Bacterial Clearance in the Sea Urchin , Strongylocentrotus

approved: Redacted for Privacy __ Dr. Christopher J. Bayne Characteristics of bacterial clearance were investigated in the purple sea urchin, Strongylocentrotus purpuratus (Echinodermata: Echinoidea). Primary clearance kinetics were determined for three bacteria, a marine Gram negative motile rod, a marine Gram positive nonmotile rod, and a fresh water fish pathogen, Aeromonas salmonicida. Various doses of live bacteria were injected into the perivisceral coelom of urchins, and coelomic fluid samples were removed for viable counts. Clearance of the two marine bacteria occurred in two stages. Reduction in viable counts was rapid and exponential during the first 90 min, followed by a period of slower clearance. The two bacteria exhibited similar clearance rates during the first 90 min, however the Gram positive bacteria were subsequently cleared more slowly than the Gram negative. Clearance of A. salmonicida was linear and not in two stages, although the initial rate of clearance was much lower than that of the marine bacteria. Attempts were made to determine whether a memory or inducible component exists in S. purpuratus' response to bacteria. After the kinetics of clearance of primary injections had been determines, the same urchins were challenged with a second injection of bacteria (9, 14, and 21 days after primary injections for the two marine bacteria, 19 days for A. salmonicida). The secondary clearance rates were not significantly different from the primary clearance rates for any of the three test bacteria. The cellfree coelomic fluid of S. purpuratus was tested in vitro for bacterial agglutination and bactericidal activity against the two marine bacteria. Declines in viable counts of bacteria were observed with both bacteria in coelomic fluid, while viable counts in sea water controls did not change. Neither previous injection of urchins with bacteria nor prior heating of coelomic fluids (100°C, 10 min) significantly'altered the viable counts. It is not clear from these experiments whether the declines in viable counts were due to bacteria killing or agglutination. However, when the coelomic fluid was tested, agglutination was not observed with either of the bacteria. The cellular responses of the urchins were also investigated. After injection of Gram negative bacteria, total numbers of circulating coelomocytes declined by 93% by 90 min after injection, and decreased slowly thereafter. By 24 hr, cell counts had risen, but not to preinjection values. All four coelomocyte cell types contributed to the overall decline. The proportions of coelomocyte types also changed during the 6 hr after injection. A fluorochrome dye, acridine orange, was used to observe phagocytosis and killing of bacteria. Clotting phagocytes trapped and engulfed bacteria. Within 2 hr after mixing bacteria and coelomocytes in vitro, dead bacteria were seen in some of