Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum

Coelomocytes were extruded from three earthworm species: Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Featuring a simple low-vacuum holding device, the proposed methodology allows the recovery of cells with minimum risk of contamination by faecal material. The viability of O. tyrtaeum coelomocytes was highly reproducible (average 93%), with an average yield of 0.92 x 10(6) viable cells per earthworm. Cell viability for L. terrestris and E. fetida averaged approximately 68% but the cell yields were higher (respectively 1.67 x 10(6) and 1.28 x 10(6)). Large inter-individual differences in cell yields were observed with L. terrestris. Flow cytometric analyses indicated species to species differences in cell populations. Coelomocytes from E. fetida were the smallest with approximately 57% of the total viable cells recovered being monitored between 2 and 10 microns. Large granulated cells (> or = 20 microns) were detected in fairly large proportions in L. terrestris and O. tyrtaeum (approximately 52 and approximately 96%, respectively) while they were less abundant in E. fetida (approximately 9%). Using the vital dye neutral red to assess functional integrity, average cellular uptakes were significantly higher for L. terrestris and O. tyrtaeum than for E. fetida (2.94, 2.66 and 0.64 micrograms/2 x 10(5) cells, respectively). In summary, the extrusion methodology herein described is applicable for the recovery of coelomocytes from a wide range of earthworm sizes and species. Moreover, this study strengthens the fact that extruded coelomocytes could be used for the evaluation of cell dysfunction and/or cell death following an in vitro and/or in vivo treatment.