Evaluation of earthworm exposure to contaminated soil by cytometric assay of coelomocytes phagocytosis in Lumbricus terrestris (Oligochaeta)

We measured non-specific immunity of the earthworm species Lumbricus terrestris by flow cytometric evaluation of coelomocytes phagocytosis activity. In vivo and in vitro phagocytosis of fluorescent microspheres by coelomocytes were quantitated 18 h following intra coelomically injections of microspheres or in vitro incubation of phagocytes with the microspheres. Then, cytometric evaluation of cell-associated fluorescence was performed using a FACScan (Becton Dickinson). Earthworms were washed, placed in extrusion medium containing 5% ethanol. Following the contractions, the coelomocytes were collected. The cells were washed and resuspended in appropriate medium. The flow cytometric acquisitions were performed immediately for in vivo phagocytosis or after 2, 18, 24 and 48 h during in vitro kinetics. Preliminary results showed significant in vivo and in vitro phagocytic activity of microspheres by coelomocytes. Application of flow cytometry for validation of cellular and functional variables in earthworms appears to be a promising approach. Thus, our data showed that cytometric determination of phagocytic activity of coelomocytes with synthetic microspheres can be applied for validation of non-specific immune response in the earthworm species, Lumbricus terrestris.