The effects of long-term exposure to cadmium on the earthworm Eisenia Fetida (Oligochaeta) : an investigation of the development of genetic metal resistance

[摘要] ENGLISH ABSTRACT: The development of potential genetically based resistance to cadmium (Cd) after long-termexposure to this metal at a sublethal concentration, was investigated in earthworm specimensbelonging to the genus Eisenia. Adult (clitellate) earthworms from a long-term laboratory Cdexposedpopulation (> 78 generations) and from other populations having no previous historyof metal exposure were exposed to increasing concentrations (0; 2.5; 5; 10 and 20 mg/l) of Cdin the form of CdSO4. Different biomarkers and molecular markers were used to determinewhether the specimens from the long-term Cd-exposed population had acclimatized or adaptedto the metal contaminated environment.Acclimation was investigated at different physiological and biochemical levels using thefollowing three biomarkers: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideassay (MTT assay) measuring mitochondrial activity and cell viability; the single cell gelelectrophoresis assay (comet assay) measuring DNA strand breaks and the biosynthesis ofmetallothioneins (MTs) that have the function of lowering metal toxicity. Earthworms from thelong-term exposed substrate as well as specimens from populations not exposed to Cd wereused.Adaptation was investigated by looking at both allozyme polymorphism at seven enzymeencoding loci and DNA polymorphism using chosen neutral and selectable genetic markers.The markers used were cytochrome c oxydase subunit I (COI) and metallothionein-2 (mt-2)respectively. This was done amongst the same pool of populations. Additionally, the DNApolymorphism study also aimed at genetically identifying the species utilized in this studythereby determining whether these earthworms belong to the species Eisenia fetida or Eiseniaandrei.MTs biosynthesis did not generate the expected data mainly due to the fact that a suitableantibody could not be obtained (discussed in Chapter 5 section 5.3.3.). Similarly, theassessment of DNA polymorphism at the chosen selectable genetic marker mt-2 did provideinformation relevant to understanding the potential development of resistance to Cd in the long-term metal contaminated group. COI sequences generated in this study were compared toE. fetida and E. andrei COI sequences available on Genbank. Consequently, specimens used inthis study were identified as possibly belonging to the species E. andrei.Allozyme polymorphism revealed no fixed genetic differences between the long-term Cdexposed laboratory culture and the rest of the populations. All the populations departed fromHardy-Weinberg equilibrium (χ2 > 5.9; p < 0.05), and showed a low mean heterozygosity perlocus (Ho ≤ 0.21), probably due to inbreeding.Cell viability and proliferation as tested by the MTT assay revealed that coelomocytes, isolatedfrom the long-term Cd-exposed group showed the highest viability (98.42%) compared tothose from other groups (+/- 80%). Kruskal-Wallis ANOVA (H2, 225=109.7165 p < 0.001)revealed that the long-term Cd-exposed laboratory culture showed a better response to acuteexposure to Cd, thus demonstrating that these worms have developed some kind of tolerance toCd.Similarly, the comet assay showed that in the long-term Cd-exposed specimens, less DNAbreaks occurred after Cd exposure than in the unexposed groups. Of all the comet parametersassessed in this study (comet tail length, tail moment and tail DNA percentage), tail DNApercentage seemed to be more sensitive although all three parameters indicated that long-termCd-exposed specimens were more resistant than unexposed specimens as shown by the numberof single strand DNA breaks induced by exposure to higher concentrations of Cd (p < 0.001).The comet and the MTT assays indicated that the earthworms with a previous history of Cdexposure have developed increased fitness towards higher doses of Cd, compared to previouslyunexposed groups. These findings mainly proved that several mechanisms could come intoplay at the physiological and biochemical level to allow the Cd exposed population toacclimatize to its chemically stressful environment. Clear genetic support for the differencesfound between the tested populations was not obtained, but needs to be investigated furtherusing Cd selectable markers such as the mt-2 gene, in order to come to a more conclusivededuction.

[发布机构] Stellenbosch University