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.

Molecular characterization and immunological response analysis of toll-like receptors from the blunt snout bream (Megalobrama amblycephala).

The innate immunity serves as the primary line of defense against pathogen invasion. Toll-like receptors (TLRs) play a pivotal role in the innate immunity by sensing specific pathogen-associated molecular patterns and activating immune responses. In this study, 14 TLRs (TLR1, 2, 3, 4, 5, 7, 8a, 8b, 9, 18, 19, 20, 21 and 22) were identified and characterized in a cyprinid fish Megalobrama amblycephala. A majority of MaTLRs possessed the typical structural features of the TLR protein family: LRR domain, TM region and TIR domain. Phylogenetic analyses confirmed the existence of six TLR families and revealed close relationships with other cyprinid orthologs. All TLRs were constitutively expressed in all eight examined tissues. After Aeromonas hydrophila challenge, the expression profiles of MaTLR mRNAs were analyzed in liver, spleen and head kidney: MaTLR1, 2, 5, 9, 21 and 22 were up-regulated, MaTLR3, 7, 8a, 8b and 19 were down-regulated, and MaTLR4, 18 and 20 expression patterns varied among tissues. These results indicate that most of MaTLRs are likely to be involved in the immune responses against bacterial infection.

Expression and functional characterization of interferon regulatory factors (irf2, irf7 and irf9) in the blunt snout bream (Megalobrama amblycephala).

Interferon regulatory factors (irfs) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irfs have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members (irf2, irf7, irf9) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irfs share a high sequence homology with other vertebrate irfs. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irfs in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irfs are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream.

Molecular identification and functional characterisation of the interferon regulatory factor 1 in the blunt snout bream (Megalobrama amblycephala).

Interferon regulatory factors (IRFs) play a key role in mediating the host response against pathogen infection and other important biological processes. This is the first report of an IRF family member in blunt snout bream Megalobrama amblycephala. The complete cDNA of M. amblycephala (Ma) IRF1 gene has 1422 nucleotides (nt.), with an open reading frame of 858 nt, encoding a polypeptide of 285 amino acids. The putative MaIRF1 polypeptide shared significant structural homology with known IRF1 homologs: a conserved IRF domain was found at the N-terminal and an IRF association domain 2 at the C-terminal. Phylogenetic analysis showed that MaIRF1 amino acid sequence clustered with other teleost IRF1s, with a grass carp ortholog exhibiting the highest similarity. MaIRF1 mRNA expression patterns were studied using quantitative real-time PCR in healthy fish tissues and after a challenge with Aeromonas hydrophila bacterium. It was constitutively expressed in all examined tissues: the highest in blood, the lowest in muscle. The expression after A. hydrophila challenge was up-regulated in liver, spleen and kidney, but down-regulated in intestine and gills. At the protein level, similar expression patterns were observed in liver and gills. Patterns differed in intestine (up-regulation), spleen (down-regulation) and kidney (expression mostly unchanged). This study indicates that MaIRF1 gene plays an important role in the blunt snout bream immune system, hence providing an important base for further studies.

The gut microbiome and degradation enzyme activity of wild freshwater fishes influenced by their trophic levels.

Vertebrate gut microbiome often underpins the metabolic capability and provides many beneficial effects on their hosts. However, little was known about how host trophic level influences fish gut microbiota and metabolic activity. In this study, more than 985,000 quality-filtered sequences from 24 16S rRNA libraries were obtained and the results revealed distinct compositions and diversities of gut microbiota in four trophic categories. PCoA test showed that gut bacterial communities of carnivorous and herbivorous fishes formed distinctly different clusters in PCoA space. Although fish in different trophic levels shared a large size of OTUs comprising a core microbiota community, at the genus level a strong distinction existed. Cellulose-degrading bacteria Clostridium, Citrobacter and Leptotrichia were dominant in the herbivorous, while Cetobacterium and protease-producing bacteria Halomonas were dominant in the carnivorous. PICRUSt predictions of metagenome function revealed that fishes in different trophic levels affected the metabolic capacity of their gut microbiota. Moreover, cellulase and amylase activities in herbivorous fishes were significantly higher than in the carnivorous, while trypsin activity in the carnivorous was much higher than in the herbivorous. These results indicated that host trophic level influenced the structure and composition of gut microbiota, metabolic capacity and gut content enzyme activity.

Molecular cloning and expression of toll-like receptor 4 (tlr4) in the blunt snout bream (Megalobrama amblycephala).

Toll-like receptors (TLRs) play a pivotal role in teleost innate immune system. In this study, Megalobrama amblycephala (ma) tlr4 gene was cloned, its putative polypeptide product characterized, and expression analysed. Matlr4 cDNA is 2862 bp long, with an open reading frame of 2364 bp encoding 787 amino acids. MaTlr4 is a typical TLR protein, including the extracellular part with nine leucine-rich repeat motifs, a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor domain. MaTlr4 has the highest level of identity (94%) and similarity (97%) with the grass carp Tlr4.2 homolog. This was also corroborated by the phylogenetic analysis, which placed MaTlr4 in a cluster with other cyprinid homologs. Matlr4 mRNA was ubiquitously expressed in all examined tissues and during all sampled developmental stages. The observed peak in matlr4 mRNA expression during gastrula and somite stages is in good agreement with its proposed role in the development of the neural system. Temporal expression patterns of matlr4 and maMyD88 mRNAs and proteins were analyzed in liver, spleen, head kidney, trunk kidney and intestine after Aeromonas hydrophila infection. And mRNA expression varied between different time-points. Both MaTlr4 and MaMyD88 protein expressions at 12 hpi were significantly enhanced in head kidney and intestine. These results indicate that matlr4 is involved in the immune response in M. amblycephala, and that it is indeed a functional homologue of tlr4s described in other animal species.

A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity.

Dioxin-like 3,3',4,4',5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200µg/L) or 30mMN-acetylcysteine (NAC)+200µg/L PCB126. After 72h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200µg/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72h exposure, 200µg/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression.