ZFIN ID: ZDB-PUB-141203-12
Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection
Jheng, Y.H., Lee, L.H., Ting, C.H., Pan, C.Y., Hui, C.F., Chen, J.Y.
Date: 2015
Source: Fish & shellfish immunology   42: 1-15 (Journal)
Registered Authors: Chen, Jyh-Yih
Keywords: Antimicrobial peptide, Artemia, Epinecidin-1, Vibrio vulnificus, Zebrafish
MeSH Terms:
  • Analysis of Variance
  • Animals
  • Animals, Genetically Modified/genetics
  • Animals, Genetically Modified/metabolism*
  • Antimicrobial Cationic Peptides/metabolism*
  • Artemia/genetics
  • Artemia/metabolism
  • Diet/veterinary
  • Disk Diffusion Antimicrobial Tests/veterinary
  • Electroporation/veterinary
  • Fish Diseases/immunology*
  • Fish Diseases/microbiology*
  • Fish Proteins/metabolism*
  • Fluorescence
  • Gene Expression Regulation/immunology*
  • Reverse Transcriptase Polymerase Chain Reaction/veterinary
  • Survival Analysis
  • Vibrio Infections/immunology
  • Vibrio Infections/veterinary*
  • Zebrafish*
PubMed: 25462461 Full text @ Fish Shellfish Immunol.
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.
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