Brachyuran crabs extrude fertilized eggs covered with glue and attach them to feathery pleopods under their tails. Eggs are brooded during embryo development and are physically and enzymatically cleaned until egg hatching. Peptide chemical signals coordinate egg hatching and larval release which is synchronized between the larvae and the female. In many crabs, release occurs in synchronous bursts that last about a minute and involves enzymes that cleave glues while eggs burst due to biological, osmotic, and physical pressure. Ovigerous pea crabs, which are oyster parasites, were removed from oysters into 0.2 micron gravity filtered aged sea water. Next, we incubated individual crabs in a sea water mixture of 3 pure proteins at 0.5 to 1 um for 20 minutes. The addition of proteins to the females with eggs about to hatch stimulated hatching. Proteomics identified peptides from pure proteins generated by maintenance proteases and hatching proteases. Based on carboxyl terminal amino acids of peptides, there is an ensemble of at least 5 endoproteases. Because we cleaved pure proteins, we know the absolute identity of the sequences around the cleavages, which means we can define enzyme specificity.
Preliminary analysis suggests the presence of serine proteases in addition to many unidentified proteases. The identification of the non-serine proteases is actively being investigated.