| effects of anthraquinone extract from rheum officinale bail on the physiological responses and hsp70 gene expression of megalobrama amblycephala under aeromonas hydrophila infection. | we evaluated the effect of dietary supplementation with anthraquinone extract (from rheum officinale bail) on the resistance to aeromonas hydrophila infection in megalobrama amblycephala. the fish were randomly divided into two groups: a control group (fed a standard diet) and a treatment group (standard diet supplemented with 0.1% anthraquinone extract) and fed for 10 weeks. we then challenged the fish with a. hydrophila and recorded mortality and changes in serum cortisol, lysozyme, alkaline p ... | 2011 | 21362482 |
| molecular cloning and expression analysis of megalobrama amblycephala transferrin gene and effects of exposure to iron and infection with aeromonas hydrophila. | transferrin (tf) plays an important function in iron homeostasis and metabolism of organisms. in this study, we identified and characterized the tf gene in megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with aeromonas hydrophila. furthermore, we studied the iron binding properties of recombinant tf. the full-length m. amblycephala tf complementary dna (cdna) (genbank accession no.: kx698308) of 2245 bp was clone ... | 2017 | 28236008 |
| 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 ... | 2016 | 26802439 |
| 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 in ... | 2015 | 25681750 |
| the megalobrama amblycephala transferrin and transferrin receptor genes: molecular cloning, characterization and expression during early development and after aeromonas hydrophila infection. | both transferrin (tf) and transferrin receptor (tfr) play vital roles in iron homeostasis, which is essential for cellular growth and survival. besides, tf and tfr are also reported to be involved in immune response against bacterial infection. in the present study, we cloned and characterized the tf and tfr genes in megalobrama amblycephala. the m. amblycephala tf gene contained 17 exons and 16 introns, encoding 651 amino acids, while the m. amblycephala tfr gene contained 18 exons and 17 intro ... | 2015 | 25530094 |
| blunt snout bream (megalobrama amblycephala) myd88 and traf6: characterisation, comparative homology modelling and expression. | 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-termin ... | 2015 | 25830478 |
| effects of light intensity on growth, immune responses, antioxidant capability and disease resistance of juvenile blunt snout bream megalobrama amblycephala. | light is necessary for many fish species to develop and grow normally since most fishes are visual feeders. however, too intense light may be stressful or even lethal. thus, this study was conducted to evaluate the effect of light intensity on growth, immune response, antioxidant capability and disease resistance of juvenile blunt snout bream megalobrama amblycephala. fish (18.04 ± 0.22 g) randomly divided into 5 groups were exposed to a range of light intensities (100, 200, 400, 800 and 1600 lx ... | 2015 | 26306857 |
| molecular cloning and expression analysis of immunoglobulin m heavy chain gene of blunt snout bream (megalobrama amblycephala). | immunoglobulins (igs), which bind antigens with high specificity, are essential molecules in adaptive immune system of jawed vertebrates. in this study, cdna encoding the secreted form of the immunoglobulin heavy chain of igm (sigm) was cloned from the mesonephros of blunt snout bream (megalabrama amblycephala) using rt-pcr and rapid amplification of cdna ends (race). the full-length cdna of sigm heavy chain gene has 1961 nucleotides encoding a putative protein of 569 amino acids, constant regio ... | 2014 | 24979225 |
| chronic stress effects of high doses of vitamin d3 on megalobrama amblycephala. | dietary vitamin d3 plays an important role in the growth of aquatic animals, but long-term excessive feeding has potential hazards. in this study, megalobrama amblycephala specimens were fed different experimental diets with 2000 iu/kg or 200,000 iu/kg of vitamin d3 for 90 days, in order to evaluate chronic stress effects of high doses of vitamin d3 on growth, immunity, and structural damage to enterohepatic tissues. the results showed that high doses of vitamin d3 did not have a significant inf ... | 2015 | 26362210 |
| cloning, characterization and mrna expression of interleukin-6 in blunt snout bream (megalobrama amblycephala). | in the present study, the interleukin-6 gene (il-6) cdna in blunt snout bream (megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. the il-6 sequence consisted of 1045 bp, including a 696 bp orf which translated into a 232 amino acid (aa) protein. the protein contained a putative signal peptide of 24 aa in length. il-6 expression analysis showed that the it is differentially expressed in various tissues under normal ... | 2016 | 26965748 |
| the effect of hyperthermia on liver histology, oxidative stress and disease resistance of the wuchang bream, megalobrama amblycephala. | this study aimed to investigate the effects of hyperthermia on serum hormones, hepatic oxidization indices, hepatic heat shock protein (hsp60, 70, and 90) mrna expression levels and liver cell ultrastructure in megalobrama amblycephala before and after high temperature stress. fish were exposed to the optimal temperature (25 ± 1 °c) or high temperature (32 ± 1 °c) and then challenged with aeromonas hydrophila. the results showed that hyperthermic stress significantly increased serum adrenocortic ... | 2016 | 27016402 |
| sigz exhibited maternal transmission in embryonic development and played a prominent role in mucosal immune response of megalabrama amblycephala. | igz is considered to be the last immunoglobulin discovered in vertebrate species. in this study, the structure of secreted form of blunt snout bream (megalabrama amblycephala) igz (sigz) heavy chain gene is vh-cζ1-cζ2-cζ3-cζ4, in which cζ4 provides the specificity to the igz isotype. the deduced amino acid sequence of sigz shows highest similarity with that of ctenopharyngodon idella (79%). the ontogeny of sigz gene expression showed a v-shape change pattern: decreased initially from unfertilize ... | 2016 | 27044330 |
| expression of heat shock protein 90 genes during early development and infection in megalobrama amblycephala and evidence for adaptive evolution in teleost. | heat shock protein 90 (hsp90) are highly conserved molecular chaperones, playing a pivotal role in cellular progress. in this study, we reported the characterization of the hsp90α and hsp90β genes in megalobrama amblycephala, the expression profiling during early development, in various healthy tissues and in response to bacterial infection, and the assessment of their adaptive evolution. the hsp90α cdna contains an open reading frame (orf) of 2193 bp encoding 731 amino acids and the hsp90β cdna ... | 2013 | 23954722 |
| 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 hom ... | 2016 | 27150048 |
| dietary carbohydrate/lipid ratios affect stress, oxidative status and non-specific immune responses of fingerling blunt snout bream, megalobrama amblycephala. | this study aimed to evaluate the effects of dietary carbohydrate/lipid (cho:l) ratios on stress, liver oxidative status and non-specific immune responses of fingerling blunt snout bream. fish were fed six isonitrogenous and isoenergetic diets containing various cho:l ratios for 10 weeks. after the feeding trial, fish were challenged by aeromonas hydrophila and survival rate was recorded for the next 10 days. the lowest plasma cortisol, lactate, aspartate aminotransferase and alanine aminotransfe ... | 2012 | 22617792 |
| identification, origin and evidence for retained functionality of two iκbα paralogs in megalobrama amblycephala. | iκbα plays an essential role in the innate immune response in mammals. we found two functional iκbα paralogs, originating from the teleost-specific genome duplication, in megalobrama amblycephala: maiκbαa and maiκbαb. their size (936/933 bp) and structure are highly analogous to known orthologs. mrna expression was analysed by qpcr in spleen, liver, kidney, intestine and gills. apart from maiκbαb in gills (<0.001-fold), both paralogs were constitutively expressed in all tissues. differential exp ... | 2016 | 27155355 |
| mannose receptor mediated phagocytosis of bacteria in macrophages of blunt snout bream (megalobrama amblycephala) in a ca(2+)-dependent manner. | mannose receptor (mr) is an important pattern-recognition receptor in macrophages and plays a critical role in immune responses. it is has been reported that mammalian macrophages are able to engulf a wide range of microorganisms mediated by ca(2+)-dependent mr binding to terminal mannose residues which are frequently found on the pathogen surfaces. however, little is known about the mr-mediated phagocytosis in macrophages of fish. in this report, the distributions of mr in the macrophage and he ... | 2015 | 25583544 |
| characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, megalobrama amblycephala. | interleukin-6 (il-6) is one of the most important multifunctional cytokines, playing essential roles in mediating the innate and adaptive immune responses. in this study, il-6 gene and its promoter from blunt snout bream, megalobrama amblycephala, were characterized, and its expression at the transcript level in healthy fish and after bacterial infection was determined by quantitative real-time pcr. the results showed that the m. amblycephala il-6 (mamil-6) cdna had an orf of 699 bp, encoding 23 ... | 2016 | 27236548 |
| molecular characterization and gene expression of ferritin in blunt snout bream (megalobrama amblycephala). | ferritins are conserved iron storage proteins that exist in most living organisms and play an essential role in iron homeostasis. in this study, we reported the identification and analysis of a ferritin middle-chain (m) subunit, maferm, from blunt snout bream, megalobrama amblycephala. the full length cdna of maferm contains a 5'-untranslated region (utr) of 152 bp, an open reading frame (orf) of 522 bp and a 3'-utr of 270 bp. the orf encodes a putative protein of 174 amino acids, which shares e ... | 2016 | 27539708 |
| 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 d ... | 2017 | 27639315 |
| 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 ... | 2017 | 27677680 |
| identification and characterization of micrornas in the liver of blunt snout bream (megalobrama amblycephala) infected by aeromonas hydrophila. | micrornas (mirnas) are small rna molecules that play key roles in regulation of various biological processes. in order to better understand the biological significance of mirnas in the context of aeromonas hydrophila infection in megalobrama amblycephala, small rna libraries obtained from fish liver at 0 (non-infection), 4, and 24 h post infection (poi) were sequenced using illumina deep sequencing technology. a total of 11,244,207, 9,212,958, and 7,939,157 clean reads were obtained from these t ... | 2016 | 27898025 |
| characterization and expression of megalobrama amblycephala toll-like receptor 22 involved in the response to aeromonas hydrophila. | the toll-like receptors (tlr) tlr22 was identified and characterized for the first time in one of the economically most important freshwater fish species in china, megalobrama amblycephala. the full-length cdna (4039 bp) of m. amblycephala tlr22 contains an open reading frame of 2706 bp, encoding a 901 amino-acid long polypeptide. the putative polypeptide contains 16 leucine-rich repeat (lrr) motifs, an lrr c-terminal, a transmembrane region and a cytoplasmic toll-interleukin-1 receptor (tir) do ... | 2017 | 27943292 |
| characterization and expression analysis of an intelectin gene from megalobrama amblycephala with excellent bacterial binding and agglutination activity. | intelectin is a recently discovered lectin that plays vital roles in the innate immune response, iron metabolism and early embryogenesis. the structure, expression pattern and function of intelectin in mammals and amphibians have been well studied, while not well known in fish. in this study, we cloned a intelectin (mamintl) gene from blunt snout bream (megalobrama amblycephala), examined its expression patterns and explored its roles in innate immune response. the mamintl cdna encoded 312 amino ... | 2017 | 28017903 |
| a nlrc3-like gene from blunt snout bream (megalobrama amblycephala): molecular characterization, expression and association with resistance to aeromonas hydrophila infection. | nlrc (the nucleotide-oligomerization domain (nod)-like receptor subfamily c) consists of teleost-specific nlrs (nod-like receptors) and plays pivotal roles in microbial recognition and regulation of innate immune response. in this study, we cloned and characterized a nlrc3-like gene (mamnlrc3-like) from blunt snout bream (megalobrama amblycephala) by using the quantitative real-time pcr method, and analyzed the correlation between its polymorphisms and resistance to aeromonas hydrophila infectio ... | 2017 | 28215743 |
| expression patterns and functional novelty of ribonuclease 1 in herbivorous megalobrama amblycephala. | ribonuclease 1 (rnase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. however, the expression patterns and potential biological function of rnase1 in herbivorous fish is not known. here, we identified rnase1 from five fish species and illuminated the functional diversification and expression of rnase1 in herbivorous megalobrama amblycephala. the five identified fish rnase1 genes all have the signature motifs of th ... | 2016 | 27213364 |
| in silico characterisation, homology modelling and structure-based functional annotation of blunt snout bream (megalobrama amblycephala) hsp70 and hsc70 proteins. | heat shock proteins play an important role in protection from stress stimuli and metabolic insults in almost all organisms. | 2015 | 26672478 |
| the complete mitochondrial genome of the hybrid of megalobrama amblycephala (♀) × megalobrama terminalis (♂). | in this study, we determined the complete mitochondrial dna sequence of the hybrid of megalobrama amblycephala (♀) × megalobrama terminalis (♂) for the first time. the complete mitochondrial genome of the hybrid bream was sequenced to be 16,623 bp in size in accordance with the female parent, m. amblycephala. the genome contained 13 protein-coding genes, 22 transfer rna genes, 2 ribosomal rna genes and 2 main non-coding regions (the control region and the origin of the light strand replication). ... | 2016 | 25484165 |
| [effect of water velocity on swimming behavior of juvenile megalobrama amblycephala]. | to investigate the swimming behavior of juvenile megalobrama amblycephala, a tracking analysis device was designed to monitor behavior indicators including velocity (v), swimming distance (sd), turn angle (ta), distance to center-point (dc) and swimming track (st) at different flow rates (0, 0.1, 0.2, 0.3 and 0.4 m/s) at 25℃. results showed that indicator values all increased with increasing flow rate. values of v, sd and ta showed no significant differences among the flow rates of 0, 0.1 and 0. ... | 2013 | 23913895 |
| characterizing the transcriptome of yellow-cheek carp (elopichthys bambusa) enables evolutionary analyses within endemic east asian cyprinidae. | the identification of genes that may be responsible for the divergence of closely related species is one of the central goals of evolutionary biology. the species of endemic east asian cyprinidae diverged less than 8millionyears ago, and the morphological differences among these species are great. however, the genetic basis of their divergence remains unknown. in this report, we investigated the transcriptome of one endemic east asian cyprinid - the yellow-cheek carp elopichthys bambusa. a compa ... | 2014 | 24973763 |
| molecular cloning and expression of two hsp70 genes in the wuchang bream (megalobrama amblycephala yih). | two complementary deoxyribonucleic acid (cdna) clones encoding heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) were isolated from the liver of wuchang bream (megalobrama amblycephala y.) using rt-pcr and rapid amplification of cdna ends (race). they were named ma-hsc70 and ma-hsp70, respectively. the cdnas were 2336 and 2224 bp in length [not including poly (a)] and contained 1950 and 1932 bp open reading frames (orfs), respectively. the orfs encoded proteins of 649 and ... | 2010 | 19944170 |
| seasonal variations of estrogenic compounds and their estrogenicities in influent and effluent from a municipal sewage treatment plant in china. | the seasonal variations of estrogenic compounds and the estrogenicities of influent and effluent were investigated by chemical analysis and in vitro assay in a municipal sewage treatment plant in wuhan (china). the levels of eight estrogenic compounds, including 17beta-estradiol (e(2)), estrone (e(1)), estriol (e(3)), diethylstilbestrol (des), 17alpha-ethinylestradiol, nonylphenol (np), 4-tert-octylphenol (op), and bisphenol a (bpa), were measured by gas chromatography-mass spectrometry. total e ... | 2008 | 18092861 |
| is aeromonas hydrophila the dominant motile aeromonas species that causes disease outbreaks in aquaculture production in the zhejiang province of china? | the significance of aeromonas hydrophila in association with disease outbreaks in aquaculture production in the zhejiang province of china was investigated. bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. pcr and traditional biochemical methods were used for identification of a. hydrophila. out of 69 motile aeromonads, 35 isolates were identified as a. hydrophila by biochemical tests. however, 6 ... | 2001 | 11592699 |
| effects of emodin and vitamin e on the growth and crowding stress of wuchang bream (megalobrama amblycephala). | this study aimed to investigate the effects of dietary emodin, high-dose vitamin e and their combination on the growth of megalobrama amblycephala and its resistance to acute crowding stress. the fish were randomly divided into four groups: a control group fed with basal diet, and three treatment groups fed with basal diet supplemented with 60 mg/kg emodin (the emodin group), 500 mg/kg vitamin e (the vit e group), and 60 mg/kg emodin together with 500 mg/kg vitamin e (the combination group). aft ... | 2014 | 25134848 |
| biochemical, sensory and microbiological attributes of bream (megalobrama amblycephala) during partial freezing and chilled storage. | bream is one of the main farmed freshwater fish species in china. this study aimed to examine the nucleotide degradation of bream during partial freezing and chilled storage and to assess the possible usefulness of nucleotide ratios (k, ki, h, p, fr and g values) as freshness indices in comparison with sensory assessment and total viable counts. | 2012 | 21834101 |
| effects of dietary fructooligosaccharide levels and feeding modes on growth, immune responses, antioxidant capability and disease resistance of blunt snout bream (megalobrama amblycephala). | this study aimed to determine the effects of fructooligosaccharide (fos) levels and its feeding modes on growth, immune response, antioxidant capability and disease resistance of blunt snout bream (megalobrama amblycephala). fish (12.5 ± 0.5 g) were subjected to three fos levels (0, 0.4% and 0.8%) and two feeding modes (supplementing fos continuously and supplementing fos two days interval 5 days) according to a 3 × 2 factorial design. at the end of 8-week feeding trial, fish were challenged by ... | 2014 | 25451000 |
| study on the immune response to recombinant hsp70 protein from megalobrama amblycephala. | the expression of heat shock protein 70 (hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. in this study, megalobrama amblycephala hsp70 promoter was cloned, and characteristic heat shock elements (hses) were identified in the promoter region. the recombinant m. amblycephala hsp70 protein (rmahsp70) was expressed and purified from escherichia coli bl21 (de3). to evaluate in vivo immune response of rmahsp70, we administered ... | 2014 | 25113416 |
| flow cytometric determination of genome size for eight commercially important fish species in china. | the genome size (c value) of eight commercially important fish species in china was measured using flow cytometry. chicken (gallus domesticus) erythrocytes were used as reference cells. when using propidium iodide (pi) as the fluorescent dye, genome sizes were 1.09 ± 0.08, 2.75 ± 0.12, 1.05 ± 0.05, 1.35 ± 0.11, 0.99 ± 0.05, 0.90 ± 0.08, 0.90 ± 0.07, and 0.88 ± 0.07 pg for japanese eel (anguilla japonica), mullet (myxocyprinus asiaticus), yellowcheek carp (elopichthys bambusa), blunt snout bream ... | 2012 | 22956044 |
| molecular cloning and expression analysis of mannose receptor in blunt snout bream (megalobrama amblycephala). | mannose receptor (mr) plays a significant role in innate immune responses to pathogens in vertebrates. here we characterized the first teleost mr from megalobrama amblycephala, named mamr and its expression patterns were investigated. the full-length mamr consists of 5,295 bp encoding a putative protein of 1,433 amino acids. the predicted amino acid sequences showed that mamr contained a signal peptide, a cysteine-rich domain, a single fibronectin type ii domain, eight tandemly arranged c-type l ... | 2014 | 24740148 |
| feeding frequency affects stress, innate immunity and disease resistance of juvenile blunt snout bream megalobrama amblycephala. | this study aimed to evaluate the effects of feeding frequency on stress, innate immunity and disease resistance of juvenile blunt snout bream megalobrama amblycephala (average weight: 9.92 ± 0.06 g). fish were randomly assigned to one of six feeding frequencies (1, 2, 3, 4, 5 and 6 times/day) following the same ration size for 8 weeks. after the feeding trial, fish were challenged by aeromonas hydrophila and cumulative mortality was recorded for the next 10 days. daily gain index of fish fed 3-5 ... | 2014 | 24636858 |
| spatio-temporal expression of blunt snout bream (megalobrama amblycephala) migd and its immune response to aeromonas hydrophila. | 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 ... | 2015 | 26557025 |
| the influence of various feeding patterns of emodin on growth, non-specific immune responses, and disease resistance to aeromonas hydrophila in juvenile wuchang bream (megalobrama amblycephala). | the present study was conducted to evaluate the effect of various feeding patterns of emodin on growth, non-specific immune response, and disease resistance to aeromonas hydrophila in juvenile wuchang bream. healthy megalobrama amblycephala (initial weight: 3.47 ± 0.032) were grown in a circulating water system for 8 weeks. five groups were studied: one control group was fed with a basal diet for eight weeks (pattern 1, p1), and three treatment groups were fed with a trial diet of 30 mg emodin k ... | 2014 | 24211853 |
| molecular cloning and expression analysis of major histocompatibility complex class i, iia and iib genes of blunt snout bream (megalobrama amblycephala). | major histocompatibility complex (mhc) plays an important role in the immune response of vertebrates. in this study, we isolated mhc class iia and iib genes from blunt snout bream (megalobrama amblycephala) by rapid amplification of cdna ends polymerase chain reaction (race-pcr). in order to study the function of the mhc genes in m. amblycephala, tissue distribution and immune response of the mhc genes to bacterial challenge were analyzed. all the characteristic features of mhc class ii chain st ... | 2014 | 23994238 |
| health risk assessment for consumption of fish originating from ponds near dabaoshan mine, south china. | mining effluents are a potential source of toxic metals in the surrounding aquatic ecosystem and pose a potential health risk to humans from fish consumption. the objective of this paper is to assess the impact of the long-term dabaoshan mining operation on heavy metal accumulation in different fish species. heavy metal accumulation (lead (pb), cadmium (cd), zinc (zn), and copper (cu)) in four tissues (liver, muscle, intestine, and gills) of five carp species (hypophthalmichthys molitrix, ctenop ... | 2013 | 23508535 |
| establishment and characterization of a fin cell line from blunt snout bream, megalobrama amblycephala. | this study established and characterized a new cell line (maf) from the fin of blunt snout bream (megalobrama amblycephala), a freshwater fish cultivated in china. maf cells proliferated well in medium 199 supplemented with 10 % fetal bovine serum at 28 °c and have been subcultured more than 95 times in almost a year. maf cells were revived at 90-95 % viability after 3-6 months of storage in liquid nitrogen. karyotyping indicated that the modal chromosome number of maf cells was 48. the maf cell ... | 2013 | 23532304 |
| molecular cloning and expression analysis of two β-defensin genes in the blunt snout bream (megalobrama amblycephala). | β-defensins are a group of cysteine-rich, cationic antimicrobial peptides that play important roles in innate immune system against pathogenic microbes invading. in this study, the part-length cdna sequences of two β-defensin genes (maβd-1, maβd-2) in blunt snout bream (megalobrama amblycephala) were identified. homology analysis showed that the cdna sequences of maβd-1 and maβd-2 had high similarities to those in common carp and zebrafish. real-time quantitative pcr results exhibited that expre ... | 2013 | 23876385 |
| prokaryotic expression and purification of soluble goldfish tgf2 transposase with transposition activity. | goldfish tgf2 transposon of hobo/activator/tam3 (hat) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of tgf2 transposase (tpase) is still poorly understood. to express the goldfish tgf2 tpase in escherichia coli, the 2061-bp coding region was cloned into pet-28a(+) expression vector containing an n-terminal (his)6-tag. the pet-28a(+)-tgf2 tpase expression cassette was transformed into rosetta 1 (de3) e. coli lines. a h ... | 2015 | 25370823 |
| baculovirus-mediated gcrv vp7 and vp6 genes expression in silkworm and grass carp. | grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in china. this study was aimed to develop a novel oral vaccine against grass carp reovirus (gcrv). gcrv vp6 and vp7 genes with β-actin promoter of megalobrama amblycephala and polyhedrin promoter (ph10) of baculovirus, respectively, were cloned into plasmid pfast™-dual to construct a vector pfast-phvp7-avp6, which was used to generate a recombinant baculovirus bacfis ... | 2016 | 27085857 |
| reproduction mode of an artificial allotetraploid carp (pisces; cyprinidae). | mature eggs of allotetraploid carp were activated by inactive sperm or crossed with normal sperms of common carp (cyprinus carpio), crucian carp (carassius auratus), chinese blunt snout bream (megalobrama amblycephala), hemiculter leucisculus and pseudorasbora parva. chromosome counts showed that all offspring of these crosses presented a mode number of 200 chromosomes (4n = 200), and their morphological traits are much like maternal. microsatelite marker and rapd patterns between allotetraploid ... | 2002 | 12627840 |
| oral vaccination of bacfish-vp6 against grass carp reovirus evoking antibody response in grass carp. | to obtain a vaccine and evaluate its immune efficacy as an oral vaccine against grass carp reovirus (gcrv), a donor vector pfastbac-fa-vp6-ph-vp6 containing two gcrv vp6 genes, one driven by the megalobrama amblycephala β-actin promoter and another one controlled by baculovirus polyhedrin promoter was constructed to generate the recombinant baculovirus bacfish-vp6. from the hemolymph of 5th instar silkworm inoculated with bacfish-vp6, a 53 kda recombinant vp6 protein could be detected. and the i ... | 2013 | 23202374 |
| mitochondrial genome variation after hybridization and differences in the first and second generation hybrids of bream fishes. | hybridization plays an important role in fish breeding. bream fishes contribute a lot to aquaculture in china due to their economically valuable characteristics and the present study included five bream species, megalobrama amblycephala, megalobrama skolkovii, megalobrama pellegrini, megalobrama terminalis and parabramis pekinensis. as maternal inheritance of mitochondrial genome (mitogenome) involves species specific regulation, we aimed to investigate in which way the inheritance of mitogenome ... | 2016 | 27391325 |
| the complete mitochondrial genome of the hybrid of megalobrama amblycephala (♀) × megalobrama skolkovii (♂). | the complete mitochondrial genome of the hybrid of megalobrama amblycephala (♀) × megalobrama skolkovii (♂) was characterized first in this study. the total length of the genome was identical to the female parent as 16 623 bp, and the overall base composition was 31.23% a, 24.69% t, 27.89% c, and 16.19% g, with a slight a + t bias. it contained 13 protein-coding genes (pcgs), 22 transfer rna genes, 2 ribosomal rna genes, and 2 main non-coding regions (the control region and the origin of the lig ... | 2016 | 26406120 |
| 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 o ... | 2016 | 26332664 |
| the complete mitochondrial genome of the hybrid of megalobrama terminalis (♀) × megalobrama amblycephala (♂). | in this study, the complete mitochondrial genome of the hybrid of megalobrama terminalis (♀) × megalobrama amblycephala (♂) was determined. the total length of the genome was 16,622 bp in accordance with the female parent, and the overall base composition was 31.13% a, 24.94% t, 27.72% c and 16.21% g, with a slight a + t bias. the genome contained 13 protein-coding genes, 22 transfer rna genes, 2 ribosomal rna genes and 2 main non-coding regions (the control region and the origin of the light st ... | 2016 | 26075479 |
| cloning and expression analysis of a heat shock protein 90 β isoform gene from the gills of wuchang bream (megalobrama amblycephala yih) subjected to nitrite stress. | heat shock protein 90 (hsp90), a highly conserved and multi-functional molecular chaperone, plays an essential role in cellular metabolism and stress response. in this study, hsp90 cdna named mahsp90 was cloned from wuchang bream (megalobrama amblycephala) gills by using rapid amplification of cdna ends. the full-length mahsp90 cdna is 2674 bp and consists of a 3',5'-untranslated region and a 2250-bp open reading frame encoding a 750-amino acid long protein. identity analysis revealed that the a ... | 2015 | 25966068 |
| 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) ... | 2015 | 25868626 |
| tc1-like transposase thm3 of silver carp (hypophthalmichthys molitrix) can mediate gene transposition in the genome of blunt snout bream (megalobrama amblycephala). | 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 salmoni ... | 2015 | 26438298 |
| inverse pcr-based method for isolating novel sines from genome. | short interspersed elements (sines) are moderately repetitive dna sequences in eukaryotic genomes. although eukaryotic genomes contain numerous sines copy, it is very difficult and laborious to isolate and identify them by the reported methods. in this study, the inverse pcr was successfully applied to isolate sines from opsariichthys bidens genome in eastern asian cyprinid. a group of sines derived from trna(ala) molecular had been identified, which were named opsar according to opsariichthys. ... | 2014 | 24122282 |
| effect of high dietary carbohydrate on the growth performance, blood chemistry, hepatic enzyme activities and growth hormone gene expression of wuchang bream (megalobrama amblycephala) at two temperatures. | the effects of high carbohydrate diet on growth, serum physiological response, and hepatic heat shock protein 70 expression in wuchang bream were determined at 25°c and 30°c. at each temperature, the fish fed the control diet (31% cho) had significantly higher weight gain, specific growth rate, protein efficiency ratio and hepatic glucose-6-phosphatase activities, lower feed conversion ratio and hepatosomatic index (hsi), whole crude lipid, serum glucose, hepatic glucokinase (gk) activity than t ... | 2015 | 25557816 |
| effect of high dietary carbohydrate on the growth performance and physiological responses of juvenile wuchang bream, megalobrama amblycephala. | an optimum dietary carbohydrate content is important for maximum fish growth. in this study, we fed wuchang bream (megalobrama amblycephala) with either control diet (30.42%) or high carbohydrate diet (52.92%) for 90 d. fish were fed to apparent satiation three times daily in an aquarium with automatic temperature control and circulated water. growth performance, plasma biochemical parameters, hepatic morphology and enzyme activities were determined. it was shown that compared to fish fed contro ... | 2013 | 25049747 |
| nitrite-induced hepatotoxicity in bluntsnout bream (megalobrama amblycephala): the mechanistic insight from transcriptome to physiology analysis. | previous studies have investigated the physiological responses to acute nitrite exposure in fish; however, little information is available for the underlying molecular mechanisms of nitrite toxicity in aquatic ecosystems. in an effort to understand the underlying mechanisms of nitrite tolerance and to illuminate global gene expression patterns modulated by nitrite toxicity, we sampled livers from juvenile megalobrama amblycephala exposed in 0.1, 15 and 30 mg l(-1) nitrite and performed short rea ... | 2014 | 24305412 |
| [cytogenetic and molecular genetic analysis of gynogenesis in megalobrama amblycephala using spermatozoa of erythroculter ilishaeformis]. | in the present study we used both cytogenetics (measurement of dna content, detection of chromosome number, observation of gonadal development)and molecular genetics(microsatellite analysis)to analyze the biological characteristics of gynogenetic m. amblycephala, which werecreatedthrough gynogenesis induced via uv-irradiated e. ilishaeformis spermatozoa to fertilize m. amblycephala eggs. the maternal genome was duplicated by cold shock in 0~4℃ cold water to form a population of m. amblycephala w ... | 2013 | 24115660 |
| identification and mrna expression of antioxidant enzyme genes associated with the oxidative stress response in the wuchang bream (megalobrama amblycephala yih) in response to acute nitrite exposure. | aquatic organisms possess cellular detoxification systems to deal with pollutants. to explore the influence of reactive oxygen species (ros) generated in response to nitrite on oxidative stress defenses and the antioxidant system in megalobrama amblycephala, the full length cdna sequences were determined for three antioxidant-related genes, namely catalase (macat), selenium-dependent glutathione peroxidase (magpx1) and cu/zn superoxide dismutase (macu/zn-sod). encoded polypeptides that exhibited ... | 2014 | 24100052 |
| hif-1alpha and -2alpha genes in a hypoxia-sensitive teleost species megalobrama amblycephala: cdna cloning, expression and different responses to hypoxia. | transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (hif) alpha subunits, i.e. hif-1alpha, -2alpha and -3alpha. in fish, the molecular constructions, expression characteristics and hypoxic regulation of hif-alpha subunits are still not well known. in this study, we identified the hif-1alpha and hif-2alpha full-length cdnas in a hypoxia-sensitive fish species wuchang bream, megalobrama amblycephala. the whole length of hif-1alpha cdna was 3,815bp, consisting of ... | 2010 | 20615476 |
| [contamination source identification study of organochlorines pesticides in 2000 chinese total diet study]. | the third chinese total diet study to estimate dietary intake of contaminants was carried out in 2000. the levels of hch and ddt residues were less significant in most of foodstuffs, but the level of hch residues of aquatic foods in south 2 and in north 1 is respectively 75.99microg/kg and 170.72microg/kg, and the ratio of gamma-hch is 90% of the total hchs residues. to identify the contamination source of gamma-hch, the individual province composite samples and the individual samples of aquatic ... | 2004 | 15461282 |
| rapid genomic changes in allopolyploids of carassius auratus red var. (♀) × megalobrama amblycephala (♂). | to better understand genomic changes in the early generations after polyploidisation, we examined the chromosomal consequences of genomic merger in allotetraploid hybrids (4 nf1) (aabb, 4n = 148) of carassius auratus red var. (rcc) (aa, 2n = 100) (♀) × megalobrama amblycephala (bsb) (bb, 2n = 48) (♂). complete loss of the paternal 5s rdna sequence and the expected number of maternal chromosomal loci were found in 4 nf1, suggesting directional genomic changes occurred in the first generations aft ... | 2016 | 27703178 |
| induced all-female autotriploidy in the allotetraploids of carassius auratus red var. (♀) × megalobrama amblycephala (♂). | following activation by uv-irradiated bsb sperm, the fertilized eggs of tetraploid hybrids (abbreviated as 4nf1) (4n = 148, aabb) of carassius auratus red var. (abbreviated as rcc) (2n = 100, aa) (♀) × megalobrama amblycephala (abbreviated as bsb) (2n = 48, bb) (♂) developed into normal live gynogenetic offspring without chromosome doubling treatment. some of these were autotriploids with three sets of red crucian carp chromosomes (abbreviated as g1) (3n = 150, aaa). g1 were all-females, and can ... | 2015 | 26242753 |
| organization and variation analysis of 5s rdna in gynogenetic offspring of carassius auratus red var. (♀) × megalobrama amblycephala (♂). | the offspring with 100 chromosomes (abbreviated as grcc) have been obtained in the first generation of carassius auratus red var. (abbreviated as rcc, 2n = 100) (♀) × megalobrama amblycephala (abbreviated as bsb, 2n = 48) (♂), in which the females and unexpected males both are found. chromosomal and karyotypic analysis has been reported in grcc which gynogenesis origin has been suggested, but lack genetic evidence. | 2015 | 25879826 |
| inheritance of the complete mitochondrial genomes in three different ploidy fishes. | diploid natural gynogenetic goldfish (2ngrcg), triploid hybrids (3nrb) and tetraploid hybrids (4nrb) are generated by distant hybridization of red common goldfish (rcg, carassius auratus red var.) and blunt snout bream (bsb, megalobrama amblycephala). in the present study, we obtained the complete mitochondrial dna (mtdna) sequences of the hybrid offspring and compared them with the homologous sequences of rcg and bsb. all mtdna sequences of these hybrids were 16,580bp in length, and the genes n ... | 2014 | 25470285 |
| the autotetraploid fish derived from hybridization of carassius auratus red var. (female) × megalobrama amblycephala (male). | the establishment of the tetraploid organism is difficult but useful in genetics and breeding. in the present study, we have artificially established an autotetraploid fish line (f2-f8) derived from the distant hybridization of carassius auratus red var. (rr, 2n = 100) (female) × megalobrama amblycephala (bb, 2n = 48) (male). the autotetraploid line (f2-f8) possess four sets of chromosomes from red crucian carp (rrrr, 4n = 200) and produce diploid ova and diploid sperm, which maintains the forma ... | 2014 | 25165120 |
| speciation of polyploid cyprinidae fish of common carp, crucian carp, and silver crucian carp derived from duplicated hox genes. | recent studies on comparative genomics have suggested that a round of fish-specific whole genome duplication (3r) in ray-finned fishes might have occurred around 226-316 mya. additional genome duplication, specifically in cyprinids, may have occurred more recently after the divergence of the teleosts. the timing of this event, however, is unknown. to address this question, we sequenced four hox genes from taxa representing the polyploid cyprinidae fish, common carp (cyprinus carpio, 2n=100), cru ... | 2010 | 20700889 |
| distant hybridization leads to different ploidy fishes. | distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. this study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. the formations of th ... | 2010 | 20596907 |
| characterization of a novel tc1-like transposon from bream (cyprinidae, megalobrama) and its genetic variation in the polyploidy progeny of bream-red crucian carp crosses. | crossing the cyprinids diploid blunt snout bream megalobrama amblycephala (bsb) and carassius auratus red var. (rcc) generated sterile triploid (3nrb) and fertile tetraploid (4nrb) hybrid offspring. utilizing inverted terminal repeats (itrs) of transposon tdr1 from danio rerio as pcr primer, the results showed that evident change in the number of tc1-like transposons in 4nrb relative to bsb occurred, whereas such change did not arise in 3nrb compared to bsb. no tc1-like transposon was found in r ... | 2009 | 19826746 |
| comparative analysis of mitochondrial control region in polyploid hybrids of red crucian carp (carassius auratus) x blunt snout bream (megalobrama amblycephala). | the entire sequences of the mitochondrial (mt)dna control region (cr) and portions of its flanking genes in the red crucian carp (rc) and blunt snout bream (bsb) as well as their polyploid hybrids (3nrb, 4nrb and 5nrb) were determined and subjected to a comparative analysis. the mtdna-crs of these five fish species ranged from 923 to 937 bp in length, they had the same flanking gene arrangement as other vertebrates and the pattern of nucleotide substitution bias was also similar to that in other ... | 2010 | 18815893 |
| factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo. | procedures to improve somatic cell nuclear transplantation in fish were evaluated. we reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (nt) embryos. exposure to 25,000 r of gamma-rays inactivated recipient eggs. single nucleus of cultured, synchronized somatic cell from gynogenet ... | 2002 | 12410600 |
| [comparative studies on gene expression of isozymes in 4 types of various ploidy crucian carp during embryogenesis]. | isozyme zymograms of esterase (est), lactate dehydrogenase (ldh), malate dehydrogenase (mdh) and superoxide dismutase (sod) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. haploid embryos of crucian carp (carassius auratus) and common carp (cyprinus carp ... | 1998 | 12016959 |
| transcriptome analysis reveals positive selection on the divergent between topmouth culter and zebrafish. | the topmouth culter (erythroculter ilishaeformis) is a predatory cyprinid fish that distributes widely in the east asia. here we report the liver transcriptome in this organism as a model of predatory fish. sequencing of 5 gb raw reads led to 27,741 unigenes and produced 11,131 annotatable genes. a total of 7093 (63.7%) genes were found to have putative functions by gene ontology analysis. importantly, a blast search revealed 4033 culter genes that were orthologous to the zebrafish. extracted fr ... | 2014 | 25267534 |
| molecular characterization and expression patterns of myogenin in compensatory growth of megalobrama amblycephala. | myogenin (myog) is a muscle-specific basic helix-loop-helix (bhlh) transcription factor that plays an essential role in regulating skeletal muscle development and growth. to investigate molecular characterization of myog and the effect of starvation/refeeding on the gene expression, we isolated the myog cdna sequence and analyzed the expression patterns using quantitative real-time polymerase chain reaction in megalobrama amblycephala. sequence analysis indicated that m. amblycephala myog shared ... | 2014 | 24440962 |
| unique nucleolar dominance patterns in distant hybrid lineage derived from megalobrama amblycephala × culter alburnus. | nucleolar dominance is an epigenetic phenomenon that occurs in interspecific hybrids and involves the expression of 45s rrna genes inherited from one progenitor due to the silencing of the other progenitor's rrna genes. in this paper, changes in the genetics and expression of 45s rrna genes in f1 and f2 hybrid progeny of blunt snout bream (bsb, megalobrama amblycephala) × topmouth culter (tc, culter alburnus) are investigated. | 2016 | 27919229 |
| global transcriptional and mirna insights into bases of heterosis in hybridization of cyprinidae. | hybrid megalobrama amblycephala × culter alburnus represents a population newly formed by interspecific crossing between two different genera. here we assessed the expression pattern of mrna and small rna in newly formed f1, f2 and their progenitors. large amounts of nonadditively expressed protein-coding genes showed parental expression level dominance (eld). interestingly, the eld pattern could inherit from f1 to f2, which guaranteed a stable appearance in progenies. the eld-b genes were found ... | 2015 | 26346824 |
| the fertility of the hybrid lineage derived from female megalobrama amblycephala × male culter alburnus. | distant hybridization can combine together the genomes of different species, which leads to changes of the offspring in phenotypes and genotypes. in this study, we successfully establish a fertile hybrid lineage by intergeneric hybridization of female blunt snout bream (bsb, megalobrama amblycephala) × male topmouth culter (tc, culter alburnus) and investigate some important biological traits of this lineage including the morphological traits, chromosomal number, karyotype, dna content, gonadal ... | 2014 | 25287724 |
| microsatellite analysis of different ploidy offspring of artificial gynogenesis in cyprinus carpio. | gynogenesis was induced by using uv-irradiated spermatozoa of blunt snout bream megalobrama amblycephala to activate eggs of common carp cyprinus carpio. the maternal genome was then duplicated by cold shock in 0 to 4° c cold water to retain the second polar body. two kinds of fry, normal fry and abnormal tortuous fry, were hatched. their dna content was measured by flow cytometry. the normal fry were identified as diploid, representing the successful gynogenesis in c. carpio whereas the abnorma ... | 2011 | 21235552 |
| induction of gynogenesis in japanese crucian carp (carassius cuvieri). | diploid gynogenesis was induced in japanese crucian carp (carassius cuvieri) eggs using uv-irradiated genetically inactive spermatozoa from mirror carp (cyprinus carpio l.) or blunt snout bream (megalobrama amblycephala), with or without cold shock. the optimal radiation dosage was 4,200 mj/cm2 and 3,600 mj/cm2 for mirror carp and blunt snout bream sperm, respectively. at this dosage and without cold shock, the yields were (32.4 +/- 3.3)% vs. (33.8 +/- 1.4)% gynogenetic haploids and (0.7 +/- 0.3 ... | 2006 | 16722335 |
| transcriptome analysis and ssr/snp markers information of the blunt snout bream (megalobrama amblycephala). | blunt snout bream (megalobrama amblycephala) is an herbivorous freshwater fish species native to china and has been recognized as a main aquaculture species in the chinese freshwater polyculture system with high economic value. right now, only limited est resources were available for m. amblycephala. recent advances in large-scale rna sequencing provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitabl ... | 2012 | 22880060 |
| effects of emodin and vitamin c on growth performance, biochemical parameters and two hsp70s mrna expression of wuchang bream (megalobrama amblycephala yih) under high temperature stress. | in order to study the effects of dietary emodin, high-dose vitamin c (vc) and their combination on growth of wuchang bream (megalobrama amblycephala y.) and its resistance to high temperature stress, 1200 healthy wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg vc, and the combination of 60 mg/kg ... | 2012 | 22281609 |