Cloning, characterization, antibacterial activity and expression of hamp in pond loach (Misgurnus anguillicaudatus) after bacterial challenge with Aeromonas hydrophila

Loaches are popular in aquaculture in China due to their flesh having a high concentration of nutrients important for human health, such as amino acids, vitamins, minerals, and taurine. As such, they are used in Chinese traditional medicine to restore health and slow aging. However, the quick expansion of intensive loach culture to meet consumer demand has been accompanied by the increased prevalence of disease outbreaks, most often caused by Aeromonas spp. bacteria. We collected and identified pathogenic bacteria causing hemorrhagic septicemia in pond loach sourced from two provinces of China (Hubei and Hunan), and found three Aeromonas species: hydrophila (the most prevalent), sobria and veronii. We performed a bacterial challenge on the pond loach (Misgurnus anguillicaudatus) using the different concentrations of A. hydrophila in order to determine the median lethal dose, and found that the LD50 value was 2.14 × 107 CFU/fish. Hepcidin protein encoded by hepcidin antimicrobial peptide (hamp) gene is a key factor for iron regulation, enhancement of the host's innate immune system and acts as antimicrobial peptide. To better understand the functions of this protein in pond loach, we cloned it, conducted in silico physicochemical characterization, and studied its expression at the mRNA level both in healthy loach, and after a challenge with A. hydrophila. We found two very similar paralogs (a and b) of the hamp 1 gene, the open reading frames of which were 276 bp, both encoding an identical 91 amino acid putative peptide. The purified recombinant hepcidin protein exhibited antibacterial activity against all six studied bacterial species at 100-3200 arbitrary units/ml and can decrease 50% of the cumulation mortality of loaches which were injected by A. hydrophila. Both hamp 1 paralogs were basally expressed in most tissues, and significantly up-regulated in the liver, spleen and kidney after the bacterial challenge. The results of this study strongly indicate that hepcidin plays a role in defense mechanisms against pathogenic bacteria and inflammation in loaches.