Egg Hatching, Peptide Pheromones, and Endoproteinases in Barnacles

The striped barnacle, <i>Amphibalanus amphitrite</i>, is a simultaneous hermaphrodite crustacean that broods eggs. The eggs are physically and enzymatically cleaned in the mantle by the barnacle to manage biofouling during incubation. There is no physiological connection between the embryos and the adult. Instead, barnacles use enzyme products as pheromones to coordinate behavioral, physiological, and biochemical processes involved in egg hatching and larval release. Known larval release pheromones are peptides generated by exogenous trypsins that act on proteins. We characterized barnacle brooding endoproteinases using a proteomic analysis of peptides generated from the hydrolysis of pure proteins that were identified by high-resolution LC electrospray MS/MS. Utilizing pure proteins permitted us to completely identify sequences around proteolytic cleavage sites. Enzyme activity was 2.22 to 2.79 times greater in barnacle and barnacle microbiome samples compared to seawater samples. Distinct enzyme patterns emerged, with higher proline- and asparagine-cutting enzymes in barnacle samples and greater proportions of elastase in seawater. There are at least 13 endoproteinases based on the C-terminus amino acids of peptides, with major contributions from serine proteases. This approach has the potential to provide exceptionally detailed information on endoproteinases in any microbiome assemblage. With a little thought, this technique can be expanded to include exoproteinases as well.