathway is involved in the immunotoxicological responses to nitrite and ammonia stress in M. rosenbergii.

Inclusion of Chlorella water extract in Oreochromis niloticus fingerling diets: Effects on growth performance, body composition, digestive enzyme activity, antioxidant and immune capacity, intestine and hepatic histomorphology and sodium nitrite stress resistance

Chlorella meal as feed ingredient in diets has been well evaluated in fish and shellfish, whereas limited information is available about Chlorella extract as feed supplement and their effects on growth performance and stress resistance of fish. This study was conducted to assess effect of Chlorella water extract (CWE) on growth performance, body composition, digestive enzyme activity, antioxidant and immune capacity, histomorphology, and sodium nitrite stress resistance of Oreochromis niloticus. In total of 1920 O. niloticus fingerlings with initial body weight of 0.9 ± 0.01 g were randomly distributed into six groups with four replicates (80 fish per replicate) and fed six diets containing 0 (C0), 2.7 (C2.7), 5.5 (C5.5), 11 (C11), 22 (C22), and 33 (C33) ml/kg dry matter of CWE for 56 d. Fish were suffered with a 24-h sodium nitrite stress test at the end of feeding trial. Results indicated that dietary CWE did not affect growth performance, body composition and histological appearance of fish, but quadratically increased (P < 0.05) protein efficiency ratio and trypsin activity as dietary CWE increasing. Inclusion of CWE in diets increased (P < 0.05) hepatic glutathione peroxidase and lysozyme activities. Fish fed CWE-containing diets had lower (P < 0.05) cumulative mortality rate but higher (P < 0.05) acid phosphatase and lysozyme activities as observed in the sodium nitrite stress test. In conclusion, dietary CWE did not affect growth performance, body composition and histological appearance, but improved protein utilization, trypsin activity, antioxidant and immune capacity, and sodium nitrite stress resistance of O. niloticus.

Effects of microcystin-LR and nitrite on the lifespan, reproduction, and heat shock responses of rotifer Brachionus calyciflorus at different temperatures

Cyanobacterial blooms aggravate with increasing temperature, and the increased concentrations of toxicants such as cyanotoxins and nitrite during bloom decay adversely affect the growth of aquatic animals. Heat-shock proteins (Hsps) are induced by a wide range of environmental stressors, including temperature and toxicants. In this study, Brachionus calyciflorus Pallas was exposed to different combined solutions of microcystin-LR (0, 10, 30, and 100 µg L−1) and nitrite (0, 1, 3, and 5 mg L−1) to evaluate their effects on the rotifer lifespan, the reproductive rate (R), and the responses of four Hsp genes at 20 °C, 25 °C, and 30 °C. Results revealed that single high doses of microcystin-LR (100 µg L−1) and nitrite (5 mg L−1) were harmful to the lifespan and reproduction of rotifers. Hormesis was induced by low doses of microcystin-LR (10–30 µg L−1) and nitrite (1–3 mg L−1). At different toxicant concentrations, the expression levels of Hsp40, Hsp60, Hsp70, and Hsp90 fluctuated, whereas reactive oxygen species (ROS) levels increased regardless of temperature. The two toxicants induced high levels of ROS production, which negatively affected the lifespan, R, and Hsp gene expression at 30 °C (p 0.05). Temperature, microcystin-LR, and nitrite had interactive effects on the lifespan, R, ROS levels, and Hsp gene expression levels (p < 0.05). The expression levels of Hsp genes are useful biomarkers of high-temperature exposure, and Hsp-mediated heat shock responses are important in microcystin-LR and nitrite stress tolerance of B. calyciflorus.

Metabolome responses of Enterococcus faecium to acid shock and nitrite stress.

Enterococcus faecium is gaining increasing interest due to its virulence and tolerance to a range of stresses (e.g., acid shock and nitrite stress in human stomach). The chemical taxonomy and basic structural features of cellular metabolite can provide us a deeper understanding of bacterial tolerance at molecular level. Here, we used hierarchical classification and molecular composition analysis to investigate the metabolome responses of E. faecium to acid shock and nitrite stress. Our results showed that considerable high biodegradable compounds (e.g., dipeptides) were produced by E. faecium under acid shock, while nitrite stress induced the accumulations of some low biodegradable compounds (e.g., organoheterocyclic compounds and benzenoids). Complete genome analysis and metabolic pathway profiling suggested that E. faecium produced high biodegradable metabolites responsible for the proton-translocation and biofilm formation, which increase its tolerance to acid shock. Yet, the presence of low biodegradable metabolites due to the nitrite exposure could disturb the bacterial productions of surface proteins, and thus inhibiting biofilm formation. Our approach uncovered the hidden interactions between intracellular metabolites and exogenous stress, and will improve the understanding of host-microbe interactions.

Effects of Ammonia and Nitrite Stress on Immune and Metabolic Responses of Japanese Sea Bass, Lateolabrax Japonicus

We investigated the effects of ammonia and nitrite stress on genes involved in metabolism and disease resistance in Japanese sea bass (Lateolabrax japonicus). Fish were exposed to pure seawater (control), 65.4 mg/L NH4+ -N, or 60.9 mg/L NO2–N for 7 days. Muscle, head-kidney, gill, spleen, and liver tissues were sampled regularly and the expression of immune and metabolism-related genes was monitored. The results indicated that the expression of immune-related genes such as heat shock proteins 70 and 90 were upregulated in the head-kidney, spleen, gill, liver, and muscle tissues under ammonia and nitrite stress. Glutamine synthetase (GS) expression increased to different degrees under ammonia and nitrite stress, while glutamate dehydrogenase (GDH) in the stress group upregulated in the gills. The expression of carbonic anhydrase (CA) was upregulated in the spleen and gill tissue under ammonia or nitrite stress. Expression of leptin (LEP) was upregulated in the muscle, gills and liver under nitrite stress; under ammonia stress, LEP expression did not fluctuate significantly in the spleen or gill. Insulin-like growth factor-1 (IGF-1) was downregulated in the spleen, while did not fluctuate obviously in the muscle and head-kidney under ammonia stress. These results indicate that ammonia and nitrite stress led to an upregulated immune related indexes response and increased metabolic activities in L. japonicus, and provides reference suggestions for response status of these health markers answering the outside environment intimidation.

Glutathione peroxidase 3 in the mud crab Scylla paramamosain: Characterization and regulation under nitrite stress

Glutathione peroxidases (GPx) are parts of the enzymatic antioxidant system that can eliminate the peroxides produced as effect of reactions of molecules with reactive oxygen species (ROS). In this study, a selenium-dependent glutathione peroxidase 3 cDNAs (designated as SpGPx3) was obtained from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpGPx3 was 639 bp, which encoded a putative protein of 212 amino acids. SpGPx3 protein contained a characteristic GPx signature motif, and an active site motif. Mud crabs were exposed to 20 mg L−1 nitrite for 72 h. Quantitative real-time PCR analysis revealed that the SpGPx3 mRNA was distributed abundantly in mud crab. The transcript levels of antioxidant enzyme genes (SpGPx3, SpSOD and SpCAT) were obviously induced after acute nitrite exposure. After knockdown of the SpGPx3 level, the mortality of mud crabs and malondialdehyde (MDA) content significantly increased under nitrite stress. These results suggested that SpGPx3 played an important role in protecting organisms against oxidative stress.

Responses of hemocyanin and energy metabolism to acute nitrite stress in juveniles of the shrimp Litopenaeus vannamei

Nitrite is a common toxic substance in culture systems of Litopenaeus vannamei, and the stress may disturb hemocyanin synthesis and energy metabolism and result in shrimp death. In the present study, nitrite at concentrations of 0 (control), 3.3 (46.2 NO2–N mg/L), 6.6 (92.4) and 9.9 mM (138.6) was used to evaluate the responses of hemocyanin level and energy metabolism in L. vannamei (5.80 ± 0.44 cm, 1.88 ± 0.38 g) for 96 h. The mortality rate at 96 h increased with nitrite concentration (50% at 9.9 mM, 40% at 6.6 mM, 30% at 3.3 mM, and 10% at 0 mM). In general, HIF-1α and hemocyanin mRNA expression in the nitrite stress groups was upregulated from 6 to 12 h and downregulated from 24 to 96 h. In the hemolymph, nitrite levels were significantly elevated in a dose-dependent manner, and exposure to nitrite stress significantly decreased the oxyhemocyanin content from 24 to 96 h. The glucose and lactate levels in the hemolymph in the nitrite stress groups were higher than those in the control group from 12 to 96 h. Compared with the control group, the shrimp in the nitrite stress groups exhibited decreased glycogen concentrations in the hepatopancreas. The triglyceride (TG) levels in the nitrite stress groups were all higher than those in the control group from 48 to 96 h. The hexokinase (HK) activity in the hepatopancreas and muscle increased in the nitrite stress groups from 48 to 96 h. In general, nitrite stress enhanced the activities of pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) in muscle from 24 to 96 h. In addition, nitrite stress decreased the activities of succinate dehydrogenase (SDH) and fatty acid synthase (FAS) from 24 to 96 h in the hepatopancreas and muscle. This study indicates that exposure to nitrite stress can enhance the accumulation of nitrite in the hemolymph and then reduce oxygenation and hemocyanin synthesis, leading to tissue hypoxia and thereby resulting in accelerated anaerobic metabolism and the inhibition of aerobic metabolism. The effects of nitrite stress on hemocyanin synthesis and energy metabolism may be one of the reasons for the mortality of L. vannamei in culture systems.

Integrated analysis of physiological, transcriptomic and metabolomic responses and tolerance mechanism of nitrite exposure in Litopenaeus vannamei

Nitrite accumulation in aquatic environments is a potential risk factor that disrupts multiple physiological functions in aquatic animals. In this study, the physiology, transcriptome and metabolome of the control group (LV-C), nitrite-tolerance group (LV-NT) and nitrite-sensitive group (LV-NS) were investigated to identify the stress responses and mechanisms underlying the nitrite tolerance of Litopenaeus vannamei. After LV-NT and LV-NS were subjected to nitrite stress, the hemocyanin contents were significantly decreased, and hepatopancreas showed severe histological damage compared with LV-C. Likewise, the antioxidant enzymes were also significantly changed after nitrite exposure. The transcriptome data revealed differentially expressed genes associated with immune system, cytoskeleton remodeling and apoptosis in LV-NT and LV-NS. The combination of transcriptomic and metabolomic analysis revealed nitrite exposure disturbed metabolism processes in L. vannamei, including amino acid metabolism, nucleotide metabolism and lipid metabolism. The multiple comparative analysis implicated that higher nitrite tolerance of LV-NT than LV-NS may be attributed to enhanced hypoxia inducible factor-1α expression to regulate energy supply and gaseous exchange. Moreover, LV-NT showed higher antioxidative ability, detoxification gene expression and enhanced fatty acids contents after nitrite exposure in relative to LV-NS. Collectively, all these results will greatly provide new insights into the molecular mechanisms underlying the stress responses and tolerance of nitrite exposure in L. vannamei.

Digital gene expression analysis in the gills of Ruditapes philippinarum after nitrite exposure

Due to the overload of pollutants from highly intensive anthropic activities, nitrite accumulates in offshore seawater and has been a long-lasting pollutant to the healthy aquaculture of the mollusk. In the present study, Ruditapes philippinarum was used as the target bivalve to receive nitrite exposure at environmental concentration for 1 and 7 days. Differentially expressed genes (DEGs) were detected and analyzed by a digital gene expression (DGE) approach to describe the toxicity of nitrite on the bivalve at the gene level. In the N1 group, 185 DEGs were generated and enriched in six Gene Ontology (GO) terms, including oxidoreductase activity, heme binding, tetrapyrrole binding, iron ion binding, metal binding and cation binding. The DEGs in the N1 group were also enriched in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, e.g., arachidonic acid metabolism and ovarian steroidogenesis. In the N7 group, 81 DEGs were generated without any GO enrichment but were enriched in five KEGG pathways, including protein processing in the endoplasmic reticulum, protein export, prion diseases, thyroid hormone synthesis and arachidonic acid metabolism. This suggested that nitrite exposure might cause adverse effects to the clams in several aspects, including oxidative damage, depressed immunity, and disorders in cell proliferation, hormone metabolism and tissue regeneration. Evaluation of oxidative stress indicated that nitrite exposure actually induced redox state imbalance by enhancing the contents of thiobarbituric acid reactive substances (TBARSs) and glutathione (GSH), and the activity of glutathione peroxidase (GSH-PX) but not superoxide dismutase (SOD). These results will provide valuable gene references for further study on the toxicology mechanism of bivalves under environmental nitrite stress.

LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species.

The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris.

Stable partial nitrification of domestic sewage achieved through activated sludge on exposure to nitrite

Partial nitrification is crucial for application of autotrophic nitrogen removal which is beneficial for treating carbon-limited wastewater. This study presents an alternative strategy for achieving partial nitrification of domestic wastewater treatment, by treating activated sludge of a nitrifying reactor on long-term nitrite exposure. Initially, the nitrifying reactor obtained complete nitrification. After the sludge of the reactor was treated with nitrite at concentration of 5–30 mg·L−1 without feeding for 32 days, the nitrite accumulation ratio (NAR) above 90% was achieved in 30 days, and then the NAR stabled at 97.9% under low temperature of 16.6 °C. Further analysis showed that the activity decay rate of ammonium-oxidizing bacteria (AOB) (0.020 d−1) was lower than that of nitrite-oxidizing bacteria (NOB) (0.035 d−1) under nitrite stress. Meanwhile, the NOB were effectively suppressed while AOB were dominant. These observations supported the feasibility of achieving stable partial nitrification by treating sludge on long-term exposure to nitrite.

Identification and characterization of differentially expressed genes in hepatopancreas of oriental river prawn Macrobrachium nipponense under nitrite stress

The oriental river prawn Macrobrachium nipponense is a highly adaptable, tolerant, and fecund freshwater prawn that inhabits a wide range of aquatic environments. The hepatopancreas of crustaceans is not only a site for secretion of digestive enzymes, and also plays important roles in several metabolic processes, such as lipid and carbohydrate metabolism. It is the main organ for the detoxification and immunity. In this study, high-throughput sequencing techniques were used to detect the effect of nitrite stress (10 mg/L nitrite-N for 48 h) on gene expression in the hepatopancreas of M. nipponense. A total of 13,769 million reads were harvested, and 94,534 transcripts were de novo assembled using Trinity software and produced 56,054 non-redundant transcripts. A total of 825 differentially expressed genes were obtained comparing 48 h nitrite stress with control group. In the analysis of GO and KEGG database, significant differences were found in 49 pathways. Immune-related pathways under nitrite stress included arginine and proline metabolism, glutamate metabolism, Jak-Stat signaling pathway, endocytosis, wnt signaling pathway, RIG-I-like receptor signaling pathway, TGF-beta signaling pathway, GnRH signaling pathway and phagosome. Apoptosis-related pathway was also significantly altered, such as lysosome and apoptosis. Remarkably, nitrite stress altered the expression patterns of key apoptosis genes (tetraspanins-like protein, LAMP, CD63, caspase 3C and Caspase 1) and immune genes (Serine proteinase-like protein, C-type lectin, daf-36, SOCS-2, alpha-2-macroglobulin), confirmed that nitrite-stress induce immune response and eventually even apoptosis. This study provided a new insight into the role of hepatopancreas in crustaceans, and further investigation will continue.

Physical Responses of Lates calcarifer to Acute Nitrite Stress

Abstract_The acute toxicity of nitrite (133.33, 266.66, 399.99mg/L) to 15 days posthatched Lates calcarifer (barramundi) was studied by semi-static bioassay method; 0 mg/L nitrite was used as control. Samples were obtained at 0, 6, 12, 24, 36, 48, 72 and 96 h respectively to measure and analyze the activity of antioxidant, digestive enzymes, and concentration of serum cortisol. Nitrite concentration and exposure time significantly affected the antioxidant and digestive enzymes activities. By the end of the experiment, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in the experimental group were significantly higher than in the control group, while peroxidase (POD) and catalase activities were significantly lower than in the control group except for in higher concentrations. Differing from POD and catalase, the acid phosphatase (ACP) activity of sub-concentration treatment was significantly higher than in the control group. Amylase activity in the nitrite stressed group was significantly lower than the control group, while the results of protease and lipase activity were reversed. Cortisol activity was delayed because of acute nitrite stress in the barramundi. Lates calcarifer showed certain short-term tolerance to nitrite stress. Results from the present study indicate that nitrite stress stimulates the responses of antioxidant and digestive enzyme activity and cortisol in fish, and these parameters can be used as physical indicators of stress.

Nitrite-Induced Oxidative Stress, Histopathology, and Transcriptome Changes in the Mud Crab (Scylla paramamosain)

Nitrite in the aquatic environment is highly toxic to aquatic animals. However, the mechanism by which the mud crab responds to nitrite-induced stress remains unclear. In this study, we investigated the physiological response and molecular mechanism in the mud crab (Scylla paramamosain) exposed to the acute nitrite exposure (20 mg/L) for 24h. The results showed that nitrite exposure induced significant changes in antioxidant enzyme activity and MDA content. Severe cytological damage was observed in the hepatopancreas. After 24h exposure to nitrite, 11,638 differentially expressed genes (DEGs) were identified by transcriptome analysis. These DEGs were involved in many pathways related to oxidative stress and immune responses. Our results also found that FoxO signaling pathway, p53 signaling pathway, and NF-kB signaling pathway participated in the anti-stress defense against nitrite stress. The study provides new insight into the understanding of nitrite-induced toxicity in the mud crab.

Dietary supplementation of selenium yeast enhances the antioxidant capacity and immune response of juvenile Eriocheir Sinensis under nitrite stress

This study elucidates the response to nitrite stress and the effect of dietary selenium supplements on the growth, antioxidant activity, immunity and transcriptome of juvenile Chinese mitten crab Eriocheir sinensis. In the control group, the crabs were fed the diet without selenium supplementation and there was no nitrite addition to the water. In the test group, the crabs were fed diets with three levels of selenium 0 (N1), 0.5 (N2) and 1.0 (N3) mg/kg in the water containing 2 mg/L NO2N as a stress factor for eight weeks. Feed conversion ratio (FCR) was improved by adding dietary selenium. There was no significant difference in specific growth rate and weight gain between N1 and the control groups, or among different selenium levels in the test group. The superoxide dismutase (SOD) activity was significantly lower, but malondialdehyde (MDA) was higher in the N1 group than those in the serum and hepatopancreas of the control group. The activities of SOD, glutathione peroxidase (GPx) and acid phosphatase increased at the medium level of selenium but decreased as the level of dietary selenium increased to 1.0 mg/kg. The serum lysozyme (LZM) activity increased but the MDA content in both serum and hepatopancreas decreased with the increase of selenium levels. The total clean reads of the crabs in the control group, N1 and N3 groups reached 390.7M and were assembled into 106 471 transcripts. Compared with the control group, 1196 gene were significantly expressed (588-up and 608-down) in the N1 group under nitrite stress. Between the N1 and N3 groups, the expression of 1537 genes (751-up and 786-down) were significantly different. KEGG pathway analysis reveals that 11 and 19 pathways were significantly different between N1 and control and between N3 and N1 groups, respectively. Transcriptome results demonstrate that nutrient metabolism is much more active in crabs fed additional selenium under nitrite stress. This study indicates that dietary selenium can improve both antioxidant capacity and immune response and alter the protein and carbohydrate metabolism of E. sinensis under nitrite stress.

Genome-wide gene expression analysis reveals novel insights into the response to nitrite stress in gills of Branchiostoma belcheri

Amphioxus has been widely used as a model for the comparative immunology of vertebrates. Studies have reported that gene expression changes in the amphioxus gill in response to biotic stress, such as microbial and their mimic challenge, but little is known about how gene expression is affected by abiotic stress in the marine environment, such as nitrite. A lack of information regarding gene expression response to abiotic stress hinders a comprehensive understanding of gill defense response in amphioxus. Here, RNA sequencing was used to carry out gene expression profiling analyses of Branchiostoma belcheri gills under nitrite stress. Six libraries were created for the control and treatment groups, including three biological replicates. In total, 2416 differently expressed genes (DEGs) were detected in response to nitrite stress, of which 1522 DEGs were up-regulated in the treatment group in comparison to the control, while the remaining 894 DEGs were down-regulated genes. Functional enrichment revealed that these DEGs are primarily involved in disease, innate immunity, xenobiotic biodegradation and metabolism, and biomolecular processes and apoptosis. We screened 11 key nitrite-responsive DEGs to detect their expression responses to nitrite stress at different time points, and validate the sequencing data using real time quantitative PCR. The results indicated that the expression of gene encoding CYP3A, POD, CASPR1, GST, MAO, DDH, and XDH/XO were induced, while those encoding MRC, GT, DNASE1L, and RIPK5 were reduced, to participate in the anti-nitrite response. This study provides a useful resource for research of molecular toxicology in amphioxus under environmental stress.

Tolerance and physiological responses of sweet flag (Acorus calamus L.) under nitrite stress during wastewater treatment

Various newly discovered novel biological nitrogen removal (BNR) routes via nitrite offer great potential for improving nitrogen removal efficiency of constructed wetlands. In these novel routes, nitrite is required as the substrate or intermediary media. Hence, in the present study, the tolerance and physiological responses of sweet flag (Acorus calamus L.) to nitrite stress were investigated. Results showed that the physiology of sweet flag was not significantly affected when exposed to nitrite up to 20 mg/L. High nitrite levels (≥30 mg/L) led to changes in its cell membrane permeability, plant photosynthesis and activities of antioxidant enzymes. Particularly, when exposed to a nitrite level of 40 mg/L, sweet flag displayed trends of significant increase in the percentage of electrolyte leakage and contents of malondialdehyde and proline, whereas the chlorophyll content and plant height decreased and antioxidant defence systems were disrupted. Therefore, for CWs with sweet flag, 30 mg/L is the recommended threshold value for nitrite.