An immunological study of predation on hatchery-reared, juvenile red drum ( Sciaenops ocellatus): preparation and assays of a “red drum-specific” protein for predator-prey experiments

Abstract

This immunological study is part of a program to test the feasibility of artificial stock enhancement using hatchery-reared red drum ( Sciaenops ocellatus) (L.) and specifically to determine the predation mortality of newly released fish. Initially, a polyvalent antiserum was produced in a goat by multiple injections of a soluble red drum extract. In comparative experiments with soluble extracts of 12 different fishes and three different invertebrates found in the same environment, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the goat polyvalent anti-serum for Western blots, an 80 kDa “red drum specific” protein was selected that was absent from the other species. The protein was present as two isoforms in fingerlings, but as one protein in older fish. The protein was purified by ammonium sulfate fractionation, and column chromatography with the Fast Protein Liquid Chromatography system. A second goat was immunized with the highly purified red drum 80 kDa protein to obtain a specific antiserum for immunoassays. Cross-reactivity in Western blots with pre-immune and immune goat sera and unrelated fish proteins was removed from the sera by solid phase adsorption. The adsorbed goat anti-80 kDa glycoprotein serum was used in predator-prey studies to identify partially digested-visually unrecognizable red drum. In controlled laboratory feeding experiments, the 80 kDa protein was detected in Western blots of stomach contents after 1 and 2 h of digestion, but not after 4 h. Western blots after 2 h showed immunoreactive breakdown products from the 80 kDa protein in stomach contents. The methods with the red drum protein appear applicable to any fish for specific identification in predator gut contents by immunological procedures.