The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet.

The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.

Transcriptome comparison reveals insights into muscle response to hypoxia in blunt snout bream (Megalobrama amblycephala).

The economic and biological significance of blunt snout bream (Megalobrama amblycephala) makes this species important to explore the underlying molecular mechanism of hypoxia response. In the present study, we compared the transcriptional responses to serious hypoxia in skeletal muscle among hypoxia tolerant (MT), sensitive (MS) and control (without hypoxia treatment, MC) M. amblycephala obtained according to the time difference of losing balance after hypoxia treatment. A total of 88,200,889 clean reads were generated and assembled into 44,493 unigenes. Transcriptomic comparison revealed 463 genes differentially expressed among different groups. A similar hypoxia-induced transcription patterns suggested a common hypoxia response involved in cell cycle, p53 signaling pathway, apoptosis, heart contraction and blood circulation. Interesting, four genes, heat shock protein beta-8 (hspb8), cysteine/serine-rich nuclear protein 1 (csrnp1), salt-inducible kinase 1 (sik1), and visinin-like 1a (vsnl1a) were up-regulated in MT Vs MC but down-regulated in MS Vs MC. Additionally, FoxO signaling pathway was significantly enriched only in MT Vs MC. These results not only provided the first insights into the mechanism that muscle tissue coped with the hypoxia stress in cyprinid species, but offered a theory base for breeding of M. amblycephala with hypoxia-resistant traits.

Molecular response and association analysis of Megalobrama amblycephala fih-1 with hypoxia.

Hypoxia is one of the most important environmental factors which affect fish growth, development and survival, but regulation mechanisms of hypoxia in fish remain unclear. Therefore, to further understand molecular functions of factor inhibiting HIF-1 (Fih-1), an essential hypoxia sensor, the full-length cDNA of fih-1 was cloned from Megalobrama amblycephala, a hypoxia-sensitive cyprinid fish. The deduced amino acid sequence showed high homology with that of other vertebrates, and all structural and functional domains were highly conserved. The mRNA level in different tissues and developmental stages indicated that M. amblycephala fih-1 expression was higher in liver and muscle, followed by gill, intestine and spleen. During embryogenesis, the fih-1 mRNA was highly expressed in the early embryonic development, then decreased to a very low level, and maintained a relative high level of expression after hatching. In most tissues, the fih-1 mRNA was down-regulated at 2 h but up-regulated at 4 h after hypoxia treatment. In addition, the promoter sequence of M. amblycephala fih-1 was obtained using thermal asymmetric interlaced PCR. Three single nucleotide polymorphism (SNP) sites were found in the cDNA and promoter sequences, and identified significant association with hypoxia trait by correlation analysis in hypoxia-sensitive group and hypoxia-tolerant group. These results demonstrated that M. amblycephala fih-1 plays important roles in embryo development and hypoxia response, which will contribute to systematic understanding of the molecular mechanisms of fish in response to hypoxia, and provide help for fish genetic breeding with hypoxia-tolerant strains or breeds.

The complete mitochondrial genome of the hybrid of Megalobrama skolkovii (♀) × Megalobrama amblycephala (♂).

In this study, we sequenced the complete mitochondrial genome of the hybrid of Megalobrama skolkovii (♀) × Megalobrama amblycephala (♂) for the first time. The complete mitochondrial genome of the hybrid bream was found to be 16 621 bp in size with a mostly conserved structural organization when compared with that of other Megalobrama species. It contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and two main non-coding regions (the control region and the origin of the light strand replication). Sequence alignment of mitochondrial genomes between the hybrid and its female parent showed that a total of 38 mutation sites in 13 genes or regions, in particular, three sense mutations in three protein-coding genes (COX1, ND4L, and ND5) with 27 mutation sites in nine protein-coding genes. This mitogenome sequence data would contribute to a better understanding of genetic mechanisms of mitochondrial DNA and phylogenetic analysis in hybrids.

Alternative splicing transcription of Megalobrama amblycephala HIF prolyl hydroxylase PHD3 and up-regulation of PHD3 by HIF-1α.

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala.

Leptin Genes in Blunt Snout Bream: Cloning, Phylogeny and Expression Correlated to Gonads Development.

To investigate the leptin related genes expression patterns and their possible function during the gonadal development in fish, the cDNA and genomic sequences of leptin, leptin receptor (leptinR), and leptin receptor overlapping transcript like-1 (leprotl1) were cloned and their expression levels were quantified in the different gonadal development stages of Megalobrama amblycephala. The results showed that the full length cDNA sequences of leptin, leptinR and leprotl1 were 953, 3432 and 1676 bp, coding 168, 1082, and 131 amino acid polypeptides, and the genomic sequences were 1836, 28,528 and 5480 bp, which respectively had 3, 15 and 4 exons, respectively. The phylogenetic analysis revealed that three genes were relatively conserved in fish species. Quantitative real-time PCR results showed that the three genes were ubiquitously expressed in all examined tissues during the different gonadal development stages. The leptin and leptinR took part in the onset of puberty, especially in female M. amblycephala, by increasing the expression levels in brain during the stage I to III of ovary. The expression levels of leptin and leptinR had significant differences between male and female in hypothalamic-pituitary-gonadal (HPG) axis tissues (p < 0.05). The leptinR had the same variation tendency with leptin, but the opposite changes of expression levels were found in leprotl1, which may resist the expression of leptinR for inhibiting the function of leptin in target organ. These findings revealed details about the possible role of these genes in regulating gonadal maturation in fish species.

Identification of MicroRNA for Intermuscular Bone Development in Blunt Snout Bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of the lower teleosts, is attracting more attention of researchers due to its particular development and lack of genetic information. MicroRNAs (miRNAs) are emerging as important regulators for biological processes. In the present study, miRNAs from IBs and connective tissue (CT; encircled IBs) from six-month-old Megalobrama amblycephala were characterized and compared. The results revealed the sequences and expression levels of 218 known miRNA genes (belonging to 97 families). Of these miRNAs, 44 known microRNA sequences exhibited significant expression differences between the two libraries, with 24 and 20 differentially-expressed miRNAs exhibiting higher expression in the CT and IBs libraries, respectively. The expressions of 11 miRNAs were selected to validate in nine tissues. Among the high-ranked predicted gene targets, differentiation, cell cycle, metabolism, signal transduction and transcriptional regulation were implicated. The pathway analysis of differentially-expressed miRNAs indicated that they were abundantly involved in regulating the development and differentiation of IBs and CT. This study characterized the miRNA for IBs of teleosts for the first time, which provides an opportunity for further understanding of miRNA function in the regulation of IB development.

Molecular characterization and mRNA expression of HIF-prolyl hydroxylase-2 (phd2) in hypoxia-sensing pathways from Megalobrama amblycephala.

HIF-prolyl-hydroxylase-2 (Phd2), a member of the iron (II) and 2-oxoglutarate-dependent dioxygenase family, is one of the key enzymes in hypoxia-sensing pathways. In this study, the phd2 cDNA sequence (1231bp), including an open reading frame (ORF) and encoding 358 amino acid residues was identified in Megalobrama amblycephala (Wuchang bream). The predicted Phd2 protein contained three conserved domains, MYND type zinc finger domain with critical regulatory activity, Fe(2+)-dependent 2OG-Fe (II) oxygenase superfamily domain with prolyl hydroxylase function, and P4Hc (prolyl 4-hydroxylase alpha subunit homologues) domain for catalyzing proline hydroxylation. The real-time PCR results showed that phd2 mRNA was ubiquitously expressed in all detected tissues with higher levels in the peripheral blood, heart and brain, and all embryogenesis stages, especially in mid-blastula stage. In larvae M. amblycephala, the expression trend of the phd2 and hypoxia-inducible factor 1 alpha (hif-1α) mRNA was opposite during hypoxia with an increase (hypoxia for 4h) and then decrease (hypoxia for 12h) for phd2. Whereas in adult fish, the phd2 mRNA appeared a transient increase under hypoxia for 4h (DO: 3.46±0.59 mg/L), and dramatically reduced with further hypoxia exposure to 12h in the peripheral blood, muscle, head kidney, liver and brain, but showed an opposite expression trend in the heart and gill. The hif-1α expression was contrary with phd2 in the peripheral blood, while it gradually decreased in the heart, but increased in the liver with continuous hypoxia treatment. Additionally, hif-1α also showed lower mRNA levels than phd2 in all detected tissues under normoxia and hypoxia conditions.

Transcriptome analysis and microsatellite discovery in the blunt snout bream (Megalobrama amblycephala) after challenge with Aeromonas hydrophila.

The blunt snout bream, Megalobrama amblycephala, is a herbivorous freshwater fish species native to China and a major aquaculture species in Chinese freshwater polyculture systems. In recent years, the bacterium Aeromonas hydrophila has been reported to be its pathogen causing great losses of farmed fish. To understand the immune response of the blunt snout bream to A. hydrophila infection, we used the Solexa/Illumina technology to analyze the transcriptomic profile after artificial bacterial infection. Two nonnormalized cDNA libraries were synthesized from tissues collected from control blunt snout bream or those injected with A. hydrophila. After assembly, 155,052 unigenes (average length 692.8 bp) were isolated. All unigenes were annotated using BLASTX relative to several public databases: the National Center for Biotechnology Information nonreduntant (Nr) database, SwissProt, Eukaryotic Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). The sequence similarity (86%) of the assembled unigenes was to zebrafish based on the Nr database. A number of unigenes (n = 30,482) were assigned to three GO categories: biological processes (25,242 unigenes), molecular functions (26,096 unigenes), and cellular components (22,778 unigenes). 20,909 unigenes were classified into 25 KOG categories and 28,744 unigenes were assigned into 315 specific signaling pathways. In total, 238 significantly differentially expressed unigenes (mapped to 125 genes) were identified: 101 upregulated genes and 24 downregulated genes. Another 303 unigenes were mapped to unknown or novel genes. Among the known expressed genes identified, 53 were immune-related genes and were distributed in 71 signaling pathways. The expression patterns of selected up- and downregulated genes from the control and injected groups were determined with reverse transcription-quantitative PCR (RT-qPCR). Microsatellites (n = 10,877), including di-to pentanucleotide repeat motifs, were also identified in the blunt snout bream transcriptome profiles. This study extends our understanding of the immune defense mechanisms of the blunt snout bream against A. hydrophila and provides useful data for further studies of the immunogenetics of this species.

Identification and characterization of microRNAs involved in growth of blunt snout bream (Megalobrama amblycephala) by Solexa sequencing.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. MicroRNAs (miRNAs) play important roles in regulation of almost all biological processes in eukaryotes. Although previous studies have identified thousands of miRNAs from many species, little information is known for miRNAs of M. amblycephala. To investigate functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates. RESULTS: In this study, we have identified 347 conserved miRNAs (belonging to 123 families) and 22 novel miRNAs in M. amblycephala. Moreover, we observed sequence variants and seed edits of the miRNAs. Of the 5,166 single nucleotide substitutions observed in two libraries, the most abundant were G-to-U (15.9%), followed by U-to-C (12.1%), G-to-A (11.2%), and A to G (11.2%). Subsequently, we compared the expression patterns of miRNAs in the two libraries (big-size group with high growth rate versus small-size group with low growth rate). Results indicated that 27 miRNAs displayed significant differential expressions between the two libraries (p < 0.05). Of these, 16 were significantly up-regulated and 11 were significantly down-regulated in the big-size group compared to the small-size group. Furthermore, stem-loop RT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in ten tissues, and the result revealed that the conserved miRNAs expressed at higher levels than the novel miRNAs, especially in brain, liver and muscle. Also, targets prediction of differentially expressed miRNAs and KEGG pathway analysis suggested that differentially expressed miRNAs are involved in growth and metabolism, signal transduction, cell cycle, neural development and functions. CONCLUSIONS: The present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile related to different growth performances. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes and the study of miRNA function and phenotype-associated miRNA identification in fish.