Characteristics of immune-competent amoebocytes non-invasively retrieved from populations of the sentinel earthworm Lumbricus rubellus (Annelida; Oligochaeta; Lumbricidae) inhabiting metal polluted field soils.

Lumbricus rubellus is a cosmopolitan earthworm devoid of riboflavin-storing eleocytes; its immune competent coelomocytes are predominantly amoebocytes. Our aim was to determine whether amoebocyte cytometrics in L. rubellus are robust biomarkers for innate immunological responses to environmental pollutants. Investigations were conducted on populations inhabiting three unpolluted and five metalliferous (mainly Pb+Zn+Cd) habitats in the UK and Poland. Inter-population differences in worm mass and amoebocyte numbers did not consistently reflect soil or tissue metal concentrations. Flow cytometry indicated that autofluorescence of the amoebocytes differs between cells from the unpolluted and metal-polluted worms, and pinocytosis of neutral red by amoebocytes was lower (especially at 15 versus 60 min incubation) in worms from the polluted Poland site compared with the reference population. To conclude, amoebocyte cytometrics and functionality are potentially useful for environmental diagnostics; deployment is contingent on better understanding potential confounders.

Riboflavin mobilization from eleocyte stores in the earthworm Dendrodrilus rubidus inhabiting aerially-contaminated Ni smelter soil.

A 6-week reciprocal transfer laboratory exposure experiment was conducted with two populations of the epigeic earthworm Dendrodrilus rubidus; one population inhabited a site approx. 200 m downwind of an active Ni smelter co-contaminated with Ni and Cu (3648 and 977 microg g(-1)d.w., respectively), the other inhabited uncontaminated soil. Worms transferred from unpolluted to Ni/Cu-polluted soil lost body mass (62%); they also had reduced (70%) total coelomocyte number, including autofluorescent eleocytes, and had significantly decreased (92%) riboflavin-derived fluorescence emission measured at 525 nm. Coelomocyte counts were low, and 525 nm emission was negligible in worms maintained on their native Ni/Cu soil. Earthworms and their coelomocytes were unaffected when transferred from Ni/Cu-polluted soil to unpolluted soil. In conclusion, exposing worms to stress-inducing factors, including metal pollution, alters the riboflavin status within the immune-competent cells of D. rubidus, but it requires further in vivo studies to establish whether the reduction in the fluorescence signal is predominantly due to depletion of riboflavin-containing eleocytes, or to riboflavin quenching, or to enzymatic conversion (and thus depletion) of stored riboflavin into its functional immune-potentiating flavin derivatives, FMN and FAD. The flavin budget of D. rubidus coelomocytes recovered by a reproducible extrusion procedure is a potentially useful biomarker for assessing sublethal stress in this early colonizer of disturbed soils.

Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution.

The effect of Pb + Zn on coelomocyte riboflavin content in the epigeic earthworm Dendrodrilus rubidus inhabiting three metalliferous soils and one reference soil was measured by flow cytometry and spectrofluorimetry. A reciprocal polluted<-->unpolluted worm transfer experiment (4-week exposure) was also performed. High proportions of autofluorescent eleocytes were counted in worms from all localities, but intense riboflavin-derived autofluorescence was detectable only in reference worm eleocytes. Other findings were: (i) fluorophore(s) other than riboflavin is/are responsible for eleocyte autofluorescence in residents of metalliferous soils; (ii) riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to metal-polluted soil; (iii) the riboflavin content of D. rubidus eleocytes is a promising biomarker of exposure; (iv) COII mitochondrial genotyping revealed that the reference population is genetically distinct from the three mine populations; (v) metal exposure rather than genotype is probably the main determinant of inter-population differences in eleocyte riboflavin status.