Antimicrobial Peptides Are Expressed during Early Development of Zebrafish (Danio rerio) and Are Inducible by Immune Challenge

Abstract

Antimicrobial peptides (AMPS) are ancestral components in the evolution of immunity from protozoans to metazoans. Their expression can be constitutive or inducible by infectious challenge. Although characterized in detail in their structure and activity, the temporal and spatial expression of AMPS during vertebrate embryogenesis is still poorly understood. In the present study, we identified selected AMPs in zebrafish, and characterized their expression during early development, and upon experimental immune challenge in adult animals, with the goal of establishing this genetically-tractable model system for further AMP studies. By mining available genomic databases, zebrafish AMP sequences homologous to AMPs from other vertebrates were selected for further study. These included parasin I and its enzyme cathepsin D, β-defensin (DB1), liver-expressed antimicrobial peptide 2 (LEAP2), bactericidal permeability-increasing protein (BPI), and chromogranin-A and -B (CgA and CgB). Specific primers were designed for RT-PCR amplification of each AMP gene of interest and amplicons between 242 bp and 504 bp were obtained from RNA extracted from adult zebrafish. Sequencing of the amplicons and alignment of their deduced amino acid sequences with those from AMPs from other vertebrate species confirmed their identity. The temporal expression of AMPs was investigated by RT-PCR analysis in fertilized oocytes, embryos, and adult individuals. Parasin I and chatepsin D transcripts were detectable immediately after fertilization, while the transcripts for CgA and CgB became evident starting at 48 h post fertilization. Mature transcripts of LEAP2 and DB1 were detectable only in the adult zebrafish, while BPI transcripts were detectable starting from the 12th day post fertilization. To explore the possible upregulation of AMP expression by infectious challenge, experiments were carried out in adult zebrafish by intraperitoneal injection of a cocktail of lipopolysaccharide and lipoteichoic acid. Except for CgA and CgB, amplicons corresponding to all tested AMPs showed stronger signals in the experimental animals as compared to the unchallenged controls. This study provided information on the early expression of AMPs in zebrafish from ontogeny to adulthood and their inducibility by microbials. This information could be useful to actuate new prophylactic strategies as an alternative to the use of antibiotics in culture.

Keywords

• zebrafish
• antimicrobial peptides
• immune system ontogeny
• lipopolysaccharide
• LPS lipoteichoic acid
• LTA