Source of alarm substances in crayfish and their preliminary chemical characterization

Abstract

We tested the hypothesis that hemolymph contains alarm substances in the crayfish Procambarus clarkii (Girard, 1852) and collected preliminary information on their chemical nature in this species. We analyzed crayfish responses in the presence of different test solutions in four experiments. The crayfish displayed an alerted behavior (i.e., feeding and locomotion were inhibited) in the presence of solutions containing different concentrations of hemolymph combined with food odor. However, hemolymph lost its bioactivity when tested 24 h after its extraction but maintained its ability to elicit alerted responses when diluted in a solution containing L-ascorbic acid. This may suggest that crayfish alarm molecules are degraded with time by oxidation. Microbial activity did not lead to the degradation of alarm substances, since hemolymph activity still declined after 24 h even if extracted and preserved in sterile conditions. Hemolymph molecules less than 5 kDa fractionated from hemolymph showed a strong bioactivity and were still bioactive after 24 h at 20 °C. As the 5 kDa fractioning eliminates all enzymes, we hypothesize that alarm substances are degraded through enzymatic reactions. Finally, we propose that alarm substances are peptides involved in the hemolymph clotting process.