Hepcidin-Expressing Fish Eggs as A Novel Food Supplement to Modulate Immunity against Pathogenic Infection in Zebrafish (Danio rerio)

Chung-Chih Tseng,Yenny Risjani,Tah-Wei Chu,Hui-Tsu Shih,Shao-Yang Hu,Ridha Danata

doi:10.3390/su12104057

Abstract

Hepcidin antimicrobial peptides are difficult to produce in prokaryotic expression systems due to their complex structure and antimicrobial activity. Although synthetic hepcidin provides an alternative to solve this issue, its high cost limits its practical application in various industries. The present study used zebrafish eggs as bioreactors to produce convict cichlid (Amatitlania nigrofasciata) hepcidin (AN-hepc) using the oocyte-specific zona pellucida (zp3) promoter. The expression plasmid pT2-ZP3-AN-hepc-ZP3-EGFP, using EGFP as a reporter of AN-hepc expression, was designed to establish the transgenic line Tg(ZP3:AN-hepc:ZP3:EGFP) for the expression of AN-hepc. The AN-hepc peptide was produced successfully in fertilized eggs, as evidenced by RT-PCR and Western blotting. The AN-hepc-expressing eggs exhibited antimicrobial activity against a variety of aquatic pathogens and antibiotic-resistant pathogens, suggesting that the AN-hepc expressed in fish eggs was bioactive. The immunomodulatory effects of AN-hepc-expressing fertilized eggs on zebrafish innate immunity were evaluated by determining the expression of indicator genes after feeding with AN-hepc-expressing fertilized eggs for two months. Zebrafish supplementation with AN-hepc-expressing fertilized eggs significantly increased the expression of innate immunity-related genes, including IL-1β, IL-6, IL-15, TNF-α, NF-κb, complement C3b, lysozyme and TLR-4a. The zebrafish administered AN-hepc-expressing eggs exhibited higher cumulative survival than fish supplemented with wild-type and control eggs after infection with Aeromonas hydrophila and Streptococcus iniae. In conclusion, the present results showed that supplementation with AN-hepc-expressing fish eggs enhanced zebrafish innate immunity against pathogenic infections, suggesting that fertilized eggs containing AN-hepc have the potential to be developed as a food supplement for improving health status in aquaculture.

Highlights

Fish diseases caused by bacterial pathogens result in high mortality of cultured fish and substantial economic loss, making them one of the major causes impeding the sustainable development of aquaculture
Prokaryotic expression systems such as E. coli bacteria are widely used for the production of heterologs; the production of the antimicrobial peptide hepcidin cannot be executed very successfully in E. coli
Unlike other cases in which recombinant proteins are ubiquitously expressed in transgenic fish [24,25,34], the present study showed that A. nigrofasciata hepcidin (AN-hepc) can accumulate during oogenesis; fertilized eggs can be used as a source to produce AN-hepc without sacrificing fish

Summary

Introduction

Fish diseases caused by bacterial pathogens result in high mortality of cultured fish and substantial economic loss, making them one of the major causes impeding the sustainable development of aquaculture. Aeromonas hydrophila and Streptococcus iniae are typical pathogens that cause red fin disease and hemorrhagic septicemia in several freshwater fish species [1,2] To address these diseases, antibiotic and antimicrobial chemicals are widely used in aquaculture for prophylactic and therapeutic purposes. The abuse of antibiotic and antimicrobial chemicals has led to the rapid spread of antimicrobial-resistant pathogens, which impacts on microbial ecology and food-related risks for human health due to residual chemicals in aquatic products. In light of this issue, alternatives to antibiotics for disease control are urgently needed. AMPs offer a promising alternative for the modulation of immunity in aquatic animals for disease control [3]

Methods

Results

Conclusion