Changes in activity and composition of the pro-teases of fish skin mucus during development of acquired resistance to glochidia larvae of fresh-water mussels

In order to metamorphose into juveniles, the larvae (glochidia) of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures of fish. When a glochidium attaches to a fish, it is potentially exposed to innate immune factors in the fish skin mucus, including proteases. Here, we examine the changes in the composition and activity of proteases in mucus of bluegill sunfish (Lepomis macrochirus) over the course of multiple infections by glochidia of Utterbackia imbecillis. Zymograms offish mucus treated with a suite of protease inhibitors revealed that the proteases in the mucus of naive fish are primarily serine proteases. However, proteases in the mucus offish infected 4 times with glochidia were primarily metalloproteases. Overall protease activity, as determined by azocasein hydrolysis, was lower in mucus from 4th infection fish as compared to naive fish and protease levels varied inversely with the humoral antibody response during the course of study. Extract from glochidia inhibits the serine proteases of fish mucus, similar to soybean trypsin inhibitor; however, the metalloproteases were not inhibited. Additionally, inhibitors of proteases decrease rates of cyst formation. This study suggests that alterations in protease activity may play a role in acquired resistance of fish against glochidia.