Project description:BACKGROUND:The blunt snout bream (Megalobrama amblycephala) is one of the most important commercial herbivorous fish in China, and dietary transition is an important event in blunt snout bream development. Gut microbiota has a vital role to host animal. However, little was known about the relationship among feeding habits transition, gut microbiota and digestive enzymes of gut content. RESULTS:In this study, 186,328 high-quality reads from nine 16S rRNA libraries were obtained using the Illumina MiSeq PE300 platform. The valid sequences were classified into 388 Operational Taxonomic Units, and a total of 223 genera, belonging to 20 phyla, were identified. The clustering result of gut bacterial communities is consistently related to the clustering result of intestinal content compositions. Proteobacteria and Firmicutes constitute the 'core' gut microbiota of blunt snout bream. Cetobacterium and Rhizobium were identified as microbiological markers of gut microbiota at zooplankton-based diet stages and diet transition stages, respectively. Moreover, thirteen potential cellulose-degrading bacteria were detected in our study. The canonical redundancy analysis (RDA) revealed that the feeding habits strongly influenced the gut microbiota and the digestive enzyme activities of gut content, while the result of PICRUSt test suggests that the metabolic capacity of gut microbiota was affected by feeding habit. CONCLUSIONS:This study provided a comprehensive survey of the gut microbiota in blunt snout bream during its dietary transition period for the first time and clearly showed that the gut microbiota was strongly affected by feeding habit. This work allows us to better understand the relationship among gut microbiota, nutrition metabolism and feeding habits in vertebrate. Further, our study provides a reference for future studies investigating the metabolic adaption of herbivorous fish to shift to a vegetarian diet during their life history.
Project description:MyD88 and TRAF6 play an essential role in the innate immune response in most animals. This study reports the full-length MaMyD88 and MaTRAF6 genes identified from the blunt snout bream (Megalobrama amblycephala) transcriptome profile. MaMyD88 is 2501 base pairs (bp) long, encoding a putative protein of 284 amino acids (aa), including the N-terminal DEATH domain of 78 aa and the C-terminal TIR domain of 138 aa. MaTRAF6 is 5474 bp long, encoding a putative protein of 542 aa, including the N-terminal low-complexity region, RING domain (40 aa), a coiled-coil region (64 aa) and C-terminal MATH domain (147 aa). Coding regions of MaMyD88 and MaTRAF6 genomic sequences consisted of five and six exons, respectively. Physicochemical and functional characteristics of the proteins were analysed. Alpha helices were dominant in the secondary structure of the proteins. Homology models of the MaMyD88 and MaTRAF6 domains were constructed applying the comparative modelling method. RT-qPCR was used to analyse the expression of MaMyD88 and MaTRAF6 mRNA transcripts in response to Aeromonas hydrophila challenge. Both genes were highly upregulated in the liver, spleen and kidney during the first 24 h after the challenge. While MyD88 and TRAF6 have been reported in various aquatic species, this is the first report and characterisation of these genes in blunt snout bream. This research also provides evidence of the important roles of these two genes in the blunt snout bream innate immune system.
Project description:Blunt snout bream (Megalobrama amblycephala) are susceptible to hepatic steatosis when maintained in modern intensive culture systems. The aim of this study was to investigate the potential roles of microRNAs (miRNAs) in diet-induced hepatic steatosis in this species. MiRNAs, small non-coding RNAs that regulate gene expression at the posttranscriptional level, are involved in diverse biological processes, including lipid metabolism. Deep sequencing of hepatic small RNA libraries from blunt snout bream fed normal-fat and high-fat diets identified 202 (193 known and 9 novel) miRNAs, of which 12 were differentially expressed between the normal-fat and high-fat diet groups. Quantitative stem-loop reverse transcriptase-polymerase chain reaction analyses confirmed the upregulation of miR-30c and miR-30e-3p and the downregulation of miR-145 and miR-15a-5p in high-fat diet-fed fish. Bioinformatics tools were used to predict the targets of these verified miRNAs and to explore potential downstream gene ontology biological process categories and Kyoto Encyclopedia of Genes and Genomes pathways. Six putative lipid metabolism-related target genes (fetuin-B, Cyp7a1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 2, 3-oxoacid CoA transferase 1b, stearoyl-CoA desaturase, and fatty-acid synthase) were identified as having potential important roles in the development of diet-induced hepatic steatosis in blunt snout bream. The results presented here are a foundation for future studies of miRNA-controlled lipid metabolism regulatory networks in blunt snout bream.
Project description:The function of IgD in fish and mammals has not been fully understood since its discovery. In this study, we have isolated and characterized the cDNA that encodes membrane-bound form of the immunoglobulin D heavy chain gene (mIgD) of blunt snout bream (Megalobrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of mIgD consisted of 3313 bp, encoding a putative protein of 943 amino acids. The structure of blunt snout bream mIgD is VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM. Multiple alignment and phylogenetic analyses indicated that blunt snout bream mIgD clusters with the homologues of cyprinid fish and that its highest identity is with that of C. idella (82%). The mIgD expression in early different developmental stages showed that the level of mIgD mRNA decreased dramatically from the unfertilized egg stage to the 32-cell stage, suggesting that mIgD mRNA was maternally transferred. As cell differentiation initially took place in the blastula stage, the mIgD expression increased significantly from the blastula stage to prelarva, which might be attributed to embryonic stem cell differentiation processes. Compared with juvenile fish, the expression and tissue distribution patterns of mIgD in adult individuals exhibited considerable variation. After the injection of Aeromonas hydrophila, mIgD expression was up-regulated in various tissues, reaching the peak expression at 5 d, 14 d or 21 d (depending on the tissue type). The present study provides a theoretical basis for further research of the teleost immune system.
Project description:This study aimed at achieving the molecular characterization of peroxisome proliferator-activated receptor-gamma coactivator 1? (PGC-1?) and exploring its modulatory roles in mitochondria biogenesis in blunt snout bream (Megalobrama amblycephala). A full-length cDNA of PGC-1? was cloned from liver which covered 3110 bp encoding 859 amino acids. The conserved motifs of PGC-1? family proteins were gained by MEME software, and the phylogenetic analyses showed motif loss and rearrangement of PGC-1? in fish. The function of PGC-1? was evaluated through overexpression and knockdown of PGC-1? in primary hepatocytes of blunt snout bream. We observed overexpression of PGC-1? along with enhanced mitochondrial transcription factor A (TFAM) expression and mtDNA copies in hepatocytes, and its knockdown led to slightly reduced NRF1 expression. However, knockdown of PGC-1? did not significantly influence TFAM expression or mtDNA copies. The alterations in mitochondria biogenesis were assessed following high-fat intake, and the results showed that it induces downregulation of PGC-1?. Furthermore, significant decreases in mitochondrial respiratory chain activities and mitochondria biogenesis were observed by high-fat intake. Our findings demonstrated that overexpression of PGC-1? induces the enhancement of TFAM expression and mtDNA amount but not NRF-1. Therefore, it could be concluded that PGC-1? is involved in mitochondrial biogenesis in blunt snout bream but not through PGC-1?/NRF-1 pathway.
Project description:Tc1-like transposons consist of an inverted repeat sequence flanking a transposase gene that exhibits similarity to the mobile DNA element, Tc1, of the nematode, Caenorhabditis elegans. They are widely distributed within vertebrate genomes including teleost fish; however, few active Tc1-like transposases have been discovered. In this study, 17 Tc1-like transposon sequences were isolated from 10 freshwater fish species belonging to the families Cyprinidae, Adrianichthyidae, Cichlidae, and Salmonidae. We conducted phylogenetic analyses of these sequences using previously isolated Tc1-like transposases and report that 16 of these elements comprise a new subfamily of Tc1-like transposons. In particular, we show that one transposon, Thm3 from silver carp (Hypophthalmichthys molitrix; Cyprinidae), can encode a 335-aa transposase with apparently intact domains, containing three to five copies in its genome. We then coinjected donor plasmids harboring 367 bp of the left end and 230 bp of the right end of the nonautonomous silver carp Thm1 cis-element along with capped Thm3 transposase RNA into the embryos of blunt snout bream (Megalobrama amblycephala; one- to two-cell embryos). This experiment revealed that the average integration rate could reach 50.6% in adult fish. Within the blunt snout bream genome, the TA dinucleotide direct repeat, which is the signature of Tc1-like family of transposons, was created adjacent to both ends of Thm1 at the integration sites. Our results indicate that the silver carp Thm3 transposase can mediate gene insertion by transposition within the genome of blunt snout bream genome, and that this occurs with a TA position preference.
Project description:Aeromonas hydrophila is the causative agent of motile aeromonad septicemia. Here, we present the complete genome sequence of highly virulent A. hydrophila strain D4, which was isolated from a diseased blunt-snout bream in China. It comprises one circular chromosome and four previously unreported plasmids with a total length of 5,275,132?bp.
Project description:PGC1? is a transcriptional coactivator that plays key roles in mitochondrial biogenesis, so exploring its molecular characterization contributes to the understanding of mitochondrial function in cultured fish. In the present study, a full-length cDNA coding PGC1? was cloned from the liver of blunt snout bream (Megalobrama amblycephala) which covered 3741 bp with an open reading frame of 2646 bp encoding 881 amino acids. Sequence alignment and phylogenetic analysis revealed high conservation with other fish species, as well as other higher vertebrates. Comparison of the derived amino acid sequences indicates that, as with other fish, there is a proline at position 176 (RIRP) compared to a Thr in the mammalian sequences (RIRT). To investigate PGC1? function, three in vitro tests were carried out using primary hepatocytes of blunt snout bream. The effect of AMPK activity on the expression of PGC1? was determined by the culture of the hepatocytes with an activator (Metformin) or inhibitor (Compound C) of AMPK. Neither AMPK activation nor inhibition altered PGC1? expression. Knockdown of PGC1? expression in hepatocytes using small interfering RNA (si-RNA) was used to determine the role of PGC1? in mitochondrial biogenesis. No significant differences in the expression of NRF1 and TFAM, and mtDNA copy number were found between control and si-RNA groups. Also, hepatocytes were cultured with oleic acid, and the findings showed the significant reduction of mtDNA copy number in oleic acid group compared to control. Moreover, oleic acid down-regulated the expression of NRF1 and TFAM genes, while PGC1? expression remained unchanged. Our findings support the proposal that PGC1? may not play a role in mitochondrial biogenesis in blunt snout bream hepatocytes.