Hemolymph clotting in crustaceans: Implications for neuropeptide extraction from invertebrate hemolymph

The hemolymph of invertebrates contains factors that facilitate clotting as a defense mechanism for injury. However, the clotting process may impair the measurement of hormone titers by interfering with the extraction of peptides. Using hemolymph from freshwater crayfish, our results demonstrate that hemolymph clotting appears to reduce both the amount of an endogenous peptide(s) (Dippu-AST 11-like) extracted from hemolymph, as well as the amount of spiked peptide tracer ([125I]-Dippu-AST 11) recovered from hemolymph. The efficacy of peptide extraction from hemolymph was improved by collecting the hemolymph into a variety of different media prior to hemolymph coagulation. Hemolymph samples collected into media containing the anticoagulant, citrate, had the highest amount of endogenous Dippu-AST 11-like peptide extracted as well as the highest percent recovery of spiked tracer.