Antimicrobial activity and proteomic analysis ofBoleophthalmus pectinirostris skin mucus

Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense, osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments, the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins, keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins, and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin, filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.