Hemocyte aggregation in the solitary ascidian Halocynthia roretzi: plasma factors, magnesium ion, and Met-Lys-bradykinin induce the aggregation.

Hemocytes of the ascidian Halocynthia roretzi undergo aggregation in hemolymph that has been collected from the body through the tunic. To investigate the mechanisms involved, we first established two methods of measuring hemocyte aggregation. In one method, hemocyte aggregation was quantified by its reduction of light scattering intensity as measured with a fluorescence spectrophotometer. In the other method, the increase of transmittance accompanying aggregation was measured with an ELISA reader. We found that ascidian plasma, Mg2+, and Met-Lys-bradykinin can induce the hemocytes of H. roretzi to aggregate. The aggregation induced by any of these three substances was inhibited by EDTA, N-ethylmaleimide, and cytochalasin B. Lipopolysaccharide had little inducing effect. We also demonstrated that, when H. roretzi plasma was treated with trypsin, low molecular weight aggregation-inducing substances were produced. These results suggest that metal ions and peptide-like substances present in the hemolymph play essential roles in the progression of hemocyte aggregation of H. roretzi.