Haemostatic and immune role of cellular clotting in the sipunculan Themiste petricola.

Sipunculans, a small phylum of coelomated marine worms closely related to polychaete annelids, lack a true circulatory system. We have previously shown that the sipunculan Themiste petricola can form a cellular clot, without congealing, of cell-free coelomic fluid. The clot is formed by the aggregation of large granular leukocytes (LGLs) and may serve not only haemostatic but immune functions, since dissimilar particles may become entrapped within it. We have now evaluated the capacity of a massive clot, induced in vitro by sea water contact, to stop coelomic fluid flow. We have further studied smaller clots induced on glass-slides either with or without the presence of bacteria placed for entrapment within the clot. The fate of clotting LGLs is cell death while forming a cohesive mass, although cytoplasmic and nuclear remnants are shed from the clot. These remnants and any bacteria that avoid clot entrapment or are detached from the clot are engulfed by non-clotting cells that include small granular leukocytes (SGLs) and large hyaline amebocytes (LHAs). Both cell types can be found other than in the clot but SGLs also occur around the clot edges heavily loaded with engulfed material. The cytoskeletal arrangement of SGLs evaluated with phalloidin-rhodamine correspond to motile cells and contrast with that of clotting LGLs that form a massive network of F-actin. Thus, the complementary roles between clotting LGLs and non-clotting SGLs and LHAs act a central immune strategy of Themiste petricola to deal with body wall injury and pathogen intrusion into the coelomic cavity.