title: Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection.
abstract: Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.
authors.affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 20224, Taiwan.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Jheng
authors.affiliation: Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan.
authors.forename: Lin-Han
authors.initials: LH
authors.lastname: Lee
authors.affiliation: Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan.
authors.forename: Chen-Hung
authors.initials: CH
authors.lastname: Ting
authors.affiliation: Department and Graduate Institute of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 811, Taiwan.
authors.forename: Chieh-Yu
authors.initials: CY
authors.lastname: Pan
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Cho-Fat
authors.initials: CF
authors.lastname: Hui
authors.affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 20224, Taiwan; Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan. Electronic address: zoocjy@gate.sinica.edu.tw.
authors.forename: Jyh-Yih
authors.initials: JY
authors.lastname: Chen
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism*; D001156:Artemia / Q000235:genetics / Q000378:metabolism; D004032:Diet / Q000662:veterinary; D052978:Disk Diffusion Antimicrobial Tests / Q000662:veterinary; D018274:Electroporation / Q000662:veterinary; D005393:Fish Diseases / Q000276:immunology* / Q000382:microbiology*; D029941:Fish Proteins / Q000378:metabolism*; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016019:Survival Analysis; D014735:Vibrio Infections / Q000276:immunology / Q000662:veterinary*; D015027:Zebrafish
chemical_list: D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; C521392:epinecidin-1, Epinephelus coioides
keywords: Antimicrobial peptide; Artemia; Epinecidin-1; Vibrio vulnificus; Zebrafish