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Erythropoietin (EPO) is a glycoprotein hormone that plays a crucial role as a downstream regulator in the Hypoxia-inducible Factor (HIF) signaling pathway, mainly involved in the regulation of erythropoiesis. The open reading frame (ORF) of EPO in blunt snout bream (Megalobrama amblycephala , BSB) was 561 bp, encoding 186 amino acids. Phylogenetic analysis revealed it clusters with homologs in others teleost. We designed two target sites (targets 1, 2) to obtain gene knockout mutants, with the mature mRNA sequence of EPO that were located in exon 2, exon 3, respectively. The mutation efficiency of targets 1, 2 was 19.60%, 24.22%, respectively, both containing 6 mutation types. F 1 EPO +/− mutants with the same mutation type were screened, and select F 2 EPO −/− mutants after mating. At the age of five months, EPO −/− fish demonstrated a significant (p < 0.05) increase in the average oxygen tension threshold for the loss of equilibrium (LOE crit) and the shedding degree of interlamellar cell masses (ILCM), whereas a significant (p < 0.05) decrease was observed in the number of red blood cells (RBC) and the concentration of hemoglobin (Hb) under hypoxia conditions, in comparison to the control group. Notably, injection of EPO mRNA rescued the downregulation of EPO expression in EPO −/− mutant, and overexpression of the EPO gene did not disturb the normal development of BSB embryos. These findings provide new insights into the potential for applying CRISPR/Cas9 in other aquaculture fish species and the breeding of hypoxia-tolerant BSB. • The selected EPO CRISPR/Cas9 knockout targets are more efficient in our study. • EPO knockout mutants EPO −/− exhibited significantly reduced hypoxia tolerance. • Injection of EPO mRNA rescued the downregulation of EPO expression of EPO −/− mutant. [ABSTRACT FROM AUTHOR]

Blunt snout bream (Megalobrama amblycephala) is a hypoxia-sensitive fish species. A sudden decrease in the dissolved oxygen (DO) content in the pond will cause massive death. Therefore, breeding of new hypoxia-tolerant strains is important for blunt snout bream aquaculture. From 2007, selective breeding under hypoxic stress was performed on the wild M. amblycephala (F 0) population from Poyang Lake, China. In 2015, the selectively bred hypoxia-tolerant F 4 population was obtained, which the LOE crit (loss of equilibrium) was 0.54 mg·L−1, 0.89 mg·L−1 and 1.28 mg·L−1 at 10 °C, 25 °C and 30 °C, respectively. And the LOE crit in the 'Pujiang No.1' breed (control group) was 0.72 mg·L−1, 1.03 mg·L−1 and 1.41 mg·L−1 at 10 °C, 25 °C and 30 °C, respectively. Compared with the control group, the hypoxia-tolerant population had a significantly (P < 0.01) lower critical oxygen tension value at which they lost equilibrium (LOE crit) at the same temperatures. Furthermore, when hypoxia-tolerant population was exposed to 4 or 7 days of hypoxia (DO = 2.0 mg·L−1) at 10 °C, the average protruding gill lamellar height and mean gill lamellar area were significantly (P < 0.01) smaller than those of the control group. Simultaneously, in order to enhance their blood oxygen-carrying capacity to adapt to the hypoxic environment, the erythrocyte count and hemoglobin concentration of the hypoxia-tolerant population were significantly increased, and were much higher (P < 0.01) than those of the control group under hypoxia treatment (DO = 2.0 mg·L−1). These data showed that the hypoxia-tolerant F 4 population had high potential for coping with hypoxic stress, and we can get a hypoxia-tolerant strain of blunt snout bream by continuous hypoxia breeding in aquaculture. • Blunt snout bream from Poyang Lake were selected through both hypoxia breeding− and population breeding. • Compared to 'Pujiang No.1', the hypoxia-tolerant F 4 population has a better hypoxia tolerance. • A hypoxia-tolerant strain of blunt snout bream by continuous hypoxia was obtained. [ABSTRACT FROM AUTHOR]

• Duplicated socs1 gene were identified in blunt snout bream. • Both socs1a and socs1b were expressed in all the throughout embryogenesis and tested tissues of blunt snout bream. • Both SOCS1a and SOCS1b inhibition of GH and JAK-STAT pathway. • Inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensively involved in many different cytokine signaling pathways, such as regulation of the immune system and growth of organism. However, the molecular and functional information on socs1 genes in freshwater fish is unclear. In the present paper, we identified and characterized the full-length closely related but distinct socs1 genes (socs 1a and − 1b) in blunt snout bream (Megalobrama amblycephala). The bioinformatic analysis results showed that duplicated socs1 s shared majority conserved motifs with other vertebrates. Both socs1a and − 1b mRNAs were detected throughout embryogenesis, and gradually increase and then constantly expressed after 16 hpf. Whole-mount in situ hybridization demonstrated that socs1a and socs1b mRNAs were detected in the brain at 12hpf and 24hpf, and in the notochord and brain at 36hpf. In adult fish, the socs1a mRNA were strongly expressed in the heart, eye, kidney, spleen and gonad, but were found to be relatively low in the intestine and liver. On the other hand, the expression of socs1b mRNA was significantly high in the muscle, eye and spleen, and relatively low in the intestine, liver, skin and heart. The results of hGH treatment experiment showed that socs1a and 1b mRNAs were upregulated markedly in the kidney, muscle and liver. Overexpression of socs1s significantly inhibit the GH and JAK/STAT factor stat3 and the inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. These results indicate that SOCS1 plays an important role in regulating growth and development. [ABSTRACT FROM AUTHOR]

The atmospheric and room temperature plasma (ARTP) mutagenesis technique was first used in the present study to produce whole-genomic mutants in blunt snout bream (Megalobrama amblycephala). The optimal ARTP mutation parameters for semen were established according to sperm motility and abnormal embryonic rate. A total of 2026 mutated offsprings were produced with normal and abnormal individuals with a ratio of approximately 1:1. Among them, 384 normal mutant fish (~20%), after three months of cultivation, demonstrated improved performance with either fast growth or hypoxia tolerance, and 89 mutant individuals with both hypoxia tolerance and fast growth (H&F). The average bodyweight of the H&F fish increased by 26.48% compared with the control group. The average oxygen tension threshold for loss of equilibrium (LOE crit) of the H&F fish was 0.45 mg/L, significantly (p < 0.05) lower than 0.86 mg/L in the control group. Meanwhile, the red blood cell count, hemoglobin concentration, and Na+/K+-ATPase enzyme activity of the H&F group increased significantly (p < 0.05), whereas, the gill tissue cell apoptosis rate decreased considerably (p < 0.05), suggesting that they are hypoxia tolerant. According to the genome resequencing analysis, 3,195,434 SNPs and 927,930 InDels were found in the H&F group, which were significantly higher than the 9127 SNPs and 2908 InDels in the control group. The average mutation rate in the H&F fishes reached 2.98 × 10−3 at the genome level, approximately 350 times that of the control group. Importantly, 3651 special non-synonymous SNP mutations were found in the H&F fish, which are located in 1223 genes. Among them, eight hypoxia- and growth-related genes with non-synonymous mutations at one or more sites were differentially expressed (DE) in H&F fish. Taken together, our results establish a novel whole-genomic mutagenesis approach using ARTP in blunt snout bream, which should be useful for future genetic breeding. • Total 2026 ARTP-mutated offsprings were produced with the optimal ARTP mutation technique. • 4% ARTP-mutant individuals (89 fish) with both hypoxia tolerance and fast growth. • The average mutation rate reached 2.98 × 10−3 at the genome level, about 350 times than that of the control group. • 3,195,434 SNPs and 927,930 InDels were found in the ARTP-mutated group, which were 350 and 320 times higher than those in control group. [ABSTRACT FROM AUTHOR]

The suppressor of cytokine signaling 3 (SOCS3) negatively regulates the responses of various immune cytokines. In this study, we identified socs3s genes of blunt snout bream. 209- and 216-aa long peptides are encoded by socs3a and socs3b genes, respectively. The socs3s mRNAs are expressed consistently during the entire process of embryonic development. Whole-mount in situ hybridization detected socs3a in the eyes and posterior somites at 12 h post fertilization (hpf), transcribed at the otic vesicle at 24 hpf, and transcribed at the eyes, brain, and otic vesicle at 36 hpf; while the socs3b mRNA was transcribed at the notochord at 12 hpf, expressed in the brain, eyes, and tailbud at 24 hpf, and detected in the brain at 36 hpf. The expression of socs3a is slightly different from that of socs3b in tissues of juvenile and adult blunt snout bream. After recombinant human growth hormone (hGH) treatment, the transcript levels of socs3s of blunt snout bream were increased in gills, spleen, kidney, and gonads. After Aerononas hydrophila infection, the mRNA levels of socs3s of blunt snout bream were significantly increased in the liver, spleen, intestine, and kidney tissues. Blunt snout bream were susceptible to various pathogenic microorganisms, we intraperitoneally injected blunt snout bream with A. hydrophila to explore the immune mechanism of socs3s. These results suggested that socs3s of blunt snout bream plays important roles in the regulation of embryonic development and tissue growth, and that socs3s may also play key roles in regulating the bacterial-induced congenital immune response. Socs3s genes has the potential to be used as targeted genes to improve the immunity against bacteria, which is conducive to the improvement of production and breeding. Unlabelled Image • Two distinct socs3 genes has the potential to be used as targeted genes to improve the immunity against bacteria. [ABSTRACT FROM AUTHOR]