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Nitrophenols are identified as the priority organic pollutants due to the chemical stability, water solubility, persistence, and toxicity to human health and the environment. Hence, removal of nitrophenols from waste water is vitally essential. In this study, amino-rich coordination polymer Cu2I2(MA)2 (MA = melamine) has been applied for efficient adsorption and catalytic reduction of nitrophenols, like 4-nitrophenol (4-NP), 2, 4-dinitrophenol (DNP) and 2, 4, 6-trinitrophenol (TNP). The effect of various parameters like contact time, initial concentrations, pH, and temperature on adsorption were investigated. The adsorption of nitrophenols fitted the pseudo-second-order kinetic model and Langmuir isotherms model well. The maximum adsorption capacities were 285.71, 232.02, and 131.57 mg g-1 for 4-NP, DNP, and TNP when initial concentrations were 50 mg L-1 at 293.15 K, respectively. The adsorption of nitrophenols is a spontaneous, endothermic, and entropy-driven process. The reduction reaction followed the pseudo-first-order kinetics, and the kinetic rate constants were 0.4413, 0.3167, and 0.17538 min-1 for 4-NP, DNP, and TNP, respectively. The effect of initial nitrophenols concentration, anions, and temperature on reduction process was investigated. The mechanism of adsorption and catalytic reduction of Cu2I2(MA)2 was studied. The results demonstrated that Cu2I2(MA)2 exhibits excellent adsorption and catalytic activity to remove nitrophenols.

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Nile tilapia is a GSD + TE (Genetic Sex Determination + Temperature Effect) fish, and high-temperature treatment during critical thermosensitive periods (TSP) can induce the sex reversal of Nile tilapia genetic females, and brain transcriptomes have revealed the upregulation of Jarid2 (Jumonji and AT-rich domain containing 2) expression after 36 °C high-temperature treatment for 12 days during TSP. It was shown that JARID2 forms a complex with polycomb repressive complex 2 (PRC2) that catalyzed H3K27me3, which was strongly associated with transcriptional repression. In this study, Jarid2b was cloned and characterized in Nile tilapia, which was highly conserved among the analyzed fish species. The expression of Jarid2b was upregulated in the gonad of 21 dpf XX genetic females after 12-day high-temperature treatment and reached a similar level to that of males. Similar responses to high-temperature treatment also appeared in the brain, heart, liver, muscle, eye, and skin tissues. Interestingly, Jarid2b expression was only in response to high-temperature treatment, and not to 17α-methyltestosterone (MT) or letrozole treatments; although, these treatments can also induce the sex reversal of genetic Nile tilapia females. Further studies revealed that Jarid2b responded rapidly at the 8th hour after high-temperature treatment. Considering that JARID2 can recruit PRC2 and establish H3K27me3, we speculated that it might be an upstream gene participating in the regulation of Nile tilapia GSD + TE through regulating the H3K27 methylation level at the locus of many sex differentiation-related genes.

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Chlorine or chloric disinfectants are cost-effective disinfectants, which are widely used to disinfect domestic and industrial water. The residual chlorine levels in some of these waters have been proven toxic to several aquatic organisms; however, the molecular mechanisms of toxicity of residual chlorine to aquatic crustaceans, including Macrobrachium nipponense, an economically important freshwater prawn native to Asian countries, have not been investigated to date. Here, M. nipponense was exposed to 0.53 mg/L of residual chlorine, and comparative transcriptomics analyses were performed to determine their response mechanisms at the molecular level. Residual chlorine caused lethal effects on prawns. Furthermore, a total of 940 differentially expressed genes (DEGs), including 501 up-regulated and 439 down-regulated genes, were identified after 48 h of residual chlorine exposure compared to the control group. After enrichment analysis of GO (Gene Ontology) functions and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, identified DEGs were demonstrated to be associated with a variety of functions including exerting "oxidoreductase activity", and participating in "oxidation-reduction process". In addition, cytochrome P450 family 1 subfamily A1 (CYP1A1), glutathione S-transferase (GST), and glucuronosyltransferase (UGT) were enriched in the pathway of metabolism of xenobiotics by cytochrome P450. Furthermore, protein-protein interaction (PPI) network analysis revealed interactions among actin beta/gamma 1 (ACTB_G1) gene encoding protein and a series of multiple functional DEGs (e.g., hexokinase (HK), fructose 1,6-biphosphate-aldolase A (ALDOA), cytochrome c (CYC), and elongation factor 1-alpha (EEF1A)) encoding proteins. This study laid a theoretical foundation for safety evaluation of chlorinated aquatic water and further investigation of the toxicity of chlorination to M. nipponense.

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BACKGROUND: The Chinese giant salamander Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci. RESULTS: In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of A. davidianus. The genetic length of LGs ranged from 17.61 cM (LG30) to 280.81 cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48 cM (LG26). The total genetic map length was 2643.10 cM with an average inter-locus distance of 0.24 cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively. CONCLUSION: We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.

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The oriental river prawn Macrobrachium nipponense is a highly adaptable, tolerant, and fecund freshwater prawn that inhabits a wide range of aquatic environments. The hepatopancreas of crustaceans is not only a site for secretion of digestive enzymes, and also plays important roles in several metabolic processes, such as lipid and carbohydrate metabolism. It is the main organ for the detoxification and immunity. In this study, high-throughput sequencing techniques were used to detect the effect of nitrite stress (10 mg/L nitrite-N for 48 h) on gene expression in the hepatopancreas of M. nipponense. A total of 13,769 million reads were harvested, and 94,534 transcripts were de novo assembled using Trinity software and produced 56,054 non-redundant transcripts. A total of 825 differentially expressed genes were obtained comparing 48 h nitrite stress with control group. In the analysis of GO and KEGG database, significant differences were found in 49 pathways. Immune-related pathways under nitrite stress included arginine and proline metabolism, glutamate metabolism, Jak-Stat signaling pathway, endocytosis, wnt signaling pathway, RIG-I-like receptor signaling pathway, TGF-beta signaling pathway, GnRH signaling pathway and phagosome. Apoptosis-related pathway was also significantly altered, such as lysosome and apoptosis. Remarkably, nitrite stress altered the expression patterns of key apoptosis genes (tetraspanins-like protein, LAMP, CD63, caspase 3C and Caspase 1) and immune genes (Serine proteinase-like protein, C-type lectin, daf-36, SOCS-2, alpha-2-macroglobulin), confirmed that nitrite-stress induce immune response and eventually even apoptosis. This study provided a new insight into the role of hepatopancreas in crustaceans, and further investigation will continue.

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Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

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The half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial induction of mitogynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole in order to assess homozygosity of gynogens and to identify WW super-female. The optimal initiation time for pressure shock of mitogynogenetic embryos was determined to be 21.5 min after insemination when water temperature is at 22-23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min. About 1,500 mitogynogenetic diploid larvae were obtained. Ten tongue sole microsatellite markers were used for homozygosity analysis of 24 mitogynogenetic larvae. Among the 24 larvae, the percentage of homozygosity ranged from 73.91% to 87.50% with an average homozygosity of 80.54%. Sex-specific simple sequence repeat (SSR) markers, CseF-SSR1, were isolated and used for identifying WW super-female mitogynogens in the tongue sole. The amplification of genomic DNA using the sex-specific SSR marker produced one DNA band of 206 bp in ZZ males, two DNA bands of 206 and 218 bp in ZW females, and one DNA band of 218 bp in WW super-females. Four WW "super-female" gynogens were observed in 39 mitogynogenetic diploids, indicating a ZW sex determination mechanism in the tongue sole. Thus, a protocol for the induction of artificial mitogynogenesis has been developed for the first time in half-smooth tongue sole, and the WW super-female diploids were identified in the mitogynogens by sex-specific SSR markers. These findings lay the foundation and provide important tool for the elaboration of sex determination mechanism, generation of WW super-females, and development of clone line and breeding of all-female stock in the half-smooth tongue sole.

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To investigate population structure and marker assisted breeding, fast isolation by AFLP of sequences containing repeats (FIASCO) and GenBank database mining were used to develop novel microsatellite markers for sea perch (Lateolabrax japonicus). Genomic DNA fragments containing SSR sequences were captured by hybridization to (GT)(13) biotin-labeled probe and were ligated to PMD18-T vector. Among 150 randomly chosen clones from the SSR-enriched library, 66 sequences contained microsatellite motif over five repeats. In addition, 540 cDNA sequences and 132 ESTs of Lateolabrax japonicus were downloaded from GenBank and screened for di-, tri- and tetra-nucleotide repeats, while 22 sequences were found to contain microsatellites. As a result, 15 microsatellite loci were shown to be polymorphic in 30 Lateolabrax japonicus individuals, with the alleles ranging from two to ten, the observed heterozygosities from 0.6000-1.0000, and the expected heterozygosities from 0.5079-0.8890. Four loci (SP17, SP52, SP94 and SP468) were deviated from HWE in the sampled population after Bonferroni's correction, and no linkage disequilibrium was found among all loci (P<0.003), whereas null alleles were detected at locus SP52 (P<0.05). Among 15 polymorphic loci, the PIC values, which can be used for related population genetics analysis, were all above 0.5, with the exception of SP17 and SP468.

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Half-smooth tongue sole, Cynoglossus semilaevis, is an ideal model to investigate the regulatory mechanisms of sexual growth dimorphism in fish species. The aim of the study was to investigate the effect of differential age of sexual maturity for females and males on growth and GH mRNA expression in C. semilaevis. The body weight differences between the sexes were not significant in C. semilaevis at age 5 months when females and males were all immature. Significant differences in body weight between the sexes were found after early sexual maturation of males at the age of 9 months. The body weight of 21-month-old females (621.4 ± 86.4g), still not immature, was even 3.28 times higher than that of the males (189.7 ± 14.4g). The cDNAs encoding GH in C. semilaevis was cloned. The GH gene is 2924bp long and consists of six exons and five introns. The results of qRT-PCR showed that GH mRNA levels of the immature females were not significantly different from that of immature males at age 5 months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9 months (P<0.05). At age 11 months, GH mRNA levels of females were even 6.4-fold higher than that of males. In conclusion, for the first time we show that early sexual maturity of males is the main cause of sexual growth dimorphism in C. semilaevis and exert significant effect on GH mRNA expression.

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Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months (P<0.05) and at 18 onths the mean body weight of the females (771.0±44.3 g) was as much as 4.9 times higher than that of males (130.6±6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females (P<0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.

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Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Its function is to present foreign peptide to the T-cell. In order to study the function and molecular polymorphism of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA were cloned from half-smooth tongue sole by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Genomic organizations, molecular polymorphism, and expression profiles of class IIA and IIB were examined to study the function in fish. As in other teleost, four exons and three introns were identified in half-smooth tongue sole class IIA gene, five exons and four introns were identified in class IIB gene. The deduced amino acid sequence of class IIA had 27.3-69.8% identity with those of mammal and teleost. Nine class IIA alleles were identified from four individuals. Four different alleles observed in a single individual may infer the existence of two loci at least. The deduced amino acid sequence of class IIB had 7.9-71.9% identity with those of other species. Fifteen class IIB alleles were identified. Six different alleles observed in a single individual may suggest that there are at least three loci in class IIB genes. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in twelve normal tissues. Challenge of half-smooth tongue sole with the pathogenic bacteria, Vibrio anguillarum, resulted in significant changes in the expression of MHC IIA and IIB mRNA in three tissues.

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The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, "tongue sole") is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.

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Molecular sex identification is important in studying sex control, sex determination, and all-female breeding in half-smooth tongue sole (Cynoglossus semilaevis). In the present study, a female-specific AFLP marker was isolated from Cynoglossus semilaevis by AFLP technique using the selective primer combination E-ACT/M-CAA. This marker was re-amplified, recovered from the agarose gels, cloned and sequenced. Bioinformatic analysis indicated that the length of the product was 791 bp, and the sequence showed no similarity to any known sequences deposited in the GenBank database using BLASTn. According to the DNA sequence of the female-specific AFLP marker, specific PCR primers were designed and PCR amplification was performed on 100 sex-known individuals of C. semilaevis (50 females and 50 males each). A specific band 324 bp in length was present in all females but absent in all males (except for one male), indicating that the female-specific AFLP marker was successfully converted into female-specific SCAR (sequence characterized amplified regions) marker. The sex analysis of 3-day-old C. semilaevis individuals using this female-specific SCAR marker indicated that the female ratio was 41.7%. The female-specific SCAR marker developed in this study allowed simple, reliable, and rapid molecular sex identification using small amounts of fin tissue without sacrifice of C. semilaevis especially at early stage of development.

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Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be > or =30 mJ/cm(2). The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20-25 min at 5 degrees C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.

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Genetic variation in the major histocompatibility complex (MHC) class IIB was tested in Japanese flounder (Paralichthys olivaceus) for survival after challenge with bacterial infection. The material consisted of 6000 Japanese flounder from 60 families challenged with Vibrio anguillarum, which causes significantly different mortality in flounder families. Five individuals from each of six high-resistance (HR) and six low-resistance (LR) families were screened for their MHC class IIB genotypes using sequence analysis. High polymorphism of MHC IIB gene and at least three loci were discovered in Japanese flounder and the rate of d(N) occurred at a significantly higher frequency than that of d(S) in PBR. Among 60 individuals, 76 alleles were discovered and 15 alleles were used to study associations between alleles and resistance to disease. We found highly significant associations between resistance towards infectious disease caused by V. anguillarum and MHC class IIB polymorphism in Japanese flounder. Some alleles appeared in both HR and LR families, while some alleles were only discovered in HR or LR families. One allele, Paol-DAB*4301, was significantly more prevalent in HR families than in LR families (P=0.023). Paol-DAB*0601, Paol-DAB*0801, Paol-DAB*2001, Paol-DAB*3803 were discovered in two HR families with high frequency. One allele, Paol-DAB*1601, was discovered in three LR families. The steady heredity of MHC class IIB alleles was observed, and the family having Paol-DAB*4301 alleles was confirmed with higher resistance to V. anguillarum. This study confirmed the association between alleles of MHC class IIB gene and disease resistance, and also detected some alleles which might be correlated with high bacterial infection resistance. The disease resistance-related MHC markers could be used for molecular marker-assisted selective breeding in the flounder.

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MHC class II (major histocompatibility complex class II) plays an important role in the immune response of vertebrates. Its function is to present antigenic peptides to the T-cell receptor. In order to study the function and molecular polymorphism of class II B gene in fish, we have isolated cDNAs encoding class II B from spleen cDNA library of red sea bream (Chrysophrys major) by using EST sequencing, and examined genomic organization, molecular polymorphism and expression of red sea bream class II B gene. As in other vertebrates, five exons and four introns were identified in red sea bream class II B gene. Seven class II B alleles were identified from seven individuals of red sea bream. The deduced amino acid sequence of red sea bream MHC class II B 1(Chma-DAB*0101) had 87.1, 85.1, 87.1, 90.4, 87.1, 90.8% identity with those of red sea bream class II B 2, 3, 4, 5, 6, 7(Chma-DAB*0201-Chma-DAB*0701), respectively, and had 75.2, 74.5, 55.9, 55.1, 34.3 and 30.4% identity with those of striped sea bass, cichlid, rainbow trout, Atlantic salmon, mouse and human, respectively. Four different class II B alleles were observed in a single individual and two different 3' untranslated region (3' UTR) sequences from this individual may infer the existence of two loci at least. Semi-quantitative RT-PCR demonstrated that high expression was detected in liver, head kidney, kidney, intestine, gill, stomach, hear and spleen, low expression in muscle and blood. Challenge of red sea bream with the pathogenic bacteria, Vibrio anguillarum, resulted in a significant decrease in the expression of MHC class II B mRNA from 5 to 72 h after infection in liver, spleen, head kidney and intestine, followed by a recovery to normal level after 96 h.

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A cDNA encoding hepcidin was isolated from a library of cDNA from spleen of red sea bream (Chrysophrys major) by expressed sequence tag analysis. The expression of the hepcidin mRNA in various tissues was examined. Challenge of red sea bream with Escherichia coli DH5alpha elevated hepcidin mRNA levels in spleen, gill, liver, and intestine.

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Nramp (natural resistance associated macrophage protein) controls aspects of innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from spleen of red sea bream (Pagrus major). The full-length cDNA of the Nramp is 4709 bp in length, including 197 bp 5'-terminal untranslated region (UTR), 1662 bp encoding region and 2850 bp 3'-terminal UTR. The 1662 nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of the amino acid sequence indicated that red sea bream Nramp consists of 12 transmembrane region (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of red sea bream Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5% and 69.5% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, mouse Nramp 1 and 2, and human Nramp1 and 2, respectively. Reverse transcription-polymerase chain reaction indicated that levels of Nramp expression were similar among head kidney, spleen, intestine and liver in non-challenged red sea bream, and that challenge of red sea bream with the pathogenic bacterium, Vibrio anguillarum, significantly elevated Nramp mRNA levels in liver and spleen in a time-dependent fashion.

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