Systemic and strict regulation of the glutathione redox state in mitochondria and cytosol is needed for zebrafish ontogeny.

BACKGROUND: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. METHODS: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. RESULTS: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. CONCLUSIONS: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. GENERAL SIGNIFICANCE: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.

FOXO3/Rab7-Mediated Lipophagy and Its Role in Zn-Induced Lipid Metabolism in Yellow Catfish (Pelteobagrus fulvidraco).

Lipophagy is a selective autophagy that regulates lipid metabolism and reduces hepatic lipid deposition. However, the underlying mechanism has not been understood in fish. In this study, we used micronutrient zinc (Zn) as a regulator of autophagy and lipid metabolism and found that Ras-related protein 7 (rab7) was involved in Zn-induced lipophagy in hepatocytes of yellow catfish Pelteobagrus pelteobagrus. We then characterized the rab7 promoter and identified binding sites for a series of transcription factors, including Forkhead box O3 (FOXO3). Site mutation experiments showed that the -1358/-1369 bp FOXO3 binding site was responsible for Zn-induced transcriptional activation of rab7. Further studies showed that inhibition of rab7 significantly inhibited Zn-induced lipid degradation by lipophagy. Moreover, rab7 inhibitor also mitigated the Zn-induced increase of cpt1α and acadm expression. Our results suggested that Zn exerts its lipid-lowering effect partly through rab7-mediated lipophagy and FA ß-oxidation in hepatocytes. Overall, our findings provide novel insights into the FOXO3/rab7 axis in lipophagy regulation and enhance the understanding of lipid metabolism by micronutrient Zn, which may help to reduce excessive lipid accumulation in fish.

Effects of dietary carbohydrate/lipid ratios on non-specific immune responses, antioxidant capacity, hepatopancreas and intestines histology, and expression of TLR-MAPK/NF-κB signaling pathway-related genes of Procambarus clarkii.

To investigate the effects of dietary carbohydrate/lipid (CHO: L) ratios on non-specific immune responses, antioxidant capacity, and expression of TLR-MAPK/NF-κB signaling pathway-related genes of red swamp crayfish (Procambarus clarkii). Four isonitrogenous and isoenergetic diets containing different CHO: L ratios were formulated. The results showed that the group with a CHO: L ratio of 5.94 had better growth performance (P < 0.05). The highest T-AOC, CAT, and SOD activities and the lowest MDA content in hemolymph and hepatopancreas were observed in the group with a CHO: L ratio of 5.94 (P < 0.05). The lowest activities of ALT, AST, ACP, AKP, and ALB in the hemolymph were observed in CHO: L ratio 5.94 group (P < 0.05), while the highest LZM activity, TP, and GLB content were observed in CHO: L 5.94 group (P < 0.05). The highest mRNA expression levels of tlr3, myd88, and mapk3, and the lowest mRNA expression levels of nf-kb α, nf-kb ß, nf-kb p105, and traf6 were observed in the CHO: L of 5.94 group (P < 0.05). The highest mRNA expression levels of immune-related genes were observed in the CHO: L of 5.94 group (P < 0.05). Overall, these results indicated that the optimum dietary CHO: L ratio is vital in promoting growth and enhancing antioxidants and immunity to maintain red swamp crayfish's intestinal and hepatopancreas health status. In conclusion, the diets with a CHO:L ratio of 5.94 (approximately 36.23% carbohydrate and 6.10% lipid) is optimal for juvenile red swamp crayfish's physiological condition and health status.

The effects of crowding stress on the growth, physiological response, and gene expression of the Nrf2-Keap1 signaling pathway in blunt snout bream (Megalobrama amblycephala) reared under in-pond raceway conditions.

We evaluated the effects of high density stress on growth performance, antioxidant parameters, and Nrf2 pathway signaling molecules after different lengths of exposure (30, 60, or 90 days) of Megalobrama amblycephala to in-pond raceway aquaculture systems (IPRS). M. amblycephala (average initial weight 2.33 ±â€¯0.15 g) were reared at two different initial densities (low density group [LD] had 534 fish/m3 and high density group [HD] had 1073 fish/m3) for 90 days. The growth performance was adversely influenced by the high stocking density. The HD group had elevated white blood cell counts, hemoglobin content, and hematocrit on days 60 and 90. The mRNA levels of NOX2 on days 60 and 90, Nrf2 on days 30, 60, and 90, Keap1 on day 30, Bach1 on days 30 and 60, SOD on day 30, and CAT on day 30 were significantly higher in the HD group than in the LD group. Similarly, higher trends were observed in the enzymatic activities of SOD on day 60, CAT on days 60 and 90, and GPx on day 60 in the HD group, compared to the LD group. Furthermore, HD bream showed an increased MDA content on days 60 and 90 compared to that of the LD group. This study demonstrates that high density-induced antioxidant defenses were involved in modifications to the enzymatic and transcriptional regulation of Nrf2-Keap1 signaling molecules and that M. amblycephala growth was reduced in a crowded IPRS.

Comparative transcriptome analysis reveals the gene expression profiling in bighead carp (Aristichthys nobilis) in response to acute nitrite toxicity.

OBJECTIVE: Nitrite exposure induces growth inhibition, metabolic disturbance, oxidative stress, organic damage, and infection-mediated mortality of aquatic organism. This study aimed to investigate the mechanism in responses to acute nitrite toxicity in bighead carp (Aristichthys nobilis, A. nobilis) by RNA-seq analysis. METHODS: Bighead carps were exposed to water with high nitrite content (48.63 mg/L) for 72 h, and fish livers and gills were separated for RNA-seq analysis. De novo assembly was performed, and differentially expressed genes (DEGs) between control and nitrite-exposed fishes were identified. Furthermore, enrichment analysis was performed for DEGs to annotate the molecular functions. RESULTS: A total of 406,135 transcripts and 352,730 unigenes were tagged after de novo assembly. Accordingly, 4108 and 928 DEGs were respectively identified in gill and liver in responses to nitrite exposure. Most of these DEGs were up-regulated DEGs. Enrichment analysis showed these DEGs were mainly associated with immune responses and nitrogen metabolism. CONCLUSIONS: We suggested that the nitrite toxicity-induced DEGs were probably related to dysregulation of nitrogen metabolism and immune responses in A. nobilis, particularly in gill.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Dynamic mRNA and miRNA expression analysis in response to hypoxia and reoxygenation in the blunt snout bream (Megalobrama amblycephala).

Adaptation to hypoxia is a complex process involving various pathways and regulation mechanisms. A better understanding of the genetic influence on these mechanisms could permit selection for hypoxia-sensitive fish. To aid this understanding, an integrated analysis of miRNA and mRNA expression was performed in Megalobrama amblycephala under four acute hypoxia and reoxygenation stages. A number of significantly differentially-expressed miRNAs and genes associated with oxidative stress were identified, and their functional characteristics were revealed by GO function and KEGG pathway analysis. They were found to be involved in HIF-1 pathways known to affect energy metabolism and apoptosis. MiRNA-mRNA interaction pairs were detected from comparison of expression between the four different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating hypoxia stress. As a unique resource for gene expression and regulation during hypoxia and reoxygenation, this study could provide a starting point for further studies to better understand the genetic background of hypoxia stress.

Regulation mechanism of oxidative stress induced by high glucose through PI3K/Akt/Nrf2 pathway in juvenile blunt snout bream (Megalobrama amblycephala).

This study was conducted to investigate the effects of oral administration of a high concentration of glucose on the respiratory burst, antioxidant status, and hepatic gene expression of heme oxygenase-1 (ho1) and PI3K/Akt/Nrf2-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala). Blunt snout bream juveniles with an initial body weight of 19.94 ± 0.58 g were orally fed with a high concentration of glucose (3 g/kg body weight). The results indicated that plasma glucose exhibited a biphasic response. Acute and persistent hyperglycemia due to the oral glucose administration significantly reduced (P < 0.05) the white blood cell count, red blood cell count, and hemoglobin content and caused oxidative stress (significantly increased alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and glucose levels) and early apoptosis of hepatocytes in the fish. Hepatic superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities increased rapidly (P < 0.05) as protection from oxidative stress and were downregulated (P < 0.05) because of persistent hyperglycemia. Blood respiratory burst was significantly reduced (P < 0.05) because of hyperglycemia and showed a trend that was opposite to that of plasma glucose. Slight upregulation of nrf2 mRNA and antioxidants acts as a compensative protection mechanism, and the downregulated PI3K/Akt pathway blocked this function of Nrf2. In conclusion, the PI3K/Akt pathway and Nrf2 mediated the antioxidative mechanism independently in the blunt snout bream juveniles subjected to the oral administration of a high glucose concentration.

De novo assembly of the blunt snout bream (Megalobrama amblycephala) gill transcriptome to identify ammonia exposure associated microRNAs and their targets.

De novo transcriptome sequencing is a robust method for microRNA (miRNA) target gene prediction, especially for organisms without reference genomes. Following exposure of Megalobrama amblycephala to ammonia (0.1 or 20 mg L(-1) ), two cDNA libraries were constructed from the fish gills and sequenced using Illumina HiSeq 2000. Over 90 million reads were generated and de novo assembled into 46, 615 unigenes, which were then extensively annotated by comparing to different protein databases, followed by biochemical pathway prediction. The expression of 2666 unigenes significantly differed; 1961 were up-regulated, while 975 were down-regulated. Among these, 250 unigenes were identified as the targets for 10 conserved and 4 putative novel miRNA families by miRNA target computational prediction. We examined expression of ssa-miRNA-21 and its target genes by real-time quantitative PCR and found agreement with the sequencing data. This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for Megalobrama amblycephala and will be useful for gene expression profile analysis and miRNA functional annotation.

Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-8 from the blunt snout bream (Megalobrama amblycephala) exposed to ammonia.

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this study, we isolated the caspase-8 gene from Megalobrama amblycephala, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The 2034 bp full-length M. amblycephala caspase-8 cDNA sequence contained an ORF of 1467 bp encoding a polypeptide of 489 amino acid residues, a 5'-UTR of 102 bp and a 3'-UTR of 462 bp. The caspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10, caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. Phylogenetic analysis revealed that M. amblycephala caspase-8 were clustered with the caspase-8 from other vertebrate. Real-time quantitative PCR analysis revealed that caspase-8 transcripts were detected in liver after exposure to ammonia. Meanwhile using Western blot analysis, caspase-8 cleaved fragment was detected and significant alteration of procaspase-8 level was found with the same ammonia treatment condition. Furthermore, the result of immunohistochemical detection showed that remarkable changes of immunopositive staining were observed after ammonia treatment. Accordingly, the results signify that caspase-8 of fish may play an essential role in ammonia induced apoptosis.