A new ultrasound protocol for extrusion of coelomocyte cells from the earthworm Eisenia fetida

There is mounting evidence that earthworms could be used as a sentinel species for soil ecotoxicity evaluation. In this aspect, phagocytosis by coelomocytes was shown to be a sensitive biomarker of exposure to xenobiotics. In this paper, we introduce a simple method for ultrasound extrusion of earthworm coelomocytes that generates a high cell yield, does not interfere with phagocytic competence, and requires a minimum of manipulations. Coelomocytes were extruded from the earthworm Eisenia fetida using this new ultrasound method and compared with ethanol and electrical extrusion. The ultrasonic extrusion showed the highest cell recovery with 3.17 +/ -0.8 x 10(6) cells per earthworm compared with 2.22 +/- 0.8 x 10(6) cells per earthworm for electrical extrusion and 1.57 +/- 0.07 x 10(6) cells per earthworm for ethanol extrusion. No significant differences in the cell viability were observed using propidium iodide and flow cytometry with viability for extrusion with ethanol of 63.8 +/- 12.7%, electrical 76.8 +/- 7.5%, and ultrasound 68.2 +/- 7.8%. To compare the potential effect of extrusion on cell quality, the cells extruded using the three methods were subjected to an 18-h in vitro exposure to methylmercury chloride (MeHgCl; CH3HgCl) with concentrations ranging from 10(-9) to 10(-4)M. The half-maximal effective concentration (EC50) for inhibition of phagocytosis occurred between 10(-7) and 10(-6)M. We found no significant differences among the extrusion methods for the phagocytic potential of the coelomocytes. This method does not harm the worms and can certainly improve collection of coelomocytes from earthworms and therefore contribute to the development of bioassays using invertebrates.