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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.

The glochidia larvae of most freshwater mussels are obligate ectoparasites on fishes, on which they metamorphose into juveniles. We have demonstrated that bluegill sunfish (Lepomis macrochirus) develop acquired resistance to glochidial larvae of Utterbackia imbecillis after 2 infections, resulting in reduction of successful metamorphosis during the 3rd and 4th infections and significantly fewer glochidia attaching to fish during the 4th infection. Since the mechanism resulting in acquired resistance is not fully understood, one possible mechanism, antibody-mediated immune response, was examined. We collected sera from fish at 5-day intervals during 4 sequential infection periods and from naive fish. Western blots of glochidial proteins probed with the sera revealed that naive fish had natural antibodies against a ~35 kDa protein in the larval profile, a protein that also was recognized by immune sera. Immunochemical studies on whole-mount glochidia probed with serum from naive fish demonstrated that the natural antibodies recognize granular structures located between the larval mantle and shell. The antibodies in naive bluegills only recognized proteins in U. imbecillis and another anodontine mussel species. The antibodies did not recognize proteins in larvae of two other mussel species from a different unionid mussel taxon, the subfamily Ambleminae. In addition to the ~35 kDa protein, other larval proteins were recognized by antibodies in sera collected during each infection period, but not from naive fish. This study suggest that serum antibodies play a role in the development of acquired resistance in fish against glochidial larvae of freshwater mussels during multiple infections.

In order to metamorphose into juveniles and subsequently mature into adults, the larvae (glochidia) of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures offish. Once attached to the fish, the glochidium is encapsulated by host fish epithelial tissue. We have recently demonstrated that bluegill sunfish (Lepomis macrochirus) develop acquired resistance to glochidial larvae of Utterbackia imbecillis after 2 infections, resulting in reduction of successful metamorphosis during the 3rd and 4th infections and significantly fewer glochidia attaching to fish during the 4th infection. Since the mechanism resulting in acquired resistance is not fully understood, one possible mechanism, differences in the nature of cyst formation on naive and resistant fish, was examined. The time course of the formation of cysts on fins of halve and resistant fish was monitored by scanning electron microscopy and by time-lapsed video microscopy. The development of cysts was scored according the relative surface area of the glochidia covered by the migration of host epithelial tissue at intervals up to 6 hours post infection. Compared to naive fish, cyst formation on resistant fish was significantly delayed. By 6 h post infection, 48.0% of the glochidia on naive fish were completely covered by cysts, compared to only 9.6% on resistant fish. Additionally, the cysts on resistant fish were morphologically irregular as compared to those on naive fish. The delay and irregularities in cyst formation on resistant fish may result in prolonged exposure of glochidia to innate immune mechanisms in fish mucus and to unsuitable external environment, resulting in increased mortality and reduced success of metamorphosis, and may represent one mechanism of acquired resistance.