Effects of hypoxia on the gill morphological structure, apoptosis and hypoxia-related gene expression in blunt snout bream (Megalobrama amblycephala).

The blunt snout bream (Megalobrama amblycephala) is a typical hypoxia-sensitive fish, and hypoxia stress leads to reduced vitality and yield during aquaculture. To explore the specific adaptation mechanism under hypoxia, the blunt snout bream was treated with hypoxia (DO = 2.0 ± 0.1 mg/L) for 24 h, followed by 3 h of recovery. Our results depicted that the gill filament structure of blunt snout bream changed after hypoxia. During hypoxia for 24 h, the gill filament structure was altered, including a more than 80% expansion of the lamellar respiratory surface area and a proportionate apoptosis decrease in interlamellar cell mass (ILCM) volume. Thus, the water-blood diffusion distance was shortened to less than 46%. During hypoxia for 24 h, the activity of ROS in gill tissue increased significantly (p < 0.05), while the mitochondrial membrane potential decreased significantly (p < 0.05). During hypoxia, mRNA expression level of anti-apoptotic gene Bcl-2 in the gills of blunt snout bream decreased significantly (p < 0.05), while the expression of pro-apoptotic gene Bax mRNA increased significantly (p < 0.05). Thus, the ratio of Bax/Bcl-2 mRNA increased in the gills of blunt snout bream to promote the activity of Caspase-3. Together, our results indicated hypoxia-induced apoptosis in the gills of blunt snout bream through the mitochondrial pathway. In addition, a decreased expression of Phd1 and an increased expression of Hif-1α in gills under hypoxia stress indicates that blunt snout bream may cope with hypoxia-induced apoptosis by enhancing the HIF pathway. These results provide new insights into fish's adaptation strategies and mechanisms of hypoxia.

Effects of high NaHCO3 alkalinity on growth, tissue structure, digestive enzyme activity, and gut microflora of grass carp juvenile.

With the gradual decrease in freshwater resources, the available space for freshwater aquaculture is diminishing. As a result, saline-alkaline water aquaculture has emerged as a crucial method to fulfill the increasing demand. This study investigates the impact of alkaline water on the growth performance, tissues (gill, liver, and kidney), digestive enzyme activity, and intestinal microbiology in grass carp (Ctenopharyngodon idella). The aquarium conditions were set with sodium bicarbonate (18 mmol/L (LAW), 32 mmol/L (HAW)) to simulate the alkaline water environment. A freshwater group was the control (FW). The experimental fish were cultured for 60 days. The findings revealed that NaHCO3 alkaline stress significantly reduced growth performance, caused alterations in the structural morphology of gill lamellae, liver, and kidney tissues, and led to decreased activity of intestinal trypsin and lipase amylase (P < 0.05). Analysis of 16S rRNA sequences demonstrated that alkalinity influenced the abundance of dominant bacterial phyla and genera. Proteobacteria showed a significant decrease under alkaline conditions, while Firmicutes exhibited a significant increase (P < 0.05). Furthermore, alkalinity conditions significantly reduced the abundance of bacteria involved in protein, amino acid, and carbohydrate metabolism, cell transport, cell decomposition, and environmental information processing. Conversely, the abundance of bacteria associated with lipid metabolism, energy metabolism, organic systems, and disease functional flora increased significantly under alkalinity conditions (P < 0.05). In conclusion, this comprehensive study indicates that alkalinity stress adversely affected the growth performance of juvenile grass carp, likely due to tissue damage, reduced activity of intestinal digestive enzymes, and alterations in intestinal microorganisms.

Functional exploration of SNP mutations in HIF2αb gene correlated with hypoxia tolerance in blunt snout bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A203A752) and its homozygous diplotype II (A203A203A752A752) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOEcrit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48 h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48 h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.

Effects of hypoxia and reoxygenation on oxidative stress, histological structure, and apoptosis in a new hypoxia-tolerant variety of blunt snout bream (Megalobrama amblycephala).

A new hypoxia-tolerant variety of blunt snout bream was obtained by successive breeding of the wild population, which markedly improved hypoxia tolerance. In this study, the hypoxia-tolerant variety was exposed to hypoxia (2.0 mg O2·L-1) for 4, 7 days. The contents of blood biochemical indicators including the number of red blood cells (RBC), total cholesterol (T-CHO), total protein (TP), triglyceride (TG), glucose (GLU), and lactic acid (LD) increased significantly (P < 0.05) under hypoxia. The glycogen content in the liver and muscle decreased significantly (P < 0.05) and the LD content in the brain, muscle and liver increased significantly (P < 0.05) under hypoxia. The levels of oxidative stress-related indicators i.e., superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) also changed significantly (P < 0.05) in the heart, liver, and intestine of the new variety under hypoxia. Additionally, hypoxia has caused injuries to the heart, liver, and intestine, but it shows amazing repair ability during reoxygenation. The apoptotic cells and apoptosis rate in the heart, liver, and intestine increased under hypoxia. Under hypoxia, the expression of the B-cell lymphomas 2 (Bcl-2) gene in the heart, liver, and intestine was significantly (P < 0.05) down-regulated, while the expression of the BCL2-associated agonist of cell death (Bad) gene was significantly (P < 0.05) up-regulated. These results are of great significance for enriching the basic data of blunt snout bream new variety in response to hypoxia and promoting the healthy development of its culture industry.

Gill remodeling increases the respiratory surface area of grass carp (Ctenopharyngodon idella) under hypoxic stress.

Seasonal changes, diurnal variations, and eutrophication result in periodic hypoxia in fish habitats, thus affecting the success of commercial aquaculture. In this study, the grass carp (Ctenopharyngodon idella) presented moderate hypoxia tolerance; they showed a medium critical oxygen tension during the loss of equilibrium. In response to 7 d of hypoxic exposure, the erythrocyte count and hemoglobin (Hb) concentration significantly increased (p < 0.01). To cope with the hypoxic environment, the grass carp underwent gill remodeling marked by reduction in the interlamellar cell mass (ILCM) and an increase in respiratory surface area. The gill remodeling under hypoxia was enabled by apoptosis induction. Although apoptotic signals were not found on ILCM cells, transferase dUTP nick end labeling (TUNEL) assay results indicated that after 1 d of hypoxic exposure, the number of TUNEL-positive cells per lamella increased until 4 d and then began to decrease. Consistent with the results of the TUNEL assay, the mRNA expression of apoptosis-related genes, caspase-3, Bax, and Bcl-2, increased at 1, 4, and 7 d of the hypoxia treatment. In addition, gill remodeling significantly (p < 0.01) decreased the concentration of sodium and chloride ions in the fish serum. These findings provide evidence that grass carps increase their respiratory surface area through gill remodeling by apoptosis in the gill filaments to acclimate to a hypoxic environment. This study expands our understanding of the morphological and physiological changes in grass carp in response to a hypoxic environment; therefore, it could be useful for maintaining grass carp production.

Chromosome-level genome assembly of grass carp (Ctenopharyngodon idella) provides insights into its genome evolution.

BACKGROUND: The grass carp has great economic value and occupies an important evolutionary position. Genomic information regarding this species could help better understand its rapid growth rate as well as its unique body plan and environmental adaptation. RESULTS: We assembled the chromosome-level grass carp genome using the PacBio sequencing and chromosome structure capture technique. The final genome assembly has a total length of 893.2 Mb with a contig N50 of 19.3 Mb and a scaffold N50 of 35.7 Mb. About 99.85% of the assembled contigs were anchored into 24 chromosomes. Based on the prediction, this genome contained 30,342 protein-coding genes and 43.26% repetitive sequences. Furthermore, we determined that the large genome size can be attributed to the DNA-mediated transposable elements which accounted for 58.9% of the repetitive sequences in grass carp. We identified that the grass carp has only 24 pairs of chromosomes due to the fusion of two ancestral chromosomes. Enrichment analyses of significantly expanded and positively selected genes reflected evolutionary adaptation of grass carp to the feeding habits. We also detected the loss of conserved non-coding regulatory elements associated with the development of the immune system, nervous system, and digestive system, which may be critical for grass carp herbivorous traits. CONCLUSIONS: The high-quality reference genome reported here provides a valuable resource for the genetic improvement and molecular-guided breeding of the grass carp.

The transcriptomic responses of blunt snout bream (Megalobrama amblycephala) to acute hypoxia stress alone, and in combination with bortezomib.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia. A new blunt snout bream strain, "Pujiang No.2", was developed to overcome this shortcoming. As a proteasome inhibitor, bortezomib (PS-341) has been shown to affect the adaptation of cells to a hypoxic environment. In the present study, bortezomib was used to explore the hypoxia adaptation mechanism of "Pujiang No.2". We examined how acute hypoxia alone (hypoxia-treated, HN: 1.0 mg·L- 1), and in combination with bortezomib (hypoxia-bortezomib-treated, HB: Use 1 mg bortezomib for 1 kg fish), impacted the hepatic ultrastructure and transcriptome expression compared to control fish (normoxia-treated, NN). RESULTS: Hypoxia tolerance was significantly decreased in the bortezomib-treated group (LOEcrit, loss of equilibrium, 1.11 mg·L- 1 and 1.32 mg·L- 1) compared to the control group (LOEcrit, 0.73 mg·L- 1 and 0.85 mg·L- 1). The HB group had more severe liver injury than the HN group. Specifically, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HB group (52.16 U/gprot, 32 U/gprot) were significantly (p < 0.01) higher than those in the HN group (32.85 U/gprot, 21. 68 U/gprot). In addition, more severe liver damage such as vacuoles, nuclear atrophy, and nuclear lysis were observed in the HB group. RNA-seq was performed on livers from the HN, HB and NN groups. KEGG pathway analysis disclosed that many DEGs (differently expressed genes) were enriched in the HIF-1, FOXO, MAPK, PI3K-Akt and AMPK signaling pathway and their downstream. CONCLUSION: We explored the adaptation mechanism of "Pujiang No.2" to hypoxia stress by using bortezomib, and combined with transcriptome analysis, accurately captured the genes related to hypoxia tolerance advantage.

Effects of hypoxia and reoxygenation on gill remodeling, apoptosis, and oxidative stress in hypoxia-tolerant new variety blunt snout bream (Megalobrama amblycephala).

Blunt snout bream plays an important role in freshwater aquaculture in China, but the development of its culture industry has been restricted by increasing hypoxia problem. Through the breeding of wild blunt snout bream populations (F0), a hypoxia-tolerant new variety (F6) was obtained. In this study, the new variety was stressed under low oxygen concentration (2.0 mg·L-1) for 4 and 7 days, the morphological structure of the gill tissue showed a striking change, the interlamellar cell mass (ILCM) volume reduced significantly (P < 0.05), and the lamellar respiratory surface area enlarged significantly (P < 0.05), compared to normoxic controls. After 7 days of oxygen recovery, gill remodeling was completely reversed. Additionally, the TUNEL-positive apoptotic fluorescence signals increased in the gills exposed to hypoxia up to 4 and 7 days; the apoptosis rate also increased significantly (P < 0.05). Under 4 and 7 days of hypoxia stress, the expression of anti-apoptotic gene Bcl-2 in the gills downregulated significantly (P < 0.05), with the significantly (P < 0.05) upregulated expression of pro-apoptotic gene Bad. Furthermore, under hypoxia stress, the activity or content of oxidative stress-related enzymes (superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione (GSH)) in gill tissue increased to varying degrees compared to normoxic controls. These results offer a new perspective into the cellular and molecular mechanism of hypoxia-induced gill remodeling in blunt snout bream and a theoretical basis for its hypoxia adaptation mechanism.

An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3' UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection.

N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3' UTR of Toll interacting protein gene (TOLLIP982T>C) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3' UTR, but TOLLIP982T>C mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3' UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor ß-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP982T>C mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP982T>C mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3' UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp.

Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV.

BACKGROUND: N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. RESULTS: This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal's responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. CONCLUSION: Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.

Identification of duplicated suppressor of cytokine signaling 3 (SOCS3) genes in blunt snout bream (Megalobrama amblycephala).

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.

Functional conservation and divergence of duplicated the suppressor of cytokine signaling 1 in blunt snout bream (Megalobrama amblycephala).

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 socs1s 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.

Identification of duplicated Cited3 genes and their responses to hypoxic stress in blunt snout bream (Megalobrama amblycephala).

The CITED3 protein is a non-DNA-binding transcriptional co-regulator involved in the regulation of various transcriptional responses against hypoxia stress. Here, we characterized two paralogs Cited3 genes (Cited3a and Cited3b) from blunt snout bream (Megalobrama amblycephala), which is a hypoxia-sensitive species. Both genes have an open reading frame of 756 and 723 bp; encoded a protein of 251 amino acid and 240 amino acid, respectively; and they shared a sequence identity of 67%. In adult fish, both Cited3a and Cited3b mRNAs were highly expressed in kidney tissues. In contrast, they were detected in the skin, muscle, and gonad at extraordinarily low levels. During embryogenesis, both Cited3a and Cited3b mRNAs were maternally deposited in eggs and fluctuated from the zygote to the 44-hpf (hours post-fertilization) larvae. Whole-mount in situ hybridization demonstrated that both Cited3a and Cited3b mRNAs were transcribed in the brain, gut, and tailbud at 12 hpf, and at the brain and gut at 24 hpf, and at the brain at 36 hpf embryos. Hypoxic treatment led to upregulated expression of the Cited3 genes during embryogenesis. Under hypoxia, both Cited3a and Cited3b genes in the kidney and brain and Cited3a genes in the liver were significantly upregulated. These results suggest that hypoxia was associated with increases in mRNA levels for both Cited3a (kidney, brain, liver) and Cited3b (kidney and liver).

Insertional mutagenesis in ChordinA induced by endogenous ΔTgf2 transposon leads to bifurcation of axial skeletal systems in grass goldfish.

The grass goldfish appeared early in the evolutionary history of goldfish, and shows heritable stability in the development of the caudal fin. The twin-tail phenotype is extremely rare, however, some twin-tail individuals were produced in the process of breeding for ornamental value. From mutations in the twin-tail goldfish genome, we identified two kinds of Tgf2 transposons. One type was completely sequenced Tgf2 and the other type was ΔTgf2, which had 858 bp missing. We speculate that the bifurcation of the axial skeletal system in goldfish may be caused by an endogenous ΔTgf2 insertion mutation in Chordin A, as ΔTgf2 has no transposition activity and blocks the expression of Chordin A. The twin-tail showed doubled caudal fin and accumulation of red blood cells in the tail. In addition, in situ hybridization revealed that ventral embryonic tissue markers (eve1, sizzled, and bmp4) were more widely and strongly expressed in the twin-tail than in the wild-type embryos during the gastrula stage, and bmp4 showed bifurcated expression patterns in the posterior region of the twin-tail embryos. These results provide new insights into the artificial breeding of genetically stable twin-tail grass goldfish families.

Identification of proteins differentially expressed in the gills of grass carp (Ctenopharyngodon idella) after hypoxic stress by two-dimensional gel electrophoresis analysis.

Two-dimensional gel electrophoresis (2-DE) was combined with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the differential proteomics of grass carp gills after hypoxic stress to better understand the roles of proteins in the hypoxic response and to explore the possible molecular mechanisms. Protein spots were obtained from a hypoxia-stressed group (372 ± 11 individuals) and a control group (406 ± 14 individuals) using the lmage Master 2D Platinum 7.0 analysis software. Fifteen protein spots were expressed differentially in the hypoxia-stressed group and varied significantly after exposure to the hypoxic conditions. In addition, these differential proteins were identified by mass spectrometry and then searched in a database. We found the expression and upregulation of the toll-like receptor 4, ephx1 protein, isocitrate dehydrogenase, L-lactate dehydrogenase, GTP-binding nuclear protein Ran, and glyceraldehyde-3-phosphate dehydrogenase; however, the expression of the keratin type II cytoskeletal 8, type I cytokeratin, ARP3 actin-related protein 3 homolog, thyroid hormone receptor alpha-A, ATP synthase subunit beta, citrate synthase, tropomyosin 2, and tropomyosin 3 were downregulated. Six proteins were found in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. We concluded that the grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress. To our knowledge, this is the first study to conduct a proteomics analysis of expressed proteins in the gills of grass carp, and this study will help increase the understanding of the molecular mechanisms involved in hypoxic stress responses in fish at the protein level.

Divergence of Genes Encoding CITED1 and CITED2 in Blunt Snout Bream (Megalobrama amblycephala) and Their Transcriptional Responses to Hypoxia.

The proteins CITED belong to a family of non-DNA-binding transcriptional co-regulators involved in the regulation of various transcriptional responses. Previous studies suggest that members of CITED family may function in response to hypoxia in mammals. however, the molecular and functional information on CITED genes in aquaculture fish is unclear. Here, we characterized and examined the transcriptional patterns of CITED1 and CITED2 genes in the hypoxia-sensitive blunt snout bream (Megalobrama amblycephala). Blunt snout bream CITED1 and CITED2 genes shared a relatively low sequence identity of 45%. CITED1 and CITED2 mRNAs were widely transcribed in adult tissues. During embryogenesis, CITED1 mRNA was significantly transcribed at 4, 24, 28, 40, and 44 hpf, whereas CITED2 mRNA levels fluctuated from the zygote to 44 hpf larval stage. Whole-mount in situ hybridization demonstrated that CITED1 and CITED2 mRNAs were detected in the brain at 12 hpf, brain and gut at 24 hpf, and brain at 36 hpf. In addition, low expression of CITED1 mRNA was detected in the tailbud at 24 hpf. The results of acute hypoxia experiment showed that CITED1 and CITED2 mRNAs were markedly upregulated in the kidney and downregulated in the liver, brain, gill, and heart under hypoxia. Embryos in hypoxic conditions at different developmental stages showed a significant increase in mRNA levels of CITED1 and CITED2. These results provide a new insight into the divergence of CITED1 and CITED2 genes and their transcriptional responses to hypoxia.

Identification of duplicated Midkine genes and their functional regulation in blunt snout bream (Megalobrama amblycephala).

Midkine (Mdk) is a heparin-binding growth factor that is involved in regulating cell growth, differentiation and migration. Here, we report the isolation and characterization of duplicated mdk genes in blunt snout bream (Megalobrama amblycephala). The mdka and -b genes encode 146 aa and 147 aa peptides, respectively, sharing a sequence identity of 64%. During embryogenesis, mdka mRNA is detectable after 12 h post-fertilization (hpf) and mdkb mRNA can be detected after 8 hpf, about 4 h prior to mdka mRNA. Whole-mount in situ hybridization demonstrated that two paralogs of mdk mRNA were detected in the brain and dorsal neural tube at 16 hpf. At 22 hpf, mdka mRNA was abundant in the brain and dorsal neural tube, whereas mdkb mRNA were transcribed in the brain and tailbud. Later, at 55 hpf, both paralogs were mainly expressed in the brain. Furthermore, both the mdk genes were highly expressed in multiple adult tissues except in the skin and a low expression of mdka in the muscle. In addition, they were differentially inhibited in the liver and intestine with exogenous recombinant human growth hormone, while their mRNA levels were up-regulated in the brain. During starvation, both the mdk genes were significantly up-regulated in the intestine, brain and liver and returned to the control levels following 6 days of refeeding. Our results suggest that duplicated mdk genes may play conserved and divergent roles in embryonic development and tissue growth regulation in blunt snout bream.

Gene duplication, conservation and divergence of Heme oxygenase 2 genes in blunt snout bream (Megalobrama amblycephala) and their responses to hypoxia.

Heme oxygenase (HO) that catalyzes the degradation of heme, is involved in responding and using oxygen in teleost fish. Physiologic heme degradation can be catalyzed by two isozymes of HO (HO-1 and HO-2). In fish, the molecular constructions, expression characteristics and hypoxic regulation of HO-2 are still not well known. Here, we report the isolation and characterization of duplicated HO-2 genes in blunt snout bream, a hypoxia sensitive fish species. Blunt snout bream HO-2a and -2b genes shared a relatively low sequence identity of 67%. The HO-2a and -2b mRNAs were widely expressed in adult tissues. During embryogenesis, HO-2a mRNAs was significantly upregulated at 16hpf and then maintained with high lever, while HO-2b mRNAs was gradually increased at 12hpf and then reduced significantly. Whole-mount in situ hybridization demonstrated that HO-2a and -2b mRNAs mainly detected in brain and eyes at different embryonic stages. The results of acute hypoxia experiment showed that both HO-2a and -2b mRNAs have significant changes in different tissues. Both HO-2a and -2b mRNAs were significantly up-regulated in the brain, but down-regulated in the gill and liver during hypoxia. Under hypoxia, HO-2a mRNA in the heart was significantly increased while HO-2b mRNA was decreased. Embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of HO-2a and -2b. These results provide new insights into the functional conservation and divergence of HO-2 genes and improve our understanding of HO-2 responses to hypoxia.

Characterization of duplicated heme oxygenase-1 genes and their responses to hypoxic stress in blunt snout bream (Megalobrama amblycephala).

The heme oxygenase (HO)-1 is a cytoprotective enzyme that can be involved in cytoprotection against hypoxia stress. In this study, we cloned duplicated HO-1a and HO-1b cDNAs in hypoxia-sensitive blunt snout bream (Megalobrama amblycephala). HO-1a and HO-1b encode peptides with 272 amino acids and 246 amino acids, respectively, and they share a low sequence identity of 55%. HO-1a and HO-1b mRNAs were maternally deposited in the zygote, and the mRNAs decreased to the lowest levels at 8 hpf. Both mRNAs were significantly (p < 0.01) expressed from 12 hpf and fluctuated but maintained a high level after 16 hpf. Using in situ hybridization, HO-1a and HO-1b mRNAs were ubiquitously expressed in embryos at 12 hpf. At 24 and 36 hpf, HO-1b transcripts were detected in the mid- and hindbrain, respectively, whereas HO-1a was mainly transcribed in the eyes and endoderm at 24 hpf and in the brain at 36 hpf. In adult fish, HO-1a was abundantly expressed in the heart, liver, gill, kidney, spleen, and brain, while HO-1b mRNA was detected mainly in the kidney. After exposure to hypoxic stress, both HO-1a and HO-1b mRNAs were upregulated significantly in the gill and liver but downregulated significantly in the brain (p < 0.01). These findings suggest that duplicated HO genes have evolved divergently and yet play overlapping biological roles in regulating the response to hypoxia in M. amblycephala.

Comparative expression and regulation of duplicated fibroblast growth factor 1 genes in grass carp (Ctenopharyngodon idella).

Fibroblast growth factor 1 (Fgf1) is known as a mitogenic factor involved in the regulation of cell growth, proliferation, and differentiation in vertebrates. Here, we report the isolation and characterization of two fgf1 genes in grass carp (Ctenopharyngodon idella). Grass carp fgf1a and fgf1b cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 50%, probably due to fish-specific gene duplication. fgf1a and fgf1b mRNAs were detected in the zygote and expressed throughout embryogenesis. Both fgf1a and fgf1b mRNAs were primarily detectable in the notochord at 12 hpf. At 24 hpf, fgf1a mRNA was mainly expressed in the gut and somites, while fgf1b transcript persisted in the notochord and was detected in the tailbud. At 36 hpf, both fgf1a and fgf1b transcripts were detected in the brain, somites, and tailbud. In addition, the fgf1a mRNA was detected at the base of the yolk sac, whereas the fgf1b mRNA was expressed in the pectoral fin. In adult fish, duplicated fgf1a and fgf1b mRNAs were distributed in most tissues. After 2-6days of starvation, both fgf1a and fgf1b mRNAs were upregulated in the muscle and liver. In the brain, fgf1a mRNA was upregulated, while fgf1b mRNA was significantly downregulated at 6days. Furthermore, both fgf1a and fgf1b mRNA levels were significantly decreased in the brain and muscle after administration of 10 or 50µg of the human growth hormone (hGH),while their mRNA levels were no significant difference in the liver. These results suggest that duplicated fgf1s may play important but divergent roles in the grass carp development.