Wuxiao Zhang

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.

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

&NA; 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 ± 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 and 12 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. Graphical abstract Figure. No caption available. HighlightsNitrite exposure induced oxidative stress and tissue damaged on the liver of Aristichthys nobilis.Upregulation of GSH‐Px, GST, GR mRNA expressions and enzyme activities maintained the dynamic balance of GSH/GSSG system.The GSH/GSSG system fighted against oxidative injury caused by nitrite stress.

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 (48.63mg/L) for 72h, 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. HIGHLIGHTSNitrite exposure caused DEGs in bighead carp gill and liver.DEGs in gill and liver responded to nitrite stress were associated with immune response.Nitrite exposure‐induced pathomechanism associated with immune responses and infections.Gills of bighead carp are more susceptible to nitrite stress than liver.

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)

ABSTRACT 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. HighlightsOral glucose administration affected blood respiratory burst in M. amblycephala.Oral glucose administration induced blood glucose fluctuation and oxidative stress.Upregulation of Nrf2 against oxidative stress injury due to hyperglycemia.Inhibition of PI3K, Akt, and HO‐1 induced inflammation due to hyperglycemia.