Fish Physiology and Biochemistry

, Volume 41, Issue 2, pp 449–461 | Cite as

Molecular cloning and functional characterization of the hepcidin gene from the convict cichlid (Amatitlania nigrofasciata) and its expression pattern in response to lipopolysaccharide challenge

  • Jing-Ruei Chi
  • Long-Si Liao
  • Rong-Guang Wang
  • Chu-Sian Jhu
  • Jen-Leih WuEmail author
  • Shao-Yang HuEmail author


The hepcidin gene is widely expressed in many fish species and functions as an antimicrobial peptide, suggesting that it plays an important role in the innate immune system of fish. In the present study, the Amatitlania nigrofasciata hepcidin gene (AN-hepc) was cloned from the liver and its expression during an immune response was characterized. The results of quantitative PCR and RT-PCR showed that the AN-hepc transcript was most abundant in the liver. The expression of AN-hepc mRNA was significantly increased in the liver, stomach, heart, intestine, gill and muscle but was not significantly altered in the spleen, kidney, brain or skin after lipopolysaccharide challenge. The synthetic AN-hepc peptide showed a wide spectrum of antimicrobial activity in vitro toward gram-positive and gram-negative bacteria. In particular, this peptide demonstrated potent antimicrobial activity against the aquatic pathogens Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, Aeromonas hydrophila and Streptococcus agalactiae. The in vivo bacterial challenge results demonstrated that the synthetic AN-hepc peptide significantly improved the survival rate of S. agalactiae- and V. vulnificus-infected zebrafish. Taken together, these data indicate an important role for AN-hepc in the innate immunity of A. nigrofasciata and suggest its potential application in aquaculture for increasing resistance to disease.


Hepcidin Amatitlania nigrofasciata Cloning Gene expression Antimicrobial activity 



We thank the Taiwan Zebrafish Core Facility at Academia Sinica (TZCAS), which is supported by a grant from the National Science Council (NSC 101-2321–B-001-026) in Taiwan, for providing the AB strain zebrafish. This research was supported by a grant from the National Science Council (NSC 102-2324-B-020-001-CC1). We appreciate Dr. Jyh-Yih Chen and Dr. Chung-Hung Liu for kindly providing the bacterial pathogens.

Supplementary material

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Supplementary material 1 (DOC 1034 kb)


  1. Bao B, Peatman E, Li P, He C, Liu Z (2005) Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection. Dev Comp Immunol 29:939–950CrossRefPubMedGoogle Scholar
  2. Chang WT, Pan CY, Rajanbabu V, Cheng CW, Chen JY (2011) Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1-5, shows antitumor activity in cancer cells. Peptides 32:342–352CrossRefPubMedGoogle Scholar
  3. Chen SL, Xu MY, Ji XS, Yu GC, Liu Y (2005) Cloning, characterization, and expression analysis of hepcidin gene from red sea bream (Chrysophrys major). Antimicrob Agents Chemother 49:1608–1612CrossRefPubMedCentralPubMedGoogle Scholar
  4. Chen JY, Lin WJ, Lin TL (2009) A fish antimicrobial peptide, tilapia hepcidin TH2-3, shows potent antitumor activity against human fibrosarcoma cells. Peptides 30:1636–1642CrossRefPubMedGoogle Scholar
  5. Cuesta A, Meseguer J, Esteban MA (2008) The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream. Mol Immunol 45:2333–2342CrossRefPubMedGoogle Scholar
  6. Douglas SE, Gallant JW, Liebscher RS, Dacanay A, Tsoi SC (2003) Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish. Dev Comp Immunol 27:589–601CrossRefPubMedGoogle Scholar
  7. Faikoh EN, Hong YH, Hu SY (2014) Liposome-encapsulated cinnamaldehyde enhances zebrafish (Danio rerio) immunity and survival when challenged with Vibrio vulnificus and Streptococcus agalactiae. Fish Shellfish Immunol 38:15–24CrossRefPubMedGoogle Scholar
  8. Gong LC, Wang H, Deng L (2014) Molecular characterization, phylogeny and expression of a hepcidin gene in the blotched snakehead Channa maculata. Dev Comp Immunol 44:1–11CrossRefPubMedGoogle Scholar
  9. Hilton KB, Lambert LA (2008) Molecular evolution and characterization of hepcidin gene products in vertebrates. Gene 415:40–48CrossRefPubMedGoogle Scholar
  10. Hirono I, Hwang JY, Ono Y, Kurobe T, Ohira T, Nozaki R, Aoki T (2005) Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus. FEBS J 272:5257–5264CrossRefPubMedGoogle Scholar
  11. Hoang VL, Kim SK (2013) Antimicrobial peptides from marine sources. Curr Protein Pept Sci 14:205–211CrossRefPubMedGoogle Scholar
  12. Hoffman LR, Déziel E, D’Argenio DA et al (2006) Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 103:19890–19895CrossRefPubMedCentralPubMedGoogle Scholar
  13. Hsieh JC, Pan CY, Chen JY (2010) Tilapia hepcidin (TH)2-3 as a transgene in transgenic fish enhances resistance to Vibrio vulnificus infection and causes variations in immune-related genes after infection by different bacterial species. Fish Shellfish Immunol 29:430–439CrossRefPubMedGoogle Scholar
  14. Hu X et al (2007) Channel catfish hepcidin expression in infection and anemia. Comp Immunol Microbiol Infect Dis 30:55–69CrossRefPubMedGoogle Scholar
  15. Hu SY, Lin PY, Liao CH, Gong HY, Lin GH, Kawakami K, Wu JL (2010) Nitroreductase-mediated gonadal dysgenesis for infertility control of genetically modified zebrafish. Mar Biotechnol (NY) 12:569–578CrossRefGoogle Scholar
  16. Huang PH, Chen JY, Kuo CM (2007) Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions. Mol Immunol 44:1922–1934CrossRefPubMedGoogle Scholar
  17. Kawashima R, Shimizu T, To M, Saruta J, Jinbu Y, Kusama M, Tsukinoki K (2014) Effects of stress on mouse beta-defensin-3 expression in the upper digestive mucosa. Yonsei Med J 55:387–394CrossRefPubMedCentralPubMedGoogle Scholar
  18. Kim YO, Hong S, Nam BH, Lee JH, Kim KK, Lee SJ (2005) Molecular cloning and expression analysis of two hepcidin genes from olive flounder Paralichthys olivaceus. Biosci Biotechnol Biochem 69:1411–1414CrossRefPubMedGoogle Scholar
  19. Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, Schulz-Knappe P, Adermann K (2000) LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 480:147–150CrossRefPubMedGoogle Scholar
  20. Lauth X, Babon HH, Stannard JA et al (2005) Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections. J Biol Chem 280:9272–9282CrossRefPubMedGoogle Scholar
  21. Liang T, Ji W, Zhang GR, Wei KJ, Feng K, Wang WM, Zou GW (2013) Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream (Megalobrama amblycephala). Fish Shellfish Immunol 35:553–563CrossRefPubMedGoogle Scholar
  22. Masso-Silva JA, Diamond G (2014) Antimicrobial peptides from fish. Pharmaceuticals (Basel) 7:265–310CrossRefGoogle Scholar
  23. Noga EJ, Ullal AJ, Corrales J, Fernandes JM (2011) Application of antimicrobial polypeptide host defenses to aquaculture: exploitation of downregulation and upregulation responses. Comp Biochem Physiol Part D Genomics Proteomics 6:44–54CrossRefPubMedGoogle Scholar
  24. Novoa B, Figueras A (2012) Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases. Adv Exp Med Biol 946:253–275CrossRefPubMedGoogle Scholar
  25. Pan CY, Peng KC, Lin CH, Chen JY (2011) Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens. Fish Shellfish Immunol 31:275–285CrossRefPubMedGoogle Scholar
  26. Pan CY, Huang TC, Wang YD, Yeh YC, Hui CF, Chen JY (2012) Oral administration of recombinant epinecidin-1 protected grouper (Epinephelus coioides) and zebrafish (Danio rerio) from Vibrio vulnificus infection and enhanced immune-related gene expressions. Fish Shellfish Immunol 32:947–957CrossRefPubMedGoogle Scholar
  27. Park CH, Valore EV, Waring AJ, Ganz T (2001) Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 276:7806–7810CrossRefPubMedGoogle Scholar
  28. Rodrigues PN, Vazquez-Dorado S, Neves JV, Wilson JM (2006) Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax). Dev Comp Immunol 30:1156–1167CrossRefPubMedGoogle Scholar
  29. Schuppler M, Loessner MJ (2010) The opportunistic pathogen Listeria monocytogenes: pathogenicity and interaction with the mucosal immune system. Int J Inflam 2010:704321CrossRefPubMedCentralPubMedGoogle Scholar
  30. Shike H, Lauth X, Westerman ME et al (2002) Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. Eur J Biochem 269:2232–2237CrossRefPubMedGoogle Scholar
  31. Tomioka H, Nakagami H, Tenma A et al (2014) Novel anti-microbial peptide SR-0379 accelerates wound healing via the PI3 kinase/Akt/mTOR pathway. PLoS ONE 9:e92597CrossRefPubMedCentralPubMedGoogle Scholar
  32. Wang KJ, Cai JJ, Cai L, Qu HD, Yang M, Zhang M (2009) Cloning and expression of a hepcidin gene from a marine fish (Pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide. Peptides 30:638–646CrossRefPubMedGoogle Scholar
  33. Yang M, Wang KJ, Chen JH, Qu HD, Li SJ (2007) Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B). Fish Shellfish Immunol 23:1060–1071CrossRefPubMedGoogle Scholar
  34. Zhao T, Zhao P, Chen D, Jadeja R, Hung YC, Doyle MP (2014) Reductions of Shiga Toxin-Producing Escherichia coli and Salmonella typhimurium on beef trim by lactic acid, levulinic acid, and sodium dodecyl sulfate treatments. J Food Prot 77:528–537CrossRefPubMedGoogle Scholar
  35. Zhou JG, Wei JG, Xu D et al (2011) Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides. Fish Shellfish Immunol 30:559–568CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Department of Biochemical Science and TechnologyNational Taiwan UniversityTaipeiTaiwan
  2. 2.Institute of Cellular and Organismic BiologyAcademia SinicaTaipeiTaiwan
  3. 3.Department of Biological Science and TechnologyNational Pingtung University of Science and TechnologyNeipu, PingtungTaiwan

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